CN101684453A - Bacterial strain for repairing chromium slag contaminated site - Google Patents
Bacterial strain for repairing chromium slag contaminated site Download PDFInfo
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- CN101684453A CN101684453A CN200910043840A CN200910043840A CN101684453A CN 101684453 A CN101684453 A CN 101684453A CN 200910043840 A CN200910043840 A CN 200910043840A CN 200910043840 A CN200910043840 A CN 200910043840A CN 101684453 A CN101684453 A CN 101684453A
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Abstract
The invention discloses a bacterial strain Pannonibacterphragmitetus BB for repairing a chromium slag contaminated site, which has the preserved number of CGMCC No. 3052. The inventor collects contaminated soil of a chromium slag scrapyard in a Hunan ferroalloy work to obtain the bacterial strain with the hexavalent chromium reducing characteristic through screening, separation and acclimatization. The bacterial strain has the function of reducing the hexavalent chromium to trivalent chromium, is used for treating chromium contamination, and has low cost and simple operation.
Description
Technical field
The present invention relates to a kind of bacterial strain that is used for environmental pollution treatment, particularly a kind of chromium restoring function bacterium.
Background technology
Sexavalent chrome is that a kind of toxicity is more carcinogenic, teratogenesis, mutagenic agent material.Pollution of chromium mainly comes from solid waste---the chromium slag of discharging and the chromium metal and the generation of chromium salt production industry of industry chromate waste waters such as plating, process hides, paint, printing and dyeing in the environment.Microorganism repairs the method for pollution of chromium because its cost is low, non-secondary pollution and simple operation and other advantages and the great interest of vast efforts at environmental protection person that causes day by day.Separation obtains efficiently, and chromium restoring function bacterium is the work that investigators endeavour to carry out always.Report in this respect is more both at home and abroad, having separated the bacterium with reduction of hexavalent chromium ability has multiplely, mainly contains desulfovibrio (Desulfovibrio vulgaris), genus bacillus (Bacillus sp.), pseudomonas (Pseudomonassp.), Shiva Salmonella (Shewanella sp.), intestinal bacteria (Escherichia coli sp) etc.But, up to now, as yet not relevant for the report of Pannonibacter phragmitetus reduction of hexavalent chromium.Therefore, the present invention repairs for hexavalent chromium polluted microorganism new Microbial resources is provided.
Summary of the invention
The object of the present invention is to provide a kind of new chromium restoring function bacterial strain, it is chromic function that this bacterial strain has hexavalent chrome reduction, and this bacterial strain is used for the improvement of pollution of chromium, and cost is low, and is simple to operate.
The contriver gathers Hunan Ferroalloy Factory's chromium slag muck puts a contaminated soil, through screening, separation, domestication, obtain a kind of bacterial strain of tool hexavalent chromium reducing characteristic, through identifying, this bacterial strain 16s rRNA gene order and Pannonibacter phragmitetus sp.16S rRNA have 99% similarity, called after Pannonibacter phragmitetus BB.This bacterial strain is submitted biological preservation on May 8th, 2009 to China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and preserving number is CGMCCNo.3052.This bacterial strain can be widely used in the chromium slag contaminated site reparation.
The process of among the present invention, screening, separating, tame this bacterial strain is: gather Hunan Ferroalloy Factory's chromium slag muck and put soil after the match, in the liquid medium within, grew 3～4 days in 30 ℃ of constant incubators; Vibration slightly, top mud is inoculated in the solid medium that contains 50～250mg/L hexavalent chromium concentration by the dilution gradient, picking different shape bacterium, in the liquid nutrient medium that contains 50～250mg/L hexavalent chromium concentration, 30 ℃ of shaking culture; Picking is single bacterium colony of purifying, progressively improves chromium concn, tames.
Described liquid nutrient medium consists of: 5g/L Sodium.alpha.-hydroxypropionate, 5g/L yeast extract paste and 2g/L sodium-chlor, all the other are water, regulate pH=9.5～9.8 with 5mol/L NaOH.Described solid medium is: 5g/L Sodium.alpha.-hydroxypropionate, 5g/L yeast extract paste and 2g/L sodium-chlor, 15g/L agar.
