CN106591168B - A kind of nicosulfuron degradation Rhodococcus ruber YMHL-1 and its application - Google Patents
A kind of nicosulfuron degradation Rhodococcus ruber YMHL-1 and its application Download PDFInfo
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Abstract
The present invention provides the remaining degradation bacteria strains YMHL-1 of nicosulfuron in a kind of elimination waste water, is identified as Rhodococcus ruber category (Rhodococcus).China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC NO.10542 were preserved on 2 9th, 2015.Main Biological is Gram's staining reaction negative, and bacterium colony is in orange colour, round opaque, rough surface, drying；Cellular morphology is spherical or rod-short, and atrichia does not move, and does not produce gemma；It is aerobic, chemoheterotrophy.Cannot liquefy gelatin, cannot hydrolyze starch, contact enzyme positive, V.P. reacting positive, clark and Lubsreaction is negative, and cannot degrade casein, not restore nitrate；Acid is produced from glucose.Glucose, fructose, citrate can be utilized, mannose, arabinose, raffinose cannot be utilized.Degradation bacteria product, which is directly applied, can be such that antibiotic residual quantity in water reduces on 90%.
Description
Technical field
The invention belongs to biological high-tech field, it is the method degradation chemical pesticide using microorganism, is suitable for Modern Agriculture
The production and processing of industry production Green non-polluted farm product, and in particular to a kind of nicosulfuron degradation Rhodococcus ruber YMHL-1 and
It is applied.
Background technique
With the improvement of people ' s living standards with the enhancing of environmental protection consciousness, the whole society is direct to pesticide residue bring
Harm and potential impact are also more and more paid close attention to, and there is an urgent need to the clean green agricultural products that non agricultural chemical residuum pollutes by people.But from
At present from the point of view of the agricultural form and demographic situation in China, farm output is reduced as the nuisanceless of cost to stop Pesticide use
Production model be it is unworkable, pesticide as disease and pest control effective means will also continue play in agricultural production it is huge
Effect.How can solve this double-barreled question in agricultural production just becomes the research hotspot of every subjects.Laboratory and reality
Application study all shows pesticide residue " library " capacity eliminated in soil of degrading using microorganism, thus reduce grain, vegetables,
Pesticide residue in the agricultural product such as tealeaves, the quality and economic value for improving agricultural product are feasible.
A large amount of uses of sulfonylurea herbicide bring many hazards to ecological environment and human health, how to remove its
Residual in environment has become the hot issue of numerous scientist's concerns.Utilize degradation bacteria and its degrading enzyme degrading pesticide of generation
Have the characteristics that it is efficient, without secondary pollution, be repair pollution by pesticides environment and remove Pesticide Residues important means it
One.
Nicosulfuron is one of most important kind, also known as rimsulfuron 25 in sulfonylurea herbicide, chemical name 2- (4,6-
Dimethoxypyridin -2- base carbamoylamino sulphonyl) N, N- dimethyl nicotinamide, structural formula is as follows:
Researched and developed by Japanese Ishihara Sangyo Kaisha, Ltd. the eighties in last century, is had to gramineae weed in corn field
Specific herbicide kind, China started to introduce and promote nicosulfuron in the initial stage nineties, and trade name Yu Nongle is 4%
The oil-suspending agent of nicosulfuron.Introduction initial stage, nicosulfuron are mainly applied to the big three provinces in the northeast of China of maize sown area, Liaoning,
Heilungkiang, Jilin some areas.Due to the advantages that nicosulfuron is high, herbicidal effect is good, low amount of application to corn safety
It is very popular.However, drug cost is big since price is higher, sales volume had once once been restricted.Until the end of the nineties, Yu Nong
It is happy to reach certain scale using area, and gradually northeastward other than other regional provinces extensions.
Although nicosulfuron has high control efficiency and preferable selectivity, however, meeting during using in farmland
Some is remained in the natural environments such as soil, atmosphere and water, these remain on natural environment and the intracorporal sulfonylureas of crop
Class herbicide can be eventually transferred to the mankind by drinking water, atmosphere and fruit etc., cause certain negative effect.
