CN104208841A - Method for degrading organophosphorus pesticide in soil by immobilized engineering bacteria - Google Patents
Method for degrading organophosphorus pesticide in soil by immobilized engineering bacteria Download PDFInfo
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- CN104208841A CN104208841A CN201410419449.1A CN201410419449A CN104208841A CN 104208841 A CN104208841 A CN 104208841A CN 201410419449 A CN201410419449 A CN 201410419449A CN 104208841 A CN104208841 A CN 104208841A
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Abstract
The invention discloses a method for degrading an organophosphorus pesticide in soil by immobilized engineering bacteria. The method comprises the following steps: (a) engineering bacteria is immobilized; (b) the immobilized engineering bacteria is appreciated; and (c) the appreciated immobilized engineering bacteria is fed in the soil for culturing; and the content of organophosphorus in the soil is measured. The method has such advantages as simplicity, easy control of operating procedures, no poison, excellent air permeability, no soil secondary pollution and quick degrading speed of the organophosphorus, and is suitable for large-scale promotion and application.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to the method for organophosphorus pesticide in a kind of immobilization engineering bacteria degraded soil.
Background technology
About there are 87 ~ 1,070,000 hm in current China
2agricultural land soil be subject to organophosphorus pesticide pollution, be planted in the crop uptake and accumulation agricultural chemicals from soil on the soil of pollution by pesticides, the normal growth not only affecting plant is grown, but also to remain in plant, to cause the residues of pesticides of main farm produce to exceed standard.Major part agricultural chemicals can fall into soil, and constantly accumulate, destroy the breeding of edaphon, the bacterial classification of sensitiveness is suppressed, the population of edaphon is tending towards unification, cause original Balance disorders, functional disturbance thus affect the circulation of soil material and energy, affect the nitrated and respiration of the ammonification of edaphon etc., destroy soil texture and physicochemical property thus, affect plant growth, cause soil texture to harden and cause soil degradation, acidifying, nutrients loss, crop yield and quality to decline.
The improvement of soil pollution mainly contains physics Treatment process, chemical harnessing technology, microbial treatment technology, plant in treating technology.Microbial treatment technology has the unrivaled superiority of physics, chemical method, and as disposal cost is low, treatment effect is good, little on the impact of environment, can not cause secondary pollution, does not destroy the soil environment needed for plant growth, process simple operation and other advantages.Microbial treatment technology accelerates the microbial degradation of pedo relict agricultural chemicals by natural or manual type, realizes the microorganism remediation of polluted soil.But adopt traditional free microorganism technology reparation to there are some defects by during organophosphorus pesticide contaminated soil, as slow in reaction speed, effectively degradation bacteria cannot be formed dominant microflora, anti-adverse environment ability, responsive to environmental change, be in a disadvantageous position when competing nutrition with indigenous bacterium, do not reach the re-set target of contaminated soil remediation, and immobilized cell technology is expected to overcome these shortcomings and drawback.
Immobilized cell technology is fixed on water-soluble or insoluble graininess, tubulose, membranaceous carrier by cell by the method for chemistry or physics, it is made to keep active and a kind of technology of energy Reusability, comparatively free bacterium has greater advantage, the adaptive capacity of cell to adverse environment can be improved significantly, can while continuous proliferation, dormancy and decline, its activity remains stable, has lot of advantages such as reducing pollution amelioration cost, economize energy consumption, simplification environment protection method.Current immobilization technology is own through being widely used in fluid media (medium), but at reparation non-fluid medium, the soil of especially organophosphorus pesticide pollution there is not yet successful report.
