CN102676439A - Serratia strain and method for disposing methidathion pesticide residues on surfaces of soil and fruits/vegetables - Google Patents
Serratia strain and method for disposing methidathion pesticide residues on surfaces of soil and fruits/vegetables Download PDFInfo
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Abstract
The invention provides a new microorganism strain capable of degrading methidathion pesticide residues, namely (Serratia sp.) SPL-2 with the Genbank accession number of JN400353, and a methidathion pesticide residue degrading bactericide developed using the degrading strain. The degrading bactericide degrades chemical pesticides by use of the microorganism technology, belonging to the technical field of environmental microbial remediation; the production technology is as follows: seedling on an inclined plane->seed liquid in a shake flask->a seed tank->a fermentation tank->a product; the preparation technology is simple, has low production cost and is easy to popularize and use in a large area; when in use, the degrading bactericide is directly sprayed, thereby being very convenient; the bactericide product can quickly and effectively degrade the residual bactericides on the surfaces of soil and fruits/vegetables so as to solve the problem of exceeding residues caused by pesticide spraying in agricultural production; and the green agricultural product without toxicity or pollution can be produced, and the quality and economic value of the agricultural product are effectively improved.
Description
Technical field
The invention belongs to environmental microorganism recovery technique field, be specifically related to the method for Serratia strain SPL-2 and processing soil and fruit and vegetable surfaces methidathion pesticide residue.
Background technology
Agricultural chemicals is that important guaranteeing role has been played in the raising the output of grain, also therefore in the agriculture prodn of countries in the world, is widely used.But the undue use of agricultural chemicals has produced serious negative impact.Pesticide residue are difficult to degraded, and people also make pesticide residue such as grain, vegetables, melon and fruit exceed standard when using agricultural chemicals to prevent disease pest and weed, and are seriously polluted, bring injury for simultaneously non-target biology.In China, influence vegetables, fruit etc. and eat raw in the many factors of agricultural product quality and safety, the most outstanding also just pesticide residue exceed standard, particularly limit with the residual problem that exceeds standard of riskiest pesticide.The whole nation increases progressively because of having eaten also generation repeatedly of drug case in the remains of pesticide fruits and vegetables by mistake day by day in recent years;
Research shows; Remains of pesticide is accumulated delay for a long time and can be caused chronic poisoning in the fruits and vegetables in human body, and is more more fearful than acute poisoning, and it mainly is to concentrate through biology; The approach of residual two aspects of fruits and vegetables brings threat to HUMAN HEALTH; So that bring out many chronic diseases, like cardiovascular and cerebrovascular disease, mellitus, hepatopathy, cancer etc.More let the people be difficult to expect and take precautions against to be agricultural chemicals accumulate in that the people is intravital, also can pass to the next generation through conceived and lactation, bring disaster to descendants's health;
The irrational a large amount of uses of agricultural chemicals have caused more and more serious adverse consequences for the mankind and ecotope, and the pollution problem of agricultural chemicals has become the focus that the whole world is paid close attention to.Therefore, the pollution problem of agricultural chemicals to environment and food studied, solved in the biological degradation of strengthening agricultural chemicals, is one of human current problem that presses for solution.Pesticide residue are distributed widely in soil, and water body in atmosphere and the agricultural-food, is difficult to utilize large-scale engineering measure decontamination, and the basic skills that the agricultural chemicals new technology is the solution pesticide residue is removed with exploitation in control pesticidal contamination source.Occurring in nature, mikrobe can be degraded to pesticidal contamination, and this is to rely on elements, while not to produce the most desirable approach of secondary pollution.Therefore utilize microbial technique to handle pesticide residue, and contaminated fruits and vegetables and water body are carried out biological prosthetic is a kind of efficient ways;
Methidathion is a kind of organic phosphorous insecticide of wide spectrum, have tag, stomach toxicity and osmosis, can infiltrate in the plant tissue, chewing type and sucking pest are all had killing effect, use very extensively.But also residual serious because of it, and get into food chain, harm humans and other animal healths, and receive extensive concern.Methidathion belongs to height toxicity sterilant, can suppress the human body cholinesterase activity, accumulate after neural system, cause feeling sick, dizzy even clouded in mind, high density exposes can cause respiratory paralysis and death.Therefore, seek the focus that a kind of method of removing methidathion pesticide residue highly effective and safe in the environment becomes concern, and the improvement that utilizes the methidathion high efficiency degradation bacterial agent to be applied to its contaminate environment has important practical significance and application prospect.
