CN101215522A - Method for separating and purifying Ochrobactrum sp. bacterium used for reducing Cr6+ - Google Patents
Method for separating and purifying Ochrobactrum sp. bacterium used for reducing Cr6+ Download PDFInfo
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- CN101215522A CN101215522A CNA2008100304105A CN200810030410A CN101215522A CN 101215522 A CN101215522 A CN 101215522A CN A2008100304105 A CNA2008100304105 A CN A2008100304105A CN 200810030410 A CN200810030410 A CN 200810030410A CN 101215522 A CN101215522 A CN 101215522A
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- ochrobactrum
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Abstract
The invention discloses a method for separation and purification of Ochrobactrum sp. with deoxidizing Cr 6+, which solves the problem of that prior microorganism is difficult to isolated culture when microorganism disposes alkali waste water with Cr 6+. Picking alkali waste soil sample with Cr 6+ which is inoculated on organic LB medium to process enriched culture generation, adopting selective liquid medium Cr-G which is improved by inventor to process passage culture, and then adopting single-layer flat-plate and improved solid medium to process streak culture. The invention is capable of realizing separation and purification of Ochrobactrum sp. with deoxidizing Cr 6+ in alkality environment, adopting single-layer solid medium and optimize culture condition, wherein culture component is simple and formation rate of bacterial colony is higher.
Description
Technical field
The present invention relates to a kind of microbial culture method, especially for Cr
6+The separation purification method of reductive Ochrobactrumsp. pure cultures of bacteria.
Background technology
Chromic content height in the waste water of discharging such as mine, smelting, inorganic salt and chromium salt factory, toxicity is big.In recent years, utilize microbiological treatment to contain Cr
6+The method of waste water is very noticeable, and biological process has simply, cheap, good effectiveness.Various microorganisms constantly are exploited, for example: ambiguous pseudomonas, Pseudomonas fluorescens, streptococcus uberis etc., though these bacterial strains have certain resistance to chromium (VI), but can not change the valence state of chromium (VI), can not reduce chromium (VI) and reduce its toxicity, not reach and administer the purpose that contains chromium (VI) waste water.And chromium (VI) is had the long action time of the bacterium of reducing power such as enterobacter cloacae, chromate reduction bacterium, reduction ratio is low.Be used for both at home and abroad at present handling and contain Cr
6+The microorganism of waste water such as Desulfovibrio, Pseudomonas, Enterobacter cloacae strain HO1, ATCC 33546, SRB etc. nearly all concentrate on the processing to the electroplating wastewater of neutrality or slant acidity, and the research of handling for the related microorganism of alkaline wastewater containing cr, chromium slag percolate especially chromium slag rarely has report.These waste water are the concentration height often, is alkalescence, and general microorganism is difficult to adapt to this system.In addition, cultivate these processing and contain Cr
6+The substratum that the Institute of Micro-biology of waste water uses, most of nutritive ingredient complexity, what have needs auxiliary separation such as somatomedin, trace element, or requires double-layer plate, anaerobic condition etc., and this has increased the spending of operation easier and expense.
Summary of the invention
The objective of the invention is to solve present microbiological treatment alkalescence and contain Cr
6+Microorganism is difficult to the problem of separation and Culture during waste water, provides a kind of solid medium separation and purification dull and stereotyped with individual layer and improvement to obtain reducing Cr
6+Ochrobactrum sp. pure cultures of bacteria method, it can be applicable to the especially processing of chromium slag etc. of alkaline wastewater containing cr, chromium slag percolate, and method is easy, colony forming efficiecy is higher.
