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Submitted to Limnology and Oceanography as a NOTE
DRAFT June 17, 1999
Photosynthetic parameters in the benthos measured in situ
with a Scuba-based fast repetition rate fluorometer
Maxim Y. Gorbunov, Paul G. Falkowski and Zbigniew S. Kolber
gorbunov@ahab.rutgers.edu; falko@imcs.rutgers.edu; zkolber@ahab.rutgers.edu
Environmental Biophysics and Molecular Ecology Program
Institute of Marine and Coastal Sciences
Rutgers, The State University of New Jersey
71 Dudley Road
New Brunswick, New Jersey 08901
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Acknowledgements
This research was supported by the Office of Naval Research under Grant # 97PR00617-00. We
would like to thank Zvy Dubinsky, Michael Lesser, and Charlie Mazel for helpful suggestions on
the instrument design, Eli Perel, Kevin Wyman, Steve Boose, Val Myrnyi, Peter Nawrot for
technical assistance, Mike Behrenfeld for helpful comments on the manuscript, and the staff of
Caribbean Marine Research Center at Lee Stocking Island for support during field campaigns.
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Abstract
Benthic photoautotrophic organisms can significantly contribute both the productivity of shallow
tropical coastal ecosystems as well as the remote sensing reflectance. Quantitative assessment of
primary production and the photosynthetic light utilization/dissipation in benthic targets is
complicated however, by taxonomic diversity, spatial heterogeneity, and natural variability in
the local nutrient, irradiance, and temperature regimes. To help overcome these problems, we
developed a diver-operated Fast Repetition Rate (FRR) Fluorometer for in situ measurements of
photosynthetic performance and fluorescence signals in corals, sea grasses, macroalgae, and
algal turfs. Using the SCUBA - FRR fluorometer, these processes can be measured in situ with
high spatial and temporal resolution. Here we describe the instrument design and characteristics
and present representative field results for the photosynthetic performance in various benthic
organisms in a tropical ecosystem.
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Benthic photoautotrophic marine organisms, including corals, sea grasses, macroalgae,
and algal turfs are among the most productive photosynthetic organisms in aquatic ecosystems
(Larkum, 1983; Falkowski and Raven, 1997). However, measurements of photophysiological
responses generally require destructive sample manipulations and are limited in spatial and
temporal coverage. Moreover, systematic sampling of each of the species comprising these local
communities is impractical, or impossible in some protected areas. To overcome these obstacles,
we developed a SCUBA-based FRR Fluorometer with an autofocussing imaging system. The
instrument permits measurements of an extensive suite of photosynthetic parameters based on
fluorescence transients induced by a sequence of brief sub-saturating flashes (Kolber et al.,
1998). Here we describe the concept of the diver-operated SCUBA-FRR instrument and present
field data that characterizes photosynthetic performance of selected classes of benthic organisms.
Instrument description - The SCUBA-based FRR instrument (Fig. 1) uses a bank of 80
high luminosity blue light-emitting diodes (LED NLPB300, Nichia Chemical Industries, Japan)
to excite chlorophyll fluorescence at 460 nm with 30 nm bandwidth. Excitation light is focused
on the target using a Fresnel lense (focal length 5 cm), with a spot size of 15 mm. A computercontrolled LED driver circuitry generates a sequence of flashlets with a pulse duration controlled
from 0.5 µs to 2 µs, and interval of 2.5 µs to 1 ms. Operating at a pulsed current of 300 mA per
LED, the instrument generates about 0.7 W/cm2 of optical power density at a distance of 3 cm
from the output window.
The fluorescence signal is collected from a central, 6 mm diameter, portion of the
illuminated target, isolated by a red cut-off filter (Schott RG665) and an interference filter
(Corion S10-680-R), and detected by an avalanche photodiode module (Hamamatsu C5460). A
small portion of the excitation light is recorded by a PIN photodiode as a reference signal. Both
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the fluorescence and reference signals are integrated over a single excitation pulse by dual
switched integrators (Burr-Brown ACF2101) and digitized by 12-bit analog-to-digital converters
(LTC1410, Linear Technology). By using an avalanche photodiode as a detector and integrating
the fluorescence signal in the analog mode, the instrument operates with a high signal-to-noise
ratio, allowing the acquisition of fluorescence transients in a single excitation sequence (see
Fig.2), a criterion of crucial importance for diving operations.
