CN102489499A - Method for controlling manganese pollution by cooperation of microorganism and plants - Google Patents

Method for controlling manganese pollution by cooperation of microorganism and plants Download PDF

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CN102489499A
CN102489499A CN2011104174723A CN201110417472A CN102489499A CN 102489499 A CN102489499 A CN 102489499A CN 2011104174723 A CN2011104174723 A CN 2011104174723A CN 201110417472 A CN201110417472 A CN 201110417472A CN 102489499 A CN102489499 A CN 102489499A
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manganese
pollution
bacterial classification
nutrient solution
microorganism
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王万能
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Chongqing University of Technology
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Chongqing University of Technology
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Abstract

The invention discloses a method for controlling manganese pollution by cooperation of microorganism and plants, which includes the following steps: 1) taking the strains R-118 and R-2; 2) inoculating the strains into culture medium of liquid LB, keeping the temperature of 28 DEG C, the rotation speed of 200-250r/min and keeping shake cultivation for one to two days; 3), culturing the strains R-118 and the R-2 by different fermentation tanks while keeping pressure in the tanks at 0.05Mpa, fermenting for 36 hours to obtain two types of strain fermentation liquor; 4) transplanting plants having high manganese adsorption capacity into soilless nutrient solution containing Mn2+, adding the two types of fermentation liquor, moving the plants to places with sufficient sunshine for 5-7 days, and detecting variation of content of manganese ion and reducing of concentration of manganese ion. The method utilizes cooperation of microorganism and plants, is capable of removing manganese pollution in environments and restoring normal functions of the polluted ecological system, is favorable for controlling manganese pollution in large scope, and further, has the advantages of low price.

