Abstract
The purpose of this experiment was to determine if there is a
relationship between lowered nitrate levels in seawater and subsequent
degradation of phycobilins in Rhodymenia pacifica when grown under
adequate light. Phycobilins, accessory photosynthetic pigments, are often
degraded under nitrate stress because of their high nitrogen content. In this
experiment, the subtidal red alga R. pacifica was placed in tanks of artificial
seawater with varying nitrate concentrations (0, 10, and 20 uM), and the
phycobilin levels of these thalli were traced over five weeks. Phycobilin
levels in the thalli in all treatments decreased significantly among weeks and
treatments. Thalli given no nitrate had lower phycobilin levels than the 10
and 20 uM treatments, whose levels decreased at approximately the same rate.
Additional thalli were grown in natural seawater with identical experimental
conditions; their phycobilin levels decreased less markedly over time. All
thalli except for those in natural seawater began to turn green at their tips
after approximately two weeks; this green tissue was very weak and degraded
when it was touched. Chlorophyll a levels of all samples remained constant
throughout the experiment, and there was no growth of any thalli.
Elemental analysis of the tissues was performed; there was a significant
increase in percent carbon in all treatments over time, but no significant
changes in percent nitrogen. In conclusion, there is a definite trend which
shows that decreased nitrate concentrations do lower phycobilin
concentrations over time. However, there presence of green thalli tissue
suggests that there may have been other factors involved as well.
Introduction
This experiment examined the relationship between phycobilin
concentration in Rhodymenia pacifica, a red algae, with nitrate levels in the
ambient seawater. Nitrogen and light intensity are the two limiting factors
for algal growth in the ocean. When nitrate concentration of the ambient
water is low, both cyanobacteria (blue-green algae) and red algae are faced with
a tradeoff. If the light intensity is very low, the algae need all of their
phycobilins to capture enough light for photosynthesis so that they may
survive. However, if the light intensity is strong enough, the algae can
afford to degrade some of their phycobilins, which are nitrogen rich accessory
photosynthetic pigments. Nitrogen from the phycobilins is used in other
metabolic functions, such as growth and regulating enzymes involved in
photosynthesis (Hanisak, 1983). Because the main absorbance ranges of
phycobilins are in regions of light where chlorophyll cannot absorb, the
degradation of these pigments affects the thallus' ability to use those other
wavelengths (Gannt, 1990). The degradation of phycobilins causes the tissue
to change from a deep red or blue-green (for red algae and cyanobacteria,
respectively) into either a lighter red-yellow or a light green-yellow,
depending on the species and the level of nitrate stress (Collier, 1992)
Several studies of cyanobacteria and red algae have examined the
effects of nitrate stress. When the cyanobacterium Synechococcus was
deprived of nitrogen, it rapidly turned to a bleached yellow as its phycobilins
were degraded. Cell growth slowed dramatically, and there was no synthesis
of new chlorophyll molecules (Collier, 1992). In the red alga Corallina
elongata, phycobiliprotein synthesis occurs under low irradiance only when
there is sufficient nitrogen present. Conditions of saturating irradiance and
limiting nitrogen resulted in degradation of the phycobilins (Vergara, 1993a).
In other work with Gelidium sesquipedale, Vergara (1993b) found that
phycobilins were an internal nitrogen reserve for the algae and that the
concentration of these pigments was related to ambient nitrate levels. It
should be noted that red algae are not capable of fixing elemental nitrogen,
they must obtain nitrogen in the form of nitrate (NÖ3) or ammonium (NH4)
(Hanisak, 1983). Absorbed nitrate and ammonium are rapidly used once
absorbed; there is less than 1 umole-cm-3 in the cytosol of red algae (Reed
1990).
There are several phycobilins present in red algae; variation in the
relative amounts of each result in different thalli colors for different species.
In nearly all species, the phycobilins are abundant enough that they mask the
presence of chlorophyll a. Unlike chlorophyll, however, these pigments are
water soluble and are attached to a protein; thus the name a phycobiliprotein.
These are grouped together as phycobilisomes on the outer surfaces of the
thylakoid membrane in chloroplasts (Bold & Wynne, 1978).
During El Nifio years, it has been observed that Rhodymenia pacifica
in shallow (7m) water changes from its deep red hue into a sickly yellow (J.
Watanabe, personal comm.). El Nifio conditions result when the nutrient
poor ocean surface layer is deeper than usual; thus when upwelling occurs,
only the surface water is recirculated. This results in warmer, nutrient poor
water along the California coast. El Nifios occur approximately every five
years, so this phenomenon is very significant in the life cycles of most marine
organisms.