Detailed technology scheme of the present invention is: the alkalescence that will gather contains Cr
6+Discarded sample is with an organic LB substratum enrichment culture generation, and with the selected liq culture medium C r-G cultivation of going down to posterity, the component of culture medium C r-G is: peptone 10.0g/L then; Yeast extract powder 5g/L; NaCl 5g/L; MgSO
47H
2O 0.2g/L; K
2HPO
40.05g/L, K
2Cr
2O
70.8～1.2g/L, the pH that adjusts substratum is 7.5～10.0, and culture temperature is 30～37 ℃;
Go down to posterity and cultivate 2～4 times, the culture that will be in logarithmic phase is got 20～100 times of an amount of dilutions, and is streak culture on solid plate.The prescription of solid plate substratum is: A: peptone 10.0g/L, yeast extract powder 5g/L, NaCl 5g/L, MgSO
47H
2O 0.2g/L and K
2HPO
40.05g/L be dissolved in distilled water, add mass percent concentration again and be 1.0～1.2% agar powder, shake up; B:K
2Cr
2O
70.8～1.2g/L is dissolved in distilled water; A and B sterilize respectively; Mix, transfer pH to 7.5～10.0, culture temperature is 30～37 ℃.
The solid medium color is the well-illuminated shape of celandine green, forms some middle canescence in media surface after 2～3 days, transparent limit is arranged, neat in edge, smooth surface, the circular bacterium colony of projection.
The present invention adopts the culture condition of the solid medium of individual layer and optimization to realize reducing in the alkaline environment Cr
6+The separation and purification of Ochrobactrum sp. bacterium.The cultivation composition is simple, need not to add any other auxiliary separation such as bacterium, trace element, somatomedin; Only make gelifying agent with common agar powder, colony forming efficiecy is higher.This method greatly reduces workload and expense, and more simple than additive method, and is with strong points.
Embodiment
Embodiment 1: separate reduction Cr
6+Ochrobactrum sp. bacterial strain Cr-1.
To join at the chromium slag specimen product 3.0g of chromium salt factory collection among the sterilized LB substratum 100mL, at 30 ℃, an enrichment culture generation under the 160r/min condition, get supernatant liquor 2ml and be inoculated into new selected liq culture medium C r-G (100ml) cultivation, the composition of selected liq culture medium C r-G substratum is: peptone 10.0g/L; Yeast extract powder 5.0g/L; NaCl 5.0g/L; MgSO
47H
2O 0.2g/L; K
2HPO
40.05g/L; K
2Cr
2O
70.8g/L.Transfer pH=7.5 with NaOH solution, culture temperature is 30 ℃, goes down to posterity to cultivate 3 times, and each inoculum size is 2ml (2%).The culture that will be in logarithmic phase is at last got 20 times of an amount of dilutions, picks an amount of diluent with transfering loop and rules on solid plate.The solid plate culture medium prescription is made up of A and two parts of B.The A composition is peptone 5.0g; Yeast extract powder 2.5g; NaCl 2.5g; MgSO
47H
2O 0.1g; K
2HPO
40.025g, be dissolved in 400ml distilled water, add agar powder 4.8g again and shake up; B composition: K
2Cr
2O
70.4g be dissolved in 100ml distilled water; Respectively at 121 ℃ of steam sterilizings, the time is 25 minutes, and is at last that A and two of B is partially mixed with A and B part, transfers pH=7.5, falls dull and stereotypedly on aseptic platform, treats can rule after plate solidifies.The plate of line was just being put 10 minutes earlier, treated that being inverted in 30 ℃ of constant incubators after bacterium liquid absorbs cultivates.The solid medium color is the well-illuminated shape of light green, forms tens canescence not of uniform size, circular single bacterium colony of intermediate projections in media surface after 3 days.
Resulting single bacterium colony is chosen among the selected liq culture medium C r-G and is cultivated.The Study on Physico-chemical of carrying out is then found: bacterial strain Cr-1 can be at 20～45 ℃, and pH is 7～11, K
2Cr
2O
7Be growth and breeding in 0～1.6g/L scope, optimum condition is 35 ℃, pH=10, K
2Cr
2O
70.6g/L, after 6 days, Cr wherein
6+Reduction efficiency reach 80%.
