Limnol.
Oceanogr.,
27(G), 1982, 1129-1140
@I 1982, by the American
Society of Limnology
and Oceanography,
Inc.
Methylammonium
uptake by Chesapeake Bay phytoplankton:
Evaluation
of the use of the ammonium analogue
for field uptake measurements L
Patricia
Museum
A. Wheeler2 und James J. McCurthy
of Comparative
Zoology,
Harvard
University,
Cambridge,
Massachusetts
02138
Abstract
The uptake of methylammonium
(MA) by Chesapeake Bay phytoplankton
was measured
in short term experiments
by using the ‘4c radioisotope.
This uptake was inversely related
to ambient NH4+ concentrations,
and the specificity
for uptake of both substrates was demonstrated by competitive
inhibition.
At stations with low ambient NH4+, MA uptake followed
saturation kinetics, but the V,,, vnlucs decreased over time during 10-60-min incubations.
Uptake rates of MA were generally lower than those of NH4+, but in short incubations
(lo15 min) the V,,, values were similar.
Although lMA and NH4+ are taken up by the same transport system, the utility of MA as an
NHdk analogue is restricted by the strong inhibition
of MA uptake by low concentrations
of
NH,+ and by the fact that in some cases MA uptake primarily
reflects membrane transport,
whereas N11,+ uptake can also reflect metabolic assimilation.
Our results show that in typical
uptake experiments,
NH4+ uptake is rate-limited
by either internal pool filling or metabolic
assimilation
rather than by the membrane transport system. As a consequence,
the kinetic
parameters reported in the literature for NIL+ uptake overestimate
K, and underestimate
V,,,
ibr the membiane transport system.
The importance of NH4+ as a nitrogen
source for marine phytoplankton
has been
clearly
established
(McCarthy
1981).
Nonetheless,
ollr knowledge
of the process by which it is taken up remains rather fragmentary
(Kleiner 1981). Accurate
determination
of NH,+ uptake kinetics by
natural assemblages of phytoplankton
is
difficult
due to our inability
to measure
uptake during very short NH4+ incubations (min) when biomass is low and to
the occurrence of rapid changes in uptake rates during the course of even brief
experiments (Glibert and Goldman 1981;
Wheeler et al. 1982a). Analysis of short
term uptake kinetics would be facilitated
by using a more sensitive tracer for uptake measurements
than is provided by
standard 15N methodology.
The 14C radioisotope
of methylammo-
’ This work was supported
2601 1 and OCE 80-22990 from
Foundation.
2 Present address: School of
gon State University,
Corvallis
by grants OCE 78the National Science
Oceanography,
97331.
Ore-
nium (MA) has been used successfLJly as
an NH,+ analogue in laboratory studies
with eucaryotic
microorganisms
(see
Wheeler 1980), bacteria (Stevenson and
Silver 1977; Kleiner
and Fitzke 1979,
1981; Gordon and Moore 1981), macrophytic algae (Smith et al. 1978; Smith and
Walker
1978; Walker et al. 1979u,b;
Wheeler 1979), and in field studies with
freshwater plankton (Vincent 1979, 1981).
MA is also used as a carbon or nitrogen
source by some bacteria (Bellion et al.
1980; Bicknell and Owens 1980) and yeast
(van Dijken and Bos 1981). We report here
on the uptake of MA by natural plankton
assemblages in the Chesapeake Bay. MA
uptake rates, uptake kinetics, and rates of
incorporation
were examined
in timecourse experiments.
The specificity
of
uptake was demonstrated by competitive
inhibition
experiments
with NH,+ and
MA, and information about the organisms
responsible for MA uptake was obtained
from size fl-actionation experiments.
The
utility of MA as an NH4+ analogue was
determined by comparison of [%JMA and
15NH4+ uptake in parallel incubations.
