CN114480446A - Method for constructing lignin degrading enzyme gene set - Google Patents
Method for constructing lignin degrading enzyme gene set Download PDFInfo
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- CN114480446A CN114480446A CN202210164778.0A CN202210164778A CN114480446A CN 114480446 A CN114480446 A CN 114480446A CN 202210164778 A CN202210164778 A CN 202210164778A CN 114480446 A CN114480446 A CN 114480446A
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- 229920005610 lignin Polymers 0.000 title claims abstract description 51
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 36
- 230000000593 degrading effect Effects 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 28
- 102000004190 Enzymes Human genes 0.000 claims abstract description 29
- 244000005700 microbiome Species 0.000 claims abstract description 13
- 238000012165 high-throughput sequencing Methods 0.000 claims abstract description 9
- 239000000758 substrate Substances 0.000 claims abstract description 8
- 230000015556 catabolic process Effects 0.000 claims abstract description 6
- 238000006731 degradation reaction Methods 0.000 claims abstract description 6
- 238000012216 screening Methods 0.000 claims abstract description 5
- 238000004458 analytical method Methods 0.000 claims abstract description 4
- 108010029541 Laccase Proteins 0.000 claims description 17
- 108020004414 DNA Proteins 0.000 claims description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 7
- 229920000642 polymer Polymers 0.000 claims description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 150000001720 carbohydrates Chemical class 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 2
- 239000007790 solid phase Substances 0.000 claims description 2
- 238000007400 DNA extraction Methods 0.000 claims 1
- 150000003384 small molecules Chemical class 0.000 claims 1
- 238000011161 development Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000002028 Biomass Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 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 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000002361 compost Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012075 bio-oil Substances 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- 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)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- 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)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/90219—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- G01N2333/90222—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3) in general
- G01N2333/90225—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3) in general with a definite EC number (1.10.3.-)
- G01N2333/90232—Laccase (1.10.3.2)
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Abstract
The invention provides a method for efficiently and conveniently constructing a lignin degrading enzyme gene set. A sample containing a large number of microorganisms in a natural or artificial environment is subjected to lignin degradation microorganism enrichment culture by taking lignin as a growth substrate under a laboratory condition. And identifying that the lignin degrading enzymes are produced or the lignin is degraded in the culture process, screening and identifying a plurality of lignin degrading enzymes by means of high-throughput sequencing and metagenomic analysis, and constructing a lignin degrading enzyme gene set. The invention greatly enlarges the source of lignin degrading enzyme screening, provides a large amount of degrading enzymes with excellent performance for constructing a lignin biorefinery, and can also promote the development of a lignocellulose full-component biorefinery.
Description
Technical Field
The invention belongs to the field of biomass lignin resource utilization, and particularly relates to a method for constructing a lignin degrading enzyme gene set by using a metagenomic technology.
Background
Renewable biomass resources are the only resources on the earth which can directly replace petroleum resources in a mode of liquid fuels and organic chemical products, and have important value in converting the biomass resources into bio-oil and aromatic chemicals under the aims of reducing the dependence of petroleum and fossil resources and realizing the environmental protection of carbon peak reaching and carbon neutralization, and the development of a biorefinery technology has economic and environmental protection feasibility. The lignin is one of the main components of lignocellulose, is the renewable aromatic high polymer with the largest source on the earth, mainly comprises C-O ether bonds, and is partially C-C bonds. The biological enzyme degradation method is concerned and paid attention to the efficient process due to the green color, but 99% of microorganisms cannot be cultured in a laboratory, so the types of enzymes excavated by the traditional method for culturing single bacteria and then separating and purifying target enzymes are very limited. The newly developed high-throughput sequencing technology can detect DNA information of all microorganisms in a sample, and can process and analyze DNA data to maximally mine genes of target enzymes.
The method firstly constructs a gene set of lignin degrading enzymes, excavates a plurality of lignin degrading enzymes with excellent performance, and provides a novel and efficient method for developing a green high-performance biocatalyst in a lignin biorefinery.
Disclosure of Invention
The invention aims to provide a method for constructing a lignin degrading enzyme gene set. The invention samples from natural/artificial environment, enriches and cultures lignin-degrading microorganisms in a laboratory by taking lignin as a substrate, obtains all DNA information of microbial communities in the sample by utilizing a high-throughput sequencing technology, screens and identifies genes of lignin degrading enzymes by utilizing a metagenomic analysis means, and constructs a lignin degrading enzyme gene set.
