CN100592076C - Preprocessing method for measuring desaturase activity of drinking water biological active carbon treatment process - Google Patents
Preprocessing method for measuring desaturase activity of drinking water biological active carbon treatment process Download PDFInfo
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- CN100592076C CN100592076C CN200810023462A CN200810023462A CN100592076C CN 100592076 C CN100592076 C CN 100592076C CN 200810023462 A CN200810023462 A CN 200810023462A CN 200810023462 A CN200810023462 A CN 200810023462A CN 100592076 C CN100592076 C CN 100592076C
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Abstract
The invention provides a dehydrogenase activity detection pretreatment method used in drinking water biological active carbon treatment technique, which is a mixed solution composed of three kinds ofreagents, under the control of adding proportionment, adding time and operational condition, the pretreatment is finished, thereafter the measuring process is selected and the specification curve is drawn. The invention has the advantages that, from the chemical angle, using organic reagent to extract and elute the adsorbed microorganism, the desorption degree is high; testing the active carbon carried dehydrogenase activity with effective proportioning and oscillation mode, and confirming the optimum organic solvent combination and proportioning for achieving the maximum measuring effect; providing the reference for similar biological carrier activity test, which is suitable for biological activity test of biological carrier in drinking water treatment technique.
Description
Technical field
The present invention relates to water-treatment technology field, specifically relate to the preprocessing method for measuring desaturase liveness of active carbon filler in a kind of drinking water activated carbon treatment process.
Technical background
Because the limitation of potable water conventional treatment process, advanced treatment process is used more and more general, wherein the biological activated carbon technology has absorption concurrently because of it and the biological treatment effect is widely used, and the biologically active of estimating bio-carrier is to judge the key of its biological treatment effect power.The dehydrogenase activity of biosome has reflected the activated state of biosome to a great extent, so the dehydrogenase activity detection is widely used in water treatment field.The measuring desaturase liveness method is since nineteen fifty-nine formally is applied in the biochemical analysis, and lot of domestic and international scholar has carried out repeatedly improving to it.Using more is 2,3,5-triphenyltetrazolium chloride-dehydrogenase activity detection method, and the reduzate triphenyl methylamine by mensuration extracts calculates the biologically active value.This method more application is in the biological sewage treatment field, and application is less in the potable water biological treatment.Existing sample pretreating method comprises ultrasonic wash-out and jolting fragmentation, and sample determination mainly comprises extraction and centrifugal.In the potable water ozone BAC process; the absorption carrier of microorganisms such as bacterium is the granular activated carbon of microcellular structure complexity; its adsorption mechanism is different from active sludge; moreover microorganism concn is higher than potable water in the sewage; particularly being higher than with the natural membrane is the treatment process of the biological generation type of charcoal bed; its suction-operated to carrier may be better than potable water; so in the ozone BAC process, adopt the method for ultrasonic wash-out or jolting fragmentation that the microorganism of absorption is come off fully, thereby the measured value after causing is very little or negative value occurs.Experimental result shows that the preprocess method in the above-mentioned dehydrogenase activity is difficult to efficiently realize the determination of activity of active carbon filler.
Summary of the invention
The object of the present invention is to provide a kind of measuring desaturase liveness method of active carbon filler of drinking water biological active carbon treatment process, the present invention is achieved through the following technical solutions:
At the low biologically active situation of drinking water biological active carbon treatment process activated charcoal, set up a kind of preprocess method of measuring the activated charcoal dehydrogenase activity based on novel organic mix reagent, promptly according to the charcoal absorption feature, the proportioning and the using method of the used organic mix reagent of pre-service have been proposed, for bio-carrier ldh assay in the potable water biological treatment provides new preprocess method.
The implementation step of the inventive method:
1. the preprocessing method for measuring desaturase liveness of drinking water biological active carbon treatment process is characterized in that this method step is as follows:
(1) active carbon filler is carried out pre-treatment: get respectively apart from charcoal bed top layer 40,80,120cm degree of depth active carbon filler 20～25g and put into 100mL tool plug triangular flask A, B, C, at first adding volume ratio is the extraction mixed liquor of 1: 2: 1 chloroform, methyl alcohol and distilled water, water bath with thermostatic control shaking table 120rpm jolting 10-15min, leave standstill 6h, in A, B, C triangular flask, add chloroform and distilled water more respectively, making the final volume ratio of chloroform, methyl alcohol and distilled water is 1.25: 1: 1, place shaking table 120rpm vibration 20min, leave standstill 6h;
(2) active carbon filler after the pre-treatment is measured absorbance respectively: the liquid of drawing respectively after the quantitative extraction of two-phase up and down places color comparison tube D, E, F, add trihydroxy aminomethane-hydrochloride buffer, sodium sulfite solution, pure water successively, and 2,3,5-triphenyl tetrazolium chloride solution, with formaldehyde as stopping reagent, add methenyl choloride after reacting completely again respectively, and measure absorbance respectively;
(3) drawing standard curve and the absorbance in the step (2) is converted into the triphenyl first growing amount of jumping up is the dehydrogenase activity value: the mensuration process of the active carbon filler extract in (2) set by step, measure 2,3, the absorbance that the triphenyl first that generates under the different sequence concentration of 5-triphenyl tetrazolium chloride is jumped up, and drawing standard curve, draw equation of linear regression, at last absorbance substitution regression equation is drawn San Ben Ji Jia Za growing amount, i.e. the dehydrogenase activity value.
