CN103937703B - One strain serratia marcescens M7a and the application in heavy-metal ion removal thereof - Google Patents
One strain serratia marcescens M7a and the application in heavy-metal ion removal thereof Download PDFInfo
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Abstract
Do you the invention discloses a strain serratia marcescens (<i>Serratia</iGr eatT.GreaT.GT<i>? marcescens</i>) M7a and the application in heavy-metal ion removal thereof, serratia marcescens M7a heavy metal ion Mn
2+there is removal effect, from soil and water, screen heavy metal ion Mn
2+there is the method for the Achromobacter xylosoxidans of removal effect, comprise the following steps: the preparation of a. metal Selective agar medium; B. the acclimating of the bacterial strain of resistance to heavy metal; C. the screening of the bacterial strain of resistance to heavy metal and multiple sieve; D. the detection of thalline heavy metal removal ability.By the substratum containing metal ion, the bacterial strain of resistance to heavy metal is carried out to the detection experiment of thalline heavy metal removal ability after acclimating and purifying, sets up rationally, the screening method of effective removal heavy metal bacterial strain, obtain the bacterial strain being applicable to needs.The present invention is applied to the improvement of heavy metal contamination, has that non-secondary pollution, processing efficiency are high, wide accommodation, low cost and other advantages.
Description
Technical field
The present invention relates to a strain microorganism and the application in heavy-metal ion removal thereof, particularly a strain serratia marcescens and the application in removal heavy metal pollutes thereof, belong to detection and the applied technical field of microorganism.
Background technology
Heavy metal is distributed widely in atmospheric sphere, biosphere, litre geosphere and hydrosphere, have permanent after being discharged into environment, and have obvious storage effect, can polluted-water soil by the final enrichment in vivo of food chain circulation, thus serious harm ecotope and HUMAN HEALTH.
The heavy metal-polluted dyeing technique of microbial treatment is that the field of development is constantly explored in new rise in the world, and the biological adsorption agent process heavy metal contamination utilizing microorganism system to prepare facts have proved a kind of the most rising Heavy Metal Pollution Control method at present.Compared with traditional treatment process, it has, and non-secondary pollution, processing efficiency are high, wide accommodation, low cost and other advantages, and at low concentrations, metal selectively is removed, Separation and Recovery.China's microbe research of resistance to heavy metal mainly concentrates on the character of microorganism and heavy metal nearly ten years affects aspect to mycetocyte, and less for bacterial screening, fungus characteristic, growth conditions research.The resistance to heavy metal population be separated at present is few, thalli growth breeding is large by the impact of habitat, removal heavy metal indifferent, can't meet the pollution situation of outside actual environment complexity, in the urgent need to enriching its population diversity, improve the removal ability of heavy metal.Therefore the research of the microorganism with efficient repair ability should be strengthened, on the basis of a large amount of resistance to heavy metal microorganism of screening, can be combined with suitable technique the engineering bacteria building efficient removal heavy metal further by after strain domestication, the heavy metal contamination for microbial treatment complex environment provides more more options.
Summary of the invention
Technical problem to be solved by this invention is, by acclimating and the screening of the bacterial strain of resistance to heavy metal, from soil and water screening can removal heavy metal serratia marcescens (
serratiamarcescens) M7a, set up the screening method of the reasonable effective bacterial strain of resistance to heavy metal, and the action effect of the removal of bacteria heavy metal filtered out is evaluated, the bacterial strain that the removal heavy metal obtaining being applicable to is effective.By being combined with suitable technique further after strain domestication, build safe, effective, the economic engineering bacteria product of efficient removal heavy metal.By product application in contaminated by heavy metals soil and water, improve the efficiency of heavily administering metallic pollution, reduce secondary pollution, reduce costs, the heavy metal contamination for microbial treatment complex environment provides more more options.
Another object of the present invention is to provide the application of above-mentioned bacterial strains in heavy-metal ion removal.
