CN105463031A - Method for cooperatively producing ethyl alcohol and methane through energy grass - Google Patents

Abstract

The invention relates to an ethyl alcohol and methane cooperative production method, and particularly discloses a method for cooperatively producing ethyl alcohol and methane through energy grass. The energy grass which is subjected to steam explosion is used as a raw material, a product obtained after enzymolysis of cellulase and beta-glucosidase is used as a substrate, batch type supplementary material high-substrate-concentration simultaneous saccharification and fermentation and alcoholic fermentation all-residue anaerobic digestion treatment are integrated for cooperative production of clean energy ethyl alcohol and methane, the main purpose is that the cellulosic ethanol conversion rate is improved as far as possible, and all ingredients, capable of being utilized by anaerobic digestion flora, in alcoholic fermentation residues are further converted and utilized, so that the energy grass holocellulos conversion rate is improved, meanwhile, multi-stage utilization of all components of the energy grass is achieved, and an effective solution is provided for solving the raw material utilization rate bottleneck problem produced when existing lignocellulose raw materials are used for producing clean energy.

Description

A method for co-producing ethanol and methane using energy grass

technical field

The invention relates to a method for the co-production of ethanol and methane, in particular to a method for co-producing ethanol and methane by using energy grass.

Background technique

Using lignocellulose as raw material and using modern biotechnology to develop bio-alternative energy has become an important part of the energy strategy of developed countries in the world today. With the gradual deepening of research on the production of clean energy from lignocellulosic raw materials, several sets of pilot production lines and demonstration plants have been built or are under construction at home and abroad, and the development of cellulosic fuel ethanol is entering the initial stage of industrialization. Among them, lignocellulosic raw materials represented by energy grasses have become more advantageous substrates for the production of renewable clean energy due to their rich cellulose, wide adaptability, strong stress resistance, fast growth, and high yield.

Energy grasses are perennial tall herbaceous plants or semi-shrubs, mostly drought-tolerant, salt-alkali-resistant, barren-resistant, and adaptable grass species, including tall herbaceous plants such as switchgrass and miscanthus crops. At present, researchers at home and abroad have conducted a lot of research on the production of clean energy ethanol from energy grasses. Different energy grasses are pretreated by silage, acid-base, steam explosion, etc., and their ethanol fermentation yields are also different. In 2015, Sun. et al. used Prairie cordgrass as a substrate, and carried out 20% (w/w) high substrate synchronous saccharification and ethanol fermentation after acid treatment, and finally obtained an ethanol yield of 205.0-275.6 g·kg-1 Viola et al. increased the cellulose conversion rate of simultaneous saccharification and fermentation of eelgrass (Eelgrass) to 90.3% through steam explosion pretreatment, and correspondingly obtained an ethanol yield of 243g·kg-1. However, although Gallego et al. treated Kinggrass (Kinggrass) with alkali and carried out step-by-step saccharification and fermentation with a substrate concentration of 10% and simultaneous saccharification and fermentation, the final ethanol yield was only 8.7-10.4 g·L-1. Similarly, the ethanol yield is only 45% when the silage method is used to carry out the step-by-step saccharification and fermentation of broomcorn millet (Panicummiliaceum L.) with a substrate concentration of 23%. However, the above-mentioned prior art fermentation ethanol production concentration is low, energy consumption is high, and it is not suitable for scale-up, and in order to carry out large-scale industrial production, there is an urgent need for a batch-type fed-batch high-substrate concentration synchronous saccharification using energy grass A process for the production of ethanol by fermentation. However, because the main components of energy grass are cellulose, hemicellulose, and lignin, the structure is hard, and the cellulose content and raw material characteristics of different energy grasses are different. As a result, not all pretreated energy grasses can be directly applied to batch feeding. High substrate concentration simultaneous saccharification and fermentation.

In addition, for the whole fermentation residue after the energy grass is fermented to produce ethanol, there is no good treatment method in the prior art, which can make better use of the available components in the residue and maximize the conversion of lignocellulose Feedstock production gets clean energy.

For example, the Chinese patent application with the publication number CN103102036A discloses a treatment method for wastewater from cellulosic ethanol production. This method is suitable for wastewater treatment after ethanol fermentation is over, and cannot increase the yield of fiber-based ethanol and cellulose conversion by this method. Rate.