In the selected mensuration process described in the step (2): after treating that layering is stable at the pre-treatment condition, draw tool plug triangular flask A, B, the C intermediate section interface organic phase 5ml of lower floor respectively, the inorganic phase 5ml in interphase upper strata, move to 50ml color comparison tube D, E, F respectively, in color comparison tube, add trihydroxy aminomethane-hydrochloride buffer (pH8.4), Na successively
2SO
3Solution (0.36%), distilled water and 2,3,5-triphenyl tetrazolium chloride solution (0.4%), volume ratio is 3: 1: 1: 1; From color comparison tube D, E, F, draw 2ml lower floor organic phase and 8ml upper strata water respectively, place color comparison tube G, H, I, add the 2ml formaldehyde fixed respectively to do sample blank, with color comparison tube D, E, F, G, H, I constant temperature water bath shaken cultivation 3h under 35-37 ℃ of condition, hunting speed 80rpm adds 3.0ml formaldehyde then and ends enzyme reaction in D, E, F color comparison tube; In D, E, F color comparison tube, add the 9ml methenyl choloride respectively again, add the 6ml methenyl choloride to G, H, I colorimetric in the shop respectively, mixing, with color comparison tube D, E, F, G, H, I after room temperature continues vibration 10min, centrifugal 5min (4000 rev/mins), take off a layer organic phase respectively, carry out colorimetric, the record light absorption value in wavelength 485nm place.
At the drawing standard curve described in the step (3): other gets 8 50mL color comparison tubes, respectively to wherein adding 2 of 7.5ml trihydroxy aminomethane-hydrochloride buffer, 2.5ml distilled water and 2.5ml variable concentrations successively, 3,5-triphenyl tetrazolium chloride series solution; Add the 1g sodium hydrosulfite more respectively, the colour developing 5min after, add the 5mL methenyl choloride respectively, 80rpm shaking out 3min, treat solution-stabilized after, take off layer organic solution respectively to the cuvette of 1cm; After stablizing 1-2min once more, measure absorbance in 752 ultraviolet grating spectrophotometers in the 485nm place, the drawing standard curve obtains equation of linear regression simultaneously; According to the absorbance that obtains, substitution equation of linear regression, and then calculate the growing amount of San Ben Ji Jia Za, the i.e. activity of dehydrogenasa.
The advantage of the inventive method is as follows:
This technology has realized the extraction of organic mix reagent to dehydrogenasa, makes that living microorganism is able to abundant wash-out in the active carbon filler, and measuring for follow-up substance that show color provides guarantee, experimental results show that effect is fairly obvious; The preprocess method of this dehydrogenase activity only need can be finished by the common micro-organisms laboratory, and complex reagent is drawn materials conveniently, and running cost is comparatively cheap, and its economic feasibility is higher; This technology also is applicable to the biologically active analysis of other fillers, and simple operating steps is easily gone, and application prospect is extensive.
Embodiment:
Development test is a research object with certain water factory's ozone-BAC process of south, by the control different experimental conditions, finally finds rational and effective pre-service and mensuration scheme.
Experiment is carried out in certain water factory, and homemade activated charcoal sampler is used in this experiment.The operational factor in activated charcoal pond is that the empty bench grafting time of touching is 15min, combined water and air backwash.Through 3 months, the technology operation tended towards stability, and formally enters the experimental phase.
Get active carbon filler 20～25g respectively apart from charcoal bed top layer 40,80, the 120cm degree of depth, put into 3 100mL tool plug triangular flasks respectively, the extraction mixed liquor (volume ratio 1: 2: 1) that adds chloroform, first alcohol and water respectively in water bath with thermostatic control shaking table 120rpm jolting 10min, leaves standstill 6h.Add chloroform and water again in triangular flask respectively, making final ratio is 1.25: 1: 1, places shaking table 120rpm vibration 20min, leaves standstill 6h.
After treating that stratified liquid is stablized in the triangular flask, draw the organic phase 5ml of intermediate section interface lower floor respectively, the inorganic phase 5ml in interphase upper strata moves to 3 50ml color comparison tube D, E, F.In color comparison tube, add 7.5ml tris-HCI buffer, 2.5mlNa successively respectively
2SO
3Solution (0.36%), 2.5ml pure water and 2.5ml2,3,5-triphenyl tetrazolium chloride solution (0.4%).
