CN107435031A - A kind of house refuse degraded compound bacteria and its application - Google Patents
A kind of house refuse degraded compound bacteria and its application Download PDFInfo
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- CN107435031A CN107435031A CN201710520704.5A CN201710520704A CN107435031A CN 107435031 A CN107435031 A CN 107435031A CN 201710520704 A CN201710520704 A CN 201710520704A CN 107435031 A CN107435031 A CN 107435031A
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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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
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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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
Abstract
The invention discloses a kind of house refuse degraded compound bacteria, it is made up of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HQY 01, bacillus (Bacillus sp) HQY 04, plan candida lambica (Candida pseudolambica) HQY 06, it is preserved in China typical culture collection center, address：China, Wuhan, Wuhan University, preservation date are on 2 16th, 2017, and deposit number is respectively CCTCC NO:M2017040、CCTCC NO:M2017042、CCTCC NO:M2017043.The house refuse degraded compound bacteria of the present invention can be used in degraded house refuse, and waste reduction rate is high, and COD, ammonia nitrogen, nitrogen removal rate are high.
Description
Technical field
The present invention relates to the biological agent in a kind of household-garbage processing process, specifically provides a kind of house refuse degraded
Compound bacteria and its application.
Background technology
At present, the processing method of China's house refuse is based on bioanalysis, because the house refuse content of organic matter is high, composition
The features such as complicated, common biological treatment effect are poor.So study the group of microorganism in life refuse biochemical processing system
Into filtering out house refuse efficient degrading bacterial strain, understand the characteristic of each bacterial strain, and carry out compounding and turn out efficient degradation life rubbish
Rubbish composite bacteria agent, makes great sense to optimization processing.
Complex micro organism fungicide, refer to made of two kinds or the two or more mutual not microorganism fungus kinds of antagonism and symbiosis
Microorganism formulation.House refuse high-effective service refers to microorganism system made of a variety of high-efficiency strains with degraded house refuse
Agent.
The content of the invention
A kind of the defects of in order to overcome the Biochemical Treatment difference of house refuse in the prior art, there is provided energy high-volume production
And the complex microorganism preparations of the efficient degradation house refuse of stable performance.Premise of the biological agent in not reforming processing equipment
Under, being capable of the parameter index such as effectively optimization processing system, the COD of house refuse of efficiently degrading, ammonia nitrogen, total nitrogen.
To achieve these goals, the technical solution adopted in the present invention is：
A kind of house refuse degraded compound bacteria, the compound bacteria is by bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens) HQY-01, bacillus (Bacillus sp) HQY-04, plan candida lambica (Candida
Pseudolambica) HQY-06 is formed.
Further, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HQY-01, in being preserved in
State's Type Tissue Collection, address：China, Wuhan, Wuhan University, preservation date are on 2 16th, 2017, deposit number
For CCTCC NO:M2017040.
Bacillus (Bacillus sp) HQY-04, is preserved in China typical culture collection center, address：In
State, Wuhan, Wuhan University, preservation date are on 2 16th, 2017, and deposit number is CCTCC NO:M2017042
Described plan candida lambica (Candida pseudolambica) HQY-06, is preserved in Chinese Typical Representative culture
Thing collection, address：China, Wuhan, Wuhan University, preservation date are on 2 16th, 2017, deposit number CCTCC
NO:M2017043
Further, in the house refuse degraded compound bacteria, the bacillus amyloliquefaciens HQY-01, bacillus HQY-
04th, it is 35 to intend the ratio between candida lambica HQY-06 living bacteria count：50：15.
House refuse degraded compound bacteria of the present invention, can be made different microbial inoculum dosage forms, such as freeze-dried powder, bacterium
Liquid, bacterium mud etc., can by by bacillus amyloliquefaciens HQY-01, bacillus HQY-04, intend candida lambica HQY-
The 06 fermented culture of difference, the fermentation culture of acquisition matches according to living bacteria count to be mixed, using microbial inoculum well known in the art
All kinds of microbial inoculum preparations are prepared in preparation method.It can be used after zymotic fluid compounding directly as compound bacteria bacterium solution, or will hair
Zymotic fluid centrifugation, take pellet frozen to dry, freeze-dried powder can be made；Or compound bacteria bacterium solution is obtained with being filtered after carrier adsorption
Bacterium mud etc.