For another example, the Chinese patent application with the publication number CN101914576A discloses a strategy for the co-production of ethanol and methane with papermaking sludge and monosodium glutamate waste liquid mixture as a substrate. The raw material papermaking sludge is mainly composed of fine fiber organic matter, and its content can be Up to 70-90%, can be easily degraded. Even if the energy grass raw material is pretreated by steam explosion, the cellulose content range is only 35-50%, which is not easy for enzymatic hydrolysis and industrial production of ethanol.

Contents of the invention

In order to solve the problems existing in the prior art, the object of the present invention is to provide a method for co-producing ethanol and methane by using energy grass to maximize the production of clean energy from lignocellulosic raw materials.

In order to realize the object of the invention, the technical scheme of the present invention is as follows:

A method for co-producing ethanol and methane using energy grass, using steam-exploded energy grass as raw material, after enzymatic hydrolysis by cellulase and β-glucosidase, performing synchronous saccharification and fermentation of ethanol with high substrate concentration, and fermenting The residue is anaerobically digested to produce methane.

The cellulose content of the energy grass after steam explosion is 30-55%, the hemicellulose content is 5-10%, the lignin content is 25-40%, and the ash content is 1-3%.

Further, the method includes the steps of:

1) Raw material pretreatment:

adding a buffer solution to the steam-exploded energy herb to make the concentration 10-30%, adjusting the pH to 5.1-5.5, adding cellulase and β-glucosidase for enzymolysis to obtain a fermentation substrate;

2) Synchronous saccharification and fermentation of ethanol with high substrate concentration:

Inoculating Saccharomyces cerevisiae into the fermentation substrate for fermentation, obtaining mash after fermentation, and distilling the mash to obtain ethanol and fermentation residue;

3) Using the fermentation residue as a digestion substrate, anaerobic digestion is performed using anaerobic sludge containing methanogens to collect methane gas.

In step 2), the fermentation is terminated when the ethanol concentration in the reaction system is stable.

Further, the enzyme equivalent of cellulase is 20 FPU/gcellulose, and the enzyme equivalent of β-glucosidase is 20 U/gcellulose. Further, the enzymolysis conditions are a temperature of 45°C-50°C, a pH of 5.1-5.5, and a time of 6-18h. The enzymolysis solution contains a large amount of glucose, which is easy to be used by contacting with Saccharomyces cerevisiae. The enzymolyzed product after enzymatic hydrolysis under the above enzymatic hydrolysis conditions is used as a fermentation substrate, which can better convert the cellulose in the raw material after steam explosion pretreatment into clean energy ethanol.

Further, the buffer is a citric acid-sodium citrate buffer containing yeast extract and peptone, and its pH value is 5.1-5.5. The role of the yeast extract in the buffer is to provide sufficient C source for the growth of yeast flora, and the buffer as a whole can provide C source and N source for the growth of yeast flora, and maintain the stable pH of the system .

The preparation method of the buffer solution is as follows: respectively prepare citric acid and sodium citrate buffer solution mother solutions, the concentration of which is 1mol/L. When using, dilute citric acid and sodium citrate mother liquor 20 times to 0.05mol/L, mix, and adjust the pH to 5.5 (because the raw material produces some small molecular acids after steam explosion, the raw material is acidic, and the pH during fermentation is 5.0, adjust the pH The use of calcium hydroxide during pH adjustment can be reduced when the adjusted buffer solution is mixed with the raw material, and calcium hydroxide is a supersaturated solution). Then add, 15g/L yeast extract, 30g/L peptone, mix well, adjust the pH to 5.1-5.5.

The present invention uses the buffer solution to provide a stable reaction system for the steam-exploded energy grass, which can make the steam-exploded energy grass easy to enzymatically hydrolyze, and is more conducive to subsequent reactions.

Further, during fermentation in step 2), adjust the concentration of the fermentation substrate (initial concentration of the substrate) to be 8%-12%, inoculate Saccharomyces cerevisiae for fermentation, and carry out batch feeding during the fermentation period, and what is added is steam explosion For energy grass, the final concentration of the fermentation substrate is 18%-22%.