1129
1130
Wheeler and McCarthy
Methods
Water samples were collected along a
transect down the center of the Chesapeake Bay aboard the RV Warfield from
31 May-11 June 1980. Stations, collection and incubation procedures, and NH,+
concentrations
(ranging from 2.61 -c- 1.14
pg-atoms *liter-l north of the Potomac to
0.07 + 0.06 south of it) are given elsewhere (Wheeler et al. 1982a). The two
groups of stations north and south of the
Potomac will be called high and low ambient NI&+ stations.
Uptake measuremen-ts-Total
volume
for each experimental
incubation
ranged
from 250 to 500 ml, and aliquots for each
time determination
of uptake ranged
from 50 to 100 ml. Uptake experiments
were initiated
by adding
l-40
@i
[ L4C]methylamine
* HCl (New England
Nuclear: sp act 50 mCi *mmol-l) supplemented with unlabeled
MA for higher
concentrations.
For each sample, duplicate aliquots of particulate material were
collected on Gelman A/E glass-fiber filters and rinsed with about 25 ml of filtered seawater. Radioactivity
was determined by liquid scintillation
spectrometry
with 5 ml of Fisher Scintifluor
per sample. Background filter retention was determined by filtering an aliquot immediately after adding the labeled substrate.
The external standard ratio was used for
quench correction.
The amount of MA
taken up (pg-atoms N *liter-‘) was calculated from the mean cpm per sample with
appropriate corrections for specific activity and counting efficiency.
Contamination of [14C]MA with ‘4c0, was estimated
to be ~0.3% f rom time-zero
measurements of respiration in laboratory experiments and has a negligible
effect on the
estimated uptake rates in these field experiments. Uptake rates were calculated
from the least-square linear regression of
amount taken up against time and are expressed as P (rho) for comparison with
NH4+ uptake rates which have been corrected for remineralization
(Gilbert et al.
1982; Wheeler et al. 1982a). Remincralization of MA was assumed to be negligible.
Incorporation
of 14C into TCA-insoluble material was determined by filtering
a duplicate sample, treating with cold 5%
TCA ~10 s, rinsing with filtered seawater, and assaying for radioactivity
as described above. We used incorporation
of
14C into TCA-insoluble
material as an indicator
of metabolic
assimilation
of
[14C]MA. Other possible metabolic products, including
CO, and formaldehyde
(Colby et al, 1979) and soluble metabolites such as methylated
amino acids
(Shaw et al. 1966), were not assayed. In
laboratory studies with a variety of algae
(Wheeler 1979, 1980, unpubl.),
respiration of 14C0, was undetectable.
Heterotrophic microorganisms,
however, tend to
respire ~30% of organic substrates taken
up
(Crawford
et al. 1974) and consequently the rates of MA uptake and metabolism by heterotrophs
present in our
samples may be underestimated.
Similarly, our estimates of MA metabolism are
underestimates
if a significant portion of
accumulated
label is present as metabolites in the TCA-soluble
material. Nonetheless, if MA is metabolized as a carbon
source, a major portion of the label should
appear in the TCA-insoluble
fraction during the incubation
periods used here.
Saturation curves were determined
by
measuring
uptake as above at 12 substrate concentrations
and for three incubation periods. Uptake rates were calculated from a single point in time and
individual
curves determined for each incubation period (Wheeler et al. 1982a).
Inhibition
experiments-Competitive
inhibition
of MA and NH,+ uptake was
measured by adding 0.1-0.5 pg-atoms
NH4+. liter-l to [ 14C]MA incubation
bottles and 1.0-100 pg-atoms MA-liter-l
to
15NH4+ incubation
bottles.
Uptake of
15NH4+ was determined
as described by
Wheeler et al. (1982a). The inhibition
constant, Ki, was calculated
from the
expression given by Neame and Richards
( 1972) :
Ki Z-L
&I
P - Pi x S-tK,
where
Pi is the inhibited
uptake rate, P
Methylammonium
1131
uptake
904N
854E
8340
GO4C
Station
& 0)
DURATION
OF INCUBATION
(h)
of MA uptake at a high amFig. 1. Time-course
bient NII,+ station, 804C (A), and a low ambient
NI14-t station, 724R-1 (B). A. MA concentrations
2.25
(XL 17.53 (.L and 43.18 (0) @g-atoms Nmliter-l. B.
