CN103484396B - New strain of streptomyces thermocarboxydus and application thereof - Google Patents
New strain of streptomyces thermocarboxydus and application thereof Download PDFInfo
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- CN103484396B CN103484396B CN201310356764.XA CN201310356764A CN103484396B CN 103484396 B CN103484396 B CN 103484396B CN 201310356764 A CN201310356764 A CN 201310356764A CN 103484396 B CN103484396 B CN 103484396B
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- carbon monoxide
- compost
- temperature
- thermophilic carbon
- monoxide streptomycete
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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Abstract
The invention discloses a new strain CF1 of streptomyces thermocarboxydus, wherein the collection number is CGMCC (China General Microbiological Culture Collection Center) No.6873, the collection date is November 23, 2012, and the collection unit is CGMCC. The invention further discloses a bacterial agent containing the strain. The new strain CF1 of the streptomyces thermocarboxydus, disclosed by the invention, is inoculated into a compost, so that the temperature of the compost can be rapidly raised in low-temperature seasons in the North, the high temperature of above 50 DEG C of the compost can be maintained for 15 days, the fermentation period is shortened, and pathogens and parasites in animal dung can be effectively killed. After the end of fermentation of the compost, the nutrient content in a composted product is high, the pH value is about 8.0, and the composted product can be used as an excellent plant organic fertilizer.
Description
Technical field
The invention belongs to soil and fertilizer field, be specifically related to a kind of agricultural microorganism preparation and application thereof.
Background technology
Along with socioeconomic high speed development, people's standard of living improves constantly, and huge variation has occurred dietary structure, and wherein animal products, in occupation of indispensable vital role in dietary structure, has promoted the fast development of livestock and poultry breeding industry thus.Recent two decades, the plant that a collection of intensive degree is more and more higher sets up in nearly outer suburbs, convenient city of transporting one after another under the active support of the government, raise quantity many by few change, a large amount of feces of livestock and poultry, because not having timely harmless treatment to be arbitrarily discarded in physical environment, has caused significant wastage and the environmental pollution of resource.Met people to meat, egg, newborn demand in, brought huge pressure to the natural ecological environment of China, cause serious pollution.
Summary of the invention
First the present invention provides thermophilic carbon monoxide streptomycete (Streptomyces thermocarboxydus) new bacterial strain CF1.
The present invention also provides the microbial inoculum that contains thermophilic carbon monoxide streptomycete CF1.
Strains separation of the present invention and screening process are as follows:
1. adopt plate dilution method in the cow dung from fresh cow dung, thermophilic fermentation stage cow dung and becoming thoroughly decomposed, to carry out microorganism separation respectively.Take the each 10g of each sample and put into respectively the triangular flask that 90mL sterilized water is housed, the 30min that vibrates on shaking table, fully disperses sample, after standing 20min, is 10
-1diluent, then serial dilution makes 10
-2-10
-8the diluent of concentration is for subsequent use.Get the diluent of the each concentration of 100 μ L, adopt conventional streak method spreader to be coated with on No. 1 solid medium of Gao Shi, three repetitions of each concentration gradient, are placed in respectively 25 DEG C, 30 DEG C, 50 DEG C constant incubators and cultivate.The single bacterium colony growing on picking flat board after 3d, is further purified cultivation, and in 4 DEG C of preservations.
No. 1 substratum of Gao Shi is Zulkovsky starch 20.0g/L, KNO
31.0g/L, K
2hPO
40.5g/L, MgSO
47H
2o 0.5g/L, NaCl 0.5g/L, FeSO
40.01g/L, water 1000ml, pH7.2-7.4, agar 1.5%-2.0%.
2. on Mierocrystalline cellulose-Congo red flat board, form the size of the distinct degree of speed, transparent circle, transparent circle and the colony diameter ratio (D/d) of transparent circle according to bacterial strain, filter out 1 plant height temperature bacterium, this bacterial strain is at the transparent circle clearly that occurs for the 2nd day of cultivating, and in cultivation, the 5th day D/d reaches 11.3.This bacterial strain bacterium colony subcircular, surperficial fine hair shape, loose, edge mycelia is tightr, gray; Base portion brown of light color.