Description

The method of the collaborative plant in treating pollution by manganese of microorganism
Technical field
The invention belongs to biological technical field, the method for the collaborative plant in treating pollution by manganese of particularly a kind of microorganism.
Background technology
Manganese and compound thereof are applied to the every field of national economy.Wherein steel and iron industry is most important field, accounts for 90%～95% with the manganese amount, mainly as deoxidier and the desulfurizing agent in ironmaking and the steelmaking process, and is used for alloying.All the other manganese of 5%～10% are used for other industrial circles, and manganese has crucial strategic position in national economy.Along with the exploitation of manganese ore, residual solid waste, the sewage of discharging etc. are to water body, and the pollution of soil environment is day and night serious.
Manganese is valence variation element, presses geochemical classification, and it is strong oxyphilic element.Manganese in the soil mainly comes from oxide and oxysalt, and organic colloid protecting action and biological agent play an important role to migration, enrichment, the deposition of manganese.In mineral, general manganese normal with iron, magnesium symbiosis, in addition, manganese is also often and coprecipitations such as zinc, copper, nickel, cobalt, calcium.The essential mineral of manganese is pyrolusite (MnO 2NH 2O), hausmannite (Mn 3O 4), manganite [MnO (OH)] and braunite (Mn 2O 3), in these mineral,, the branch of α, β, r is arranged again along with the difference of crystallization configuration, in addition, also have a lot of manganese-bearing minerals.
The soil improvement function alfalfa of alfalfa is perennial leguminous herbaceous plant, and its root system is dark, and biomass is high and many, has a large amount of secretion; Radicula and rhizobium upgrade very fast, and along with the prolongation of the alfalfa plantation time limit, topsoil soils is organic, available nutrient content increases gradually; In addition, alfalfa adaptability is strong, and this has further confirmed its critical role in barren, degenerated soil are improved.
To the improvement of heavy metal pollution of soil, at present commonly used have chemical methodes such as physical method such as soil moved in to improve the original method, leaching method, absorption fixation and biological reducing method, complexing extraction.But often quantities is big, investment is expensive for these methods, and it is big that difficulty is implemented in large-area pollution.Contaminated soil as to 1km carries out engineering control, the expending up to 800～2,400 ten thousand dollars of every 1m degree of depth soil body.
Bioremediation technology can weaken and even eliminate the toxicity of environmental contaminants, reduces the pollutant risk.Physico-chemical process with traditional is compared, and it is low that biological restoration has a cost, do not produce secondary pollution, the ecological coordination and being easy to by advantages such as public's acceptance.
Summary of the invention
The method that the purpose of this invention is to provide the collaborative plant in treating soil pollution by manganese of a kind of microorganism; This method utilizes microorganism to work in coordination with plant; Stable and remove the pollution by manganese in the environment, recover the normal function of the contaminated ecosystem, help administering pollution by manganese on a large scale.The inventive method also has the low advantage of price.
The technical scheme that realizes is:
The method of the collaborative plant in treating pollution by manganese of microorganism has following steps:
1) gets bacterial classification R-118 and R-2;
2) picking bacterial classification R-118 and R-2 are inoculated in the liquid LB culture medium, temperature is 28 ℃, the rotating speed of 200-250r/min, and concussion was cultivated 1-2 days, obtained bacterial classification R-118 and R-2 after cultivating;
3) will use different fermentation tank culture respectively with R-2 through the bacterial classification R-118 after cultivating, a jar internal pressure is 0.05Mpa, ferments 36 hours, obtains R-118 and R-2 strain fermentating liquid;
4) will adsorb the stronger plant transplantation of pollution by manganese to containing Mn 2+Nothing soil nutrient solution in, add R-118 or R-2 strain fermentating liquid, move under the sufficient condition of illumination, grow detection manganese ion content 5-7 days.
The described Mn that contains of step 4) 2+The collocation method of nothing soil nutrient solution be: get NH 4NO 30.3g, KH 2PO 40.19g, KNO 30.08g draw 10ml behind the dissolving mixing, add water to 1L, add 1.512g MnSO again 4Mixing.
Contain Mn in the step 4) 2+Nothing soil nutrient solution and the volume ratio of strain fermentating liquid be 75:1.
The stronger plant of absorption pollution by manganese of the present invention is pulse family, crucifer, like alfalfa, rape etc.
The preparation method of said bacterial classification R-118 and R-2 is: 1) get the manganese ore surface mass, add deionized water and stirring, process former bacteria suspension, former bacteria suspension is uniformly coated on the solid-state PYCM culture medium flat plate; 2) under 25～28 ℃ of constant temperature, cultivated 1～2 day picking list bacterium colony line purifying, 25～28 ℃ of cultivations in solid-state PYCM culture medium; Gained manganese oxidizing microorganisms bacterial strain to be selected, at liquid PYCM culture medium, shaking speed is 200r/min; Temperature is 28 ℃, cultivates 2-4 days, measures the content of manganese ion in the nutrient solution; Filter out the manganese oxidizing microorganisms, obtain R-118 and R-2 manganese oxidizing microorganisms bacterial classification.