The present invention also provides application of the described house refuse degraded compound bacteria in house refuse of degrading.
Further, the house refuse refers to the small stores discarded object containing organic components.Mainly waste paper, useless
Duds thing bed necessaries, fiber, bamboo and wood (such as discarded chopsticks, chopping block), kitchen dish slag etc..Chemically composition on, mainly with cellulose,
Phosphorus, protein, starch, lipid and inorganic salts.
Further, the method for the application is：House refuse degraded compound bacteria and house refuse are added into garbage disposer
In, degradation reaction, waste reduction rate more than 90% are stirred at normal temperatures.
Further, the time of degradation reaction is 1~3 day (preferably 24~48 hours), and waste reduction rate is dirty up to more than 90%
Water water outlet COD is further discharged after water treatment facilities processing is up to standard in below 3000mg/L.
Further, the injected volume of the house refuse degraded compound bacteria adds effective total viable count for per kilogram house refuse
For (1~10) × 107Cfu compound bacteria (preferably 3 × 107cfu)
The waste reduction rate=(undecomposed mass of residue after input rubbish gross mass-degraded)/input rubbish gross mass
× 100%.The part of waste reduction is large amount of sewage.
House refuse is mainly degraded to large amount of sewage.
House refuse obtains large amount of sewage and the residue that can not be decomposed on a small quantity after compound bacteria is degraded, and sewage equally passes through
Compound bacteria is crossed to degrade, cod values are from more than the 10000mg/L degradeds of rubbish to 500~3000mg/L or so, but still be unable to reach
Discharge standard, gained sewage are subsequently handled through water treatment facilities again.Handled using the water treatment facilities of routine.
The house refuse of the present invention can carry out continuous processing with garbage disposer, when first time launching house refuse, press
It is (1~10) × 10 to add effective total viable count according to per kilogram rubbish7Cfu house refuse compound bacteria, is then collected daily
House refuse is continuously launched, and daily garbage throwing quality is between the 50%~150% of the rubbish quality that first time launches, no longer
New compound bacteria is launched, sewage water outlet COD periodic detections, in below 3000mg/L, the sewage of discharge is directly entered water process system
System, water processing system are discharged after handling to discharge standard, garbage disposer continuous processing 2~3 months, and shutdown is emptied and can not dropped
The residue of solution.Garbage disposer carries out new circulation again.
Further, when launching house refuse compound bacteria, when compound bacteria is bacterium solution form, generally requires while add absorption
Carrier, bacterium solution is set to be adsorbed on carrier, the carrier can be rice husk, stalk, wheat bran, wood chip etc..
House refuse can be efficiently handled using the house refuse degraded compound bacteria of the present invention, and microorganism therein is to have
Machine thing is that food carries out metabolic activity, is finally discharged in the form of water and gas, waste reduction rate is high, and obtained residue is few, institute
Obtain a small amount of residue and stay in filler of the cavity as microorganism, and compound bacteria is to the COD in garbage leachate and ammonia nitrogen degradation rate
Very high, obtained water outlet is subsequently handled through conventional water treatment system and can discharged.
The beneficial effects of the present invention are：Microorganism in house refuse high-efficiency degradation composite bacteria agent belongs to environment-friendly
Four quasi-microorganisms of type.And with the species composition of specific processing house refuse, such as HQY-04 bacillus, to life
The degradation treatment excellent of rubbish.Other compositions are natural biological culture medium in sample, safe and convenient to use, nontoxic, no corruption
Erosion, it is harmless to people and animals and animals and plants；There is biological degradability simultaneously, ecological safety, no negative environmental consequences, be conventional micro- life
Thing composite bacteria agent product.
Embodiment
Substantive features of the present invention can be embodied from the following examples, but these embodiments are only used as explanation, rather than
Limit the invention.
Embodiment 1
Bacterial strain screening and identification：
(1) bacterial screening：Bacterial strain in house refuse reactor is screened using selective medium；
(2) strain secondary screening：Screen of effective and efficient bacteria is carried out respectively to the single bacterial strain of screening；
(3) strain is enriched with：Liquid enrichment culture is carried out to high-efficiency strain；
(4) composite bacteria is built：Bacterial strain is combined and tested；
(5) the strain idenfication in compound bacteria：Molecular biology identification is carried out to bacterial strain.
