CN101628296A - Method for remedying salmonella-polluted soil by utilizing antagonistic microbe agent - Google Patents
Method for remedying salmonella-polluted soil by utilizing antagonistic microbe agent Download PDFInfo
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Abstract
The invention discloses a method for remedying salmonella-polluted soil by utilizing antagonistic microbe agent, comprising the following steps: separating the salmonella-polluted soil by utilizing an aseptic filter paper plate method to obtain antagonistic bacterium suitable for the soil environment and having the function of inhibiting or killing the salmonella; fermenting antagonistic microbe strains through the following processes: inoculum inoculation, first-stage culture, second-stage culture and inoculation and second-stage extensive culture to obtain antagonistic bacteria liquid; spraying the antagonistic bacteria liquid to the salmonella-polluted soil, and then uniformly raking the soil. In the method, the microbial ecology principle is applied, and the function of inhibiting or killing the antagonistic bacterium of the salmonella which is obtained from the soil separated and screened from the soil is utilized to remedy the in-situ microbes for the salmonella-polluted soil, not only the damage of the physical remediation to the soil structure and the soil organism is avoided, but also the secondary pollution caused by the chemical remediation is avoided, therefore, the method is a low-cost and environmentally friendly remediation way.
Description
One, technical field
The invention belongs to microorganism reparation field in the soil pollution recovery technique, utilize the antagonistic microbe microbial inoculum to repair the method for restoring microorganism ecology of salmonella-polluted soil during particularly a kind of edaphon pollutes.
Two, background technology
Salmonella (Salmonella) is to cause that the enteric bacteria sexuality dyes the important pathogen with food posioning.In the world, the salmonella poisoning incident sharply rises in recent years, wherein many poisonings cause owing to having eaten salmonella-polluted food and having drunk salmonella-polluted water, thereby people pay close attention to more to the pathogenic soil fungi pollution that causes food and water pollution.Studies show that, a large amount of without the people, animal of dealing carefully with, poultry manure be manured into soil and life, aquaculture wastewater to be discharged into soil environment be the principal element that increases the salmonella-polluted soil risk, people, animal, poultry manure are applied to the time-to-live in soil that not only can increase salmonella in the soil as fertilizer, and the salmonellas that exist in a large number in the soil can increase the risk of livestock and poultry breeding industry simultaneously.In 4-6 ℃ of scope, most of pathogens can survive about 30 days in the soil, but the salmonella time-to-live is longer relatively.In numerous pathogens, salmonella is in 20-30 ℃ soil, and is more much longer than other pathogen time-to-live.After Islam etc. discover that salmonella enters soil with dirt filling or the improper use of feces of livestock and poultry, in pedotheque, still can detect the salmonella of survival after 203-231 days, still can detect the existence of salmonella in the soil after 203 days at salmonella-polluted soil upper seeding wheel radish 84 days and sowing carrot.Bibliographical information summer is also arranged along with the salmonella that feces of livestock and poultry is applied in the soil can survive nearly 300 days, the salmonella in the survival soil also can enter into surface water body with face of land runoff, polluted surface water.Salmonella-polluted in the soil can cause that human body intestinal canal infects by causing food pollution and pollution of waterhead, and then influences public health security.Therefore, suppress or kill in salmonella in the soil environment, repairing polluted soil, the reduction soil environment salmonella survival quantity, for the outburst that reduces the salmonella infection disease with spread significant.But at home and abroad there is no the relevant report that carries out the microorganism reparation for salmonella-polluted soil.
Three, summary of the invention
Goal of the invention: the present invention is directed to above-mentioned technological deficiency, a kind of method of utilizing the antagonistic microbe microbial inoculum to repair salmonella-polluted soil is provided, this method utilization microbial ecology principle, inhibition of utilization salmonella antagonism bacterium of separation screening in soil or killing action carry out the original position microorganism to salmonella-polluted soil and repair, both avoided the infringement of physics reparation, again the secondary pollution of having avoided chemical reparation to cause to soil texture and geobiont.
