CN116987598A - Composite microbial agent for efficiently degrading lignocellulose and application thereof - Google Patents
Composite microbial agent for efficiently degrading lignocellulose and application thereof Download PDFInfo
- Publication number
- CN116987598A CN116987598A CN202310877975.1A CN202310877975A CN116987598A CN 116987598 A CN116987598 A CN 116987598A CN 202310877975 A CN202310877975 A CN 202310877975A CN 116987598 A CN116987598 A CN 116987598A
- Authority
- CN
- China
- Prior art keywords
- lignocellulose
- degrading
- trichoderma
- composite microbial
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000002131 composite material Substances 0.000 title claims abstract description 35
- 230000000593 degrading effect Effects 0.000 title claims abstract description 24
- 230000000813 microbial effect Effects 0.000 title claims abstract description 15
- 241001557886 Trichoderma sp. Species 0.000 claims abstract description 20
- 241000222393 Phanerochaete chrysosporium Species 0.000 claims abstract description 19
- 241000609240 Ambelania acida Species 0.000 claims abstract description 17
- 239000010905 bagasse Substances 0.000 claims abstract description 17
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims description 36
- 108090000790 Enzymes Proteins 0.000 claims description 36
- 229940088598 enzyme Drugs 0.000 claims description 36
- 108010059892 Cellulase Proteins 0.000 claims description 13
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 229920005610 lignin Polymers 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 108010029541 Laccase Proteins 0.000 claims description 7
- 229940106157 cellulase Drugs 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- 101710112457 Exoglucanase Proteins 0.000 claims description 6
- 108010054320 Lignin peroxidase Proteins 0.000 claims description 6
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 6
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 6
- 238000000855 fermentation Methods 0.000 claims description 6
- 230000004151 fermentation Effects 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 6
- 108010059896 Manganese peroxidase Proteins 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 241000223259 Trichoderma Species 0.000 claims description 3
- 108010062085 ligninase Proteins 0.000 claims 2
- 108010084185 Cellulases Proteins 0.000 claims 1
- 102000005575 Cellulases Human genes 0.000 claims 1
- 238000012262 fermentative production Methods 0.000 claims 1
- 239000002068 microbial inoculum Substances 0.000 abstract description 22
- 238000006731 degradation reaction Methods 0.000 abstract description 19
- 230000015556 catabolic process Effects 0.000 abstract description 18
- 244000005700 microbiome Species 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 4
- 238000004064 recycling Methods 0.000 abstract description 4
- 239000002154 agricultural waste Substances 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 239000010815 organic waste Substances 0.000 abstract description 3
- 230000000694 effects Effects 0.000 description 33
- 239000000243 solution Substances 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000002910 solid waste Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000002028 Biomass Substances 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 230000008485 antagonism Effects 0.000 description 5
- 239000001913 cellulose Substances 0.000 description 5
- 229920002678 cellulose Polymers 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 4
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 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 description 3
- 239000008272 agar Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000002195 synergetic effect Effects 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 229920002488 Hemicellulose Polymers 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000001384 succinic acid Substances 0.000 description 2
- ABDKAPXRBAPSQN-UHFFFAOYSA-N veratrole Chemical compound COC1=CC=CC=C1OC ABDKAPXRBAPSQN-UHFFFAOYSA-N 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- NGFMICBWJRZIBI-JZRPKSSGSA-N Salicin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O1)c1c(CO)cccc1 NGFMICBWJRZIBI-JZRPKSSGSA-N 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- NGFMICBWJRZIBI-UHFFFAOYSA-N alpha-salicin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UHFFFAOYSA-N 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 239000008380 degradant Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- FEWJPZIEWOKRBE-LWMBPPNESA-N levotartaric acid Chemical compound OC(=O)[C@@H](O)[C@H](O)C(O)=O FEWJPZIEWOKRBE-LWMBPPNESA-N 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000010813 municipal solid waste Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000011197 physicochemical method Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- NGFMICBWJRZIBI-UJPOAAIJSA-N salicin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UJPOAAIJSA-N 0.000 description 1
- 229940120668 salicin Drugs 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
