Uncoupling of Bacterioplankton and Phytoplankton Production in Fresh Waters Is Affected by Inorganic Nutrient Limitation JIANHUA LE, JOHN D

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1994, p. 2086-2093 Vol. 60, No. 6 0099-2240/94/$04.00 + 0 Copyright ©D 1994, American Society for Microbiology Uncoupling of Bacterioplankton and Phytoplankton Production in Fresh Waters Is Affected by Inorganic Nutrient Limitation JIANHUA LE, JOHN D. WEHR,* AND LORI CAMPBELL Louis Calder Center, Fordham University, Armonk, New York 10504 Received 13 January 1994/Accepted 1 April 1994

Pelagic bacterial production is often positively correlated, or coupled, with primary production through utilization of autotrophically produced dissolved organic carbon. Recent studies indicate that inorganic N or P can directly limit both bacterial and phytoplanktonic growth. Our mesocosm experiments, with whole communities from mesotrophic Calder Lake, test whether this apparent bacterial-algal coupling may be the result of independent responses to limiting inorganic nutrients. In systems without N additions, numbers of bacteria but not phytoplankton increased 2- to 2.5-fold in response to P fertilization (0 to 2.0 ,umol of P per liter); this resulted in uncoupled production patterns. In systems supplemented with 10 ,umol of NH4NO3 per liter, P addition resulted in up to threefold increases in bacteria and two- to fivefold increases in total phytoplankton biomass (close coupling). P limitation of pelagic bacteria occurred independently of phytoplankton dynamics, and Downloaded from regressions between bacterial abundance and phytoplankton chlorophyll a were nonsignificant in all systems without added N. We describe a useful and simple coupling index which predicts that shifts in phytoplankton and bacterioplankton growth will be unrelated (A bacteria/A phytoplankton -> either +x0 or - 0) in systems with inorganic N/P (molar) ratios of <-40. In systems with higher N/P ratios (>40), the coupling index will approach 1.0 and close coupling between bacteria and phytoplankton is predicted to occur. aem.asm.org Recent investigations have shown that a significant propor- ton or phytoplankton production solely may lead to their tion of the carbon fixed by phytoplankton is processed through dynamics becoming uncoupled. a microbial food web in which planktonic bacteria assimilate Studies which have identified quantitative relationships be- and funnel extracellular dissolved organic carbon (DOC) to tween the abundance of pelagic bacteria and phytoplankton at FORDHAM UNIV on May 11, 2010 heterotrophic and mixotrophic protists and on to larger meta- chlorophyll a may overlook the separate but direct influences zoans (2, 20, 21, 24, 38, 41). Studies of a variety of marine and of N- and P-limited growth on each component. Cross-system freshwater ecosystems have further shown that the abundance analyses in the literature are useful for identifying certain and production of planktonic, heterotrophic bacteria are pos- correlations (e.g., references 6 and 15), but such comparisons itively correlated with phytoplankton chlorophyll a levels and may be unable to identify specific causative factors, such as production (2, 6, 15, 24). The slopes of regressions between temperature, different nutrient requirements, or trophic dy- direct counts of pelagic bacteria and phytoplankton chloro- namics (8, 18, 19). Indeed, Bird and Kalff (6) have mentioned phyll a levels have also been used to describe the nature of that despite strong quantitative links between bacteria and these dynamics (6, 19, 33). All of these relationships have been phytoplankton in many ecosystems, a considerable amount of interpreted to be the result of a close metabolic coupling scatter in these relationships still needs to be explained. between bacterioplankton and phytoplankton. Indeed, organic Covariance among several factors (studied by correlation) can carbon exudates from phytoplankton are thought to be a further lead to misleading relationships between the abun- critical limiting resource for bacterial growth (7, 8, 15, 16, 31). dance of bacteria and phytoplankton biomass (5). For exam- In contrast, a few studies have shown that inorganic nutri- ple, an analysis of studies of many lakes revealed that bacterial ents, or nonphytoplankton DOC, may also limit the growth of abundance, the chlorophyll a level, and the aqueous total P heterotrophic bacteria. Some regression models describing level covary, but the correlation between bacteria and phyto- whole communities in lakes have found a stronger relationship plankton is not as strong as that between bacteria and the total between bacterial production and dissolved Pi (DIP) than with P level (18). Because our studies have indicated that levels of phytoplankton (18, 28). Recent studies have also demonstrated DIP may regulate the production of pelagic bacteria in fresh that bacterial abundance and production in fresh water can be water (36, 37), phosphorus may be one critical factor that directly limited by levels of DIP (17, 36, 37). Marine bacterio- drives the apparent coupling between bacteria and phytoplank- plankton growth can apparently be limited by inorganic N ton. deficiency; bacteria may also account for a large portion of In this study, experiments were conducted with large meso- ammonium uptake in the sea (42). In the freshwater tidal cosms containing whole plankton communities from mesoeu- region of the Hudson River (New York State), bacterial trophic Calder Lake. A range of N and P concentrations and production is apparently driven by DOC derived from macro- N/P ratios were tested in a factorial design (two factors tested phytes and allochthonous sources (19). These studies all simultaneously and in combination) to examine coupling be- suggest that specific factors which affect either bacterioplank- tween the biomass of planktonic bacteria and that of phytoplankton under nutrient-limited and nutrient-surplus conditions. We hypothesize that (i) the growth of both bacteria and * Corresponding author. Mailing address: Louis Calder Center, phytoplankton can be stimulated directly and separately by N Fordham University, Drawer K, Armonk, NY 10504. Phone: (914) or P loading, (ii) increases in bacterial growth can occur 273-3078. Fax: (914) 273-2167. Electronic mail address (Internet): independently of those of phytoplankton, and (iii) correlation [email protected]. between bacterial abundance and phytoplankton (as chloro- 2086 VOL. 60, 1994 EFFECT OF INORGANIC N AND P ON BACTERIAL-ALGAL GROWTH 2087 phyll a) will be affected by the different abilities of these UV-2A filter (excitation, 330 nm; barrier, 420 nm) at a organisms to obtain Pi. We also aimed to develop a simple magnification of x1,250 (29). A minimum of 300 cells were model which may be used to predict and understand patterns counted from each sample. of coupled and uncoupled production in pelagic bacteria and Water chemistry. Soluble-reactive phosphorus (SRP) was phytoplankton more broadly in aquatic ecosystems. analyzed via the antimony-ascorbate-molybdate method (1, 10). NH4+-N was measured by the phenol-hypochlorite MATERIALS AND METHODS method, and NO3- was determined with sulfanilamide-N-1- naphthylethylenediamine dihydrochloride, following reduction Site description. Calder Lake is a small (3.9-ha) mesoeutro- to N02 in a Cd-Cu column (1, 11, 12). All of the methods phic, dimictic lake located in southern New York State; this is were modified for automated analysis and run on a TrAAcs- also the site of Fordham University's biological field station. 800 analyzer (Bran + Luebbe Technologies, Inc.). The lake has an average depth of 2.8 m and a maximum depth Statistical analyses. To determine nutrient effects on both of 6.7 m. DIP concentrations range from <0.03 jimol/liter to bacterial abundance and phytoplankton chlorophyll a, a two- 0.84 jimol/liter (<1.0 to 26 jig of P per liter), and total P way analysis of variance was run with the SYSTAT 5.1 program concentrations range from 0.12 jimol/liter to 2.74 jimol/liter (43), with factors A (N addition) and B (P addition) as (3.7 to 85 jig of P per liter [39, 40]). The DOC concentration independent variables. Response variables included chloro- tends to be constant (relative variation of usually <10%) on phyll a in three size fractions (micro-, nano-, and picoplank- different days and among depths, with an average of 3.7 ton), total chlorophyll a, and bacterial density. Variables were mg/liter (36, 37). first tested for assumptions of normality and homogeneity of

