WO2011022895A1

WO2011022895A1 - Rapid measurement method for biochemical oxygen demand (bod) by using saccharomyces cerevisiae as biological recognition elements

Info

Publication number: WO2011022895A1
Application number: PCT/CN2009/074694
Authority: WO
Inventors: 王建龙; 胡俊; 孙中华
Original assignee: 清华大学; 江苏清大同和环境工程技术有限公司
Priority date: 2009-08-31
Filing date: 2009-10-29
Publication date: 2011-03-03

Abstract

A rapid measurement method for biochemical oxygen demand (BOD) by using Saccharomyces cerevisiae as biological recognition elements is disclosed. The method includes: selecting Saccharomyces cerevisiae as biological recognition elements; preparing the biological recognition elements by Saccharomyces cerevisiae immobilized in polyvinyl alcohol (PVA); and rapidly measuring BOD with Saccharomyces cerevisiae immobilized in PVA. The method can be applied to rapid BOD measurement for natural water body, industrial wastewater and sea water.

Description

Method for rapid determination of B0D using Saccharomyces cerevisiae as a biometric component

FIELD OF THE INVENTION The present invention relates to the field of environmental pollution monitoring, and relates to rapid monitoring of organic pollution in water bodies, specifically by using immobilized Saccharomyces cerevisiae as a biometric component of a BOD rapid measuring instrument, broadening the measurement range of organic pollutants in wastewater, and improving A method for rapidly determining BOD using Saccharomyces cerevisiae as a biometric element for determining the stability and reproducibility of the results and prolonging the life of the biometric element. BACKGROUND OF THE INVENTION There are many kinds of organic pollutants in water, and it is difficult to separately determine the quantitative values of various components. Therefore, in the water quality evaluation process, biochemical oxygen demand (BOD) is an important parameter, which refers to a certain volume. The amount of dissolved oxygen consumed by the organic matter in the water body by oxidation under the action of microorganisms, and the unit is mg/L. As can be seen from the above definition, B0D represents the degree of contamination of organic pollutants in water. Since B0D can indirectly indicate the content of microbial oxidized organic matter, B0D has become one of the most widely used monitoring indicators in the field of water pollution control. At present, both domestic and foreign use are 5 days 20 . c culture method to determine the B0D value in the water sample, which includes the steps of water sample collection, water sample oxygenation, culture, and determination. Simply put, the water sample is sealed in a test bottle at 20 ± l. C culture for 5 days, and then determine the dissolved oxygen before and after the sample culture, the difference between the two is 5d biochemical oxygen demand B0D5. There are many shortcomings in this method, such as long measurement period, complicated operation, poor reproducibility, and unsuitable on-site monitoring. Therefore, there is an urgent need for a new method that is simple, accurate, fast, highly automated, and widely applicable to determine B0D.

The BOD biosensor rapid tester uses the principle of biosensors to identify microorganisms. Material, when the sample enters the measurement chamber, the organic matter in the water sample contacts the microorganism and is decomposed by the microorganism. Microorganisms consume dissolved oxygen in the water during the decomposition of organic matter, resulting in a decrease in dissolved oxygen concentration. The corresponding transducer can detect changes in dissolved oxygen concentration and produce corresponding signals. The magnitude of this signal change has a certain linear relationship with the B0D concentration in the sample. By processing the signal, the B0D value of the water sample can be obtained. Since the measurement process is carried out at a relatively high temperature (generally 20-30.c) and the water sample is aerated, the microbial reaction rate is accelerated, and the measurement time of B0D is greatly shortened. This is the basic principle of the biosensor B0D rapid tester.

In 1977, Japanese scholar Karube et al. developed the B0D analyzer using the principle of microbial sensors. The instrument consists of an immobilized soil flora and an oxygen electrode, and the BOD value of the wastewater can be measured within 15 minutes. However, due to the destruction of the immobilized microbial membrane by the microbial enzyme, the sensor lost its activity after 10 days. Since then, Clark et al. have used a porous cellulose acetate membrane to immobilize microorganisms to make BOD sensors, which are mainly used to measure high-concentration wastewater, and can be used continuously for 17 days. Based on the principle of biosensor, B0D measurement method and measuring instrument are characterized by fast, simple and convenient testing, and easy to carry. Therefore, since 1977, B0D rapid measuring instrument has obtained extensive research and made great progress.

