CN105670966A - High-temperature-resistant garden waste decomposing bacterium ST4 strain and application thereof - Google Patents
High-temperature-resistant garden waste decomposing bacterium ST4 strain and application thereof Download PDFInfo
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Abstract
The invention discloses a high-temperature-resistant cellulose degrading bacterium strain Streptomyces albidoflavus of which the collection number is CGMCC No.12136. The strain can produce abundant enzymes at the high temperature of 40-70 DEG C, has the characteristics of high cellulase activity, high temperature resistance and high cellulose degradation efficiency, can be used as a microbial inoculant in a high-temperature composting system, and is suitable for garden waste composting.
Description
Technical field
The invention belongs to field of environmental biotechnology, be specifically related to the cellulosic thermoduric bacteria of high-efficiency degradation and the application in garden waste During High-Temperature Composting thereof filtered out from garden waste compost.
Background technology
Garden waste refers to that ornamental plant naturally withers and falls or manually prunes produced deadwood, fallen leaves, grass cuttings, residual flower, trees and shrub beta pruning and other plant residue etc., is mainly composed of cellulose and the hemicellulose of difficult degradation. In recent years, the speed increase of the annual 8%-10% of China's garden waste, bringing large drag forces to City Green process, current China processes the mode of garden waste and is mainly burning and fills up, and both processing modes not only waste Renewable resource and also create serious environmental pollution. Therefore, how rationally garden waste is effectively processed so that it is recycling is one of hot issue of everybody common concern at present.
Utilize composting technology garden waste to be become thoroughly decomposed and make organic fertilizer and seedling medium etc., it it is one of effective outlet of its recycling, but garden waste contains the material of the difficult degradations such as substantial amounts of lignin, cellulose, cause that composting efficiency is low, the cycle is long, greatly limit the recycle value of garden waste. It is thus desirable to add specific microorganism in compost, to accelerate garden waste digest process. Cellulose-degrading bacteria is that a class can produce born of the same parents' outer fiber element enzyme cellulose macromolecule is hydrolyzed into the microorganism of glucose, it is possible to the degradation speed of accelerating fibers cellulosic material. In nature, produce cellulosic microbe species a lot, study at present and what apply more is fungus, such as Trichoderma spp., Penicillium, aspergillus, Rhizopus etc. Antibacterial and actinomycetic research and application are all less. In composting process, cellulose degradation occurs mainly in the megathermal period, and fungus is main under mesophilic condition, enzyme is lived maximum, along with the rising of composting process temperature, the enzymatic activity of fungus number of viable and generation thereof is substantially reduced, and which limits the utilization in compost of cellulase-producing mycete. The screening of high-temperature fibre element degradation bacteria and application are the effective measures solving the problems referred to above.
A lot of cellulose-degrading bacterias are both for the agricultural wastes such as corn straw, Caulis et Folium Oryzae, wheat straw, seldom have screening and the research on utilization of the degradation bacteria being specifically designed for garden waste cellulosic material. Chinese patent " plant height temperature cellulose-degrading bacteria and an application thereof " (number of patent application: 201410018582.6) discloses the high temperature fiber element degradation bacteria ground bacillus that a strain separates from garden waste megathermal period compost sample, cellulase activity is 7.8U/ml, and this bacterium is antibacterial.About separating from garden waste compost, the high-temperature fibre element actinomycetic report of degraded is less.
Summary of the invention
The technical problem to be solved is: provide a high temperature resistant Streptomycesalbidoflhaving of strain, this bacterium can in 40-70 DEG C of high temperature big volume production enzyme, cellulase activity is high, there is high temperature resistant, efficient degradation cellulosic nature, microbial bacterial agent can be it can be used as to be applied in During High-Temperature Composting system, it is adaptable to garden waste compost.
Present invention provide the technical scheme that a strain high-temperature fibre element degradation bacteria Streptomycesalbidoflhaving (Streptomycesalbidoflavus) ST4, deposit number is CGMCCNo.12136, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Streptomycesalbidoflhaving of the present invention (Streptomycesalbidoflavus) on February 18th, 2016, for the preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC institute, (preservation address was ST4: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode: 100101), its preserving number is CGMCCNo.12136, survives after testing.
