CN115521957A - Lignocellulose whole cell standing saccharification technology - Google Patents
Lignocellulose whole cell standing saccharification technology Download PDFInfo
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- 238000005516 engineering process Methods 0.000 title claims abstract description 39
- 239000000758 substrate Substances 0.000 claims abstract description 49
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims abstract description 19
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims abstract description 19
- 239000001099 ammonium carbonate Substances 0.000 claims abstract description 19
- 238000003756 stirring Methods 0.000 claims abstract description 19
- 230000001954 sterilising effect Effects 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 12
- 239000007787 solid Substances 0.000 claims abstract description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 9
- 239000008103 glucose Substances 0.000 claims abstract description 9
- 230000003301 hydrolyzing effect Effects 0.000 claims abstract description 8
- 238000005507 spraying Methods 0.000 claims abstract description 5
- 238000000855 fermentation Methods 0.000 claims abstract description 4
- 230000004151 fermentation Effects 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 33
- 239000001963 growth medium Substances 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 21
- 238000011218 seed culture Methods 0.000 claims description 17
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 12
- 229920000324 Cellulosome Polymers 0.000 claims description 11
- 210000000166 cellulosome Anatomy 0.000 claims description 11
- 239000002994 raw material Substances 0.000 claims description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 10
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 10
- 230000003698 anagen phase Effects 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 9
- 239000007789 gas Substances 0.000 claims description 8
- 241000193448 Ruminiclostridium thermocellum Species 0.000 claims description 7
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 7
- 239000010902 straw Substances 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 6
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 6
- 239000011790 ferrous sulphate Substances 0.000 claims description 6
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 6
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 6
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 6
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 6
- 241000209140 Triticum Species 0.000 claims description 4
- 235000021307 Triticum Nutrition 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 claims description 4
- 102000004366 Glucosidases Human genes 0.000 claims description 3
- 108010056771 Glucosidases Proteins 0.000 claims description 3
- 229920002488 Hemicellulose Polymers 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 229920005610 lignin Polymers 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 229910052979 sodium sulfide Inorganic materials 0.000 claims description 3
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 claims description 3
- 230000001360 synchronised effect Effects 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000009825 accumulation Methods 0.000 claims description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 2
- 235000018417 cysteine Nutrition 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims 1
- 238000005265 energy consumption Methods 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract 2
- 230000000052 comparative effect Effects 0.000 description 18
- 229910001873 dinitrogen Inorganic materials 0.000 description 6
- 230000036983 biotransformation Effects 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 229920002678 cellulose Polymers 0.000 description 4
- 239000001913 cellulose Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- 239000011942 biocatalyst Substances 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- AEQDJSLRWYMAQI-UHFFFAOYSA-N 2,3,9,10-tetramethoxy-6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinoline Chemical compound C1CN2CC(C(=C(OC)C=C3)OC)=C3CC2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000176 sodium gluconate Substances 0.000 description 1
- 235000012207 sodium gluconate Nutrition 0.000 description 1
- 229940005574 sodium gluconate Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/145—Clostridium
-
- 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
Abstract
The invention provides a lignocellulose whole cell standing saccharification technology, which comprises four steps of (1) pretreatment, (2) seed liquid preparation, (3) saccharification system preparation and (4) saccharification. In the step (3), the substrate and the seed liquid are synchronously added into the saccharification system, and the substrate and the cells in the seed liquid can be effectively promoted to be effectively mixed by spraying the seed liquid to the solid substrate. Meanwhile, when a fermentation tank is adopted to prepare the saccharification system, sterile ammonium bicarbonate is supplemented into the system after sterilization. And (4) standing and hydrolyzing (saccharifying) at the temperature of 55-65 ℃ to obtain a sugar solution containing glucose. The lignocellulose whole-cell standing saccharification technology not only eliminates the requirement of the lignocellulose whole-cell saccharification technology on stirring, solves the problem of high energy consumption in the prior art, but also overcomes the technical bias, realizes the remarkable improvement of the sugar yield, and produces the unpredictable technical effect.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a lignocellulose whole-cell saccharification method, in particular to a lignocellulose whole-cell standing saccharification technology without stirring.
