1. Introduction

As a primary producer in water, phytoplankton possess less than 1% of the total vegetation biomass globally, but its productivity accounts for about 45% of total plant productivity and 40% of global total carbon sequestration [1]. The phytoplankton photosynthesis is the basis of the material circulation and ecosystem energy flow, and affects the global biogeochemical cycle and climate change fundamentally. The rapid and accurate measurement of phytoplankton photosynthetic rate is of great significance to water ecological environment monitoring, marine resource assessment and global climate change prediction.

In the photosynthesis process, the photosynthetic tissues are driven by light produce oxygen and electrons by cracking water molecules. The electron transmitted by reaction center and multistep electron acceptors finally generate reduction force and participate in photosynthetic carbon sequestration. In the black and white bottle method, ¹⁴C tracer method and other traditional gas exchange methods, the photosynthetic rate is calculated by measuring the photosynthetic oxygen evolution rate or the photosynthetic carbon fixation rate, which require “sampling-incubation-offline analysis”. These methods have the disadvantages of time-consuming, low efficiency and complicated procedure, and cannot meet the rapid observation demand in ecological and environmental monitoring. In 1992, Falkowski et al. [2] first proposed the photosynthesis analysis method using chlorophyll fluorescence kinetics. The method employs the fluorescence of reaction center as the probe to analyze the light absorption, transmission and conversion efficiency of cells and the phytoplankton photosynthetic capacity can be obtained directly and rapidly. This method possesses the advantages of rapid measurement, non-pretreatment, non-pollution and non-invasion, and thus it’s an advanced tool for the rapid sensing of phytoplankton photosynthesis in natural conditions. However, the chlorophyll fluorescence kinetics is affected by many photosynthetic chemical processes, leading to a complex theoretical model. Thus there would be large errors if the complex theoretical model is directly used to fit the fluorescence kinetics curve. In recent decades, in order to improve the measurement accuracy of photosynthetic parameters, Schreiber and Kolber et al. [3,4] put forward Pulse Amplitude Modulation (PAM), Fast Repetition Rate (FRR) and some other kinds of photosynthesis analysis techniques using chlorophyll fluorescence successively. However, these techniques are mainly used to measure the local photosynthetic parameters and cannot measure the photosynthetic rate.

In this paper, a photosynthetic rate measurement method based on Tunable Pulsed Light Induced Fluorescence kinetics (TPLIF) was proposed. The phytoplankton photosynthetic rate was evaluated by measuring photosynthetic electron transport rate. The validity of the fluorescence kinetics method was verified by comparing with the photosynthetic oxygen evolution method.

2. Principle and method

The energy flow of phytoplankton photosynthesis is shown in Fig. 1. Driven by light energy, the water molecules are cracked and electrons are produced. The electrons transmitted by reaction center and multistep electron acceptors finally generate reduction force and participate in Calvin cycle photosynthetic carbon sequestration. From the perspective of energy transport, photosynthetic rate is essentially photosynthetic electron transport rate ${\text{P}}_{\text{e}}$. According to “Bio-Optical” model [5], photosynthetic electron transport rate depends on the ambient light intensity $E$, light energy absorption efficiency of photosystem II (PSII) $A_{\text{PSII}}$, efficiency of charge separation and conversion $T_{\text{e}}$, as defined in Eq. (1).

 

Fig. 1 Diagram of the energy flow in phytoplankton photosynthesis (modified from [5]).

Download Full Size | PDF

In order to accurately obtain the two key elements of photosynthetic electron transfer rate ${\text{P}}_{\text{e}}$, light energy absorption efficiency of photosystem II (PSII) $A_{\text{PSII}}$ and efficiency of charge separation and conversion $T_{\text{e}}$, a photosynthetic rate measurement method based on TPLIF was studied. High light pulse is employed to stimulate the dark-adapted phytoplankton cells, and during the Q_A single-turnover period (photochemical reaction inactivated), the photosynthetic electron transport chain is blocked by a large number of electrons produced by photosynthetic reaction center. As a consequence, the fluorescence increases rapidly from the minimum fluorescence ${\text{F}}_{\text{o}}$ to the maximum fluorescence ${\text{F}}_{\text{m}}$, that is the fast phase fluorescence. The high light pulse is then turned off and the photochemical reaction process is activated. The fluorescence decays exponentially, and the decrease of the fluorescence, that is the relaxation fluorescence, is recorded using weak pulse light, as shown in Fig. 2.