When the initial substrate concentration and the final substrate concentration meet the above conditions, a relatively stable reaction system can be maintained, thereby better utilizing cellulose for ethanol production.

Furthermore, during the fermentation period, feeding is fed 3-4 times, and the amount of each feeding needs to be calculated according to the formula described in the claims . The function of feeding is to increase the substrate equivalent, so that greater ethanol production can be obtained at the end of fermentation.

Preferably, the Saccharomyces cerevisiae is deposited in the General Microorganism Center of China Committee for the Collection of Microbial Cultures, with the date of preservation on May 6, 2008, and the preservation number is CGMCC No.2660. The strain has a better ability to ferment ethanol.

The inoculum amount of Saccharomyces cerevisiae is 1-3×10 ⁸ /mL. If the concentration of the inoculated flora is low, the ethanol yield will be affected; to maintain a stable reaction system.

Further, the conditions of the anaerobic digestion are 30°C-35°C, pH 6.8-7.4. Under these conditions, the progress of anaerobic digestion can be accelerated.

Preferably, the anaerobic sludge containing methanogens has a pH of 6.5-7.0, a soluble COD value of less than 20,000 mg/L, a propionic acid content of less than 1,000 mg/L, and a total solid TS content of 100-200 g/Kg. Sludge with volatile solid VS content of 100-200g/Kg and VS/TS content of 80-85%. The amount of inoculated anaerobic sludge is 150mL of anaerobic sludge for every 10mL of fermentation residue

Further, the energy grass is selected from one or more of wheatgrass, elymus, miscanthus, stipa, grass, switchgrass, pennisetum, and eelgrass.

Preferably, the energy grass is one or more of switchgrass, wheatgrass and needlegrass.

The beneficial effects of the present invention are:

The invention provides a method for using energy grass to co-produce ethanol and methane to maximize the production of clean energy from lignocellulosic raw materials.

The invention uses steam-exploded energy grass as a raw material, and the product hydrolyzed by cellulase and β-glucosidase as a substrate, and performs simultaneous saccharification and fermentation with high substrate concentration in batch feeding and production of all residues from ethanol fermentation Anaerobic digestion treatment is integrated to carry out the joint production of ethanol and methane two clean energy sources. The main goal is to improve the conversion rate of cellulosic ethanol as much as possible and at the same time, through anaerobic digestion technology, further convert and utilize all available ethanol fermentation residues. Anaerobic digestion of the components used by the bacteria group, so as to improve the conversion rate of the whole cellulose of the energy grass, and realize the multi-level utilization of the whole component of the energy grass an effective solution.

In addition, the present invention has the following advantages when applied to industrial production:

1. The same production line can simultaneously obtain two kinds of clean energy, ethanol and methane, both liquid fuel and gas fuel, which can meet different energy demands.

2. In the process of producing clean energy, the utilization rate of energy grass cellulose can be improved through joint production, and the available components can be converted into clean energy that is urgently needed in today's world as much as possible.

3. Through the scientific research strategy of co-production of ethanol and methane, the multi-level utilization of all components of energy grass can be realized, providing a reasonable basis for the industrial production of clean energy.

Description of drawings

Fig. 1 is a diagram of cellulose conversion of high substrate simultaneous saccharification fermentation in Examples 1-4 of the present invention.

Figure 2 is the experimental results of the methane potential test of Examples 1-4 of the present invention.

Fig. 3 is the mass balance analysis result of ethanol and methane cogeneration in Example 1-4 of the present invention.

detailed description

Preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are given for the purpose of illustration only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The preparation method of the buffer used in the following examples is:

Prepare citric acid and sodium citrate buffer mother solutions respectively, the concentration of which is 1mol/L. Before use, dilute the mother liquor of citric acid and sodium citrate 20 times to 0.05mol/L, and mix the two until the pH is 5.1-5.5. Yeast extract and peptone were then added at concentrations of 15 g/L and 30 g/L, respectively.

Cellulase was purchased from SIGMAC2730-50mL, and β-glucosidase was purchased from SIGMA49290-250MG.

