CN106755130A - It is a kind of to utilize the blue or green fresh method containing sugared Ethanol Production by Biomass Fermentation - Google Patents

Abstract

The invention relates to a method for producing ethanol by fermenting fresh and fresh sugar-containing biomass, which comprises adding pretreatment residue of lignocellulosic solid content of fresh and fresh sugar-containing biomass to the sugar-containing juice of fresh and fresh sugar-containing biomass, A step in which cellulase and ethanol-producing strains are fermented to obtain ethanol-rich fermented mash; wherein, the fresh sugar-containing biomass source sugar-containing juice and the green fresh sugar-containing biomass source lignocellulosic solid content are passed through the green fresh sugar-containing biomass Biomass raw materials are obtained by pressing. The method realizes the simultaneous utilization of free sugars and cellulose sugars in fresh sugar-containing plant raw materials, improves the utilization rate of biomass raw materials, and reduces the cost of raw materials for ethanol production; at the same time, the fermentation method improves the yield of fermentation products. The concentration and production efficiency of ethanol in the medium solve the problems of low ethanol concentration and production efficiency in the traditional fiber ethanol fermentation process.

Description

A method for producing ethanol by fermenting green and fresh sugar-containing biomass

technical field

The invention belongs to the technical fields of fermentation engineering and biomass energy, and in particular relates to a method for producing ethanol by fermenting green and fresh sugar-containing biomass.

Background technique

With the development of science and technology economy, the dependence of human society on fossil energy is increasing day by day. The use of fossil fuels has problems such as high consumption and high pollution. Under the dual background of energy security and environmental protection, it is particularly necessary to develop sustainable, renewable, efficient, low-cost, convenient and safe energy sources. Biomass energy can effectively replace traditional fossil fuels and realize green and renewable energy. It is recognized as the only solution that can replace liquid fuels. Biofuel ethanol can reduce the consumption of gasoline and make gasoline burn more fully, thereby reducing the emission of harmful gases.

Using agricultural waste or energy crops grown on marginal land as raw materials to produce fuel ethanol has become an important development direction of new energy strategies in many countries, and its industrialization is of great significance to the sustainable development of society. At present, due to the limitation of fermentation technology, when the sugar in the fresh biomass raw material is used for ethanol production, the utilization rate of the solid biomass residue of fresh straw is low, resulting in waste of resources, making ethanol production uneconomical. In addition, the sugar in fresh straw resources needs to be concentrated and preserved, which further increases the cost of ethanol raw materials.

Compared with the direct production of ethanol from the sugar in fresh plants, the fermentation of cellulosic ethanol using lignocellulosic plants as raw materials is more difficult. Since lignin and hemicellulose in lignocellulosic raw materials tightly wrap cellulose to form a complex structure, it is necessary to pretreat lignocellulose to change the natural structure of cellulose and reduce the crystallinity and polymerization degree of cellulose. This is beneficial to the combination and hydrolysis of cellulase and substrate.

Simultaneous saccharification fermentation realizes the combination of fiber hydrolysis and ethanol fermentation. The glucose concentration in the enzymatic solution is always kept in a low range, which will not cause feedback inhibition on cellulase, which improves the saccharification efficiency and reduces the production of cellulase. The dosage and the required reaction equipment are also relatively reduced. But the fiber solid residue has water absorption, and the excessively high feeding amount makes the fermentation substrate viscosity increase, and the mass transfer of the fermentation system is hindered. Generally speaking, the charging amount of the lignocellulosic solid raw material is less than 20% in the batch fermentation process, which also As a result, the ethanol concentration of the fermentation product is low, therefore, it is difficult to achieve high fiber conversion rate and high ethanol product concentration at the same time. In addition, the toxic substances that appear during lignocellulose pretreatment will have adverse effects on microbial fermentation and cellulase activity.

At present, the high cost of raw materials for ethanol production, low concentration of fermentation products, low ethanol production efficiency, and high separation costs make lignocellulosic ethanol technology difficult to commercially apply.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for producing ethanol by fermenting green and fresh sugar-containing biomass in view of the deficiencies in the prior art. Biomass raw materials reduce the cost of raw materials for production, and at the same time solve the problems of low ethanol yield, low concentration and high separation cost in the traditional synchronous saccharification and ethanol fermentation process.

