CN105567610B - One plant of high temperature resistant garden waste decomposer ST5 and its application - Google Patents
One plant of high temperature resistant garden waste decomposer ST5 and its application 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
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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
Abstract
The invention discloses one plant of common streptomycete of high-temperature fibre element degradation bacteria heat (Streptomyces thermovulgaris), deposit number is CGMCC No.12137.Bacterium of the present invention a large amount of producing enzymes, cellulase activity height can have high temperature resistant, efficient degradation cellulosic nature in 40-70 DEG C of high temperature, can be applied in During High-Temperature Composting system as microbial bacterial agent, be suitable for garden waste compost.
Description
Technical field
The invention belongs to field of environmental biotechnology, and in particular to the plant height effect filtered out from garden waste compost
The thermoduric bacteria of degraded cellulose and its application in garden waste During High-Temperature Composting.
Background technique
Garden waste refers to that ornamental plant withers and falls naturally or manually trim generated deadwood, fallen leaves, grass cuttings, residual flower, tree
Wood and shrub beta pruning and other plant residues etc., main component are cellulose and hemicellulose difficult to degrade.In recent years, China garden
The speed increase of the annual 8%-10% of woods waste brings large drag forces to City Green process, and China handles gardens at present
The mode of waste predominantly burn and fill up, both processing modes not only waste renewable resource also create it is serious
Environmental pollution.Therefore, how garden waste is rationally effectively treated, making its resource utilization is the heat of everybody current common concern
One of point problem.
Organic fertilizer and seedling medium etc. is made by garden waste is decomposed using composting technology, is its resource utilization
One of effective outlet, however lead to heap fertilizer efficiency containing substances difficult to degrade such as a large amount of lignin, celluloses in garden waste
Rate is low, the period is long, greatly limits the recycle value of garden waste.Therefore it needs that specific micro- life is added into compost
Object, to accelerate garden waste digest process.Cellulose-degrading bacteria is that one kind can generate extracellular cellulase, and cellulose is big
Molecule is hydrolyzed into the microorganism of glucose, can be with the degradation speed of accelerating fibers cellulosic material.In nature, the micro- of cellulose is generated
There are many biological species, and study and apply at present more is fungi, such as trichoderma, Penicillium, aspergillus, rhizopus.Bacterium
Research and application with actinomyces is less.Cellulose degradation occurs mainly in the megathermal period in composting process, and fungi mainly exists
Under medium temperature condition, enzyme activity is maximum, and with the rising of composting process temperature, the enzymatic activity of fungi number of viable and its generation drops significantly
Low, which limits the utilizations in compost of cellulase-producing mould.The screening of high-temperature fibre element degradation bacteria and application are
The effective measures to solve the above problems.
Many cellulose-degrading bacterias both for agricultural wastes such as corn stover, straw, wheat straws, it is few specifically for
The screening of the degradation bacteria of garden waste cellulosic material and research on utilization.Chinese patent " a plant height temperature cellulose-degrading bacteria and
It is applied " it (number of patent application: 201410018582.6) discloses one plant and is separated from garden waste megathermal period compost sample
High temperature fiber element degradation bacteria ground bacillus, cellulase activity be 7.8 U/ml, the bacterium be bacterium.About discarded from gardens
The report that high-temperature fibre element degradation actinomyces are separated in object compost is less.
Summary of the invention
The technical problems to be solved by the present invention are: providing one plant of common streptomycete of high temperature heat-resistant, which can be at 40-70 DEG C
A large amount of producing enzymes in high temperature, cellulase activity is high, has high temperature resistant, efficient degradation cellulosic nature, can be as microorganism
Microbial inoculum is applied in During High-Temperature Composting system, is suitable for garden waste compost.
Present invention provide the technical scheme that one plant of common streptomycete of high-temperature fibre element degradation bacteria heat
(Streptomycesthermovulgaris) ST5, deposit number is CGMCC No.12137, is preserved in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center.
The common streptomycete of heat of the present invention (Streptomycesthermovulgaris) ST5 is in 2 months 2016 18
Day, (preservation address was: court, Beijing for the preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC institute
The institute 3 of positive area's North Star West Road 1, postcode: 100101), deposit number is CGMCC No. 12137, is survived through detection.