Background
Lignocellulose is the biomass raw material with the largest reserve in nature, the high-efficiency biotransformation from lignocellulose to bio-based chemicals and energy is realized, the increasingly severe fossil energy crisis can be continuously relieved, and the environmental pollution caused by improper treatment of agricultural and forestry wastes can be effectively avoided, so that the requirement of developing green circular economy in China is met. However, due to the complex structure and composition of lignocellulose, the difficulty of biotransformation is great; the biggest bottleneck is higher than the cellulose crystallization area, so that the cellulose crystallization area is difficult to degrade, the enzymolysis efficiency is low, and the biotransformation cost is always high. In order to achieve efficient use of lignocellulose, it is imperative to achieve efficient hydrolytic saccharification of lignocellulosic substrates.
Integrated biological saccharification is a new strategy for lignocellulose biotransformation, microorganisms capable of realizing in-situ enzyme production such as Clostridium thermocellum (Clostridium thermocellum) and the like are used as high-efficiency whole-cell catalysts, low-cost fermentation sugar is used as a target product, and the types and markets of lignocellulose biotransformation products are greatly widened by coupling a downstream fermentation process. Previous work by the inventors was around integrated biological saccharification, as disclosed in patent nos. ZL201810939181.2, ZL201810939517.5, ZL201810939518.X, ZL201810939182.7, ZL201810939294.2, ZL201810939170.4 and ZL201810939296.1, which disclose the use of integrated biological saccharification technology to achieve conversion of lignocellulose into products such as sodium gluconate, fats, pigments, biofuels, etc.
Currently, lignocellulosic whole cell saccharification protocols use live cells of clostridium thermocellum as biocatalysts. Clostridium thermocellum is a strictly anaerobic bacterium that achieves efficient degradation of lignocellulosic substrates by producing extracellular cellulosomes. As is well known to those skilled in the art, a cellulosome is a supramolecular protein complex with a large molecular weight and a poor ability to migrate during degradation ("A biological nanomachine at work: fetching the cellulose derivative crystalline cellulose. ACS Central Science 2020.6. Therefore, whether the mixing of the biocatalyst and the substrate is sufficient during the saccharification greatly influences the saccharification efficiency. At present, it is common practice to solve this problem by achieving thorough mixing of the cellulosome and the lignocellulosic substrate by means of agitation. However, in the reaction system of the current lignocellulose whole cell saccharification technology, the solid content of lignocellulose is usually 5-20%; at this time, stirring causes a sharp increase in energy consumption.
Disclosure of Invention
Based on the current situation of high energy consumption in the whole-cell saccharification process of lignocellulose in the prior art, the invention provides a whole-cell catalyst inoculation and saccharification method without stirring. The method not only eliminates the requirement of the lignocellulose whole cell saccharification technology on stirring and reduces energy consumption, but also realizes the remarkable improvement of the sugar yield and produces unpredictable technical effects.
The technical scheme of the invention is as follows: the lignocellulose whole cell standing saccharification technology comprises the following steps:
(1) Pretreatment: pretreating a lignocellulosic feedstock to obtain a lignocellulosic substrate having a lignin content of no more than 10% and a hemicellulose content of no more than 20%, and then washing with water to a pH =7.0-8.0. The lignocellulose raw materials are corn straws, wheat straws, pasture and xylose residues; the pretreatment is an alkaline process.
(2) Preparing a seed solution: under anaerobic condition, inoculating the strain capable of producing fibrosome into seed culture medium, and culturing at 55-65 deg.C and 100-200rpm to logarithmic growth metaphase to obtain seed liquid. The described cellulosome-producing strain is Clostridium thermocellum recombinant strain for expressing glucosidase. The seed culture medium is as follows: 0.6-2.9 g/L dipotassium phosphate, 0.3-1.5g/L potassium dihydrogen phosphate, 1.0g/L ammonium bicarbonate, 0.1g/L calcium chloride, 0.5g/L magnesium chloride, 0.5mg/L ferrous sulfate, 1g/L cysteine, 3g/L yeast powder, 0.7g/L lignocellulose substrate obtained in the step (1), and pH 7.5.
