CN105713931B - Method for producing ethanol by continuous enzymolysis and fermentation of lignocellulose - Google Patents
Method for producing ethanol by continuous enzymolysis and fermentation of lignocellulose Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for producing ethanol by continuous enzymolysis and fermentation of lignocellulose, which comprises the following steps: (1) Pretreating a lignocellulose raw material to obtain a pretreated raw material; (2) Continuously adding the pretreated raw materials, cellulase and water into an enzymolysis tank for pre-enzymolysis, and controlling the dry matter concentration of an enzymolysis system to be 18-36 wt%; (3) Continuously feeding the pre-enzymolysis feed liquid into a fermentation tank, and adding temperature-resistant saccharomyces cerevisiae for synchronous saccharification and fermentation; (4) Fully mixing fermented mash with nonionic surfactant in a pipeline mixer, and then entering a settling tank; (5) The lower concentrated mixed solution after sedimentation in the sedimentation tank enters a product separation unit, and the supernatant is circulated back to the enzymolysis tank to participate in enzymolysis again. The method can realize continuous production, improve the activity and the utilization rate of the enzyme, improve the yield of fermentation products, and reduce the enzymolysis and fermentation cost while ensuring the recycling of the enzyme.
Description
Technical Field
The invention belongs to the field of biomass energy, and particularly relates to a method for producing ethanol by continuous enzymolysis and fermentation of lignocellulose.
Background
Energy consumption continues to rise as world population grows and the degree of industrialization of various countries increases. Petroleum is the primary resource to meet energy demands, but petroleum resources are limited, scientists predict that crude oil production will drop from 258 hundred million barrels (35.25 hundred million tons) to 50 hundred million barrels in 2009 in 2050. The renewable biological energy source is used as a renewable transportation fuel, can effectively reduce the room temperature effect, slow down the environmental pollution, change the current unequal petroleum supply and demand relationship, keep continuous supply, and has obvious advantages compared with mineral fuel. Compared with other bioenergy sources such as biodiesel, the production of fuel ethanol has been on a considerable scale (about 1700 ten thousand tons worldwide in 2002), and mainly sugar-containing substances are produced by a fermentation method. The 10% absolute ethyl alcohol (E10) is mixed into the gasoline, so that the energy pressure can be relieved, the octane number can be increased, and the tail gas emission quality can be improved. Fuel ethanol has been widely used as a transportation fuel as a petroleum substitute in the united states and brazil.
The government of China has paid attention to the problems of energy diversification and environmental pollution, and adopts encouraging measures such as financial subsidy, tax reduction and the like to greatly promote the technology and industry development of diversification alternative petroleum energy. The development process of biodiesel, fuel ethanol and other biological liquid fuels is greatly promoted by the export of renewable energy method and national long-term science and technology development planning schema. By 2020, the consumption of the biofuel is about 15% of the total traffic fuel, and the biofuel industry with international competitiveness is established, which brings good development opportunity to the fuel ethanol industry in China. At present, 9 provinces of China are covered with ethanol gasoline in China, the capacity of the existing fuel ethanol is 152 ten thousand tons, and the actual production of the fuel ethanol in 2010 is more than 180 ten thousand tons, and corn and wheat are used as main raw materials. The number of people in China is small, cultivated land resources are short, grain supply is tension, the production of fuel ethanol by taking corn and wheat as raw materials threatens the national grain safety, and causes chain reactions such as price rise of agricultural products, so that the new construction and energy expansion project of fuel ethanol by taking grain as raw materials are strictly controlled in China.
Lignocellulose belongs to a non-grain raw material and is abundant in resources relative to sugar and starch crops. It can be derived from agricultural waste such as wheat straw, corn stalk, corncob, soybean residue, bagasse, etc.; industrial waste such as pulp and paper mill fibrous residue, sawdust, etc.; forestry waste; urban waste such as waste paper, packaging paper and the like. It is estimated that lignocellulose raw material accounts for 50% of 100-500 hundred million tons of world biomass, the annual output of crop straw in China can reach 7 hundred million tons, and a large amount of waste and unused fiber (small branches, bark, leaves, scraps, waste paper and the like) has about five hundred million tons per year, and only 1 hundred million tons of the biomass is used for annual production of 2000 ten thousand tons of fuel ethanol. Therefore, the development of a new process for producing ethanol by lignocellulose has good prospect.