For this reason, the present invention provides a kind of method that utilizes green and fresh sugar-containing biomass to ferment and produce ethanol, and it comprises adding the fresh and fresh sugar-containing biomass source lignocellulosic solid content to fresh and fresh sugar-containing biomass source sugar-containing juice The step of degrading treatment residues, cellulase and ethanol-producing strains for fermentation to obtain ethanol-rich fermented mash; wherein, the sugar-containing juice of fresh and fresh sugar-containing biomass sources and the solid content of lignocellulosic fibers of fresh and fresh sugar-containing biomass sources are both It is obtained by pressing fresh and fresh sugar-containing biomass raw materials.

According to the present invention, the fresh and fresh sugar-containing biomass raw material contains sugar-containing juice and lignocellulosic solids.

In some embodiments of the invention, the lignocellulosic solids include cellulose and/or hemicellulose.

In the present invention, the fresh and fresh sugar-containing biomass raw materials include one or more of fresh and fresh corn stalks, fresh and fresh sorghum stalks, fresh and fresh sugarcane stalks and fresh and fresh sugar beet tubers with water contents above 40 wt%.

According to the present invention, the degradation treatment residue of the lignocellulosic solid content of the fresh sugar-containing biomass source is added in the form of mixing with the sugar-containing juice of the fresh sugar-containing biomass source, or added to the sugar-containing juice of the fresh sugar-containing biomass source. The sugar in the juice is fed in batches or continuously before it is completely degraded by fermentation.

In some preferred embodiments of the present invention, the degradation treatment residue of the lignocellulosic solid content of the fresh sugar-containing biomass source is degraded in batches before the sugar in the sugar-containing juice of the fresh sugar-containing biomass source is completely degraded by fermentation. Added as supplementary feed.

In the present invention, the ethanol-producing strain is one or more of bacteria that can metabolize ethanol, fungi that can metabolize ethanol, and genetically engineered bacteria that can metabolize ethanol; preferably the ethanol-producing strain is one that can metabolize ethanol. Ethanol fungi.

In the present invention, the cellulase includes one or more of endoglucanase, exoglucanase, β-glucosidase and xylanase.

In the present invention, the total sugar content in the sugar-containing juice of the fresh sugar-containing biomass source is less than 300 g/L.

In some embodiments of the present invention, the ethanol-producing strain is added in the form of seed liquid, or in the form of active dry bacteria powder;

When the ethanol-producing strain is added in the form of seed liquid, the added amount of the seed liquid is 1v%-20v% based on the total volume of the fresh sugar-containing biomass mixture, preferably the added amount of the seed liquid is green The total volume of the fresh sugar-containing biomass mixture is 5v%-10v%;

When the ethanol-producing bacterial strain is added in the form of active dry bacterial powder, the added amount of the active dry bacterial powder is 0.005w%-0.1w% based on the total weight of the green and fresh sugar-containing biomass mixture.

In some embodiments of the present invention, the added amount of the cellulase is 1-60 FPU/g based on the total weight of the degradation treatment residue of the fresh sugar-containing biomass source lignocellulosic solid content; The addition amount is 5-40 FPU/g based on the total weight of the degradation treatment residue of the fresh sugar-containing biomass source lignocellulosic solid content, and the addition amount of cellulase is preferably based on the green fresh sugar-containing biomass source lignocellulosic solid content The total weight of the degradation treatment residue of the product is 5-20FPU/g.

According to the present invention, the fresh and fresh sugar-containing biomass source lignocellulosic solids are degraded in the form of powder.

In some embodiments of the present invention, the particle size of the powder is >30 mesh; preferably, the particle size of the powder is >60 mesh.

In the present invention, the method of degradation treatment is one or more of alkali method, acid method, hydrothermal method, organic solvent method, ionic liquid method, steam explosion method, ultrasonic method, ball milling method, high pressure homogenization method and biological method One, the method of preferred degradation treatment is acid method and/or alkali method.

In some embodiments of the present invention, the fermentation temperature is 20-60°C, preferably the fermentation temperature is 30-50°C, and more preferably the fermentation temperature is 35-45°C.

Description of drawings

The present invention will be described below in conjunction with the accompanying drawings.

Fig. 1 is the specific operation flowchart of the method of the present invention

detailed description

In order to make the present invention easy to understand, the present invention will be described in detail below.