Summary of the invention
It is an object of the invention to be degraded using the method for microorganism for the practical problem and demand in production practices
It is phonetic to play reparation cigarette to reduce nicosulfuron genetoxic caused by environment for nicosulfuron residual in soil and water body
It sulphur grand contaminated soil, the effect preserved the ecological environment.The microbial inoculum preparation process of the bacterial strain is simple, at low cost, high-efficient, without secondary
Pollution, has a good application prospect.
The present invention provides a kind of elimination nicosulfuron remaining degradation bacteria, and bacterial strain YMHL-1 is Gram's staining reaction yin
Property bacterium, be preserved on 2 9th, 2015 China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as
CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), it protects
Number CGMCC NO.10542 is hidden, identified is Rhodococcus ruber (Rhodococcus ruber).Biological characteristics are gram dye
Color is negative, and bacterium colony is in orange colour, round opaque, rough surface, drying；Cellular morphology be spherical or rod-short, atrichia, no
Movement, does not produce gemma；It is aerobic, chemoheterotrophy.Cannot liquefy gelatin, cannot hydrolyze starch, contact enzyme positive, V.P. reaction sun
Property, clark and Lubsreaction is negative, and cannot degrade casein, not restore nitrate；Acid is produced from glucose.Glucose, fruit can be utilized
Sugar, citrate, cannot utilize mannose, arabinose, raffinose.The bacterium can be carried out with the general Zymolysis Equipment of fermentation industry
Production.
The present invention provides application of the Rhodococcus ruber YMHL-1 in nicosulfuron degradation.
The present invention provides the microbial inoculums for containing the Rhodococcus ruber YMHL-1.
The present invention also provides the methods of Rhodococcus ruber YMHL-1 production microbial inoculum.
The method of the Rhodococcus ruber YMHL-1 production microbial inoculum, comprising: inclined-plane kind-shaking flask induction-seeding tank induction hair
Ferment-production tank-product, the packaging dosage form of the product are liquid bacterial agent or solid absorption microbial inoculum.
The method of the production microbial inoculum, specifically:
1) test tube species of Rhodococcus ruber YMHL-1 are inoculated in the LB culture medium containing 100mg/L nicosulfuron, are vibrated
It cultivates to logarithmic phase；
2) the cultured strain of step 1) is inoculated with by 5% inoculum concentration into 500 liters of seeding tanks, culture to logarithmic growth
Phase, i.e. seed liquor；
3) the step 2) seed liquor is subjected to fermented and cultured, the training used of production tank by 10% inoculum concentration access production tank
It supports base and seed tank culture base phase is same, culture solution after fermentation is microbial inoculum.
LB culture medium prescription described in step 1) are as follows: nicosulfuron 0.1g/L, NaCl 10.00g/L, peptone
10.00g/L, yeast powder 5.00g/L, pH7.0.
Culture medium prescription used in seeding tank described in step 2) are as follows: nicosulfuron 0.1%, glucose 0.5%, (NH4)2SO41%, K2HPO40.2%, MgSO40.05%, NaCl 0.01%, CaCO30.3%, yeast extract 0.02%, pH value
7.2-7.5。
In the incubation for producing tank described in the step 2) seeding tank and step 3), the ventilatory capacity of filtrated air is 1:
0.6-1.2, mixing speed be 220 revs/min, cultivation temperature be 37 DEG C, step 2 and 3 entire process flow incubation time be 48-
60 hours.
Application of the microbial inoculum in nicosulfuron degradation.
Rhodococcus ruber YMHL-1 of the present invention can make the residual quantity of nicosulfuron in water body reduce by 90% or more, reduce
Nicosulfuron is enriched in the environment and influences the risk of environment Tiny ecosystem.Prepared microbial inoculum has production and application at low cost, makes
With conveniently, the good advantage of removal effect is suitble to promote the use of a large area in nicosulfuron contaminated soil and water body.The present invention
For preserving the ecological environment, the health of the mankind is protected, quality of water environment is improved and has great importance safely.
