CN101429486B - Brood cell bacillus MK3-1 for purification of heavy metal pollution and uses thereof - Google Patents
Brood cell bacillus MK3-1 for purification of heavy metal pollution and uses thereof Download PDFInfo
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- CN101429486B CN101429486B CN2008101979019A CN200810197901A CN101429486B CN 101429486 B CN101429486 B CN 101429486B CN 2008101979019 A CN2008101979019 A CN 2008101979019A CN 200810197901 A CN200810197901 A CN 200810197901A CN 101429486 B CN101429486 B CN 101429486B
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Abstract
The invention belongs to the technical field of environmental microorganisms, and particularly relates to Bacillus sp MK3-1 with manganese oxidation capability and application of the Bacillus sp MK3-1 in the purification of manganese pollution. The invention is characterized in that the Bacillus sp MK3-1 which has oxidation effect on soluble bivalent manganese is obtained through the separation from soil with the manganese pollution. The strain can oxidize bivalent manganese ions in the environment of the manganese pollution into a manganese oxide precipitate which can not be dissolved into water so as to remove manganese in the environment. The strain is named the Bacillus sp MK3-1 and belongs to a manganese-oxidizing bacterium, and CCTCC NO of the strain is M208237. The preliminary study shows that the strain has good application prospect in the treatment of heavy metal water body pollution of the environment.
Description
Technical field
The invention belongs to the environmental microorganism technical field, be specifically related to a strain divalent manganesetion is had oxygenizement brood cell bacillus MK 3-1 screening and aspect purification of heavy metal pollution in purposes.
Background technology
Heavy metal generally extensively is present in occurring in nature with natural concentration, but it is because human increasing to exploitation, smelting, processing and the commercial manufacturing activities of heavy metal, cause many heavy metals such as manganese, arsenic, mercury, cadmium, cobalt etc. to enter in atmosphere, water, the soil, cause the serious environmental pollution.Wherein, pollution by manganese is comparatively common in heavy metal contamination.
At pollution by manganese, this has the problem of important society and economic implications just to have established " underground water deferrization and demanganization " as far back as China departments of science and technology in the early days of foundation, and it is theoretical and use and 1. natural oxidation method successively occurred: the flow process that comprises a series of complexity such as aeration, oxidizing reaction, precipitation, filtration.This method is comparatively effective for removing iron, and for demanganization, only depend on aeration to be difficult to phreatic pH value is brought up to the required pH of natural oxidation demanganization〉9.5 higher level, need add alkali (as lime) to improve pH value in the water, make technical process complicated more, water pH value after the processing is too high, needs could normally to use after the acidifying, has further increased management difficulty and working cost; 2. contact oxidation method: mainly be that underground water will be entered filtering layer through behind the aeration, make the manganic compound of high price be adsorbed on filter material surface and form filter membrane.This filter membrane has contact action, and the new active filter film of the oxidized formation of energy was the process of an autocatalysis after the mn ion of the solubilised state in the water body was adsorbed on the filter membrane.The defective that this method exists is that required time is long, cost height, and the filter membrane instability that forms, and efficient is not high; 3. biological oxidation process: utilize microorganism with soluble bivalent manganese (Mn
2+Thereby) be oxidized to insoluble manganese oxides precipitate and filter and remove.In addition, the biological manganese oxidation style is in removing environment in the pollution by manganese, the biological manganese oxide compound that generates has very strong absorption, displacement and precipitating action to other heavy metals (as As, Ni, Cu, Co, Cd, Cr etc.), the As (III) of strong toxicity can also be oxidized to weak As (V) the back absorption of toxicity and remove (because As (III) neutral, As (V) is electronegative).Therefore, the biological manganese oxidation has very high using value in the improvement of water, heavy metal pollution of soil with in repairing.
Manganese at occurring in nature with II, III, three kinds of valence states of IV exist, under anaerobic and low pH condition, Mn (II) chemically reactive is very strong, on the contrary, under aerobic and high pH value condition, the chemically reactive height of trivalent and tetravalence manganese.Bivalent manganese is main dissolving form, in solution usually with MnCl
2, MnOH
+Exist etc. form.Brood cell bacillus MK 3-1 of the present invention can be oxidized to divalent manganesetion insoluble manganese oxides precipitate, thereby manganese and other heavy metal are removed from sewage, but also can cooperate emerging microorganism embedding techniques, reach effective purification to manganese, arsenic and other Heavy-metal Polluted Environment.
