CN102703341B - Urease-producing microorganisms and method for solidifying heavy metals in foundation using same - Google Patents
Urease-producing microorganisms and method for solidifying heavy metals in foundation using same Download PDFInfo
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Abstract
Urease-producing microorganisms of Sporosarcina antarctica UR53, Sporosarcina koreensis UR47, Sporosarcina sp. UR31 and Bacillus lentus UR41 were preserved in China General Microbiological Culture Collection Center (CGMCC) on March 16, 2012 with the preservation numbers of CGMCC NO.5916, CGMCC NO.5915, CGMCC NO.5913 and CGMCC NO.5914, respectively. The method for solidifying heavy metals in the foundation by using the urease-producing microorganisms comprises the steps of: carrying out fermentation culture on the four microorganisms and microorganisms of Sporosarcina pasteuril and Terrabacter tumescens in a fermentation culture medium to obtain microorganism liquid, adding reaction liquid urea to the microorganism liquid, and then adding to the solution containing heavy metals to form a mixed solution, so that microorganism-heavy metal flocculates are formed in the mixed solution and then water-insoluble heavy metal carbonates are further formed. The method for solidifying the heavy metals in the foundation by using the microorganisms has the advantages of short solidifying time, good effect, low cost and no secondary pollution to environment.
Description
Technical field
The invention belongs to microorganism and curing heavy metal technical field thereof, be specifically related to the method for heavy metal in urease-producing microorganism and curing ground thereof.
Background technology
Heavy metal contamination refers to due to the mankind's production and activity, causes heavy metal content in environment apparently higher than its background value, because heavy metal ion has prolonged stay and nondegradable characteristic, ecotope to be caused to very big destruction.Along with large-scale urban sprawl and construction, many buildings are near discarded factory and refuse tip, and in ground, heavy metal contamination is serious.Heavy metal element can finally enter in organism by food chain, destroys organism metabolism normally, and serious harm HUMAN HEALTH, has become very important environmental problem.
Traditional heavy metal treatment process mainly comprises: chemical precipitation method, ion exchange method, evaporation concentration method, electrolytic process, gac and silica gel adsorption and membrane separation process etc., but these methods exist remove thoroughly, the shortcoming such as somewhat expensive, generation toxic sludge or other waste materials.Therefore, heavy metal treatment technology and the technique of research and development efficient and environment-friendly type become one of focus of research.
The development of modern biotechnology, comes into one's own microbial treatment heavy metal contamination gradually.Microorganism treatment is to utilize the biomaterials such as bacterium, fungi, algae and life Metabolic activity thereof to remove and/or accumulation heavy metal, thereby reduces the concentration of Mobility of Heavy Metals In Soil Environment ion.With traditional treatment technology, compare and have clear superiority, as low in its processing cost, treatment effect is good, and biochemical treatment after stain thing residual quantity can reach very low level, thereby this technology becomes the recovery technique that has development potentiality and market outlook most.
Summary of the invention
The problem existing in order to solve above-mentioned prior art, the object of the present invention is to provide the method for heavy metal in urease-producing microorganism and curing ground thereof, adopt microorganism of the present invention to solidify heavy metal in ground, have that set time is short, effective, cost is low, and can not cause secondary pollution to environment.
In order to achieve the above object, the technical solution adopted in the present invention is:
Urease-producing microorganism: circle spore gemma sarcina Sporosarcina antarctica UR53, Korea S gemma sarcina Sporosarcina koreensis UR47, gemma sarcina Sporosarcina sp.UR31 and bacillus lentus Bacillus lentus UR41 have been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 16th, 2012, deposit number is respectively CGMCC No.5916, CGMCC No.5915, CGMCC No.5913, CGMCC No.5914, CGMCC is called for short at this preservation center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.
Described round spore gemma sarcina Sporosarcina antarctica UR53, Korea S gemma sarcina Sporosarcina koreensis UR47, gemma sarcina Sporosarcina sp.UR31 and bacillus lentus Bacillus lentus UR41 are all ellipse bar, there is gemma, without pod membrane, Gram-positive.At NH
4-YE is yeast extract 20g/L, and on ammonium sulfate 10g/L flat board, bacterium colony is rounded, and surface wettability is smooth, neat in edge, and bacterium colony size is 1-2mm, and it is faint yellow that bacterium colony is, and this bacterium all can grow under the scope of the substratum temperature of 4 ℃-37 ℃ and pH7-9.5.
