CN102031233A - Method for preparing fermentation strains of microbial organic fertilizer - Google Patents
Method for preparing fermentation strains of microbial organic fertilizer Download PDFInfo
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- CN102031233A CN102031233A CN 201010538124 CN201010538124A CN102031233A CN 102031233 A CN102031233 A CN 102031233A CN 201010538124 CN201010538124 CN 201010538124 CN 201010538124 A CN201010538124 A CN 201010538124A CN 102031233 A CN102031233 A CN 102031233A
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Abstract
The invention provides fermentation strains Brevibacterium sp D1 (with the perservation number of CCTCC No: M 2010250) and bacillus amyloliquefaciens S4 ( with the preservation number of CCTCC No: M 2010249) of a microbial organic fertilizer of tobacco straws; the fermentation strains Brevibacterium sp D1 and the bacillus amyloliquefaciens S4 are separated and screened from the tobacco straws under a high-temperature and heavy-nicotine condition, and can exist in the presence of the nicotine; and after being cultivated and proliferated, the ermentation strains Brevibacterium sp D1 and the bacillus amyloliquefaciens S4 are added into a waste tobacco straw mixture, and are subjected to solid fermentation under a natural condition to convert the tobacco straw wastes into the tobacco straw microbial organic fertilizer. The tobacco straw microbial organic fertilizer can replace other fertilizers to produce the tobacco, and has the advantages of increasing yield of the tobacco leaves, increasing production values of the tobacco leaves, improving smoking quality of the tobacco leaves, and simultaneously removing pollution to the environment by the tobacco straw wastes.
Description
Technical field
What the present invention relates to is agro-ecology organic fertilizer production technology, and particularly a kind of solid state fermentation prepares the process for preparing strain thereof of tabacco straw biological organic fertilizer.
Background technology
Tobacco is a kind of important cash crop, has a very important role in national economy.In China's tobacco planting production process, for a long time based on chemical fertilizer, cause soil organism quantity to fall sharply, the coacervate quality and quantity descends, and air permeability is degenerated, and influences the output and the quality of leaf tobacco production.In addition, behind tobacco leaf picking, abandon a large amount of discarded tabacco straws in vega, maximum duration can reach five months, increases next year vega disease and pest, pollutes the vega ecotope.According to the industry regulation, should concentrate the tabacco straw burning disposal, but can increase CO like this
2Quantity discharged causes air environmental pollution.Be a kind of must not and for it way.
Use general agricultural crop straw solid-fermented technique in the prior art fermentation of tobacco straw is become the biological organic fertilizer method, but because of tabacco straw contains a large amount of nicotine, has a large amount of harmful germs and viruses again, this method is not preferably selected microbial strains, and effect is bad.Fermenting bacteria commonly used is not suitable for surviving under the condition with a large amount of nicotine materials, and the conversion of can not effectively fermenting is so existing method is difficult to address the above problem.If can seek a kind of anti-strong nicotine high temperature microbe, have the fermenting bacteria that under the condition of a large amount of nicotine materials, survives, carry out solid state fermentation, discarded tabacco straw is changed into a kind of safe biological organic fertilizer, part instead of chemical composite fertilizer is backfilling into vega.Disease and pest, Soil degradation, environment protection, the utilization of resources and quality of tobacco problem in the tobacco production are considered in the lump, and be used.Help improving environment like this, widely.
Summary of the invention
The objective of the invention is to propose a kind of preparation method of the solid state fermentation bacterial classification that can under the heavy nicotine condition of high temperature, survive.With the solid state fermentation bacterial classification of the anti-nicotine of this heatproof through cultivate and expand numerous after, join in the depleted tabacco straw mixture, carry out solid state fermentation under field conditions (factors), the tabacco straw waste is converted into the tabacco straw biological organic fertilizer.
