CN105624260B - A method of using saccharomycete evaluating oilfield drilling fluid bio-toxicity - Google Patents
A method of using saccharomycete evaluating oilfield drilling fluid bio-toxicity Download PDFInfo
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Abstract
The invention discloses a kind of methods using saccharomycete evaluating oilfield drilling fluid bio-toxicity, including (1) to activate saccharomycete and cultivate to stationary phase, and obtained yeast bacteria culture fluid is centrifuged；(2) discontinuous density gradient centrifugation is carried out after hanging saccharomycete weight after above-mentioned centrifugation, is collected the discontinuous density gradient solution that top layer includes young age saccharomycete or spore, is synchronized culture；(3) using the saccharomycete obtained after above-mentioned synchronous culture as evaluation strain, or freeze dried powder is made in saccharomycete and is used.The present invention is using the uniform saccharomycete of the form that synchronous culture obtains as bioorganism, can within the shorter reaction time sensitive efficient, stable and accurate evaluating oilfield drilling fluid bio-toxicity, can satisfy demand to Fast Evaluation oil field drilling fluids bio-toxicity in oil field development and production.
Description
Technical field
The present invention relates to the evaluation methods of drilling fluid bio-toxicity, and in particular to a kind of to use the drilling well of saccharomycete evaluating oilfield
The method of liquid bio-toxicity.
Background technique
Oil field drilling fluids are the circulation flushing media used in drilling in oil well drilling process, mainly by liquid phase, solid phase
It is formed with chemical treatments.It is (contaminated product emulsion, anti-that liquid phase can be water (fresh water, salt water), oily (crude oil, diesel oil) or emulsion
Phase emulsion) etc.；Solid phase includes useful solid phase (bentonite, weighting material) and useless solid phase (rock) etc.；Chemical treatments packet
Include inorganic, organic and high-molecular compound etc..
With the sustainable development of petroleum industry, the ingredient of oil field drilling fluids becomes increasingly complex, and bio-toxicity is to environment
Influence is also more and more obvious.Therefore, using bio-toxicity as evaluation index, strengthen oil field drilling fluids source control measure, after avoiding
Continuous environmental pollution, it has also become current problem in the urgent need to address.
Currently, there is no unified oil field drilling fluids bio-toxicity detection method and grade scale both at home and abroad.American petroleum
It can recommend to execute " drilling fluid bio-toxicity measures standard " using the quasi- oppossum shrimp of Brazil as bioorganism, due to the bioorganism
In China's distribution-free and fragile easily dead, Chinese scholar is dedicated to finding new bioorganism species.
Chinese national standard " the offshore oil exploration and exploitation pollutant bio-toxicity method of inspection " (GB/T18420.2-
2001) mention the small young shrimp using shrimp age less than 10d or the artemia larvae just hatched be as bioorganism, but this method be applied to it is oily
Drilling fluid bio-toxicity evaluation in field has the following problems: (1) testing source of species inconvenience, the condition of selecting is not easy to grasp, and shrimp age is wanted
Ask harsh, it is desirable that technical staff carries out operation experiments in specialized laboratory, and inconvenience promotes and applies；(2) time-consuming, experiment needs every time
96h or more and at high cost；(3) operating process is complicated, and experimental technique is not easy to grasp, and is unable to field application.
China National Petroleum Corporation's company standard " Oilfield Chemicals, the classification of drilling fluid bio-toxicity and detection side
Method-Luminous bacteria " (Q/SY111-2007) be according to photobacteria luminosity and survival thalline quantity it is linear positively related
Characteristic show that the bio-toxicity of oil field drilling fluids is horizontal but more to the lethality of photobacteria by CALCULATING OILFIELD drilling fluid
The dark brown that number oil field drilling fluids are presented has interference in the luminescence band of photobacteria, needs to carry out school one by one to each sample
It could be used after just.
CN102031280A discloses a kind of method using marine microalgae evaluating oilfield drilling fluid bio-toxicity, this method
It is required that handling drilling fluid sample using natural sea-water, require to mix oil field drilling fluids and marine microalgae during the test
Bio-toxicity rank can be just obtained after closing 7~8h.The method is not suitable for due to the features such as test material is limited, evaluation cycle is longer
In the bio-toxicity of Fast Evaluation oil field drilling fluids.
