CN106755286A - A kind of method that utilization Vibrio-qinghaiensis sp. Q67 tests oil extraction waste water bio-toxicity - Google Patents
A kind of method that utilization Vibrio-qinghaiensis sp. Q67 tests oil extraction waste water bio-toxicity Download PDFInfo
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- CN106755286A CN106755286A CN201611171167.XA CN201611171167A CN106755286A CN 106755286 A CN106755286 A CN 106755286A CN 201611171167 A CN201611171167 A CN 201611171167A CN 106755286 A CN106755286 A CN 106755286A
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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 method that utilization Vibrio-qinghaiensis sp. Q67 tests oil extraction waste water bio-toxicity.It is test waste water with oil extraction waste water, it is biological to indicate with Vibrio-qinghaiensis sp. Q67, it is carrier with 96 hole microwell plates；By 96 hole microwell plate analysis methods, its luminous intensity is detected using microplate spectrophotometer；Luminous inhibiting rate is calculated using the luminous intensity of experimental group and control group, Zn is set（NO3）2As toxicity reference substance, so as to judge the bio-toxicity of oil extraction waste water.The inventive method is tested and analyzed using microplate method to the toxicity of oil extraction waste water, by the luminous inhibiting rate for calculating photogen, so as to judge the comprehensive toxicity size of oil extraction waste water, solve the problems, such as that conventional physical and chemical index can not represent oil extraction waste water bio-toxicity, quickly, toxicity reference substance Zn sensitive, easy to utilize and selected（NO3）2Experimental result stabilization, cheap, toxicity is medium.
Description
Technical field
The invention belongs to wastewater biological toxicity detection field, more particularly to a kind of oil recovery using Vibrio-qinghaiensis sp. Q67 test is given up
The method of aquatic toxicity.
Background technology
Oil extraction waste water be oil field in oil recovery process, except returned as re-injection, technique mix or the industrial water such as other purposes with
Outward, the waste water of outer row is needed.Wherein contain the macromolecule persistent organic pollutants such as petroleum-type, surfactant.Oil extraction waste water has
There is the characteristics of salinity is high, and water temperature is high, the object to contacting has certain corrosivity, in addition, also containing certain in waste water
The bactericide 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, are subject to
Extensive concern.The detection method of current wastewater biological toxicity has：Photobacterium Phosphoreum Toxicity is tested, algae toxicity experiment, flea class poison
Property experiment, toxicity test of fishes, birds toxicity test etc..Wherein, photogen is biological as the instruction that wastewater toxicity is checked, and has
Fast and convenient advantage.
The content of the invention
It is an object of the invention to provide a kind of method that utilization Vibrio-qinghaiensis sp. Q67 tests oil extraction waste water bio-toxicity.
Thinking of the present invention：It is test waste water with oil extraction waste water, is carrier with 96 hole microwell plates；By 96 hole micropore plate analysis
Method, its luminous intensity is detected using Synergy H2 microplate spectrophotometers；It is luminous with control group using experimental group
Intensity calculates luminous inhibiting rate, setting Zn (NO3)2As toxicity reference substance, so as to judge the bio-toxicity of waste water.
Concretely comprise the following steps：
(1) the muddy oil extraction waste water that will be adopted back is obtained the oil extraction waste water after treatment, then by it with 0.45 μm of membrane filtration
It is placed in 4 DEG C of refrigerator and preserves stand-by, it is assumed that the concentration of the oil extraction waste water after treatment 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 hole microwell plate of White-opalescent 96 for pre-setting oil extraction waste water concentration gradient
In, make oil extraction waste water and the reaction system of the bacterium solution that works, it is 200 μ L per hole cumulative 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 solution and 100 μ L ultra-pure waters, make work per hole
Bacterium solution fully reacts 15min with oil extraction waste water, then determines 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 averages.