Summary of the invention
The object of the invention is to overcome the defective of existing environment heavy metal contamination purification techniques, separates to obtain a strain manganese oxidation bacterium (bacillus).This bacterial strain can be oxidized to the divalent manganesetion of solvable water in the environment insoluble manganese oxides precipitate, thereby it is removed from sewage, the biological manganese oxide compound that generates also has absorption, displacement or oxygenizement to other heavy metals, can be applicable to purify the water body of manganese heavy metal contamination.
The present invention is achieved through the following technical solutions:
The inventor separates, screens a strain manganese oxidation bacterium (bacillus), this bacterial strain is named as MK3-1, belong to bacillus (Bacillus sp), this bacterial strain of bacillus (Bacillus sp) MK3-1 delivers Chinese typical culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on November 25th, 2008, and its preserving number is CCTCC NO:M208237.
The screening scheme of brood cell bacillus MK 3-1 of the present invention is referring to shown in the accompanying drawing 1.Take the topsoil sample of Tianjin City, China Xiqing District malleable iron factory manganese raw material silo earlier, add the finite concentration (detailed description of seeing below, down together) MnCl2 carries out enrichment culture, again the soil sample of enrichment culture is diluted and be coated with contain 20mmol/L hydroxyethyl croak piperazine second thiosulfonic acid (N '-a-hydroxythylpiperazine-N '-ethanesulfanic acid, be called for short HEPES, transfer pH to 7.5) and 0.15mmol/L MnCl
2K solid medium flat board (reference literature: L.G.van Waasbergen et al.Geneticanalysis of the marine manganese-oxidizing Bacillus sp.strain SG-1:protoplast transformation, Tn917mutagenesis, and identification of chromosomal loci involved in manganese oxidation.J Bacteriol, 1993,175:7594-7603), 28 ℃ of incubators are cultivated a week.Picking surface for tan bacterium colony rule mono-clonal, use LBB method (W.E.Krumbeinet al.A new method for the detection and enumeration of manganese oxidizing and reducing microorganisms.Helgol.Wiss.Meeresunters.1973 again, 25:347-356) detect to determine whether being manganese oxidation bacterium, again detected manganese oxidation bacterium is cooked relevant evaluation work such as 16S ribosomal RNA gene (16S rDNA), morphology and functional gene, finally obtained brood cell bacillus MK 3-1.
More detailed technological step is seen giving an example of " embodiment ".
Positively effect of the present invention is:
At present, since human increasing to exploitation, smelting, processing and the commercial manufacturing activities of heavy metal, cause many heavy metals such as manganese, arsenic, mercury, cadmium, cobalt etc. to enter in atmosphere, water, the soil, cause the serious environmental pollution.Separation screening of the present invention to brood cell bacillus MK 3-1 divalent manganesetion can be oxidized to manganese oxides precipitate and remove.Can also cooperate emerging microorganism entrapping method to administer pollution by manganese.And the biological manganese oxide compound has very strong absorption, displacement and precipitating action to other heavy metals, even the As (III) of strong toxicity can also be oxidized to weak As (V) the back absorption removal (because As (III) neutral, As (V) is electronegative) of toxicity.Therefore, the biological manganese oxidation has very high using value in the improvement of water, heavy metal pollution of soil with in repairing.The isolating manganese oxidation of the present invention bacterium MK3-1 has the ability that potential is removed other heavy metal, and this bacterium separation screening in the pollution by manganese environment arrives, and the pollution by manganese environment is had stronger adaptability, is expected to playing a significant role aspect the pollution by manganese purifying.
Description of drawings
Fig. 1: be technological line figure of the present invention.
Fig. 2: being brood cell bacillus MK 3-1 of the present invention identifies the photo as a result of its manganese oxidisability with the LBB method, among the figure: a: distilled water+LBB; B: the substratum+LBB that does not connect bacterium; C: the substratum+LBB that connects brood cell bacillus MK 3-1.
Fig. 3: be the stereoscan photograph of brood cell bacillus MK 3-1 of the present invention, magnification and scale are indicated in the drawings, among the figure: a:Bacillus sp.MK3-1 cell, b: the Bacillus sp.MK3-1 cell that is adsorbing the biological manganese oxide compound.