The common streptomycete of present invention heat (Streptomycesthermovulgaris) ST5, it is from Changping County, Beijing area apple
The strain that separates in the sample of orchard garden waste compost high temperature mid-term acquisition, being numbered is bacterial strain ST5, which can be
Cellulase and degraded cellulose are generated under the conditions of 40-70 DEG C.Bacterial strain on Gao Shi I culture medium when cultivating, aerial hyphae ash
Color, substrate mycelium dark brown, does not generate soluble pigment.Under microscope, thallus is in mycelioid, and Gram's staining is positive, spore
Sub- oval, surface is smooth, fibrillae of spores helical form, flexible.In conjunction with bacterium colony morphological features and it is based on bacterial 16 S rDNA base
Because of the Phylogenetic Analysis of sequence, be accredited as hot common streptomycete (Streptomycesthermovulgaris)。
The method of bacterial strain of the present invention production cellulase, by the bacterium be inoculated in fermentation medium (medium component:
CMC-Na 0.5%, peptone 1%, wheat bran 3%, NaCl 2%, KH2PO40.1%g, MgSO4•7H2O 0.02%, (NH4)2SO4
0.3%, pH 7.0-7.6) in, turn to cultivate 3-4d in shaking table in temperature 50 C, rpm150, centrifuging and taking supernatant, measures cellulose
Enzymatic activity.
It is the During High-Temperature Composting decomposing agent of main composting material that the present invention also provides a kind of suitable for garden waste, this is decomposed
Agent is obtained in a manner of liquid fermentation, and bacterial concentration is 5 × 109CFU/mL, wheat bran and maize flour 2:1 mixing are adsorbed as bacterium solution
Agent is mixed with adsorbent 1:1 by bacterium solution, is fabricated to solid-state microbial inoculum.
Meanwhile the present invention also provides the common streptomycete of heat (Streptomycesthermovulgaris) ST5 bacterial strain
Application in the compost maturity using garden waste as main material, with garden waste for main composting material, addition is dynamic
Object excrement and water, make mixed material carbon-nitrogen ratio 25-40:1, moisture content 50-60%, and solid-state decomposing agent compares by the 5% of weight of material
Example is inoculated into compost material, carries out During High-Temperature Composting.
The invention has the following advantages:
The common streptomycete of heat of the present invention (Streptomycesthermovulgaris) ST5 is from garden waste
The strain of degraded cellulose is filtered out in compost, more cellulase can be generated in 40-70 DEG C of temperature range, and enzyme activity is up to
28.02 U/mL have high temperature resistant, efficient degradation cellulosic nature.Enzyme activity is maximum under mesophilic condition for opposite fungi, and bacterium
The lower problem of producing enzyme vigor, the common streptomycete of heat not only adapts to hot environment, but also can generate higher cellulase, to fiber
Cellulosic material has good degradation capability, so the common streptomycete of heat (Streptomycesthermovulgaris) can more fit
Answer composting process complex environment.
Solid-state decomposing agent is made in the high-temperature fibre element degradation bacteria that the present invention obtains to be added to based on garden waste
Want in the compost of material, compared with the control not being inoculated with, can be improved compost enter the megathermal period time, extend the megathermal period continue
Time megathermal period temperature reduces composting C/N ratio, to accelerate compost maturity process.It can be answered as microbial bacterial agent
For being suitable for garden waste compost in During High-Temperature Composting system.
The present invention be directed to the decomposing agents of the strain of garden waste composting material screening and preparation, can be discarded for gardens
The resource utilization of object provides a reasonable, effective approach, realizes the purpose for reducing environmental pollution, resource circulation utilization.
Detailed description of the invention
The common streptomycete of Fig. 1 heat (Streptomycesthermovulgaris) isolate and purify picture
The common streptomycete of Fig. 2 present invention heat (Streptomycesthermovulgaris) 16S rDNA gene order.
Fig. 3 by the common streptomycete of heat (Streptomycesthermovulgaris) and close bacterial strain 16SrDNA
Gene order carries out the phylogenetic tree picture constructed when homology Blast is compared.
Heap temperature changes in Fig. 4 composting process.
Carbon-nitrogen ratio (C/N ratio) changes in Fig. 5 composting process.