 

Fig. 2 Measurement process of tunable pulsed light induced fluorescence kinetics.

Download Full Size | PDF

Based on the photosynthesis energy flow theory, the fast phase fluorescence $\text{F}(t)$ and relaxation fluorescence $\text{G}\left(t\right)$ can be formulated by Eqs. (2) and (3) respectively. From these two equations, minimum fluorescence ${\text{F}}_{\text{o}}$, maximum fluorescence ${\text{F}}_{\text{m}}$, functional absorption cross section ${\sigma }_{\text{PSII}}$, QA average reoxidation time ${\tau }_{\text{QA}}$ and other photosynthetic fluorescence parameters that dominate the process of photosynthesis can be inverted [6].

According to “Bio-Optical” model, the light energy absorption efficiency of PSII $A_{\text{PSII}}$ depends on functional absorption cross section ${\sigma }_{\text{PSII}}$ and light-harvesting efficiency ${\text{Φ}}_{\text{T}}$. The efficiency of charge separation and conversion $T_{\text{e}}$ depends on light-harvesting efficiency ${\text{Φ}}_{\text{T}}$, photochemical quenching ${\text{Φ}}_{\text{P}}$ and PQ average reduction rate ${\text{Φ}}_{\text{QA}}$, as shown in Eqs. (4) and (5). Photochemical quenching ${\text{Φ}}_{\text{P}}$ and PQ average reduction efficiency ${\text{Φ}}_{\text{QA}}$ can be evaluated by photosynthetic fluorescence parameters, as shown in Eqs. (6) and (7), where ${\text{F}}_{\text{m}}^{\text{'}}$ and ${\text{F}}_{\text{o}}^{\text{'}}$ are maximum fluorescence and minimum fluorescence under light adaptation respectively.

Combining Eqs. (4)–(7) to Eq. (1), a photosynthetic rate analysis model based on TPLIF is established. The photosynthetic rate evaluated by photosynthetic electron transport rate ${\text{P}}_{\text{e}}$ can be calculated in Eq. (8).

3. Apparatus and experiment

For evaluating photosynthetic rate, the photosynthetic electron transport rate ${\text{P}}_{\text{e}}$, photosynthetic oxygen evolution rate ${\text{P}}_{\text{O}2}$ and photosynthetic carbon fixation rate ${\text{P}}_{\text{CO}2}$ are in consistent [7]. In this paper, the photosynthetic electron transport rate ${\text{P}}_{\text{e}}$ measured by TPLIF is compared with the photosynthetic oxygen evolution rate ${\text{P}}_{\text{O}2}$ measured by liquid-phase oxygen measurement system (Hansatec, Chlorolab2) to verify the validity of the fluorescence kinetics measurement method of photosynthetic rate.

3.1 TPLIF measurement system

The system consists of four parts: excitation-emission optical system, tunable fast-pulsed light source, fluorescence detection module and computer control system, as shown in Fig. 3. The excitation-emission optical system adopts orthogonal optical path structure and is combined with the filter system, the interference of excitation light to the fluorescence signal is deeply inhibited. The tunable fast-pulsed light source is composed of LEDs array with a wavelength of 465nm. The excitation intensity is accurately regulated by changing power supply voltage, frequency and duty ratio of driving pulse. A saturated intensity that is higher than 30000 μmol quanta/m²/s (hereinafter referred μmol quanta/m²/s to as L) is used to regulate photosynthetic electron transfer process, and a weak intensity of several L is employed to record the fluorescence change process [8]. Photomultiplier tube (Hamamatsu, h6779-01) is used as photoelectric detector in the fluorescence detection module. The fast phase fluorescence signal is acquired by high speed AD without distortion following I/V conversion, variable gain and low pass filtering. The relaxation fluorescence signal is sensitively detected by integral amplification and high resolution AD. The computer control system is mainly used for the synchronous control of pulsed light excitation and fluorescence detection, as well as the collection of fluorescence kinetic curves and calculation of photosynthetic electron transport rate ${\text{P}}_{\text{e}}$.