Streptomycesalbidoflhaving of the present invention (Streptomycesalbidoflavus) ST4, it being the strain separated from the sample of apple orchard, Changping County, Beijing district garden waste compost high temperature collection in mid-term, be numbered bacterial strain ST4, this bacterium can produce cellulase degraded cellulose under 40-70 DEG C of condition. Bacteria colony white when bacterial strain is cultivated in Gao Shi I culture medium, aerial hyphae micro white, substrate mycelium is light yellow, does not produce soluble pigment. Under microscope, thalline is mycelioid, and Gram’s staining is positive, spore oval, and fibrillae of spores is flexible, and 1-3 encloses spiral. In conjunction with bacterium colony morphological features with based on the Phylogenetic Analysis of bacterial 16 S rDNA gene order, be accredited as Streptomycesalbidoflhaving (Streptomycesalbidoflavus).
Bacterial strain of the present invention produces the method for cellulase, and this bacterium is inoculated in fermentation medium (medium component: CMC-Na0.5%, peptone 1%, wheat bran 3%, NaCl2%, KH2PO40.1%g, MgSO4·7H2O0.02%, (NH4)2SO40.3%, pH7.0-7.6) in, turn cultivation 3-4d in shaking table, centrifuging and taking supernatant in temperature 50 C, rpm150, measure cellulase activity.
The present invention also provides for that a kind of this decomposing agent obtains in liquid fermentation mode suitable in the During High-Temperature Composting decomposing agent that garden waste is main composting material, and bacterial concentration is 5 × 109CFU/mL, wheat bran and Semen Maydis flour 2:1 mix as bacterium solution adsorbent, mix with adsorbent 1:1 by bacterium solution, are fabricated to solid-state microbial inoculum.
Meanwhile, the present invention also provide for described Streptomycesalbidoflhaving (Streptomycesalbidoflavus) application in garden waste for the compost maturity of main material of the ST4 bacterial strain, with garden waste for main composting material, add animal wastes and water, making mixed material carbon-nitrogen ratio is 25-40:1, moisture content 50-60%, solid-state decomposing agent is inoculated in compost material in 5% ratio of weight of material, carries out During High-Temperature Composting.
The method have the advantages that
Streptomycesalbidoflhaving of the present invention (Streptomycesalbidoflavus) ST4 is the strain filtering out degraded cellulose from garden waste compost, can produce more cellulase in 40-70 DEG C of temperature range, enzyme work, up to 27.58U/mL, has high temperature resistant, efficient degradation cellulosic nature. Relative fungus enzyme under mesophilic condition is lived maximum, and the problem that Production by Bacteria enzyme activity is relatively low, Streptomycesalbidoflhaving had both adapted to hot environment, can produce again higher cellulase, cellulosic material is had good degradation capability, so Streptomycesalbidoflhaving is suitable for composting process complex environment.
The high-temperature fibre element degradation bacteria that the present invention obtains is made solid-state decomposing agent and adds to garden waste for the compost of main material, compared with the comparison do not inoculated, compost can be improved enter the time of megathermal period, extend persistent period megathermal period megathermal period temperature, reduce composting C/N ratio, thus accelerating compost maturity process. Microbial bacterial agent can be it can be used as to be applied in During High-Temperature Composting system, it is adaptable to garden waste compost.
The present invention be directed to the decomposing agent of the strain of garden waste composting material screening and preparation, it is possible to the recycling for garden waste provides an approach reasonable, effective, it is achieved reduce the purpose of environmental pollution, resource circulation utilization.
Accompanying drawing explanation
Fig. 1 Streptomycesalbidoflhaving (Streptomycesalbidoflavus) separate purification picture
Fig. 2 Streptomycesalbidoflhaving of the present invention (Streptomycesalbidoflavus) 16SrDNA gene order.
Fig. 3 by Streptomycesalbidoflhaving (Streptomycesalbidoflavus) and the phylogenetic tree picture that builds when the gene order of close bacterial strain 16SrDNA carries out homology Blast comparison.
Heap temperature change in Fig. 4 composting process.
Carbon-nitrogen ratio (C/N ratio) change in Fig. 5 composting process.
Detailed description of the invention
It is further elucidated with the present invention below by the detailed description of detailed description of the invention, but is not limitation of the present invention, only illustrate.