(4) setting Zn (NO3)2As toxicity reference substance, with ultra-pure water by Zn (NO3)2Dilution various concentrations gradient, then
Add work bacterium solution fully to react 15min, its luminous intensity is determined using SynergyH2 microplate spectrophotometers, calculate hair
Xanthophyll cycle rate and with the luminous inhibiting rate of Logistic function pairs and Zn (NO in Origin9.03)2Concentration is fitted, and sets up dense
Degree-effect curve.
(5) using Synergy H2 microplate spectrophotometers detection microwell plate luminous intensity, using experimental group with compare
The luminous intensity of group calculates luminous inhibiting rate E of the various concentrations oil extraction waste water to Vibrio-qinghaiensis sp. Q67, dense with oil extraction waste water difference
It is abscissa to spend, with the corresponding luminous inhibiting rate of various concentrations as ordinate, using the Logistic function pairs in Origin9.0
Various concentrations oil extraction waste water carries out nonlinear fitting with luminous inhibiting rate E.
In formula：I0It is the RLU average values of blank sample, I is the RLU average values of 9 Duplicate Samples of each concentration；Control is 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, have
Body is as follows：
Zn(NO3)2Concentration C ＜ 0.29mg/L when, toxicity level is for micro- malicious or nontoxic；Zn(NO3)2Concentration 0.29mg/
During L≤C ＜ 1.11mg/L, toxicity level is low toxicity；Zn(NO3)2Concentration 1.11mg/L≤C ＜ 1.60mg/L when, toxicity level
It is poisoning；Zn(NO3)2Concentration 1.60mg/L≤C ＜ 2.91mg/L when, toxicity level is high poison；Zn(NO3)2Concentration C >=
During 2.91mg/L, toxicity level is severe toxicity.
The inventive method is tested and analyzed using microplate method to the toxicity of oil extraction waste water, by calculating the luminous of photogen
Inhibiting rate, so as to judge the comprehensive toxicity size of oil extraction waste water, solving conventional physical and chemical index can not represent that oil extraction waste water is biological
The problem of toxicity, toxicity reference substance Zn (NO quick, sensitive, easy to utilize and selected3)2Experimental result stabilization,
Cheap, toxicity is medium.
Brief description of the drawings
Fig. 1 is the sample-adding design drawing of 96 hole microwell plates used in the embodiment of the present invention, wherein, B is control group, containing 100 μ
The μ L bacterium solutions of L ultra-pure waters+100；C1~C8It is followed successively by oil extraction waste water 8 concentration gradients from high to low.
Fig. 2 is concentration effect curve of the zinc nitrate to the luminous inhibiting rate of Vibrio-qinghaiensis sp. Q67 in the embodiment of the present invention.
Fig. 3 is concentration-effect of each process section oil extraction waste water to the luminous inhibiting rate of Vibrio-qinghaiensis sp. Q67 in the embodiment of the present invention
Curve.
Specific embodiment
Embodiment：
Waste water sample to be measured in the present embodiment takes from each handling process section of certain oil Weizhou terminal wastewater treatment plant oil extraction waste water, five
Individual sample point is respectively：Raw water, ABR waters, the water outlet of ABR ponds, SBR waters and water outlet, are carried out as follows to the sample of each sample point
Experiment：
(1) so that each process section oil extraction waste water is as test waste water and carries out pre-treatment：
After oil extraction waste water is through pipeline to terminal wastewater treatment plant, using anaerobic baffled reactor and activated sludge process joint work
Skill biochemical treatment, the waste water after being processed through biochemical process is delivered to clear water reserviors and is then discharged out outside factory.In order to avoid muddy waste water is to reality
The influence of result is tested, by testing sample with 0.45 μm of membrane filtration, 4 DEG C of Refrigerator stores are stand-by, it is assumed that the oil extraction waste water after treatment
Concentration be unit 1.
(2) culture Vibrio-qinghaiensis sp. Q67 reaches exponential phase, as work bacterium solution.
