CN114908133B - Method for improving reducing sugar produced by corn straw hydrolysis through biological combined ammoniation pretreatment - Google Patents
Method for improving reducing sugar produced by corn straw hydrolysis through biological combined ammoniation pretreatment Download PDFInfo
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- CN114908133B CN114908133B CN202210412143.8A CN202210412143A CN114908133B CN 114908133 B CN114908133 B CN 114908133B CN 202210412143 A CN202210412143 A CN 202210412143A CN 114908133 B CN114908133 B CN 114908133B
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- 240000008042 Zea mays Species 0.000 title claims abstract description 66
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 title claims abstract description 66
- 235000002017 Zea mays subsp mays Nutrition 0.000 title claims abstract description 66
- 235000005822 corn Nutrition 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 27
- 235000000346 sugar Nutrition 0.000 title claims abstract description 24
- 230000007062 hydrolysis Effects 0.000 title claims abstract description 20
- 238000006460 hydrolysis reaction Methods 0.000 title claims abstract description 20
- 239000010902 straw Substances 0.000 title claims abstract description 12
- 240000001462 Pleurotus ostreatus Species 0.000 claims abstract description 35
- 235000001603 Pleurotus ostreatus Nutrition 0.000 claims abstract description 35
- 238000000855 fermentation Methods 0.000 claims abstract description 12
- 230000004151 fermentation Effects 0.000 claims abstract description 12
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000001099 ammonium carbonate Substances 0.000 claims abstract description 9
- 235000012501 ammonium carbonate Nutrition 0.000 claims abstract description 9
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 9
- 238000003860 storage Methods 0.000 claims abstract description 5
- 238000010563 solid-state fermentation Methods 0.000 claims abstract description 4
- 239000000843 powder Substances 0.000 claims description 41
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- 108010059892 Cellulase Proteins 0.000 claims description 29
- 239000007788 liquid Substances 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 25
- 229940106157 cellulase Drugs 0.000 claims description 21
- 238000005303 weighing Methods 0.000 claims description 13
- 235000007685 Pleurotus columbinus Nutrition 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 11
- 230000001954 sterilising effect Effects 0.000 claims description 11
- 230000000694 effects Effects 0.000 claims description 10
- 239000007974 sodium acetate buffer Substances 0.000 claims description 10
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 230000003301 hydrolyzing effect Effects 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 229920002678 cellulose Polymers 0.000 abstract description 10
- 239000001913 cellulose Substances 0.000 abstract description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 abstract description 6
- 239000002028 Biomass Substances 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 4
- 239000002351 wastewater Substances 0.000 abstract description 4
- 235000014633 carbohydrates Nutrition 0.000 abstract description 3
- 150000001720 carbohydrates Chemical class 0.000 abstract description 3
- 239000003112 inhibitor Substances 0.000 abstract description 3
- 238000005406 washing Methods 0.000 abstract description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 abstract description 2
- 239000003513 alkali Substances 0.000 abstract description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000002994 raw material Substances 0.000 description 4
- 229920002488 Hemicellulose Polymers 0.000 description 3
- 239000002029 lignocellulosic biomass Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 230000007797 corrosion Effects 0.000 description 2
- 238000005260 corrosion Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 229920005610 lignin Polymers 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108010084185 Cellulases Proteins 0.000 description 1
- 102000005575 Cellulases Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 238000012824 chemical production Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
-
- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/59—Biological synthesis; Biological purification
Abstract
The invention discloses a method for improving reducing sugar produced by corn straw hydrolysis by biological combined ammoniation pretreatment. Relates to the technical field of biomass energy. The method comprises the following steps: solid state fermentation of oyster mushrooms; and (5) ammoniation pretreatment. Compared with the traditional biological pretreatment, the method shortens the fermentation time, improves the enzymolysis rate and reduces the loss of carbohydrate. The ammonium carbonate is adopted as an ammoniating agent, is different from ammonia water, has lower price, lower toxicity and corrosiveness, does not need special equipment in storage and transportation, and has higher safety. Compared with the alkali pretreatment with sodium hydroxide as the main component, the method has the advantages of milder condition, no need of washing, no generation of a large amount of wastewater, low requirement on equipment and more environmental friendliness. The method has the advantages of little loss of cellulose components, high yield of reducing sugar, no inhibitor generation and more contribution to subsequent fermentation.
