CN110987910A - Method for detecting comprehensive content of heavy metals in soil by using luminous bacteria and application of method - Google Patents
Method for detecting comprehensive content of heavy metals in soil by using luminous bacteria and application of method Download PDFInfo
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Classifications
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
- G01N21/763—Bioluminescence
Abstract
The invention discloses a method for detecting the comprehensive content of heavy metals in soil by using luminous bacteria and application thereof, belonging to the field of environmental detection by using a biological method. Removing impurities from the collected soil sample, air-drying, grinding and sieving, adding water to prepare a soil suspension, and obtaining a sample to be detected after shaking, standing, filtering and centrifuging; the method adopts Vibrio fischeri (Vibrio fischeri) with lower cost in luminous bacteria, determines the relation between the relative luminous intensity of the Vibrio fischeri and the concentration of the heavy metal ions, and establishes a mathematical model according to the relation, so that each relative luminous intensity has a corresponding heavy metal ion toxicity interval, thereby achieving the purpose of conveniently and rapidly detecting the heavy metal pollution of the soil. The method has the advantages of simple operation, single equipment, low cost and wide applicability, and has important significance in soil pollution detection.
Description
Technical Field
The invention belongs to the field of environmental detection by a biological method, and particularly relates to a method for detecting the comprehensive content of heavy metals in soil by using luminous bacteria and application thereof.
Background
Soil is a very important component in nature and plays an essential role in maintaining ecological balance, energy circulation and the like. Urban soil is an important factor influencing urban environment and also seriously influences human health. Domestic sewage and industrial wastewater are the main sources of urban soil pollution; among them, the heavy metal ions such as copper, nickel, zinc, lead, cadmium and the like can damage the ecological environment in the soil, so that the ecological imbalance is caused, and the human society and the human health are endangered. For example, the heavy metal ion nickel can cause lung cancer, nasal cancer, laryngeal cancer and prostate cancer; research shows that cadmium can cause kidney and lung diseases and lead to bone fragility; lead is a carcinogen and can lead to death when exposed to excessive amounts; even non-abundant lead can interfere with neurological development, particularly in children, and can lead to permanent learning and behavior impairment. Statistical investigations of HQ with non-carcinogenic risks indicate that children are more sensitive to heavy metal carcinogenesis because they are more likely to come into contact with or ingest heavy metals orally; also, large doses of copper and zinc can cause gastrointestinal discomfort, and high doses of copper can cause liver damage. With the rapid development of cities, more and more factories are built around urban areas or even nearby residential areas, the types of harmful metal ions contained in wastewater are more and more complex, and despite the clear regulation of industrial wastewater discharge in China, a lot of factories can discharge untreated or incompletely treated industrial wastewater to further pollute urban water and urban soil.
The biological toxicity test is to systematically determine the influence or harm of an organism when a contaminant or an environmental factor is present by using a biological reaction. Luminescent bacteria are a class of bacteria that are capable of emitting visible fluorescence at wavelengths between 450 and 490nm under normal physiological conditions. The common genera include genus Heterobrevibacterium, Photorhabdus, Shewanella and Vibrio. The method is characterized in that the luminous intensity is weakened after the inhibition of heavy metal ions, and the change degree and the concentration of a tested substance are in a relevant relationship in a certain range, so that the concentration of the metal ions and the toxicity can be judged according to the luminous intensity of luminous bacteria. Meanwhile, the luminous bacteria have the advantages of low detection cost, high speed and convenient operation, so that the luminous bacteria detection method has wider application prospect.
At present, a method for detecting whether industrial wastewater discharge reaches the standard to cause soil heavy metal pollution by popularizing luminous bacteria from water pollution detection to soil pollution detection does not exist, and a method for effectively and rapidly carrying out comprehensive toxicity detection on heavy metals and quantitatively describing the comprehensive content of heavy metal ions in soil does not exist.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a method for detecting the comprehensive content of heavy metal in soil by using luminous bacteria and application thereof, and solves the problems in the background technology.
