Estuarine, Coastal and Shelf Science 147 (2014) 78e86
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Estuarine, Coastal and Shelf Science
journal homepage: www.elsevier.com/locate/ecss
Dissolved inorganic and organic nitrogen uptake in the coastal North
Sea: A seasonal study
Alessia Moneta a, *, Bart Veuger a, Pieter van Rijswijk a, Filip Meysman a, b,
Karline Soetaert a, Jack J. Middelburg a, c
a
b
c
Department of Ecosystem Studies, Royal Netherlands Institute for Sea Research NIOZ-Yerseke, PO Box 140, 4400 AC Yerseke, The Netherlands
Laboratory of Analytical and Environmental Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium
Faculty of Geosciences, Utrecht University, PO Box 80021, 3508 TA Utrecht, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 August 2013
Accepted 25 May 2014
Available online 9 June 2014
Nitrogen incorporation into total particulate suspended matter, hydrolysable amino acids and bacterial
biomarker D-Alanine was assessed seasonally in the coastal North Sea using 15N-labeled ammonium,
nitrate, nitrite and 15N- and 13C-labeled urea, glycine, leucine, phenylalanine, and two complex pools of
dissolved organic matter (DOM) derived from algal and bacterial cultures (A-DOM, B-DOM). We investigated: 1) uptake rates for the various substrates and their contribution to total N uptake; 2) microbial
preferences for the different N sources; 3) the coupling of C and N uptake from organic substrates; 4) the
contribution of bacteria to the total microbial uptake of these substrates, and 5) the role of a complex
pool of organic matter for plankton nutrition. Seasonality in the preferences for N substrates was
observed, with A-DOM and B-DOM being preferred in autumn and winter whereas NHþ
4 was preferentially taken up in spring and summer. C and N uptake was coupled for all the organic substrates, except
urea that was mainly used as a nitrogen source in summer and spring. Bacterial contribution to the
uptake of A-DOM and B-DOM was, on an annual average, the lowest among the N-substrates. This
suggests an important role for phytoplankton in the incorporation of complex organic matter and the
importance of DOM for phytoplankton nutrition.
© 2014 Elsevier Ltd. All rights reserved.
Keywords:
nitrogen uptake
phytoplankton
bacteria
dissolved organic nitrogen
stable isotopes and biomarkers
D-alanine
The Netherlands
Wadden sea
Marsdiep tidal inlet (53.001833 N,
4.789201 E)
1. Introduction
Nitrogen (N) is an essential element for growth of phytoplankton and heterotrophic bacteria and is often limiting in marine
and coastal waters. It can be present in dissolved inorganic forms
(DIN: NHþ
4 , NO3 , NO2 ) or as dissolved organic nitrogen (DON), a
complex pool whose chemical composition varies spatially and
temporally (Benner, 2002). The chemical composition of this heterogeneous DON pool is difficult to characterize and only a small
fraction (<20%) of the individual compounds can be typically
identified (Benner, 2002). This fraction includes urea, amino acids,
amino sugars, humic and fulvic substances, as well as nucleic acids.
DON typically accounts for 10e20% of the total dissolved nitrogen
in coastal waters and ~80% in open ocean systems, but its role in
phytoplankton nutrition was considered negligible (Antia et al.,
1991; Benner, 2002; Berman and Bronk, 2003).
* Corresponding author.
E-mail address: [email protected] (A. Moneta).
http://dx.doi.org/10.1016/j.ecss.2014.05.022
0272-7714/© 2014 Elsevier Ltd. All rights reserved.
However, more than fifty years ago the ability of phytoplankton
to take up DON was demonstrated by Hattori (Antia et al., 1991).
Moreover, there is increasing evidence that natural communities of
phytoplankton and bacteria can rely on a much greater range of N
substrates than traditionally believed (Andersson et al., 2006;
Bradley et al., 2010a; Van Engeland et al., 2013). The DON contribution to the total N uptake range from ~20% to up to 80% and
phytoplankton can rely more on organic N when competition with
bacteria for DIN is high. However, these studies have used a limited
range of simple organic substrates, mainly urea and amino acids,
whereas a more complex and heterogeneous pool of DON is
available in marine ecosystems. Few studies have examined
whether natural planktonic microbial communities can utilize this
large, complex pool (Bronk and Glibert, 1993; Veuger et al., 2004;
Van Engeland et al., 2013).
Another important aspect that remained understudied is the
relative importance of phytoplankton versus bacteria in total microbial N assimilation. Early works from the 1970's (Goering et al.,
1970; Eppley et al., 1977) already suggested bacterial use of DIN
showing that heterotrophs were important in the total uptake of
A. Moneta et al. / Estuarine, Coastal and Shelf Science 147 (2014) 78e86
DIN in different systems. More recent studies showed that a large
fraction of the bacterial production is supported by NHþ
4 and that
bacteria could switch to NHþ
and
NO
when
the
supply
of dis3
4
solved free amino acids is insufficient. Moreover, bacteria appear
able to outcompete phytoplankton in the uptake of NHþ
4 at low
concentrations (Hoch and Kirchman, 1995; Middelboe et al., 1995;
Kirchman, 2000; Jørgensen, 2006). The assessment of the relative
contribution of phytoplankton versus bacteria to N uptake, remains
a challenging issue. Methods applied so far are size fractionation,
flow-cytometric sorting, the use of specific metabolic inhibitors, or
a combination of these (Veuger et al., 2004; Bradley et al., 2010b;
Trottet et al., 2011). However, each suffers from limitations such
as overlapping sizes fractions and limited efficiency and specificity
of inhibitors (Glibert et al., 1991; Bradley et al., 2010b; Trottet et al.,
2011). Recently, the use of stable isotope tracers in combination
with biomarkers, has enabled better discrimination between the
algal and bacterial contribution to N and C uptake in natural systems. The use of the hydrolysable amino acid D-alanine, a structural
component of bacteria, has proven a powerful tool to estimate the
bacterial contribution to the uptake of N by benthic microorganisms (Veuger et al., 2005; 2007).