Fig. 4: be the colonial morphology photo of brood cell bacillus MK 3-1 of the present invention, among the figure: a: in the K substratum; B: in 0.15mmol/L K substratum
Fig. 5: be brood cell bacillus MK 3-1 manganese oxidation graphic representation of the present invention, Fig. 5-a:Bacillus sp.MK3-1 in the K substratum to oxidation/removal curve of Mn (II); Fig. 5-b makes oxidation/removal curve of Bacillus sp.MK3-1 Mn (II) in the MnCl2 of the 0.15mmol/L aqueous solution of embedding microbial inoculum.Annotate: Fig. 5-a:
: cultivate and concentrate remaining Mn (II) content;
: cultivate and concentrate Mn (II) content that is adsorbed;
Mn in the Mn oxide (II) content;
Blank (not connecing the MnCl2K substratum of the 0.15mmol/L of bacterium); Fig. 5-b
The cell concentration of MK3-1;
0.15mmol/L the concentration of remaining Mn (II) in the MnCl2 aqueous solution;
PH;
Blank (not connecing the MnCl2 aqueous solution of the 0.15mmol/L of bacterium)
Embodiment
Embodiment 1: separate from pollution by manganese soil and evaluation manganese oxidation brood cell bacillus MK 3-1
(1) sample is taked: in late June, 2007 is gathered the topsoil sample of Tianjin City, China Xiqing District malleable iron factory manganese raw material silo.
(2) example enrichment: get the soil sample of 100g, in soil sample, add aseptic MnCl
2It is 989.55mg/Kg that solution makes its final concentration, stirs evenly gently to put in 28 ℃ of incubators to cultivate a week, notes adding sterilized water, guarantees sample humidity.
(3) manganese oxidation bacterium separates: accurately take by weighing through MnCl
2The soil sample 10g of enrichment puts in 28 ℃ of shaking tables and vibrates half an hour in the triangular flask that the 90ml stroke-physiological saline solution is housed, and gets 1ml more successively and joins in the 9ml stroke-physiological saline solution and progressively be diluted to 10
-2, 10
-3, 10
-4, get respectively 0.1ml coating contain 20mmol/L hydroxyethyl croak piperazine second thiosulfonic acid (N '-a-hydroxythylpiperazine-N '-ethanesulfanic acid, be called for short HEPES, transfer pH to 7.5) and 0.15mmol/L MnCl
2K solid medium flat board (reference literature: L.G.van Waasbergen et al.Geneticanalysis of the marine manganese-oxidizing Bacillus sp.strain SG-1:protoplast transformation, Tn917mutagenesis, and identification of chromosomal loci involved in manganese oxidation.J Bacteriol, 1993,175:7594-7603), 3 flat boards of each extent of dilution coating, put in 28 ℃ of incubators and cultivate a week, with the surface is that tan bacterium colony is decided to be doubtful manganese oxidation bacterium, puts in 4 ℃ of refrigerators flat board stand-by.K solid culture based formulas is as follows: yeast extract paste 0.5g, and peptone 2g, agar 15g,, artificial seawater 750mL replenishes distilled water to 1L (artificial seawater: NaCl 17.53g, KCl 0.75g, MgSO
47H
2O 12.32g, CaCl
21.11g, replenish distilled water to 1L).
(4) line separates: the bacterium colony line that the doubtful manganese oxidation bacterium picking that obtains in the step (3) is different, guarantee to obtain mono-clonal, and the K culture medium flat plate is used in line, treats to put after bacterium grows in 4 ℃ of refrigerators stand-by and a in-80 ℃ of refrigerators with the preservation of glycerine freeze pipe.
(5) bacterial strain manganese oxidizing property is identified: the mono-clonal that obtains in the step (4) is transferred to contains 20mmol/L HEPES (pH7.5) and 0.15mmol/LMnCl
2In the K liquid nutrient medium, put shaking culture in 28 ℃ of shaking tables, treat to use when the substratum color becomes brown Berbelin blue-I (LBB, N.N '-Dimethylamino-p, p '-triphenyhnethane-o ＂-sulphonicacid) is its oxidisability of ratio reaction detection of 1:3 in substratum and LBB volume ratio.If changing, solution colour do not illustrate that then this bacterium does not have the manganese oxidisability, if color is become dark sapphirine and illustrated that then this bacterium has manganese oxidisability (seeing accompanying drawing 2) by light blue.(LBB is the staining agent of redox agent, Mn
+ 3+ LBB (reduced)〉Mn
+ 2+ LBB (oxidized), oxidized LBB are dark sapphirine).K liquid culture based formulas does not just contain agar with the K solid medium.The compound method of LBB is as follows: spend the night with 4 ℃ of dark places of 40mmol/L aqueous acetic acid of 100ml and dissolve the LBB of 40mg.