Wherein, (1) described bacterial screening, the present invention provide following methods to step：
With sterile liquid-transfering gun pipette 10.0mL incinerator for treating domestic refuse biochemical system mixing liquid be added to fill 90.0mL without
In the conical flask of bacterium water, and mix.Then the 1.0mL dilutions are taken in the test tube (- 10 for filling 9.0mL sterilized waters-2) in fully
Mix, then draw 1.0mL in the test tube (- 10 of another 9.0mL sterilized water from this test tube with sterile liquid-transfering gun-3) in, mix
Close uniformly, 10 are made by that analogy-2、10-3、10-4、10-5……10-8、10-9、10-10The bacterium solution of different dilution factors.Then it is coated with
To bacteria culture media, fungi culture medium, certain time is cultivated under corresponding conditionses.Wherein described bacteria culture media composition：Albumen
Peptone 1%, sodium chloride 0.5%, yeast extract 1%, water 100ml, pH 7.0, agar powder 1.5% is added in solid medium.Wherein institute
State fungal culture based component：Yeast extract 1%, peptone 2%, glucose 2%, agar powder 1.5% is added in solid medium.Respectively
The condition of medium sterilization is：0.10-0.15MPa, 121 DEG C sterilize 30 minutes.
Bacteria culture media condition of culture is：37 DEG C of solid medium cultures 24 hours
Fungi culture medium condition of culture is：35 DEG C of solid medium cultures 48 hours
Wherein, (2) described strain secondary screening, the present invention provide following methods to step：
With the different bacterium colony of form on oese picking culture medium plate, line separation is carried out.Repeat until growing
Single bacterium colony, it is inoculated on culture test tube inclined-plane, culture is standby, 4 DEG C of preservation bacterial strains.15 kinds of bacterial strains are isolated, and observe bacterium
Strain colony characteristicses and strain microscopy situation, finally filter out 11 kinds of bacterial strains do culture it is standby.
Above bacterial strain carries out secondary screening, and picking inclined-plane bacterial strain respectively adds 10ml sterilized waters, suspension is made, and then draws 1ml bacterium
Liquid is in the sterilized house refuse nutrient solutions of 99ml, 30 DEG C, 120r/min shaking table cultures, is then noted by 0.25% dosage
Enter into house refuse nutrient solution sterilized 150ml and do experiment, 30 DEG C, 120r/min shaking table cultures.
COD, ammonia nitrogen value are determined per 8h, therefrom the high and more stable bacterial strain of screening degradation rate, totally 6 kinds.
The house refuse nutrient solution is to simulate the nutrient solution that each material concentration content configures in real life rubbish, composition
For organic matter 25%, total nitrogen 3%, phosphorus pentoxide 4%, potassium oxide 1%, pure water (constant volume), arsenic 0.01%, lead 0.01%, chromium
0.03%.
Wherein, (3) described strain enrichment, the present invention provide following methods to step：
6 kinds of inclined-plane bacterial strains that picking screens respectively add 10ml sterilized waters, and suspension is made, then draw 1ml bacterium solutions in
In house refuse nutrient solution sterilized 99ml, 30 DEG C, 120r/min shaking table cultures.
The house refuse nutrient solution is same as above.
Wherein, (4) described composite bacteria structure, the present invention provide following methods to step：
The bacterial strain for being enriched with acquisition is combined by certain volume ratio, being injected into 150ml by 0.25% dosage goes out
Cross in the house refuse nutrient solution of bacterium and do experiment, 30 DEG C, 120r/min shaking table cultures.Measure COD, ammonia nitrogen value per 12h.Wherein go
Reach 91.67% except one group of 48h of rate highest COD clearances can reach 96.16%, ammonia nitrogen removal frank
Wherein, the strain idenfication of step (5) described compound bacteria, step are as follows：
One group of combination of clearance highest in selecting step (4), three kinds of bacterial strains therein are identified, identification includes thin
Bacterium and the molecular biology identification of fungi are as follows respectively：
(1) molecular biological variety identification method of bacterium：The Liquid Culture of strain, by the strain to be tested after secondary screening, from line
The ring of picking one on flat board, is inoculated into 10ml/50ml LB fluid nutrient mediums, 37 DEG C, 180rpm constant-temperature table overnight incubations；Treat
The extraction and checking of bacterial strain complete genome DNA are surveyed, the liquid culture that will have been activated, genomic DNA is extracted using phenol chloroform method.