The technical scheme adopted by the invention for solving the technical problems is as follows: the method for detecting the comprehensive content of the heavy metals in the soil by the luminous bacteria comprises the following steps:
1) sample pretreatment: removing impurities from the collected soil sample, air-drying, grinding, sieving, adding water to prepare a soil suspension, and oscillating, standing, filtering and centrifuging to obtain a colorless and transparent supernatant which is a sample to be detected;
2) the Vibrio fischeri acted on the samples: diluting newly recovered vibrio fischeri with a sodium chloride solution to obtain a bacteria diluent, mixing the bacteria diluent with a sample to be detected, and reacting for 15 min;
3) and (3) detecting the luminous intensity: detecting the luminous intensity of the Vibrio fischeri on the mixed sample to be detected by using an ultra-weak light detector to obtain the luminous intensity of the sample;
4) data processing: constructing a relation model of the luminous intensity of the vibrio fischeri and the concentration of the soil heavy metal ions, and comparing the calculated relevant luminous intensities in the step 3) to obtain the concentration range of the soil heavy metal ions in the sample to be detected.
In a preferred embodiment of the invention, in the step 1), impurities in the collected soil sample are removed, the soil sample is air-dried for 3-7 days at the temperature of 30-35 ℃, ground and sieved by a 100-mesh sieve, and the soil and water after sieving are mixed according to the weight ratio of 1: 5, shaking the shaking table for 8 hours, standing for 16 hours to obtain a supernatant, filtering with a 20-micron ultrafiltration membrane, and centrifuging the filtrate at the rotation speed of 5000-.
In a preferred embodiment of the invention, in the step 2), the freeze-dried vibrio fischeri is recovered, and the newly recovered vibrio fischeri is diluted by 2% sodium chloride solution according to the volume ratio of 1:4 to obtain bacteria diluent; and mixing the bacterial dilution with a sample to be detected in a volume ratio of 1: 90.
In a preferred embodiment of the present invention, in step 3), the AOL-1 type full-spectrum ultramicro-weak photodetector is used to detect the luminescence intensity of Vibrio fischeri by cyclic voltammetry, the initial potential is 0.2, the high potential is 1.25, the low potential is 0.2, the scanning direction is positive, and the sensitivity is 1. e-0.005。
In a preferred embodiment of the present invention, in step 3), the luminescence intensity of the blank group is detected, wherein the blank group is obtained by mixing the bacterial dilution with 2% sodium chloride solution in a volume ratio of 1:90, and reacting for 15 min.
In a preferred embodiment of the present invention, in step 4), the relationship model is a relationship between the relative luminescence intensity of vibrio fischeri and the soil toxicity, the relative luminescence intensity is a percentage value of the luminescence intensity of the sample and the luminescence intensity of the blank group, and the soil toxicity is linearly related to the concentration of the heavy metal ions.
In a preferred embodiment of the present invention, in step 4), a model of a relationship between the relative luminescence intensity of vibrio fischeri and the soil toxicity is constructed by using a response surface analysis method, wherein the type and ion concentration of the metal ions are used as input variables, and the relative luminescence intensity of vibrio fischeri when different types of metal ions are mixed at different concentrations is used as a response variable.
