CN104560817B - Thermophilic bacillus licheniformis UTM102 for producing phytase and application of thermophilic bacillus licheniformis UTM102 - Google Patents
Thermophilic bacillus licheniformis UTM102 for producing phytase and application of thermophilic bacillus licheniformis UTM102 Download PDFInfo
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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
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- 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
- C12R2001/07—Bacillus
- C12R2001/10—Bacillus licheniformis
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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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
- 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
- C12N1/20—Bacteria; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The invention provides a thermophilic bacillus licheniformis UTM102 for producing phytase and an application of the thermophilic bacillus licheniformis UTM102. The biological collection number of the thermophilic bacillus licheniformis UTM102 is CGMCC (China General Microbiological Culture Collection Center) No.9508. The thermophilic bacillus licheniformis UTM102 can perform aerobic composting fermentation in organic solid wastes such as sewage sludge, household garbage, animal carcasses, livestock manure, crop straw and the like, can adapt to high-temperature environment and be multiplied massively in the organic solid wastes; a pile is high in heating speed and temperature, can be quickly composted, can degrade organic matter more quickly, minimization and harmlessness are realized more completely, and the organic solid wastes can be converted into biological fertilizers which contain massive bacillus licheniformis, can inhibit plant pathogens, can promote growth of plants and can improve the yield of crops. The bacillus licheniformis UTM102 has good strain performance and remarkable economic efficiency.
Description
Technical field
The present invention relates to fermentation arts, in particular it relates to a kind of thermophilic bacillus licheniformis for carrying phytase gene
Bacterial strain UTM102 and its degrade and organic solid castoff and prepare the purposes of bio-feritlizer.
Background technology
Substantial amounts of organic solid castoff can be produced during exhausting and production.Such as at town domestic sewage
Substantial amounts of sludge is produced during reason, it contains a large amount of harmful levels of pathogens, content of organics enriches in addition, easily degenerate smelly causing sternly
The secondary pollution of weight.And the discarded object in agricultural zootechnical production process, such as tangerine bar, feces of livestock and poultry and spoil have turned into
Serious pollution sources, first pollution source has been turned into certain areas.
Organic solid castoff main composition in town and country is wood fibre metallic substance.Lignocellulosic is by cellulose, half
The crystal-like structure of cellulose and lignin composition, with lignocellulose degradation is efficiently to utilize using During High-Temperature Composting aerobic fermentation
The effective ways of organic solid castoff.Using the synergy of multiple-microorganism, make the organic nutrient of various complexity, conversion
It is nutrient and humus soluble, easily absorb.While (80~100 DEG C) diseases killed in materials of high temperature produced by compost
Bacterium, worm's ovum.Prepare bio-feritlizer by microbial fermentation organic solid castoff has critical role in developing eco-agriculture,
The reluctant problem of discarded object is not only solved, while changing harmful to treasure.Will be with phytase activity, and with various high temperature resistants
The bacillus licheniformis of biomass by hydrolyzation enzyme apply to the treatment of organic solid waste material, and prepare growth-promoting sexual function bio-feritlizer
It is not reported.
The content of the invention
It is an object of the invention to provide a kind of thermophilic bacillus licheniformis UTM102 bacterial strains of phytase generating and its application.
From nature hot spring bed mud is picked up from, using culture medium is tamed, (glucose 10g, beef extract 3g, yeast are extracted the present invention
Thing 5g, peptone 10g, sodium chloride 5g, distilled water 1000ml) at 40 DEG C, through more for acclimating, UTM102 being obtained after directed screening
Bacterial strain, it has degraded organic solid castoff characteristic.The bacterial strain is in August in 2014 13 days in Chinese microorganism strain preservation
Administration committee's common micro-organisms center (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science
Institute of microbiology of institute, postcode 100101) preservation, Classification And Nomenclature is bacillus licheniformis Bacillus licheniformis,
Preserving number is CGMCC No.9508.
The UTM102 that the present invention is provided is Gram-positive, cell 0.8 × (2.0~3.5), direct rod shape, Dan Sheng, produce it is near
Middle raw ellipse gemma, sporangiocyst expands；(glucose 10g, peptone 10g, yeast extract on the culture medium containing glucose proteins peptone
5g, sodium chloride 5g, agar 15g, distilled water 1000ml, pH7.0) the smooth positive garden of bacterium colony, slightly dash forward, eggshell yellow, growth it is rapid, 30
~40 DEG C culture 24 hours after colony diameter about 2~3mm, 40~60 DEG C of optimum growth temperature.Contact enzyme positive, oxidizing ferment sun
Property, VP experiments it is positive.
