CN103969303B - Method for rapidly determining BOD (Biochemical Oxygen Demand) by taking collagenous fiber loaded saccharomyces cerevisiae as biosensor identification element - Google Patents
Method for rapidly determining BOD (Biochemical Oxygen Demand) by taking collagenous fiber loaded saccharomyces cerevisiae as biosensor identification element Download PDFInfo
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- CN103969303B CN103969303B CN201410204412.7A CN201410204412A CN103969303B CN 103969303 B CN103969303 B CN 103969303B CN 201410204412 A CN201410204412 A CN 201410204412A CN 103969303 B CN103969303 B CN 103969303B
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Abstract
The invention discloses a method for rapidly determining BOD (Biochemical Oxygen Demand) by taking collagenous fiber loaded saccharomyces cerevisiae as a biosensor identification element, belonging to the field of biosensors. The method comprises a process for preparing a collagenous fiber loaded saccharomyces cerevisiae biosensitive membrane by selecting collagenous fiber loaded saccharomyces cerevisiae as the biological identification element, a method for rapidly determining BOD by utilizing the collagenous fiber loaded saccharomyces cerevisiae as the biological identification element, and a method for storing and activating the immobilized biosensitive membrane. According to the method, microorganism immobilizing materials in the microbial sensor field are widened; the microbial membrane has the advantages of good water permeability, good mass transfer effect, high saccharomyces cerevisiae activity, strong stability and the like, the manufacturing cost of a BOD microbial sensor is greatly lowered due to the good water permeability and the good mass transfer effect, and meanwhile, the sensor has relatively large measuring range. According to the BOD microbial sensor formed by the membrane, the response range to GGA (glucose glutamic acid) standard liquid is BOD5 10-250mg/L, the response time is 3-15 minutes, and the microbial membrane can be continuously used by more than 150 times.
Description
Technical field
The invention belongs to field of biosensors, there is provided for the collagen fiber supported of biological sensor of oxygen amount needed biochemically
The preparation of saccharomyces cerevisiae bio-sensitive film and its using method.
Background technology
BOD（Biochemical oxygen demand (BOD)）It is to represent the dissolved oxygen consumed during Organic substance in water Jing microbial biochemical action breaks
Amount, is a highly important index in water quality monitoring.The higher content of organics represented in water of BOD values is higher, and water body is dirty
Dye degree is more serious.Conventional BOD values determination methods are biochemical oxygen demand (BOD) inoculation dilution method (BOD on the 5th5Method), i.e., sample is 20
5d, the dissolved oxygen before and after determination sample culture are cultivated under conditions of ± 1 DEG C, both differences are biochemical oxygen demand (BOD) on the 5th.Due to this
Method has analytical cycle long, complex operation, poor reproducibility, and the deficiency such as unsuitable field monitoring, domestic and international researcher proposes high temperature
The detection water sample BOD's such as method, ultraviolet spectroscopy, inspection pressure type Coulomb meter method, activated sludge aeration edman degradation Edman, microbiological sensor method
Method, wherein microbiological sensor method have and determine cycle is short, certainty of measurement height, favorable reproducibility, can continuously monitor on-line
Advantage.
Microbiological sensor is mainly made up of immobilized microorganism and chromacoder, and wherein microbe-sensitive film is to passing
The performances such as sensitivity, stability, service life, measurement range, the response time of sensor have conclusive impact, are BOD micro-
The core content of biology sensor research.For many years, scholars have done many trials to this, have explored absorption method, interlayer
The microorganism immobilization methods such as method, cross-linking method and investment, commonly used immobilization material has polyacrylamide gel, polyethylene
Alcohol, cellulose acetate film, isocyanates, amino silane etc..But these method generally existings cell seepage, suppress cytoactive,
The deficiencies such as film blocking, so as to have impact on the performance such as stability, service life, measurement range of sensor.
The microbial film prepared by above-mentioned material also has following both sides：
First, because the permeability for commonly using immobilization material is poor, the dissolved oxygen content of oxygen electrode measurement is remote in sensor
Much smaller than the true dissolved oxygen content in test water sample so that electric signal reduces the 3-6 order of magnitude.So during composition sensor
The support of dissolved oxygen instrument in higher sensitivity is needed, this cost for allowing for integral sensors is substantially increased；
Second, the measurement range of BOD microbiological sensors is micro- with fixation by the quantity and activity of fixation of microbial cell
The impact of biomaterial mass-transfer performance.In the past material used in research was carried with sacrificing fixation of microbial cell activity mostly
The mass-transfer performance of high immobilization quantity and material, it is impossible to balance or solve the contradiction between them very well, causes sensor to measure
Scope is narrower, typically only to tens milligrams per liter, less than the BOD levels of municipal sewage, needs to carry out detected sample during measurement
Dilution.