The cost of cellulase in the hydrolysis process of cellulase, the energy consumption in the distillation process of ethanol and the continuity of the enzymolysis process are all the restriction factors of industrialization of cellulosic ethanol. CN200810189465.0 discloses a method for producing cellulosic ethanol, which comprises the following steps: (1) Adding a culture medium containing cellulose and/or hemicellulose raw materials into a fermentation reaction kettle; (2) Adding cellulase into a fermentation reaction kettle, and inoculating candida vinosa; (3) And (3) carrying out synchronous saccharification and fermentation under the combined action of cellulase and candida viticola, and separating to obtain the cellulosic ethanol. CN200810101314.5 discloses a method for preparing ethanol from a cellulose-containing feedstock, which comprises steam explosion of the cellulose-containing feedstock; mixing the obtained steam explosion product with enzyme, and performing enzymolysis; fermenting and hydrolyzing to obtain the product. In order to reduce the cost of the cellulase, the cellulase in the enzymolysis fermentation liquor is recycled and reused as a better method. CN200910212693 discloses a method for enzymatic hydrolysis of wood fibre raw material, which uses pretreated wood fibre as raw material, adopts staged enzymolysis and ultrafiltration to recycle cellulase and beta-glucosidase. However, the cost for recycling the cellulase by using the ultrafiltration membrane is high, and the operation process is complicated.
The gap enzymolysis technology can be used for production, but has low enzymolysis efficiency, can only hydrolyze with low dry matter concentration, has low concentration of hydrolyzed sugar, causes low concentration of final ethanol, affects the energy consumption of subsequent distillation, and has the defects of low equipment utilization rate, large occupied area, large total motor stirring power and the like.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for producing ethanol by continuous enzymolysis and fermentation of lignocellulose. The method can realize continuous production, improve the activity and the utilization rate of the enzyme, improve the yield of fermentation products, and reduce the enzymolysis and fermentation cost while ensuring the recycling of the enzyme.
The invention relates to a method for producing ethanol by continuous enzymolysis and fermentation of lignocellulose, which comprises the following steps:
(1) Pretreating a lignocellulose raw material to obtain a pretreated raw material;
(2) Continuously adding the pretreated raw materials, cellulase and water into an enzymolysis tank for pre-enzymolysis, and controlling the dry matter concentration of an enzymolysis system to be 18-36 wt%;
(3) Continuously feeding the pre-enzymolysis feed liquid into a fermentation tank, and adding temperature-resistant saccharomyces cerevisiae for synchronous saccharification and fermentation;
(4) Fully mixing fermented mash with nonionic surfactant in a pipeline mixer, and then entering a settling tank;
(5) The lower concentrated mixed solution after sedimentation in the sedimentation tank enters a product separation unit, and the supernatant is circulated back to the enzymolysis tank to participate in enzymolysis again.
In the method of the present invention, the mash after the simultaneous saccharification and fermentation in step (3) is first subjected to cell disruption treatment, for example, a high-speed stirring bead grinding disruption method, a high-pressure homogenizing disruption method, a ultrasonic disruption method, an enzymatic disruption method, etc., preferably an enzymatic disruption method is adopted: if the snailase and/or lysozyme can be adopted, the snailase and/or lysozyme are added in a continuous flow mode, the addition amount of the snailase is controlled to be 1-2mg/g dry matter, and the addition amount of the lysozyme is controlled to be 0.2-1.0mg/g dry matter. The feed liquid after cell disruption treatment is mixed with nonionic surfactant in a pipeline mixer and then enters a settling tank for settling.
The lignocellulose raw material in the step (1) comprises all biomass raw materials containing cellulose, such as straw, wood dust, energy plants (such as switchgrass) and waste paper, and the like, preferably corn straw. The pretreatment mode can adopt all physical, chemical and thermochemical technologies capable of improving the enzymolysis performance of lignocellulose, including mechanical crushing, radiation, microwaves, acid treatment, alkali treatment, steam explosion pretreatment, solvent pretreatment, or combined pretreatment of the methods, and the like, and preferably adopts dilute acid steam explosion combined pretreatment.