MA concentrations
0.04 (x), 1.17 (O), and 20.83 (0)
pg-atoms N sliter-l.
the noninhibited
uptake rate, K, the halfsaturation constant for uptake, I the concentration
of the inhibitor,
and S the
concentration
of the substrate. Half-saturation constants were determined in independent
experiments
on the same or
the previous clay. The values of S and I
for MA were the amounts added initially,
since there were no significant
changes
over 1-2 h. Ammonium concentrations for
S and I changed rapidly and, for each time
point, were estimated from calculated
uptake rates in the same or parallel incubation bottles.
Size fractionation-The
size class of
organisms responsible for MA uptake was
determined
by both prefiltration
and
postfiltration
procedures. In prefiltration
Fig. 2. Rate of uptake of MA normalized to standard concel2trations
of 0.05 (x),
1.0 (a), and 20.0
(0) pg-atoms N *liter-’ using data from Table 1 closest to the standard concentration.
experiments, water samples were filtered
before incubation
through 102-, 35-, and
lo-pm Nitex mesh or l-pm Nuclepore
filters. Uptake for each size class was determined as the difference between suecessive fractions. In postfiltration
experiments, samples were collected on 8- or
l-pm Nuclepore filters and Gelman A/E
glass-fiber
filters after incubation
with
MA. Uptake was calculated as above.
Results
Uptake kinetics-Uptake
of MA was
concentration-dependent
and increased
linearly
with time for up to 1 h, r2 =
0.96 -C 0.04 (Fig. 1, Table 1). At four low
ambient
NH4+ stations (744-1, 744-2,
724R-1, 724R-2) somedeparture from linearity was apparent and is reflected in the
lower coefficient
of determination
(f2 =
0.88 -+ 0.02). Uptake rates, normalized to
standard concentrations
of 0.05, 1.0, and
20.0 pg-atoms N *liter-l
for comparison
(Fig. 2), were significantly
lower at high
ambient NH,+ than at low ambient NH4+
stations. At high ambient NH,+ stations,
52% of the MA supplied was taken up
during the first 2 h of incubation,
At low
ambient NH,+ stations, ~15, 10, and 1%
of MA supplied was taken up in 2 h at
MA concentrations
of 0.04-0.08, 1.2-1.5,
1132
Wheeler and McCarthy
B
z
a
-
I
15 min
60 min
I
E
30 min
60 min
.3
D
IO min
F
MA
Concentration
(pg-atom
Fig. 3. Uptake rate (P) as a function of MA concentration.
panel. A,B,C-Station
744-l; I),E,F-station
707+.
4.0 T
r
.
T
z
.!I
-
3.0 -
;
E
0
0
&
a
C
2.0 -
a
aI
Y
+0
9
/
/
z I.O- 1’
c0)
f.?
I//
0
0
o$
’
0
IO
MA
20
Concentration
30
40
(pg-atom
I
I
50
60
N liter-‘).
l
Fig. 4. Expansion of Fig. 3A showing uptake at
highest MA concentrations
(dashed line-diffusive
component of uptake for NH,).
F
N= liter-’
Duration
)
of incubation
indicated
in each
and 17-21 pg-atoms N. liter-l. MA uptake
followed saturable uptake kinetics at two
of the three stations tested (Fig. 3), but
for the l-min incubation,
nearly linear
uptake kinetics were observed at high
(>20 pg-atoms Nmliter-l) concentrations
(Fig. 4). At station 744-1, uptake rates
were too low to permit kinetic analysis.
The apparent maximal uptake rate (P,,,)
decreased 50-75% during the l-h incubation (Table 2). Half-saturation
constants ranged from 1.9 to 7.5 pg-atoms N.
liter-l and did not vary with length of incubation.
