Stoichiometry of the Microbial Loop: J. Matthew Haggerty

Stoichiometry of the Microbial Loop

J. Ma5hew Haggerty SDSU/UC Davis

Dinsdale/Eisen Lab

What is the Microbial Loop?

Complicated By Diversity

Diverse Nutrient Regimes

Diverse ProkaryoJc Community

SelecJve Grazing from a Diverse Protozoan Community

Nutrients in the Ocean Deep-Sea Research II 49 (2002) 463–507

Iron cycling and nutrient-limitation patterns in surface watersof the World Ocean

J. Keith Moorea,*, Scott C. Doney, David M. Gloverb, Inez Y. Fungc

aNational Center for Atmospheric Research NCAR, P.O. Box 3000, Boulder, CO 80307, USAbWoods Hole Oceanographic Institution, 573 Woods Hole Road, Woods Hole, MA 02543, USA

cUniversity of California Berkeley, 301 McCone Hall, Berkeley, CA 94720, USA

Accepted 17 June 2001

Abstract

A global marine ecosystem mixed-layer model is used to study iron cycling and nutrient-limitationpatterns in surface waters of the world ocean. The ecosystem model has a small phytoplankton size classwhose growth can be limited by N, P, Fe, and/or light, a diatom class which can also be Si-limited, and adiazotroph phytoplankton class whose growth rates can be limited by P, Fe, and/or light levels. The modelalso includes a parameterization of calcification by phytoplankton and is described in detail by Moore et al.(Deep-Sea Res. II, 2002).

The model reproduces the observed high nitrate, low chlorophyll (HNLC) conditions in the SouthernOcean, subarctic Northeast Pacific, and equatorial Pacific, and realistic global patterns of primaryproduction, biogenic silica production, nitrogen fixation, particulate organic carbon export, calciumcarbonate export, and surface chlorophyll concentrations. Phytoplankton cellular Fe/C ratios and surfacelayer dissolved iron concentrations are also in general agreement with the limited field data. Primaryproduction, community structure, and the sinking carbon flux are quite sensitive to large variations in theatmospheric iron source, particularly in the HNLC regions, supporting the Iron Hypothesis of Martin(Paleoceanography 5 (1990) 1–13). Nitrogen fixation is also strongly influenced by atmospheric irondeposition.

Nitrogen limits phytoplankton growth rates over less than half of the world ocean during summermonths. Export of biogenic carbon is dominated by the sinking particulate flux, but detrainment andturbulent mixing account for 30% of global carbon export. Our results, in conjunction with other recentstudies, suggest the familiar paradigm that nitrate inputs to the surface layer can be equated withparticulate carbon export needs to be expanded to include multiple limiting nutrients and modes of export.r 2001 Elsevier Science Ltd. All rights reserved.

*Corresponding author.E-mail address: [email protected] (J.K. Moore).

0967-0645/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved.PII: S 0 9 6 7 - 0 6 4 5 ( 0 1 ) 0 0 1 0 9 - 6

•  Comprehensive nutrient compilaJon of nutrients (N,C,P,Fe,Si,CaCO3,Chl)

•  Analysis predicts regions of nutrient limitaJon for different funcJonal groups.

Diatom LimitaJon

Deep-Sea Research II 49 (2002) 463–507






Abstract






macronutrients and the initially high iron concentrations in this region (see Januaryconcentrations in Fig. 2, and MET). Most field evidence suggests that Fe limitation does notplay a strong role in this region (Martin et al., 1993; Sunda, 1997), and high chlorophyllconcentrations are seen throughout the summer in the SeaWiFS data. There are several possibleexplanations for Fe limitation in the model. Advection from nearby continental sources may playa role in delivering iron to this region (Martin et al., 1993; Fung et al., 2000). The atmosphericsource may be underestimated. The dust flux to the North Atlantic is much higher in MAH99

Fig. 8. Nutrient-limitation patterns for the diatoms (A), the small phytoplankton (B), and the diazotrophs (C) duringsummer months. Areas where all nutrient cell quotas are >97% of the maximum cell quota values are arbitrarilydefined as nutrient-replete. Also shown is the percentage of total ocean area where each nutrient is limiting growth.

J.K. Moore et al. / Deep-Sea Research II 49 (2002) 463–507 477

Small Phytoplankton LimitaJon







Abstract









Diazotroph LimitaJon







Abstract









What about co-‐limiJng nutrients

LETTERS

NATURE GEOSCIENCE DOI: 10.1038/NGEO667

60° N

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Figure 1 | Cruise tracks and maps of annual mean excess nutrientdistributions. a, Cruise tracks for the AMT-17 cruise and hydrographicsections (WOCE A10, GEOSECS and the 1981 occupation of 36� N,including the Gulf Stream (GS)) used in the analysis of deep ocean nutrientdistributions and transport. b, Climatological surface P⇤ (= DIP�DIN/rn/p)distribution with the AMT-17 cruise track and a contour corresponding to0.1 µmol kg�1 P⇤ superimposed. c, Distribution of N⇤ (= �rn/p P⇤) on the�✓ = 26.8 surface corresponding to the thermocline/SAMW (ref. 24) withWOCE A10 (white) and GS 36� N (black) sections indicated.

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� +NO2� and DIP (note logarithmic scale).

g, P⇤ (= DIP�DIN/rn/p), DOP⇤ (= DOP�DON/rn/p) and TDP⇤

(= P⇤ +DOP⇤). h, N⇤ (= DIN� rn/p DIP) at the nutricline, defined asDIN = 1 µmol l�1.

south. Moreover, DOP⇤ was more negative in the northern gyre(Fig. 2g and Supplementary Fig. S1c). Marked gradients were thusobserved in surface TDP⇤, with positive values in the southerngyre and negative values in the north (Fig. 2g), which were spa-tially correlated with the region of enhanced N2 fixation (Fig. 2,Supplementary Fig. S1).

These data clearly do not support a proximal role for surface-water P availability as a control on the spatial distribution of N2fixation across inter-basin scales in the Atlantic. Higher (TD)P and(TD)P⇤ concentrations observed in the South Atlantic probablyresult from exchanges with low N/P (negative N⇤) thermoclinewaters (Fig. 2h) as well as lateral transport of N-deficient watersgenerated in the Benguela current system4 (Fig. 1b). The severelyN-deficient waters were shown to result in N limitation of bulkcommunity productivity (see Supplementary Fig. S2). Clearly some

868 NATURE GEOSCIENCE | VOL 2 | DECEMBER 2009 | www.nature.com/naturegeoscience

LETTERS

PUBLISHED ONLINE: 1 NOVEMBER 2009 | DOI: 10.1038/NGEO667

Large-scale distribution of Atlantic nitrogenfixation controlled by iron availabilityC. Mark Moore1,2*, MatthewM. Mills3, Eric P. Achterberg2, Richard J. Geider1, Julie LaRoche4,Mike I. Lucas5, Elaine L. McDonagh2, Xi Pan2, Alex J. Poulton2, Micha J. A. Rijkenberg2,David J. Suggett1, Simon J. Ussher6 and E. Malcolm S. Woodward7

Oceanic fixed-nitrogen concentrations are controlled by thebalance between nitrogen fixation and denitrification1–4. Anumber of factors, including iron limitation5–7, can restrictnitrogen fixation, introducing the potential for decouplingof nitrogen inputs and losses2,5,8. Such decoupling couldsignificantly affect the oceanic fixed-nitrogen inventory andconsequently the biological component of ocean carbon storageand hence air–sea partitioning of carbon dioxide2,5,8,9. However,the extent to which nutrients limit nitrogen fixation in theglobal ocean is uncertain. Here, we examined rates of nitrogenfixation and nutrient concentrations in the surfacewaters of theAtlantic Ocean along a north–south 10,000 km transect duringOctober and November 2005. We show that rates of nitrogenfixation were markedly higher in the North Atlantic comparedwith the South Atlantic Ocean. Across the two basins,nitrogen fixation was positively correlated with dissolvediron and negatively correlated with dissolved phosphorusconcentrations. We conclude that inter-basin differences innitrogen fixation are controlled by iron supply rather thanphosphorus availability. Analysis of the nutrient content ofdeep waters suggests that the fixed nitrogen enters NorthAtlantic Deep Water. Our study thus supports the suggestionthat iron significantly influences nitrogen fixation5, andthat subsequent interactions with ocean circulation patternscontribute to the decoupling of nitrogenfixation and loss2,4,8.

