The distribution and dynamics of nitrogen and microbial plankton in
southern Lake Michigan during spring transition 1999-2000

Wayne S. Gardner^1^, Peter J. Lavrentyev^2^, Joann Cavaletto^3^, Mark
McCarthy^1^, B.J.Eadie^3^, Thomas H. Johengen^4^, and James B. Cotner^5^

# 

# ^1^ Present address: The University of Texas at Austin, Marine Science Institute, 

#  750 Channel View Drive, Port Aransas, Texas 78373, USA

^2^ Department of Biology, The University of Akron, Akron, Ohio 44325,
USA

^3^ National Oceanic and Atmospheric Administration, Great Lakes
Environmental Research Laboratory, 2205 Commonwealth Blvd., Ann Arbor,
Michigan 48105, USA

^4^Cooperative Institute for Limnology and Ecosystem Research (CILER),

The University of Michigan, Ann Arbor, Michigan 48109, USA

^5^Department of Ecology, Evolution and Behavior, University of
Minnesota, St. Paul, MN 55108

January 13, 2003

**Abstract**

Ammonium and amino acid fluxes were examined as indicators of N and
microbial food web dynamics in southern Lake Michigan during spring.
Either ^15^NH~4~^+^ or a mixture of ^15^N-labelled amino acids (both at
4 μM N final concentration) was added to Lake Michigan water. Net fluxes
were measured over 24 h under natural light and dark conditions using
deck-top incubators and compared to microbial food web characteristics.
Isotope dilution experiments showed similar light and dark NH~4~^+^
regeneration rates at lake (6 nM N h^-1^ vs. 5 nM N h^-1^) and
river-influenced (20 nM N h^-1^ vs. 24 nM N h^-1^) sites. Ammonium
uptake rates were similar to regeneration rates in dark bottles. Dark
uptake (attributed mainly to bacteria) accounted for \~70% of total
uptake (bacteria plus phytoplankton) observed under natural light at
most lake sites but only \~30% of total uptake at river-influenced sites
in or near the St. Joseph River mouth (SJRM). Cluster analysis grouped
stations having zero, average, or higher than average N-cycling rates.
Discriminant analysis indicated that Chl concentration, biomass of
oligotrich ciliates, and total P concentration could explain 66% of the
variation in N-cycling rates on average. Heterotrophic bacterial N
demand was equal to about one third of the observed NH~4~^+^
regeneration rate. Overall, our results suggest that, with the exception
of the SJRM stations, bacterial uptake and protist grazing mediated much
of the N dynamics during spring transition. Since NH~4~^+^ is more
available to bacteria than NO~3~^-^, regenerated NH~4~^+^ may influence
the biochemical energetics of the lake by enhancing the degradation of
N-poor organic matter in this NO~3~^-^ -replete ecosystem.

#### Introduction

Lake Michigan is a large, temperate lake with pronounced seasonal
changes in physical dynamics that are driven by temperature changes. The
lake is stratified thermally in the summer but is isothermal in the
winter and spring with enhanced internal mixing. These physical changes
affect particle and nutrient distributions, which, along with seasonal
light and temperature variations, affect food web dynamics (Eadie et al.
2002). Various studies during stratification development and occurrence
have clarified our understanding of food web dynamics during this period
(see conceptual model of Scavia and Fahnenstiel 1987). Strong temporal
and spatial disequilibrium of bacterial production, with respect to the
amount of autotrophic fixation, have been reported for Lake Michigan
(Scavia and Laird 1987). Abundant assemblages of protists (ciliates and
flagellates) occur in the pelagic zone of the Great Lakes (Taylor and
Haynen 1987; Carrick and Fahnenstiel 1990; Fahnentsiel et al. 1998).
These eukaryotic microorganisms have high growth rates (Carrick et al.
1992; Lavrentyev et al. 1995), can exert considerable grazing pressure
on bacteria (Gardner et al. 1986; Hwang and Heath 1997) and
picophytoplankton (Fahnenstiel et al. 1991), form direct trophic links
to planktonic crustaceans (Carrick et al. 1991; LeBlanc et al. 1997),
and recycle phosphorus (Taylor 1984) and trace elements (Twiss et al.
1996).

Little information is available about biological processes during the
isothermal winter-spring period when lake physics are unstable, but
microbial processes may be active. Seasonal observations in Lake
Constance, Germany (Müller et al. 1991; Gaedke and Straile 1994) and
small temperate lakes (Lavrentyev and Maslevtsov 1988; Sommaruga and
Psenner 1995) indicated the presence of sizable microbial populations,
including protists, during winter and early spring. These observations
suggest that microbial food web (MFW) processes can be active in
temperate lakes during the winter-spring transition despite low water
temperatures and sub-optimal light regime.

Coastal turbidity plumes, from resuspension of near-shore sediments
during storms, may affect nutrient and food web dynamics (Eadie et al.
2002). For example, elevated concentrations of bacteria and loricated
ciliates occurred in a turbidity plume along the eastern shore of Lake
Michigan after a severe storm in early April 1996 (Eadie et al. 2002).
During a major resuspension event in March 1998, bacterial production
was high (64% of summertime rates; Cotner et al. 2000). The Episodic
Events Great Lakes Experiment (EEGLE) was funded jointly by The National
Science Foundation and National Oceanic and Atmospheric Administration
to provide information on effects of recurring plumes on the dynamics of
various processes during winter/spring in southern Lake Michigan.