Because the phytoremediation of pollution by manganese soil is to utilize the absorption of the excess absorption and accumulation manganese of manganese excess accumulator plant or hyperaccumulative plant to reduce or remove soil pollution by manganese thing.Plant has three kinds of modes to the reparation in manganese metallic pollution site: plant is fixed, phytovolatilization and plant absorbing, removes manganese ion in the environment through these three kinds of modes.Microorganism mainly is that the manganese in the soil is fixed, shifted or transforms to the reparation of pollution by manganese, changes the Environmental Chemistry behavior of manganese in soil, can promote poisonous, harmful substance to detoxify or reduction toxicity, thereby reaches the purpose of biological restoration.Therefore, the present invention has utilized microorganism recovery technique and the synergistic principle of phytoremediation technology,
Applicant's experimental result shows, the method for the invention, and plant (alfalfa) can tolerate manganese content soil with high, not only can normal growth, and can promote growth.Coordination of manganese plant microbial adsorption capacity:
Figure 2011104174723100002DEST_PATH_IMAGE001
alfalfa and strain R-118>
Figure 647061DEST_PATH_IMAGE002
alfalfa and strain R-2>
Figure 2011104174723100002DEST_PATH_IMAGE003
alfalfa>
Figure 3349DEST_PATH_IMAGE004
strain R-118>
Figure 2011104174723100002DEST_PATH_IMAGE005
strain R-2.Be based on the new method of the collaborative plant in treating pollution by manganese of microorganism,
Manganese ion content shows in the nutrient solution through measuring, and this effect of testing the collaborative plant in treating soil pollution by manganese of said microorganism is obvious, is applicable on a large scale and promotes the use of.
Description of drawings
Fig. 1 is the calibration curve of manganese;
Fig. 2 is for containing Mn 2+Nothing soil nutrient solution in Mn2 +Curve over time.
The specific embodiment
Agents useful for same of the present invention all adopts commercially available chemical pure product.
One. set up calibration curve
Figure 720769DEST_PATH_IMAGE006
formaldoxime reagent: 8.0 gram hydroxylamine hydrochlorides are dissolved in the 50.0mL water; Add 4.0mL formaldehyde (37%) again, constant volume is in the 100mL volumetric flask.
EDTA solution (0.015mol/L): take by weighing the 5.6g disodium ethylene diamine tetraacetate, constant volume is in the 1000mL volumetric flask.
Figure 868034DEST_PATH_IMAGE004
hydroxylamine hydrochloride solution (5%): accurately take by weighing the 5.0g hydroxylamine hydrochloride, constant volume is in the 100mL volumetric flask.
Cook titer (Mn with manganese sulfate 2+).Accurately take by weighing 0.7684g MnSO 4H 2O, constant volume shakes up subsequent use in the 250mL volumetric flask with being transferred to behind the deionized water dissolving.Reagent dosage and addition sequence that the formaldoxime method is measured manganese content are: get solution 2.00mL to be measured earlier in the volumetric flask of 50mL; Add about 10 mL of water; Add 0.50mL ammoniacal liquor (1:1) solution, 0.60mL formaldoxime, 0.20mL hydroxylamine hydrochloride (5%), 1.00mL EDTA (0. 015mol/L) more successively; Constant volume, shake up, colour developing 10min measures absorbance in 375nm.
Titer is diluted 1,4,8,10 respectively, and 20 times, promptly manganese content is respectively 1g/L 0.5g/L
0.25g/L 0.125g/L 0.1g/L 0.05g/L. gets 2ml respectively then in the 50ml volumetric flask, surveys absorbance with the formaldoxime method, is reference with the blank.
The result shows that manganese content has good linear relationship in the 0.1g/L-1g/L scope.Regression equation: y=0.823x+0.1123 R2=0.9996. calibration curve is as shown in Figure 1.
Two. the preparation of strain fermentating liquid
1. the preparation of bacterial classification R-118 and R-2:
1) gets the manganese ore surface mass, add deionized water and stirring, process former bacteria suspension, former bacteria suspension is uniformly coated on the solid-state PYCM culture medium flat plate;
2) under 25～28 ℃ of constant temperature, cultivated 1～2 day picking list bacterium colony line purifying, 25～28 ℃ of cultivations in solid-state PYCM culture medium; Gained manganese oxidizing microorganisms bacterial strain to be selected, at liquid PYCM culture medium, shaking speed is 200r/min; Temperature is 28 ℃, cultivates 2-4 days, measures the content of manganese ion in the nutrient solution; Filter out and obtain two kinds of manganese oxidizing microorganisms, called after R-118 and R-2 bacterial classification.
2.LB medium component: peptone 1g; Yeast extract 0.5g; NaCl 1g; Agar 1.8g; Distilled water 100ml; PH transfers to 7.0;
3. a small amount of bacterial classification inoculation of picking uses in order to fermentation to fluid nutrient medium from the inclined-plane with bacterial classification R-118 and R-2.Then, flask is fixed on the shaking table, carries out the microorganism concussion and cultivate.Be fixed on flask on the shaking table with the rotary speed movement of 200-250r/min, temperature remains on 28 ℃ and cultivated 1-2 days.
4. the strain fermentation jar is cultivated
Two kinds of bacterial classifications are added different fermentation tank culture respectively, and a jar internal pressure remains on 0.05Mpa.Ferment after 36 hours, the strain fermentating liquid running into tank is used as field trial.
Three. the plantation of plant
1) plantation of alfalfa
The earth (taking from the Chongqing University of Technology campus) that will fetch earth is removed impurity, is divided into 2 parts, and every part of weight is 1000g, in 2 porcelain dishes of packing into.