The production and remineralization of organic material inthe ocean typically results in a nitrogen (N) to phosphorus(P) ratio (rn/p) of ⇠16:1 (refs 1, 3, 4, 10). However, thenet influences of dinitrogen (N2) fixation and fixed-nitrogenloss by denitrification and/or anaerobic ammonium oxidation(anammox), generate deviations of the dissolved N/P ratio awayfrom this typical value2,4,11. Removal of NO3

� in sediments andsuboxic water-column oxygen-minimum zones (OMZs) reducesthe availability of fixed-N relative to P. An excess of P is thusgenerated, which may subsequently favour diazotrophic growthand hence ultimately replacement of the lost N (refs 1, 3,4). This feedback mechanism probably maintains the marine Ninventory and couples it to the P inventory3,4,12, at least on longtimescales2. However, diazotrophic growth may be limited byfactors other than the availability of excess P (refs 2, 5, 8, 13).For example, the apparent dominance of oceanic N2 fixationby facultative diazotrophic cyanobacteria7,14 seems to constrain

1Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK, 2National Oceanography Centre, University of Southampton, EuropeanWay, Southampton SO14 3ZH, UK, 3Department of Environmental Earth System Science, Stanford University, Stanford, California 94305, USA, 4MarineBiogeochemistry, Leibniz-Institut für Meereswissenschaften, D-24105 Kiel, Germany, 5Department of Zoology, University of Cape Town, Rondebosch7701, South Africa, 6School of Earth, Ocean and Environmental Sciences, University of Plymouth, Plymouth PL4 8AA, UK, 7Plymouth Marine Laboratory,Prospect Place, The Hoe, Plymouth PL1 3DH, UK. *e-mail: [email protected].

high rates to well-lit N-limited surface waters6,13. Furthermore,iron (Fe) limitation of diazotrophs5–7 may further contribute todecoupling of N inputs and losses, generating the potential forperturbations in the N inventory as a result of past or future climatechange2,5,8,9,15. Although limited correlative evidence supports theFe-limitation hypothesis16–18, direct experimental tests are rare6.Moreover, recent interpretation of nutrient distributions calls fortight spatial coupling of N2 fixation and denitrification, suggestingthat theN/P feedbackmechanismoperates efficiently in themodernocean, potentially implying a limited role for Fe limitation4.

On ameridional transect between 37� N and 35� S in the AtlanticOcean (Fig. 1), we observed maximum surface and euphotic-zone integrated N2 fixation rates north of the Equator (Fig. 2a,b,Supplementary Fig. S1a). N2 fixation by unicellular (<20 µm)diazotrophs contributed a background of 16± 7 µmolNm�2 d�1

throughout the transect, whereas N2 fixation by the larger sizefraction, dominated by the diazotrophic filamentous cyanobacteriaTrichodesmium spp. (Fig. 2c), peaked between ⇠5 and 15� Nat ⇠200 µmolm�2 d�1. Although diazotroph distributions mayvary seasonally13,14, data available at present suggest that theobserved inter-hemispheric contrast is broadly persistent17. Inturn, Trichodesmium abundance and N2 fixation rates were bothpositively correlated with dissolved iron (see SupplementaryTable S1) and dissolved aluminium, a non-nutrient tracer ofatmospheric dust input (Fig. 2d,e).

Surface concentrations of dissolved inorganic N (DIN) were lowthroughout the transect and indistinguishable between northernand southern gyres (5 ± 3 nmol l�1 NO3

� + NO2� (Fig. 2f) and

17±8 nmol l�1 NH4+). In contrast, dissolved inorganic P (DIP) was

over an order ofmagnitude higher in the surfacewaters of the south-ern gyre (210± 60 nmol l�1) than the potentially biolimiting con-centrations (9±4 nmol l�1) observed in the northern gyre (Fig. 2f).Dissolved organic P (DOP) concentrations were also significantlylower in the northern sub-tropical gyre (0.19 ± 0.02 µmol l�1)than in the south (0.28 ± 0.02 µmol l�1), whereas DON aver-aged ⇠6 µmol l�1 in both gyres. Excess DIP was calculated asP⇤ =DIP�DIN/rn/p. Similarly, the related term N⇤ (= �rn/p P⇤)provides a measure of excess DIN relative to DIP (ref. 11) andDOP⇤ (= DOP⇤ �DON⇤/rn/p) and TDP⇤ (= P⇤ +DOP⇤) can beused to indicate the excess of the organic and total dissolved Ppools respectively. Assuming rn/p = 16:1 (refs 1, 10), P⇤ increasedfrom <0.016 µmol l�1 in the northern gyre to ⇠0.2 µmol l�1 in the

NATURE GEOSCIENCE | VOL 2 | DECEMBER 2009 | www.nature.com/naturegeoscience 867

The ComplicaJons




BGD8, 6227–6263, 2011

Seasonal variabilityin seston

stoichiometry

H. Frigstad et al.

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Biogeosciences Discuss., 8, 6227–6263, 2011www.biogeosciences-discuss.net/8/6227/2011/doi:10.5194/bgd-8-6227-2011© Author(s) 2011. CC Attribution 3.0 License.

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This discussion paper is/has been under review for the journal Biogeosciences (BG).

Please refer to the corresponding final paper in BG if available.

Seasonal variation in marine C:N:Pstoichiometry: can the composition ofseston explain stable Redfield ratios?H. Frigstad1,2, T. Andersen3, D. O. Hessen3, L.-J. Naustvoll4, T. M. Johnsen5, andR. G. J. Bellerby6,2,1

1Geophysical Institute, University of Bergen, Allegaten 70, 5007 Bergen, Norway2Bjerknes Centre for Climate Research, University of Bergen, Allegaten 55, 5007 Bergen,Norway3Department of Biology, University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway4Institute of Marine Research (IMR), Flødevigen Research Station, Nye Flødevigveien 20,4814 His, Norway5Norwegian Institute for Water Research (NIVA), Thormøhlensgate 53D, 5006 Bergen, Norway6Uni Research, University of Bergen, Allegaten 55, 5007 Bergen, Norway

Received: 7 June 2011 – Accepted: 14 June 2011 – Published: 30 June 2011

Correspondence to: H. Frigstad ([email protected])

Published by Copernicus Publications on behalf of the European Geosciences Union.

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6263

DeviaJon in Microbial Redfield RaJo

Quick Rundown on Cyanobacteria

•  Known for their role as nitrogen fixers •  Spurred iron ferJlizaJon experiments •  The primary nitrogen fixer is Trichodesmium •  Prochlorococcus and Synechococcus are not nitrogen fixers but primary producers

•  Ber1lsson highlighted the role of C:P ra1os limi1ng carbon sequestra1on

Trichodesmium

•  Nitrogen fixaJon is iron limited in 75% of the worlds oceans

•  Iron limitaJon results in loss of photosyntheJc pigments

•  Use five Jmes as much iron •  20-‐50% iron used as nitrogenase •  Phosphate limitaJon seen at N:P raJo > 40

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Limnol. Oceanogr., 51(4), 2006, 1777-1790 ? 2006, by the American Society of Limnology and Oceanography, Inc.

Flexible elemental stoichiometry in Trichodesmium spp. and its ecological implications

Angelicque E. White1 and Yvette H. Spitz College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, Oregon 97331-5503

David M. Karl School of Ocean and Earth Science and Technology, University of Hawaii, Honolulu, Hawaii 96822

Ricardo M. Letelier College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, Oregon 97331-5503

Abstract We conducted laboratory experiments to assess the bioelemental plasticity of cultures of Trichodesmium

IMS101 under phosphorus (P)-replete, P-restricted, and light-limited conditions. The results reveal a high degree of stoichiometric flexibility. Specifically, Trichodesmium IMS101 is capable of growth with carbon (C) : nitrogen (N) : P ratios of C585_56: N90o+10 P1, approximately six times higher than would be predicted by the Redfield reference ratio (C106 N16 : PI), thus signifying low cellular P quotas relative to C and N. Luxury consumption of P occurs rapidly after periods of prolonged P restriction, under both light and dark conditions, resulting in substantial increases in P quotas and reductions of C:N:P ratios (C96__8 :N16-1: P1). Comparisons of laboratory culture data to our field observations from the Northwest Atlantic and the North Pacific indicate that, while natural populations of Trichodesmium exhibit persistently low P content relative to C and N (C290_15: N53+-3: P1), the highest and lowest C: P and N: P ratios recorded in the laboratory are rarely observed in nature. We have also performed laboratory experiments intended to simulate the energetic and nutritional extremes that would occur as naturally migrating populations of Trichodesmium sink out of the euphotic zone into P-rich regions of the upper disphotic zone. The duration of dark survival for this isolate is on the order of 3-6 d, after which time cells are unable to recover from light deprivation. This finding provides a constraint on the temporal scale of vertical migration.