In the step (2), the retention time of the enzymolysis feed liquid is controlled to be 8-96 hours, preferably 8-24 hours. According to the effective volume of the enzymolysis tank, the adding rate of the pretreatment raw materials and water is controlled, so that the dry matter concentration (the sum of the mass of soluble solids and insoluble solids and the total mass of the system, hereinafter the sum of the mass of soluble solids and the total mass of insoluble solids) of the enzymolysis system is 20-30wt%. The cellulase adopts enzyme protein or enzyme protein mixture of all hydrolyzable lignocellulose components, can be produced on line in factories, and can also be commercially available commercial cellulases, such as Norwesinase or Zealand enzyme. The addition amount of the cellulase is controlled so that the ratio of the cellulase to the cellulose in the pretreated feedstock is 5-25IU/g cellulose. Controlling the pH of the pre-enzymolysis to be 4.5-5.5, preferably 4.8-5.2; the temperature is 45-55deg.C, preferably 48-52deg.C.
The synchronous saccharification and fermentation in the step (3) refers to a process that residual cellulose after the pre-enzymolysis is continuously hydrolyzed while fermenting glucose to produce ethanol or other products. The temperature-resistant saccharomyces cerevisiae adopts a strain which is known at present and can utilize lignocellulose raw materials to ferment and produce ethanol or other products, preferably the temperature-resistant saccharomyces cerevisiae which can tolerate 36-42 ℃ is used, more preferably the temperature-resistant saccharomyces cerevisiae (Saccharomyces cerevisiae) FE-B described in CN200910204295.3 is used, and the preservation number of the strain is CGMCC No. 2735. The seed liquid of the zymophyte is prepared by adopting a conventional culture mode in the field, and the inoculation amount of the seed liquid is 1-5 v percent. In the invention, the pH value is not required to be regulated after enzymolysis, synchronous saccharification and fermentation is directly carried out, the fermentation temperature is controlled to be 35-42 ℃, and the fermentation residence time is controlled to be 12-72h. The nitrogen source added into the synchronous saccharification and fermentation system can be one or more selected from yeast extract, peptone, corn steep liquor, ammonium sulfate or urea, etc., preferably urea, and the addition amount is 0.02 wt% -0.2 wt% of the total mass of the system.
The nonionic surfactant in the step (4) is one or more of Tween20, tween80, PEG, SDS and the like, preferably Tween80 or PEG, and the addition amount of the nonionic surfactant is controlled to be 2-20mg/g dry matter. The fermentation mash is mixed with nonionic surfactant in a pipeline mixer in proportion, and then enters a settling tank for settling, and the combination of lignin and nonionic surfactant helps the cellulase adsorbed on the solid surface in the fermentation mash to be desorbed into the liquid, so that the content of the cellulase in the liquid phase is increased, and the cellulase is recycled back to the enzymolysis tank to participate in the enzymolysis reaction again along with the supernatant, so that the cellulase recycling amount is increased.
In the cellulosic ethanol fermentation mash, cellulase exists in both solid and liquid phases, and no matter what way is adopted to separate and purify the product later, the cellulase can be lost and deactivated to a certain extent. To achieve partial reuse of enzymes, the beer is typically circulated in 3 ways: ① Directly recycling part of fermentation liquor; ② After solid-liquid separation, the fermentation liquor is recycled; ③ After solid-liquid separation, the fermentation liquor is recycled in solid phase. However, the solid residue particles in the fermented mash are below tens of micrometers, most of the solid residue particles are only a few micrometers, the solid-liquid separation is very difficult, and the combination of plate-frame filtration and centrifugation is generally adopted, but the energy consumption is higher; if natural sedimentation is directly carried out, the treatment period is longer, the effect is poor, and the enzyme can not be effectively recycled. The invention mixes the fermented liquor with nonionic surfactant, then enters a settling tank for settling, the temperature of the settling tank is controlled to be 35-55 ℃, and the retention time is controlled to be 0-72 h. Pumping the settled concentrated mixed solution into a product separation unit from the lower part of a settling tank, and circularly feeding the supernatant into an enzymolysis tank, wherein the volume ratio of the concentrated mixed solution and the circulated supernatant of the product separation unit is controlled to be 1:3-3:1. The clear liquid at the upper part of the settling tank is circulated into the enzymolysis tank to replace part or all of water to be added to continuously participate in the enzymolysis reaction, and part of nonionic surfactant is circulated back to the enzymolysis tank along with the supernatant liquid, so that the enzymolysis efficiency is improved.