 

Fig. 3 Diagram of tunable pulsed light induced fluorescence kinetics measurement system.

Download Full Size | PDF

3.2 Comparative experiment for photosynthetic rate

Photosynthetic oxygen evolution rate measurement: The oxygen evolution was measured by Chlorolab2 every 120 seconds, and the total oxygen evolution rate was obtained by calculating the slope of oxygen evolution amount of adjacent moments. The net photosynthetic oxygen evolution rate ${\text{P}}_{\text{O}2}$ was obtained by deducting the respiration rate (i.e., oxygen consumption rate under dark adaptation).

Photosynthetic electron transport rate measurement: the fluorescence kinetics curve was measured every 5 seconds, and the instantaneous photosynthetic electron transport rate was calculated from the curve. The instantaneous photosynthetic electron transport rates were averaged every 120 seconds to get a photosynthetic electron transport rate ${\text{P}}_{\text{e}}$ that corresponding to ${\text{P}}_{\text{O}2}$ that measured by Chlorolab2.

Comparative experiments: Chlorella pyrenoidosa was used as the experimental subject. Under different DCMU stress conditions, different culture light and different nutrient (nitrogen) concentrations, the Chlorolab2 and TPLIF system were used to measure the photosynthetic oxygen evolution rate ${\text{P}}_{\text{O}2}$ and photosynthetic electron transport rate ${\text{P}}_{\text{e}}$ respectively. Each sample was measured under eight ambient light intensities, and the results were analyzed, as shown in Fig. 4. In order to ensure the consistency of experimental samples and conditions, the following steps were taken. Firstly, the samples came from the same source. Secondly, the sample pools and ambient light sources used in Chlorolab2 and TPLIF system were respectively of the same specification, and the ambient light sources of the two instruments were synchronized by computer.

 

Fig. 4 Procedure of comparative experiment for phytoplankton photosynthetic rate.

Download Full Size | PDF

4. Results and discussion

4.1 DCMU stress experiment

DCMU is an electron transport inhibitor that can inhibit the photosynthesis by inhibiting the Q_A reoxidation. Sample groups with DCMU concentrations of 30μmol/L, 50μmol/L, 80μmol/L, 100μmol/L were prepared by adding DCMU to the Chlorella pyrenoidosa solution. The Chlorella pyrenoidosa in the solution is in logarithmic phase. A control group without DCMU was also prepared. Each sample group contained three parallel samples. The Chlorolab2 and TPLIF system were used to record the photosynthetic oxygen evolution rate and the electron transport rate respectively under the eight ambient light intensities of 9L, 19L, 29L, 40L, 62L, 74L, 87L,100L for each sample group.

As shown in Fig. 5(a) and Fig. 5(b), as the concentration of DCMU increases, the phytoplankton photosynthesis is increasingly stressed. The photosynthetic rate curves and photosynthetic electron transfer rate curves have the same variation trend and the correlation coefficient R² between ${\text{P}}_{\text{O}2}$ and ${\text{P}}_{\text{e}}$ is 0.934, as shown in Fig. 5(d). Under an ambient light intensity of 100L, ${\text{P}}_{\text{O}2}$ and ${\text{P}}_{\text{e}}$ decrease obviously when the DCMU concentration is lower than 80μmol/L, while when the DCMU concentration is higher than 80μmol/L, ${\text{P}}_{\text{O}2}$ and ${\text{P}}_{\text{e}}$ almost remain unchanged, indicating that the DCMU stress reaches a threshold, as shown in Fig. 5(c).

 

Fig. 5 Results of phytoplankton photosynthetic rate under DCMU stress (a)Photosynthetic oxygen evolution rate curves (b)Photosynthetic electron transport rate curves (c)Change of PO2 and Pe versus DCMU concentration (d)Correlation between PO2 and Pe.