(6) classification of manganese oxidation bacterium is identified: the one, and utilize 16S rDNA to identify, (the PCR concrete grammar is authorized patent of invention (patent No.: ZL 200510120584.7, denomination of invention: a kind of small quality fast extraction method for soil total DNA) referring to Hua Zhong Agriculture University promptly to adopt prokaryotic organism 16S rDNA universal primer 27F (5 ' AGAGTTTGATCMTGGCTCAG3 ') and 1492R (5 ' GGYTACCTTGTTACGACTT3 ') to be PCR.Increase its 16S rDNA and order-checking, with international NCBI GenBank (www.ncbi.nlm.nih.gov) Nucleotide database comparison, nucleotide homology is 98%, is accredited as bacillus Bacillus again; The 2nd, utilize scanning electron microscope identification of morphology (seeing accompanying drawing 3), gramstaining analysis and growth characteristics to identify.
The mycology feature is as follows: the thalline direct rod shape, be about 1.94 μ m, and wide about 1.29 μ m, gram-positive microorganism, suitable growth temperature 28-30 ℃, appropriate pH 7.6-7.8 is not containing MnCl
2The K solid medium on, bacterium colony is circular, oyster white, projection, surface wettability, the edge is irregular.Containing the MnCl of 0.15mmol/L
2On the K substratum, the bacterium colony surface is brown (seeing accompanying drawing 4); The 3rd, the many copper oxidase gene relevant with the manganese oxidation identified, with degenerate pcr (Chris A.et al.Enzymatic manganese (II) oxidation by metabolically dormant spores ofdiverse Bacillus species.Appl Environ Microbiol, 2002,68:874-880) isolate the gene of one section 903bp, this sequence is delivered to NCBI GenBank database, and its number of registration is FJ389508.
The method for preserving of brood cell born of the same parents bacterium MK3-1 of the present invention:
Brood cell born of the same parents bacterium MK3-1 can make short term storage after the cultivation in the last 28 ℃ of cultivations of K liquid or solid substratum (it is the same to fill a prescription) under 4 ℃.If long-term preservation, can use glycerine freeze pipe or lyophilize pipe (referring to Zhao Bin, He Shaojiang. the microbiology experiment. first version. the .2002:202 of Science Press-205) preservation strain is proper.
Embodiment 2: the manganese oxidation curve of brood cell bacillus MK 3-1
Prepare MnCl
2Concentration is respectively each three bottles of the K substratum of 0mmol/L and 0.15mmol/L, inoculates back 28 ℃, and the 150r/min shaking table is cultivated.The sample preparation step is as follows: 1. get 10mL bacterium liquid every 24h, and the centrifugal 10min of 12000r/min, supernatant liquor is a remaining manganese in the substratum with the membrane filtration of 0.2 μ m, the Mn in the filtrate (II); 2. the centrifugal back thalline CuCl of 40mmol/L
2Handle more than the 10h, treat Cu
2+Thoroughly behind the Mn2+ that adsorbs in the displacement bacterium liquid, constant volume is to 10mL, the centrifugal 10min of 12000r/min, and with the membrane filtration of supernatant with 0.2 μ m, the Mn in this filtrate (II) is the manganese that is adsorbed; 3. centrifuged deposit is handled more than the 10h with the oxammonium hydrochloride of 20mmol/L, and oxammonium hydrochloride is reduced into Mn with the manganese in the Mn oxide
2+, constant volume centrifugal 10min of 12000r/min behind the 10mL, with the membrane filtration of supernatant with 0.2 μ m, the Mn (II) in this moment filtrate is the manganese in the Mn oxide; 4. manganese content in each filtrate of aas determination.The manganese oxidation curve of the brood cell bacillus MK 3-1 of drawing is seen accompanying drawing 5-a.Shown the demanganization ability of Bacillus sp.MK3-1 in substratum by Fig. 5-a.This bacterium manganese oxidation just began at first day, and 67.53 ± 2.04% manganese was removed at the 4th day, wherein 50.51% ± 1.67% had generated Mn oxide.In the time of the 7th day, the demanganization amount reaches maximum value, removed to cultivate and concentrated 96.80% ± 2.64% manganese, wherein 77.56% ± 2.64%, Mn oxide, cultivating and concentrating the final concentration of manganese is 0.48mg/L ± 0.02, meets (HJ/T357-2007) related request of " electrolytic metal manganese industry cleaner production assessment indicator system (trying) " (People's Republic of China's national development and reform council bulletin 2007 No. 63) and " cleaner production standard electrolytic manganese industry ".And in the blank assay, manganese content does not have considerable change in the substratum, illustrates that this experiment manganese oxidation is the result of microbial process.