Technical scheme: technical solution of the present invention is: a kind of microbial ecology method of utilizing antagonistic microbe microbial inoculum original position to repair salmonella-polluted soil, and method step is: utilize the antagonistic bacterium of aseptic filter paper sheet flat band method from being obtained by separation the salmonella-polluted soil being suitable for soil environment, salmonella being had inhibition or killing action; The antagonistic microbe bacterial strain ferments through following flow process: seed liquor inoculation → one-level cultivation → secondary is cultivated inoculation → secondary and is enlarged cultivation, obtains antagonistic bacterium bacterium liquid; Antagonistic bacterium bacterium liquid sprays in salmonella-polluted soil, sprays the back rake and spares.
Salmonella-polluted soil antagonistic microbe in-situ remediation method, method step is: on LB bacteria culture media flat board, at first evenly be coated with the pathogen inoculation liquid, dip in respectively with the bacterium circular filter paper sheet that went out then and get target antagonistic bacterium nutrient solution, be attached on the flat board that was coated with pathogen, in incubator, cultivated 1-3 days under 28 ℃ of conditions, select and have obvious inhibition zone bacterial strain, this bacterium is carried out record, preserve; Antagonistic microbe composite bacteria agent capable preparation: with the aseptic inoculation ring bacterial classification is preserved on the inclined-plane, be seeded to shake up in the triangular flask aseptic liquid nutrient medium bacteria suspension, 30 ℃, 200r/min cultivates 24h, cultivation obtains primary seed solution; Primary seed solution is inoculated in the fermentation flask secondary by itself and 1: 10 inoculum concentration of fermentation medium volume ratio to be enlarged and cultivates 48h and obtain zymotic fluid; Utilize complex microbial inoculum that salmonella-polluted soil is carried out the microorganism reparation, divide contrast and add the test of 2 processing layouts of antagonistic microbe composite bacteria agent capable earth culture, place under the natural conditions and cultivate, add processing of antagonistic microbe composite bacteria agent capable and contrast and all establish 4 repetitions, regularly take soil sample to measure salmonella survival quantity in the soil, the quantity dynamic change of salmonella in the monitoring contaminated soil; According to greenhouse earth culture microbial bacterial agent repairing effect, the using microbe restorative procedure carries out the reparation of antagonistic microbe composite bacteria agent capable microorganism in situ in the field salmonella-polluted soil, fermented and cultured antagonistic microbe bacterium liquid is applied in the salmonella-polluted field soil, 3 repetitions are established in each contrast and processing, after applying the antagonistic microbe composite bacteria agent capable, regularly gather contaminated soil 0-10cm top layer soil sample, adopt five point sampling methods, whether the quantity dynamic change of the salmonella in the monitoring contaminated soil reaches the effect of administering salmonella-polluted soil with monitoring.
Beneficial effect: screening is adapted to the efficient salmonella antagonistic bacterium of soil environment; Antagonistic bacterium is cultivated by fermentation and is mixed with the antagonistic microbe composite bacteria agent capable; The antagonistic microbe microbial inoculum sprays in salmonella-polluted soil, brings into play its inhibition or kills salmonella in the soil, thereby reduce the salmonella quantity of surviving in the soil environment, reduces the Ecological Environment Risk that salmonella-polluted soil brought.Eliminate the means that pathogen is all adopted physics or chemistry traditionally, but these two kinds of approach all there is limitation in the contaminated soil reparation.This method proposes to use the microbial ecology principle first; inhibition of utilization salmonella antagonism bacterium of separation screening in soil or killing action carry out the original position microorganism to salmonella-polluted soil and repair; both avoided the infringement of physics reparation to soil texture and geobiont; the secondary pollution of having avoided chemical reparation to cause again; be a kind of low cost, eco-friendly reparation approach; can keep edaphon diversity and population balance, protection soil ecology function.