Download Full Size | PDF

4.2 Culture light intensity experiment

Light is the energy source of photosynthesis. Sample groups were cultured under seven light intensities of 60L, 100L, 150L, 200L, 300L, 500L, and 700L respectively. Each sample group contained three parallel samples. After 5-day culture, The Chlorolab2 and TPLIF system were used to respectively record the photosynthetic oxygen evolution rate and the electron transport rate under the eight ambient light intensities of 9L, 19L, 29L, 40L, 62L, 74L, 87L, 100L for each sample group.

It shows in Fig. 6 that the photosynthetic oxygen evolution rate and photosynthetic electron transport rate possess the same variation trend under all the seven culture light intensities. The photosynthetic rate reaches its maximum under 150L culture light intensity, but begins to decrease under higher culture light intensity because of photoinhibition, which is consist with reported results [9]. The correlation coefficient R² between ${\text{P}}_{\text{O}2}$ and ${\text{P}}_{\text{e}}$ is 0.957.

 

Fig. 6 Results of phytoplankton photosynthetic rate under different culture light intensities (a)Photosynthetic oxygen evolution rate curves (b)Photosynthetic electron transport rate curves (c)Change of P_O2 and Pe versus DCMU concentration (d)Correlation between P_O2 and Pe.

Download Full Size | PDF

4.3 Nutrient (nitrogen) culture experiment

Nitrogen and phosphorus are essential nutrients for the phytoplankton, and nitrogen is crucial for the production of chlorophyll. Six sample groups with nutrient (nitrogen) concentrations of 16mg/L, 32mg/L, 64mg/L, 128mg/L, 228mg/L, 512mg/L were prepared respectively by regulating the nitrogen concentration in BG11 culture medium using NaNO₃. Chlorolab2 and TPLIF system were used respectively to record the photosynthetic oxygen evolution rate and the electron transport rate under the eight ambient light intensities of 9L, 19L, 29L, 40L, 62L, 74L, 87L, 100L for each sample group.

As shown in Fig. 7, the photosynthetic oxygen evolution rate and photosynthetic electron transport rate also possess the same variation trend under all nutrient (nitrogen) concentrations, and the correlation coefficient R² between ${\text{P}}_{\text{O}2}$ and ${\text{P}}_{\text{e}}$ is 0.955. Under nitrogen deficiency (16mg/L) condition, the photosynthetic rate is low, indicating the photosynthesis is limited. The photosynthetic rate increased significantly with the increase of nitrogen concentration (32mg/L, 64mg/L). However, the photosynthetic rate begins to decrease when the nitrogen concentration is higher than 128mg/L. The reason for the decrease of photosynthetic rate under high nitrogen concentration is unknown and needs further study.

 

Fig. 7 Results of phytoplankton photosynthetic rate under different nutrient concentrations (a) Photosynthetic oxygen evolution rate curves (b) Photosynthetic electron transport rate curves (c) Change of P_O2 and Pe versus DCMU concentration (d)Correlation between P_O2 and Pe.

Download Full Size | PDF

5. Conclusion

On the basis of “Bio-Optical” model, a photosynthetic rate measurement method based on tunable pulsed light induced fluorescence kinetics was put forward in this paper. The phytoplankton photosynthetic rate was evaluated by photosynthetic electron transport rate. Comparative experiment results showed that the photosynthetic electron transport rate measured by fluorescence kinetic method under different conditions of DCMU, culture light and nutrients (nitrogen) were consistent with the photosynthetic oxygen evolution rate measured by oxygen evolution method, and the correlation coefficients R² were above 0.934.

The proposed method possesses the advantages of rapid measurement, non-pretreatment, non-pollution and non-invasion, and breaks through the limitation of gas exchange method, which requires “sampling-incubation-offline analysis”. It is an effective method to measure the phytoplankton photosynthetic rate in situ, thus possesses a broad prospect for applications in water ecological environment monitoring, marine resource assessment and global climate change prediction.