Method for detecting content change of E-DOM and I-DOM released by algae under exposure of PAHs and derivatives thereof

Abstract

The invention discloses a method for detecting the content change of E-DOM and I-DOM released by algae under exposure of PAHs and derivatives thereof, and the method comprises the following steps: comparing the fluorescence characteristic difference of E-DOM and I-DOM of algae, and forming quantitative recognition by using four main fluorescence components; the method can be used for determining the content change of E-DOM and I-DOM released by algae under the exposure of PAHs including pyrene and 1-nitropyrene and derivatives thereof, further exploring the dynamic change of persistent organic pollutants on dissolved organic matters released by algae, and has important significance on eutrophication treatment and environmental protection.

Description

Technical Field

[0001] The present invention specifically relates to a method for detecting changes in the contents of E-DOM and I-DOM released by algae under exposure to PAHs and their derivatives. Background Art

[0002] Algal dissolved organic matter (A-DOM) is the key link between biogenic carbon and inorganic carbon, and has an important impact on the bioavailability and environmental behavior of pollutants (such as heavy metals and organic pollutants). It can directly participate in the dynamic cycle of organic carbon in water bodies, and some active and semi-active components can be used by microorganisms and converted into CO 2 and CH 4 A-DOM is released back into the atmosphere in the form of DOM, while the other part of the difficult-to-degrade components are stored in the lake water and sediments in the form of relatively inert organic matter. A-DOM includes extracellular DOM (E-DOM) released by living algal cells and intracellular DOM (I-DOM) leaked by damaged cells. The content of the two in the water body is an important factor reflecting the growth of algae.

[0003] Polycyclic aromatic hydrocarbons (PAHs) are small molecule organic pollutants that can have toxic effects on algae. Among them, pyrene (Pyr), a four-ring PAH with a symmetrical structure, is a common pollutant in various environmental media and has a certain effect on algal growth and the release of A-DOM. 1-Nitropyrene (1-NPyr) differs from Pyr in that it has one nitro functional group substitution, and has different effects on algal growth, photosynthesis, and metabolite release.

[0004] At present, there is no effective method to explore the effects and differences of PAHs such as Pyr and 1-NPyr and their derivatives on the properties of E-DOM and I-DOM released by microalgae such as S. costatum without interference, making it difficult to study the role of such pollutants in affecting the environmental behavior of pollutants. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for detecting changes in the content of E-DOM and I-DOM released by algae under exposure to PAHs and their derivatives, thereby solving the problems in the above-mentioned background technology.

[0006] The technical solution adopted by the present invention to solve the technical problem is: a method for detecting the changes in the E-DOM and I-DOM content released by algae under exposure to PAHs and their derivatives is provided, comprising the following steps:

[0007] (1) extracting E-DOM and I-DOM and mixing them in different proportions, and drawing a standard curve of the spectral ratio composed of four fluorescent components of E-DOM and I-DOM and the change of I-DOM content;

[0008] (2) preparing algae cell filtrate exposed to PAHs and their derivatives to be tested, and measuring its three-dimensional fluorescence spectrum; performing parallel factor analysis on the three-dimensional fluorescence spectrum and calculating the ratio of the fluorescence intensities of the four fluorescent components; and obtaining the relative content changes of E-DOM and I-DOM in the exposed sample by comparing with the standard curve of step (1).

[0009] Preferably, the four fluorescent components are tyrosine-like C1, tryptophan-like C2, humic acid-like C3 and fulvic acid-like C4, and the spectral ratio is (C1+C2) / (C3+C4).

[0010] Preferably, the PAHs and their derivatives include Pyr and 1-NPyr.

[0011] Preferably, the microalgae include Alexandrium tamarense, Chlorella vulgaris and Skeletonema costatum.

[0012] Preferably, in step (1), the method for extracting E-DOM and I-DOM comprises the following steps:

[0013] ① Select microalgae in the exponential growth stage, take 40 mL of algae solution in a conical flask, centrifuge at 4000 g for 10 min, and filter the supernatant through a glass fiber filter with a pore size of 0.45 μm to obtain the filtrate containing E-DOM;

[0014] ② Use 40 mL of sterilized Milli Q water to resuspend the algal cells after centrifugation in step ①, then freeze at -20°C for 2 h, thaw at 30°C, repeat freezing and thawing 3 times, and finally filter through a 0.45 μm glass fiber filter to obtain I-DOM filtrate.

[0015] Preferably, in step (1), the extracted E-DOM and I-DOM filtrates are diluted to the same DOC concentration, mixed in equal proportions according to a volume ratio, and the mixed solution is subjected to three-dimensional fluorescence spectrum measurement; the measurement conditions are: excitation wavelength range 200-450nm, 5nm spacing; emission wavelength range 250-550nm, 2nm spacing; excitation and emission slit widths are both 5nm, and the residence time is 0.01s.