Species of the colony-forming marine cyanobacterial genus Trichodesmium have been well described as promi- nent dinitrogen (N2) fixing organisms (diazotrophs) in the oligotrophic tropical and subtropical regions of the global ocean (Capone 2001; Karl et al. 2002). The notoriety of this genus has only increased in recent years as revisions of abundance estimates and N2 fixation rates have empha- sized Trichodesmium as a significant source of new nitrogen to otherwise nitrogen (N)-deficient ecosystems (LaRoche and Breitbarth 2005). More than just a delivery vehicle for reactive N (NR), Trichodesmium spp. are also noted for their ability to episodically form large surface accumula- tions, thereby transiently dominating primary productivity and N cycling (Bowman and Lancaster 1965; Karl et al. 1992; Capone et al. 1998).

Trichodesmium is ecologically significant in oligotrophic oceanic regimes, such as the North Atlantic and North

1 Corresponding author ([email protected]). Acknowledgments

We thank A. Ashe and S. Laney for their assistance. Thanks also to D.G. Capone and E.J. Carpenter for helpful discussion and to two anonymous reviewers who provided valuable criticism. National Science Foundation (NSF) OCE03-26616 (D.M.K./ R.M.L.), NSF OCE99-81313 (D.M.K.), NSF OCE 0001407 (R.M.L.), NASA Earth System Science Fellowship grant NNG05GP79H (A.E.W.), and the Gordon and Betty Moore Foundation's Initiative in Marine Microbiology (D.M.K.) provided support for this project.

Pacific gyres, where the process of biological N2 fixation has been documented to seasonally enhance the net transport of carbon (C) and nitrogen (N) out of the euphotic zone (Karl et al. 1997; Capone et al. 2005). Given that Trichodesmium-based productivity is by nature sto- chastic, a composite understanding of the role of this genus in elemental cycling will require characterization of physiological variability rather than just the biological averages. Specifically, if we can define the range of physiological and stoichiometric flexibility and begin to understand the underlying functionality driving deviations from average elemental composition, we will improve our capability to predict potential responses of oligotrophic marine ecosystems to the stresses imposed by a changing environment.

Assuming a fixed elemental stoichiometry for marine biota, e.g., the Redfield reference ratio (Redfield 1958), requires that the internal chemical content of cells is strictly regulated. From this perspective, variable stoichiometry must be the consequence of the modulation of the relative proportions or the governing transformation rates of intracellular pools of biomolecules. In the North Pacific Subtropical Gyre (NPSG), a fundamental characteristic of increased Trichodesmium productivity is a systematic alter- nation of the particulate and dissolved elemental pools relative to the canonical Redfield ratios (C06 : N16 : P1) occurring as a primary result of the excess production of NR through N2 fixation (Karl et al. 1997; Dore et al. 2002). Particularly during bloom periods, the stoichiometric

777

White et al.

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lines show Redfield PC: PP (106), PN: PP (16) stoichiometry, and the critical PN: PP ratio (PN: PPcritical = 50). Histograms for each axis are also shown. Field data conform to a normal distribution [p values for the Kolomogorov-Smirnov and Lilliefors tests for normality (Zar 1999) are not significant at the 0.05 level]. Laboratory data adhere to a log normal distribution.

to light (Fig. 6A, Table 1). In contrast, samples isolated from P-restricted growth remained viable for 5-6 d (Fig. 6B-C, Table 1). If we speculate that this coastal isolate is representative of Trichodesmium in blue water, our results indicate that migrating populations would be able to persist for a minimum period of 3 to 6 d in total darkness and perhaps longer in dim light.

The ability to survive and consume P, in dark, nutrient- rich environments, at rates sufficient to support growth does not confirm vertical migration as a source of P to the euphotic zone, but rather implies that there is potential for this process to occur. The actual feasibility of phosphocline-scale migrations depends on whether the rate of Trichodesmium density change is sufficient to facilitate vertical migration to and from the depth of the phosphocline. Direct determination of colony density, via density centrifugation measurements or some comparable method, along with measures of parameters relevant to Stokes equation (e.g., colony size), would more closely constrain the actual range of vertical velocities achievable by natural populations of Trichodesmium. These measures will be crucial to a precise understanding of the potential for phosphocline-scale vertical migration in natural populations. Alternately, vertical migration is likely advantageous because movement alone, whether up or down, may enhance P contact and facilitate increased P uptake relative to nonmotile cells.

Ecological implications-In the oligotrophic biomes of the world's oceans, Trichodesmium spp. are ubiquitous and

key for the addition of reactive N. For these reasons, and for their role in elemental cycling, there has been a substantial research effort devoted to understanding the ecology and physiology of this genus (see Karl et al. 2002 and references therein). Nonetheless, there are still many unanswered questions. In regions such as the NPSG where the system has been hypothesized to be in a period of NR sequestration and phosphorus control of plankton rate processes (Karl et al. 2001), the physiological plasticity of Trichodesmium can have a profound impact on the composition of particulate and dissolved elemental pools and, furthermore, may play a regulatory role in net export of particulate matter from the upper ocean.

In the laboratory portion of this study, we show that Trichodesmium is able to grow with significantly reduced intracellular quotas of phosphorus. Conversely, when exposed to P following periods of P restriction, cultured populations appear to engage in luxury consumption of P. In sum, this organism is able to appreciably alter its biochemical composition in response to environmental fluctuations in light and nutrient availability. Comparison of these results to stoichiometric observations of natural populations indicates that the extremes of biochemical composition generated in the laboratory are rarely observed in nature. However, analysis of field data establishes Trichodesmium spp. as a genus typically possessing low proportions of intracellular P relative to either C or N. The ability to maintain net productivity with reduced P quotas is clearly an adaptation to the oligotrophic habitat. Given that natural populations only rarely appear to be pushed

1788

•  Highlight the extreme deviaJons that can be seen in Trichodesmium C891: N125: P1

•  Low demand for P

•  Highlights the strong differences seen in lab extremes and field: Believes extreme stoichometric variaJon is limited by colimitaJon

LIMNOLOGY AND

OCEANOGRAPHY September 2003

Volume 48 Number 5

Limnol. Oceanogr., 48(5), 2003, 1721-1731 ? 2003. by the American Society of Limnology and Oceanography, Inc.

Elemental composition of marine Prochlorococcus and Synechococcus: Implications for the ecological stoichiometry of the sea S. Bertilsson' and 0. Berglund2 Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

D. M. Karl School of Ocean and Earth Science and Technology, University of Hawaii, Honolulu, Hawaii 96822

S. W. Chisholm Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Abstract The elemental composition of marine cyanobacteria is an important determinant of the ecological stoichiometry

in low-latitude marine biomes. We analyzed the cellular carbon (C), nitrogen (N), and phosphorus (P) contents of Prochlorococcus (MED4) and Synechococcus (WH8103 and WH8012) under nutrient-replete and P-starved conditions. Under nutrient-replete conditions, C, N, and P quotas (femtogram cell-') of the three strains were 46 ? 4, 9.4 ? 0.9, and 1.0 + 0.2 for MED4; 92 + 13. 20 + 3, and 1.8 ? 0.1 for WH8012; and 213 + 7, 50 + 2, 3.3 + 0.5 for WH8103. In P-limited cultures, they were 61 + 2, 9.6 ? 0.1, and 0.3 ? 0.1 for MED4; 132 ? 6, 21 + 2, and 0.5 ? 0.2 for WH8012; and 244 ? 21, 40 + 4, and 0.8 + 0.01 for WH8103. P limitation had no effect on the N cell quota of MED4 and WH8012 but reduced the N content of WH8103. The cellular C quota was consis- tently higher in P-limited than in nutrient-replete cultures. All three strains had higher C: P and N: P ratios than the Redfield ratio under both nutrient-replete and P-limited conditions. The C:N molar ratios ranged 5-5.7 in replete cultures and 7.1-7.5 in P-limited cultures; C:P ranged 121-165 in the replete cultures and 464-779 under P limitation; N:P ranged 21-33 in the replete cultures and 59-109 under P limitation. Our results suggest that Prochlorococcus and Synechococcus may have relatively low P requirements in the field, and thus the particulate organic matter they produce would differ from the Redfield ratio (106C: 16N: IP) often assumed for the production of new particulate organic matter in the sea.

