ABSTRACT
In situ instruments report a daily periodicity in fluorescence (f) and beam
attenuation (c) in the ocean. The daily changes are hypothesized to result from changes in
phytoplankton physiology. To determine the relation between f,c, chlorophyll (chl), and
photosynthetically active radiation (PAR), time series measurements were made on cultures
of phytoplankton which were exposed to different light conditions. F normalized to chl
(f*) varied inversely to PAR. c* displayed daily changes that did not correlate to PAR. The
f response is well above experimental noise and its amplitude is two times as strong as the
in situ signal. The c* response is not significantly outside of the experimental error, and
it's amplitude is within the range of the in situ signal. Absorption (a) was also measured.
A* did not correlate to PAR or time, but steadily decreased as cultures grew older.
conclude c' varies due to b* (scattering per chl.) with a circadian rhythm, and f' varies in
response to PAR. This is consistent with in situ data. Further, I present what is believed to
be the first reported empirical method to measure chl from both f and PAR.
INTRODUCTION
Stimulated fluorescence and beam attenuation may be powerful tools to measure in
situ chlorophyll and biomass in the open ocean. If perfected, these methods would
provide oceanographers with several orders of magnitude more data to study primary
productivity and algal ecology. However, these signals are not yet reliable tools for
ecological parameters. This is due in part to diel oscillations of the signals which are
hypothesized to result from changes in phytoplankton physiology (Dickey 1992; Stegman
et al 1992; Stramska and Dickey 1992). Understanding and predicting these diel cycles is
an essential step in the determination of accurate algal biomass from shipboard and moored
fluorometers (Prezelin and Ley 1980; Mauzerall 1972; Abbott 1982).
Stimulated fluorescence in the ocean has been correlated to phytoplankton
populations (Prezelin and Ley 1980; Keifer 1973a; Abbot et al. 1982). Recently, a diel
pattern of this signal has been observed from drogues, ships, and moorings in the
Sargasso Sea, Monterey Bay, and the equatorial Pacific. (Dickey 1992; Chavez pers.
comm.; Abbot et al. 1990) The daily changes in fluorescence may be due to a
combination of phytoplankton physiology, growth, grazing, mixing, demography, and
dissolved organic sources of variability. A model of the diel patter due to physiology
would decouple signals due to physiology from signals due to actual changes in biomass.
In this paper, cultures of phytoplankton exposed to sunlight consistantly had a
daily minimum in fluorescence equal to 50% of their daily maximum (a factor of 2
difference between night and day). Beam attenuation decreased to 80% of its maximum (a
factor of 1.25 between night and day). These data are consistent with in situ
observations, and provide the basis for an empirical determination of chlorophyll from
strobe-stimulated fluorescence and PAR.
Theory:
Fluorescece is a measure of photons which were absorbed by pigments but
whose energy was not shunted into photosynthesis. When plants absorb light, there are
three sinks for the energy: vibrations, fluorescence and redox reactions. Vibrations (heat)
are insignificant, and redox reactions are synonymous with photosynthesis (Arnon 1968).
In the steady state, conservation of energy requires:
Eabsorbed - Efluoresced + Ephotosynthesis
Thus, changes in fluorescence must be correlated with changes in absorption and
photosynthesis (Gentry et al 1989; Kolber et. al. 1990; Falkowski et al 1986, 1988;
Mauzerall 1972; Bannister and Weidmann 1984)
The emitted photon has a discrete wavelength which depends on the available
quantum states of the pigment molecules. The most intense fluorescence emission from
phytoplankton is at 685 nm, corresponding to the Stokes shifted decay of an electron
excited by a 670 nm photon - right in the middle of a peak in the absorption spectra of
Chl.
Beam attenuation is a measure of how many photons starting out toward a target do
not make it there. Attenuated light is either absorbed or scattered. Thus, beam attenuation
is the sum of absorption and scattering (Table 1)
c =a+b
If one could collect all the scattered and unattenuated light that passed through sample, one