To sum up, it in field drilling exploitation and production process, needs to find a kind of reality that can be evaluated as bio-toxicity
Biology is tested, can be under oil field drilling fluids effect, bio-toxicity that is quick, effective and accurately measuring oil field drilling fluids, from
And set up oil field drilling fluids bio-toxicity fast appraisement method.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of side using saccharomycete evaluating oilfield drilling fluid bio-toxicity
Method.The uniform saccharomycete of the form that the present invention uses synchronous culture to obtain, can be in the shorter reaction time as bioorganism
The bio-toxicity of interior sensitive efficient, stable and accurate evaluating oilfield drilling fluid can satisfy in oil field development and production to quick
The demand of evaluating oilfield drilling fluid bio-toxicity.
The method that the present invention uses saccharomycete evaluating oilfield drilling fluid bio-toxicity, including following content:
(1) saccharomycete is activated and is cultivated to stationary phase, obtained yeast bacteria culture fluid is centrifuged；
(2) discontinuous density gradient centrifugation is carried out after hanging saccharomycete weight after above-mentioned centrifugation, collecting top layer includes children
The discontinuous density gradient solution of age saccharomycete or spore, synchronizes culture；
(3) saccharomycete obtained after above-mentioned synchronous culture is directlyed adopt as evaluation strain, or freeze-drying is made in saccharomycete
Pulvis uses；
(4) it is corrected using bio-toxicity sensibility of the standard toxicant to saccharomycete, while determining oil drilling
Bio-toxicity graded index of the liquid to saccharomycete；
(5) saccharomycete is diluted to the yeast bacteria suspension of certain cell concentration, and prepares oil field drilling fluids by concentration gradient
Test sample；
(6) yeast bacteria suspension and oil field drilling fluids test sample are contacted into certain time, each concentration is detected by decoration method
Lethality of the oil field drilling fluids test sample to saccharomycete；
(7) medium effective concentration (EC of the CALCULATING OILFIELD drilling fluid test sample to saccharomycete50), it is classified according to bio-toxicity
Index determines oil field drilling fluids bio-toxicity grade.
In the method for the present invention, the saccharomycete be selected from saccharomyces cerevisiae (Saccharomyces cerevisiae), grape
Juice yeast (Saccharomyces uvarum), Hansenula yeast (Hansenula), torulopsis (Toruiopsis), false silk ferment
Female (Candida), pichia (Pichia), rhodotorula (Rhodotorula), cotton disease needle spore yeast (Nematspora gossypii) or geotrichum candidum (Geotrichum candidum) etc., preferably saccharomyces cerevisiae (Saccharomyces cerevisiae)。
In the method for the present invention, the activation and culture of saccharomycete are using mode known to skilled person.It can specifically adopt
With the following methods: preparing saccharomycete seed liquor first, be then inoculated in saccharomycete seed liquor newly according to 10% inoculum concentration (v/v)
Fresh culture medium expands culture, and 25~35 DEG C of cultivation temperature, 100~150rpm of shaking speed, culture to stationary phase terminates.It will
Above-mentioned yeast bacteria culture fluid is centrifuged under 500~1000g centrifugal force and collects thallus.
In the method for the present invention, prepare discontinuous density gradient centrifugation medium can for glycerol, 1,3-PD, sucrose,
One or more of lactose, trehalose, skimmed milk, preferably trehalose.When selecting trehalose as centrifugation medium, discontinuously
Density gradient solution the preparation method comprises the following steps: in centrifugal bottle from bottom to top successively paving plus descending concentrations aqueous trehalose solution, sea
The mass concentration of algae sugar aqueous solution is 5%~70%, and every layer of concentration reduces 5%~30% step by step.The present invention can be spread plus 3-5 layers, excellent
3 layers of aqueous trehalose solution are selected, every layer of volume is identical or different, and the aqueous trehalose solution volume of preferably top layer is other layers
1.5~3 times, trehalose discontinuous density gradient solution is made with this.The preparation of yeast liquid to be centrifuged: step (1) is taken to collect
Yeast thallus, then suspended again with a small amount of deionized water, the yeast liquid after resuspension be layered on to the trehalose prepared not
Gradient of continuous density solution top layer is centrifuged 5~10min under 50~100g centrifugal force, collects top layer's discontinuous density gradient
Young age saccharomycete wherein included or spore are synchronized culture by solution.Synchronous cultivation temperature is 25~35 DEG C, shaking speed
For 100~150rpm.In synchronous culture, the molasses of 5~30g/L can be added, facilitate the activity stabilized of saccharomycete and mention
Its high tolerance.
