Ammonium uptake by heterotrophic bacteria in the Delaware estuary and adjacent coastal waters

Transcription

1 Limnol. Oceanogr., 40(5), 1995, , by the American Society of Limnology and Oceanography, Inc. Ammonium uptake by heterotrophic bacteria in the Delaware estuary and adjacent coastal waters Matthew P. Hochl and David L. Kirchman University of Delaware, College of Marine Studies, Lewes Abstract Uptake of NH4+ by heterotrophic bacteria and the relative importance of NH,+ and dissolved free amino acids (DFAA) as nitrogen sources for bacterial production were examined in the Delaware estuary and adjacent coastal waters during 1988 and Although total uptake of NH,+ and bacterial production were -4-fold higher in 1988 than in 1990, percent NH,+ uptake by bacteria in the upper and lower estuary was similar for both years. Bacterial uptake rates were highest at the mouth of the bay in summer and represented lo-25% of total uptake of NH,+. Less than 5% of NHd+ uptake was by bacteria at salinities <207& In contrast to NH4+ uptake, DFAA uptake was greatest in the upper estuary and often exceeded nitrogen requirements for bacterial growth. Bacteria accounted for 15-35% of total NH,+ uptake at coastal and offshore stations in About 50% of bacterial nitrogen demand was supported by NH4+ at the mouth of the bay and in coastal waters during summer, when DFAA concentrations were generally lowest. Although DFAA concentration and uptake did not explain all variability, they appeared to explain large-scale features in NH,+ uptake by heterotrophic bacteria. Ammonium uptake by bacteria was lowest in the estuary, where DFAA concentrations and uptake were highest; at an offshore station, where DFAA concentrations and uptake were low, relative NH,+ uptake by bacteria was highest. These and other results suggest that NH,+ uptake by bacteria is relatively high in oligotrophic water and low in eutrophic systems, which has important implications on the role of heterotrophic bacteria in the N cycling of marine environments. Uptake and regeneration of NH4+ by heterotrophic marine bacteria may partly control the supply of NH4+-N to primary production. The few studies that have directly measured NH4+ uptake by bacteria-size particles (< 1.O pm) in situ demonstrate that the percent of total NH4+ uptake by picoplankton can be large; however, there is considerable variability between and within environments. In the subarctic Pacific (Kirchman et al. 1989), North Atlantic (Kirchman et al. 1994; Harrison and Wood 1988), Antarctic (Probyn and Painting 1985) and South Atlantic (Probyn 1985), uptake of NH4+ by picoplankton ranges from 30 to 80% of total NH4+ uptake. In contrast, < 30% of total NH4+ uptake is by < 1.O-pm size organisms in the eutrophic waters of upper Chesapeake Bay (Gilbert 1982) and Georges Banks (Harrison and Wood 1988). In Long Island Sound, NH4+ uptake attributable to picoplankton increases from < 10% in winter to -40% in summer (Fuhrman et al. 1988; Suttle et al. 1990). It is important to note that not all uptake of NH4+ in the picoplankton size fraction is by heterotrophic bacteria. Perhaps because of difficulties in separating uptake by heterotrophic bacteria from that by other organisms, we l Present address: Texas A&M University % U.S. EPA- GBERL, Gulf Breeze, -Florida Acknowledgments We thank R. G. Keil for analyzing DFAA concentrations and turnover during 1990, T. Fisher and J. H. Sharp for the use of their mass spectrometers, and Capt. D. McCabe and crew of the RV Cape Henlopen. This work was supported by NOAA Sea Grant 86AA-D- SG040 alrd NSF 9 l do not understand when and why uptake of NH4+ by heterotrophic bacteria is large relative to phytoplankton uptake. Uptake vs. regeneration of NH4+ by heterotrophic bacteria is restrained by the elemental balance of C and N (e.g. Goldman et al. 1987). Unfortunately, the complex and unknown composition of dissolved organic matter (DOM) in seawater precludes the in situ application of elemental balance models for predicting the role of bacteria in NH4+ cycling. Specifically, we do not know the C : N ratio of bacterial substrates nor the growth efficiencies of bacteria that use these substrates. We might be able to use C and N mass balance to examine NH4+ cycling if we knew more about the DOM supporting bacterial production. Although many compounds potentially can be used by bacteria, amino acids have been studied the most. Dissolved free amino acids (DFAA) can support a high percentage of bacterial production in some aquatic ecosystems (Jorgensen 1987; Billen and Fontigny 1987). In other waters, NH4+, as well as DFAA uptake, is important; the sum of the two accounts for >90% of bacterial growth (Keil and Kirchman 199 1; Jorgensen et al. 1993). Other dissolved organic N (DON) compounds, such as DNA, dissolved combined amino acids (DCAA), and NO,-are less important (< 10%) sources of N for bacteria in these experiments (Keil and Kirchman 199 1; Jorgensen et al. 1993), although there are exceptions (e.g. Tupas and Koike 199 1). These estimates of DFAA and NH4+ uptake are from long incubations with the picoplankton size fraction (seawater cultures). Based on C and N mass balance, we can expect some relationship between DFAA and NH4+ uptake. Kirchman and Hodson (1986) predicted that the concentration and

2 NH4+ uptake by bacteria 887 production of extracellular DFAA would affect rates of NH4+ assimilation by marine bacteria. In fact, additions of DFAA can inhibit NH4+ uptake by assemblages of marine bacteria (Fuhrman et al. 1988; Kirchman et al. 1989); but what happens in natural marine systems is still unclear. Although seawater culture experiments have improved our understanding of bacterial assemblages, the findings cannot be directly extrapolated to in situ conditions if only because incubations of seawater cultures are very long. Only one study (Kirchman et al. 1994) has examined uptake rates of both DFAA and NH4+ by picoplankton, and more measurements are needed to understand NH4+ uptake by heterotrophic bacteria. The Delaware estuary is an ideal site for investigating the relationship between DFAA and bacterial NH4+ uptake because of the large range in amino acid and NH4+ concentrations, which vary spatially and seasonally (Coffin 1989; Pennock 1987). In this study, we measured NH4+ and DFAA uptake by bacteria in the estuary and adjacent coastal waters to determine the contribution of total uptake of NH4+ by bacteria, to estimate the relative importance of NH4+ and DFAA for supporting bacterial nitrogen demand, and to test the hypothesis that NH4+ is used by bacteria when DFAA abundance is low (Kirchman et al. 1989; Keil and Kirchman 199 1). Materials and methods Field site and sample collection -Ammonium uptake was measured in and 1990 at four stations along a transect that followed the spine of the estuary from near Wilmington ( N, O W) to coastal waters adjacent to Cape May ( N, W). Stations are reported as kilometers upstream from the mouth of the estuary ( N, O W). A map ofthe estuary with station locations is given elsewhere (Hoch and Kirchman 1993). Mean (+ SD) salinities for these stations during the study were Ym at 100 km, o/oo at 65 km, 17.9&1.67%0 at 40 km, !