Summary of the invention
The objective of the invention is practical problems and needs, a kind of methidathion sterilant degradation bacteria is provided to pesticide residual contamination in the present agriculture prodn.Another object of the present invention has provided uses the methidathion degradation bacteria to prepare the method for water-in-oil microbial inoculum; Thereby slowly keep the supply of bacterial classification nutritive substance through slow releasing function; Prolong the quality guaranteed period of product greatly, use this microbial inoculum product that field methidathion residual quantity is reduced more than 85%, and low production cost; Easy to use, have vast market prospect.
Technical scheme
The present invention provides a kind of methidathion degradation bacteria, and this bacterial strain is the serratia (Serratia sp.) of Gram-negative, and main biological characteristics is G-; Facultative anaerobic bacteria; Size is 0.7-0.9 μ m * 1.0-1.2 μ m, is shaft-like, no gemma; Peritrichous is dynamic, and single or short chain shape is arranged.Bacterium colony after cultivating 18h on the LB solid medium takes on a red color, and convex is opaque, moistening, irregular; Can utilize glucose, semi-lactosi, sucrose, SANMALT-S, sorbyl alcohol; Can reduce nitrate salt; Utilize Citrate trianion can make gelatin hydrolysis; Methyl red, oxydase, phenylalanine(Phe) reaction experiment are negative; VP reaction, catalase reaction show positive; Three pool iron agar tests do not produce hydrogen sulfide.The Genbank accession number of this bacterial strain 16SrDNA is JN400353.The righttest growth pH of this bacterium is 7.2-8.0, and optimum growth temperature is 37 ℃.This degradation bacteria can be produced through the general fermentation equipment of fermentation industry, and with low cost, microbial inoculum directly sprays easy to use, is easy to promote large-scale application;
The technical process of using above-mentioned methidathion degradation bacteria to produce the microbial inoculum product is: inclined-plane kind → medicine bottle seed liquor → seeding tank → fermentor tank → microbial inoculum product;
Detailed implementation step of the present invention is:
(1) single colony inoculation of picking methidathion residue degrading bacterium Serratia sp. SPL-2 is in the test tube slant;
(2) the inclined-plane kind with methidathion residue degrading bacterium SPL-2 is inoculated in the LB culture media shaking vase, and shaking culture is to logarithmic phase;
(3) change above-mentioned cultured bacterium liquid over to seeding tank by 15% volume inoculum size, continue to be cultured to logarithmic phase;
The seed liquor that (4) will reach logarithmic phase is produced cultivation in the jar by 15% inoculum size access;
(5) fermentation is processed the water-in-oil formulation with inoculum after accomplishing, and is distributed into the liquid dosage form product with plastic barrel or packing bottle;
The culture medium prescription of LB described in the step 2 is: yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L, pH:7.0-7.2;
The substratum of seeding tank and fermentor tank is identical in the step 3 and 4, fills a prescription to be: glucose 5g/L, NH
4NO
310g/L, K
2HPO
42 g/L, MgSO
47H
2O 0.5 g/L, Na
2HPO
42 g/L, CaCO
33g/L, NaCl 1.0 g/L, yeast extract paste 0.5 g/L, pH:7.0-7.2;
The air flow of sterile air is 1:0.8-1.5 in the culturing process of seeding tank and fermentor tank, and stirring velocity is 150-220 rev/min, and culture temperature is 28-35 ℃, and omnidistance incubation time is 48-72 hour, finishes 6,000,000,000/ml of back thalline;
The oil phase that adds in the water-in-oil formulation that step 5 is developed is No. 10 white oils, and emulsifying agent is span80, and solubility promoter is a tween 80.Fermented liquid is processed the nutritive substance that can make behind the water-in-oil wherein to be wrapped in around the degradation bacteria suspending stabilizedly; Provide the nutrition substrate to keep the activity of bacterial classification through slowly-releasing; Prevent the deposition of degradation bacteria; And oil phase can provide suitable moist environment to degradation bacteria, thereby prolongs the quality guaranteed period of microbial inoculum product greatly.Directly be sprayed onto fruit and vegetable surfaces during use, thereby, improve the residual degradation rate of fruit and vegetable surfaces farming, also can prevent the generation of phenomenons such as fruits and vegetables dehydration, brown stain simultaneously because water in oil water retention characteristic can make nutraceutical slow release keep the activity of bacterium.