Description of drawings
Fig. 1: the colonial morphology figure of bacterial strain of the present invention;
Fig. 2: strain morphology figure of the present invention;
Fig. 3: the Phylogenetic Analysis tree of bacterial strain of the present invention;
Fig. 4: with the reduction effect figure of bacterial strain reduction Cr of the present invention (VI).
Embodiment:
Embodiment 1: separation and purification and the domestication of chromium reduction bacterium of the present invention
Gather Hunan Ferroalloy Factory's chromium slag muck and put soil after the match, take by weighing 10g chromium dregs earth in the sterilized Erlenmeyer flask of 150mL, add 10mL sterilising liq substratum (5g/L Sodium.alpha.-hydroxypropionate, 5g/L yeast extract paste and 2g/L sodium-chlor, all the other are water, regulate pH=9.5～9.8 with 5mol/L NaOH), in 30 ℃ of incubators, cultivated 4 days.Get 1mL mud subsequently and become 10 by gradient dilution
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, with liquid-transfering gun from 10
-6, 10
-7With 10
-8Getting 0.1mL in the diluent respectively places and contains 250mg/L Cr (VI) (with K
2Cr
2O
7Solution is as the chromium source) solid medium, solid medium consists of: 5g/L Sodium.alpha.-hydroxypropionate, 5g/L yeast extract paste and 2g/L sodium-chlor, 15g/L agar.Fully smoothen with the triangle rod, flat board is inverted in 30 ℃ of incubators cultivated 4 days.With the bacterium colony of aseptic inoculating needle picking different shape, streak inoculation is on the solid medium that contains 250mg/L Cr (VI), and the solid medium component is the same.Be inverted, cultivated 4 days in 30 ℃ of incubators.
Respectively picking single bacterium colony of purifying contain in 250mg/L Cr (VI) liquid nutrient medium in above-mentioned, liquid nutrient medium is formed: 5g/L Sodium.alpha.-hydroxypropionate, 5g/L yeast extract paste and 2g/L sodium-chlor, 250mg/L Cr (VI) is (with K
2Cr
2O
7Solution is as the chromium source), all the other are water, when the substratum color when initial yellow becomes blue grey, Cr in the nutrient solution (VI) concentration is lower than detection limit.Detection limit is meant with spectrophotometry can't detect Cr (VI) in the substratum.
Choose this bottle and transfer, progressively improve Cr (VI) concentration,, cultivate for every kind and repeat 2～3 times from 250mg/L, 300mg/L, 400mg/L, 500mg/L.After domestication finishes, get nutrient solution 10mL and be linked in the 100mL nutrient solution, 30 ℃, the 150rpm liquid that vibrated is cultivated, and takes out 4 ℃ of preservations.
Embodiment 2: bacterial strain bacterial characteristics and biological assay
A little drops on the slide glass to get bacterium liquid, makes thalline adhere to 15min, puts into fixedly 1h of 2.5% glutaraldehyde solution, alcohol gradient 30%～70% is dewatered step by step, isoamyl acetate displacement 30min, critical point drying, ion sputtering, sem observation is also taken pictures, and sees accompanying drawing 1.
1. morphological character
(1) colony morphology characteristic: have the liquid nutrient medium of chromium reduction bacterium to pass through streak plate growth, be inoculated in the solid medium that contains the 250mg/L hexavalent chromium concentration, flat board is inverted in 30 ℃ of constant incubators cultivated 3～4 days, observe its colonial morphology, on the flat board that Cr (VI) arranged, bacterium colony is blue grey, circular, neat in edge, central protrusion is seen accompanying drawing 1.
(2) thalline feature: this bacterial strain belongs to Gram-negative bacteria, and thalline is shaft-like, the afterbody flaggellation, and thalline moves about, and surface irregularity and have rope to adhere to is referring to accompanying drawing 2.
2. biochemical characteristic
Reduction nitrate is nitrite, and tween 80 hydrolysis, phosphoesterase are hydrolyzed to positive reaction; Methyl red, indoles generation, 3-ketone group lactose, H2S generation, cellulose hydrolysis, urease activity, gelatinum hydrolysis, starch are hydrolyzed to negative reaction.