3. utilize 16SrDNA-PCR technology to carry out molecular biology identification to this bacterial strain, measure the sequence of 16SrDNA, sequence is as shown in SEQ ID No.1, with the sequence homology of the 16SrDNA of the thermophilic carbon monoxide streptomycete of other announcement be 99%, be accredited as thermophilic carbon monoxide streptomycete (Streptomyces thermocarboxydus), called after CF1, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 23rd, 2012, be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.6873.
Thermophilic carbon monoxide streptomycete CF1 of the present invention can be prepared into microbial inoculum, wherein, the viable count of thermophilic carbon monoxide streptomycete CF1 can be 5,000,000,000/gram more than, be preferably 10,000,000,000/gram more than.
The present invention also provides the preparation method of described bacteria agent, and it,, for ferment thermophilic carbon monoxide streptomycete CF1 in liquid fermentation medium, is bacteria agent by the fermented liquid obtaining.
Wherein, preferred fermention medium is No. 1 liquid nutrient medium of Gao Shi.
Preferably, described preparation method can specifically comprise the steps:
By No. 1 culture medium slant of thermophilic carbon monoxide streptomycete CF1 inoculation Zhi Gaoshi, cultivate 48-72h in 50 DEG C of constant temperatures and obtain inclined-plane seed;
By No. 1 liquid nutrient medium of inclined-plane seed inoculation Zhi Gaoshi, in 50 DEG C of constant temperatures, 120-200r/min speed oscillation is cultivated 48-72h and is obtained liquid seeds;
By liquid seeds, with 0.5-10%(V/V, every 100ml liquid nutrient medium is inoculated 0.5-10ml liquid seeds) inoculum size, be inoculated in the productive culture base in liquid fermentation tank, carry out bacterial classification production, in continuing under aeration condition, 50 DEG C of constant temperature culture 3-4 days.After cultivation finishes, bacterial classification is contained in the container of sterilizing, is placed in shady and cool dry place and preserves.
Preferably, can also the thermophilic carbon monoxide streptomycete CF1 fermented liquid of preparation be carried out to lyophilize according to this area ordinary method, thereby prepare the bacteria agent of lyophilized powder form.
Further, the present invention also provides a kind of composite fungus agent for animal excrement fermentation, and it comprises subtilis, bacillus megaterium and thermophilic carbon monoxide streptomycete CF1 claimed in claim 1.
Wherein, the viable count of described subtilis is 1,000,000,000/gram more than, described bacillus megaterium viable count is 2,000,000,000/gram more than, the viable count of described thermophilic carbon monoxide streptomycete CF1 is 2,000,000,000/gram more than.
Bacillus megaterium of the present invention, subtilis are all commercially available, as purchased from Chinese industrial microbial strains preservation administrative center, bacillus megaterium CICC 20611, subtilis CICC 10090.Certainly the invention is not restricted to this two kinds of bacterial strains.
In heap body, inoculate the new bacterial strain CF1 of thermophilic carbon monoxide streptomycete of the present invention, can improve rapidly heap temperature at northern low temperature season, and can make to pile the more than 50 DEG C high temperature of body maintenance 15 days, shorten fermentation period, can effectively kill pathogen and parasite in animal excrement.After compost fermentation finishes, it is high that compost finishes rear nutrient content, and pH8.0 left and right can be used as good plant organic fertilizer and uses.
The composite fungus agent that inoculation comprises the new bacterial strain CF1 of thermophilic carbon monoxide streptomycete of the present invention in heap body, not only can improve rapidly heap temperature at northern low temperature season, pliotherm period is long, fermentation period shortens, and compost finishes rear nutrient content loss less, the nutrient content of product significantly improves, and significantly increases than the product nutrient content of the commercially available EM microbial inoculum of inoculation and Natural compost.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
Unless otherwise noted, " part " described in the present invention refers to " weight part "; Bacillus megaterium bacterial strain CICC 20611, subtilis CICC 10090 bacterial strains are purchased from Chinese industrial microbial strains preservation administrative center.