Beneficial effect of the present invention
1 the present invention provides a kind of degradation bacteria of organophosphorus pesticide methidathion, and field soil and fruits and vegetables are repaired experimental result and shown, the degradation rate of methidathion is reached more than 85%;
2 use these methods to produce the methidathion degradation bacterial agent to have a production cost low, easy to use, the advantage of good degrading effect is adapted at grain vegetables fruit production export base and pollution-free food production base and uses on a large scale.This invention is for preserving the ecological environment, and the protection people health increases additional value of farm products and has great significance.
Embodiment
Embodiment 1
Gather field, the peach garden topsoil that farming Quan Yi district, Chengdu uses the methidathion sterilant for successive years.Take by weighing the pesticidal contamination soil sample 5g of collection, be added to 100ml excessively behind 20 mesh sieves and contain in the sterilization basis salt culture medium of 50mg/l methidathion, the prescription of basic salt culture medium is (being g/L): NH
4NO
31.0, MgSO
47H
2O 0.5, (NH
4)
2SO
40.5, KH
2PO
40.5 NaCl 0.5, K
2HPO
41.5, PH:7.0-7.2, in 37 ℃, the 180r/min shaking culture.Get 5ml bacterium liquid behind the 7d and insert 37 ℃ of cultivations down in the same substratum, the concentration of methidathion is progressively brought up to 400mg/l in the process that goes down to posterity, and goes down to posterity 6 times, and approximately 7d goes down to posterity 1 time.The pesticide degradation bacteria bacterium liquid of enrichment is made 10 times of gradients (10 with sterilized water
-7, 10
-8, 10
-9) dilution, get the 0.1mL bacteria suspension respectively and be coated on the inorganic salt solid medium that contains 50 mg/L methidathions 37 ℃ of cultivations; When bacterium colony appears in flat board, with single bacterium colony on inorganic salt agricultural chemicals flat board, rule separation, purifying bacterial strain.Single bacterium colony inclined-plane that growth is fast, bacterium colony is regular, go down to posterity stable is preserved.With the 4 strain bacterium activation that primary dcreening operation obtains, insert in the salt culture medium of 100mg/l methidathion basis, 37 ℃, 180r/min cultivates 10d, surveys its degradation rate.Through multiple sieve, obtain the highest bacterial strain SPL-2 of degradation rate.Through the 16SrDNA sequential analysis, find that the sequence similarity of bacterial strain SPL-2 and Serratia sp. FS014 is 99%, with the sequence similarity of Serratia sp. HA1 also be 99%, GenBank accession number JN400353.Therefore SPL-2 should belong to serratia, and main biological characteristics is G-, facultative anaerobic bacteria, and size is 0.7-0.9 μ m * 1.0-1.2 μ m, is shaft-like, no gemma, peritrichous is dynamic, and single or short chain shape is arranged.Bacterium colony after cultivating 18h on the LB solid medium takes on a red color, and convex is opaque, moistening, irregular; Can utilize glucose, semi-lactosi, sucrose, SANMALT-S, sorbyl alcohol; Can reduce nitrate salt; Utilize Citrate trianion can make gelatin hydrolysis; Methyl red, oxydase, phenylalanine(Phe) reaction experiment are negative; VP reaction, catalase reaction show positive; Three pool iron agar tests do not produce hydrogen sulfide;
Embodiment 2
The inclined-plane of getting the Serratia of Gram-negative among the embodiment 1 is inoculated in the 100mlLB substratum, and LB substratum (g/l) is filled a prescription as follows: peptone 10, yeast extract 5; NaCl 10; PH 7.0-7.2, constant-temperature shaking culture obtains bacterium liquid to logarithmic phase.Above-mentioned cultured bacterium liquid is changed over to 121 ℃ of high pressure moist heat sterilizations and is cooled to the seeding tank about 35 ℃ by 15% volume inoculum size, and the seed tank culture based formulas is: glucose 5g/L, NH