The DNA extracted examines extraction result with 1% agarose gel electrophoresis；16sPCR and checking, using the DNA extracted as
Template, 16srDNA fragments are expanded using bacterial universal primers 27F and 1492R.The PCR primer expanded is coagulated with 1% agarose
Gel electrophoresis verify expanding effect.
27F：AGAGTTTGATCCTGGCTCAG
1492R：GGTTACCTTGTTACGACTT
LB Liquid Culture based formulas is：Tryptone (Tryptone) 10g/L, yeast extract (Yeast
Extract) 5g/L, sodium chloride (NaCl) 10g/L, solvent are deionized water, pH value 7.0.
The steam sterilizing 20min under 15psi (1.05kg/cm2) high pressure.
(2) molecular biological variety identification method of fungi：The Liquid Culture of strain, by the strain to be tested after secondary screening, from line
The ring of picking one on flat board, is inoculated into 10ml/50ml YPD fluid nutrient mediums, 30 DEG C, 180rpm constant-temperature table overnight incubations；
The extraction of strain to be tested complete genome DNA, the liquid culture that will have been activated, genomic DNA, extracting are extracted using phenol chloroform method
The DNA arrived examines extraction result with 1% agarose gel electrophoresis；18sPCR and checking, using fungi universal primer NS1 and
FR1 expands 18srDNA fragments.Amplified fragments are run into 1% agarose gel electrophoresis checking expanding effect afterwards.
NS1:GTAGTCATATGCTTGTCTC
FR1AICCATTCAATCGGTAIT
YPD Liquid Culture based formulas is：1%Yeast Extract (yeast extract), 2%Peptone (peptone), 2%
Dextrose (glucose) (glucose), solvent are deionized water, pH value 7.0.Under 15psi (1.05kg/cm2) high pressure
Steam sterilizing 20min.
By the above method, present invention screening obtains three kinds high to house refuse degradation efficiency of bacterial strain, three kinds of bacterial strains
16S rDNA complete sequences are respectively as shown in SEQ ID.NO1, SEQ ID.NO2, SEQ ID.NO3.The 16S rRNA sequences of three kinds of bacterial strains
Row analysis result, with reference to physiological and biochemical test result, after the comparison of NCBI gene pools, it is respectively to solve starch bud to determine three kinds of bacterial strains
Spore bacillus (Bacillus amyloliquefaciens), bacillus (Bacillus sp), intend candida lambica
(Candida pseudolambica), and bacillus amyloliquefaciens HQY-01 is named as, bacillus HQY-04, intend Lang Bike
Candida HQY-06
SEQ ID.NO1：
ATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGG
GTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCA
AACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCAC
CAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGG
AGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGA
TCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGC
CACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGC
TCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGA
GTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGC
GACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACG
CCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTG
GGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTC
GAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAG
AGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTT
GATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGT
CAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGG
TTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGT
AATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTA
ACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCT
SEQ ID.NO2：
ATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACAC
GTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATGG
TTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGC
TCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTA
CGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTT
CGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGA
AAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAA
GGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAAC
TTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGA
AGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTC
CACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCG
CCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGG
GCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC
CCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATG
ACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGC
GAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGC
TAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTT
TGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAA
SEQ ID.NO3：
TTCGGCGGCTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGG
CGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAG
TCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGT
TCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGT
TTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACT
TAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTAT
CTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGC
TTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAG
CTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATC
CTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCA
CATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGAC
CCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGG
ACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAG
GTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGC
GGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCT
CAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAG
CTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAAGCA
TCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCC
GCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCT
Embodiment 2
The degradation efficiency of the composite bacteria agent of different formulations and single microbial inoculum contrasts
By screen three kinds of strains B. amyloliquefaciens HQY-01, bacillus HQY-04, intending youth's ratio can false silk
Yeast HQY-06 carries out fermented and cultured respectively：It is inoculated into nutrient solution, at 35 DEG C, frequency of oscillation is trained under conditions of being 120r/min
Support 4 days.