Concrete steps are:
1, strains separation screening: from the tabacco straw of the regional vega of difference, sampling joins glucose, the peptone that contains nicotine respectively, and yeast extract paste in the liquid nutrient medium of inorganic salt, under 50～60 ℃ of high temperature, was cultivated 48 hours.With the nutrient solution dilution, coat nicotine-containing peptone, in the agar culture dish of yeast extract paste, under 25～35 ℃, cultivated 24～36 hours.Picking list bacterium colony is inoculated into respectively and simultaneously in the liquid or solid substratum of same numbering of peptone, yeast extract paste, inorganic salt, under 30 ℃, cultivates 24～30 hours.Extract centrifugal supernatant nutrient solution, add Streptomycin sulphate, penicillin and carboxymethyl cellulose, under 50 ℃, be incubated 18 hours, measure reducing sugar content, separate S
4And D
1Bacterial strain.
2, strain classification is identified and preservation
Measure S according to uncle's Jie Shi handbook (the 7th edition) and 16SrRNA gene order
4And D
1Carrying out the classification of bacterial strain identifies.
S
4Bacterium: according to S
4The colonial morphology of bacterium, cellular form, physiological and biochemical test and 16SrRNA gene sequencing result, S
4Bacterium is bacillus amyloliquefaciens (Bacillus amyloliquefaciens S
4).
This bacterial strain is preserved in the Chinese typical culture collection center of Chinese Wuhan City, Hubei Province Wuhan University on September 26th, 2010, and name is called bacillus amyloliquefaciens S
4, preserving number: CCTCC NO.M 2010249).
The bacteria characteristic of this bacterial strain.Bacterium colony is coarse, and is opaque, expansion, and little band is yellow, and cell dia＜1 μ m is shaft-like, and uniform coloring does not have the folder film, motion, the leather Albert'stain Albert is positive; Form gemma, the gemma ellipse, middle life is not expanded, no parasporal crystal, catalase and oxydase are positive, the VP test is positive, and methyl red test is positive, and anaerobic growth can utilize glucose, wood sugar, seminose, L-arabinose, and produce acid, hydrolyzed starch decomposes casein; Can not utilize lactose, Citrate trianion, nitrate reduction test are all positive; 5 ℃ of growths down, 7%NaCI growth down, PH5.7 growth, VPPH＜6 positives, VPPH＞7 feminine genders.
This bacterium can grow under the condition that nicotine exists.
D
1Bacterium: according to D
1The colonial morphology of bacterium, cellular form, physiological and biochemical test and 16SrRNA gene sequencing result, D
1Bacterium is tyrothricin (Brevibacterium spD1).
This bacterial strain is preserved in the Chinese typical culture collection center of Chinese Wuhan City, Hubei Province Wuhan University on September 26th, 2010, and name is called tyrothricin D
1, preserving number: CCTCC NO.M 2010250).
The bacteria characteristic of this bacterial strain.Bacterium colony is coarse, and is opaque, expansion, dirty white, cell is shaft-like, uniform coloring, gramstaining is positive, no gemma, catalase is positive, oxydase is negative, can utilize glycerine, L-arabinose, ribose, the D-wood sugar, semi-lactosi, glucose, fructose, N.F,USP MANNITOL, rhamnosyl, the D-tagatose, L-arabinose alcohol, gluconate, 2-ketone group-gluconate and growth, nitrate reduction is positive, can not utilize the D-pectinose, red tinea sugar, the L-wood sugar, sorbose, sorbyl alcohol, inositol, melampyrin, ribitol, lactose, maltose, close disaccharides, sucrose, trehalose, raffinose, Xylitol, D-lyxose, D-Fucose L-Fucose, L-arabinose alcohol, and glycogen, not hydrolyzed starch, α methyl D-seminose, N-acetyl-grape amine, Vitamin B17, black bearberry glucoside, Beta-methyl-D-xyloside, 5-ketone group-gluconic acid test is all negative.
This bacterium can grow under the condition that nicotine exists.
3, seed culture.With D
1Bacterium and S
4Bacterium adds substratum MD, its composition and ratio such as table 1 as bacterial classification in seed triangular flask or fermentor tank.