The marine cyanobacterium Prochlorococcus is a small photosynthetic prokaryote (diameter, 0.5-0.8 /tm) that is

'Corresponding author ([email protected]). Present address: De- partment of Evolutionary Biology/Limnology, Uppsala University, SE-75236 Uppsala, Sweden.

2 Present address: Department of Ecology/Limnology, Lund Uni- versity, SE-223 62 Lund, Sweden.

Acknowledgments We thank John Waterbury for kindly making the axenic Syne-

chococcus strains available to us, Tom Gregory for skillful analysis of total P using the MRP method, Crystal Shih for laboratory work in a preliminary study of C:N:P in Prochlorococcus, and Lisa Moore and Stephanie Shaw for initial discussions on experimental design.

This research was supported in part by the K & A Wallenberg foundation (postdoctoral fellowships to S.B. and O.B.) and in part by the U.S. National Science Foundation (grants to S.W.C. and D.M.K.) and the Department of Energy (grant to S.W.C.).

ubiquitous in the euphotic zone of tropical and subtropical oceans (40?S-40?N; Chisholm et al. 1988; Partensky et al. 1999). Abundances in warm surface waters are typically > 105 cells ml-', and cells are found as deep as 200 m below the surface (Campbell et al. 1994; Partensky et al. 1999). These features make Prochlorococcus the numerically dominant oxygenic phototroph in the oceans. Its close relative, Synechococcus, is also important in subtropical ecosystems. Although concentrations are typically an order of magnitude lower than those of Prochlorococcus (Campbell et al. 1994; DuRand et al. 2001), their larger average cell size (diameters of 0.6-2.1 /xm; Herdman et al. 2001) makes them approximately equal in terms of global photosynthetic biomass. Sy- nechococcus cells are usually limited to the upper 100 m of the water column (Partensky et al. 1999) but have a broader latitudinal distribution than Prochlorococcus; they are not limited to warm oceanic waters but can be encountered at temperatures as low as 2?C (Shapiro and Haugen 1988).

1721

C:N:P in Prochlorococcus and Synechococcus 1723

l ooo Despite the apparent morphological similanties and close phylogenetic relationships, pigmentation is very different in the three strains; for example, Prochlorococcus lack phyco- biliproteins but instead synthesize divinyl-chlorophyll a and b (Partensky et al. 1999) and the Synechococcms strains differ in their relative content of phycouribilin and phycoery- thrin (Rocap 2002). Furthermore, Synechococcus WH8103 is motile, whereas the other two strains lack any form of swimming motility (Waterbury 1986; Rocap 2002).

All cultures were maintained at 22°C in constant light at 30-40 ,umol quanta m 2 S-l. Cool-white fluorescent lamps were used for all incubations. Batch cultures were grown in 25 ml Sargasso Sea-based medium added to 50-ml borosil- icate glass tubes with 25 mm inner diameter (Moore et al. 2002). Filter-sterilized and autoclaved seawater was amended with filter-sterilized stock solution of NH4Cl to a final concentration of 800 jumol L-', and a stock solution of NaH2PO4 was added to either 50 or 1 ,umol L-l to obtain media with molar N: P ratios of 16 and 800 respectively. A filter-sterilized trace-metal stock solution was added to the following final concentrations: 1.17 ,umol L-1 ethylene dia- minetetraacetic acid, 8 nmol L-l zinc, 5 nmol L-l cobalt, 90 nmol L-l manganese, 3 nmol L-' molybdenum, lO nmol L-l selenium 10 nmol L-' nickel, and 1.17 gmol L-l iron.

All cultures were acclimated to the iradiance conditions and the respective growth medium (P-replete and P-limited) for a minimum of three transfers before the start of the experiment. Eighteen replicate cultures were set up for each medium and organism combination, and the growth of each culture was monitored on a daily basis by noninvasive in vivo chlorophyll fluorescence using a Turner lOAU fluorom- eter (Turner Designs). The P-replete cultures were harvested during the midexponential growth phase (after 7-9 d), and the P-limited cultures were harvested during the early stationary growth phase (12-14 d) (Fig. 2). Nine replicate cultures (three pooled samples of three tubes) were collected on combusted Whatman GF/F filters (2 h at 450QC) for the analysis of particulate C and N, and six single cultures were harvested in a similar way for the analysis of particulate P. All filtrations were done under low vacuum (<10 kPa) in an acid-washed and rinsed filtration funnel made of glass. Additional filtrations with 25-75 ml of 0.2-,um-filtered media from the harvested cultures were done as controls for adsorption of dissolved nutrients to the Whatman GF/F filters. All filters were dried at 60°C (1-2 d) and stored in the dark in a desiccator until analysis. The three remaining cultures for each treatment were used to monitor growth of the various cultures after the end-point sampling for particulate C, N, and P.

To determine the number of cells captured on each filter, l-ml samples were collected before harvesting the cultures and in all filtrates for subsequent analysis by flow cytometry and epifluorescence microscopy. Samples were preserved in cryovials by first adding 0.2-,am-filtered glutaraldehyde to a final concentration of ().125Wo followed by rapid freezing and subsequent storage in liquid nitrogen. The filtration ef- ficiency (percentage of cells captured on filters) was >99No for Synechococcus WH8103, >97% for Synechococcus WH8012 and >95% for Prochlorococcus MED4, indicating a highly efficient harvesting of cells by low vacuum filtration

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Fig. 2. Growth curves for (A) Prochlorococcus MED4, (B) Sy- nechococcms WH8012 and (C) Synechococcms WH8103 based on in vivo chlorophyll fluorescence. Parallel growth curves are shown for cyanobacteria growing in P-replete (squares) and P-limited (tri- angles) medium. Black symbols indicate fluorescence of cultures used for CNP analysis (n- 15), and white symbols indicate fluorescence of triplicate cultures used for monitoring growth after harvesting cells for CNP analysis. Error bars represent the standard deviation (often hidden in symbol). Cultures used for CNP analyses were harvested after 4-12 d, as indicated by the disappearance of the solid symbols in the graphs. The triplicate cultures used to monitor growth in the P-limited cultures were amended with P during the late stationary growth phase, to assess P limitation. The growth rate in the exponential growth phase (indicated by brackets) is shown for each treatment (r2 > 0.97, n > 4).

C:N: P in Prochlorococcus and Synechococcus

Table 3. Elemental ratios (molar C:N, C:P, and N:P) for marine phytoplankton cultures, bacteria, and particulate organic matter. Arithmetic means are presented, with standard deviation between brackets.

Sample C:N C:P N: P Reference Prochlorococcus Med4

P-replete 5.7 (0.7) 121 (17) 21.2 (4.5) This study P-limited 7.4 (0.2) 464 (28) 62.3 (14.1)

Synechococcus WH8012 P-replete 5.4 (1.1) 130 (19) 24.0 (3.6) This study P-limited 7.5 (0.8) 723 (61) 96.9 (37)

Synechococcus WH8103 P-replete 5.0 (0.2) 165 (8) 33.2 (5.2) This study P-limited 7.1 (0.9) 779 (122) 109 (10.5)

Exponential* Prochlorococcus (6 strains) 8.5-9.9 156-215 15.9-24.4 Heldal et al. 2003

Exponential* Synechococcus WH8103 10 150 15 Heldal et al. 2003 WH7803 8.9 113 13.3

Exponential Synechococcus WH7803 7 115 16.4 Cuhel and Waterbury 1984

Marine phytoplankton culturest Average 7.7 (2.6) 75 (31) 10.1 (3.9) Geider and La Roche 2002 Range 4-17 27-135 5-19

Marine particulate mattert Average 7.3 (1.7) 114 (45) 16.4 (6.2) Geider and La Roche 2002 Range 3.8-12.5 35-221 5-34

Marine bacterial cultures Exponential 3.8-6.3 26-50 5.5-7.9 Vrede et al. 2002 P-limited 7.7-12 176-180 16-19

* Cells cultured in PCR-S11 medium (Partensky et al. 1999), sampled in exponential phase. t Meta-analysis of nutrient-replete eukaryotic marine phytoplankton cultures. t Meta-analysis of marine particulate matter.

oceanic environments (Indian Ocean, Atlantic Ocean, South- ern Ocean, and Mediterranean Sea) demonstrated an average C:P ratio of 103 and an average N:P ratio of 16.4, even though there was a considerable variability between the different sites (Copin-Montegut and Copin-Montegut 1983). Hence, it appears that either marine picocyanobacteria are not well represented in the analyzed particulate fractions or that other organisms or particles rich in P counterbalance the signal from the low-P cyanobacteria. Both of these expla- nations are possible, but neither seems likely for the vast low-nutrient open-ocean gyres that dominate our planet.