Description
Technical Field
The invention relates to the technical field of biomass energy, in particular to a method for improving reducing sugar produced by corn straw hydrolysis through biological combined ammoniation pretreatment.
Background
Currently, energy shortage has become a critical problem in the world, and biomass energy is one of the important schemes for solving energy problems as a renewable and recyclable energy source available worldwide. Lignocellulosic biomass has become a major source of biofuels due to its low cost, wide sources, large reserves, and renewable resources. Lignocellulosic biomass is composed primarily of cellulose, hemicellulose, and lignin, wherein cellulose and hemicellulose are potential sources of fermentable sugars, and lignin intertwines between cellulose and hemicellulose to form a natural physical barrier, thereby reducing accessibility of cellulose to cellulose by cellulases. In order to achieve high value utilization of lignocellulosic biomass, it is necessary to employ efficient pretreatment methods to overcome the recalcitrance of natural biomass and increase the accessibility of cellulose to cellulase hydrolysis.
The pretreatment methods commonly employed mainly include three types, namely chemical, physical and biological pretreatment. Biological pretreatment is paid attention to because of the characteristics of environmental protection, simple operation and the like, but the defects of long fermentation period, high carbohydrate loss, low sugar yield and the like of the treatment technology still exist. The alkaline pretreatment in the chemical pretreatment has the defects of environmental pollution, inhibitor generation, equipment corrosion and the like.
Therefore, how to provide a method for improving the yield of reducing sugar by hydrolysis of corn straw is a problem which needs to be solved by the person skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a method for improving reducing sugar produced by corn stalk hydrolysis by biological combined ammoniation pretreatment. Solves the problems of long time consumption, low efficiency, high compound loss, corrosion resistance equipment required in the traditional treatment, complicated and complex wastewater production and post-treatment process, environmental pollution and the like in the biological pretreatment.
Ammonia has low toxicity and corrosiveness, high volatility and easy recycling, and can improve the enzymolysis rate of cellulose by changing the crystallinity and the morphology of cellulose. The ammoniation agent adopted by the invention has more advantages in the aspects of safety, transportation, storage and the like, and the yield of reducing sugar can be obviously improved by treating the corn straw with a small amount of ammonium carbonate after the corn straw is subjected to oyster mushroom solid state fermentation. In addition, the invention reduces the discharge cost of a large amount of washing wastewater, does not need large-scale equipment, has simple operation and is more beneficial to commercial production.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for improving reducing sugar produced by corn stalk hydrolysis by biological combined ammoniation pretreatment comprises the following steps:
(1) Solid state fermentation of oyster mushrooms: weighing a certain amount of corn stalk powder, regulating the moisture content, sterilizing, inoculating oyster mushroom seed liquid, fermenting, culturing and drying to obtain corn stalk powder subjected to biological pretreatment;
(2) Ammoniation pretreatment: weighing a certain amount of corn stalk powder subjected to biological pretreatment, an ammoniating agent and water, uniformly stirring, storing under a closed condition, drying after ammoniation is finished to obtain corn stalk powder subjected to ammoniation pretreatment, and adding cellulase for hydrolysis to obtain a reducing sugar solution.
Further, the step (1) is to adjust the water content by deionized water; the fermenting culture and storage equipment is a constant temperature incubator;
and (3) sealing conditions: is placed in a tightly pressed and closed container.