This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 18th, 2014, be called for short CGMCC, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number: CGMCCNo.8829, Classification And Nomenclature: serratia marcescens.
For solving the problems of the technologies described above, the invention provides a strain serratia marcescens M7a, described serratia marcescens M7a heavy metal ion Mn
2+there is removal effect.
For solving the problems of the technologies described above, present invention also offers and a kind ofly from soil and water, screen the method that heavy metal ion has removal effect, comprising the following steps:
A. the preparation of metal Selective agar medium;
B. the acclimating of the bacterial strain of resistance to heavy metal;
C. the screening of the bacterial strain of resistance to heavy metal and multiple sieve;
D. the detection of thalline heavy metal removal ability.
Described step a may further include: calculate Manganous chloride tetrahydrate [MnCl
2× 4H
2o] in the massfraction of metal ion, be dissolved in deionized water the storage solution being made into 10g/L.Preparation basic medium: beef-protein medium (g/L)-extractum carnis 3.0, peptone 10.0, NaCl5.0.After basic medium sterilizing, metal storage solution is made an addition in substratum according to concentration requirement.
Described step b may further include: get 10g environment collected specimens and add enrichment culture in 90mL liquid beef-protein medium, and 37 DEG C of 150r/min cultivate 24h; Get 1mL enrichment culture liquid and be inoculated in fresh liquid substratum containing 0.1g/L different metal ion respectively, 37 DEG C of 150r/min cultivate; After metal Selective agar medium muddiness, therefrom get in the liquid nutrient medium that 1mL nutrient solution is inoculated in containing 0.2g/L metal ion, analogize raising concentration of metal ions step by step.According to the difference of thalline to each metal tolerance degree, the tolerance growth conditions choosing higher concentration repeatedly tames cultivation.
Described step c may further include: get the streak inoculation of the highest tolerable concentration culture in containing on the solid plate of respective metal ion with transfering loop, cultivate 24 ~ 72h for 37 DEG C.According to flat-plate bacterial colony form respectively picking list bacterium colony inoculate and verify to liquid metal Selective agar medium, circulate three times, to obtain the good pure bacterial strain of metal tolerance.
Described steps d may further include: be inoculated in the liquid metal Selective agar medium of corresponding proper concn respectively by the list of the resistance to heavy metal bacterium colony that screening obtains, and 37 DEG C of 150r/min cultivate 24 ~ 48h.The centrifugal 5min of 12000r/min, gets supernatant ICP-MS method and measures concentration of metal ions.
ICP-MS instrument test condition: reflective power 1550w, sampling depth 7mm, carrier gas flux 1.04L/min, spray chamber temperature 2 DEG C.The integral time of Mn is 0.30S, replication 3 times.Pulling speed 0.30rps, promote time 45S, steady time 30S.
Heavy metal removing rate calculation formula is:
P=[（C
0-C）/C
0]×100%
P: clearance (%);
C
0: concentration of metal ions before bacterium process;
C: concentration of metal ions after bacterium process.