Compared with the prior art, the invention has the following advantages:
1. The nonionic surfactant is added into the fermented mash, and is easy to combine with lignin particles, so that the cellulase adsorbed on the surfaces of the solid particles is facilitated to be desorbed again, and the nonionic surfactant is dissolved in a liquid phase, so that part of cellulase is circulated back to an enzymolysis tank along with the supernatant to participate in enzymolysis reaction again, and the recovery amount and the utilization rate of the enzyme are improved.
2. The enzymatic hydrolysis of cellulose is a heterogeneous reaction in which the cellulase first diffuses to the surface of the substrate cellulose to be adsorbed and then hydrolyzes the cellulose to fermentable sugars. The non-ionic surfactant remaining in the supernatant after settling is recycled back to the enzymatic tank with the supernatant, which can increase the soluble and fermentable sugar conversion levels of cellulose.
3. The fermentation mash is subjected to cell disruption treatment, so that the consumption of cellulase and nonionic surfactant can be reduced; and the sedimentation rate of solid particles can be quickened, the sedimentation residence time is shortened, and the equipment investment cost is reduced. In addition, the extract produced by breaking yeast cells can be used as a nitrogen source for ethanol fermentation along with the circulation of fermentation liquid, so that the fermentation cost is further reduced.
4. The nonionic surfactant is added into the fermented mash, so that the activity and the utilization rate of the enzyme are fully improved, the yield of fermentation products is improved, and the enzymolysis and fermentation cost is reduced while the enzyme is ensured to be recycled. The method realizes continuous production, improves the utilization rate of equipment and saves time.
Drawings
FIG. 1 is a process flow diagram of the present invention;
wherein, 1-enzymolysis tank, 2-synchronous saccharification fermentation tank, 3-cell dissolution tank, 4-pipeline mixer, 5-sedimentation tank and 6-product separation unit.
FIG. 2 is a schematic diagram of the construction of the settling tank of the present invention.
Detailed Description
The process according to the invention is further illustrated by the following examples. In the invention, wt% is mass fraction and v% is volume fraction.
The lignocellulose raw material used in the embodiment of the invention is corn dry straw, wherein 38.2 wt% of cellulose, 22.1 wt% of hemicellulose, 20.2 wt% of lignin and 3.9 wt% of ash are crushed by a crusher until the particle size is 1-5mm. The pretreatment is carried out by adopting dilute acid steam explosion, the reaction temperature is 190 ℃, the reaction time is 5min, the solid-to-liquid ratio is 1:1.2, and the concentration of dilute sulfuric acid is 2.0wt%. After exiting the steam explosion apparatus, the pH was adjusted to 5.0 with NaOH, wherein the dry matter concentration was 45 wt%, the cellulose content in the dry matter was 38: 38 wt%, and the pretreated dry matter recovery was 95: 95 wt%.
The temperature-resistant saccharomyces cerevisiae (Saccharomyces cerevisiae) FE-B is adopted, and the preservation number of the strain is CGMCC No. 2735. The yeast seed liquid culture medium is 2 wt% glucose, 2 wt% peptone and 1 wt% yeast extract, and the yeast seed liquid culture medium is sterilized at 115 ℃ for 30min for standby. Preparing yeast seed liquid and carrying out total 3-stage culture: scraping 1-2 loops of bacterial mud from an inclined plane by using an inoculating loop, inoculating the bacterial mud into a 25 mL seed culture medium placed in a 100 mL triangular flask, and performing shake culture for 24 hours at 37 ℃ and 100 r/m; second-stage culture the whole first-stage culture solution is inoculated into 500mL seed culture medium in a 1L fermentation tank, and cultured for 24h at 37 ℃ and 100 r/m; third stage culture the second stage broth was inoculated entirely into 25L seed medium in a 50L fermenter, at 37℃at 100 r/min, and cultured at 24 h.