Another aspect of microbial N-assimilation is the use of N versus
C from organic substrates. While organic N is primarily used to
support growth, the coupled organic C may either be used for
growth (assimilation) or be used as a source of energy (respiration).
Few studies have studied the relative use of C and N from organic
substrates by natural communities (Bronk and Glibert, 1993; Fan
and Glibert, 2005; Andersson et al., 2006; Veuger and
Middelburg, 2007). These have demonstrated that DON and DIN
uptake by natural marine communities, as well as competition for
different substrates, vary both spatially and temporally, highlighting the need for further investigations (Middelburg and
Nieuwenhuize, 2000a, b; Veuger et al., 2004; Fouilland et al.,
2007; Bradley et al., 2010a; Bronk et al., 2007).
To the best of our knowledge, in this study, we are the first one
investigating monthly over a whole year the uptake of DIN, urea,
dissolved free amino acids (DFAA) and two complex DON pools by
phytoplankton and bacteria in the water column of the coastal
North Sea. The use of 15N and 13C labeled organic substrates and
stable isotope analysis of bulk suspended particulate matter (SPM)
and of hydrolysable amino acids (HAA) therein allowed us to
investigate 1) the importance of various DIN and DON substrates in
total microbial N assimilation, 2) microbial preferences for the
various N substrates, 3) the coupling between N and C from organic
N-substrates, 4) the role of algae versus bacteria in total microbial N
assimilation, and 5) the potential importance of uncharacterized
DON as source of N for microbial growth.
2. Materials and methods
2.1. Study site and sampling
Water was sampled from the Marsdiep from the pier of the
Royal Netherlands Institute for Sea Research on the island of Texel
(53.001833 N, 4.789201 E), starting in October 2009 until October
2010. The Marsdiep is the most southwestern tidal inlet of the
Wadden Sea and is connecting the Western Wadden Sea to the
Southern North Sea. Even though it is an outflow channel, at high
e and Hegeman, 1993).
tide it receives coastal North Sea water (Cade
Samples were always collected at high tide, meaning that we
sampled coastal North Sea water. Due to strong tidal currents, these
waters are fully mixed (i.e. sampled water represents an average for
the whole water column).
A pump and hose apparatus was used to collect surface water in
10 L white plastic containers, one for each treatment described
79
below, and the 15N- and 13C-containing substrates were added
immediately after sampling. Due to time, budget and other logistic
constraints, replicates were unfortunately not performed.
2.2. Incubations
15
15
15
NHþ
4 ( NH4Cl, 99%), NO3 (Na NO3, 98%) and NO2 (Na NO2,
13
98%) were used as inorganic substrates, whereas urea ( C, 99%; 15N2,
98%), L-glycine (U13C2, 98%; 15N, 98%), L-leucine (U13C6, 98%; 15N,
98%) and L-phenylalanine (U13C9, 98%; 15N, 98%) (all from Cambridge
Isotope Laboratories) were used as simple, well-defined organic
substrates with different structural complexity. In addition, two
pools of complex DOM were derived from an axenic algal culture and
a bacterial culture grown on 15N and 13C labeled substrates. For the
algae derived DOM (referred to as A-DOM), an axenic culture of the
diatom Skeletonema costatum was grown in artificial sea water
containing F2 medium using 30% NaH13CO3 (13C, 99%) and 15%
Na15NO3 (15N, 98%). For the bacteria derived DOM (B-DOM), a bacterial sample isolated from waters of the Eastern Scheldt (a marine
coastal bay in the southwest of the Netherlands) was grown on the
modified medium M63 (Miller, 1972) with 15% NH4Cl (15N, 99%) and
15% D-glucose (U13C6, 99%). After approximately 3 weeks of incubation, algal and bacterial material was harvested through filtration
and suspended in Milli-Q water inside centrifuge tubes. To eliminate
all the DIN, Devarda's Alloy (to reduce NOx to NHþ
4 ) and MgO (to
convert all NHþ
to
NH
)
were
added
to
the
tubes,
which
were then
3
4
shaken for 48 h to remove all gaseous NH3 from the water. This DIN
removal procedure has been tested extensively as reported in
Veuger et al. (2004). Following three consecutive steps of centrifugation and collection of the supernatant, organic material was
filtered onto 0.2 mm polycarbonate filters (Millipore) to isolate only
the dissolved fraction. Final isotope enrichments of A-DOM and BDOM were 12.4% and 16.7%, respectively for N (15N atom %) and 5.8%
and 14.7%, respectively for C (13C atom %).