The nutrient solution contains following component (containing in per volume nutrient solution)：Peptone 10g/L, sodium chloride 5g/L, ferment
Female cream 10g/L, glucose 10g/L, PH are transferred to 7.
Fermentation obtains bacillus amyloliquefaciens HQY-01, bacillus HQY-04, intends candida lambica HQY-06's
Viable count is respectively 2.5*10 in nutrient solution7CFU/mL、2.5*107CFU/mL、4.5*107CFU/mL
6 parts of house refuse nutrient solution 100mL is taken, 1*10 is added according to every 100mL7CFU quantity is separately added into solution starch
Bacillus HQY-01 nutrient solutions, bacillus HQY-04 nutrient solutions, intend candida lambica HQY-06 nutrient solutions, compound bacteria
Liquid A, composite bacteria liquid B, composite bacteria liquid C, composite bacteria liquid D.
House refuse nutrient solution is the nutrient solution for simulating each material concentration content configuration in real life rubbish, and composition is to have
Machine matter 25%, total nitrogen 3%, phosphorus pentoxide 4%, potassium oxide 1%, pure water (constant volume), arsenic 0.01%, lead 0.01%, chromium
0.03%.
Bacillus amyloliquefaciens HQY-01, bacillus HQY-04 in wherein composite bacteria liquid A, intend candida lambica
The ratio between HQY-06 living bacteria count is 35：50：15
The ratio between bacillus amyloliquefaciens HQY-01, bacillus HQY-04 living bacteria count is 40 in composite bacteria liquid B：60
Bacillus HQY-04 in composite bacteria liquid C, it is 80 to intend the ratio between candida lambica HQY-06 living bacteria count：
20
Bacillus amyloliquefaciens HQY-01 in composite bacteria liquid D, intend candida lambica HQY-06 living bacteria count it
Than for 70:30
30 DEG C, 120r/min shaking table cultures, measure COD, ammonia nitrogen value, total values of nitrogen might per 12h, and calculate 48h COD conversions
Rate, ammonia nitrogen, nitrogen removal rate, acquired results such as table 1 below：
Table 1
Table 1 as can be seen that composite bacteria liquid A COD, ammonia nitrogen, total nitrogen removal efficiency highest, be significantly better than single culture and
Other composite bacteria liquids.
The composite bacteria preparation of embodiment 3 is used for the degraded of house refuse：
1st, the fermentation of composite bacteria preparation
Fermentation comprises the following steps：By screen three kinds of bacterial strains：Bacillus amyloliquefaciens HQY-01, bacillus
HQY-04, plan candida lambica HQY-06 are inoculated into nutrient solution respectively, and at 35 DEG C, frequency of oscillation is 120r/min bar
Cultivated 4 days under part.
The nutrient solution contains following component (containing in per volume nutrient solution)：：Peptone 10g/L, sodium chloride 5g/L, ferment
Female cream 10g/L, glucose 10g/L, PH are transferred to 7.
Gained bacillus amyloliquefaciens HQY-01, bacillus HQY-04, intend candida lambica HQY-06 culture
Viable count is respectively 2.5*10 in liquid7CFU/mL、2.5*107CFU/mL、4.5*107CFU/mL
According to bacillus amyloliquefaciens HQY-01, bacillus HQY-04, intend the effective of candida lambica HQY-06
The ratio between viable count is 35：50：15, three kinds of nutrient solutions are mixed, obtain composite bacteria liquid.Total living bacteria count in composite bacteria liquid
For 3*107CFU/mL
2nd, composite bacteria agent adds after fermenting
Test site：Wide stale garbage terminal；House refuse primary categories：The resident family on periphery, the house refuse of enterprise.
Problem describes：House refuse amoun is excessive, collects not in time or weather temperature is high, will grow flies, influence week
The hygienic conditions on side.
