CN110396483A - One plant height temperature straw degradative bacterium B-8 and its microbial inoculum and application - Google Patents
One plant height temperature straw degradative bacterium B-8 and its microbial inoculum and application Download PDFInfo
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Abstract
The invention discloses a plant height temperature straw degradative bacterium B-8, classification naming is native bacillusGeobacillus sp., China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC NO.17301 are preserved on March 22nd, 2019.Bacterium B-8 of the present invention can generate degradation circle on the Congo red culture medium of CMC-, can grow on the culture medium using agricultural crop straw as sole carbon source.Bacterium B-8 can generate the enzyme in relation to degraded cellulose and hemicellulose, convert the macromolecular substances in agricultural crop straw to the small-molecule substance utilized for plant absorption, realize the resource utilization of agricultural wastes under hot conditions.After experiment shows rice straw inoculation B-8 microbial inoculum, 75 DEG C, 170 r/m liquid fermentation and culture 7d, the degradation rate of rice straw are 17.67%.The invention also discloses bacterium B-8 in the application of agricultural crop straw of degrading.
Description
Technical field
The invention belongs to Agricultural resources and environment fields, particularly belong to solid waste resource recovery using field, are related to one plant
High temperature straw degradative bacterium B-8 and its microbial inoculum, the bacterium B-8 and its microbial inoculum are for discarded crop material production organic fertilizer of degrading
Material realizes agricultural wastes recycling.
Background technique
China is large agricultural country, generates a large amount of agricultural wastes waste, such as wheat, rice, corn farming every year
Object stalk.On the one hand, these stalks contain nutrient necessary to a large amount of plant growths, but it is mainly in a manner of cellulose
In the presence of cannot directly be utilized by plant since molecular weight is larger.On the other hand, the application of recent decades, chemical fertilizer seriously destroys
Soil environment health, reduces beneficial microorganism diversity in soil.How agricultural wastes to be efficiently used, while
Yield reduces applying quantity of chemical fertilizer under the premise of not reducing be one of problem instantly.Discarded straw are handled using cellulose-degrading bacteria
Stalk has the characteristics that environmental protection, energy conservation, efficient, and meets the requirement of sustainable development.Therefore, some fibre is screened from environment
Plain degradation bacteria gets up these resource rational utilizations, will be a pith in China's strategy of sustainable development.
Composting Process is under the action of microorganism, and the macromolecular substances of decomposition more difficult in raw material are decomposed into can be micro-
The carbon source of biological utilisation.Therefore, the decomposition for accelerating cellulose in composting process is to promote composting efficiency, shorten composting cycle and mention
One of high humified critical issue.Studies have shown that compost hot stage (general > 55 DEG C) is lignin, cellulose and half fiber
Tie up the destruction of the molecules such as element, fracture and one of decomposition, the important stage of pathogen reduction.Therefore, it is inoculated with high temperature cellulose-decomposting bacteria,
The compost time can be effectively shortened, raising efficiency.The high temperature cellulose-decomposting bacteria reported at present is concentrated mainly on 55 DEG C~65 DEG C
Between, and 70 DEG C or more are rarely reported.
Summary of the invention
It, can efficient-decomposition under the high temperature conditions the purpose of the present invention is to provide a kind of high temperature straw degradative bacterium B-8
Agriculture waste stalk, so that waste straw recycling in farmland, it is ensured that sustainable agriculture shapes up.
The purpose of the present invention is what is be achieved through the following technical solutions:
One plant height temperature straw degradative bacterium B-8, classification naming is native bacillus (Geobacillus sp.), in 2019
On March 4, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address: Beijing
The institute 3 of Chaoyang District North Star West Road 1, deposit number are CGMCC NO.17301.
The biological characteristics of bacterium B-8 are as follows: at 65~75 DEG C, cultivated on NA culture medium, initial stage bacterium colony is white, finally
Bacterium colony main body is in faint yellow, and soft, surface is smooth, and edge is white.
Bacterium B-8 can grow on the Congo red culture medium of CMC- and generate transparent hydrolysis circle.The Congo red culture medium of CMC-
Formula are as follows: sodium carboxymethylcellulose (CMC-Na) 2.0g, (NH4)2SO42.0g, KH2PO41.0g, MgSO4·7H2O 0.5g,
Congo red 0.2g, agar 20g, deionized water 1000mL, pH are naturally, 121 DEG C of high pressure sterilization 20min.
Another object of the present invention provides the high temperature straw degradative bacterium B-8 in terms of agricultural crop straw of degrading
Using.
It is a further object to provide a kind of microbial inoculums containing the high temperature straw degradative bacterium B-8.
