A method and device for measuring primary productivity of phytoplankton

Abstract

The present invention provides a method and device for measuring phytoplankton primary productivity, comprising the following steps: obtaining spectra of calibration samples at different temperatures; establishing a calibration plane based on the spectra, using water environment temperature and spectral baseline intensity as independent variables and fluorescence peak intensity as a dependent variable; obtaining the corrected fluorescence peak intensity of the calibration sample based on the calibration plane; establishing a calibration curve based on the corrected fluorescence peak intensity of the calibration sample and primary productivity; obtaining a fluorescence spectrum of a sample to be measured; obtaining the corrected fluorescence peak intensity of the sample to be measured based on the calibration plane; and obtaining the phytoplankton primary productivity of the sample to be measured from the corrected fluorescence peak intensity of the sample to be measured based on the calibration curve. The present invention can improve the accuracy of chlorophyll fluorescence measurements and further improve the measurement accuracy of phytoplankton primary productivity observed using laser-induced chlorophyll fluorescence.

Description

Technical Field

[0001] The present invention relates to the technical field of phytoplankton detection, in particular to a method and device for measuring the primary productivity of phytoplankton. Background Art

[0002] The ocean is the world's largest and most important ecosystem. In ecology, producers are autotrophic organisms that can convert simple inorganic substances into organic matter, while primary productivity is the total amount of organic matter produced per unit area and per unit time by plant communities in an ecosystem. Phytoplankton are the primary producers in aquatic environments. While their biomass accounts for only 1%-2% of global plant life, they contribute approximately 50% of global primary productivity, comparable to the carbon sequestration of terrestrial plants. Marine phytoplankton convert atmospheric carbon dioxide into organic compounds through photosynthesis, directly or indirectly providing food for nearly all other marine life. They not only provide the majority of atmospheric oxygen, but their fossil remains are also converted into petroleum through geological processes. Phytoplankton are a significant "carbon reservoir" in the ocean, playing a crucial role in the circulation of carbon dioxide between the atmosphere and the biosphere, and playing a crucial role in regulating the carbon cycle in both the atmosphere and the ocean. Because of the importance of marine phytoplankton, the study of its primary productivity has become one of the focuses of research on the air-sea interface exchange process. However, the study of the air-sea interface exchange process relies on high-precision and high-resolution field measurement data, and it is necessary to solve the problem of accurate in-situ online measurement of phytoplankton primary productivity. Therefore, it is necessary to develop instruments to measure and evaluate the primary productivity of phytoplankton in the ocean surface water.

[0003] Currently, the main methods for measuring phytoplankton primary productivity include plant growth analysis, traditional gas exchange, remote sensing, chlorophyll fluorescence, and fluorescence kinetics. The plant growth method is time-consuming and cannot be repeated. Traditional gas exchange methods, including the black and white bottle method, isotope tracer method, and liquid oxygen electrode method, require complex pre-processing, expensive analytical instruments, complex and time-consuming operation, and are prone to secondary pollution. Remote sensing methods for studying primary productivity over large areas cannot accurately measure the productivity required by current high-resolution air-sea flux exchange research. Fluorescence kinetics provides accurate measurements but relies on precise primary productivity measurement models, many model parameters of which are still under investigation and computationally complex. The chlorophyll fluorescence method has a simple principle model, fast measurement speed, and can be used for real-time measurement. Although the assimilation coefficient is not constant with environmental changes, under the offshore measurement conditions used in studies of air-sea interface exchange, the influence of nutrients near the coast is small, the offshore water quality is relatively stable, and the assimilation coefficient is relatively stable. Therefore, laser-induced fluorescence can be used to measure chlorophyll concentration and thus derive the primary productivity of phytoplankton in surface waters.

[0004] Measuring phytoplankton primary productivity in surface waters faces a challenge: accuracy degradation due to temperature fluctuations. During the measurement process, internal circuits, lasers, detectors, and batteries generate heat. Underwater instruments are enclosed, so heat easily accumulates inside the instrument, affecting optoelectronic devices and causing signal drift due to temperature fluctuations. Therefore, overcoming the loss of accuracy caused by temperature fluctuations is a pressing challenge.

[0005] A search revealed a Chinese invention patent application with publication number CN109490270A, which discloses a device and method for measuring phytoplankton primary productivity based on chlorophyll fluorescence. The patent proposes using simulated ambient lighting to measure fluorescence kinetic parameters under light-acclimated conditions. The patent employs an underwater photosynthetically active radiation measurement unit and a simulated ambient light source to generate controllable lighting conditions similar to those in natural underwater environments. Fluorescence kinetic curves are then measured during the intervals between simulated illumination exposures. This patent utilizes fluorescence kinetic parameter measurement technology and fails to consider the impact of ambient temperature on fluorescence measurement. Summary of the Invention

[0006] In view of the defects in the prior art, the purpose of the present invention is to provide a method and device for measuring the primary productivity of phytoplankton.