Our research effort was directed at estimating N-cycling rates and
relating them to microbial characteristics in southern Lake Michigan
during spring transition when particle resuspension is prevalent.
Although Lake Michigan is considered P-limited (Scavia and Fahnenstiel
1987), studying N dynamics can provide insights about plankton dynamics
at the community and population levels (Haga et al. 1995; Miller et al.
1995). Nitrogen can serve as a "metabolic currency" to assess microbial
processes in aquatic ecosystems (Legendre and Rassoulzadegan 1995). We
expected that ammonium (NH~4~^+^) and/or amino acid uptake and
regeneration would reflect MFW characteristics. For example, community
NH~4~^+^ regeneration reflects heterotrophic activity by bacteria,
microbial grazers, or other secondary producers, whereas NH~4~^+^ uptake
is mediated by phytoplankton and bacteria. In addition to diatoms, we
hypothesized that non-diatom autotrophic and heterotrophic MFW organisms
were significant contributors to MFW dynamics in spring. We further
hypothesized that MFW processes would be enhanced at the edge of the
recurrent coastal plume, or near river mouths, where light levels and
nutrient concentrations would be optimized for primary and secondary
production.

In this paper, we report light and dark regeneration and potential
uptake rates for NH~4~^+^ and net amino acid flux in southern Lake
Michigan. These results are compared to MFW structure and other
information to gain insights about the autotrophic and heterotrophic
factors controlling N and microbial dynamics during the spring
transition.

**Methods**

**Sampling strategy and water column parameters.** Samples were
collected from the RV Lake Guardian during March 8-15 and June 3-7,
1999, and March 10-15 and May 14-20, 2000. Sampling locations are
indicated on Figure 1. Most sampling sites were located on
nearshore-offshore transects, but a station in the St. Joseph River
mouth (SJRM) provided information about N cycling and microbe
characteristics at the mouth of an important tributary delivering
materials into southern Lake Michigan. Temperature and turbidity were
measured at each station with a SeaBird STE-911 multi-sensor unit. Total
suspended materials (TSM) concentrations were determined gravimetrically
using a Mettler AT250 balance. Two-L aliquots of lake water were
filtered through pre-rinsed, dried, combusted and pre-weighed 45mm GF/F
filters (unless the filter became clogged sooner). Concentrations were
determined to the nearest 0.1 mg l^-1^ based on the volume filtered.
Nutrient and chloride concentrations were measured using standard
automatic colorimetric procedures on an Auto Analyzer II as detailed in
Davis and Simmons (1979). Samples for dissolved nutrient analysis were
filtered through a 0.2 μm nylon filter after collection and frozen until
analyses. Total phosphorus (P) and total dissolved P samples were stored
in acid-cleaned Pyrex test-tubes in the refrigerator, digested in an
autoclave after addition of potassium persulfate (5% final
concentration; Menzel and Corwin 1965), and analyzed for SRP with Auto
Analyzer.

**Ammonium and amino acid flux experiments.** Unfiltered lake water was
fortified with 0.40 μM PO~4~, except for the March 1999 cruise, which
did not include P-addition experiments. ^15^Nitrogen-labeled NH~4~^+^ or
an amino acid mixture (Algal Amino Acid mixture-U-^15^N, 96-99/NLM-2161,
Cambridge Isotope Laboratories, Inc.) was added to either untreated or
P-fortified lake water, to give a final concentration of 4 μM added N.
Seventy-ml aliquots from each of the 4 treatments was dispensed into
each of 6 tissue culture bottles (Corning 25 cm^2^ Polystyrene Tissue
Culture Bottles) for a total of 24 incubation bottles per experiment.
Initial (T-0) samples were collected, filtered through a rinsed syringe
filter (0.2 μm pore size), and analyzed in the shipboard laboratory for
NH~4~^+^ and primary amines (Gardner and St. John 1991). Remaining
filtrates from ^15^NH~4~^+^ samples were frozen in 8-ml Wheaton vials
for direct analysis of atom % ^15^NH~4~^+^ in the laboratory by high
performance liquid chromatography (HPLC; Gardner et al. 1995). Three
replicate bottles from each treatment were wrapped with aluminum foil
(dark treatments). All treatment bottles were placed in a deck-top
incubator maintained at *in situ* temperature with a Neslab temperature
controller and incubated for 24 h under natural light conditions
(Lohrenz et al. 1988). Twenty-four hour incubation intervals were
selected to provide sufficient time for measurable concentration changes
to occur and make light-dark comparisons consistent among samples
collected at different times of the day. At the end of the incubations,
samples were collected and filtered, and the filtrates were analysed
immediately for NH~4~^+^ and primary amines. Remaining filtrates were
frozen for atom % ^15^NH~4~^+^ analysis.

