A method for estimating marine carbon sequestration based on phytoplankton live chlorophyll fluorescence technology

Abstract

This invention discloses a method for estimating marine carbon fixation based on phytoplankton live chlorophyll fluorescence technology. The method includes the following steps: 1) culturing a single phytoplankton species to obtain an algal culture medium; 2) determining the PSIIactive content of the algal culture medium using FRR chlorophyll fluorescence induction technology; and 3) using isotope... 14 C) The photosynthetic carbon fixation of algal solutions was determined using tracer technology; 3) The PSIIactive content was combined with the photosynthetic carbon fixation amount to establish a model relating the photosynthetic carbon fixation rate based on PSIIactive and changes in light intensity. Using PSIIactive as a bridge, a method for estimating marine carbon fixation based on phytoplankton chlorophyll fluorescence technology was established. This invention has the advantages of being simple to operate, economical, and rapid, solving the problems of high cost, environmental pollution, and harm to human health in marine phytoplankton carbon fixation measurement.

Description

Technical Field

[0001] This invention belongs to the field of marine primary productivity measurement technology, specifically relating to a method for estimating marine carbon sequestration based on phytoplankton live chlorophyll fluorescence technology. Background Technology

[0002] The ocean is the Earth's largest carbon sink, and its absorption of atmospheric CO2 can alleviate a series of environmental problems caused by rising CO2 levels. Marine phytoplankton utilize light energy to drive photosynthesis, fixing CO2 and synthesizing organic matter. This carbon fixation process accelerates the dissolution of atmospheric CO2 into seawater, driving the ocean's absorption of atmospheric CO2. During predation or death, phytoplankton release organic carbon into the water, and this organic carbon, combined with the growth and reproduction of marine bacteria and viruses (carbon fixation processes), further increases marine carbon absorption. Therefore, phytoplankton carbon fixation provides the impetus for the ocean's function as a carbon sink, and accurately quantifying this biological carbon fixation is key to estimating the ocean's carbon fixation and storage capacity.

[0003] Currently, the most commonly used and sensitive method for measuring carbon sequestration, i.e., primary productivity, in marine phytoplankton is radiocarbon isotopes (RFI). 14 C) Tracer method; however, the use of this method is greatly limited due to its high cost, environmental pollution, and harm to human health. The determination of primary productivity using the photosynthetic oxygen release method is limited by the detection limit, requiring a high concentration of phytoplankton, which is extremely low in the ocean, especially in the open ocean. Therefore, this method is greatly limited in determining in-situ primary productivity.

[0004] The intensity of chlorophyll fluorescence signals from living phytoplankton can characterize the concentration of phytoplankton (Chl a) in water, and it is highly sensitive, easy to operate, economical, and rapid. However, due to changes in environmental factors such as light, the level of Chl a content in in-situ water does not accurately correlate with the level of carbon fixation in the water; that is, the intensity of the chlorophyll fluorescence signal (Chl a content) cannot accurately reflect the level of primary productivity. Furthermore, the intracellular Chl a content of phytoplankton varies greatly, resulting in significant differences in the carbon fixation rate based on Chl a. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a method for estimating marine carbon sequestration based on phytoplankton chlorophyll fluorescence technology. This method is simple to operate, economical, and rapid, and solves the problems of high cost, environmental pollution, and harm to human health associated with the determination of marine phytoplankton carbon sequestration.

[0006] To achieve the goal of simple, economical, and rapid estimation of marine primary productivity, this invention provides the following technical solution:

[0007] A method for estimating marine carbon sequestration based on phytoplankton live chlorophyll fluorescence technology includes the following steps:

[0008] (1) Cultivate a single species of phytoplankton to obtain an algal culture medium;

[0009] (2) The PSIIactive content of the algal solution was determined using the FRR chlorophyll fluorescence induction technique; isotope analysis was performed. 14 C-tracer technology was used to determine the photosynthetic carbon fixation of algal solutions;

[0010] (3) Combine the PSIIactive content with the photosynthetic carbon fixation amount to establish a relationship model between the PSIIactive photosynthetic carbon fixation rate and light intensity change. Using PSIIactive as a bridge, establish a method for estimating marine carbon fixation based on phytoplankton live chlorophyll fluorescence technology.

[0011] In step (1), the single phytoplankton species includes single phytoplankton species isolated and cultured from in situ phytoplankton populations or single phytoplankton species purchased from algal culture banks.