15
N enriched substrates were targeted to be added at a tracer
level of 10% of ambient concentrations. The latter were based on data
from previous years, which were retrieved from the DONAR database (Rijkswaterstaat, 2009) via the Waterbase website (http://live.
waterbase.nl). Added 15N in the urea and, on some occasions, in the
DIN incubations was higher than 10% of the ambient concentrations,
but we will discuss in more detail the implications of this in the
sections 3.2 and 4.1. DFAA concentrations were always very low
(<0.1 mM) which made labeling at true trace level very difficult. For
this reason labeled DFAA were added at concentrations of
0.1 mmol L1. Finally the unknown chemical composition of the
natural DON pool made it not possible to estimate percentages of 15N
addition for our complex DOM tracers. Final concentrations of 15N in
the incubations, C:N ratios, mean percentages of label addition and
the incubation time for each tracer, are summarized in Table 1.
Sample containers were mixed manually at three times:
immediately after addition of the tracers, after approximately 2 h of
incubation, and immediately before water filtration. Incubations
were performed in a water bath at ambient light and temperature
and lasted 4e6 h (see Table 1). Incubations were ended by filtration
on pre-combusted (450 C for 4 h) GF/F filters which were rinsed
with filtered sea water and then immediately frozen and stored
at 20 C until further analysis. Approximately 2 L of water was
filtered (GF/F) for extraction of hydrolysable amino acids from
suspended particulate matter, while about 1 L was filtered on preweighed GF/F filters for bulk SPM analysis.
2.3. Analytical methods
Concentrations and isotopic ratios of particulate organic C and N
were measured using a Thermo EA 1112 elemental analyzer
80
A. Moneta et al. / Estuarine, Coastal and Shelf Science 147 (2014) 78e86
Table 1
Final concentrations in mmol L1 of 15N for the labelled substrates used in the
experiment, C:N ratio of the organic substrates, mean relative label enrichment (in
%) and incubation time, in hours, for each tracer. A-DOM and B-DOM indicate algal
and bacterial derived dissolved organic matter respectively.
Substrate
NHþ
4
NO
2
NO
3
Urea
Glycine
Phenylalanine
Leucine
A-DOM
B-DOM
15
N
concentration
(mmol L1)
C:N ratio
organic
substrates
Mean relative
label
enrichment
(%)
Incubation
time (hours)
0.2
0.1
2.0
0.5
0.1
0.1
0.1
0.2
0.2
e
e
e
0.5
2
9
6
5.5
4.4
10 ±
19 ±
56 ±
50 ±
100
100
100
e
e
4.3
4.6
4.7
5.0
5.1
5.3
5.5
5.7
5.9
14
15
110
46
Up, which was intended as a proxy for substrate preference, was
calculated from values of 15N uptake rates and concentrations of
added 15N as:
Up ¼ Us ½concadded
where [concadded] is the amount of 15N added with each tracer
(nmol L1).
The bacterial contribution to 15N uptake was determined
following Veuger et al. (2007) from the ratio between 15N incorporated into the bacterial specific amino acid D-Ala versus L-Ala,
which is an abundant component of the biomass of every
microorganism.
As in Veuger et al. (2007), bacterial contribution to total 15N
uptake was calculated by:
Excess 15 N D=L Ala 0:01 =ðbacterial D=L Ala 0:01Þ
100;
coupled to a Thermo Delta V Advantage isotope ratio mass spectrometer with a Conflo II interface (EA-IRMS). Hydrolysable amino
acids were extracted according to Veuger et al. (2005, 2007) with
some minor modifications. Briefly, filters were placed inside glass
vials closed with Teflon-lined screw caps, and then hydrolyzed in
6 M HCl at 110 C for 20 h. After hydrolysis, samples were purified
by cation exchange chromatography, derivatized with isopropanol
and pentafluoropropionic anhydride, and further purified by solvent extraction. HAA concentrations were measured by gas
chromatography-flame ionization detection (GC-FID) and the isotopic ratio of the different HAA by gas chromatographycombustion-isotope ratio mass spectrometry (GC-c-IRMS)
(Veuger et al., 2005, 2007).
Temperature, salinity, chlorophyllea and nutrient data were
kindly provided by Dr. Katja Philippart (NIOZ-Texel). Urea concentrations were measured spectrophotometrically after reaction with
diacetylmonoxim. DFAA were measured by high performance
liquid chromatography (HPLC) after acid hydrolysis and derivatization with ortho-phthaldialdehyde (OPA) and N-isobutyryl-Lcysteine (IBLC).
where 0.01 represents the racemization background. This value
was needed to correct for the abiotic formation of D-ala from Lalanine (L-ala) which occurs during acid hydrolysis of organic
material (see Veuger et al., 2007). The racemization background of
0.01 was obtained empirically by using the lowest measured excess
15
N D/L-Ala ratio (for substrate A-DOM). This value is lower than
typical values in previous work on sediments and appears to be
specific for water-column SPM samples. Bacterial D/L-Ala is the D/
L-Ala ratio of bacterial biomass to which the value of 0.05 was
assigned. This value represents the lower end of the range of values
for benthic bacterial communities (0.05e0.1, Veuger et al., 2007),
where highest values are associated with high abundances of Gram
positive bacteria and/or cyanobacteria, which were both assumed
to be negligible in the present study.
Bacterial biomass in mg C L1 was calculated from concentrations of the bacteria specific polar lipids derived fatty acids i15:0,
ai15:0 and i14:0 (data not shown) using specific conversion factors
as in Van Engeland et al. (2013).