Urease-producing microorganism is solidified the method for heavy metal in ground, comprises the steps:
Step 1: by microorganism Sporosarcina pasteurii, Terrabacter tumescens, circle spore gemma sarcina Sporosarcina antarctica UR53, Korea S gemma sarcina Sporosarcina koreensis UR47, gemma sarcina Sporosarcina sp.UR31 and the single bacterium colony of bacillus lentus Bacillus lentus UR41 respectively under 25 ℃ of-37 ℃ of conditions in fermention medium fermentation culture 12-60 hour obtain six kinds of bacterium liquid;
Step 2: adding respectively concentration in six kinds of bacterium liquid that obtain to step 1 is the reaction solution urea soln of 0.01-2mol/L, it is 1:1-1:20 that bacteria liquid amasss with urea soln volume ratio, in the heavy metal solution that again the bacterium liquid that adds reaction solution urea soln to be joined respectively containing heavy metal concentration be 0.1g/L-5g/L, form mixing solutions, heavy metal solution is long-pending than being 1:1-1:100 with the bacteria liquid containing reaction solution urea soln, make to form in mixing solutions microorganism-heavy metal flocs unit, and then generate water-fast heavy metal carbonate.
Described in step 1, microorganism Sporosarcina pasteurii comes from US mode culture collection warehousing (American type culture collection), is numbered ATCC11859; Described microorganism Terrabacter tumescens comes from China Committee for Culture Collection of Microorganisms's common micro-organisms center, is numbered AS.1.2690
Described in step 1, fermention medium comprises yeast extract 10-20g/L, ammonium sulfate or ammonium chloride 10g/L, and pH is 7-9.5.
Compared to the prior art, tool has the following advantages in the present invention:
1, the microorganism of screening energy curing heavy metal from existing urease-producing bacterial strain, soil sample and water sample, and unknown bacterium is carried out to Molecular Identification, the microorganism strains that can effectively administer heavy metal contamination for obtaining provides candidate resource;
Under the effect of the urase that 2, urea produces in Institute of Micro-biology, be decomposed into ammonium root and carbonic acid gas, make simultaneously bacterium around pH raise, impel and in solution, form microorganism-heavy metal flocs unit, further generate water-fast heavy metal carbonate, thereby heavy metal ion is cured; There is the curing time of administering of heavy metal contamination short, effective, within 48 hours, just can make heavy metal ion curing degree reach more than 90%;
3, the cost of nutritive medium used is low;
4, in the present invention the microorganism that utilizes be microorganism itself that exist in ground (soil) or the culture of certain microorganism wherein, nutritive substance is also crude substance, can not cause secondary pollution to environment.
Accompanying drawing explanation
Fig. 1 is the urease-producing bacterial strain phylogenetic tree according to 16S rDNA sequence construct.
Fig. 2 (uses OD for cultivating the biomass of bacterial strain
600value representation) and urease activity, X-coordinate is different types of urease-producing bacterial strain, and ordinate zou is biomass and urease activity.
Fig. 3 is the precipitation removal rate to different sorts heavy metal, and X-coordinate is the urease-producing bacterial strain of different kind, the curing removal rate of ordinate zou heavy metal.
Fig. 4 is heavy metal precipitation curve, and X-coordinate is the time, and take hour is unit, the curing removal rate of ordinate zou heavy metal.
Fig. 5 is the stereoscan photograph of the cured article of heavy metal; Wherein
Fig. 5 a is the stereoscan photograph of the cured article of heavy metal nickel;
Fig. 5 b is the stereoscan photograph of the cured article of heavy metal copper;
Fig. 5 c is the stereoscan photograph of the cured article of heavy metal lead;
The attach most importance to stereoscan photograph of cured article of cobalt metal of Fig. 5 d;
Fig. 5 e is the stereoscan photograph of the cured article of heavy metal zinc;
Fig. 5 f is the stereoscan photograph of the cured article of heavy metal cadmium.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Embodiment:
Urease-producing has the separation screening of curing heavy metal ability bacterial strain
1, sample collecting
Sporosarcina pasteurii comes from US mode culture collection warehousing (American type culture collection), be numbered ATCC11859, Terrabacter tumescens comes from China Committee for Culture Collection of Microorganisms's common micro-organisms center, is numbered AS.1.2690.