& at 10 km, and o/oo at -20 km. During July 1990, we also sampled at two oceanic stations that were 250 km ( N, W) and 465 km ( N, W) offshore. Surface water (<2-m depth) samples were collected in 1 O-liter Niskin bottles. Polycarbonate bottles or carboys were acid washed and rinsed twice with sample prior to subsampling and incubation. Dissolved compounds and biomass -Ammonium was assayed with the hypochlorite reaction and dissolved organic N (DON) with the persulfate digestion technique (Parsons et al. 1984). Dissolved primary amines (DPA) were measured as an estimate of DFAA by the o-phthalaldehyde method (Parsons et al. 1984) with glycine as a standard; NH4+ fluorescence was measured in standard curves and subtracted out of DPA estimates. Concentrations of DFAA were determined by precolumn derivatization with o-phthalaldehyde and reverse-phase HPLC with fluorometric detection (Mopper and Lindroth 1982). The organic solvent was a 3 : 1 mix of acetonitrile and methanol, and the gradient was modified according to Nagata and Kirchman (1991). Automated injection of samples helped reduce blanks and improved detection limits to 0.1 nm per amino acid per 360 ~1 injection of sample, and the percent deviation from the mean of duplicate HPLC runs was within 10%. Only protein amino acids were considered in estimating DFAA. Chlorophyll a was measured fluorometrically in acetone extracts of particles collected on GF/F filters (Parsons et al. 1984). Bacterial abundance was determined by acridine orange direct counts (Hobbie et al. 1977). Bacterial nitrogen demand and DFM incorporation - Bacterial nitrogen demand was estimated from incorporation rates of [14C]leucine (Leu). Leucine (sp act > 1.1 kbq nmol- l; New England Nuclear) was added to 10 ml of seawater at a final concentration of 20 nm. This concentration was previously demonstrated to be saturating for Delaware estuary samples (Kirchman and Hoch 1988). Incubations were run in triplicate, plus a formaldehydekilled control, in the dark for min at in situ temperature. Incubations were stopped by adding ice-cold 5% trichloracetic acid (TCA). Samples were filtered onto 0.45-pm Millipore HA filters and rinsed with 3 ml of icecold 5% TCA and 3 ml of ice-cold 70% ethanol. Filters were then radioassayed. Incorporation rates of Leu were multiplied by the mean (+ SD) conversion factor for experiments run from July 1986 to September 1988, x 1016 cells mol-l Leu (+ SD) (Hoch and Kirchman 1993). Bacterial N demand was calculated using 5.4 fg N cell-l (Lee and Fuhrman 1987). DFAA assimilation was measured by adding an L- [3H]amino acid mixture (0.84 kbq nmol-l total amino acid; Amersham) to 10 ml of seawater to a final concentration of 20 nm total amino acids for estuarine and coastal stations and 10 nm offshore. Triplicate samples plus a formaldehyde-killed control were incubated for 15 min in the dark at ambient seawater temperature and then stopped by filtration onto 0.45~pm Millipore HA filters. Filters were rinsed once with 5 ml of 0.22~pm filtered seawater and the filters radioassayed. Uptake rates were estimated by multiplying turnover (radioactivity incorporated per hour) by the DFAA concentration. Previous studies have shown that this approach for estimating DFAA uptake gives rates similar to the sum of uptake rates for individual amino acids (Jorgensen 1987). To convert uptake of DFAA to nitrogen equivalents, we used 1.3 moles nitrogen per mole total amino acids, which was the weighted average for the amino acid mixture. NH4+ uptake and 15N analysis -Uptake rates of NH,+ were calculated for the total and the < 1.O-pm (estuarine stations) or <0.8-pm (stations at the mouth of the bay and offshore) size fractions. Surface water was incubated in 20-liter polycarbonate carboys with 15NH4+ (99% 15N) for 4 h at 60% surface light intensity in a deck incubator. The added 15NH4+ was lo-70% of the ambient NH4+ concentration. After incubation, samples were gravity filtered through 142-mm-diameter Nuclepore polycarbonate membrane filters (0.8- or l.o-pm pore size). Total

3 888 Hoch and Kirchman Table 1. Summary of measurements for examining phytoplankton and bacteria in the small size fraction used to estimate NH,-+ uptake by heterotrophic bacteria. Station (km)* nt Chl a % in small size fraction* ( ko.05) 2.7(f 1.8) (+ 1.6) (&0.3) 5.2(f6.8) l.l(ko.3) 1.6(+1.1) (+3.0) (f0.7) 5.9(&5.1) (+2.0) 6.9(+2.4) C0, % Phyto : Bact uptake Bacteria biomass$ 41(*8) 64(+ 16) 46(+ 14) 59(& 10) 70( + 13) 79(& 13) 78(+5) (&2.2) 9.4(+8.6) 3.3(+3.2) 5.5(*2.9) 4.9(*5-o) 7.8(?3.7) 5.5(+6.6) * Distance upstream from the mouth of the bay. t Number of times each station was sampled. $ Percent of total (unfiltered) seawater Chl a, net 14C0, uptake, and bacterial abundance in < 1.O-pm size fractions for stations > 20 km upstream from the mouth of the bay and <0.8- pm size fractions for stations nearest the mouth of the bay and offshore. 0 Phytoplankton biomass in the small size fraction is reported as percent of bacterial biomass in the size fraction. particulate material and particles in the <0.8- or < 1.Opm size fraction were collected on duplicate, baked (500 C; 1 h) 25-mm-diameter Whatman GF/F filters and rinsed twice with 10 ml of 0.22~pm-filtered seawater. Isotope ratio mass spectroscopy was used to determine the atom% 15N and concentration of particulate organic N (PON). The dried samples from 1988 cruises were combusted by a micro-dumas technique, and N2 was analyzed on a AEI MS- 1 OS mass spectrometer. Samples from 1990 cruises were analyzed on a Europa ANCA- MS mass spectrometer. For both instruments, PON was determined from the mass 28 and 29 peak areas, with anhydrous ethylenediaminetetraacetate (EDTA) as a standard. Efficiency of size fractionation was calculated from bacterial abundance in the unfiltered seawater and 0.8- or 1.O-pm filtrate. Efficiency of retention of bacteria-size particles was calculated from the bacterial abundance in the 0.8- or l.o-pm size fraction and the GF/F filtrate. These filtration efficiencies were then used to correct PON estimates