As mentioned above, the high cost of raw materials for ethanol production, the low production efficiency of ethanol in the traditional simultaneous saccharification and fermentation ethanol process, and the low concentration of ethanol in the fermentation mash make lignocellulosic ethanol production technology difficult for commercial application. The inventors have found that the sugar-containing juice and the fiber raw material in the fresh sugar-containing biomass are not simultaneously utilized in the existing technology due to the limitation of the fermentation technology, resulting in a low utilization rate of the raw material and making the raw material cost of ethanol relatively low. High, low production efficiency, low ethanol concentration in fermentation mash.

The inventors have further studied and found that the semi-synchronous saccharification and fermentation method, that is, first saccharifying a part of the lignocellulosic substrate into the liquid phase, and then inserting the fermentation strain to continue the synchronous saccharification and fermentation, can have the advantages of both free and synchronous saccharification and fermentation , to solve the problem of insufficient fermented sugar in the initial stage of fermentation in the traditional synchronous saccharification ethanol production process, and obtain higher ethanol yield. Correspondingly, by pre-adding a certain amount of free sugar in the initial fermentation medium, combined with the conventional simultaneous saccharification and fermentation of fiber, the fermentation effect similar to that of the semi-synchronous saccharification method can also be obtained. For the first time, the inventors used green and fresh sugar-containing biomass as raw material to carry out the fermentation production of ethanol. The present invention is made based on the above method.

Therefore, the method for producing ethanol by fermentation of green and fresh sugar-containing biomass involved in the present invention can be understood as the application of fresh and fresh sugar-containing biomass in the fermentation and production of ethanol. The specific operation flow chart of the method is shown in Figure 1. It includes adding the degradation treatment residue of the lignocellulosic solid content of the green and fresh sugar-containing biomass source to the sugar-containing juice of the fresh sugar-containing biomass source, cellulase and ethanol-producing strains to ferment to obtain the fermented mash rich in ethanol Step; wherein, the fresh sugar-containing biomass source sugar-containing juice and the green fresh sugar-containing biomass source lignocellulosic solid content are both obtained by pressing the green fresh sugar-containing biomass raw material.

In some embodiments, the concentration of ethanol in the ethanol-rich fermented mash is 68.83-98.1 g/L.

In some embodiments, the fermentation temperature is 20-60°C, preferably the fermentation temperature is 30-50°C, more preferably the fermentation temperature is 35-45°C.

In some preferred embodiments, after mixing the fresh fresh sugar-containing biomass source sugar juice with the green fresh sugar-containing biomass source lignocellulosic solid content, add urea, potassium dihydrogen phosphate and anhydrous magnesium sulfate before use Ethanol fermentation.

According to the present invention, the ethanol-producing bacterial strain is added in the form of seed liquid, or in the form of active dry bacteria powder;

In some embodiments of the present invention, the ethanol-producing bacterial strain is added in the form of seed liquid, and the amount of the seed liquid added is 1v%-20v% based on the total volume of the green and fresh sugar-containing biomass mixture; in the present invention In some preferred embodiments, the amount of the seed liquid added is 5v%-10v% based on the total volume of the green and fresh sugar-containing biomass mixture.

In other embodiments of the present invention, when the ethanol-producing bacterial strain is added in the form of active dry bacterial powder, the added amount of the active dry bacterial powder is 0.005w% based on the total weight of the green and fresh sugar-containing biomass mixture -0.1w%.

In the present invention, the ethanol-producing strain is one or more of bacteria capable of metabolizing ethanol, fungi capable of metabolizing ethanol, and genetically engineered bacteria capable of metabolizing ethanol.

In some embodiments, the ethanologenic strain is Saccharomyces cerevisiae H5 strain.

In some embodiments, the added amount of the cellulase is 1-60 FPU/g based on the total weight of the degradation treatment residue of the fresh sugar-containing biomass source lignocellulosic solid content; preferably the added amount of the cellulase is The total weight of the degradation treatment residue of the green and fresh sugar-containing biomass source lignocellulosic solid content is 5-40FPU/g, and the addition of cellulase is preferably based on the degradation of the fresh and fresh sugar-containing biomass source lignocellulosic solid content The total weight of the treatment residue is 5-20 FPU/g.

It should be understood that the time for adding the cellulase is when the residual sugar concentration in the fermentation broth is lower than 5 g/L.

In the present invention, the cellulase includes one or more of endoglucanase, exoglucanase, β-glucosidase and xylanase.