Example 1
According to the flow shown in FIG. 1, the effective volume of the vertical enzymolysis tank is 240L, the effective volume of the synchronous saccharification fermentation tank is 480L, and the effective volume of the sedimentation tank is 240L. The pretreated corn stalks, cellulase (purchased from NoveXin biotechnology Co., ltd., model Ctec2, filter paper enzyme activity 135 IU/g) and water are continuously added into an enzymolysis tank in proportion to carry out pre-enzymolysis, wherein the adding rate of the corn stalks is 3.3333 kg/h, the adding rate of tap water is 1.4322 kg/h, the adding rate of the cellulase is 0.0844 kg/h (equivalent to 20I U/g cellulose after adding), the dry matter concentration in an enzymolysis system is 30wt%, the temperature in the enzymolysis tank is 52 ℃, the pH is 5.2, the stirring rate is 50 r/min, and the residence time is 48 h. Simultaneously, the enzymolysis liquid is pumped into the synchronous saccharification and fermentation tank from the enzymolysis tank at the speed of 5 kg/h, and the FE-B seed liquid 24L is only connected into the synchronous saccharification and fermentation tank at one time in the initial stage of system startup. The nitrogen source was urea to prepare a10 wt% mother liquor which was added continuously at a rate of 0.06 kg/h (equivalent to 0.12 wt% after addition). The temperature in the simultaneous saccharification and fermentation tank is 37 ℃, the stirring speed is 100 r/min, and the residence time is 96 h. The mash after simultaneous saccharification and fermentation was mixed with Tween80 in a pipeline mixer, tween80 solution was formulated as a10 wt% mother liquor and added at a rate of 0.09 kg/h (equivalent to 6 mg/g dry matter after addition).
The mixed beer was pumped at a rate of 5 kg/h into a settling tank as shown in FIG. 2 for settling, maintaining the temperature of the settling tank at 40℃ and the residence time at 24 h. The supernatant was recycled back to the enzymatic tank at 1.4322 kg/h and the concentrated mixture was fed to the product separation unit at 3.5678 kg/h. After the circulation starts, the addition of tap water to the enzymolysis tank is stopped.
The total amount of the pretreated corn stalks is 1600 kg, and the corn stalks are continuously operated for 20 days. The glucose and ethanol concentrations were measured by liquid chromatography. 144 After h, the system is stable in operation, the concentration of glucose in the sampling detection enzymolysis liquid is 10.8 wt%, and the glucose yield is 59.7 wt%. The ethanol concentration in the effluent of the fermentation tank is 9.6 wt percent (including the accumulation of ethanol as the clear liquid enters the enzymolysis tank, and the same applies below), and the yield of glucose in the enzymolysis and simultaneous saccharification and fermentation of the whole system is 81.9 wt percent calculated by the yield of glucose to ethanol being 90 wt percent. The total amount of cellulase used was 40.5 kg.