- B09B3/60—Biochemical treatment, e.g. by using enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01013—Manganese peroxidase (1.11.1.13)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01014—Lignin peroxidase (1.11.1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
-
- 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
- C12R2001/645—Fungi ; Processes using fungi
-
- 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
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Mycology (AREA)
- Botany (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Environmental & Geological Engineering (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a composite microbial agent for efficiently degrading lignocellulose and application thereof, relates to the field of microorganisms, and belongs to the field of agricultural intensive production technology. The composite microbial inoculum comprises Phanerochaete chrysosporium and Trichoderma sp.LH-413. Phanerochaete chrysosporium and Trichoderma sp.LH-413 are mutually cooperated, the degradation capacity of the composite microbial inoculum is improved compared with that of single strain after cooperation, filter paper can be degraded into paste within 48 hours, the degradation rate reaches 58.02%, and the bagasse degradation rate is 28.28%. The composite microbial inoculum provided by the invention has high-efficiency lignocellulose degradation capability, can promote the recycling of lignocellulose high-content organic waste, and provides technical basis for the production and development of green treatment modes of agricultural waste.
Description
Technical Field
The invention relates to the technical field of microbial agents, belongs to the technology of agricultural intensive production, and in particular relates to a composite microbial agent for efficiently degrading lignocellulose and application thereof.
Background
With the development of agricultural production, the total amount of agricultural solid waste such as forestry waste, agricultural and livestock waste and household garbage is also increasing. Because of the problems of large base number, insufficient importance degree and the like, the recovery rate of agricultural wastes is low, and resource waste exists. Meanwhile, the solid waste occupies a large amount of land area, secondary pollution can occur when the solid waste is stacked for a long time, and the surrounding atmosphere can be damaged when the solid waste is serious, so that the soil is polluted. The reasonable utilization of the agricultural solid waste has important environmental significance and accords with the sustainable development strategy.
The agricultural solid waste is rich in a large amount of lignocellulose, and the lignocellulose biomass mainly consists of three interweaved polymer components of cellulose, hemicellulose and lignin, and exists as a natural resistant biological compound. Because of the heterogeneous and crystalline structural characteristics of lignocellulose biomass, the recycling treatment difficulty is high, the utilization rate is low, hemicellulose is the matrix which is most easily degraded in lignocellulose, lignin is most difficult to degrade, and cellulose is inferior. Lignocellulosic conversion technologies, including mainly physical, chemical, physicochemical and biological methods, are becoming a research focus in this field. Bioconversion and degradation are one of the important ways to realize lignocellulose resource utilization, and have a plurality of advantages, including mild reaction conditions, green and pollution-free properties, environmental friendliness and the like.
There are a number of lignocellulose-degrading microorganisms in nature, including bacteria, fungi and actinomycetes, which produce related lignocellulose-degrading enzymes for degrading biomass to release monosaccharides and other compounds. Fungi are considered as the main degradants of lignocellulose substances, and have the characteristics of strong decomposing capacity and higher enzyme activity. The pure culture of the microorganism is utilized to degrade the lignocellulose biomass, the operation is simple, the economy is good, but the pure culture of the microorganism degrades enzyme types singly and the enzyme activity is low, so that the treatment efficiency is low, the treatment time is long, and the industrialized application of lignocellulose is seriously affected. Thus, degradation of lignocellulose needs to be accomplished by synergy between different microorganisms. The composite bacterial system is a microbial community which is selected from the natural world, is constructed by manual selection and consists of a plurality of microorganisms and has synergistic effect. The utilization of composite bacterial systems has become one of the hot spots in the research of the resource utilization of lignocellulose biomass. The development and utilization of biotechnology to degrade and transform lignocellulose raw materials are not only effective ways for recycling utilization, but also have great significance for solving environmental pollution and energy crisis.