For solving the problems of the technologies described above, invention further provides a strain serratia marcescens M7a, comprising following gene order:
1tttgctccccgggtgacgagcggcggacgggtgagtaatgtctgggaaactgcctgatgg
61agggggataactactggaaacggcagctaataccgcataacgtcgcaagaccaaagaggg
121ggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggta
181atggctcacctaggcgacgatccctagctggtctgagaggatgaccagccacactggaac
241tgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgca
301agcctgatgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcag
361cgaggaggaaggtggtgagcttaatacgctcatcaattgacgttactcgcagaagaagca
421ccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaatt
481actgggcgtaaagcgcacgcaggcggtttgttaagtcagatgtgaaatccccgggctcaa
541cctgggaactgcatttgaaactggcaagctagagtctcgtagaggggggtagaattccag
601gtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctgg
661acgaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagataccctggta
721gtccacgctgtaaacgatgtcgatttggaggttgtgcccttgaggcgtggcttccggagc
781taacgcgttaaatcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattg
841acgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaacctt
901acctactcttgacatccagagaactttccagagatggattggtgccttcgggaactctga
961gacaggtgctgcatggttgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaa
1021cgagcgcaacccttatcctttgttgccagcggttcggccgggaactcaaaggagactgcc
1081agtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtaggg
1141ctacacacgtgctacaatggcgtatacaaagagaagcgacctcgcgagagcaagcggacc
1201tcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatc
1261gctagtaatcgtatatcagaatgctacggtgaatacgttcccgggccttgtacacaccgc
1321ccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggagggcgc
1381ttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacct
1441gcggc。
For solving the problems of the technologies described above, the present invention has reoffered a strain serratia marcescens M7a, adopts when identifying with the following method, comprises following gene order:
1tttgctccccgggtgacgagcggcggacgggtgagtaatgtctgggaaactgcctgatgg
61agggggataactactggaaacggcagctaataccgcataacgtcgcaagaccaaagaggg
121ggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggta
181atggctcacctaggcgacgatccctagctggtctgagaggatgaccagccacactggaac
241tgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgca
301agcctgatgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcag
361cgaggaggaaggtggtgagcttaatacgctcatcaattgacgttactcgcagaagaagca
421ccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaatt
481actgggcgtaaagcgcacgcaggcggtttgttaagtcagatgtgaaatccccgggctcaa
541cctgggaactgcatttgaaactggcaagctagagtctcgtagaggggggtagaattccag
601gtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctgg
661acgaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagataccctggta
721gtccacgctgtaaacgatgtcgatttggaggttgtgcccttgaggcgtggcttccggagc
781taacgcgttaaatcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattg
841acgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaacctt
901acctactcttgacatccagagaactttccagagatggattggtgccttcgggaactctga
961gacaggtgctgcatggttgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaa
1021cgagcgcaacccttatcctttgttgccagcggttcggccgggaactcaaaggagactgcc
1081agtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtaggg
1141ctacacacgtgctacaatggcgtatacaaagagaagcgacctcgcgagagcaagcggacc
1201tcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatc
1261gctagtaatcgtatatcagaatgctacggtgaatacgttcccgggccttgtacacaccgc
1321ccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggagggcgc
1381ttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacct
1441gcggc。
Authentication method: pcr amplification adopts bacterial 16 S rDNA universal primer: 27f (5'-AGAGTTTGATCMTGGC-TCAG-3') and 1541r (5'-AAGGAGGTGATCCAGCC-3').PCR reaction system (50 μ L): MgCl
2(1.5mM), genomic dna (10ng), dNTP(200 μM), primer (0.4 μM) and
taqarchaeal dna polymerase (1.25U).Reaction conditions: 94 DEG C of sex change 30s, 50 DEG C of annealing 30s, 72 DEG C extend 1.5min, totally 30 circulations.Reclaim the specification sheets operation of test kit (TIANgelMidiPurificationKit, Dp209, TIANGEN) by PCR, purified pcr product, checking order is completed by Shanghai biotechnology company limited.