(3) Preparing a saccharification system: the lignocellulose substrate obtained in the step (1) is firstly sterilized at 126 ℃ for 30-40 minutes and then independently sterilized, the substrate is spread in a multilayer way in the sterilization process, and the thickness of each layer of substrate is not more than 1cm, so that the sterilization efficiency is improved, the sterilization time is shortened, and the pollution probability of mixed bacteria and spores in the substrate is reduced. Subsequently, the lignocellulosic substrate is transferred under aseptic conditions to a fermentor containing a pre-sterilized saccharification medium. The substrate is mixed at a speed of 10-50 rpm during the substrate addition process to prevent the substrate from being locally accumulated. And (3) inoculating and uniformly mixing the seed liquid obtained in the step (2) in a synchronous spraying manner to obtain a saccharification system. Finally, the inoculation amount of the seed liquid in the system is 10% (v/v), and the solid content of the lignocellulose substrate is 5-20% (w/v). Then nitrogen is introduced into the gas layer of the system to replace air, and sterile ammonium bicarbonate is supplemented by 0.2-1.0 g/L.
Wherein, the saccharification culture medium is as follows: dipotassium hydrogen phosphate 0.6g/L, potassium dihydrogen phosphate 0.3g/L, ammonium bicarbonate 0.5g/L, calcium chloride 0.1g/L, magnesium chloride 0.5g/L, ferrous sulfate 0.5mg/L, sodium sulfide 0.5g/L, corn steep liquor 4g/L, and pH 7.5. The pre-sterilization conditions of the saccharification culture medium are as follows: sterilized at 121 ℃ for 20 minutes. The substrate and the seed liquid are synchronously added into the saccharification system, and the substrate and the cells in the seed liquid can be effectively promoted to be effectively mixed by spraying the seed liquid to the solid substrate. Since ammonium bicarbonate is easily lost during non-hermetic sterilization due to the formation of carbon dioxide and ammonia gas, when a saccharification system is prepared using a fermenter, sterile ammonium bicarbonate is replenished into the system after sterilization.
(4) Saccharification: and (3) standing and hydrolyzing (saccharifying) the saccharification system prepared in the step (3) at the temperature of 55-65 ℃ to obtain a glucose-containing sugar solution. When the sugar solution concentration in the system is not changed any more for two consecutive days, the saccharification is finished. The standing saccharification can reduce the energy consumption cost in the production process, and has very important significance for large-scale industrial production. Meanwhile, the inventors unexpectedly found that the saccharification system prepared according to the steps and conditions described in the step (3) can realize rapid sugar accumulation under the standing condition, and the sugar yield is remarkably improved. The inventors speculate that this is because stationary saccharification can slow down the metabolic level of the strain and reduce the level of the cells converting the substrate into metabolic end products such as acetic acid, ethanol, carbon dioxide and the like.
The invention has the beneficial effects that:
(1) According to the lignocellulose whole-cell saccharification technology, the saccharification stage does not need material stirring, the problem of high energy consumption in the prior art is solved, and the lignocellulose whole-cell saccharification technology has extremely important significance for industrial production.
(2) By adopting the lignocellulose whole-cell saccharification technology, the saccharification system prepared by specific steps and conditions overcomes the technical prejudice that the saccharification step in the prior art needs stirring, obtains higher sugar yield and produces unpredictable technical effects.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: the lignocellulose whole cell standing saccharification technology comprises the following steps:
(1) Pretreatment: pretreating a lignocellulosic feedstock to obtain a lignocellulosic substrate having a lignin content of no more than 10% and a hemicellulose content of no more than 20%, and then washing with water to a pH =7.0-8.0. The lignocellulose raw material is corn straw; the pretreatment is an alkaline method.
