**5. Phytoplankton**

260 Artificial Photosynthesis

In principle, photosynthesis generates three phenomena which may be detected by photoacoustics, with adequate setups. The thermal expansion of tissue, liquids and gases due to light energy converted to heath is termed as the **photothermal** signal. That is generated always, when photosynthetic tissue, or cell is exposed to a light pulse, since never is all of the light absorbed by plant tissue stored as products of the process. The unused fraction of the absorbed light energy is converted to heat, resulting in measurable pressure transient. When a leaf is illuminated by a pulse of light, the resulting photosynthetic photolysis of water causes the evolution of a burst of gaseous oxygen. That process leads to an increase in pressure, a change which is readily detected by a microphone as the **photobaric** signal. For detailed definitions and description see review by Malkin (1996). In addition to these two, absorption of light by components of the photosynthetic apparatus, such as PSI, or PSII, is accompanied by change in its spatial conformation and volume

Photoacoustic methods provide unique capabilities for photosynthesis research. The pulsed photoacoustic technique gives a direct measurement of the enthalpy change of photosynthetic reactions (Carpentier et al., 1984). A microphone may detect the photoacoustic waves *via* the thermal expansion in the gas phase. This method allows *in vivo*  measurements of the photosynthetic thermal efficiency, or energy storage, and of the optical cross-section of the light harvesting systems (Carpentier et al., 1985, Buschmann, 1990). Photoacoustic spectroscopy first emerged as a technique for photosynthesis research in the pioneering works of Cahen and Malkin (Malkin and Cahen 1979). Oxygen evolution by leaf tissue can be measured photoacoustically with a time resolution that is difficult to achieve

Photoacoustic measurements can achieve microsecond time resolution and allow determination of fast induction phenomena in isolated reaction centers, photosystems,

Fig. 4. Setup for photoacoustic photosynthesis measurements in air phase on leaf discs and algae collected on filters (Cha and Mauzarall 1992). Both the sample and reference twin

chambers are connected by an air passage to hearing aid microphones.

**3. Photoacoustics and photosynthesis** 

by other methods (Canaani et al. 1988; Malkin 1996).

thylakoid membranes intact cells and leaf tissue (Fig. 4).

change, or **electrostriction.**

**4. A leaves** 

A simple technique based on photoacoustic measurements allowed us to determine the biomass, as well as the efficiency of photosynthesis, for different taxa of phytoplankton in situ (Dubinsky et al., 1998).

The experimental system is shown schematically in Figure 5 and 6 (Dubinsky et al. (1998), Mauzerall et al. (1998), and Pinchasov et al. (2005)).

Fig. 5. Photoacoustic phytoplankton cell (Dubinsky et al., 1998). The reddish algal culture is of *Porphyridium cruentum* and the laser pulse at 560nm.

The laser pulse is incident upon the suspension of algae, whose pigments absorb part of the laser beam. A variable fraction of the absorbed light pulse is stored in the photochemical products of photosynthesis. The remainder of the absorbed light is converted to heat, producing an acoustic wave that is intercepted by a detector (for details see Pinchasov *et al*. 2005). The signal contains a noisy background and later reflections from the walls of the vessel as well as from impedance mismatch within the detector.

Photoacoustics: A Potent Tool for the Study of Energy Fluxes in Photosynthesis Research 263

As seen in Fig. 7, all three algal species showed a sharp decrease in efficiency; by ~50±5% in the P limited, and ~60±5% in the N limited cultures, as compared to the nutrient replete controls (=100%). Fig. 8 shows the light energy storage efficiency under different ambient

Fig. 7. The effect of nutrient limitation on relative photosynthetic efficiency. For each species photosynthetic energy efficiency of the nutrient replete control was taken as 100%. Controls (clear columns) were grown in nutrient replete media, whereas in the -P (gray) and -N (horizontal hatch) cultures, phosphorus and nitrogen were omitted from the medium.

(Pinchasov et al., 2005).

irradiance levels, resulting in an energy-storage curve for *Isochrysis galbana.* 

Fig. 6. Schematic representation of the experimental setup: L - Minilite Q – Switched Nd:YAG Laser, 532 nm, S – beam-shaping slits, BS – beam splitter, PAC – photoacoustic cell with suspension of algae (30 ml), D - stainless-steel photoacoustic detector, containing a 10-mm diameter resonating ceramic disc (BM 500, Sensor, Ontario, Canada), P – low-noise Amptek A-250 preamplifier, A – SRS 560 – low noise amplifier, PD – photodiode, TR - trigger signal, B – background light source, quartz-halogen illuminator (Cole Parmer 4971), O – Tektronix TDS 430A oscilloscope, C – computer.

