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Seasonal variation in the regulation of phytoplankton
salt-marsh estuary
by nitrogen and grazing in a
Alan J. Lewitus
Belle W. Baruch Institute for Coastal Research, Baruch Marine Laboratory, University
Georgetown, South Carolina 29442
of South Carolina, PO. Box 1630,
Eric T. Koepjler
Coastal Carolina University,
Marine Science Department, Conway, South Carolina 29526
James T. Morris
Department of Biological
South Carolina 29208
Sciences, Belle Baruch Institute for Coastal Research, University
of South Carolina, Columbia,
Abstract
In North Inlet, a tidally dominated salt-marsh estuary near Georgetown, South Carolina, the summer chlorophyll
maximum correlates with an annual peak in ambient NH,+ concentration. This relationship suggests that phytoplankton population growth during the summer bloom is limited by factors other than nutrient supply, because
NH,+ is the major inorganic nitrogen source available to phytoplankton in North Inlet, and phosphorus should not
be limiting (N : P is generally -7). We tested the hypothesis that phytoplankton population growth during the bloom
was controlled by grazing. Natural samples were incubated in treatments designed to differentiate between nutrient
and grazing effects, and time-course changes in total phytoplankton biomass and phototrophic community composition were followed. Marked seasonal differences were observed in the relative contribution of pica-, nano-, or
microplankton to phytoplankton community biomass, as well as the mechanisms controlling phytoplankton population growth. During the summer bloom, phototrophic picoplankton (mostly Synechococcus spp.) and nanoplankton
(mostly flagellates) were relatively abundant, and phytoplankton population growth was unaffected by NH,’ addition, but was greatly stimulated by dilution that reduced microzooplankton grazing pressure. During the winter,
when diatoms dominated the phytoplankton, the response to dilution was relatively minor, while NH,’ addition
significantly stimulated the growth of various phytoplankton groups and total chlorophyll. The results indicate a
seasonal transition in microbial food-web trophic structure and regulation in North Inlet estuary. During the summer,
microzooplankton grazing is an important factor regulating phytoplankton population growth during the nanoflagellate-prevalent bloom, whereas in the winter, a diatom-dominated community is limited by nutrient supply.
Salt-marsh estuaries along the southeastern Atlantic Coast
of the United States characteristically contain dense stands
of cordgrass, Spartina alterniJlora, which has long been recognized as the dominant primary producer in these systems.
However, other primary producers (e.g. macroalgae, edaphic
algae, phytoplankton) can be significant sources of organic
matter in these estuaries (Sellner et al. 1976; Pomeroy et al.
1981; Dame et al. 1986; Sullivan and Moncreiff 1988;
Pinckney and Zingmark 1993~). For example, in North Inlet
estuary (near Georgetown, South Carolina), where phytoplankton alone reportedly contributed up to one-fourth of the
total system primary production (Pinckney and Zingmark
1993b; based on comparisons with aboveground Spartina
production), the sum of algal contributions to primary productivity may surpass vascular plant production on an annual
basis (Vemberg 1993). Furthermore, trophic transfer efficiencies of these primary producers may be greater than
from vascular plants. For example, although Spartina production exceeds algal production in the Sapelo Island, Georgia, salt marsh (Pomeroy et al. 1981), stable isotope measurements indicated approximately equal contributions of
vascular plant and algal carbon to the estuarine fauna
(Haines 1976; Peterson and Howarth 1987; Sullivan and
Moncreiff 1990), suggesting that a greater percentage of algal carbon is transported to higher trophic levels. Despite
the potential importance of phytoplankton to trophic dynamics in these warm, temperate salt marshes, regulation of phytoplankton communities in these estuaries is poorly understood.
North Inlet, a bar-built, high-salinity salt-marsh estuary,
represents an end-member of estuarine types in that it is
tidally dominated with little freshwater input. NH,’ is consistently the major inorganic nitrogen source in North Inlet,
which results from restricted freshwater input and the combined influences of oyster nutrient regeneration, subtidal
Acknowledgments
The authors thank Lucie Aplan, Julie Brader, Doug Carter, Kenneth Hayes, Leroy Humphries III, Gloria Lyons, Kevin Malone,
Mark Osberg, J. Vince Osborne, Scott Sellers, and Bonnie Willis
for technical assistance. This research was funded by grants OCE
93-15663 and DEB 95-09057 from the National Science Foundation, and Estuarine Habitat Research Program grant 92 from the
National Oceanic and Atmospheric Administration. Data also were
obtained from the NSF Long-Term Ecological Research program
(grant DEB 92-11774) and the NOAA-funded Winyah Bay/North
Inlet National Estuarine Research Reserve program (grant DIR9119725). Contribution 1160 of the Belle W. Baruch Institute for Marine Biology and Coastal Research.