Detailed description of the invention
The violet staining photo of Fig. 1 Rhodococcus ruber YMHL-1 of the present invention；
Influence of Fig. 2 inoculum concentration to Rhodococcus ruber YMHL-1 degradation effect of the present invention；
Influence of Fig. 3 pH to Rhodococcus ruber YMHL-1 degradation effect of the present invention.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Without departing substantially from spirit of that invention
In the case where essence, to modifications or substitutions made by the method for the present invention, step or condition, all belong to the scope of the present invention.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
The screening and separation of 1 nicosulfuron of embodiment degradation Rhodococcus ruber YMHL-1
Activated sludge 3.0ml obtained in the purification tank for liquid waste from certain insecticide factory's workshop of production nicosulfuron is taken to add
Enter in the 100ml LB culture medium that nicosulfuron concentration is 100mg/L, formula are as follows: every liter of 10.00g/L containing NaCl, peptone
10.00g/L, yeast powder 5.00g/L, pH 7.0, the shaken cultivation under the conditions of 220r/min, 37 DEG C connect every 3 days by 3%
Kind amount is transferred in fresh minimal medium, continuous switching 5 times.
Enrichment bacterium solution 1.0ml obtained above is taken, is added in 9.0ml sterile water, is made into 10-1Pregnant solution, then draw
1.0ml prepare 10-1Pregnant solution be added 9.0ml sterile water in, mix well and be made into 10-2Pregnant solution, and so on, it is right
Pregnant solution carries out gradient dilution.And so on, gradient dilution is carried out to pregnant solution.Draw the dilution 0.1ml coating of each gradient
It is that 100mg/L inorganic salts solid medium (is formulated are as follows: every liter of 1.50g K in the concentration containing nicosulfuron2HPO4、0.50g
KH2PO4、0.20g MgSO4·7H2O、1.00g NaCl、1.00g(NH4)2SO4, 12.5g agar, pH 7.0) on, 37 DEG C of cultures
5 days.From picking single colonie on above inorganic salts solid medium after 5 days, it is small that 24 are cultivated in the LB liquid medium of 5.0ml
Shi Hou, LB liquid medium formula are as follows: NaCl 10.00g/L, peptone 10.00g/L, yeast powder 5.00g/L, pH7.0 in
It is centrifuged 2min under conditions of 12000r/min, removes supernatant, the sterile water that 3.0ml is added shakes up, still in 12000r/min's
Under the conditions of be centrifuged 2min, according to said method with it is sterile washing twice after, be added 3.0ml sterile water the bacterium is resuspended.Draw the 1.0ml bacterium
Liquid is added in nicosulfuron-inorganic salt liquid culture medium that 100ml nicosulfuron concentration is 100mg/L, in 220r/min, 37 DEG C
Under the conditions of after shaken cultivation 72h, survey its degradation effect with high performance liquid chromatography.The higher one plant of bacterial strain of degradation efficiency is saved,
Carry out subsequent experimental.
Degradation bacteria strains are inoculated on LB solid medium, are formulated are as follows: nicosulfuron 0.1g/L, NaCl10.00g/L,
Peptone 10.00g/L, yeast powder 5.00g/L observe colonial morphology after 12.5g/L, pH7.0,30 DEG C of constant temperature incubation 48h of agar,
Bacterium colony is in orange colour, round opaque, rough surface, drying；Thalli morphology (1000X) is observed with optical microscopy, such as Fig. 1 institute
Show, cellular morphology is spherical or rod-short, and atrichia does not move, and does not produce gemma.The Physiology and biochemistry of bacterial strain is identified according to " common
Bacterial system identification handbook " it carries out.It is accredited as Rhodococcus ruber category (Rhodococcus)；Name are as follows: YMHL-1.