In the method for the present invention, the yeast bacteria suspension for carrying out bio-toxicity evaluation can be straight using synchronous culture yeasts bacterium solution
It connects dilution to obtain, can also redissolve to obtain by saccharomycete freeze dried powder.Since oil field drilling fluids are frequently necessary to evaluate at the scene, because
This is preferably prepared into saccharomycete freeze dried powder.Freeze dried powder, detailed process is made in synchronous culture yeasts bacterium solution are as follows: by saccharomycete
Synchronous culture solution is centrifuged under 500~1000g centrifugal force and collects thallus, with the freezing drying protective agent of the wt% of 20 wt%~50
The thallus being collected into is suspended by aqueous solution again, then is sub-packed in peace and cuts open in pipe and be freeze-dried, first maintained at 4 DEG C 6~8h,
6~8h is maintained in -20 DEG C of refrigerators again, finally in -80 DEG C of 24~48h of maintenance；Freeze dryer temperature -50 is controlled when freeze-drying
~-80 DEG C, 1~10pa of vacuum degree, 24~48h of sublimation drying have until slight crack occur in sample surfaces with freeze-drying inside pipe wall
Obscission.In the present invention, freezing drying protective agent be one or more of glycerol, lactose, trehalose, skimmed milk, preferably
Trehalose.After freeze-drying, (survival rate is living thin in saccharomycete to the survival rate of saccharomycete in the freeze dried powder that finally prepares
Born of the same parents' number accounts for the percentage of total cell number) it is not less than 70%.
In the method for the present invention, saccharomycete freeze dried powder redissolves or saccharomycete synchronizes in the yeast bacteria suspension after culture solution dilution
Cell concentration range is 104~108A/mL.Saccharomycete redissolves or dilution uses deionized water, physiological saline or phosphate-buffered
Liquid, preferably deionized water.
In the method for the present invention, yeast bacteria suspension and oil field drilling fluids test sample time of contact are 1~10min.
In the method for the present invention, dyeing processing is carried out using Lv Shi alkaline methylene blue dye liquor；It is aobvious using blood counting chamber and optics
Micro mirror carries out microscopy counting to saccharomycete living cells and dead cell and calculates lethality.
In the method for the present invention, bio-toxicity graded index is determined with standard toxicant mercury chloride.
In the method for the present invention, if the oil field drilling fluids bio-toxicity including pH influence need to be measured, test should not be adjusted
Sample pH；If the oil field drilling fluids bio-toxicity excluded including pH influence need to be measured, test sample pH value should be adjusted to 7.0.
In the method for the present invention, the concentration gradient for preparing oil field drilling fluids test sample is 100mg/L、101mg/L、102mg/L、103mg/
L、104mg/L、105Mg/L and 106mg/L；The dilution of oil field drilling fluids sample is deionized water.
Compared with prior art, the beneficial effects of the present invention are:
1, it is centrifuged by discontinuous density gradient and obtains saccharomycete activity stabilized, that form is uniform with synchronous culture, it will
The saccharomycete or its freeze dried powder are evaluated for oil field drilling fluids bio-toxicity, the experimental results showed that it is raw to oil field drilling fluids
Object toxicity assessment result is consistent with Luminous bacteria, can be used for the Fast Evaluation of drilling fluid bio-toxicity.
2, freeze dried powder is convenient for saccharomycete long-term storage, ensure that the survival rate of saccharomycete not less than 70%, also makes the present invention
Method, which realizes, once to be prepared, is used for multiple times, and the time of later period oil field drilling fluids bio-toxicity evaluation experimental is saved.
3, there is the method for the present invention the efficient, test result that is quick on the draw to stablize accurate, the easy procedure of step, strong applicability etc.
Advantage can satisfy the demand in oil field development and production to Fast Evaluation oil field drilling fluids bio-toxicity.With existing evaluation side
Method is compared, and the time foreshortens within 1h；And it can directly use saccharomycete freeze dried powder, used valuator device cost
It is low, be convenient for carrying, can oil drilling scene carry out oil field drilling fluids bio-toxicity evaluation experimental, have procedure application before
Scape.