Embodiment 2: separate reduction CR
6+Ochrobactrum sp. bacterial strain Cr-2
Go down to posterity to selected liq culture medium C r-G from enrichment culture, step is with embodiment 1, K
2Cr
2O
7Be 0.9g/L, medium pH=9.0, its culture temperature is 30 ℃ still, goes down to posterity and has cultivated 2 times, will be in the logarithmic phase nutrient solution at last and dilute 60 times, carries out the dull and stereotyped coating of solid medium with embodiment 1 step and separates K
2Cr
2O
7Still be 0.4g, agar powder concentration is 1.1%, transfers pH=9.0, and culture temperature is made as 30 ℃.Respectively at 121～125 ℃ of steam sterilizings, the time is 20 minutes with A and B part.Evenly be coated with the bacterium liquid of dilution with glass stick, left and right sides media surface formed more than 60 grey, intermediate projections, circular bacterium colony in streak culture 2 days, but this colony diameter little than embodiment 1.
Resulting single bacterium colony chosen among the selected liq culture medium C r-G cultivate.The Study on Physico-chemical of carrying out is then found: bacterial strain Cr-2 can be at 20～45 ℃, and pH is 7～11, K
2Cr
2O
7Be growth and breeding in 0～1.6g/L scope, optimum condition is 35 ℃, pH=10, K
2Cr
2O
70.6g/L, after 5 days, Cr wherein
6+Reduction efficiency reach 78%.
Embodiment 3: separate reduction Cr
6+Ochrobactrum sp. bacterial strain Cr-3
Go down to posterity to selected liq culture medium C r-G from enrichment culture, step is with example 1, but the sample of gathering is about 0.5 meter, chromium slag muck edge, the soil sample 4.0g that 10cm is dark, and with the K among the selected liq culture medium C r-G
2Cr
2O
7Be adjusted into 1.0g/L, medium pH=9.0, its culture temperature is 30 ℃, goes down to posterity to cultivate 3 times, inoculum size is 2mL (2%), will be in the logarithmic phase nutrient solution at last and dilute 80 times, carries out the solid medium plate streaking with embodiment 1 mode and separates K
2Cr
2O
7Be set at 0.5g, agar powder concentration is 1.1%, transfers pH=9.0, and culture temperature is made as 37 ℃.Respectively at 121～125 ℃ of steam sterilizings, the time is 20 minutes with A and B part, and left and right sides media surface formed tens grey, circular single bacterium colony of intermediate projections in streak culture 2～3 days.
Resulting single bacterium colony is chosen among the selected liq culture medium C r-G and is cultivated.Carrying out Study on Physico-chemical then finds: bacterial strain Cr-3 can be at 20～45 ℃, and pH is 7～11, K
2Cr
2O
7Be growth and breeding in 0～1.6g/L scope, optimum condition is 35 ℃, pH=11, K
2Cr
2O
70.6g/L, after 5 days, Cr wherein
6+Reduction efficiency reach 85%.
Embodiment 4: separate reduction Cr
6+Ochrobactrum sp. bacterial strain Cr-4
Go down to posterity to selected liq culture medium C r-G from enrichment culture, step is with example 1, but the sample of gathering is about 0.5 meter, chromium slag muck edge, the soil sample 4.0g that 10cm is dark, and the K among the selected liq culture medium C r-G
2Cr
2O
7Be 1.0g/L, medium pH=9.5, its culture temperature is 30 ℃, goes down to posterity to cultivate 3 times, inoculum size is 1mL (1%), will be in the logarithmic phase nutrient solution at last and dilute 40 times, carries out the solid medium plate streaking with embodiment 1 mode and separates K
2Cr
2O
7Be set at 0.5g, agar powder concentration is 1.2%, transfers pH=9.5, and culture temperature is made as 37 ℃.Respectively at 121～125 ℃ of steam sterilizings, the time is 20 minutes with A and B part, and left and right sides media surface formed more than 20 grey not of uniform size in streak culture 2～3 days, intermediate projections, smooth surface, circular single bacterium colony, colony diameter is bigger, maximum reached at 2.5mm.