Disclosure of Invention
The invention provides a compound microbial agent for efficiently degrading lignocellulose and application thereof, which aims to solve the problems of low treatment efficiency and long treatment time caused by single pure culture degrading enzyme types and low enzyme activity of microorganisms.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention discloses a composite microbial inoculum for efficiently degrading lignocellulose, which comprises Phanerochaete chrysosporium and Trichoderma sp.LH-413, wherein Trichoderma sp.LH-413 is classified and named Trichoderma sp, and is preserved in China Center for Type Culture Collection (CCTCCM) 2023749.
The volume ratio of the Phanerochaete chrysosporium to the Trichoderma sp.LH-413 is 1:1.
The concentration of the spore liquid of the Phanerochaete chrysosporium and the Trichoderma sp.LH-413 is 1.0-1.5X10 6 And each ml.
The lignocellulose degrading composite microbial inoculum is applied to the fermentation production of cellulase and/or lignin enzyme.
The lignocellulose degrading complex bacteria are fermented by an enzyme producing medium, and the cellulase and/or lignin enzyme comprises endoglucanase, filter paper enzyme, exoglucanase, beta-glucosidase, laccase, lignin peroxidase and manganese peroxidase.
The enzyme-producing culture medium is prepared by the following method of 5g/L bagasse and 1g/L (NH) 4 ) 2 SO 4 ，1g/LKH 2 PO 4 ,0.5g/LMgSO 4 ·7H 2 O,0.1g/L yeast extract, 0.1g/LCaCl 2 Sterilizing at 121deg.C for 20min.
The lignocellulose degrading composite microbial inoculum is applied to degradation of lignocellulose.
The lignocellulose is lignocellulose in bagasse.
The beneficial effects obtained by the invention are as follows:
the composite microbial inoculum prepared by the invention has high-efficiency lignocellulose degradation capability, can degrade natural cellulose such as bagasse and the like, can degrade filter paper after 12 hours, causes the filter paper to expand and bend downwards, and degrades the filter paper into paste after 48 hours. The endoglucanase activity of the composite microbial inoculum is 7177.15U/L, the filter paper enzyme activity is 2628.49U/L, the exoglucanase activity is 1798.02U/L, the beta-glucosidase activity is 3959.86U/L, the laccase activity is 678.70U/L, the lignin peroxidase activity is 16558.90U/L, and the manganese peroxidase activity is 1851.90U/L. The composite microbial inoculum can enable the degradation rate of bagasse to reach 28.28%. The composite microbial inoculum can promote the degradation of lignocellulose, accelerate the decomposition process, shorten the degradation period, achieve the effect of economy, low cost and long-term throwing, can be used for preparing cellulase and/or lignin degrading enzyme, and provides technical basis for developing and promoting the recycling utilization of lignocellulose high-content organic waste and for producing and developing an agricultural waste green treatment mode.
The Phanerochaete chrysosporium and Trichoderma sp.LH-413 strain have no antagonism and good synergistic effect, and the synergistic effect can effectively accelerate the degradation process of lignocellulose, so that a strain foundation is laid for fully utilizing cheap natural lignocellulose as an artificial carbon source in high-content agricultural organic wastes of lignocellulose, and particularly the lignocellulose in bagasse.
Drawings
FIG. 1 is a colony growth morphology of Trichoderma sp.LH-413 on solid medium.
FIG. 2 is a morphological diagram of colony growth of Phanerochaete chrysosporium on solid medium.
FIG. 3 is a graph showing the results of an antagonistic test on Phanerochaete chrysosporium and Trichoderma sp.LH-413.
FIG. 4 is a graph showing the disintegration results of the filter paper of the composite microbial inoculum of Phanerochaete chrysosporium and Trichoderma sp.LH-413.
FIGS. 5 to 6 show comparison of enzyme-producing activities of Phanerochaete chrysosporium and Trichoderma sp.LH-413 complex inoculant with single strain.