[0012] The method of this invention is based on research on the following single phytoplankton species including Thalassiosirapseudonana, T. punctigera, Synechococcus sp., Prochlorococcus marinas, and Ostreococcus tauri.

[0013] In step (2), the FRR chlorophyll fluorescence induction technique is used to determine the PSIIactive content of the algal solution. Specifically, the FRR rapid repeat fluorescence technique is used to determine the basic fluorescence signal F of the algal solution under light adaptation. O 'and the effective absorption cross section σ of PSII under light-adapted conditions' PSII ', calculate F O ' / σ PSII 'Value, F O ' / σ PSII 'Represents the PSIIactive content per unit volume, i.e., PSIIactive = a × F O ' / σ PSII ', where a is the correction factor.

[0014] In step (2), the use of isotopes 14 The C-tracer technique was used to determine the photosynthetic carbon fixation of algal solutions. Specifically, the following steps were taken: Adding... 14 Phytoplankton were cultured using C-labeled materials. After cultivation, phytoplankton were collected using a Whatman GF / F filter membrane, and then acidified and dried to remove unassimilated phytoplankton. 14 C. Add scintillation fluid and use a liquid scintillation counter to measure the concentration of assimilated organic matter.14 The radioactivity of C is CPM, and the photosynthetic carbon fixation C = [(CPM)] (l) -CPM (d) ) / C e ]×I f ×DIC / A, where CPM (l) CPM is the number of samples treated with light per minute. (d) C is the count per minute for samples treated in darkness. e For counting efficiency, I f The isotopic difference factor is DIC, where DIC is the total dissolved inorganic carbon, and A is the added carbon. 14 The number of μCi in C multiplied by 2.2 × 10 6 .

[0015] The specific steps of step (3) are as follows:

[0016] S1. The total photosynthetic oxygen production rate of the algal solution was determined by the photosynthetic oxygen release method, and the chlorophyll a content of the algal solution, i.e., Chl a content, was determined by the fluorescence method. The quantitative relationship between PSIIactive content and Chl a content was calculated, i.e., PSIIactive / Chl a=A'×B'×C'×D'×E', where A' is the total photosynthetic oxygen production rate of the algal solution, B' is the time consumed for each flash, C' is the 4 electrons required to produce 1 O2 molecule, D' is the 1 electron accepted by 1 PSII molecule in one flash cycle, and E' is the concentration of Chl a in the algal solution.

[0017] S2. Calculate the amount of carbon fixation per unit of chlorophyll a per unit time based on the photosynthetic carbon fixation C measured in step (2) and the Chl a content measured in step S1, and obtain the carbon fixation rate AsN = C / (Chl a × time).

[0018] S3, based on 14 Based on the carbon fixation rate of phytoplankton under different light intensities determined by C isotope tracing technology, a model was established to show the relationship between carbon fixation rate and light intensity variation: AsN=E / (aE) 2 +bE+c), where a, b, c are correction coefficients, and E is the light intensity;

[0019] S4. Based on the quantitative relationship between PSIIactive content and Chl a content determined in step S1, substitute PSIIactive / Chl a into AsN=E / (aE) 2 +bE+c), establish a relationship model between the change in photosynthetic carbon fixation rate and the change in light intensity based on PSIIactive: AsN'=E / (aE 2 +bE+c), that is, [PSIIactive]×R=E / (aE 2 +bE+c), where R is a fixed coefficient;

[0020] S5. Using PSIIactive as a bridge, establish the following method for determining water carbon sequestration based on fluorescence technology:

[0021]

[0022] Where t is time, Z is depth, and Zeu is the depth to which 1% of the surface light intensity reaches, i.e., the depth of the true light layer.

[0023] F O ' / σ PSII 'An alternative to PSIIactive, establishing a method for estimating marine carbon sequestration based on phytoplankton live chlorophyll fluorescence technology.'