The activation of strain and inoculation：- 30 DEG C of Q67 freezing dry powders equipped with Qinghai Vibrion bacterial strain of preservation are taken out (purchased from north
Jing Binsong photon technologies limited company) ampoule bottle, be placed in about 10~15min in 4 DEG C of refrigerators, wiped with cotton ball soaked in alcohol outer
After week sterilization, ampoule bottle is cut with emery wheel in superclean bench, add 300 μ L Q67 freeze-dried powder resuscitation fluids, slight oscillatory to enter
Row activation, then pipettes strain liquid to culture dish flat board with the pipettor that range is 100 μ L, and culture dish then is positioned over into perseverance
In warm incubator, 22 DEG C of cultures (summer 18h, winter 30h) grow bacterium colony and obtain F1Generation, then with oese from F1Choosing colony
It is seeded on plating medium, inversion is put in constant incubator, 22 DEG C of cultures (summer 18h, winter 30h) grow bacterium colony acquisition
F2In generation, cultivate after the same method to F3Generation.It is stand-by that taking-up is positioned over 4 DEG C of Refrigerator stores.
Test the culture of strain：From the ring bacterium colony of picking one on 4 DEG C of Q67 solid plates of preservation, the training of 50mL liquid is transferred to
Support in base, be placed in shaken cultivation case and cultivate, 22 DEG C, shaken cultivation under 120rpm, until Q67 reaches exponential phase, it is stand-by.
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 extract, 5.0g pancreas eggs
White peptone and 3.0g glycerine.Each composition of culture medium is weighed, heating is dissolved in 1L ultra-pure waters.PH value is adjusted with 1mol/L NaOH solutions
To 8.5~9.0, it 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, cools down standby after 4 DEG C of Refrigerator stores.
Solid medium is prepared：Take the above-mentioned fluid nutrient medium 300ml for having prepared, 1.5%~2% (4.5~6g) fine jade
Cosmetics is added in the triangular flask of 1L, while the culture dish brown paper wrapping of clean drying is tightly put into height together with culture medium
In pressure autoclave, 120 DEG C, high pressure steam sterilization 25min take out, are placed in the superclean bench of 10~20min of ultraviolet sterilization
In, it is slightly cold after prepare solid plate culture medium, culture medium is fallen in culture dish, be made flat board after sprawling uniform, cooling, be put into
In 4 DEG C of refrigerators preserve, it is standby.
(3) concentration of the oil extraction waste water after hypothesis treatment is unit 1 (100%), sets concentration gradient C1~C8Respectively 1,
0.9th, 0.8,0.7,0.6,0.5,0.4,0.3, work bacterium solution 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 work bacterium solution, wherein per hole cumulative volume be 200 μ L (100 μ L's
The oil extraction waste water of work bacterium solution and 100 μ L various concentrations, blank is the work bacterium solution and 100 μ L ultra-pure waters of 100 μ L) specific sample-adding
Method is as shown in Figure 1.Work bacterium solution is set fully to react 15min with oil extraction waste water, then with Synergy H2 microwell plate light splitting light
Degree meter determines 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)2Dilution various concentrations gradient, then
Add work bacterium solution fully to react 15min, its luminous intensity is detected using Synergy H2 microplate spectrophotometers；Utilize
The luminous inhibiting rate of Logistic function pairs and Zn (NO in Origin9.03)2Concentration is fitted, and sets up concentration effect curve.
By extremely toxic substance HgCl in conventional method2As toxicity reference substance, but because its be extremely toxic substance be not only difficult into
Row experiment, and environmental pollution is easily caused, Zn2+Because its experimental result stabilization, it is cheap, toxicity is medium and be picked as poison
Property object of reference.
(5) using Synergy H2 microplate spectrophotometers detection microwell plate luminous intensity, using experimental group with compare
The luminous intensity of group calculates luminous inhibiting rate E of the various concentrations oil extraction waste water to Vibrio-qinghaiensis sp. Q67, dense with oil extraction waste water difference
It is abscissa to spend, with the corresponding luminous inhibiting rate of various concentrations as ordinate, using the Logistic function pairs in Origin9.0
Various concentrations oil extraction waste water carries out nonlinear fitting with luminous inhibiting rate E, its fitting R2>=0.97.Compare identical suppression simultaneously
Zn (NO under rate3)2Concentration, so as to judge the comprehensive toxicity size of oil extraction waste water.
