CN106755286B - A method of testing oil extraction waste water bio-toxicity using Vibrio-qinghaiensis sp. Q67 - Google Patents
A method of testing oil extraction waste water bio-toxicity using Vibrio-qinghaiensis sp. Q67 Download PDFInfo
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- CN106755286B CN106755286B CN201611171167.XA CN201611171167A CN106755286B CN 106755286 B CN106755286 B CN 106755286B CN 201611171167 A CN201611171167 A CN 201611171167A CN 106755286 B CN106755286 B CN 106755286B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/64—Geomicrobiological testing, e.g. for petroleum
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
Abstract
The invention discloses a kind of methods for testing oil extraction waste water bio-toxicity using Vibrio-qinghaiensis sp. Q67.It is test waste water with oil extraction waste water, is instruction biology with Vibrio-qinghaiensis sp. Q67, using 96 hole microwell plates as carrier；By 96 hole microwell plate analysis methods, its luminous intensity is detected using microplate spectrophotometer；Luminous inhibiting rate is calculated using experimental group and the luminous intensity of control group, sets Zn（NO3）2As toxicity reference substance, to judge the bio-toxicity of oil extraction waste water.The method of the present invention is detected analysis using microplate method to the toxicity of oil extraction waste water, by the luminous inhibiting rate for calculating photogen, to judge the comprehensive toxicity size of oil extraction waste water, solve the problems, such as that conventional physical and chemical index cannot indicate oil extraction waste water bio-toxicity, quickly, toxicity reference substance Zn sensitive, easy to utilize and selected（NO3）2Experimental result is stablized, and cheap, toxicity is medium.
Description
Technical field
The invention belongs to wastewater biological toxicity detection field, more particularly to a kind of recovered the oil using Vibrio-qinghaiensis sp. Q67 test is given up
The method of aquatic toxicity.
Background technology
Oil extraction waste water is oil field in oil recovery process, except returned as re-injection, technique mix or the industrial waters such as other purposes with
Outside, the outer waste water arranged is needed.Wherein contain the macromolecules persistent organic pollutants such as petroleum-type, surfactant.Oil extraction waste water has
There are salinity height, the high feature of water temperature that there is certain corrosivity to the object of contact, in addition to this, in waste water also contains certain
The fungicide of amount.Existing《Integrated wastewater discharge standard》The physical and chemical index limit of discharge of wastewater is defined in (GB 8978-1996)
Value, but it is not directed to the discharge index of wastewater toxicity.At this stage, the bio-toxicity of oil extraction waste water and the harm to environment, by
Extensive concern.The detection method of wastewater biological toxicity has at present：Photobacterium Phosphoreum Toxicity is tested, algae toxicity experiment, flea class poison
Property experiment, toxicity test of fishes, birds toxicity test etc..Wherein, the instruction biology that photogen is examined as wastewater toxicity, has
Fast and convenient advantage.
Invention content
The object of the present invention is to provide a kind of methods for testing oil extraction waste water bio-toxicity using Vibrio-qinghaiensis sp. Q67.
Thinking of the present invention：It is test waste water with oil extraction waste water, using 96 hole microwell plates as carrier；Pass through 96 hole micropore plate analysis
Method detects its luminous intensity using Synergy H2 microplate spectrophotometers；Utilize shining for experimental group and control group
Intensity calculates luminous inhibiting rate, setting Zn (NO3)2As toxicity reference substance, to judge the bio-toxicity of waste water.
The specific steps are：
(1) by 0.45 μm of membrane filtration of the muddy oil extraction waste water adopted back, the oil extraction waste water that is made that treated, then by it
Be placed in 4 DEG C of refrigerator preserve it is for use, it is assumed that the concentration of treated oil extraction waste water is unit 1.
(2) culture Vibrio-qinghaiensis sp. Q67 reaches exponential phase, Vibrio-qinghaiensis sp. Q67 bacterium solution is obtained, as work
Bacterium solution.
