Limnol. Oceanogr., 34(6), 1989, 1025-1033
Q 1989, by the American
Society of Limnology
and Oceanography,
Inc.
Diel periodicity in ammonium uptake and regeneration in
the oceanic subarctic Pacific: Implications for
interactions in microbial food webs
Patricia A. Wheeler
College of Oceanography, Oregon State University, Corvallis 9733 1
David L. Kirchman
College of Marine Studies, University of Delaware, Lewes 19958
Michael R. Landry
Department of Oceanography and Hawaii Institute of Geophysics,
University of Hawaii, Honolulu 96822
Steven A. Kokkinakis
College of Oceanography, Oregon State University
Abstract
Diel periodicity in NH,’ uptake and regeneration in the nutrient-rich environment of the oceanic
subarctic Pacific was examined. Surface water was incubated in large shipboard microcosms that
allowed repeated sampling of the planktonic community for NH,+ cycling rates, bacterial production
rates, and population densities of the dominant autotrophs and heterotrophs. Changes in NH,+
concentrations and isotopic enrichments indicated that regeneration took place exclusively at night.
Nitrogen uptake rates and bacterial production were maximal during the day. Abundance of <2pm autotrophic flagellates reached a maximum at the end of day or beginning of night, whereas
abundance of zooflagellates in the <2-~m and I-IO-pm size classes showed a twofold variation
with maximal abundances occurring at the beginning of day. These contrasting diel patterns suggest
short-term, cyclic disequilibrium in production and grazing within the microbial food web. Hence,
analysis of relationships among the various components of microbial populations in this type of
environment will require sampling on much shorter time scales than has been attempted in past
Ammonium
is an important
nitrogen
source for phytoplankton
in both oligotrophic and eutrophic waters. Experimental
studies generally report a close coupling between NH,+ uptake and regeneration rates
(e.g. Caperon et al. 1979; Glibert 1982; Probyn 1987; Probyn and Lucas 1987), but exceptions have been noted. Harrison et al.
( 1983) found that regeneration rates often
exceeded uptake rates in the Middle Atlantic Bight. In contrast, Paasche and Kris-
tiansen (1982) and LaRoche (1983) concluded that NH,+ regeneration was not
sufficient to meet the uptake requirements
of phytoplankton.
Diel periodicity in either
ammonium uptake or regeneration would
result in an apparent discrepancy between
the two rates for incubations of ~24 h. An
extensive study of diel pcriodicity in NH,+
uptake and regeneration was carried out in
the Sargasso Sea (Glibert 1982). Glibert
concluded that NH,+ uptake and regeneration were equivalent “within our present
analytical resolution.” This conclusion was
Acknowledgments
largely based on absence of significant
Support for this research was provided by NSF grants
changes in NH,+ concentration,
not on a
OCE 86-13878, OCE 86-14170, and OCE 86-14401.
comparison of rate measurements. When
We thank the captain and crew of RV T. G. Thompboth uptake and regeneration rates were
son for their assistance. Joyce Lehner-Fournier and
Bruce Monger were responsible for sampling the mimeasured directly, uptake exceeded regencrocosms, and Joyce Lehner-Fournier made all the eration for samplings at 0300 and 1100
population counts. R. Keil helped with measuring bachours, and regeneration (with one excepterial production and primary amine concentrations.
tion) exceeded uptake for samplings at 2 100
P. M. Glibert and two anonymous reviewers provided
comments on an earlier draft of the manuscript.
hours. These results suggest a diel difference
1025
1026
Wheeler et al.
in the coupling of the two processes which
is generally masked by insufficient analytical resolution in oligotrophic waters.
Nutrient levels are high throughout the
year in the oceanic subarctic Pacific (Anderson et al. 1969), and there is no evidence
of nitrogen-limited
phytoplankton
growth
(Miller et al. 1988). Rapid growth of phytoplankton is indicated by carbon uptake
and microscopic estimates of total phytoplankton carbon (Booth et al. 1988). Grazing pressure by microzooplankton
is hypothesized
to maintain
phytoplankton
biomass at low and relatively constant levels
throughout the year (Frost 1987; Miller et
al. 1988). Despite high concentrations
of
N03- in the oceanic subarctic Pacific, recycled NH,+ is the dominant nitrogen source
accounting for about 50% of the total nitrogen used in spring and 75% in late summer
(Wheeler 1985).