The anaerobic sludge containing methanogenic bacteria has a pH of 7.0, a soluble COD value of 19200mg/L, a propionic acid content of 850mg/L, a total solid TS content of 128g/Kg, and a total volatile solid VS content of 107g/Kg. And the activated sludge with VS/TS content of 83.5% was taken from Beijing Dahongmen Sewage Treatment Plant.

Other materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

In the present invention, required measurement parameter and measurement method thereof are as follows:

1: Determination method of glucose, ethanol, volatile acid content: high performance liquid chromatography (Agilent 1260). The chromatographic column was Hi-PlexH (300mm×7.7mm), the column temperature was 60°C, the Agilent differential detector was used, the detector temperature was 40°C, the mobile phase was dilute sulfuric acid (0.005mol/L), and the flow rate was 0.6mL·min ^-1 . The sample volume is 5 μL.

2: Measuring method of methane gas: NaOH drainage gas collection method

3: Soluble COD measurement method: COD is measured by Hach COD analyzer. First, the sample needs to be diluted. Take 2ml of the sample, add 1ml of potassium dichromate standard solution, 3ml of sulfuric acid-silver sulfate solution, and observe whether it becomes Green, if it turns green, the sample needs to be diluted until it does not change color. After dilution, add 2ml of sample, 1ml of potassium dichromate standard solution, and 3ml of sulfuric acid-silver sulfate solution to the COD measuring tube. At the same time, a control group was set up (2ml of distilled water was used instead of the sample). Mix well and digest in a COD digester at 150°C for 2 hours. After the digestion is completed, when the temperature drops to 120°C, take out the COD measuring tube and cool it down to room temperature. Colorimetric measurement: first select the corresponding measurement program, wipe the surface of the test tube clean, set it to zero with the control group, and then measure the COD value of the sample group respectively to obtain data.

4: Determination method of total solid TS and volatile solid VS: adopt standard method APHA, 1998.

Table 1 - Table 4 involved in the following embodiments are as follows:

Table 1 Energy Grass Component Analysis Table ( %, DM)

Table 2 Determination of Fermentation Residue and Inoculum Components

Table 3 Analysis table of parameter determination after methane potential test

Table 4 Analysis of parameter removal rate results before and after methane potential test

Example 1 Agropyroncristatum combined fed-batch high-substrate synchronous saccharification and fermentation with anaerobic digestion technology to produce clean energy ethanol methane.

1. Experimental materials

The raw material of steam-exploded wheatgrass comes from Tianjin University of Science and Technology, and its composition determination results are shown in Table 1 .

2. Experimental method

After the steam-exploded wheatgrass (Agropyroncristatum) raw material is obtained, it is dried in an oven, and the final water content is 6.4%. Weigh 10.68g of steam explosion raw material into a 250mL Erlenmeyer flask, then add 89.32mL of buffer solution to make the concentration reach 10%, stir well with a glass rod, add Ca(OH) ₂ , adjust the pH to 5.5, seal and store at 105°C , sterilized under the condition of 10min. After the sterilization, after the temperature dropped to room temperature, cellulase and β-glucosidase were added to carry out enzymolysis at 50°C and 200rpm. After 18 hours of enzymolysis, wait until the temperature dropped to At room temperature, the fermentation substrate was obtained.

Saccharomyces cerevisiae (CGMCCNo.2660) was added for ethanol fermentation, the inoculum size was 1×1010/mL, and the initial concentration of the fermentation substrate was ¹⁰ %. The amount of each feed was 4.53g, and the concentration of the fermentation substrate was increased to 20%, until 96h, and the ethanol fermentation experiment was ended. The fermented mash is distilled to obtain ethanol, and the fermentation residue is used for future use.

Then, add 10mL of fermentation residue and 150mL of anaerobic sludge into the anaerobic bottle, set up a control group and add 10mL of distilled water and 150mL of anaerobic sludge, because the anaerobic sludge itself will also produce gas, so a control group must be set up to judge End time of anaerobic digestion, and methane production from residues. Finally, fill with _N2 to remove the air at the top of the anaerobic bottle, and seal the bottle. Gas collection was carried out by NaoH drainage method every day, and methane gas production was measured with a 10mL gas collection tube.