It is (27f) using primer：5 '-AGA GTT TGA TCC TGG CTC AG-3 ' and (1492r)：5′-GGT TAC
CTT GTT ACG ACT T-3 ' expand the 16S rRNA genes of the bacterial strain through PCR, and the sequence obtained after sequencing is shown in sequence table SEQ
ID No:1.Sequence will be obtained to be submitted to EzTaxon databases and compare, as a result show UTM102 bacterial strains the gene and
The similitude highest of Bacillus licheniformis, is 99.3%, and this sequence is to identify the principal character foundation of the bacterial strain.
Using universal primer UP-1S:5 '-GAA GTC ATC ATG ACC GTT CTG CA-3 ' and UP-2Sr:5’-AGC
AGG GTA CGG ATG TGC GAG CC-3 ' expand the gyrB genes of the bacterial strain through PCR, and the gene order after sequencing is shown in sequence
Table SEQ ID No:2.The sequence of acquisition is submitted to NCBI GeneBank databases and carries out sequence alignment with Blast programs, knot
The gyrB gene orders and Bacillus licheniformis similitude highests of fruit display UTM102 bacterial strains, with reference to the bacterial strain
Physio-biochemical characteristics be differentiate the bacterial strain secondary feature.Determine that the bacterial strain is bacillus licheniformis Bacillus
licheniformis。
The invention provides a kind of bacillus licheniformis (Bacillus licheniformis) bacterial strain UTM102, its preservation
Numbering is CGMCC No.9508.
The bacillus licheniformis UTM102 that the present invention is provided has beta glucan restriction endonuclease, cellobiase, zytase
And phytase activity, its encoding gene is shown in sequence table SEQ ID No:3~SEQ ID No:6.
The invention provides the microbial inoculum containing bacillus licheniformis UTM102.
The invention provides bacillus licheniformis UTM102 or its microbial inoculum organic solid castoff biodegradation process
In application.
The invention provides a kind of method for preparing the compost Inoculant containing bacillus licheniformis UTM102, including following step
Suddenly：
(1) zymotic fluid is prepared：Lichem bacillus strain UTM102 activated spawns are inoculated in the liquid of glucose proteins peptone
In body culture medium, fermenting and producing is carried out；
(2) zymotic fluid packing turns into liquid bacterial agent, and bacteria powder is obtained through dehydrating.
In the above method, step (1) takes a small amount of lawn and is seeded to dress from 4 DEG C of bacterial strain UTM102 inclined-planes of the present invention of preservation
There are culture medium (glucose 10g, peptone 10g, yeast extract 5g, sodium chloride 5g, distilled water of the 40ml containing glucose proteins peptone
1000ml, pH7.0) 250ml triangular flasks.35~40 DEG C of cultivation temperature, 160-220 revs/min of shaking speed, culture 1~2
My god, OD600Stop culture during more than 1.8, this is shake-flask seed liquid.
Shake-flask seed liquid is seeded to culture medium (glucose 10g, peptone 10g, yeast extract containing glucose proteins peptone
5g, sodium chloride 5g, distilled water 1000ml, pH7.0) seeding tank in cultivated.35~40 DEG C of cultivation temperature, throughput is 1:
0.1~0.5 (v/v), mixing speed is 100~400 revs/min, incubation time 30~48 hours.Work as OD600It is to stop more than 1.8
Only cultivate, this is seeding tank seed liquor.
Seeding tank seed liquor is pressed into 0.1~5% inoculum concentration, inoculation carries out fermentation training in the fermentation tank containing above-mentioned culture medium
Support.35~40 DEG C of cultivation temperature, throughput is 1:0.2~0.6 (v/v), mixing speed is 60~200 revs/min, incubation time
20~40 hours.Work as OD600Stop culture when being more than 1.8, this is the zymotic fluid of bacterial strain.
Microbial inoculum the invention provides lichem bacillus strain UTM102 or containing it is in organic solid waste compost
Application in fermentation.
The organic solid castoff is in downflow sludge, house refuse, crop material, spoil or feces of livestock and poultry
One or more.