Example 2
According to the flow shown in FIG. 1, the effective volume of the vertical enzymolysis tank is 360L, the effective volume of the synchronous saccharification fermentation tank is 360L, and the effective volume of the sedimentation tank is 240L. The pretreated corn stalks, cellulase (purchased from Shandong Zea Biotechnology Co., ltd., model 1300, 100 IU/g of filter paper enzyme activity) and water are continuously added into an enzymolysis tank in proportion for pre-enzymolysis, wherein the adding rate of the corn stalks is 2.2222 kg/h, the adding rate of tap water is 2.6015 kg/h, the adding rate of the cellulase (equivalent to 20 IU/g of cellulose after adding) is 0.0563 kg/h, the dry matter concentration in an enzymolysis system is 20 wt%, the temperature in the enzymolysis tank is 48 ℃, the pH is 4.8, the stirring rate is 50 r/min, and the retention time is 72 h. Simultaneously, the enzymolysis liquid is pumped into the synchronous saccharification and fermentation tank from the enzymolysis tank at the speed of 5 kg/h, and the FE-B seed liquid 18L is only connected into the synchronous saccharification and fermentation tank at one time in the initial stage of system startup. The nitrogen source is urea, 10 wt% of mother liquor is prepared, and the nitrogen source is continuously added at the rate of 0.06 kg/h. The temperature in the simultaneous saccharification and fermentation tank is 37 ℃, the stirring speed is 100 r/min, and the residence time is 72 h. The mash after simultaneous saccharification and fermentation was mixed with PEG8000 in a pipeline mixer, PEG8000 was formulated as a10 wt% mother liquor and added at a rate of 0.06 kg/h (equivalent to 4 mg/g dry matter after addition).
The mixed beer was pumped into a settling tank at a rate of 5 kg/h for settling, maintaining the temperature of the settling tank at 40℃ and a residence time of 24: 24 h. The supernatant was recycled back to the enzymatic tank at 2.6015 kg/h and the concentrated mixture was fed to the separation unit at 2.3985 kg/h. After the circulation starts, the addition of tap water to the enzymolysis tank is stopped.
Taking the total amount of the pretreated corn stalks to be 1066.7 kg, and continuously running for 20 days. 144 After h, the system is stable in operation, the concentration of glucose in the sampling detection enzymolysis liquid is 7.2 wt%, and the glucose yield is 68.2 wt%. The concentration of ethanol in effluent liquid of the fermentation tank is 6.6 wt percent, and the yield of glucose by enzymolysis and simultaneous saccharification and fermentation of the whole system is 82.6 wt percent calculated by the yield of glucose to ethanol being 90 wt percent. The cellulase is used in an amount of 27.0 kg.
Example 3
The process flow and process conditions were the same as in example 1, except that: the cellulase addition rate in the enzymolysis tank is 0.0633 kg/h (corresponding to 15 IU/g cellulose after addition), and the tap water addition rate is 1.4533 kg/h. The fermented mash is prepared by firstly adopting snailase to carry out cell disruption on saccharomycetes, wherein the retention time of a cell dissolution tank is 8 hours, the snailase is prepared into 10 wt percent aqueous solution, the adding rate is 0.03 kg/h (which is equivalent to 2 mg/g dry matter after adding), the temperature is 37 ℃, and the pH is 6.0. Then the mixture enters a pipeline mixer to be mixed with Tween80, 10 wt percent of Tween80 solution is fed at the rate of 0.06 kg/h, so that the Tween80 and the fermentation mash after cell disruption are uniformly mixed (the addition is equivalent to 4 mg/g dry matter). The mixed mash was pumped at a rate of 5 kg/h into a settling tank as shown in FIG. 2 for settling, maintaining the temperature of the settling tank at 40℃and the residence time at 24 h. The supernatant was recycled to the enzymatic tank at 1.4322 kg/h and the concentrated mixture was fed to the separation unit at 3.5678 kg/h. After the circulation is started, urea solution is stopped from being fed into the fermentation tank, and the tap water rate in the enzymolysis tank is adjusted to 0.0811 kg/h.
The total amount of the pretreated corn stalks is 1600 kg, and the corn stalks are continuously operated for 20 days. 144 After h, the system is stable in operation, the concentration of glucose in the sampling detection enzymolysis liquid is 11.8 wt%, and the glucose yield is 65.2 wt%. The concentration of ethanol in the effluent of the fermentation tank is 10.1 wt percent, and the yield of glucose of enzymolysis and synchronous saccharification and fermentation of the whole system is 86.7 wt percent calculated by the yield of glucose to ethanol of 90 percent. The cellulase was used in an amount of 30.4 kg.