3. Experimental results

After 96 hours of ethanol fermentation, the highest ethanol yield reached 15.24 g·kg ^-1 , and the corresponding cellulose conversion rate reached 30.49%, as shown in Figure 1( A). It can be known from the methane potential test that after 30 days, the final cumulative methane production reached 562.4mL, and the methane yield was 360.05mL·gVS ^-1 , the results are shown in Figure 2( A). From the mass balance analysis, it can be known that 7.5g of ethanol, 17.44g of methane, and 24.94g of clean energy can be obtained for every 100g of wheatgrass after batch-type high-substrate simultaneous saccharification and fermentation. The conversion rate of whole cellulose can reach 91.42% ( see Table 4 ), which is 2.0 times of ethanol production alone.

4 Conclusion

Through the strategy of combining batch high-substrate synchronous saccharification and fermentation with anaerobic digestion technology, the production of clean energy ethanol and methane from lignocellulosic raw materials can be realized, and the utilization rate of whole cellulose of wheatgrass (Agropyroncristatum) and its whole components can be improved. use.

Experimental example 1

After the fermented mash was distilled in Example 1 to obtain the fermented residue, the components of the fermented residue and the inoculum were determined, and the results are shown in Table 2 .

It can be seen from Table 2 that the ethanol fermentation residue contains a large amount of small molecule acid, the concentration of COD is high, and there is more cellulose, which is more suitable for anaerobic fermentation. A control group was established at the beginning of the experiment in the experimental group, which was distilled water with the same volume as the fermentation residue and the same inoculum amount inserted into the methanogenic bacteria. Fill with _N2 to remove air from the top of the anaerobic bottle, and seal the bottle. Gas production was determined by drainage method at a fixed time every day. After the experiment, the composition analysis was carried out, and the results are shown in Table 3 .

It can be seen from Table 3 that after anaerobic digestion, the cellulose content, COD content, hemicellulose content, small molecular acid content, etc. in the residue are greatly reduced compared with Table 2 .

Example 2 Elymus dahuricusturcz batch fed-batch high-substrate synchronous saccharification and fermentation combined with anaerobic digestion technology to produce clean energy ethanol methane.

1. Experimental materials

The raw material of steam-exploded Elymus is from Tianjin University of Science and Technology, and its composition determination results are shown in Table 1 .

2. Experimental method

After the steam-exploded Elymus dahuricusturcz raw material is obtained, it is dried in an oven, and the final water content is 4.4%. Weigh 10.46g of steam explosion raw material into a 250mL Erlenmeyer flask, then add 89.54mL of buffer solution to make the initial substrate concentration reach 10%, stir well with a glass rod, add Ca(OH) ₂ , adjust the pH to 5.5, seal it in Sterilize at 105°C for 10 minutes. After the sterilization is completed, wait until the temperature drops to room temperature, add cellulase and β-glucosidase at 50°C and 200 rpm for enzymolysis. After 18 hours of enzymolysis, wait for the temperature Cool down to room temperature to obtain a fermentation substrate.

Add Saccharomyces cerevisiae (CGMCCNo.2660) for ethanol fermentation, the inoculum size is 1×1010/mL, the initial concentration of the fermentation substrate is ¹⁰ %, and the raw material of Elymus chinensis by steam explosion is supplemented at 12, 24, 36, and 48 hours during the fermentation period , the amount of each feed is 4.41g, the concentration of the fermentation substrate is increased to 20%, until 96h, the ethanol fermentation experiment is ended. The fermented mash is distilled to obtain ethanol, and the fermentation residue is used for future use.

Then, add 10mL of fermentation residue and 150mL of anaerobic sludge into the anaerobic bottle, set up a control group and add 10mL of distilled water and 150mL of anaerobic sludge, because the anaerobic sludge itself will also produce gas, so a control group must be set up to judge End time of anaerobic digestion, and methane production from residues. Finally, fill with _N2 to remove the air at the top of the anaerobic bottle, and seal the bottle. Gas collection was carried out by NaoH drainage method every day, and methane gas production was measured with a 10mL gas collection tube.

