CN103966202A - Improved freeze thawing extraction method of total DNA (deoxyribonucleic acid) in activated sludge and application thereof - Google Patents

Abstract

The invention discloses an improved freeze thawing extraction method of total DNA (deoxyribonucleic acid) in activated sludge and an application thereof. A 'CTAB (Cetyltrimethyl Ammonium Bromide)+freeze thawing+lysozyme+protease method' DNA extraction method is improved by fully utilizing the advantages of a gradient repeated freeze-thawing method and the improved method and is applied to high-efficiency extraction of the DNA in the activated sludge in a high-salinity oil producing wastewater biological treatment system. The improved freeze thawing extraction method is characterized in that CTAB is mainly used for improving a conventional DNA extraction buffer solution, required DNA fragments are effectively separated by using a gradient freeze-thawing and dissolving-out method, and then lysozyme and a protease method are used for completing high-efficiency extraction of the DNA from the activated sludge in a high-salinity oil producing wastewater purification system. The improved freeze thawing extraction method has the advantages of high separation and purification degree and simple and stable operation process and avoids the influence of high-salinity oily wastewater on an extraction process, and meanwhile, the practice shows that the improved freeze thawing extraction method has a good separation function for various unpredictable impurities in the high-salinity oily sludge.

Description

Freeze thawing Improvement type extracting method and the application thereof of total DNA in active sludge

Technical field

The invention belongs to environmental science, particularly freeze thawing Improvement type extracting method and the application thereof of total DNA in a kind of active sludge.

Background technology

The water that the oil-water mixture of extraction produces after oily water separation below natural rock is just oil extraction waste water.In oil extraction waste water, contain many organic and inorganicss, and residing geographical position, different oil fields, geologic framework, the production life of well in oil field, the kind difference of recover petroleum hydrocarbon, the kind of the oil extraction waste water producing is also different.In addition, due to sneaking into of the sewage such as drilling waste water, well flushing waste water, sanitary sewage, make the composition of oil extraction waste water more complicated.Under normal circumstances, oil extraction waste water has following characteristics: (1) oleaginousness is high.Conventionally the crude content in oil extraction waste water can reach 1000-2000mg/L, more than the oleaginousness in the waste water in part oil field even can reach 5000mg/L.According to being divided into oil slick, dispersed oil, oil in water emulsion and dissolving oil containing the particle diameter of oil particles in waste water.Particle diameter is greater than the oil slick of 100 μ m and the dispersed oil of 10-100 μ m and accounts for 90% left and right of total oil content amount in waste water, and the oil in water emulsion of 0.1-10 μ m accounts for 10%, and the dissolving oil content that is less than 0.1 μ m is very low.(2) saltiness is high.The contained salt of oilfield produced water is mainly inorganic salt, and the general different oil field even saltiness of different blocks, oil reservoir is all different, and from several thousand to tens0000 milligrams per liters are not etc.Contained inorganic ion mainly comprises: Ca ²⁺, Mg ²⁺, K ⁺, Na ⁺, Fe ²⁺, Cl ^?, SO ₄ ², HCO ₃ ^?and CO ₃ ²deng.(3) containing suspended solids.Be generally the particle of 1-100 μ m, mainly comprise clay particle, flour sand and fine sand etc.(4) containing bacterium.Generally in the majority with saprophytic microorganism and sulphate reducing bacteria.(5) some is also containing tensio-active agent.In addition, oil extraction waste water also has the feature of high temperature (40-80 DEG C) and high pH value.

The method that the processing of oil extraction waste water can adopt Physical, chemical method, biological process, embrane method and combine.In these treatment processs biological treatment because of its working cost low, the feature such as easy and simple to handle and being widely used.Conventionally can be divided into the biological treatment of oil extraction waste water processing: activated sludge process, biomembrance process, biological contact oxidation pond, oxidation pond etc.Activated sludge process is modal technology in the biodegradable sewage of processing, the method can be divided into biological and two stages of physics to the processing of waste water, is mainly the materials such as organism, ammonium salt and phosphoric acid salt in the microbiological deterioration waste water in active sludge in the biology stage.What in this technology, treatment effect is played to key effect is exactly the activity of Microbial Communities in Activated Sludge, and microbic activity is determined by structure of community and the feature of Microbial Communities in Activated Sludge.Microflora in active sludge is very abundant, is mainly made up of bacterium, fungi, ancient bacterium, actinomycetes, algae, protozoon and metazoan etc., and wherein bacterium most importantly, accounts for greatly 95% of whole active sludge microorganism group.Therefore, bacterium is the important leverage that the materials such as organism in system for activated sludge wastewater treatment, ammonium salt and phosphoric acid salt are effectively removed.But in strain identification, screening and the genetic engineering bacterium to bacterium synthetic, the high efficiency extraction of DNA be its gordian technique, therefore, scholars carry out the research of DNA extraction method in a large number both at home and abroad.Adopt cetyl trimethylammonium bromide (CTAB)+gradient multigelation method+N,O-Diacetylmuramidase+protease method to carry out the research of bacterial classification DNA extraction, be beneficial in analysis oil extraction waste water treatment system active sludge bacterium, fungi in kind and quantitative variation.Result of study can be follow-up active sludge screening efficient degreasing bacterial strain, structure efficient degreasing composite flora provides theoretical foundation, simultaneously to instructing actual production operation significant.