(2) Preparing a seed solution: inoculating the strain producing the cellulosome into a seed culture medium under the anaerobic condition, and culturing to the middle logarithmic growth phase under the temperature condition of 55 ℃ and the rotating speed condition of 200rpm to obtain a seed solution. The cellulosome-producing strain is a clostridium thermocellum recombinant strain for expressing glucosidase. The seed culture medium is as follows: 0.6g/L of dipotassium hydrogen phosphate, 0.3g/L of potassium dihydrogen phosphate, 1.0g/L of ammonium bicarbonate, 0.1g/L of calcium chloride, 0.5g/L of magnesium chloride, 0.5mg/L of ferrous sulfate, 1g/L of cysteine, 3g/L of yeast powder, 0.7g/L of the lignocellulose substrate obtained in the step (1), and pH 7.5.
(3) Preparing a saccharification system: and (2) sterilizing the lignocellulose substrate obtained in the step (1) at 126 ℃ for 30-40 minutes, and sterilizing separately, wherein the substrate is spread in a multilayer way in the sterilization process, and the thickness of each layer of substrate is not more than 1cm. Subsequently, the lignocellulosic substrate is transferred under aseptic conditions to a fermentor containing a pre-sterilized saccharification medium. The mixing was carried out at a rate of 10 revolutions per minute during the addition of the substrate. And (3) simultaneously inoculating and uniformly mixing the seed liquid obtained in the step (2) in a synchronous spraying manner to obtain a saccharification system. The inoculation amount of the seed liquid in the final system is 10% (v/v), and the solid content of the lignocellulose substrate is 5% (w/v). Then, nitrogen gas was introduced into the gas layer of the system to displace air, and sterile ammonium bicarbonate was supplied in an amount of 0.2g/L.
Wherein, the saccharification culture medium is as follows: dipotassium hydrogen phosphate 0.6g/L, potassium dihydrogen phosphate 0.3g/L, ammonium bicarbonate 0.5g/L, calcium chloride 0.1g/L, magnesium chloride 0.5g/L, ferrous sulfate 0.5mg/L, sodium sulfide 0.5g/L, corn steep liquor 4g/L, and pH 7.5. The pre-sterilization conditions of the saccharification culture medium are as follows: sterilized at 121 ℃ for 20 minutes.
(4) Saccharification: and (4) standing the saccharification system prepared in the step (3) at the temperature of 55 ℃ for hydrolysis (saccharification) to obtain a sugar solution containing glucose. When the sugar solution concentration in the system is not changed any more for two consecutive days, the saccharification is finished.
Comparative example 1: adopts the conventional lignocellulose whole cell saccharification technology
Unlike example 1, the saccharification of step (4) was carried out under the condition of a stirring rotation speed of 100 revolutions per minute.
Example 2: lignocellulose whole cell standing saccharification technology
In contrast to the embodiment 1, the process,
(1) Pretreatment: the same as in example 1.
(2) Preparing a seed solution: under the anaerobic condition, the cellulosome-producing strain is inoculated in a seed culture medium and cultured to the middle logarithmic growth phase under the temperature condition of 60 ℃ and the rotating speed condition of 170rpm to obtain seed liquid. The seed culture medium contains 2.9g/L dipotassium phosphate and 1.5g/L potassium dihydrogen phosphate.
(3) Preparing a saccharification system: the final solids content of the lignocellulosic substrate was 10% (w/v). Then, nitrogen gas was introduced into the gas layer of the system to displace air, and sterile ammonium bicarbonate was supplied in an amount of 0.5g/L.
(4) Saccharification: and (3) standing and hydrolyzing (saccharifying) the saccharification system prepared in the step (3) at the temperature of 60 ℃ to obtain a glucose-containing sugar solution. When the sugar solution concentration in the system is not changed any more for two consecutive days, the saccharification is finished.
Comparative example 2: adopts the conventional lignocellulose whole cell saccharification technology
Unlike example 2, the saccharification of step (4) was carried out under a stirring speed of 150 rpm.