3. physio-biochemical characteristics
Can utilize D-wood sugar, D-glucose, L-pectinose, sucrose, lactose to produce acid, can utilize Citrate trianion, D-fructose, D-semi-lactosi, the negative reaction of maltose, the appropriate pH of growth is 9-10, and optimal temperature is 30 ℃, and non-sodium chloride can be grown.
The physio-biochemical characteristics of table 1 bacterial strain
4.16s rRNA characteristic and phylogenetic tree analysis
The bacterium colony that encircles purifying with the direct picking one of transfering loop is in being equipped with the cell pyrolysis liquid transfer pipet, and the lysate consumption is 1mL (consisting of of cell pyrolysis liquid: 100 μ L or 10% sodium laurylsulfonate, 20 μ L 40%NaOH, 880 μ L sterilized waters).Broken wall reels off raw silk from cocoons, and directly carries out 16sr RNA bacterium colony PCR as template behind the broken wall.Reclaim the test kit operation by the DNA of TIANGEN company and carry out pcr amplification purpose fragment rubber tapping recovery, fragment after sepharose DNA reclaims is further purified by the operation of the DNA of TIANGEN company purification kit is undertaken, target DNA segment (product is reclaimed in rubber tapping) is connected with the pGM-T carrier, to connect product and carry out the thermal shock conversion, transform the mensuration that the white colony that obtains on the flat board carries out nucleotide sequence after the picking incubated overnight.
Listed 16s rRNA gene order is carried out nucleotide homology relatively among 16s rRNA gene order and the Genbank, with Pannonibacter phragmitetus 16S rRNA 99% similarity is arranged, with this bacterial strain called after Pannonibacter phragmitetusBB.The Phylogenetic Analysis tree of this bacterial strain sees accompanying drawing 3.
Embodiment 3: chromium reduction bacterium reduction Cr (VI) effect
Bacterial reduction Cr (VI) condition: nutrient media components: 5g/L Sodium.alpha.-hydroxypropionate, 5g/L yeast extract paste, 2g/L sodium-chlor; Regulate pH=9.5～9.8 with 5mol/L NaOH; 30 ℃ of temperature.
With the bacterium colony of transfering loop picking purifying in the Erlenmeyer flask that the 100mL sterilising medium is housed, in 30 ℃, the 150rpm shaking table is cultivated 24h, then, pipette the bacterium liquid of this overnight growth of 10mL with the sterilization transfer pipet, be inoculated into hexavalent chromium concentration and be respectively 25,50,100,150,200,300,400 and the 100mL sterilising medium of 500mg/L in (with K
2Cr
2O
7Solution is as the source), in 30 ℃, 150rpm, shaking table cultivate 72h, during, respectively after cultivation 0,6,12,24,36,48,60 and 72h, with the sampling of sterilization syringe, centrifugal, supernatant liquor carries out remaining chromic mensuration, and the result that each period is measured Cr (VI) as shown in Figure 4.
Claims (5)
1. one kind is used for the bacterial strain that chromium slag contaminated site is repaired, and described bacterial strain is Pannonibacterphragmitetus BB, and CGMCC No.3052 has the ability that sexavalence Cr can be reduced to trivalent Cr.
2. bacterial strain as claimed in claim 1, it is characterized in that this bacterium is fit to culture condition and is: substratum contains the 5g/L Sodium.alpha.-hydroxypropionate, 5g/L yeast extract paste, 2g/L sodium-chlor; PH=9.5～9.8; 30 ℃ of temperature.
3. the method for the described bacterial strain of separation and Culture claim 1 is characterized in that may further comprise the steps: gather Hunan Ferroalloy Factory's chromium slag muck and put soil after the match, in Sodium.alpha.-hydroxypropionate yeast extract paste liquid nutrient medium, grew 3～4 days in 30 ℃ of constant incubators; Vibration slightly, top mud is inoculated in the solid medium that contains 50～250mg/L hexavalent chromium concentration by the dilution gradient, picking different shape bacterium, in the substratum that contains 50～250mg/L hexavalent chromium concentration, 30 ℃ of shaking culture; Picking is single bacterium colony of purifying, progressively improves chromium concn, tames.