**Low-level ^15^NH~4~-addition uptake experiments.** Duplicate bottle
experiments were conducted in daylight and dark (duct tape covered),
except for samples collected in the evening or night, which were
incubated in the dark only. Two-liter portions of lake water were placed
in light and dark polycarbonate bottles. The water was spiked with
^15^NH~4~^+^ (0.5 μM final concentration) and mixed. Water from an
initial T-0 bottle was filtered through a 47 mm combusted (4 hours at
450 °C) glass fiber filter. Experimental bottles were incubated for at
least 6 h at ambient temperature and light in the deck-top incubator,
and experimental water was filtered to collect particles for T-1
measurements. One liter was filtered for experiments conducted in year
1999, and two liters were filtered in 2000. One-N HCl was applied to all
filters to remove inorganic material. The filters were stored frozen in
a small Petri dish until analysis of ^15^N:^14^N (Dugdale and Goering
1967; Glibert and Capone 1993) using a VG PRISM stable isotope mass
spectrometer.

Microbial plankton measurements. Microzooplankton (ciliates,
heterotrophic dinoflagellates, and rotifers), heterotrophic
nanoflagellates (HNF), and chlorophyll *a* (Chl) were sampled using 10-l
Niskin bottles mounted on a SeaBird carousel water sampler.
Heterotrophic nanoflagellates, preserved with 1% formaldehyde (final
concentration), were stained with DAPI on 0.8 μm black polycarbonate
filters and counted under an Olympus IX-70 microscope at 1,000x (Sherr
et al. 1993). Microzooplankton, preserved in 1% (final concentration)
acid Lugol's iodine, were counted after settling 25 to 100-ml aliquots
in a chamber for at least 18 hours. The entire chamber was scanned at
200-400x. Two to 3 liters of lake water were filtered through a 25 μm
Nitex mesh using gravity reverse flow to quantify rotifers, which often
were less abundant than protists. The resulting 100-ml sample was
preserved, stored, and processed as described above. Linear dimensions
of 30 to 90 individual protists and rotifers (fewer for less abundant
taxa) were measured at 400-600x and converted to volumes using
appropriate geometric shapes to estimate plankton biomass. Ciliate and
rotifer volumes were converted to carbon following Putt and Stoecker
(1989) and Fahnenstiel et al. (1998), respectively. Dinoflagellate and
HNF volumes were corrected for shrinkage using a factor of 1.3
(Montagnes et al. 1994) and converted to carbon (Menden-Deuer and
Lessard 2000). Bacterial secondary productivity (BSP) was measured with
tritiated leucine incorporation (20-nM final concentration) and a
theoretical conversion factor (3.1 kg C per mol leucine incorporated
(Kirchman 1993). Bacterial abundance and size were measured with
epifluorescence microscopy (Hobbie eta l. 1977). A SPOT digital camera
was used to photograph 500-1000 cells at each sampling site. Image
analysis was performed with Image Pro Plus software. Cell carbon was
estimated from cell volume assuming an ellipsoidal shape and applying a
conversion of 0.25 x 10^-12^ g C μm^-3^ (Psenner 1993; Cotner et al.
2000).

Chl was collected by filtering 100-ml aliquots of lake water through a
45-mm Whatman GF/F filter under low vacuum pressure and freezing the
filters in polypropylene tubes until extraction with N,
N-dimethylformamide (DMF; Speziale et al. 1984). DMF was added to the
tubes and heated in a water bath at 65°C for 15 min. After agitation and
centrifugation, the supernatant was analyzed on a fluorometer using the
acid correction method (Strickland and Parsons 1972; Speziale et al.
1984). The reported values are means from duplicate analyses. The
established coefficient of variation for this procedure is less than 4%.

Statistical comparisons. Statistical analyses, using Systat 8.0 (SPSS
1998) and JMP 4.0 (SAS 2000) software, were performed to compare
N-cycling data to MFW characteristics. Descriptive statistics showed
that N-cycling rate data were not distributed normally (skewness
coefficient values ranged from 1.6 to 3.5 for N dark regeneration and N
light uptake, respectively), which is typical for small data sets. Due
to some negative values, these data could not be log transformed.

The data were compared with heuristic statistics, which, in contrast to
linear techniques, do not rely on the normal distribution assumption. A
hierarchical cluster tree was derived for the uptake and regeneration
rates using the Ward linkage algorithm. Stepwise interactive
discriminant analysis was performed using the obtained clusters as a
grouping variable and other chemical and biological parameters as
explanatory variables. The jackknifed classification matrix was
calculated to cross-validate the results of disciminant analysis.
Group-means of variables included in the model were analyzed by the
two-group t-test.

**Results**

**Site description and characteristics.** Temperature, TSM, and
nutrients varied among stations and sampling times (Table 1). Mean water
temperatures (^o^C) for the different cruises were 1.5 ­[+]{.underline}
0.21 (March 1999), 3.5 ­[+]{.underline} 0.34 (March 2000), 10.2
­[+]{.underline} 1.6 (June 1999), and 11.0 ­[+]{.underline} 1.10 (May
2000). Nutrient and particle concentrations were higher in or near SJRM
than most open lake stations. TSM ranged from 0.7 to 21 mg l^-1^ among
sites. Particulate organic materials expressed as carbon (POC) ranged
from 13 to 67 μM C (with the highest value being offshore from Chicago)
and had C:N ratios ranging from 5 to 13. Lake Michigan has high nitrate
(NO~3~^-^) levels during the winter-spring period (e.g. 17-30 μM for
stations not influenced directly by the river; Table 1). By contrast,
ambient NH~4~^+^ levels ranged from 0.1 to about 1.0 μM except at SJRM
where concentrations reached 4.3 μM. Most SRP concentrations were less
than 0.05 μM but higher values were observed at stations near SJRM.
Chloride ion, indicative of coastal or riverine influence, ranged from
11 to 13 mg l^-1^ at most stations but were elevated at SJRM and,
occasionally, at nearshore stations along the St. Joseph transect.