when bacterial uptake of NH4+ was calculated. The effect of isotope dilution was estimated by means of the model of Kanda et al. (1987) where NH4+ regeneration equaled NH4+ uptake. Phototrophic uptake of NH4+ in the bacterial size fraction for 1990 samples was estimated from the incorporation of 14C02 into protein (DiTullio and Laws 1983). Incorporation of 14C0, into protein was measured in the total and bacterial size fraction. We added 63 kbq of 14C0, to station water in l-liter polycarbonate bottles and incubated the bottles in natural sunlight with a dark control. Incubation condition, size fractionation, and filtration were identical to those with 15NH4+ (see above), except incubations were 8-10 h. Replicate GF/F filters with biomass were extracted in 5% TCA at 80 C for 45 min to precipitate proteins and hydrolyze nucleic acids. The GE/F filter and extract were then filtered onto pm Millipore HA filters and rinsed twice with 5 ml of ice-cold 5% TCA and then with 3 ml of ice-cold 70% ethanol to remove lipids. Incorporation rates of 14C02 into protein were converted to units of N-incorporation rates for whole cells by the calculation outlined by DiTullio and Laws (1983) which assumed an N : C ratio of 0.3 for protein and protein N as 85% of cell N for phytoplankton under N limitation. Because N-replete phytoplankton have a lower percentage of protein N, 67% of cell N (Dortch et al. 1984), we used this lower value in calculating picophytoplankton N uptake for stations within the Delaware estuary where concentrations of nitrogen nutrients were high (BO.5 PM NH4+; ~5 PM N03-). Results Uptake of NH4+ by heterotrophic bacteria vs. picophytoplankton -To assess the contribution of picophytoplankton to NH,+ uptake in the bacterial size fraction, we used three different approaches: abundance of autofluorescent cells, Chl a concentration, and 14C02 incorporation into protein. First, we did not observe autofluorescent cells in the bacterial size fraction, which suggested that this fraction was mostly heterotrophic bacteria. Second, the percent of Chl a in the bacterial size fraction was % (+ SD; n = 30) for all stations in both 1988 and 1990 (Table 1). There was no correlation between percent NH4+ uptake and percent Chl a in the bacterial size fraction in 1988 (r = 0.33; n = 13). This relationship was significant in 1990 when we used uncorrected uptake rates (r = 0.57; n = 15; P < 0.05) but was not significant (r = 0.4 1; n = 17) once these data were corrected for picophytoplankton NH4+ uptake (see below). Therefore, there does not seem to be a phytoplankton biomass-specific relationship with NH,+ uptake in the small size fraction in data, and we can correct

4 NH,+ uptake by bacteria 889 Table 2. Apparent uptake of NH,+ by bacteria (V, nm N h-l, mean %C.V. + SE was %) during 1990 was corrected for the efficiency of size fractionation (I;), isotope dilution (ID), picophytoplankton nitrogen demand (P, nm N h- I), and the retention efficiency of GF/F filters (R, which was 1.7). Efficiency of size fractionation is the inverse of the fraction of total bacteria in the small size fraction (see Table I). May Jun Jul Sta- Corrections tion (km)* ID P Uptake of NH,+ by bacteria (nm N h-l) U*F* (U.F.ID u U-P U.F - P ID - P - P)R * Distance upstream from the mouth of the bay. for it in 1990 data. The third and most informative control for phototrophic uptake of inorganic nitrogen in the bacterial size fraction was incorporation of 14C02 into protein (Table 1). Again, the percent of 14C02 incorporated in the bacterial size fraction was low ( %). There was no correlation between percent 14C02 incorporation in the small size fraction and either percent NH4+ uptake (r = 0.37; n = 17) or percent Chl a (r = 0.16; n = 17) in the small size fraction. In contrast to Chl a, bacterial abundance in the <0.8- and < 1.O-pm size fractions was 40-l 00% of that in the unfiltered seawater (Table 1). This efficiency of size fractionation was highest offshore (> 95%) and lowest in the middle and upper estuary (40-60%). By assuming C : Chl a = 50 and 20 fg C per bacteria cell, we converted Chl a concentrations and bacterial abundances for the small size fractions to units of C biomass. Phytoplankton biomass was only % (+ SD; n = 30) of bacterial biomass in the small size fraction (Table 1). On average, GF/F filters used for 15N analysis retained 6Ok 7% (+ SD; n = 30) of the bacteria in the <0.8- and < l.o-pm size fraction. We used the 14C02 incorporation into protein (DiTullio and Laws 1983) to estimate N uptake by picophytoplankton (P) in the bacterial size fraction. Picophytoplankton uptake of nitrogen averaged 2.2 nm h-l and was always ~7 nm h-l (Table 2). These values were subtracted from NH4+ uptake measured for the small size fraction. In effect, we assumed that all N required by picophytoplankton was supported only by NH4+. As a result, some estimates of picophytoplankton NH4+ uptake were greater than the measured rate of NH4+ uptake in the small size fraction. Efficiency of size fractionation (Table 1) was used to correct for the loss of bacterial size PON during size fractionation. We assumed that bacterial abundance traces all PON of this size class and that the atom%15n of PON collected on GF/F filters is the same as bacterial size PON lost during filtration through 0.8- or 1.0~pm filters. This correction (F) increases uptake in the small size fraction (U) by as much as 3-fold. Correcting for isotope dilution (ID), based on the model of Kanda et al. (1987), increased uptake by only -20% (1.23k1.08; n = 17). After adjusting for both isotope dilution and efficiency of size fractionation (We Fe ID), most estimates of NH4+ uptake by the small size fraction were greater than the estimate of picophytoplankton uptake (Table 2). If we assume that losses of PON through GF/F filters used for 15NH4+ and 14C02 uptake were equal and that picophytoplankton were not preferentially collected on GF/F filters compared to bacteria, then the retention efficiency (R) can be applied after correcting bacterial uptake of NH4+ (U) for the efficiency of size fractionation (F), isotope dilution of the l 5NH4+ pool (ID), and picophytoplankton uptake of NH4+ (P). This increased estimates by 1.7-fold (Table 2). After applying all of these adjustments, the resulting rates were similar to rates originally measured for the small size fraction, which suggests that subtraction of picophytoplankton uptake is roughly balanced by correcting for filtration artifacts and isotope dilution. Therefore, we re- port the measured rates of uptake in the small size fraction as NH4+ uptake by heterotrophic bacteria.