2.4. Data treatment
3. Results
Incorporation of 13C and 15N into total particulate organic matter
(POM) and HAA is expressed as excess (above background) 13C and
15
N calculated as the difference between the fractions of the sample
(Fs) and the control (Fc):
3.1. Environmental conditions
Excess ¼ Fs Fc ;
where F ¼ 13C/(13C þ 12C) or 15N/(15N þ 14N).where 13C and
were calculated from delta values.
The 13C and 15N uptake rate (Us) was calculated as:
15
N
Us ¼ Excess ½conc=T;
where [conc] is the C or N concentration (nmol L1) in POM or HAA
on the filter and T is the incubation time (h). Total N uptake rates
(14N þ 15N) were calculated from our data on isotope incorporation
and from the ambient concentrations of DIN, urea, glycine (GLY),
phenylalanine (PHE) and leucine (LEU) as:
Total N uptake rates ¼ Us þ ½Us *100=ð% enrichmentÞ
with (% enrichment) the percentage of added enriched substrates.
Isotope dilution due to NHþ
4 and NO3 regeneration was quantified
using the Kanda et al. (1987) model assuming balanced uptake and
regeneration.
Water temperature followed a typical seasonal pattern, with the
lowest temperature in January (1.4 C) and highest in July (19.4 C).
Salinity ranged between 24.7 in September 2010 and 31.2 in June
2010 (Fig. 1). Chlorophyll-a showed a strong spring bloom between
AprileJune (max value 48 mg L1), with baseline concentrations of
5e10 mg L1 in early spring and autumn, while lowest values were
reached in December (0.8 mg L1) (Fig. 1). Bacterial biomass followed chlorophyll-a.
NHþ
4 was highest in autumn and early winter, reaching nearly
15 mmol L1 in October 2009. Concentrations rapidly declined to a
minimum of 0.4 mmol L1 in March 2010, and steadily increased
from spring to autumn (Fig. 2). NO
2 concentrations were one order
of magnitude lower than NHþ
4 concentrations, but followed more or
less the same pattern. The seasonal cycle of NO
3 acted opposite to
that of NHþ
4 and NO2 (Fig. 2). NO3 concentrations were low in
October 2009 (5 mmol L1), increased till a maximum in February
2010 (47 mmol L1), before decreasing again to a minimum of
0.5 mmol L1 in summer. Decreases of NHþ
4 concentrations during
autumn and winter were accompanied with higher NO
3 concentrations, probably due to nitrification.
Urea, GLY, PHE and LEU showed no clear seasonal trend, but had
a common peak in February 2010 (Fig. 2). GLY concentrations were
A. Moneta et al. / Estuarine, Coastal and Shelf Science 147 (2014) 78e86
Fig. 1. Environmental conditions and microbial biomass. Values of temperature ( C),
salinity, concentration of chlorophyll-a (mg L1) and bacterial biomass (mg C L1) during
the study.
10 times higher than those of PHE and LEU. Total DFAA concentrations were generally very low compared to the other N substrates, ranging from 0.04 mmol L1 in December and September to
0.5 mmol L1 in February. DON showed some seasonal variations,
with concentrations ranging between 10 and 24 mmol L1 and
highest values during late spring and summer (Fig. 2).
3.2. Nitrogen uptake
DFAA uptake rates are potential rates because added concentrations were one to two orders of magnitude higher than ambient
concentrations. For the two complex pools of organic matter only
potential uptake rates could be calculated, because the composition
of the ambient DON pool is unknown.
Highest mean annual uptake rates were measured for NHþ
4
(169 ± 185 nmol L1 h1), followed by, in order, NO
3 , urea, A-DOM,
B-DOM, NO
2 , GLY and LEU with much lower values (29 ± 47,
26 ± 33, 11 ± 2, 8 ± 2, 5 ± 5, 4 ± 4 and 4 ± 5 nmol L1 h1
respectively). Lowest mean uptake rates were measured for PHE
81
(2 ± 2 nmol L1 h1). Isotope dilution factors based on Kanda et al.
(1987) ranged from 1.000013 to 1.09 for NHþ
4 and 0.99 to 1.05 for
þ
NO
3 , indicating that our NH4 and NO3 rates were not significantly
affected by isotope dilution given other uncertainties.
The uptake rates for NHþ
4 , NO3 , urea and the three DFAA showed
similar seasonal trends with low uptake in autumn and winter, and
high rates in spring and summer. However, NO
3 , urea, GLY and PHE
uptake peaked during the phytoplankton bloom in May (171, 101, 12
and 7 nmol L1 h1, respectively), while NHþ
4 and LEU uptake rates
were highest later in summer (630 and 19 nmol L1 h1 in July and
in June 2010, respectively). Lowest values were measured in
1 1
November for NHþ
h , respectively),
4 and GLY (1.6 and 0.1 nmol L
1 1
in December for NO
(1
nmol
L
h
),
in
January
for urea and LEU
3
(0.3 and 0.4 nmol L1 h1, respectively) and in February for PHE
(0.3 nmol L1 h1). The uptake rate of NO
2 was typically low
(range: 0.03e14 nmol L1 h1), showed no seasonal trend, and was
in the same order of magnitude as potential uptake rates of DFAA
(Fig. 3). Potential uptake rates of A-DOM did not exhibit large
seasonal or systematic variations, but those of B-DOM increased
from values around 5 nmol L1 h1 in winter to >10 nmol L1 h1 in
May and September 2010.