[0024] The present invention has the following beneficial effects: The present invention provides a method for estimating marine primary productivity (carbon sequestration) based on phytoplankton live chlorophyll fluorescence technology, which has the advantages of simple operation, economy and speed, and solves the problems of high cost, environmental pollution and harm to human health in marine phytoplankton carbon sequestration measurement. Attached Figure Description

[0025] Figure 1 This is a technical roadmap for estimating marine carbon sequestration (primary productivity) based on phytoplankton live chlorophyll fluorescence technology. As shown in the figure: 1. In-situ phytoplankton community; 2. Isolation and culture; 3. Single phytoplankton species; 4. Purchase from algal strain bank; 5. Fluorescence technology; 6. 14 7. C isotope tracing technology, 8. PSIIactive content, 9. Photosynthetic carbon fixation, 10. Relationship between PSIIactive photosynthetic carbon fixation rate and light intensity variation, 11. Establishing a new method for estimating marine primary productivity based on chlorophyll fluorescence technology using PSIIactive as a bridge.

[0026] Figure 2 This is a comparison of the photosynthetic carbon fixation of phytoplankton T. pseudonaenana determined by the method of this invention (chlorophyll fluorescence method) and conventional methods.

[0027] Figure 3 This is a comparison of the photosynthetic carbon fixation of phytoplankton Synechococcus sp. determined by the method of this invention (chlorophyll fluorescence method) and conventional methods. Detailed Implementation

[0028] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0029] Example 1

[0030] A method for estimating marine carbon sequestration based on phytoplankton live chlorophyll fluorescence technology includes the following steps:

[0031] (1) A single phytoplankton species, *Thalassiosirapseudonana*, was cultured in a semi-continuous culture medium (f / 2 medium, 12h:12h light-dark cycle) in an indoor light incubator to obtain algal solutions; the light gradient was set to 30 (close to the light compensation point), 150 (optimal growth light intensity), and 300 μmol photons m -2 s -1 (Produces light inhibition), nitrogen nutrient concentrations were set to 500, 50, and 5 μmol L. -1 The temperature was set to 18, 21 and 24°C to obtain algal solution samples adapted to different environments.

[0032] (2) The PSIIactive content of the algal solution was determined using the FRR chlorophyll fluorescence induction technique: The basic fluorescence signal F of the algal solution under light adaptation was determined using the FRR rapid repeat fluorescence technique. O 'and its effective absorption cross section σ of PSII under light-adapted conditions PSII ', calculate F O ' / σ PSII 'Value, F O ' / σ PSII 'Represents the PSIIactive content per unit volume, i.e., PSIIactive = a × F O ' / σ PSII ', where a is the correction factor;

[0033] (3) Use of isotopes 14 C-ray tracer technique for determining photosynthetic carbon fixation in algal solutions: A certain amount of radiation is added to the algal solution. 14 Temperature-controlled incubation was performed using C-labeled phytoplankton [temperature was controlled to the incubation temperature using a circulating water temperature control system (MaXircu CR-12, DAIHAN Scientific, Korea)]. After incubation, phytoplankton were collected using a Whatman GF / F filter membrane, acidified, and dried to remove unassimilated phytoplankton. 14 C. Add 5 mL of scintillation solution and determine the concentration of assimilated organic matter using a liquid scintillation counter (Tri-Carb 4810TR, PerkinEmer, USA). 14 Radioactivity of C (CPM), photosynthetic carbon fixation (C, μg C / L) = [(CPM) (l) -CPM (d) ) / C e ]×I f ×DIC / A, where CPM (l) CPM is the number of samples treated with light per minute. (d) C is the count per minute for samples treated in darkness. e For counting efficiency, I fThe isotopic difference factor is DIC, where DIC is the total dissolved inorganic carbon, and A is the added carbon. 14 The number of μCi in C multiplied by 2.2 × 10 6 ;