could measure of absorption. Similarly, if one excluded all scattered light, one can
measure beam attenuation. The spectrophotometer measures all of a and part of b,
because it collects some but not all of the scattered light.
Measured absorption = a + x*b where Oxx1.
If all of the scattered light is collected, x = 1; if none of the scattered light is collected,x -
0. x depends on the volume scattering cross section and the geometry of the detector
(Bricaud 1983; Roach 1974). The Shibada technique for measureing absorption with a
spectrophotometer uses a diffusing plate to redirect forward scattered light. (Mitchell and
Kiefer 1987; Shibada 1954; ) Approximate attenuation can be measured by positioning
the sample far from the detector so that the apperture eclipses most of the scattered light. If
a and c are measured, b is known as well. (Keifer pers. comm.; Chavez pers. comm.)
a and b depend on phytoplankton physiology: Changes in absorption indicate
changes in pigment concentration, quantum state, and package effect. Changes in
scattering evidence changes in particle size, density, and index of refraction.
Materials and Methods:
Dunalliela , Phaeodactylum , and Amphidinium were grown in sterile enriched
sea water. Cultures in their exponential phase were subjected to four different lighting
conditions (Table 2). To assess the relationship between phytoplankton populations and
optical phenomena, five properties were measured: Chlorophyll a (hereafter referd to
simply as chlorophyll). fluorescence, attenuation, absorption and PAR.
Chlorophyll concentration was quantified using acetone extractions and a Turner
fluorometer (Holm Hansen et al. 1965). Absorption was measured with a 10 cm cuvette
and a diffusing plate close to the detector of a diode array spectrophotometer (Shibada
1954; Mitchell 1988). Attenuation was measured using a 1 cm cuvette far from the
detector (Bricaud 1983). Fluorescence was measured using a Sea Tech strobe stimulated
fluorometer. PAR was measured with a photodiode callibrated to detect quanta of light.
Over a 48 hour time series, fluorescence was measured 22 times, chlorophyll was
measured 11 times, and spectra were measured 5 times. The sampeling rate was not
constant.
Fluorescence, absorbtion and attenuation were normalized to chlorophyll and
analyzed on a UNIX workstation. The spectra were smoothed and integrated.
A Sea Tech fluorometer and a Sea Tech transmissometer have been recording data
at 15 minute intervals from a mooring in Moneterey Bay since January 16, 1992. The
fluorometer is the same instrument as used with the cultures. The transmissometer
opperates at 660 nm and returns a voltage. Voltage is converted into beam c by the
following equation:
C =-4*log(VDC/5)
The mooring also has a PAR sensor which samples every 10 minutes. However, there is
no instrument to measure chlorophyll on the mooring.
Results:
The mooring fluorometer shows long term (3- 15 day) events which are
hypothesized to represent spring phytoplankton blooms. (Figure 1) There is a short
term (1 day) signal superimposed on this signal. (Figure 2). The diel cycle has a
maximum at night (usually either one hour after sun set or one hour before sunrise) and a
minimum near noon.
The amplitude of the diel cycle correlates with the mean fluorescence value; when
the mean signal is high, the variations (the difference between daily maximum and
minimum) are large e.g. the value of the mean correlates with the envelope of the raw -
mean timeseries (Figure 3). The minimum value is consistently half the maximum value
for each day (+- 10%). This is true even when the daily oscillations grow larger during
the phytoplankton bloomes e.g. the envelope for the raw/mean time series is nearly
constant (Figure 3)
The raw/mean fluorescence timeseries has an inverse functional relationship with
PAR both within days and between days (Figure 4). The raw/mean timeseries is
hypothesized to represent f* in situ, because mean fluorescence has been shown to
correlate with chlorophyll (Chavez 1991). This relationship is apparent in cloudy days as
the fluorescence is higher than on sunny days. Sometimes the fluorescence cycle is
perturbed for one or more days after a cloudy day regardless of PAR. (Figure 2)
The beam attenuation increased during the spring bloom, yet it was also high in