The gene order recorded is:
1tttgctccccgggtgacgagcggcggacgggtgagtaatgtctgggaaactgcctgatgg
61agggggataactactggaaacggcagctaataccgcataacgtcgcaagaccaaagaggg
121ggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggta
181atggctcacctaggcgacgatccctagctggtctgagaggatgaccagccacactggaac
241tgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgca
301agcctgatgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcag
361cgaggaggaaggtggtgagcttaatacgctcatcaattgacgttactcgcagaagaagca
421ccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaatt
481actgggcgtaaagcgcacgcaggcggtttgttaagtcagatgtgaaatccccgggctcaa
541cctgggaactgcatttgaaactggcaagctagagtctcgtagaggggggtagaattccag
601gtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctgg
661acgaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagataccctggta
721gtccacgctgtaaacgatgtcgatttggaggttgtgcccttgaggcgtggcttccggagc
781taacgcgttaaatcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattg
841acgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaacctt
901acctactcttgacatccagagaactttccagagatggattggtgccttcgggaactctga
961gacaggtgctgcatggttgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaa
1021cgagcgcaacccttatcctttgttgccagcggttcggccgggaactcaaaggagactgcc
1081agtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtaggg
1141ctacacacgtgctacaatggcgtatacaaagagaagcgacctcgcgagagcaagcggacc
1201tcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatc
1261gctagtaatcgtatatcagaatgctacggtgaatacgttcccgggccttgtacacaccgc
1321ccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggagggcgc
1381ttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacct
1441gcggc。
Picking each bacterial classification pure growth list bacterium colony, is inoculated in BUG culture medium flat plate, 33 DEG C of constant temperature culture 24h, switching 1-2 generation.Aseptically, use Inoculatorz cotton swab from BUG flat board, pick diameter and be about the colony inoculation of 3mm in IF-A inoculation liquid, turbidity is adjusted to 90-98%.Bacteria suspension is poured in V-type application of sample tank, use 8 road pipettors bacteria suspension is sequentially added into microwell plate institute porose in, every hole 100 μ L.Microwell plate 33 DEG C of constant temperature culture 16-24h, use BiologGenIIIMicroStation automatic microbe identification systems to identify.
Biolog microbial identification system utilizes microorganism to carry out the difference of respiratory metabolism to different carbon source, and BiologGenIII microwell plate carries out 94 kinds of phenotype tests (71 kinds of utilization of carbon source, 23 kinds of chemosensitivity tests) to microorganism.By the differences in turbidity (turbidity) detecting in microbial metabolism colour-change (absorbancy) that the redox materials that produces and four azole materials (as TTC, TV) react and cause and cause due to microorganism growth, generating feature finger printing, compare with reference culture spectrum data storehouse, can qualification result be drawn, see Fig. 4.
For solving the problems of the technologies described above, the present invention separately provides a strain serratia marcescens M7a and the application in removal heavy metal pollutes thereof.
The technique effect that the present invention is useful is: by the preparation of metal Selective agar medium; The acclimating of the bacterial strain of resistance to heavy metal; The screening of the bacterial strain of resistance to heavy metal and multiple sieve; The detection experiment of thalline heavy metal removal ability, sets up the screening method of reasonable, effective removal heavy metal bacterial strain from soil and water, obtains the bacterial strain being applicable to needs.The present invention is applied to the improvement of heavy metal contamination, has that non-secondary pollution, processing efficiency are high, wide accommodation, low cost and other advantages.
Below in conjunction with drawings and Examples, the present invention is described in detail.
Accompanying drawing explanation
Fig. 1 is for possessing removal Mn
2+the bacterial strain plate morphology of ability;
Fig. 2 is that bacterial strain M7a is to different concns Mn
2+energy for growth and removal effect;
Fig. 3 is that bacterial strain M7a is to different concns Mn
2+removal ability compare;
Fig. 4 is the Biolog qualification result of bacterial strain M7a.
Embodiment
Heavy-metal ion removal Mn
2+bacterial strain screening
1 experiment material
1.1 experimental strain
Sample source
From Weifang, sunshine, the area such as Nanjing gather soil sample or the water sample of different location, and pedotheque gets the veneer of soil of 1-4cm in the many places on sampling ground, mixes respectively.Sample amounts to 27 parts.
1.2 laboratory apparatus
Vertical pressure steam sterilizer (Japanese HirayamaHVE-50); Airbath shaking table (German IKAKS4000ic); Analytical balance (Switzerland MettlerToledoPL602-S); Constant incubator (German MMMIncucell111); Liquid-transfering gun (German Eppendorf); Table model high speed centrifuge (German Eppendorf5417R); Inductivity coupled plasma mass spectrometry (ICP-MS, U.S. Agilent7700X) etc.