FIG. 7 shows the degradation rate of Phanerochaete chrysosporium and Trichoderma sp.LH-413 complex inoculant compared to bagasse treated with a single strain.
Detailed Description
The invention is further illustrated by the following detailed description of specific embodiments, which is not intended to be limiting, but is made merely by way of example.
Phanerochaete chrysosporium (Phanerochaete chrysosporium) is a commercially available product. Trichoderma sp.LH-413, classified under the name Trichoderma sp, deposited with the China center for type culture Collection, accession number: cctcm 2023749, preservation address: beijing, china.
Example 1 antagonism experiment between strains
The culture medium used in this example was prepared as follows:
PDA solid medium: 200g of peeled potatoes, 20g of glucose and 20g of agar are weighed. Cutting potato into small cubes, boiling in water for about 30min, filtering with gauze, adding glucose and agar powder into the filtrate, stirring with glass rod until glucose and agar powder are completely dissolved, adding ultrapure water to 1000mL, and sterilizing at 121deg.C under moist heat for 20min.
Phanerochaete chrysosporium (Phanerochaete chrysosporium) and Trichoderma sp.LH-413 were subjected to a plate antagonism test and were plated on PDA solid medium for 5 days to observe the growth conditions of each strain and the influence of each other. The two adjacent strains grow well, and the junction of the bacterial colonies is fused and connected or grows into one bacterial colony, so that the two strains are judged to be compatible; if the growth vigor of two adjacent strains is equal, then one colony grows slowly, and a gap is left at the junction, the two strains are judged to be partially compatible; if there is a significant difference in growth trend between the two strains, one colony surrounds the other or there is a significant demarcation line at the junction, then antagonism of the two strains is determined. To examine whether each strain can be used for constructing a composite bacterial strain.
From the results, the two strains have no antagonism and do not interfere with each other to influence the growth of the strains, so the strain can be prepared into a composite bacterial agent.
EXAMPLE 2 construction of Complex microbial systems
Respectively inoculating Phanerochaete chrysosporium and Trichoderma sp.LH-413 to PDA solid culture medium, and culturing in an incubator at 28deg.C for 5 days to activate strain; scraping spores on the activated plate culture medium, and adjusting the concentration of spore liquid to 1.0-1.5X10 6 And mixing the two components according to the volume ratio of 1:1 to obtain the composite microbial inoculum. Inoculating the microbial inoculum into a liquid enzyme production culture medium, continuously culturing for 5 days at 28 ℃ by a shaking table at 150rpm, and determining the lignocellulose activity of the composite microbial inoculum.
Example 3 Complex bacterial enzyme Activity assay
Preparation of crude enzyme solution
The prepared composite microbial inoculum and single strain are respectively inoculated into enzyme-producing culture medium (5 g/L bagasse, 1g/L (NH) 4 ) 2 SO 4 ，1g/LKH 2 PO 4 ,0.5g/LMgSO 4 ·7H 2 O，0.1g/L yeast extract, 0.1g/LCaCl 2 Sterilizing at 121deg.C for 20 min), culturing at 28deg.C in shaking table at 150rpm for 5 days, centrifuging the strain fermentation broth 8000r/min for 10min, and collecting supernatant as crude enzyme solution.
Determination of cellulose-related enzyme Activity
The endoglucanase activity, the filter paper enzyme activity, the exoglucanase activity and the beta-glucosidase activity are respectively measured by a DNS method.
1.0mL of 1% carboxymethyl cellulose (prepared by using sodium citrate buffer solution with pH of 4.8 and 0.05 mol/L) and 1.0mL of 1% salicin (prepared by using acetic acid-sodium acetate buffer solution with pH of 4.8) are respectively taken as substrates, 0.5mL of enzyme solution which is properly diluted is added, the mixture is uniformly mixed, the mixture is incubated at 50 ℃ for 30min, a test tube is taken out, and 3mL of LDNS color reagent is immediately added, uniformly mixed and the enzyme reaction is stopped. The sample was boiled in a water bath for 10min, cooled in water to maintain color stability, and the endoglucanase activity and beta-glucosidase activity were calculated by measuring absorbance at 540nm using the inactivated enzyme solution as a blank.