In the present study, we examined diel
periodicity in NH,+ uptake and regeneration in the nitrogen-rich environment of the
oceanic subarctic Pacific. Both NH,+ concentrations and 15N-isotopic enrichment
were monitored during a 48-h. time-course
experiment.
We also measured bacterial
production and the abundances of the dominant autotrophic and heterotrophic
picoand nanoplankton in order to relate changes
in NH,+ cycling to other microbial processes. Experimental incubations were conducted in large, shipboard microcosms that
allowed repeated sampling of the planktonic
communities.
Methods
Experimental work was conducted in June
1987 as part of the SUPER (subarctic Pacific ecosystem research) Program. Shipboard incubations were conducted in 60Liter microcosms made from polyethylene
drum liners (J. F. Shelton Co.). The microcosm drums and their operation are described by Landry and Lehner-Fournier
(in
press). Briefly the drums were closed with
a screwtop cap after filling and were sampled through a silicone umbilical cord connecting through the cap to a rigid tube with
a flared outlet at the center of the drum. The
silicone tubes had stopcocks near the sampling end to completely close the containers
when they were not being sampled. The microcosms were screened to the ambient light
level at 10-m depth (about 50% incident).
To provide gentle mixing, the containers
were continuously rotated (about 1 rpm) in
a water-cooled incubator.
The microcosm drums were soaked with
dilute HCl and rinsed with Milli-Q water
and seawater before use. The containers were
gently filled with seawater collected from
10-m depth at 0130-0430 hours from GoFlo bottles. Isotope additions to the four
microcosms were: treatments 1, 2, and 3,
+0.5 PM 99.7 atom% 15NH4+; treatment
4, + 10 PM 98.8 atom% 15N03-. Isotopes
were mixed into the microcosm containers
as lthe filling process neared completion. Initial samples were taken at 0500 hours.
Thereafter, at 4-h intervals for the next 48
h e:ach container was sampled for nutrients
(NH,+, N03-, NO*-, POd3-, Si04-, and primary amines), particulate
nitrogen, and
plankton population
abundances (phytoplankton, heterotrophic Protozoa, and bacteria). Samples were also taken at each time
to determine N03- and NH,+ assimilation
and thymidine and leucine incorporation
rates and for analysis of the atom% enrichment of dissolved NH4+. Results are plotted
1 h after sample withdrawal
to allow for
incubation
time and sample preparation
time.
Nutrients (except NH4+) were determined
by standard autoanalyzer techniques (Atlas
et al. 19’7 1). Ammonium
concentrations
were determined manually with the phenolhypochlorite
method (Stickland and Parsons 1972) scaled down to 10 ml. Primary
amines were measured by o-phthalaldehyde fluorescence with glycine as a standard
(Parsons et al. 1984). Particulate nitrogen
was determined via persulfate digestion of
particulate nitrogen (from 500-ml samples)
collected on 25-mm GF/F filters and suband
sequent :N03- analysis (Strickland
Parsons 1972). Slides for enumeration of
pica- and nanoplankton populations by epifluorescence microscopy were prepared from
15-ml water samples filtered onto 0.2~pm
Nuclepore filters following the DAPI-staining procedure (Porter and Feig 1980).
INitrogen uptake rates were determined
by measuring rates of assimilation of 15N-
NH,+ uptake and regeneration
labeled N03- and NH,+ into particulate nitrogen. Isotopic enrichment of particulate
nitrogen was measured by emission spectrometry. Samples were prepared by a dry
Dumas combustion as described by Whecler and Kirchman
(1986). The emission
spectrometer was calibrated with Jasco prepared standards, and the atom% enrichment of each sample was determined from
the mean of three peak scans. The mean SD
for 27 sets of triplicate readings was 0.011
atom% 15N with a mean C.V. of 0.9%. The
mean SD for 12 sets of duplicate combustion tubes prepared from the same filter
sample was 0.030 atom% 15N with a C.V.
of 2.7%. The accuracy of 15N standards dried
onto GF/F filters and analyzed by emission
spectrometry is within 1% (Dudek et al.