Metabolism
and size fractionutionAlthough
MA uptake rates were low at
high ambient NH, + stations, the fraction
of 14C label taken up that was incorporated into TCA-insoluble
material was large
(0.61 -I 0.10). c onversely, at low ambient
NH4+ stations, where uptake rates were
high, the fraction incorporated
(0.14 +
0.02) was low (Table 3). In postfiltration
experiments
(after 3-24 h of labeling),
about 25% of the label appeared in each
of the 35-8-p and 8-1-p size classes,
whereas about 50% appeared in the <l-p
size class (Fig. 5). In prefiltration
exper-
Methylummonium
1133
uptake
NIL,+ concentraTable 1. Linear regressions for time-course (O-l h) of MA uptake. [NH,+]*-Ambient
of determination,
P-uptake
r2- coefficient
tion, n-nundxr
of samples, I-ordinal
intercept, m-slope,
rate.
sta.
WI,+l,
[MAI
n
I
m
r2
P
(pg-atoms
liter-‘.h-‘)
N.
PMA: bHI+
904N
1.52
1.84
16.84
4
3
0.001
0.008
0.004
0.038
0.98
0.90
0.005
0.046
0.02
0.27
854E
2.78
6.42
16.84
41.84
3
2
3
0.001
0.003
0.009
0.002
0.008
0.019
0.94
1.00
0.003
0.011
0.028
0.03
0.13
-
834G
4.14
4.34
17.53
43.40
5
3
3
0.002
0.007
0.003
0.001
0.021
0.009
0.94
0.98
0.91
0.003
0.028
0.013
0.02
0.13
-
804C
2.02
2.26
17.53
43.18
5
3
3
0.001
0.005
0.011
0.007
0.016
0.060
0.99
0.99
0.99
0.008
0.021
0.071
0.01
0.06
-
744-l
0.06
0.04
0.38
5.38
17.53
4
5
5
5
0
0.001
0.002
0.010
0.003
0.007
0.020
0.042
0.99
0.97
1.00
0.91
0.003
0.007
0.021
0.052
0.16
0.25
724R- 1
0.04
0.04
1.17
20.83
4
4
4
0
0.001
0.015
0.003
0.037
0.515
0.98
0.97
0.87
0.003
0.038
0.166
0.10
0.25
1.94
7074
CO.03
0.04
1.17
20.83
4
4
3
-0.002
-0.003
0.001
0.002
0.024
0.054
0.97
0.98
0.98
0.002
0.021
0.055
0.03
0.26
0.36
724R-2
CO.03
0.09
1.47
21.70
4
4
4
0
0.002
0.001
0.006
0.015
0.054
1.00
0.92
0.88
0.006
0.017
0.117
0.02
-
0.08
0.04
1.20
21.00
3
4
4
0
0
0.004
0.001
0.007
0.033
0.90
0.97
0.86
0.001
0.008
0.037
0.08
0.19
744-2
iments, <lo% of the label was found in
the <l-p size class when the incubation
period was ~2 h (Fig. 6).
Specificity
of uptakeThe substrate
specificity
of the observed
uptake is
clearly indicated by the mutually
comTable 2.
uptake.
Sta.
744- 1
7074
Kinetic
parameters
Duration
of
incltbation
(min)
1
15
60
10
30
60
* Estimated
by assuming
that
cstitwtc
of P,,, for metliated
petitive inhibition
of MA and NH,+ uptake. Submicromolar
concentrations
of
NH4+ caused a strong inhibition
of MA
uptake at concentrations
of 0.04 and 1.17
pg-atoms’ N-liter-’
(Fig. 7). Much higher
concentrations
of MA were required to
+ 1 SE for MA saturation
(pg-atoms
in NII,+
uptake
to parameters
P max
P,,,-cSE
N.liter-‘.h-‘)
K,kSE
(pg-atoms
N’liter-‘)
0.572kO.121
0.367+-0.031
0.148kO.Ol.5
0.191-+0.016
0.104_+0.013
0.085+0.006
linear incrcarc
tnmsport.