Example 3: lignocellulose whole cell standing saccharification technology
In contrast to the embodiment 1, the process,
(1) Pretreatment: the lignocellulose raw material is wheat straw;
(2) Preparing a seed solution: under the anaerobic condition, the strain capable of producing the fibrosome is inoculated in a seed culture medium and cultured to the middle logarithmic growth phase under the temperature condition of 55 ℃ and the rotating speed condition of 170rpm to obtain a seed solution.
(3) Preparing a saccharification system: sterile ammonium bicarbonate 0.4g/L was supplemented.
(4) Saccharification: the same as in example 1.
Comparative example 3: adopts the conventional lignocellulose whole cell saccharification technology
Unlike example 3, the saccharification of step (4) was carried out under the condition of a stirring rotation speed of 100 revolutions per minute.
Example 4: lignocellulose whole cell standing saccharification technology
In contrast to the embodiment 1, the process,
(1) Pretreatment: the lignocellulose raw material is wheat straw;
(2) Preparing a seed solution: inoculating the strain producing the cellulosome into a seed culture medium under the anaerobic condition, and culturing to the middle logarithmic growth phase under the temperature condition of 60 ℃ and the rotating speed condition of 200rpm to obtain a seed solution. The seed culture medium contains 2.9g/L dipotassium phosphate and 1.5g/L potassium dihydrogen phosphate.
(3) Preparing a saccharification system: mixing was carried out at a rate of 50 revolutions per minute during the substrate addition. The final solids content of the lignocellulosic substrate was 10% (w/v). Then, nitrogen gas was introduced into the gas layer of the system to displace air, and sterile ammonium bicarbonate was supplied thereto at 1.0g/L.
(4) Saccharification: and (4) standing the saccharification system prepared in the step (3) at the temperature of 60 ℃ for hydrolysis (saccharification) to obtain a sugar solution containing glucose. When the sugar solution concentration in the system is not changed any more for two consecutive days, the saccharification is finished.
Comparative example 4: adopts the conventional lignocellulose whole cell saccharification technology
Unlike example 4, the saccharification of step (4) was carried out under the condition of a stirring rotation speed of 150 revolutions per minute.
Example 5: lignocellulose whole cell standing saccharification technology
In contrast to the embodiment 1, the process of the invention,
(1) Pretreatment: the lignocellulose raw material is pasture;
(2) Preparing a seed solution: inoculating the strain producing the fibrosome into a seed culture medium under the anaerobic condition, and culturing to the middle logarithmic growth phase under the temperature condition of 60 ℃ and the rotating speed condition of 170rpm to obtain a seed solution.
(3) Preparing a saccharification system: sterile ammonium bicarbonate 0.5g/L was supplemented.
(4) Saccharification: and (3) standing and hydrolyzing (saccharifying) the saccharification system prepared in the step (3) at the temperature of 60 ℃ to obtain a glucose-containing sugar solution. When the sugar solution concentration in the system is not changed any more for two consecutive days, the saccharification is finished.
Comparative example 5: adopts the conventional lignocellulose whole cell saccharification technology
Unlike example 5, the saccharification of step (4) was carried out under the condition of a stirring rotation speed of 100 revolutions per minute.
Example 6: lignocellulose whole cell standing saccharification technology
In contrast to the embodiment 1, the process of the invention,
(1) Pretreatment: the lignocellulose raw material is pasture;
(2) Preparing a seed solution: inoculating the strain producing the cellulosome into a seed culture medium under the anaerobic condition, and culturing to the middle logarithmic growth phase under the temperature condition of 65 ℃ and the rotating speed condition of 100rpm to obtain a seed solution. The seed culture medium contains 2.9g/L dipotassium phosphate and 1.5g/L potassium dihydrogen phosphate.
(3) Preparing a saccharification system: the final solids content of the lignocellulosic substrate was 10% (w/v). Then, nitrogen gas was introduced into the gas layer of the system to displace air, and sterile ammonium bicarbonate was supplied in an amount of 0.5g/L.
(4) Saccharification: and (3) standing and hydrolyzing (saccharifying) the saccharification system prepared in the step (3) at the temperature of 65 ℃ to obtain a glucose-containing sugar solution. When the sugar solution concentration in the system is not changed any more for two consecutive days, the saccharification is finished.