January and February. The daily cycles in care less consistent than the fcycles, c
oscillates over the course of the day, and during the bloom the daily variations are larger.
yet they are not as clearly related to PAR as fis. (Figure 2)
Ten different time series were made with cultures: Dunalliela in the dark (DD).
incubator (DI, windowsill (DW), sunlight (DS); Phaeodactylum: (PD), (PW), (PD.
and (PS); and Amphidinium: (AD), and (Al) (Table 3) (Figures 5 to 11).
The cultures grew at different rates under each condition. In general, chlorophyll
concentration grew at an exponential rate when cultures were exposed to light, and not at
all when they were kept dark. The rate of growth was not proportional to light intensity
The best environment for Dunalliela was the window sill, and Phaeodactylum grew
quickest in the incubator. Amphidinium died in the direct sunlight, and grew best in thi
incubator. These rates may be related to the change in fluorescence on the days after a
cloudy day
Normalized fluorescence (f*) was constant for DD, PD, AD, DI, AI, There is a
small change in f* (inverse to PAR) in PI, PW, and DW. There is a clear inverse
relationship between f“ and PAR for DS and PS. Raw f correlated well with chlorophyl
for specemins in the dark and in the incubator, but not for samples in the sun (Figure 12).
f* for Dunalliela = .013 volts/ (ug chl/1) when it is dark adapted (i.e. when the f
has adapted to dark conditions). f* for phaodactylum = 035 volts when it is dark
adapted. It took 20 minutes for Dunalliela and 30 minutes for Phaodactylum to adapt to
changes in PAR (Figure 13).
The PS f* timeseries is more of a square shape than DS. This observation, and
the fact that the PI f“ timeseries changed with PAR, while the DI timeseries did not.
suggest different species have different sensitivities to PAR and that the fluorescence
response to PAR can be saturated
The normalized absorbtion spectra for each species had charactaristic shapes. The
spectrum for Dunalliela is similar to Phaeodactylum and Amphidinium except that it is
higher at the longer wavelenths. This offset appears in the c'spectrum as well. This is
consistent with the observation that Dunalliela appears green to the human eve and
Amphidinium and Phaeodactylum appear red-brown. Absorbtion spectra from all
species in all different light conditions decreased with time irrespective of PAR or time of
day (Figure 14 a). The exceptions to this decreasing trend are in PW and PS.
Measurements ofc do not have a decreasing trend over time as the absorption
spectra do. Instead, on average, c changes over the time of day inversely with respect
to expected daylight; it appears to be correlate with time of day, not PAR (Figure 14 b)
(Table 4). For combined timeseries of Dunalliela this trend is larger than two
standard deviations. For Phaeodactylum the trend is not outside one standard deviation.
(Figure 15).
Discussion
A similar diel cycle in fluorescence is observed in cultures and in the field. In the
cultures chlorophyll does not oscillate over the day as fluorescence does. This supports
the hypothesis that diel variations are primarily due to changes in phytoplankton
physiology, not growth, grazing or mixing.
Normalized fluorescence varies inversely to PAR, regardless of time of day. This
suggests the cycle in fluorescence is related to PAR, not circadian rhythms. The
observation that f“ takes an average of 20 minutes to adapt to changes in PAR, means
a timeseries of f* lags approximately 20 minutes behind a timeseries of PAR. Thus,
the correlation between f* and PAR can be made tighter by taking a running average of
PAR over 20 minutes and shifting PAR ahead in time by 20 minutes. Qualitatively
correcting for this lag time makes a small but noticable improvement in the uncertainty of
f* as a function of PAR (Figure 4)
These data provide the basis for an empirical way to measure mean fluorescence
from instantaneous fluorescence
corrected f = (.0005)*(shifted and smoothed PAR)"(mean f) + f
This is mathematically equivalent to adding a constant fraction of the irradiance peaks to
the fluorescence troughs in a time series of f*. The coefficient .0005 comes from the slope
of f* vs PAR in situ (Figure 4). This coefficient was only qualitatively determined. A
statistically derrived higher order fit to Figure 4 may provide a better correction