3. Experimental results

After 96 hours of ethanol fermentation, the highest ethanol yield reached 13.04 g·kg ^-1 , and the corresponding cellulose conversion rate reached 20.75%, as shown in Figure 1( B). According to the methane potential test, after 30 days, the cumulative methane production reached 546.7mL, and the methane yield was 382.04mL·gVS ^-1 , the results are shown in Figure 2( B). From the mass balance analysis, it can be known that 6.5g of ethanol, 16.21g of methane, and 22.71g of clean energy can be obtained for every 100g of Elymus chinensis through batch-type high-substrate synchronous saccharification and fermentation. The conversion rate of whole cellulose can reach 95.84% ( Table 4 ), which is 3.6 times of ethanol production alone.

4 Conclusion

Through the strategy of combining batch high-substrate synchronous saccharification and fermentation with anaerobic digestion technology, the production of clean energy ethanol methane from lignocellulosic raw materials can be realized, and the utilization rate of whole cellulose from Elymus dahuricusturcz and its full components can be improved. level utilization.

Experimental example 2

After the fermented mash was distilled in Example 2 to obtain the fermented residue, the components of the fermented residue and the inoculum were determined, and the results are shown in Table 2 .

It can be seen from Table 2 that the residue of ethanol fermentation contains a large amount of small molecule acid, the concentration of COD is high, and there is more cellulose, which is more suitable for anaerobic fermentation.

A control group was established at the beginning of the experiment in the experimental group, which was distilled water with the same volume as the fermentation residue and the same inoculum amount inserted into the methanogenic bacteria. Fill with _N2 to remove air from the top of the anaerobic bottle, and seal the bottle. Gas production was determined by drainage method at a fixed time every day. After the experiment, the composition analysis was carried out, and the results are shown in Table 3 .

It can be seen from Table 3 that after anaerobic digestion, the cellulose content, COD content, hemicellulose content, small molecular acid content, etc. in the residue are greatly reduced compared with Table 2 .

Example 3 Stipabaicalensis roshev batch fed-batch high-substrate synchronous saccharification and fermentation combined with anaerobic digestion technology to produce clean energy ethanol methane

1. Experimental materials

The raw materials of steam-exploded Stipa grass come from Tianjin University of Science and Technology, and the composition determination results are shown in Table 1 .

2. Experimental method

After obtaining the steam-exploded Stipa grass (Stipabaicalensis roshev) raw material, it is dried in an oven, and the final water content is 21.4%. Weigh 12.72g of steam explosion raw material into a 250mL Erlenmeyer flask, then add 87.28mL of buffer solution to make the initial substrate concentration reach 10%, stir well with a glass rod, add Ca(OH) ₂ , adjust the pH to 5.5, seal it in Sterilize at 105°C for 10 minutes. After the sterilization is completed, wait until the temperature drops to room temperature, add cellulase and β-glucosidase to carry out enzymatic hydrolysis at 50°C and 200 rpm, and wait for 18 hours after enzymatic hydrolysis. Cool down to room temperature to obtain a fermentation substrate.

Saccharomyces cerevisiae (CGMCCNo.2660) was added for ethanol fermentation, the inoculum size was 1×1010/mL, and the initial concentration of the fermentation substrate was ¹⁰ %. The feeding amount is 4.41g each time, and the concentration of the fermentation substrate is increased to 20%, until 96h, and the ethanol fermentation experiment is ended. The fermented mash is distilled to obtain ethanol, and the fermentation residue is used for future use.

Then, add 10mL of fermentation residue and 150mL of anaerobic sludge into the anaerobic bottle, set up a control group and add 10mL of distilled water and 150mL of anaerobic sludge, because the anaerobic sludge itself will also produce gas, so a control group must be set up to judge End time of anaerobic digestion, and methane production from residues. Finally, fill with _N2 to remove the air at the top of the anaerobic bottle, and seal the bottle. Gas collection was carried out by NaoH drainage method every day, and methane gas production was measured with a 10mL gas collection tube.

3. Experimental results

After 96 hours of ethanol fermentation, the highest ethanol yield reached 15.78 g·kg ^-1 , and the corresponding cellulose conversion rate reached 29.53%, as shown in Figure 1 ( C). According to the methane potential test, after 30 days, the final cumulative methane production reached 566.7mL, and the methane yield was 258.65mL·gVS ^-1 , the results are shown in Figure 2( C). From the mass balance analysis, it can be known that 8.0 g of ethanol, 16.80 g of methane, and 24.80 g of clean energy can be obtained for every 100 g of Elymus chinensis through batch high-substrate synchronous saccharification and fermentation. The conversion rate of whole cellulose can reach 85.98% ( Table 4 ), which is 2.0 times of ethanol production alone.