Draw 2ml lower floor organic phase and 8ml upper strata water in the mixed liquor from above-mentioned 3 color comparison tubes respectively, place color comparison tube G, H, the I of other 3 correspondences, add the 2ml formaldehyde fixed respectively to do blank.With 6 color comparison tubes constant temperature water bath shaken cultivation 3h under 37 ℃ of conditions, hunting speed 80rpm adds 3.0ml formaldehyde then respectively and ends enzyme reaction in D, E, F.
In D, E, F, add the 9ml methenyl choloride more respectively, in G, H, I, add the 6ml methenyl choloride respectively,, mixing.With 6 color comparison tubes after room temperature continues vibration 10min, centrifugal 5min (4000rpm).Take off a layer organic phase respectively, carry out colorimetric, the record light absorption value in wavelength 485nm place; According to the light absorption value that obtains, the reference standard curve is brought equation of linear regression into, and then calculates the growing amount of San Ben Ji Jia Za, the i.e. activity of dehydrogenasa.
The drawing standard curve: other gets in 8 50mL color comparison tubes, add 7.5ml trihydroxy aminomethane-hydrochloride buffer, 2.5ml distilled water and 2.5ml concentration successively to it and be respectively 2 of 0,5,10,15,20,40,60,80 μ g/ml, 3,5-triphenyl tetrazolium chloride series solution.Add the 1g sodium hydrosulfite again, behind the colour developing 5min, add the 5mL methenyl choloride respectively, shaking out 3min.Treat solution-stabilized after, take off layer organic solution respectively to the cuvette of 1cm.After stablizing 1-2min once more, measure absorbance in 752 ultraviolet grating spectrophotometers in the 485nm place, the drawing standard curve obtains equation of linear regression.
Experimental result shows, uses existing biological activity determination method, in the process of test, the light absorption value that blank light absorption value is higher than sample often occurs, that is the dehydrogenase activity that obtains is a negative value under the lower situation of biologically active; And after stating this method in the use, result that twice parallel experiment obtains such as following table, obtaining equation of linear regression by typical curve simultaneously is y=534.97x-14.74 (R
2=0.9849), y represents the growing amount of triphenyl methylamine in the formula, i.e. the activity value of dehydrogenasa, the absorbance that x representative test records, R
2Represent the related coefficient of typical curve.
Twice parallel experiment dehydrogenase activity result of table 1 activated carbon process charcoal bed fillers
| Numbering | Apart from the charcoal headroom height | Absorbance x 1 | Activity value y 1 | Absorbance x 2 | Activity value y 2 |
| A | 40cm | 0.075 | 25.383 | 0.066 | 20.568 |
| B | 80cm | 0.045 | 9.334 | 0.050 | 12.009 |
| C | 120cm | 0.034 | 3.449 | 0.033 | 2.914 |
Claims (3)
1. the preprocessing method for measuring desaturase liveness of drinking water biological active carbon treatment process is characterized in that this method step is as follows:
(1) active carbon filler is carried out pre-treatment: get respectively apart from charcoal bed top layer 40,80,120cm degree of depth active carbon filler 20～25g and put into 100mL tool plug triangular flask A, B, C, at first adding volume ratio is the extraction mixed liquor of 1: 2: 1 chloroform, methyl alcohol and distilled water, water bath with thermostatic control shaking table 120rpm jolting 10-15min, leave standstill 6h, in A, B, C triangular flask, add chloroform and distilled water more respectively, making the final volume ratio of chloroform, methyl alcohol and distilled water is 1.25: 1: 1, place shaking table 120rpm vibration 20min, leave standstill 6h;
(2) active carbon filler after the pre-treatment is measured absorbance respectively: the liquid of drawing respectively after the quantitative extraction of two-phase up and down places color comparison tube D, E, F, add trihydroxy aminomethane-hydrochloride buffer, sodium sulfite solution, pure water successively, and 2,3,5-triphenyl tetrazolium chloride solution, with formaldehyde as stopping reagent, add methenyl choloride after reacting completely again respectively, and measure absorbance respectively;
(3) drawing standard curve and the absorbance in the step (2) is converted into San Ben Ji Jia Za growing amount is the dehydrogenase activity value: the mensuration process of the active carbon filler extract in (2) set by step, measure 2,3, the absorbance of the San Ben Ji Jia Za that generates under the different sequence concentration of 5-triphenyl tetrazolium chloride, and drawing standard curve, draw equation of linear regression, at last absorbance substitution regression equation is drawn San Ben Ji Jia Za growing amount, i.e. the dehydrogenase activity value.