Comparative example 6: adopts the conventional lignocellulose whole cell saccharification technology
Unlike example 6, the saccharification of step (4) was carried out under the condition of a stirring rotation speed of 150 revolutions per minute.
Example 7: lignocellulose whole cell standing saccharification technology
In contrast to the embodiment 1, the process of the invention,
(1) Pretreatment: the lignocellulose raw material is xylose residue;
(2) Preparing a seed solution: under the anaerobic condition, the strain capable of producing the fibrosome is inoculated in a seed culture medium and cultured to the middle logarithmic growth phase under the temperature condition of 55 ℃ and the rotating speed condition of 150rpm to obtain a seed solution.
(3) Preparing a saccharification system: the mixing was carried out at a rate of 40 revolutions per minute during the addition of the substrate. The final solids content of the lignocellulosic substrate was 20% (w/v). Then, nitrogen gas was introduced into the gas layer of the system to displace air, and sterile ammonium hydrogen carbonate was supplied in an amount of 1.0g/L.
Comparative example 7: adopts the conventional lignocellulose whole cell saccharification technology
Unlike example 7, the saccharification of step (4) was carried out under the condition of a stirring rotation speed of 100 revolutions per minute.
Example 8: lignocellulose whole cell standing saccharification technology
In contrast to the embodiment 1, the process,
(1) Pretreatment: the lignocellulose raw material is xylose residue;
(2) Preparing a seed solution: under the anaerobic condition, the strain capable of producing the fibrosome is inoculated in a seed culture medium and cultured to the middle logarithmic growth phase under the temperature condition of 60 ℃ and the rotating speed condition of 200rpm to obtain a seed solution. The seed culture medium contains 2.9g/L dipotassium phosphate and 1.5g/L potassium dihydrogen phosphate.
(3) Preparing a saccharification system: mixing was carried out at a rate of 50 revolutions per minute during the substrate addition. The final solids content of the lignocellulosic substrate was 5% (w/v). Then, nitrogen gas was introduced into the gas layer of the system to displace air, and sterile ammonium bicarbonate was supplied in an amount of 0.2g/L.
(4) Saccharification: and (3) standing and hydrolyzing (saccharifying) the saccharification system prepared in the step (3) at the temperature of 60 ℃ to obtain a glucose-containing sugar solution. And when the sugar solution concentration in the system is not changed any more for two consecutive days, the saccharification is finished.
Comparative example 8: adopts the conventional lignocellulose whole cell saccharification technology
Unlike example 8, the saccharification of step (4) was carried out under the condition of a stirring rotation speed of 150 revolutions per minute.
TABLE 1 saccharification efficiencies of examples 1-8 and comparative examples 1-8
| Standing saccharification | The sugar content is% | Conventional stirring saccharification | The sugar content is% | Increase in sugar yield |
| Example 1 | 95.20％ | Comparative example 1 | 80.40％ | 14.80％ |
| Example 2 | 90.90％ | Comparative example 2 | 82.00％ | 8.90％ |
| Example 3 | 90.80％ | Comparative example 3 | 82.80％ | 8.00％ |
| Example 4 | 92.70％ | Comparative example 4 | 79.30％ | 13.40％ |
| Example 5 | 91.60％ | Comparative example 5 | 82.30％ | 9.30％ |
| Example 6 | 93.90％ | Comparative example 6 | 82.00％ | 11.90％ |
| Example 7 | 94.70％ | Comparative example 7 | 88.30％ | 6.40％ |
| Example 8 | 99.10％ | Comparative example 8 | 87.90％ | 11.20％ |
As can be seen from Table 1, in examples 1 to 8 of the present application, which employ the whole lignocellulose cell standing saccharification technique, the sugar yield was 90.8% to 99.1%. The sugar yield of comparative examples 1 to 8 using the conventional lignocellulose whole cell saccharification technique was 79.3% to 88.3%. Therefore, compared with the conventional lignocellulose whole cell saccharification technology, the lignocellulose whole cell standing saccharification technology has the advantage that the sugar yield is improved by 6.40-14.80%. In conclusion, according to the lignocellulose whole-cell standing saccharification technology, the saccharification stage does not need material stirring, so that the technical bias that stirring is needed in the saccharification step in the prior art is overcome, a higher sugar yield is obtained, and an unpredictable technical effect is generated. In addition, the standing saccharification technology solves the requirement of high energy consumption of the prior art, and has extremely important significance for industrial production.