Preferably: the grain diameter of the corn stalk powder in the step (1) is 20-40 meshes;
adjusting the moisture content to 80%;
and (3) sterilization: autoclaving is adopted: the pressure is 100kPa, the temperature is 121 ℃, and the sterilization time is 40 minutes;
the strain of oyster mushroom: the preservation number of Pleurotus ostreatus (Pleurotus ostreatus) is CGMCC 5.930;
further, the Pleurotus ostreatus (Pleurotus ostreatus) has a collection number of CGMCC 5.930 and is purchased from China general microbiological culture Collection center.
The preparation method of the oyster mushroom seed liquid comprises the following steps: inoculating Pleurotus ostreatus to PDA culture medium, activating for 7d, inoculating 3 bacterial cakes with diameter of 0.5cm to PDA liquid culture medium, and culturing at 25deg.C at rotation speed of 160r/min in dark place for 7d; when in use, homogenate is carried out for 5 to 10 seconds;
the mass ratio of the oyster mushroom seed liquid to the oyster mushroom seed liquid is 10%;
fermentation temperature: constant temperature is 28 ℃ and time is 14-28 days.
Preferably: the mass ratio of the corn stalk powder and the ammoniation agent of the biological pretreatment in the step (2) is 1:0.05 to 0.2 percent, and the solid content of the corn stalk powder which is biologically pretreated is 30 to 70 percent;
and (3) storing: constant temperature is 30 ℃ for 7-11 days;
the cellulase: 0.01g/mL of cellulase solution;
cellulase enzyme activity: 100FPU/g;
the hydrolysis also needs to add acetic acid-sodium acetate buffer solution with pH value of 4.8 and 0.05 mol/L;
the volume ratio of the cellulase solution to the acetic acid-sodium acetate buffer solution is 3:7, preparing a base material;
corn stalk powder and cellulase after ammoniation pretreatment in mass ratio: 0.2g:30mg;
conditions of hydrolysis: hydrolyzing in a water bath shaker at 50deg.C and rotational speed of 150r/min for 48h.
Further, the solid content of the biological pretreatment corn stalk powder=the biological pretreatment corn stalk powder mass/(the biological pretreatment corn stalk powder mass+the water mass);
the enzyme activity of the cellulase is 100FPU/g, 15FPU cellulase (namely 15 FPU/g) is needed for each gram of corn straw during enzymolysis, when the cellulase is used, the solution prepared into 0.01g/mL is added into an enzymolysis system, namely 3mL (0.01 g/mL) of cellulase solution and 7mL of pH value of 4.8 (0.05 mol.L) are added into 0.2g of corn straw -1 ) Acetic acid-sodium acetate buffer solution of (a).
Preferably: the ammoniating agent in the step (2) is ammonium carbonate.
Preferably: and (2) after hydrolysis, further comprises centrifugation: 10000r/min, time 10min.
The invention also provides application of the method in biological energy, environmental protection and chemical production.
Compared with the prior art, the invention discloses a method for improving the reducing sugar produced by corn straw hydrolysis by biological combined ammoniation pretreatment, which has the following technical effects:
1. compared with the traditional biological pretreatment, the method shortens the fermentation time, improves the enzymolysis rate and reduces the loss of carbohydrate.
2. The ammonium carbonate is adopted as an ammoniating agent, is different from ammonia water, has lower price, lower toxicity and corrosiveness, does not need special equipment in storage and transportation, and has higher safety.
3. Compared with the alkali pretreatment with sodium hydroxide as the main component, the method has the advantages of milder condition, no need of washing, no generation of a large amount of wastewater, low requirement on equipment and more environmental friendliness.
4. The method has the advantages of little loss of cellulose components, high yield of reducing sugar, no inhibitor generation and more contribution to subsequent fermentation.
Detailed Description
The following description will clearly and fully describe the technical solutions of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention discloses a method for improving reducing sugar produced by corn straw hydrolysis through biological combined ammoniation pretreatment.