636
Phytoplankton
4
IS
3
10
2
5
I
regulation
Table
(triplicate
hexmide
alone or
637
1. Cross-matrix of eight standard incubation treatments
cultures per treatment). Antibiotics addition (Ab), cycloaddition (Cyc), or dilution treatment (Dil) were applied
in combination with NH,’ addition.
Substrate
addition
None
None
NH,’
Control
NH,’
Competition/predation
treatment
Antibiotics
Cycloheximide
Dilution
Ab
Ab + NH,’
CYC
Cyc + NH,’
Dil
Dil + NH,’
0
0
MJJASONDJFMAM
Month (1981-1991)
30
20
10
0
1
M
J
J
1.
A
:l
1
SONDJF
1994
M
Month
A
M
1995
Fig. 1. A. Monthly mean concentrations and standard errors of
Chl a (m), NH,,+ (O), NO,- (0), and PO,‘- (A) from water samples collected at 1000 h daily from 198 1 to 1991 from Oyster Landing. Data provided by the NSF Long-Term Ecological Research
program. B. The mean and standard deviation of NH,+ (0) and Chl
a (m) from water collected biweekly from May 1994 through May
1995 from Oyster Landing at or near a morning slack low tide.
Data provided by the NOAA North Inlet/Winyah Bay National Estuarine Research Reserve (NERR) monitoring program. Arrowheads indicate the sampling periods used in this study.
phyll, we hypothesized
that phytoplankton
population
growth during the summer bloom in North Inlet is controlled
by grazing. The idea that grazing can affect phytoplankton
production in warm, temperate salt-marsh estuaries is not
new (e.g. Pomeroy et al. 1981). Based on the abundance of
heterotrophic protists, as well as their high rates of bacterivory (on natural populations and cultured bacterioplankton)
and algivory (on laboratory cultures of phytoplankton),
Sherr et al. (1991) speculated that grazing by ciliates and
nanoflagellates could explain low nanophytoplankton abundance during the winter in Sapelo Island estuary. Also, Litaker et al. (1988, 1993) found that microzooplankton grazing was an important factor in the reduction of Chl a
concentrations from spring to summer in the Newport River,
North Carolina, estuary. These studies, however, used grazing as an explanation for relatively low phytoplankton abundance, and not as a major factor limiting population growth
during primary bloom formation.
We used bioassay experiments to determine the relative
effects of nutrients and microzooplankton grazing on phytoplankton population growth in North Inlet estuary. The
results demonstrated seasonal variability in the mechanism
by which phytoplankton population growth is controlled,
with nutrient limitation predominating during the winter and
early spring, and regulation of growth by microzooplankton
grazers during the summer phytoplankton bloom.
Methods
and episodic runoff from forested wetlands (Wolaver et al. 1988; Whiting and Childers 1989; Dame et al.
1989; Childers et al. 1993). The N:P atom ratio of North
Inlet waters is -7 throughout the year, well below the Redfield ratio, suggesting that nitrogen is potentially limiting to
phytoplankton. However, the seasonal patterns of NH,+ and
chlorophyll in North Inlet argue against nitrogen-limited
growth, at least during the annual phytoplankton bloom,
which occurs during the stmrmer (Fig. 1). In contrast to estuaries where the phytoplankton bloom is nitrogen limited,
and where an inverse relationship between nitrogen and
chlorophyll occurs during the bloom (Fichez et al. 1992;
Magnien et al. 1992; Litaker et al. 1993), the seasonal distribution of the potential limiting nutrient, NH,+, is positively correlated with that of chlorophyll. This relationship suggests that the regeneration and input of NH,+ to the estuary
exceed the NH,+ assimilation capacity of the phytoplankton
community; that is, the phytoplankton bloom is limited by
factors other than inorganic nutrient supply.
Based on the relationship between nutrients and chlorodrainage,
Water was collected in triplicate bottles from the Oyster
Landing site in North Inlet (Houser and Allen 1996) at 1 h
after slack low tide (at -0800 h) on 11 July 1994, 12 September 1994, 8 November 1994, and 20 February 1995. After transport to the laboratory, the water was dispensed into
250-ml sterile tissue culture flasks and incubated under various treatments (see below) designed to differentiate between
microzooplankton grazing and nutrient effects. The samples
were incubated for 72 h on shaker tables under controlled
temperature and light conditions. Temperature was adjusted
to simulate the ambient condition at Oyster Landing (28.O”C
in July, 28.8”C in September, 16S”C in November, 11.O”C
in February), and irradiance was set at 80 pEinst m ? s ’ at
a light/dark cycle simulating ambient conditions.