The contribution of DIN, urea and DFAA to their summed uptake
rates showed that NHþ
4 contributed most throughout the year (on
average 71 ± 25%) except in November and May when highest
contribution came from NO
2 (75%) and NO3 (56%), respectively.
Urea and DFAA never contributed more than 20% (on average 6 ± 6%
and 2 ± 2%, respectively), but their contribution increased from
March (for DFAA) and April (for urea) to June (Fig. 4).
Substrate preference estimation showed that A-DOM and BDOM were preferred N sources in autumn and winter (Fig. 5). In
spring and summer, the preference for DIN, urea and DFAA strongly
increased. Microbial preference was highest for NHþ
4 during spring
and summer. The three DFAA were similarly preferred throughout
the year, with the only exception of June, when LEU was the
preferred N source among all the tracers. DFAA were preferred over
DIN and urea in October 2009, December and January, then again
from August to October 2010. Urea was never the preferred N
substrate but there is a clear seasonal trend with very low preferences in winter and autumn, an increase during spring and
decrease during autumn (Fig. 5).
Fig. 2. Ambient concentrations of nitrogen substrates. The graph shows concentrations (mmol L1) of dissolved inorganic nitrogen, urea, total DFAA (dissolved free amino acids),
glycine, leucine, phenylalanine and DON (dissolved organic nitrogen) during the seasonal study.
82
A. Moneta et al. / Estuarine, Coastal and Shelf Science 147 (2014) 78e86
Fig. 3. Nitrogen uptake rates (nmol L1 h1) into total particulate organic matter. For glycine, phenylalanine, leucine, algal and bacterial derived dissolved organic matter, shown
uptake rates represents potential uptake rates (see Section 3.2 for details).
Bacteria contributed the most, on average, to the uptake of N
from PHE (66 ± 22%), followed by LEU (57 ± 17%), GLY (56 ± 19%)
and NO
2 (45 ± 22%). Lower contributions were obtained for the
þ
uptake of N from NO
3 , NH4 , urea and B-DOM (28 ± 21%, 26 ± 17%,
25 ± 23% and 20 ± 9%, respectively). Annual mean bacterial
contribution to N uptake was lowest from A-DOM (10 ± 8%).
The highest bacterial contributions to total 15N uptake were
found in autumn and spring, reaching almost 98% in March for PHE.
Lower contributions were observed in summer with minimum
values in July (Fig. 7). Bacterial uptake of DIN reached up to 61%, 85%
and 74% of the total, for respectively NHþ
4 in February, NO2 in
March and NO
in
October
2010.
Lowest
contributions
were
3
measured in May for NHþ
4 (17%) and NO3 (10%) and in July for NO2
(20%) (Fig. 7). 15N-urea incorporation was highest in December
(79%), still important in October (38% and 27% respectively in 2009
and 2010) and then lowest from March to May (1.2e9%). Minimum
and maximum bacterial contributions to the incorporation of 15N
from A-DOM and B-DOM were found, respectively, in December
(0.2%) and June (31%) for the former and in August (10%) and May
(39%) for the latter (Fig. 7).
Fig. 4. Contribution of NHþ
4 , NO2 , NO3 , urea and DFAA (dissolved free amino acids) to
their summed uptake rate. Values are expressed as percentage of the total uptake.
Fig. 5. Microbial preferences for nitrogen substrates. Preferences are expressed as 15Nuptake rate divided by added amount of 15N.
3.3. Nitrogen assimilation into total hydrolysable amino acids
(THAA)
15
N incorporation into THAA is shown as a fraction (%) of 15N
taken up into bulk SPM (Fig. 6). Percentages of N assimilation into
THAA did not show seasonal variations for any of the substrates, for
this reason only averaged values and standard deviations are
shown. On an annual basis, 15N from LEU showed the highest
percentage (43 ± 17%) of incorporation, followed by PHE (41 ± 11%),
B-DOM (28 ± 10%), A-DOM (26 ± 11%) and GLY (21 ± 7%). 15N
incorporation from NHþ
4 was, on average, the lowest, with values of
16 ± 7%, but similar to that of urea, NO
2 and NO3 with 17 ± 6%,
18 ± 13%, 19 ± 9%, respectively.
3.4. Bacterial contribution to total
15
N uptake
A. Moneta et al. / Estuarine, Coastal and Shelf Science 147 (2014) 78e86
Fig. 6. Nitrogen assimilation into microbial biomass. Annual average percentages of
the total 15N taken up into bulk suspended particulate matter which was incorporated
into total hydrolyzable amino acids. GLY: glycine, PHE: phenylalanine, LEU: leucine,
ADOM: algae derived dissolved organic matter, BDOM: bacteria derived dissolved
organic matter.
3.5. C and N uptake from organic substrates
Incorporation of 13C versus 15N into total POM from the organic
substrates was generally coupled (Fig. 8). The only exception was
urea, which did not show a clear linear relationship between
assimilated C and N. 13C:15N ratios for SPM were almost always
below the 13C:15N ratios of the added substrates.
C and N from the three DFAA were taken up at a very constant
ratio which was only slightly different from the ratio of the tracers
except for PHE which showed relatively high N-uptake. Mean C:N
ratios into total POM, taken up from GLY, PHE and LEU were 1.5, 4.8
and 5.3, respectively. Only in November, C was taken up preferentially over N from both GLY and PHE.