Embodiment 1 high-temperature fibre element degradation bacteria strains screens
Collected specimens from apple orchard, Changping County, Beijing district garden waste compost high temperature initial stage, high temperature mid-term, high temperature post material, Yanqing County of Beijing Plain afforestation afforestation garbage deposit; Weigh above-mentioned fresh sample 10g be put in equipped with 10 beades and fill the conical flask of 90ml sterilized water, be placed in the shaking table of 30 DEG C of 150rpm and shake 30min, make sample fully scatter, at 50 DEG C, stand enrichment culture 24h. Drawing 1ml supernatant with aseptic straw to join in the test tube containing 9ml sterilized water, this is 10-1Sample diluting liquid, then from 10-1Taking 1ml in sample to join in 9ml sterilized water, this is 10-2Sample diluting liquid, by that analogy, obtains 10-3、10-4、10-5、10-6Sample diluting liquid, then draws the 10 of 100 μ l with pipettor-3、10-4、10-5、10-6On Cellulose and congo red differential medium, (culture medium consists of sample diluting liquid: K2HPO40.5g, microcrystalline Cellulose 1.88g, MgSO40.25g, gelatin 2.0g, Congo red 0.5g, agar 16g, distilled water 1000ml, pH7.0), with spreader, diluent is spread evenly across whole flat board, is placed in 50 DEG C of incubators and cultivates 3 days. Selecting the bacterium colony having obvious transparent circle on cellulose Congo red flat board, be numbered, then carry out the line separation purification purebred bacterial strain of acquisition repeatedly, receive on inclined-plane by the strain after separating, 4 DEG C of preservations carry out subsequent experimental.
The pure bacterial strain being preserved in 4 DEG C is transferred to carboxymethyl cellulose culture medium (medium component: CMC-Na15.0g, NH4NO31.0g, yeast extract 1.0g, MgSO4·7H2O0.5g, KH2PO41.0g, distilled water 1000ml, agar 16g, pH7.0) on flat board, activation culture at 50 DEG C, then provoke the single bacterium colony on flat board to be transferred on cellulose Congo red flat board, be placed in 50 DEG C of incubators and cultivate, after 72h, measure colony diameter d and transparent circle diameter D, calculate its ratio H, i.e. H=D/d, H-number is more big, is worth the bigger expression cellulolytic ability of this bacterial strain more strong. Identify that the size forming transparent circle in culture medium primarily determines that its cellulase-producing activity according to cellulose Congo red.
Pass through aforesaid operations, obtain many strains cellulose-degrading bacteria, the strain wherein separated in the sample of apple orchard, Changping County, Beijing district garden waste compost high temperature collection in mid-term, called after ST4's, it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 18th, 2016, it is referred to as CGMCC(unit address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode: 100101), deposit number is CGMCCNo.12136.ST4 bacterial strain carrying out optical microscope and Gram’s staining is identified, bacteria colony white when this bacteria strain is cultivated in Gao Shi I culture medium, aerial hyphae micro white, substrate mycelium is light yellow, does not produce soluble pigment. Under microscope, thalline is mycelioid, and Gram’s staining is positive, spore oval, and fibrillae of spores is flexible, and 1-3 encloses spiral, sees Fig. 1.
Embodiment 2ST4 bacterial strain molecular biology identification
The Streptomycesalbidoflhaving that screening is obtained carries out Molecular Identification, carry out according to following steps: single colony inoculation of picking bacterium is in liquid Gao Shi I culture medium, 30 DEG C, 120r/min shaking table shaken cultivation, culture fluid is taken out at 2d, the centrifugal 1min of 5000r/min takes supernatant, according to bacterial genomes DNA extraction kit (Tian Gen biochemical technology company limited provide), extract bacterium colony DNA; The DNA of bacteria extracted is carried out pcr amplification by universal primer 27F and 1492R; 27F sequence is 5 '-AGAGTTTGATCCTGGCTCAG-3 '; 1492R sequence is 5 '-AAGGAGGTGATCCAGCCGCA-3 '; PCR primer is carried out sequence, and sequencing result BLAST in ncbi database carries out sequence analysis, and carries out tetraploid rice.
16SrDNA gene order (the referring to Fig. 2) length of Potsdam bacillus brevis is 1263bp, gene order is submitted on Genbank, carries out tetraploid rice, then adopt MEGA6.0 Software on Drawing phylogenetic tree, see Fig. 3, so that it is determined that the kind of bacterial strain. Result show this sequence and Streptomycesalbidoflhaving (Streptomycesalbidoflavus) 16SrDNA gene order similarity up to 100%, in combination with colony morphology characteristic, physiological and biochemical property, thalline microscopic features determine ST4 bacterial strain be Streptomycesalbidoflhaving (Streptomycesalbidoflavus).