The triplicate samples were incubated under eight standard treatments (Table 1). The potential stimulation of NH,’
(4 PM) on phytoplankton growth was examined in the presence or absence of two metabolic inhibitors, or in combination with a 20: 1 dilution treatment. The concentration of
Lewitus et al.
0.03 -f
a
0.02 --
1
f
i
0.01 --
0
0.00
I
0.01
1
0.1
Fraction
unfiltered
water
Fig. 2. The apparent growth rate, [t ln(Pp,,-l)]- I, of phytoplankton vs. the fraction of unfiltered seawater for water collected
in July, where I is the incubation time (72 h), PO is the initial chlorophyll concentration, and P, is the chlorophyll concentration after
72 h.
added NH,’ fell within the range of naturally occurring levels (Fig. l), and therefore should not be inhibitory to phytoplankton growth. Metabolic inhibitors were used to evaluate the net effect on phytoplankton growth of reducing the
growth of trophic components that may act as competitor or
predator. The inhibitors included an antibiotic mixture (19
mg liter-’ penicillin, 30 mg liter’ streptomycin, and 60 mg
liter’ neomycin), which was used to depress the growth of
bacterioplankton (a potential competitor for nutrients), or a
eukaryotic inhibitor (10 mg liter-l cycloheximide), used to
decrease the growth of potential grazers of cyanobacteria.
Taylor and Pace (1987) found that 50 mg liter’ cycloheximide did not affect the growth of Synechococcus cultures
after 1 week of incubation.
The dilution treatment was used as a means of reducing
microzooplankton grazing pressure on phytoplankton (Landry and Hassett 1982). A portion of the water from each
sample bottle was filtered using a 0.2-pm polycarbonate
membrane filter, the filtrate was dispensed into culture flasks,
and whole-water aliquots from the remaining sample water
were then transferred into the flasks at 5% of the filtrate
volume. The principle of the dilution technique is that encounter rates between microzooplankton and phytoplankton
prey (and therefore grazing pressure on phytoplankton prey)
are reduced by dilution. Grazing rate can be derived as the
negative slope of the linear relationship between apparent
phytoplankton growth rate (estimated from the rate of
change in chlorophyll over the incubation period) and the
fraction of unfiltered water (e.g. Fig. 2). From repeated experiments involving serial dilution of Oyster Landing water,
we found that the apparent phytoplankton growth rate over
72 h was maximal at 20: 1 dilution, indicating that this treat-
ment achieved the maximum reduction of microzooplankton
grazing pressure on phytoplankton.
The incubating water was sampled daily for Chl a and
phytoplankton composition and abundance. Chl a was determined by fluorometry (Sequoia-Turner model 450 fluorometer with NB440 excitation and SC665 emission filters)
following extraction in 90% acetone by a freeze-thaw method (Glover and Morris 1979). Phytoplankton abundances
were determined on fixed samples (1% glutaraldehyde).
Three size categories were delineated: microplankton (>20
pm), nanoplankton (2-20 pm), and picoplankton (<2 pm).
The phototrophic microplankton were counted either by settling overnight in 24-welled plates (2.85 ml well I), and
counting over the entire well or half of the well area with
an inverted microscope, or they were counted using a Sedgwick-Rafter counting chamber and a compound microscope.
By using epifluorescence attachments, chlorophyll autofluorescence was determined to differentiate heterotrophic and
phototrophic microplankton. Phototrophic nanoplankton and
picoplankton were quantified by epifluorescent examination
of DAPI-stained samples filtered onto 0.2-pm black membrane filters (Porter and Feig 1980). Alternating between ultraviolet and green excitation allowed identification of the
cyanobacterial component of the picoplankton.
Means were compared with a t-test with a significance
level of 0.05.
Results
The seasonal distributions of chlorophyll and NH, + during
the study period were typical of this site in that peak abundances occurred during July-August, followed by sharp declines during late summer and autumn, and minimal concentrations during the winter (Fig. 1). Thus, sampling dates
occurred at the height of the phytoplankton bloom when
NH,’ concentrations were near maximal (July), on the
downslope of the phytoplankton bloom when NH,+ levels
were decreasing (September), and in winter (November) and
early spring (February) when chlorophyll and NH,’ concentrations were relatively low.