The use of piezoelectric films acoustically coupled to a liquid sample and a pulsed laser light source increased the time resolution of the photoacoustic technique to the microsecond scale (Nitsch et al., 1988; Mauzerall et al., 1995). Photoacoustic thermodynamic studies have been carried out on isolated photosynthetic reaction centers from bacteria *Rb. sphaeroides* (Arata & Parson, 1981), on PS I from cyanobacteria (Delosme et al., 1994), and on PS II from *Chlamydomonas reinhardtii* (Delosme et al., 1994).

The resulting electric signal PA, is stored and subsequently analyzed on a computer. Thus, the light energy storage efficiency f is determined following Eq. 1.

$$\mathbf{f} = \begin{pmatrix} \mathbf{PA}\_{\text{light}} \text{ -PA}\_{\text{dark}} \end{pmatrix} \begin{pmatrix} \mathbf{PA}\_{\text{light}} \end{pmatrix} \tag{1}$$

PAdark is the photoacoustic signal generated by the weak laser pulse in the dark and PAlight is the signal produced under the same pulse obtained under saturating (~3000 µmole photons m-2 s-1) continuous white light from a quartz-halogen illuminator (Pinchasov 2006).

We illustrate the application of the method by determining the effects of photoacclimation, nutrient limitation and lead poisoning on phytoplankton cultures from different taxa.

### **6. The effect of nutrient limitation on photosynthesis**

We were able to follow the effects of the key environmental parameter, nutrient status, on the photosynthetic activity of phytoplankton. The nutrients examined were nitrogen, phosphorus and iron (Pinchasov at al., 2005).

The algae for these cultures were harvested by centrifugation from the nutrient-replete media in which they were grown, and resuspended in media from which N or P was omitted. Cultures were followed over two weeks and compared for their photosynthetic energy storage efficiency.

Fig. 6. Schematic representation of the experimental setup: L - Minilite Q – Switched

Amptek A-250 preamplifier, A – SRS 560 – low noise amplifier, PD – photodiode, TR - trigger signal, B – background light source, quartz-halogen illuminator (Cole Parmer 4971), O – Tektronix TDS 430A oscilloscope, C – computer.

*Chlamydomonas reinhardtii* (Delosme et al., 1994).

phosphorus and iron (Pinchasov at al., 2005).

energy storage efficiency.

the light energy storage efficiency f is determined following Eq. 1.

**6. The effect of nutrient limitation on photosynthesis** 

Nd:YAG Laser, 532 nm, S – beam-shaping slits, BS – beam splitter, PAC – photoacoustic cell with suspension of algae (30 ml), D - stainless-steel photoacoustic detector, containing a 10-mm diameter resonating ceramic disc (BM 500, Sensor, Ontario, Canada), P – low-noise

The use of piezoelectric films acoustically coupled to a liquid sample and a pulsed laser light source increased the time resolution of the photoacoustic technique to the microsecond scale (Nitsch et al., 1988; Mauzerall et al., 1995). Photoacoustic thermodynamic studies have been carried out on isolated photosynthetic reaction centers from bacteria *Rb. sphaeroides* (Arata & Parson, 1981), on PS I from cyanobacteria (Delosme et al., 1994), and on PS II from

The resulting electric signal PA, is stored and subsequently analyzed on a computer. Thus,

PAdark is the photoacoustic signal generated by the weak laser pulse in the dark and PAlight is the signal produced under the same pulse obtained under saturating (~3000 µmole photons

We illustrate the application of the method by determining the effects of photoacclimation, nutrient limitation and lead poisoning on phytoplankton cultures from different taxa.