The excitation protocols and data acquisition are controlled by an embedded Digital
Signal Processing circuitry based on an ADSP-2181 microprocessor (Analog Devices),
interfaced to a PC/104 computer board (486DX4 100 MHz, Ampro Computers). Up to 2,000
fluorescence transients can be stored in the on-board Flash Memory Card (SanDisk PCMCIA
Type II, 40 Mb). A compact black and white video camera (Marshall Electronics) is
incorporated into the instrument, allowing the diver to monitor the target in real-time. A frame
grabber (CX-100-30, Imagenation Vision System Specialists) captures the image simultaneously
with the fluorescence measurements. A LCD screen installed on the front panel displays both the
video signal from the CCD camera and the RGB signal from the on-board PC. A pair of
orthogonal, off-axis IR laser diodes (wavelength 780 nm, optical power 5 mW) is incorporated in
the viewfinder to precisely control the distance to target. Using an underwater keyboard
comprising twelve piezo-keys (Tschudin & Heid Inc., #7020), a diver manipulates the instrument
and annotates the acquired data. Photosynthetically available radiation (PAR), temperature, and
depth are simultaneously measured by a LiCor 2 underwater quantum meter, a thermistor, and a
pressure gauge, all incorporated within the instrument.
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Results - SCUBA-FRR fluorometers were used during the Coastal Benthic Optical
Properties (CoBOP) field program at Lee Stocking Island, in the Bahamas, during May 1998 and
1999 and January 1999 to study a variety of benthic targets, including corals, seagrass, algal turfs
and sediments. Over two thousand measurements were obtained both in situ and in laboratory
sea tables. Typical FRR fluorescence profiles measured on zooxanthellate corals, the seagrass,
Thallasia testudinum, and surficial sediment are shown in Fig. 2. The corresponding
photosynthetic parameters calculated using the procedure described in (Kolber et al., 1998) are
presented in Table 1. These results indicate that different benthic photoautotrophs have
characteristic photosynthetic signatures. Zooxanthellae, the symbiotic dinoflagellates living in
coelenterate hosts, are characterized by moderate functional absorption cross-sections, but low
quantum yields of photochemistry (Fv/Fm = 0.39). The algal turf, comprising in this region of
cyanobacteria, dinoflagellates and diatoms, have the highest PSII and high photochemical
activity (Fv/Fm = 0.50). Finally, seagrasses have small functional cross-sections and very high
quantum yields of photochemistry in PSII (Fv/Fm up to 0.70 - 0.73). Such a high level of
variability in photosynthetic parameters among benthic objects reflect differences in the
molecular structures of the photosynthetic apparatus, as well as in a higher level of morphology.
FRR fluorometry permitted us to explore environmental factors that potentially affect the
rates of photosynthetic energy conversion (Kolber et al., 1990; Falkowski and Kolber, 1995;
Kolber et al., 1993, 1994; Behrenfeld et al., 1999), such as nutrient availability and ambient light.
Nutrient availability and ambient irradiance are the major natural factors directly controlling
photosynthetic activity in the ocean (Falkowski and Kolber, 1995). Under nutrient-replete
conditions the Fv/Fm ratio averages at 0.65 in wide variety of phytoplankton species, independent
of growth irradiance (Falkowski and Kolber, 1995). Nutrient (e.g. nitrogen or iron) limitation
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leads to a characteristic decline in Fv/Fm, accompanied by a rise in the Fo fluorescence level
(Kolber et al., 1988). In zooxanthellae isolated from corals and cultivated on nutrient-replete
media, we observed a range of Fv/Fm value from 0.62 to 0.66, similar tonutrient-replete
phytoplankton (Kolber et al., 1988). Measurements in a wide variety of zooranthellate corals,
however, revealed an average Fv/Fm of 0.39 ± 0.07 (n = 350), well below the level characteristic
for nutrient-replete cells. The low efficiency of photochemical energy conversion in PSII is
likely due to nitrogen deficiency of zooxanthellae in hospitace (Falkowski et al., 1993). In
contrast to symbiotic zooxanthellae, seagrasses display quantum yields of photochemistry in PSII
in a range of 0.70 ± 0.04, suggesting an absence of nutrient limitation. The highest level of
variability in the quantum yields of photochemistry in PSII was observed in macroalgae: Fv/Fm
ratio varied from 0.50 up to 0.75, reflecting a high level of variability in nutrient status of
macroalgae in benthic environment.