2, the Isolation and screening of urease-producing bacterial strain
Urease-producing strains separation gathers pedotheque from Tsing-Hua University flower nursery.
Bacterium has urea decomposing enzyme, can produce a large amount of ammonia by decomposing urea, makes substratum be alkalescence, aobvious red.This experiment utilizes this characteristic, test sample is first at 37 ℃, under 5M high concentration urea condition after enrichment culture 24h, kill the various microbial nutrition somatocyte that can not tolerate and utilize high concentration urea, again the nutrient solution after processing is carried out to gradient dilution, coating urase screening and culturing is dull and stereotyped, at 37 ℃, cultivate, the bacterial strain that picking reddens substratum color, the separated single bacterium colony of ruling, the microorganism obtaining is urease-producing microorganism, and utilize 16S rDNA method to identify, difference called after circle spore gemma sarcina Sporosarcina antarctica UR53, Korea S gemma sarcina Sporosarcina koreensis UR47, gemma sarcina Sporosarcina sp.UR31, bacillus lentus Bacillus lentus UR41, Fig. 1 is the urease-producing bacterial strain phylogenetic tree according to 16S rDNA sequence construct.
The substratum heavy metals immobilization of urease-producing bacterial strain
1, strain culturing
Preparation fermention medium: yeast extract 10-20g/L, ammonium sulfate or ammonium chloride 10g/L, pH is 7-9.5, pack 100ml fermention medium in 500ml culturing bottle sterilizing, the single bacterium colony Sporosarcina of picking pasteurii from flat board respectively, Terrabacter tumescens, circle spore gemma sarcina Sporosarcina antarctica UR53, Korea S gemma sarcina Sporosarcina koreensis UR47, gemma sarcina Sporosarcina sp.UR31 and bacillus lentus Bacillus lentus UR41 are inoculated in respectively in fermention medium, at 30 ℃ of temperature, cultivate, rotating speed is 150rpm-250rpm.Cultivate and collect bacterium liquid after 16 hours, detect the biomass of six kinds of bacterium liquid and (use OD
600value representation) and urease activity, detected result as shown in Figure 2.
2, heavy metal solidifies
Get respectively six kinds of bacterium liquid 1ml, add the urea soln of equal-volume 0.5mol/L to make mixing solutions, six kinds of Duplicate Samples of every kind of bacterium solution preparation, then the mixing solutions that is 2ml by volume to join respectively volume be 0.5ml, the heavy metal solution NiCl that concentration is 2g/L
2, CuCl
2, PbCl
2, CoCl
2, ZnCl
2and CdCl
2in, result shows, all urease-producing bacterial strains to the curing clearance of above six heavy metal species all more than 88%, UR47 is the highest to the curing clearance of copper and lead, UR31 is the highest to the curing clearance of cobalt and zinc, Terrabacter tumescens is the highest to the curing clearance of nickel and cadmium, and result as shown in Figure 3.
In experiment, also get respectively bacterium liquid: Korea S gemma sarcina Sporosarcina koreensis UR47, gemma sarcina Sporosarcina sp.UR31, Terrabacter tumescens, the urea soln that adds respectively different concns, bacteria liquid is long-pending is respectively 1:1 with urea soln volume ratio, 1:10, 1:20, making urea final concentration is 0.25mol/L, get two samples containing the Korea S gemma sarcina Sporosarcina koreensis UR47 solution of urea soln, adding respectively concentration is the copper solutions of 0.5g/L, concentration is the lead solution of 5g/L, two samples are 1:10 and 1:100 with the volume ratio of copper solutions and lead solution respectively, get two samples containing the gemma sarcina Sporosarcina sp.UR31 solution of urea soln, add respectively cobalt and zinc solution, get two samples containing the Terrabacter tumescens solution of urea soln, add respectively nickel and cadmium solution, the concentration of metallic solution and volume ratio add identical with Korea S gemma sarcina Sporosarcina koreensis UR47 and metallic solution.Result shows that, under different bacterium liquid, urea, concentration of heavy metal ion condition, the deposition of heavy metal ion is all more than 88%.