Summary of the invention
the technical problem solved:for prior art problem, the object of the present invention is to provide the method for organophosphorus pesticide in a kind of immobilization engineering bacteria degraded soil, good degrading effect, avoids causing secondary pollution to soil.
technical scheme:for solving prior art problem, the technical scheme that the present invention takes is:
A method for organophosphorus pesticide in immobilization engineering bacteria degraded soil, comprises the following steps:
a.the immobilization process of engineering bacteria, LB fluid nutrient medium culturing engineering bacterium is adopted to make bacteria suspension, engineering bacterium fermentation bacterial classification is obtained again after liquid fermentation medium is cultivated, with engineering bacterium fermentation bacterial classification for immobilization object, adopt immobilized cell technology, with polyvinyl alcohol, sodium alginate, active carbon, engineering bacteria is embedded, form immobilization engineering bacteria;
B. immobilization engineering bacteria is inoculated into 50 ~ 80ml liquid fermentation medium/250ml conical flask to cultivate, temperature is 28 ~ 32 DEG C, be cultivate 6 ~ 12h, the immobilization engineering bacteria must bred by sterile gauze isolated by filtration in the constant temperature oscillation case of 150 ~ 250r/ min at rotating speed;
C. the immobilization engineering bacteria of propagation is pressed soil weight 3 ~ 10% and throw grain ratio, be uniformly distributed in in examination soil, again 100 ~ 600mg/kg organophosphorus pesticide is added in sterilized water, add by regulating during soil moisture content, regulate water content to reach 40 ~ 60% of saturated soil water content, after cultivating, sampling detects organic phosphorous content in soil.
As preferably, described engineering bacteria be bacillus subtilis (
bacillus subtilis) bacterial strain, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, deposit number: CGMCC No. 9272, preservation date: on 06 05th, 2014, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
In above-mentioned immobilization engineering bacteria degraded soil organophosphorus pesticide method in the concrete steps of immobilization process of step a engineering bacteria be:
(1) LB fluid nutrient medium 28-32 DEG C of culturing engineering bacterium 12-24h is adopted to make bacteria suspension, be inoculated in the liquid fermentation medium containing 100 ~ 600mg/L parathion-methyl, liquid amount is 50 ~ 80 ml/250ml conical flasks, shake up, 28 ~ 32 DEG C, 150 ~ 250 r/ min constant-temperature shaking culture 24 ~ 48 h, obtain engineering bacterium fermentation bacterial classification;
(2) polyvinyl alcohol cold water swelling after add in 95 DEG C of water and dissolve, polyvinyl alcohol and active carbon are mixed in sodium alginate soln and make three's final concentration reach 100 ~ 120 g/L, 8 ~ 10 g/L and 9 ~ 11 g/L respectively, by engineering bacterium fermentation bacterial classification and liquid fermentation medium sterile gauze isolated by filtration, after bacterial classification washed with de-ionized water, according to bacterial classification: polyvinyl alcohol-sodium alginate-Actidose mass volume ratio is that the standard of 1:1 ~ 2:1 is got bacterial classification and added in polyvinyl alcohol-sodium alginate-Actidose and mix;
(3) CaCl of above-mentioned mixed liquor instillation 8 ~ 10g/L is drawn with syringe
2forming diameter in solution is 0.3 ~ 0.5cm immobilization particle, and after room temperature fixes 1 ~ 3h, 4 ~ 6h fixed by 4 DEG C of refrigerators, then after using deionized water rinsing 2 ~ 4 times, directly to add in liquid fermentation medium propagation or 4 DEG C of refrigerations for subsequent use.
As preferably, in step b, the cultivation temperature of immobilization engineering bacteria is 30 DEG C.
As preferably, in the immobilization process of engineering bacteria, polyvinyl alcohol final concentration is 120 g/L, and sodium alginate final concentration is 10 g/L, and active carbon final concentration is 11 g/L, CaCl
2the concentration of solution is 10g/L.
beneficial effect
The method of organophosphorus pesticide in immobilization engineering bacteria degraded soil of the present invention, there is method easy, operation sequence is easy to control, nontoxic, aeration is good, can not cause the secondary pollution of soil, the advantages such as degrading organic phosphor speed is fast, compared with non-immobilization engineering bacteria, immobilization engineering bacteria is stable, degradation rate is high and degradation speed is fast.