Summary of the invention

The object of this invention is to provide freeze thawing Improvement type extracting method and the application thereof of total DNA in a kind of active sludge.

Thinking of the present invention: make full use of the advantage of gradient multigelation method, improvement " CTAB+ freeze thawing+N,O-Diacetylmuramidase+protease method " DNA extraction method, and be applied in the high efficiency extraction of activated sludge DNA of high salinity oil extraction biological treatment of waste water system.

Mechanism of the present invention: utilize CTAB to improve traditional DNA extraction damping fluid, utilize the method for gradient freezing stripping effectively to separate required DNA fragmentation, then apply N,O-Diacetylmuramidase and protease method and complete the DNA high efficiency extraction of active sludge in high salinity oil extraction waste water purification system.

Concrete steps are:

(1) evenly obtain target dna source body mud 20 ~ 30mL, its water ratio is greater than 98%, be placed in 100mL centrifuge tube, carry out centrifugal pre-treatment 5 ~ 8min with the rotating speed of 6000 ~ 8000r/min, abandoning supernatant, cross quick filter paper collecting precipitation material stand-by, collected sedimentable matter is less than 1mL.

(2) the collected sedimentable matter of step (1) is joined in the tool plug centrifuge tube of 2mL, and add the DNA extraction damping fluid of 1 ~ 2mL, stand-by after vortex oscillation device vibration 5 ~ 10min; The PVPP that the NaCl of Tris-HCl, the 1.5 ~ 3.0mol/L of EDTA, 100 ~ 150mmol/L that described DNA extraction damping fluid contains 100 ~ 150mmol/L, the CTAB that mass percent concentration is 2 ~ 5% and mass percent concentration are 1 ~ 3%, and use the HCl of 0.1 ~ 0.5mol/L and NaOH dilute solution to regulate the pH value to 8.0 of DNA extraction damping fluid.

(3) material step (1) being made is put into-80 DEG C of refrigerator and cooled and is frozen 6 ~ 8min, then put into 65 DEG C of water-bath water-bath 10 ~ 15min, put into again-60 DEG C of freezing 10 ~ 15min of Ultralow Temperature Freezer, then put into 65 DEG C of water-bath water-bath 10 ~ 15min, then put into-40 DEG C of freezing 25 ~ 30min of Ultralow Temperature Freezer, then put into 65 DEG C of water-bath water-bath 10min; During each water-bath after freezing, all require to put upside down to mix 1 ~ 2 time, complete gradient increased temperature freezing-water-bath is thawed and to make sample for subsequent use.

(4) in the sample making to step (3), add the N,O-Diacetylmuramidase that 80 ~ 100 μ L concentration are 10mg/mL, be placed in 37 DEG C of water-baths and cultivate 10 ~ 15min, turn upside down and mix once every 2 ~ 3min, then adding 10 ~ 15 μ L concentration is the SDS that 10mg/mL Proteinase K and 150 ~ 200 μ L mass percent concentrations are 10%, 65 DEG C of water-bath 10min, then directly put into-80 DEG C of refrigerator quick-frozen 6 ~ 8min, 28 ~ 32min finally thaws in 65 DEG C of warm water; Repeat this freeze thawing step 3 time, make sample for subsequent use.

(5) sample step (4) being made is at normal temperatures with the centrifugal 10 ~ 15min of rotating speed of 6000 ~ 8000r/min, get in the centrifuge tube that supernatant liquor is sub-packed in 2mL, adding isopyknic volume ratio is the saturated phenol-chloroform mixed solution of Tris of 1:1, turn upside down and mix, centrifugal 10 ~ 15min under the rotating speed of 8000 ~ 10000r/min, complete extracting, this extraction steps repeats 2 times, makes supernatant liquor for subsequent use.