The preparation method of microbial inoculum of the present invention, comprising: bacterial strain B-8 is inoculated into NB culture medium, temperature 65~
75 DEG C, under 170~200r/m of revolving speed culture to logarithmic phase；The bacterium B-8 of above-mentioned culture to logarithmic phase is connect by inoculum concentration 1% again
Kind cultivates 7d, centrifugal filtration under the conditions of 65~75 DEG C of temperature, 170~200r/m of revolving speed in rice straw fluid nutrient medium
Thallus and impurity are removed to get microbial inoculum.
NB (Nutrient Broth) culture medium: beef extract 3.0g, sodium chloride 5.0g, peptone 10.0g, deionized water
1000mL, pH7.3 ± 0.1,121 DEG C of sterilizing 15min.
The rice straw fluid nutrient medium: rice straw powder 20g, (NH4)2SO41.4g, KH2PO42.0g
MgSO4·7H2O 0.3g, CaCl2·2H2O 0.4g, tryptone 1g, FeSO4·7H2O 0.0075g, MnSO4·H2O
0.0025g, ZnSO40.002g, CoCl20.003g, deionized water 1000mL, pH are naturally, 121 DEG C of high pressure sterilization 20min.
The rice straw powder is that the rice straw of water content≤1% is crushed, 40 meshes excessively are made.
Application of the microbial inoculum of the present invention containing high temperature straw degradative bacterium B-8 in terms of agricultural crop straw of degrading.
The application, comprising: agricultural crop straw impregnates 30min using 1%NaOH solution, and 50 DEG C of drying shred；It will be through
The agricultural crop straw and liquid culture medium for crossing processing mix, and microbial inoculum are inoculated with, in 65~75 DEG C of temperature, 170~200r/ of revolving speed
It is cultivated under m；Wherein, the ratio of treated agricultural crop straw, liquid culture medium and microbial inoculum be 1:50:0.5 (g:g:
mL)。
The liquid culture medium: (NH4)2SO41.4g, KH2PO42g, MgSO4·7H2O 0.3g, CaCl2·
2H2O 0.4g, tryptone 1g, FeSO4·7H2O 0.0075g, MnSO4·H2O 0.0025g, ZnSO40.002g, CoCl2
0.003g, deionized water 1000mL, pH are naturally, 121 DEG C of high pressure sterilization 20min.
The agricultural crop straw is the agricultural crop straws such as rice straw, wheat stalk, corn.
High temperature of the present invention is 65~75 DEG C.
Beneficial effects of the present invention:
Bacterium B-8 of the present invention can generate hydrolysis circle on the Congo red culture medium of CMC-, can be in agricultural wastes straw (water
The stalks such as rice, wheat, corn) be sole carbon source culture medium on grow.
Bacterium B-8 of the present invention can generate the enzyme of degraded cellulose and hemicellulose, including endoglucanase, circumscribed
Dextranase, beta-glucosidase provide the foundation, it is ensured that straw degradative bacterium for different stalk substrates of efficiently quickly degrading
Small organic molecule material is made after the fermentation of discarded agricultural stalk is decomposed under the high temperature conditions, degrade 7d, can make stalk substrate
Degradation rate reaches 17.67%, accelerates straw-returning decomposed, can not only turn waste into wealth, protect environment, can also promote sustainable
Development, has broad application prospects.
Detailed description of the invention
Fig. 1 is the colonial morphology of bacterium B-8.
Fig. 2 is the hydrolysis circle that bacterium B-8 is generated on the Congo red plate of CMC-.
Fig. 3 is the phylogenetic evolution tree of bacterium.
Fig. 4 is comparison diagram before and after rice straw is degraded, and A is the rice straw before degradation, and B is the rice straw after degrading 7 days
Stalk.
Biological deposits information
B-8, classification naming are native bacillus (Geobacillus sp.), and it is micro- that China is preserved on March 4th, 2019
Biological inoculum preservation administration committee common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in
Institute of microbiology, the academy of sciences, state, deposit number are CGMCC NO.17301.
Specific embodiment
Embodiment 1
The separation and identification of straw degradative bacterium B-8
Straw degradative bacterium bacterial strain B-8 is that stalk accumulation place screens to obtain from Weifang City, Shandong Province.Utilize gradient dilution
Rubbing method diluted sample suspension will be applied to the Congo red culture medium flat plate of CMC-, 75 DEG C of culture screen fibre element degradations step by step
Bacterium.Select that generate transparent circle on the Congo red culture medium flat plate of CMC- larger and grow faster bacterial strain purifies and separates, by them
It is seeded to using rice straw as in the rice straw medium of sole carbon source, 75 DEG C are cultivated, more each strain growth speed and life
Long situation, finally obtain can efficient degradation rice straw under the high temperature conditions bacterium bacterial strain B-8.