Accompanying drawing explanation
The effect of Fig. 1 immobilization engineering bacteria degraded parathion;
The effect of Fig. 2 immobilization engineering bacteria degrading methamidophos;
Fig. 3 difference throws the impact of the immobilization engineering bacteria comparison degraded parathion of grain;
Fig. 4 difference throws the impact of the immobilization engineering bacteria comparison degrading methamidophos of grain.
Detailed description of the invention
The following examples can make the present invention of those skilled in the art comprehend, but do not limit the present invention in any way.
The source explanation of biomaterial of the present invention
1, engineering bacteria: bacillus subtilis (
bacillus subtilis) bacterial strain, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, deposit number: CGMCC No. 9272, preservation date: on 06 05th, 2014, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
2, culture medium
LB fluid nutrient medium (g/L): tryptone 10, yeast extract 5, NaCl 10, pH 7.0 ~ 7.2;
Liquid fermentation medium (g/L): glucose 10, NH
4cl 2, KH
2pO
40.5, K
2hPO
41.5, MgSO
40.2, NaCl 0.5, parathion-methyl 0.1, pH 7.0 ~ 7.2.
Embodiment 1
The present embodiment illustrates the immobilization process of engineering bacteria.
(1) preparation of engineering bacterium fermentation bacterial classification:
Inoculation engineering bacteria list bacterium colony is in LB fluid nutrient medium, and in 30 DEG C, after 250 r/min shaken cultivation are spent the night, culture continues at 30 DEG C, and 300 r/min shaken cultivation are to OD
600=0.8, add derivant IPTG to final concentration 0.2 mmol/L, continue to cultivate 3h, collected by centrifugation thalline, and with 0.1mol/L phosphate buffer (pH 7.0) cleaning, in 3000 r/min, 4 DEG C are centrifugal, thalline repeated washing, centrifugal 3 times, obtain bacteria suspension; Bacteria suspension is inoculated into 50ml liquid fermentation medium/250ml conical flask according to 4% inoculum concentration (v/v) and cultivates, 30 DEG C, 250 r/min shaken cultivation 12h, liquid spawn is cultured to 240ml liquid fermentation medium/500ml conical flask according to above-mentioned inoculum concentration expansion, obtains engineering bacterium fermentation bacterial classification.
(2) add after PVA cold water swellable in 95 DEG C of water and dissolve, PVA and active carbon are mixed in SA solution, three's final concentration is made to be respectively 100 g/L, 9 g/L and 10 g/L, with sterile gauze isolated by filtration engineering bacterium fermentation bacterial classification and fermentation medium, after engineering bacterium fermentation bacterial classification washed with de-ionized water, according to engineering bacterium fermentation bacterial classification: PVA-SA-Actidose=1:1(w/v) ratio, get 0.3g thalline, add 300ml PVA-SA-Actidose mixing.
(3) draw with No. 5 syringes the CaCl that above-mentioned thalline and PVA-SA-Actidose are added dropwise to 9 g/L
2must about 2500 immobilization engineering bacteria particles in solution, particle diameter is about 0.5cm, and after room temperature fixes 1h, 4h fixed by 4 DEG C of refrigerators, and after particle deionized water rinsing 2 times, 4 DEG C of refrigerations are for subsequent use.
Embodiment 2
The present embodiment illustrates the increment of immobilization engineering bacteria.
Immobilization engineering bacteria is inoculated into 50ml liquid fermentation medium/250ml conical flask to cultivate, temperature is 30 DEG C, is to cultivate 6 h, the immobilization engineering bacteria must bred by sterile gauze isolated by filtration in the constant temperature oscillation case of 250 r/ min at rotating speed.
Embodiment 3
The present embodiment compares immobilization engineering bacteria and on-fixed engineering bacteria to the effect of organophosphorus pesticide in soil of degrading
.