Specific embodiment
Come that the present invention is furture elucidated below by the detailed description of specific embodiment, but is not to limit of the invention
System, only illustrates.
The screening of 1 high-temperature fibre element degradation bacteria strains of embodiment
From Changping County, Beijing area apple orchard garden waste compost high temperature initial stage, high temperature mid-term, high temperature post material, Beijing
Sample is acquired in Yanqing Plain afforestation afforestation waste deposit；Above-mentioned fresh sample 10g is weighed to be put in equipped with 10
Grain bead simultaneously fills in the conical flasks of 90 ml sterile waters, is placed in the shaking table of 30 DEG C of 150 rpm and shakes 30 min, makes
Sample sufficiently scatters, and stands enrichment culture for 24 hours at 50 DEG C.With aseptic straw draw 1 ml supernatant be added to containing 9ml without
In the test tube of bacterium water, this is 10-1Sample diluting liquid, then from 10-11ml is taken to be added in 9ml sterile water in sample, this is
10-2Sample diluting liquid, and so on, obtain 10-3、10-4、10-5、10-6Then sample diluting liquid draws 100 μ l with pipettor
10-3、10-4、10-5、10-6 Sample diluting liquid is in (culture medium composition are as follows: K on Cellulose and congo red differential medium2HPO40.5g,
Microcrystalline cellulose 1.88g, MgSO4 0.25g, gelatin 2.0g, Congo red 0.5g, agar 16g, distilled water 1000ml, pH
7.0) dilution, is spread evenly across entire plate with spreader, is placed in 50 DEG C of incubators and cultivates 3 days.It selects in fiber
There is the bacterium colony of obvious transparent circle on the Congo red plate of element, be numbered, then crossed repeatedly and isolate and purify acquisition pure strain,
Strain after separation is connected on inclined-plane, 4 DEG C of preservations carry out subsequent experimental.
By the pure bacterial strain for being preserved in 4 DEG C be transferred to carboxymethyl cellulose culture medium (medium component: CMC-Na 15.0g,
NH4NO31.0g, yeast extract 1.0g, MgSO4•7H2O 0.5g, KH2PO41.0g, distilled water 1000ml, agar 16g, pH
7.0) on plate, then activation culture at 50 DEG C is provoked the single colonie on plate and is transferred on the Congo red plate of cellulose, is placed in
It being cultivated in 50 DEG C of incubators, colony diameter d and transparent loop diameter D is measured after 72h, calculates its ratio H, i.e. H=D/d, H value is bigger,
It is stronger to be worth the larger ability for indicating the bacterial strain decomposition of cellulose.According to formation transparent circle on the Congo red identification culture medium of cellulose
Size primarily determines its cellulase-producing activity.
By aforesaid operations, more plants of cellulose-degrading bacterias are obtained, wherein in Changping County, Beijing area apple orchard garden waste
The strain separated in the sample of compost high temperature mid-term acquisition, is named as ST5, and the micro- life of China was deposited on 2 18th, 2016
Object culture presevation administration committee common micro-organisms center is referred to as CGMCC (unit address: the Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica, postcode: 100101), and deposit number CGMCC
No.12137.Optical microscope and Gram's staining identification are carried out to ST5 bacterial strain, which cultivates on Gao Shi I culture medium
When, aerial hyphae grey, substrate mycelium dark brown does not generate soluble pigment.Under microscope, thallus is in mycelioid, gram
Stained positive, spore oval, surface is smooth, fibrillae of spores helical form, flexible, sees Fig. 1.
2 ST5 bacterial strain molecular biology identification of embodiment
Molecular Identification is carried out to the common streptomycete ST5 of heat that screening obtains, is followed the steps below: picking bacterium
Single colonie be inoculated in liquid Gao Shi I culture medium, 30 DEG C, 120r/min shaking table shaken cultivation, 2d take out cultivate
Liquid, 5000r/min centrifugation 1min takes supernatant, according to bacterial genomes DNA extraction kit (the limited public affairs of Tiangeng biochemical technology
Department provides), extract bacterium colony DNA；Universal primer 27F and 1492R carries out PCR amplification to the DNA of bacteria of extraction；27F
Sequence is 5 '-AGA GTT TGA TCC TGG CTC AG-3 '；1492R sequence is 5 '-AAG GAG GTG ATC CAG CCG
CA-3′；PCR product is subjected to sequence, sequencing result BLAST in NCBI database carries out sequence analysis, and
Carry out tetraploid rice.