Gained list bacterium colony chosen among the selected liq culture medium C r-G cultivate.The Study on Physico-chemical of carrying out is then found: bacterial strain Cr-4 can be at 20～45 ℃, and pH is 7～11, K
2Cr
2O
7Be growth and breeding in 0～1.6g/L scope, optimum condition is 35 ℃, pH=11, K
2Cr
2O
70.8g/L, after 5 days, Cr wherein
6+Reduction efficiency reach 75%.
Claims (2)
1. separation and purification Cr
6+The method of reductive Ochrobactrum sp. bacterium is characterized in that may further comprise the steps:
(1) alkalescence that will gather contains Cr
6+Discarded sample is with an organic LB substratum enrichment culture generation, then with the selective medium Cr-G cultivation of going down to posterity; The component of described culture medium C r-G is: peptone 10.0g/L, yeast extract powder 5g/L, NaCl 5g/L, MgSO
47H
2O 0.2g/L, K
2HPO
40.05g/L, K
2Cr
2O
70.8～1.2g/L, the pH that adjusts substratum is 7.5～10, and culture temperature is 30～37 ℃;
(2) culture that will be in logarithmic phase is got 20～100 times of an amount of dilutions, and is streak culture on solid plate; Described prescription is: A: peptone 10.0g/L, yeast extract powder 5g/L, NaCl 5g/L, MgSO
47H
2O 0.2g/L and K
2HPO
40.05g/L be dissolved in distilled water, add mass percent concentration again and be 1.1～1.2% agar powder, shake up; B:K
2Cr
2O
70.8～1.2g/L is dissolved in distilled water; A and B sterilize respectively, mix, and transfer pH to 7.5～10, and culture temperature is 30～37 ℃.
2. the method for claim 1 is characterized in that: the K in the solid plate substratum in liquid nutrient medium Cr-G in the described step (1) and the step (2)
2Cr
2O
7Add-on be respectively 0.8g/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100304105A CN101215522A (en) | 2008-01-02 | 2008-01-02 | Method for separating and purifying Ochrobactrum sp. bacterium used for reducing Cr6+ |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100304105A CN101215522A (en) | 2008-01-02 | 2008-01-02 | Method for separating and purifying Ochrobactrum sp. bacterium used for reducing Cr6+ |
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|---|---|
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531593A (en) * | 2015-01-15 | 2015-04-22 | 南京工业大学 | Staphylococcus equorum and application of staphylococcus equorum in heavy metal ion degradation |
| CN108300678A (en) * | 2018-03-12 | 2018-07-20 | 桂林理工大学 | A kind of preparation method and application of the Leersia Sw endogenetic bacteria with reduction of hexavalent chromium |
| CN109593667A (en) * | 2017-09-30 | 2019-04-09 | 天津科技大学 | The identification of Cr (VI) reducing bacteria and its cultural method |
-
2008
- 2008-01-02 CN CNA2008100304105A patent/CN101215522A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531593A (en) * | 2015-01-15 | 2015-04-22 | 南京工业大学 | Staphylococcus equorum and application of staphylococcus equorum in heavy metal ion degradation |
| CN104531593B (en) * | 2015-01-15 | 2017-05-17 | 南京工业大学 | Staphylococcus equorum and application of staphylococcus equorum in heavy metal ion degradation |
| CN109593667A (en) * | 2017-09-30 | 2019-04-09 | 天津科技大学 | The identification of Cr (VI) reducing bacteria and its cultural method |
| CN108300678A (en) * | 2018-03-12 | 2018-07-20 | 桂林理工大学 | A kind of preparation method and application of the Leersia Sw endogenetic bacteria with reduction of hexavalent chromium |
| CN108300678B (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 |
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Open date: 20080709 |