The relative abundances of three phytoplankton size-fractions changed with sampling period (Fig. 3). Nano- and picosize classes dominated the total phytoplankton population
during summer, and reached their highest population densities in July and September, respectively. However, in terms
of biovolume (estimated from linear measurements and assuming simple geometric forms), the microplankton and nanoplankton fractions dominated in July, when the picoplankton made up only 1% of the total phototrophic community
biomass (Fig. 3). The relative contribution of picoplankton
to total biovolume was greatest in September. The abundance
of all size-fractions decreased sharply from September to
November, and the relative importance of microplankton to
phytoplankton community biovolume increased. In February, microplankton made up 24% of total phytoplankton
abundance and 77% of community biovolume. Overall, these
data indicate a seasonal shift in the relative contribution of
phototrophic components from predominantly smaller ((20
pm) phytoplankton in the summer to larger (>20 pm) phytoplankton in the winter-early spring.
Phytoplankton regulation
6.E+04
t
‘L
E
;
4.E+04
8
5
;
2.E+04
:
O.E+OO
.-
m
2.E+05
July
Sep
Nov
Feb
W Microplankton
El Nanoplankton
0 Picoplankton
Fig. 3. The mean and standard deviation of (A) abundances and
(B) biomass of phototrophic microplankton (black), nanoplankton
(white), and picoplankton (gray) at Oyster Landing during the sam-
ple periods.
The phototrophic microplankton were composed almost
entirely (>97%) of diatoms (data not shown). The abundance of Cylindrotheca closterium peaked in July, when it
made up 53% of microplankton, and decreased sharply in
successive sampling periods. This diatom was not observed
in February. The phototrophic nanoplankton were dominated
by a mixed assemblage of flagellates, comprising 76% (July)
to 97% (February) of total nanoplankton abundance. The
remainder of the phototrophic nanoplankton were diatoms,
primarily small pennates (e.g. Nitzschia spp.) and Thalassiosira spp. (data not shown). The phototrophic picoplankton
were predominantly (from 95 to 100%) Synechococcus spp.
NH,’ additions resulted in significantly higher 72-h chlo-
639
rophyll concentrations in all sampling periods but July (Fig.
4). The greatest response was observed in November, when
NH,+-enriched flasks contained 7 1% more chlorophyll than
did controls after 72 h. In July, the addition of NH, + to flasks
containing either whole water or diluted water did not affect
chlorophyll levels (Fig. 5A), which indicates that phytoplankton population growth was insensitive to NH, ’ , even
under conditions of reduced microzooplankton grazing pressure. In September and November, additions of NH,’ had
positive effects on chlorophyll in all treatments (control, Fig.
4B,C; dilution, Fig. 5B,C; or antibiotic, Fig. 6B,C), but in
February, only NH,’ addition to whole water had a statistically significant effect on 72-h chlorophyll (Fig. 4D).
In comparing the effect of dilution on chlorophyll concentration, a distinct seasonal trend was observed (Fig. 7).
Relative to whole-water controls, the dilution treatment resulted in a much greater increase in chlorophyll in July. For
example, after 72 h, chlorophyll concentrations were 23-fold
greater than initial levels in the dilution treatment compared
to a 2-fold increase in whole water (Fig. 7C). In fact, chlorophyll in the July diluted samples increased by 168% from
48 to 72 h, while chlorophyll in the whole-water controls
decreased by 27% during the same period (Fig. 7B,C). Similar differences between dilution and control samples were
observed during September, although the increase in chlorophyll in the dilution treatment was less than that in July.
Chlorophyll in September control samples decreased from
24 to 72 h, while chlorophyll in diluted samples increased
during this time. In November, dilution stimulated an increase in chlorophyll relative to controls up to 48 h (Fig.
7A,B), but the 72-h change in chlorophyll did not differ
significantly between treatments (Fig. 7C). In contrast to July
and September, chlorophyll in November whole-water controls increased throughout the experimental period. In February, chlorophyll increased to a greater extent in controls
than in the diluted samples up to 48 h (Fig. 7A,B), and the
72-h increase was similar in each treatment (Fig. 7C). Overall, dilution stimulated chlorophyll production to the greatest
extent in July and September and to a lesser extent in November, and did not stimulate chlorophyll production in February.
In general, the seasonal patterns of NH,’ and dilution effects on chlorophyll were consistent with the effects on phytoplankton abundance (Table 2). In July, NH, ’ addition did
not significantly affect the abundance of nanoplankton or
most microplankton. Asterionella and Chaetoceros populations, however, were stimulated by NH,‘, but these groups
were only -10% of total microplankton abundance. Picoplankton were reduced significantly by NH, ’ addition in
July. NH,’ addition resulted in significant increases in phytoplankton abundance in the other sampling periods; however, whereas phototrophic pica- or nanoplankton growth
was enhanced in September, phototrophic microplankton
were stimulated in November and February. Interestingly,
Asterionella was consistently stimulated by NH., ’ addition
in all sampling periods.