Solution method：Treatment in situ is carried out, is added in garbage disposal all-in-one with pending garbage bulk than 1:1 rice husk
(absorption carrier using rice husk as bacterium solution), 3*10 is launched according to per kilogram rubbish7CFU composite bacteria liquid, bacterium solution stir with rice husk
After mixing fully, house refuse is launched into processing all-in-one, stirring reaction under normal temperature, degradation reaction 24 hours, rubbish subtracts
Dose rate reaches more than 90%.
And continuous processing can be carried out, the rubbish collected daily is continuously launched, and daily garbage throwing quality is thrown in first time
Between the 50%~150% of the rubbish quality put, it is not necessary to launch new compound bacteria again, sewage water outlet COD periodic detections, substantially
In below 3000mg/L, the follow-up water processing system of gained water outlet discharges after handling to discharge standard.Can continuous processing 2-3
Month, the remaining residue that can not be degraded is shut down and removed once for regular 2-3 months in all-in-one.Then new circulation is carried out again.
As a result：House refuse can be efficiently handled using composite bacteria preparation, microorganism therein is using organic matter as food
Metabolic activity is carried out, is finally discharged in the form of water and gas, waste reduction rate is high, and a small amount of residue stays in cavity as microorganism
Filler, and high to COD, ammonia nitrogen, total nitrogen degradation rate, the sewage subsequent treatment of discharge is easy, using the sewage disposal system of routine
System can qualified discharge.
SEQUENCE LISTING
<110>Qingyuan bio tech ltd of Zhejiang China
<120>A kind of house refuse degraded compound bacteria and its application
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1420
<212> DNA
<213> Bacillus amyloliquefaciens
<400> 1
atgcaagtcg agcggacaga tgggagcttg ctccctgatg ttagcggcgg acgggtgagt 60
aacacgtggg taacctgcct gtaagactgg gataactccg ggaaaccggg gctaataccg 120
gatggttgtt tgaaccgcat ggttcaaaca taaaaggtgg cttcggctac cacttacaga 180
tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggcg acgatgcgta 240
gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg 300
aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag 360
tgatgaaggt tttcggatcg taaagctctg ttgttaggga agaacaagta ccgttcgaat 420
agggcggtac cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg 480
cggtaatacg taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg ctcgcaggcg 540
gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt ggaaactggg 600
gaacttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat gcgtagagat 660
gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct gaggagcgaa 720
agcgtgggga gcgaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct 780
aagtgttagg gggtttccgc cccttagtgc tgcagctaac gcattaagca ctccgcctgg 840
ggagtacggt cgcaagactg aaactcaaag gaattgacgg gggcccgcac aagcggtgga 900
gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca tcctctgaca 960
atcctagaga taggacgtcc ccttcggggg cagagtgaca ggtggtgcat ggttgtcgtc 1020
agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt 1080
gccagcattc agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg 1140
atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggacaga 1200
acaaagggca gcgaaaccgc gaggttaagc caatcccaca aatctgttct cagttcggat 1260
cgcagtctgc aactcgactg cgtgaagctg gaatcgctag taatcgcgga tcagcatgcc 1320
gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgag agtttgtaac 1380
acccgaagtc ggtgaggtaa ccttttagga gccagccgct 1420
<210> 2
<211> 1427
<212> DNA
<213> Bacillus sp.