curves and comparison
from
difbion
or pool
KS
MA/N&+
5.16k3.19
7.49k1.11
5.24k1.17
3.75-+ 1.69
1.90+- 1.45
2.30k0.74
results
for NH4+
1.17*
0.95
0.49
0.87”
0.57
0.53
expansion
:md using
18.1
13.1
16.6
9.9
did
intcrccpt
as nn
1134
Wheeler and McCarthy
Size
Fractions
(pm )
[ MA1
(pg-atom
Nmliter-I
)
20.8
724 R-2 4h
Station
Fig. 5. Size fractionation
percent of total uptake.
--
707
(p 3h
Duration
of
of MA uptake
determined
inhibit NH,+ uptake at similar concentrations (Fig. 8). The inhibition
constant (KJ
is about 0.3 ,ug-atoms N 8liter-’ for NH4+
and 5.3 for MA (Tables 4, 5).
Compurison of MA and NH*+ uptakeMA uptake rates were generally
much
lower than NH4+ uptake rates. The average value of PI~n:P~~4+ (for 1-2-h rates)
was 0.09 +- 0.10 at high ambient NH4+
stations and 0.23 -I 0.09 at low ambient
NH4+ stations (Table l), At low ambient
NH,+ stations, maximal uptake rates for
MA and NH4+ determined during shorter
incubation
periods were close (Table 2),
but the ratio between them decreased as
the duration of the incubation period in-
707
4, 24h
Incubation
by postfiltration
(h)
at stations
724K2
and 7074 as
creased. For those incubation
periods
with saturation kinetics, the half-saturation constant for MA uptake was an average of 17 times greater than that for
NH4+ uptake. The fraction of MA incorporated into macromolecules
was 80% of
the fraction of NH4+ incorporated
into
macromolecules
at high and 20% at low
ambient NH4+ stations (Table 3).
Discussion
All Chesapeake Bay plankton assemblages that we examined in this study took
up MA. The rate of uptake of MA was
inversely
related to the ambient NH,+
concentration.
The low rates of uptake
Methylammonium
Size
Fractions
1135
uptake
(pm)
WI
(pg-atom
0.5
Duration
Fig. 6.
2.0
1.0
of
Incubation
Table 3. Mean percentage of [‘“C]MA taken up
that was subsequently
incorporated
into TCA-insoluble material.
Wb+I
sto.
904N
8543
(pg-atom
litcr-‘)
1.52
2.78
Mean&SE
724R-1
7074
724R-2
744-2
Mean&SE
0.04
co.03
co.03
0.04
Fraction
1
4.0
(h)
As Fig. 5, but by prefiltration
north of the Potomac probably reflect inhibition
of MA uptake by high ambient
NH4+. This explanation
is supported by
the strong inhibitory
effect of submicromolar NH4+ concentrations
on MA uptake at the low ambient NH,+ stations.
Furthermore,
the mutually
competitive
inhibition
of MA and NH,+ uptake indicates the specificity
of uptake of the two
substrates.
NII,+ transport systems, which also take
up MA, have been reported in bacteria
Neliter-’
at station 724R-2.
(Stevenson and Silver 1977; Kleiner and
Fitzke 1979, 1981), fungi (Hackette et al.
1970), yeast (Roon et al. 1975, 1977), and
100 c
80 g
.-+
5
z
5
8
60-
40-
incorporated
N
MA
MA/NII,+
0.54~014
0.6820.32
0.78
0.77
0.61+0.10
0.77-co.01
0.15+-0.04
0.11~0.03
0.15+0.03
0.17kO.17
0.21
0.19
0.19
0.20
0.14kO.02
0.20+0.01
Fig. 7. Inhibition
of MA uptake by NH,+ at station 724R-1. MA concentration
(pug-atoms Nalitcr-I)
was 0.04 (0) or 1.17 (0).