The raw materials and equipment not mentioned in the examples are conventional commercially available raw materials and equipment, for example: the Pleurotus ostreatus (Pleurotus ostreatus) with a preservation number of CGMCC 5.930 is purchased from China general microbiological culture Collection center; and will not be described in detail herein.
The preparation method of the oyster mushroom seed liquid comprises the following steps: inoculating Pleurotus ostreatus to PDA culture medium, activating for 7d, inoculating 3 bacterial cakes with diameter of 0.5cm to PDA liquid culture medium with puncher, and culturing at 25deg.C and rotation speed of 160r/min in dark place for 7d; when in use, the slurry is homogenized for 5 to 10 seconds.
Example 1
Weighing 10g of corn stalks of 20-40 meshes, adding deionized water into a 250mL conical flask to adjust the water content to 80%, sterilizing by high-pressure steam (the pressure is 100kPa, the temperature is 121 ℃) for 40min, inoculating 1mL of oyster mushroom liquid seed liquid, placing the inoculated oyster mushroom liquid seed liquid into a constant-temperature incubator of 28 ℃ for 21 days, and drying after fermentation is finished to obtain corn stalk powder subjected to biological pretreatment;
according to 50% of solid content { solid content=mass of corn stalk powder of biological pretreatment/(mass of corn stalk powder of biological pretreatment+water mass) }, 10% (w/w) of ammonium carbonate of the mass of corn stalk powder of biological pretreatment is dissolved in required water and then mixed with corn stalk powder of biological pretreatment, and the mixture is placed in a closed container after being stirred uniformly, stored in a constant temperature incubator at 30 ℃ for 11 days, and dried after the completion;
weighing 0.2g of corn stalk after ammoniation pretreatment, 3mL of 0.01g/mL of cellulase solution (cellulase enzyme activity: 100 FPU/g) and 7mL of pH value of 4.8 (0.05 mol.L) -1 ) The acetic acid-sodium acetate buffer solution of (2) was placed in a 50mL centrifuge tube, hydrolyzed in a 150r/min water bath shaker at 50℃for 48h, the product was centrifuged (10000 r/min, time 10 min), and the yield of reducing sugar was measured by the DNS method at 311mg/g.
Example 2
Weighing 10g of corn stalks of 20-40 meshes, adding deionized water into a 250mL conical flask to adjust the water content to 80%, sterilizing by high-pressure steam (the pressure is 100kPa, the temperature is 121 ℃) for 40min, inoculating 1mL of oyster mushroom liquid seed liquid, placing the inoculated oyster mushroom liquid seed liquid into a constant-temperature incubator of 28 ℃ for 14 days, and drying after fermentation is finished to obtain corn stalk powder subjected to biological pretreatment;
according to 50% of solid content { solid content=mass of corn stalk powder of biological pretreatment/(mass of corn stalk powder of biological pretreatment+water mass) }, 15% (w/w) of ammonium carbonate of the mass of corn stalk powder of biological pretreatment is dissolved in required water and then is mixed with corn stalk powder of biological pretreatment, after being stirred uniformly, the mixture is placed in a closed container, stored in a constant temperature incubator at 30 ℃ for 7 days, and after the completion, the mixture is dried;
weighing 0.2g of corn stalk after ammoniation pretreatment, 3mL of 0.01g/mL of cellulase solution (cellulase enzyme activity: 100 FPU/g) and 7mL of pH value of 4.8 (0.05 mol.L) -1 ) The acetic acid-sodium acetate buffer solution of (2) was placed in a 50mL centrifuge tube, hydrolyzed in a 150r/min water bath shaker at 50℃for 48h, the product was centrifuged (10000 r/min, time 10 min), and the yield of reducing sugar was 301mg/g by the DNS method.