5 890 Hoch and Kirchman n r i ai.- - l, 0 Midbay 180 A 2 0, 0 Midbay 2 %I-- A, A Mouth hn 3 I \ l A + go-- 0 *= I I f O- C m A C JFMAMJJASO 0, W Midbay A, V Mouth Fig. 1. Concentrations of Chl a (A), bacteria (B), NH4+ (C), and dissolved organic nitrogen (DON) and dissolved primary amines (DPA) (D) for stations at 65 km upstream (midbay) and 10 km upstream (mouth) during 1988 (closed symbols) and summer 1990 (open symbols). Different symbols are used for 1988 DPA data m-midbay; v-mouth). Seasonal variability of NH4+ uptake- Biomass and dissolved nitrogen concentrations at the mouth of the Delaware estuary ( 10 km upstream) and midbay (6 5 km upstream) during 1988 (Fig. 1) were similar to previous years (Pennock and Sharp 1986; Pennock 1987). Uptake a D t k lo- -*AA\ JFMAMJJASO Fig. 2. NH,+ uptake by the total plankton (A), bacterial uptake ofnh,+ (B), the percent of total NH,+ uptake by bacteria (C). Symbols as in Fig. 1, and the mean %C.V. + SE for total uptake was 6.4f 1.3%. of NH4+ by the total plankton in 1988 was generally the same at both stations (Fig. 2A), although Chl a concentrations were often different (Fig. 1A). The average summer uptake (+ SD) for both stations in summer 1990 was 39 * 14 nm NH4+ h- 1 compared with nm NH4+ h-l in A mmonium concentrations were highest from January through March in 1988 and < 2 PM during spring and summer months (Fig. 1C). Greater uptake rates and lower concentrations of NH4+ from April to September suggest that turnover of the NH4+ pool was more rapid compared with earlier in Ammonium uptake by heterotrophic bacteria in the estuary was highest (20-30 nm h-l) in August and September at the mouth of the bay (Fig. 2B) but was low (< 5 nm h-l) at both stations earlier in About 1 O-30%

6 NH4+ uptake by bacteria 891 of total NH4+ uptake was by bacteria in August and September 1988 (Fig. 2C). Percent NH4+ uptake by bacteria was higher at the mouth of the bay than at midbay. Bacterial uptake was a larger fraction of total NH4+ uptake (-23%) at the mouth of the bay in May 1990 than it was in May 1988 when < 5% of uptake was by bacteria. During January and March 1988, only - 3 and 9% NH4+ uptake was by bacteria at the midbay and mouth stations, respectively. Estimates of bacterial production were converted from units of carbon to nitrogen for determining the percent of bacterial nitrogen demand supported by NH4+ uptake (Fig. 3). When all 1988 data were considered, bacterial nitrogen demand correlated with total NH4+ uptake (r = 0.75, n = 13, P < 0.01). Because NH,+ is a primary source of nitrogen for phytoplankton (Pennock 1987), this correlation reflects coupling of bacterial and primary production in the estuary, which were also correlated (r = 0.69, n = 13, P < 0.05). Hoch and Kirchman (1993) presented a more complete data set for bacterial and phytoplankton parameters during In 1988, NH4+ supplied as much as 35-50% of bacterial nitrogen demand during March and 20-50% at the mouth during August and September (Fig. 3B); during May, only - 10% of the nitrogen requirements for bacteria in the estuary was fulfilled by NH4 +. This estimate was similar to estimates made for Long Island Sound in spring (Fuhrman et al. 1988). Ammonium supported more bacterial growth at the mouth of the bay than at midbay, where DON was higher (Fig. 1D). NH4 + did not become an important source of nitrogen for bacteria at midbay until September, when DON decreased from 75 to 30 PM. Spatial variability of NH4+ and DFAA uptake-in 1988, bacterial NH4+ uptake and the importance of NH4+ as a source of bacterial nitrogen differed between midbay and the mouth. Therefore, in summer 1990 we increased our sampling within the estuary and included a coastal station. Chl a concentration was lowest in coastal waters and generally highest toward the river (i.e km from the estuary mouth) (Fig. 48). Bacterial abundance varied between 2 and 6 x 1 O6 ml- l (Fig. 4B), which did not covary with Chl a concentrations (P > 0.05). A source of NH4+ was apparent in the middle estuary, where concentrations were 3-5 PM in May and July (Fig. 4C). DFAA concentration was highest in May (720+ 1,150 nm) and lowest in July ( nm) (Fig. 4D). Phytoplankton dominated NH4+ uptake throughout the estuary in summer 1990, but uptake by bacteria increased toward the mouth of the bay (Fig. 5). Total NH4+ uptake was greatest toward the river (100 km upstream from the mouth) for all three 1990 cruises (Fig. 5A). Generally, total uptake was greater in July than in May and June, Bacterial NH4+ uptake was greatest at the mouth of the bay for all summer months ( nm NH4+ h-l) and at the coastal station in July (11.6 nm NH4+ h-l). Throughout most of the,estuary, bacterial NH4+ uptake was 15% of total uptake, but it was 13-33% of the total uptake at the mouth of the bay and at the coastal station (Fig. 5B). These averages for summer 1990 were similar e c !z w + z *o 20 sm 10 8 JFMAMJJASO Fig. 3. Bacterial demand for nitrogen (A) and percent of bacterial demand for nitrogen supported by uptake of NH,+ by bacteria (B). Symbols as in Fig. 1. to those for 1988 at estuarine stations but were -2-fold higher than the 1988 annual average at the mouth of the bay (Fig. 6). At the two offshore stations the bacteria accounted for 3 1 and 36% of total NH4+ uptake (Table 3). The average percent NH4+ uptake by bacteria for estuarine, coastal (stations closest to the mouth of the bay), and oceanic stations increased by -50% when data for the small size fraction were corrected for lost PON during filtration and picophytoplankton uptake (Fig. 6). Percent NH4+ uptake by bacteria from summer 