4NO
310g/L, K
2HPO
42 g/L, MgSO
47H
2O 0.5 g/L, Na
2HPO
42 g/L, CaCO
33g/L, NaCl1.0 g/L, yeast extract paste 0.5 g/L, pH:7.0-7.2; Continue to be cultured to logarithmic phase, the air flow of sterile air is 1:1.5 in the culturing process, and stirring velocity is 200 rev/mins, and culture temperature is 35 ℃.The seed liquor that will reach logarithmic phase is produced a jar cultivation by 15% inoculum size access; The used substratum of fermentor tank is identical with seeding tank; Postvaccinal leavening temperature is controlled at 28-35 ℃, and the air flow of sterile air is 1:0.8-1.5, and stirring velocity is 150-220 rev/min; Omnidistance fermented incubation time is 48-72 hour, and thalline quantity reaches 6,000,000,000/ml after the fermentation ends.Fermentation is processed water-in-oil formulation with inoculum according to certain oil-water ratio after accomplishing under the effect of emulsifying agent, be distributed into liquid dosage form with plastic barrel or packing bottle then;
Application examples 1
The loquat of choosing dragon's fountain production is an experimental subjects, sprays certain density methidathion, treat that the agricultural chemicals drop disappears after, its agricultural chemicals initial content is surveyed in sampling.Experimental group is sprayed degradation bacterial agent SPL-2, and control group does not spray degradation bacterial agent.Through 7 days degraded, its pesticide residue content was surveyed in sampling.The content of methidathion is 6.26 mg/kg on the preceding sample of degraded, and degraded back experimental group content is 85.9% for the 0.88mg/kg degradation rate, and the content of control group methidathion is 4.89 mg/kg, and degradation rate is merely 21.9%, and the experimental group degradation rate is 3.9 times of control group.(seeing table 1);
Table 1. Serratia under laboratory condition to the residual degraded of loquat surface methidathion
| Loquat is handled | Before the degraded (mg/kg) | Degraded back (mg/kg) | Degradation rate (%) | The multiple that improves |
| The methidathion contrast | 6.26 | 4.89 | 21.9 | ? |
| Add methidathion degradation bacterial agent SPL-2 | 6.26 | 0.88 | 85.9 | 3.9 |
Application examples 2
One mu of field choosing dragon's fountain plantation peach is an experimental subjects; Wherein get half mu as contrast; Other half mu is the degradation bacterial agent treatment group, at the week back collection soil sample sample of spraying insecticide, adopts the gas chromatography combined with mass spectrometry method to measure methidathion pesticide residue content (being measured by Sichuan University's life science institute Biological resources and test center of key lab of the ecotope Board of Education); Treatment group is sprayed the water-in-oil degradation bacterial agent by development among the embodiment 2 then, and concentration is 6 * 10
9Cfu/ml, totally 30 L are sprayed in the peach soil equably, collection soil is returned the content that Sichuan University surveys its methidathion behind the degraded 8d, control group with clear water as contrast.Through measuring; The content of methidathion is 7.6mg/kg in the preceding soil of degraded; The content of degradation bacterial agent treatment group degraded back methidathion is 1.1mg/kg; Its degradation rate is 85.5%, and the degradation rate of control group methidathion is 44.7% behind the 8d, can find out that degradation bacterial agent is remarkable to the residual degraded of methidathion in the soil;
The degraded residual in field experiment of table 2. Serratia to methidathion in the soil
| Processing mode | Contrast | Handle |
| Pesticide residue concentration (mg/L) | 4.2 | 1.1 |
| Degradation rate | 44.7% | 85.5% |
Application examples 3