C and N from the DOM were taken up in a ratio of, on average,
1.9 ± 0.1 from the A-DOM treatment, which is much lower than the
C:N ratio of the added A-DOM (5.6), and 3.0 ± 0.3 for the B-DOM,
which is closer to the C:N ratio of the added B-DOM (4.2) (Fig. 8).
4. Discussion
83
labeled substrate beyond the tracer level (<10%) because this might
impact rate measurements. Excess 15N in POM was always lower
than the added amount of 15N, except for NHþ
4 in May and LEU in
June, when all the added 15N was recovered in the POM at the end
of the incubation. Therefore, apart from these two occasions, 15N
labeled substrates were never completely exhausted during the
incubations. Isotope dilution for NHþ
4 and NO3 due to regeneration
was limited.
In a seasonal study of N uptake by natural communities, it is
difficult to meet the tracer-level labeling condition, because one has
no data yet of the ambient nutrient levels at the time of incubation.
Here we used the average nutrient concentrations from previous
years, to predict the amount of labeled tracers to add. For this
reason, 15N in the urea and in some of the DIN incubations was
higher than 10% of the ambient concentrations (Table 1). However,
uptake rates were not higher in months in which enrichments
where higher than 10%. Therefore we assume that our results are
reliable with respect to seasonal trends and differences among
substrates.
The use of 15N labeled DOM to assess microbial uptake involves
some assumptions (Bronk and Glibert, 1993). One assumption is
that A-DOM and B-DOM were uniformly labeled; i.e. 15N enrichment was equal for the different constituents of the complex DOM
substrate. Another assumption is that the labeled DOM pools have a
similar bioavailability and/or composition as the natural DOM
pools. The nature of our A-DOM and B-DOM could be influenced by
the method of substrate production, which could make it either
more or less bio-available. Another crucial issue is whether the
composition of our complex DOM resembled the natural one. None
could be tested, but we believe that our approach provides
instructive insight on the actual use of a complex DON pool by
natural communities.
GF/F filters are traditionally applied in study on phytoplankton.
However, as reported in Bronk et al. (2007), GF/F filters were proved
to retain up to 93% of the bacterial community in different marine
ecosystems. This should be particularly true in shallow-water,
turbid systems as investigated here where the fraction of particles attached bacteria is high.
4.1. Methodology
4.2. Total N uptake
Isotopic labeling incubations should (1) avoid limitation of
substrates, (2) limit isotope dilution, and (3) prevent the addition of
1 1
1 1
NHþ
h ), NO
h ), NO
3 (1e171 nmol L
2
4 (1.6e630 nmol L
-1 1
(0.03e14 nmol L h ) and urea uptake rates are similar to those
Fig. 7. Bacterial contribution to total 15N uptake. Values are expressed as percentages from each substrate per month. Asterisks indicate values which were not possible to measure
for analytical reasons (i.e. concentration of D-Alanine was below the limit of detection).
84
A. Moneta et al. / Estuarine, Coastal and Shelf Science 147 (2014) 78e86
Fig. 8. 13C vs 15N uptake rates (nmol L1 h1) from organic substrates into suspended particulate matter. Solid lines indicate the expected relation between C and N uptake if the
whole molecule was taken up; the dotted line is the linear regression fit to the data (if significant).
from other coastal studies (Glibert et al., 1991; Berg et al., 2003;
Andersson et al., 2006; Filippino et al., 2009, 2011).
Potential uptake rates of GLY, LEU and PHE, are very similar and
imply high turnover of DFAA pool, consistent with Fuhrman (1990)
and Suttle et al. (1991). Our results are comparable with the ones
calculated by Berg et al. (2003) in the Baltic Sea and by Mulholland
et al. (2011) for phytoplankton cultures and natural estuarine
communities. They are in the lower range of values obtained using
a mixture of several DFAA (Veuger et al., 2004; Andersson et al.,
2006; Filippino et al., 2011). With ranges of values of 7.9e14 and
5e12 nmol L1 h1, respectively, uptake rates for A-DOM and BDOM are at the lower range of values reported for A-DOM uptake in
different environments (Bronk and Glibert, 1993; Veuger et al.,
2004).
Apart from NO
2 uptake rates, all the other N substrates rates
increased between February and March, corresponding to the chl-a
and bacterial biomass peak in March. Highest uptake rates for NO
3,
GLY, PHE and urea were measured in May, when both phyto
plankton and bacteria were blooming, whereas NHþ
4 and NO2 , LEU,
A-DOM and B-DOM peaked later in the year, respectively in July,
June (LEU and A-DOM) and September (Fig. 3). Similarly, in the
marine end of the Scheldt estuary Andersson et al. (2006) found
maximum uptake for NHþ
4 in July and for NO3 in April, when the
highest concentration of chl-a had been measured. Microbial
preferences for NHþ
4 were highest in May, even though it was still
the absolute preferred substrate in July. NHþ
4 uptake rates peaked in
July rather than in May, likely because of a combination of both
preference and availability. Maximum uptake rates in June for LEU
were probably linked to the highest preference for this N-source in
this month.