<400> 2
atacatgcaa gtcgagcgga cagatgggag cttgctccct gatgttagcg gcggacgggt 60
gagtaacacg tgggtaacct gcctgtaaga ctgggataac tccgggaaac cggggctaat 120
accggatgct tgtttgaacc gcatggttca aacataaaag gtggcttcgg ctaccactta 180
cagatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcaacgatg 240
cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc cagactccta 300
cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg 360
tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtaccgttc 420
gaatagggcg gtaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca 480
gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggctcgca 540
ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac 600
tggggaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg aaatgcgtag 660
agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga cgctgaggag 720
cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt aaacgatgag 780
tgctaagtgt tagggggttt ccgcccctta gtgctgcagc taacgcatta agcactccgc 840
ctggggagta cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg 900
tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct 960
gacaatccta gagataggac gtccccttcg ggggcagagt gacaggtggt gcatggttgt 1020
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt 1080
agttgccagc attcagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt 1140
ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga 1200
cagaacaaag ggcagcgaaa ccgcgaggtt aagccaatcc cacaaatctg ttctcagttc 1260
ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg cggatcagca 1320
tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg 1380
taacacccga agtcggtgag gtaacctttt aggagccagc cgccgaa 1427
<210> 3
<211> 1413
<212> DNA
<213> Candida pseudolambica
<400> 3
ttcggcggct ggctcctaaa aggttacctc accgacttcg ggtgttacaa actctcgtgg 60
tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga 120
ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa ctgagaacag 180
atttgtggga ttggcttaac ctcgcggttt cgctgccctt tgttctgtcc attgtagcac 240
gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt 300
ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag atcaagggtt 360
gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 420
acctgtcact ctgcccccga aggggacgtc ctatctctag gattgtcaga ggatgtcaag 480
acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg 540
cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcg gagtgcttaa 600
tgcgttagct gcagcactaa ggggcggaaa ccccctaaca cttagcactc atcgtttacg 660
gcgtggacta ccagggtatc taatcctgtt cgctccccac gctttcgctc ctcagcgtca 720
gttacagacc agagagtcgc cttcgccact ggtgttcctc cacatctcta cgcatttcac 780
cgctacacgt ggaattccac tctcctcttc tgcactcaag ttccccagtt tccaatgacc 840
ctccccggtt gagccggggg ctttcacatc agacttaaga aaccgcctgc gagcccttta 900
cgcccaataa ttccggacaa cgcttgccac ctacgtatta ccgcggctgc tggcacgtag 960
ttagccgtgg ctttctggtt aggtaccgtc aaggtaccgc cctattcgaa cggtacttgt 1020
tcttccctaa caacagagct ttacgatccg aaaaccttca tcactcacgc ggcgttgctc 1080
cgtcagactt tcgtccattg cggaagattc cctactgctg cctcccgtag gagtctgggc 1140
cgtgtctcag tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat cgttgccttg 1200
gtgagccgtt acctcaccaa ctagctaatg cgccgcgggt ccatctgtaa gtggtagccg 1260
aagccacctt ttatgtttga accatgcggt tcaaacaagc atccggtatt agccccggtt 1320
tcccggagtt atcccagtct tacaggcagg ttacccacgt gttactcacc cgtccgccgc 1380
taacatcagg gagcaagctc ccatctgtcc gct 1413
Claims (10)
- The compound bacteria 1. a kind of house refuse is degraded, it is characterised in that the compound bacteria is by bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) HQY-01, bacillus (Bacillus sp) HQY-04, plan candida lambica (Candida Pseudolambica) HQY-06 is formed；Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HQY-01, is preserved in Chinese Typical Representative culture Collection, address：China, Wuhan, Wuhan University, preservation date are on 2 16th, 2017, and deposit number is CCTCC NO: M2017040；Bacillus (Bacillus sp) HQY-04, is preserved in China typical culture collection center, address：China, it is military The Chinese, Wuhan University, preservation date are on 2 16th, 2017, and deposit number is CCTCC NO:M2017042；Described plan candida lambica (Candida pseudolambica) HQY-06, it is preserved in Chinese Typical Representative culture guarantor Tibetan center, address：China, Wuhan, Wuhan University, preservation date are on 2 16th, 2017, and deposit number is CCTCC NO: M2017043。
- The compound bacteria 2. house refuse as claimed in claim 1 is degraded, it is characterised in that in the house refuse degraded compound bacteria, Bacillus amyloliquefaciens HQY-01, bacillus HQY-04, the ratio between the living bacteria count of intending candida lambica HQY-06 are 35：50：15.
- 3. application of the house refuse degraded compound bacteria in house refuse of degrading as described in one of claim 1~2.
- 4. application as claimed in claim 3, it is characterised in that the method for the application is：By house refuse degraded compound bacteria and House refuse is added in garbage disposer, stirs degradation reaction, waste reduction rate more than 90% at normal temperatures.
- 5. application as claimed in claim 4, it is characterised in that the injected volume of the house refuse degraded compound bacteria is per kilogram It is (1~10) × 10 that house refuse, which adds effective total viable count,7Cfu compound bacteria.