Liquid degradation microbial inoculum among the investigation embodiment 2 is to the residual removal effect of land for growing field crops fruit and vegetable surfaces methidathion farming.Choose peach and French beans as experimental subjects, the fruits and vegetables sample in first acquisition test place detects before the spray medicine, does not find the residual of target agricultural chemicals, with the accuracy of guaranteeing to test.Spray certain density target agricultural chemicals methidathion then, treat that the agricultural chemicals drop slowly disappears on the blade, collected specimens is measured the starting point concentration of agricultural chemicals after one day.The water-in-oil degradation bacteria liquid that the fermented liquid that using this laboratory afterwards provides is processed is sprayed to the sub-district crossed of spray medicine, and control group does not spray degradation bacterial agent, executes the bacterium time to be selected in and to carry out 5 o'clock to 6 o'clock afternoon to avoid the sunlight direct projection to reduce its activity.Behind the spray bacterium, carried out sampling determination pesticide residue concentration on the the 2nd, 4,6,8 day, by the serpentine method sample determination methidathion content of gathering.Before the degraded on peach and the French beans blade residual content of methidathion be respectively 6.63mg/kg and 9.91mg/kg, and in different treatment on peach and the French beans degradation speed of methidathion evident difference is arranged.In not spraying the sample of bacterium, the degradation rate of methidathion on peach and French beans has only 63.9% and 76.8%.And in the sample that has added degradation bacteria; Corresponding methidathion degradation rate is respectively 95.3% and 98.0%; Degradation process meets the first order kinetics model; The transformation period of methidathion on peach and French beans is 4.6 (day) and 3.7 (day) in the contrast, and in test group the corresponding transformation period be respectively 1.8 (day) and 1.3 (day) obviously faster than not connecing the bacterium sample;
Transformation period that table 3. methidathion is degraded on peach and French beans and degradation rate
| Handle | Before the degraded (mg/kg) | Degraded back (mg/kg) | Transformation period | Speed improves doubly speed | Degradation rate | Efficient improves doubly speed |
| Peach | 6.63 | 2.39 | 4.6 | ? | 63.9 | ? |
| Peach+bacterium SPL-2 | 6.63 | 0.31 | 1.8 | 2.56 | 95.3 | 1.49 |
| French beans | 9.91 | 2.29 | 3.7 | ? | 76.8 | ? |
| French beans+bacterium SPL-2 | 9.91 | 0.20 | 1.3 | 2.85 | 98.0 | 1.28 |
Utilizing microbiological deterioration and handle agricultural-food, is frontier, new way that pesticide residue control is handled, and many developed countries and enterprise of major company drop into huge fund to be engaged in the research and development of pesticide residue efficient degrading bacteria.Laboratory and real application research all show and utilize the mikrobe pesticide residue in the fruits and vegetables of degrading, and can effectively improve the quality and the economic worth of agricultural-food.
Claims (4)
1. a methidathion pesticide residual degrading bacteria is characterized in that this mikrobe is the SPL-2 bacterial strain of the serratia (Serratia sp.) of Gram-negative, and main biological characteristics is G-; Facultative anaerobic bacteria; Size is 0.7-0.9 μ m * 1.0-1.2 μ m, is shaft-like, no gemma; Peritrichous is dynamic, and single or short chain shape is arranged; Bacterium colony after cultivating 18h on the LB solid medium takes on a red color, and convex is opaque, moistening, irregular; Can utilize glucose, semi-lactosi, sucrose, SANMALT-S, sorbyl alcohol; Can reduce nitrate salt; Utilize Citrate trianion can make gelatin hydrolysis; Methyl red, oxydase, phenylalanine(Phe) reaction experiment are negative; VP reaction, catalase reaction show positive; Three pool iron agar tests do not produce hydrogen sulfide; The Genbank accession number of this bacterial strain 16SrDNA is JN400353.