The Congo red culture medium of CMC-: sodium carboxymethylcellulose (CMC-Na) 10.0g, (NH4)2SO42.0g, KH2PO4
1.0g, MgSO4·7H2O 0.5g, Congo red 0.2g, agar 20g, deionized water 1000mL, pH are naturally, 121 DEG C of high pressure sterilizations
20min。
Rice straw medium: rice straw powder 10g, KH2PO43.0g, NaNO33.0g, CaCl20.5g,
MgSO4·7H2O 0.5g, FeSO4·7H2O 0.0075g, MnSO4·H2O 0.0025g, ZnSO4·7H2O 0.002g, CoCl2
0.003g, agar 20g, deionized water 1000mL, pH are naturally, 121 DEG C of high pressure sterilization 20min.The rice straw powder is
Rice straw be dried to 1% or less water content, crushing, cross 40 meshes be made.
Such as Fig. 1, B-8 biological characteristics are as follows: at 75 DEG C, cultivated on NA culture medium, initial stage bacterium colony is white, final bacterium colony
Main body (culture 12h after) is in faint yellow, and soft, surface is smooth, and edge is white.
As Fig. 2, bacterium B-8 generate hydrolysis circle on the Congo red culture medium of CMC-.
16S sequencing is carried out using bacterial universal primers 27F and 1492R, gained sequence compares through BLAST, discovery with
Geobacilluszalihae strain WSUCF-019B has 99% similarity.It chooses and its similar some bacterial strain is drawn
Phylogenetic evolution tree (such as Fig. 3) identifies bacterial strain B-8 for native bacillus in conjunction with bacterium colony and morphological feature
(Geobacillus sp.)。
Embodiment 2
Bacterial strain B-8 is inoculated into NB culture medium, 8~12h is cultivated under the conditions of 75 DEG C of temperature, revolving speed 170r/m to logarithm
Phase；The bacterium B-8 by culture to logarithmic phase is inoculated in rice straw fluid nutrient medium by 1% inoculum concentration again, in temperature 75
DEG C, 7d is cultivated under the conditions of revolving speed 170r/m, centrifugal filtration removes thallus and impurity, obtains crude enzyme liquid, measures inscribe in crude enzyme liquid
Enzyme, beta-glucosidase, zytase, xylobiase are living, are shown in Table 1.
Rice straw fluid nutrient medium: rice straw powder 20g, (NH4)2SO41.4g, KH2PO42g, MgSO4·7H2O
0.3g, CaCl2·2H2O 0.4g, tryptone 1g, FeSO4·7H2O 0.0075g, MnSO4·H2O 0.0025g, ZnSO4
0.002g, CoCl20.003g, deionized water 1000mL, pH are naturally, 121 DEG C of high pressure sterilization 20min；Wherein, rice straw powder
End is that rice straw is dried to that 1% or less water content, crushings, to cross 40 meshes obtained.
The enzyme activity of 1 high temperature straw degradative bacterial strain bacterium B-8 of table
From table 1 it follows that bacterium B-8 can generate the enzyme of a large amount of degraded celluloses and hemicellulose, it is efficient
Different stalk substrates of quickly degrading provide the foundation.
Embodiment 3
Using the technique study bacterium B-8 of triangular flask liquid fermentation to the degradation effect of rice straw:
The preparation of microbial inoculum: bacterial strain B-8 is inoculated into NB culture medium, cultivates 8 under the conditions of 75 DEG C of temperature, revolving speed 170r/m
~12h is to logarithmic phase；The bacterium B-8 by culture to logarithmic phase is inoculated in rice straw fluid nutrient medium by 1% inoculum concentration again
In, 7d is cultivated under the conditions of 75 DEG C of temperature, revolving speed 170r/m, centrifugal filtration removes thallus and impurity, obtains microbial inoculum.
Rice straw pretreatment: rice straw impregnates the wax on 30min removal stalk surface using 1%NaOH solution, makes
Cellulose-decomposing bacterium preferably contacts with the cellulose in stalk, and 50 DEG C of drying are shredded to 2-3cm long.
Liquid culture medium: (NH4)2SO41.4g, KH2PO42g, MgSO4·7H2O 0.3g, CaCl2·2H2O
0.4g, tryptone 1g, FeSO4·7H2O 0.0075g, MnSO4·H2O 0.0025g, ZnSO40.002g, CoCl2
0.003g, deionized water 1000mL, pH are naturally, 121 DEG C of high pressure sterilization 20min.