In a preferred embodiment of the present inventionThe soil toxicity can be divided into three toxicity intervals, and the toxicity intervals are divided by defined toxicity coefficients. Adding Pb2+:Cu2+:Ni2+The toxicity coefficient was defined as 1 at a concentration ratio of 0.2:0.35:0.45 mg/L. Therefore, Pb2+:Cu2+:Ni2+The toxicity coefficient of 10 times of the concentration ratio is 10, the toxicity coefficient of 0.3 time of the concentration ratio is 0.3, the toxicity coefficient of 0.5 time of the concentration ratio is 0.5, and so on. Wherein the toxicity coefficient is in the I-grade toxicity range of 0-10, and the heavy metal ion content corresponding to the range is 10-2mg/L; the toxicity coefficient is in the range of 10-15 and II grade toxicity, and the content of corresponding heavy metal ions is 10-1mg/L; the toxicity coefficient is in the grade III toxicity range of 15-25, and the content of the corresponding heavy metal ions is 2 multiplied by 101mg/L。
Compared with the background technology, the technical scheme has the following advantages:
1. according to the scheme, a corresponding model of the heavy metal content and the relative luminous intensity of the luminous bacteria is constructed, the detection method for judging the heavy metal pollution degree of the soil by detecting the luminous intensity of the luminous bacteria is realized, the operation is simple, the equipment is single, the cost is low, the applicability is wide, and the method has important significance in soil pollution detection;
2. the vibrio fischeri adopted in the scheme has strong vitality, high propagation speed and low price, has stable performance when being used as a characterization reagent for detecting the toxicity of the single heavy metal ion in the sewage, and has high experimental repeatability and strong reliability;
3. the method of the scheme applies the soil detection kit to achieve the purpose of conveniently and rapidly detecting the heavy metal pollution of the soil, and has great application prospect in reagent application and commercial promotion.
Drawings
FIG. 1 is a graph showing the relationship between the relative luminescence intensity and toxicity coefficient of Vibrio fischeri.
FIG. 2 is a: the relative luminous intensity simulated by the response surface is sorted from strong to weak; b, the concentration of each ion is reduced by 2 times; c, the concentration of each ion is reduced by 1 time; d each ion concentration was amplified by a factor of 10.
Detailed Description
Example 1
The method for detecting the comprehensive content of the heavy metals in the soil by using the photobacteria comprises the following steps:
1) sample pretreatment:
collecting soil near a certain plastic plant, and removing impurities, wherein the impurities comprise gravels, stones, wood sticks, weeds, plant residual roots, insect corpses and stones, and new organisms such as manganese nodule, lime nodule and the like;
and spreading the soil after removing the impurities on a drying plate or a wood plate lined with clean white paper for natural air drying, and keeping the drying plate from being exposed to the sun strictly. When the soil reaches a semi-dry state, the large pieces of soil are smashed so as not to be hardened into hard pieces, and gravels and animal and plant residues are picked up at any time in the air drying process. The air drying chamber ensures the drying and ventilation, the air drying temperature is 30-35 ℃, and the air drying time is 3-7 days;
grinding the air-dried soil by using a grinding rod, firstly sieving the ground soil by using a nylon sieve with the aperture of 2mm, repeatedly grinding the ground soil by using a mortar, sieving the ground soil for 3 to 4 times until only a small amount of sand particles exist, and then sieving the ground soil by using a 100-mesh fine sieve;
and (3) mixing the sieved soil with soil: water 1: preparing a soil suspension by the volume ratio of 5, shaking the shaking table for 8 hours, standing for 16 hours to obtain a supernatant, filtering by using a 20-micron ultrafiltration membrane, and centrifuging the filtrate at the rotating speed of 7000rpm for 3min to obtain a colorless transparent solution as a sample to be detected;
2) the Vibrio fischeri acted on the samples: resuscitating lyophilized powder of Vibrio fischeri purchased from Toosendan Biotechnology Limited liability company of Zhejiang department, diluting newly resuscitated Vibrio fischeri with 2% sodium chloride solution at a ratio of 1:4 to obtain bacteria diluent; mixing the bacteria diluent with the pretreated sample in a ratio of 1:90, wherein the reaction time is 15 min;
3) and (3) detecting the luminous intensity:
① setting blank group, mixing the diluted bacteria solution with 2% sodium chloride solution at a ratio of 1:90 for 15min, detecting the luminous intensity of Vibrio fischeri with AOL-1 type full spectrum ultra-weak light detector, and recording the luminous intensity of blank group;
② detecting the luminous intensity of Vibrio fischeri in blank group and mixed sample by AOL-1 full-spectrum ultra-weak light detector, and recording the luminous intensity of blank group and sample;
when in use, cyclic voltammetry is adopted, the initial potential is 0.2, the high potential is 1.25, the low potential is 0.2, the scanning direction is positive, and the sensitivity is 1. e-0.005(ii) a Firstly, performing ambient light detection by using an AOL-1 type full-spectrum ultra-weak light detector, wherein the detection time is 30 seconds, and after the ambient light detection is finished, sequentially putting a blank group and a sample to be detected into the AOL-1 type full-spectrum ultra-weak light detector for detection, wherein the detection time is 30 seconds;
4) data processing:
①, constructing a relation model of the luminous intensity of the vibrio fischeri and the concentration of the heavy metal ions in the soil:
a linear relation model of the comprehensive ion concentration and the relative luminous intensity from strong to weak is fitted by adopting a response surface analysis method, the ion concentration in the linear model is amplified and reduced in an equal proportion, the absolute values of the slopes under different multiples are the same, and the linear relation is established at random, as shown in figure 2.
Defining the toxicity coefficient: adding Pb2+:Cu2+:Ni2+The toxicity coefficient was defined as 1 at a concentration ratio of 0.2:0.35:0.45 mg/L.
Therefore, the 10-fold amplification toxicity coefficient is 10, the 0.3-fold amplification toxicity coefficient is 0.3, the 0.5-fold amplification toxicity coefficient is 0.5, and so on, the model is obtained, as shown in FIG. 1
②, comparing the calculated relevant luminous intensity in the step 3) to obtain the concentration range of the soil heavy metal ions in the sample to be detected;
wherein the content of the first and second substances,
referring to FIG. 1, when the relative luminescence intensity of the sample is in the range of 0.283% -0.335%, the relative luminescence intensity of the sample is brought into a graph of the relationship between the concentration of mercury ions and the relative luminescence intensity of Vibrio fischeri; if the concentration of mercury ions corresponding to the relative luminous intensity of the sample is obtained to be more than 0.09mg/L, the sample is considered to belong to the grade III toxicity interval in the invention, and if the concentration of mercury ions corresponding to the relative luminous intensity of the sample is less than or equal to 0.09mg/L, the sample is considered to belong to the grade I toxicity interval in the invention;
in this example, the relative luminescence intensity of the obtained sample is 0.726%, corresponding to the toxicity interval II, the total toxicity of the soil is equivalent to the heavy metal ion content of 101And the grade is that plastic foam leftovers are mixed in the soil outside the factory during sampling, and plastic products are difficult to degrade in the soil, so that the soil has high toxicity and certain rationality.
Example 2
Example 2 differs from example 1 in that:
in this example, soil near a factory with a certain production stoppage is collected as a test sample, gravel, stones, weeds and plant roots are removed, the sample is ground, and then ground and sieved for 3 times after passing through a 2mm sieve until only a small amount of sand particles are stopped, and then the sample is sieved through a 100-mesh fine sieve. And (3) mixing the sieved soil with soil: water 1: 5, shaking the shaking table for 8 hours, standing for 16 hours to obtain a supernatant, filtering with a 20-micron ultrafiltration membrane, and centrifuging the filtrate at the rotating speed of 7000rpm for 3 minutes to obtain a colorless transparent solution as a sample to be detected.
In this example, the relative luminous intensity of the obtained sample is 0.773%, corresponding to the toxicity interval II, the total toxicity of the soil is equivalent to the heavy metal ion content of 101And (4) grading.
Example 3
Example 3 differs from example 1 in that:
in this example, soil near a metal processing plant was collected as a test sample, gravel, stones, weeds, and plant roots were removed, and the sample was ground, sieved through a 2mm sieve and then ground again, and sieved 5 times until only a small amount of sand was present, and then sieved through a 100-mesh fine sieve. And (3) mixing the sieved soil with soil: water 1: 5, shaking the shaking table for 8 hours, standing for 16 hours to obtain a supernatant, filtering with a 20-micron ultrafiltration membrane, and centrifuging the filtrate at a rotating speed of 9000rpm for 3min to obtain a colorless transparent solution as a sample to be detected.
In this example, the relative luminous intensity of the obtained sample is 0.553%, corresponding to the toxicity interval II, the total toxicity of the soil is equivalent to the heavy metal ion content of 101And (4) grading.
Example 4
Example 4A soil detection kit was designed using the method of the present invention.
The reagent kit comprises 2mL of Vibrio fischeri freeze-dried powder, 2mL of Vibrio fischeri resuscitation fluid, 30mL of 2% sodium chloride solution and two ultrafiltration membranes with the diameter of 20 micrometers, and one part of a specification. The specification contains a toxicity detection model diagram as shown in figure 1 and a relative luminous intensity calculation formula.
Method for operating reagent kit
1) And (3) air-drying and sieving a soil sample to be detected, and mixing the soil: water 1: 5, preparing soil suspension, shaking the shaking table for 8 hours, and standing for 16 hours. Filtering the supernatant after standing through a 20-micron filter membrane, centrifuging the filtrate at 9000rpm for 3min at 5000-.
2) Taking the Vibrio fischeri freeze-dried powder and the resuscitation solution, and placing the Vibrio fischeri freeze-dried powder and the resuscitation solution at room temperature for balancing for 15 min. And (3) injecting 2mL of resuscitation solution into a freeze-dried powder reagent bottle, and standing for 10 min. After resuspension is finished, diluting the vibrio fischeri with a 2% sodium chloride solution in a ratio of 1:4 to obtain a bacterium diluent.
3) 2ml of soil solution to be tested is taken as a blank control. And mixing 200 mul of diluted bacterium liquid and 1800 mul of soil solution to be detected. The kit can detect 80 parts of samples in total, and comprises 70 parts of soil samples to be detected and 10 parts of blank control.
In order to ensure the detection accuracy, the kit needs to ensure that the contact time of the diluted bacterium liquid and a sample to be detected is within 15-20min when in use, an AOL-1 type full-spectrum ultra-weak light detector or other weak light detection instruments can be used for detecting the luminous intensity of the vibrio fischeri, and the optimal detection time is 30 s.
(4) After the luminous intensity of the vibrio fischeri is obtained, the relative luminous intensity of the sample is calculated according to the calculation formula of the relative luminous intensity in the step 4) of the embodiment 1, and the relative luminous intensity is corresponded to the model of the invention, so that the content range of the heavy metal ions in each part of soil to be detected can be judged.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.
Claims (10)
1. A method for detecting the comprehensive content of heavy metals in soil by using luminous bacteria is characterized by comprising the following steps:
1) sample pretreatment: removing impurities from the collected soil sample, air-drying, grinding, sieving, adding water to prepare a soil suspension, and oscillating, standing, filtering and centrifuging to obtain a colorless and transparent supernatant which is a sample to be detected;
2) the Vibrio fischeri acted on the samples: diluting newly recovered vibrio fischeri with a sodium chloride solution to obtain a bacteria diluent, mixing the bacteria diluent with a sample to be detected, and reacting for 15 min;
3) and (3) detecting the luminous intensity: detecting the luminous intensity of the Vibrio fischeri on the mixed sample to be detected by using an ultra-weak light detector to obtain the luminous intensity of the sample;
4) data processing: constructing a relation model of the luminous intensity of the vibrio fischeri and the concentration of the soil heavy metal ions, and comparing the calculated relevant luminous intensities in the step 3) to obtain the concentration range of the soil heavy metal ions in the sample to be detected.
2. The method for detecting the comprehensive content of the heavy metals in the soil by the luminous bacteria according to claim 1, which is characterized in that: in the step 1), removing impurities from the collected soil sample, air-drying for 3-7 days at the temperature of 30-35 ℃, grinding, then screening by a 100-mesh sieve, and mixing the screened soil and water in a proportion of 1: 5, shaking the shaking table for 8 hours, standing for 16 hours to obtain a supernatant, filtering with a 20-micron ultrafiltration membrane, and centrifuging the filtrate at the rotation speed of 5000-.
3. The method for detecting the comprehensive content of the heavy metals in the soil by the luminous bacteria according to claim 1, which is characterized in that: in the step 2), recovering freeze-dried vibrio fischeri, and diluting newly recovered vibrio fischeri with 2% sodium chloride solution according to the volume ratio of 1:4 to obtain bacteria diluent; and mixing the bacterial dilution with a sample to be detected in a volume ratio of 1: 90.
4. The method for detecting the comprehensive content of the heavy metals in the soil by the luminous bacteria according to claim 1, which is characterized in that: in the step 3), an AOL-1 type full-spectrum ultra-weak light detector is adopted to detect the luminous intensity of the vibrio fischeri by a cyclic voltammetry method, the initial potential is 0.2, the high potential is 1.25, the low potential is 0.2, the scanning direction is positive, and the sensitivity is 1. e-0.005。
5. The method for detecting the comprehensive content of the heavy metals in the soil by the luminous bacteria according to claim 1, which is characterized in that: and 3) detecting the luminous intensity of a blank group, wherein the blank group is obtained by mixing the bacterium diluent with a 2% sodium chloride solution in a volume ratio of 1:90 and reacting for 15 min.
6. The method for detecting the comprehensive content of the heavy metals in the soil by the luminous bacteria according to claim 5, wherein the method comprises the following steps: in the step 4), the relation model is a relation between the relative luminous intensity of the vibrio fischeri and the soil toxicity, the relative luminous intensity is a percentage value of the luminous intensity of the sample and the luminous intensity of the blank group, and the soil toxicity is linearly related to the concentration of the heavy metal ions.
7. The method for detecting the comprehensive content of the heavy metals in the soil by the luminous bacteria according to claim 6, wherein the method comprises the following steps: in the step 4), the type and the ion concentration of the metal ions are used as input variables, the relative luminous intensity of the Vibrio fischeri under the condition that different types of metal ions are mixed at different concentrations is used as a response variable, and a response surface analysis method is adopted to construct a relation model between the relative luminous intensity of the Vibrio fischeri and the soil toxicity.
8. The method of claim 1The method for detecting the comprehensive content of the heavy metals in the soil by the luminous bacteria is characterized by comprising the following steps: the soil toxicity is divided into three toxicity intervals, the toxicity intervals are divided by defined toxicity coefficients, and Pb is divided2+:Cu2+:Ni2 +Is defined as a toxicity coefficient of 1 at a concentration ratio of 0.2:0.35:0.45mg/L, Pb2+:Cu2+:Ni2+The concentration ratio is synchronously amplified or reduced by corresponding to the toxicity coefficient of the same multiple, the toxicity coefficient is set to be in a grade I toxicity interval from 0 to 10, the toxicity coefficient is set to be in a grade II toxicity interval from 10 to 15, and the toxicity coefficient is set to be in a grade III toxicity interval from 15 to 25.
9. The method for detecting the comprehensive content of the heavy metals in the soil by the luminous bacteria according to claim 8, wherein the method comprises the following steps: the content of heavy metal ions corresponding to the grade I toxicity interval is 10-2mg/L; the content of heavy metal ions corresponding to the II-grade toxicity interval is 10-1mg/L; the content of heavy metal ions corresponding to the III-grade toxicity interval is 2 multiplied by 101mg/L。
10. The application of the method for detecting the comprehensive content of the heavy metals in the soil by the luminous bacteria as claimed in any one of claims 1 to 9, wherein the method comprises the following steps: is applied to a soil detection kit.
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