Collagenous fibres are a kind of natural macromolecular materials, are cleaned Animal Skins such as ox, sheep, pig, alkali process, splitting, de-
Make after the process of the operations such as alkali, dehydration, drying, grinding, it is good, permeable with good mechanical property, resistance to aeration intensity height, mass transfer effect
Property and water retention property is good, with low cost, good biocompatibility, soda acid wide accommodation the advantages of.Jing after metal ion-modified, glue
Fibrillation obtains preferable resistance to biological degradability and heat endurance on the basis of original advantage is kept.After metal ion-modified
Collagenous fibres have stronger adsorption capacity to microorganism, there are some researches show the immobilized wine brewing ferment of collagenous fibres after modified
Mother has compared with the more preferable fermenting property of free saccharomyces cerevisiae, illustrates that collagenous fibres immobilized brewing yeast has higher biology living
Property.
The problem that this patent exists for existing BOD biology sensors, with collagenous fibres fixed yeast cell as response
Element prepares BOD microbiological sensors, and Jing tests obtain the technology of preparing and using method of microbe-sensitive film, and determine the biography
The performances such as measurement range, response time, the service life of sensor.
The content of the invention
It is an object of the invention to provide one kind is quickly determined using collagen fiber supported saccharomyces cerevisiae as biological identification element
The method of BOD, it is characterised in that the method includes selecting collagen fiber supported saccharomyces cerevisiae as biological identification element, prepares
The method of collagen fiber supported saccharomyces cerevisiae bio-sensitive film, by the use of collagen fiber supported saccharomyces cerevisiae as biological identification element
Rapid assay methods to BOD, and the activation method of the store method of the immobilized brewing yeast and the microbe-sensitive film；
The collagen fiber supported saccharomyces cerevisiae is used as follows the reason for biological identification element：
1) collagenous fibres are used as a kind of natural macromolecular material, with good mechanical property, resistance to aeration intensity it is high, permeable and
Water retention property is good, mass transfer effect is good, with low cost, good biocompatibility, soda acid wide accommodation the advantages of；
2) collagenous fibres Jing after metal ion-modified are on the basis of the original premium properties of collagenous fibres is kept, more
For heat resistance well and resistance to biological degradability；
3) collagenous fibres after metal ion changes show stronger absorption property, and metal ion zirconium to microorganism
（Zr）Modified collagenous fibres have the fermenting property higher compared with free saccharomyces cerevisiae as carrier immobilized brewing yeast, i.e., more
High biologically active；
4) good water penetration causes the BOD microbiological sensors prepared by the material without the need for high-precision dissolved oxygen instrument,
The equipment cost of sensor can be substantially reduced；
5) good mass transfer effect causes the BOD microbiological sensors prepared by the material to have wider measurement range,
Reduce the dilution in actual measurement；
6) collagen fiber supported saccharomyces cerevisiae has long service life, immobilized microorganism preservation and activation method simple
The characteristics of.
The metal ion zirconium（Zr）The fibre immobilized carrier of modified collagen（ZICF）Prepared by existing method（Invention is special
Profit：Collagen fiber supported metal ion adsorption material and its production and use, the patent No.： 200410040145.0）.
The preparation method of the immobilized brewing yeast：Weigh dry weight 0.5g ZICF and be placed in sterilized water and soak 5-12h.
Saccharomyces cerevisiae is Jing after liquid PDA culture medium activation culture（30 DEG C, concussion and cultivate 30-36h）, receive Jing after centrifugation, sterile water wash
Collect cell and bacteria suspension is obtained in physiological saline, and adjust bacteria suspension concentration for 106-109cfu/mL.Add after ZICF is filtered
In above-mentioned bacteria suspension, with sterile water wash to detecting without free bacterium after concussion absorption 5h, collagenous fibres immobilization wine brewing ferment is obtained final product
It is female.
The preparation method of the bio-sensitive film：The collagenous fibres immobilization that weight in wet base is weighed every time for 0.60-0.90g is made wine
Yeast, immobilized brewing yeast is placed between the mesh nylon wire of two-layer 200, is allowed to form one layer of collagen fiber membrane, i.e. microorganism
Film, is close to the film on the pellicle before dissolved oxygen electrode head when using.
The rapid assay methods by the use of collagen fiber supported saccharomyces cerevisiae as bio-sensitive film to BOD：During measurement,
Dissolved oxygen electrode head with immobilized brewing yeast is placed in and fills 0.07mol/L disodium hydrogen phosphates, potassium dihydrogen phosphate buffering
In the measuring cup of liquid, aeration is carried out to buffer solution with the aeration intensity of 24L/h, measurement temperature is 30 DEG C, pH is 6, inorganic salts from
Sub- concentration range is 0-3.5%；Oxygen to be dissolved（DO）Value reaches and adds sample, immobilized microorganism to decompose in sample after stablizing
Organic matter causes oxygen in water to drop to another stationary value, the difference of oxygen dissolving value twice is tried to achieve in front and back, by calibration curve meter
The BOD values of water sample are surveyed in calculation.