1990 correlated with the ratio of bacterial abundance and Chl a concentration (r = 0.77, n = 17, P < 0.01) and with the ratio of bacterial production and phytoplankton production (r = 0.67, n = 17, P < 0.01). Bacterial nitrogen demand (Fig. 7A) was only partially supported by NH 4+. The largest contribution by NH4+ was at the estuary mouth (45-55% of bacterial N demand) in May and July and in coastal water (85%) in July (Fig. 7B). Less than 15% of bacterial nitrogen demand was supported by NH,+ at estuarine stations (40,65, and 100 km upstream from the mouth). In contrast to NH4+ uptake, DFAA assimilation often exceeded the nitrogen requirements for bacterial production (Fig. 7C). In May, DFAA assimilation exceeded bacterial nitrogen demand by as much as 38-fold, assuming there was no excretion of DFAA nitrogen as NH4 +. DFAA assimilation was % of nitrogen demand during June. Only in July were amino acids insufficient to support all of the nitrogen A A

7 892 Hoch and Kirchman n i al May l - A- -A June n - -4 July r e- 0 May 60 t A- -A June n n - -. t n July I I 3 0 i 0 n i -E UI 0, i 8 m f t -3 l / \ \- -A \ \ l 1 \ \ \ i\ \ X 03 I \ -30 Disk7ce3LJpstr~am (9kom) Fig. 5. Total NH,+ uptake (A) and percent of total NH,+ uptake by bacteria (B) in the estuary and adjacent coastal water during summer 1990 (symbols as in Fig. 4). The mean %C.V. 31 SE for total uptake was O% ative to the sum concentration of NH4+ and DFAA. During summer 1990, DFAA was the preferred source of nitrogen (NH4 +-RPI < l), except at the mouth of the bay in June and 465 km offshore in July (NH4+-RPI > 1) (Table 4). O Ei ha measured/l988 measured/l990 corrected/l990 Fig. 4. Concentrations of Chl a (A), bacterial abundance (B), NH4+ (C), and dissolved free amino acids (DFAA) (D) during May, June, and July Distance upstream is relative to the mouth of Delaware Bay. required for bacterial growth at all stations (~20% of bacterial N demand). NH4+ met this nitrogen deficit only at the coastal station (20 km from the mouth) where NH4+ plus DFAA nitrogen uptake represented 96% of demand. We calculated relative preference indices (RPI) (Mc- Carthy et al. 1977) for NH4+ and DFAA nitrogen. The NH4+-RPI compares NH4+ uptake to DFAA uptake rel- I I- estuarine coastal oceanic Fig. 6. The percent of total uptake of NH,+ attributable to bacteria in estuarine and adjacent coastal water of Delaware Bay and for two oceanic sites in the Atlantic Ocean. Data from 1990 after correcting for PON lost during filtration and uptake of nitrogen by picophytoplankton are also presented. Error bars are f SE and the number of samples (n) is given in parentheses.

8 NH4+ uptake by bacteria 893 Table 3. NH,+ and DFAA concentrations and uptake rates for bacteria at two offshore stations. Location Uptake (nm N h-l) Concentration Bac- Bac- NH,+ DFAA Total teria teria (PM) (nm) NH,+ NH4+ DFAA N, W N, 73 O.ll W The importance of NH4+ as a nitrogen source for bacteria can be more simply illustrated by the fraction of NH4+ plus DFAA uptake by bacteria accounted for by NH4+ uptake alone [i.e. NH4+ uptake/(nh,+ + DFAA uptake)]. This proportion increased from May to July and was highest at coastal and offshore stations throughout summer. At DFAA concentrations below - 10 nm, NH4+ uptake was ~50% of NH4+ plus DFAA uptake. Above this concentration, DFAA was the dominant source of nitrogen used by bacteria. Ammonium uptake as a fraction of NH4+ plus DFAA uptake correlated with DFAA concentration (r = 0.82; P < 0.05). However, this relationship cannot be evaluated statistically because DFAA concentration was used to calculate DFAA uptake. Apparently, at low DFAA concentrations the bacteria, although preferring amino acids, used a larger percentage of NH4+ than of DFAA nitrogen for growth. During summer 1990 in the estuary and adjacent shelf waters, there were three potential sources of bacterial nitrogen investigated: NH4+, DFAA, and DCAA (present study; Keil and Kirchman 1993; Keil 1991). Concentrations of all three N sources decreased from eutrophic waters of the upper estuary to offshore (Fig. 8A); similarly, the summed uptake of the three sources also decreased by about an order of magnitude (Fig. SB). Bacteria in surface waters became less dependent on DFAA as a source of N and more dependent on NH4+ along this trophic gradient from the estuary to the ocean, with DCAA (measured as protein uptake; Keil and Kirchman 1993; Keil 199 1) being a minor source of N in all waters (Fig. 8B). One problem with size fractionation is the possible contamination of the bacterial size fraction with photoautotrophic microorganisms. Both unicellular cyanobacteria (e.g. Synechococcus) and free-living prochlorophytes are abundant in marine waters. Based on the similar proportions of inorganic nitrogen uptake and photoautotrophic activity and biomass of < l-pm size plankton, Harrison and Wood (198 8) inferred that picophytoplankton were responsible for most of the NH4+ uptake in the < l- pm size fraction, which was -40% of total uptake. In contrast, most of the biomass in bacterial size fractions collected from the Delaware estuary and coastal waters was nonphototrophic bacteria. Potential uptake of NH4+ by picophytoplankton in our, u a- l May c 60 A- -A June +-E *n Irn =be 04 I 60-- le4 \ \ \. Discussion Estimating NH,+ uptake by heterotrophic bacteria vs. picophytoplankton -Several methods have been used to measure the contribution of heterotrophic bacteria to NH4+ uptake in marine systems, including the use of metabolic inhibitors (Wheeler and Kirchman 1986) or size fractionation (Kirchman et al. 1989) to distinguish bacterial and phytoplankton 15NH4+ uptake. Because preliminary work in the Delaware estuary found metabolic inhibitors to be problematic, we elected to use postincubation size fractionation. Some of the advantages and disadvantages of this technique have been discussed previously (Kirchman et al. 1989), but no study has examined all possible errors. 130 I : Distance Upstream (km) Fig. 7. Bacterial nitrogen demand (A) and percent of bacterial nitrogen demand supported by NH,- - uptake by bacteria (B) and dissolved free amino acid (DFAA) uptake (C) during summer 1990 (symbols as in Fig. 4). I!O