Be sprinkled upon alfalfa seed uniformly in the above-mentioned soil, the surface covers one deck soil again, waters an amount of water.The temperature of illumination box is controlled at 20 ℃, the observation of plant growth conditions that waters every day, and record.In the process of plant growth, branch of moisturizing in time and nutriment.
2) contain Mn 2+The preparation of nothing soil nutrient solution
Accurately take by weighing NH with assay balance 4NO 30.3g, KH 2PO 40.19g, KNO 30.08g be dissolved in the 100ml volumetric flask behind the mixing, draw 10ml and add in the 1L beaker, add water to 1L, obtain soilless culture nutrient fluid.Accurately take by weighing 1.512g MnSO again 4Be dissolved in the soilless culture nutrient fluid, obtain containing Mn 2+Nothing soil nutrient solution.Gained contains Mn 2+Nothing soil nutrient solution, average mark installs in 6 dixie cups.
3) plantation
Be simulation pollution by manganese environment and detection easily, present embodiment adopts the soilless culture alfalfa.
Transplant after the said plant of step 1) grows to stronger root system, water an amount of water during transplanting earlier, make that soil is wetting to be beneficial to plant and to take out and do not damage root system.After taking out plant, water is cleaned the earth on the root system, and plant is fixed on the foam, and 5 strain plants are an experimental considerations unit, transplants and advances to be loaded with to contain Mn 2+The dixie cup of nothing soil nutrient solution in.Be placed under the sufficient condition of illumination and grow.Notice that the plant gap can not be excessive in each cup.
Four. alfalfa and microorganism absorption manganese detect
1. what at first will prepare contains Mn 2+Soilless culture nutrient fluid draw 2ml, record initial OD=0.456 with the formaldoxime method.Calculate Mn with calibration curve 2+Content does, 0.4179g/L. shakes up soilless culture liquid then, divides to install in 6 dixie cups, and each dixie cup 150ml contains Mn 2+Soilless culture nutrient fluid.Carry out alfalfa and bacterium adsorption test at last.Method is following:
Figure 690496DEST_PATH_IMAGE006
Number dixie cup is for containing Mn 2+Soilless culture nutrient fluid (doing blank);
Figure 957530DEST_PATH_IMAGE005
Number dixie cup is for containing Mn 2+Soilless culture nutrient fluid in add the R-118 strain fermentating liquid (abbreviating bacterium R-118 down as) of 2ml;
Figure 530331DEST_PATH_IMAGE004
Number dixie cup is for containing Mn 2+Soilless culture nutrient fluid in add 2ml R-2 strain fermentating liquid (abbreviating bacterium R-2 down as);
Figure 199210DEST_PATH_IMAGE002
Number dixie cup is that alfalfa is transplanted and added the Mn that contains of 2ml R-118 strain fermentating liquid 2+Soilless culture nutrient fluid in (abbreviate down alfalfa and bacterium R-118);
Figure 192574DEST_PATH_IMAGE001
Number dixie cup is that alfalfa is transplanted and added the Mn that contains of 2ml R-2 strain fermentating liquid 2+Soilless culture nutrient fluid in (abbreviate down alfalfa and bacterium R-2);
Figure 884586DEST_PATH_IMAGE003
Number dixie cup moves russian fenugreek herb and plants and purplely containing Mn 2+Soilless culture nutrient fluid in (abbreviate down alfalfa).
2. each dixie cup is moved to and let its growth under the sufficient condition of illumination, every mistake one day is surveyed its OD value with getting the 2ml nutrient solution with the formaldoxime method, treats requiredly to stop after respectively organizing data stabilization.
Note, when drawing nutrient solution, earlier nutrient solution is shaken up.
3. experimental result
From Fig. 2, can draw alfalfa and microorganism absorption manganese ability:
(1) Mn in the blank 2+Content increases gradually, and the reason that causes is because in open environment, the evaporation of moisture causes the concentration of manganese ion in the nutrient solution to increase.
(2) draw the ability of absorption manganese:
Figure 621598DEST_PATH_IMAGE001
alfalfa and bacterium R-118 by every curve minimum point>
Figure 82666DEST_PATH_IMAGE002
alfalfa and bacterium R-2>
Figure 512511DEST_PATH_IMAGE003
alfalfa>
Figure 691819DEST_PATH_IMAGE004
bacterium R-118>
Figure 966943DEST_PATH_IMAGE005
bacterium R-2.
(3) bacterium R-118 and R-2 reach minimum back manganese ion concentration sharply increases explanation: As time goes on, the condition that is fit to growth changes, and bacterial classification is dead gradually.Mn 2+Again be discharged into that concentration uprises again gradually in the nutrient solution.
(4) bacterial classification R-2 begins death to occur than bacterium R-118 at first, explains that the composition in the nutrient solution is more suitable for bacterium R-118 growth.
(5) content of the collaborative microorganism fungus kind absorption of alfalfa manganese is maximum, but is not equal to the amount of both additions separately.
(6) alfalfa not only has and adsorbs the manganese ability preferably, and can be over time with Mn 2+Again be discharged in the environment.
Conclusion: two experimental results through top can find out that alfalfa can tolerate manganese content soil with high, not only can normal growth, and can promote growth; The ability of absorption manganese: the content of the collaborative bacterial classification absorption of alfalfa manganese is maximum; Wherein,
Figure 49562DEST_PATH_IMAGE001
alfalfa and bacterium R-118>
Figure 384728DEST_PATH_IMAGE002
alfalfa and bacterium R-2>
Figure 113650DEST_PATH_IMAGE003
alfalfa>
Figure 130147DEST_PATH_IMAGE004
bacterium R-118>
Figure 628125DEST_PATH_IMAGE005
bacterium R-2.