Table 1 strain liquid substratum
The seed culture condition: insert the female kind in inclined-plane behind the medium sterilization, under 28～30 ℃, shaking culture 16～22 hours;
4, expand numerous cultivation, in fermentor tank, add substratum MD, back 3～8% volume ratios according to fermentation tank culture medium of sterilization insert makes seed after above-mentioned culture mixes, expand numerous culture condition: tank pressure is 0.4～0.6Mpa, the jar temperature is 28～30 ℃, and air flow quantity is 0.3～0.6 (v/v/min), and agitator speed is 200rpm, expand numerous cultivation 18～24 hours, when the OD of nutrient solution value stops fermentation 0.02 the time for OD600nm descends;
5, product is cultivated, in large fermentation tank, add substratum MD, the culture that numerous cultivation is expanded according to 3～8% volume ratios access of fermentation tank culture medium in the sterilization back is made liquid seeds, the product culture condition: tank pressure is 0.4～0.6Mpa, the jar temperature is 28～30 ℃, air flow quantity is 0.3～0.6 (v/v/min), and agitator speed is 140～200rpm, and product was cultivated 18～24 hours; When the OD of nutrient solution value stops fermentation 0.02 the time for OD600nm descends;
6, above-mentioned fermenting culture is concentrated 1～8 times through membrane filtration, detect liquid microbial inoculum; Or with above-mentioned fermenting culture after membrane filtration concentrates 1～8 times, add dispersion agent, further dry solid-state microbial inoculum product.
Show by agricultural and tobacco field experiment, utilize this patent D
1Bacterium and S
4Bacterium is biological fermented bacterium, carries out the tabacco straw biological organic fertilizer of fermentative production on tabacco straw, uses by part instead of chemical composite fertilizer, can increase the output and the output value of flue-cured tobacco and burley tobaccos, improves the inner quality of tobacco leaf.
Bacterial classification of the present invention can use separately, also can unite use, and the ratio of uniting use is: D
1Bacterium: S
4Bacterium=1～4: 1
The present invention has following advantage
The bacterial classification D of A, seed selection of the present invention
1Bacterium and S
4Bacterium can be suitable for the tabacco straw solid state fermentation, and with two kinds of bacterial strains according to a certain percentage, adding is stirred in the tabacco straw, can carry out biological fermentation to it, changes biological organic fertilizer into.
B, the present invention have found D
1Bacterium and S
4The preparation method of bacterium, and optimized its working condition, for the suitability for industrialized production of tabacco straw biological organic fertilizer microbial inoculum and biological tobacco fertilizer is laid a good foundation.
C, by D
1Bacterium and S
4Bacterium is the basis, utilize tabacco straw, can produce and be fit to the biological organic fertilizer that tobacco produces, its effect can increase yield of tobacco, increase the tobacco leaf output value, improve the tobacco leaf quality of smokeing panel test, and has potential and improve tobacco and produce disease and pest phenomenon, the structure of improving the soil and reduce the environmental pollution effect.
D, the present invention have provided a technological line for discarded tabacco straw comprehensive utilization.
Embodiment
Embodiment 1:
Spawn culture: get above-mentioned D
11 of tube and S
41 of tube, access fills in the 250ml seed bottle of substratum respectively, and it consists of described substratum: glucose 1%, peptone 0.8%, yeast extract paste 0.5%, sodium-chlor 0.4%, all the other are water.PH is transferred to 7.8.
Under 28 ± 1 ℃ and oscillating condition, 250ml seed flask culture 20 hours, with D
1Bacterium liquid and S
4After the bacterium liquid balanced mix,, insert again in the 1000ml fermentation flask according to 6% volume ratio kind amount; Under 1 ℃ of 28 scholar and oscillating condition, the 1000ml fermentation flask was cultivated 24 hours, measure the OD600nm value, obtain fermenting agent.
Embodiment 2:
Spawn culture: get above-mentioned D
13 of tubes and S
41 of tube, access fills in the 250ml seed bottle of substratum respectively, and it consists of described substratum: glucose 0.8%, peptone 0.6%, yeast extract paste 0.3%, sodium-chlor 0.2%, KH
2PO
40.1%, all the other are water.PH is transferred to 6.7.