The invention provides a method for constructing a lignin degradation laccase gene set, which comprises the following specific steps:
(1) taking a sample rich in microorganisms from a natural or artificial environment;
(2) taking the sample obtained in the step (1), and enriching lignin degrading microorganisms in the obtained sample under a laboratory condition; the method specifically comprises the following steps: adding natural lignin serving as a microbial growth substrate into the obtained sample, and providing other conditions for maintaining the normal growth of the microbes;
(3) enriching and culturing the product obtained in the step (2) for a period of time, extracting a crude enzyme solution in the product obtained in the step (2), stopping enriching and culturing when detecting that lignin degrading enzyme exists in the crude enzyme solution or detecting that lignin is degraded, and storing an enriched sample at a low temperature;
(3.1) wherein the lignin degrading enzyme is detected by selecting various specific substrates to carry out protein concentration test;
(3.2) the degradation of the lignin is to detect small molecular polymers or monomolecular compounds after the lignin structure is broken through an instrument;
(4) taking the enriched sample in the step (3), and extracting DNA in the sample;
(5) entering the DNA in the step (4) into a high-throughput sequencing platform, and collecting the measured DNA data information;
(6) carrying out metagenomics analysis on the DNA data obtained in the step (5);
(6.1) cleaning data, and removing redundant data and pollution data;
(6.2) splicing the short sequences generated in the step (6.1) into long sequences;
(6.3) transcribing the long DNA sequence produced in step (6.2) into an amino acid sequence;
(6.4) comparing the amino acid sequence in step (6.3) with a carbohydrate database, and screening out sequences having correlation with the lignin degrading enzyme.
In the present invention, the sample in step (1) may be in a solid phase or a liquid phase.
In the present invention, the laboratory enrichment conditions in step (2) may be various conditions that enable normal growth of microorganisms at any temperature.
In the invention, the lignin degrading enzyme in the step (3) is detected by selecting various specific substrates to carry out protein concentration test.
In the invention, the degradation of the lignin in the step (3) is to detect small molecular polymers or monomolecular compounds after the lignin structure is broken through an instrument.
In the present invention, the method for extracting DNA from the sample in step (4) is not limited.
In the invention, the high-throughput sequencing platform in the step (5) is not limited.
In the invention, the metagenomic analysis software type in the step (6) is not limited.
The invention has the beneficial effects that: the invention provides a method for efficiently and conveniently constructing a large amount of lignin degrading enzymes, which does not depend on screening and culturing of strains in a laboratory, greatly expands the sources of the lignin degrading enzymes, provides a large amount of degrading enzymes with excellent performance for constructing a lignin biorefinery, can also promote the development of a lignocellulose full-component biorefinery, and can generate great ecological effect, economic effect and social benefit.
Detailed Description
The following examples are intended to further illustrate the invention and are not intended to limit the invention.
Example 1
The method comprises the steps of taking high-temperature compost residues back from a high-temperature compost treatment unit of a municipal waste treatment plant, mixing the residues with water, placing the mixture in a laboratory constant-temperature incubator, controlling the temperature to be 60-90 ℃, keeping natural ventilation and a certain rotating speed in the incubator, and keeping an aerobic environment in a sample by assisting manual stirring on the basis of the natural ventilation. Supplementing water to the sample to enable the water content to be about 40%, wherein the adding proportion of the lignin is that the lignin: mixed liquor =1:10, while supplementing a certain amount of basic carbon source and nitrogen source. Culturing for 5-10 days. 5ml of the culture medium was taken out of the mixture, and the crude enzyme solution on the upper layer was extracted by stirring and centrifugation and was kept at 60 ℃.
The enzyme activity was determined as ABTS method. Laccase activity is detected in the experimental group, the culture is stopped, and a certain sample is taken for low-temperature storage. Extracting DNA in a sample by using a DNA kit, entering an Illumina high-throughput sequencing platform, and setting the sequencing depth to be more than 10G; collecting a measured original sample DNA sequence read length data set; carrying out primary cleaning, removing redundant and polluted data by using MetaWrap integrated software, and assembling short sequences (reads) into long sequences (contigs); and performing data binning on the long sequence to obtain a plurality of bins. According to default parameters, bins are compared with CAZyme (carbohydrate database) to screen out laccase genes; and classifying all laccase gene sequences into a document to build a gene set of the lignin degrading enzyme laccase.
The result shows that DNA sequences of microbial communities in 20G samples are detected by an Illumina high-throughput sequencing platform, and 64 genes of laccase are obtained through data analysis and processing.
Example 2:
the surface layer muddy water mixture is taken from the natural hot spring, is cultured by a shaking table at the temperature of 60 ℃ and the rpm/min, and is stirred and mixed evenly by hand at intervals of several hours. The concentration of the suspended sludge in the mixed liquid is controlled to be 140-200 mg/L. Water (mixed solution of glucose and ammonium sulfate) is added every 6 to 8 hours. The total amount of lignin added was 20 g. 0.1 mol/L-1 copper sulfate solution 5ml is supplemented every day. A control group was set, and only a mixed solution of glucose and ammonium sulfate was added. The crude laccase solution activity was determined as in example 1. On day 10, the strongest laccase activity was measured at 260. + -. 62.89U/L.