The method that microbial inoculum with lichem bacillus strain UTM102 or containing it carries out compost to organic solid castoff,
Comprise the following steps：
(1) UTM102 or its microbial inoculum that will be equivalent to total material weight in wet base 0.1%~0.5% are inoculated in organic solid castoff
In, mixing carries out aerobic composting fermentation after mixing thoroughly；
(2) it is 45%~65% the moisture of material to be adjusted into moisture content with moisture conditioner, and the carbon-nitrogen ratio of this material is 10
~60:1, heap body keeps oxygen content to be about 8%~15%；
(3) heap body ferments 12-20 days, and fermentation terminates；Leftover materials or landfill or burning or packing of sieving obtain bio-fertilizer
Material.
In above-mentioned compost method, the highest temperature reaches 103~105 DEG C in the course of fermentation of step (2)；Step (3) is through 12-20
It aerobic fermentation material moisture is down to 30~35%, and the most of organic matter in discarded object is decomposed, and terminates fermentation.
Microbial inoculum the invention provides lichem bacillus strain UTM102 or containing it answering in organic fertilizer is prepared
With.
Microbial inoculum the invention provides lichem bacillus strain UTM102 or containing it answering in plant growth is promoted
With.
The present invention is separated to one plant from hot spring bed mud has phytase, cellobiase, beta glucan restriction endonuclease, wood poly-
The bacterial strain of carbohydrase isoreactivity, provides one kind to prepare bio-feritlizer using organic solid castoff and has crops growth-promoting effect concurrently
And degraded organic solid castoff makes the microorganism of the innoxious characteristic of its minimizing.UTM102 contains phytase, the plant of its secretion
Phytic acid (salt) can be degraded to inositol and Phos by sour enzyme, while discharge the other materials combined with phytic acid (salt) participating in
In the energetic supersession of plant and substance metabolism process, so as to promote plant growth.UTM102 bacterial strains can be in downflow sludge, life rubbish
Aerobic composting fermentation is carried out in the organic solid wastes such as rubbish, spoil, feces of livestock and poultry, agricultural crop straw, in organic materials
The bacterium colony ecosystem of stabilization is formed, a large amount of quick breedings, the organic matter in organic solid castoff of effectively degrading makes its fast
Speed reaches minimizing, innoxious；Organic solid castoff can be changed into using the microbial bacterial agent prepared by the present invention simultaneously
Bio-feritlizer, a large amount of bacillus licheniformis contained in this fertilizer can suppress phytopathogen, and rush can be played to plant growth
Enter effect, the yield of crop can be improved.
Brief description of the drawings
Fig. 1 is bacterial strain UTM102 systematic evolution trees of the present invention.
Fig. 2 is the microphotograph of bacterial strain UTM102 of the present invention.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention
In the case of essence, the modification or replacement made to the inventive method, step or condition belong to the scope of the present invention.
If not specializing, the conventional meanses that technological means used is well known to those skilled in the art in embodiment.
The separation of the bacillus licheniformis UTM102 of embodiment 1 and identification
1 gram of hot spring bed mud sample is taken to be placed in the 250ml triangular flasks equipped with many small beades and 50ml sterilized waters, 40
Shaken 1 hour on DEG C constant-temperature table, stand 30 minutes.Aseptically Aspirate supernatant 1ml, accesses and has been sterilized equipped with 50ml
Domestication culture medium (glucose 10g, beef extract 3g, yeast extract 5g, peptone 10g, sodium chloride 5g, 1000ml distilled water)
250ml triangular flasks in, 40 DEG C culture 3 days (160 revs/min of rotating speed).With same method, 1ml pregnant solutions, passage are drawn again
Cultivated for 3 generations.
The bacteria suspension 1ml after 3 generations of culture is taken, is added in 9ml sterilized waters, 10 are configured to by 10 times of dilution methods-1, 10-2,
10-3, 10-4, 10-5, 10-6, 10-7Dilution factor.Each dilution factor respectively takes 0.1ml and coats 3 identification trainings containing phytic acid calcium
Support on the flat board of base.The sterilized water for taking 0.1ml is operated by the same method, is compared.40 DEG C are cultivated 3 days.Selection bacterium colony is well dispersed
Flat board, from the big single bacterium colony of upper picking degraded circle.And on identification culture medium, line repeatedly is isolated and purified 3 times, is seeded in containing Portugal
(glucose 10g, peptone 10g, yeast extract 5g, sodium chloride 5g, agar 15g, distilled water on the culture medium of grape glycoprotein peptone agar
1000ml, pH7.0) inclined-plane culture to abundant, preserves at 4 DEG C, standby.