Facilities, experimental design, and sampling. Experiments variances; a loglo transformation was required to reduce Downloaded from were conducted in 24 large cylindrical fiberglass tanks (Exper- skewness. Regressions between bacterial abundance and total imental Lake Facility), each filled to a volume of 5,400 liters chlorophyll a, with or without N addition, were used to (1.9-m diameter by 2.1-m depth [41]). The tanks are located evaluate factors affecting coupling. Regression equations were about 100 m from Calder Lake. Lake water containing zoo- derived from loglo-transformed data in order to meet normal- plankton, phytoplankton, and bacteria was pumped (-1-m ity assumptions. This approach also enabled direct compari- depth) directly into Experimental Lake Facility tanks from a sons with prior studies (6, 19). In our regressions, we assume a site near the deepest part of the lake during the night. A 2 x functional relationship between bacterial numbers and phyto- aem.asm.org 4 factorial design was used with two N levels and four P levels, plankton, especially because temperature effects were mini- each replicated three times. The two N treatments included (i) mized by using data from experiments conducted during the no N added and (ii) 10 jimol of NH4NO3 added per liter. The month of June. We recognize, however, that both variables four P treatments included (i) no P added or (ii) 0.5, (iii) 1.0, were measured with error. Therefore, a second set of slopes and (iv) 2.0 jimol of K2HPO4 added per liter. Nutrient pulses (based on the geometric mean) were computed according to a at FORDHAM UNIV on May 11, 2010 were applied on day 0 (- 4 h prior to sampling) and repeated model II regression, as described by Ricker (30) and Sokal and on day 14. Rohlf (34). For all hypothesis tests, effects were judged to be Water was collected from Experimental Lake Facility tanks significant if the probability of an event having occurred with a peristaltic pump from a 1-m depth at four weekly because of chance (P) was <0.05. intervals (days 0, 7, 14, and 21) and stored in the dark and cold (-5°C) until returned to the laboratory (-2 h). Autotrophic RESULTS microplankton (algal and cyanobacterial cells or colonies, >20 jim to 200 jim) were first filtered in situ with 47-mm Nalgene Nutrient chemistry. Average concentrations of SRP, NH4+, in-line filter units fitted with 20-jim Nitex disks (stored on ice and NO3 in mesocosms on four sampling days according to until returned to the laboratory [-2 h]). The remaining experimental treatments are presented in Table 1. The chem- fractions were also filtered in the laboratory later (described ical pulses were applied twice-A h before sampling on day 0 below). Chemistry samples (collected in duplicate) were also and 14 days after the first sampling. Two sets of samples were filtered in line (Whatman GF/F filters) during collection by taken before (day 14b) and after (day 14a) the pulse on day 14. peristaltic pump from -1-m depth; half were preserved to pH Differences between target P concentrations and measured P <2.0 with concentrated H2S04. Whole lake water was col- varied within ±+10% to ±20%. Addition of 10 jimol of lected in an otherwise identical manner for bacterial samples. NH4NO3 per liter resulted in measured NO3--N concentra- Phytoplankton and bacteria. Smaller size fractions of phy- tions increasing from 0.3 jimol/liter to about 11.6 jimol/liter on toplankton were separated in the laboratory by using polycar- day 0. These higher levels were depleted within the water bonate filters under a vacuum of '100 mm Hg (-13 kPa). A column to below the detection limit (<0.07 jimol/liter) by day series of twelve 1,000-ml polysulfone filtration units were 7 in tanks with P added (0.5 to 2.0 jimol/liter). However, no linked in series to filter 12 samples simultaneously. Nanoplank- change in NO3f-N concentration was observed in systems ton (20 jim to >2 jim) were collected on 2.0-jim-pore-size without added P. In contrast, nearly all systems experienced a filters; picoplankton (2 jim to >0.2 jim) passed through large depletion of NH4'-N from the water column regardless 2.0-jim filters and were collected on 0.2-jim-pore-size filters of P treatment. Only one treatment community, those receiv- (Poretics Corp.). Filters and 20-,um Nitex disks were placed in ing no added P but with 10 jimol of NH4NO3 added, had large, extraction vials containing 5 ml of neutral 90% acetone, but lower, rates of NH4'-N depletion ("50% removed by day ground with a glass pestle, and then extracted cold (4°C) in the 7). dark for 18 h. Extracts were centrifuged and measured for For the purposes of this study, we define the average chlorophyll a concentrations spectrophotometrically and cor- depletion rate of SRP as the per day rate of change in P rected for pheophytin a (1, 25). concentration at each weekly period. The P depletion rates Bacteria were filtered onto 0.2-jim-pore-size prestained varied from 0.0 jimol/liter/day to 0.03 jimol/liter/day in systems (Irgalan) black filters, preserved with 2% glutaraldehyde, without N added but were enhanced up to 0.18 jimol/liter/day stained with 4',6-diamidino-2-phenylindole (DAPI), and (5.7 times greater) in systems with N added (Table 2). At the counted with a Nikon epifluorescence microscope with a start and at the end of the experiment, pairwise contrasts (i.e., 2088 LE ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 1. Average nutrient concentrations in Calder Lake mesocosms with different N and P additions in five sampling periods Treatment" Avg nutrient concn (,umol/liter + SD [n = 3]) on dayb: Variable N P 0 7 14b 14a 21 0 0.0 SRP 0.04 ± 0.01 0.03 + 0.04 <0.02 <0.02 0.13 + 0.01 0 0.5 0.37 + 0.02 0.16 + 0.00 <0.02 0.62 + 0.05 0.56 + 0.05 0 1.0 0.81 ± 0.06 0.60 + 0.05 0.42 + 0.06 1.47 + 0.08 1.44 + 0.08 0 2.0 1.70 ± 0.05 1.48 + 0.02 1.28 + 0.05 3.33 + 0.05 3.20 + 0.03 20 0.0 0.03 + 0.01 <0.02 <0.02 <0.02 0.14 0.02 20 0.5 0.34 +0.02 <0.02 <0.02 0.45 ± 0.02 0.17 +0.02 20 1.0 0.79 ± 0.02 0.07 + 0.02 <0.02 0.88 + 0.09 0.28 + 0.13 20 2.0 1.76 + 0.04 0.47 ± 0.06 0.22 + 0.06 2.15 + 0.09 1.33 ± 0.09 0 0.0 NH4+-N 10.6 + 0.54 0.16 + 0.19 0.89 + 0.11 0.76 + 0.10 1.29 + 0.36 0 0.5 14.8 + 0.66 0.34 + 0.07 0.42 + 0.06 0.68 ± 0.12 0.48 + 0.12 0 1.0 6.70 + 0.65 0.36 + 0.20 0.65 + 0.08 0.61 ± 0.06 0.47 + 0.11 0 2.0 9.68 + 0.54 0.26 + 0.18 0.61 + 0.06 0.62 + 0.05 0.35 + 0.02 20 0.0 9.67 + 0.60 6.69 + 0.02 7.17 + 0.36 20.2 + 0.29 14.9 + 0.31 20 0.5 14.7 + 0.28 0.69 +0.20 8.41 + 1.11 20.9 ± 0.70 9.57 + 0.51 20 1.0 6.08 + 0.66 0.47 + 0.07 5.57 + 0.17 17.9 ± 2.43 3.35 + 0.15