Example 3
Weighing 10g of corn stalks of 20-40 meshes, adding deionized water into a 250mL conical flask to adjust the water content to 80%, sterilizing by high-pressure steam (the pressure is 100kPa, the temperature is 121 ℃) for 40min, inoculating 1mL of oyster mushroom liquid seed liquid, placing the inoculated oyster mushroom liquid seed liquid into a 28 ℃ constant-temperature incubator for 28 days, and drying after fermentation is finished to obtain corn stalk powder subjected to biological pretreatment;
according to 50% of solid content { solid content=mass of corn stalk powder of biological pretreatment/(mass of corn stalk powder of biological pretreatment+water mass) }, 15% (w/w) of ammonium carbonate of the mass of corn stalk powder of biological pretreatment is dissolved in required water and then is mixed with corn stalk powder of biological pretreatment, after being stirred uniformly, the mixture is placed in a closed container, stored in a constant temperature incubator at 30 ℃ for 11 days, and after the completion, the mixture is dried;
weighing 0.2g of corn stalk after ammoniation pretreatment, 3mL of 0.01g/mL of cellulase solution (cellulase enzyme activity: 100 FPU/g) and 7mL of pH value of 4.8 (0.05 mol.L) -1 ) The acetic acid-sodium acetate buffer solution of (2) was placed in a 50mL centrifuge tube, hydrolyzed in a 150r/min water bath shaker at 50℃for 48h, the product was centrifuged (10000 r/min, time 10 min), and the yield of reducing sugar was 374mg/g by the DNS method.
Example 4
Weighing 10g of corn stalks of 20-40 meshes, adding deionized water into a 250mL conical flask to adjust the water content to 80%, sterilizing by high-pressure steam (the pressure is 100kPa, the temperature is 121 ℃) for 40min, inoculating 1mL of oyster mushroom liquid seed liquid, placing the inoculated oyster mushroom liquid seed liquid into a 28 ℃ constant-temperature incubator for 28 days, and drying after fermentation is finished to obtain corn stalk powder subjected to biological pretreatment;
according to 50% of solid content { solid content=mass of corn stalk powder of biological pretreatment/(mass of corn stalk powder of biological pretreatment+water mass) }, 10% (w/w) of ammonium carbonate of the mass of corn stalk powder of biological pretreatment is dissolved in required water and then mixed with corn stalk powder of biological pretreatment, and the mixture is placed in a closed container after being stirred uniformly, stored in a constant temperature incubator at 30 ℃ for 9 days, and dried after the completion;
weighing 0.2g of corn stalk after ammoniation pretreatment, 3mL of 0.01g/mL of cellulase solution (cellulase enzyme activity: 100 FPU/g) and 7mL of pH value of 4.8 (0.05 mol.L) -1 ) The acetic acid-sodium acetate buffer solution of (2) was placed in a 50mL centrifuge tube, hydrolyzed in a 150r/min water bath shaker at 50℃for 48h, the product was centrifuged (10000 r/min, time 10 min), and the yield of reducing sugar was determined by the DNS method to be 328mg/g.
Comparative experiments
1. Compared with the raw materials which are not subjected to ammoniation pretreatment:
0.2g of corn stalk and 3mL of 0.01g/mL cellulase solution (cellulase enzyme activity: 100 FPU/g) are weighed and put into a 50mL centrifuge tube, hydrolyzed for 48 hours in a water bath shaker at 50 ℃ and 150r/min, the product is centrifuged, and the supernatant is sucked to measure the yield of the reducing sugar of 205mg/g by a DNS method.
2. Adjusting the process steps and the raw material proportion:
weighing 10g of corn stalks of 20-40 meshes, adding deionized water into a 250mL conical flask to adjust the water content to 80%, sterilizing by high-pressure steam (pressure 100kPa, temperature 121 ℃) for 40min, inoculating 1mL of oyster mushroom liquid seed liquid, placing the mixture into a constant-temperature incubator of 28 ℃ for 28 days, and drying after fermentation is finished to obtain the corn stalk powder subjected to biological pretreatment.