To determine the relative effect of dilution on the growth
of phototrophic components, we compared the 72-h abundance in the dilution treatment with a projected abundance,
which estimates the value predicted if the 72-h rate of
Lewitus et al.
640
100 I
80 --
*
A
July
50
40
30
60 --
20
10
0
80 --
Q
=
50
r"
g
40
2
30
f
20
B
September
November
November
10
0
20
1.5 --
15
1 -10
5
0
24
Time (h)
48
72
Fig. 4. The time-course change in mean and standard deviation of Chl a concentrations of whole-water controls and NH,+enriched samples during (A) July, (B) September, (C) November,
or (D) February. Note that the ordinate range differs between panNH,‘.
els. 0 ----Cl,
control; W -m,
/
0
24Time
I
(h) 48
72
Fig. 5. The time-course change in mean and standard deviation
of Chl a concentrations of diluted and NH,,+-enriched diluted samples during (A) July, (B) September, (C) November, or (D) February. Note that the ordinate range differs between panels. O-0,
dilution + NH,‘.
dilution; n -¤,
Phytoplankton
641
regulation
100
2400
A
24 h
80
60
40
20
0
80 -- September
Itl
6
2400
1900
a
1400
z
0 1
E-
50
900
C
-- November
30-20 --
40
I
3
2400
L
C
72 h
15 --
February
July
O-
0
I
I,
24
Time (h)
72
48
Fig. 6. The time-course change in mean and standard deviation
of Chl a concentrations of antibiotic-treated and NH,+-enriched antibiotic-treated samplesduring (A) July, (B) September,(C) November, or (D) February. Note that the ordinate range differs between
0, antibiotics; U,
antibiotics + NH,‘.
panels. IJ-
change in diluted samples were equivalent to that of wholewater control samples:
Projected abundance = D&C,,,
- C,,,>/C,, + Dr,,,
where D,, is the initial abundance in the dilution
treatment,
Sep
Nov
Feb
Fig. 7. The mean and standard deviation of the rate of change
in Chl a concentration in July whole water controls and diluted
samples over incubations of (A) 24 h, (B) 48 hr, or (C) 72 h. 0,
whole water; n , dilution.
C,,, is the abundance in the whole-water control at 72 h, and
C,, is the initial abundance in the whole-water control. The
dilution treatment is considered to have an effect on phytoplankton growth if the observed 72-h abundance is significantly greater than the projected abundance. Dilution stimulated the growth of phototrophic nano- and microplankton
in July and all three size-fractions in September (Table 2).
Only a few phytoplankton groups were stimulated by dilution in November, and, with the exception of picoplankton,
642
Lewitus
et al.
Table 2. Comparison of the effects of NH,+ addition or dilution on the abundances (cells ml-‘) of various components of the phytoplankton community after 72-h incubations. Values under NH,+ additions represent the mean (n = 3) fractional difference in abundance
between NH,+-enriched and whole-water control cultures, or (NH,+ treatment - Wbole-water control) X (Whole-water control) ‘. Dilution
values represent the mean (n = 3) fractional difference belween the observed abundance in dilution cultures and the predicted abundance
if the rate of change in diluted cultures over 72 h were equivalent to that of whole water control cultures, or (Dilution treatment - Projected
abundance) X (projected abundance)-‘, where projected abundance is based on the 72-h change in whole water controls. Underlined values
indicate significant differences between treatment and whole water controls (NH,’ addition) or treatment and projected abundance (dilution)
(nd (t,) or nd (t,,) indicates that no cells were detected at 0 or 72 h, respectively).
NH,+ addition relative to control
Group/subgroup
Jul
Picoplankton
Nanoplankton
Nanoflagellate
Nano-diatoms
Microplankton
0.20
0.32
-0.28
Cylindrotkeca
Cyclotella
Skeletonema
Asterionella
Nitzschia
Chaetoceros
Ampkiprora
0.24
0.22
-0.08
15
3.2
2.0
0.98
Sep
-0.37
-8.6
0.81
0.68
-1.9
0.49
Nov
Feb
h
Z.E+05
0.22
-0.23
0.30
0.27
0.54
-0.095
-0.12
2.0
20
ii
26
0.43
0.78
0.31
-0.44
-0.076
0.82
-8.9
0.050
0.81
-0.16
-0.30
0.067
-1.9
4.4
1.3
-9.7
-0.41
nd (t,,>
0
0.20
-1.2
-0.90
30
-7.0
=Cycloheximide
z
2%
E
5
1
q Control
E
1 .E+OS
-0
5
2
:
;i
S.E+04
3
E
2
O.E+OO
July
Sw
Nov
Jul
-0.29
the stimulatory effect was much less pronounced. Although
the growth of nano-size diatoms, Cyclotella, and Amphiprora was enhanced by dilution in February, these groups were
a minor component of the phytoplankton community (totaling -10% at the end of the experiment). Whereas Asterionella responded strongly to NH,’ addition, Amphiprora was
stimulated by dilution in all sampling periods, suggesting
selective grazing on this genus.