[0007] According to one aspect of the present invention, a method for measuring phytoplankton primary productivity is provided, the method comprising:

[0008] Obtain spectra of calibration samples at different temperatures;

[0009] Based on the spectrum, a calibration plane is established with the water environment temperature and the spectrum baseline intensity as independent variables and the fluorescence peak intensity as the dependent variable;

[0010] Based on the calibration plane, obtaining the corrected fluorescence peak intensity of the calibration sample;

[0011] A calibration curve was established based on the corrected fluorescence peak intensity of the calibration sample and the primary productivity;

[0012] Obtaining the fluorescence spectrum of the sample to be tested;

[0013] Obtaining a corrected fluorescence peak intensity of the sample to be tested based on the calibration plane and the fluorescence spectrum of the sample to be tested;

[0014] According to the calibration curve, the phytoplankton primary productivity of the sample to be tested is inverted from the corrected fluorescence peak intensity of the sample to be tested.

[0015] Optionally, the calibration sample is a chlorophyll a alcohol extract that is uniform and stable and has a set concentration.

[0016] Optionally, establishing a calibration plane based on the spectrum, with the water environment temperature and the spectrum baseline intensity as independent variables and the fluorescence peak intensity as the dependent variable, includes:

[0017] For a calibration sample with a certain concentration of C1, the spectral signal intensity at the set wavelength is selected as the spectral baseline intensity. The water environment temperature and the spectral baseline intensity are used as independent variables, and the fluorescence peak intensity is used as the dependent variable to establish a correction plane. The correction plane formula is: f=k1T+k2B+m, T is the water environment temperature, B is the spectral baseline intensity, f is the fluorescence peak intensity, m is a constant, k1 and k2 are the coefficients of the two-variable linear equation obtained based on the calibration sample.

[0018] Optionally, obtaining the corrected fluorescence peak intensity of the calibration sample based on the correction plane includes:

[0019] The parameters of any point of the calibration sample with a concentration of C1 on the calibration plane are expressed as (T0, B0, f0), then a certain temperature T is selected. s and a spectral baseline B s As a benchmark, the parameters (T0, B0, f0) are corrected to (T s , B s , f c1 ), then the fluorescence peak intensity of the C1 calibration sample with known concentration at any temperature and baseline is f c1 =k1(T s -T0)+k2(B s -B0)+f0; Therefore, a series of calibration samples are obtained that have been calibrated to temperature T s and spectral baseline B s Corrected fluorescence peak intensity f c1 , f c2 …f cn , where C1, C2…C n Chlorophyll a concentration in a series of solutions used as calibration samples.

[0020] Optionally, the principle for selecting the set wavelength is to ensure that during normal measurement, the wavelength positions of chlorophyll a fluorescence and the fluorescence or scattered light of other substances in the complex water body to be measured do not overlap with the selected wavelength point.

[0021] Optionally, establishing a calibration curve based on the corrected fluorescence peak intensity of the calibration sample and the primary productivity includes:

[0022] The corrected fluorescence peak intensity f c and chlorophyll a concentration C a The linear relationship between c =k3C a, where k3 is the slope of the linear fitting line between fluorescence intensity and concentration; according to the primary productivity of phytoplankton in surface water (within 1m), P S The relationship between the concentration of chlorophyll a and the S =QC a , C a is the chlorophyll a content in the surface water, Q is the assimilation coefficient, and the assimilation coefficient of the surface water is generally taken as 3.7; therefore, the relationship between the primary productivity of the calibration sample and the corrected fluorescence peak intensity is P S =Qf c / k3 is the calibration formula.

[0023] Optionally, obtaining the fluorescence spectrum of the sample to be tested includes: obtaining the fluorescence peak intensity f from the fluorescence spectrum of the sample to be tested 0_test .

[0024] Optionally, the method of obtaining the corrected fluorescence peak intensity of the sample to be tested based on the correction plane includes: the corrected fluorescence peak intensity is f c_test =k1(T s -T0)+k2(B s -B0)+f 0_test .

[0025] Optionally, the phytoplankton primary productivity of the sample to be tested is inverted from the corrected fluorescence peak intensity of the sample to be tested according to the calibration curve, including S =Qf c_test / k3, and obtain the primary productivity of the sample to be tested.