[0034] (4) The PSIIactive content of the algal solution was determined by the photosynthetic oxygen release method: 50 mL of algal solution under the above different environmental conditions was centrifuged (4000 g, 10 min) at the culture temperature and concentrated to 5 mL. Then, 2.5 mL of the concentrated algal solution was placed in a 1 cm × 1 cm cuvette equipped with an oxygen electrode (Pyroscience, Germany) and a temperature control device. The cuvette was then placed in the measuring device of a fluorescence spectrometer (L3500, Photon Systems Instruments, Czech Republic). This fluorescence spectrometer has a built-in LED light source and can provide an intensity of 90,000 μmol photons m -2 s -1 A flash of light at a time interval of 2 μs is used. Before turning on the fluorescence light source to measure photosynthetic oxygen release, the concentrated sample needs to be placed in the dark for 3-5 minutes for dark adaptation until the sample chamber temperature reaches a constant value. Then, turn on the weak light (50 μmol photons m). -2 s -1 To ensure smooth electron transfer between PSII and PSI when the flash is activated, preliminary experiments were conducted to determine the required saturation light intensity for a single phytoplankton species, *Thalassirapseudonana*, to adapt to different growth environments. Using 3000 flashes over 150 seconds, the change in O2 concentration (net photosynthetic oxygen production rate) in the algal solution was measured. The light source was then turned off, and the respiration oxygen consumption rate of the cells under dark conditions was measured. The total photosynthetic oxygen production rate of the algal solution was obtained by dividing the sum of the O2 concentration changes measured under light and dark conditions by the measurement time. After the assay, phytoplankton cells were collected by centrifugation, and the Chl a content in the concentrated sample was quantitatively determined: First, the fluorescence values ​​of a series of standard Chl a concentrations were measured using a fluorometer, and a standard curve was plotted; then, a certain volume of algal solution or concentrate was filtered through a Whatman GF / F filter membrane (25 mm in diameter), 90% acetone saturated with MgCO3 was added, and the mixture was sonicated (10 min) and extracted in the dark at -20℃ (24 h). After extraction, the solution was centrifuged, and the supernatant was used to measure the fluorescence value of the algal cell extract using a Turner fluorometer. The Chl a concentration of the algal solution was calculated based on the standard curve.

[0035] Based on the above-measured total photosynthetic oxygen production rate and Chl a concentration, the quantitative relationship between PSIIactive content and Chl a was estimated:

[0036] PSIIactive / Chl a=A'(mol O2 L -1 s-1 )×B'(5×10 -2 s flash cycle -1 )×C'(4mole - mol O2 -1 )×D'(1flash cycle mol PSII mol e --1 )×E'(1mol Chl a L -1 ) -1 Where A' is the total photosynthetic oxygen production rate of the algal solution, B' is the time consumed for each flash, C' is the number of electrons required to produce one O2 molecule, D' is the number of electrons accepted by one PSII molecule in one flash cycle, and E' is the concentration of Chl a in the algal solution.

[0037] (5) Calculate the photosynthetic carbon fixation rate (AsN, assimilation coefficient) based on the photosynthetic carbon fixation C measured in step (3) and the Chl a concentration measured in step (4), that is, the amount of carbon fixed per unit chlorophyll a per unit time (μg C μg Chl a). -1 h -1 );

[0038] (6) Based on 14 Based on the carbon fixation rate of phytoplankton under different light intensities determined by C isotope tracing technology, a model was established to show the relationship between carbon fixation rate and light intensity variation: AsN=E / (aE) 2 +bE+c), where a, b, c are correction coefficients, and E is the light intensity;

[0039] (7) Based on the quantitative relationship between PSIIactive content and Chla determined in step (4), substitute PSIIactive / Chla into AsN=E / (aE) 2 +bE+c), establish a model based on the relationship between changes in photosynthetic carbon fixation rate and light intensity changes using PSIIactive: AsN'=E / (aE 2 +bE+c), that is, [PSIIactive]×R=E / (aE 2 +bE+c), where R is a fixed coefficient;

[0040] (8) Using PSIIactive as a bridge, the following method for determining carbon sequestration in marine waters based on fluorescence technology is established:

[0041]

[0042] Where t is time, Z is depth, and Zeu is the depth to which 1% of the surface light intensity reaches, i.e., the depth of the true light layer.

[0043] F O ' / σ PSII'An alternative to PSIIactive, establishing a method for estimating marine carbon sequestration based on phytoplankton live chlorophyll fluorescence technology.'

[0044] (9) The comparison results of photosynthetic carbon fixation of phytoplankton T. pseudodonana based on the method of this invention (chlorophyll fluorescence method) and conventional methods are shown in [reference needed]. Figure 2 .

[0045] Figure 2 In section A), the concentration of 30 μmol photonsm was determined using the method of this invention (horizontal axis, fluorescence method) and the conventional method (vertical axis). -2 s -1 Light intensity, different temperatures (18, 21, and 24 °C), and nitrogen nutrient concentrations (500, 50, and 5 μmol L⁻¹) -1 A) The relationship between the carbon fixation of *T. pseudodonana* grown under different conditions; B) The determination of carbon fixation at 18°C ​​for different nitrogen nutrients (500 and 50 μmol L) using fluorescence method (horizontal axis) and conventional method (vertical axis). -1 ) and illumination (30, 150 and 300 μmol photons m -2 s -1 The relationship between carbon fixation of *T. pseudodonana* grown under [a specific environment] is shown in the figure. The slopes of the linear regressions are 1.140 and 1.096, respectively, which are close to 1. This indicates that the carbon fixation measured using the method of this invention is close to the carbon fixation measured using conventional methods, thus proving that the method is feasible.