Four, description of drawings
Fig. 1 is aseptic filter paper sheet flat band method screening salmonella Antagonistic Fungi schematic diagram. As can be seen from the figure: use Aseptic filter paper sheet flat band method filters out from soil has antagonism or inhibiting antagonistic bacterium to salmonella, The antagonistic bacterium that screens on the flat board is to showing preferably antagonism or inhibition work for examination salmonella cause of disease bacterium With, choose the bacterial strain of the big and effect stability of inhibition zone for the preparation of microbial inoculum through repeated screening.
Fig. 2 is the antagonistic bacterium microbial inoculum application example effect of preparation. This application example is the sand in hot-house culture Use in the door Salmonella contaminated soil, as can be seen from the figure: first three sky after spraying microbial inoculum, it is right not spray According to and spray composite bacteria agent capable process between the salmonella amount of survival do not have significant difference, but after 7 days, spray Salmonella quantity significantly descends in the soil of complex microbial inoculum, is lower than 2-3 order of magnitude of contrast, and explanation adds The composite bacteria agent capable that enters shows preferably antagonism or killing action to salmonella in soil environment.
Fig. 3 and Fig. 4 are the effect example of using composite bacteria agent capable reparation salmonella-polluted soil in the field. Fig. 3 is the effect on slight salmonella-polluted soil, and Fig. 4 is at the height salmonella-polluted soil Effect. As can be seen from the figure: salmonella-polluted soil was spraying the antagonistic microbe composite bacteria agent capable 10 days After, the antagonistic microbe composite bacteria agent capable that sprays has shown the obvious inhibitory action of salmonella, afterwards spray in 30 days Execute that salmonella quantity contrast descends more than 10 times in the soil of antagonistic microbe composite bacteria agent capable, and afterwards spray in 40 days Execute that salmonella quantity contrast descends more than 100 times in the soil of antagonistic microbe composite bacteria agent capable, show in the field Between spray the antagonistic bacterium microbial inoculum and can play the effect that suppresses the salmonella breeding, reduces its amount of survival. This The microbial ecology restorative procedure that utilizes antagonistic microbe microbial inoculum original position to repair pathogen contamination soil has cost Low, effect is relatively good, long, eco-friendly advantage of the time of playing effectiveness, and actual using value is arranged, can Effectively reduce the potential Ecological Environment Risk of pathogen contamination soil, reach protection public health security purpose.
Five, the specific embodiment
Embodiment 1:
1, the separation of antagonistic bacterium bacterial strain and screening
1.1 culture medium
Salmonella differential medium: magnesium chloride peacock green enriched medium, HE agar medium
Bacteria culture media: LB (Luria-Bertani) culture medium: peptone 10g, yeast extract 5g, NaCl 10g, distilled water 1000ml, 7.0,121 ℃ of sterilizations of pH 20min; Solid medium adds agar powder 1.2-1.5%.
1.2 for examination salmonella bacterial strain
Reference culture: Bacterium enteritidis (Salmonella enteritidis), salmonella typhimurium (Salmonella typhimurium).
The salmonella bacterial strain that separates in the natural environment: separate from feces of livestock and poultry contaminated soil sample.
1.3 separating screening method:
Bacterial isolates separates: take by weighing the bright sample of 5g soil respectively in the 50ml sterilized water, vibrated 45 minutes, the classification dilution (10 then
-3-10
-8) be coated on bacterium LB culture medium.After treating that bacterium colony grows, select single colony inoculation, cultivate 24h for 28 ℃ in 96 orifice plates that the LB fluid nutrient medium is housed in advance.