The combined effect of preference and availability on N uptake
rates can also be seen in Fig. 4 showing the contributions of DIN,
urea and DFAA to their summed uptake rates. Highest contribution
came from NHþ
4 throughout the year, but in November and March,
þ
when the main contributors were NO
2 and NO3 , respectively. NH4
was the preferred inorganic substrate in every month except in
November, when NO
2 was preferred over all the substrates apart
from the complex DON (Fig. 5). The low contribution of NHþ
4 in
March was probably because ambient concentrations reached the
annual minimum. Previous studies conducted on microbial N uptake in estuarine systems confirmed that the combination of affinity for the substrate and substrate availability explain the
proportion in which different sources contribute to total N uptake
(Veuger et al., 2004; Andersson et al., 2006). DFAA was the
preferred N source in these studies, but compared to other Nsubstrates they were taken up in important proportion only when
their ambient concentrations were high enough to support N microbial demand. Despite high preferences for DFAA in autumn and
winter, their ambient concentrations were too low to result in high
contribution in this part of the year. However, DFAA and urea
contributions increased in spring, due to an increase in affinity for
these substrates and a decrease of NHþ
4 ambient concentrations
(Figs. 5 and 2 respectively).
4.3.
15
N incorporation into THAA
THAA constitute an important fraction of the biomass of all organisms; 15N incorporation into THAA could thus be regarded as a
measure of microbial assimilation. For each substrate we compare
the percentage of 15N incorporated into THAA relative to total POM.
Cowie and Hedges (1992), reported that N present as amino acids in
phytoplankton and bacterial biomass accounts for 40e80% and
50e56% of total N, respectively. Our percentages of 15N assimilated
into THAA from incubations with DFAA, A-DOM and B-DOM are in
the above reported range, but surprisingly 15N assimilation from
DIN and urea was really low (on average <20%). There are two
possible explanations. One, the incubation time was too short for
DIN and urea to be assimilated into amino acids to a large extent,
because due to the order in which the samples were filtered
(Table 1), the incubations with DFAA or DOM lasted longer than
those of urea and DIN. Moreover, in the case of DFAA or DOM, the N
is already reduced or in amide form, which presumably also reduces the assimilation time. Nevertheless, the difference in incubation times is not large thus it is unlikely that this might be the
main or only reason for these results. The second possible and more
interesting explanation, is that 15N from DIN and urea was preferentially assimilated into other macromolecules than hydrolysable
amino acids. This last assumption could be supported by a study
A. Moneta et al. / Estuarine, Coastal and Shelf Science 147 (2014) 78e86
measuring 15N uptake into total particulate N and assimilation into
trichloroacetic acid insoluble material (i.e. macromolecules such as
proteins, DNA, and RNA). Results showed that already after 4 h of
incubation with labeled NHþ
4 phytoplankton from the Chesapeake
Bay were able to assimilate between 40 and 100% of the 15N taken
up into the trichloroacetic acid extractable fraction (Wheeler et al.,
1982). Nevertheless, we can only speculate on the possibility that
15
N could actually have been assimilated in molecules different
from hydrolysable amino acids (i.e. non-hydrolysable amino acids,
nucleic acids or amino sugars).
4.4. Bacterial contributions to total microbial N uptake
The possibility to discriminate between substrate use by bacteria versus phytoplankton is fundamental to ecological studies. If
their contributions are not effectively separated, this could lead to
biased estimates. For example, the estimation of bacterial productivity on the base of LEU and thymidine uptake is not without
problems, as autotrophs have been shown to take up LEU and
thymidine at high rates even at low ambient concentrations
(Mulholland et al., 2011). Similarly, the conventional calculation of
the f-ratio (i.e. ratio of new over regenerated primary production)
from the ratio of nitrate uptake over the total DIN uptake is biased,
when bacteria substantially contribute to the total DIN uptake
(Fouilland et al., 2007; Mulholland et al., 2011).
Traditional methods to separate bacteria from phytoplankton
metabolic rates in ecological studies (i.e. size fractionation, flow
cytometric sorting and the use of specific procaryotic inhibitors),
are prone to limitations (Glibert et al., 1991; Bradley et al., 2010b;
Trottet et al., 2011). Both size-based separation and flowcytometric sorting are not 100% effective due to size overlap and
because a large number of bacterial cells are often attached to
larger phytoplankton cells or to abiotic particles (Bronk et al., 2007;
Bradley et al., 2010a,b). In addition, in highly turbid systems filters
tend to be clogged easily and quickly, increasing the retention capacity of cells smaller than the mesh size. Partitioning based on
metabolic inhibitors are uncertain due to limited specificity and
variable efficiency (Trottet et al., 2011).
Stable isotopes tracer incorporation in the prokaryotic
biomarker D-alanine (Veuger et al., 2005) enables discriminating
between bacterial and algal 13C and 15N incorporation in a natural
sample. This method has so far been applied mainly in benthic
studies (Veuger et al., 2006; 2007), and we are aware of only
another recent study successfully applying it to water column SPM
samples (Van Engeland et al., 2013).
Using values of 15N incorporation into D-alanine, it is possible to
estimate the bacterial contribution to the total microbial 15N uptake. Lower DIN concentrations and higher bacterial and phytoplankton biomass during the spring/summer bloom lead to
competition for bio-available N. Under these circumstances, one
would expect phytoplankton to take up reduced N sources, such as
urea and DFAA, to complement DIN uptake (Mulholland et al.,
1998; Berg et al., 2003; Andersson et al., 2006). Our results support this hypothesis. The bacterial contribution to the uptake of
DFAA in spring and summer was lower than in the rest of the year
and was the highest from October 2009 to April 2010 (z70e98%),
when competition with algae was low (Fig. 7). Bacteria contributed substantially to urea uptake only in October and December
(38e79%). This is in agreement with our hypothesis that during
months of higher microbial biomass and lower DIN concentrations, DFAA and urea could be a source of N for phytoplankton
rather than bacteria.