It is above-mentioned identify culture medium formula be：Phytic acid calcium 0.1%~0.5%, glucose 3~5%, NH4NO30.5~
0.8%th, MnSO4·4H2O 0.002~0.005%, FeSO4·7H2O0.003~0.005%, MgSO4·7H2O 0.03~
0.06%th, KC1 0.03~0.07%, 0.04% bromocresol green solution 1.5~2.0% (v/v), agar 1.5~2%, pH5.0
~6.0.
As template, PCR expands its 16S rRNA gene order to DNA with bacterial strain of the present invention, and primer is (27f)：5′-AGA
GTT TGA TCC TGG CTC AG-3 ' and (1492r)：5′-GGT TAC CTT GTT ACG ACT T-3′.PCR reaction intervals
Sequence is：95 DEG C of predegenerations 5 minutes, 95 DEG C are denatured 30 seconds, and 53 DEG C are annealed 45 seconds, and 72 DEG C extend 90 seconds, 30 circulations, 72 DEG C of extensions
10 minutes.Amplified production is sequenced through after electrophoresis detection its purity, and sequencing result is shown in sequence table SEQ ID No:Shown in 1.Obtain
16S rRNA gene orders pass through EzTaxon databases (http://www.ezbiocloud.net/) compare, with bacterial strain
The similitude highest of Bacillus licheniformis, homology is 99.3%.Fig. 1 is based on UTM102 bacterial strains and its close
Bacterial strain 16S rRNA gene orders, using the systematic evolution tree (maximum likelihood method) of Mega5.0 phyletic evolution software buildings, explanation
Its Phylogenetic is nearest with bacillus licheniformis.
Using universal primer UP-1S:5 '-GAA GTC ATC ATG ACC GTT CTG CA-3 ' and UP-2Sr:5’-AGC
AGG GTA CGG ATG TGC GAG CC-3 ' expand the gyrB genes of the bacterial strain through PCR, and sequencing result is shown in sequence table SEQ ID
No:2 sequences for obtaining are submitted to NCBI GeneBank databases carries out sequence alignment with Blast programs, as a result shows
The gyrB gene orders of UTM102 bacterial strains and Bacillus licheniformis similitude highests.
It is Bacillus the identification of strains with reference to strain morphology feature (Fig. 2) and physiological and biochemical property
Licheniformis, is named as UTM102, and general in China Committee for Culture Collection of Microorganisms in August in 2014 13 days
Logical microorganism center (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode
100101) preservation, Classification And Nomenclature is bacillus licheniformis Bacillus licheniformis, and preserving number is CGMCC
No.9508。
The bacterial strain UTM102 cellulase activities of embodiment 2 are detected and its related gene sequence
By bacillus UTM102 dibbling methods dibbling sodium carboxymethylcellulose culture medium (sodium carboxymethylcellulose 5g,
KH2PO4Lg, agar 17g, NaNO3 3g、KCL 0.5g、MgSO4 0.5g、FeSO40.01g, distilled water 1000ml, pH 5.5
~6.0) on, 40 DEG C cultivate 48 hours.Using 0.2% congo red staining 30 minutes, dye liquor is washed away with distilled water, then be with concentration
The NaC1 of 1mol/L soaks 1 hour, finally fixes color with 5% acetate solution.Water white transparency circle is formed in periphery of bacterial colonies to show
This bacterium eccrine fiber element enzyme.Cellulase is many enzymatic mixtures, and it is made up of cellobiase, beta glucan restriction endonuclease etc..