There is no particular limitation on the source of cellulase in the present invention, for example, cellulase can be commercially available or have the microorganism that produces cellulase ability; Preferably said microorganism includes Trichoderma reesei, Penicillium decumbens and One or more of Saccharomyces cerevisiae modified by protein surface display technology.

In the present invention, the fresh sugar-containing biomass raw material contains sugar-containing juice and wood fiber solid content. In some embodiments, for example, the fresh sugar-containing biomass raw material includes one or more of fresh corn stalks, fresh sorghum stalks, fresh sugarcane stalks and fresh beet tubers with a water content above 40 wt%. Various.

In the present invention, the sugar-containing juice of fresh and fresh sugar-containing biomass sources can be directly used for fermentation, or can be fermented after being diluted or concentrated, but the sugar-containing juice of fresh and fresh sugar-containing biomass sources used for fermentation The total sugar content of <300g/L.

In the present invention, the lignocellulosic solid content includes cellulose and/or hemicellulose. In some embodiments, the degradation treatment residue of the fresh sugar-containing biomass source lignocellulosic solids is added in the form of mixing with the fresh sugar-containing biomass source sugar-containing juice, or in batches or batches before the fermentation is terminated. The method of continuous feeding is added. Preferably, the degradation treatment residue of the fresh and fresh sugar-containing biomass source lignocellulosic solid content is added in the form of fed batches before the fermentation is terminated.

In the present invention, the green and fresh sugar-containing biomass source lignocellulosic solids are degraded in the form of powder. In some embodiments, for example, the particle size of the powder is >30 mesh; preferably, the particle size of the powder is >60 mesh.

In the present invention, the method of degradation treatment is one or more of alkali method, acid method, hydrothermal method, organic solvent method, ionic liquid method, steam explosion method, ultrasonic method, ball milling method, high pressure homogenization method and biological method One, the method of preferred degradation treatment is acid method and/or alkali method.

According to some embodiments of the present invention, the method further includes a step of separating and purifying the ethanol-rich fermented mash to obtain an ethanol product.

In some embodiments of the present invention, the separation and purification method includes one or more of ordinary distillation, vacuum rectification, liquid-liquid extraction, pervaporation, steam permeation, steam stripping, vacuum extraction and adsorption .

According to the present invention, the separation and purification are carried out after the anaerobic fermentation is terminated, or in-situ separation is carried out coupled with the anaerobic fermentation process.

In some specific embodiments of the present invention, the method for producing ethanol by fermentation of fresh sugar-containing biomass comprises the following steps:

(1) Raw material squeezing: squeeze fresh and fresh sugar-containing biomass raw materials to realize solid-liquid separation of fresh and fresh sugar-containing biomass raw materials, and obtain fresh and fresh sugar-containing biomass source sugar-containing juice and fresh and fresh sugar-containing biomass source woody fiber solids;

(2) Treatment of fiber residues: dry and pulverize the fresh and fresh sugar-containing biomass source lignocellulosic fibers, and then use a pretreatment method to destroy the structure of fiber residues to obtain fresh and fresh sugar-containing biomass source lignocellulosic solids. Degradation treatment residues of substances, in order to achieve the purpose of effective enzymatic hydrolysis;

(3) Preparation of seed liquid: amplifying and culturing ethanol-producing strains in synthetic medium and/or natural medium to obtain seed liquid for fermentation;

(4) Production of ethanol by fermentation: mix the sugar-containing juice of the fresh and fresh sugar-containing biomass source with the degradation treatment residue of the lignocellulosic solid content of the green and fresh sugar-containing biomass source, add cellulase, and insert the seed liquid of the ethanol-producing strain Fermentation is carried out to obtain a fermented mash rich in ethanol;

In some preferred embodiments, after mixing the fresh fresh sugar-containing biomass source sugar juice with the green fresh sugar-containing biomass source lignocellulosic solid content, add urea, potassium dihydrogen phosphate and anhydrous magnesium sulfate before use Ethanol fermentation.

(5) Separation and purification of ethanol: Separation and purification of fermented mash rich in ethanol to obtain ethanol products. The separation and purification methods include one or more of ordinary distillation, vacuum rectification, liquid-liquid extraction, pervaporation, steam permeation, steam stripping, vacuum extraction and adsorption. The separation and purification can be performed after the anaerobic fermentation is terminated, or coupled with the anaerobic fermentation process for in-situ separation.

The term "lignocellulosic solids" in the present invention is also referred to as "fibrous residue".