In formula：I0It is the average value of blank 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 that each handling process section waste water of certain oil Weizhou terminal wastewater treatment plant has certain noxious material,
There is certain inhibitory action to Vibrio-qinghaiensis sp. Q67 luminous intensity, toxicity size is：Raw water>ABR ponds>ABR goes out>SBR ponds>Go out
Water.Toxicity level is：
Raw water is shown as high poison；ABR ponds are shown as poisoning；ABR goes out to be shown as low toxicity；SBR ponds are shown as low toxicity；Water outlet shows
It is shown as low toxicity.
Therefore, oil extraction waste water has certain effect after biochemical treatment, but certain harm is still suffered to water environment.
The present embodiment shows significant test effect：Oil extraction waste water is tested with biology-Vibrio-qinghaiensis sp. Q67 is indicated
Toxicity, by calculating size of the different process section waste water to the luminous inhibiting rate of Vibrio-qinghaiensis sp. Q67, toxicity of reaction waste etc.
Level, such that it is able to effectively, quickly and accurately judge the comprehensive toxicity size of each process section waste water.
Claims (1)
1. a kind of method that utilization Vibrio-qinghaiensis sp. Q67 tests oil extraction waste water bio-toxicity, it is characterised in that concretely comprise the following steps：
(1) the muddy oil extraction waste water that will be adopted back is obtained the oil extraction waste water after treatment with 0.45 μm of membrane filtration, is then placed on
Preserve stand-by in 4 DEG C of refrigerator, it is assumed that the concentration of the oil extraction waste water after treatment 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 hole microwell plate of White-opalescent 96 for pre-setting oil extraction waste water concentration gradient, is made
Make the reaction system of oil extraction waste water and work bacterium solution, be 200 μ L per hole cumulative 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 solution and 100 μ L ultra-pure waters, make work bacterium solution per hole
15min is fully reacted with oil extraction waste water, then its luminous intensity RLU, phase is determined with Synergy H2 microplate spectrophotometers
Under the conditions of, each oil extraction waste water concentration carries out 9 repetitions and tests, and averages；
(4) setting Zn (NO3)2As toxicity reference substance, with ultra-pure water by Zn (NO3)2Dilution various concentrations gradient, is subsequently adding work
Make bacterium solution and fully react 15min, its luminous intensity is determined using Synergy H2 microplate spectrophotometers, calculate luminous suppression
Rate and with the luminous inhibiting rate of Logistic function pairs and Zn (NO in Origin9.03)2Concentration is fitted, and sets up concentration-effect
Answer curve；
(5) using Synergy H2 microplate spectrophotometers detection microwell plate luminous intensity, using experimental group and control group
Luminous intensity calculates luminous inhibiting rate E of the various concentrations oil extraction waste water to Vibrio-qinghaiensis sp. Q67, is with oil extraction waste water various concentrations
Abscissa, it is different using the Logistic function pairs in Origin9.0 with the corresponding luminous inhibiting rate of various concentrations as ordinate
Concentration oil extraction waste water carries out nonlinear fitting with luminous inhibiting rate E；
In formula：I0It is the RLU average values of blank sample, I is the RLU average values of 9 Duplicate Samples of each concentration；Control is identical luminous
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 is for micro- malicious or nontoxic；Zn(NO3)2Concentration 0.29mg/L≤C
During ＜ 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 >=
During 2.91mg/L, toxicity level is severe toxicity.
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| 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 |
| 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 |
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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 |
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| 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 |
| 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 |
| CN114397418A (en) * | 2022-01-21 | 2022-04-26 | 浙江清华长三角研究院 | Logistic fitting-based water quality comprehensive toxicity and suspected toxic substance testing 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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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 |