algorithm. The mean f must be included in the correction because the magnitued of the
in situ diel oscillations varies directly with the mean (Figure 3). It should be noted that
this is just a step in the direction of a real-time empirical correction, because this model
requires the time averaged f. Yet this correction tightens the relationship between f and
mean f by a factor of 2 (Figure 16).
This is useful because mean f correlates well to chlorophyll concentration (Chavez
1991) and chlorophyll concentration correlate to rates of primary productivity (Balch et.
al 1992). Primary productivity rates are the key element in the models of the biological
carbon budget in the ocean. Moreover, primary productivity in the ocean may be the
major sink for atmospheric carbon dioxide. Thus, instantaneous fluorescence
measurements may provide useful data for current issues in global oceanography if
correction algorithms such as the one presented here are made more consistant. Despite
the many unknowns, this correction algorithm already correlates instantaneous
fluorescence with carbon budgets by a factor of 2 more certainty.
Specifically, this correction algorithm is useful for transects made with a
fluorometer, because one cannot observe a full daily cycle in each parcel of water from a
moving ship. Even if an accurate mean is not known, the emperical relationship to PAR
discussed thus far will indicate the direction of error due to physiological changes in
phytoplankton during in situ observations of f.
The observed perturbation of fluorescence one and two days after a cloudy day may
represent real changes in chlorophyll concentration. This is likely because all
phytoplankton cultures chlorophyll concentrations increased faster when they were not in
direct sun. Thus, on a cloudy day, the increased fluorescence signal is likely to be a
physiological response to reduced PAR, while high f values on the next sunny day are
hypothesized to represent actual increases in chlorophyll.
Thus far, I have discussed ways to correct for the diel cycle in fluorescence. Yet
this cycle may contain useful data. For example, changes in the diel fluorescence cycles
may identify species. The culture results support this hypothesis. Fluorescence changes
in Phaeodactylum were to be more sensitive to PAR than in Dunalliella in the incubator
and unlike Dunalliela the fluorecence response for Phaeodactylum in the direct sun did
not change significantly for PAR above 1000 mE/m2.
These species-specific responses to light may explain some of the more fine points
of the in situ results. For example, the in situ f* (f divided by mean f) does not
quite have a constant amplitude: it is largest at the end of the first of the spring blooms.
This may reflect a higher population of more sensitive phytoplankton, such as
Phaodactylum , at that time in the Monterey bay. Yet, this is a difficult problem,
because many changes: sensitivity, threshholds, and growth rates are all superimposed
on each other. Thus, it is difficult at this point to make significant claims about in situ
species distributions with these remote bio-optical data.
High attenuation in the bay during January and Feburary is hypothesized to result
from suspended sediment load, not phytoplankton, because it does not correlate with the
increase in mean fluorescence, the increase in amplitude of diel fluorescence changes, or
the increase in amplitude of diel transmissivity changes. This is plausable because Winter
is the stormy season in Monetery Bay, and more turbulent waters suspend more sediment.
However, the lack of large diel cycles in attenuation does not necessacerily indicate lack
of phytoplankton because, in the cultures Dunalliela exhibited diel changes in attenuation
while Phaodactylum did not.
In the cultures a* did not change with light adaptation. Thus, E(absorbed) for a
constant amount of PAR (e.g. the fluorometer strobe light) did not change. But
E(fluoresced) did change with light adaptation. Thus, by concervation of energy, a
change in the efficiency of photosynthesis (photons used for photosynthesis/ photons
absorbed) must account for the changes in fluorescence. The changes in a* were two
orders of magnitude smaller than the changes in c*.
c* did change with a circadian rhythm. c* varies inversely to expected PAR (not
actual PAR) in Dunalliela but not significantly in Phaeodactylum. By inference