1.3 experiment reagent
Manganous chloride tetrahydrate [MnCl
2× 4H
2o] chemical reagent is analytical pure.Calculate the massfraction of metal ion, be dissolved in deionized water the storage solution being made into 10g/L respectively.
1.4 substratum
Basic medium: beef-protein medium (g/L)-extractum carnis 3.0, peptone 10.0, NaCl5.0.
Metal Selective agar medium: after basic medium sterilizing, makes an addition to metal storage solution in substratum according to concentration requirement.
2 experimental techniques
The preparation of 2.1 metal Selective agar medium
Calculate Manganous chloride tetrahydrate [MnCl
2× 4H
2o] in the massfraction of metal ion, be dissolved in deionized water the storage solution being made into 10g/L.Preparation basic medium: beef-protein medium (g/L)-extractum carnis 3.0, peptone 10.0, NaCl5.0.After basic medium sterilizing, metal storage solution is made an addition in substratum according to concentration requirement.
2.2. the acclimating of the bacterial strain of resistance to heavy metal
Get 10g environment collected specimens and add enrichment culture in 90mL liquid beef-protein medium, 37 DEG C of 150r/min cultivate 24h; Get 1mL enrichment culture liquid and be inoculated in fresh liquid substratum containing 0.1g/L different metal ion respectively, 37 DEG C of 150r/min cultivate; After metal Selective agar medium muddiness, therefrom get in the liquid nutrient medium that 1mL nutrient solution is inoculated in containing 0.2g/L metal ion, analogize raising concentration of metal ions step by step.According to the difference of thalline to each metal tolerance degree, the tolerance growth conditions choosing higher concentration repeatedly tames cultivation.
The screening of 2.3 bacterial strains of resistance to heavy metal and multiple sieve
Get the streak inoculation of the highest tolerable concentration culture in containing on the solid plate of respective metal ion with transfering loop, cultivate 24 ~ 72h for 37 DEG C.According to flat-plate bacterial colony form respectively picking list bacterium colony inoculate and verify to liquid metal Selective agar medium, circulate three times, to obtain the good pure bacterial strain of metal tolerance.
2.4. the detection of thalline heavy metal removal ability
Be inoculated in the liquid metal Selective agar medium of corresponding proper concn respectively by the list of the resistance to heavy metal bacterium colony that screening obtains, 37 DEG C of 150r/min cultivate 24 ~ 48h.The centrifugal 5min of 12000r/min, gets supernatant ICP-MS method and measures concentration of metal ions.
ICP-MS instrument test condition: reflective power 1550w, sampling depth 7mm, carrier gas flux 1.04L/min, spray chamber temperature 2 DEG C.The integral time of Zn is 0.30S, replication 3 times.Pulling speed 0.30rps, promote time 45S, steady time 30S.
Heavy metal removing rate calculation formula is:
P=[（C
0-C）/C
0]×100%
P: clearance (%);
C
0: concentration of metal ions before bacterium process;
C: concentration of metal ions after bacterium process
2.5 times and concentration of metal ions are on the impact of clearance
Mn
2+select 0.2g/L, 0.4g/L, 0.6g/L, the substratum of 0.8g/L, 1.0g/L totally 5 groups of metal concentrations, from picking list colony inoculation flat board to the low metal concentration substratum of 100mL, get 1mL bacterium liquid after 37 DEG C of shaking culture 24h and be forwarded to higher first order concentration substratum, class is pushed into maximum concentration step by step.Each concentration every 24h sampling and measuring bacterium liquid OD
600, centrifuging and taking supernatant measures solution metal content, monitors 6 ~ 7 days continuously.
2.6 bacterial strains are to the highest tolerable concentration research of respective metal ion
According to acquired time and concentration of metal ions on the impact of clearance, strengthen metal concentration step by step, the substratum of high one-level metal concentration of transferring with 1% inoculum size after substratum muddiness, until without growth phenomenon.Every first order concentration samples after cultivating 6 days, and centrifuging and taking supernatant measures solution metal content.