Biological characteristics are presented in Table 2. Chlorophyll
concentration and bacterial biomass and production rates were higher at
or near SJRM than other stations. Chlorophyll concentrations ranged from
0.7 to 18 μg Chl l^-1^ (0.7 to 2.8 μg Chl l^-1^ excluding SJRM).
Bacterial biomass ranged from 10 to 104 μg C l^-1^ (10 to 40 μg C l^-1^
excluding SJRM). Mean bacterial biomass for the same samples ranged from
23 ± 4 μg C l^-1^ in March to 28 ± 4 μg C l^-1^ in June 1999 and were
not significantly different among the 4 cruises. Bacterial production
rates were less than 6 μg C l^-1^d^-1^ at most stations but were higher
at some stations in the St. Joseph transect. Other heterotrophic
organisms, considered for biomass comparison with N-cycling rates, were
heterotrophic nanoflagellates, oligotrichs, and other microbial grazers
including non-oligotrichous ciliates, heterotrophic dinoflagellates, and
rotifers (Table 2). Total grazer biomass ranged from about 0.4 μg C
l^-1^ off Chicago in both March cruises to 54 μg C l^-1^ at SJRM in May
2000. Excluding SJRM and Chicago stations, total grazer biomass ranged
from 2 to 24 μg C l^-1^ (Table 2).

**Ammonium uptake and regeneration rates.** Light NH~4~^+^ uptake rates
in small bottles (high-level NH~4~^+^ additions) were of the same order
of magnitude as rates from large bottles (low-level additions) except
near SJRM, which showed higher rates for high- versus low-level
additions (Figure 2). In dark bottles, a linear relationship was
observed between the two methods with progressively lower rates from the
low-level addition experiments as rates increased for the high-level
additions, again with differences emphasized at SJRM.

Potential uptake rates in March of both years ranged from about zero to
30 nM h^-1^ except for light SJRM samples, which had uptake rates \~60
nM h^-1^ (Figure 3). Light uptake rates were higher than dark at SJRM
but not other stations. Ammonium regeneration rates were low and
comparable in March with standard error (SE) bars often overlapping zero
except at SJRM, where rates were higher in March 2000.

In May and June, NH~4~^+^ uptake rates ranged from about zero to 30 nM
h^-1^ at all sites except SJRM, where mean light uptake rates were about
160 nM h^-1^ in May 2000 and 110 nM h^-1^in June 1999 (Figure 4).
Ammonium regeneration rates for the same samples ranged from near zero
to 29 nM h^-1^ (Figure 4). Most sites had positive mean regeneration
rates but some SE overlapped with zero. Sites near SJRM had higher
NH~4~^+^ regeneration rates, but differences among sites were not as
pronounced as for uptake. The effects of darkness, P additions, and
interactions between the two processes were not uniform for either
uptake or regeneration rates. Both light and P-addition effects on
NH~4~^+^ uptake rates were most pronounced at stations near SJRM, where
regeneration rates also were elevated. In contrast to uptake results,
NH~4~^+^ regeneration rates were not much different for light vs. dark
and P addition experiments near SJRM.

**Ammonium cycling rates and their relationship to chemical/biological
sampling-site characteristics.** Data from all experiments and
treatments were combined to observe relationships between NH~4~^+^
uptake and regeneration rates in light and dark bottles. The
relationship was not useful for comparisons among stations because
multiple treatments were done on water from the same stations, but it
provided a useful tool to examine light effects and explain outlying
data. In dark bottles, NH~4~^+^ uptake and regeneration rates were
related (R^2^ = 0.836, y = 1.015 + 0.0030; p \<0.001) and near equal on
average. Light-bottle comparisons of uptake and regeneration rates were
similar except that uptake was higher than regeneration at SJRM and at
J15 in June 1999. These outliers were separated from the other lake data
to form general conclusions about mean N cycling rates in "lake" and
"river-influenced" regions, respectively, but were included in the
statistical comparisons of N-cycling data to MFW characteristics.

The mean dark uptake rate at the "lake" sites (i.e. without outlier
sites) was 7 ± 1 nM N h^-1^ compared to a mean light uptake rate of 10 ±
1 nM N h^-1^ (n = 52). The mean dark NH~4~^+^ regeneration rate (5 ± 1
nM N h^-1^) did not differ significantly from the mean rate under
natural light (6 ± 1 nM N h^-1^) at "lake" sites. The mean NH~4~^+^
regeneration rate at "river-influenced" sites was higher than "lake"
sites but also did not differ significantly between light (20 ± 2 nM N
h^-1^) and dark (24 ± 5 nM N h^-1^) bottles.