20 2.0 1.86 + 0.69 0.56 ± 0.23 1.92 + 0.69 14.4 + 1.36 4.53 + 0.41 Downloaded from 0 0.0 NO3+-N 0.34 + 0.01 <0.07 <0.07 <0.07 0.14 + 0.04 0 0.5 0.31 + 0.02 <0.07 <0.07 <0.07 <0.07 0 1.0 0.31 + 0.04 <0.07 <0.07 <0.07 0.07 + 0.1 0 2.0 0.29 + 0.04 <0.07 <0.07 <0.07 <0.07 20 0.0 11.5 + 0.31 11.5 ± 0.28 11.6 ± 0.29 15.3 + 0.04 15.2 ± 0.71 20 0.5 11.4 + 0.19 <0.07 0.07 + 0.11 10.2 + 0.44 8.67 ± 0.32 20 1.0 11.6 + 0.34 <0.07 <0.07 10.3 ± 0.10 10.7 + 0.62 aem.asm.org 20 2.0 11.6 0.34 <0.07 <0.07 10.0 0.34 4.29 0.61 "Treatment additions were 0 or 10 plmol of NH4NO3 per liter for N and 0.), 0.5, 1.0, and 2.0 p.mol of K2HPO4 per liter for P. "Two chemical pulses were applied during the experiment: (i) 4 h before day 0 sampling and (ii) on day 14. Two sets of samples were taken on day 14: (i) before (day 14b) and (ii) after (day 14a) the second nutrient pulse. Treatments with average concentrations below the analytical detection limits are listed as <. at FORDHAM UNIV on May 11, 2010