Specific embodiment
In the present invention, using blood counting chamber and optical microscopy to the living cells after yeast bacteria concentration, dyeing and extremely carefully
Born of the same parents carry out microscopy counting and calculate lethality.
1, yeast bacteria suspension cell concentration is calculated
Usually there are two types of specifications for blood counting chamber, and one is there is 16 middle lattice in block plaid, it is small that every 1 middle lattice are divided into 25 again
Lattice；Another kind is that have 25 middle lattice in block plaid, and each middle lattice are divided into 16 small lattice again.
Firstly, saccharomycete is prepared into certain density bacteria suspension with deionized water, it is outstanding to draw a drop saccharomycete with suction pipe
Liquid penetrates into yeast bacteria suspension slowly in blood count room in the edge of blood counting chamber coverslip.When being counted under microscope,
16 lattice × 25 lattice blood counting chamber is such as used, the yeast count of 4 middle lattice should be counted；Such as use 25 lattice × 16 lattice hemocytometer
Number plate, should count the yeast count of 5 middle lattice.
Yeast count number contained in every 1mL yeast bacteria suspension is calculated according to the following formula.
16 lattice × 25 lattice blood counting chamber Counting Formulas:
Yeast count/100 × 400 × 10 in every small lattice in yeast count=100 1mL4
25 lattice × 16 lattice blood counting chamber Counting Formulas:
Yeast count/80 × 400 × 10 in every small lattice in yeast count=80 1mL4
The present invention uses 16 lattice × 25 lattice blood counting chambers, calculates bacterium in the yeast bacteria suspension of preparation by the above method
Bulk concentration.
2, the saccharomycete death rate is calculated
Saccharomycete has stronger reducing power due to metabolism, and methylene blue can be made to become from the oxidized form of blue
Colourless reduced form, so the living cells of yeast is colourless because having reducing power to present, and dead cell or the faint aging of metabolism
Cell then because extremely weak without this reducing power or reducing power into the cell, and is dyed blue or light blue by methylene blue.
Lv Shi alkaline methylene blue dyeing liquor is dissolved in 95% ethyl alcohol of 30mL the preparation method is as follows: weigh methylene blue 0.3g
In.Potassium hydroxide 0.001g is weighed again, it will be in its molten 100mL deionized water.Above two solution mix and is sufficiently mixed
It is spare after even.
The calculation method of saccharomycete lethality:
(1) 1mL yeast bacteria suspension and 4mL deionized water is taken to stand 5~10min after mixing.
(2) 50 μ L Lv Shi alkaline methylene blue dyeing liquors are added dropwise in blood counting chamber center with microsyringe, then 50 μ L is added dropwise
Mixed liquor described in (1) step, close the lid Coplin 3min after mixing.
(3) carry out microscopy using optical microscopy, according to cellular colours to living cells (colourless) and dead cell (blue) into
Row is counted and is recorded.
(4) the saccharomycete death rate (%)=dead cell blue number/(be in colourless viable count+dead cell blue number) ×
100%.The method of the present invention and effect are illustrated below by embodiment.
Embodiment 1
Selection saccharomyces cerevisiae (Saccharomyces cerevisiae) FE-B screens by Fushun Petrochemical Research Institute, it protects
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number CGMCC
NO.2735。
Saccharomycete activation and the nutrient media components content for expanding culture are as follows: yeast extract 10g/L, peptone 20g/L, grape
Sugared 20g/L.
The nutrient media components content of the synchronous culture of saccharomycete is as follows: yeast extract 10g/L, peptone 20g/L, glucose 20g/
L, molasses 10g/L.
1, for the acquisition of examination saccharomycete
(1) it takes 5 oese saccharomyces cerevisiaes to be seeded to from culture presevation inclined-plane to sterilize equipped with 120mL through 121 DEG C, 20min
250mL triangular flask in, 32 DEG C of cultivation temperature, shaking speed 120rpm, culture 48h obtain saccharomyces cerevisiae seed liquor.
Saccharomyces cerevisiae seed liquor is inoculated in 500mL shaking flask according to 10% inoculum concentration (v/v) to expand culture, culture medium
Volume 250mL, condition of culture are same as above.Take 500mL centrifugal bottle by the yeast bacteria culture fluid in stationary phase 700gUnder centrifugal force
It is centrifuged 10min, the yeast thallus being collected into is suspended again with 50mL deionized water.