Thus, the primary purpose of this experiment was to determine
whether bleaching of Rhodymenia thalli occurred as a response to nitrate
limitation or as a response to other factors, including increased temperature
and/or light. In particular, by maintaining water at the same temperature as
ambient seawater and giving the thalli sufficient light, the effects due to
nitrate stress alone could be measured.
Rhodymenia is a slow growing subtidal red algae which is common off
the coast of Central California. Thalli of this species grow to several
centimeters in length, and by the end of the summer they are overgrown
with small invertebrates such as bryozoans and polychaetes. Rhodymenia
grows in large clusters attached to rocks on the ocean floor. Rhodymenia
thalli were collected and then grown in artificial seawater with differing
amounts of nitrate so that the concentration of phycobilins over time could
be determined through simple extractions of the pigments. The most
abundant phycobiliprotein by far in Rhodymenia is R-phycoerythrin; thus,
the levels of this pigment were tracked over time. By measuring factors such
as chlorophyll content, growth, and percent carbon and nitrogen, as well as R
phycoerythrin over the course of a month, it was hoped that trends in some
or all of these would become apparent as thalli were subjected to varying
levels of dissolved nitrate.
Materials and Methods
Algae Collection and Culture
Rhodymenia pacifica thalli were placed in twelve clear plastic tanks
" and these tanks were
which had base dimensions of approximately 13",
covered with clear thin plastic sheets. Each tank contained approximately
twenty grams wet weight of thalli, immersed in three liters of either artificial
seawater with one of three known concentrations of nitrate (0, 10, 20 uM) or
natural seawater with ambient levels of nitrate (approx. 20 uM). There were
two replicates per treatment.
Thalli were collected during April 1994 by using SCÜBA at depths of
6.5m (referred to as "pale") and 10m ("dark") from the Hopkins Marine Life
Refuge (HMLR) in Pacific Grove, CA. There was a slight but noticeable
difference in the colors of the thalli at different depths; shallow (pale)
specimens were a lighter red than their deeper (dark) counterparts. Thus,
these were placed in separate tanks. Dark thalli were placed in tanks with 0,
10, and 20 uM NÖg as well as in natural seawater. Pale thalli were placed in
tanks with 0 or 20 uM NO3. The artificial seawater was made from Sigma’s
Sea Salts and Provasoli’s enrichment solution (Carolina Biological Supply,
Technical Report). At the end of the month, additional thalli were harvested
from 6.5m and 10m at approximately the same locations as experimental
thalli to determine their pigment concentrations. During the spring, ambient
levels at the HMLR range from 20 to 25 uM NO3 (Watanabe & Phillips,
unpubl. data). Three times, prior to daily addition of nitrate, water samples
were taken from selected experimental tanks and frozen for later nitrate
analysis. All tanks were immersed in ambient-temperature running natural
seawater; this ensured that the tanks' water temperature was never more
than 2°C higher than the surrounding water, which varied from 12 to 13°C.
The artificial seawater was changed every four to five days, with nitrate added
daily. The average nitrate uptake rate was estimated to be half of the Vmax
for Gracilaria, a red algae, from Hanisak (1983), and all nitrate in a tank was
assumed to have been used in twenty-four hours. These rates were used to
estimate quantities of nitrate that were required to maintain the desired
experimental conditions. Each morning, nitrate was added to tanks so that
the initial concentrations were 40 uM or 20 uM. Thus, the daily average
concentrations were 20 uM or 10 uM. Small air stones were placed inside
each tank to maintain adequate mixing of the water. Shade cloth was placed
on top of all of the tanks so that the maximum light intensity that the algae
experienced was not more than 400 uEm-2s-1, which is slightly lower than the
maximum light intensity at 10m deep over the past three years at HMLR
(Watanabe & Phillips, unpubl. data). Most thalli in the tanks had small
amounts of fouling organisms growing on them; sections of the thalli with
these organisms were not used in any of the procedures listed in the
following sections.
Extraction of Pigments
Tissue samples were taken weekly from each tank and analyzed for
chlorophyll and phycobiliprotein content in the following manner. For
chlorophyll extractions, approximately 1.5g of tissue was frozen in liquid
nitrogen, ground in a mortar and pestle, and then ground for ten to fifteen
minutes in a ground glass tissue grinder with 4ml acetone. The resulting
mixture was spun until the supernatant was no longer cloudy. It was
analyzed in a Hewlett Packard 8452A diode array spectrophotometer, and the
concentration of chlorophyll was determined according to Jeffrey and
Humphrey (1975). Phycobiliproteins were extracted from 0.25 g of tissue and
then frozen in liquid nitrogen, ground in a mortar and pestle, and then
ground with 4ml of O.1M phosphate buffer, pH 6.8 until no color remained in
the tissue fragments. Concentration of R-phycoerythrin, the main