The store method of the immobilized brewing yeast：Collagenous fibres immobilized brewing yeast is in 0.07 mol/L phosphoric acid hydrogen
In disodium, potassium phosphate buffer, pH be preserve 4 months under the conditions of 6,4 DEG C after its activity it is still good.
The activation method of the immobilized brewing yeast：By the immobilized brewing yeast being stored under the conditions of 4 DEG C in 30 DEG C of bars
24h is placed in original solution under part.GGA solution after composition BOD sensors to concentration for 50mg/L is repeatedly measured 8-10 time, DO
Continuous 5 relative deviations of drop-out value are less than 5%, that is, complete the activation of immobilized brewing yeast.
Description of the drawings
Fig. 1 is BOD5For the calibration curve of the GGA solution of 10-250mg/L scopes.
Specific embodiment
Example 1：Using collagen fiber supported saccharomyces cerevisiae proposed by the present invention as biological identification element, to variable concentrations
The measure of GGA (glucose glutamic acid) solution.
It is respectively configured BOD5Concentration be 5,10,25,50,75,125,150,175,200,225,250,275,300mg/L
GGA solution, with collagen fiber supported saccharomyces cerevisiae as biological identification element combined with dissolved oxygen electrode composition BOD microorganisms
Sensor, determines the GGA solution of above-mentioned concentration.Condition determination is：30 DEG C of temperature, pH=6, aeration intensity 24h/L, magnetic agitation
Rotor speed makes dissolved oxygen show value in 4-7mg/L.
Measurement result shows there is good linear relationship between DO drop-out values and sample BOD concentration, in BOD5Concentration
In the range of 10-250mg/L, linearly dependent coefficient is more than 0.995, meets the required precision of BOD measurements.
Example 2：Using collagen fiber supported saccharomyces cerevisiae proposed by the present invention as biological identification element, it is in 30 DEG C, pH
6, to BOD under the conditions of aeration intensity 24L/h5It is worth the GGA solution for 30mg/L, 50mg/L, 100 mg/L to be measured, determines and pass
The result reappearance of sensor, as a result as shown in table 1：
The sensor reappearance measurement result of table 1
| GGA | 30 mg/L | 50mg/L | 100mg/L |
| For the first time | 0.63 | 0.69 | 0.89 |
| Second | 0.63 | 0.77 | 0.88 |
| For the third time | 0.67 | 0.73 | 0.79 |
| 4th time | 0.69 | 0.73 | 0.83 |
| 5th time | 0.69 | 0.74 | 0.94 |
| 6th time | 0.68 | 0.69 | 0.85 |
| 7th time | 0.70 | 0.70 | 0.87 |
| 8th time | 0.67 | 0.79 | 0.91 |
| Averagely (mg/L) | 0.67 | 0.73 | 0.87 |
| RSD | 2.8% | 3.6% | 4.6% |
By being analyzed to data in table, sensor has preferable reappearance, and maximum relative deviation 4.6% is full
The quick certainty of measurement of sufficient BOD is required.
Example above is only that embodiments of the present invention are described, and not the scope of the present invention is limited
It is fixed, for a person skilled in the art, described above can be improved or be deformed, but all these is improved or become
Shape all should fall into the protection domain that the claim of the present invention determines.
Claims (6)
1. a kind of method that BOD is quickly determined as biological identification element using collagen fiber supported saccharomyces cerevisiae, it is characterised in that
The method includes preparing collagen fiber supported saccharomyces cerevisiae, preparing collagen fiber supported saccharomyces cerevisiae bio-sensitive film and utilize glue
Fibrillation loads quick measure of the saccharomyces cerevisiae as biological identification element to BOD, and the collagen fiber supported saccharomyces cerevisiae
Store method and the collagen fiber supported saccharomyces cerevisiae activation method；
It is described by the use of collagen fiber supported saccharomyces cerevisiae as biological identification element to the quick measure of BOD as follows：
During measurement, the collagen fiber supported saccharomyces cerevisiae bio-sensitive film for preparing is invested on the electrode tip of dissolved oxygen, then will
Electrode tip is placed in the measuring cup for filling 0.07mol/L disodium hydrogen phosphates, potassium phosphate buffer, strong with the aeration of 24L/h
Degree carries out aeration to buffer solution, and measurement temperature is 30 DEG C, pH is 6, and inorganic ion concentration range is 0-3.5%, oxygen to be dissolved
Value reaches and adds sample, the organic matter that collagen fiber supported saccharomyces cerevisiae decomposes in sample to cause oxygen in water to decline after stablizing
To another stationary value, the difference of oxygen dissolving value twice is tried to achieve in front and back, the BOD values for surveying water sample are calculated by calibration curve.
