CN106434456A - Thermophile bacterium A.rupiensis ZQ2 and application thereof - Google Patents
Thermophile bacterium A.rupiensis ZQ2 and application thereof Download PDFInfo
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- 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
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Abstract
The invention provides a thermophile bacterium A.rupiensis ZQ2. The preservation number of the strain of the thermophile bacterium A.rupiensis ZQ2 is GDMCC No.60056. The invention further provides an inoculant of the thermophile bacterium A.rupiensis ZQ2 and a preparation method and application of the inoculant. The thermophile bacterium A.rupiensis ZQ2 has the advantages that as the thermophile bacterium A.rupiensis ZQ2 is screened out at 60 DEG C, the thermostability is good, the carbon source utilization rate is high, the metabolic activity is not inhibited by the thermophilic composting temperature, and the adaptability is high. Meanwhile, by applying the inoculant to organic solid waste thermophilic composting treatment, the composting fermentation period can be greatly shortened, the composting treatment efficiency can be improved, and the treatment cost can be reduced.
Description
Technical field
The invention belongs to field of environment microorganism is and in particular to a kind of thermophilic microorganism and its application.
Background technology
Aerobic compost is using thermophilic bacteria group, decomposing organic matter, discharges heat energy simultaneously, and organic carbon is converted into class
The process of humus.At present, aerobic compost technology has become the important means of organic solid waste resource.Traditional aerobic compost is general
It is to be fermented using the indigenous microorganism in compost material, its sweat is substantially thermophilic microorganism becomes dominant microflora
Process.In composting process thermophilic microorganism become dominant microflora process longer, therefore lead to that fermentation time is long, odor pollution is tight
Weight and the low problem of rotten degree.In composting process starting stage microbe inoculation microbial inoculum, increase functional microorganism quantity, Ke Yiti
High composting efficiency and product quality.
At present, the microbial inoculant that aerobic compost adopts is mainly mesophilic aerobic bacteria, and wherein applying most is hay
Bacillus cereuss (Bacullus subtilis) and Bacillus licheniformis (B.licheniformis)；When fermentation temperature is more than 50
DEG C, strain activity is substantially suppressed.The Introduction To Cn Patent of Application No. 201010514582.7 is a kind of will be false for Aerugo single
Born of the same parents bacterium, bacillus subtilises, Bacillus licheniformis, Bacillus coagulans, Lactobacillus plantarum, Bacillus pumilus and coral promise
Block Salmonella and Trichoderma viride, Paracoccus denitrificans, Aspergillus flavus, Phanerochaete chrysosporium and azotobacter chroococcum are prepared into two kinds of heaps
Fertile Inoculant, for the different phase of compost fermentation.Patent No. CN 101486969A, CN101186879A,
The Chinese patents such as CN101696391A also disclose multiple bacillus cereuss, actinomycetes, yeast, lactic acid bacteria, photosynthetic bacteria, fixed nitrogen
The compost composite bacteria agent capable of the combination preparation such as bacterium, mycete.
Thermophilic Bacteria is the microorganism that a class is lived in hot environment, is widely present in geothermal spring, submarine volcano mouth and
The unique thermostability of artificial thermal and hydric environment such as hot water pipeline and high temperature aerobic composting, its cell and pheron and adaptable therewith
Molecular structure, makes the important microbe resource that can develop.At present, existing part Thermophilic Bacteria is used for aerobic compost
Report.Utilize Thermophilic Bacterium Calditerricola yamamurae as patent CN 102851247 A discloses one kind
The method that UTM801 is applied to organic solid waste During High-Temperature Composting.Compared with conventional microbiological Inoculant, inoculate in aerobic composting process
Thermophilic bacteria agent has higher microbial metabolic activity and organic matter degradation rate, has in solid organic waste substances process field
Wide application prospect and scientific value.
Anoxybacillus is the new genus that Pikuta in 2000 divides out from bacillus, starts to be accredited as tight
The thermophilc anaerobe of lattice, 2003 is facultative anaerobe by Authenticate afresh.Patent CN102732465B discloses one kind
The method that A.pushchinoensis UTM601 is used for compost Inoculant.But there are no Anoxybacillus.rupiensis to exist
The open report of application on organic solid waste compost.