- 6. the application stated such as claim 4 or 5, it is characterised in that obtain large amount of sewage and a small amount of nothing after the house refuse degraded The residue that method is decomposed, the cod values of sewage are degraded to below 3000mg/L through compound bacteria, and heel row up to standard is handled through water treatment facilities Put.
- A kind of 7. bacillus amyloliquefaciens (Bacillus for the house refuse degraded compound bacteria being used to prepare described in claim 1 Amyloliquefaciens) HQY-01, China typical culture collection center, address are preserved in：China, Wuhan, Wuhan are big Learn, preservation date is on 2 16th, 2017, and deposit number is CCTCC NO:M2017040.
- A kind of 8. bacillus (Bacillus sp) for the house refuse degraded compound bacteria being used to prepare described in claim 1 HQY-04, it is preserved in China typical culture collection center, address：China, Wuhan, Wuhan University, preservation date are 2017 2 The moon 16, deposit number is CCTCC NO:M2017042.
- A kind of 9. plan candida lambica (Candida for the house refuse degraded compound bacteria being used to prepare described in claim 1 Pseudolambica) HQY-06, China typical culture collection center, address are preserved in：China, Wuhan, Wuhan University, protect It is on 2 16th, 2017 to hide the date, and deposit number is CCTCC NO:M2017043.
- The compound bacteria agent 10. house refuse made of house refuse degraded compound bacteria as claimed in claim 1 is degraded, the bacterium Agent includes freeze-dried powder, bacterium solution or bacterium mud.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111196632A (en) * | 2020-01-15 | 2020-05-26 | 宁波大学 | Rhodotorula mucilaginosa for removing total nitrogen in sewage and application thereof |
| CN114350556A (en) * | 2021-12-31 | 2022-04-15 | 浙江华庆元生物科技有限公司 | Fruit and vegetable wastewater recycling microbial inoculum and application thereof in preparation of microbial compound fertilizer |
| CN114540219A (en) * | 2021-12-31 | 2022-05-27 | 浙江华庆元生物科技有限公司 | Tail vegetable wastewater recycling microbial inoculum and application thereof in preparation of plant enzymes |
| CN114606154A (en) * | 2021-12-31 | 2022-06-10 | 浙江华庆元生物科技有限公司 | Tail vegetable wastewater recycling microbial inoculum and application thereof in preparation of plant nutrient solution |
| CN117187125A (en) * | 2023-08-31 | 2023-12-08 | 上海纺织建筑设计研究院有限公司 | In-situ degradation deodorizing strain for waste landfill aged garbage and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111196632A (en) * | 2020-01-15 | 2020-05-26 | 宁波大学 | Rhodotorula mucilaginosa for removing total nitrogen in sewage and application thereof |
| CN111196632B (en) * | 2020-01-15 | 2022-02-22 | 宁波大学 | Rhodotorula mucilaginosa for removing total nitrogen in sewage and application thereof |
| CN114350556A (en) * | 2021-12-31 | 2022-04-15 | 浙江华庆元生物科技有限公司 | Fruit and vegetable wastewater recycling microbial inoculum and application thereof in preparation of microbial compound fertilizer |
| CN114540219A (en) * | 2021-12-31 | 2022-05-27 | 浙江华庆元生物科技有限公司 | Tail vegetable wastewater recycling microbial inoculum and application thereof in preparation of plant enzymes |
| CN114606154A (en) * | 2021-12-31 | 2022-06-10 | 浙江华庆元生物科技有限公司 | Tail vegetable wastewater recycling microbial inoculum and application thereof in preparation of plant nutrient solution |
| CN114540219B (en) * | 2021-12-31 | 2024-03-12 | 浙江华庆元生物科技有限公司 | Tail vegetable wastewater recycling microbial inoculum and application thereof in preparation of plant ferment |
| CN117187125A (en) * | 2023-08-31 | 2023-12-08 | 上海纺织建筑设计研究院有限公司 | In-situ degradation deodorizing strain for waste landfill aged garbage and application thereof |
| CN117187125B (en) * | 2023-08-31 | 2024-03-29 | 上海纺织建筑设计研究院有限公司 | In-situ degradation deodorizing strain for waste landfill aged garbage and application thereof |
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| CN107435031B (en) | 2020-05-19 |
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