(3) bacterium solution that works is added to the 96 hole microwell plate of White-opalescent for pre-setting oil extraction waste water concentration gradient
In, the reaction system of oil extraction waste water and work bacterium solution is made, is 200 μ L per hole total volume, experimental port is per hole containing 100 μ L work bacterium
The oil extraction waste water of liquid and 100 μ L various concentrations, blank control wells, containing 100 μ L work bacterium solutions and 100 μ L ultra-pure waters, make work per hole
Bacterium solution fully reacts 15min with oil extraction waste water, then measures its luminous intensity with Synergy H2 microplate spectrophotometers
RLU, under the same terms, each oil extraction waste water concentration carries out 9 repetitions and tests, and is averaged.
(4) setting Zn (NO3)2As toxicity reference substance, with ultra-pure water by Zn (NO3)2Various concentration gradient is diluted, then
Work bacterium solution is added and fully reacts 15min, its luminous intensity is measured using SynergyH2 microplate spectrophotometers, calculates hair
Xanthophyll cycle rate is simultaneously shone inhibiting rate and Zn (NO with the Logistic function pairs in Origin9.03)2Concentration is fitted, and is established dense
Degree-effect curve.
(5) use Synergy H2 microplate spectrophotometers detect microwell plate luminous intensity, using experimental group with compare
The luminous intensity of group calculates luminous inhibiting rate E of the various concentration oil extraction waste water to Vibrio-qinghaiensis sp. Q67, dense with oil extraction waste water difference
Degree is that abscissa utilizes the Logistic function pairs in Origin9.0 using the corresponding luminous inhibiting rate of various concentration as ordinate
Various concentration oil extraction waste water carries out nonlinear fitting with luminous inhibiting rate E.
In formula：I0For the RLU average values of blank control sample, I is the RLU average values of 9 Duplicate Samples of each concentration；It compares identical
Zn (NO under luminous inhibiting rate3)2Concentration, according to toxicity reference substance Zn (NO3)2Judge the comprehensive toxicity size of oil extraction waste water, has
Body is as follows：
Zn(NO3)2Concentration C < 0.29mg/L when, toxicity level be it is micro- poison or it is nontoxic；Zn(NO3)2Concentration 0.29mg/
When L≤C < 1.11mg/L, toxicity level is low toxicity；Zn(NO3)2Concentration 1.11mg/L≤C < 1.60mg/L when, toxicity level
For poisoning；Zn(NO3)2Concentration 1.60mg/L≤C < 2.91mg/L when, toxicity level is high poison；Zn(NO3)2Concentration C >=
When 2.91mg/L, toxicity level is severe toxicity.
The method of the present invention is detected analysis using microplate method to the toxicity of oil extraction waste water, by calculating shining for photogen
Inhibiting rate, to judge the comprehensive toxicity size of oil extraction waste water, oil extraction waste water biology cannot be indicated by solving conventional physical and chemical index
The problem of toxicity, toxicity reference substance Zn (NO quick, sensitive, easy to utilize and selected3)2Experimental result is stablized,
Cheap, toxicity is medium.
Description of the drawings
Fig. 1 is the sample-adding design drawing of the 96 hole microwell plates used in the embodiment of the present invention, wherein B is control group, contains 100 μ
+ 100 μ L bacterium solutions of L ultra-pure waters；C1~C8It is followed successively by 8 concentration gradients of oil extraction waste water from high to low.
Fig. 2 is that zinc nitrate shines to Vibrio-qinghaiensis sp. Q67 the concentration effect curve of inhibiting rate in the embodiment of the present invention.
Fig. 3 is that each process section oil extraction waste water shines to Vibrio-qinghaiensis sp. Q67 concentration-effect of inhibiting rate in the embodiment of the present invention
Curve.
Specific implementation mode
Embodiment：
Waste water sample to be measured in the present embodiment is derived from certain each treatment process section of oil Weizhou terminal wastewater treatment plant oil extraction waste water, and five
A sample point is respectively：Raw water, ABR waters, the water outlet of the ponds ABR, SBR waters and water outlet carry out the sample of each sample point as follows
Experiment：
(1) it is test waste water with each process section oil extraction waste water and carries out pre-treatment：
Oil extraction waste water combines work after pipeline to terminal wastewater treatment plant, using anaerobic baffled reactor and activated sludge process
Skill biochemical treatment, through biochemical process, treated that waste water is delivered to clear water reserviors is then discharged out outside factory.In order to avoid muddy waste water is to reality
The influence for testing result, by 0.45 μm of membrane filtration of sample to be tested, 4 DEG C of refrigerators preserve for use, it is assumed that treated oil extraction waste water
Concentration be unit 1.