The beneficial effects of the present invention are:

(1) The method realizes simultaneous utilization of juice free sugars and cellulose sugars in green and fresh sugar-containing plant raw materials, improves the utilization rate of biomass raw materials, and reduces the cost of raw materials for ethanol production.

(2) Adopting the above-mentioned fermentation method, due to the addition of the sugar-containing juice of the fresh and fresh sugar-containing biomass source at the initial stage of fermentation, it is helpful to obtain a higher cell proliferation rate and ethanol yield in the early stage of ethanol fermentation. The addition of sugar-containing juice from sugar biomass sources can effectively increase the concentration of substrate carbon source in the fermentation process, so that the concentration of ethanol in the fermentation product can be further increased, which solves the problem of low production efficiency of traditional synchronous saccharification ethanol fermentation and low ethanol concentration in mash The problem.

Example

In order to make the present invention easier to understand, the present invention will be further described in detail below in conjunction with examples, and these examples are for illustrative purposes only, and do not limit the scope of application of the present invention. The raw materials or components used in the present invention can be prepared by commercial channels or conventional methods unless otherwise specified.

In following embodiment, the detection method of main process parameter and index is as follows:

(1) HPLC method for measuring sugar: use high-performance liquid chromatography (HPLC) to separate and then analyze sugar using a sugar analysis column. After pH adjustment of the sample to be analyzed, 1.00 g was taken, centrifuged at 8000 rpm for 10 minutes, and the supernatant was diluted 100 times for liquid phase analysis. The sugar analysis column used was Aminex87P analysis column from Bio Rad Company, and the liquid phase analysis conditions were: flow rate 0.6mL/min, column temperature 65°C, sample volume 20μL; standard retention time was used for qualitative analysis; standard curve external standard was used method for quantitative analysis.

(2) Composition analysis of degradation treatment fiber residue: first extract the sample to be analyzed with ethanol Soxhlet extraction to remove resin, pigment, etc., then use 72% concentrated sulfuric acid and 4% dilute sulfuric acid to hydrolyze the sample to be analyzed in two steps to make The components in lignocellulose are hydrolyzed into readily quantifiable substances. Among them, cellulose and hemicellulose are hydrolyzed to form monosaccharides, which are quantitatively analyzed by high performance liquid chromatography, while lignin is quantitatively analyzed by burning method.

(3) Determination of ethanol concentration by gas chromatography: the analytical column used is cbp-20 chromatographic column. The analysis conditions are: column temperature 180°C, sample injection temperature 90°C, N ₂ as carrier, H ₂ as combustion gas, detector as FID, sample volume 1.0 μL; standard retention time is used for qualitative analysis; Standard method for quantitative analysis.

(4) The cellulase compound solution described in the examples was provided by Shandong Kangdien Biotechnology Co., Ltd. The method for measuring the enzyme activity of the test paper is as follows: take an appropriate diluted enzyme solution, hydrolyze Whatman No. 1 filter paper at a constant temperature of 50°C for 60 minutes, and define the enzyme amount of 1 ug of reducing sugar produced by degrading the filter paper per minute, which is an FPU enzyme activity; among them, the measuring method of reducing sugar Use DNS colorimetric method or HPLC method.

Example 1: Ethanol was produced by fermenting green and fresh sweet sorghum stalks as raw materials.

(1) Pressing of fresh and fresh sweet sorghum stalks: take 1 kg of fresh and sweet sorghum stalks grown in the field, and after peeling, obtain about 654 ml of sugar-containing juice and fiber residue after repeated pressing three times; Contains about 144g/L of free sugar. In addition, as measured by ICP-MS and Kjeldahl method, the contents of inorganic ions and nitrogen and phosphorus contained in the sugary juice are shown in Table 1:

Table 1 Inorganic ions and nitrogen and phosphorus contents in sugary juice from fresh and fresh sweet sorghum straw

element Content (mg/L) K 262.76 Na 17.6 Ca 143.86 Mg 263.45 Fe 2.07 mn 0.34 P 128.69 N 1480

(2) Degradation treatment of fiber residues: Dry the remaining about 294g fiber residues at 105°C overnight to dryness to obtain 216g of absolute-dry fiber residues; use a pulverizer to crush the fiber residues to 50 mesh, and then dry the absolute-dry fiber residues Add the solid-liquid ratio of 1:10 into 2% NaOH solution for soaking, keep warm at 121°C for 80 minutes, and then realize solid-liquid separation by filtration under reduced pressure; take the filter residue and wash it with tap water until the pH is neutral. Dry overnight at 105°C to absolute dryness to obtain fiber residue after alkali degradation treatment, the main components of which are shown in Table 2