2.7 identification of strains
16SrDNA gene order PCR reacts: upstream primer: AGAGTTTGATCATGGCTCAG, downstream primer: TACGGTTACCTTGTTACGACTT.Reaction conditions: 94 DEG C of 5min; 94 DEG C of 45s, 58 DEG C of 30s, 72 DEG C of 45s, totally 30 circulations; 72 DEG C of 10min.
The gene order recorded is:
1tttgctccccgggtgacgagcggcggacgggtgagtaatgtctgggaaactgcctgatgg
61agggggataactactggaaacggcagctaataccgcataacgtcgcaagaccaaagaggg
121ggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggta
181atggctcacctaggcgacgatccctagctggtctgagaggatgaccagccacactggaac
241tgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgca
301agcctgatgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcag
361cgaggaggaaggtggtgagcttaatacgctcatcaattgacgttactcgcagaagaagca
421ccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaatt
481actgggcgtaaagcgcacgcaggcggtttgttaagtcagatgtgaaatccccgggctcaa
541cctgggaactgcatttgaaactggcaagctagagtctcgtagaggggggtagaattccag
601gtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctgg
661acgaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagataccctggta
721gtccacgctgtaaacgatgtcgatttggaggttgtgcccttgaggcgtggcttccggagc
781taacgcgttaaatcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattg
841acgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaacctt
901acctactcttgacatccagagaactttccagagatggattggtgccttcgggaactctga
961gacaggtgctgcatggttgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaa
1021cgagcgcaacccttatcctttgttgccagcggttcggccgggaactcaaaggagactgcc
1081agtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtaggg
1141ctacacacgtgctacaatggcgtatacaaagagaagcgacctcgcgagagcaagcggacc
1201tcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatc
1261gctagtaatcgtatatcagaatgctacggtgaatacgttcccgggccttgtacacaccgc
1321ccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggagggcgc
1381ttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacct
1441gcggc。
3 experimental results and analysis
3.1 heavy metal Mn
2+the screening of resistant strains
Mn
2+resistant strains: through containing Mn
2+substratum acclimation and screening and repeatedly purifying checking after, have 25 strain bacteriums at Mn
2+usual dispatch still can be kept to grow when reaching the concentration of more than 0.6g/L, be labeled as M2, M3, M4, M5, M6, M7a, M7c, M8a, M8b, M9, M10a, M10b, MnB4, MnB5a, MnB5c, MnB6, MnB7, MnB10, MnB11, MnC1, MnC2, MnC3, MnC4a, MnC4b, MnC6 respectively.
3.2 heavy metal Mn
2+the mensuration of removal ability
Mn
2+tolerance bacterium has 25 strains, wherein has 15 strains to show Mn
2+removal ability in various degree, although all the other 10 strains can have Mn
2+environment in grow, but the Mn in solution supernatant
2+content has no obvious minimizing.Table 1 lists 15 strains to be had except Mn
2+the bacterial strain situation of ability, the plate morphology of its pure culture as shown in Figure 1.