The shaking flask degradation experiment of 2 Rhodococcus ruber YMHL-1 of embodiment
Influence of 2.1 inoculum concentrations to YMHL-1 degradation efficiency
In the minimal medium of 100mg/L nicosulfuron, formula are as follows: every liter of K containing 0.50g2HPO4、0.50g
KH2PO4、0.20g MgSO4·7H2O、1.00g NaCl、1.00g(NH4)2SO4, pH 7.0, by 1%, 2%, 3%, 4%,
5%, inoculum concentration is inoculated with YMHL-1 in culture solution, in 37 DEG C of shaken cultivations, is measured by sampling within 72 hours.It as a result can be with by Fig. 2
To find out, when being inoculated with by 1%, degradation rate 91%, with the raising of inoculum concentration, nicosulfuron degradation rate has a small increase,
In view of thalli growth and economic reason, requirement can reach using 1% inoculum concentration.
Influence of 2.2 pH to YMHL-1 degradation efficiency
PH value is to influence another important environmental factor of microorganism growth and existence, microorganism growth have one it is suitable
PH value range, more than the pH value range of suitable growth, most of microbe cannot all be grown well.
In the minimal medium of the nicosulfuron containing 100mg/L, formula are as follows: every liter contains 100mg/L, 1.50g
K2HPO4、0.50g KH2PO4、0.20g MgSO4·7H2O、1.00g NaCl、1.00g(NH4)2SO4, pH 7.0 connects by 1%
In culture solution, pH value is adjusted to 5.0,6.0,7.0,8.0,9.0,10.0 respectively by kind of amount inoculation YMHL-1, in 30 DEG C of shaken cultivations,
It is measured by sampling within 72 hours.As a result as seen from Figure 3, the too high or too low degradation that will affect YMHL-1 to nicosulfuron of pH, model
Enclosing has preferable degradation effect in 6-9, can satisfy the demand of most of soil environment, and optimal pH range is 7.
Preparation of the embodiment 3 containing Rhodococcus ruber YMHL-1 microbial inoculum
Embodiment 1 is screened and the original seed of isolated nicosulfuron degradation Rhodococcus ruber YMHL-1 activates on culture dish,
And degradation property is measured, it is inoculated in spare on test tube slant.Test tube species are inoculated in the LB culture medium containing 200ml containing nicosulfuron
In 1000ml shaking flask, LB culture medium prescription are as follows: nicosulfuron 100mg/L, yeast powder 5.00g/L, peptone 10.00g/L, NaCl
10.00g/L, pH 7.0, constant-temperature shaking culture to logarithmic phase prepare inoculation seeding tank.500 liters of seeding tank, 400 liters of inventory,
Culture medium prescription are as follows: nicosulfuron 0.1%, glucose 0.5%, (NH4)2SO41%, K2HPO40.2%, MgSO40.05%,
NaCl 0.01%, CaCO30.3%, yeast extract 0.02%, pH value 7.2-7.5.121 DEG C of high pressure moist heat sterilizations after feeding intake,
After being cooled to 33 DEG C, above-mentioned cultured shaking flask strain is inoculated with by 5% inoculum concentration into 500 liters of seeding tanks, culture to logarithm
Growth period, mixing speed are 220 revs/min, and filtrated air intake is 1:0.8.Produce tank used medium ingredient and seeding tank
Culture medium is identical, and 5 tons of capacity, 4.5 tons of inventory.Production tank 1.1kg/cm after feeding intake2Pressure under, 121 DEG C of high pressures are damp and hot
Sterilizing is cooled to 35 DEG C hereinafter, logical filtrated air keeps germ-free condition spare after sterilizing.The seed liquor for reaching logarithmic phase is pressed
10% inoculum concentration access production tank culture, the production tank temperature degree after inoculation are controlled at 35 DEG C, produce nothing in the incubation of tank
The ventilatory capacity of bacterium air is 1:1.2, and mixing speed is 240 revs/min, and entire process flow incubation time is 60 hours.Fermentation knot
Thalline quantity reaches 1,000,000,000/ml or more after beam.Culture solution goes out tank and directly uses plastic barrel or Packaging Bottle point after fermentation
It dresses up liquid dosage form or solid fungicide dosage form is distributed into packaging bag using adsorption by peat.