Table 2 Composition of fiber residues of green and fresh sweet sorghum straw after alkaline degradation treatment

Element Percentage (%) Glucoside 54.03 Xyloside 21.54 Arabinoside 0.43 Galactoside 0.13 Mannoside 0.05 Klason lignin 4.33 Ash 0.08

(3) Preparation of fermented liquid: 118.5 g of fiber residue after alkaline degradation treatment is crushed to below 60 mesh, fully mixed with the sugar-containing juice prepared in step (1), placed in a fermenter, without adding Under the condition of other exogenous nutrients, it was sterilized at 121° C. for 25 minutes to obtain a fermentation broth for later use.

(4) Preparation of seed liquid: the ethanol-producing strain is the high-temperature resistant Saccharomyces cerevisiae H5 screened by evolutionary engineering in the laboratory. The composition of the strain seed synthetic medium was (g/L): 0.8 sucrose, 20 glucose, 5 soybean peptone, 3 yeast powder, tap water, and the pH was adjusted to 5.5 with 1 mol/L HCl. The strain was inoculated into the seed synthetic medium, and the shake flask was cultured at 35° C. and 80 rpm for 20 h to obtain the seed liquid.

(5) Production of ethanol by fermentation: insert the seed liquid into the fermentation liquid with a 10% inoculation amount, and at the same time, add 20 FPU/g of compound cellulase liquid for degrading and processing fiber residue; Fermentation 72h, after fermentation finishes, get fermentation mash and centrifuge 10 minutes under 10000rpm; Get the supernatant after centrifugation and measure the residual sugar in the supernatant with HPLC method, measure the ethanol output in the supernatant with gas chromatography, The measurement results are shown in Table 3.

Table 3 Results of ethanol fermentation of fresh and sweet sorghum stalks

Test items Fermentation result Residual sugar concentration (g/L) 0.3 (for the total concentration of fructose, glucose, and sucrose, excluding xylose) Ethanol concentration (g/L) 88.4 Ethanol yield (g/L h) 1.23 Conversion rate of fresh sweet sorghum straw to ethanol (g/kg) 57.5

Example 2: Production of ethanol by fermenting green and fresh sweet sorghum stalks as raw materials.

Steps (1), (2) are the same as in Example 1.

(3) Preparation of fermented liquid: 118.5 g of fiber residue after alkali degradation treatment is pulverized to 60 meshes, divided into 4 parts, and one part is fully mixed with the sugary juice prepared in step (1), and placed In addition, 1.7g/L urea, 1g/L potassium dihydrogen phosphate, and 2g/L anhydrous magnesium sulfate were added to the fermenter, and sterilized at 121°C for 25min to obtain a fermented liquid for later use.

(4) Preparation of seed liquid: the ethanol-producing strain is the high-temperature resistant Saccharomyces cerevisiae H5 screened by evolutionary engineering in the laboratory. The composition of the strain seed synthetic medium was (g/L): 0.8 sucrose, 20 glucose, 5 soybean peptone, 3 yeast powder, tap water, and the pH was adjusted to 5.5 with 1 mol/L HCl. The strain was inoculated into the seed synthetic medium, and the shake flask was cultured at 37° C. and 200 rpm for 20 h to obtain the seed liquid.

(5) Production of ethanol by fermentation: insert the seed liquid into the fermentation liquid with an inoculation amount of 5%, and at the same time, add 25 FPU/g of compound cellulase liquid for degrading and processing fiber residue; Carry out ethanol fermentation, when fermenting to 12, 24, 36h, add successively other 3 aliquots of alkali-treated fiber residues packed in step (3), and degrade the enzyme addition amount of fiber residues with 25FPU/g, Add the corresponding compound cellulase liquid, until 72h fermentation is terminated, take the fermentation liquid and centrifuge at 10000rpm for 10 minutes; take the centrifuged supernatant and measure the residual sugar in the liquid by HPLC, and measure the ethanol in the liquid by gas chromatography Yield, measurement result is as shown in table 4:

Table 4 Results of Ethanol Fermentation of Green and Fresh Sweet Sorghum Straw

Example 3: Fermentation of fresh corn stalks as raw material to produce ethanol.