(6) supernatant liquor step (5) being made is transferred in the centrifuge tube of 1 ~ 2mL, be incorporated as the Virahol of 0.6 ~ 0.8 times of volume of supernatant liquor, turn upside down and mix, room temperature leaves standstill 1 ~ 1.5h, centrifugal 10 ~ 15min under the rotating speed of 10000-120000r/min, discard supernatant liquor, by 50 ~ 70% the washing with alcohol precipitation of precooling 2 ~ 3 times, centrifugal 2 ~ 5min under the rotating speed of 8000 ~ 10000r/min, with the continuous rinsing of dehydrated alcohol 2 times, abandon after supernatant liquor, air-dry under room temperature, add 30 μ L sterilized waters to make target sample, target sample is preserved at-20 DEG C.

The inventive method is applied to the extraction of the total DNA of high salinity oil extraction Waste Water Treatment active sludge.

Adopt the inventive method to there is separation and purification degree to the extraction of total DNA in high salinity oil extraction Waste Water Treatment active sludge high, operation process is simple and stable, avoid the impact of high salinity oily(waste)water on leaching process, meanwhile, the method all has good separation function to multiple unpredictable impurity in mud under high salinity oil-containing background.

Brief description of the drawings

Fig. 1 is the agarose detection figure to total DNA Different Extraction Method in the active sludge of SBR pond in the embodiment of the present invention.

Wherein M is λ-DNA marker, total DNA result that swimming lane 1 extracts for method two, total DNA result that swimming lane 2 extracts for method 1.

Embodiment

embodiment:

Get Zhanjiang Branch of China National Offshore Oil (China) Co., Ltd. Weizhou SBR of terminal process factory pond mud 20mL, join in the centrifuge tube of 100mL, carry out centrifugal pre-treatment 5min with the rotating speed of 8000r/min, remove supernatant liquor, collecting precipitation 0.95mL joins in 2mL tool plug centrifuge tube, and to add 1mLpH be that 8.0 DNA extraction damping fluid is (containing 100mmol/L EDTA, 100mmol/L Tris, 1.5mol/L NaCl, 2% CTAB and 1% PVPP), with after vortex oscillation device vibration 5min, be placed in-80 DEG C of refrigerator and cooled and freeze 6min, then put into 65 DEG C of water-bath water-bath 10min, put into again-60 DEG C of refrigerator and cooled and freeze 10min, then put into 65 DEG C of water-bath water-bath 10min, put into again-40 DEG C of refrigerator and cooled and freeze 25min, then put into 65 DEG C of water-bath water-bath 10min, in course of defrosting, all put upside down and mix 2 times.

In the sample making, add the N,O-Diacetylmuramidase that 100 μ L concentration are 10mg/mL, be placed in 37 DEG C of water-baths and cultivate 10min, turn upside down and mix once every 2min, then add the SDS that Proteinase K that 13 μ L concentration are 10mg/mL and 200 μ L mass percent concentrations are 10%, 65 DEG C of water-bath 10min, then-80 DEG C of quick-frozen 8min, 65 DEG C of 30min that thaw again, repeat this freeze thawing step 3 time, the sample making is at normal temperatures with the centrifugal 10min of rotating speed of 8000r/min, get in the centrifuge tube that supernatant liquor is sub-packed in 2mL, adding isopyknic volume ratio is the saturated phenol-chloroform mixed solution of Tris of 1:1, turn upside down and mix, centrifugal 10min under the rotating speed of 10000r/min, complete extracting, repeat this extraction steps 2 times, the supernatant liquor making is transferred in the centrifuge tube of 1.5mL, be incorporated as the Virahol of 0.6 times of volume of supernatant liquor, turn upside down and mix, room temperature leaves standstill 1h, centrifugal 10min under the rotating speed of 120000r/min, discard supernatant liquor, by 70% the washing with alcohol precipitation of precooling 2 ~ 3 times, centrifugal 2min under the rotating speed of 10000r/min, with the continuous rinsing of dehydrated alcohol 2 times, abandon after supernatant liquor, room temperature is air-dry, add 30 μ L sterilized waters to make target sample, at target sample-20 DEG C, preserve.