**Statistical comparison of microbial food web characteristics and
ammonium cycling rates.** Hierarchical cluster analysis for all stations
and treatments without P additions resulted in a cluster tree for uptake
and regeneration rates consisting of three major groups (A, B, C) and
one outlying station, SJRM-May 2000 (D; Figure 5). These clusters
grouped similar observations of N cycling rates. The first cluster (A)
combined stations where rates were not significantly different from
zero. The second cluster (B) included sites with \"average\" rates (i.e.
significantly above zero but not different from average values). The
third cluster (C) combined sites with rates that were above average, and
the fourth cluster (D) consisted of the SJRM station with much higher
rates (Figure 6).

Results of multivariate discriminant analysis, which used cluster order
as a grouping variable, are presented in Table 3. On average, the
combination of Chl, biomass of oligotrich ciliates, and total P (TP)
correctly predicted 81% (66% after the jackknifed cross-validation) of
the observed cycling rates.  The other variables (temperature, Cl^-^,
NH~4~^+^ and NO~3~^-^ concentrations, C:N ratios, bacterial biomass and
production) did not show significant relationships to NH~4~^+^ cycling
rates. Both Chl concentration and oligotrich biomass followed patterns
similar to N-cycling rates among the clusters (Figure 7). Comparison of
group means suggested that Chl helped distinguish between the
\"zero\" and \"average\" clusters (A vs. B, p =0.02), and oligotrich
biomass distinguished more between the \"average\" and \"high\"
categories (B vs. C, p=0.014).  Although TP was not significantly
different among the groups, it helped improve the model by ca. 10%
(based on the jackknife; Table 3). 

**Net amino acid fluxes.** In March 1999, net amino acid flux ranged
from uptake of 20 nM N h^-1^ to production of 20 nM N h^-1^ (Table 4).
In March 2000, net amino acid flux overlapped with zero for one or more
treatments at all sites except SJRM and J15, which had uptake rates of 5
and 30 nM N h^-1^, respectively, and C15,which had production rates of
20 nM N h^-1^ (Table 4). Except for J15 with uptake rates of 15-20 nM N
h^-1^, amino acid flux was \<10 nM N h^-1^ in either direction in June
1999 (Table 5). Significant uptake was observed once in May 2000 (30 nM
N h^-1^ at SJRM; Table 5). Significant production (12 to 60 nM N h^-1^)
occurred at three sites in May, but rates at the other four sites were
low and often overlapped with zero. Increases in NH~4~^+^ regeneration
in response to amino acid additions were not observed at any sites.

**Discussion**

**Methodological considerations.** Although NO~3~^-^ is the dominant
form of inorganic N in Lake Michigan, we focused on NH~4~^+^ and amino
acid dynamics because they are active N forms mediated by MFW organisms
and occur at low concentrations since they are often taken up as rapidly
as they are produced. Ammonium is preferred by phytoplankton and
bacteria (Kirchman 2001) and often is the dominant N form produced by
heterotrophic mineralization of organic N (Campbell 1973). Amino acids
are labile organic N and C sources produced by phytoplankton exudation
(Bronk and Glibert 1993, Bronk et al. 1994) or grazing (Nagata and
Kirchman 1991) and are incorporated into the food web by bacterial
uptake (Fuhrman 1990, Kirchman et al. 1989). Alternatively,
extra-cellular enzymes released by N-starved phytoplankton may
de-aminate amino acids to provide an NH~4~^+^ N-source (Palenik and
Morel 1990). We assumed that phytoplankton production of amino acids
would occur in the light, or soon after light exposure, but bacterial
uptake rates would be similar in the light and dark.

The addition of ^15^NH~4~^+^ to water samples allowed measurement of
NH~4~^+^ concentration and isotope ratio changes over time (Gardner et
al. 1995) and calculation of potential NH~4~^+^ uptake and regeneration
rates (Blackburn 1979; Caperon et al. 1979). Since concentrations of
added NH~4~^+^ were higher than ambient concentrations, uptake rates
must be considered to be "potential" rather than "actual" rates.
However, regeneration rates should not be affected directly by NH~4~^+^
additions. The differences between potential and actual uptake rates
should have been minimized for the low-level (0.5 μM) additions where
added concentrations were similar to natural levels (0.1 -- 0.7 μM N).
Our small-bottle experiments were conducted over 24-h incubation
intervals to obtain measurable concentration changes over a light-dark
cycle. The large-bottle experiments with tracer additions were run for
only about 6 h to keep the tracer from being removed quantitatively.
However, even the 6-h period was too long for tracer additions at SJRM
in May with high biomass and N demand. A feasible explanation for those
exceptions is that high biomass at SJRM caused ^15^NH~4~^+^ to be
removed before samples were collected at the first time point. Such
early removal results in an underestimation of calculated uptake rate
that is based on the length of the sampling interval. The high-level
addition experiments gave reasonable estimates of potential NH~4~^+^
uptake and were more robust for long incubation times because added
^15^NH~4~^+^ was not depleted from solution between time points. The
small magnitude of the observed rates, the similarities between results
from high and tracer level additions at lake stations, and the agreement
between dark regeneration and uptake suggest that measured potential
uptake rates may have approached actual uptake rates in our experiments.
The incubation period of 24 h was needed to obtain light-dark
comparisons among stations sampled at different times of the day. We
believe that bottle effects over the 24 h incubation period were likely
minimal considering the low temperatures and high substrate additions.