4 Conclusion

Through the strategy of combining batch high-substrate synchronous saccharification and fermentation with anaerobic digestion technology, the production of clean energy ethanol methane from lignocellulosic raw materials can be realized, and the utilization rate of whole cellulose from Stipabaicalensis roshev and its whole components can be improved. use.

Experimental example 3

After the fermented mash was distilled in Example 3 to obtain the fermented residue, the components of the fermented residue and the inoculum were determined, and the results are shown in Table 2 .

It can be seen from Table 2 that the ethanol fermentation residue contains a large amount of small molecule acid, the concentration of COD is high, and there is more cellulose, which is more suitable for anaerobic fermentation.

A control group was established at the beginning of the experiment in the experimental group, which was distilled water with the same volume as the fermentation residue and the same inoculum amount inserted into the methanogenic bacteria. Fill with _N2 to remove air from the top of the anaerobic bottle, and seal the bottle. Gas production was determined by drainage method at a fixed time every day. After the experiment, the composition analysis was carried out, and the results are shown in Table 3 .

It can be seen from Table 3 that after anaerobic digestion, the cellulose content, COD content, hemicellulose content, small molecular acid content, etc. in the residue are greatly reduced compared with Table 2 .

Example 4 Production of clean energy ethanol methane by combining batch-type fed-batch high-substrate synchronous saccharification and fermentation with anaerobic digestion technology

1. Experimental materials

The raw material of steam-exploded grass came from Tianjin University of Science and Technology, and its composition determination results are shown in Table 1 .

2. Experimental method

After obtaining the steam-exploded grass (Triarrhenasacchariflora) raw material, it is dried in an oven, and the final water content is 12.8%. Weigh 11.47g of steam explosion raw material into a 250mL Erlenmeyer flask, then add 88.53mL of buffer solution to make the initial substrate concentration reach 10%, stir well with a glass rod, add Ca(OH) ₂ , adjust the pH to 5.5, seal it in Sterilize at 105°C for 10 minutes. After the sterilization is completed, wait until the temperature drops to room temperature, add cellulase and β-glucosidase to carry out enzymatic hydrolysis at 50°C and 200 rpm, and wait for 18 hours after enzymatic hydrolysis. Cool down to room temperature to obtain a fermentation substrate.

Saccharomyces cerevisiae (CGMCCNo.2660) was added for ethanol fermentation, the inoculum size was 3×10 ¹⁰ cells/mL, and the initial concentration of fermentation substrate was 10%. The secondary feeding amount was 4.41g, and the concentration of the fermentation substrate was increased to 20%, until 96h, and the ethanol fermentation experiment was ended. The fermented mash is distilled to obtain ethanol, and the fermentation residue is used for future use.

Then, add 10mL of fermentation residue and 150mL of anaerobic sludge into the anaerobic bottle, set up a control group and add 10mL of distilled water and 150mL of anaerobic sludge, because the anaerobic sludge itself will also produce gas, so a control group must be set up to judge End time of anaerobic digestion, and methane production from residues. Finally, fill with _N2 to remove the air at the top of the anaerobic bottle, and seal the bottle. Gas collection was carried out by NaoH drainage method every day, and methane gas production was measured with a 10mL gas collection tube.

3. Experimental results

After 96 hours of ethanol fermentation, the highest ethanol yield reached 14.96 g·kg ^-1 , and the corresponding cellulose conversion rate reached 25.11%, as shown in Figure 1( D). According to the methane potential test, after 30 days, the final cumulative methane production reached 443.9mL, and the methane yield was 309.12mL·gVS ^-1 , the results are shown in Figure 2( D). From the mass balance analysis, it can be known that 7.5g of ethanol, 13.33g of methane, and 20.83g of clean energy can be obtained for every 100g of Elymus chinensis through batch-type high-substrate synchronous saccharification and fermentation. The conversion rate of whole cellulose can reach 85.08% ( Table 4 ), which is 2.4 times of ethanol production alone.