Primer betaglu-f is designed according to beta glucan incision enzyme gene:5’-CGA TGT TGTTCA TGC CGG CT-
3 ' and betaglu-r:5 '-TTG CCA GCG TGT GTG ACAGC-3 ', enter by template of bacterial strain UTM102 DNA of the present invention
Performing PCR is reacted, and PCR reaction conditions are 95 DEG C of predegenerations 5 minutes, and 95 DEG C are denatured 30 seconds, and 55 DEG C are annealed 45 seconds, and 72 DEG C extend 90
Second, 30 circulations, 72 DEG C extend 10 minutes, obtain the fragment of about 2.0kb, are sequenced and will be transported in NCBI GeneBank databases
Sequence alignment is carried out with Blast programs, genetic fragment coding beta glucan restriction endonuclease, its gene order is shown in sequence table SEQ ID
No:3。
According to the degenerate primer Cellobiase-f that cellobiase genes are designed:5’-GAA GGCATT CCT TAT
CAT TC-3 ' and Cellobiase-r:5 '-ACG GTC ATA CTC AGCGTA AG-3 ', and with bacterial strain UTM102 of the present invention
DNA is template, carries out pcr amplification reaction, and PCR response procedures are：95 DEG C of predegenerations 5 minutes, 95 DEG C are denatured 30 seconds, 53 DEG C of annealing
45 seconds, 72 DEG C extended 90 seconds, 35 circulations, and 72 DEG C extend 10 minutes, and the nucleotides piece of about 2kb sizes is obtained by electrophoresis detection
Section, is sequenced and will carry out sequence alignment with Blast programs in NCBI GenBank databases, the sequence fragment encoding fiber two
Carbohydrase gene, its gene order is shown in sequence table SEQ ID No:4.
The bacterial strain UTM102 xylanase activities of embodiment 3 are detected and its gene order
Beech xylan (Sigma) (being accurate to 0.001 gram) accurately is weighed, with 50mmol/L NaAc-HAc (pH 5.0)
Buffer solution is made into 1% beech xylan solution (best matching while using, glycan is easily decomposed in acid condition)；1ml is taken in addition
Supernatant (the fermentation medium of UTM102 cultures：Glucose 5g, peptone 15g, yeast extract 5g, sodium chloride 5g, distilled water
1000ml, pH7.0), with 50mmol/L NaAc-HAc (pH 5.0) buffers into debita spissitudo enzyme liquid, it is ensured that light inhale
It is received between 0.2~0.6.Then the beech xylan solutions of 0.9ml 1% are taken, the enzyme liquid for adding 0.1ml UTM102 to be prepared,
After 30 minutes being incubated in 55 DEG C of water-baths, plus 1ml distilled water and 2ml DNS reagents.Boiling water bath 5 minutes, are cooled to after mixing
Room temperature, plus distilled water constant volume is to 25ml.Its OD value is determined at wavelength 540nm with spectrophotometer after mixing.Simultaneously with having gone out
The enzyme liquid that the supernatant (100 DEG C are boiled 10 minutes) of enzyme living is prepared makees blank.UTM102 bacterial strains can be hydrolyzed after measured
Beech xylan, illustrates that it has the activity of zytase.
According to the degenerate primer Xylanase-f that xylanase gene is designed:5 '-GCG CTG ACCTAT AAC G-3 ' and
Xylanase-r:5 '-CGC TCA CAG TGG ATT C-3 ', and with bacterial strain UTM102DNA of the present invention as template, enter performing PCR
Amplified reaction, PCR response procedures are：95 DEG C of predegenerations 5 minutes, 95 DEG C are denatured 30 seconds, and 56 DEG C are annealed 45 seconds, and 72 DEG C extend 90
Second, 35 circulations, 72 DEG C extend 10 minutes, obtain the nucleotide fragments of about 1.3kb sizes by electrophoresis detection, are sequenced and will be
NCBI GeneBank databases carry out sequence alignment with Blast programs, obtain the sequence fragment encoding xylanase gene,
Its gene order is shown in sequence table SEQ ID No:5.
The bacterial strain UTM102 phytase activities of embodiment 4 are detected and its gene order
It is on Phytate Ca medium solid plate, being formulated by the streak inoculation of UTM102 bacterial strains：Phytic acid calcium 0.5%, grape
Sugar 5%, NH4NO30.8%th, MnSO4·4H2O 0.005%, FeSO4·7H2O 0.005%, MgSO4·7H2O 0.06%,
KC1 0.07%, 0.04% bromocresol green solution 2.0% (v/v), agar 1.5~2%, pH5.0~6.0.What UTM102 was produced
Phytic acid calcium degraded is produced transparent hydrolysis circle by phytase.
