New Type of Alga Photosynthesis Activity Modulation Fluorometer in

Situ Liu Jing1*2. Liu Wenqing2. Zhao Nanjing2*. Zhang Yujun2. Yin Gaofang2. Dai Pangda2. Ma Mingjun2.

Wang Chunlong2. Zhang Wei2. Duan Jingbo2. Fang Li2 1.School of Physical Science. University of Science and Technology of China.

Hefei 230026. China 2.Key Lab.of Environmental Optics & Technology,Anhui Institute of Optics and Fine

Mechanics,Chinese Academy of Sciences,Hefei 230031,China

ABSTRACT

A developed in situ measuring system uses light emitted diodes (LED) and laser diodes(LD) as light sources is employed for the recording of algae chlorophyll fluorescence induction kinetics (Kautsky-effect). Photosynthesis activity as an important parameter is obtained in the system which measures the chlorophyll fluorescence yield. Minimal fluorescence is excited by the brief but really weak light pulses from LED as measure light while photosynthesis is happened, variable fluorescence which means there is energy conversion through photosynthesis is also excited by a LED but the beam is more stronger, LD induced saturation fluorescence stands for maximum fluorescence. The system could works for alga chlorophyll photosynthesis activity continuous measure in situ through efficiently mechanical and optical design.

Keywords: photosynthesis activity, modulation, algae, vivo-fluorescence, fluoreometer, in situ

1. INTRODUCTION

Alga can never grow without energy provided by photosynthesis. it blooms under some special environment conditions, alga breeds very fast in a short term because it have strong ability of photosynthesis, the photosynthesis activity can represent the potential of alga’s growth[1]. In combination with other environmental parameters, the occurrence of algal blooms in a short term can be predicted accurately. The research of photosynthesis activity is widely used in areas of aquatic biology[2], waters ecology, limnology, algal stress resistance and blooms warning, it can reveal the character of alga population dynamic change, temporal and spatial variation of photosynthesis in field water and alga blooms. As early as 1834,first record of chlorophyll fluorescence was made by Brewster, when a bunch of strong sunlight shines on the ethanol extract of laurel leaves, the color of the solution turns from green to its complementary color—red. Later in 1852 Stokes recognized this phenomenon was caused by light emission and used the word “fluorescence” firstly. Chlorophyll fluorescence as photosynthesis probe was mainly due to Kautsky and U. Schreiber, Kautsky effect reveal relationship between photosynthesis and chlorophyll fluorescence. In 1931, Kautsky and his coworkers observed and recorded chlorophyll fluorescence induction phenomenon[3], after that researchers did intensive work based on Kautsky Effect[4], and gradually formed a photosynthetic fluorescence induction theory which widely used in photosynthesis research[5]. In 1983, Ulrich Schreiber designed a pulse amplitude modulation(PAM) fluorescence analyzer[6], the detector only record the fluorescence which have the same frequency with the modulation measure light, so the spectrometer can

6th International Symposium on Advanced Optical Manufacturing and Testing Technologies: Optical Test and Measurement Technology and Equipment, edited by Yudong Zhang, Libin Xiang, Sandy To,

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measure fluorescence in all physiological conditions. The emergence of modulation technique make chlorophyll fluorescence measurements become easy in the field[7].

2. METHODS

2.1 Principal of algae photosynthesis activity modulation fluorometer Intracellular chlorophyll molecules get energy by absorb photons directly or indirectly light quanta through the light-harvesting pigment(LHC) ,then transition from the ground state to excited states. In the higher excitation state of the chlorophyll molecule is very unstable, in a very short of time(nearly several hundred femtoseconds, fs), energy radiation to the surrounding environment by vibrational relaxation and return to the lowest excited state. Chlorophyll molecules at low excitation state can release the energy by the follow ways: a, release a photon resisting in fluorescence; b, directly dissipate as heat; c, energy transfer to the nearby molecules and finally to the reaction center where photochemical reaction happened[8]. when photosynthetic organisms is in the normal physiological state, the energy absorbed by antenna pigments are mainly used for photochemical reaction, only very little part of the energy are transferred into fluorescence, the detail information can be seen in the under Fig 1.