across identical P treatments) of P depletion rates between size-fractionated chlorophyll a levels were not significantly N-surplus and N-limited systems indicate that rates were different across all N and P treatments at the start of the between 40% (0.0 F.mol of P added) and 500% (2 ,umol of P experiment (day 0). On days 7 and 14, total and size-fraction- added) greater in N-surplus communities. ated chlorophyll a concentrations increased in response to P Effects of nutrients on biomass of phytoplankton and abun- additions, but only in mesocosms supplied with added dance of bacteria. The chlorophyll a densities measured in NH4NO3. In contrast, bacterioplankton abundance increased size-fractionated phytoplankton and the abundance of bacteria following P addition regardless of N treatment. In all systems in different treatments are presented in Fig. 1. Total and (with and without N addition), bacterial numbers increased by between about 2 x 106 to 5 x 106 cells per ml (more than a 100% increase) along the experimental P gradient. Among TABLE 2. Average daily depletion rate of DIP in mesocosms mesocosms supplied with 10 ,umol of NH4NO3 per liter, the P receiving different N and P treatment combinations stimulation effect on different phytoplankton size fractions for three periods during the study differed according to day. There was a positive response to P treatment (threefold increase) by picoplankton chlorophyll a Depletion rate of DIP (nmol of P/liter/day) from": by day 7 (coefficient of variation, -24% among treatments). Treatment" Day 0 to Day 7 to Day 14a to The nanoplankton chlorophyll a level increased 2.5-fold in day 7 day 14b day 21 response to P addition by day 7 (coefficient of variation, 14% No N added among treatments) and increased nearly 4-fold by day 14 p (coefficient of variation, "18% among treatments). Larger 0.0 3.1 1 2.2 4.1 1 5.7 Undetectable" cells and colonies comprising the microplankton exhibited an 0.5 31.0 1 2.1 21.6 + 0.7 8.0 + 3.7 increase in chlorophyll a level later in the experiment, but the 1.0 30.0 ± 0.0 26.0 + 0.0 4.6 + 0.0 effect averaged roughly a fivefold increase (coefficient of 2.0 32.3 1 5.4 28.3 1.3 + 18.4 + 11.3 variation, - 13% among treatments). On day 21, both total and N added p size-fractionated phytoplankton chlorophyll a levels declined 0.0 4.3 + 1.3 0.5 1 0.7 Undetectable from previous weeks (Fig. 1 [day 21]). However, there was still 0.5 46.0 1 1.1 0.6 1 0.8 39.5 1 1.9 a marked positive relationship between bacterial abundance 1.0 103.5 1 4.6 9.8 1 3.1 86.3 ± 8.3 and P treatments in systems without added N; the bacterial 2.0 183.7 1 3.2 35.9 1 4.9 117.1 1 1.9 growth response to these P additions was more than threefold. Two-way analysis of variance indicated that N, P, and aTreatment additions were t) or 10 ,umol of NH4NO3 per liter of N, and 0.0, combined N-P treatments had and different 0.5, 1.0, and 2.0 p.mol of K2HPO4 per liter for P (n = 3 for each). several significant "Data are averages ± standard deviation. influences on the plankton. The effects among different size Undetectable, concentrations below the analytical limit of detection. classes occurred on different days and extended over different VOL. 60, 1994 EFFECT OF INORGANIC N AND P ON BACTERIAL-ALGAL GROWTH 2089

6E1 O 5 7 and 14. In contrast, N additions had a significant effect on Day bacterial abundance on day 14 but no significant effects on day 5 . 4 0, 7, or 21. P additions had sustained, significant effects on the

- abundance of bacteria on all days after day 0. The interaction T . -. 0) of N and P had a significant effect only on day 21, a period in

2 2 which phytoplankton were unaffected by the combination of N CL and P. 0.r Relationship between bacterial abundance and phytoplank- 0 0L- ton biomass. To examine relationships between bacterial

-C 0.0 0.5 1.0 2.0 0.0 0.5 1.0 D2.0 0.0 0.5 1.0 2.0 0.0 0.5 1.0 2.0 C. abundance and phytoplankton biomass, two regressions be- o 50-2 4 i t ^ 0 -mC C) _ *1 2 i w i |CD tween bacterial numbers and total chlorophyll were run sepa- 0 61 rately: mesocosms with and without N addition (Fig. 2). Day 14 Day 21 I -Q Bacterial numbers were not significantly correlated with total CL phytoplankton chlorophyll a level when N was not added (Fig. 0 4k 2A); less than 1% of the total variation in bacterial numbers 05~ .3 6j3 3 was explained by algal biomass. In contrast, there was a - significant positive regression between these two components 2 - I m~~~~~~~~~~~~C (P < 0.002) in mesocosms supplemented with 10 pLmol of I1- NH4NO3 per liter (Fig. 2B). While the relationship is noisy,