Rice straw 3.00g, liquid culture medium 150mL after each 250mL triangular flask packing pretreatment.Processing group connects
Kind 1.5mL microbial inoculum, mixing is placed on 75 DEG C of constant-temperature table 170r/m cultures, to be inoculated with equivalent sterile water as a control group.Periodically
The metamorphosis for observing rice straw after 7d, washes away the thallus on stalk, drying to constant weight, measures its degradation rate.
After inoculation for 24 hours, processing group culture solution starts to become cloudy, at this point, rice straw starts to soften, disintegration dispersion.After 7d
(Fig. 4 B), the stalk degradation of substrates rate of processing group are 17.67%；And there is no completely still stalk without significant change in control group
Apparent decomposition, the degradation rate of stalk substrate are 8.34%, 1/2 of degradation rate less than processing group.It follows that inoculation drop
Solution bacterium B-8 can accelerate the decomposition and the wherein conversion of ingredient of stalk under the high temperature conditions, accelerate the decomposition and conversion of substrate.
Claims (8)
1. a plant height temperature straw degradative bacterium B-8, classification naming was native bacillus (Geobacillus sp.), in 2019 3
It is preserved within 4th China Committee for Culture Collection of Microorganisms's common micro-organisms center the moon, deposit number is CGMCC NO.17301.
2. application of the straw degradative bacterium B-8 described in claim 1 in terms of agricultural crop straw of degrading.
3. a kind of microbial inoculum containing high temperature straw degradative bacterium B-8 described in claim 1.
4. the preparation method of microbial inoculum as claimed in claim 3, characterized by comprising: bacterial strain B-8 is inoculated into NB culture medium,
It cultivates at 65~75 DEG C of temperature, 170~200r/m of revolving speed to logarithmic phase；Again by the bacterium B-8 of culture to logarithmic phase by inoculation
Amount 1% is inoculated in rice straw fluid nutrient medium, cultivates 7d under the conditions of 65~75 DEG C of temperature, 170~200r/m of revolving speed, from
The heart is filtered to remove thallus and impurity to get microbial inoculum.
5. the preparation method of microbial inoculum according to claim 4, it is characterised in that the rice straw fluid nutrient medium: water
Rice straw powder 20g, (NH4)2SO41.4g, KH2PO42g, MgSO4·7H2O 0.3g, CaCl2·2H2O 0.4g, tryptose
Peptone 1g, FeSO4·7H2O 0.0075g, MnSO4·H2O 0.0025g, ZnSO40.002g, CoCl20.003g, deionized water
1000mL, pH are naturally, 121 DEG C of high pressure sterilization 20min.
6. the preparation method of microbial inoculum according to claim 5, it is characterised in that the rice straw powder is water content
≤ 1% rice straw is crushed, 40 meshes excessively are made.
7. application of the microbial inoculum as claimed in claim 3 in terms of agricultural crop straw of degrading.
8. application according to claim 8, it is characterised in that the agricultural crop straw be rice straw, wheat stalk,
Corn stover.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111705010A (en) * | 2020-02-19 | 2020-09-25 | 东北林业大学 | Application of halomonas in synthesis of biodegradable mulching film by using straws and kitchen waste grease |
| CN116083280A (en) * | 2022-10-25 | 2023-05-09 | 西北农林科技大学 | Clostridium for degrading straw cellulose, application and preparation thereof |
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| CN102174424A (en) * | 2010-12-06 | 2011-09-07 | 中国农业科学院农业资源与农业区划研究所 | Microbial bacterial agent for degrading straws |
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| CN102174424A (en) * | 2010-12-06 | 2011-09-07 | 中国农业科学院农业资源与农业区划研究所 | Microbial bacterial agent for degrading straws |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111705010A (en) * | 2020-02-19 | 2020-09-25 | 东北林业大学 | Application of halomonas in synthesis of biodegradable mulching film by using straws and kitchen waste grease |
| CN111705010B (en) * | 2020-02-19 | 2024-02-09 | 东北林业大学 | Application of halomonas in synthesizing biodegradable mulching film by utilizing straw and kitchen waste grease |
| CN116083280A (en) * | 2022-10-25 | 2023-05-09 | 西北农林科技大学 | Clostridium for degrading straw cellulose, application and preparation thereof |
| CN116083280B (en) * | 2022-10-25 | 2024-04-26 | 西北农林科技大学 | Clostridium for degrading straw cellulose, application and preparation thereof |
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