The immobilization engineering bacteria of propagation is thrown grain ratio by soil weight 5%, be uniformly distributed in in examination soil, being added with machine phosphorus insecticide makes its final concentration be 100 mg/kg, cultivate in 30 DEG C of constant incubators, period, the water-holding capacity of soil remained on 60%, sampled, according to the sample preparation of Accelerate solvent extraction (ASE) method after cultivating 12h, 24h and 36h, cyclohexane extracts the agricultural chemicals in each soil sample, and high performance liquid chromatography detects parathion and methamidophos residue amount in soil.Inoculate without immobilized engineering bacteria with step in the same way and do not connect bacterium in contrast, testing result is as shown in table 1, wherein, CK representative does not connect the blank sample of bacterium, on-fixed refers to the engineering bacteria without immobilization process, immobilization refers to immobilization engineering bacteria of the present invention, degradation rate (%)=(1-processing sample residual quantity/control sample residual quantity) × 100%.
Efficient liquid phase measures parathion chromatographic condition: chromatographic column Hypersil C18 post; Mobile phase: V (methyl alcohol): V (water) :=80:20; Flow velocity: 1.0 ml/ min; Determined wavelength 280 nm, column temperature: 35 DEG C; Sample size: 20 μ 1.
Efficient liquid phase measures acephatemet chromatographic condition: chromatographic column Hypersil C18 post; Mobile phase: V (methyl alcohol): V (water) :=30:70; Flow velocity: 1.5 ml/ min; Determined wavelength 215 nm, column temperature: 30 DEG C; Sample size: 15 μ 1.
Table 1 CK, on-fixedization and immobilization engineering bacteria are degraded the effect of organophosphorus pesticide in soil:
36h after throwing grain, the degradation rate of immobilization engineering bacteria parathion reaches 96.29%, and acephatemet is almost all degraded; Without immobilization process engineering bacteria after throwing grain 36h, its parathion and methamidophos degradation rate reach 82.61% and 79.47% respectively.Along with the increase of time, all the time higher than non-immobilization engineering bacteria, there is significant difference therebetween and reflect that the engineering bacteria after immobilization is in the stable performance of degraded in soil organophosphorus pesticide very intuitively by Fig. 2 and Fig. 3 in the degradation rate of immobilization engineering bacteria parathion and acephatemet.Although non-immobilization engineering bacteria also has good degradation capability to the organophosphorus pesticide such as parathion, acephatemet, its degradation rate and degradation speed are still lower than the engineering bacteria after immobilization.
Embodiment 4
The present embodiment illustrates that the difference of immobilization engineering bacteria throws the impact of organic phosphorus pesticide degradation effect in grain comparison soil.
The immobilization engineering bacteria of propagation is pressed respectively soil weight 3%, 5%, 7%, 10% throws grain ratio, is uniformly distributed in in examination soil.In the present embodiment 3%, 7%, 10% throw the experimental technique of grain ratio and determination step all in the same manner as in Example 3, complete 3%, 5%, 7%, 10% degradation rate throwing grain ratio in embodiment 3 to measure simultaneously, measurement result is in table 2, wherein in table, CK representative does not add the blank sample of bacterial strain, and 3%, 5%, 7%, 10% represents different throwing grain ratio.
Table 2 difference throws the immobilization engineering bacteria of grain ratio to the degradation effect of organophosphorus pesticide in soil:
?when immobilization engineering bacteria throwing grain ratio is 5%, the degradation rate of 36h immobilization engineering bacteria parathion reaches 96.29%, and the degradation rate of acephatemet is 99.18%, all throws the degradation rate of grain ratio higher than 3%, 7% and 10%.Along with the increase of time, when to throw grain ratio be 5%, the degradation rate of immobilization engineering bacteria parathion and acephatemet all the time higher than throwing grain than (the seeing Fig. 3, Fig. 4 respectively) that be 3%, 7% and 10%, due to throwing grain lower time, immobilization engineering bacteria bacterial classification concentration is lower, affects organic phosphorus pesticide degradation efficiency; Throw grain higher, there will be growth inhibition, nutriment sharply consumes, and affects the growth of immobilization engineering bacteria, makes organic phosphorus pesticide degradation rate and declines.