more than half of the variation in bacterial abundance was Downloaded from

0.0 0.5 1.0 2.0 0.0 0.5 1.0 2.0 0.0 0.5 1.0 2.0 0.0 0.5 1.0 2.0 explained by its dependence on phytoplankton chlorophyll a. no N + N noN +N These relationships were compared with the results of earlier studies (Table 4). Three of them find that the intercept Phosphorus addition ([trmol/ L) of this relationship is very near 6.3 ("2 x 10" cells per ml), FIG. 1. Effects of Pi (as K2HPO4) and N (as NH4NO3) additions on although the slopes appear to differ. A significant model I bacterial abundance and phytoplankton chlorophyll a levels in Calder regression of Calder Lake bacterial density versus chlorophyll Lake mesocosms. Each graph indicates a different sampling date. a level was possible only for N-fertilized mesocosms. However, aem.asm.org Error bars indicate + standard deviation (n = 3; note differences in model II (geometric mean) slopes for both sets of Calder Lake scales for phytoplankton). Solid areas, picoplankton; shaded areas, communities were similar. The slope for N-fertilized commu- nanoplankton; open areas, microplankton; 0, bacteria. nities averaged about 30% lower than was observed among several are not Quebec lakes (6), but these values significantly at FORDHAM UNIV on May 11, 2010 different (P > 0.05). periods (Table 3). For example, N fertilization elicited significant effects on microplankton chlorophyll a on day 7; this DISCUSSION effect continued through day 21. Overall, N pulses resulted in the largest significant effects on picoplankton, but these effects Our results clearly show that bacterial abundance in fresh were observed only on day 7. Analysis of variance also indi- waters can be stimulated by inorganic nutrients. We have cated that all treatments (N, P, and N x P interaction) had found that bacterial production can be stimulated by P addi- significant effects on total phytoplankton chlorophyll a on days tion alone without being coupled to increases in phytoplankton

TABLE 3. Two-way analysis of variance testing the effects of fertilization on phytoplankton chlorophyll a level and bacterial abundance in Calder Lake mesocosms" Effect of N and P addition and N x P interaction at": Plankton category Factor Day 0 Day 7 Day 14 Day 21 F ratio P value F ratio P value F ratio P value F ratio P value Microphytoplankton N 0.48 0.497 16.09 0.001 33.70 <0.001 32.73 <0.001 P 3.38 0.044 0.21 0.889 5.92 0.006 0.58 0.663 N x P 1.77 0.193 1.02 0.411 13.03 <0.001 1.06 0.394 Nanophytoplankton N 0.80 0.384 35.57 <0.001 49.65 <0.001 4.02 0.062 P 1.20 0.342 1.68 0.211 18.63 <0.001 1.39 0.281 N x P 1.39 0.281 3.14 0.055 16.17 <0.001 0.86 0.483 Picophytoplankton N 0.62 0.442 152.24 <0.001 0.66 0.427 0.17 0.687 P 0.55 0.656 8.36 0.001 0.46 0.717 1.22 0.335 N X P 1.92 0.166 9.15 0.001 0.47 0.709 0.49 0.693 Total phytoplankton N 0.08 0.785 138.74 <0.001 20.08 <0.001 0.08 0.777 P 0.62 0.612 7.13 0.003 4.75 0.015 2.18 0.130 N X P 1.63 0.222 8.84 0.001 5.56 0.008 0.19 0.899 Planktonic bacteria N 0.12 0.733 0.16 0.692 18.26 <0.001 4.72 0.045 P 0.82 0.504 21.62 <0.001 14.95 <0.001 5.40) 0.009 N x P 2.54 0.093 1.44 0.269 0.66 0.589 6.84 0.004 Results compare the effects of N addition (O or 10 ,umol of NH4NO3 per liter), P addition (), 0.5, 1, or 2 p.mol of K2HPO4 per liter), and N x P interaction on three size classes of phytoplankton chlorophyll a (micrograms per liter) and bacterial abundance (cells per milliliter [see Materials and Methods for details regarding assumptions]). " All P values of <0.(5 are given in boldface. 2090 LE ET AL. APPL. ENVIRON. MICROBIOL.