**Microbial food web composition.** The results of this study indicate
that planktonic protists, particularly ciliates, are an abundant
component of plankton during late winter and early spring in southern
Lake Michigan. Although microzooplankton biomass can reach 25 μg C l^-1^
in the epilimnion or deep chlorophyll layer in offshore Lake Michigan
during late stratification (Carrick and Fahnenstiel 1990), the
depth-weighted averages were comparable between winter-spring (this
study) and summer stratification (Fahnenstiel et al. 1998). The relative
biomass of ciliates in Lake Michigan appears to be high in early spring
when cladocera are virtually absent and copepod numbers are low
(Barbiero et al. 2001). Combined with their high specific growth rates
at low temperatures (Carrick et al. 1992; Kovalcik 2001), these data are
consistent with the enhanced role of microzooplankton during the
winter-spring transition. Heterotrophic nanoplankton are at their
seasonal minimum in offshore Lake Michigan during winter-early spring
(Carrick et al. 1991). Nanoflagellates peaked near SJRM in late
spring-early summer (May-June), but remained low (\< 2 μg C l^-1^) at
mid-transect and in offshore waters in this study. This result can be
explained by predation pressure from microzooplankton-sized ciliates,
which is the primary factor controlling nanoflagellate populations early
spring (Weisse 1991).

In offshore Lake Michigan, microbial plankton had an almost uniform
distribution during the winter-spring transition. Along the lake
margins, spatial variability was profound reflecting the complexity of
physical processes in the southern basin. Similar trends have been
described in Lake Ontario (Taylor and Haynen 1987). The greatest and
most variable concentrations of microzooplankton, nanoflagellates, and
bacteria occurred nearshore at St. Joseph. Because these stations are
immediately offshore the St. Joseph River confluence, this variability
may reflect the co-influence of tributary inflows and sediment
resuspension on coastal assemblages.

Heterotrophic or autolithotrophic, rather than autotrophic, organisms
may dominate in winter/early spring because "nephloid layer" organic
materials are distributed throughout the water column (Cotner et al.
2000). Riverine inputs of nutrients and organic matter also are
amplified in the spring when river flow is high. These plumes of
resuspended and riverine particles reduce available light for
phytoplankton (Fahnenstiel et al. 2000) but can provide labile organic
substrates and other nutrients to heterotrophic bacteria during spring
(Cotner et al. 2000; Eadie et al. 1984).

**Nitrogen fluxes and microbial food web characteristics.** Not
including the outlying SJRM sites, NH~4~^+^ uptake (mean = 10 nM N
h^-1^) and regeneration rates (6 nM N h^-1^) under natural light
conditions were low relative to other published rates in coastal waters
(Lipshultz et al. 1986; Selmer 1988; Hanson et al. 1990; Cotner and
Gardner 1993; Gardner et al. 1993, 1997). This difference is not
surprising for cold waters with low primary production rates. The SJRM
sites had higher rates and exhibited greater response to light or added
P than lake sites, which is an indication of high microbial activity and
phytoplankton importance.

Spring process rates were compared with microbial characteristics at
various sites and times to gain insights about the activity and function
of the microbial community during this transition period. Except for
effects of the St Joseph River, our data did not distinguish
hypothesized regional influences related to the recurrent coastal plume.
However, the plume-effect hypothesis could not be tested rigorously
because resuspension events during our cruises were moderate relative to
the large plume of 1998 (Cotner et al. 2000). Our data do provide
insights about N-cycling during the spring transition period in Lake
Michigan.

The lack of a temporal pattern in N cycling rates is of interest because
mean temperatures ranged from 1.5 to 11.0 ^o^C during the cruises. We
expected that May and June results would be affected by spring diatoms
(Fahnenstiel and Scavia et al. 1987), but such trends were not apparent.
A feasible explanation for this apparent lack of diatom effects on
NH~4~^+^ cycling rates is that diatoms can use NO~3~^-^ rather than
NH~4~^+^ as their primary N source. In addition, based on soluble silica
depletion, epilimnetic diatom production accounted for \< 20% of total
primary production even when diatoms dominated (Laird et al. 1988).

Discriminant analysis identified variables (Chl, OLIG, and TP) that
helped explain differences among clusters of stations with different N
cycling rates. This comparison is consistent with the idea that observed
regeneration was driven by protists, such as oligotrichs.
Microzooplankton-sized oligotrich ciliates are major consumers of
primary production in offshore southern Lake Michigan during
winter-spring (Kovalcik 2001). In addition, small oligotrichs, like
*Rimostrombidium brachykinetum,* often are abundant at locations
characterized by elevated bacterial and picophytoplankton abundance.
Dense populations (compared to background abundance at other stations)
of small (\<3 μm) picophytoplankton (*Synechococcus*-like cyanobacteria)
occurred at most sites where regeneration rates were enhanced (Hersha
2002). Although their contribution to total phytoplankton is not as high
during spring transition as during summer stratification (Fahnenstiel et
al. 2000), they grow faster than diatoms and cryptophytes and are
consumed by ciliates at high rates (Kovalcik 2001). The resuspension
zone near St. Joseph was characterized by enhanced bacterivory rates
(Hersha 2002). In contrast, shallow sites away from the river often had
low populations of cyanobacteria and ciliates and corresponding low
N-cycling rates. 