Content of the invention
It is an object of the present invention to provide a kind of Thermophilic Bacteria A.rupiensis ZQ2 and its application in compost.
The present invention, from megathermal period sludge composting, through high temperature enrichment, isolates and purifies and obtains Thermophilic Bacteria A.rupiensis ZQ2
Bacterial strain.This bacterial strain is stored in Guangdong Province's Culture Collection, preserving number on July 8th, 2016：GDMCC
No.60056.
The identification mark of described Thermophilic Bacteria A.rupiensis ZQ2 is：
(1) it is Gram-positive, aerobic, thalline is in elongated rod shape；60 DEG C of active growth, and do not give birth on 75 DEG C at 30 DEG C
Long.
48h is cultivated, bacterium colony is rounded, regular edges, center projections, diameter 2～3mm at (2) 60 DEG C.
(3) utilize primer (27f)：5 '-AGA GTT TGA TCC TGG CTC AG-3 ' and (1492r)：5′-GGTTAC
CTT GTT ACG ACT T-3 ' expands the 16S rRNA gene of this bacterial strain through PCR, is submitted to EzTaxon data by obtaining sequence
Compare in storehouse, result shows the similarity highest of this bacterium and Anoxybacillus rupiensis R270, is 99.3%.
The present invention also provides the Inoculant containing described Thermophilic Bacteria A.rupiensis ZQ2, containing A.rupiensis ZQ2
Viable count is 106-108CFU/g.
The preparation method of ZQ2 Inoculant, comprises the following steps：
(1) seed liquor preparation：2- is cultivated at A.rupiensis ZQ2 slant strains are seeded to 60 DEG C on LB solid-state inclined-plane
3d；The strain of activation is inoculated in the culture of LB liquid culture medium.
(2) fermentation liquid preparation：Seed liquor is seeded to the fermentation cylinder for fermentation of sterilizing.
(3) Inoculant：The materials such as Testa Tritici, straw, Testa oryzae, analysis for soybean powder are mixed, by 5-10% inoculation fermentation liquid, ferment,
It is dried, screening is obtained the Inoculant of Thermophilic Bacteria A.rupiensis ZQ2.
Wherein, described in step (1), the method for actication of culture is：Take the A.rupiensis ZQ2 slant strains of 4 DEG C of preservations
It is seeded on LB inclined-plane and cultivates to abundant, 60 DEG C of constant incubators are cultivated 2-3d.
Described in step (1), the method for seed liquor is：It is forwarded in LB fluid medium with inoculating loop picking lawn, in water
2-3d is cultivated on bath shaking table.Condition of culture is temperature 50-65 DEG C, rotating speed 120-200rpm/min.
Fermentation liquid described in step (2) preparation method be：In 5-10% ratio, seed liquor is seeded to the fermentation of sterilizing
In tank LB fluid medium, in temperature 50-65 DEG C, rotating speed 120-200rpm/min condition bottom fermentation, obtain fermentation liquid.
Described in step (3), Inoculant preparation method is：The materials such as the materials such as Testa Tritici, straw, Testa oryzae, analysis for soybean powder are taken to mix
Close, after mix homogeneously, inoculation fermentation liquid carries out solid fermentation 3-5d, obtains after drying, screening.Described Testa Tritici, straw, Testa oryzae,
The mixing quality ratio of analysis for soybean powder is：(1-2)∶(1-3)∶(1-3)∶(1-2).
The present invention also provides Inoculant the answering in organic solid waste composted of described Thermophilic Bacteria A.rupiensis ZQ2
With：By weight 1%-5%, the Inoculant of Thermophilic Bacteria A.rupiensis ZQ2 to organic solid waste mixed material is carried out sending out
Ferment.Described organic solid waste initial aqueous rate is 55-65%, C/N is 25-35, pH 6-8.