DNA was extracted as in example 1, sequenced, cleaned, assembled and analyzed, and 68 lignin degrading enzyme laccase gene sets were obtained from the 20G DNA data.
Claims (8)
1. A method for constructing a lignin-degrading laccase gene set is characterized by comprising the following specific steps:
(1) taking a sample rich in microorganisms from a natural or artificial environment;
(2) taking the sample obtained in the step (1), and enriching lignin degrading microorganisms in the obtained sample under a laboratory condition; the method specifically comprises the following steps: adding lignin as a microorganism growth substrate in the obtained sample, and providing other conditions for maintaining the normal growth of the microorganism;
(3) enriching and culturing the product obtained in the step (2) for a period of time, extracting a crude enzyme solution from the product obtained in the step (2), stopping enriching and culturing when detecting that lignin degrading enzyme exists in the crude enzyme solution or detecting that lignin is degraded, and storing an enriched sample at a low temperature;
(3.1) wherein the lignin degrading enzyme is detected by selecting various specific substrates to carry out protein concentration test;
(3.2) the degradation of the lignin is to detect small molecular polymers or monomolecular compounds after the lignin structure is broken through an instrument;
(4) taking the enriched sample in the step (3), and extracting DNA in the sample;
(5) entering the DNA in the step (4) into a high-throughput sequencing platform, and collecting the measured DNA data information;
(6) carrying out metagenomics analysis on the DNA data obtained in the step (5);
(6.1) cleaning data, and removing redundant data and pollution data;
(6.2) splicing the short sequences generated in the step (6.1) into long sequences;
(6.3) transcribing the long DNA sequence produced in step (6.2) into an amino acid sequence;
(6.4) comparing the amino acid sequence in step (6.3) with a carbohydrate database, and screening out sequences having correlation with the lignin degrading enzyme.
2. The method for constructing a lignin-degrading laccase gene set according to claim 1, wherein the sample in step (1) may be in solid phase or liquid phase.
3. The method for constructing a lignin-degrading laccase gene set according to claim 1, wherein the laboratory enrichment conditions in step (2) can be any conditions that allow the normal growth of microorganisms at any temperature.
4. The method for constructing a lignin-degrading laccase gene set according to claim 1, wherein the lignin-degrading enzymes detected in step (3) are protein concentration tests with specific substrates.
5. The method for constructing lignin-degrading laccase gene set according to claim 1, wherein in the step (3), the lignin is degraded by detecting small molecule polymer or single molecule compound after lignin structure breakage by instrument.
6. The method for constructing a lignin-degrading laccase gene set according to claim 1, wherein the DNA extraction method in the sample in step (4) is not limited.
7. The method for constructing a lignin-degrading laccase gene set according to claim 1, wherein the high throughput sequencing platform in step (5) is unlimited.
8. The method for constructing a lignin-degrading laccase gene set according to claim 1, wherein the metagenomic analysis software in step (6) is not limited in kind.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101652381A (en) * | 2007-01-30 | 2010-02-17 | 维莱尼姆公司 | Be used to handle the enzyme of lignocellulose, encode their nucleic acid and methods for making and using same thereof |
| CN105462999A (en) * | 2015-12-08 | 2016-04-06 | 江西省农业科学院农业应用微生物研究所 | Method for screening beta-glucosaccharase gene from mildewed sugarcane leaves based on metagenomic technology |
| CN105821033A (en) * | 2016-05-11 | 2016-08-03 | 清华大学 | Extracting method of cellulose degradation flora metagenome |
| CN107653243A (en) * | 2017-11-15 | 2018-02-02 | 中国农业科学院农业基因组研究所 | A kind of method that microorganism macro genome DNA is extracted from intestinal contents |
| CN109658980A (en) * | 2018-03-20 | 2019-04-19 | 上海交通大学医学院附属瑞金医院 | A kind of screening and application of excrement gene marker |
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2022
- 2022-02-23 CN CN202210164778.0A patent/CN114480446A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101652381A (en) * | 2007-01-30 | 2010-02-17 | 维莱尼姆公司 | Be used to handle the enzyme of lignocellulose, encode their nucleic acid and methods for making and using same thereof |
| CN105462999A (en) * | 2015-12-08 | 2016-04-06 | 江西省农业科学院农业应用微生物研究所 | Method for screening beta-glucosaccharase gene from mildewed sugarcane leaves based on metagenomic technology |
| CN105821033A (en) * | 2016-05-11 | 2016-08-03 | 清华大学 | Extracting method of cellulose degradation flora metagenome |
| CN107653243A (en) * | 2017-11-15 | 2018-02-02 | 中国农业科学院农业基因组研究所 | A kind of method that microorganism macro genome DNA is extracted from intestinal contents |
| CN109658980A (en) * | 2018-03-20 | 2019-04-19 | 上海交通大学医学院附属瑞金医院 | A kind of screening and application of excrement gene marker |
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