Fig.1 Light absorption and emission by chlorophyll

(ref. by Lincoln Taiz and Eduardo Zeiger,2006[9])

Fluorometer which use to measure the yield of the photosynthesis is able to apply at least 3 part of light source:measuring light, actinic light and saturation pulse. measuring light is low enough so when it is beaming, no photochemical reaction happened, the signal collected by the detector is the fluorescence emission by the sample itself, the intensity of measuring light is usually less than 10 umol.m-2s-1, actinic light(also called ACT Light) is usually generated by one or several LEDs, which intensity ranging from hundreds to thousands. Several type of light source (etc

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red, blue, green and white) could be used to induce chlorophyll fluorescence, Changes in chlorophyll fluorescence can be detected by the amplitude of modulation signal which induced by measuring light. Part of the excitation light could be reflected or scattered by the sample, the reflected light can be cut-off by short-wave filter ,the highly selective amplification system was used to avoid the interference of non-fluorescent signal. The third part of the light is saturation pulse(also called SAT Pulse) which was set to sustain about 0.4 to 2 seconds, it’s intensity could reach nearly 10000 umol.m-2s-1 . In this paper, blue laser diode made by NICHIA was used as saturating pulse light source. Algal photosynthesis activity detection is based on the method of fluorescence measurements and chlorophyll fluorescence induction kinetics characteristic. Chlorophyll molecules fluorescence is excited by light, accompanied with heat dissipation. Fluorescence of algae in vivo is almost entirely derived from the photosynthesis system II (PSII) of chlorophyll a, algal cells absorb light for photosynthesis (P), part of the energy dissipate into the environment (D) in the form of heat , and the remaining energy is emitted in the form of fluorescence (F). According to the law of conservation of energy , P + D + F = 1. Therefore, the maximum photosynthetic quantum yield of algae in the PS II and the actual quantum yield can be calculate by the fluorescence, which excited by different light source. The algae photosynthesis activity can be characterize by the yield. Figure 2 shows the process of the measurement.

Fig.2 Measurement process diagram of the algae fluorescence quantum yield The process shows in Fig. 2 can be described in the follow 5 steps: 1) After fully dark-adapted, open the weak measuring light, which is low enough to stimulate algal cytochrome fluorescence, at the same time there is no photochemical reaction carry out in the algal cells. 2) Open the SAT Pulse, which intensity is so high that the photosynthesis of the algae cells is completely inhibited, fluorescence rapidly increased to the maximum, then the maximum fluorescence can be measured. 3) Open the ACT Light, normal photosynthesis can be carry out in this period, the fluorescence rapidly increase to the maximum in the very beginning. Then due to the effects of photosynthesis and heat dissipation, the fluorescence gradually decreased to a stable value. The light energy absorbs by algal cells reach dynamic equilibrium in photosynthesis, the emission fluorescence and thermal dissipation, this state is known as light adaptation. 4) Open the SAT Pulse periodically. Fluorescence increase to a maximum value rapidly, which present the maximum fluorescence after light adaption.

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5) Calculate the maximum yield and actual yield use F0, Fm, F0’and Fm’. 2.2 Instrumentation for measuring system Fig 3 shows a block diagram of a set-up for measuring fluorescence induction curve. Microcontrol center is the key part of the measuring system, which include power supply function and mainly parameter settings. Light intensity can be set into several levels, for example intensity of measuring light directly determines the instrument’s measurement range with gain settings in signal detection part , as is well-know that the measurement range is a very important indicator of the instrument. Many useful information can get from the fluorescence induction curve under different intensity of actinic light and saturation pulse.

Fig .3 Block diagram of the set-up to measure fluorescence induction curve

Light source 470nm LED and 440nm LD are used in the instrument, the emission spectra is shown in the follow Fig.4 LED and LD can be switched on and off in less than microseconds, that rapid induction kinetics can be benefit from it. Another important thing is the size of LED and LD[10], make it possible for using in the field. In the meantime, LED and LD can be modulated up to very high frequencies, that would do some help for signal detection. Conventional lamp are more suitable particularly where large areas have to illuminated, but it is so huge that make using in the field very difficult. LD is a pretty good choice for SAT Pulse, for its power reach nearly 1500mW, heat issue must be resolved properly, otherwise it can be damaged easily. The follow Fig.4 shows the emission spectra of light source.

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Focus and filter systems Compared to the ambient light and the strong excitation light, chlorophyll fluorescence is very weak. Therefore the wavelength of the excitation light must be chosen not to overlap the fluorescence. In the system, a short-wave cut-off filter is used for avoiding ambient light, a bandpass filter for protecting the photomultiplier tube. The transmission spectra of the filter is shown in the follow Figure 5.