(1) Pressing of fresh corn stalks: take 1 kg of fresh corn stalks grown in the field and have harvested mature corn kernels, after peeling, obtain about 574ml of sugary juice and fiber residue after three repeated pressings; determined by HPLC method, the Sugary juice contains about 97g/L of free sugar. In addition, as measured by ICP-MS and Kjeldahl method, the contents of inorganic ions and nitrogen and phosphorus contained in the sugary juice are shown in Table 5:

Table 5 Inorganic ions and nitrogen and phosphorus contents in fresh corn stalk sugary juice

element Content (mg/L) K 362.48 Na 11.04 Ca 238.68 Mg 468.10 Fe 3.30 mn 1.14 P 154.86 N 1128.20

(2) Degradation treatment of fiber residue: dry the remaining about 380g fiber residue at 105°C overnight to dryness to obtain 256g of absolute dry fiber residue; crush the absolute dry fiber residue to 40 mesh, with a solid-to-liquid ratio of 1: Add the ratio of 10 to 2% dilute sulfuric acid for soaking, keep it warm at 121°C for 60 minutes, and then realize solid-liquid separation by vacuum filtration; take the filter residue and wash it with tap water until the pH is neutral, and dry it at 105°C overnight to absolute Dry to obtain fiber residue after acid degradation treatment, the main components of which are shown in Table 6.

Table 6 Composition of green and fresh corn stalk fiber residue after acid degradation treatment

(3) Preparation of fermented liquid: crush 256g of the fiber residue after acid degradation treatment to below 80 mesh, and take 57.4g of it with the sugar-containing juice prepared in step (1) according to the ratio of solid-to-liquid ratio of 1:10 Mix well and put into the fermenter; in addition, add 2g/L urea, 1.5g/L potassium dihydrogen phosphate, 1g/L anhydrous magnesium sulfate to the fermenter, and sterilize at 121°C for 25 minutes to obtain fermentation liquid for use.

(4) Preparation of seed liquid: the ethanol-producing strain is the high-temperature resistant Saccharomyces cerevisiae H5 screened by evolutionary engineering in the laboratory. The composition of the seed synthetic medium of the strain is (g/L): 0.8 sucrose, 20 glucose, 5 soybean peptone, 3 yeast powder, tap water, and adjust the pH to 5.5 with 1 mol/L HCl. The strain was inoculated into the seed synthetic medium, and the shake flask was cultured at 35° C. and 200 rpm for 20 h to obtain the seed solution.

(5) Production of ethanol by fermentation: insert the seed liquid into the fermentation broth with an inoculum amount of 10%, and carry out ethanol fermentation at a rotating speed of 200rpm and 35°C. During the fermentation process, the residual sugar in the fermentation broth is monitored in real time. When the total residual sugar concentration of the solution is lower than 5g/L, add 15FPU/g compound cellulase solution for degrading and treating fiber residue, and continue fermentation until 72h fermentation is terminated, take the fermentation solution and centrifuge at 10000rpm for 10 minutes; take the centrifuged Supernatant liquid is measured the residual sugar in the liquid with HPLC method, measures the ethanol output in the liquid with gas chromatography, and measurement result is as shown in table 7:

Table 7 Results of ethanol fermentation of fresh corn stalks

Test items Fermentation result Residual sugar concentration (g/L) 0.2 (for the total concentration of fructose, glucose, and sucrose, excluding xylose) Ethanol concentration (g/L) 68.83 Ethanol yield (g/L h) 0.96

It should be noted that the above-mentioned embodiments are only used to explain the present invention, and do not constitute any limitation to the present invention. The invention has been described with reference to typical embodiments, but the words which have been used therein are words of description and explanation rather than words of limitation. The present invention can be modified within the scope of the claims of the present invention as prescribed, and the present invention can be revised without departing from the scope and spirit of the present invention. Although the invention described therein refers to specific methods, materials and examples, it is not intended that the invention be limited to the specific examples disclosed therein, but rather, the invention extends to all other methods and applications having the same function.

Claims (10)

1. a kind of using the blue or green fresh method containing sugared Ethanol Production by Biomass Fermentation, it includes containing syrup containing sugared biomass sources to green grass or young crops is fresh The blue or green fresh degradation treatment residue containing sugared biomass sources wood fibre Solid content, cellulase and producing and ethanol bacterial strain are added in liquid to be carried out The step of fermentation obtains the fermentation liquid rich in ethanol；Wherein, it is blue or green fresh containing sugared biomass sources liquid containing syrup and blue or green fresh containing sugared biological Matter source wood fibre Solid content is obtained by blue or green fresh squeezing containing sugared biomass material.