(because c= a + b) b* also changes inversely to expected PAR. Thus, cell swelling,
change in refractive index or cell division (change from fewer larger cells to more smaller
cells) might explain the change in beam attenuation, because these are the factors that
affect b“. Further, pigment density, quantum state of the pigments, and optical depth of
the phytoplankton cannot be the factors that affect the diel cycle in c*, because thier net
affect would have affected a* on a diel pattern.
However, a* did change over time, though not with a diel cycle. a* decreased;
i.e.: the observed percent change in a is less than the percent change in chl at each
successive measurement in the experiment regardless of PAR or time of day.
(al - a2)a (cl-c2)c
Three working hypotheses may explain how a* decreases over time. 1) some absorption
may be due to things other than chlorophyll in the sample which are not in the blank.
This is possible because the blank was enriched filtered sea water, not filtered culture.
Thus, there may be biogenic solutes or particles in the cultures which do not contain
chlorophyll but do absorb light. 2) The relationship of a to chl may be non linear,
This may be true, because when the cultures are thicker there is more scattered light, and
scattered light travels through a longer path length in the cuvette, therefore, it has more
opportunity to be absorbed. This is especially true in the 10 cm cuvette (as opposed to the
1 cm cuvette used for attenuation). 3) older plants may absorb less light per chlorophyll.
This is likely, because as Dunalliela grew older, it was observed to have more
chloroplasts per plant, and these chloroplasts may eclipse each other.
Conclusion
In situ diel variations in fluorescence are primarily due to changes in phytoplankton
physiology, specifically an increased quantum yeild of photosynthesis due to increases
in PAR. An empirical correction based on this hypothesis explains half of the daily
variation in fluorescence
Absorption decreases as cultures grow older, but it does not vary enough to
affect attenuation. Attenutaion exhibits a circadian rhythm in Dunalliela but not in
Phaodactylum. By inference, scattering changes inversely to expected PAR. Thus,
cell swelling, change in refractive index or cell division might explain the change in beam
attenuation.
Chlorophyll measurements from in situ fluorescence can be more accurate by a
factor of two by using the emperical equation: corrected(f) = raw(f) +.0005* (smoothed
and shifted PAR)“mean(f). Further work is needed to accurately determine chlorophyl
from remote bio-optical parameters, but this relationship is likely to be refined by
correlating fluorescence, PAR, and attenuation on daily and instantaneous time scales.
When this is done, the carbon budget for open ocean water masses may be better
monitored with the aid of a fluorometer. It is also possible that attenuation and
fluorescence will be able to identify species of phytoplankton by remote techniques
Acknowledgements:
Francisco Chavez was a superb mentor for this project. Thank you, Francisco, for
letting me choose the direction of this projet, guiding me whenever I got stuck, and
always being ready to pick up the pieces and look at them as a consistant whole. Keep
hanging in there!
Kurt Buck, the man who lives with phytoplankton, was a well-spring of
knowledge regarding observations and species-specific habits.
Jason Smith shared his sophistocated culture and knowledge of phytoplankton.
Tim Pennington - thanks for taking me on the boat.
Annie Reese - thanks for proofreading this project through to the very end.
REFERENCES
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Baker, E.T. and Lavelle, J.W. 1984. The effect of partical size on the light attenuation
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of photosystem II during steady-state photosynthesis in eucaryotic algae. Biochem.
Biophys. Acta 933 (1988) 432 -443.
Falkowski, P.G., Wyman, K. Ley, A.C., and Mauzerall, D.C. 1986. Relationship of
steady state photosynthesis to fluorescence in eucariotic algae. Biochem. Biophys. Acta
849 (1986) 183-192.
Gently, B., Briantais, J.-M., and Baker, N.R. 1989. The relationship between the quantum
yeild of photosynthetic electron transport and quenching of chlorophyll fluorescence.