1136
Wheeler and McCarthy
Table 5. Inhibition
of NH4+ uptake.
[NII,+]
constants
[MAI
(I*p-atoms
liter-‘)
Sta.
for MA inhibition
804C
4.53
6.47
1~100
15-100
744-2
0.04a
0.04b
l-40
l-40
K,* 2 SE
N.
Mean&SE
* Assmning
”
20
40
60
[ 1(pg-atom
MA
80
N.liter“)
phytoplankton
(Wheeler 1980). Although
the large fraction of MA uptake found in
the <l-p size class in the postfiltration
experiments
suggests significant
uptake
by free-living
bacteria, the long incubation period (3-24 h) and possible artifacts
of the postfiltration
procedure (McCarthy
et al. 1974; Wheeler et al. 1977) preclude
an unambiguous
interpretation
of these
results. The prefiltration
experiments
clearly indicate that free-living
bacteria
were not the dominant microorganisms
responsible for MA uptake in incubations
of ~2 h. It is harder to attribute MA uptake specifically to either phototrophic
or
heterotrophic
microorganisms
in the larger size classes, The competitive
inhibition of MA and NH*+ uptake provides indirect evidence that MA is being taken
up as a nitrogen rather than a carbon
constants
for NI14+ inhibiK,kSE
[NIL+1
Sh.
724R- 1
7074
744-2
MeankSE
(pg-atoms
0.14-0.46
0.57-1.19
0.50-1.12
10.35k6.77
2.59 5.33k3.52
K, I’or NII4+
is 0.1 pg-atom
N.litcr-‘.
100
Fig. 8. Inhibition
of NI14+ uptake by MA. NH4+
concentration
(pg-atoms N-liter-I)
was 4.53 at stdtion 804C (0) and 0.04 at station 744-2 (0).
‘Tnblc 4. Inhibition
tion of MA uptake.
3.24kO.65
5.14+ 1.85
N. liter-‘)
0.29eO.36
0.14kO.09
0.37kO.23
0.2720.12
source. Thus, at low ambient stations, uptake of MA can be characterized
as follows: rapid uptake by organisms >I pm,
saturable kinetics with P,,, dependent on
duration of incubation,
strong inhibition
by added NH4+, and no extensive incorporation of the accumulated 14C. We conclude that rapid MA uptake at low ambient NH,+ stations is due to the same
organisms responsible
for NH4+ uptake,
but in contrast to NH4+ (Wheeler et al.
1982u), MA is not metabolized
rapidly.
The low rates of MA uptake at high ambient NH4+ stations may reflect incomplete inhibition
of MA uptake by ambient
may reflect low
NKs+, or alternatively,
rates of heterotrophic
utilization
of MA as
a carbon source. The latter explanation is
supported by the high percentage of l4c
incorporation
at these stations.
The use of [14C]MA as an NH,+ analogue
for the purpose of estimating rates of NH4+
uptake requires determination
of the relationship between MA and NH4+ uptake
rates. The time-course data show that the
rates of uptake for MA were 9% of the
rates for NH,+ at high ambient NH4+ stations and 23% at low ambient NH4+ stations. However, in both cases MA uptake
may be depressed due to NH4+ inhibition, so that it is more appropriate to compare the kinetic parameters for uptake.