The present embodiment experiment showed, that through two-year DNA separation and purification level is compared with common methods excellence, and extraction level is higher, and specific experiment numerical value is in table 1.

The detected result of total DNA that table 1 extracts different methods with ultramicron ultraviolet spectrophotometer

Note: the extracting method that method one is the present embodiment; Method two is tradition " CTAB+ freeze thawing+N,O-Diacetylmuramidase+protease method " DNA extraction method.

Claims (2)

1. a freeze thawing Improvement type extracting method of total DNA in active sludge, is characterized in that concrete steps are:

(1) evenly obtain target dna source body mud 20 ~ 30mL, its water ratio is greater than 98%, be placed in 100mL centrifuge tube, carry out centrifugal pre-treatment 5 ~ 8min with the rotating speed of 6000 ~ 8000r/min, abandoning supernatant, cross quick filter paper collecting precipitation material stand-by, collected sedimentable matter is less than 1mL;

(2) the collected sedimentable matter of step (1) is joined in the tool plug centrifuge tube of 2mL, and add the DNA extraction damping fluid of 1 ~ 2mL, stand-by after vortex oscillation device vibration 5 ~ 10min; The PVPP that the NaCl of Tris-HCl, the 1.5 ~ 3.0mol/L of EDTA, 100 ~ 150mmol/L that described DNA extraction damping fluid contains 100 ~ 150mmol/L, the CTAB that mass percent concentration is 2 ~ 5% and mass percent concentration are 1 ~ 3%, and use the HCl of 0.1 ~ 0.5mol/L and NaOH dilute solution to regulate the pH value to 8.0 of DNA extraction damping fluid;

(3) material step (1) being made is put into-80 DEG C of refrigerator and cooled and is frozen 6 ~ 8min, then put into 65 DEG C of water-bath water-bath 10 ~ 15min, put into again-60 DEG C of freezing 10 ~ 15min of Ultralow Temperature Freezer, then put into 65 DEG C of water-bath water-bath 10 ~ 15min, then put into-40 DEG C of freezing 25 ~ 30min of Ultralow Temperature Freezer, then put into 65 DEG C of water-bath water-bath 10min; During each water-bath after freezing, all require to put upside down to mix 1 ~ 2 time, complete gradient increased temperature freezing-water-bath is thawed and to make sample for subsequent use;

(4) in the sample making to step (3), add the N,O-Diacetylmuramidase that 80 ~ 100 μ L concentration are 10mg/mL, be placed in 37 DEG C of water-baths and cultivate 10 ~ 15min, turn upside down and mix once every 2 ~ 3min, then adding 10 ~ 15 μ L concentration is the SDS that 10mg/mL Proteinase K and 150 ~ 200 μ L mass percent concentrations are 10%, 65 DEG C of water-bath 10min, then directly put into-80 DEG C of refrigerator quick-frozen 6 ~ 8min, 28 ~ 32min finally thaws in 65 DEG C of warm water; Repeat this freeze thawing step 3 time, make sample for subsequent use;

(5) sample step (4) being made is at normal temperatures with the centrifugal 10 ~ 15min of rotating speed of 6000 ~ 8000r/min, get in the centrifuge tube that supernatant liquor is sub-packed in 2mL, adding isopyknic volume ratio is the saturated phenol-chloroform mixed solution of Tris of 1:1, turn upside down and mix, centrifugal 10 ~ 15min under the rotating speed of 8000 ~ 10000r/min, complete extracting, this extraction steps repeats 2 times, makes supernatant liquor for subsequent use;

(6) supernatant liquor step (5) being made is transferred in the centrifuge tube of 1 ~ 2mL, be incorporated as the Virahol of 0.6 ~ 0.8 times of volume of supernatant liquor, turn upside down and mix, room temperature leaves standstill 1 ~ 1.5h, centrifugal 10 ~ 15min under the rotating speed of 10000-120000r/min, discard supernatant liquor, by 50 ~ 70% the washing with alcohol precipitation of precooling 2 ~ 3 times, centrifugal 2 ~ 5min under the rotating speed of 8000 ~ 10000r/min, with the continuous rinsing of dehydrated alcohol 2 times, abandon after supernatant liquor, air-dry under room temperature, add 30 μ L sterilized waters to make target sample, target sample is preserved at-20 DEG C.

2. the application of the freeze thawing Improvement type extracting method of total DNA in active sludge according to claim 1, is characterized in that the method is applied to the extraction of total DNA in high salinity oil extraction Waste Water Treatment active sludge.