Claims (5)

1. the method for the collaborative plant in treating pollution by manganese of a microorganism is characterized in that following steps are arranged:
1) gets bacterial classification R-118 and R-2;
2) picking bacterial classification R-118 and R-2 are inoculated in the liquid LB culture medium, temperature is 28 ℃, the rotating speed of 200-250r/min, and concussion was cultivated 1-2 days, obtained bacterial classification R-118 and R-2 after cultivating;
3) will use different fermentation tank culture respectively with R-2 through the bacterial classification R-118 after cultivating, a jar internal pressure is 0.05Mpa, ferments 36 hours, obtains R-118 and R-2 strain fermentating liquid;
4) will adsorb the stronger plant transplantation of manganese to containing Mn 2+Nothing soil nutrient solution in, add R-118 or R-2 strain fermentating liquid, move under the sufficient condition of illumination, grow 5-7 days, detect the manganese ion content, the manganese ion concentration reduction.
2. require 1 described method according to patent, it is characterized in that, the described Mn that contains of step 4) 2+The collocation method of nothing soil nutrient solution be: get NH 4NO 30.3g, KH 2PO 40.19g, KNO 30.08g draw 10ml behind the dissolving mixing, add water to 1L, add 1.512g MnSO again 4Mixing.
3. require 1 described method according to patent, it is characterized in that, contain Mn in the step 4) 2+Nothing soil nutrient solution and the volume ratio of strain fermentating liquid be 75:1.
4. require 1 described method according to patent, it is characterized in that, the stronger plant of said absorption pollution by manganese is a legume.
5. require 1 described method according to patent, it is characterized in that, the preparation method of said bacterial classification R-118 and R-2 is: 1) get the manganese ore surface mass, add deionized water and stirring, process former bacteria suspension, former bacteria suspension is uniformly coated on the solid-state PYCM culture medium flat plate; 2) under 25～28 ℃ of constant temperature, cultivated 1～2 day picking list bacterium colony line purifying, 25～28 ℃ of cultivations in solid-state PYCM culture medium; Gained manganese oxidizing microorganisms bacterial strain to be selected, at liquid PYCM culture medium, shaking speed is 200r/min; Temperature is 28 ℃, cultivates 2-4 days, measures the content of manganese ion in the nutrient solution; Filter out the manganese oxidizing microorganisms, obtain R-118 and R-2 manganese oxidizing microorganisms bacterial classification.
CN2011104174723A 2011-12-14 2011-12-14 Method for controlling manganese pollution by cooperation of microorganism and plants Pending CN102489499A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106180175A (en) * 2016-07-11 2016-12-07 董晓 A kind of method planting Brassica campestris L restoration of soil polluted by heavy metal
CN110142285A (en) * 2019-05-24 2019-08-20 上海交通大学 Trichoderma asperellum-alfalfa renovation of heavy metal polluted soil with combined method

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106180175A (en) * 2016-07-11 2016-12-07 董晓 A kind of method planting Brassica campestris L restoration of soil polluted by heavy metal
CN106180175B (en) * 2016-07-11 2019-04-16 绿艺园林建设有限公司 A method of plantation rape restoration of soil polluted by heavy metal
CN110142285A (en) * 2019-05-24 2019-08-20 上海交通大学 Trichoderma asperellum-alfalfa renovation of heavy metal polluted soil with combined method
CN110142285B (en) * 2019-05-24 2021-02-12 上海交通大学 Method for remedying heavy metal contaminated soil by combination of trichoderma asperellum and alfalfa

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Application publication date: 20120613