Our data for NH4+ uptake
kinetics
(Wheeler et al. 1982a) indicate very rapid
linear uptake kinetics in l- and 5-min incubations and saturable kinetics with a
relatively constant V,,, in 10-60-min incubations. Uptake kinetics for MA contrast sharply with those for NH4+ in two
Methylammonium
ways: although there was some evidence
of linear uptake of MA in the l-min incubation, the increase was linear only at
high concentrations
(~20 pg-atoms N *liter-r); and although saturation kinetics
were obtained for all longer incubation
periods, there was a significant decrease
duration of inin Knax with increasing
cubation,
We have postulated that the initial linear uptake kinetics for NH4+ result from
either a diffusive component of uptake or
from internal pool expansion and isotopic
equilibration
of external
and internal
NH,+ (Wh-eeler et al. 1982u). The diffusing molecular species is probably the unprotonated base. As calculated from the
respective pK, values, 9.25 and 10.6, NH3
is about 20 times more abundant than
CH3NH2 (MA) for equivalent
total concentration
of charged plus uncharged
species. Consequently,
the slope of the
diffusive component of uptake would be
proportionally
greater for NH3. However,
from the data available (Fig. 4) the slopes
appear to be similar, and this is an argument against a simple diffusive
component of uptake. Rapid uptake before pool
equilibration
for both substrates
thus
seems to be a more plausible explanation
for the initial linear uptake kinetics, The
discrepancy
between MA and NH,+ uptake rates at the lowest concentrations
reflects the much higher affinity
of the
transport system for NII,+ and possibly
inhibition
of MA uptake at low concentrations by ambient NH4+.
We interpret these results in conjunction with the evidence for substrate specificity of the uptake system as follows.
Phytoplankton
have very efficient membrane transport systems for NH4+, which
can also transport MA. The activity of
these transport systems is sufficient to establish an equilibrium
between the external and intracellular
concentrations
within the first few minutes of incubation. Thereafter, net NH4+ uptake continues as a result of metabolic assimilation
of the NH4+, whereas the apparent rate of
MA uptake decreases. This view is supported by recent laboratory
investigations. The elevated internal NH4+ pools
uptake
1137
observed during periods of NH4+ uptake
by nitrogen-limited
diatoms (DeManche
et al. 1979; Dortch 1982) indicate that
transport is more rapid than assimilation.
We have detected similar pools in the
diatom Thalassiosira
weissflogii,
the dinoflagellate Amphidinium
carterae, and
the coccolithophore
Hymenomonas
carterae (Wheeler et al. 1982b). Additional
evidence for limitation
of NH,+ uptake
by the rate of assimilation
is provided by
the close correspondence
of net NH,+ uptake and the activity of the primary NH4+
assimilatory
enzyme, glutamine
synthetase, in Chlamydomonas
reinhnrdii
(Cullimore and Sims ‘1981a).
Noteworthy
are results of laboratory
experiments
(Wheeler et al. 1982a) suggesting that large internal NH4+ pools and
isotopic exchange could lead to apparent
“elevated”
initial uptake rates that do not
reflect net uptake. Isotopic
exchange
alone however, does not account for the
initial
NH4+ uptake rates observed in
Chesapeake Ray (Wheeler et al. 198%)
or in the laboratory experiments of Goldman and Glibert (1982). Clearly, the extent to which tracer uptake reflects net
uptake or isotopic exchange depends on
preconditioning
and experimental
conditions and needs to be investigated more
thoroughly.
In principle,
NH,+ uptake rates could
be calculated from measured MA uptake
rates by correcting for the difference in
affinity for the two substrates. In practice,
however, the utility of MA as an NH,+
analogue for uptake measurements is restricted in two ways: because the transport system has a higher affinity for NH4+,
MA uptake rates must be corrected for
inhibiting
effects of ambient NH,+; and
because MA is not always metabolized to
the same extent as NH4+, uptake of the
two substrates will reflect different processes unless carefully restricted to measure only transport activity. Differences
in metabolic assimilation of the two substrates may have caused the discrepancy
between V maxfor MA transport and NH,+
uptake reported by Vincent (1981: table
III).
The decrease in net uptake of MA over
1138
Wheeler and McCarthy
time reported here was also apparent in
the results of laboratory experiments with
Cyclotella
cryptica
(Wheeler
1980;
Wheeler and Hellebust
1981) and Clostridium
pasteurianum
(Kleiner
and
Fitzke 1981). A difference between uptake rates of metabolized
substrates and
nonmetabolized
analogues has likewise
been found for sugar transport in algae
(Komor and Tanner 1976) and appears to
be due to a kinetic control of the uptake
of metabolized
substrates and a thermodynamic control of the uptake of nonmetabolized
substrates (Raven 1980).