9 894 Hoch and Kirchman Table 4. Comparison of NH4+ uptake and DFAA nitrogen uptake by heterotrophic bacteria in the Delaware estuary and adjacent stations in the Atlantic Ocean during summer ,. r0.25 Relative preference % N uptake Station indext as NH4+$ (km)* May Jun Jul May Jun Jul * Distance upstream from the mouth of the bay. t RPI compares NH4+ and DFAA N uptake rates weighted by their concentrations. RPI is defined as [NH,+ uptake/(nh,+ + DFAA) uptake] + [NH,+/(NH4+ + DFAA)]. Preference for NH4+ is inferred by values > 1. $ Percent uptake as NH,+ is defined as 100 x [NH,+ uptake/ (NH,+ + DFAA) uptake] = L! a" 20 Sum of NHq+ DFAA DCM N Uptake samples was estimated from measurements of 14COZ incorporation into protein in the bacterial size fraction. The assumptions we used in converting [14C]protein synthesis rate to cell nitrogen demand were conservative for three reasons. First, some of the [14C]protein in the bacterial size fraction results from release of [ 14C]DOM from larger organisms and its subsequent assimilation by heterotrophic bacteria or it results from breakage of larger phytoplankton during postincubation size fractionation (Ward 1984). Second, for the estuarine samples, the value used for percent cell nitrogen in protein was 67% instead of 85%, as was used in other studies (e.g. Fuhrman et al. 1988). Third, we assumed that NH,+ was the only source of nitrogen for picophytoplankton. Nitrate uptake is - 30% of NH4+ plus N03- uptake by total plankton in the midbay and mouth of Delaware Bay during summer (Pennock 1987). However, most N03- uptake was probably by phytoplankton larger than 1 pm (Harrison and Wood 1988). Together, these assumptions resulted in an overestimate of picophytoplankton NH4+ uptake. As a result, some of the bacterial NH4+ uptake values were negative after subtracting out estimates of picophytoplankton uptake; only one value was negative for bacterial uptake when estimates were corrected for loss of bacteria-size PON (Table 2). It is necessary to perform this type of assessment when measuring uptake of inorganic nitrogen by picoplankton communities in order to define the relative contributions of phototrophic and heterotrophic uptake; otherwise, biased interpretation of total picoplankton uptake may result. We are confident that our estimates of NH4+ uptake by the small (co.8 or < 1.O pm) size fraction in this study reflect uptake by heterotrophic bacteria. Nitrogen sources for heterotrophic marine bacteria - Bacterial NH4+ uptake varied seasonally and spatially in the Delaware estuary, and NH4+ accounted for a large OJ-- estuarine coastal oceanic Fig. 8. A. Average concentrations of NH4+, DFAA, and DCAA for estuarine, coastal, and oceanic stations collected in summer 1990 (note different units for DFAA). B. Average NH4+, DFAA, or DCAA nitrogen uptake by bacteria as a percentage of the sum of these nitrogen uptake rates for 1990 data, and the summed uptake (nm N h- l) by bacteria for these three nitrogen sources (A). Error bars are + SE and the number of samples (n) for 1990 is given in Fig. 6. percent of bacterial nitrogen requirements in spring and fall 1988 (30-50%) and in summer 1990 near the mouth of the bay (50-90%). In contrast, at all other times and locations in the estuary, NH4+ was a minor source of nitrogen for bacteria (< 20%). Uptake of NH4+ by bacteria correlated with bacterial production over seasonal scales. However, NH4+ uptake by bacteria was variable during summer 1990, when bacterial production was nearly constant throughout the estuary. Factors other than bacterial production obviously impact rates of NH4+ uptake by bacteria, such as the supply and C : N ratio of labile DOM, including DFAA. In contrast to NH4+, estimates of N assimilation from DFAA uptake in the estuary during May and June 1990 often exceeded the nitrogen requirement for bacterial production. The additional contribution of N from DCAA uptake (Keil and Kirchman 1993) exacerbates this nitrogen budget problem by - 15%. Amino acid catabolism can account for a large percent of NH4+ regeneration when the uptake of amino acids is greater than bacterial nitrogen demand (Kirchman et al. 1989). Billen and Fontigny (1987) suggested that the spring phytoplankton bloom in Belgian coastal waters is supported by NH,+ regenerated from DFAA uptake in excess of bacterial nitrogen demand. However, during spring in the Chesa-