2. press the preprocessing method for measuring desaturase liveness of the described drinking water biological active carbon treatment process of claim 1, it is characterized in that in the mensuration process that the pre-treatment condition is selected described in the step (2): after treating that layering is stable, draw tool plug triangular flask A, B, the C intermediate section interface organic phase 5ml of lower floor respectively, the inorganic phase 5ml in interphase upper strata, move to 50ml color comparison tube D, E, F respectively, in color comparison tube, add tris-HCI buffer, the 0.36%Na of pH8.4 successively
2SO
3Solution, distilled water and 0.4% 2,3,5-triphenyl tetrazolium chloride solution, volume ratio is 3: 1: 1: 1; From color comparison tube D, E, F, draw 2ml lower floor organic phase and 8ml upper strata water respectively, place color comparison tube G, H, I, add the 2m1 formaldehyde fixed respectively to do sample blank, with color comparison tube D, E, F, G, H, I constant temperature water bath shaken cultivation 3h under 35-37 ℃ of condition, hunting speed 80rpm adds 3.0ml formaldehyde then and ends enzyme reaction in D, E, F color comparison tube; In D, E, F color comparison tube, add the 9ml methenyl choloride respectively again, in G, H, I, add the 6ml methenyl choloride respectively, mixing, with color comparison tube D, E, F, G, H, I after room temperature continues vibration 10min, centrifugal 5min, 4000 rev/mins, take off layer organic phase respectively and carry out colorimetric estimation, the record light absorption value in wavelength 485nm.
3. press the preprocessing method for measuring desaturase liveness of the described drinking water biological active carbon treatment process of claim 1, it is characterized in that at the drawing standard curve described in the step (3): other gets 8 50mL color comparison tubes, respectively to wherein adding 2 of 7.5ml trihydroxy aminomethane-hydrochloride buffer, 2.5ml distilled water and 2.5ml variable concentrations successively, 3,5-triphenyl tetrazolium chloride series solution; Add the 1g sodium hydrosulfite more respectively, the colour developing 5min after, add the 5mL methenyl choloride respectively, 80rpm shaking out 3min, treat solution-stabilized after, take off layer organic solution respectively to the cuvette of 1cm; After stablizing 1-2min once more, measure absorbance in 752 ultraviolet grating spectrophotometers in the 485nm place, the drawing standard curve obtains equation of linear regression simultaneously; According to the absorbance that obtains, substitution equation of linear regression, and then calculate the growing amount of San Ben Ji Jia Za, the i.e. activity of dehydrogenasa.
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| WO2010074052A1 (en) | 2008-12-22 | 2010-07-01 | ポーラ化成工業株式会社 | Melanin production inhibitor |
| CN101936909B (en) * | 2010-08-31 | 2012-05-23 | 天津工业大学 | Enzyme activity testing method for micro-biological degradation of benzene ring |
| CN107084934A (en) * | 2017-06-16 | 2017-08-22 | 哈尔滨工业大学 | A kind of method of in-site detecting drinking water biological active carbon TTC dehydrogenase activities |
| CN109470637B (en) * | 2018-10-08 | 2020-04-17 | 浙江大学 | Method for determining activity of ethanol dehydrogenase |
| CN109580510A (en) * | 2018-12-04 | 2019-04-05 | 天津大学 | A kind of activated sludge TTC measuring desaturase liveness method |
| CN111620441B (en) * | 2020-06-11 | 2022-09-16 | 南京中洲环保科技有限公司 | Synchronous sewage nitrification and denitrification control method and device |
Citations (3)
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| CN1096582A (en) * | 1993-06-11 | 1994-12-21 | 周春生 | Digested sludge dehydrogenase activity normal temperature extraction and determination method |
| CN1598578A (en) * | 2004-07-22 | 2005-03-23 | 哈尔滨工业大学 | Method for investigating TTC-dehydrogenase activity for drinking water treated biologic |
| CN1707243A (en) * | 2004-06-07 | 2005-12-14 | 中国科学技术大学 | Method for measuring dehydrogenase activity of sludge |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1096582A (en) * | 1993-06-11 | 1994-12-21 | 周春生 | Digested sludge dehydrogenase activity normal temperature extraction and determination method |
| CN1707243A (en) * | 2004-06-07 | 2005-12-14 | 中国科学技术大学 | Method for measuring dehydrogenase activity of sludge |
| CN1598578A (en) * | 2004-07-22 | 2005-03-23 | 哈尔滨工业大学 | Method for investigating TTC-dehydrogenase activity for drinking water treated biologic |
Non-Patent Citations (2)
| Title |
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| 生物活性炭法深度处理纺织废水二级出水中生物活性的研究. 田晴等.环境污染治理技术与设备，第7卷第3期. 2006 |
| 生物活性炭法深度处理纺织废水二级出水中生物活性的研究. 田晴等.环境污染治理技术与设备，第7卷第3期. 2006 * |
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