16S rDNA gene order (the referring to Fig. 2) length of hot common streptomycete is 1418bp, and gene order is mentioned
It is sent on Genbank, carries out tetraploid rice, then use 6.0 Software on Drawing phylogenetic tree of MEGA, Fig. 3 is seen, thus really
Determine the kind of bacterial strain.The result shows that the sequence and the common streptomycete of heat (Streptomycesthermovulgaris) 16S
RDNA gene order similarity is up to 100%, true in combination with colony morphology characteristic, physiological and biochemical property, thallus microscopic features
Determine ST5 bacterial strain for the common streptomycete of heat (Streptomycesthermovulgaris)。
The common streptomycete ST5 growth measurement of 3 heat of embodiment
By hot common streptomycete (Streptomycesthermovulgaris) ST5 is inoculated into CMC fluid nutrient medium
(CMC-Na 15.0g, NH4NO31.0g, yeast extract 1.0g, MgSO40.5g, KH2PO41.0g, distilled water 1000mL), often
Culture solution is taken every 2h, connection is continuous to be sampled to 48h, measures the OD600 value in each period, using incubation time as abscissa, each sample point
OD600 value is ordinate, draws the growth curve of the bacterium, the i.e. growth measurement of the bacterium.From measurement result it can be seen that 0-8h is
Period of delay, 9 ~ 16h be logarithmic growth phase, 16 ~ be for 24 hours stationary phase, > 26h be decline phase.The growth of the bacterial strain of logarithmic phase is fast
In fast, energetic therefore later enzymatic production experiment, 12 hours fermentation liquids of Ying Xuanyong are that seed liquor is inoculated with.
The common streptomycete ST5 cellulase-producing vitality test of 4 heat of embodiment
By the common streptomycete of logarithmic phase heat (Streptomycesthermovulgaris) ST5 by 1% inoculum concentration be inoculated with
To fermentation medium (medium component: CMC-Na 0.5%, peptone 1%, wheat bran 3%, NaCl 2%, KH2PO40.1%,
MgSO4•7H2O 0.02%, (NH4)2SO40.3%), pH 7.0-7.6) in, turn to cultivate 3- in shaking table in temperature 50 C, rpm150
Culture 8000r/min is centrifuged 5min by 4d, and taking supernatant is crude enzyme liquid, measures cellulase activity with DNS method.Take 1
ML supernatant (blank is replaced with 1 ml distilled water) in test tube, is placed in 50 DEG C of water-baths, preheats 2min, is then added 4
The substrate solution that ml has been preheated at 50 DEG C takes out after clock reaction 5min, and 4mL DNS developing solution is added, places after shaking up
Heat 5 min in boiling water bath, be immediately placed in cold bath cooling after taking-up, be settled to 20ml with distilled water, after mixing
Absorbance is measured at 540 nm wavelength.Convert the producing enzyme vigor of the bacterial strain after reference standard curve.Enzyme activity unit is according to the world
Unit stipulative definition, i.e., in 1 mL system, catalyzing cellulose hydrolysis is generated needed for 1 μm of ol glucose in 1 min
Enzyme amount be an enzyme activity unit (U/mL).The cellulase activity of ST5 bacterial strain is 28.02 U/mL after measured.
The preparation of 5 solid-state decomposing agent of embodiment
(1) bacterial strain activates: take 4 DEG C of preservation inclined plane inoculatings of microorganism of the present invention to high family name I solid plate culture medium,
12h is cultivated in 50 DEG C of permanent case realizes bacterial strain activation.
(2) prepared by seed liquor: strain plate 1 in step (1) through slant activation is seeded to the sterile Gao Shi of 1 L
Seed liquor is obtained after cultivating 12h in I fluid nutrient medium, under the conditions of 50 DEG C of 150rpm shaking tables.
(3) preparation of fermentation seed liquid: above-mentioned seed liquor is by 6-10%(v/v) inoculum concentration be seeded to sterilized fermentation
In tank, expansion fermented and cultured is carried out.Under conditions of temperature 50 C, 120 rpm of frequency of oscillation, it must ferment after cultivating 48h
Seed liquor.