Embodiment 3: the mensuration of the brood cell bacillus MK 3-1 of embedding manganese oxidation capacity in simulation pollution by manganese water body
Embedding microbial inoculum (MK3-1) demanganization ability measurement operation step: 1. wheat bran pre-treatment: wheat bran oven dry back is by mix sterilization with water 1:1 (w/v); 2. thalline (MK3-1) is received (being that the dried wheat bran of 10g meets 1mL, the bacterium liquid of 1OD) in the wheat bran by 1% (g/ μ l) inoculum size, and 28 ℃ leave standstill cultivation 24h, and 50 ℃ of cryodryings obtain solid fungicide; 3. use polypropylene (PP) non-woven fabrics to make the rectangle embedding bag of 10cm * 20cm,, make the embedding microbial inoculum by the actual needs solid fungicide of packing into; 4. the embedding microbial inoculum is put in the glass jar of 20cm * 25cm * 30cm specification, is 10L in the cylinder, the MnCl of 0.15mmol/L
2The aqueous solution, experiment is carried out in 28 ℃ of thermostatic chambers; 5. get water sample one time every 24h, measure manganese content, pH and bacterial content.The removal curve of drawing is seen accompanying drawing 5-b.By the embedding microbial inoculum made with the PP non-woven fabrics embedding that contains wheat bran of Bacillus sp.MK3-1 as can be seen among Fig. 5-b, at the MnCl of 0.15mmol/L
2The demanganization ability is still stable in the aqueous solution, and demanganization speed is accelerated along with the increase of amount of bacteria.Reach peak value the 4th day removal amount, 84.80% ± 0.31% manganese is removed.Making the manganese final concentration of the aqueous solution is 0.87mg/L ± 0.02, meets (HJ/T357-2007) related request of " electrolytic metal manganese industry cleaner production assessment indicator system (trying) " (People's Republic of China's national development and reform council bulletin 2007 No. 63) and " cleaner production standard electrolytic manganese industry ".In sum, fixed thalline (MK3-1) manganese oxygen ability is stable, can effectively remove manganese in the aqueous solution.In addition, this method is fixing thalline effectively not only, and the remaining organic matter of wheat bran can make thalline still can breed after dropping into water body in the solid fungicide, thereby this entrapping method can be saved the microbial inoculum usage quantity.Selected polypropylene non-woven fabric among the present invention is drawn materials easily, and cost is low, makes simply, and therefore this embedding techniques may will obtain excellent popularization.
Claims (3)
1. the genus bacillus of a strain purification of heavy metal pollution (Bacillus sp) MK3-1 is deposited in Chinese typical culture collection center (CCTCC), and its preserving number is CCTCC NO:M208237.
2. the described genus bacillus of claim 1 is characterized in that described genus bacillus (Bacillus sp) MK3-1 can be oxidized to manganese oxides precipitate with bivalent manganese.
3. the described genus bacillus MK3-1 of claim 1 is in the application that bivalent manganese is oxidized to aspect the manganese oxides precipitate.
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| CN101974448B (en) * | 2010-09-07 | 2012-05-23 | 新疆农业科学院微生物应用研究所 | Acinetobacter and application thereof in biological treatment of heavy metal ions |
| CN102517231B (en) * | 2011-12-14 | 2013-06-19 | 重庆理工大学 | Manganese oxidizing microbe separating and screening method |
| CN103421702B (en) * | 2012-06-28 | 2015-03-11 | 华中农业大学 | Bacteria Lysinibacillus sp. for adsorbing gold and antimony |
| CN108384731B (en) * | 2018-02-05 | 2020-09-29 | 华中农业大学 | Manganese oxidizing bacteria and screening method and application thereof |
| CN114643275B (en) * | 2022-02-23 | 2024-02-20 | 青岛理工大学 | Method for in-situ remediation of arsenic in soil by bacteria and application |
| CN115532809A (en) * | 2022-09-27 | 2022-12-30 | 西南科技大学 | Process for treating heavy metal pollution of soil |
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Assignee: Yueyang city Yuanbang Er Kang Biotechnology Co. Ltd. Assignor: Huazhong Agricultural University Contract record no.: 2012430000046 Denomination of invention: Brood cell bacillus MK3-1 for purification of heavy metal pollution and uses thereof Granted publication date: 20110504 License type: Exclusive License Open date: 20090513 Record date: 20120425 |
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