According to the degenerate primer Phytase-f that phytase gene is designed:5 '-CGC AGC ATCCTT ATG G-3 ' and
Phytase-r:5 '-TCT GAT TGG CTG GTT G-3 ', and with bacterial strain UTM102DNA of the present invention as template, enter performing PCR expansion
Increase reaction, PCR response procedures are：95 DEG C of predegenerations 5 minutes, 95 DEG C are denatured 30 seconds, and 55 DEG C are annealed 45 seconds, and 72 DEG C extend 90 seconds,
30 circulations, 72 DEG C extend 10 minutes, and the nucleotide fragments of about 1kb sizes are obtained by electrophoresis detection, and sequencing simultaneously will be in NCBI
GenBank databases carry out sequence alignment with Blast programs, obtain sequence fragment coding phytase gene, its gene sequence
Row are shown in sequence table SEQ ID No:6.
The preparation of the bacterial strain UTM102 liquid bacterial agents of embodiment 5 and bacteria powder
Take a small amount of lawn and be seeded to from 4 DEG C of bacterial strain UTM102 inclined-planes of the present invention of preservation and contain glucose proteins equipped with 40ml
The 250ml tri- of the culture medium (glucose 10g, peptone 10g, yeast extract 5g, sodium chloride 5g, distilled water 1000ml, pH7.0) of peptone
Angle bottle.37 DEG C of cultivation temperature, 180 revs/min of shaking speed is cultivated 2 days, OD600Stop culture during more than 1.8, this is shaking flask kind
Sub- liquid.
Shake-flask seed liquid is seeded to culture medium (glucose 10g, peptone 10g, yeast extract containing glucose proteins peptone
5g, sodium chloride 5g, distilled water 1000ml, pH7.0) seeding tank in cultivated.37 DEG C of cultivation temperature, throughput is 1:0.3
(v/v), mixing speed is 200 revs/min, incubation time 38 hours.Work as OD600It is to stop culture more than 1.8, this is seeding tank
Seed liquor.
Seeding tank seed liquor is pressed into 0.1~5% inoculum concentration, inoculation carries out fermentation training in the fermentation tank containing above-mentioned culture medium
Support.37 DEG C of cultivation temperature, throughput is 1:0.4 (v/v), mixing speed is 100 revs/min, incubation time 30 hours.Work as OD600
Stop culture when being more than 1.8, this is the zymotic fluid of bacterial strain.
Zymotic fluid packing turns into liquid bacterial agent, and zymotic fluid obtains bacteria powder through dehydrating.
The microbial inoculum compost fermentation treatment domestic sludge that embodiment 6 is prepared using bacterial strain UTM102
By domestic sludge: moisture conditioner (1: 0.8) prepare material (scale of construction ratio), if without with addition 1% embodiment 5
2 experimental groups of obtained UTM102 microbial inoculums.Compost primary condition is moisture 63.1%, and C/N values are 35：1, sent out using aerobic compost
Fermenting process.The compost fermentation time is 20 days, and period is daily in heap body difference meter record temperature.Compost 0 day, 5 days, 10 days, 15
It turning and is measured by sampling sample moisture content to fall in the way of groove.Both temperature and moisture result of variations are shown in Table 1.
The compost temperature of table 1 and moisture situation of change
Add bacterium solution group moisture to be less than 40% during compost fermentation 15 days, reach national Sludge landfill standard, and be not added with bacterium
Liquid group takes 20 days and can be only achieved standard of landfill；The former loss of weight about 60%, the latter's loss of weight about 50%.
Embodiment 7 prepares bio-feritlizer using UTM102 microbial inoculum compost fermentations
With the maize straw after feces of livestock and poultry and crushing as main material, 400kg swine excrements and 600kg maize straws and
UTM102 microbial inoculums pulvis mixing obtained in 1kg embodiments 5 is mixed thoroughly, and regulation moisture to moisture content is about 55%, is placed in fermentation tank
Carry out aerobic composting fermentation.It is simultaneously control with the experiment without microbial inoculum of the present invention.Period is every 3~5 days in the mode of falling groove
Turning is once.When the moisture of material is less than 30%, and heap temperature terminates fermentation when no longer rising.This material is through sieving
Bio-feritlizer.Carbon-nitrogen ratio and germination index analysis in table 2 to prepared bio-feritlizer.
Table 2 is inoculated with influence of the UTM102 microbial inoculums to compost maturity
The above is only the preferred embodiment of the present invention.It should be pointed out that for the ordinary skill people of the art
For member, on the premise of the technology of the present invention principle is not departed from, some improvements and modifications can also be made, these improvements and modifications
Also should be regarded as protection scope of the present invention.