Respectively taking 50mg of filter paper and 50mg of absorbent cotton as substrates, adding 1.5mL of enzyme solution which is diluted properly, uniformly mixing, incubating the mixture at 50 ℃ for 30min, taking out a test tube, immediately adding 3mLDNS color-developing agent, uniformly mixing, and stopping enzyme reaction. The sample was boiled in a water bath for another 10min, cooled in water to maintain color stability, and the filter paper enzyme activity and exoglucanase activity were calculated by measuring absorbance at 540nm using the inactivated enzyme solution as a blank.
As a result, the endoglucanase activity, the filter paper enzyme activity, the exoglucanase activity and the beta-glucosidase activity of the composite microbial inoculum are higher than those of single bacteria, and reach 7177.15U/L, 2628.49U/L, 1798.02U/L and 3959.86U/L respectively.
Determination of lignin-related enzyme Activity
The lignin key enzyme activities (laccase, lignin peroxidase, manganese peroxidase) were determined.
Laccase: the laccase enzyme activity was calculated by using 0.5mL of 0.5 mol/LABSS as substrate, 2mL of a solution of citric acid-sodium citrate with pH of 5.0 as buffer, adding 1mL of crude enzyme solution to start the reaction at 25℃and detecting the change of absorbance value of the first 3min at 420 nm.
Lignin peroxidase: 1.5mL of 0.1mol/L tartaric acid buffer (pH 3.0); 1.0mL of 10mmol/L veratrole; crude enzyme solution 0.4mL;10mmol/LH 2 O 2 The reaction was started and at 30 ℃. The change in absorbance value at 310nm wavelength was measured by an ultraviolet spectrophotometer for the first 3 minutes of the reaction.
Manganese peroxidase: 2.0mL of 0.05mol/L succinic acid buffer (succinic acid) (pH 4.5); 15mmol/LMnSO 4 0.5mL; crude enzyme solution 0.4mL;10mmol/LH 2 O 2 The reaction was started at 0.1mL and at 30 ℃. The change in absorbance value of the mixture at a wavelength of 240nm during the first 3 minutes of the reaction was detected by an ultraviolet spectrophotometer.
As a result, the laccase activity and lignin peroxidase activity of the composite microbial inoculum are higher than those of single bacteria, namely 678.70U/L and 16558.90U/L respectively. It can be seen that the complex microbial inoculum increases the activity of the lignin cellulase.
EXAMPLE 4 Filter paper strip disintegration test
The composite bacterial agent and the single strain are respectively inoculated into a 250mL triangular flask filled with 50mL of filter paper strip disintegration culture medium, shake cultivation (28 ℃ and 130 r/min) is carried out by taking the non-bacterial liquid treatment as a control, the disintegration condition of the filter paper strip is regularly observed and recorded, the filter paper is centrifugally collected after 7-10d, and the filter paper is dried and weighed at 80 ℃ to calculate the degradation rate of the filter paper.
The result shows that Trichoderma sp.LH-413 starts to degrade on the 3 rd day and the 7 th day is almost degraded into paste, the composite microbial inoculum can degrade the filter paper after 12 hours, the filter paper is expanded and bent downwards, and the filter paper is degraded into paste after 48 hours, so that the degradation rate reaches 58.02%. Therefore, the effect of the composite microbial inoculum on degrading cellulose is quite remarkable.
Example 5 liquid fermentation of Complex microbial inoculants to degrade Natural lignocellulosic bagasse
And (3) carrying out a liquid fermentation and degradation bagasse experiment on the composite microbial inoculum, and measuring the degradation capability of the strain on natural lignocellulose substances by using the bagasse weight loss rate.