1986).
Ammonium
regeneration was measured
by isotopic dilution. Ammonium in filtrate
samples was converted to indophenol (Dudek et al. 1986), which was recovered by a
solid-phase extraction (Selmer and Siirensson 1986) with a modification
of the rinse
and elution solvents. Indophenol was dried
onto 47-mm GF/F filters and analyzed for
isotopic composition with the same procedure used for labeled particulate nitrogen.
The mean SD for 34 sets of triplicate scans
was 0.025 atom% 15N, for a C.V. of 0.4%.
The mean SD for 15 sets of duplicate extractions from the same filtrate sample was
0.059 atom% 15N, for a C.V. of 1.3%.
Bacterial production was determined from
the incorporation of [3H-methyl]thymidine
and [3H]leucine into the cold, trichloroacctic acid (TCA)-insoluble
fraction (Fuhrman
and Azam 1980; Kirchman et al. 1985).
Briefly, 5 nM [3H]thymidine
or 12 nM
[3H]leucine were added to duplicate 25-ml
subsamples. After 1 h of incubation, subsamples were filtered through Nuclepore filters (0.2~pm pore size) and rinsed twice with
cold 5% TCA.
Since no significant differences were observed among the three NH,+ treatments,
results were pooled across sampling times
and the mean and standard error are reported. Similarly, no significant differences
were observed among the four treatments
for primary amine concentrations, bacterial
production, and abundances of autotrophic
1027
and heterotrophic
microorganisms.
Thus,
for these parameters results from the four
microcosms were pooled for each sampling
time, and the mean and standard error are
given.
Specific uptake rates were calculated as
the rate of increase of atom% enrichment
in particulate nitrogen divided by the mean
atom% enrichment of dissolved NH4+ (see
below). Since isotopic dilution was discontinuous, uptake rates were calculated for
specific time intervals. No significant isotopic dilution occurred between 0600 and
1800 hours, so the mean atom% enrichment
of NH4+ during this interval was used to
calculate uptake rates. Significant isotopic
dilution occurred between 1800 and 0200
hours; consequently the mean of the logarithm of NH,+ enrichment (Glibert et al.
1982) was used to calculate uptake rates
during this period. Assumption of a linear
rather than exponential decrease in NH4+
enrichment would lead to < 10% difference
in the estimated uptake rates. Absolute uptake rates were calculated as the specific uptake rate times the mean PN value for each
time interval. Ammonium regeneration was
calculated with equations presented by
Blackburn (1979).
Growth rates and mortality rates were estimated from the regression of the natural
log of cell abundance against time. Mean
abundances for the four treatments were
used, and the standard error of the slope is
reported when three or four time points were
available for the calculation. Daily rates were
calculated by multiplying
hourly rates by
the number of hours in the relevant time
interval. It should bc noted that this calculation provides a minimal estimate of
growth and mortality since only net changes
are considered.
Results
Ammonium concentrations, uptake, and
regeneration - Ammonium
concentrations
decreased during the first day to about 60%
of initial levels and then increased again
during the night to N 92% of initial levels
(Fig. IA). The mean increase in NH,+ during the first night was 0.128+0.024
PM
(mean and SE, n = 3). During the second
Wheeler et ~1.
1028
change significantly during the 48-h sampling period (data not shown).
z
80
The mean atom% enrichment
of dis‘”
solved
NH,+
for
the
three
NH,+
treatments
s
60
0.6
2s
ranged from 80 to 96 atom% 15N but showed
5
J
s
40
0.4 +
no significant change during the first light
d
period. During the first dark period, the
E
20
0.2
mean NH4+ enrichment decreased from 96
I
I
’
-I
0
0
to 7 1 atom% 15N (Fig. 1). A similar isotopic
an
dilution (from 74 to 53 atom% 15N) occurred during the second dark period, but
was not accompanied by a further increase
in NH,+ concentration. These results indicatc that net uptake of NH,+ occurred during both daylight periods and the second
darlk period, while a net increase in NH,+
concentration occurred only during the first
dark period.