[0016] Preferably, in step (1), the three-dimensional fluorescence spectrum data of the mixed solution is analyzed by parallel factor analysis to obtain the individual fluorescent components C1, C2, C3 and C4 in E-DOM and I-DOM, and the changes in the spectral ratios in solutions with different contents of I-DOM are recorded.

[0017] Preferably, in step (1), the I-DOM content is in the range of 0-100%.

[0018] Preferably, in step (2), the minimum detection percentage of I-DOM is 2%.

[0019] Preferably, the standard curve of step (1) is y=5.2642e^(0.0255x), where x is the I-DOM content, y is the ratio of (C1+C2) / (C3+C4), and R 2 =0.9795,p<0.05.

[0020] Compared with the background technology, this technical solution has the following advantages:

[0021] 1. The present invention extracts and compares the spectral characteristics and differences of E-DOM and I-DOM of the selected microalgae, proposes a spectral ratio parameter composed of four fluorescent components of E-DOM and I-DOM, and establishes a method for in-situ quantitative identification of E-DOM and I-DOM released by algae through the spectral ratio and I-DOM content, which can effectively avoid the mutual interference of E-DOM and I-DOM, accurately identify the content ratio of the two, and can be used as a reliable indicator for in-situ analysis of changes in the content of I-DOM and E-DOM released by algae under pollutant exposure; the established method confirms that all concentrations of 1-NPyr and Pyr above medium concentration inhibit E-DOM secretion. The minimum concentrations of Pyr and 1-NPyr that cause I-DOM leakage are 1.0×10 -8 mol L -1 and 0.5×10 -8 mol L -1 , 1-NPyr has a more significant effect on the properties of E-DOM and I-DOM released by algal cells;

[0022] 2. The inspection method of the present invention is simple to operate and can conveniently and quickly monitor the dynamic changes of the corresponding content in the sample;

[0023] 3. The present invention can further explore the effects of PAHs and their derivatives on the release of E-DOM and I-DOM by algae, and has great significance for the in situ detection of the effects of persistent organic pollutants on the properties of dissolved organic matter released by microalgae, the environmental behavior and related mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a standard curve diagram in the examples, wherein the abscissa "% I-DOM (DOC)" represents the DOC concentration ratio of I-DOM. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0026] Example

[0027] In this embodiment, the detection method for the change of E-DOM and I-DOM content released by algae under the exposure of PAHs and their derivatives is selected from Skeletonema costatum, and the specific steps are as follows:

[0028] (1) Drawing of E-DOM and I-DOM standard curves

[0029] ① Select Skeletonella costatum that has been cultured for 6 days, take 40 mL of the algae solution in the conical flask, centrifuge at 4000 g for 10 min, and filter the supernatant through a glass fiber filter with a pore size of 0.45 μm. The obtained solution contains E-DOM.

[0030] ② For the extraction of I-DOM, the algal cells after centrifugation were resuspended in 40 mL of sterilized Milli Q water, then frozen at -20°C for 2 h, thawed at 30°C, and repeated freezing and thawing for 3 times. Finally, the I-DOM solution was obtained by filtering through a 0.45 μm glass fiber filter.

[0031] ③ After all E-DOM and I-DOM samples were diluted to the same dissolved organic carbon concentration, they were mixed in equal proportions according to the volume ratio and their fluorescence spectra were measured to compare the characteristic fluorescence spectra of E-DOM and I-DOM.

[0032] The three-dimensional fluorescence spectrum was measured by fluorescence spectrometer. The measurement conditions were as follows: excitation wavelength range 200-450nm, 5nm spacing; emission wavelength range 250-550nm, 2nm spacing; excitation and emission slit widths were both 5nm, and the dwell time was 0.01s.

[0033] Parallel factor analysis was used to analyze the three-dimensional fluorescence spectrum data of the samples to obtain the individual fluorescent components (C1, C2, C3 and C4) in E-DOM and I-DOM, and the changes in the spectral ratio parameters in solutions with different contents of I-DOM were recorded. A standard curve of the obtained spectral ratio parameters and the change in the content of I-DOM was drawn.

[0034] (2) In situ quantitative determination of the relative changes in the content of E-DOM and I-DOM released by Skeletonella costatum under exposure to Pyr and 1-NPyr

[0035] The exposure concentrations of Pyr and 1-NPyr were prepared to be 0 and 0.25×10 -8 mol L -1 , 0.5×10 -8 mol L -1, 1.0×10 -8 mol L -1 , 2.0×10 -8 mol L -1 The Skeletonema costatum filtrate is subjected to three-dimensional fluorescence spectrum measurement by using a fluorescence spectrometer.

[0036] The three-dimensional fluorescence spectrum of A-DOM was split into four main fluorescence components by parallel factor analysis. The fluorescence ratio method was established to calculate the changes in the contents of E-DOM and I-DOM released by Skeletonema costatum (Tables 1 and 2).