Claims (10)
1. bacillus licheniformis (Bacillus licheniformis) bacterial strain UTM102, its deposit number is CGMCC
No.9508。
2. lichem bacillus strain UTM102 as claimed in claim 1, it is characterised in that it can be produced in beta glucan
Enzyme cutting, cellobiase, zytase, phytase.
3. lichem bacillus strain UTM102 as claimed in claim 2, it is characterised in that the beta glucan restriction endonuclease is compiled
Code nucleotide sequence contains SEQ ID NO:Nucleotide sequence or its complementary nucleotide sequence described in 3；Cellobiase is encoded
Nucleotide sequence contains SEQ ID NO:Nucleotide sequence or its complementary nucleotide sequence described in 4；Zytase encoding nucleoside
Acid sequence contains SEQ ID NO:Nucleotide sequence or its complementary nucleotide sequence described in 5；Phytase coding nucleotide sequence
Contain SEQ ID NO:Nucleotide sequence or its complementary nucleotide sequence described in 6.
4. the microbial inoculum of lichem bacillus strain UTM102 described in claim 1 is contained.
5. a kind of method for preparing the compost Inoculant containing bacillus licheniformis UTM102, it is characterised in that comprise the following steps：
(1) zymotic fluid is prepared：Lichem bacillus strain UTM102 activated spawns are inoculated in the liquid training of glucose proteins peptone
Support in base, carry out fermenting and producing；
(2) zymotic fluid packing turns into liquid bacterial agent or obtains pulvis through dehydrating；
The deposit number of the bacillus licheniformis UTM102 is CGMCC No.9508.
6. any described lichem bacillus strain UTM102 of claim 1-3 or the microbial inoculum described in claim 4 are organic
Application in solid waste compost fermentation.
7. application as claimed in claim 6, it is characterised in that the organic solid castoff be downflow sludge, house refuse,
One or more in crop material, spoil or feces of livestock and poultry.
8. application as claimed in claim 6, it is characterised in that comprise the following steps：
(1) UTM102 or its microbial inoculum that will be equivalent to total material weight in wet base 0.1%~0.5% are inoculated in organic solid castoff,
Mixing carries out aerobic composting fermentation after mixing thoroughly；
(2) it is 45%~65% the moisture of material to be adjusted into moisture content with moisture conditioner, and the carbon-nitrogen ratio of this material is 10~60:
1, it is 8%~15% that heap body keeps oxygen content；
(3) heap body ferments 12-20 days, and fermentation terminates；Leftover materials or landfill or burning or packing of sieving obtain bio-feritlizer.
9. prepared by any described lichem bacillus strain UTM102 of claim 1-3 or the microbial inoculum described in claim 4
Application in organic fertilizer.
10. any described lichem bacillus strain UTM102 of claim 1-3 or the microbial inoculum described in claim 4 are promoting
Application in plant growth.
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| CN105695354B (en) * | 2016-02-19 | 2019-09-13 | 大地绿源环保科技(北京)有限公司 | The technique and application of superhigh temperature aerobic composting fermentation processing municipal sludge |
| CN110951649B (en) * | 2019-12-26 | 2021-03-26 | 江苏大道生物环境科技有限公司 | Bacillus for treating industrial excess sludge and application method thereof |
| CN112175875A (en) * | 2020-10-13 | 2021-01-05 | 蛋壳城矿环保科技发展(广州)有限公司 | Preparation method and application of ultrahigh-temperature aerobic composite biological agent |
| CN114717125B (en) * | 2021-01-04 | 2023-06-20 | 山东农业大学 | Thermophilic bacillus licheniformis AMCC101380 and application thereof in high-temperature composting of tail vegetables |
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| CN101892182A (en) * | 2010-06-07 | 2010-11-24 | 中国农业大学 | Bacillus licheniformis and application thereof in promotion of cellulose degradation |
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| CN101892182A (en) * | 2010-06-07 | 2010-11-24 | 中国农业大学 | Bacillus licheniformis and application thereof in promotion of cellulose degradation |
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| 一株嗜热地衣芽孢杆菌及其产高温淀粉酶的初步研究;李秋鹏等;《工业微生物》;20100630;第40卷(第3期);第34页右栏第1.1.3节、第36页第2.2节 * |
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