Daylight NH,+ uptake rates averaged
10.9+0.3 nmol liter-l h-l, and dark NH,+
uptake rates averaged 5.6 +0.4 (mean and
SE, Table 1). Daylight N03- uptake rates
(9.lkO.7 nmol liter-l h-l) were similar to
NH,+ uptake rates, but night uptake rates
(2.7 rtO.1 nmol liter-l h-l) were significantly
lower (mean and SE, Table 1). Uptake rates
increased during the second light period, but
TIME OF DAY
because
of depletion of NH4+ and approach
Fig. 1. A. Mean concentration (A) and atom% 15N
of equilibrium values for N03-, dark uptake
of dissolved NH,+ 0 for three microcosms with NH,+
enrichment. B. Net NH,+ uptake (W) and total NH4+ rates for the second dark period could not
assimilation (A). C. Total NH,+ depletion from net be accurately determined. As shown by isochanges in chemical concentration m and calculated
topic dilution,
regeneration of NH,+ ocas the difference between isotopic measurements of
curred
only
during
the dark and exceeded
uptake and regeneration rates (A). Error bars indicate
dark uptake by a factor of three (Table 1).
standard errors (n = 3); black bars indicate dark period.
Ammonium uptake rates determined by
24-h period, however, NH,+ concentrations
assimilation of 15NH,+ and by net changes
decreased at a constant rate through the day in NH,+ concentration were similar for the
and night. The other inorganic nutrients
first 14 h of the incubation (Fig. 1B). Once
(N03-, NO;?-, POd3-, and Si04-) did not
NH,+ regeneration began, however, the net
Table 1. Summary of uptake and regeneration rates (nmol liter-’ h-l), means, and SE with n given in
parentheses. (Not determined, - .)
.-
N source
Ammonium
Nitrate
--
Time
interval
0600-2250
2250-1045
1045-1905
1905-0630
0600-2250
2250-1045
1045-l 905
1905-0630
Substrate
uptake
3.0.9+0.3(4)
5.6+_0.4(3)
116.6+0.7(3)
9.1 f0.7(4)
2.7&O. l(3)
21.7k5.0(3)
-
* The lowest rate of regeneration
that could be detected in this study was 0.4 nmol
.I Regeneration
rate is for the interval 2250-0600
hours.
liter-’
h-l.
Regeneration
rate
<0.4*
16.1-+0.3-f(3)
co.4
16.0-+8.2(3)
-
-
NHik uptake and regeneration
change in NH,+ concentration
underestimates uptake (Fig. 1B). The difference between NH4+ uptake and regeneration (as determined isotopically) should be equivalent
to the net change in NH4+ concentration.
Calculated and measured changes in NH,+
concentration
show close correspondence
(Fig. lC), thus validating the isotopic rate
measurements.
Bacterial production-With
the exception of one high value during the first night,
primary amines were high (1144 10 nM,
mean and SE, n = 4) during the day and
low (50+ 11 nM, mean and SE, n = 4) at
night (Fig. 2A). The patterns for both diel
cycles were similar. Thus net release of primary amines was maximal at noon, in sharp
contrast to the net release of NH4+, which
was highest during the first dark period.
There was no difference between primary
amine concentrations
in microcosms receiving NH,+ and N03-.
Bacterial production measured by leucine
and thymidine incorporation
followed the
same pattern observed for concentrations of
primary amines, i.e. rates were highest at
1400 and lowest at 2300 hours in the dark
(Fig. 2). The diel pattern was more evident,
however, and production rates were significantly higher during the second 24 h (Fig.
2B, C).
Abundances of pica- and nanoplankton Bacterial numbers ranged from 6.2 to 10.2
x 1O5 cells ml-l.
Highest
numbers
(7.8OkO.24 X lo5 cells ml-l, mean and SE,
n = 4) occurred between 1400 and 2200
hours but were not significantly higher than
the overall mean (7.77f0.14
x lo5 cells
ml-l).