(2) culture Vibrio-qinghaiensis sp. Q67 reaches exponential phase, as work bacterium solution.
The activation and inoculation of strain：The Q67 freezing dry powders equipped with Qinghai Vibrion bacterial strain of -30 DEG C of preservations are taken out (purchased from north
Jing Binsong photon technologies limited liability company) ampoule bottle, be placed in about 10~15min in 4 DEG C of refrigerators, wiped with cotton ball soaked in alcohol outer
Week disinfection after, in superclean bench with grinding wheel cut ampoule bottle, be added 300 μ L Q67 freeze-dried powder resuscitation fluids, slight oscillatory, into
Then row activation is pipetted on strain liquid to culture dish tablet with the pipettor that range is 100 μ L, culture dish is then positioned over perseverance
In warm incubator, 22 DEG C of cultures (summer 18h, winter 30h) grow bacterium colony and obtain F1Then in generation, uses oese from F1Choosing colony
It is seeded on plating medium, inversion is put in constant incubator, and 22 DEG C of cultures (summer 18h, winter 30h) grow bacterium colony acquisition
F2In generation, is cultivated after the same method to F3Generation.Taking-up is positioned over 4 DEG C of refrigerators and preserves for use.
Test the culture of strain：One ring bacterium colony of picking on the Q67 solid plates preserved from 4 DEG C is transferred to the training of 50mL liquid
It supports in base, is placed in shaken cultivation case and cultivates, 22 DEG C, shaken cultivation under 120rpm, until Q67 reaches exponential phase, for use.
Fluid nutrient medium is prepared：13.6mg KH2PO4、35.8mg Na2HPO4·12H2O、250.0mg MgSO4·7H2O、
610.0mg MgCl2·6H2O、33.0mg CaCl2、1.34g NaHCO3, 1.54g NaCl, 5.0g yeast extracts, 5.0g pancreas eggs
White peptone and 3.0g glycerine.Each ingredient of culture medium is weighed, heating is dissolved in 1L ultra-pure waters.PH value is adjusted with 1mol/L NaOH solutions
It to 8.5~9.0, is sub-packed in 250mL conical flasks, every bottle of about 50mL is wrapped up with brown paper, 120 DEG C of high pressure steam sterilizations
25min is saved backup after cooling in 4 DEG C of refrigerators.
Solid medium is prepared：Take the above-mentioned fluid nutrient medium 300ml prepared, 1.5%~2% (4.5~6g) fine jade
Cosmetics is added in the triangular flask of 1L, while the culture dish of clean drying being wrapped up with brown paper and is tightly put into togerther height with culture medium
It presses in autoclave, 120 DEG C, high pressure steam sterilization 25min take out, are placed in the superclean bench of 10~20min of ultraviolet sterilization
In, prepare solid plate culture medium after slightly cold, culture medium fallen in culture dish, sprawl it is uniform, cooling after tablet is made, be put into
It is preserved in 4 DEG C of refrigerators, is spare.
(3) assume that the concentration of treated oil extraction waste water is unit 1 (100%), setting concentration gradient C1~C8Respectively 1,
0.9,0.8,0.7,0.6,0.5,0.4,0.3, the bacterium solution that works is added to and pre-sets the white impermeable of waste strength gradient
In the microwell plate of bright 96 hole, make the reaction system of oil extraction waste water and the bacterium solution that works, wherein per hole total volume be 200 μ L (100 μ L's
The oil extraction waste water for the bacterium solution and 100 μ L various concentrations of working, blank are the work bacterium solution and 100 μ L ultra-pure waters of 100 μ L) specifically it is loaded
Method is as shown in Figure 1.So that work bacterium solution is fully reacted 15min with oil extraction waste water, then Synergy H2 microwell plates is used to be divided light
Degree meter measures its luminous intensity RLU.In order to reduce experimental error, under the same terms, each concentration carries out 9 repetitions and tests.
(4) setting Zn (NO3)2As toxicity reference substance, with ultra-pure water by Zn (NO3)2Various concentration gradient is diluted, then
Work bacterium solution is added and fully reacts 15min, its luminous intensity is detected using Synergy H2 microplate spectrophotometers；It utilizes
The luminous inhibiting rate of Logistic function pairs in Origin9.0 and Zn (NO3)2Concentration is fitted, and establishes concentration effect curve.