(2) discontinuous density gradient is centrifuged
Successively paving adds 50% aqueous trehalose solution 150mL, 20% aqueous trehalose solution from bottom to top in 1L centrifugal bottle
Trehalose discontinuous density gradient solution is made with this in 250mL, 5% aqueous trehalose solution 350mL.
Again the yeast liquid after suspending is layered on the trehalose discontinuous density gradient solution top layer prepared, in 60g
It is centrifuged 5min under centrifugal force, collects top layer's discontinuous density gradient solution.
(3) synchronous culture
The discontinuous density gradient solution inoculum being collected into is synchronized in 3L shaking flask according to 10% inoculum concentration (v/v)
Culture, culture volume 1.2L, condition of culture obtain the synchronous culture solution of saccharomyces cerevisiae with (1) step.
2, the bio-toxicity of the saccharomycete of above-mentioned acquisition is classified
The bio-toxicity sensibility of different bioorganisms is different, in order to confirm saccharomycete bio-toxicity graded index, adopts
It is corrected (GB/T 15441-1995), is tied with toxic sensitivity of the standard toxicant mercury chloride to saccharomycete and photobacteria
Fruit is as shown in table 1.
Toxicity tests result of 1 mercury chloride of table to saccharomyces cerevisiae and photobacteria
From toxic sensitivity experimental result it is found that saccharomyces cerevisiae is more stronger than the toxic sensitivity of photobacteria, mercury chloride pair
Saccharomyces cerevisiae 5min EC50With photobacteria 15min EC50Quite.
The whether toxic standard of oil field drilling fluids bio-toxicity is judged, there is document report (Li Xiuzhen, 2004) photobacteria
15min EC50For 25000mg/L.According to correction experimental result, that is, think saccharomyces cerevisiae 5min EC50It is greater than or equal to
When 25000mg/L, determine that the bio-toxicity of oil field drilling fluids is nontoxic；The biology poison of oil field drilling fluids when less than 25000mg/L
Property to be toxic.
3, the bio-toxicity using above-mentioned saccharomycete solution for oil field drilling fluids is evaluated
(1) oil field drilling fluids sample is prepared
5 kinds of oil field drilling fluids number of bio-toxicity to be evaluated is respectively ZJY-1, ZJY-2, ZJY-3, ZJY- in the present invention
4 and ZJY-5.It takes 1 part of oil field drilling fluids that 9 parts of deionized waters are added by volume, makes it uniformly with whirlpool oscillator concussion 10min
Afterwards, middle layer suspension is taken after being centrifuged at 5000rpm with centrifuge, according to 100mg/L、101mg/L、102mg/L、
103mg/L、104mg/L、105Mg/L and 106Mg/L concentration gradient prepares the test sample of every kind of oil field drilling fluids.
(2) saccharomycete is contacted with test oil field drilling fluid sample
8 test tubes are taken, 1mL yeast liquid is added in every test tube, wherein the conduct pair of 4mL deionized water is added in the 1st test tube
According to remaining 7 test tube is added sequentially 7 concentration samples of the oil field drilling fluids prepared in 4mL (1) step, after mixing
Stand 5min.
(3) Lv Shi alkaline methylene blue dyeing liquor dyes
50 μ L Lv Shi alkaline methylene blue dyeing liquors are added dropwise in blood counting chamber center with microsyringe, then 50 μ L the are added dropwise
(2) mixed liquor in step, close the lid Coplin 3min after mixing.
(4) saccharomycete EC50It calculates and oil field drilling fluids bio-toxicity grade determines
Microscopy is carried out using optical microscopy, living cells (colourless) and dead cell (blue) are counted, each branch test tube
The saccharomycete death rate to subtract the saccharomycete death rate of control tube be saccharomycete lethality under each concentration of oil field drilling fluids.
Go out saccharomycete lethality and oil field drilling fluids concentration relationship curve using Software on Drawing such as Microsoft Excel,
Corresponding oil field drilling fluids concentration when showing that saccharomycete lethality is 50% according to relation curve, the as oil field drilling fluids
Saccharomycete EC50。
The saccharomycete EC that will be obtained50Corresponding saccharomycete bio-toxicity is classified experimental result, determines the life of the oil field drilling fluids
Object toxicity.