(4) it the preparation of solid-state decomposing agent: is adsorbed after wheat bran is mixed with maize flour according to 2:1 mass ratio as bacterium solution
Bacterium solution obtained in agent, with step (3) is mixed with adsorbent volume mass ratio 1:1 by bacterium solution, is fabricated to solid-state decomposing agent, heap
After setting 1 week, it can be used for During High-Temperature Composting.
The compost effect test of 6 decomposing agent of embodiment
With garden wastes for main composting material, animal wastes and water are added, mixed material carbon-nitrogen ratio 25-40 is made:
1, moisture content 50-60%, solid-state decomposing agent are inoculated into compost material in 5% ratio of weight of material, During High-Temperature Composting are carried out, with not
Add the material of decomposing agent for control, when heap temperature rises to 50 DEG C, start turning, the megathermal period, every turning in 2 days was primary, cooling
Phase, turning was primary weekly, not in turning after temperature drops to 40 DEG C.In composting process, become by the daily temperature of measurement heap body
Change, composting material carbon nitrogen (C/N) is than variation, influence of the investigation addition decomposing agent to garden-waste compost decomposition progress.
Heap temperature variation is as shown in Figure 4 in composting process.As shown in Figure 4, the compost treatment of decomposing agent is inoculated in compost
3d temperature rises to 50 DEG C or more, and 50 DEG C or more of megathermal period continues 24d, and temperature is begun to decline later.And it is not inoculated with
Compost treatment temperature just rise to 50 DEG C or more in compost 4d, the processing than inoculation postpones 1d and reaches 50 DEG C or more high temperature
Phase, and 50 DEG C or more duration megathermal period are 17d, fewer than the processing of inoculation 7d are inoculated with the heap body megathermal period of processing
Maximum temperature illustrates to accelerate compost after being inoculated with decomposing agent and enter time megathermal period and high temperature also above processing is not inoculated with
Duration phase.C/N variation is as shown in Figure 5 in composting process.As shown in Figure 5, with the progress of compost, it is inoculated with decomposing agent and not
The lasting reduction of the processing C/N ratio of inoculation, and thus the processing decline degree being inoculated with illustrates higher than processing is not inoculated with, addition is originally
Decomposing agent made from bacterium can accelerate composting process, improve the decomposed effect of garden waste compost.
Claims (5)
1. the hot commonly streptomycete of one plant of high-temperature fibre element degradation bacteria (Streptomyces thermovulgaris), deposit number
For CGMCC No.12137.
2. it is a kind of suitable for garden waste be main composting material During High-Temperature Composting decomposing agent, it is characterised in that: the decomposing agent
It is obtained in a manner of liquid fermentation, raw material is bacterium solution and adsorbent, and bacterium solution mixes in proportion with adsorbent, is fabricated to solid-state bacterium
Agent, wherein bacterium solution be the common streptomycete of heat described in claim 1 (Streptomyces thermovulgaris) bacterium solution, it inhales
Attached dose is wheat bran and maize flour.
3. During High-Temperature Composting decomposing agent as claimed in claim 2, it is characterised in that: the bacterial concentration is 5 × 109CFU/mL,
Wheat bran and maize flour 2:1 mixing are used as bacterium solution adsorbent, mix by bacterium solution with adsorbent 1:1, are fabricated to solid-state microbial inoculum.
4. application of the During High-Temperature Composting decomposing agent in compost maturity as described in Claims 2 or 3.
5. application as claimed in claim 4, it is characterised in that: with garden waste for main composting material, add animal excreta
Just and water, make mixed material carbon-nitrogen ratio 25-40:1, moisture content 50-60%, solid-state decomposing agent presses the 2.5%-5% ratio of weight of material
Example is inoculated into compost material, carries out During High-Temperature Composting.
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| JPH09268088A (en) * | 1996-04-01 | 1997-10-14 | Hiroaki Anada | Chaff compost and its production |
| CN103570448A (en) * | 2012-08-09 | 2014-02-12 | 河南省恒隆态生物工程股份有限公司 | Large-scale innocent treatment technique of tobacco stems |
| CN103820361A (en) * | 2014-01-15 | 2014-05-28 | 深圳市铁汉生态环境股份有限公司 | High-temperature cellulose degradation bacterium and application thereof |
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