10 NH4+ uptake by bacteria 895 peake Bay plume, DFAA uptake in excess of bacterial N demand represents < 5% of total NH4+ regeneration and <2% of the N required for phytoplankton production (Fuhrman 1990; Glibert et al ). Possibly, degradation of amino acids was the source of high NH4+ concentrations in the middle of Delaware estuary during May Upstream from the NH4+ maximum was a large peak in concentrations of DFAA (3.6 PM) and DCAA (1.1. PM; Keil and Kirchman 1993), and DFAA uptake in excess of bacterial nitrogen demand was 15-fold greater than the total NH4+ uptake rate, which suggests an accumulation of NH4+. Amino acids radiolabeled with 14C or 3H obviously do not directly trace the flow of nitrogen. Using 15N- and 14C-labeled glycine and glutamate, Schell (1974) found that glycine N is incorporated preferentially over glycine C and that the opposite is true for glutamate. Therefore, when a mix of radiolabeled amino acids is used, nitrogen uptake may be overestimated or underestimated depending on the specific amino acid. Tenfold greater uptake of amino acid nitrogen compared to nitrogen required for bacterial production in the Delaware estuary may be explained by some other mechanism in addition to preferential use of amino acid C by bacteria. One possibility is assimilation of amino acids by phytoplankton, including deamination and uptake of NH4+ by phytoplankton (Palenik and Morel 1990) with subsequent uptake of released radiolabeled a-keto acids by bacteria. Simultaneous uptake of NH4+ and DFAA is well documented for cultures of aquatic bacterial assemblages (Kirchman et al. 1989; Keil and Kirchman 199 1; Jarrgensen et al. 1993; Hoch et al. 1994), but what remains an enigma is concurrent NH4+ uptake and regeneration by bacteria. Tupas and Koike (199 1) and Fuhrman et al. (1988) pointed out that the assemblage of marine bacteria is essentially a consortium, with some strains assimilating NH4+ and others regenerating NH4+. An alternative hypothesis is that simultaneous regeneration and uptake of NH,+ results from 15N isotope equilibrium across bacterial cell membranes due to passive diffusion of NH3 (Hoch et al. 1992). NH4+ labeled with 15N could get incorporated into cell biomass despite the net excretion of NH4+ due to amino acid deamination. Explaining NH,+ uptake by bacteria -One approach for explaining the regulation of NH4+ uptake by heterotrophic bacteria is based on C and N mass balance between bacterial requirements for growth (assimilation and respiration) and the DOM used as substrate by bacteria (DOM,) (Goldman et al. 1987). The problem in using this approach is that DOMs is not well known and thus neither is the C : N ratio of DOM, (C : N,). In order to examine the regulation of NH4+ uptake by heterotrophic bacteria in natural marine environments, it is necessary to focus on a few compounds whose concentration and turnover can be measured. We examined DFAAs because they can support much bacterial growth (Jorgensen 1987; Billen and Fontigny 1987) and because they directly and indirectly regulate the nitrogen metabolism of marine bacteria (Fuhrman et al. 1988; Kirchman et al. 1989). Therefore, DFAAs are likely to affect NH4+ uptake. Although DFAA concentrations and uptake may explain differences in NH4+ uptake by bacteria over large spatial scales, i.e. the estuarine to oceanic gradient (Fig. 8), there is much variation in NH4+ uptake by bacteria that DFAA concentration and uptake do not explain. The concentration and uptake of other labile components of dissolved organic C (DOC) is probably also quite important. One possibility is monosaccharides such as glucose, the addition of which can stimulate NH4+ uptake in marine waters (Kirchman et al. 1990; Keil and Kirchman 199 1; Hoch et al. 1994). The concentrations of polysaccharides can be quite high (Pakulski and Benner 1994), and monosaccharides can support a large fraction of bacterial production in open-ocean waters (Rich and Kirchman unpubl. data). Uptake of NH,+ attributed to bacteria along estuarine to oceanic gradients - One of the most interesting outcomes of this study is that the percent of total NH4+ uptake attributed to bacteria increases along the trophic gradient from estuarine to oceanic conditions (Fig. 6). Bacteria account for <5% of total uptake in the upper and middle regions of the Delaware estuary, and this percentage increases to - lo-20% at coastal stations in summer. Similarly, Glibert (1982) found - 30% of total NH4+ uptake in the < 1 -pm size fraction near the mouth of Chesapeake Bay compared to < 10% at stations in upper Chesapeake Bay and the Potomac estuary. Our estimate of percent uptake by bacteria is % for the oceanic stations in the Gulf Stream during July, which is comparable to maximum values for the North Atlantic (Kirchman et al. 1994) and the subarctic Pacific (Kirchman et al. 1989). Changes in the proportion of NH4+ uptake by bacteria may simply be a function of differences in bacteria-phytoplankton trophic structure or may be due to some specific ecophysiological response that affects bacterial substrate supply and quality. Spatial variability in the percent NH4+ uptake by bacteria during summer 1990 is partly explained (- 50%) by ratios of bacterial to phytoplankton biomass or production. Therefore, if heterotrophic bacteria use NH4+ in a constant ratio with other nitrogen sources, then percent uptake of NH4+ by bacteria would increase with increasing ratios of bacteria to phytoplankton production and biomass from the upper estuary to offshore. However, the ratio of NH4+ uptake to uptake of dissolved amino acids (both DFAA and DCAA) is not constant in the Delaware estuary and western Atlantic. As average concentrations of these compounds decrease (roughly in similar proportions) from the upper estuary to offshore, the uptake of NH4+ increases relative to the uptake of dissolved amino acids (Fig. S), suggesting that the availability and quality of substrates is also important in explaining variability in the percent NH4+ uptake by bacteria. The importance of NH4 + for supporting bacterial nitrogen requirements increases along the trophic gradient