2. method according to claim 1, it is characterised in that the green grass or young crops it is fresh containing sugared biomass material contain liquid containing syrup and Wood fibre Solid content；It is preferred that the wood fibre Solid content includes cellulose and/or hemicellulose；It is preferred that the green grass or young crops is fresh containing sugar Biomass material includes blue or green stalk of fresh corn of the water content in more than 40wt%, blue or green fresh broomcorn straw, blue or green fresh sugarcane bar and green grass or young crops One or more in fresh and sweet dish stem tuber.

3. method according to claim 1 and 2, it is characterised in that the green grass or young crops is fresh to be contained admittedly containing sugared biomass sources wood fibre The degradation treatment residue of thing is added in the way of mixing containing sugared biomass sources liquid containing syrup with green grass or young crops is fresh, or fresh containing sugared biomass in green grass or young crops Sugar in the liquid containing syrup of source is added before being fermented degraded completely in the way of fed-batch or continuous feeding；It is preferred that the green grass or young crops is fresh Degradation treatment residue containing sugared biomass sources wood fibre Solid content is sent out in the fresh sugar containing in sugared biomass sources liquid containing syrup of green grass or young crops Added in the way of fed-batch before ferment degraded completely.

4. the method according to claim any one of 1-3, it is characterised in that the producing and ethanol bacterial strain is can be metabolized generation The bacterium of ethanol, the one kind or many that the fungi of generation ethanol can be metabolized and can be metabolized in the genetic engineering bacterium for producing ethanol Kind；It is preferred that the producing and ethanol bacterial strain is that can be metabolized the fungi for producing ethanol.

5. the method according to claim any one of 1-4, it is characterised in that the cellulase includes endo-glucanase glycosides One or more in enzyme, exoglucanase, beta-glucosidase and zytase.

6. the method according to claim any one of 1-5, it is characterised in that the green grass or young crops is fresh containing sugared biomass sources liquid containing syrup In total sugar content ＜ 300g/L.

7. the method according to claim any one of 1-6, it is characterised in that the producing and ethanol bacterial strain is in the form of seed liquor Add, or added in the form of active dry bacterium powder；

When the producing and ethanol bacterial strain in the form of seed liquor when to add, the addition of the seed liquor is fresh mixed containing sugared biomass with green grass or young crops The total volume meter of compound is 1v%-20v%, and the addition of preferably described seed liquor is with the fresh totality containing sugared biomass mixture of green grass or young crops Product is calculated as 5v%-10v%；

When the producing and ethanol bacterial strain in the form of active dry bacterium powder when to add, the addition of the active dry bacterium powder is fresh containing sugar with green grass or young crops The gross weight meter of biomass mixture is 0.005w%-0.1w%.

8. the method according to claim any one of 1-7, it is characterised in that the addition of the cellulase contains so that green grass or young crops is fresh The gross weight meter of the degradation treatment residue of sugared biomass sources wood fibre Solid content is 1-60FPU/g；Preferred cellulose enzyme plus It is 5-40FPU/g to enter amount with the gross weight meter of the fresh degradation treatment residue containing sugared biomass sources wood fibre Solid content of green grass or young crops, enters one The addition of preferred cellulose enzyme is walked with the gross weight of the fresh degradation treatment residue containing sugared biomass sources wood fibre Solid content of green grass or young crops It is calculated as 5-20FPU/g.

9. the method according to claim any one of 1-8, it is characterised in that the green grass or young crops is fresh containing sugared biomass sources wood fibre Solid content carries out degradation treatment in the way of powder；It is preferred that the mesh of particle diameter ＞ 30 of the powder；The further preferred powder Particle diameter>60 mesh；It is preferred that the method for degradation treatment is alkaline process, acid system, hydro-thermal method, organic solvent method, ionic liquid, steam blasting One or more in method, ultrasonic method, ball-milling method, high-pressure homogenization and bioanalysis, the method for further preferred degradation treatment is Acid system and/or alkaline process.

10. the method according to claim any one of 1-9, it is characterised in that the temperature of the fermentation is 20-60 DEG C, excellent The temperature of ferment is published for 30-50 DEG C, further preferably the temperature of fermentation is 35-45 DEG C.