Cycloheximide was used to further examine the effect of
reduced grazing pressure on phototrophic picoplankton (Synechococcus) abundance (Fig. 8). When compared to wholewater controls, cycloheximide addition had no effect on picoplankton population density after 72 h in July, whereas it
significantly increased abundance in September and Novem-
r
Dilution relative to projected abundance
Feb
Fig. 8. The mean and standard deviation of phototrophic picoplankton abundance after 72 h in whole-water controls and cycloheximide-treated samples during the sampling periods.
u
ii
a3L
13
nat,J
14
not,,)
-3.8
Sep
Nov
-3.8
-7.7
17
15
-w
-0.13
-0.21
0.57
11
0.50
13
ii
i3
33
1.8
9.6
8.5
-
0.11
0.42
-0.37
1.4
0.34
-0.041
-6.9
Feb
-0.53
0.72
0.46
11
).62
nd Cl,,)
15
378
0.44
0.86
- 1.0
-269
ber and decreased abundance in February. Thus, the results
were consistent with the effect of dilution on picoplankton
abundance (Table 2). Seasonal differences in the cycloheximide effect may not represent real changes in grazing pressure, because the inhibitory effect of cycloheximide on
growth and grazing activity varies with eukaryotic species
(Sanders and Porter 1986; B. E Sherr et al. 1986; Taylor and
Pace 1987). However, the likelihood that the cyanobacterial
responses to cycloheximide reflects relative seasonal changes
in grazing pressure is supported by the similar patterns in
dilution response (see also Campbell and Carpenter 1986).
Discussion
In temperate estuaries, the relative proportions of new and
regenerated nitrogen often vary seasonally, and these
changes are associated with modifications in microbial community composition and food-web structure (Furnas 1982;
Officer et al. 1984; Flynn and Butler 1986; Marshall and
Lacouture 1986). Relatively high NH,’ levels during the
summer consistently are linked to a food-web structure in
which small, flagellated phytoplankton outnumber larger diatoms, and material and energy fluxes are mediated principally through bacteria-nanophytoplankton-protozoa
consortia (Parsons et al. 1978; Glibert et al. 1982; Caron et al.
1988; Malone et al. 1988; Glibert et al. 1992; Goldman and
Dennett 1992). The concomitant increase in NH,’ and chlorophyll during the summer in North Inlet was accompanied
by a pronounced increase in phototrophic nanoflagellate
abundance and relative contribution to biomass (Fig. 3). The
summer-to-winter transition from excess NH,‘, when summer NH,’ supply exceeds demand and a nanoflagellate phytoplankton community prevails, to low NH, ’ and a diatom-
Phytoplankton
dominated community is consistent with a seasonal change
from a microbial loop to a microphytoplankton-based
food
web.
The seasonal difference in microbial food-web type is supported not only by the relative change in nutrient supply and
phytoplankton community composition, but in the contrasting mechanisms regulating
phytoplankton
population
growth. Our results indicate a relative transition from the
predominant influence of microzooplankton grazing in the
summer to that of nutrient supply in the winter and early
spring.
July: Regulation by grazers-Phytoplankton
population
growth was unresponsible to increased NH,+ supply during
the July bloom due to the importance of regulatory factors
other than NH,‘. Light availability has been inferred as a
growth-limiting factor in North Inlet and other turbid, shallow, warm temperate salt-marsh estuaries (Williams 1972;
Pomeroy et al. 1981; Vernberg 1993) but co-regulation by
grazing has rarely been considered (Pomeroy et al. 1981;
Sherr et al. 1991; Litaker et al. 1993). Our results, based on
a comparison of time-course responses of diluted treatments
vs. whole-water controls, demonstrate a dramatic stimulation
of chlorophyll production by dilution in July. With the exception of picoplankton, the population growth of all phototrophic groups was stimulated by dilution. The results suggest a strong influence of microzooplankton
grazers in
limiting the phytoplankton bloom during the summer.