phycobiliprotein in Rhodymenia, was determined from absorption data from
the spectrophotometer (Lobban et al, 1988; Beer & Eschel, 1985). After several
weeks, it became clear that pigment loss was occurring mostly at the tips of
the thalli, rather than uniformly across the thalli. Thus, samples for week
four were taken specifically from the tips.
Other Procedures
One plant from each tank was tagged and its blotted wet mass was
taken weekly to determine growth rates. At the end of the month, the
condition of all remaining thalli was determined by estimating by eye the
percent of algae which had turned from a thick, deep red into a thin, sickly
green in each tank. This was to provide an independent measurement of the
degradation of phycobilins by Rhodymenia. At weeks zero, two, and four,
one to two grams of tissue were dried overnight at 60’C, ground, and placed in
a Leeman Labs 240XA elemental analyzer to determine the percent
composition of carbon, hydrogen, and nitrogen for each sample.
Data Analysis
Data were analyzed by a two-way ANÖVA with time and NO3
concentration as fixed factors.
Results
Growth and Appearance of Thalli
Over the course of a month, there were several noticeable changes in
the appearance of Rhodymenia thalli in tanks with artificial seawater.
Although the thalli in the two tanks with natural seawater did not show any
visible differences in color or texture; thalli in all other tanks began to turn
green at the growing tips. The green tissue then slowly spread down the
thalli. This green tissue was much thinner and disintegrated easily when
touched. Consequently, the tissue samples which were weighed each week
remained constant or decreased in mass as the green tissue broke off of the
thalli (Fig. 1). As a result, there was little or no growth of thalli during the
course of the experiment.
At the end of the experiment, each tank was analyzed for percent of
thalli which had turned a sickly green (Fig. 2). It should be noted, however
that none of the algae was green at the beginning of the experiment. During
the course of the experiment, the dark and the pale thalli which had not yet
turned green became indistinguishable in color. The dark thalli's color faded
slightly so that it became the same shade as the pale thalli
Assays of Chlorophyll and R-Phycoerythrin Content
During the first four weeks, chlorophyll a levels all remained constant
at approximately 0.O6mg-g-1 fresh weight of tissue (Fig. 3). Data from dark
and pale thalli have been averaged in these figures unless otherwise noted.
There were no significant differences, neither among nitrate treatments nor
among weeks (ANÖVA, p».62).
R-Phycoerythrin levels did change over the course of the experiment.
Initially, the dark tissue had more pigments than the pale colored tissue (1.95
vs 1.41 mg/g fresh weight). However, concentrations in all thalli decreased,
including those in the natural seawater (Fig 4). There were significant
differences between the nitrate treatments as well as between the weeks
(Pk.005). Samples harvested from the ocean at the end of the experiment
had nearly identical phycobilin levels to those obtained initially (dark thalli
had an average of 2.025 mg/g wt, and pale an average of 1.597 mg/g wt).
Thus, there was no similar ocean trend, for either the pale or the dark thalli.
Composition of Algal Tissue
The percentage of carbon in the thalli gradually increased over the
month of the experiment (Fig 5). ANOVA results indicated that there was a
significant difference between weeks (Pk.05), but not between nitrate
treatments. Samples taken from the ocean at the end of the experiment had
similar percent composition to those taken at the start, for both carbon and
nitrogen (24.36% carbon for dark and 22.99% for pale thalli; 2.955% nitrogen
for dark and 2.805% for pale thalli).
Levels of nitrogen in the thalli did not change significantly during the
experiment, either between weeks or treatments (Fig. 6). However, when the
pale tissue is examined separately, it is obvious that the thalli in the 20 uM
NO3 treatment increased in percent nitrogen, while that in the 0 uM NO3
tanks hardly increased at all (Fig 7). Although this difference was not
10
statistically significant (p20.18), probably because sample sizes were limited, it
is worth noting.
Discussion
Ambient nitrate levels and sufficient light affect the concentration of
phycobilins in the thalli of Rhodymenia pacifica (Fig. 4). It is clear that,
under reduced nitrate, their phycobilin concentration decreases. Those thalli
which did not receive any nitrate at all had the lowest levels by the end of the
experiment. This suggests that the bleaching in R. pacifica observed during
El Ninnos is at least partly caused by breakdown of phycobilins in response to
lowered ambient nitrate levels with sufficient light. Thalli which were pale
at the beginning of the experiment demonstrated this the most. Due to their
initial lower phycobilin levels, they must have been much more susceptible
to decreased nitrate concentrations. The dramatic decrease in pigment levels
suggests that they were being degraded very rapidly in the thalli to obtain the
nitrogen for needed metabolic functions.