[0026] According to another aspect of the present invention, a device for measuring phytoplankton primary productivity is provided, for implementing the above-mentioned method for measuring phytoplankton primary productivity, the device comprising:

[0027] An instrument housing, wherein a light window and a flow-through light-shielding sample chamber are sequentially provided at one end of the instrument housing, the light window being located at the front end of the device and embedded in the instrument housing, and the flow-through light-shielding sample chamber being sleeved on the outside of the light window;

[0028] A light source module, a probe, a spectrometer and a circuit module are provided inside the instrument housing, wherein the light source module and the spectrometer are connected to the probe respectively, and the circuit module is connected to the spectrometer;

[0029] The light source module emits incident light which passes through the probe and the light window in turn and enters the sample to be tested in the flow-through light-shielding sample chamber, and irradiates the phytoplankton. The detection light including the backward fluorescence generated by the sample to be tested and part of the incident light is transmitted through the light window and the probe in turn and enters the spectrometer to obtain a fluorescence spectrum. The circuit module analyzes the fluorescence spectrum to obtain the primary productivity of phytoplankton.

[0030] Optionally, the flow-through light-shielding sample chamber is a cup-shaped structure, the cup mouth of the cup-shaped structure is buckled at the light window, and a plurality of evenly distributed flow openings are provided on the cup body and the cup bottom of the cup-shaped structure.

[0031] Optionally, the circuit module includes:

[0032] The signal modulation unit is used to trigger and synchronize the light source module and the spectrometer, so that the spectrometer starts exposure at a preset time after the light source module is turned on and stabilized;

[0033] A communication unit, used to implement communication functions;

[0034] A core control unit is connected to the signal modulation unit and the communication unit respectively;

[0035] A power supply unit is used to supply power to the circuit module.

[0036] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0037] 1. The method and device for measuring the primary productivity of phytoplankton provided by the present invention can correct the fluorescence peak intensity deviation caused by fluctuations in internal and external environmental temperatures, thereby improving the accuracy of chlorophyll fluorescence measurements. This provides a new solution to the problem of measurement signal drift and inaccuracy caused by the closed design of underwater sensors, heating of photoelectric devices, and changes in external environmental temperature, thereby further improving the measurement accuracy of phytoplankton primary productivity observation using laser-induced chlorophyll fluorescence.

[0038] 2. The phytoplankton primary productivity measurement method and device provided by the present invention use a spectrometer as the core and can perform temperature correction based on the spectral baseline and the external ambient temperature of the instrument, thereby accurately measuring the primary productivity indicator of the amount of carbon dioxide consumed by phytoplankton photosynthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0040] Figure 1 Schematic diagram of the process of measuring the primary productivity of phytoplankton in one embodiment of the present invention;

[0041] Figure 2 This is a schematic diagram of the structure of a phytoplankton primary productivity measuring instrument according to one embodiment of the present invention;

[0042] Figure 3 Schematic diagram of the structure of a flow-through light-shielded sample chamber of a phytoplankton primary productivity measuring instrument in one embodiment of the present invention;

[0043] Figure 4 Schematic diagram of a flow chart of a temperature correction method for a phytoplankton primary productivity measuring instrument based on a spectral baseline and the ambient temperature outside the instrument in one embodiment of the present invention;

[0044] Figure 5 is the original fluorescence spectrum collected in one embodiment of the present invention;

[0045] Figure 6 is a graph showing changes in the baseline over time in the original fluorescence spectrum in one embodiment of the present invention;

[0046] Figure 7 is a graph showing changes in the fluorescence peak over time in the original fluorescence spectrum in one embodiment of the present invention;

[0047] Figure 8 A three-dimensional schematic diagram of a correction plane established in one embodiment of the present invention;

[0048] Figure 9 A comparison chart of the correction results of the temperature correction method, the single baseline correction method, and the single temperature correction method in one embodiment of the present invention;

[0049] The reference numerals in the figure are respectively represented as: 1 is the power supply interface, 2 is the communication interface, 3 is the connecting line, 4 is the optical fiber, 5 is the light window, 6 is the flow port, 7 is the phytoplankton, 8 is the flow-through light-shielding sample chamber, 9 is the incident light, 10 is the detection light, 11 is the instrument housing, 12 is the cup mouth, and 13 is the cup bottom. DETAILED DESCRIPTION

[0050] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0051] In view of the problem that the sensor is affected by temperature and causes signal drift during the measurement of phytoplankton primary productivity, which leads to an urgent need to improve the fluorescence measurement accuracy, an embodiment of the present invention provides a method for measuring phytoplankton primary productivity, referring to Figure 1 , the method comprising:

[0052] S1. Obtain spectra of calibration samples at different temperatures;

[0053] S2. Based on the spectrum, a calibration plane is established with the water environment temperature and the spectrum baseline intensity as independent variables and the fluorescence peak intensity as the dependent variable;

[0054] S3. Based on the calibration plane, obtain the corrected fluorescence peak intensity of the calibration sample;

[0055] S4. establishing a calibration curve based on the corrected fluorescence peak intensity of the calibration sample and the primary productivity;

[0056] S5, obtaining the fluorescence spectrum of the sample to be tested;

[0057] S6. Obtaining a corrected fluorescence peak intensity of the sample to be tested based on the calibration plane and the fluorescence spectrum of the sample to be tested;

[0058] S7. According to the calibration curve, the phytoplankton primary productivity of the sample to be tested is inverted from the corrected fluorescence peak intensity of the sample to be tested.