[0046] Example 2

[0047] A method for estimating marine carbon sequestration based on phytoplankton live chlorophyll fluorescence technology includes the following steps:

[0048] (1) A single phytoplankton species, *Synechococcus* sp., was cultured in an indoor light incubator using a semi-continuous f / 2 medium (12h:12h light-dark cycle) to obtain algal solution; the light gradient was set to 30 (close to the light compensation point), 75 (optimal growth light intensity), and 120 μmol photons m -2 s -1 (Produces light inhibition), nitrogen nutrient concentrations were set to 500, 50, and 5 μmol L. -1 The temperature was set to 18, 21 and 24°C to obtain algal solution samples adapted to different environments.

[0049] (2) The PSIIactive content of the algal solution was determined using the FRR chlorophyll fluorescence induction technique: The basic fluorescence signal F of the algal solution under light adaptation was determined using the FRRf rapid repeat fluorescence technique. O 'and the effective absorption cross section σ of PSII under light-adapted conditions' PSII', calculate F O ' / σ PSII 'Value, F O ' / σ PSII 'Represents the PSIIactive content per unit volume, i.e., PSIIactive = F O ' / σ PSII ';

[0050] (3) Use of isotopes 14 C-ray tracer technique for determining photosynthetic carbon fixation in algal solutions: A certain amount of radiation is added to the algal solution. 14 Temperature-controlled culture was performed using C-labeled phytoplankton [temperature was controlled close to the growth temperature using a circulating water temperature control system (MaXircu CR-12, DAIHAN Scientific, Korea)]. After culture, phytoplankton were collected using a Whatman GF / F filter membrane, and the plants were acidified and dried to remove unassimilated phytoplankton. 14 C. Add 5 mL of scintillation solution and determine the concentration of assimilated organic matter using a liquid scintillation counter (Tri-Carb 4810TR, PerkinEmer, USA). 14 Radioactivity of C (CPM), photosynthetic carbon fixation (C, μg C / L) = [(CPM) (l) -CPM (d) ) / C e ]×I f ×DIC / A, where CPM (l) CPM is the number of samples treated with light per minute. (d) C is the count per minute for samples treated in darkness. e For counting efficiency, I f The isotopic difference factor is DIC, where DIC is the total dissolved inorganic carbon, and A is the added carbon. 14 The number of μCi in C multiplied by 2.2 × 10 6 ;

[0051] (4) The PSIIactive content of the algal solution was determined by the photosynthetic oxygen release method: 50 mL of algal solution under the above different environmental conditions was centrifuged (4000 g, 10 min) at the culture temperature and concentrated to 5 mL. Then, 2.5 mL of the concentrated algal solution was placed in a 1 cm × 1 cm cuvette equipped with an oxygen electrode (Pyroscience, Germany) and a temperature control device. The cuvette was then placed in the measuring device of a fluorescence spectrometer (L3500, Photon Systems Instruments, Czech Republic). This fluorescence spectrometer has a built-in LED light source and can provide an intensity of 90,000 μmol photons m -2 s -1A flash of light at a time interval of 2 μs is used. Before turning on the fluorescence light source to measure photosynthetic oxygen release, the concentrated sample needs to be placed in the dark for 3-5 minutes for dark adaptation until the sample chamber temperature reaches a constant value. Then, turn on the weak light (50 μmol photons m). -2 s -1 To ensure smooth electron transfer between PSII and PSI when the flash is activated, preliminary experiments were conducted to determine the required saturated light intensity for a single phytoplankton species, *Synechococcus sp.*, to adapt to different growth environments. Using 3000 flashes over 150 seconds, the change in O2 concentration (net photosynthetic oxygen production rate) in the algal solution was measured. The light source was then turned off, and the respiration oxygen consumption rate of the cells under dark conditions was measured. The total photosynthetic oxygen production rate of the algal solution was obtained by dividing the sum of the O2 concentration changes measured under light and dark conditions by the measurement time. After the assay, phytoplankton cells were collected by centrifugation, and the Chl a content in the concentrated sample was quantitatively determined: First, the fluorescence values ​​of a series of standard Chl a concentrations were measured using a fluorometer, and a standard curve was plotted; then, a certain volume of algal solution or concentrate was filtered through a Whatman GF / F filter membrane (25 mm in diameter), 90% acetone saturated with MgCO3 was added, and the mixture was sonicated (10 min) and extracted in the dark at -20℃ (24 h). After extraction, the solution was centrifuged, and the supernatant was used to measure the fluorescence value of the algal cell extract using a Turner fluorometer. The Chl a concentration of the algal solution was calculated based on the standard curve.