However, why the thalli in 10 and 20 uM NÖ3 treatments had similar
phycobilin concentrations throughout the experiment is unclear.
Rhodymenia may absorb nitrate from the water at a faster rate than
estimated; thus both treatments may have been nitrate stressed. Nitrate was
added daily, and it is possible that the thalli absorbed it so quickly that after a
few hours no nitrate was left in the water. Water samples taken twenty-four
hours after nitrate was added were frozen and will be analyzed. Results from
these samples will demonstrate if all the nitrate was absorbed each day or not.
If Rhodymenia absorb nitrate faster than expected from the original nitrate
calculations, this would explain the lowered phycobilin levels in all of the
artificial seawater treatments. It is surprising, however, that even the thalli
in natural seawater showed decreased phycobilin levels over time.
Because all tissue samples that were weighed weekly remained
constant or decreased in mass, the algae must have been under some degree
of stress; although the tissue grows slowly, some increase in mass would have
been expected. Stress was also obvious from the thalli which were turning
green. This change in tissue was similar to the normal changes of nitrate
deprivation; in the ocean thalli become blotched with green or yellow tissue,
and in this experiment the tissue became green and disintegrated very easily.
Green sections had, obviously, little or no detectable phycobilins. In the
natural seawater tanks there was little green tissue; this suggests that the
thalli were not stressed to the same degree as those in the artificial seawater.
Possibilities include nitrate stress as well as the lack of another nutrient
needed for algal growth, since green tissue appeared in much larger quantities
in the artificial seawater tanks. However, there is no straightforward way to
determine what exactly was different in the artificial seawater without further
experimentation.
The significant increase in percent carbon among weeks is consistent
with algae which are nitrogen limited (Hanisak 1983). However, it is not
known if the additional carbon was incorporated into carbohydrates or used
for producing new structural tissue. When the measured growth rates are
considered (Fig. 1), it becomes clear that the thalli were deteriorating, not
growing. Thus, the increase in carbon was most likely incorporated into
carbohydrates. All of these factors discussed demonstrate that, under
sufficient light and limiting nitrate, phycobilin levels in Rhodymenia pacifica
1
will decrease. Thus, during an El Nino, at least part of the observed
phycobilin decrease is due to these conditions.
Acknowledgments
I would like to thank my advisor, Jim Watanabe, for advice on my
project throughout the quarter. Special thanks goes to the Monterey Bay
Aquarium, in particular Roger Phillips, for his support as well as the use of
his lab space, spectrophotometer, elemental analyzer. Deb Robertson’s
insights on experimental design and pigment extraction were of great help to
me. Stuart Thompson was kind enough to let me use his lab for the
preparation of the artificial seawater. In conclusion, I am grateful to all those
who went diving with me to collect the Rhodymenia.
13
Literature Cited
Beer, S. & A. Eshel. 1985. Determining phycoerythrin and phycocyanin
concentrations in aqueous crude extracts of red algae. Aust. J. Mar.
Freshw. Res. 36, 785-792.
Bold, H. & M. Wynne, 1978 Introduction to the Algae: Structure and
Reproduction. Englewood Cliffs: Prentice-Hall, Inc. p. 450-452.
Collier, J. & A. Grossman, 1992. Chlorosis induced by nutrient deprivation in
Synechococcus sp. strain PCC 7942: Not all bleaching is the same. Journal of
Bacteriology. 174 (14): 4718-4726.
Gannt, E. Pigmentation and Photoacclimation. 1990. In,Biology of the Red
Algae, eds. K. Cole & R. Sheath. pp. 203-219. Cambridge University Press.
Hanisak, D. The Nitrogen Relationships of Marine Macroalgae. 1983. In,
Nitrogen in the Marine Environment,, eds E.J. Carpenter & D.G. Capone
pp. 699-730. Academic Press.
Jeffrey & Humphrey. 1975. Biochem. Biophys. Phlanz. 167:191-194.
Lobban, C, Chapman, D & B. Kremer. 1988. Experimental Phycology: A
Laboratory Manual. pp. 127-128. New York: Cambridge University Press.
Reed, R. Solute Accumulation and Ösmotic Adjustment. 1990. In,Biology of
the Red Algae, eds. K. Cole & R. Sheath. pp. 203-219. Cambridge University
Press.
Vergara, J. 1993a. Effects of nitrate availability and irradiance on internal
nitrogen constituents in Corallina elongata. Journal of Phycology.
29(3):285-293.
Vergara, J., Niell, F. & M. Torres. 1993b. Culture of Gelidium sesquipedale
(Clem.) Born. et Thur. chemostat system: Biomass production and
metabolism responses affected by nitrogen flow. Journal of Applied
Phycology. 5(4): 405-415.
Figure Legends
Figure 1: Weight of Samples of Rhodymenia pacifica
Figure 2: Percent of Green Thalli in Different Nitrate Treatments
Figure 3: Chlorophyll Levels in Rhodymenia pacifica
Figure 4: R-Phycoerythrin Levels in Rhodymenia pacifica
Figure 5: Percent Carbon in Rhodymenia pacifica
Figure 6: Percent Nitrogen in Rhodymenia pacifica
Figure 7: Percent Nitrogen in Paler Rhodymenia pacifica
Figure 1