The primary dcreening operation of salmonella antagonistic bacterium, adopt aseptic filter paper sheet flat band method: on LB bacteria culture media flat board, at first evenly be coated with the pathogen inoculation liquid in flat board, soak in the bacterium liquid that the diameter 6mm circular filter paper sheet of bacterium of will going out is then cultivated in 96 hole microwell plates, filter paper is attached on the flat board that was coated with pathogen at last, cultivated 1-3 days for 28 ℃, check filter paper inhibition zone situation on every side.If any obvious inhibition zone, illustrate that this bacterium has antagonism to pathogen.Choosing has inhibiting different shape bacterium colony to the pathogen growth, and record, takes pictures, and back 4 ℃ of preservations are numbered in separation and purification.
The multiple sieve of efficient antagonistic bacterium: use the same method antagonistic strain that primary dcreening operation is obtained of profit carries out repeatedly multiple sieve, and choosing at last all has obvious antagonism to all for the examination salmonella, and the big stable bacterial strain of fungistatic effect of inhibition zone prepares microbial inoculum.
2, antagonistic microbe composite bacteria agent capable preparation:
Antagonistic microbe fungicide preparation flow process: seed liquor inoculation → one-level cultivation → secondary is cultivated inoculation → secondary and is enlarged cultivation → bacterium liquid mixing → composite bacteria agent capable
Concrete steps are: with the aseptic inoculation ring with some inclined-planes preserve bacterial classification inoculations shake up to the triangular flask aseptic liquid nutrient medium bacteria suspension, 30 ℃, 200r/min cultivates 24h, cultivation obtains primary seed solution; Primary seed solution is inoculated in the fermentation flask secondary by itself and 1: 10 amount of fermentation medium volume ratio to be enlarged and cultivates 48h and obtain zymotic fluid, with the second order fermentation liquid of different strains equal proportion mixed preparing composite microbial bacteria liquid by volume, as have only the zymotic fluid of a strain, then needn't prepare.The preservation in low temperature environment as far as possible of bacterium liquid.
3, the microbial inoculum application case 1: the test of greenhouse earth culture
Test method: got the wind desiceted soil of 20 mesh sieves, and added fresh pig manure by the amount of wind desiceted soil quality 2%, mixing becomes salmonella-polluted soil; Soil is sub-packed in the basin alms bowl, and (microbial inoculum contains the bacterium order of magnitude 10 to 100ml/kg soil to spray the antagonistic microbe microbial inoculum (single microbial inoculum or composite bacteria agent capable all can) of preparation then
10Individual/ml), adding running water adjusting water content again is 60% of maxmun field capacity, places under the greenhouse natural conditions and cultivates.Gather the soil of 0d, 1d, 3d, 5d and 7d, measure the salmonella quantity of survival.Adopt MPN method (most probable number, counting method is cultivated in dilution) to increase bacterium through magnesium chloride peacock green selective medium, then with the check of HE selective medium, the reacting positive person looks into the MPN table and determines salmonella survival quantity for containing salmonella.
4, the microbial inoculum application case 2: salmonella-polluted soil field original position repairing test
Field test is selected in the vegetable plot, according to what of fresh pig manure applied amount the salmonella-polluted degree of soil is divided into slight pollution and highly polluted.The amount of slight pollution soil application pig manure is 20 tons/hectare, and highly polluted soil application amount is 40 tons/hectare, and rake was even after fresh pig manure was added to soil, and the composite bacteria agent capable with preparation sprays application in the soil then, and the amount of spraying is 1500ml/m
2If atmospheric temperature is lower, can adopt the method that covers plastic sheeting for farm use to make the soil insulation, the 0th, 5,10,15,20,30,40 day collection soil 0-10cm top layer, sub-district, field soil sample after applying microbial inoculum, adopt five point sampling methods, detect the salmonella survival quantity in the soil immediately.