4. method as claimed in claim 3 is characterized in that: described liquid nutrient medium consists of: 5g/L Sodium.alpha.-hydroxypropionate, 5g/L yeast extract paste, 2g/L sodium-chlor, and all the other are water; Described solid medium is: 5g/L Sodium.alpha.-hydroxypropionate, 5g/L yeast extract paste, 2g/L sodium-chlor and 15g/L agar.
5. method as claimed in claim 3 is characterized in that: Cr (VI) gradient concentration is respectively during described domestication: 250mg/L, 300mg/L, 400mg/L, 500mg/L.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127517A (en) * | 2010-12-20 | 2011-07-20 | 中南大学 | Strain with heavy metal tolerance and applications thereof |
| CN103981142A (en) * | 2013-12-05 | 2014-08-13 | 青岛理工大学 | Method for continuously culturing alkaline waste repair bacterial liquid containing Cr (VI) |
| CN104550222A (en) * | 2014-11-27 | 2015-04-29 | 中南大学 | Microbial leaching and chemical fixing joint repairing method for heavily polluted soil of chromium slag yard |
| CN107309270A (en) * | 2017-06-23 | 2017-11-03 | 河南大学 | Application of one plant of BB bacterium in reduction red mud pH value |
| CN108441441A (en) * | 2018-03-12 | 2018-08-24 | 桂林理工大学 | A kind of preparation method and application of the Leersia Sw endogenetic bacteria with reduction of hexavalent chromium |
| CN110144308A (en) * | 2019-05-14 | 2019-08-20 | 大江环境股份有限公司 | It is a kind of to be resistant to high salt, efficient degradation nitrate denitrifying bacterium and its preparation and purposes |
| CN111268810A (en) * | 2020-03-20 | 2020-06-12 | 微米环创生物科技(北京)有限公司 | Nitrogen and phosphorus removal microbial community and application thereof |
| CN114682624A (en) * | 2022-04-29 | 2022-07-01 | 中南大学 | Iron-based composite material coupled hexavalent chromium reduction microorganism chromium pollution remediation reagent and preparation method and application thereof |
-
2009
- 2009-07-03 CN CN200910043840A patent/CN101684453A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127517A (en) * | 2010-12-20 | 2011-07-20 | 中南大学 | Strain with heavy metal tolerance and applications thereof |
| CN103981142A (en) * | 2013-12-05 | 2014-08-13 | 青岛理工大学 | Method for continuously culturing alkaline waste repair bacterial liquid containing Cr (VI) |
| CN104550222A (en) * | 2014-11-27 | 2015-04-29 | 中南大学 | Microbial leaching and chemical fixing joint repairing method for heavily polluted soil of chromium slag yard |
| CN107309270A (en) * | 2017-06-23 | 2017-11-03 | 河南大学 | Application of one plant of BB bacterium in reduction red mud pH value |
| CN108441441A (en) * | 2018-03-12 | 2018-08-24 | 桂林理工大学 | A kind of preparation method and application of the Leersia Sw endogenetic bacteria with reduction of hexavalent chromium |
| CN108441441B (en) * | 2018-03-12 | 2021-06-15 | 桂林理工大学 | Preparation method and application of Leersia hexandra endophytic bacteria capable of reducing hexavalent chromium |
| CN110144308A (en) * | 2019-05-14 | 2019-08-20 | 大江环境股份有限公司 | It is a kind of to be resistant to high salt, efficient degradation nitrate denitrifying bacterium and its preparation and purposes |
| CN110144308B (en) * | 2019-05-14 | 2022-06-07 | 大江环境股份有限公司 | High-salt-tolerance denitrifying bacterium capable of efficiently degrading nitrate, and preparation and application thereof |
| CN111268810A (en) * | 2020-03-20 | 2020-06-12 | 微米环创生物科技(北京)有限公司 | Nitrogen and phosphorus removal microbial community and application thereof |
| CN111268810B (en) * | 2020-03-20 | 2022-03-08 | 微米环创生物科技(北京)有限公司 | Nitrogen and phosphorus removal microbial community and application thereof |
| CN114682624A (en) * | 2022-04-29 | 2022-07-01 | 中南大学 | Iron-based composite material coupled hexavalent chromium reduction microorganism chromium pollution remediation reagent and preparation method and application thereof |
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Open date: 20100331 |