[0052] Based on the above measurements, the quantitative relationship between PSIIactive content and Chl a was estimated:

[0053] PSIIactive / Chl a=A'(mol O2 L -1 s -1 )×B'(5×10 -2 s flash cycle -1 )×C'(4mole - mol O2 -1 )×D'(1flash cycle mol PSII mol e --1 )×E'(1mol Chl a L -1 ) -1 Where A' is the total photosynthetic oxygen production rate of the algal solution, B' is the time consumed for each flash, C' is the number of electrons required to produce one O2 molecule, D' is the number of electrons accepted by one PSII molecule in one flash cycle, and E' is the concentration of Chl a in the algal solution.

[0054] (5) Calculate the carbon fixation rate (AsN, assimilation coefficient) based on the photosynthetic carbon fixation C measured in step (3) and the Chl a concentration measured in step (4), that is, the amount of carbon fixed per unit chlorophyll a per unit time (μg C μg Chl a). -1 h -1 );

[0055] (6) Based on 14 Based on the carbon fixation rate of phytoplankton under different light intensities determined by C isotope tracing technology, a model was established to show the relationship between carbon fixation rate and light intensity variation: AsN=E / (aE) 2 +bE+c), where a, b, c are correction coefficients, and E is the light intensity;

[0056] (7) Based on the quantitative relationship between PSIIactive content and Chla determined in step (4), substitute PSIIactive / Chla into AsN=E / (aE) 2 +bE+c), establish a model based on the relationship between changes in photosynthetic carbon fixation rate and light intensity changes using PSIIactive: AsN'=E / (aE 2 +bE+c), that is, [PSIIactive]×R=E / (aE 2 +bE+c), where R is a fixed coefficient;

[0057] (8) Using PSIIactive as a bridge, the following method for determining carbon sequestration in marine waters based on fluorescence technology is established:

[0058]

[0059] Where t is time, Z is depth, and Zeu is the depth to which 1% of the surface light intensity reaches, i.e., the depth of the true light layer.

[0060] F O ' / σ PSII 'An alternative to PSIIactive, establishing a method for estimating marine carbon sequestration based on phytoplankton live chlorophyll fluorescence technology.'

[0061] (9) The comparison results of photosynthetic carbon fixation of phytoplankton Synechococcus sp. based on the method of this invention (chlorophyll fluorescence method) and conventional methods are shown in [the table]. Figure 3 .

[0062] Figure 3 In section A), the concentration of 30 μmol photonsm was determined using the method of this invention (horizontal axis, fluorescence method) and the conventional method (vertical axis). -2 s -1 Light intensity, different temperatures (18, 21, and 24 °C), and nitrogen nutrient concentrations (500, 50, and 5 μmol L⁻¹) -1A) The relationship between the carbon fixation of Synechococcus sp. grown under different nitrogen nutrient levels (500 and 50 μmol L) and the carbon fixation of Synechococcus sp. grown under different nitrogen nutrient levels (500 and 50 μmol L) at 24 °C, determined by fluorescence method (horizontal axis) and conventional method (vertical axis). -1 ) and illumination (30, 75 and 120 μmol photons m -2 s -1 The relationship between carbon fixation and the amount of carbon fixed by *Synechococcus sp.* grown under the conditions of [insert specific conditions here]. The slopes of the linear regressions in the figure are 0.897 and 1.072, respectively, which are close to 1. This indicates that the amount of carbon fixation measured using the method of this invention is close to the amount of carbon fixation measured using conventional methods, thus proving that the method is feasible.