The preparation of embodiment 1 is thermophilic carbon monoxide drappus microbial inoculum
By No. 1 culture medium slant of thermophilic carbon monoxide streptomycete CF1 inoculation Zhi Gaoshi, cultivate 72h in 50 DEG C of constant temperatures and obtain inclined-plane seed;
By No. 1 liquid nutrient medium of inclined-plane seed inoculation Zhi Gaoshi, in 50 DEG C of constant temperatures, 180r/min speed oscillation is cultivated 72h and is obtained liquid seeds;
By liquid seeds with 5%(V/V, i.e. every 100ml liquid nutrient medium inoculation 5ml liquid seeds) inoculum size, be inoculated in the productive culture base in liquid fermentation tank, carry out bacterial classification production, in continuing under aeration condition, 50 DEG C of constant temperature culture 4 days.Fermented liquid after cultivation can be used as microbial inoculum and uses.
Described No. 1 solid culture based component of Gao Shi is: in every 1000ml distilled water, add Zulkovsky starch 20.0g, KNO
31.0g, K
2hPO
40.5g, MgSO
47H
2o 0.5g, NaCl 0.5g, FeSO
40.01g, agar 20.0g, adjusting pH is 7.2-7.4.Be distributed in test tube, every pipe 5-10ml, tampon beyond the Great Wall, autoclave sterilization 20min, is put into inclined-plane afterwards.
Described No. 1 liquid culture based component of Gao Shi is: in every 1000ml distilled water, add Zulkovsky starch 20.0g, KNO
31.0g, K
2hPO
40.5g, MgSO
47H
2o 0.5g, NaCl 0.5g, FeSO
40.01g, regulating pH is 7.0.Be distributed in 500ml triangular flask every bottle of 150ml, tampon beyond the Great Wall, autoclave sterilization 20min.
Embodiment 2
Fresh cow dung is mixed with the ratio of dry weight 6:1 with crushing straw, or according to the suitable resize ratio of the physico-chemical property of cow dung, sawdust, the C/N that makes mixture is 30, water content is 60%.
The microbial inoculum that embodiment 1 is produced is inoculated in heap material mixture according to mass ratio 5 ‰, and regularly turning ventilation, carries out fermented manure fermentation.Simultaneously taking the heap body that do not access microbial inoculum of the present invention as contrast.
Inoculate compared with the cow dung of microbiobacterial agent of the present invention and the cow dung of Natural compost, heat-up rate is fast, and heap body top temperature is high, and fermentation period is short, its C/N declines rapidly, and these results show in cow dung, to inoculate microbiobacterial agent of the present invention can accelerate the degree of becoming thoroughly decomposed of cow dung compost.
Inoculate the cow dung of microbiobacterial agent of the present invention and the cow dung compost of Natural compost and finish the nutrient content of rear windrow by comparative analysis, after the cow dung compost of discovery inoculation microbiobacterial agent of the present invention finishes, its full nitrogen, full phosphorus, full potassium and organic content are all significantly higher than the cow dung of Natural compost, and this shows to inoculate microbial inoculum of the present invention in cow dung and is more conducive to Nutrient Transformation and preservation in compost.Inoculate the cow dung of microbiobacterial agent of the present invention and the cow dung of the Natural compost difference in the degree of becoming thoroughly decomposed and the each index of nutrient content in table 1 and table 2.