Light represents another environmental factor directly controlling photosynthetic
efficiency in natural ecosystems. Variations in ambient irradiance induce complex
photoadaptive responses in a photosynthetic apparatus, directed to optimize light utilization at
low irradiances, while limiting the adverse effects of overexcitation at high irradiances (Baker
and Bowyer, 1994; Falkowski and Raven, 1997). These processes can be quantified using FRR
fluorescence. Fig. 3A shows FRR fluorescence profiles measured on the zooxanthellate coral M.
faveolata at various levels of ambient irradiance. Corresponding light-induced changes in the
photosynthetic parameters are presented in Fig. 3B. As the ambient light intensity increased,
both actual (F’) and maximum (Fm’) fluorescence yields decreased due to non-photochemical
quenching of chlorophyll fluorescence (the prime character indicates the measurements are made
under ambient light). The variable fluorescence component (F’ = Fm’ - F’) also declined due to
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combined influence of photochemical and non-photochemical quenching, reducing the quantum
yield of photochemistry in PSII (F’/Fm’). The non-photochemical quenching, representing
thermal deactivation of the absorbed excitation energy in the light-harvesting antennae, also led
to a reduction in PSII. Under supra-optimal irradiance, when photosynthesis is saturated,
photoinhibition of PSII may have also contributed to the light-induced decline in the
fluorescence yields. These observations, together with our in situ measurements suggest that
under bright light ( 1000 mol quanta m-2 s-1), e.g. in shallow waters, up to 20-40 % of the
reaction centers of PSII in symbiotic corals may be transiently down regulated.
Fluorescence-derived parameters measured as a function of irradiance allow calculation
of the rates of photosynthetic electron transport and reconstruction of photosynthesis versus
irradiance (P-E) curves (Kolber and Falkowski, 1993). We express the rate of electron transport
through PSII as follows:
Pf = E PSII F’/Fm’
(1)
Fitting the Pf versus E curve (Fig. 3B) with a model dependence Pf = f(E, , Pmax) permits
retrieval of important photosynthetic parameters, namely, , the initial slope of the Pf-E curve,
and Pmax, the maximum rate of electron transport (Webb et al., 1974; Jassby and Platt, 1976; Platt
et al., 1980; Falkowski and Raven, 1997). The light-saturation parameter, Ek, which represents
an optimal irradiance on the P-E curve, is defined as:
Ek = Pmax / 
(2)
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The maximum turnover rate of photosynthetic electron transport (1/) can be assessed as the
product (Falkowski, 1992):
1/ = Ek × PSII
(3)
As an example, using the FRR fluorescent data presented in Fig. 3, the following values of the
photosynthetic parameters were calculated for PSII reaction centers for M. faveolata :  = 135
Å2 e quanta-1, Pmax = 115 electrons s-1, Ek = 140 mol quanta m-2 s-1, and 1/ = 240 s-1. For T.
testudinum the following values were obtained:  = 152 Å2 e quanta-1, Pmax = 415 electrons s-1,
Ek = 475 mol quanta m-2 s-1, and 1/ = 530 s-1.
Measuring a comprehensive suite of photosynthetic parameters, SCUBA-FRR
fluorometry can be used to monitor the physiological status of coral reefs in situ and for studying
the dynamics of reef response to environmental changes. The precision and robustness of
fluorescent measurements achieved in the instrument allows one to follow minute variations in
photosynthetic processes in real-time, making this technology an efficient diagnostic tool for
monitoring the physiological status of coral reefs and other benthic environments. By analogy
with active fluorescent techniques applied to phytoplankton and terrestrial vegetation, we
anticipate that the SCUBA-FRR technology will permit an early indication of harmful
modifications or stresses to benthic photosynthetic organisms, prior to the appearance of
macroscopic changes in the organisms. For example, the instrument can quantify changes in
photosynthetic performance of zooxanthellate corals and other benthic organisms in response to
such phenomena as temperature induced coral bleaching, eutrophication, anthropogenic
pollution, UV exposure, etc. Additionally, understanding the patterns of diel variability of
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fluorescence-derived photosynthetic parameters provides a basis for modeling daily primary
production in coastal environment.
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REFERENCES
Baker, N.R. and Bowyer, J.R. (1994) Photoinhibition of photosynthesis from molecular
mechanisms to the field. BIOS Scientific Publishers Ltd., Oxford.