3, heavy metals immobilization speed
Analyze respectively Korea S gemma sarcina Sporosarcina koreensis UR47 to copper and lead, gemma sarcina Sporosarcina sp.UR31 is to cobalt and zinc, Terrabacter tumescens to the curing clearance of nickel and cadmium over time, result as shown in Figure 4, the solidification process of this six heavy metal species all mainly occurs in and adds first 20 minutes of bacterium liquid, reaches maximum value at 48 hours.
Analysis to formed heavy metals immobilization thing
1, the analysis of heavy metals immobilization thing
Sedimentable matter is carried out to XRD analysis, and formed heavy metals immobilization thing is heavy metal carbonate.Throw out is carried out to electron microscopic observation, result as shown in Figure 5, Fig. 5 a wherein, Fig. 5 d is heavy metal Ni and Co cured article, is hexahedron shape, length of side 10-40 μ m; Fig. 5 b, Fig. 5 f is the cured article of heavy metal Cu and Cd, is spherical, diameter 5-10 μ m; Fig. 5 c and Fig. 5 e are the cured articles of heavy metal Pb and Zn, are needle-like length at 20-50 μ m.
2, the acidproof property analysis of heavy metals immobilization thing
The formed heavy metals immobilization thing of microorganism, is exposed in air, can be subject to the threat of acid rain in environment, therefore, need to detect its acidproof character.The acidproof character of testing formed heavy metals immobilization thing with the sulfuric acid of a series of pH values, pH value is respectively: 0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0, and 5.5.From the sulfuric acid of pH 5.5, start test, drip sulfuric acid to the sand post of bonding, with magnifying glass, examine two minutes, observe and whether have bubble formation.If there is no bubble formation, can tolerate the acid of this pH, then with the sulfuric acid of next pH value, continue experiment, until there is bubble formation, acid-fast ability is for producing the pH value of the previous sulfuric acid of bubble.The tolerance acid pH value that the results are shown in Table 1, six heavy metal species cured article is 2.0, can be in acid rain environment stable existence.
The acidproof experimental result of table 1 heavy metals immobilization thing (+: without Bubble formation-: have Bubble formation)
Claims (4)
1. urease-producing microorganism, it is characterized in that: described urease-producing microorganism is circle spore gemma sarcina Sporosarcina antarctica UR53, on March 16th, 2012, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.5916, CGMCC is called for short at this preservation center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.
2. urease-producing microorganism according to claim 1, is characterized in that: described round spore gemma sarcina Sporosarcina antarctica UR53 is ellipse bar, has gemma, without pod membrane, and Gram-positive; At NH
4-YE is yeast extract 20g/L, and on ammonium sulfate 10g/L flat board, bacterium colony is rounded, and surface wettability is smooth, neat in edge, and bacterium colony size is 1-2mm, it is faint yellow that bacterium colony is, and under the scope that this bacterium is 7-9.5 at the substratum temperature of 4 ℃-37 ℃ and pH, all can grow.
3. the method that described in claim 1, urease-producing microorganism is solidified heavy metal in ground, is characterized in that: comprise the steps:
Step 1: by the single bacterium colony of described round spore gemma sarcina Sporosarcina antarctica UR53 under 25 ℃ of-37 ℃ of conditions in fermention medium fermentation culture 12-60 hour obtain bacterium liquid;
Step 2: adding concentration in the bacterium liquid obtaining to step 1 is the reaction solution urea soln of 0.01-2mol/L, it is 1:1-1:20 that bacteria liquid amasss with urea soln volume ratio, in the heavy metal solution that again the bacterium liquid that adds reaction solution urea soln to be joined containing heavy metal concentration be 0.1g/L-5g/L, form mixing solutions, heavy metal solution is long-pending than being 1:1-1:100 with the bacteria liquid containing reaction solution urea soln, make to form in mixing solutions microorganism-heavy metal flocs unit, and then generate water-fast heavy metal carbonate.
4. method according to claim 3, is characterized in that: described in step 1, fermention medium comprises yeast extract 10-20g/L, ammonium sulfate or ammonium chloride 10g/L, and pH is 7-9.5.
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