Claims (5)
1. the method for organophosphorus pesticide in immobilization engineering bacteria degraded soil, is characterized in that, comprise the following steps:
a.the immobilization process of engineering bacteria, LB fluid nutrient medium culturing engineering bacterium is adopted to make bacteria suspension, engineering bacterium fermentation bacterial classification is obtained again after liquid fermentation medium is cultivated, with engineering bacterium fermentation bacterial classification for immobilization object, adopt immobilized cell technology, with polyvinyl alcohol, sodium alginate, active carbon, engineering bacteria is embedded, form immobilization engineering bacteria; B. immobilization engineering bacteria is inoculated into 50 ~ 80ml liquid fermentation medium/250ml conical flask to cultivate, temperature is 28 ~ 32 DEG C, be cultivate 6 ~ 12h, the immobilization engineering bacteria must bred by sterile gauze isolated by filtration in the constant temperature oscillation case of 150 ~ 250r/ min at rotating speed; C. the immobilization engineering bacteria of propagation is pressed soil weight 3 ~ 10% and throw grain ratio, be uniformly distributed in in examination soil, again 100 ~ 600mg/kg organophosphorus pesticide is added in sterilized water, add by regulating during soil moisture content, regulate water content to reach 40 ~ 60% of saturated soil water content, after cultivating, sampling detects organic phosphorous content in soil.
2. the method for organophosphorus pesticide in immobilization engineering bacteria degraded soil according to claim 1, is characterized in that: described engineering bacteria be bacillus subtilis (
bacillus subtilis) bacterial strain, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, deposit number: CGMCC No. 9272, preservation date: on 06 05th, 2014, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
3. the method for organophosphorus pesticide in immobilization engineering bacteria degraded soil according to claim 1, it is characterized in that, the concrete steps of the immobilization process of step a engineering bacteria are:
(1) LB fluid nutrient medium 28-32 DEG C of culturing engineering bacterium 12-24h is adopted to make bacteria suspension, be inoculated in the liquid fermentation medium containing 100 ~ 600mg/L parathion-methyl, liquid amount is 50 ~ 80 ml/250ml conical flasks, shake up, 28 ~ 32 DEG C, 150 ~ 250 r/ min constant-temperature shaking culture 24 ~ 48 h, obtain engineering bacterium fermentation bacterial classification;
(2) polyvinyl alcohol cold water swelling after add in 95 DEG C of water and dissolve, polyvinyl alcohol and active carbon are mixed in sodium alginate soln and make three's final concentration reach 100 ~ 120 g/L, 8 ~ 10 g/L and 9 ~ 11 g/L respectively, by engineering bacterium fermentation bacterial classification and liquid fermentation medium sterile gauze isolated by filtration, after bacterial classification washed with de-ionized water, according to bacterial classification: polyvinyl alcohol-sodium alginate-Actidose mass volume ratio is that the standard of 1:1 ~ 2:1 is got bacterial classification and added in polyvinyl alcohol-sodium alginate-Actidose and mix;
(3) CaCl of above-mentioned mixed liquor instillation 8 ~ 10g/L is drawn with syringe
2forming diameter in solution is 0.3 ~ 0.5cm immobilization particle, and after room temperature fixes 1 ~ 3h, 4 ~ 6h fixed by 4 DEG C of refrigerators, then after using deionized water rinsing 2 ~ 4 times, directly to add in liquid fermentation medium propagation or 4 DEG C of refrigerations for subsequent use.
4. the method for organophosphorus pesticide in immobilization engineering bacteria degraded soil according to claim 1, is characterized in that: in step b, the cultivation temperature of immobilization engineering bacteria is 30 DEG C.
5. the method for organophosphorus pesticide in immobilization engineering bacteria degraded soil according to claim 3, it is characterized in that: polyvinyl alcohol final concentration is 120 g/L, sodium alginate final concentration is 10 g/L, and active carbon final concentration is 11 g/L, CaCl
2the concentration of solution is 10g/L.
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| CN106381276A (en) * | 2016-08-30 | 2017-02-08 | 河海科技工程集团有限公司 | Bacillus subtilis cultured by biochar and preparation method and application thereof |
| CN113214840A (en) * | 2021-05-12 | 2021-08-06 | 淮阴师范学院 | Preparation method and application of biodegradable carbon particles |
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