The basic components of B0D biosensors mainly include biometric components and transducers. The biometric component is the core component of the B0D biosensor and contains the microorganisms needed for biochemical reactions. Therefore, biometric components largely determine the performance and accuracy of the B0D sensor. A lot of research has been done on the selection of B0D sensor identification components at home and abroad. At present, the microorganisms used as B0D biosensor identification originals are Clostridium butyricus, Pseudomo s sp., Ansemila anomala, Trichosporon. Cut an i urn) and so on. Biosensors can shorten the BOD measurement cycle of water bodies, saving manpower, material and financial resources, and more importantly It can reflect the pollution degree of surface water, industrial wastewater and domestic sewage in time, and provide scientific decision-making basis for the management and decision-making departments to grasp the status of water pollution (especially when the accident occurs), and also for wastewater treatment of industrial enterprises, sewage treatment plant. Process design, control, improved processing efficiency, etc. provide timely parameters. Therefore, it is a very necessary and urgent task to develop a biosensor B0D rapid tester with independent intellectual property rights that is suitable for China's national conditions and has superior performance and reasonable price. It is a rapid monitoring of organic pollution in water bodies and the operation of wastewater treatment plants in China. Control provides technical support and generates greater economic, environmental and social benefits. After years of research, we have invented a B0D rapid measurement method (invention patent: biochemical oxygen demand biosensor, patent number: ZL 00 1 32125. 0) and a method for preparing microbial particles by immobilization technology (invention patent: a Method for immobilizing enzyme/microorganism of polyvinyl alcohol, Patent No.: ZL 00 1 32126. 9; Patent of invention: A method for inhibiting water-soluble swelling of polyvinyl alcohol-immobilized microbial particles, Patent No.: ZL 2006 1 0011608. X) , and developed a B0D rapid tester. The invention proposes a bio-identification component for measuring BOD by using Saccharomyces cerevisiae, and strives to broaden the measurement range for various wastewaters to meet the needs of more industries. In addition, PVA immobilized Saccharomyces cerevisiae is used to prepare bioactive components with high activity and selectivity to improve the stability of biological response and ensure the consistency of identification components, thereby improving the commercialization degree of B0D sensors. SUMMARY OF THE INVENTION The object of the present invention is to provide a method for rapidly determining BOD using Saccharomyces cerevisiae as a bio-identification element, characterized in that the method comprises selecting Saccharomyces cerevisiae as a bio-identification element, preparing a bio-identification element by using PVA immobilized Saccharomyces cerevisiae, and utilizing Rapid determination of B0D by PVA-immobilized Saccharomyces cerevisiae biorecognition elements; the selection of Saccharomyces cerevisiae as a bio-identification element is the selection of Saccharomyces cerevisiae The main reasons for identifying materials are as follows:

1) Saccharomyces cerevisiae is a highly active aerobic microorganism with strong ability to assimilate carbohydrates. It has been widely used in the comprehensive treatment of high-concentration organic industrial wastewater.

2) Saccharomyces cerevisiae itself has no toxic effects and will not cause secondary pollution to the environment;

3) The culture conditions of Saccharomyces cerevisiae are not high, and they grow rapidly, so it is suitable for obtaining;

4) Saccharomyces cerevisiae can be dried and stored for a long time, and can maintain the vitality of the enzyme for a long time;

5) Saccharomyces cerevisiae cells have the characteristics of osmotic pressure resistance and can be applied to the monitoring of marine pollution. The process of preparing biometric components by using PVA immobilized Saccharomyces cerevisiae: suspending Saccharomyces cerevisiae cells in deionized water at a concentration of 10-20 g/100mL (w/v), and then mixed with the dissolved PVA solution to obtain a uniform mixture, the final concentration of PVA is 8-10% (w / v), the mixture is dropped into 500mL of saturated boric acid solution After fully 12 to 12 hours, the cross-linking reaction was taken out and rinsed 3 times with deionized water to obtain immobilized Saccharomyces cerevisiae granules as bio-identification elements, which were placed in a refrigerator for storage; the PVA-immobilized Saccharomyces cerevisiae biorecognition element pair Rapid determination of B0D: During measurement, the immobilized microbial particles are mixed with the sample to be tested in the measurement chamber, stirred and aerated with a micro-aeration tube to saturate dissolved oxygen, and the process of immobilizing microbial particles in decomposing organic pollutants Decomposes biodegradable organic pollutants in water, and consumes dissolved oxygen in water, causing dissolved oxygen concentration to decrease, resulting in dissolved oxygen Level is reduced to a new stable state; dissolved oxygen according to the difference between the two stable states, a standard curve can be calculated by the measured values of water samples B0D. The two steady state states that the first steady state is set to a predetermined measurement temperature when the water sample reaches a predetermined measurement temperature, and the second steady state is set to a range of three consecutive current value readings having a variation range of less than 0.001 μΑ. The immobilized microbial particles are immobilized Saccharomyces cerevisiae particles. The beneficial effects of the present invention are compared with the biometric components of the currently used B0D rapid tester. The brewing yeast proposed by the present invention makes up for its many shortcomings:

(1) Saccharomyces cerevisiae has high metabolic activity and strong ability to decompose organic pollutants, which broadens the measurement range of various wastewaters;

(2) Depending on the degree of contamination of the water sample, changing the amount of immobilized microbial particles can adjust the effective measurement range;

(3) The immobilized microbial particles have a longer service life, and the activation and preservation of microorganisms are simpler and more convenient;

(4) Immobilized Saccharomyces cerevisiae cells are resistant to osmotic pressure. Therefore, Saccharomyces cerevisiae is used as a biometric identification component for rapid monitoring of organic pollution in marine waters. DRAWINGS