2. utilize the described husky Lei Shi degradation bacteria strains of claim 1 to be developed into methidathion residue degrading microbial inoculum, it is characterized in that, concrete preparation technology is following:
(1) single colony inoculation of picking methidathion residue degrading bacterium Serratia sp. SPL-2 is in the test tube slant;
(2) the inclined-plane kind with methidathion residue degrading bacterium SPL-2 is inoculated in the LB culture media shaking vase, and shaking culture is to logarithmic phase;
(3) change above-mentioned cultured bacterium liquid over to seeding tank by 10-15% volume inoculum size, continue to be cultured to logarithmic phase; The used culture medium prescription of seeding tank is: glucose 5g/L, NH
4NO
310g/L, K
2HPO
42 g/L, MgSO
47H
2O 0.5 g/L, Na
2HPO
42 g/L, CaCO
33g/L, NaCl 1.0 g/L, yeast extract paste 0.5 g/L, pH:7.0-7.2;
The seed liquor that (4) will reach logarithmic phase is inoculated in the production jar by the inoculum size of 10-15% and is cultivated; It is the same with seeding tank to produce jar used culture medium prescription;
(5) air flow of sterile air is 1:0.8-1.5 in the culturing process of seeding tank and production jar, and stirring velocity is 150-220 rev/min, and culture temperature is 28-35 ℃, and omnidistance incubation time is 48-72 hour, finishes 6,000,000,000/ml of back thalline;
(6) fermentation is processed the water-in-oil formulation with inoculum after accomplishing; Oil phase preparation: get 6 parts of span-80 and 94 parts of No. 10 white oil mixing autoclavings are subsequent use; Water preparation: the sterilization tween 80 of adding 4% and fully dissolving in the good ferment product of activation; Emulsification preparation: earlier the oil phase impouring is put in the vertical colloid mill; Under stirring at low speed, slowly add water (oil phase and water are in the ratio thorough mixing of 1:3); High-speed stirring 3min-5min then, quick emulsification becomes the water-in-oil shape, is distributed into the water-in-oil liquid dosage form with plastic barrel or packing bottle.
3. the preparation method of the residual high efficiency degradation bacterial agent of methidathion according to claim 2; It is characterized in that: when the said fermented liquid of step (6) is processed the water-in-oil product; Need to add a certain proportion of span80 as emulsifying agent; Its HLB value is 4.3, can make immiscible profit two miscible each other, and forms homodisperse invert emulsion system; Microbiobacterial agent receives such environmental effects and loss of activity after being administered in the environment easily, the most important thing is the supply of nutritive element and moisture in these factors; Fermented liquid is processed the nutritive substance that can make behind the water-in-oil wherein to be wrapped in around the degradation bacteria suspending stabilizedly; Provide the nutrition substrate to keep the activity of bacterial classification through slowly-releasing; Prevent the deposition of degradation bacteria; And oil phase can provide suitable moist environment to degradation bacteria, thereby prolongs the quality guaranteed period of microbial inoculum product greatly; Directly be sprayed onto fruit and vegetable surfaces during use, thereby, improve the residual degradation rate of fruit and vegetable surfaces farming, also can prevent the generation of phenomenons such as fruits and vegetables dehydration, brown stain simultaneously because water in oil water retention characteristic can make nutraceutical slow release keep the activity of bacterium.
4. Serratia strain SPL-2 as claimed in claim 1 is in the application of handling on soil and the fruit and vegetable surfaces methidathion pesticide residue.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119168A (en) * | 2016-07-22 | 2016-11-16 | 湖北大学 | One strain has rich phosphorus, degrading organic phosphor and the Serratieae of suppression plant pathogenic fungi |
| CN107019140A (en) * | 2017-05-22 | 2017-08-08 | 青岛天工智造创新科技有限公司 | Degradating machine |
| CN110804568A (en) * | 2019-11-14 | 2020-02-18 | 重庆理工大学 | Complex microbial inoculant and application thereof |
| CN110804567A (en) * | 2019-11-14 | 2020-02-18 | 重庆理工大学 | Serratia marcescens capable of degrading chlorobenzene and application thereof |
-
2012
- 2012-06-26 CN CN2012102110420A patent/CN102676439A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119168A (en) * | 2016-07-22 | 2016-11-16 | 湖北大学 | One strain has rich phosphorus, degrading organic phosphor and the Serratieae of suppression plant pathogenic fungi |
| CN106119168B (en) * | 2016-07-22 | 2019-06-18 | 湖北大学 | One plant has rich phosphorus, degrading organic phosphor and the Serratieae for inhibiting plant pathogenic fungi |
| CN107019140A (en) * | 2017-05-22 | 2017-08-08 | 青岛天工智造创新科技有限公司 | Degradating machine |
| CN110804568A (en) * | 2019-11-14 | 2020-02-18 | 重庆理工大学 | Complex microbial inoculant and application thereof |
| CN110804567A (en) * | 2019-11-14 | 2020-02-18 | 重庆理工大学 | Serratia marcescens capable of degrading chlorobenzene and application thereof |
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Application publication date: 20120919 |