By extremely toxic substance HgCl in conventional method2As toxicity reference substance, but because its for extremely toxic substance be not only difficult into
Row experiment, and it is be easy to cause environmental pollution, Zn2+Because its experimental result is stablized, cheap, toxicity is medium and is picked as poison
Property object of reference.
(5) use Synergy H2 microplate spectrophotometers detect microwell plate luminous intensity, using experimental group with compare
The luminous intensity of group calculates luminous inhibiting rate E of the various concentration oil extraction waste water to Vibrio-qinghaiensis sp. Q67, dense with oil extraction waste water difference
Degree is that abscissa utilizes the Logistic function pairs in Origin9.0 using the corresponding luminous inhibiting rate of various concentration as ordinate
Various concentration oil extraction waste water carries out nonlinear fitting with luminous inhibiting rate E, is fitted R2>=0.97.Compare identical inhibition simultaneously
Zn (NO under rate3)2Concentration, to judge the comprehensive toxicity size of oil extraction waste water.
In formula：I0For the average value of blank control RLU, I is the RLU average values of 3 Duplicate Samples of each concentration.
The class of pollution and bio-toxicity grade scale
Experimental result shows, certain each treatment process section waste water of oil Weizhou terminal wastewater treatment plant there are certain noxious material,
There is certain inhibiting effect to Vibrio-qinghaiensis sp. Q67 luminous intensity, toxicity size is：Raw water>The ponds ABR>ABR goes out>The ponds SBR>Go out
Water.Toxicity level is：
Raw water is shown as high poison；The ponds ABR are shown as being poisoned；ABR goes out to be shown as low toxicity；The ponds SBR are shown as low toxicity；Water outlet is aobvious
It is shown as low toxicity.
Therefore, oil extraction waste water has the effect of certain after biochemical treatment, but there are still certain harm to water environment.
The present embodiment shows significant test effect：Oil extraction waste water is tested with instruction biology-Vibrio-qinghaiensis sp. Q67
Toxicity, by calculating different process section waste water to the size of the luminous inhibiting rate of Vibrio-qinghaiensis sp. Q67, the toxicity etc. of reaction waste
Grade, so as to effectively, quickly and accurately judge the comprehensive toxicity size of each process section waste water.
Claims (1)
1. a kind of method for testing oil extraction waste water bio-toxicity using Vibrio-qinghaiensis sp. Q67, it is characterised in that the specific steps are：
(1) by 0.45 μm of membrane filtration of the muddy oil extraction waste water adopted back, then the oil extraction waste water that is made that treated places it in
It is preserved in 4 DEG C of refrigerator for use, it is assumed that the concentration of treated oil extraction waste water is unit 1；
(2) culture Vibrio-qinghaiensis sp. Q67 reaches exponential phase, obtains Vibrio-qinghaiensis sp. Q67 bacterium solution, as work bacterium
Liquid；
(3) work bacterium solution is added in the 96 hole microwell plate of White-opalescent for pre-setting oil extraction waste water concentration gradient, is made
The reaction system for making oil extraction waste water and the bacterium solution that works, is 200 μ L per hole total volume, experimental port per hole containing 100 μ L work bacterium solutions and
The oil extraction waste water of 100 μ L various concentrations, blank control wells, containing 100 μ L work bacterium solutions and 100 μ L ultra-pure waters, make work bacterium solution per hole
15min is fully reacted with oil extraction waste water, then measures its luminous intensity RLU, phase with Synergy H2 microplate spectrophotometers
With under the conditions of, each oil extraction waste water concentration carries out 9 repetitions and tests, and is averaged；
(4) setting Zn (NO3)2As toxicity reference substance, with ultra-pure water by Zn (NO3)2Various concentration gradient is diluted, work is then added
Make bacterium solution and fully react 15min, its luminous intensity is measured using Synergy H2 microplate spectrophotometers, calculates the inhibition that shines