1 -

—

g
Week

20uM NO3
—
1OUM NOS
—n
OuM NO3
—g seawater
Figure 2

20
10
NO3 leve

ae
Pale
2
0
0

OE
Source
NO3
Week
NO3 x Week
Error
Figure 3
0.08
0.06 -
0.04:
0.02


000 +
Week
Analysis of Variance
Degrees of
Mean-Square F-Ratio
Freedom
0.00000947
0.608
0.00000161
0.103
0.198
0.00000308
24
0.00001557

0.616
0.902
0.947
E OuM NO3
E 1OUM NO3
20uM NO3
seawater
Figure 4
5
82
EE
A


Week
Analysis of Variance
Degrees of
Source
Mean-Square
Freedom
NO3
0.376
Week
0.759
NO3 x Week
0.142
Error
0.064
32
Post-Hoc Tests
Week 4: Natural seawater vs. 0 uM NÖ3 (p=.039)
0 uM NO3:
Week 1
Week 2
Week 4
p=0.00
p=0.027

F-Ratio
5.879
11.583
2.223

0.003
0.000
0.047
Week 3
p=0.016
D OUM NO3
E 1OuM NO3
20uM NO3
seawater
Source
NO3
Week
NO3 x Week
Error
Figure 5


Week
Analysis of Variance
Degrees of
Mean-Square F-Ratio
Freedom
0.780
0.932
4.762
3.987
1.110
1.326
1.194
24
0.517
0.032
0.385
E OUM NO3
E 1OuM NO3
20uM NO3
seawater
Source
NO3
Week
NO3 x Week
Error
Figure 6


Week
Analysis of Variance
Degrees of
Mean-Square F-Ratio
Freedom
1.875
0.098
1.197
0.062
0.109
2.097
0.052
24

0.161
0.319
0.091
□ OUM NO3
E 1OUM NO3
20uM NO3
seawater
Source
NO3
Week
NO3 x Week
Error
Figure 7
3.6

3.5 -
3.4 -

3.3

3.2
3.1
3.0
2.9+
4
Week
Analysis of Variance
Degrees of
Mean-Square F-Ratio
Freedom
0.095
2.532
0.035
0.940
0.011
0.281
0.037
P
0.187
0.387
0.624
—
— OUM NO3
— 20uM NO3