7.0 potential phytoplankton DOC, bacterioplankton continued to A No Nitrogen Added be stimulated by N and P additions. Most of the DOC available to planktonic bacteria in lakes 6.8 - r2= 0.007, NS and open oceans is thought to be produced by phytoplankton (3, 7, 13, 15, 16). Thus, the relatively constant, high concentra- 6.6 - c5n tions of DOC in mesoeutrophic Calder Lake (spring to sum- mer, 260 to 325 ,u.mol/liter [36, 37]) and similar levels in 6.4 k mesotrophic Dillon Lake, Colo. (125 to 210 ,umol/liter [28]), as o 0 well as oligotrophic Mirror Lake, N.H. (200 to 270 p.mol/liter 6.2 - 0 0 [23]), suggest that DOC levels may exceed the amount re- E quired for bacterial growth. In heterotrophic systems such as cn o 0 the Hudson River, allochthonous, rather than autochthonous, = 6.0 - sources of DOC apparently meet bacterial demands (19). a.)0 Collectively, these data and the present experimental results 5.8 - imply that phytoplankton-derived DOC may not be the sole 0).0 0.1 0.2 0.3 0.4 0.5 cause for the quantitative link between phytoplankton and 'D~0 bacteria. Inorganic nutrient conditions may also play a central .0cU 7.0 - role in the widely observed phenomenon of algal-bacterial a) B +101iMNH4NO3 coupling. C.)=3 - < 0.002 6.8 r2=0.539 P It is unreasonable, however, to ignore many previous find- Downloaded from ings which show that algal exudates are important sources of Cu 6.6 - 0 carbon for pelagic, heterotrophic bacteria (3, 7, 13, 14, 16, 31). C) One possible explanation for data which depart from DOC- 0 based models of bacterium-phytoplankton coupling lies in 6.4 - variations in substrate lability. Bacterial growth yields varied -j widely (8 to 60%) in eutrophic Lake Fredericksborg (Den- 6.2 - 0 mark) as a function of the lability of DOC seasonally (26). The aem.asm.org possible causes for changes in concentrations of labile DOC 6.0 - were not clear, however, because labile DOC concentrations in that system were not significantly correlated with phytoplankton chlorophyll a concentrations. Experiments in mesotrophic 5.8 at FORDHAM UNIV on May 11, 2010 Lake Schohsee (Germany) demonstrated that total DOC 0.0 0.2 0.4 0.6 0.8 1.0 levels were greatest in N-P-enriched mesocosms and that the contribution of carbohydrates and amino acids (high-quality Log Chlorophyll a ([g / L) DOC) to the DOC pool was also greater (14). In the present FIG. 2. Relationship between bacterial abundance and phytoplank- Calder Lake experiments, additions of N may have resulted in ton chlorophyll a concentrations in Calder Lake mesocosms without higher-quality DOC being excreted by phytoplankton, which (A) and with (B) 10-,umol/liter NH4NO3 additions. Each point repre- would then result in the establishment of a closer coupling sents the mean value of three replicate measurements on different between phytoplankton and bacterial production. sampling dates and among different nutrient treatments. In systems Since both algal production and bacterial production in without N added, the relationship has a slope insignificantly different fresh water also respond directly to P availability (17, 37), we from 0. The line in panel B was fitted by least-squares regression with may also hypothesize that algal-bacterial coupling is indirect, 95% confidence limits (outer curved lines; see text for details and assumptions). derived from a mutual dependence on Pi. This hypothesis makes it necessary to examine possible common factors that may regulate both bacteria and phytoplankton simultaneously but independently. In general, phytoplankton require dissolved and without N addition. Further proof can be seen in a inorganic N and DIP approximating the Redfield ratio of 16:1, nonsignificant regression between bacterial numbers and phy- but it is not yet clear whether the inorganic requirements of toplankton chlorophyll a level in systems without added N (Fig. bacterioplankton are similar (but see Discussion in references 2A). A significant, positive regression between bacterial abun- 37 and 41). The first way we tested this idea was by running dance and phytoplankton chlorophyll a level in mesocosms regressions between the system-level P depletion rate (per day with N added (Fig. 2B) suggests that bacterial-algal coupling rate of SRP decline) and (i) bacterial numbers or (ii) phyto- may not be primarily based on DOC supplied by phytoplank- plankton chlorophyll a concentration, separated by N treat- ton, as suggested previously by Cole and others (15). Such ment (Fig. 3). A steeper slope implies that more rapid P linkages may be, at least in part, a response to different depletion may result from greater microbial (bacteria or inorganic nutrient regimens. Unexplained declines in plankton phytoplankton) production levels. Our results show that in chlorophyll a levels (especially algal nanoplankton on day 21 systems without added N, a significant regression (P < 0.01) [Fig. 1]) may be the result of grazing pressure brought on by exists between water-column P depletion rates and bacterial increasing numbers of large cladocerans in the mesocosms numbers only (Fig. 3A); there was no significant dependence (12a). Because many zooplankton species are known to graze on phytoplankton biomass (as chlorophyll a [Fig. 3B]). One size selectively, cladocerans, ciliates, and small heterotrophic may conclude that under low-N conditions, P dynamics are protists may also affect apparent algal-bacterial coupling. regulated more by bacterioplankton than phytoplankton activ- Between day 14 and day 21, more than half of the phytoplank- ity; the two components of the microbial loop are uncoupled. ton chlorophyll a was removed from the water column in tanks However, in mesocosms with N added, P depletion rates are supplied with higher levels of N and P, presumably via grazing significantly correlated with both bacterial abundance and and/or sedimentation (41). Despite a probable decline in phytoplankton biomass (Fig. 3C and D). The results also show VOL. 60, 1994 EFFECT OF INORGANIC N AND P ON BACTERIAL-ALGAL GROWTH 2091

TABLE 4. Regression results describing the relationship between bacterial density and phytoplankton chlorophyll a level in Calder Lake mesocosm experiments' Water source x V n2 Intercept Slope -2> Model 11 slope Calder Lake No N added Chlorophyll a DSDC 16 6.33 (+0. 11) 0.14 (1+0.42) 0.0)07 0.626 N added Chlorophyll a DSDC 16 6.26 (+0.05) t).39 (1-0.09) 0.539** 0.528 Quebec lakes' Chlorophyll a AODC 13 6.28 (10.19) 0.57 (10.27) 0.66*** 0.700 Hudson River" Chlorophyll a AODC 17 6.78 (10.09) -0.11 (+0.09) 0.(9 0.359 Results are divided into the two main N treatments (without and with addition of 1) ILmol of NH4NO3). The variables tested were chlorophyll a concentration (micrograms per liter [x]) and bacterial numbers counted by DAPI-stained direct counts (DSDC) or acridine orange direct counts from prior studies (AODC). Variables were log transformed to more closely meet normality assumptions. The first set of values for they intercept and slope ( +95% confidence limits are given in parentheses) were based on model I regression assumptions (on the basis of the presumed dependence of bacterial growth on phytoplankton-produced DOC). A modcl 11 regression (geometric mean) estimate of the slope is provided for comparison (on the basis of the recognition that hoth variables are measured with crror). e**, P < .01; 0.r(n***, P < . Results from reference 6. "Results from reference 19.