Separation, screening and microbial bacterial agent application case by the antagonistic bacterium bacterial strain can draw to draw a conclusion:
The present invention utilizes aseptic filter paper sheet flat band method to screen bacterial isolates to the effective antagonism of salmonella, the microbial bacterial agent that utilizes these antagonistic strain fermented and cultured to be mixed with adds in the salmonella-polluted soil, can significantly reduce in the contaminated soil salmonella quantity up to 2-3 the order of magnitude, depression effect improves along with increasing of soil Salmonella pathogen contamination degree, thereby greatly reduced the Ecological Environment Risk that salmonella-polluted soil brought, reached the purpose that reduces salmonella-polluted potential hazard and ensure public health security.Result's proof utilizes the antagonistic microbe microbial inoculum to be applied to the original position repairing method of microorganism of pathogen salmonella-polluted soil; both avoided the infringement of physics reparation to soil texture and geobiont; the secondary pollution of having avoided chemical reparation to cause again; have that rehabilitation cost is low, environmental friendliness, can keep edaphon diversity and population balance; the advantage of protection soil ecology function is a kind of repairing method of microorganism of administering the salmonella-polluted soil actual application value that has.
Claims (2)
1. microbial ecology method of utilizing antagonistic microbe microbial inoculum original position to repair salmonella-polluted soil is characterized in that method step is:
A. utilize the antagonistic bacterium of aseptic filter paper sheet flat band method from being obtained by separation the salmonella-polluted soil being suitable for soil environment, salmonella being had inhibition or killing action;
B. the antagonistic microbe bacterial strain ferments through following flow process: seed liquor inoculation → one-level cultivation → secondary is cultivated inoculation → secondary and is enlarged cultivation, obtains antagonistic bacterium bacterium liquid;
C. antagonistic bacterium bacterium liquid sprays in salmonella-polluted soil, sprays the back rake and spares.
2. salmonella-polluted soil antagonistic microbe in-situ remediation method according to claim 1 is characterized in that method step is:
A. on LB bacteria culture media flat board, at first evenly be coated with the pathogen inoculation liquid, dip in respectively with the bacterium circular filter paper sheet that went out then and get target antagonistic bacterium nutrient solution, be attached on the flat board that was coated with pathogen, in incubator, cultivated 1-3 days under 28 ℃ of conditions, select and have obvious inhibition zone bacterial strain, this bacterium is carried out record, preserve;
B. antagonistic microbe composite bacteria agent capable preparation: with the aseptic inoculation ring bacterial classification is preserved on the inclined-plane, be seeded to shake up in the triangular flask aseptic liquid nutrient medium bacteria suspension, 30 ℃, 200r/min cultivates 24h, cultivation obtains primary seed solution; Primary seed solution is inoculated in the fermentation flask secondary by itself and 1: 10 inoculum concentration of fermentation medium volume ratio to be enlarged and cultivates 48h and obtain zymotic fluid;
C. utilize complex microbial inoculum that salmonella-polluted soil is carried out the microorganism reparation, divide contrast and add the test of 2 processing layouts of antagonistic microbe composite bacteria agent capable earth culture, place under the natural conditions and cultivate, add processing of antagonistic microbe composite bacteria agent capable and contrast and all establish 4 repetitions, regularly take soil sample to measure salmonella survival quantity in the soil, the quantity dynamic change of salmonella in the monitoring contaminated soil;
D. according to greenhouse earth culture microbial bacterial agent repairing effect, the using microbe restorative procedure carries out the reparation of antagonistic microbe composite bacteria agent capable microorganism in situ in the field salmonella-polluted soil, fermented and cultured antagonistic microbe bacterium liquid is applied in the salmonella-polluted field soil, 3 repetitions are established in each contrast and processing, after applying the antagonistic microbe composite bacteria agent capable, regularly gather contaminated soil 0-10cm top layer soil sample, adopt five point sampling methods, whether the quantity dynamic change of the salmonella in the monitoring contaminated soil reaches the effect of administering salmonella-polluted soil with monitoring.
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| CN108027849B (en) * | 2015-12-07 | 2020-03-20 | 齐默尔根公司 | Improvement of microbial strains by using HTP genome engineering platform |
| US10647980B2 (en) | 2015-12-07 | 2020-05-12 | Zymergen Inc. | Microbial strain improvement by a HTP genomic engineering platform |
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