Autotrophic
flagellates ~2 pm in size
showed a pronounced daily periodicity in
abundance (Fig. 3A). Minimal growth rates
(estimated from the increase in cell numbers
during the daylight period) were 0.75 and
0.73 d-l for the 2 d, while mortality rates
(estimated from decreases in cell numbers
during the night) were 0.72 and 0.58 d-l for
the 2 d (Table 2). Autotrophic flagellates in
the l-lo-pm
size class did not show diel
periodicity in abundance, but a decrease occurred during the first 12 h and was followed
by a continuous increase for the next 36 h
(data not shown). Growth and mortality
300 ,-
0600
180
TIME OF DAY
Fig. 2. A. Concentration of primary amines. B.
Leucine incorporation. C. Thymidine incorporation.
Symbols represent mean for four microcosms and error
bars indicate standard errors (n = 4); black bars indicate dark period.
rates for the l-l O-pm autotrophic flagellates
were 0.30 and 0.22 d-l (Table 2).
Zooflagellates in the < 2- and l-l O-pm
size classes showed similar patterns, with a
decline in numbers during the day and increases in numbers at night (Fig. 3B, C).
Growth rates for the zooflagellates (from the
nighttime increase in abundance) ranged
from 0.26 to 1.19 d-l, while estimates of
mortality rates during daylight ranged from
0.31 to 1.17 d-* (Table 2).
Discussion
The large r5NH,+ addition (0.5 PM) to
three of the microcosms represented a significant increase over ambient levels, but
allowed a 48-h monitoring of uptake and
regeneration rates for three parallel incubations with natural microbial communities. Standard practice for determination of
Wheeler et al.
1800
0600
TIME OF DAY
Fig. 3. A. Abundance of c2-pm autotrophic flagellates. B. Abundance of <2-pm zooflagellates. C.
Abundance of l-l O-km zooflagellates. Symbols represent mean for four microcosms and error bars indicate standard errors (n = 4); black bars indicate dark
period.
“‘in situ” rates is to keep isotopic additions
to I 10% of ambient concentrations whenever possible. In the high-nutrient subarctic
waters, however, the large isotopic addition
did not significantly alter metabolic rates of
and N03- upphytoplankton. Ammonium
take rates were similar during the light period, suggesting that either nitrogen source
can be used to supply phytoplankton
nitrogen requirements. Finally, regeneration rates
reported here are similar to short-term rate
measurements made for bottle incubations
with 0.1 PM l 5NH,+ additions (Kirchman
et al. 1989). Thus, these data can be considered representative of in situ, water-column rates.
Diel periodicity in ammonium remineralization has been difficult to demonstrate
in open ocean studies, where low NH,+ con-
centrations preclude accurate measurement
of isotope dilution (Glibert et al. 1982). Although considerable variability was evident
in the 15N data for the first 8 h of our experiment, the subsequent results clearly indicate a rapid regeneration of NH4+ between
1800 and 0200 hours and insignificant regeneration from 0200 to 1800 hours. Caperon et al. (1979) also presented evidence
that regeneration of NH,+ was faster at night
than during the day, but added that bottle
effects may contribute to the difference. Diel
periodicity in NH4+ uptake and regeneration has important implications
for comparison of uptake and regeneration rates.
Paa.sche and Kristiansen
(1982) and LaRoche ( 1983) found that regeneration was
insufficient to balance uptake rates in Oslofjord and Bedford Basin, respectively.
Probyn (1987) found NH,+ assimilation and
regeneration to be roughly in balance in the
Benguela upwelling region, though notable
deviations were evident at low rates, where
uptake exceeded regeneration. Uptake and
regeneration were roughly in balance in the
oligotrophic Agulhas Bank regions (Probyn
and Lucas 1987). In these studies only daytime measurements of uptake and regeneration were made, and this bias may be the
reason that regeneration rates were less than
uptake rates. If diel periodicity
occurs in
either one or both processes, then accurate
daily averages will require repeated shortter:m measurements throughout the diel period.
Ammonium can be released by mesozooplankton, microzooplankton,
and bacteria.
We did not attempt size fractionation
of
NH,+ regeneration during this study, but
changes in abundances of the dominant picoand nanoplankton
can be compared with
isotopic rate measurements to examine relationships among the members of the microbial community. Maximal bacterial production
co-occurs
with
the highest
phytoplankton
activity (NH,+ and N03uptake) and the highest levels of dissolved
primary amines. Short-term variations (< 24
h) in bacterial production and in situ concentrations of dissolved compounds such as
fre:e amino acids (DFAA) are complex. Bacterial production has been observed to peak
during the day in freshwater (Jorgensen
NH,+ uptake and regeneration
1031
Table 2. Summary of mean growth and mortality rates (d- ‘, &SE) for pica- and nanoplankton (n = 4).