Our light-dark NH~4~^+^ cycling-rate data and comparisons with microbe
characteristics support the conclusion that N uptake by bacteria and
regeneration by ciliates may be important mechanisms for NH~4~^+^
cycling during the spring transition. The results suggest that
heterotrophic and nitrifying bacteria may be responsible for about 70%
of the total NH~4~^+^ uptake observed at "lake" sites, agreeing with the
conclusion that bacterial activity is the dominant force driving MFW
dynamics in regions influenced by resuspension (Cotner et al. 2000).
However, our light-dark comparison may be biased by the fact that
nitrification may be inhibited by light (Ward 2000) during daylight
hours in light but not dark bottles. Previous work in southern Lake
Michigan indicated that nitrification rates were higher in dark than
light bottles (Personal communication, Harvey Bootsma, University of
Wisconsin at Milwaukee).

Comparing uptake to regeneration rates for all sites and treatments
revealed that potential uptake was higher than regeneration in some
light bottles. However, in dark bottles, NH~4~^+^ uptake and
regeneration rates were correlated (p \<0.001) and about equal in
magnitude. This 1:1 relationship between dark uptake and regeneration
suggests that bacterial uptake and community regeneration were coupled
tightly and may indicate that the amount of NH~4~^+^ taken up was
regulated by the production rate.

The SJRM and June 1999 J15 "river-influenced" samples, with high Chl
values, had three times higher uptake rates in the light (100 ± 17 nM N
h^-1^, n = 6) than dark (32 ± 4 nM N h^-1^, n = 6), except for the May
2000 J15 site, which had low Chl levels and N-cycling rates similar to
other lake samples. That site was the only one where heterotrophic
nanoflagellates dominated the biomass. Overall, the comparison of light
vs. dark bottles revealed that photoautotrophic (light minus dark)
uptake was small relative to rates for heterotrophic or nitrifier uptake
(dark) or regeneration rates in lake samples but important at two sites
in the river mouth region.

**Possible importance of ammonium as an N substrate for bacteria during
spring resuspension.** An intriguing question considered in this study
was: "What nutrients or other factors limit microbial activity during
spring transition?" Added amino acids or P did not enhance microbial
dynamics as reflected by N cycling rates at most sites in our study. Net
amino acid fluxes were small at most stations indicating that the
community did not have a large demand for either organic C or N supplied
as amino acids. Native microbial populations must not have been adapted
to high concentrations or inputs of amino acids. Even in the summer, a
lag of more than 24 h was observed before amino acids were taken up at
significant rates in Lake Michigan (Gardner et al. 1986, 1987, 1989). In
contrast, rapid uptake of amino acids occurred within a few hours in
mid-salinity regions of the Mississippi River plume (Gardner et al.
1993, 1996). Phosphorus additions did not cause NH~4~^+^ or amino acid
uptake rates to increase except near SJRM, where the P effect was
moderate. Soluble reactive P concentrations were low in most of the
unspiked lake samples, but available P, associated with particles, is
abundant in surface waters during the spring transition period (Eadie et
al. 1984).

Nitrogen limitation initially seemed unlikely because dissolved NO~3~^-^
was abundant, but our experiments suggest that NH~4~^+^ could be an
important factor. The degree of use of NO~3~^-^ as a source of N for
natural-water bacteria is not well understood, but energetically, it is
an inefficient source of N for bacterial assimilation (Kirchman 2001).
In contrast, NH~4~^+^ may enhance microbial degradation of N-poor
organic matter (e.g. Goldman et al. 1987; Gardner et al. 1996) because
NH~4~^+^ requires five times less energy for assimilation than NO~3~^-^
(Vallino et al. 1996). Available NH~4~^+^ regenerated in the water
column by microbes in response to autotrophic production could provide
reduced N for heterotrophic bacteria that derive energy from
partially-degraded organic materials with high C:N ratios. Reduced N
forms, such as NH~4~^+^ or amino acids, may enhance degradation of
organic residues with high C:N ratios (e.g. Amon et al. 2001). The C:N
ratios of the particles in our samples (7.5 to 13.9, mean 9.3 ± 0.3)
were higher than normal Redfield ratios for bacteria or phytoplankton
(4.6 to 7; Kirchman 2001).

Our amino acid addition experiments argue against C limitation of
bacterial growth but are consistent with the idea that bacteria and
organic C dynamics could be enhanced by NH~4~^+^ regeneration. Rapid
uptake of added amino acids and/or production of ^15^NH~4~^+^, expected
if bacteria were limited by organic C (Gardner et al. 1996), were not
observed in any of the experiments where labeled amino acids were added.
The absence of ^15^NH~4~^+^ accumulation at SJRM where significant
amounts of amino acids were removed suggests that any NH~4~^+^ produced
was taken up immediately.