Claims (6)
1. The lignocellulose whole-cell standing saccharification technology is characterized in that: the method comprises the following steps:
(1) Pretreatment: pretreating a lignocellulose raw material to obtain a lignocellulose substrate with the lignin content of not more than 10% and the hemicellulose content of not more than 20%, and then washing the lignocellulose substrate with water until the pH is =7.0-8.0;
(2) Preparing a seed solution: inoculating a cellulosome-producing strain into a seed culture medium under an anaerobic condition, and culturing to a middle logarithmic growth phase under the conditions of a temperature of 55-65 ℃ and a rotating speed of 100-200rpm to obtain a seed solution; the cellulosome-producing strain is a clostridium thermocellum recombinant strain for expressing glucosidase;
(3) Preparing a saccharification system: sterilizing the lignocellulose substrate obtained in the step (1) independently, and then conveying the lignocellulose substrate into a fermentation tank containing a pre-sterilized saccharification culture medium under an aseptic condition; simultaneously inoculating and uniformly mixing the seed liquid obtained in the step (2) in a synchronous spraying manner to obtain a saccharification system; wherein the inoculation amount of the seed liquid in the system is 10% (v/v), and the solid content of the medium lignocellulose substrate is 5-20% (w/v);
(4) Saccharification: standing and hydrolyzing the saccharification system prepared in the step (3) at the temperature of 55-65 ℃ to obtain a sugar solution containing glucose; and when the sugar solution concentration in the system is not changed any more for two consecutive days, the saccharification is finished.
2. The lignocellulose whole cell saccharification technology as recited in claim 1, wherein: in the step (3), in the substrate adding process, stirring at the speed of 10-50 rpm to reduce the local accumulation of the substrate; meanwhile, nitrogen is introduced into a gas layer of the saccharification system to replace air, and sterile ammonium bicarbonate is supplemented by 0.2-1.0 g/L.
3. The lignocellulose whole cell saccharification technology as recited in claim 2, wherein: the lignocellulose substrate sterilization of the step (3) is specifically operated as follows: sterilizing at 126 deg.C for 30-40 min, spreading the substrate in multiple layers, wherein the thickness of each layer of substrate is not more than 1cm, thereby improving the sterilization efficiency; the pre-sterilization conditions of the saccharification culture medium are as follows: sterilized at 121 ℃ for 20 minutes.
4. The lignocellulose whole cell saccharification technology as recited in claim 2, wherein: the seed culture medium is as follows: 0.6-2.9 g/L of dipotassium phosphate, 0.3-1.5g/L of monopotassium phosphate, 1.0g/L of ammonium bicarbonate, 0.1g/L of calcium chloride, 0.5g/L of magnesium chloride, 0.5mg/L of ferrous sulfate, 1g/L of cysteine, 3g/L of yeast powder, 0.7g/L of the lignocellulose substrate obtained in the step (1) and pH 7.5.
5. The lignocellulose whole cell saccharification technology of claim 2, characterized in that: the saccharification culture medium comprises: dipotassium hydrogen phosphate 0.6g/L, potassium dihydrogen phosphate 0.3g/L, ammonium bicarbonate 0.5g/L, calcium chloride 0.1g/L, magnesium chloride 0.5g/L, ferrous sulfate 0.5mg/L, sodium sulfide 0.5g/L, corn steep liquor 4g/L, pH 7.5.
6. The lignocellulose whole cell saccharification technology of any one of claims 1-5, characterized in that: the lignocellulose raw materials are corn straws, wheat straws, pasture and xylose residues; the pretreatment is an alkaline method.
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