[0059] In the measurement method of this embodiment of the present invention, steps S1 to S4 are the modeling process of the temperature-corrected primary productivity prediction method, and steps S5 to S7 are the prediction process of the temperature-corrected primary productivity prediction method. The calibration sample used is a homogeneous and stable chlorophyll a alcohol extract with a set concentration. Different concentrations of the calibration sample correspond to different primary productivity levels, and the concentration of the calibration sample must cover the instrument's measurement range.

[0060] In some embodiments, step S2 includes: for a calibration sample with a certain concentration of C1, selecting the spectral signal intensity at a set wavelength as the spectral baseline intensity, taking the water environment temperature and the spectral baseline intensity as independent variables, and taking the fluorescence peak intensity as the dependent variable, establishing a correction plane, and the correction plane formula is: f=k1T+k2B+m, T is the water environment temperature, B is the spectral baseline intensity, f is the fluorescence peak intensity, m is a constant, and k1 and k2 are coefficients of a two-variable linear equation obtained based on the calibration sample.

[0061] In some embodiments, step S3 includes the parameters of any point on the calibration plane of the calibration sample with a concentration of C1 being represented by (T0, B0, f0), and a certain temperature T is selected. s and a spectral baseline B s As a benchmark, the parameters (T0, B0, f0) are corrected to (T s , B s , f c1 ), then the fluorescence peak intensity of the known concentration calibration sample at any temperature and baseline is f c1 =k1(T s -T0)+k2(B s -B0)+f0. Therefore, a series of different known chlorophyll a concentrations (C1, C2…C n ) solution is the calibration sample, and a series of calibration samples can be obtained. s and spectral baseline B s Corrected fluorescence peak intensity (f c1 , f c2 …fcn ), so that the effect of temperature change on the fluorescence peak change can be corrected.

[0062] In the above embodiment, the wavelength selection principle is to ensure that during normal measurement, the wavelength positions of chlorophyll a fluorescence and the fluorescence or scattered light of other substances in the complex water body to be measured do not overlap with the selected wavelength point.

[0063] In some embodiments, step S4 includes:

[0064] The corrected fluorescence peak intensity f c and chlorophyll a concentration C a The linear relationship between c =k3C a , where k3 is the slope of the linear fitting line between fluorescence intensity and concentration; according to the primary productivity of phytoplankton in surface water (within 1m), P S The relationship between the concentration of chlorophyll a and the S =QC a , C a is the chlorophyll a content in the surface water, Q is the assimilation coefficient, and the assimilation coefficient of the surface water is generally taken as 3.7; therefore, the relationship between the primary productivity of the calibration sample and the corrected fluorescence peak intensity can be obtained as P S =Qf c / k3 is the calibration formula, and the calibration curve can be obtained according to this formula.

[0065] In S5, the fluorescence peak intensity f is obtained from the fluorescence spectrum of the sample to be tested. 0_test , then the corrected fluorescence peak intensity in S6 is f c_test =k1(T s -T0)+k2(B s -B0)+f 0_test , in S7 according to the calibration formula P S =Qf c_test / k3, and obtain the primary productivity of the sample to be tested.

[0066] Based on the same inventive concept, another embodiment of the present invention provides a phytoplankton primary productivity measurement device for implementing the above-mentioned phytoplankton primary productivity measurement method, referring to Figure 2The device includes an instrument housing 11, one end of which is provided with a light window 5 and a flow-through light-shielding sample chamber 8 in sequence, the light window 5 is located at the front end of the device and embedded in the instrument housing 11, and the flow-through light-shielding sample chamber 8 is sleeved on the outside of the light window 5; a light source module, a probe, a spectrometer and a circuit module are provided inside the instrument housing 11, the light source module and the spectrometer are respectively connected to the probe, and the circuit module is connected to the spectrometer; the light source module emits incident light 9 which passes through the probe and the light window 5 in sequence and enters the sample to be tested in the flow-through light-shielding sample chamber 8, and irradiates the phytoplankton 7, and the detection light 10 containing the backward fluorescence generated by the sample to be tested and part of the incident light is transmitted through the light window 5 and the probe in sequence into the spectrometer to obtain a fluorescence spectrum, and the circuit module analyzes the fluorescence spectrum to obtain the primary productivity of phytoplankton.