Although the great disparity in results between nutrient
amendment and dilution treatments on phytoplankton population growth in July suggests that microzooplankton grazing is an important regulatory factor, the experimental protocol limits our understanding of the nature of this control.
Because the prevalence of small flagellates and microzooplankton is indicative of a microbial loop community structure, nutrient flow and prey-predator interactions are bound
to be complex and reciprocal. Although the net positive response of phytoplankton populations to dilution may be a
result of reduced grazing pressure directly on phytoplankton,
other explanations for the stimulatory effect are possible. For
example, dilution may have selected against grazers of small
bacterivores, possibly resulting in the reduction of bacteria,
a potential competitor for nutrients. Another limitation of the
dilution method is the exclusion of macrozooplankton and
benthic macrofaunal grazers. Oyster reefs, which are important sites of material cycling in North Inlet, especially in the
summer, can have both negative (as direct grazers) and positive (as nutrient regenerators) effects on phytoplankton
growth (Dame et al. 1992; Dame and Libes 1993).
September: Transition period-Results
from the September samples indicate that the microbial food web was controlled by intense grazing pressure from microzooplankton,
as would be expected during the period following a summer
nanoplankton bloom when zooplankton abundances and
grazing rates are relatively high (E. B. Sherr et al. 1986;
Childers and McKellar 1987; Sherr et al. 1992). In wholewater controls, no net population growth was evident after
72 h, while the growth of all phototrophic groups was stimulated significantly by dilution. However, increases in pho-
regulation
643
totrophic picoplankton and nanoplankton abundances also
resulted from NH,+ addition, in contrast to the findings in
July. Ambient NH,+ concentration decreased by -50% from
July to September. Although the abundance of phototrophic
microplankton was not affected by NH,’ enrichment of
whole water, NH,+ addition to diluted water resulted in significant increases in total microplankton abundance (by 1.6fold) and that of some individual genera (e.g. Skeletonema,
Chaetoceros, and Cyclindrotheca increased by 1.4-, 2.4-,
and 1.4-fold, respectively). These results contrast those from
July, when NH,+ addition to diluted samples failed to cause
increases in any phototrophic component. Thus, September
was a transitional period when the net growth of phototrophs
in the microbial food web was co-regulated by grazing and
nutrients.
November and February: Nutrient limitation-The
greatest seasonal effect of NH,+ on phytoplankton was observed
in the November samples. Chlorophyll concentrations were
enhanced by NH,’ addition to whole-water, dilution, or antibiotic treatments. The NH,+ effect was selective, as phototrophic microplankton and nano-size diatoms were stimulated, but not small flagellates and picoplankton. The lack
of an NH,+ effect on the nanoflagellates and picoplankton is
peculiar given the relatively low ambient nutrient concentrations and the expectation that these phytoplankton would
have relatively high NH,+ uptake affinities. One possible explanation is that relatively low temperatures suppressed nanoflagellate and picoplankton metabolic activities, and enhanced the competitive ability for NH,’ uptake by the
diatoms (Eppley 1972; Goldman and Ryther 1976; Waterbury et al. 1986).
In November, the dilution treatment resulted in a significantly greater increase in chlorophyll over 24 and 48 h, similar to the patterns observed in July and September samples.
In contrast to the summer results, however, chlorophyll did
not increase in diluted November samples from 48 to 72 h.
Also in contrast to the earlier months, chlorophyll increased
over 72 h in November whole-water controls. The 72-h accumulation of only three groups was increased by dilution
in November (i.e. picoplankton, nano-size diatoms, and Amphiprora sp.). Thus, although microzooplankton grazing appeared to exert some control over phytoplankton population
growth in November, this influence was minor when compared to the July or September results.
As in November, NH,+ additions significantly increased
72-h chlorophyll concentrations in February, and the effect
was specific to diatoms. In contrast to all other seasons, however, dilution had no effect on chlorophyll production over
any interval. In fact, chlorophyll decreased over 24 h in the
dilution treatment, but increased in whole water over the
same period. Based on these results, microzooplankton grazing appeared to be an insignificant factor in controlling phytoplankton population growth during the February period.
However, the influence of larger grazers (macrozooplankton,
benthic macrofauna) on phytoplankton was not addressed in
our experiments. Although the population densities and metabolic activities of these groups are seasonally low during
the winter and early spring in North Inlet (Lonsdale and
Coull 1977; Dame et al. 1986, 1989, 1992; Houser and Allen
644
1996), their influence on phytoplankton
period cannot be discounted.