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Fig.5 Transmission spectra of filter used in the instrument. Filter 1 is used to avoid excitation light and ambient

light, filter 2 is used to ensure only fluorescence can be photomultiplier tube.

Signal detection system Fluorescence signal is nearly synchronous with the excitation light, it’s intensity is much lower, even much lower than the ambient light. The weak signals can be submerged by the noise like ambient light and scatter light, most important thing is to extract the signal in a strong noise background. Lock in amplifier(LIA) can solve this problem, photomultiplier tube catch the fluorescence and turn it into electronic signal, the LIA system enlarge the signal which have the same frequency with the reference signal. The signal noise ratio can be enhanced significantly.

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3. RESULTS AND DISCUSSIONS

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Fig.6 Induct curve induced by measuring light, actinic light and saturation pulse

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A experiment was carry out with alga solution, which concentration of chlorophyll a is 150ug/L, after adaptation about more than 5 minutes, open the measuring light, the light is so weak that there is only fluorescence emission but no photosynthesis happened. Obviously the fluorescence intensity is only related with the concentration of cytochrome in alga when excitation light is stable, so the fluorescence is also stable. The induct curve induced by measuring light can be seen in Fig.6-A. Then increase the intensity of excitation light, once it exceed nearly 6 μmolm-2s-1 the induct curve changed like Fig.6-B, this phenomenon represent some part of energy have been transferred to photochemical, the intensity of fluorescence reach the maximum value in the very beginning, and then continued to decline until the yield of photosynthesis maintained at a stable level. Continue to increase the intensity until nearly 2000 μmolm-2s-1 the induct curve is also the one with the maximum value increased only. When the excitation intensity is high enough, the induct curve changed like Fig.6-C, the fluorescence maintain at a level in a very short period, then decreased rapidly. the reason can be give like this: self-protection mechanism of alga works, light is too strong that alga have to close the photosynthesis systems. A completely induct curve is measured in situ in a waterwork, the activity of the alga in the water is about 0.58, it means nearly 58 percent of energy, caught by the light harvest complex is used to photosynthesis.

4. CONCLUSION

A fluorometer is designed for alga photosynthesis activity measurement. In this paper, a newly type of light source like laser diode was introduced as saturation pulse, a briefly mechanism structure was employed of measure in situ. More details about the principal and instrumentation of measuring system also were shown. Induct curve induced by measuring light, actinic light and saturation pulse light were got throughout measurement in situ, and a completely induct curve was discussed.

5.ACKNOWLEDGEMENTS

This work was supported by a grant from the National High Technology Research and Development Program of China (863 Program) (No. 2009AA063005), National key scientific and technological projects (No. 2009ZX07420-008-005) ， Natural Science Foundation of Anhui province (No. 11040606M26) and Foundation of Director of Anhui Institute of Optics and Fine Mechanics (No. Y03AG31144). I am very grateful to many colleagues for their contributions to our studies, without which this paper could not have been written.

REFERENCES

[1] G. H. Krause Chlorophyll fluorescence and photosynthesis: the basics. Annu.Rev. Plant Physiol. Plant Mol. Biol.1991,42:313-349 [2] Suggett D.J., Prasil O., Borowitzka M.A., Chlorophyy a Fluorescence in Aquatic Sciences. 2011,Springer Publishers [3] Govindjee.Sixty-three years Since Kautsky: Chlorophyll a Fluorescence. Aust.J. Plant Physiol.1995,22,131-160 [4] U. Schreiber, U. Schliwa. A solid-state, portable instrument for measurement of chlorophyll luminesence induction in plants. Photosynthesis Research,1987,11:173-182

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[5] George C. Papageorgious, Govindjee. Chlorophyll a fluorescence A signature of photosynthesis. Springer Publishers,2004,280-312 [6] Ulrich Schreiber. Chlorophyll fluorescence yield changes as a tool in plant physiology I. The measuring system. Photosynthesis Research,1983,4:361-373 [7] U. Schreiber, U. Schliwa and W. Bilger. Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new of modulation fluorometer. Photosynthesis Research,1986,10:51-62 [8] Warren L. Butler. Energy transfer between photosystem II units in a connected package model of the photochemical apparatus of photosynthesis. Proc. Natl. Acad. Sci. USA,1980,77(8) 4697-4701 [9] Lincoln Taiz and Eduardo Zeiger, Plant Physiology,2006,Springer Publishers [10] Yordan Kostov. Govind Rao. Low-cost optical instrumentation for biomedical measurements. Rev. Sci. Instrum.2000,71,4361-4373

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