Claims (9)
- Rhodococcus ruber (Rhodococcus ruber) YMHL-1, deposit number CGMCC 1. a kind of nicosulfuron is degraded NO.10542。
- 2. application of the Rhodococcus ruber YMHL-1 described in claim 1 in nicosulfuron degradation.
- 3. the microbial inoculum containing Rhodococcus ruber YMHL-1 described in claim 1.
- 4. a kind of production method of microbial inoculum described in claim 3 characterized by comprising inclined-plane kind-shaking flask induction-seed Tank induction fermentation-production tank-product, the packaging dosage form of the product are liquid bacterial agent or solid absorption microbial inoculum.
- 5. production method according to claim 4, which is characterized in that specifically comprise the following steps:1) test tube species of Rhodococcus ruber YMHL-1 are inoculated in the LB culture medium containing 100mg/L nicosulfuron, shaken cultivation To logarithmic phase；2) into 500 liters of seeding tanks, culture to logarithmic growth phase, i.e., the cultured strain of step 1) is inoculated with by 5% inoculum concentration Seed liquor；3) the step 2) seed liquor is subjected to fermented and cultured by 10% inoculum concentration access production tank, produces tank used medium Same with seed tank culture base phase, culture solution after fermentation is microbial inoculum.
- 6. according to the method described in claim 5, it is characterized in that, LB culture medium prescription described in step 1) are as follows: the phonetic sulphur of cigarette Grand 0.1g/L, NaCl 10.00g/L, peptone 10.00g/L, yeast powder 5.00g/L, pH7.0.
- 7. according to the method described in claim 5, it is characterized in that, culture medium prescription used in seeding tank described in step 2) Are as follows: nicosulfuron 0.1%, glucose 0.5%, (NH4)2SO41%, K2HPO40.2%, MgSO40.05%, NaCl 0.01%, CaCO30.3%, yeast extract 0.02%, pH value 7.2-7.5.
- 8. according to the method described in claim 5, it is characterized in that, production tank described in the step 2) seeding tank and step 3) Incubation in, the ventilatory capacity of filtrated air is 1:0.6-1.2, and mixing speed is 220 revs/min, and cultivation temperature is 37 DEG C, step Rapid 2 and 3 entire process flow incubation time is 48-60 hours.
- 9. application of the microbial inoculum of the described in any item method preparations of claim 5-8 in nicosulfuron degradation.
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| CN112218647B (en) | 2019-04-24 | 2023-07-04 | 辽宁天安生物制药股份有限公司 | Use of rhodococcus ruber products in the treatment of thermal injury |
| US20230069441A1 (en) | 2020-01-21 | 2023-03-02 | Liaoning Greatest Bio-Pharmaceutical Co., Ltd. | Use of rhodococcus ruber cell wall skeleton in regenerative medicine |
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| CN104480043A (en) * | 2014-12-12 | 2015-04-01 | 中国农业科学院农业资源与农业区划研究所 | Rhodococcus sp. capable of degrading four herbicides and application thereof |
| CN105274036A (en) * | 2015-12-01 | 2016-01-27 | 东北农业大学 | Complex microbial inoculant capable of efficiently degrading chlorimuron-ethyl and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104480043A (en) * | 2014-12-12 | 2015-04-01 | 中国农业科学院农业资源与农业区划研究所 | Rhodococcus sp. capable of degrading four herbicides and application thereof |
| CN105274036A (en) * | 2015-12-01 | 2016-01-27 | 东北农业大学 | Complex microbial inoculant capable of efficiently degrading chlorimuron-ethyl and application thereof |
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