Under 3 ℃ and oscillating condition,, in the 1000ml fermentation flask, insert D according to 2% volume ratio kind amount respectively then 250ml seed flask culture 18 hours
1Bacterium liquid and 2.5% volume ratio kind amount insert S
4Bacterium liquid; 33 ± ℃ and oscillating condition under, the 1000ml fermentation flask was cultivated 18 hours, measure the OD600nm value, obtain fermenting agent.
Embodiment 3:
Spawn culture: get above-mentioned D
14 of tubes and S
41 of tube, access fills in the 500ml seed bottle of substratum respectively, and it consists of described substratum: glucose 0.85%, peptone 0.7%, yeast extract paste 0.4%, sodium-chlor 0.3%, KH
2PO
40.2%, all the other are water.PH is transferred to 7.5.
Under 35 scholars ℃ and oscillating condition, 500ml seed flask culture 16 hours, with D
1Bacterium liquid and S
4Bacterium liquid according to 5% volume ratio kind amount, inserts the automatic fermentor tank of 100L again and carries out enlarged culturing after mixing, control fermentor tank pressure 0.5mpa, and 2 ℃ of jar temperature, air flow 0.4 (v/v/min), stirring velocity 200rpm, incubation time 18 hours obtains zymocyte liquid.
Above-mentioned fermenting culture is concentrated 4 times through membrane filtration, detect liquid microbial inoculum.
Embodiment 4:
Spawn culture: get above-mentioned D
13 of tubes and S
42 of tubes, access fills in the 500ml seed bottle of substratum respectively, and it consists of described substratum: glucose 0.7%, peptone 0.8%, yeast extract paste 0.4%, sodium-chlor 0.4%, KH
2PO
40.1%, all the other are water.PH is transferred to 7.2.Under 1 ℃ of 32 scholar and oscillating condition, 500ml seed flask culture 18 hours.
Bacterial classification expands numerous cultivation: add substratum MD in fermentor tank, back 8% volume ratio according to fermentation tank culture medium of sterilization inserts makes seed after above-mentioned culture mixes, expand numerous culture condition: tank pressure is 0.5Mpa, the jar temperature is 28 ℃, air flow quantity is 0.4 (v/v/min), agitator speed is 200rpm, expands numerous cultivation 18 hours, stops fermentation when the OD of nutrient solution value is OD600nm decline 0.02nm;
Product is cultivated, in large fermentation tank, add substratum, the culture that numerous cultivation is expanded according to the 6% volume ratio access of fermentation tank culture medium in the sterilization back is made liquid seeds, the product culture condition: tank pressure is 0.4Mpa, the jar temperature is 30 ℃, air flow quantity is 0.6 (v/v/min), and agitator speed is 170rpm, and product was cultivated 20 hours; When being OD600nm decline 0.02nm, the OD of nutrient solution value stops fermentation;
Above-mentioned fermenting culture after membrane filtration concentrates 8 times, is added dispersion agent, further dry solid-state microbial inoculum product.
Claims (4)
1. biological organic fertilizer fermented bacterium D1 tyrothricin, its deposit number is: CCTCC No.M 2010250.
2. biological organic fertilizer fermented bacterium S4 bacillus amyloliquefaciens, its deposit number is: CCTCC No.M2010249.