[0067] In some embodiments, the flow-through light-shielding sample chamber 8 is a cup-shaped structure, the cup mouth 12 of the cup-shaped structure is buckled at the light window 5, and a plurality of evenly distributed flow ports 6 are opened on the cup body and the cup bottom 13 of the cup-shaped structure. Specifically, the cup body has 6 evenly distributed flow ports 6 near the light window 5, and 4 evenly distributed flow ports 6 are opened at the cup bottom 13. Thus, it ensures both smooth water flow and relative closedness during the measurement process, effectively avoids direct sunlight, blocks and effectively weakens the external environment stray light and interference light entering the instrument, and realizes in-situ online measurement.

[0068] In some embodiments, the circuit module includes a signal modulation unit, a communication unit, a core control unit, and a power supply and self-contained storage unit. The signal modulation unit is used to trigger and synchronize the light source module and the spectrometer, causing the spectrometer to begin exposure at a preset time after the light source module is turned on and stabilized. The communication unit is used to implement communication functions. The core control unit is connected to the signal modulation unit and the communication unit, respectively. The power supply unit supplies power to the circuit module. Specifically, the signal modulation unit ensures that the light source module and the spectrometer operate synchronously. The light source module emits laser light, the spectrometer receives the fluorescence spectrum and transmits the fluorescence spectrum data to the signal modulation unit. The signal modulation unit transmits the spectrum data to the core control unit via the communication unit. The core control unit analyzes the spectrum data to obtain a primary productivity value. The device also includes a self-contained storage module. The core control unit stores the primary productivity value and the fluorescence spectrum in the self-contained storage unit.

[0069] In the above embodiment, a power module is also provided in the instrument housing 11, and the power module supplies power to the measuring device; a power supply interface 1 and a communication interface 2 are provided on the instrument housing 11, and the water environment temperature is measured by an external temperature sensor and overall analyzed through the communication interface 2 and the circuit module.

[0070] In a preferred embodiment, continue to refer to Figure 2 and Figure 3, the light source module is a laser, more preferably, the light source module is a 405nm laser, rated voltage 12V, power consumption 150mW. The probe is a laser probe, and the laser probe contains a variety of functional filters such as a reflective filter, a bandpass filter, a cut-off filter, and a converging lens. The spectrometer adopts a visible CCD spectrometer, and the spectrometer is a visible band micro spectrometer with a spectral range of 350-750nm and a resolution of 1nm. Of course, in some other embodiments, spectrometers of other specifications containing phytoplankton chlorophyll characteristic fluorescence spectrum response bands can also be used, including but not limited to visible spectrometers, ultraviolet-visible spectrometers, visible near-infrared spectrometers, ultraviolet-visible near-infrared spectrometers, etc. The laser and the spectrometer are respectively connected to the laser probe via optical fibers 4. The circuit module is electrically connected to the spectrometer via a connecting line 3; the circuit module includes a core control module (STM32), a signal modulation module (FPGA), a communication module (RS485), a power supply and self-capacitive storage module, etc. An FPGA-based digital pulse generation circuit triggers and synchronizes the light source and CCD. After the light source is turned on for a period of time (e.g., 100 milliseconds) and the light intensity stabilizes, the spectrometer's CCD begins exposure. The exposure time error is controlled to sub-microseconds, thus ensuring the stability of the measurement results. During the measurement process, after setting the integration time for a single measurement, the laser will turn on early and turn off later to ensure a stable detection signal within the integration time. An automatic TTL-to-RS485 communication module is used to communicate with the host computer, ensuring the high reliability of the communication module in long-distance transmission scenarios. A master control module based on an STM32 microcontroller provides overall control of all modules. The power module uses a built-in battery, which is connected to the laser via a cable. More preferably, the built-in battery is a rechargeable lithium battery pack with a battery capacity of 6400mAh, which can ensure the self-contained instrument can operate stably in situ online for a period of time (3 weeks). Light window 5 is a circular acrylic light window. The instrument housing 11 is a cylindrical stainless steel housing. Other materials and thicknesses of housings may also be selected according to requirements and the water depth measured by the instrument, including but not limited to stainless steel, aluminum alloy, plastic, etc. The flow-through light-shielding sample chamber 8 is a cup-shaped sample chamber. The cup mouth 12 in the cup-like structure of the flow-through light-shielding sample chamber 8 can be buckled on the light window 5 and fixed. The detailed scheme and screw hole positions for fixing the flow-through light-shielding sample chamber 8 to the instrument housing 11 are not unique, and the fixing mode and screw hole positions can be adjusted according to requirements. The cup body has 6 evenly distributed flow ports 6 near the light window 5, and 4 evenly distributed flow ports 6 at the cup bottom 13. The flow ports 6 of the flow-through light-shielding sample chamber 8 are used to ensure smooth water flow, and the instrument can realize in-situ online measurement. The design of the cup bottom 13 in the cup-like structure of the flow-through light-shielding sample chamber 8 makes it possible to optimize the cup body length of the flow-through light-shielding sample chamber 8. Selecting a suitable cup body length can significantly improve the fluorescence acquisition signal-to-noise ratio to a certain extent.The design of the flow-through light-shielded sample chamber 8 ensures relative enclosure during measurement, effectively preventing direct sunlight and effectively reducing the amount of ambient stray light and interference light entering the instrument. The power supply port 1 and communication port 2 are two watertight connectors used for communication and power supply, respectively.