(5) experimental result
After 5 kinds of oil field drilling fluids such as ZJY-1, ZJY-2, ZJY-3, ZJY-4 and ZJY-5 are evaluated with saccharomycete, respectively
The classification of oil field drilling fluids bio-toxicity is as shown in table 2.According to saccharomycete EC50As a result, 5 kinds of oil field drilling fluids bio-toxicity sequences
Are as follows: ZJY-2 > ZJY-4 > ZJY-3 > ZJY-5 > ZJY-1.
2 saccharomycete of table evaluates each oil field drilling fluids toxicity tests
Embodiment 2
1, for the acquisition of examination saccharomycete with embodiment 1, the difference is that above-mentioned bacterium solution is prepared into freeze dried powder, then
Evaluation for bio-toxicity.Specific preparation process is as follows:
(1) it is centrifuged: by the synchronous culture solution of 200mL saccharomycete 700gIt is centrifuged 10min under centrifugal force, obtains yeast thallus.
(2) it is suspended: yeast thallus obtained in (1) being suspended with the aqueous trehalose solution of 20mL 20%(wt), is obtained
To saccharomycete suspension.
(3) it dispenses: 1mL saccharomycete suspension is drawn into the ampoul tube for saving strain with liquid-transfering gun, with de- after packing
Rouge tampon seals bottleneck.1ml deionized water is respectively charged into several ampoul tubes again as control tube.
(4) pre-freeze: ampoul tube is first maintained into 6h at 4 DEG C, then maintains 6h in -20 DEG C of refrigerators, is finally maintained at -80 DEG C
For 24 hours, it after freezing completely, is dried in vacuo.
(5) vacuum freezedrying: ampoul tube is put into freeze dryer, starting frozen vacuum dryer refrigeration system setting
- 55 DEG C of freeze dryer temperature, vacuum degree 10pa, sublimation drying 36h, until slight crack occur in sample surfaces, with freeze-drying inside pipe wall
There is obscission, is completely dried in control tube.
(6) it seals: ampoul tube being vacuumized using vacuum system, while with alcohol blast burner to being melted at ampoul tube neck mouth
Envelope.
(7) survival rate detects: randomly selecting 5 ampoul tubes, redissolves saccharomycete freeze dried powder with deionized water, make
Detection saccharomycete freeze-dried powder survival rate is all larger than 70% after being handled with Lv Shi alkaline methylene blue dyeing liquor, and it is raw to can be used for oil field drilling fluids
The experiment of object toxicity assessment.
2, the redissolution of freeze dried powder
1 ampoul tube is taken, is redissolved with 5mL deionized water concussion 1min, 5min is stood after redissolution makes saccharomycete recover
It is stand-by afterwards.
3, the bio-toxicity using the saccharomycete of above-mentioned redissolution for oil field drilling fluids is evaluated.Specific method is the same as embodiment 1.
The experimental results are shown inthe following table:
3 saccharomycete freeze dried powder of table evaluates each oil field drilling fluids toxicity tests
Seen from table 3, consistent with the evaluation result for directlying adopt yeast liquid using saccharomycete freeze dried powder, it will not influence
Drilling fluid bio-toxicity evaluation result.
Embodiment 3
Difference from Example 1 is to use candida tropicalis as bioorganism.
Selection candida tropicalis (Candida tropicalis) mutant strain PF-UV-56 studies by Fushun petrochemical industry
Institute's screening, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number is
CGMCC NO.0356。
1, for the acquisition of examination saccharomycete: specific method is the same as embodiment 1.
2, the bio-toxicity of the saccharomycete of above-mentioned acquisition is classified
(GB/T is corrected using toxic sensitivity of the standard toxicant mercury chloride to saccharomycete and photobacteria
15441-1995), the results are shown in Table 4.
Toxicity tests result of 4 mercury chloride of table to candida tropicalis and photobacteria
From toxic sensitivity experimental result it is found that candida tropicalis is stronger than the toxic sensitivity of photobacteria, mercury chloride
To candida tropicalis 10min EC50With photobacteria 15min EC50Quite.According to correction experimental result, that is, think tropical false
Silk yeast 10min EC50When more than or equal to 25000mg/L, determine that the bio-toxicity of oil field drilling fluids is nontoxic；It is less than
The bio-toxicity of oil field drilling fluids is toxic when 25000mg/L.