Example 4
The process flow and process conditions were the same as in example 2, except that: the cellulase addition rate in the enzymolysis tank is 0.0422 kg/h (corresponding to 15I U/g cellulose after addition), and the tap water addition rate is 2.6156 kg/h. The synchronous saccharification and fermentation mash is firstly crushed by snailase, the retention time in a cell dissolution tank is 8h, the snailase is prepared into 1 g/L aqueous solution, the adding rate is 0.02 kg/h (equivalent to 2 mg/g dry matter after adding), the temperature is 37 ℃, and the pH is 6.0. Then, the mixture was put into a pipe mixer and mixed with PEG8000, and 10 wt% of PEG8000 solution was fed at a rate of 0.04 kg/h to uniformly mix PEG8000 with the beer (equivalent to 4 mg/g dry matter after the addition). The mixed mash was pumped at a rate of 5 kg/h into a settling tank as shown in FIG. 2 for settling, maintaining the temperature of the settling tank at 40℃and the residence time at 24 h. The supernatant was recycled to the enzymatic tank at 2.6015 kg/h and the concentrated mixture was fed to the separation unit at 2.3985 kg/h. After the circulation is started, urea is stopped from being fed into the fermentation tank, and the tap water rate in the enzymolysis tank is regulated to be 0.0741 kg/h.
The total amount of the treated corn stalks was 1066.7 kg and the operation was continued for 20 days. 144 After h, the system is stable in operation, the concentration of glucose in the sampling detection enzymolysis liquid is 7.4%, and the glucose yield in the pre-enzymolysis stage is 70.1 wt%. The ethanol concentration in the effluent of the fermentation tank is 7.1 wt percent, and the yield of glucose by enzymolysis and simultaneous saccharification and fermentation of the whole system is 88.8 wt percent calculated by the yield of glucose to ethanol being 90 percent. The cellulase is used in an amount of 20.3 kg.
Example 5
The process flow and process conditions were the same as in example 3, except that: and (3) performing cell disruption on the fermented mash by adopting a high-pressure homogenizing breaker, wherein the disruption pressure is 50 MPa in a continuous operation mode, and the outlet temperature is controlled to be 20 ℃. After the system runs stably, the concentration of glucose in the sampling detection enzymolysis liquid is 11.3 wt percent, and the glucose yield is 62.4 wt percent. The ethanol concentration in the effluent of the fermentation tank is 10.0 wt percent, and the yield of glucose in the enzymolysis and synchronous saccharification and fermentation of the whole system is 84.3 wt percent calculated by the yield of glucose to ethanol being 90 percent. The cellulase was used in an amount of 30.4 kg.
Comparative example 1
The process flow and process conditions were the same as in example 1, except that: and the fermentation liquor after fermentation is not fed into a settling tank, and is directly recycled to the enzymolysis tank according to the proportion of 2:5, and is partially fed into a product separation unit. After the system runs stably, the concentration of glucose in the sampling detection enzymolysis liquid is 9.8 wt percent, and the glucose yield is 54.2 wt percent. The ethanol concentration in the effluent of the fermentation tank is 8.5 wt percent, and the yield of glucose in the enzymolysis and synchronous saccharification and fermentation of the whole system is 77.1 wt percent calculated by the yield of glucose to ethanol being 90 percent. The cellulase is used in an amount of 40.5 kg.
Comparative example 2
The process flow and process conditions were the same as in example 1, except that: and (3) enabling fermentation liquor after fermentation to enter a solid-liquid separation tank for solid-liquid separation, enabling separated solid phase to enter a product separation unit, enabling a part of separated liquid phase to be recycled back to the enzymolysis tank (recycling ratio is 2:5), and enabling a part of separated liquid phase to enter the product separation unit. After the system runs stably, the concentration of glucose in the sampling detection enzymolysis liquid is 9.6 wt percent, and the glucose yield is 53.1 wt percent. The concentration of ethanol in the effluent of the fermentation tank is 8.9 wt percent, and the yield of glucose in the enzymolysis and synchronous saccharification and fermentation of the whole system is 75.9 percent calculated by the yield of the glucose to the ethanol being 90 percent. The cellulase is used in an amount of 40.5 kg.