[0071] The above embodiment of the present invention adopts chlorophyll fluorescence measurement technology with a spectrometer as the core, and can perform temperature correction based on the spectral baseline and the external ambient temperature of the instrument, thereby improving the measurement accuracy of phytoplankton primary productivity observation using laser-induced chlorophyll fluorescence.

[0072] Regarding the validation of the above-mentioned phytoplankton primary productivity measuring instrument based on spectral baseline and temperature correction, Figure 4 A flow chart of the temperature correction method is given, and the specific process is as follows:

[0073] (1) Prepare solution and obtain standard primary productivity value

[0074] First, a uniform, stable, and constant concentration of chlorophyll alcohol extract for calibration is prepared. Wash the fresh spinach leaves, chop them into small pieces, place them in a mortar, and add a small amount of calcium carbonate, a small amount of quartz sand, and 95% ethanol. After sufficient grinding, first filter the mixture with 4 layers of medical gauze to remove residual leaf tissue fibers and quartz sand. Then place the filtered solution in a dark environment at 4°C for static precipitation for 1 hour. Only the upper clear liquid after precipitation is taken and passed through a medium-speed filter paper with a pore size of 15μm to finally obtain the chlorophyll alcohol extract. The prepared chlorophyll alcohol extract is placed in a UV-visible spectrophotometer (such as TU-1901, Beijing Puxi General Instrument Co., Ltd.) to measure its absorbance spectrum. According to the concentration measurement relationship of chlorophyll a in 95% ethanol proposed by Lichtenthaler et al. using a spectrophotometer: C a =13.95A 665 -6.88A 649 , where C a is the concentration of chlorophyll a in the solution being tested, A 665 is the absorbance of the solution being tested at 665 nm, A 649 The concentration of chlorophyll a in the prepared chlorophyll alcohol extract can be calculated based on the absorbance of the solution at 649 nm.

[0075] A chlorophyll alcohol extract of known concentration was diluted with 95% ethanol to prepare a 3.65ug / L chlorophyll solution.

[0076] The chlorophyll a concentration was measured by spectrophotometer. According to the experimental requirements, the solution with known chlorophyll a concentration was diluted and used for the experiment. S ) and the relationship between chlorophyll a concentration PS =QC a Calculate the standard primary productivity value of the sample tested.

[0077] (2) Temperature variation experiment

[0078] The phytoplankton primary productivity meter was placed from room temperature (23°C) into 41°C hot water. Fluorescence measurements were initiated, and spectra were acquired every 50 seconds, with the spectra and temperature recorded simultaneously. The water temperature was measured by an external temperature sensor and analyzed using the communication interface and circuit module.

[0079] As the water temperature naturally cooled from 41°C to room temperature (23°C), the phytoplankton primary productivity meter continuously collected spectra, simultaneously recording the changes in water temperature over time.

[0080] After the water environment cooled to room temperature (23°C), hot water was injected into the water, rapidly raising the temperature to 41°C over a relatively short period of time. Spectra were continuously collected. During the water injection and temperature increase process, the changes in water temperature over time were also recorded.

[0081] (3) Temperature correction and result analysis

[0082] The spectral signal intensity at a certain wavelength (such as 370nm) is selected as the baseline intensity. The principle for selecting this wavelength point is to ensure that during normal measurement, the wavelength position of chlorophyll a fluorescence and the fluorescence or scattered light of other substances in complex water bodies does not overlap with this wavelength point.

[0083] The water environment temperature T and the spectral baseline B are used as independent variables, and the fluorescence peak intensity f is used as the dependent variable to establish a correction plane. The correction plane formula is: f = k1T + k2B + m

[0084] Select T s =25℃, B s =3400 as the reference, and use the calibration plane formula to calibrate the fluorescence peak intensity of any sample. Specifically, the information of a sample can be expressed as (T0, B0, f0). First, calculate m0 = -k1T0 - k2B0 + f0 using the calibration plane formula, and then calculate the peak intensity of the fluorescence of the selected sample according to the selected reference temperature T s and the reference spectrum baseline B s , calculate the corrected fluorescence peak intensity f c =k1T s +k2B s +m0=k1(T s -T0)+k2(B s -B0)+f0. Calculate and compare the original fluorescence peak intensity f and the corrected fluorescence peak intensity f cThe fluctuation range and relative standard deviation are used to evaluate the calibration effect. Figure 5-7 shown.