We were able to follow the effects of the key environmental parameter, nutrient status, on the photosynthetic activity of phytoplankton. The nutrients examined were nitrogen,

The algae for these cultures were harvested by centrifugation from the nutrient-replete media in which they were grown, and resuspended in media from which N or P was omitted. Cultures were followed over two weeks and compared for their photosynthetic

m-2 s-1) continuous white light from a quartz-halogen illuminator (Pinchasov 2006).

f = (PAlight -PAdark)/PAlight (1)

As seen in Fig. 7, all three algal species showed a sharp decrease in efficiency; by ~50±5% in the P limited, and ~60±5% in the N limited cultures, as compared to the nutrient replete controls (=100%). Fig. 8 shows the light energy storage efficiency under different ambient irradiance levels, resulting in an energy-storage curve for *Isochrysis galbana.* 

Fig. 7. The effect of nutrient limitation on relative photosynthetic efficiency. For each species photosynthetic energy efficiency of the nutrient replete control was taken as 100%. Controls (clear columns) were grown in nutrient replete media, whereas in the -P (gray) and -N (horizontal hatch) cultures, phosphorus and nitrogen were omitted from the medium. (Pinchasov et al., 2005).

Photoacoustics: A Potent Tool for the Study of Energy Fluxes in Photosynthesis Research 265

Fig. 9. The effect of iron concentration on photosynthetic efficiency. Controls (clear columns) were grown in iron replete media containing 0.6 mg L-1. The iron concentration in the ironlimited cultures was (hatched columns, from left to right), 0 mg L-1, 0.03 mg L-1, 0.09 mg L-1,

In our experiments, the exposure of the Cyanobacteria *S. leopoliensis* to different concentrations of lead resulted in some major changes in chlorophyll concentration and

Figure 11 shows the changes in photosynthetic efficiency following lead application. The reduction of photosynthesis reached ~50% and ~80% with 25 ppm and 200 ppm correspondingly. It is important to emphasize that these results are similar in trend to the

and 0.18 mg L-1, respectively (Pinchasov et al., 2005).

photosynthesis Fig. 10 (Pinchasov et al., 2006).

Fig. 8. The effect of nutrient limitation on the photosynthesis – irradiance relationship of *Isochrysis galbana*. Nutrient replete control (-Δ-), phosphorus limited (-◊-),and nitrogen limited (-□-).

The maximal storage in the nutrient replete control was taken as 100% (Pinchasov et al., 2006).

For the iron limitation experiments the algae were cultured in iron-replete media, under the same conditions as in the nitrogen and phosphorus depletion experiments. The photoacoustic experiments were conducted after two weeks in these media. As the iron is progressively depleted, the ability of the three species to store energy decreases Fig. 9.

Fig. 8. The effect of nutrient limitation on the photosynthesis – irradiance relationship of *Isochrysis galbana*. Nutrient replete control (-Δ-), phosphorus limited (-◊-),and nitrogen

The maximal storage in the nutrient replete control was taken as 100% (Pinchasov et al.,

For the iron limitation experiments the algae were cultured in iron-replete media, under the same conditions as in the nitrogen and phosphorus depletion experiments. The photoacoustic experiments were conducted after two weeks in these media. As the iron is progressively depleted, the ability of the three species to store energy decreases Fig. 9.

limited (-□-).

2006).

Fig. 9. The effect of iron concentration on photosynthetic efficiency. Controls (clear columns) were grown in iron replete media containing 0.6 mg L-1. The iron concentration in the ironlimited cultures was (hatched columns, from left to right), 0 mg L-1, 0.03 mg L-1, 0.09 mg L-1, and 0.18 mg L-1, respectively (Pinchasov et al., 2005).

In our experiments, the exposure of the Cyanobacteria *S. leopoliensis* to different concentrations of lead resulted in some major changes in chlorophyll concentration and photosynthesis Fig. 10 (Pinchasov et al., 2006).

Figure 11 shows the changes in photosynthetic efficiency following lead application. The reduction of photosynthesis reached ~50% and ~80% with 25 ppm and 200 ppm correspondingly. It is important to emphasize that these results are similar in trend to the

Photoacoustics: A Potent Tool for the Study of Energy Fluxes in Photosynthesis Research 267

With increasing lead concentration and duration of exposure, inhibition of photosynthesis increases. Since the photoacoustic method yields photosynthetic energy storage efficiency results that are independent of chlorophyll concentration, it means that the observed decrease in efficiency is not due to the death of a fraction of the population, but rather due to the impairment of photosynthetic function in all cells, possibly due to inactivation of

Fig. 11. The effect of lead on relative photosynthetic efficiency of *Synechococcus leopoliensis* 

Fig. 12. An experimental photoacoustic setup, where the pulses previously obtained by a laser were produced by red light emitting diodes. The cuvette with the algal culture faces the LED array, whereas the microphone, visible on the left, is horizontal, placed at the rear

increasing fractions of the photosynthetic units.