First, particulate matter is an operational definition, and most analyses of particulate C: N: P ratios have used filters with nominal pore sizes of 0.7-1.2 /Lm, which may be more or less efficient in capturing the smaller unicellular micro- organisms such as Prochlorococcus and Synechococcus. In agreement with earlier studies (Chavez et al. 1995), our results show that gentle filtration through a glass-fiber filter with a nominal pore size of 0.7 gxm efficiently captures even small Prochlorococcus cells (diameter, 0.7 /urm).

Second, next to marine picocyanobacteria, heterotrophic prokaryotes represent a dominant biomass component of marine food webs (Campbell et al. 1994; Gasol et al. 1997). Studies of indigenous freshwater bacterioplankton and cul-

tured aquatic bacteria have suggested that these organisms may be enriched in phosphorus (Table 3; Fagerbakke et al. 1996; Vrede et al. 2002), which suggests that they would be able to counterbalance the high C: P and N: P ratios in cyanobacteria. This hypothesis is supported by the often tight spatial and temporal coupling between these two groups of organisms in oceanic systems (Li et al. 1992); for example, cyanobacteria and other primary producers supply the energy and nutrients required for heterotrophic growth of bacteria, "the microbial loop" (Azam et al. 1983). However, a recent study of indigenous aquatic bacteria collected in the Sargasso Sea (Gundersen et al. 2002) suggested that the cellular ratios of C:N:P in marine bacteria are close to the Redfield ratio of 106:16:1, and, if these ratios are representative for bacterioplankton in a wider range of open- ocean systems, the balancing effect would be minimal.

The cellular C: N in the nutrient-replete cultures of Pro- chlorococcus and Synechococcus varied between 5 and 5.7 (Table 3). These ratios are within the observed range of C: N for nutrient-replete cultures of marine eukaryotic phytoplankton (Table 3) and are also consistent with reported values of C: N in two different marine Svnechococcus strains (range, 4.4-8.7; Biddanda and Benner 1997; Lourenco et al. 1998). The largest proportion of cellular N in phytoplankton

1727

LIMNOLOGY AND


Volume 48 Number 5














1721

•  Over half the phosphate is Jed up in the DNA •  P replete cultures are dividing quickly and operaJng with low levels of carbon

•  P limited cultures much more carbon and nitrogen dense

•  Discusses the value of slow growth and low proporJons of geneJc machinery to surviving oligotrophic condiJons and phage

•  Cyanobacteria seem to have a disJnct low phosphate signature

•  Findings conflict with marine parJculate ma5er studies

•  BerJlsson refers to Copin-‐Montegut 1983 but also seen in Frigstad

LIMNOLOGY AND


Volume 48 Number 5














1721

BGD8, 6227–6263, 2011


stoichiometry

H. Frigstad et al.

Title Page



Tables Figures

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Fig. 10. Calculated N:P, C:P and C:N of live autotrophs for Arendal and Jomfruland with linesshowing annual mean ratio and the Redfield ratios. Error bars show the SE.

6262

Divergence in the Picoplankton

•  Stoichiometric variability exists across the microbial community

•  Includes funcJonal differences (auto and heterotrophs)

•  Within classificaJons of autotrophic cyanobacteria •  And even within a genus as a result of nutrient limitaJon

Moving Microbial Stoichiometry Up the Food Chain




Growth Phase and Elemental Stoichiometry of Bacterial Prey Influences CiliateGrazing Selectivity

DAVID F. GRUBER,a,b STEVEN TUORTOa and GARY L. TAGHONa

aInstitute of Marine and Coastal Sciences, Rutgers, The State University of New Jersey, 71 Dudley Road, New Brunswick, New Jersey 08901,and

bDepartment of Natural Sciences, City University of New York, Baruch College, P. O. Box A-0506, 17 Lexington Avenue, New York,New York 10010

ABSTRACT. Protozoa are known to selectively graze bacteria and can differentiate prey based on size and viability, but less is knownabout the effects of prey cellular composition on predator selectivity. We measured the effect of growth phase and elemental stoichiometryof Escherichia coli on grazing by two ciliates, Euplotes vannus and Cyclidium glaucoma. Bacterial cells of a single strain were trans-formed with green and red fluorescent protein and harvested from culture at differing growth stages. Cells in exponential growth phase hadlow carbon:phosphorus (39) and nitrogen:phosphorus (9) ratios, while cells from stationary phase had high carbon:phosphorus of 104 andnitrogen:phosphorus of 26. When offered an equal mixture of both types of bacteria, Cyclidium grazed stationary phase, high car-bon:phosphorus, high nitrogen:phosphorus cells to 22% of initial abundance within 135 min, while Euplotes reduced these cells to 33%.Neither ciliate species decreased the abundance of the exponential phase cells, lower carbon:phosphorus and nitrogen:phosphorus, relativeto control treatments. Because protozoa have higher nitrogen:phosphorus and carbon:phosphorus ratios than their prokaryotic prey, thisstudy raises the possibility that it may be advantageous for protozoa to preferentially consume more slowly growing bacteria.

Key Words. Bacteria growth phase, microbial loop protozoa grazing, selective grazing.

PROTOZOA play a major ecological role in aquatic environ-ments due to their effectiveness in consuming a wide range of

prey size classes and types, which include other protozoa, phyto-plankton, and bacteria (Azam et al. 1983; Fenchel 1980a; Porter etal. 1985; Sherr and Sherr 2002; Sherr, Sherr, and Pedros-Alio1989). Grazing by bacterivorous protozoa is of considerable im-portance given that protozoa not only constitute a major source ofmortality to both heterotrophic and autotrophic bacteria (Cole1999; Pace 1986), but they are also food for other zooplankton,serving an important link in the microbial web (Kiorboe 1998;Sherr and Sherr 2002, 2007; Stoecker and Capuzzo 1990). Bac-terivory has been demonstrated to strongly influence prokaryoticdiversity and alter genotypic, phenotypic, and metabolic compo-sition of bacterial communities (Hahn and Hofle 2001; Hahn,Moore, and Hofle 1999; Jurgens and Matz 2002; Matz and Jurgens2003; Matz et al. 2002b; Simek et al. 1997); viruses also play animportant role in bacterial community composition (Fuhrman1999; Fuhrman and Noble 1995; Herndl et al. 2005). While wedo not address viruses in this study, an understanding of themechanisms that influence grazing by phagotrophic protozoa isimportant in determining how bacterivory influences microbialfood webs.

Grazing is most often quantified by measuring changes in bac-terial abundance over time (Enzinger and Cooper 1976; McCam-bridge and McMeekin 1980) or by monitoring grazing rates onbacterial analogs, mainly fluorescent microspheres or fluorescent-ly labeled bacteria (Borsheim 1984; McManus and Fuhrman1988; Sherr, Sherr, and Fallon 1987). The use of bacterial ana-logs in microbial grazing studies has multiple limitations. Fluo-rescently labeled bacteria are often non-viable cells that have beenchemically modified by the staining process in ways that haveunknown effects on their detection by protozoa and their palat-ability as prey. Also, microspheres are commonly composed ofplastic microbeads with no food value to consumers. This could beproblematic as predators have been reported to discriminate be-tween motile vs. non-motile (Gonzalez, Sherr, and Sherr 1993)and live vs. dead prey (Landry et al. 1991). Molecular approaches,

such as length heterogeneity analysis by PCR (Suzuki 1999), havealso been used to assess protistan bacterivory of filtered and non-filtered seawater samples.

There are multiple factors that can influence grazing. Growthrates of both ciliates (Taylor and Berger 1976) and flagellates(Sherr, Sherr, and Berman 1983) have been demonstrated to varydepending on the species of bacteria grazed. Different bacterialspecies are grazed at varying efficiencies (Mitchell, Baker, andSleigh 1988). In addition, protozoa actively select bacteria basedon various prey attributes, such as cell size and cell surface prop-erties (Gurijala and Alexander 1990; Monger, Landry, and Brown1999; Sanders 1988; Tso and Taghon 1999).