Figure 1 is a standard curve for seawater in the range of 5-50 mg/L for B0D ₅ . BEST MODE FOR CARRYING OUT THE INVENTION The technical solution of the present invention will be further described below in conjunction with the examples: Example 1 Using the Saccharomyces cerevisiae proposed by the present invention as a biological identification element for rapid determination of BOD, the measurement of a standard BOD sample was carried out. Standard GGA solutions with B0D concentrations of 5, 20, 25, 40, 50, 75, 100, 125, 150, 175, 200 mg/L were prepared under the following conditions: temperature control at 30 ± 1 °C, aeration It is 200-500 mL/min. The immobilized Saccharomyces cerevisiae particles were used as biometric materials, and the concentration of the above B0D standard solution was measured using a dissolved oxygen electrode. The measurement results show that there is a good linear relationship between the change of DO value and the B0D value of the sample, linear The correlation coefficient is greater than 0.99, which satisfies the rapid measurement requirements of BOD. Example 2 The Saccharomyces cerevisiae proposed by the present invention was used as a biometric component for rapid determination of B0D, and a standard GGA solution having a B0D concentration of 50 mg/L was prepared under the following conditions: temperature control at 30 ± 1 ° C, dilution with deionized water, The reproducibility of the measurement results of the aeration volume of 200 mL/mino is shown in Table 1. Table 1 Reproducibility of immobilized S. cerevisiae response to GGA solution with B0D ₅ of 50 mg/L

Number of measurements 1 2 3 4 5 6 7 8 Initial concentration (mg/L) 6. 24 6. 17 6. 33 6. 74 6. 7 6. 72 6. 89 6. 76 Equilibrium concentration (mg/L) 5. 94 5. 8 5. 98 6. 4 6. 4 6. 38 6. 54 6. 32 Response time (min) 12 15 15 10 15 15 12 13 Reduced dissolved oxygen value 0. 3 0. 37 0. 35 0. 34 0. 3 0. 34 0. 35 0. 34

(mg/L) The analysis of the data in the table shows that the reproducibility test is 8 times, the average value of the dissolved oxygen reduction value is 0. 34 mg / L, the maximum relative deviation is 10. 7 %, meet the B0D fast measurement requirements. Example 3 The Saccharomyces cerevisiae proposed by the present invention was used as a biometric material for rapid determination of BOD, and a seawater B0D standard sample was measured. The configuration steps of the seawater B0D standard sample were completely the same as those of the GGA solution (Example 1), and only the deionized water used in the configuration was replaced with standard seawater. This standard seawater is taken 50 m below the sea level in the distant sea, then sand filtration, and finally UV disinfection. The measurement conditions are as follows: the temperature is controlled at 30 ± 1 °C, diluted with deionized water, and the aeration amount is 200-500 mL/min. The measurement results are shown in Fig. 1. It can be seen that there is a good linear relationship between the change of the DO value and the B0D value of the sample, and the linear correlation coefficient is greater than 0.99, which satisfies the B0D fast measurement requirement. Example 4 Using the Saccharomyces cerevisiae proposed by the present invention as a biometric material for rapid determination of BOD, BOD measurement of actual domestic sewage samples was carried out. The domestic sewage used in the experiment was taken from the domestic sewage of the student area of Tsinghua University. The test method of the GGA standard solution is used to measure the domestic sewage, and the B0D value of the domestic sewage is 48.5 mg/L, and the result of the five-day standard dilution method is 42. 4 mg / L. It can be seen that with the Saccharomyces cerevisiae of the present invention as a biometric material for rapid determination of BOD, the B0D rapid measurement value and the five-day B0D standard dilution measurement value are similar in the measurement of domestic sewage.

Claims

Claim

A method for rapidly measuring BOD using Saccharomyces cerevisiae as a biorecognition element, the method comprising: selecting Saccharomyces cerevisiae as a biorecognition element, preparing a bio-component device using PVA immobilized Saccharomyces cerevisiae, and immobilizing Saccharomyces cerevisiae using PVA Rapid determination of BOD by biometric elements; The selection of Saccharomyces cerevisiae as a biometric element is the main reason for selecting Saccharomyces cerevisiae as a rapid determination of B0D4 recognition elements as follows:

5) Saccharomyces cerevisiae cells have the characteristics of osmotic pressure resistance and can be applied to the monitoring of marine pollution. The process of preparing biometric components by using PVA immobilized Saccharomyces cerevisiae: floating Saccharomyces cerevisiae cells in a certain amount of deionized water (concentration range) 10-20 g/100mL (w/v) is), and then mixed with the dissolved PVA solution to obtain a uniform mixture. The final concentration of PVA is 8-10% (w/v), and the i mixture is dropped into the saturation. In 500 mL of boric acid solution, the mixture was fully cross-linked for 10-12 hours, and then washed out with deionized water for 3 times to obtain immobilized Saccharomyces cerevisiae particles as biometric components, which were placed in a refrigerator for cold standby;