that depletion of dissolved P from the water column was does not stimulate the growth of phytoplankton. Our data Downloaded from greatly enhanced by the addition of N. P depletion rates were suggest that bacteria are not superior competitors for low P roughly three times greater for equivalent bacterial densities supplies in mixed communities. We observed three consecutive (Fig. 3A and C). weeks of apparent P limitation in bacteria, while phytoplank- We interpret these phenomena to be the result of differences ton groups experienced only one or two such periods (Fig. I in algal versus bacterial responses to N and P deficiencies in and Table 3). Bacterioplankton may simply be unable to store

lake water. When N was not added, phytoplankton experi- polyphosphates as efficiently as most algal species, making aem.asm.org enced an apparent N deficiency (day 7), but bacteria did not. them dependent on a more continuous supply of DIP. We Because bacteria have high affinities for inorganic N (42), they observed that bacteria respond positively and significantly to P may be able to obtain enough N (perhaps as dissolved organic addition, apparently regardless of external dissolved inorganic N) from occasional pulses (e.g., lake snow) but may be limited N pools. When N is added, both bacteria and phytoplankton by adequate supplies of DIP. In our experiments, addition of P can respond to P addition, and so their growth appears at FORDHAM UNIV on May 11, 2010 without N stimulates the growth of planktonic bacteria but coupled. This suggests that N and P conditions are mutual factors affecting algal-bacterial coupling, which elicit independent but parallel increases in bacterial and phytoplanktonic levels. No_.Nitroaen...-j Added An analysis of data from the literature representing many r2= 0.329 .A r2=0.043 B lakes found a less consistent relationship between chlorophyll : 1.2 - o NS p< 0.01 o o o -J O 0 / a concentrations and bacterial production among contrasting > 0.8 0 00 ...- systems. Chlorophyll a levels could explain only about 16% of ol o o 8 i ° the total variability in bacterial production (18). Some longer- '/ F °o ° o~~~~ .° 0.4 oOC term studies of single lakes have found significant positive a) / 00 00 o relationships between bacteria and phytoplankton (32), while O 0.0 06. aCD other single-lake studies have found no significant bacterium- chlorophyll relationship (27, 28). Other factors clearly must be -0.4 involved, which act differently on algae and bacteria (e.g., 0 1 2 3 4 5 6 0 1 2 3 4 protist and metazoan grazers). We propose that inorganic N +10 .tmol NH4NO3/ L and P may be two key factors that link bacterial and phyto- rl=0.285 plankton production and may account for some of the unex- _ 6 r2= 0.147 C D P< 0.05 . * P< 0.01 * plained variation (residuals) found in past studies. Coupling bacterial and phytoplankton production can de-

0) pend on both N and P status. In systems with a low N/P ratio 0.* ('40; N limited), bacteria are more responsive to pulses of Pi, 0. a) 2 and hence the two components become uncoupled. In systems a) , .s~- / ....0 with higher N/P ratios, both components of the community

_,., should respond to P dynamics. The necessity of greater N in O- this dynamic suggests that a high N/P ratio (>40) is necessary 0 1 2 3 4 5 6 0 2 4 6 8 10 12 14 for a close coupling between bacteria and phytoplankton in Bacterial Phytoplankton fresh water. We have proposed a simple index which describes Density (1 06cells/mL) Chlorophyll-a (p.g/L) the strength of this coupling. The coupling index (CI) is defined as the ratio of percent change in bacterial abundance FIG. 3. Relationship between bacterial numbers or phytoplankton or biomass to the percent change in phytoplankton biomass a concentrations and P rates in mesocosms chlorophyll depletion over the same period, expressed as follows: CI = % rate of A without (A and B) and with (C and D) 10-[.mol/liter NH4NO3 rate of A The range of values for CI additions. Lines were fitted by least-squares regression with 95%/c bacteria/% phytoplankton. confidence limits (outer curved lines). The relationship between should be -< < CI < +x. When bacterial and phytoplank- phytoplankton chlorophyll a level and P depletion in systems without tonic growth are closely linked, CI will be small and vary only N addition has a slope insignificantly different from 0. between -1 and + 1. We predict that the variation in CI will be 2092 LE ET AL. APPL. ENVIRON. MICROBIOL.