The physical and chemical properties of prey and their role inselective grazing have been extensively studied in phytoplanktonand metazoan zooplankton (DeMott 1995), but less so in bacteriaand protozoa (Matz, Deines, and Jurgens 2002a). Protozoan se-lective feeding appears to be an important mechanism determin-ing community structure of planktonic food webs and influencingrates of organic matter remineralization and nutrient cycling. Thepurpose of this study was to examine if bacterivorous protozoaselectively graze prey based on the growth phase of bacterial cellswithin a population containing a single species of bacteria. Wealso measured C:N:P stoichiometry, cell dimensions, and proteincontent of the bacteria to determine what attributes may causeselection, so that inferences can be made regarding the nutritionalvalue of bacteria at different growth phases. To accomplish this,we utilized bacterial prey that were labeled with either red fluo-rescent protein (RFP) or green fluorescent protein (GFP), to differ-entiate between growth phases. The use of GFP to label bacteria ingrazing studies has been shown to be a successful method to mea-sure bacterial grazing without using an external stain (Ishii et al.2002), and this is the first study to employ several colored fluo-rescent proteins to examine selective protozoa grazing.

MATERIALS AND METHODS

Bacteria containing plasmids for RFP and GFP. A singleEscherichia coli strain was transformed with a gene for RFP. Thisfluorescent protein was originally cloned from the corallimorphDiscosoma sp. (Matz et al. 1999). The RFP coding sequence frompDsRed (Clontech, Palo Alto, CA) was excised by PvuII/StuI di-gestion (positions 55 to 1,041), digested with alkaline phospha-tase, and the resulting 986-bp fragment was inserted into vector

Corresponding Author: David F. Gruber, Department of Natural Sci-ences, City University of New York, Baruch College, 17 LexingtonAvenue, New York, New York 10010—Telephone number: 646 6606236; FAX number: 646 660 6201; e-mail: [email protected]

466

J. Eukaryot. Microbiol., 56(5), 2009 pp. 466–471r 2009 The Author(s)Journal compilation r 2009 by the International Society of ProtistologistsDOI: 10.1111/j.1550-7408.2009.00428.x

•  Grazers: Euplotes and Cyclidium •  Grazed: Fluorescently labeled E. coli

–  Glowing Green 8 and 72 hours Glowing Red 72 and 72 hours –  8: 39:9:1 72: 104:26:1

Growth Phase and Elemental Stoichiometry of Bacterial Prey Influences CiliateGrazing Selectivity

DAVID F. GRUBER,a,b STEVEN TUORTOa and GARY L. TAGHONa

aInstitute of Marine and Coastal Sciences, Rutgers, The State University of New Jersey, 71 Dudley Road, New Brunswick, New Jersey 08901,and

bDepartment of Natural Sciences, City University of New York, Baruch College, P. O. Box A-0506, 17 Lexington Avenue, New York,New York 10010

ABSTRACT. Protozoa are known to selectively graze bacteria and can differentiate prey based on size and viability, but less is knownabout the effects of prey cellular composition on predator selectivity. We measured the effect of growth phase and elemental stoichiometryof Escherichia coli on grazing by two ciliates, Euplotes vannus and Cyclidium glaucoma. Bacterial cells of a single strain were trans-formed with green and red fluorescent protein and harvested from culture at differing growth stages. Cells in exponential growth phase hadlow carbon:phosphorus (39) and nitrogen:phosphorus (9) ratios, while cells from stationary phase had high carbon:phosphorus of 104 andnitrogen:phosphorus of 26. When offered an equal mixture of both types of bacteria, Cyclidium grazed stationary phase, high car-bon:phosphorus, high nitrogen:phosphorus cells to 22% of initial abundance within 135 min, while Euplotes reduced these cells to 33%.Neither ciliate species decreased the abundance of the exponential phase cells, lower carbon:phosphorus and nitrogen:phosphorus, relativeto control treatments. Because protozoa have higher nitrogen:phosphorus and carbon:phosphorus ratios than their prokaryotic prey, thisstudy raises the possibility that it may be advantageous for protozoa to preferentially consume more slowly growing bacteria.

Key Words. Bacteria growth phase, microbial loop protozoa grazing, selective grazing.

PROTOZOA play a major ecological role in aquatic environ-ments due to their effectiveness in consuming a wide range of

prey size classes and types, which include other protozoa, phyto-plankton, and bacteria (Azam et al. 1983; Fenchel 1980a; Porter etal. 1985; Sherr and Sherr 2002; Sherr, Sherr, and Pedros-Alio1989). Grazing by bacterivorous protozoa is of considerable im-portance given that protozoa not only constitute a major source ofmortality to both heterotrophic and autotrophic bacteria (Cole1999; Pace 1986), but they are also food for other zooplankton,serving an important link in the microbial web (Kiorboe 1998;Sherr and Sherr 2002, 2007; Stoecker and Capuzzo 1990). Bac-terivory has been demonstrated to strongly influence prokaryoticdiversity and alter genotypic, phenotypic, and metabolic compo-sition of bacterial communities (Hahn and Hofle 2001; Hahn,Moore, and Hofle 1999; Jurgens and Matz 2002; Matz and Jurgens2003; Matz et al. 2002b; Simek et al. 1997); viruses also play animportant role in bacterial community composition (Fuhrman1999; Fuhrman and Noble 1995; Herndl et al. 2005). While wedo not address viruses in this study, an understanding of themechanisms that influence grazing by phagotrophic protozoa isimportant in determining how bacterivory influences microbialfood webs.

Grazing is most often quantified by measuring changes in bac-terial abundance over time (Enzinger and Cooper 1976; McCam-bridge and McMeekin 1980) or by monitoring grazing rates onbacterial analogs, mainly fluorescent microspheres or fluorescent-ly labeled bacteria (Borsheim 1984; McManus and Fuhrman1988; Sherr, Sherr, and Fallon 1987). The use of bacterial ana-logs in microbial grazing studies has multiple limitations. Fluo-rescently labeled bacteria are often non-viable cells that have beenchemically modified by the staining process in ways that haveunknown effects on their detection by protozoa and their palat-ability as prey. Also, microspheres are commonly composed ofplastic microbeads with no food value to consumers. This could beproblematic as predators have been reported to discriminate be-tween motile vs. non-motile (Gonzalez, Sherr, and Sherr 1993)and live vs. dead prey (Landry et al. 1991). Molecular approaches,

such as length heterogeneity analysis by PCR (Suzuki 1999), havealso been used to assess protistan bacterivory of filtered and non-filtered seawater samples.

There are multiple factors that can influence grazing. Growthrates of both ciliates (Taylor and Berger 1976) and flagellates(Sherr, Sherr, and Berman 1983) have been demonstrated to varydepending on the species of bacteria grazed. Different bacterialspecies are grazed at varying efficiencies (Mitchell, Baker, andSleigh 1988). In addition, protozoa actively select bacteria basedon various prey attributes, such as cell size and cell surface prop-erties (Gurijala and Alexander 1990; Monger, Landry, and Brown1999; Sanders 1988; Tso and Taghon 1999).

The physical and chemical properties of prey and their role inselective grazing have been extensively studied in phytoplanktonand metazoan zooplankton (DeMott 1995), but less so in bacteriaand protozoa (Matz, Deines, and Jurgens 2002a). Protozoan se-lective feeding appears to be an important mechanism determin-ing community structure of planktonic food webs and influencingrates of organic matter remineralization and nutrient cycling. Thepurpose of this study was to examine if bacterivorous protozoaselectively graze prey based on the growth phase of bacterial cellswithin a population containing a single species of bacteria. Wealso measured C:N:P stoichiometry, cell dimensions, and proteincontent of the bacteria to determine what attributes may causeselection, so that inferences can be made regarding the nutritionalvalue of bacteria at different growth phases. To accomplish this,we utilized bacterial prey that were labeled with either red fluo-rescent protein (RFP) or green fluorescent protein (GFP), to differ-entiate between growth phases. The use of GFP to label bacteria ingrazing studies has been shown to be a successful method to mea-sure bacterial grazing without using an external stain (Ishii et al.2002), and this is the first study to employ several colored fluo-rescent proteins to examine selective protozoa grazing.