[0085] like Figure 5 As shown in the figure, it is the original fluorescence spectrum collected in the temperature correction experiment, the horizontal axis is the wavelength, and the vertical axis is the fluorescence intensity. Figure 5 It can be seen from the figure that during the continuous heating and cooling process, the chlorophyll a fluorescence spectrum will fluctuate in a large intensity range, causing great interference to the measurement accuracy.

[0086] like Figure 6 The following is a graph showing the baseline variation over time in the original fluorescence spectrum during the temperature correction experiment. The horizontal axis is time and the vertical axis is fluorescence intensity. Figure 6 It can be seen that during the first stage of heating, the baseline intensity gradually decreases with increasing temperature; during the second stage of cooling, the baseline intensity gradually increases with decreasing temperature; during the third stage of heating, the baseline intensity once again gradually decreases with increasing temperature.

[0087] like Figure 7 As shown in Figure 2, the curve of the fluorescence peak in the original fluorescence spectrum changes with time in the temperature correction experiment. Figure 7 It can be seen that although the three heating-cooling-heating stages can be distinguished, the fluorescence peak intensity decreases as the temperature rises and increases as the temperature drops. However, the increase or decrease of the fluorescence peak intensity is not stable. Figure 5 , it can be seen that the fluorescence peak intensity cannot be simply corrected by the baseline, especially when the external ambient temperature changes over a large range and drastically.

[0088] According to the primary productivity prediction formula P S =Qf c / k3, when Q and k3 are both determined, improve the accuracy of fluorescence peak intensity and correct from f to f c , can increase primary productivity P S The measurement accuracy is improved. The effect of the primary productivity prediction method with temperature correction in the above embodiment of the present invention is now evaluated from the perspective of the correction effect of the fluorescence peak. The three-dimensional schematic diagram of the correction plane obtained by the temperature correction method in the above embodiment is as follows Figure 8 As shown in , the fluorescence intensity experimental results obtained by applying this correction plane correction are compared with the fluorescence intensity correction results obtained by the single baseline correction and single temperature correction methods, as shown in Figure 9 As shown. Figure 9As can be seen, the uncorrected fluorescence peak fluctuation range caused by ambient temperature changes and CCD baseline drift is 53%, the fluorescence peak fluctuation range after single baseline correction is 26%, and the fluorescence peak intensity fluctuation range after single temperature correction is 15%. The fluctuation range of the comprehensive temperature correction method based on spectral baseline and temperature in the above embodiment is 8.8%, and the relative standard deviation is less than 2%, which is much better than the uncorrected and other single correction methods. The above experiment demonstrates that the temperature correction method of the above embodiment of the present invention can effectively correct for temperature-induced fluorescence signal drift, thereby improving the fluorescence measurement accuracy of the phytoplankton primary productivity meter and further improving the measurement accuracy of phytoplankton primary productivity observation using laser-induced chlorophyll fluorescence.

[0089] The method and device for measuring the primary productivity of phytoplankton in the above-mentioned embodiments are based on laser-induced chlorophyll fluorescence and can correct temperature changes. They can solve the difficult problem of measuring the primary productivity of phytoplankton in surface water bodies, improve the measurement accuracy of chlorophyll fluorescence in water bodies, and propose a new solution to the problem of inaccurate measurement caused by measurement signal drift due to the closed design of underwater sensors, heating of photoelectric devices, and changes in external environmental temperature. This further improves the measurement accuracy of phytoplankton primary productivity observed using laser-induced chlorophyll fluorescence.

[0090] The above describes specific embodiments of the present invention. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various modifications or variations within the scope of the claims without affecting the essence of the present invention. The above preferred features may be used in any combination as long as they do not conflict with each other.