2. according to claim 1 the side of BOD quickly determined as biological identification element using collagen fiber supported saccharomyces cerevisiae
Method, it is characterised in that described collagenous fibres are the collagenous fibres after metal ion modified zirconia, are designated as ZICF.
3. according to claim 2 the side of BOD quickly determined as biological identification element using collagen fiber supported saccharomyces cerevisiae
Method, it is characterised in that the preparation method of the collagen fiber supported saccharomyces cerevisiae is：Weigh dry weight 0.5g ZICF be placed in it is aseptic
5-12h is soaked in water；Saccharomyces cerevisiae Jing liquid PDA culture medium activation cultures, the condition of activation culture is concussion and cultivate at 30 DEG C
30-36h, cell is then collected Jing after centrifugation, sterile water wash bacteria suspension is obtained in physiological saline, and adjusts bacteria suspension concentration
For 106-109cfu/mL；During above-mentioned bacteria suspension is added after ZICF is filtered, with sterile water wash to without free after concussion absorption 5h
Bacterium detects, and obtains final product collagen fiber supported saccharomyces cerevisiae.
4. according to claim 1 the side of BOD quickly determined as biological identification element using collagen fiber supported saccharomyces cerevisiae
Method, it is characterised in that the preparation method of the collagen fiber supported saccharomyces cerevisiae bio-sensitive film is：Weighing weight in wet base every time is
The collagen fiber supported saccharomyces cerevisiae of 0.60-0.90g, by collagen fiber supported saccharomyces cerevisiae be placed in the mesh nylon wire of two-layer 200 it
Between, it is allowed to form one layer of collagen fiber membrane, i.e. microbial film, the film is close to into the pellicle before dissolved oxygen electrode head when using
On.
5. according to claim 1 the side of BOD quickly determined as biological identification element using collagen fiber supported saccharomyces cerevisiae
Method, it is characterised in that the store method of described collagen fiber supported saccharomyces cerevisiae is：In 0.07mol/L phosphate buffers
Middle preservation, pH is 6,4 DEG C of temperature.
6. according to claim 1 the side of BOD quickly determined as biological identification element using collagen fiber supported saccharomyces cerevisiae
Method, it is characterised in that the activation method of described collagen fiber supported saccharomyces cerevisiae is：The collagen being stored under the conditions of 4 DEG C is fine
Dimension load saccharomyces cerevisiae places in original solution 24h under the conditions of 30 DEG C；To concentration for 50mg/L's after composition BOD sensors
GGA solution is repeatedly measured 8-10 time, and continuous 5 relative deviations of DO drop-out values are less than 5%, that is, complete collagen fiber supported wine brewing ferment
Female activation.
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| CN104267082A (en) * | 2014-10-14 | 2015-01-07 | 四川农业大学 | Method for rapidly measuring BOD (biochemical oxygen demand) by collagenous fiber immobilized escherichia coli serving as biosensor recognition element |
| CN104531825A (en) * | 2014-12-23 | 2015-04-22 | 安徽助成信息科技有限公司 | Glucose microbial electrode membrane process |
| CN105842315B (en) * | 2016-03-17 | 2018-09-11 | 中国科学院电子学研究所 | A kind of electrode sensitive layer for biochemical oxygen demand (BOD) detection |
| CN109115848B (en) * | 2018-07-17 | 2020-11-06 | 浙江理工大学 | Method for preparing sensor for measuring microbial biochemical oxygen demand |
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| CN1593746A (en) * | 2004-07-07 | 2005-03-16 | 四川大学 | Collagen fiber solid borne metallic ion adsorbing material, preparation method and purpose thereof |
| CN101639472A (en) * | 2009-08-31 | 2010-02-03 | 清华大学 | Reactor type BOD rapid measuring method and measuring instrument |
| CN101644702A (en) * | 2009-09-02 | 2010-02-10 | 清华大学 | Method for rapid BOD determination using Saccharomyces cerevisiae as biometric elements |
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|---|---|---|---|---|
| CN1593746A (en) * | 2004-07-07 | 2005-03-16 | 四川大学 | Collagen fiber solid borne metallic ion adsorbing material, preparation method and purpose thereof |
| CN101639472A (en) * | 2009-08-31 | 2010-02-03 | 清华大学 | Reactor type BOD rapid measuring method and measuring instrument |
| CN101644702A (en) * | 2009-09-02 | 2010-02-10 | 清华大学 | Method for rapid BOD determination using Saccharomyces cerevisiae as biometric elements |
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