MATERIALS AND METHODS

Bacteria containing plasmids for RFP and GFP. A singleEscherichia coli strain was transformed with a gene for RFP. Thisfluorescent protein was originally cloned from the corallimorphDiscosoma sp. (Matz et al. 1999). The RFP coding sequence frompDsRed (Clontech, Palo Alto, CA) was excised by PvuII/StuI di-gestion (positions 55 to 1,041), digested with alkaline phospha-tase, and the resulting 986-bp fragment was inserted into vector

Corresponding Author: David F. Gruber, Department of Natural Sci-ences, City University of New York, Baruch College, 17 LexingtonAvenue, New York, New York 10010—Telephone number: 646 6606236; FAX number: 646 660 6201; e-mail: [email protected]

466

J. Eukaryot. Microbiol., 56(5), 2009 pp. 466–471r 2009 The Author(s)Journal compilation r 2009 by the International Society of ProtistologistsDOI: 10.1111/j.1550-7408.2009.00428.x

declined significantly in the Euplotes treatment (t9.5 5 5.36,P 5 0.0004), to 33% of initial abundance (Table 1).

In the experiment where both E. coli-GFP and E. coli-RFP wereharvested after 72 h, the C:N:P (mean ! 95% confidence level) ofE. coli-GFP was 114 (! 13):28 (! 3.1):1 (! 0.2), not significantlydifferent than that of E. coli-RFP, 104 (! 2.3):25 (! 0.6):1 (! 0.1). Initial abundance was 1.32E106 (! 6.5E104) ml" 1

for E. coli-GFP and 1.25E106 (! 5.7E104) ml" 1 for E. coli-RFP. The abundance of E. coli-GFP declined significantly in theCyclidium treatment (t18 5 3.05, P 5 0.0068) to 76% of initialabundance (Fig. 2). The abundance of E. coli-GFP also declinedsignificantly in the Euplotes treatment (t13.6 5 8.80, Po0.00001)to 27% of initial abundance. The abundance of E. coli-RFP de-clined significantly in the Cyclidium treatment (t18 5 3.46,P 5 0.0028) to 68% of initial abundance. The abundance of E.coli-RFP also declined significantly in the Euplotes treatment(t13.5 5 7.88, Po0.00001) to 29% of initial abundance.

There were no significant differences in cell length, width, andvolume (P 5 0.79, 0.52, and 0.67, respectively) between preytypes at either growth stage (Table 2). Protein content was sub-stantially higher after 8 h of growth compared with 72 h for bothprey types (GFP, P 5 0.0001; RFP, P 5 0.0023). When compar-ing prey types, there was no significant difference in protein con-

tent between the two at 72 h (P 5 0.51), while protein wassignificantly higher for E. coli-GFP than for E. coli-RFP at 8 h(P 5 0.0097).

DISCUSSION

It is well documented that bacterivorous protozoa selectivelygraze based on cell size of bacteria (Gonzalez, Sherr, and Sherr1990; Posch et al. 2001). While size-based selectivity has receivedthe most attention, ciliates and flagellates use additional criteria todiscriminate among prey, such as prey mobility (Gonzalez et al.1993), cell surface hydrophobicity (Monger et al. 1999), and C:Nratio (John and Davidson 2001; Tso and Taghon 1999). Little re-search has been done on how growth-phase elemental stoichiome-try of prokaryotic prey impacts protozoa grazing selection.

Generally, larger bacterial cells are more rapidly grazed by cil-iates (Epstein and Shiaris 1992; Gonzalez et al. 1990; Simek andChrzanowski 1992). Ciliates have a maximum, minimum, andoptimal prey-size range depending solely on their anatomicalfeeding structure (Fenchel 1980b; Verni and Gualtieri 1997). Ahigher contact probability for larger particles has been assumed tobe a primary mechanism for prey selectivity (Fenchel 1987) withmorphological limitations of feeding also recognized as an im-portant determinant (Boenigk and Arndt 2000). Sherr, Sherr, andMcDaniel (1992) theorized that selective grazing of dividing cellsmay be an important factor in maintaining taxonomic and meta-bolic diversity within bacterioplankton assemblages. Because thelarger-sized bacteria are under greater grazing pressure and thelarger cells in the assemblage are mainly cells that are activelygrowing and dividing (Krambeck and Krambeck 1984), it hasbeen indirectly surmised that protozoa are selectively grazing onthe faster growing cells (Gonzalez et al. 1990; Krambeck 1988;Sherr et al. 1992) and are cropping the production rather than thestanding stock of suspended bacteria. Cell dimensions were sim-ilar for all prey types in the present study. Therefore, size was notconsidered a selective factor.

The data from our study indicate that prey size and species be-ing equal, protozoa selectively graze the older standing stock (i.e.not actively growing), rather than the faster-growing cells. Re-moval of non-growing bacterial stock is possibly a mechanismthat stimulates activity of the actively growing bacteria by less-ening competition for resources, such as space and nutrients, andkeeping the population in a state of perpetual youth. This is in

Fig. 1. Change in bacterial abundance in the grazing experimentwhere Escherichia coli-GFP was harvested after 8 h and E. coli-RFP af-ter 72 h. Symbols are the average for each treatment, error bars are 95%confidence levels. Vertical dashed line denotes no change in abundance.GFP, green fluorescent protein; RFP, red fluorescent protein.

Table 1. Initial and final abundance of bacteria (# 106 cells/ml) forboth the grazer, as well as the control treatments in both experiments.

Treatment E. coli-GFP E. coli-RFP

Initial Final 1 Initial Final 1

E. coli-GFP harvested after 8 h and E. coli-RFP after 72 hControl 1.26 (0.209) 1.24 (0.156) 1.16 (0.214) 1.15 (0.332)Cyclidium sp. 1.19 (0.163) 1.16 (0.346) 1.08 (0.173) 0.237 (0.088)E. vannus 1.28 (0.137) 1.26 (0.116) 1.09 (0.121) 0.361 (0.123)

E. coli-GFP and E. coli-RFP harvested after 72 hControl 1.29 (0.205) 1.29 (0.169) 1.31 (0.165) 1.23 (0.183)Cyclidium sp. 1.41 (0.154) 1.08 (0.136) 1.29 (0.185) 0.881 (0.184)E. vannus 1.25 (0.124) 0.334 (0.065) 1.15 (0.125) 0.336 (0.044)

Data are means for n 5 10 (SD).E. coli, Escherichia coli; E. vannus, Euplotes vannus.

Fig. 2. Change in bacterial abundance in the grazing experimentwhere both Escherichia coli-GFP and E. coli-RFP were harvested after72 h. Symbols are the average for each treatment, error bars are 95% con-fidence levels. Vertical dashed line denotes no change in abundance. GFP,green fluorescent protein; RFP, red fluorescent protein.

468 J. EUKARYOT. MICROBIOL., 56, NO. 5, SEPTEMBER–OCTOBER 2009

72 hr

72 hr

72 hr

8 hr

•  Marine nutrients •  Controls on nutrient cycling •  Role of microbial taxa maybe something on parJculate Red field raJos

•  PredaJon on microbial communiJes •  Cyanobacteria and how there red field raJo is different

•  Difference influences predaJon •  Ecological role •  Control on nutrient cycling

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Iron and growth in Trichodesmium spp.

Table 2. Fe: C: N: P for Trichodesmium from the north coast of Australia. All samples collected in November 1999; date indicates day of month. Paired samples for Fe:P and C:N were collected (see text) in duplicate in most cases, except on 17 November 1999 (n = 3) and where no standard deviation is reported (n = 1).

Latitude Fe: C (SD) Fe: P (SD) C: P (SD) N: P (SD) Date collections Longitude (/,mol: mol) (mmol: mol) (mol: mol) (mol: mol)

Zodiac 1 11?12.75'S 139?19.47'E 67.5 (5.0) 1.06 (0.09) 158 (25) 21(7) 2 10?32.73'S 136?34.15'E 29.6 (2.1) 0.69 (0.07) 235 (7) 33 (1) 5 11?49.82'S 128?42.64'E 28 (11) 0.51 253 38 6 12?49.22'S 125?53.54'E 78 (13) 1.58 (0.19) 203 (8) 31.6 (0.5) 8 14?06.22'S 123?09.46'E 21.4 0.23 105 14

12 14?47.22'S 122?10.31'E 27.9 0.32 115 20 13 13?33.57'S 124?31.13'E 18.5 0.14 79 17 14 12?43.43'S 127?33.64'E 18.0 (3.4) 0.29 (0.06) 159 (2) 28 (1) 17 12?04.65'S 128?56.72'E 24.0 (4.1) 0.74 (0.13) 310 (20) 52 (7) 19 9049.60'S 130?06.69'E 40.0 (8.5) 0.79 232 36

Mean (SE) 35 (21) 0.64 (0.44) 185 (74) 29 (12) R/V Ewing collections

2 10?32.73'S 136?34.15'E 143 82) 6 12?49.22'S 125053.54'E 222

quantities of cellular iron in excess of that needed to support maximum growth (Figs. 2B, 3A). This ability for luxury uptake may be an important adaptive strategy for Tricho- desmium in near-surface oceanic waters, where iron inputs are often sporadic. The majority of the Fe flux to the open ocean can be supplied by aeolian dust deposition (i.e., calculated as 95% at a station in the N. Pacific Gyre; Martin and Gordon 1988), which is highly seasonal in the North Pacific and North Atlantic Oceans. Superposed on this broad seasonality are episodic dust deposition events. For example, at Midway Island, about 50% of the annual deposition of iron to the mixed layer occurred during a 3-week period (Uematsu et al. 1985). In contrast, the flux of iron due to upwelling in high nutrient low chlorophyll (HNLC) areas is believed to be less sporadic. In oligotrophic waters, luxury uptake of Fe may confer a selective advantage to Tricho- desmium to help offset the high Fe demand of diazotrophy.