Rate is simultaneously shone inhibiting rate and Zn (NO with the Logistic function pairs in Origin9.03)2Concentration is fitted, and establishes concentration-effect
Answer curve；
(5) it uses Synergy H2 microplate spectrophotometers to detect microwell plate luminous intensity, utilizes experimental group and control group
Luminous intensity calculates luminous inhibiting rate E of the various concentration oil extraction waste water to Vibrio-qinghaiensis sp. Q67, is with oil extraction waste water various concentration
Abscissa, it is different using the Logistic function pairs in Origin9.0 using the corresponding luminous inhibiting rate of various concentration as ordinate
Concentration oil extraction waste water carries out nonlinear fitting with luminous inhibiting rate E；
In formula：I0For the RLU average values of blank control sample, I is the RLU average values of 9 Duplicate Samples of each concentration；Compare identical shine
Zn (NO under inhibiting rate3)2Concentration, according to toxicity reference substance Zn (NO3)2Judge the comprehensive toxicity size of oil extraction waste water, specifically such as
Under：
Zn(NO3)2Concentration C < 0.29mg/L when, toxicity level be it is micro- poison or it is nontoxic；Zn(NO3)2Concentration 0.29mg/L≤C
When < 1.11mg/L, toxicity level is low toxicity；Zn(NO3)2Concentration 1.11mg/L≤C < 1.60mg/L when, during toxicity level is
Poison；Zn(NO3)2Concentration 1.60mg/L≤C < 2.91mg/L when, toxicity level is high poison；Zn(NO3)2Concentration C >=
When 2.91mg/L, toxicity level is severe toxicity.
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| CN108226493A (en) * | 2018-01-10 | 2018-06-29 | 中国科学院广州地球化学研究所 | A kind of industrial wastewater bio-toxicity detection method based on genetic recombination photogen |
| CN108507999A (en) * | 2018-03-26 | 2018-09-07 | 成都飞航智库科技有限公司 | One kind being applied to bio-toxicity detection method in biotechnology |
| CN109975228A (en) * | 2019-05-10 | 2019-07-05 | 苏州华能检测技术有限公司 | A kind of wastewater toxicity detection method |
| CN110484591A (en) * | 2019-08-25 | 2019-11-22 | 桂林理工大学 | A method of sulfa antibiotics bio-toxicity is tested using Vibrio-qinghaiensis sp. Q67 |
| CN110376146A (en) * | 2019-08-25 | 2019-10-25 | 桂林理工大学 | A method of sulfa antibiotics bio-toxicity is tested using scenedesmus obliquus |
| CN112082961A (en) * | 2020-08-28 | 2020-12-15 | 桂林理工大学 | Method for testing toxicity of microbial plastics by using scenedesmus obliquus |
| CN112540162A (en) * | 2020-11-25 | 2021-03-23 | 江苏雅信昆成检测科技有限公司 | Water quality biotoxicity detection method |
| CN114397418B (en) * | 2022-01-21 | 2023-10-24 | 浙江清华长三角研究院 | Logistic fitting-based water quality comprehensive toxicity and suspected toxic substance testing method |
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| CN102175606A (en) * | 2011-01-19 | 2011-09-07 | 西安建筑科技大学 | Method for detecting acute biological toxicity of sewage |
| CN103045524A (en) * | 2013-01-18 | 2013-04-17 | 朱文杰 | Vibrio qinghaiensis Q67B and separation, screening and application thereof |
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| CN101915759B (en) * | 2010-07-20 | 2012-11-07 | 同济大学 | Vibrio qinghaiensis Q67 based long-term microplate toxicity analyzing method of environmental pollutant |
| CN105588831A (en) * | 2016-01-04 | 2016-05-18 | 中国科学院城市环境研究所 | Method for detecting acute toxicity of rare earth tailing pond surrounding groundwater pollution by using freshwater luminescent bacteria |
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| CN102175606A (en) * | 2011-01-19 | 2011-09-07 | 西安建筑科技大学 | Method for detecting acute biological toxicity of sewage |
| CN103045524A (en) * | 2013-01-18 | 2013-04-17 | 朱文杰 | Vibrio qinghaiensis Q67B and separation, screening and application thereof |
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Application publication date: 20170531 Assignee: Guangxi Zhongpinzhi Environmental Monitoring Co.,Ltd. Assignor: GUILIN University OF TECHNOLOGY Contract record no.: X2022450000033 Denomination of invention: A Method for Testing Biotoxicity of Oil Production Wastewater by Vibrio Qinghai Q67 Granted publication date: 20181106 License type: Common License Record date: 20221117 |
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