Organisms
Autotrophic flagellates, < 2 pm
Autotrophic flagellates, I-10 pm
Zooflagellates, < 2 pm
Zooflagellates, I- 10 pm
Time
interval
0600-1400
0200-l 000
1000-2200
2200-0600
0600-l 800
0600-0600*
0600-l 800
0200-I 000
1000-l 800
1800-0600
0600-l 800
0200-0600
1000-1400
Growth
rate
Mortality
rate
0.75kO.12
0.72f0.01
0.73kO.05
0.58+0.19
0.22kO.03
0.3o-to.03
1.17+0.17
1.19-to.32
0.40+0.07
0.26kO.04
0.76kO.05
0.4220.15
0.3 1-+NA(n = 2)
* Second 24-h interval.
198 7, but see Riemann and Sondergaard
1984) and in coastal marine waters (Fuhrman et al. 1985). Likewise, DFAA concentrations are sometimes maximal during the
day in lakes (Jorgensen 1987) and coastal
marine waters (Carlucci et al. 1984), but
exceptions have been observed (Mopper and
Lindroth 1982). The coupling between phytoplankton production, levels of free amino
acids, and bacterial production
may be
clearer in open ocean waters, such as the
subarctic
Pacific where phytoplankton
abundance is low and bacterial production
may bc limited by the availability
of dissolved organic carbon and nitrogen. Daytime peaks in bacterial production in our
experiments would seem to indicate that
bacteria do not play a significant role in
nighttime ammonium regeneration.
Phytoplankton
biomass in the oceanic
subarctic Pacific is dominated by cells < 10
pm in diameter (Booth 1988), and within
this size range autotrophic and heterotrophic cells are about equally abundant (Fig.
3). Changes in cell numbers reflect the balance between growth and mortality rates and
must be interpreted carefully since we do
not have independent estimates of the two
components. Increases in the <2-pm autotrophs in the light indicate a mean minimal growth rate of 0.71 d-l. Decreases in
abundance of <2-pm zooflagellates during
the same period indicate a mortality rate of
0.72 d-l. If grazing pressures on autotrophic
and heterotrophic plankton in the <2-pm
size class are equivalent, a mean gross growth
rate of 1.4 d-l is obtained from the summation of the net increase in numbers of
autotrophic cells plus the net decrease in
zooflagellates. Both autotrophic and heterotrophic nanoplankton
in this study were
growing at close to maximal rates expected
for the temperature of surface water in the
subarctic Pacific during May (Booth et al.
1988). Despite these fast growth rates,
abundances were maintained at a relatively
constant level over a diel cycle, presumably
by grazing.
Can the diel changes in pica- and nanoplankton abundances be related to the observed periodicity
in NH,+ remineralization? Microzooplankton
are important
grazers in the subarctic Pacific (Miller et al.
1988; Parsons and Lalli 1988) and presumably are responsible for a significant portion
of both grazing pressure on the nanoplankton and NH4+ regeneration. The isotopic
dilution results indicate that excretion occurs between 1800 and 0200 hours, while
the major increase in zooflagellate abundance occurs between 0200 and 1000 hours.
We postulate that grazing and excretion by
the zooflagellates occur simultaneously, but
that a lag exists between maximal grazing
activity and the increase in zooflagellate
numbers. Although the length of the cell
division period has not been reported for
zooflagellates, Heinbokel (1988) has found
that oceanic ciliates have a division period
of about 8 h and that cell division usually
occurs around midnight.
The oceanic subarctic Pacific appears to
1032
Wheeler et al.
R. W. EPPLEY, A. HAGSTR~M, AND F. AZAM.
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--,
F. LIPSCHULTZ, J. J. MCCARTHY, AND M. A.
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Submitted: 21 June 1988
Accepted: I7 ‘April 1989
Revised: 7 June 1989