Biochem. Biophys. Acta. 990 (1989) 87 -92.
Keifer, D.A. 1973. Fluorescence Properties of Natural Phytoplankton Populations. Mar.
Bio. 22, 263-269.
Kolber, Z., Wyman, K.D. and Falkowski, P.G., 1990. Natural variability in
photosynthetic energy conversion efficiency: a field study in the Gulf of Maine. Limlnol
Oceanogr. 35 (1) 72 -79
Mauzerall, D. 1972. Light-induced changes in Chorella, and the primary photoreactions
for the production of oxygen. Proc. Nat. Acad. Aci. USA. 69 (6) 1358 - 1362.
Mitchell, B.G., and Kiefer, D.A. 1988. Chlorophyll a specific absorption and
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Figure Legends:
Figure 1. Time series of surface PAR, fluorescence at 10 meters depth and transmissivity
at 10 meters from the mooring in Monterey Bay from January 16 to May 20, 1992.
Figure 2. Time series of surface PAR, fluorescence at 10 meters depth and transmissivity
at 10 meters from the mooring in Monterey Bay from May 7 to May 14, 1992.
Figure 3. Raw, mean, raw - mean, and raw/mean timeseries for fluorescence measured
by the mooring in Monterey bay from March 20 to May 5, 1992.
Flgure 4. raw/mean fluorescence vs. PAR measured by the mooring in Monterey bay
from March 20 to May 5, 1992.
Figure 5. Fluorescence vs. mean fluorescence (determined by a one day running
average). [Mean fluorescence correlates well with chlorophyll (Chavez 1991).)
Figure 6. Time series of fluorescence, chlorophyll, fluorescence normalized to
chlorophyll and PAR for Dunalliela in the sun (DS)
Figure 7. Time series of fluorescence, chlorophyll, fluorescence normalized to
chlorophyll and PAR for Phaodactylum in the sun (PS)
Figure 8. Time series of fluorescence, chlorophyll, fluorescence normalized to
chlorophyll and PAR for Dunalliela in the incubator (DI)
Figure 9. Time series of fluorescence, chlorophyll, fluorescence normalized to
chlorophyll and PAR for Phaodactylum in the incubator (PI)
Figure 10. Time series of fluorescence, chlorophyll, fluorescence normalized to
chlorophyll and PAR for Dunalliela in darkness (DD)
Figure 11. Time series of fluorescence, chlorophyll, fluorescence normalized to
chlorophyll and PAR for Phaodactylum in darkness (PD)
Figure 12 a. Chlorophyll concentration vs. raw fluorescence for a two day timeseries of
phytoplankton cultures in direct sun
Figure 12 b. Chlorophyll concentration vs. raw fluorescence for a two day timeseries of
phytoplankton cultures in an incubator
Figure 12 c. Chlorophyll concentration vs. raw fluorescence for a two day timeseries of
phytoplankton cultures in darkness
Figure 13. Time series of fluorescence and par for Phaodactylum , Dunalliela , and
Amphidinium moved from darkness to direct sun at 11:00 am.
Figure 14 a. Absorption spectra for Dunalliela in the incubator.
Figure 14 b. Attenuation spectra for Dunalliela in the incubator.
Figure 15 a. Mean and standard deviation for attenuation vs. expected light in Dunalliela.
Figure 15 b. Attenuation vs. expected light for each specemin of Dunalliela.
Figure 15 c. Attenuation vs. expected light for each specemin of Phaeodactylum.
Figure 16 a. Mean fluorescence vs. raw fluorescence measured by the mooring in
Monterey bay from March 20 to April 14.
Figure 16 b. Mean fluorescence vs. emperically corrected fluorescence measured by the
mooring in Monterey bay from March 20 to April 14.
Table 1
SYMBOL
chl
PAR
MEANING
UNITS
absorption
meter -1
scattering
meter -
meter -1
attenuation
raw fluorescence
volts
chlorophyll
ug/l
absorption / chlorophyll
m2/mgchl
scattering / chlorophyll
m2/ mg chl
attenuation / chlorophyll
m2/ mgchl
fluorescence / chlolophyll
m2/ mg chl
Photosynthetically active radiation mE/ m
Table 2
LOCATION
Darkness
Incubator
Window.
Sunlight
LIGHT (maximum)
mEm?
35
mEm?
750 mE/m2
1800 mE/m2
Table 3
SPECIMEN
ABBREVIATION
DS
Dunalliela in the sun
Dunalliella in the incubator
DD
Dunalliella in the dark
PS
Phaeodactylum in the sun
Phaeodactylum in the incubator
Phaeodactylum in the dark
PD
Amphidinium in the incubator
PD
Amphidinium in the dark
Table 4 a
Hyothesis: c is high inversely proportional to expected light
Supporting Data Refuting Data
Specemin
DD
DW
PD
PW
Table 4 b
Hypothesis: c is directly proportional to expected light
Supporting Data
Specemin
Refuting Data
DD
DW
PD
PW
Figure 1.
2.0 x103 -
1.6 x103.
1.2 x103 -
8.0 x102 -
4.0 x102 -
-0.0 x10°
4.0 -
2.0 -
-.0 -
5.00 -
3.00
bhtee