6 3. Berman, T., and B. Kaplan. 1984. Diffusion chamber studies of 0 carbon flux from living algae to heterotrophic bacteria. Hydrobio- logia 108:127-134. 4 0 0 4. Billen, G. 1984. Heterotrophic utilization and regeneration of nitrogen, p. 313-355. In J. E. Hobbie and P. J. Williams (ed.), a) _ 2 - 0 he tooo Bact APhyto Heterotrophic activity in the sea. Plenum Publishing Corp., New York. -c c (a 5. Bird, D. F., and C. M. Duarte. 1989. Bacteria-organic matter C 0 < i0 relationship in sediments: a case of spurious correlation. Can. J. 0o 0 co Fish. Aquat. Sci. 46:904-908. -2 co 6. Bird, D. F., and J. Kalff. 1984. Empirical relationships between -' bacterial abundance and chlorophyll concentration in fresh and 0 -4 marine waters. Can. J. Fish. Aquat. Sci. 41:1015-1023. 0 7. Bj0rnsen, P. K. 1988. Phytoplankton exudation of organic matter: why do healthy cells do it? Limnol. Oceanogr. 33:151-154. -6 8. Bj0rnsen, P. K., B. Riemann, J. Pock-Steen, T. G. Nielsen, and 0 50 100 150 200 250 300 350 S. J. Horsted. 1989. Regulation of bacterioplankton production and cell volume in a eutrophic estuary. Appl. Environ. Microbiol. N:P ratio 55:1512-1518. FIG. 4. Relationship between the empirically derived bacterial- 9. Bloem, J., M. Starink, M.-J. B. Bar-Gilissen, and T. E. Cappen- phytoplanktonic CI (expressing closeness of bacterial and phytoplank- berg. 1989. Protozoan grazing, bacterial activity, and mineraliza- tonic growth activities) and inorganic N/Pa ratios in experimental tion in two-stage continuous cultures. Appl. Environ. Microbiol. Downloaded from mesocosms containing Calder Lake communities. CI is a rate function 54:3113-3121. expressing the ratio of daily percent changes in bacterial abundance 10. Bran+Luebbe Analyzing Technologies. 1986. Ortho-phosphate in divided by the daily percent change in phytoplankton chlorophyll a water and seawater. Industrial method no. 812-86T. Bran+Luebbe level at each sampling interval (see Materials and Methods for details). Analyzing Technologies, Buffalo Grove, Ill. 11. Bran+Luebbe Analyzing Technologies. 1986. Ammonia in water and seawater. Industrial method no. 804-86T. Bran+Luebbe Analyzing Technologies, Buffalo Grove, Ill. large (either positive or negative) in systems with low N/P 12. Bran+Luebbe Analyzing Technologies. 1987. Nitrate/nitrite in aem.asm.org ratios and limited in systems with high N/P ratios. We tested water and seawater. Industrial method no. 818-87T. Bran+Luebbe this hypothesis by plotting CI against the N/P ratio in all Analyzing Technologies, Buffalo Grove, Ill. mesocosms over the 4-week experimental period (n = 72); the 12a.Cavassi, K., and J. D. Wehr. Unpublished data. results match our predictions fairly closely (Fig. 4). Relatively 13. Chr6st, R. J., and M. A. Faust. 1983. Organic carbon release by little variation in CI (values of 1.0) is observed in systems phytoplankton: its composition and utilization by bacterioplank- at FORDHAM UNIV on May 11, 2010 with N/P ratios >-40. Prior experiments indicate that these ton. J. Plankton Res. 5:477-493. conditions favor the dominance of smaller phytoplankton 14. Chr6st, R. J., and H. Rai. 1993. Ectoenzyme activity and bacterial secondary production in nutrient-impoverished and nutrient-en- species, particularly the cyanobacterium Synechococcus sp. (35, riched freshwater mesocosms. Microb. Ecol. 25:131-150. 39, 40). 15. Cole, J. J., S. Findlay, and M. L. Pace. 1988. Bacterial production Among studies which have examined the presence or lack of in fresh and saltwater ecosystems: a cross-system overview. Mar. algal-bacterial coupling, attention has been given to the resid- Ecol. Progr. Ser. 43:1-10. ual (or unexplained) variation in this relationship. Some 16. Cole, J. J., G. E. Likens, and D. L. Strayer. 1982. Photosyntheti- studies suggest that nutrient levels, variations in allochthonous cally produced dissolved organic carbon: an important source for carbon levels, and even methodological errors may be involved planktonic bacteria. Limnol. Oceanogr. 27:1080-1090. (6, 18, 19, 22). Data from other systems would be similarly 17. Coveney, M. F., and R. G. Wetzel. 1992. Effects of nutrients on rate useful to test preliminary ideas concerning our CI. Our exper- specific growth of bacterioplankton in oligotrophic lake water cultures. Appl. Environ. Microbiol. 58:150-156. iments demonstrate directly that N limitation can uncouple 18. Currie, D. J. 1990. Large scale variability and interactions among this linkage and result in different trajectories for the growth of phytoplankton, bacterioplankton, and phosphorus. Limnol. bacterioplankton and phytoplankton along P gradients. It is Oceanogr. 35:1437-1455. possible that N regeneration, as mediated by grazing zooplank- 19. Findlay, S., M. L. Pace, D. Lints, J. J. Cole, N. F. Caraco, and B. ton and protozoans (4, 9, 20), may be the short-term stimulus Peierls. 1991. Weak coupling of bacterial and algal production in between turnover periods which links phytoplankton and bac- a heterotrophic ecosystem: the Hudson River estuary. Limnol. terial production. Oceanogr. 36:268-278. 20. Hessen, D., T. Anderson, and F. Bj0rn. 1992. Zooplankton con- ACKNOWLEDGMENTS tribution to particulate phosphorus and nitrogen in lakes. J. Plankton Res. 14:937-947. This study was supported in part by the NSF (grant DIR-9002145) 21. Hobbie, J. E., and P. J. Williams (ed.). 1984. Heterotrophic activity and the Louis Calder Center of Fordham University. in the sea. Plenum Publishing Corp., New York. We gratefully acknowledge T. Baron and J. Kalish for assistance in 22. Karner, M., D. Fuks, and G. J. Herndl. 1992. Bacterial activity maintaining the Experimental Lake Facility tanks and K. Cavassi for along a trophic gradient. Microb. Ecol. 24:243-257. generous assistance in the field. We also thank T. Toolan for valuable 23. Likens, G. E., J. S. Eaton, and N. M. Johnson. 1985. Physical and comments on an earlier version of the manuscript and an anonymous chemical environment, p. 89-108. In G. E. Likens (ed.), An reviewer who provided comments on this paper. ecosystem approach to aquatic ecology. Mirror Lake and its environment. Springer-Verlag, New York. REFERENCES 24. Linley, E. A. S., R. C. Newell, and M.I. Lucas. 1983. Quantitative 1. American Public Health Association. 1985. Standard methods for relationships between phytoplankton, bacteria and heterotrophic the analysis of water and wastewater, 16th ed. American Public microflagellates in shelf waters. Mar. Ecol. Progr. Ser. 12:77- Health Association, Washington, D.C. 89. 2. Azam, F., T. Fenchel, J. G. Field, and J. S. Gray. 1983. The 25. Lorenzen, C. J. 1967. Determination of chlorophyll and pheopig- ecological role of water-column microbes in the sea. Mar. 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