The invention has the beneficial effects as follows：The Thermophilic Bacteria A.rupiensis ZQ2 that the present invention provides is to filter out at 60 DEG C
Come, heat stability is good, utilization of carbon source rate is wide, and metabolic activity is not suppressed by During High-Temperature Composting temperature, strong adaptability；Meanwhile, will
Inoculant of the present invention is applied to organic solid waste During High-Temperature Composting and processes, and is substantially shorter the fermentation period of compost, improves compost treatment
Efficiency, reduces processing cost.
Specific embodiment
Following examples are used for the present invention is described, but are not limited to the scope of the present invention.
The separation of embodiment 1 ZQ2
Weigh 10g megathermal period sludge composting sample and be placed in the 250mL conical flask equipped with 10 beades and 10mL sterilized water
In, it is placed in 60 DEG C on shaking bath, 160rpm/min cultivates 4gh, draw the cultured liquid medium of 10mL and be inoculated in 90mL
In the fluid medium of sterilizing, it is placed in 60 DEG C on shaking bath, 160rpm/min cultivates 48h, so send out culture 4 times again, then
Draw 1mL culture fluid to carry out plating dilutions and coat LB flat board, be placed in 60 DEG C of cultures in incubator.Daily observation, waits to grow
After bacterium colony, examine colony characteristicses, determine dissimilar bacterial strain, isolated and purified using line is carried out on LB culture medium flat plate,
From flat board, colony morphology characteristic confirms to obtain pure bacterium after thalli morphology is consistent with many visuals field under microscope after simple dyeing, and continues
Continue Secondary Culture 5 generation at 60 DEG C, eliminate the unstable bacterial strain of growth, obtain Thermophilic Bacteria A.rupiensis ZQ2 (hereinafter referred to as
ZQ2).ZQ2 is gone to LB medium slant, cultivates to abundant standby in 4 DEG C of preservations.
Wherein, the compound method of LB culture medium is：Yeast extract 5.0g, bacto peptone 10.0g, deionized water
1000mL, pH 7.2, sterilize at 121 DEG C 25min.
Embodiment 2 ZQ2 identifies
By Thermophilic Bacteria ZQ2 of the present invention obtained by embodiment 1 on LB culture medium flat plate, at 60 DEG C, cultivate 48h, bacterium colony is in
Circle, regular edges, center projections, diameter 1～2mm.
Physiological and biochemical test shows, ZQ2 Gram-positive, spore can be produced, in stable phase, cell about 0.4-
0.7-0.8-1.2um, the scope of growth temperature is 30-70 DEG C, and optimum growth temperature is 60 DEG C.Catalase reaction is in sun
Property it is impossible to gelatin hydrolysate is it is impossible to degraded nitrate, indole reaction is negative, and phenylalanine deaminase reaction is negative.Being capable of profit
With arabinose, cellobiose, galactose, trehalose, xylose etc..
With the DNA of bacterial strain ZQ2 of the present invention as template, PCR expands its 16S rRNA sequence, and primer is (27f)：5′-AGA
GTT TGA TCC TGG CTC AG-3 ' and (1492r)：5′-GGT TAC CTT GTT ACG ACTT-3′.PCR response procedures
For：95 DEG C of denaturations 5min, 95 DEG C of degeneration 30s, 53 DEG C of annealing 45s, 72 DEG C of extension 90s, 30 circulations, 72 DEG C of extension 10min.
Amplified production is sequenced after its purity of electrophoresis detection, then by the base sequence obtaining in the world such as GenBank nucleic acid sequence data
Carry out homologous sequence search (Blast search) in storehouse, find out in this bacterial strain and data base homology highest type strain or
It is preserved in the bacterial strain of ATCC HuoDSMDeng world DSMZ.According to comparison result find, this bacterial strain with
Anoxybacillus rupiensis R270 16S rRNA sequence tool highest homology, similarity is up to 99.10%.
In sum, 16S rRNA homologous sequence compares and relevant physiological biochemical characteristic is it may be determined that bacterial strain ZQ2 of the present invention
Belong to Anoxybacillus rupiensis, be named as A.rupiensis subsp.Rupiensis ZQ2, and in 2016 7
The moon is stored in Guangdong Province Culture Collection (address on the 8th：5th floor, Xianlie Middle Road, Guangzhou City 100 No. 59 building of compound), protect
Tibetan number is GDMCC No.60056.