[0063] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for estimating marine carbon sequestration based on phytoplankton live chlorophyll fluorescence technology, characterized in that, Includes the following steps: (1) Cultivate a single species of phytoplankton to obtain an algal culture medium; (2) The PSIIactive content of the algal solution was determined using the FRR chlorophyll fluorescence induction technique; isotope analysis was performed. 14 C-tracer technology was used to determine the photosynthetic carbon fixation of algal solutions; (3) Combine PSIIactive content with photosynthetic carbon fixation amount to establish a relationship model between PSIIactive photosynthetic carbon fixation rate and light intensity change, and use PSIIactive as a bridge to establish a method for estimating marine carbon fixation based on phytoplankton live chlorophyll fluorescence technology. In step (2), the FRR chlorophyll fluorescence induction technique is used to determine the PSIIactive content of the algal solution. Specifically, the FRR rapid repeat fluorescence technique is used to determine the basic fluorescence signal F of the algal solution under light adaptation. O 'and the effective absorption cross section σ of PSII under light-adapted conditions' PSII ', calculate F O ' / σ PSII 'Value, F O ' / σ PSII 'Represents the PSIIactive content per unit volume, i.e., PSIIactive = a × F O ' / σ PSII ',a is the correction factor; In step (2), the use of isotopes 14 The C-tracer technique was used to determine the photosynthetic carbon fixation of algal solutions. Specifically, the following steps were taken: Adding... 14 Phytoplankton were cultured using C-labeled materials. After cultivation, phytoplankton were collected using a Whatman GF / F filter membrane, and then acidified and dried to remove unassimilated phytoplankton. 14 C. Add scintillation fluid and use a liquid scintillation counter to measure the concentration of assimilated organic matter. 14 The radioactivity of C is CPM, and the photosynthetic carbon fixation C = [(CPM)] (l) -CPM (d) ) / C e ]×I f ×DIC / A, where CPM (l) CPM is the number of samples treated with light per minute. (d) C is the count per minute for samples treated in darkness. e For counting efficiency, I f The isotopic difference factor is DIC, where DIC is the total dissolved inorganic carbon, and A is the added carbon. 14 The number of μCi in C multiplied by 2.2 × 10 6 ; The specific steps of step (3) are as follows: S1. The total photosynthetic oxygen production rate of the algal solution was determined by the photosynthetic oxygen release method, and the chlorophyll a content of the algal solution, i.e., Chl a content, was determined by the fluorescence method. The quantitative relationship between PSIIactive content and Chl a content was calculated, i.e., PSIIactive / Chl a=A'×B'×C'×D'×E', where A' is the total photosynthetic oxygen production rate of the algal solution, B' is the time consumed for each flash, C' is the 4 electrons required to produce 1 O2 molecule, D' is the 1 electron accepted by 1 PSII molecule in one flash cycle, and E' is the concentration of Chl a in the algal solution. S2. Calculate the amount of carbon fixation per unit of chlorophyll a per unit time based on the photosynthetic carbon fixation C measured in step (2) and the Chl a content measured in step S1, and obtain the carbon fixation rate AsN = C / (Chl a × time). S3, based on 14 Based on the carbon fixation rate of phytoplankton under different light intensities determined by C isotope tracing technology, a model was established to show the relationship between carbon fixation rate and light intensity variation: AsN=E / (aE) 2 +bE+c), where a, b, c are correction coefficients, and E is the light intensity; S4. Based on the quantitative relationship between PSIIactive content and Chl a content determined in step S1, substitute PSIIactive / Chl a into AsN=E / (aE) 2 +bE+c), establish a relationship model between the change in photosynthetic carbon fixation rate and the change in light intensity based on PSIIactive: AsN'=E / (aE 2 +bE+c), that is, [PSIIactive]×R=E / (aE 2 +bE+c), where R is a fixed coefficient; S5. Using PSIIactive as a bridge, establish the following method for determining water carbon sequestration based on fluorescence technology: Where t is time, Z is depth, and Zeu is the depth to which 1% of the surface light intensity reaches, i.e., the depth of the true light layer. F O ' / σ PSII 'An alternative to PSIIactive, establishing a method for estimating marine carbon sequestration based on phytoplankton live chlorophyll fluorescence technology.' 2. The method according to claim 1, characterized in that, In step (1), the single phytoplankton species includes single phytoplankton species isolated and cultured from in situ phytoplankton populations or single phytoplankton species purchased from algal culture banks.

3. The method according to claim 2, characterized in that, The monotypic phytoplankton species mentioned include Thalassiosirapseudonana, T. Punctigera, Synechococcus sp., Prochlorococcus marinas, and Ostreococcus tauri.

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