On average, bacteria contributed most to the uptake of N from
DFAA (60%), especially PHE, throughout the year, whereas the
annual mean bacterial contribution to DIN and urea uptake was
85
never higher than 50%. In a seagrass ecosystem Van Engeland
et al. (2013) found that bacteria were responsible for the majority of DFAA uptake whereas their contribution to DIN and urea
uptake was not higher than 20%. The absolute lowest mean
annual contributions (<20%), in our study, were directed to the
uptake of A-DOM and B-DOM, with a slightly higher contribution
to the uptake of B-DOM. This may be due to a higher similarity in
molecular composition between B-DOM and bacterial cellular
components, which may have made its assimilation easier. As it
was mentioned in Section 4.1, it is not known how the composition and bioavailability of our DON pools reflects the bulk
ambient DON, thus it is not possible to draw any conclusions on
the possibility for phytoplankton to access and take up all the
bulk DON. However, this result is interesting because this is the
first evidence of higher autotrophic versus heterotrophic contribution to the uptake of a complex DON pool. Finally, it might be
that microbes capable of heterotrophic growth other than bacteria (i.e. Thaumarchaeota) were present in our incubations.
Recently, Veuger et al. (2013) reported that Thaumarchaeota peak
during winter in Dutch coastal water and can use DFAA and urea
as sources of N.
4.5. N and C utilization from organic sources
The use of dual labeled organic substrates allowed us to study
whether these substrates were utilized to meet both C and N demand for growth at the same time or if their uptake was decoupled.
Our results confirm previous observations that microbes use DFAA
and urea as a source of N or C or both (Middelboe et al., 1995;
Jørgensen et al., 1999; Mulholland et al., 2003; Andersson et al.,
2006). There was consistency in the ratio in which C and N were
taken up, for all except urea. Urea was used as a source of N from
April to July, when NHþ
4 concentrations are at an annual minimum
and N requirements are higher due to increased planktonic
biomass. This is in accordance with findings by Andersson et al.
(2006). Other studies showed that urea is a major source of N
rather than C but it is still uncertain if the missing fraction of C is
actually respired or released outside the cell immediately after urea
uptake (Tamminen and Irmisch, 1996; Veuger et al., 2007; Bradley
et al., 2010b).
The C:N incorporation ratios into POM in the GLY and LEU incubations were similar to the C:N ratios of the substrates, while the
C:N ratio after incubation with PHE was different and closer to that
of bacterial cells (Fig. 8). This partial decoupling of C and N suggests
that PHE was not just assimilated as intact molecule, but rather
used as a substrate for synthesis of proteinaceous biomass (with a
C:N of 4.8) with the excess C being lost.
C and N from A-DOM and B-DOM were taken up at a constant
ratio throughout the year. In the case of A-DOM, the C:N ratio was
very different from that of the original tracer. This suggests that a
N-rich subset of the complex substrate mixture was taken up or
that this material was intensively reworked/degraded and then
used for selective incorporation of N. This complex DOM could have
undergone biological and photochemical reactions releasing more
labile fractions that were readily incorporated (Bushaw et al., 1996;
Berman and Bronk, 2003; Mulholland et al., 2003; Bronk et al.,
2007). Surprisingly, the ratio in which C and N were taken up
from A-DOM was lower than the ratio required to meet phytoplankton and bacterial growth needs (Fig. 8). It is likely, as we
mentioned before, that we might have modified the bioavailability
of the A-DOM during the production, making it easier or more
energetically favorable to take up a sub-fraction of it. B-DOM, on
the contrary, appeared to be taken up as it was originally added in
the incubations, and the slightly lower C:N ratio is possibly due to
respiration.
86
A. Moneta et al. / Estuarine, Coastal and Shelf Science 147 (2014) 78e86
5. Conclusions
This study showed that phytoplankton can contribute more
than bacteria to the uptake of DON with percentages up to 80% of
the total uptake. Moreover it showed that, in summer, urea and
DIN, particularly NHþ
4 and NO3 , were as important and even more
than complex DON and DFAA to bacterial nutrition. Finally, it
showed that C and N uptake from organic substrates appeared to be
tightly coupled all year long except for A-DOM which was mainly
used as a source of N probably following intense reworking of its
constituents.
Acknowledgments
We thank Peter van Breugel, Marco Houtekamer, and the other
people of the analytical lab of NIOZ-Yerseke for the big help with
the EA-IRMS and the GC-c-IRMS analyses. Part of the data presented in this manuscript was collected as part of the project ‘Integrated Network for Production and Loss Assessment in the
Coastal Environment’ (IN PLACE), coordinated by the Royal
Netherlands Institute for Sea Research (Royal NIOZ). This project is
financed by the Division for Earth and Life Sciences (ALW), which is
part of the Netherlands Organisation for Scientific Research (NWO),
in the framework of the ‘National Programme Sea and Coast
Research e Changing Carrying Capacity’. This research was supported by the Netherlands Organisation of Scientific Research
(NWO) via its Marine and Coastal program (NICYCLE, project no.
839.08.334).
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