Comparative example 3
The process flow and process conditions were the same as in example 3, except that: only cell disruption was performed without adding nonionic surfactant. 144 After h, the system is stable in operation, the concentration of glucose in the sampling detection enzymolysis liquid is 11.6 wt%, and the glucose yield is 64.1 wt%. The ethanol concentration in the effluent of the fermentation tank is 9.7 wt percent, and the yield of glucose in the enzymolysis and synchronous saccharification and fermentation of the whole system is 83.1 wt percent calculated by the yield of glucose to ethanol being 90 percent. The cellulase was used in an amount of 30.4 kg.
Comparative example 4
The process flow and process conditions were the same as in example 4, except that: only cell disruption was performed without adding nonionic surfactant. 144 After h, the system is stable in operation, the concentration of glucose in the sampling detection enzymolysis liquid is 7.0 wt%, and the glucose yield is 66.3 wt%. The ethanol concentration in the effluent of the fermentation tank is 6.8 wt percent, and the yield of glucose by enzymolysis and simultaneous saccharification and fermentation of the whole system is 84.6 wt percent calculated by the yield of glucose to ethanol being 90 percent. The cellulase is used in an amount of 20.3 kg.
Claims (8)
1. A method for producing ethanol by continuous enzymolysis and fermentation of lignocellulose comprises the following steps:
(1) Pretreating a lignocellulose raw material to obtain a pretreated raw material;
(2) Continuously adding the pretreated raw materials, cellulase and water into an enzymolysis tank for pre-enzymolysis, and controlling the dry matter concentration of an enzymolysis system to be 18-36 wt%;
(3) Continuously feeding the pre-enzymolysis feed liquid into a fermentation tank, and adding temperature-resistant saccharomyces cerevisiae for synchronous saccharification and fermentation; performing cell disruption treatment on the mash after synchronous saccharification and fermentation; cell disruption adopts an enzymatic disruption method, and snailase and/or lysozyme are/is added in a continuous flow manner; controlling the adding amount of snailase to be 1-2mg/g dry matter, and controlling the adding amount of lysozyme to be 0.2-1.0mg/g dry matter;
(4) Fully mixing fermented mash with nonionic surfactant in a pipeline mixer, and then entering a settling tank; the nonionic surfactant is one or more of Tween20, tween80, PEG and SDS; controlling the temperature of the sedimentation tank to be 35-55 ℃ and the residence time to be 24-72 h;
(5) The lower concentrated mixed solution after sedimentation in the sedimentation tank enters a product separation unit, and the supernatant is circulated back to the enzymolysis tank to participate in enzymolysis again.
2. A method according to claim 1, characterized in that: in the step (1), the pretreatment adopts dilute acid steam explosion combined pretreatment.
3. A method according to claim 1, characterized in that: in the step (2), the retention time of the enzymolysis feed liquid is controlled to be 8-96h, and the dry matter concentration of the enzymolysis system is controlled to be 20-30 wt%.
4. A method according to claim 1 or 3, characterized in that: controlling the addition amount of the cellulase in the step (2) so that the ratio of the cellulase to the cellulose in the pretreated raw material is 5-25IU/g cellulose; the pH of the pre-enzymolysis is 4.5-5.5, and the temperature is 45-55 ℃.
5. A method according to claim 1, characterized in that: in the step (3), the temperature-resistant saccharomyces cerevisiae uses temperature-resistant saccharomyces cerevisiae (Saccharomyces cerevisiae) FE-B described in CN200910204295.3, and the preservation number of the strain is CGMCC No. 2735.
6. A method according to claim 1 or 5, characterized in that: the inoculation amount of the temperature-resistant saccharomyces cerevisiae seed liquid is 1-5 v%; and after enzymolysis, the pH is not required to be regulated, synchronous saccharification and fermentation is directly carried out, the fermentation temperature is controlled to be 35-42 ℃, and the fermentation residence time is controlled to be 12-72h.
7. A method according to claim 1, characterized in that: the nonionic surfactant in the step (4) is Tween80 or PEG, and the adding amount of the nonionic surfactant is controlled to be 2-20mg/g dry matter.
8. A method according to claim 1, characterized in that: and (5) controlling the volume ratio of the concentrated mixed solution to the circulating supernatant of the product separation unit to be 1:3-3:1.
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