The preparation of embodiment 3 ZQ2 Inoculant
Concrete grammar is：
(1) shake-flask culture：From ZQ2 inclined-plane, picking lawn one ring accesses in the conical flask equipped with LB fluid medium, and 60
DEG C, 200rpm/min shaking table concussion and cultivate 36h.
(2) ferment tank：Above-mentioned cultured ZQ2 fermentation liquid is accessed in 10L fermentation tank, 60 DEG C, 200rpm/min
Fermentation culture 36h；Fermentation medium used is LB fluid medium, and its formula is (g/L)：Yeast extract 5g, tryptone
10g, NaCl 10g, water 1000mL, pH 7.2.
(3) solid fermentation：Ratio in mass ratio for 1: 1: 1: 1, by Testa Tritici, straw, Testa oryzae, analysis for soybean powder mixing, adjusts water
Divide to 50%, pH7.0, high temperature sterilize, cooling, the inoculum concentration by 10% accesses above-mentioned fermentation liquid, and 60 DEG C carry out solid fermentation.
(4) fermentation post processing：Solid medium after above-mentioned fermentation is by drying, comminution by gas stream, sieves, as ZQ2 connects
Plant agent.
(5) product quality inspection：Product quality inspection method is according to agricultural microbial agent standard (GB 20287-2006)
Detected.Testing result shows, living bacteria count >=2 × 108CFU/g in above-mentioned preparation, and product is qualified.
The application of embodiment 4 ZQ2 Inoculant
The application effect of ZQ2 Inoculant of the present invention is described with municipal sludge aerobic compost below.
After taking the preliminary mix homogeneously of municipal sludge, wood flour, straw by weight 1: 1: 1, add embodiment by total material 2%
The ZQ2 Inoculant of preparation, aerobic composting fermentation 25d in 3.Separately set and do not inoculate ZQ2 high temperature Inoculant as comparison.Wherein, conventional
Process, every 5d or temperature carry out turning more than 60 DEG C；Inoculation is processed, and every 5d carries out turning.By compost temperature change, C/N ratio,
Seed germination index judges the rotten degree of compost.
Table 1 is the impact to compost temperature for the ZQ2 Inoculant.As it can be seen from table 1 after compost inoculation ZQ2, maximum temperature can
Reach 75 DEG C, reach the maximum temperature time for 4d after fermentation, the megathermal period, its maximum temperature and megathermal period held time up to 10d
It is above compareing, and reaches the time of maximum temperature also early than comparison, illustrate that ZQ2 Inoculant can be obviously promoted compost early stage
Fermentation calefaction, and improve compost temperature, and also high-temperature duration is long, and compost reaction effect is good.
Table 2 is the impact to compost maturity for the ZQ2 Inoculant.From table 2 it can be seen that after compost inoculation ZQ2, composting C/N
Reduce.With compare, germination index reduce, and germination index be more than 80% time substantially do sth. in advance, ZQ2 Inoculant is described
Mud can be obviously promoted, shorten the compost fermentation cycle, promote compost quick composting, improve the innoxious and stabilisation of mud.
The impact to compost temperature for the table 1.ZQ2 Inoculant
The impact to compost maturity for the table 2.ZQ2 Inoculant
Claims (4)
1. a kind of Thermophilic Bacteria A.rupiensis ZQ2, its bacterial strain preserving number is GDMCC No.60056.
2. a kind of Inoculant of Thermophilic Bacteria A.rupiensis ZQ2 it is characterised in that：Thermophilic Bacteria A.rupiensis ZQ2 connects
Planting agent is to be prepared by fermentation with the Thermophilic Bacteria A.rupiensis ZQ2 described in claim 1, containing A.rupiensis
ZQ2 viable count is 106-108CFU/g.