Embodiment 3 Streptomycesalbidoflhaving growth measurement
Streptomycesalbidoflhaving ST4 is inoculated into (CMC-Na15.0g, NH in CMC fluid medium4NO31.0g, yeast extract 1.0g, MgSO40.5g, KH2PO41.0g, distilled water 1000mL), take culture fluid every 12h, connection is continuous is sampled to 120h, measures the OD600 value in each period, and with incubation time for abscissa, the OD600 value of each sample point is vertical coordinate, draws the growth curve of this bacterium, i.e. the growth measurement of this bacterium. From measurement result can be seen that 0-24h to be period of delay, 24 ~ 72h be exponential phase, 72 ~ 96h be stable phase, > 96h is decline phase. The growth of the bacterial strain of logarithmic (log) phase is rapid, energetic, and in therefore later enzymatic production experiment, the fermentation liquid that should select 48h is that seed liquor is inoculated.
Embodiment 4 Streptomycesalbidoflhaving cellulase-producing vitality test
Logarithmic (log) phase Streptomycesalbidoflhaving ST4 is inoculated into fermentation medium (medium component: CMC-Na0.5%, peptone 1%, wheat bran 3%, NaCl2%, KH by the inoculum concentration of 1%2PO40.1%, MgSO4·7H2O0.02%, (NH4)2SO40.3%), pH7.0-7.6) in, turn cultivation 3-4d in shaking table in temperature 50 C, rpm150, by centrifugal for culture 8000r/min 5min, take supernatant and be crude enzyme liquid, measure cellulase activity by DNS method. Take 1mL supernatant (replacement of blank 1ml distilled water) in test tube, it is placed in 50 DEG C of water-baths, preheating 2min, it is subsequently adding 4ml at the substrate solution of 50 DEG C of preheatings, takes out after clock reaction 5min, add 4mLDNS nitrite ion, it is positioned in boiling water bath after shaking up and heats 5min, it is immediately placed in psychrolusia after taking-up and cools down, be settled to 20ml with distilled water, after mixing, measure absorbance at 540nm wavelength place.Convert after reference standard curve the product enzyme activity of this bacterial strain. Enzyme activity unit is according to iu stipulative definition, and namely in 1mL system, in 1min, catalyzing cellulose hydrolysis generates the enzyme amount needed for 1 μm of ol glucose is an enzyme activity unit (U/mL). The cellulase activity of ST4 bacterial strain is 27.58U/mL after measured.
The preparation of embodiment 5 solid-state decomposing agent
(1) bacterial strain activation: take microorganism of the present invention 4 DEG C and preserve inclined plane inoculating Zhi Gaoshi I solid plate culture medium, cultivates 12h in the permanent case of 50 DEG C and realizes bacterial strain activation.
(2) prepared by seed liquor: be seeded in the aseptic Gao Shi I fluid medium of 1L by strain flat board 1 through slant activation in step (1), cultivates after 12h to obtain seed liquor when 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 in sterilized fermentation tank, be enlarged fermentation culture. When temperature 50 C, frequency of oscillation 120rpm, after cultivating 48h, obtain fermentation seed liquid.
(4) preparation of solid-state decomposing agent: as bacterium solution adsorbent after Testa Tritici is mixed according to 2:1 mass ratio with Semen Maydis flour, bacterium solution with preparing in step (3), mixes with adsorbent volume mass ratio 1:1 by bacterium solution, is fabricated to solid-state decomposing agent, after banking up 1 week, can be used for During High-Temperature Composting.
The compost effect test of embodiment 6 decomposing agent
With garden waste for main composting material, add animal wastes and water, making mixed material carbon-nitrogen ratio is 25-40:1, moisture content 50-60%, and solid-state decomposing agent is inoculated in compost material in 5% ratio of weight of material, carries out During High-Temperature Composting, with the material that is not added with decomposing agent for comparison, when heap temperature rises to 50 DEG C, starting turning, the turning of every 2 days of megathermal period is once, cooldown period turning weekly once, when temperature after dropping to 40 DEG C not in turning. In composting process, pile the variations in temperature of body every day, composting material carbon nitrogen (C/N) than change by measuring, investigate and add the decomposing agent impact on garden-waste compost decomposition progress.