(Pinchasov et al., 2006).

window of the cuvette.

decrease in chlorophyll concentration. Most of the decrease seen after the first 24 hours already took place in the first 40 min, and probably even earlier.

With increasing lead concentration and duration of exposure, inhibition of photosynthesis increases. Since the photoacoustic method yields photosynthetic energy storage efficiency results that are independent of chlorophyll concentration, it means that the observed decrease in efficiency is not due to the death of a fraction of the population, but rather due to the impairment of photosynthetic function in all cells, possibly due to inactivation of increasing fractions of the photosynthetic units.

Fig. 10. Relative photosynthetic efficiency of *Synecococcus leopoliensis* (Cyanobacteria) versus irradiance after 7 days of exposure to lead. (■) MFM medium and 0 ppm, (∆) phosphorus free medium (MFM-P) (●) MFM-P and 25 ppm, (□) MFM-P and 50 ppm, (�) MFM-P and 100 ppm, (○) MFM-P and 200 ppm (Pinchasov et al., 2006).

decrease in chlorophyll concentration. Most of the decrease seen after the first 24 hours

With increasing lead concentration and duration of exposure, inhibition of photosynthesis increases. Since the photoacoustic method yields photosynthetic energy storage efficiency results that are independent of chlorophyll concentration, it means that the observed decrease in efficiency is not due to the death of a fraction of the population, but rather due to the impairment of photosynthetic function in all cells, possibly due to inactivation of

Fig. 10. Relative photosynthetic efficiency of *Synecococcus leopoliensis* (Cyanobacteria) versus irradiance after 7 days of exposure to lead. (■) MFM medium and 0 ppm, (∆) phosphorus free medium (MFM-P) (●) MFM-P and 25 ppm, (□) MFM-P and 50 ppm, (�) MFM-P and 100

ppm, (○) MFM-P and 200 ppm (Pinchasov et al., 2006).

already took place in the first 40 min, and probably even earlier.

increasing fractions of the photosynthetic units.

With increasing lead concentration and duration of exposure, inhibition of photosynthesis increases. Since the photoacoustic method yields photosynthetic energy storage efficiency results that are independent of chlorophyll concentration, it means that the observed decrease in efficiency is not due to the death of a fraction of the population, but rather due to the impairment of photosynthetic function in all cells, possibly due to inactivation of increasing fractions of the photosynthetic units.

Fig. 11. The effect of lead on relative photosynthetic efficiency of *Synechococcus leopoliensis*  (Pinchasov et al., 2006).

Fig. 12. An experimental photoacoustic setup, where the pulses previously obtained by a laser were produced by red light emitting diodes. The cuvette with the algal culture faces the LED array, whereas the microphone, visible on the left, is horizontal, placed at the rear window of the cuvette.

Photoacoustics: A Potent Tool for the Study of Energy Fluxes in Photosynthesis Research 269

3. The effects of any environmental stressor, such as temperature, nutrient limitation, high/dim light and pollutants on the photosynthetic capacity of phytoplankton can be

4. Future work is likely to seek the replacement of lasers by LED sources, allowing the development of portable systems suited for field work, including submersible profilers.

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Recently Gorbunov et al., (submitted) were able to conduct photoacoustic measurements on *Chlamydomonas reihardtii* and to determine the allocation of energy to either photosystem by using PSI or PSII deficient mutants. In these experiments the brief exciting pulses hitherto produced by lasers were generated by red light emitting diodes (Fig. 12), and the saturating, continuous light was provided by blue LEDs (Fig. 13).

Fig. 13. The same setup as in fig 12. The blue LEDs provide the saturating, continuous light.

Recently Chengyi Yan et al. (submitted) were able to conduct photoacoustic measurements on *Chlamydomonas reihardtii* and to determine the allocation of energy to either photosystem by using PSI or PSII deficient mutants. In these experiments the brief exciting pulses hitherto produced by lasers were generated by red light emitting diodes (Fig. 12), and the saturating, continuous light was provided by blue LEDs (Fig. 13).

In these experiments the authors also estimated the contribution of electrostriction to the photoacoustic signal by comparing results at room temperature with ones measured at 4 oC, the temperature at which the photobaric signal is eliminated, and electrostiction is singled out.