Behrenfeld, M.J. and Kolber, Z. S. (1999) Widespread iron limitation of phytoplankton in the
South Pacific ocean. Science, 283:840-843
Falkowski, P.G., Dubinsky, Z., Muscatine, L., McCloskey, L. (1993) Population control in
symbiotic corals. BioSci. 43: 606-611
Falkowski, P. G. and Kolber, Z. S. (1995) Variations in chlorophyll fluorescence yields in
phytoplankton in the world oceans. Aust. J. Plant Physiol. 22: 341-355
Falkowski and Raven (1997) Aquatic photosynthesis, pp. 375. Blackwell Scientific Publishers,
Oxford
Jassby, A.D. and Platt, T. (1976) Mathematical formulation of the relationship between
photosynthesis and light for phytoplankton. Limnol. Oceangr. 21:540-547
Kolber, Z., Zehr, J., Falkowski, P.G. (1988) Effects of growth irradiance and nitrogen limitation
on photosynthetic energy conversion in Photosystem II. Plant Physiol 88: 72-79
Kolber, Z. and Falkowski, P.G. (1993) Use of active fluorescence to estimate phytoplankton
photosynthesis in situ. Limnol. and Oceanogr. 38: 1646-1665
Kolber, Z. S., Barber, R. T., Coale, K. H., Fitzwater, S. E., Greene, R. M., Johnson, K. S.,
Lindley, S., Falkowski, P. G. (1994) Iron limitation of phytoplankton photosynthesis in
the equatorial Pacific Ocean. Nature 371: 145-149
Kolber, Z., Prasil, O., and Falkowski, P.G. (1998) Measurements of variable chlorophyll
fluorescence using fast repetition rate techniques: defining methodology and
experimental protocols. Biochem Biophys Acta 13 67: 88-106
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Larkum, A.W.D. (1983) The primary productivity of plant communities on coral reefs, In:
Perspectives on Coral Reefs, D.J. Barnes ed., B.Clouston, Australia.
Platt, T., Gallegos, C.L., and Harrison, W.G. (1980) Photoinhibition of photosynthesis in natural
assemblages of marine phytoplankton. J. Mar. Res. 38:687-701
Webb, W.L., Newton, M., and Starr D. (1974) Carbon dioxide exchange of Alnus rubra: a
mathematical model. Oecologica 17:281-291
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Table 1. Symbols and abbreviations used throughout the text.
FRR
Fast Repetition Rate (fluorometry)
PSII
Photosystem II
PSII
functional absorption cross section for PSII (Å2)
Fo, Fm
Minimum and maximum yields of chlorophyll-a fluorescence measured after dark
adaptation (relative units)
Fv
Variable fluorescence ( = Fm - Fo);
Fv/Fm
Maximum quantum yield of photochemistry in PSII, dimensionless
p
“Connectivity factor” defining the exciton energy transfer between individual
photosynthetic units, dimensionless
Qa
time constant for photosynthetic electron transport on the acceptor side of PSII
(Qa reoxidation) (s)
F’, Fm’
Actual and maximum yields of chlorophyll-a fluorescence measured at ambient
light (relative units)
F’/Fm’
Quantum yield of photochemistry in PSII measured under ambient light
E
Irradiance (mol quanta m-2 s-1)
Pf
Rate of photosynthetic electron transport through PSII (e s-1)
Pmax
Maximum rate of electron transport (e s-1)

Initial slope of the Pf-E curve (Å2 e quanta-1)
Ek
Light-saturation parameter (mol quanta m-2 s-1)
1/
Maximum turnover rate of photosynthesis (s-1)
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Table 2. Fluorescent and photosynthetic parameters calculated from the FRR profiles presented
in Fig.2.
Fv/Fm
PSII (Å2)
Fm (a.u.)
p
Qa (s)
Coral
0.38 ± 0.01
410 ± 20
610 ± 5
0.15
530 ± 100
Sediment
0.55 ± 0.01
530 ± 20
350 ± 2
0.30
475 ± 80
Seagrass
0.73 ± 0.01
190 ± 6
455 ± 5
0.55
560 ± 50
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Figure captures.
Figure 1. Block diagram of the SCUBA-based Fast Repetition Rate Fluorometer.
Figure 2. Chlorophyll fluorescence transients measured with the FRR protocol on the
zooxantellate coral Montastraea cavernosa, the seagrass Thallasia testudinum and algal turf on
sediment. On the first phase (saturation protocol) a series of 64 sub-saturating flashlets of 1.5 s
duration was used to cumulatively saturate PSII within 150 s. A magnitude of the rise in
fluorescence yield is determined by the quantum yield of photochemistry in PSII (Fv/Fm), while
the rate of the fluorescence rise is proportional to the functional absorption cross section of PSII
(PSII). Upon cessation of the saturation protocol, the fluorescence yield decreases, reflecting
kinetics of electron transfer on the acceptor side of PSII. A series of the weak flashes of 0.5 s
duration at 100 to 800 s intervals was used to monitor the relaxation kinetics.
Figure 3. (A) FRR fluorescent transients measured in the coral Montastraea faveolata at various
levels of ambient light. (B) Light-induced changes in photosynthetic parameters calculated from
the FRR profiles.