The most significant finding of our short
term measurements
in the Chesapeake
Bay is that membrane transport can be
extremely
rapid relative to subsequent
rates of metabolism. We agree with Conway et al. (1976) that most reported V,,,
values for NH4+ uptake underestimate the
capacity of the NH4+ transport (or permease) system. However,
we disagree
with their conclusion that K, is underestimated. Valid estimation of K, cannot be
inferred
from “truncated”
hyperbolae,
and our results (Wheeler et al. 1982u)
suggest that K, increases with length of
incubation.
Hence, K, values from standard Z-4-h incubations
are likely to be
overestimates of actual values. Better estimates of the kinetic parameters for the
transport process could have a significant
effect on the interpretation
and prediction of phytoplankton
responses to various nutrient regimes. For example, precise measurement of the K, value for NH4+
transport would provide an indication
of
the actual NH4+ concentration
in phytoplankton microenvironments.
A relatively high value would suggest that phytoplankton may have to obtain NH,+ from
micropatches of elevated concentrations,
whereas a very low one would suggest
that enough NH4+ could be obtained from
low (<3O ng-atoms N *liter-l), but rather
constant, concentrations
by continuous
transport activity.
If the latter is true,
minimum
K, values are probably set by
diffLlsion limitation
(Gavis 1976; Button
1978; Raven 1980). Similarly,
better estimates of V,,, values for NH,+ transport
are needed to evaluate the significance of
enhanced short term uptake responses,
Underestimates of V maxfor transport could
lead to a significant
error in estimating
the amount of NH,+ that could be taken
up by phytoplankton
during very short
(seconds to minutes) exposures to an elevated nutrient
supply (Jackson 1980;
Williams and Muir 1981).
Both membrane transport (Pellcy and
Bannister 1979; Wheeler 1980) and assimilation
of NH 4+ (Edge and Ricketts
1978; Tischner
and IIiittermann
1980;
Cullimore and Sims 1981b) are enhanced
in response to nitrogen limitation.
The
extent to which the kinetics of transport
or assimilation
dominate the actual rate
of uptake depends on the nutritional
history of the cells, the external concentration of the nutrient, and the duration of
exposure. Species dominance can be affected by temporal variations in nutrient
supply (Turpin and Harrison 1979), and
we suggest that the physiological
basis of
the competitive
advantage in one situation or another may lie in the relationships between the kinetic parameters for
the transport and assimilation
processes.
Similarly,
species-specific
relationships
between
the kinetics
of transport and
subsequent
metabolic
assimilation
may
explain the variations in the duration of
enhanced
uptake for different
species
(Goldman and Glibert 1982).
Defining the rate-limiting
step for uptake in the wide variety of temporal and
spatial scales of nitrogen availability
that
may be encountered
by a marine phytoplankter would be invaluable
in assessing and predicting the dynamics of NH4+
uptake. The use of MA as an NH4+ analogue provides a convenient
way to determine the characteristics
and regulation of the NH 4+ transport system. We
have attempted similar, although less extensive,
uptake
studies
with IMA in
oceanic waters where low biomass necessitated the use of l-liter samples and
longer (l-4 h) incubation periods. In view
of the results presented here, the open
ocean measurements of MA uptake probably underestimated
transport activity.
Short term uptake measurements
in
oceanic waters might be improved if sam-
Methylammonium
ple volume were increased and the length
of the incubation
decreased.
Additional
metabolic
studies of the
regulation
of NH,+
assimilation
are
needed to determine
the role of subsequent metabolic processes in adaptations
to, and competition
for, available nitrogen supplies.
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Submitted: 4 January 1982
Accepted: 20 May 1982