[0037] Table 1 Changes in fluorescence intensity of E-DOM and I-DOM in groups exposed to different concentrations of Pyr

[0038]

[0039] Table 2 Changes in fluorescence intensity of E-DOM and I-DOM in groups exposed to different concentrations of 1-NPyr

[0040]

[0041] From the above table, we can see that in the four main growth stages of algae, low concentration (0.25×10 -8 mol L -1 ) had similar effects on the E-DOM and I-DOM content of S. costatum. As the exposure concentration of Pyr and 1-NPyr continued to increase, the release of E-DOM by algae cells was inhibited on the second day, and the maximum inhibition rates were 34.8% and 60.5% on the sixth day, respectively. -8 mol L -1 Under these experimental conditions, the E-DOM content of Pyr and 1-NPyr in the stable period (10th day) and the decay period (20th day) was significantly lower than that in the control group (p < 0.05), and it was obviously concentration-dependent. -8 mol L -1 The 1-NPyr is 0.5-2.0×10 -8 mol L -1 It can be seen that the method of the present invention can effectively avoid the mutual interference between the two and accurately measure the changes in the content of E-DOM and I-DOM released by microalgae under the exposure of pollutants.

[0042] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting changes in the content of E-DOM and I-DOM released by algae under exposure to PAHs and their derivatives, characterized in that: The steps include: (1) extracting E-DOM and I-DOM and mixing them in different proportions, and drawing a standard curve of the spectral ratio composed of four fluorescent components of E-DOM and I-DOM and the change of I-DOM content; (2) preparing algae cell filtrate from microalgae exposed to PAHs and their derivatives to be tested, and measuring its three-dimensional fluorescence spectrum; Performing parallel factor analysis on the three-dimensional fluorescence spectrum and calculating the ratio of the fluorescence intensities of the four fluorescent components; By comparing with the standard curve in step (1), the relative content changes of E-DOM and I-DOM in the exposed samples were obtained.

2. The method for detecting changes in the content of E-DOM and I-DOM released by algae under exposure to PAHs and their derivatives according to claim 1, characterized in that: The four fluorescent components are tyrosine-like C1, tryptophan-like C2, humic acid-like C3 and fulvic acid-like C4, and the spectral ratio is (C1+C2) / (C3+C4).

3. The method for detecting changes in the content of E-DOM and I-DOM released by algae under exposure to PAHs and their derivatives according to claim 1, characterized in that: The PAHs and their derivatives include Pyr and 1-NPyr.

4. The method for detecting changes in the content of E-DOM and I-DOM released by algae under exposure to PAHs and their derivatives according to claim 1, characterized in that: The microalgae include Alexandrium tamarense, Chlorella vulgaris and Skeletonema costatum.

5. The method for detecting changes in the content of E-DOM and I-DOM released by algae under exposure to PAHs and their derivatives according to claim 1, characterized in that: In step (1), the method for extracting E-DOM and I-DOM comprises the following steps: ① Select microalgae in the exponential growth stage, take 40 mL of algae solution in a conical flask, centrifuge at 4000 g for 10 min, and filter the supernatant through a glass fiber filter with a pore size of 0.45 μm to obtain the filtrate containing E-DOM; ② Use 40 mL of sterilized Milli Q water to resuspend the algal cells after centrifugation in step ①, then freeze at -20°C for 2 h, thaw at 30°C, repeat freezing and thawing 3 times, and finally filter through a 0.45 μm glass fiber filter to obtain I-DOM filtrate.

6. The method for detecting changes in the contents of E-DOM and I-DOM released by algae under exposure to PAHs and their derivatives according to claim 5, characterized in that: In step (1), the extracted E-DOM and I-DOM filtrates are diluted to the same DOC concentration, mixed in equal proportions according to a volume ratio, and the mixed solution is subjected to three-dimensional fluorescence spectrum measurement; the measurement conditions are: excitation wavelength range 200-450nm, 5nm spacing; emission wavelength range 250-550nm, 2nm spacing; excitation and emission slit widths are both 5nm, and the residence time is 0.01s.

7. The method for detecting changes in the content of E-DOM and I-DOM released by algae under exposure to PAHs and their derivatives according to claim 6, characterized in that: In step (1), the three-dimensional fluorescence spectrum data of the mixed solution is analyzed by parallel factor analysis to obtain the individual fluorescent components C1, C2, C3 and C4 in E-DOM and I-DOM, and the changes in the spectral ratios in solutions with different contents of I-DOM are recorded.

8. The method for detecting changes in the content of E-DOM and I-DOM released by algae under exposure to PAHs and their derivatives according to claim 1, characterized in that: In step (1), the I-DOM content ranges from 0 to 100%.

9. The method for detecting changes in the content of E-DOM and I-DOM released by algae under exposure to PAHs and their derivatives according to claim 1, characterized in that: In step (2), the minimum detection percentage of I-DOM is 2%.

10. The method for detecting changes in the content of E-DOM and I-DOM released by algae under exposure to PAHs and their derivatives according to claim 1, characterized in that: The standard curve of step (1) is y=5.2642e^(0.0255x), where x is the I-DOM content, y is the ratio of (C1+C2) / (C3+C4), and R 2 =0.9795,p<0.05.

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