3. a kind of preparation method of the Inoculant of Thermophilic Bacteria A.rupiensis ZQ2：It is characterized in that：Described in claim 2
The preparation process of the Inoculant of Thermophilic Bacteria A.rupiensis ZQ2 is as follows：
(1) seed liquor preparation：2-3d is cultivated at A.rupiensis ZQ2 slant strains are seeded to 60 DEG C on LB solid-state inclined-plane；
The strain of activation is inoculated in the culture of LB liquid culture medium；
(2) fermentation liquid preparation：Seed liquor is seeded to the fermentation cylinder for fermentation of sterilizing；
(3) Inoculant：The materials such as Testa Tritici, straw, Testa oryzae, analysis for soybean powder are mixed, by 5-10% inoculation fermentation liquid, ferment, be dried,
Screening is obtained the Inoculant of Thermophilic Bacteria A.rupiensis ZQ2；
Wherein, described in step (1), the method for actication of culture is：Take the A.rupiensis ZQ2 slant strains inoculation of 4 DEG C of preservations
Cultivate to LB inclined-plane to abundant, 60 DEG C of constant incubators are cultivated 2-3d；
Described in step (1), the method for seed liquor is：It is forwarded in LB fluid medium with inoculating loop picking lawn, shake in water-bath
2-3d is cultivated on bed；Condition of culture is temperature 50-65 DEG C, rotating speed 120-200rpm/min；
Fermentation liquid described in step (2) preparation method be：In 5-10% ratio, seed liquor is seeded to the fermentation tank LB of sterilizing
In fluid medium, in temperature 50-65 DEG C, rotating speed 120-200rpm/min condition bottom fermentation, obtain fermentation liquid；
Described in step (3), Inoculant preparation method is：Take the materials such as the materials such as Testa Tritici, straw, Testa oryzae, analysis for soybean powder to mix, mix
Conjunction is uniformly inoculated fermentation liquid afterwards and is carried out solid fermentation 3-5d, obtains after drying, screening；Described Testa Tritici, straw, Testa oryzae, Semen Glyciness
The mixing quality ratio of powder is：(1-2)∶(1-3)∶(1-3)∶(1-2).
4. application in organic solid waste composted for the Inoculant of the Thermophilic Bacteria A.rupiensis ZQ2 described in right 2.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108660087A (en) * | 2017-09-28 | 2018-10-16 | 湖北省农业科学院畜牧兽医研究所 | A kind of complex micro organism fungicide and its preparation method and application for reducing the release of Chicken Manure Compost ammonia |
| CN109294951A (en) * | 2018-10-15 | 2019-02-01 | 广东省生态环境技术研究所 | The application of one plant of false Xanthomonas campestris and its microorganism formulation in terms of biological compost |
| CN109609412A (en) * | 2019-01-15 | 2019-04-12 | 河北省科学院生物研究所 | A kind of Thermophilic Bacteria Bacillus smithii Ths1 and its application |
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| CN102732465A (en) * | 2012-05-14 | 2012-10-17 | 大地绿源环保科技(北京)有限公司 | Thermophilic facultative aerobic bacteria UTM601 and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660087A (en) * | 2017-09-28 | 2018-10-16 | 湖北省农业科学院畜牧兽医研究所 | A kind of complex micro organism fungicide and its preparation method and application for reducing the release of Chicken Manure Compost ammonia |
| CN108660087B (en) * | 2017-09-28 | 2021-08-10 | 湖北省农业科学院畜牧兽医研究所 | Compound microbial agent for reducing release of chicken manure compost ammonia gas and preparation method and application thereof |
| CN109294951A (en) * | 2018-10-15 | 2019-02-01 | 广东省生态环境技术研究所 | The application of one plant of false Xanthomonas campestris and its microorganism formulation in terms of biological compost |
| CN109294951B (en) * | 2018-10-15 | 2021-09-14 | 广东省生态环境技术研究所 | Pseudoxanthomonas and application of microbial preparation thereof in biological composting |
| CN109609412A (en) * | 2019-01-15 | 2019-04-12 | 河北省科学院生物研究所 | A kind of Thermophilic Bacteria Bacillus smithii Ths1 and its application |
| CN109609412B (en) * | 2019-01-15 | 2021-10-01 | 河北省科学院生物研究所 | Thermophilic bacterium Bacillus smithii Ths1 and application thereof |
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