3, the bio-toxicity using above-mentioned saccharomycete solution for oil field drilling fluids is evaluated
In addition to saccharomycete and test oil field drilling fluid sample time of contact are 10min, remaining method is the same as embodiment 1.It will
After 5 kinds of oil field drilling fluids such as ZJY-1, ZJY-2, ZJY-3, ZJY-4 and ZJY-5 are evaluated with saccharomycete, each oil field drilling fluids
Bio-toxicity classification is as shown in table 5.According to saccharomycete EC50As a result, 5 kinds of oil field drilling fluids bio-toxicity sequences are as follows: ZJY-2 >
ZJY-4> ZJY-3> ZJY-5> ZJY-1。
5 saccharomycete of table evaluates each oil field drilling fluids toxicity tests
Embodiment 4
Difference from Example 3 is using candida tropicalis freeze dried powder.
1, for the acquisition of examination saccharomycete with embodiment 1, the difference is that above-mentioned bacterium solution is prepared into freeze dried powder, then
For the evaluation of bio-toxicity, specific preparation process is with embodiment 2, wherein detection candida tropicalis freeze-dried powder survival rate is greater than
70%, it can be used for oil field drilling fluids bio-toxicity evaluation experimental.
2, the bio-toxicity using the candida tropicalis of above-mentioned redissolution for oil field drilling fluids is evaluated.Specific method is the same as real
Apply example 3.The experimental results are shown inthe following table:
6 saccharomycete freeze dried powder of table evaluates each oil field drilling fluids toxicity tests
It is by table 6 as it can be seen that consistent with the evaluation result for directlying adopt yeast liquid using saccharomycete freeze dried powder, it will not influence
Drilling fluid bio-toxicity evaluation result.
Comparative example 1
By above-mentioned 5 kinds of oil field drilling fluids according to China National Petroleum Corporation's company standard " Oilfield Chemicals, drilling well
The classification of liquid bio-toxicity and detection method-Luminous bacteria " (Q/SY111-2007) carry out bio-toxicity evaluation and classification, as a result
As shown in table 3.Each oil field drilling fluids bio-toxicity sequence as seen from table are as follows: ZJY-2 > ZJY-4 > ZJY-3 > ZJY-5 > ZJY-
1, evaluation result is consistent with the method for the present invention.
7 photobacteria of table evaluates each oil field drilling fluids toxicity tests
Comparative example 2
With embodiment 1, the difference is that: discontinuous density gradient centrifugation and synchronous culture are not used, by routine culture
Saccharomycete to the random growth of stationary phase is directly used in toxicity assessment.The experimental results showed that very due to saccharomycete form span
Greatly, and there is thallus budding bunchiness to form mycelia or cenobium, oil field drilling fluids bio-toxicity evaluation experimental can not be applied to.
Comparative example 3
With embodiment 1, the difference is that: it is centrifuged using discontinuous density gradient, takes the middle layer containing saccharomycete not
Gradient of continuous density solution is used for toxicity assessment.The experimental results showed that due to not having using subsequent synchronisation of the present invention
Culture, the tolerance of saccharomycete is not strong enough, and after freeze-drying, survival rate drops to 60% or so, therefore to a certain extent
Influence toxicity assessment effect.