Ammonium regeneration and uptake rates were about equal in most of our
experiments, indicating a close coupling between these two processes. To
compare potential bacterial requirements with our observed NH~4~^+^
regeneration rates, we estimated mean N demand from measured bacterial
growth rates (Table 2) and compared it to dark NH~4~^+^ regeneration
rates. Assuming that bacteria have a molar C:N ratio of five (Kirchman
2001) and a mean growth rate of 3.7 (SE =1.0, N=33) μg C l^-1^ day^-1^
(average bacterial production rate from Table 2) , the calculated N
demand was 2.6 nM N h^-1^ compared to mean dark NH~4~^+^ regeneration of
9.4 nM N h^-1^ for the same stations. This calculation implies that
NH~4~^+^ regeneration provides more than sufficient N for heterotrophic
bacterial production with the remainder available for uptake by
nitrifiers or autotrophs. Nitrifying bacteria, which obtain energy from
NH~4~^+^ and fix inorganic C, also contribute to the bacterial base of
microbial populations (Lavrentyev et al. 1997), but their biomass
production would be low because nitrifiers convert most NH~4~^+^ to
NO~3~^-^ rather than into biomass (Ward 2000).

We conclude that microbial and N dynamics are active and interactive
during spring transition. Ammonium regenerated in the water column may
provide reduced N needed to supplement resuspended or river-derived
organic C for bacterial production, which drives much of the food web
during spring transition. These results contrast to the stratified
summer period when P and light availability, as well as top-down
grazing, are major forces limiting autotrophic production and consequent
food web processes (Scavia and Fahnenstiel 1987). More studies are
needed to define the role of NH~4~^+^ vs. NO~3~^-^ (and organic N) in
the degradation of organic residues with high C:N ratios.

**Acknowledgements**

We thank Paul Kovalik and Deb Herhsa for laboratory and field
assistance, Hank Vanderploeg and other EEGLE colleagues for field
assistance and advice, Greg Lang for data handling contributions, Harvey
Bootsma and two anonymous referees for reviewing an earlier version of
the manuscript, and the Captain and Crew of the E.P.A. R/V LAKE GUARDIAN
for help with sample collections and efficient operations. This research
was supported by NSF (OCE-9726681, OCE-9817805) and NOAA (46290000) as
part of the Episodic Events Great Lakes Experiment program administered
through the Cooperative Institute of Limnology and Ecosystem Research.

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# List of Tables

Table 1. Concentrations of total suspended materials (TSM), total P
(TP), total dissolved P (TDP), particulate P (PP), soluble reactive P
(SRP); NO~3~^-^, NH~4~^+^, particulate organic N (PON), PON; POC ratio,
temperature and Cl^-^ in March 1999, June 1999, March 2000, May 2000,

Table 2. Chlorophyll concentrations, biomass for bacteria, heterotrophic
nanoflagellates (HNF), oligotrichs (OLIG), other ciliates, and total
microbial grazers, and bacterial secondary production (BSP) in samples
from southern Lake Michigan and the SJRM during March 1999, March 2000,
May 2000, and June 1999.

Table 3. Result of discriminant analysis using clusters (Fig 6) as a
grouping variable. Multivariate statistics: Wilk's lambda = 0.391; F =
5.39, p = 0.0002.

Table 4. Net amino acid fluxes ([+]{.underline} SE) in amino
acid-fortified incubation bottles for waters sampled in March 1999 and
March 2000

Table 5 Net amino acid fluxes ([+]{.underline} SE) in amino
acid-fortified incubation bottles for waters sampled in May 2000, and
June 1999.

List of Figures

Figure 1. Map of sampling locations in southern Lake Michigan. Specific
samples are indicated in the text by sequential listing of the transect,
station depth, and sample depth.

Figure 2. Comparison of ammonium removal rates in small bottles with
high-level (4 μM) ammonium substrate additions to those collected on
particles from the large bottles with low-level (0.5 μM) additions

Figure 3. Potential uptake and regeneration rates ([+]{.underline} SE)
for ammonium at southern Lake Michigan sites sampled in March 1999 and
March 2000

Figure 4. Potential uptake and regeneration rates ([+]{.underline} SE)
for ammonium at southern Lake Michigan sites sampled in May 2000 and
June 1999

Figure 5. The results of hierarchical cluster analysis of N cycling
rates.  The plot beneath the dendrogram has a point for each cluster
join. The ordinate is the distance that was bridged to join the clusters
at each step.  The distance between each two clusters is the ANOVA sum
of squares between the two clusters added up over all the variables.

 

Figure 6. Box plots of nitrogen cycling rates arranged in clusters (see
Figure 5). LR - light regenration, DR - dark regeneration, LU - light
uptake, DU - dark uptake. The center vertical line marks the median of
the sample. The length of each box shows the range within which the
central 50% of the values fall, with the box edges at the first and
third quartiles. Fences define outside and far outside values. The
whiskers show the range of observed values that fall within the inner
fences. Values between the inner and outer fences are plotted with
asterisks.

Figure 7. Box plots of explanatory variables from the discriminant
analysis (Table 3). The center vertical line marks the median of the
sample. See Figure 6 for explanation of symbols.

# 

#  

#