Inoculating the composite microbial inoculum and single strain into bagasse culture medium (10 g/L bagasse, 1g/L (NH) 4 ) 2 SO 4 ，1g/LKH 2 PO 4 ,0.5g/LMgSO 4 ·7H 2 O,0.1g/L yeast extract, 0.1g/LCaCl 2 ) After the culture was completed, the residual cells and calcium carbonate were removed by shaking culture in a shaker at 28℃and 150rpm, and then repeatedly rinsed with distilled water. The culture was centrifuged at 5000rpm for 10 minutes and the pellet was dried to constant weight in a dry box. The relative degradation rate of bagasse was determined with reference to a lignocellulose-degrading liquid medium without seed.
The result shows that compared with single bacteria, the composite bacterial agent obviously improves the degradation rate of bagasse to 28.28 percent.
Claims (8)
1. A composite microbial agent for efficiently degrading lignocellulose is characterized in that: comprises Phanerochaete chrysosporium and Trichoderma sp.LH-413, wherein Trichoderma sp.LH-413 is classified and named Trichoderma sp, and is preserved in China Center for Type Culture Collection (CCTCCM) 2023749.
2. The lignocellulose degrading composite microbial agent as set forth in claim 1, wherein the volume ratio of Phanerochaete chrysosporium to Trichoderma sp.LH-413 is 1:1.
3. The lignocellulose degrading complex bacterial agent as set forth in claim 1 or 2, wherein the Phanerochaete chrysosporium and Trichoderma sp.LH-413 spore liquid concentrations are 1.0-1.5X10 6 And each ml.
4. Use of the lignocellulose degrading composite microbial inoculant of any one of claims 1-3 for the fermentative production of cellulases and/or lignin enzymes.
5. The use of the lignocellulose degrading complex microbial inoculant according to claim 4 for producing cellulase and/or ligninase by fermentation, wherein the use is as follows: the lignocellulose degrading complex bacteria are fermented by an enzyme producing medium, and the cellulase and/or lignin enzyme comprises endoglucanase, filter paper enzyme, exoglucanase, beta-glucosidase, laccase, lignin peroxidase and manganese peroxidase.
6. The use of the lignocellulose degrading complex microbial inoculant according to claim 4 for producing cellulase and/or ligninase by fermentation, wherein the use is as follows: the enzyme-producing culture medium is prepared by the following method of 5g/L bagasse and 1g/L (NH) 4 ) 2 SO 4 ，1g/LKH 2 PO 4 ,0.5g/LMgSO 4 ·7H 2 O,0.1g/L yeast extract, 0.1g/LCaCl 2 Sterilizing at 121deg.C for 20min.
7. Use of a lignocellulose degrading composite bacterial agent according to any one of claims 1-3 for degrading lignocellulose.
8. The use of the lignocellulose degrading composite microbial agent of claim 7 for degrading lignocellulose, wherein: the lignocellulose is lignocellulose in bagasse.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310877975.1A CN116987598B (en) | 2023-07-18 | 2023-07-18 | Composite microbial agent for efficiently degrading lignocellulose and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310877975.1A CN116987598B (en) | 2023-07-18 | 2023-07-18 | Composite microbial agent for efficiently degrading lignocellulose and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116987598A true CN116987598A (en) | 2023-11-03 |
| CN116987598B CN116987598B (en) | 2024-07-09 |
Family
ID=88531307
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310877975.1A Active CN116987598B (en) | 2023-07-18 | 2023-07-18 | Composite microbial agent for efficiently degrading lignocellulose and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116987598B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140080193A1 (en) * | 2011-03-31 | 2014-03-20 | Kirk Schnorr | Methods For Enhancing The Degradation Or Conversion Of Cellulosic Material |
| CN106434386A (en) * | 2016-11-24 | 2017-02-22 | 郑州大学 | Microorganism and microbial inoculum for degrading straws as well as preparation method and application of microbial inoculum |
| CN111278986A (en) * | 2017-10-30 | 2020-06-12 | 帝斯曼知识产权资产管理有限公司 | Methods for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars |
| CN115820510A (en) * | 2022-12-21 | 2023-03-21 | 安徽省农业科学院农业工程研究所 | Cellulose degradation composite microbial flora preparation and application thereof in straw degradation |
-
2023
- 2023-07-18 CN CN202310877975.1A patent/CN116987598B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140080193A1 (en) * | 2011-03-31 | 2014-03-20 | Kirk Schnorr | Methods For Enhancing The Degradation Or Conversion Of Cellulosic Material |
| CN106434386A (en) * | 2016-11-24 | 2017-02-22 | 郑州大学 | Microorganism and microbial inoculum for degrading straws as well as preparation method and application of microbial inoculum |
| CN111278986A (en) * | 2017-10-30 | 2020-06-12 | 帝斯曼知识产权资产管理有限公司 | Methods for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars |
| CN115820510A (en) * | 2022-12-21 | 2023-03-21 | 安徽省农业科学院农业工程研究所 | Cellulose degradation composite microbial flora preparation and application thereof in straw degradation |
Non-Patent Citations (1)
| Title |
|---|
| 张尧;仝蒙;胡旭旭;章云;胡鹏举;: "混合菌种发酵秸秆生产蛋白饲料的研究进展", 中国畜牧兽医文摘, no. 10, 15 October 2016 (2016-10-15), pages 210 - 211 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116987598B (en) | 2024-07-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105567612A (en) | Preparation and application of garden waste compound degrading microbial agent | |
| CN101967452B (en) | Fermentable silk spore yeast strains and application for preparing microbial oil thereof | |
| US8728777B2 (en) | High performance metabolic bacteria | |
| CN108753640B (en) | Microbial compound microbial inoculum for efficiently degrading straw lignin | |
| CN111518731B (en) | Bacillus subtilis with antagonistic effect for degrading cellulose at low temperature and application thereof | |
| Wu et al. | Microbial hydrolysis and fermentation of rice straw for ethanol production | |
| CN103194392A (en) | Complex microbial inoculant for degrading straw and method for degrading straw by using same | |
| CN112410246B (en) | Microbial composition for degrading livestock and poultry manure and application thereof | |
| CN114561327B (en) | Cellulose degradation composite microbial inoculant, and preparation method and application thereof | |
| Saxena et al. | Simultaneous saccharification and fermentation of waste newspaper to ethanol | |
| CN116987598B (en) | Composite microbial agent for efficiently degrading lignocellulose and application thereof | |
| CN102031276A (en) | Method for producing hydrogen by fermenting organism and alkali mat composite pretreated straw fiber raw erial at two steps | |
| CN112592862A (en) | Preparation method and application of aerobic fermentation salt-tolerant composite microbial agent | |
| CN109486863B (en) | Method for degrading wood fibers in crop straws by using microbial agent | |
| CN103468592A (en) | Fungus with straw degradation capability and application thereof in composting | |
| CN114317381B (en) | T-level microorganism composite microbial agent and preparation method and application thereof | |
| CN115029281A (en) | Preparation method of high-temperature-resistant cellulose degradation microbial inoculum and application of high-temperature-resistant cellulose degradation microbial inoculum in compost | |
| CN112795597B (en) | Method for promoting straw rapid hydrolysis by domesticating paddy soil | |
| CN110452852B (en) | Farmer market biomass waste fermentation inoculant and preparation method thereof | |
| CN112442460A (en) | Formula of composite microbial flora preparation for improving aerobic dynamic composting efficiency | |
| CN110835610B (en) | Composite microbial inoculum suitable for degrading straw and preparation method thereof | |
| CN109988729B (en) | Normal-temperature compound strain system and application thereof in degradation of lignocellulose | |
| CN102899361A (en) | Method for producing methane by using lignocellulosic materials | |
| CN109762769B (en) | Anaerobic straw degradation microbial inoculum and preparation method and application thereof | |
| CN114015619A (en) | Straw fermentation composite bacterium preparation and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |