Quantitative detection method of cadmium sulfide in tetrahymena culture system

Abstract

This invention belongs to the field of cadmium sulfide detection technology, specifically relating to a quantitative detection method for cadmium sulfide in a Tetrahymena culture system. The invention involves adding cadmium ions to Tetrahymena to form a Tetrahymena culture system sample, followed by ultrafiltration after disruption to separate cadmium ions into free cadmium ions (Cd). 2+ The sample was divided into two parts: cadmium sulfide (CdS) and cadmium sulfide (CdS). The cadmium content in the separated liquid sample was determined using a high-sensitivity detection technique (such as inductively coupled plasma mass spectrometry), thus obtaining the CdS content. 2+ The method not only provides information on the composition of cadmium in Tetrahymena but also dynamically monitors the efficiency of cadmium ion conversion to cadmium sulfide, providing reliable data support for assessing the remediation effect of Tetrahymena on cadmium pollution. Furthermore, this method has advantages such as ease of operation, high sensitivity, and good repeatability.

Description

Technical Field

[0001] This invention belongs to the field of cadmium sulfide detection technology, specifically relating to a quantitative detection method for cadmium sulfide in a Tetrahymena culture system. Background Technology

[0002] Cadmium is a highly toxic heavy metal pollutant with a long biological half-life. Cadmium in water is easily absorbed by crops such as rice and accumulates through the food chain, eventually reaching animals and humans. Because cadmium is metabolized slowly in organisms, even small and continuous ingestion can gradually accumulate in the human body, leading to serious diseases such as kidney damage. Even at low concentrations, cadmium pollution in water can cause long-term and irreversible harm to human health and the environment. Therefore, effective remediation measures, such as phytoremediation, chemical passivation, and biosorption, must be implemented to reduce cadmium pollution in aquatic environments and protect human health and ecological security.

[0003] Currently, microbial remediation technology has attracted significant attention from researchers in the field of heavy metal pollution control due to its substantial advantages such as cost-effectiveness, high specificity, and environmental friendliness. This technology can not only reduce cadmium toxicity through the metabolic activities of microorganisms, but also enhance remediation efficiency through genetic engineering and other means to modify organisms. As a model protist for toxicological research, Tetrahymena has shown some potential in heavy metal environmental response and remediation. Studies have shown that Tetrahymena can remove highly toxic cadmium ions (Cd) from the environment through related metabolic pathways (including cysteine ​​synthesis and glutathione metabolism). 2 +) can be converted into low-toxicity cadmium sulfide (CdS), thus significantly reducing the toxicity of cadmium (Lv Hongrui. Study on the synthetic pathway of thermohymena Cysteine ​​and its molecular mechanism for alleviating heavy metal stress [D]. Shanxi University, 2021; Jiawei Tu, Tian Li, Zihan Gao, Jie Xiong, Wei Miao. Construction of CdS-Tetrahymena thermophila hybrid system by efficient cadmium adsorption for dye removal under light irradiation [J]. Journal of Hazardous Materials, 2022, 439, 129683). In addition, Tetrahymena can also adsorb heavy metals through multiple mechanisms such as endocytosis and metal-binding proteins, further reducing the bioavailability of cadmium in the environment. Therefore, Tetrahymena not only has high efficiency and environmental friendliness in cadmium pollution remediation, but also provides an important foundation for the development of bioremediation technologies.

[0004] Therefore, it is essential to use the concentration of cadmium sulfide (CdS) generated by cadmium accumulation in Tetrahymena as a key indicator to assess the cadmium ion conversion rate of Tetrahymena. However, currently, there are no commercially available dedicated instruments or methods for directly detecting CdS content in Tetrahymena, which limits in-depth research on the assessment of cadmium conversion rate in Tetrahymena. To address this issue, it is urgent to develop and establish a quantitative analytical method for detecting CdS content in Tetrahymena culture systems, which can provide reliable data support for the assessment of cadmium conversion rate in Tetrahymena. Summary of the Invention

[0005] To address the technical problems existing in the prior art, this invention proposes a quantitative detection method for cadmium sulfide in a Tetrahymena culture system. This method involves adding cadmium ions to Tetrahymena to form a Tetrahymena culture system sample, followed by ultrafiltration after disruption to separate cadmium ions into free cadmium ions (Cd). 2 The sample was divided into two parts: cadmium (Cd) and cadmium sulfide (CdS). The cadmium content in the separated liquid sample was determined using a high-sensitivity detection technique (such as inductively coupled plasma mass spectrometry), thus yielding the Cd content. 2 The method not only provides information on the composition of cadmium in Tetrahymena, but also dynamically monitors the efficiency of cadmium ion conversion to cadmium sulfide, providing reliable data support for assessing the remediation effect of Tetrahymena on cadmium pollution. Furthermore, this method has advantages such as ease of operation, high sensitivity, and good repeatability.

[0006] A method for quantitative detection of cadmium sulfide content in a Tetrahymena culture system, comprising the following steps:

[0007] (1) Culture and treatment of Tetrahymena

[0008] Tetrahymena was inoculated into SPP medium containing 1% penicillin and streptomycin (preferably with an initial density of 3125 cells / mL) and incubated in a constant temperature shaker at 20–35°C until a Tetrahymena culture system in the stable late stage was obtained (preferably incubated at 135 r / min for 2 days, at which point the density was approximately 1,500,000 cells / mL); the Tetrahymena culture system was washed with Tris buffer (preferably 10 mM, pH 7.4 Tris buffer) and resuspended in SPP medium containing 1% penicillin and streptomycin to obtain the Tetrahymena culture system;

[0009] (2) Sample preparation and processing:

[0010] (2-1) Add a certain concentration of CdCl2 solution to the Tetrahymena culture system obtained in (1), and place it in a constant temperature shaker incubator at 20-35℃ and shake it slowly at a uniform speed; collect Tetrahymena from the culture system as samples as needed;

[0011] (2-2) The sample was ultrasonically broken up using an ultrasonic disruptor. An equal volume of EDTA-2Na solution was added to the broken sample and mixed well. EDTA-2Na was used to fully chelate the cadmium ions adsorbed in the sample.

[0012] (3) Sample ultrafiltration: After the sample was treated with EDTA-2Na, ultrapure water was added to the sample and then the sample was added to an ultrafiltration tube (15ml, 50kDaMWCO, catalog number UFC905096) for ultrafiltration. After ultrafiltration was completed, the ultrafiltrate in the centrifuge tube was collected.

[0013] (4) Sample digestion: The sample treated with EDTA-2Na in (2-2) and the ultrafiltrate collected in (3) were mixed with electronic grade nitric acid and added to the digestion tube. The tube was then capped and tightened, and placed in a microwave digester for digestion. Then, the tube was placed in an acid removal apparatus to remove acid. After washing with nitric acid, the resulting solution was collected.

[0014] (5) Sample content determination: The digested sample was filtered through a 0.22 μm aqueous filter membrane. The cadmium content in the filtered sample was determined by inductively coupled plasma mass spectrometry. The determination results of the ultrafiltrate sample and the sample treated with EDTA-2Na (2-2) correspond to the cadmium ion content and total cadmium content in the system, respectively. The cadmium sulfide content and cadmium conversion rate were obtained by formulas (1-1) and (1-2), where the cadmium sulfide content is the amount of cadmium converted to cadmium sulfide from the total cadmium: Cadmium sulfide content (mg / L) = Total cadmium content (mg / L) - Cadmium ion content (mg / L) (1-1)

[0015]

[0016] Furthermore, the concentration required to ensure sufficient chelation of the EDTA-2Na solution in (2-2) is determined by the following method:

[0017] The quantitative detection method described above is followed, except that step (2) is replaced with the following steps:

[0018] (2a) Sample preparation and processing:

[0019] Collect Tetrahymena from (1) as a sample, use an ultrasonic disruptor to disrupt it, add a certain concentration of CdCl2 solution to the disrupted sample, mix well, and then add an equal volume of a certain concentration of EDTA-2Na solution and mix well.

[0020] Based on the concentration of the CdCl2 solution, different concentration ranges of EDTA-2Na solution were added to perform the above operation. The percentage of cadmium ion content to total cadmium content at different concentrations was calculated, i.e., the cadmium recovery rate. The concentration at which the cadmium recovery rate reached 95% was taken as the minimum concentration for sufficient chelation.

[0021] Furthermore, in step (2), the CdCl2 solution is added to the Cd... 2+ The concentration is 5 mg / L-10 mg / L.

[0022] Furthermore, in (2a) Cd 2+ When the concentration is 5 mg / L, add 60–80 μM EDTA-2Na solution (preferably 60 μM), Cd 2+ When the concentration is 10 mg / L, add 120–180 μM EDTA-2Na solution (preferably 120 μM).

[0023] Furthermore, the SPP culture medium is prepared as follows: the solvent is ultrapure water, and the solutes and their concentrations are as follows: glucose 2g / L, 20 g / L peptone, 1 g / L yeast extract, and 0.03 g / L ferric citrate; sterilize at 121°C for 15 min before use.

[0024] Furthermore, the tetrahymena is Tetrahymena thermophila SB210.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] This invention establishes a quantitative detection method for cadmium sulfide in a Tetrahymena culture system. The method involves adding cadmium ions to Tetrahymena to form a Tetrahymena culture system sample. After the system is broken up at different fixed time points as required, ultrafiltration is used to separate cadmium from the sample into Cd. 2+ The sample is divided into two parts: CdS and CdS. The dynamic Cd content is calculated by measuring the cadmium content of the separated sample. 2+ By analyzing CdS content, this method not only provides information on the composition of cadmium in Tetrahymena, but also dynamically monitors the efficiency of cadmium ion conversion to cadmium sulfide, providing reliable data support for assessing the remediation effect of Tetrahymena on cadmium pollution. Attached Figure Description

[0027] Figure 1 The main flowchart of the quantitative detection method of cadmium sulfide in the Tetrahymena culture system provided by the present invention is shown below.

[0028] Figure 2 The graph shows the relationship between EDTA-2Na concentration and cadmium recovery rate under different cadmium ion concentrations (Figure a: cadmium ion concentration of 5 mg / L, Figure b: cadmium ion concentration of 10 mg / L).

[0029] Figure 3This is a linear relationship between the added cadmium ion concentration and the actual cadmium ion concentration measured in the sample under different total cadmium concentrations (Figure a: total cadmium concentration of 5 mg / L, Figure b: total cadmium concentration of 10 mg / L).

[0030] Figure 4 This is a trend graph showing the cadmium conversion rate of Tetrahymena thermophila at different time points and under different cadmium ion concentrations in Example 3. Detailed Implementation

[0031] The following examples are only used to illustrate the implementation method of the present invention, but they should not be construed as limiting the scope of protection of the present invention.

[0032] The tetrahymena used was Tetrahymena thermophila SB210, provided by Cornell University (https: / / tetrahymena.vet.cornell.edu / ).

[0033] Preparation of SPP medium: The solvent is ultrapure water, and the solutes and their concentrations are as follows: glucose 2g / L, 20 g / L peptone, 1 g / L yeast extract, and 0.03 g / L ferric citrate; sterilize at 121°C for 15 min before use.

[0034] The method for preparing cadmium chloride mother liquor is as follows: Dissolve 0.10157g of cadmium chloride hemihydrate (pentahydrate) powder in 50mL of water (final cadmium concentration is 1000mg / L), and filter it with a 0.22μm filter membrane (Millex-GP 0.22μm) to remove bacteria and particles.

[0035] The preparation method for EDTA-2Na stock solution is as follows: Dissolve 3.7224g of ethylenediaminetetraacetic acid disodium dihydrate powder in 500mL of water (final concentration of 20mM), and filter with a 0.22μm filter membrane to remove bacteria and particles.

[0036] The preparation method for Tris buffer is as follows: Weigh 1.2114 g of tris(hydroxymethyl)aminomethane (Tris) powder and dissolve it in 1 L of water (final concentration is 10 mM). Adjust the pH to 7.4 with hydrochloric acid and sterilize at 120 °C for 15 min.

[0037] The basic design principle of this invention is as follows:

[0038] The EDTA-2Na solution can desorb cadmium adsorbed on Tetrahymena cell fragments through chelation, and ultrafiltration can separate the desorbed cadmium into free cadmium ions (Cd). 2 The sample is divided into two parts: cadmium (CdS) and cadmium sulfide (CdS). The cadmium content in the separated liquid sample is then determined using a high-sensitivity detection technique, thereby calculating the cadmium content converted into CdS.

[0039] To ensure the reliability of the results from the Tetrahymena culture system, a crucial factor is the concentration of the EDTA-2Na solution. Insufficient EDTA-2Na leads to incomplete desorption of adsorbed cadmium; excessive EDTA-2Na, besides desorbing cadmium ions, also chelates a small amount of cadmium from cadmium sulfide, affecting the determination of cadmium composition in the experimental samples. Therefore, Example 1 investigated the optimal EDTA-2Na concentration under different cadmium ion concentrations.

[0040] Example 1: Establishment of Tetrahymena culture system and investigation of EDTA-2Na concentration conditions

[0041] (1) Culture and treatment of Tetrahymena thermophila: The thermophila thermophila were inoculated into SPP medium containing 1% penicillin and streptomycin at an initial density of 3125 cells / mL. After culturing in a constant temperature shaker at 30℃ for 2 days at 135 r / min, a thermophila thermophila culture system in the stable late stage was obtained (Tetrahymena density was about 1,500,000 cells / mL). The thermophila thermophila culture system was washed twice with Tris buffer (10 mM, pH 7.4) and resuspended in fresh SPP medium containing 1% penicillin and streptomycin.

[0042] (2) Sample preparation and treatment: Tetrahymena were collected from the culture system as samples. The samples were ultrasonically broken up using an ultrasonic disruptor (model Ningbo Xinzhi JY92-IIN, 650W, amplitude rod 10mm) (50% power, 10min). CdCl2 stock solution (cadmium concentration 1000mg / L) was added to the broken samples until the desired final cadmium concentration was 5mg / L or 10mg / L. After mixing, an equal volume of EDTA-2Na solution was added and shaken for 10min to mix.

[0043] (3) Sample ultrafiltration: Take 500 μL of the sample treated with EDTA-2Na, add 2500 μL of ultrapure water, mix, and add the mixture to the upper filter of the ultrafiltration tube (15 ml, 50 kDa MWCO, catalog number UFC905096). Centrifuge at 3000×g for 10 min using a centrifuge with a swing-type rotor. Add 2000 μL of ultrapure water to the ultrafiltration tube again, centrifuge once more, and collect all the ultrafiltrate in the centrifuge tube after ultrafiltration is completed.

[0044] (4) Sample digestion: 2 mL of the untreated sample and the ultrafiltrate collected after ultrafiltration were mixed with 5 mL of electronic-grade 2% nitric acid and added to a digestion tube. The tube was then capped and tightened, and placed in a microwave digester. Digestion was performed according to a specific temperature program (heating time: 40 min; digestion temperature: 180℃; holding time: 15 min). After digestion, the digestion tube was removed, the cap was opened, and the tube was placed in an acid-removing apparatus at 160℃ until the solution was reduced to less than 0.2 mL. The digestion tube was then removed, and 2% nitric acid was added to rinse the tube. All solution was collected, transferred to a 15 mL centrifuge tube, and brought to a final volume of 10 mL with 2% nitric acid.

[0045] (5) Sample content determination: The digested sample was filtered through a 0.22 μm aqueous filter membrane (Millex-GP 0.22 μm), and the cadmium content in the filtered sample was determined directly using inductively coupled plasma mass spectrometry. The cadmium ion content and total cadmium content in the system could be obtained from the determination results of the ultrafiltrate sample and the unfiltered sample, respectively.

[0046] This experiment set up two cadmium ion concentration systems and eight corresponding EDTA-2Na concentration systems, for a total of 16 groups, with three replicates in each group. The specific concentration settings are shown in Table 1.

[0047] Table 1 Cadmium ion concentration system and EDTA-2Na concentration settings

[0048]

[0049] Experimental results are as follows Figure 2 The results showed that when the cadmium ion concentration was 5 mg / L and no EDTA-2Na was used, the cadmium recovery rate (the percentage of cadmium ion content to total cadmium content) was only about 30%. When the EDTA-2Na concentration was set to 10 μM, 20 μM, 30 μM, and 50 μM, the cadmium recovery rate did not reach 100%, while when the EDTA-2Na concentration was set to 60 μM, 70 μM, and 80 μM, the cadmium recovery rate was close to 100%. In the subsequent Example 2 experiment, in order to achieve saturation of cadmium recovery rate, the EDTA-2Na concentration used was 60 μM when the cadmium ion or total cadmium concentration was 5 mg / L.

[0050] Experimental results Figure 2 b shows that when the cadmium ion concentration is 10 mg / L and no EDTA-2Na is used, the cadmium recovery rate is only about 20%. When the EDTA-2Na concentration is set to 40 μM, 60 μM, 80 μM, and 100 μM, the cadmium recovery rate does not reach 100%, while when the EDTA-2Na concentration is set to 120 μM, 160 μM, and 180 μM, the cadmium recovery rate is close to 100%. In the subsequent Example 2 experiment, in order to achieve saturation of cadmium recovery rate, the EDTA-2Na concentration of 10 mg / L was 120 μM.

[0051] Example 2 Methodological Investigation

[0052] 2.1 Examination of Linear Relationships

[0053] To examine the practicality of this method, Tetrahymena were cultured and treated according to the method in Example 1. The difference was that during sample preparation and treatment, the CdCl2 mother liquor was replaced with a certain amount of CdCl2 and cadmium sulfide (CdS) added to the broken sample, with total cadmium concentrations of 5 mg / L and 10 mg / L, respectively. Subsequently, ultrafiltration, digestion, and content determination were performed according to the method in Example 1. Finally, the linear relationship between the added cadmium ion concentration and the actual measured cadmium ion concentration was compared.

[0054] To obtain a certain total cadmium concentration, firstly, a cadmium sulfide (CdS) suspension was prepared: after thoroughly mixing the cadmium chloride solution and the sodium sulfide solution, the precipitate was washed three times with ultrapure water and then resuspended in ultrapure water. The suspension was then crushed using an ultrasonic disruptor to obtain a homogeneous cadmium sulfide suspension. The suspension was digested according to the method in Example 1, and its concentration was determined. Finally, it was diluted to a CdS mother liquor with a cadmium concentration of 1000 mg / L.

[0055] Secondly, CdCl2 (Cd) containing different concentrations of cadmium ions was prepared. 2+ A mixture of cadmium sulfide (CdS) and cadmium sulfide (CdS) with total cadmium concentrations of 5 mg / L and 10 mg / L, respectively, is shown in Tables 2 and 3. When the total cadmium concentration is 5 mg / L, the concentration of CdS in section A1 is 0 mg / L. 2+ 5 mg / L CdS (in the following examples, the expression of cadmium sulfide (CdS) concentration refers to the concentration of cadmium in the mixture within CdS), that is, add 5 μL of CdS stock solution (1000 mg / L) and 0 μL of CdCl2 (Cd concentration 1000 mg / L) to the broken Tetrahymena thermophila culture system, and bring the volume to 10 mL. Numbered B1-H1, Cd 2+ The concentrations were 0.5, 1, 2, 3, 4, 4.5, 4.8, and 5 mg / L, and the CdS concentrations were 4.5, 4, 3, 2, 0.5, 0.2, and 0 mg / L, respectively. When the total cadmium concentration was 10 mg / L, the CdS concentration in component B2-H2 was... 2+ The concentrations were 0, 1, 2, 4, 6, 8, 9, and 10 mg / L, and the CdS concentrations were 10, 9, 8, 6, 4, 2, 1, and 0 mg / L. There were 16 groups of samples in total, with 3 replicates per group.

[0056] Table 2. Different cadmium concentration settings under a total cadmium concentration of 5 mg / L.

[0057]

[0058] Table 3. Different cadmium concentration settings under a total cadmium concentration of 10 mg / L.

[0059]

[0060]

[0061] The linear relationship was investigated by plotting the concentration of cadmium ions added to the sample (preset cadmium ion concentration) on the x-axis and the actual measured cadmium ion concentration (actual cadmium ion concentration) on the y-axis. Figure 3 Total cadmium concentration 5 mg / L, Figure 3 b. With a total cadmium concentration of 10 mg / L, the results showed that within the corresponding linear range, the correlation coefficient (R) was... 2 The values ​​were 0.9957 and 0.9964, respectively, indicating a good linear relationship between cadmium ion concentrations in both systems. However, as... Figure 2 As shown in a, in the preset Cd 2+ When the concentration is less than 3 mg / L (i.e., the preset CdS concentration is greater than 2 mg / L), EDTA-2Na will chelate a small portion of the cadmium in CdS, thus reducing the measured Cd concentration. 2+ The concentration will be slightly higher than the preset concentration; within the preset Cd 2+ When the concentration is greater than or equal to 3 mg / L (i.e., the preset CdS concentration is less than or equal to 2 mg / L), the measured Cd 2+ The concentration is not much different from the preset concentration.

[0062] 2.2 Repeatability Examination

[0063] To examine the repeatability of this method, Tetrahymena were cultured and treated according to the method in Example 1. Sample preparation and treatment were performed on samples numbered D1, E1, D2, E2, and F2 in Example 2.1. Three copies of each sample number were prepared. An equal volume of EDTA-2Na solution of the corresponding concentration was added, and the samples were shaken and mixed for 10 min before ultrafiltration, sample digestion, and sample content determination to obtain the cadmium ion content in the filtrate. The repeatability of the method was examined by calculating the relative standard deviation (RSD) between the three sets of results.

[0064] The repeatability results are shown in Table 4. The RSD range of cadmium content in the filtrate of each sample is 1.18% to 4.13%, and the RSD values ​​are all less than 5%, indicating that the method has good repeatability.

[0065] Table 4 Results of Repeatability Experiments

[0066]

[0067] 2.3 Stability Assessment

[0068] To examine the stability of this method, Tetrahymena were cultured and treated according to the method in Example 1. Samples prepared and processed as described in Example 2.1 (numbered D1, E1, D2, E2, and F2) were placed at 4°C. After 0 h, 12 h, and 24 h, equal volumes of EDTA-2Na solution of corresponding concentrations were added, and the mixture was shaken and mixed for 10 min. Subsequent ultrafiltration, sample digestion, and sample content determination were then performed to obtain the cadmium ion content in the filtrate. The stability of the method was assessed by calculating the relative standard deviation (RSD) between the sample determination results at the three time points.

[0069] The test results and relative standard deviations are shown in Table 5. The stability results show that the RSD of cadmium content in the filtrate of each sample ranges from 0.88% to 3.54%, and the RSD values ​​are all less than 5%, indicating that the samples have good stability within 24 hours at 4℃.

[0070] Table 5. Results of the stability experiment

[0071]

[0072] Example 3: Evaluation of cadmium conversion rate under different treatments with Tetrahymena thermophila.

[0073] The above method was applied to Tetrahymena thermophila to detect its cadmium conversion rate at different time points. Tetrahymena culture and treatment were performed according to the method in Example 1, except that in the sample preparation and treatment, CdCl2 stock solution (1000 mg / L) was added to the Tetrahymena to the desired final concentration (5 mg / L or 10 mg / L), and then the mixture was placed in a constant temperature shaker incubator at 30–35°C with a uniform slow shaking speed of 50 r / min. The Tetrahymena culture system was collected as samples at different time points (including 0 h, 3 h, 6 h, 12 h, 24 h, and 48 h) for ultrasonic disruption, and an equal volume of EDTA-2Na (5 mg / L CdCl2) was added. 2+ Add an equal volume of 60 μM EDTA-2Na and 10 mg / L Cd at the specified concentration. 2+ An equal volume of 120 μM EDTA-2Na was added at the specified concentration. Subsequently, ultrafiltration, digestion, and content determination of the sample were performed according to the method described in Example 1. This sample treatment allows for accurate measurement of the cadmium conversion rate of Tetrahymena thermophila at different time points.

[0074] The cadmium sulfide content is calculated using Formula 1-1 (cadmium sulfide content is the amount of cadmium in total cadmium that is converted into cadmium sulfide).

[0075] Cadmium sulfide content (mg / L) = Total cadmium content (mg / L) - Cadmium ion content (mg / L) (1-1)

[0076] The cadmium conversion rate was calculated using formula 1-2.

[0077]

[0078] Experimental results are as follows Figure 4 The results showed that the conversion rate of cadmium sulfide by Tetrahymena thermophila increased over time, eventually stabilizing, exhibiting a time-dependent trend. 2+ At a concentration of 5 mg / L, the conversion rate of cadmium approached saturation after 12 hours, reaching 91.5%; when Cd 2+ At a concentration of 10 mg / L, the conversion rate of cadmium approaches saturation within 24 hours, reaching 78.3%.

[0079] In summary, this invention provides a quantitative detection method for cadmium sulfide in a Tetrahymena culture system, which has good linearity, repeatability, and stability. This method is suitable for quantitatively determining the cadmium sulfide content and detecting the cadmium conversion rate in a Tetrahymena culture system.

[0080] It should be noted that, in this application, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus.

Claims

1. A method for quantitatively detecting the amount of cadmium sulfide in a Tetrahymena culture system, characterized by, The method comprises the following steps: (1) culture and treatment of Tetrahymena Tetrahymena is inoculated into SPP culture medium containing 1% of penicillin and is cultured in a constant-temperature shaking incubator at 20-35 DEG C to obtain a Tetrahymena culture system in a stable late stage, and then is washed and resuspended in SPP culture medium containing 1% of penicillin to obtain a Tetrahymena culture system; (2) preparation and treatment of a sample: (2-1) a certain concentration of CdCl2 solution is added to the Tetrahymena culture system obtained in (1), and is placed in a constant-temperature shaking incubator at 20-35 DEG C; then Tetrahymena is collected from the culture system as a sample according to requirements; (2-2) the sample is broken by an ultrasonic cell disruptor, and an equal volume of EDTA-2Na solution is added to the broken sample and mixed to chelate cadmium ions adsorbed in the sample by EDTA-2Na, so as to obtain a sample treated by EDTA-2Na; (3) sample ultrafiltration: after the sample treated by EDTA-2Na is added with ultrapure water, it is added into a 50k ultrafiltration tube for ultrafiltration, and after the ultrafiltration is completed, the ultrafiltrate is collected; (4) sample digestion: the sample treated by EDTA-2Na in (2-2) and the ultrafiltrate collected in (3) are mixed with electronic-grade nitric acid, and then are placed in a microwave digestion instrument for digestion, and then are placed in an acid removal instrument for acid removal, and after being washed with nitric acid, the obtained solution is collected; (5) determination of the content of the sample: the digested sample is filtered by a water phase filter membrane, the filtered sample is determined for the content of cadmium in the sample by an inductively coupled plasma mass spectrometer, the determination results of the ultrafiltrate sample and the sample treated by EDTA-2Na in (2-2) correspond to the content of cadmium ions and the total content of cadmium in the system respectively, and the content of cadmium sulfide and the conversion rate of cadmium are obtained by formulas (1-1) and (1-2), wherein the content of cadmium sulfide is the amount of the total cadmium converted into cadmium sulfide: (1-1) (1-2)； In the (2-2), when the EDTA-2Na solution is ensured to fully chelate the cadmium ions adsorbed in the sample, the concentration of the EDTA-2Na solution is determined by the following method: According to the above quantitative detection method, the difference is that (2) is replaced by the following steps: (2a) preparation and treatment of a sample: The Tetrahymena in (1) is collected as a sample, and is broken by an ultrasonic cell disruptor, and a certain concentration of CdCl2 solution is added to the broken sample and mixed, and then an equal volume of a certain concentration of EDTA-2Na solution is added and mixed; According to the concentration of the CdCl2 solution, different concentrations of EDTA-2Na solution are set to carry out the above operation, and the percentage of the content of cadmium ions and the total content of cadmium at different concentrations, i.e. the cadmium recovery rate, is calculated, and the concentration when the cadmium recovery rate is 95% is taken as the minimum concentration for full chelation.

2. The quantitative detection method according to claim 1, characterized by, The Cd in the step (2) after adding CdCl2 solution 2+ concentration of 5 mg / L-10 mg / L.

3. The quantitative detection method according to claim 2, characterized in that, Cd in (2a) 2+ When the concentration of Cd is 5 mg / L, add 60 μM~80 μM EDTA-2Na solution. 2+ When the concentration of Cd is 10 mg / L, add 120 μM~180 μM EDTA-2Na solution.

4. The quantitative detection method according to claim 1, characterized by, The preparation method of the SPP culture medium is as follows: the solvent is ultrapure water, and the solutes and their concentrations are as follows: glucose 2 g / L, proteose peptone 20 g / L, yeast extract 1 g / L, and ferric citrate 0.03 g / L; the mixture is sterilized at 121 DEG C for 15 min and is ready for use.

5. The quantitative detection method according to claim 1, characterized in that, The Tetrahymena is Tetrahymena thermophila SB210.

6. The method according to claim 1, wherein the method is characterized by, The method (2-1) further comprises collecting the tetrahymena from the culture system as samples at fixed time points according to requirements, other steps remain unchanged, and the cadmium conversion rate of the samples at different time points is measured to realize dynamic quantitative measurement of the cadmium sulfide content.

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