a
60.
Julian Day in 1992
120.
2.0 x103
1.6 x103
1.2 x103
8.0 x102
40 x102
00 2100
4.0-
2.0 -
-.0 -
4.50
4.00
3.50 -
128.0
Hn
E

130.0
PAR at the surface
Surface fluorometer

Surface transmissometer

132.0
Julian Day in 1992
M
h
W
134.0
Figure 2.
Figure 3.
6.0
raw data
9 4.0
2.0

Mhnst
-.0
1 day running average
4.0 -
20


-.0 -
raw data - 1 day running average
2.0 —
0 oh
o

2.0
-4.0
3.00
raw data / running average
2.00
31.00




-.00 -
80.
90.
100.
110.
Julian Day in 1992
120.
Flgure 4.
3.00 —
F3 days 82 to 126 (all)
fmean(f)
2.00

4
k.
..
**

...*+
.


1.00
1.4.
4



...:



*






.







*

:
*.....*
*...
. . . * ...**
-00 +
2.0 x103
1.0 x103
-0.0 x10°
PAR
Figure 5.
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.* *
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2.

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:






-00 +
2.0
3.0
1.0
raw(f)
10
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14 2102
1.0 x10-2-
6.0 X10-3
200.
100.
0. —
Dunalliela in direct sun
Raw Fluor
* *x
Chlorophyll
*

Normalized Fluor (f*)
**
12
PAR

Fluorescence and CHlorophyll Timéserie.

x
Figure 6.
1.00
80.
40.
0. -L
8.0 x10-2
§ 40 2102
-0.0 x10° —
200.
100.
0. -
Figure 7.
Phaodactylum in direct sun
Raw Fluor
* *

Chlorophyll
*
* *

Normalized Fluor (f*)

X„ *
PAR
Fluorescence and CHlorophyll Timéseries
** *
10
150.
50.
4.0 x10-2 -
2.0 x10-2
-0.0 x10°
80
40
00
Dunalliela in incubator
Raw Fluor
r
Chlorophyll

Normalized Fluor (f*)


„ n
5 -

PAR
Fluorescence and CHlorophyll Timéseries
Figure 8.
1.00 -
100.
4.0 X10
§ 20 2102
-0.0 x100 -
.80
40
-.00 —
Figure 9.
Phaodactylum in incubator
Raw Fluor




Chlorophyll
*


Normalized Fluor (f*


PAR

Fluorescence and CHlorophyll Timéseries
4.00
2.00
400.
3 200.
0. —
2.5 x10-2-
1.5 x10-2
5.0 x10-3 -
80 -
40
-.00 -
Figure 10.
Dunalliela in Darkness
Raw Fluor
Chlorophyll

Normalized Fluor (f*)
+
+

--- --- - -
PAR
— ——- — — — — —



Fluorescence and CHlorophyll Timéseries
4.00
2.00 —
20
5 40 210 2
2.0 x10-2—
-0.0 x100
80 -
40
-.00 —
Figure 11.
Phaeodactylum in Darkness
Raw Fluor

Chlorophyll
Normalized Fluor (f*)
* .

——— — — — —-
PAR
— — - -—— — ——



Fluorescence and CHlorophyll Timéserie
Figure 12 a.
Figure 12 b.
200 -
100.—
Figure 12 c.
200. -
§ 100.
-00
FLO vs. CHL in direct sun



Dunalliela

Phaeodactylum
ge
raw FLUORESCENCE (voltage)
FLO vs. CHL in the incubator
Dunalliela
Amphodinium






Taw FLUORESCENCE (voltage)
FLO vs. CHL in the dark
Amphodinium

Dunalliela
Phaeodactylum

raw FLUORESCENCE (voltage)
Figure 13.

Phaodactylun
2.00 -

Dunalliella

X
Amphidinium
00
8.00


TIME (hours)

PAR




10.00
Figure 14 a.
4.0 x10-2 -
-0.0 x10° -
300
Figure 14 b.
4.0 x10-3
00 r10
-4.0 x10-3 —
300
1
DI 3
DI 5

fne

R

DI 5

—

514


D1 3
DI 2
700
wavelength



wavelength
4.0 x10-2 -
2.0 x10-2 -

DARK
-.00
Dunal
la
Expected light
LIGHT
2.00

Figure 15 a.
Figure 15 b.
8.0 x10-2 —
6.0 x10-2 -
5 4.0 x10-2-
2.0 x10-2 -
-0.0 x10° -
8.0 x10-2 -
6.0 x10-2 -
5 4.0 x10-2 -
2.0 x10-2 -
-0.0 x10° -


DUNALLIELA
DD


DI
OFF
ON
Expected PAR

DS

DW
OFF
ON


Expected PAR
Figure 15 c.
2.0 x10-3 -
PD
1.6 x10-3 -
1.2 x10-3 -
8.0 x10—4 -
4.0 x10-4-

-0.0 x10° -

12 -
5 06
04 -
00 OFF O
Expected PAR
PHAODACTYLUM



PW
ON
OFF
Expected PAR
Figure 16 a.
Figure 16 b.
2.00
1.00-
00 -
2.00 -
1.00
-.00 —
++
snt i ti

.


+
n i



(


1.0
3.0
2.0
Raw F
++











1.0
2.0
3.0
Corrected F