11 896 Hoch and Kirchman from the eutrophic Delaware estuary to mesotrophic coastal waters and offshore to the oligotrophic Gulf Stream. In contrast, amino acids as sources of N for bacterial growth and as potential sources for regenerated NH4+ appear to decrease as N deficiency increases. As NH4+ becomes more important for supporting bacterial growth, the contribution of bacteria to total NH4+ uptake also increases from the Delaware estuary to the Gulf Stream. Increasing percent of NH4+ uptake with decreasing eutrophic conditions indicates an important shift in the role of heterotrophic bacteria in nitrogen cycling of marine surface waters. We presume that labile sources of DOC (e.g. monosaccharides) are utilized by bacteria in coastal and oceanic waters to maintain an elemental balance of C and N for bacterial growth and respiration when the supply of amino acids or other labile DON alone is inadequate to achieve this steady state stoichiometry. References BILLEN, G., AND G. FONTIGNY Dynamics of a Phaeocyctis-dominated spring bloom in Belgian coastal waters. 2. Bacterioplankton dynamics. Mar. Ecol. Prog. Ser. 37: COFFIN, R. B Bacterial uptake of dissolved free and combined amino acids in estuarine waters. Limnol. Oceanogr. 34: DITULLIO, G. R., AND E. A. LAWS Estimates of phytoplankton N uptake based on 14C0, incorporation into protein. Limnol. Oceanogr. 28: DORTCH, Q., J. R. CLAYTON, JR., S. S. THORESEN, AND S. I. AHMED Species differences in accumulation of nitrogen pools in phytoplankton. Mar. Biol. 81: FUHRMAN, J. A Dissolved free amino acid cycling in an estuarine outflow plume. Mar. Ecol. Prog. Ser. 66: , S.G. HOFUUGAN, AND D.G.CAPONE Use of 13N as tracer for bacterial and algal uptake of ammonium from seawater. Mar. Ecol. Prog. Ser. 45: 27 l-278. GLIBERT, P. M Regional studies of daily, seasonal and size fraction variability in ammonium remineralization. Mar. Biol. 70: ,~.GARSIDE, J.A. FIJHRMAN,AND M.R.RoMAN Time-dependent coupling of inorganic and organic nitrogen uptake and regeneration in the plume of the Chesapeake Bay estuary and its regulation by large heterotrophs. Limnol. Oceanogr. 36: GOLDMAN, J. C., D. A. CARON, AND M. R. DENNETT Regulation of gross growth efficiency and ammonium regeneration in bacteria by substrate C : N ratio. Limnol. Oceanogr. 32: 1239-l 252. HARRISON, W. G., AND L. J. E. WOOD Inorganic nitrogen uptake by marine picoplankton: Evidence for size partitioning. Limnol. Oceanogr. 33: HOBBIE, J. E., R. J. DALEY, AND S. JASPER Use of Nuclepore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbial. 33: 1225-l 228. HOCH, M.P.,M.L. FOGEL, AND D.L. KIRCHMAN Isotope fractionation during ammonium uptake by marine microbial assemblages. Geomicrobiol. J. 12: ,AND Isotope fractionation associated with ammonium uptake by a marine bacterium. Limnol. Oceanogr. 37: , AND D. L. KIRCHMAN Seasonal and inter-annual variability in bacterial biomass and production in a temperate estuary. Mar. Ecol. Prog. Ser. 98: JORGENSEN, N. 0. G Free amino acids in lakes: Concentrations and assimilation rates in relation to phytoplankton and bacterial production. Limnol. Oceanogr. 32: , N. KROER, R. B. COFFIN, X.-H. YANG, AND C. LEE Dissolved free amino acids, combined amino acids, and DNA as sources of carbon and nitrogen to marine bacteria. Mar. Ecol. Prog. Ser. 98: KANDA, J.,E.A. LAWS, T. SAMO,AND A. HATTORI An evaluation of isotope dilution effect from conventional data sets of 15N uptake experiments. J. Plankton Res. 9: I&IL, R. G The biogeochemistry of dissolved combined amino acids in marine waters. Ph.D. thesis, Univ. Delaware. 141 p, -, AND D. L. KIRCHMAN Contribution of dissolved free amino acids and ammonium to the nitrogen require- - ments of heterotrophic bacterioplankton. Mar. Ecol. Prog. Ser. 73: l-10. -,AND Dissolved combined amino acids: Chemical form and utilization by marine bacteria. Limnol. Oceanogr. 38: 1256-l 270. KIRCHMAN, D.L.,H. W.DUCKLOW,J.J. MCCARTHY,AND C. GARSIDE Biomass and nitrogen uptake by heterotrophic bacteria during the spring phytoplankton bloom in the North Atlantic Ocean. Deep-Sea Res. 41: AND M. P. HOCH Bacterial production in the Delaware Bay estuary estimated from thymidine and leucine incorporation rates. Mar. Ecol. Prog. Ser. 45: 169-l 78. -, AND R. E. HODSON Metabolic regulation of amino acids uptake in marine waters. Limnol. Oceanogr. 31: , R. G. KEIL, AND P. A. WHEELER The effect of amino acids on ammonium utilization and regeneration by heterotrophic bacteria in the subarctic Pacific. Deep-Sea Res. 36: 1763-l ,AND Carbon limitation of ammonium uptake by heterotrophic bacteria in the subarctic Pacific. Limnol. Oceanogr. 35: LEE, S., AND J. A. FUHRMAN Relationships between biovolume and biomass of naturally derived marine bacterioplankton. Appl. Environ. Microbial. 53: 1298-l 303. MCCARTHY, J. J., W. R. TAYLOR, AND J. L. TAFT Nitrogenous nutrition of the plankton in the Chesapeake Bay. 1. Nutrient availability and phytoplankton preferences. Limnol. Oceanogr. 22: 996-l MOPPER, IS., AND P. LINDROTH Diel and depth variations in dissolved free amino acids and ammonium in the Baltic Sea determined by shipboard HPLC analysis. Limnol. Oceanogr. 27: NAGATA, T., AND D. L. KIRCHMAN Release of dissolved free and combined amino acids by bacterivorous marine flagellates. Limnol. Oceanogr. 36: PAKULSKI, J. D., AND R. BENNER Abundance and distribution of carbohydrates in the ocean. Limnol. Oceanogr. 39: PALEMK, B., AND F. M. M. MOREL Amino acid utilization by marine phytoplankton: A novel mechanism. Limnol. Oceanogr. 35: PARSONS, T.R.,Y. MAITA,ANDC. M. LALLI Amanual of chemical and biological methods for seawater analysis. Pergammon. PENNOCK, J. R Temporal and spatial variability in phy-