The impact of table 1 microbiobacterial agent of the present invention on compost maturity degree
Compost initial stage microbial inoculum promotes to decompose free nutritive substance in heap body, can make heap temperature increase rapidly, and then promote again the breeding of microorganism, temperature rise and microbial reproduction are played to benign cycle effect, promoting temperature and reach top temperature, when temperature reaches the highest, is that microbial reaction is the fiercest time, a large amount of organic matters are degraded, and carbon source is converted into CO
2, decline and cause piling weight.Temperature is the external manifestation of heap body internal-response situation.Temperature rise is faster, high-temperature duration is longer, and heap body inside is fully reacted more fully, is more conducive to the release of the nutrient being fixed.More abundant at highest temperature elementary reaction, the free nutritive substance and the degradable organic substance that are consumed will be fewer, can not cause fertilizer after fertilising, to occur burning seedling phenomenon.Large amount of organic is after hot stage is degraded, and the after-ripening stage is decomposed in the back to only have a small amount of degradable material.The material that decompose early stage is more much more abundant, just more few more mitigation that after-ripening is decomposed, and temperature will be lower, and outlet temperature can approach room temperature.High-temperature duration is long simultaneously, can kill thoroughly the pathogen in animal excrement.Therefore, the thermophilic carbon monoxide streptomycete CF1 of the present invention's screening is a kind of good high temperature bacterium.
Compost finishes rear timely sampling, drills through 40～50cm place sample under heap surface layer with little earth boring auger, and air-dry rear grinding is measured organic, full nitrogen, full phosphorus, potassium content entirely, in table 2.
The nutrient content of table 2 compost sample
Embodiment 3 is for the composite fungus agent of animal excrement fermentation
The subtilis seed liquor 10mL that gets incubated overnight transfers into 500ml fermention medium, and 28 DEG C ferment 20 hours, and rotating speed is 180rpm.
Consisting of of fermentation of bacillus subtilis substratum: sucrose 10g/L, peptone 5g/L, extractum carnis 3g/L, yeast soaks powder 1g/L, bitter salt 0.5g/L, manganous sulfate 0.0005g/L, pH7.0.
The bacillus megaterium seed liquor 20mL that gets incubated overnight transfers into 500ml fermention medium, under 30 DEG C of conditions, cultivates 48h.Rotating speed is 180rpm.
Bacillus megaterium fermention medium consists of: extractum carnis 5g/L, peptone 5g/L, sodium-chlor 5g/L, pH7.0.
Prepare the fermented liquid of thermophilic carbon monoxide streptomycete CF1 by the method for embodiment 1.
The fermented liquid of the fermented liquid of the fermentation of bacillus subtilis liquid of preparation, bacillus megaterium and thermophilic carbon monoxide streptomycete CF1 is mixed by weight 1:1:2, obtain the composite fungus agent for animal excrement fermentation.
Fresh cow dung is mixed with the ratio of dry weight 6:1 with sawdust, or according to the suitable resize ratio of the physico-chemical property of cow dung, sawdust, the C/N that makes mixture is 30, water content is 60%.
Inoculate aforesaid composite fungus agent, inoculum size is 0.8%, and with the commercially available positive contrast of EM microbial inoculum, not inoculate microbial inoculum as negative control.The results are shown in Table 3 and table 4.
The impact of table 3 complex micro organism fungicide of the present invention on compost maturity degree
The nutrient content of table 4 compost sample
As shown in Table 3, the composite fungus agent that inoculation comprises thermophilic carbon monoxide streptomycete CF1 of the present invention, heap body can complete fermentation in 23 days, shifts to an earlier date 10-12 days complete fermentation than inoculation commercially available EM microbial inoculum and Natural compost, and the high temperature extended period is long, the degree of becoming thoroughly decomposed after compost finishes is good.As shown in Table 4, the composite fungus agent that inoculation comprises thermophilic carbon monoxide streptomycete CF1 of the present invention, compost finishes organic content in rear windrow, full nitrogen, full phosphorus and full potassium content all higher than the windrow of inoculating commercially available EM microbial inoculum and Natural compost.Its organic content of composting production of the composite fungus agent that wherein inoculation comprises thermophilic carbon monoxide streptomycete CF1 of the present invention and total nutrient content are all higher than the regulation in national standard (organic fertilizer NY-525-2012), and inoculate the composting production of commercially available EM microbial inoculum, just be up to state standards (organic fertilizer NY-525-2012), the product of Natural compost be not up to state standards (organic fertilizer NY-525-2012).