0.2g of corn stalk powder subjected to biological pretreatment, 3mL of 0.01g/mL cellulase solution (cellulase enzyme activity: 100 FPU/g) and 7mL of acetic acid-sodium acetate buffer solution with pH value of 4.8 (0.05 mol.L-1) are weighed, placed into a 50mL centrifuge tube, hydrolyzed for 48 hours in a water bath shaker at 50 ℃ and 150r/min, the product is centrifuged, and the supernatant is sucked to measure the yield of reducing sugar to be 210mg/g through a DNS method.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (2)
1. A method for improving reducing sugar produced by corn stalk hydrolysis through biological combined ammoniation pretreatment is characterized by comprising the following steps:
(1) Solid state fermentation of oyster mushrooms: weighing a certain amount of corn stalk powder, regulating the moisture content, sterilizing, inoculating oyster mushroom seed liquid, fermenting, culturing and drying to obtain corn stalk powder subjected to biological pretreatment;
(2) Ammoniation pretreatment: weighing a certain amount of corn stalk powder subjected to biological pretreatment, an ammoniating agent and water, uniformly stirring, storing under a closed condition, drying after ammoniation is finished to obtain corn stalk powder subjected to ammoniation pretreatment, and adding cellulase for hydrolysis to obtain a reducing sugar solution;
the grain diameter of the corn stalk powder in the step (1) is 20-40 meshes;
said adjusting the moisture content to 80%;
the sterilization: autoclaving is adopted: the pressure is 100kPa, the temperature is 121 ℃, and the sterilization time is 40 minutes;
the strain of the oyster mushroom comprises the following steps: the preservation number of Pleurotus ostreatus (Pleurotus ostreatus) is CGMCC 5.930;
the preparation method of the oyster mushroom seed liquid comprises the following steps: inoculating Pleurotus ostreatus to PDA culture medium, activating for 7d, inoculating 3 bacterial cakes with diameter of 0.5cm to PDA liquid culture medium, and culturing at 25deg.C at rotation speed of 160r/min in dark place for 7d; when in use, homogenate is carried out for 5 to 10 seconds;
the mass ratio of the oyster mushroom seed liquid to the oyster mushroom seed liquid is 10%;
fermentation temperature: constant temperature is 28 ℃ for 14-28 days;
the mass ratio of the corn stalk powder and the ammoniating agent of the biological pretreatment in the step (2) is 1:0.05 to 0.2 percent, and the solid content of the corn stalk powder which is biologically pretreated is 50 percent;
the storage is as follows: constant temperature is 30 ℃ for 7-11 days;
the cellulase: 0.01g/mL of cellulase solution;
the cellulase enzyme activity: 100FPU/g;
the hydrolysis also needs to be added with acetic acid-sodium acetate buffer solution with the pH value of 4.8 and 0.05 mol/L;
the volume ratio of the cellulase solution to the acetic acid-sodium acetate buffer solution is 3:7, preparing a base material;
the mass ratio of the corn stalk powder and the cellulase after the ammoniation pretreatment is as follows: 0.2g:30mg;
the conditions of the hydrolysis: hydrolyzing in a water bath shaker at 50 ℃ and a rotating speed of 150r/min for 48 hours;
the ammoniation agent in the step (2) is ammonium carbonate.
2. The method for improving the hydrolysis production of reducing sugar by corn straw by biological combined ammoniation pretreatment according to claim 1, which is characterized by comprising the following steps: and (2) after hydrolysis, further comprises centrifugation: 10000r/min, time 10min.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2565433A1 (en) * | 2006-10-25 | 2008-04-25 | Iogen Energy Corporation | Inorganic salt recovery during processing of lignocellulosic feedstocks |
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| CN107177637A (en) * | 2017-06-15 | 2017-09-19 | 沈阳农业大学 | The methods and applications pre-processed using microorganism to maize straw hydrolysis and saccharification |
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