Claims

1. A method for measuring phytoplankton primary productivity, characterized in that: include: Obtain spectra of calibration samples at different temperatures; Based on the spectrum, a calibration plane is established with the water environment temperature and the spectrum baseline intensity as independent variables and the fluorescence peak intensity as the dependent variable; Based on the calibration plane, obtaining the corrected fluorescence peak intensity of the calibration sample; A calibration curve was established based on the corrected fluorescence peak intensity of the calibration sample and the primary productivity; Obtaining the fluorescence spectrum of the sample to be tested; Obtaining a corrected fluorescence peak intensity of the sample to be tested based on the calibration plane and the fluorescence spectrum of the sample to be tested; According to the calibration curve, the phytoplankton primary productivity of the sample to be tested is inverted from the corrected fluorescence peak intensity of the sample to be tested; wherein: The calibration sample is a chlorophyll a alcohol extract that is uniform and stable and has a set concentration; The method of establishing a calibration plane based on the spectrum, taking the water environment temperature and the spectrum baseline intensity as independent variables and the fluorescence peak intensity as the dependent variable, comprises: For a certain concentration The calibration sample is selected, the spectral signal intensity at the set wavelength is selected as the spectral baseline intensity, the water environment temperature and the spectral baseline intensity are used as independent variables, and the fluorescence peak intensity is used as the dependent variable to establish a correction plane. The correction plane formula is: , is the water environment temperature, is the spectral baseline intensity, is the fluorescence peak intensity, is a constant, and It is the coefficient of the linear equation of two variables obtained based on the calibration sample; The obtaining of the corrected fluorescence peak intensity of the calibration sample based on the correction plane includes: The concentration is The parameter of any point of the calibration sample on the calibration plane is expressed as ( , , ), then select a temperature and a spectral baseline As a benchmark, the parameters ( , , ) is corrected to ( , , ), then the known concentration of any temperature and baseline The fluorescence peak intensity of the calibration sample is Therefore, a series of temperature-corrected and spectral baseline Corrected fluorescence peak intensity ,in, Chlorophyll a concentration in a series of solutions for calibration samples; The calibration curve is established based on the corrected fluorescence peak intensity of the calibration sample and the primary productivity, comprising: Corrected fluorescence peak intensity and chlorophyll a concentration The linear relationship is obtained ,in is the slope of the linear fitting line between fluorescence intensity and concentration; According to the primary productivity of phytoplankton in surface waters The relationship between the concentration of chlorophyll a and , is the chlorophyll a content in surface water, is the assimilation coefficient; The relationship between the primary productivity of the calibration sample and the corrected fluorescence peak intensity is: , which is the calibration formula; The method of obtaining the fluorescence spectrum of the sample to be tested includes: obtaining the fluorescence peak intensity from the fluorescence spectrum of the sample to be tested. ; The method of obtaining the corrected fluorescence peak intensity of the sample to be tested based on the correction plane includes: the corrected fluorescence peak intensity is .

2. The method for measuring phytoplankton primary productivity according to claim 1, wherein: The principle for selecting the set wavelength is to ensure that during normal measurement, the wavelength positions of chlorophyll a fluorescence and the fluorescence or scattered light of other substances in the complex water body to be measured do not overlap with the selected wavelength point.

3. The method for measuring phytoplankton primary productivity according to claim 1, wherein: The method of inverting the primary productivity of phytoplankton of the sample to be tested from the corrected fluorescence peak intensity of the sample to be tested according to the calibration curve includes: , and obtain the primary productivity of the sample to be tested.

4. A phytoplankton primary productivity measuring device, used to implement the phytoplankton primary productivity measuring method according to any one of claims 1 to 3, characterized in that: include: An instrument housing, wherein a light window and a flow-through light-shielding sample chamber are sequentially provided at one end of the instrument housing, the light window being located at the front end of the device and embedded in the instrument housing, and the flow-through light-shielding sample chamber being sleeved on the outside of the light window; A light source module, a probe, a spectrometer and a circuit module are provided inside the instrument housing, wherein the light source module and the spectrometer are connected to the probe respectively, and the circuit module is connected to the spectrometer; The light source module emits incident light which passes through the probe and the light window in turn and enters the sample to be tested in the flow-through light-shielding sample chamber, and irradiates the phytoplankton. The detection light including the backward fluorescence generated by the sample to be tested and part of the incident light is transmitted through the light window and the probe in turn and enters the spectrometer to obtain a fluorescence spectrum. The circuit module analyzes the fluorescence spectrum to obtain the primary productivity of phytoplankton.

5. The phytoplankton primary productivity measuring device according to claim 4, characterized in that: The flow-through light-shielding sample chamber is a cup-shaped structure, the cup mouth of the cup-shaped structure is buckled at the light window, and a plurality of evenly distributed flow openings are provided on the cup body and the cup bottom of the cup-shaped structure.

6. The phytoplankton primary productivity measuring device according to claim 4, characterized in that: The circuit module includes: The signal modulation unit is used to trigger and synchronize the light source module and the spectrometer, so that the spectrometer starts exposure at a preset time after the light source module is turned on and stabilized; A communication unit, used to implement communication functions; A core control unit is connected to the signal modulation unit and the communication unit respectively; A power supply unit is used to supply power to the circuit module.

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