Lewitus et al.
dynamics during this
Summary and implications-Seasonal
changes in the effects of NH,+ (minimal in July, maximal in November) can
be considered in light of the seasonality of ambient NH,’
concentration. Wintertime accumulations of NH,+ in marsh
and upland soils are introduced into North Inlet waters during spring runoff events and subsurface drainage. Nitrogen
excretion from oyster reefs combines with these inputs to
produce a summer maximum in NH,+ concentration. NH,’
concentration decreased sharply from July to September, and
was maintained at a minimal level during November and
February. The increased response of phytoplankton to NH,+
enrichment from summer to winter indicates that, during
nonbloom periods, nitrogen can limit phytoplankton population growth in North Inlet.
During the July phytoplankton bloom, phototrophic nanoplankton (mostly flagellates) and picoplankton (Synechococcus) dominated phytoplankton composition numerically,
and equaled microplanktonic diatom biomass. In fact, chlorophyll concentrations and nanoplankton abundance were
much higher in July than in February, but phototrophic microplankton abundance was lower in July, and microplankton biovolume did not vary between the periods, suggesting
that the bloom resulted primarily from an increased standing
stock of phototrophic nanoplankton. Also note that the samples in this study were collected just after slack low tide,
and therefore the relative contribution of benthic diatoms to
the phytoplankton community in these samples should be
greater than that in water collected during other tidal stages.
Given their relatively high biomass and potential for rapid
growth, small flagellates and Synechococcus likely play a
potentially important role in organic matter production in
North Inlet.
Material and energy transfer from primary producers to
macrofauna is often considered inherently inefficient through
the microbial loop because of the myriad of interacting trophic compartments and the prevalence of positive feedback
pathways. It is perhaps paradoxical therefore that the North
Inlet estuary maintains a productive faunal community, given that phytoplankton presumably are an important source
of carbon for that community, while the bulk of phytoplankton production occurs during an annual bloom that is characterized by microbial loop dynamics. However, North Inlet,
like many other warm, temperate salt-marsh estuaries, typically receives NH,+ pulses of varying frequencies, and the
high capacity for rapid nutrient turnover, also inherent in
microbial loops, may increase the transfer efficiency of materials and energy to higher trophic levels under transient
nutrient conditions (Berman and Stone 1994). The degree to
which faunal production depends on this type of microbial
processing is a pressing question, especially in view of the
expected impact of continued coastal development on nutrient loading patterns in southeastern U.S. salt-marsh estuaries.
Because information is lacking on microbial food-web dynamics in the coastal waters just outside North Inlet estuary,
it is unknown whether the structure and control of microbial
communities observed at our site is also characteristic of the
local nearshore environment. Although North Inlet has a relatively high flushing rate (e.g. -40% of total water volume
is lost on an ebbing spring tide; Kjerfve et al. 1991) and is
a net annual exporter of nutrients to the coastal ocean (Dame
et al. 1986), a net import and rapid turnover of phytoplankton within the estuary is thought to occur (Dame et al. 1986).
Nearshore phytoplankton populations probably do not have
the same controls as estuarine communities, given the likelihood that nutrient availability would be lower, and light
availability higher, in coastal waters.
The relative predominance of grazing over nutrient control
of phytoplankton population growth during the bloom in
North Inlet deviates from the common and well-founded
conclusion that estuarine phytoplankton blooms generally
are nutrient limited (Nixon et al. 1986). However, North Inlet
differs from most well-studied estuaries in the influence of
terrestrial runoff. A comparative study of 40 estuarine systems showed that North Inlet had an exceptionally low mean
annual chlorophyll : dissolved inorganic nitrogen ratio, relative to other “microtidal”
estuaries (Monbet 1992). The annual concentrations of dissolved inorganic nitrogen and
POd3- in North Inlet are low compared to most estuaries
studied, where NO,- loadings are important and where phytoplankton blooms are nitrogen limited (e.g. Nixon 1982;
Rudek et al. 1991). The key consideration, therefore, in explaining fundamental differences in phytoplankton bloom
dynamics (e.g. microbial food-web structure and regulation)
between North Inlet and other estuaries is nutrient quality
(i.e. the relative proportion of regenerated and “new” nitrogen) and loading rates. It is tempting to speculate that phytoplankton bloom regulation in North Inlet (i.e. trophic control rather than nitrogen limitation)
may be typical of
estuaries characterized by a predominant influence of tidal
exchange processes over terrigenous exchange, and a prevalence of NH,+ over NO,- as the major form of available
nitrogen.
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Received: 25 April 1997
Accepted: 1 December 1997
Amended: 8 January 1998