3. the preparation method of a biological organic fertilizer fermented bacterium is characterized in that preparation process is:
1), seed culture, as bacterial classification, in the seed triangular flask, carry out seed culture with claim 1, claim 2 described bacterial classification D1 bacterium and S4 bacterium, in the seed triangular flask, add substratum MD, its composition and ratio such as following table:
The seed culture condition: insert the female kind in inclined-plane behind the medium sterilization, under 28～30 ℃, shaking culture 16～22 hours;
2), expand numerous cultivation, in fermentor tank, add substratum MD, back 3～8% volume ratios according to fermentation tank culture medium MD of sterilization insert makes seed after above-mentioned culture mixes, expand numerous culture condition: tank pressure is 0.4～0.6Mpa, the jar temperature is 28～30 ℃, and air flow quantity is 0.3～0.6 (v/v/min), and agitator speed is 200rpm, expand numerous cultivation 18～24 hours, when the OD of nutrient solution value stops fermentation 0.02 the time for OD600nm descends;
3), product is cultivated, in large fermentation tank, add substratum MD, the culture that numerous cultivation is expanded according to 3～8% volume ratios access of fermentation tank culture medium in the sterilization back is made liquid seeds, the product culture condition: tank pressure is 0.4～0.6Mpa, the jar temperature is 28～30 ℃, air flow quantity is 0.3～0.6 (v/v/min), and agitator speed is 140～200rpm, and product was cultivated 18～24 hours; When the OD of nutrient solution value stops fermentation 0.02 the time for OD600mn descends;
4), above-mentioned fermenting culture is concentrated 1～8 times through membrane filtration, detect liquid microbial inoculum; Or with above-mentioned fermenting culture after membrane filtration concentrates 1～8 times, add dispersion agent, further dry solid-state microbial inoculum product.
4. a kind of biological organic fertilizer fermented bacterium according to claim 1 or 2 can use separately, perhaps unites use, and the ratio of uniting use is; D
1Bacterium: S
4Bacterium=1～4: 1.
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|---|---|---|---|
| CN 201010538124 CN102031233B (en) | 2010-10-27 | 2010-10-27 | Method for preparing fermentation strains of microbial organic fertilizer |
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|---|---|---|---|
| CN201210125426.0A Division CN102943051B (en) | 2010-10-27 | 2010-10-27 | Preparation method of bacillus amyloliquefaciens S4 for fermentation of tobacco straw bio-organic fertilizer |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642718A (en) * | 2013-11-22 | 2014-03-19 | 贵州省烟草科学研究院 | Tobacco residue solid organic waste decomposing inoculant and preparation method thereof |
| CN113213996A (en) * | 2021-03-29 | 2021-08-06 | 湖北省烟草科学研究院 | Root promoting agent for promoting growth of cigar root system and preparation and use methods thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05252928A (en) * | 1992-03-11 | 1993-10-05 | Japan Tobacco Inc | Method for preventing generation of mold on leaf tobacco |
| CN1377958A (en) * | 2002-04-30 | 2002-11-06 | 高明金葵子植物营养有限公司 | Microbial fermentation agent for treating waste alkohol liquid and its preparing method |
| CN1952116A (en) * | 2006-04-18 | 2007-04-25 | 兰州大学 | Bacillusamyloliquefaciens strain and application thereof |
-
2010
- 2010-10-27 CN CN 201010538124 patent/CN102031233B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05252928A (en) * | 1992-03-11 | 1993-10-05 | Japan Tobacco Inc | Method for preventing generation of mold on leaf tobacco |
| CN1377958A (en) * | 2002-04-30 | 2002-11-06 | 高明金葵子植物营养有限公司 | Microbial fermentation agent for treating waste alkohol liquid and its preparing method |
| CN1952116A (en) * | 2006-04-18 | 2007-04-25 | 兰州大学 | Bacillusamyloliquefaciens strain and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 《环境科学》 20010930 席北斗等 高效复合微生物菌群在垃圾堆肥中的应用 全文 1-4 第22卷, 第5期 2 * |
| 《食品工业科技》 20041231 张志玲等 利用废次烟草生产生物有机肥的工艺研究 全文 1-4 , 2 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642718A (en) * | 2013-11-22 | 2014-03-19 | 贵州省烟草科学研究院 | Tobacco residue solid organic waste decomposing inoculant and preparation method thereof |
| CN103642718B (en) * | 2013-11-22 | 2016-09-14 | 贵州省烟草科学研究院 | A kind of cigarette foam solid organic castoff decomposing microbial inoculum and preparation method thereof |
| CN113213996A (en) * | 2021-03-29 | 2021-08-06 | 湖北省烟草科学研究院 | Root promoting agent for promoting growth of cigar root system and preparation and use methods thereof |
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