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (7)
- A thermophilic carbon monoxide streptomycete ( streptomyces thermocarboxydus) bacterial strain CF1, its preserving number is CGMCC No.6873.
- 2. contain the bacteria agent of bacterial strain described in claim 1.
- 3. bacteria agent according to claim 2, wherein the viable count of thermophilic carbon monoxide streptomycete CF1 be 5,000,000,000/gram more than.
- 4. the method for the bacteria agent described in preparation claim 2 or 3, is characterized in that, the thermophilic carbon monoxide streptomycete CF1 claimed in claim 1 that ferments in liquid fermentation medium, and the fermented liquid obtaining is bacteria agent.
- 5. method as claimed in claim 4, it also comprises described thermophilic carbon monoxide streptomycete CF1 fermented liquid is carried out to lyophilize, the lyophilized powder being prepared into is bacteria agent.
- 6. for the composite fungus agent of animal excrement fermentations, it comprise subtilis ( bacillus subtilis), bacillus megaterium ( bacillus megaterium) and thermophilic carbon monoxide streptomycete CF1 claimed in claim 1.
- 7. microbial inoculum as claimed in claim 6, it is characterized in that, the viable count of described subtilis is 1,000,000,000/gram more than, described bacillus megaterium viable count is 2,000,000,000/gram more than, the viable count of described thermophilic carbon monoxide streptomycete CF1 is 2,000,000,000/gram more than.
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| CN104531557B (en) * | 2014-12-04 | 2017-10-03 | 江苏省血吸虫病防治研究所 | One plant generation ovicidal trait material sub- scarlet streptomycete and its application |
| CN104988095B (en) * | 2015-07-03 | 2019-01-22 | 北京农学院 | One plant of thermophilic carbon monoxide streptomycete low temperature subspecies Dstr3-3 and its application |
| CN105567607B (en) * | 2016-03-03 | 2019-11-01 | 北京林业大学 | One plant of high temperature resistant garden waste decomposer ST3 and its application |
| CN105567612B (en) * | 2016-03-05 | 2019-08-02 | 北京林业大学 | A kind of degradation composite bacteria agent preparation of garden waste and application |
| CN106007847A (en) * | 2016-05-17 | 2016-10-12 | 许祚员 | Efficient bio-organic fertilizer for pomegranate trees and preparation method of fertilizer |
| CN109136156B (en) * | 2018-10-10 | 2021-10-22 | 皖西学院 | Solid complex microbial inoculum, preparation method thereof and application thereof in dendrobium officinale cultivation |
| CN109517754A (en) * | 2018-11-20 | 2019-03-26 | 上海交通大学 | A method of high temperature bacterial strain is isolated and purified using common biochemical equipment |
| CN111378604B (en) * | 2020-03-17 | 2022-06-10 | 山东中农普宁药业有限公司 | Thermoactinomyces hopeiensis and application thereof |
| CN114196583B (en) * | 2021-12-15 | 2023-11-17 | 天津科技大学 | Streptomyces thermophilus, synergistic biological agent containing same and application thereof |
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| KR100904632B1 (en) * | 2007-07-30 | 2009-06-29 | 경상대학교산학협력단 | Production and Application of subtilisin-like proteinase inhibitor by Streptomyces thermocarboxydus C12 |
| CN101348389B (en) * | 2008-07-25 | 2011-04-06 | 李海泉 | Solid fermentation substrate |
| CN102061270B (en) * | 2010-04-20 | 2012-05-02 | 怀化学院 | Streptomyces thermocarboxydus strain and cultivating method thereof |
| CN101880182A (en) * | 2010-06-25 | 2010-11-10 | 东北农业大学 | Low-temperature fast fermentation agent for animal dung in cold regions as well as preparation method and application thereof |
| CN103103151B (en) * | 2013-01-18 | 2014-12-24 | 南京林业大学 | Streptomyces thermocarboxydus NL-1 as actinomycete capable of efficiently eroding limestone and application of Streptomyces thermocarboxydus NL-1 |
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