In composting process, heap temperature changes as shown in Figure 4. As shown in Figure 4, the compost treatment of inoculation decomposing agent rises to more than 50 DEG C in compost 4d temperature, and the megathermal period of more than 50 DEG C continues 23d, and temperature begins to decline afterwards. And nonvaccinated compost treatment temperature just rises to more than 50 DEG C at compost 4d, postpone 1d than the process of inoculation and arrive 50 DEG C of high temperature above phases, and 50 DEG C of persistent period high temperature above phase are 18d, 5d fewer than the process of inoculation, the maximum temperature of the heap body megathermal period that inoculation processes is also above not inoculating process, compost can be accelerated after inoculation decomposing agent is described and enter time megathermal period and persistent period megathermal period. In composting process, C/N changes as shown in Figure 5. As shown in Figure 5, along with the carrying out of compost, inoculation decomposing agent and the process C/N ratio do not inoculated all continue to reduce, and the process decline degree inoculated is higher than not inoculating process, thus illustrate, add the prepared decomposing agent of this bacterium and can accelerate composting process, improve the effect of becoming thoroughly decomposed of garden waste compost.
Claims (5)
1. a strain high-temperature fibre element degradation bacteria Streptomycesalbidoflhaving (Streptomycesalbidoflavus), preserving number is CGMCCNo.12136.
2. one kind is applicable to the During High-Temperature Composting decomposing agent that garden waste is main composting material, it is characterized in that: this decomposing agent obtains in liquid fermentation mode, its raw material is bacterium solution and adsorbent, it is mixed in proportion by bacterium solution and adsorbent, be fabricated to solid-state microbial inoculum, wherein bacterium solution be Streptomycesalbidoflhaving described in claim 1 (Streptomycesalbidoflavus) bacterium solution, adsorbent is wheat bran and Semen Maydis flour.
3. During High-Temperature Composting decomposing agent as claimed in claim 2, it is characterised in that: described bacterial concentration is 5 × 109CFU/mL, wheat bran and Semen Maydis flour 2:1 mix as bacterium solution adsorbent, mix with adsorbent 1:1 by bacterium solution, are fabricated to solid-state microbial inoculum.
4. During High-Temperature Composting decomposing agent application in compost maturity as described in Claims 2 or 3.
5. apply as claimed in claim 4, it is characterized in that: with garden waste for main composting material, add animal wastes and water, making mixed material carbon-nitrogen ratio is 25-40:1, moisture content 50-60%, solid-state decomposing agent is inoculated in compost material in the 2.5%-5% ratio of weight of material, carries out During High-Temperature Composting.
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Cited By (4)
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| CN106588330A (en) * | 2016-12-09 | 2017-04-26 | 重庆市黑土地白蚁防治有限公司 | Method for reusing garden wastes |
| CN110156501A (en) * | 2019-07-04 | 2019-08-23 | 山西农业大学 | A kind of organic fertilizer and preparation method thereof based on garden waste and mushroom bran production |
| ES2772548A1 (en) * | 2018-12-20 | 2020-07-07 | Univ Valladolid | RECYCLING OF WASTE FROM THE CEREAL INDUSTRY TO OBTAIN PRODUCTS WITH ANTIMICROBIAL CHARACTERISTICS THROUGH COMPOSTATION (Machine-translation by Google Translate, not legally binding) |
| CN111676172A (en) * | 2020-07-15 | 2020-09-18 | 德州学院 | Multifunctional oligotrophic cellulose degrading bacterium compound microbial inoculum and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106588330A (en) * | 2016-12-09 | 2017-04-26 | 重庆市黑土地白蚁防治有限公司 | Method for reusing garden wastes |
| ES2772548A1 (en) * | 2018-12-20 | 2020-07-07 | Univ Valladolid | RECYCLING OF WASTE FROM THE CEREAL INDUSTRY TO OBTAIN PRODUCTS WITH ANTIMICROBIAL CHARACTERISTICS THROUGH COMPOSTATION (Machine-translation by Google Translate, not legally binding) |
| CN110156501A (en) * | 2019-07-04 | 2019-08-23 | 山西农业大学 | A kind of organic fertilizer and preparation method thereof based on garden waste and mushroom bran production |
| CN111676172A (en) * | 2020-07-15 | 2020-09-18 | 德州学院 | Multifunctional oligotrophic cellulose degrading bacterium compound microbial inoculum and application thereof |
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