All of the Fe:C ratios measured from the north coast of Australia were at least 18 utmol mol-~ (Table 2), in concor- dance with our diazotrophic culture data showing a main- tenance total cellular Fe:C of -16 /umol mol-~ (Fig. 4). This quantity is estimated from the intracellular iron value divided by the ratio of intracellular to total cellular iron (i.e., 13.5 /umol:mol divided by 0.83). About half of the populations sampled on this cruise had Fe: C contents below the 38 ,/mol mol-~ Fe:C required for a moderately Fe-limited diazotrophic growth rate in cultures of 0.1 d- (Fig. 4). A comparison of the growth rate versus Fe:C relationship measured in our diazotrophic cultures with the observed relationship between N2 fixation rates and colony Fe: C in field populations suggests that iron may be limiting in these populations. Some in situ Trichodesmium Fe: C values exceed those required by cultures to attain identical rates of N2 fixation. This observation has at least two possible explana- tions. The field populations may have stored excess Fe and were not limited by this nutrient at the time of collection. The Fe:C ratio is regulated by the balance between iron

uptake and growth, and, thus, populations can achieve high Fe: C ratios during periods of low specific growth rate due to other limiting factors. This additional iron can then be used to support diazotrophy once other limitations subside. Alternately, the extracellular contributions of Fe toward the field Fe: C contents are unknown, suggesting that there may be physiological limitation despite the apparent excess of measured Fe.

The Fe: C ratios in Trichodesmium reported here are con- siderably lower than previous reports for this region (Ber- man-Frank et al. 2001). Although we cannot offer a conclu- sive explanation for these differences, the higher previously measured values may have resulted from iron contamination, a potential problem in any iron analyses of natural marine samples (Martin and Knauer 1973; Collier and Edmond 1984). The clean techniques we used should have decreased, and hopefully eliminated, contamination by Fe. The results from our shipboard collections (during which we did not employ any special precautions, except that colonies were picked in the clean hood and digested in our clean lab) support this supposition.

There is a general consistency between the effects of added iron in our field incubation experiments and the measured Fe: C of natural populations. The enhancement of N2 fixation in Fe-amended treatments (Fig. 6C) relative to controls from the 8 November 1999 incubations is consistent with the low Trichodesmium Fe: C ratio (Table 2) and dissolved Fe at this station. The lack of an iron effect in the 6 November 1999 incubations (Fig. 6A) is also consistent with the high colony Fe: C content and dissolved Fe at this station. However, these incubation results do not prove iron limitation in the ambient 8 November population. First, F,, F, at t = 1.5 d was not significantly different among treatments; only after an additional 2 d did the control (no Fe added) incubations show a decline in F,IF,, (Fig. 6D). These results could suggest that iron was not initially limiting in the control but rather became limiting with time

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C:N: P in Prochlorococcus and Synechococcus

Table 3. Elemental ratios (molar C:N, C:P, and N:P) for marine phytoplankton cultures, bacteria, and particulate organic matter. Arithmetic means are presented, with standard deviation between brackets.

Sample C:N C:P N: P Reference Prochlorococcus Med4

P-replete 5.7 (0.7) 121 (17) 21.2 (4.5) This study P-limited 7.4 (0.2) 464 (28) 62.3 (14.1)

Synechococcus WH8012 P-replete 5.4 (1.1) 130 (19) 24.0 (3.6) This study P-limited 7.5 (0.8) 723 (61) 96.9 (37)

Synechococcus WH8103 P-replete 5.0 (0.2) 165 (8) 33.2 (5.2) This study P-limited 7.1 (0.9) 779 (122) 109 (10.5)

Exponential* Prochlorococcus (6 strains) 8.5-9.9 156-215 15.9-24.4 Heldal et al. 2003

Exponential* Synechococcus WH8103 10 150 15 Heldal et al. 2003 WH7803 8.9 113 13.3

Exponential Synechococcus WH7803 7 115 16.4 Cuhel and Waterbury 1984

Marine phytoplankton culturest Average 7.7 (2.6) 75 (31) 10.1 (3.9) Geider and La Roche 2002 Range 4-17 27-135 5-19

Marine particulate mattert Average 7.3 (1.7) 114 (45) 16.4 (6.2) Geider and La Roche 2002 Range 3.8-12.5 35-221 5-34

Marine bacterial cultures Exponential 3.8-6.3 26-50 5.5-7.9 Vrede et al. 2002 P-limited 7.7-12 176-180 16-19

* Cells cultured in PCR-S11 medium (Partensky et al. 1999), sampled in exponential phase. t Meta-analysis of nutrient-replete eukaryotic marine phytoplankton cultures. t Meta-analysis of marine particulate matter.

oceanic environments (Indian Ocean, Atlantic Ocean, South- ern Ocean, and Mediterranean Sea) demonstrated an average C:P ratio of 103 and an average N:P ratio of 16.4, even though there was a considerable variability between the different sites (Copin-Montegut and Copin-Montegut 1983). Hence, it appears that either marine picocyanobacteria are not well represented in the analyzed particulate fractions or that other organisms or particles rich in P counterbalance the signal from the low-P cyanobacteria. Both of these expla- nations are possible, but neither seems likely for the vast low-nutrient open-ocean gyres that dominate our planet.

First, particulate matter is an operational definition, and most analyses of particulate C: N: P ratios have used filters with nominal pore sizes of 0.7-1.2 /Lm, which may be more or less efficient in capturing the smaller unicellular micro- organisms such as Prochlorococcus and Synechococcus. In agreement with earlier studies (Chavez et al. 1995), our results show that gentle filtration through a glass-fiber filter with a nominal pore size of 0.7 gxm efficiently captures even small Prochlorococcus cells (diameter, 0.7 /urm).

Second, next to marine picocyanobacteria, heterotrophic prokaryotes represent a dominant biomass component of marine food webs (Campbell et al. 1994; Gasol et al. 1997). Studies of indigenous freshwater bacterioplankton and cul-

tured aquatic bacteria have suggested that these organisms may be enriched in phosphorus (Table 3; Fagerbakke et al. 1996; Vrede et al. 2002), which suggests that they would be able to counterbalance the high C: P and N: P ratios in cyanobacteria. This hypothesis is supported by the often tight spatial and temporal coupling between these two groups of organisms in oceanic systems (Li et al. 1992); for example, cyanobacteria and other primary producers supply the energy and nutrients required for heterotrophic growth of bacteria, "the microbial loop" (Azam et al. 1983). However, a recent study of indigenous aquatic bacteria collected in the Sargasso Sea (Gundersen et al. 2002) suggested that the cellular ratios of C:N:P in marine bacteria are close to the Redfield ratio of 106:16:1, and, if these ratios are representative for bacterioplankton in a wider range of open- ocean systems, the balancing effect would be minimal.

The cellular C: N in the nutrient-replete cultures of Pro- chlorococcus and Synechococcus varied between 5 and 5.7 (Table 3). These ratios are within the observed range of C: N for nutrient-replete cultures of marine eukaryotic phytoplankton (Table 3) and are also consistent with reported values of C: N in two different marine Svnechococcus strains (range, 4.4-8.7; Biddanda and Benner 1997; Lourenco et al. 1998). The largest proportion of cellular N in phytoplankton

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•  Also saw how low P abundance was different from other autotrophic communiJes

Stoichiometry of the Microbial Loop: J. Matthew Haggerty

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