Table 1 possesses removal Mn
2+the strain characteristics of ability
| Strain number | Mn 2+Clearance | Flat-plate bacterial colony form | |
| 1 | M2 | 40.9% | White colony, smooth surface, moistening, neat in edge |
| 2 | M3 | 8.6% | White colony, smooth surface, moistening, neat in edge |
| 3 | M4 | 19.3% | White colony, smooth surface, moistening, neat in edge |
| 4 | M5 | 3.2% | The tiny bacterium colony of white, smooth surface, moistening, neat in edge |
| 5 | M6 | 17.8% | Brick-red bacterium colony, smooth surface, moistening, neat in edge |
| 6 | M7a | 36.9% | Brick-red bacterium colony, smooth surface, moistening, neat in edge |
| 7 | M8b | 9.9% | White colony, smooth surface, moistening, neat in edge, toughness |
| 8 | M9 | 42.1% | White colony, smooth surface, moistening, neat in edge |
| 9 | M10a | 6.6% | White colony, smooth surface, moistening, neat in edge |
| 10 | M10b | 3.7% | Brick-red bacterium colony, smooth surface, moistening, neat in edge |
| 11 | MnC1 | 15.2% | White colony, surface flat, smooth, moistening, out-of-shape |
| 12 | MnC2 | 18.6% | White small colonies, smooth surface, moistening, neat in edge |
| 13 | MnC3 | 15.3% | White colony, surface flat, smooth, moistening, neat in edge |
| 14 | MnC4a | 10.1% | White colony, surface flat, smooth, moistening, out-of-shape |
| 15 | MnC4b | 9.8% | White small colonies, smooth surface, moistening, neat in edge |
3.3 times and concentration of metal ions are on the impact of clearance
Bacterial strain M7a is at Mn
2+when concentration is 0.2g/L, increase in time, clearance rises rapidly, and thalli growth is vigorous, bacterium liquid OD
600reached the highest at the 3rd day, after this OD
600fall after rise, but metal removal rate continues to rise to about 90% and keeps stable, not affecting by thalline decline, may be that cellular lysate does not affect soluble metal content, and (Fig. 2 a) due to this bacterium and metal generation irreversible effect.
Mn
2+when concentration is 0.4g/L, bacterial strain M7a grows more rapid, bacterium liquid OD
600namely maximum is reached at second day.Remove metal ability comparatively strong, more than 90%, keep stable, do not affect (Fig. 2 b) by thalline decline.
Mn
2+when concentration is 0.6g/L, bacterial strain M7a reaches higher concentration after growing one day, thereafter by the overlaying influence of meta-bolites, at the 5th day OD
600reach maximum.Remove metal ability very strong, at the 3rd day clearance more than 99%, and keep stable (Fig. 2 c).
Mn
2+when concentration is 0.8g/L, bacterial strain M7a grows still vigorous, by the overlaying influence of meta-bolites, and OD
600there are two peaks in curve the 4th day and the 6th day.Remove metal ability very strong, namely reach 99% at the 3rd day clearance, keep stable (Fig. 2 d) thereafter.
Mn
2+when concentration is 1.0g/L, bacterial strain M7a grows still vigorous, by the overlaying influence of meta-bolites, and OD
600there are two peaks in curve the 3rd day and the 5th day.Remove metal ability very strong, at the 3rd day clearance close to 99%, stablize thereafter and remain on more than 99.5% (Fig. 2 e).
By five groups of concentration Mn
2+the highest clearance compare, as shown in Figure 3, bacterial strain M7a is to Mn
2+removal ability is very strong and speed of action is fast.The growth of M7a and the feature of removal metal are the OD when high metal concentration
600curve occurs bimodal, and being better than low concentration group to the removal ability of higher concentration metal, may be because the inducing and acclimating effect of low-concentration metallic to thalline improves removal ability equally.
All above-mentioned be primary implementation method of the present invention, not setting restriction does not implement this novel method and/or product innovation with other forms.Those skilled in the art will utilize this important information, to foregoing amendment, to realize similar implementation status.But, all based on amendment of the present invention or transformation novel method, belong to the right of reservation.
The above is only preferred embodiment of the present invention, and be not restriction the present invention being made to other form, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the Equivalent embodiments of equivalent variations.But everyly do not depart from technical solution of the present invention content, any simple modification, equivalent variations and the remodeling done above example according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.
Claims (2)
1. a strain serratia marcescens (
serratiamarcescens) M7a, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCCNo.8829.
2. a serratia marcescens as described in claim 1 (
serratiamarcescens) M7a is at heavy-metal ion removal Mn
2+in application.
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