Claims (8)
1. a kind of method using saccharomycete evaluating oilfield drilling fluid bio-toxicity, it is characterised in that include the following steps:
(1) saccharomycete is activated and is cultivated to stationary phase, obtained yeast bacteria culture fluid is centrifuged；
(2) discontinuous density gradient centrifugation is carried out after hanging saccharomycete weight after above-mentioned centrifugation, collecting top layer includes young age ferment
The discontinuous density gradient solution of female bacterium or spore, synchronizes culture；The medium of discontinuous density gradient centrifugation is trehalose,
The layer-by-layer aqueous trehalose solution of paving plus descending concentrations from bottom to top in centrifugal bottle, the mass concentration of aqueous trehalose solution is 5%~
70%, every layer of concentration reduces 5%~30% step by step；Paving plus 3~5 layers of aqueous trehalose solution, every layer of volume is identical or different, is made with this
At trehalose discontinuous density gradient solution；The cultivation temperature of the synchronous culture is 25~35 DEG C, shaking speed is 100~
150rpm；
(3) using the saccharomycete obtained after above-mentioned synchronous culture as evaluation strain, or freeze dried powder is made in saccharomycete and is used；
(4) it is corrected using bio-toxicity sensibility of the standard toxicant to saccharomycete, while determining oil field drilling fluids pair
The bio-toxicity graded index of saccharomycete；
(5) saccharomycete is prepared into yeast bacteria suspension, cell concentration range is 10 in bacteria suspension4~108A/mL, and by concentration ladder
Degree prepares oil field drilling fluids test sample；
(6) yeast bacteria suspension is contacted with oil field drilling fluids test sample, each concentration oil field drilling fluids is detected by decoration method and are surveyed
Lethality of the test agent to saccharomycete；Yeast bacteria suspension and oil field drilling fluids test sample time of contact are 5~10min；
(7) medium effective concentration EC of the CALCULATING OILFIELD drilling fluid test sample to saccharomycete50, true according to bio-toxicity graded index
Determine oil field drilling fluids bio-toxicity grade；
The saccharomycete be saccharomyces cerevisiae (Saccharomyces cerevisiae) FE-B, deposit number is CGMCC No.
2735, or for candida tropicalis (Candida tropicalis) mutant strain PF-UV-56, deposit number is CGMCC No.
0356。
2. according to the method for claim 1, it is characterised in that: the activation and incubation of saccharomycete specifically: make first
Standby saccharomycete seed liquor, is then inoculated in fresh culture for saccharomycete seed liquor according to 10% inoculum concentration (v/v) and carries out expansion training
It supports, 25~35 DEG C of cultivation temperature, 100~150rpm of shaking speed, culture to stationary phase terminates；Above-mentioned yeast bacteria culture fluid is existed
It is centrifuged under 500~1000g centrifugal force and collects thallus.
3. according to the method for claim 1, it is characterised in that: paving plus 3 layers of aqueous trehalose solution, the seaweed syrup of top layer
Liquor capacity is 1.5~3 times of other layers.
4. according to the method for claim 1, it is characterised in that: then the yeast thallus for taking step (1) to collect is gone with 50mL
Ionized water suspends again, and the yeast liquid after resuspension is layered on to the trehalose discontinuous density gradient solution top layer prepared, In
It is centrifuged 5~10min under 50~100g centrifugal force, collects top layer's discontinuous density gradient solution, is synchronized culture.
5. according to method described in claim 1 or 4, it is characterised in that: in synchronous culture, the molasses of 5~30g/L are added.
6. according to method described in claim 1 or 4, it is characterised in that: freeze dried powder is made in synchronous culture yeasts bacterium solution, is had
Body process are as follows: the synchronous culture solution of saccharomycete is centrifuged under 500~1000g centrifugal force and is collected thallus, with 20wt%~50wt%
Freezing drying protective agent aqueous solution the thallus being collected into is suspended again, then be sub-packed in peace and cut open in pipe and be freeze-dried, first
6~8h is maintained at 4 DEG C, maintains 6~8h in -20 DEG C of refrigerators again, finally in -80 DEG C of 24~48h of maintenance；It is freeze-dried time control
- 50~-80 DEG C of freeze dryer temperature processed, 1~10pa of vacuum degree, 24~48h of sublimation drying, until sample surfaces are split
Trace has obscission with freeze-drying inside pipe wall.
7. according to the method for claim 6, it is characterised in that: freezing drying protective agent is glycerol, lactose, trehalose, takes off
One or more of rouge cream.
8. according to the method for claim 7, it is characterised in that: freezing drying protective agent is trehalose.
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Non-Patent Citations (3)
| Title |
|---|
| 发光细菌法测定钻井液综合生物毒性;李欣;《油气田环境保护》;19990331;第9卷(第1期);29-30 * |
| 有机化学品对酵母菌生物毒性的检测技术;高大文和王鹏;《哈尔滨商业大学学报(自然科学版)》;20041231;第20卷(第6期);706-709,714 * |
| 酿酒酵母单细胞生长动力学的初步研究;李伟军等;《化工冶金》;20000430;第21卷(第2期);149-153 * |
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