4 Conclusion

Through the strategy of combining batch high-substrate synchronous saccharification and fermentation with anaerobic digestion technology, the production of clean energy ethanol and methane from lignocellulosic raw materials can be realized, and the utilization rate of whole cellulose of grass (Triarrhenasacchariflora) and its multi-stage utilization of whole components can be improved. .

Experimental example 4

After the fermented mash was distilled in Example 4 to obtain the fermented residue, the components of the fermented residue and the inoculum were determined, and the results are shown in Table 2 .

It can be seen from Table 2 that the ethanol fermentation residue contains a large amount of small molecule acid, the concentration of COD is high, and there is more cellulose, which is more suitable for anaerobic fermentation.

A control group was established at the beginning of the experiment in the experimental group, which was distilled water with the same volume as the fermentation residue and the same inoculum amount inserted into the methanogenic bacteria. Fill with _N2 to remove air from the top of the anaerobic bottle, and seal the bottle. Gas production was determined by drainage method at a fixed time every day. After the experiment, the composition analysis was carried out, and the results are shown in Table 3 .

It can be seen from Table 3 that after anaerobic digestion, the cellulose content, COD content, hemicellulose content, small molecular acid content, etc. in the residue are greatly reduced compared with Table 2 . Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. one kind utilizes the method for energy grass co-producing ethanol and methane, it is characterized in that, with the energy grass after steam explosion for raw material, after cellulase and beta-glucosidase enzymolysis, carry out high concentration of substrate simultaneous saccharification and fermentation ethanol, and fermentation residue is carried out Anaerobic Digestion to produce methane.

2. according to claimmethod described in 1, is characterized in that, described method comprises the steps:

1) raw materials pretreatment:

In the energy grass after steam explosion, add damping fluid, make its concentration be 10%-30%, adjustment pH is 5.1-5.5, adds cellulase and beta-glucosidase carries out enzymolysis, obtains fermentation substrate;

2) high concentration of substrate simultaneous saccharification and fermentation ethanol:

In described fermentation substrate, inoculate yeast saccharomyces cerevisiae ferment, after fermentation ends, obtain mash, distillation is carried out to mash and obtains ethanol and fermentation residue;

3) with described fermentation residue for digestion substrate, utilize and carry out anaerobic digestion containing the anaerobic sludge of methanogen, collect methane gas.

3. according to claimmethod described in 1 or 2, is characterized in that, described enzymatic hydrolysis condition is temperature 45 C-50 DEG C, pH5.1-5.5, time 6-18h.

4. according to claimmethod described in 3, is characterized in that, the citric acid-sodium citrate damping fluid containing yeast extract and peptone, its pH value is 5.1-5.5.

5. according to claimmethod described in 1 or 2, it is characterized in that, step 2) in fermentation time, adjustment fermentation substrate concentration is 8%-12%, inoculation yeast saccharomyces cerevisiae ferments, carry out batch feed supplement between yeast phase, what needs were added is the raw material obtained after steam explosion pre-treatment, makes the final concentration of fermentation substrate be 18%-22%.

6. want the method described in 5 according to right, it is characterized in that, feed supplement 3-4 time.

7. want the method described in 6 according to right, it is characterized in that, described yeast saccharomyces cerevisiae is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date on May 6th, 2008, and deposit number is CGMCCNo.2660.

8. according to claimmethod described in 1 or 2, is characterized in that, the condition of described anaerobic digestion is 30 DEG C-35 DEG C, pH6.8-7.4.

9. according to claimmethod described in 8, it is characterized in that, the described anaerobic sludge containing methanogen is pH6.5-7.0, solubility COD value is less than 20000mg/L, propionic acid content is lower than 1000mg/L, total solids TS content is 100-200g/Kg, and total volatile solid(s) (TVS) VS content is 100-200g/Kg, and VS/TS content is the mud of 80-85%.

10. according to claimmethod described in 1 ~ 9 any one, is characterized in that, described energy grass be selected from wheatgrass, lyme grass, Chinese silvergrass, needle Rhizoma Imperatae, reed, switchgrass, Herba penniseti and eelgrass one or more.