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Processes in nutrient based phytoplankton ecology Turpin, David Howard 1980

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PROCESSES IN NUTRIENT BASED PHYTOPLANKTON ECOLOGY by DAVID HOWARD TURPIN B.Sc. , The Univ e r s i t y of B r i t i s h Columbia, 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES i n the DEPARTMENT OF BOTANY and the DEPARTMENT OF OCEANOGRAPHY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Apr i l " 1980-c ) David Howard Turpin, 1980 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced d e g r e e at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS -6 i i ABSTRACT Fluctuations i n the free i n t r a c e l l u l a r amino acid pools following an ammonia perturbation to ammonium l i m i t e d Skeletonema costatum and Gymnodiniwn simplex provides evidence which suggests that the enzyme glutamine synthetase (EC.6.3.1.2) acts as the primary ammonium a s s i m i l a t i n g enzyme i n marine phyto-plankton under nitrogen l i m i t a t i o n . L i m i t i n g nutrient patchiness (ammonium) i s examined as a f a c t o r a f f e c t i n g both phytoplankton physiology and competition. I t i s shown that temporal patchiness i n the supply of the l i m i t i n g nutrient sets up period-i c i t i e s i n c e l l u l a r carbon f i x a t i o n and in vivo c h l o r o p h y l l a fluorescence. Populations grown i n a patchy l i m i t i n g nutrient environment appear better adapted to take up nutrient pulses than do populations grown under conditions of homogeneous d i s t r i b u t i o n s of the l i m i t i n g n u t r i e n t s . I t i s also shown that the patchiness of the l i m i t i n g nutrient e f f e c t s the outcome of species competition with the winners being those species best able to optimize uptake under that p a r t i c u l a r patchy regime. A t h e o r e t i c a l framework i s developed to explore the e f f e c t s of l i m i t i n g nutrient patchiness on phytoplankton growth. This work shows that the degree of patchiness i n the environment can a f f e c t i n d i v i d u a l growth rates and thus a l t e r community structure even though there i s no change i n the average i ambient nutrient concentration. In addition the apparent K g for growth, for patch adapted populations, may be lowered s i g n i f i c a n t l y by making the d i s t r i b u t i o n of the nutrient patchy with respect to time. A q u a l i t a t i v e model i s proposed r e l a t i n g nutrient supply, l i g h t and temperature and t h e i r e f f e c t s on phytoplankton community structure. TABLE OF CONTENTS i i i Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES V 1 1 : L ACKNOWLEDGMENTS x v INTRODUCTION 1 1) Marine phytoplankton and food chain ecology 1 2) Nitrogen i n the marine ecosystem 3 2.1) Nitrogen c y c l i n g i n the sea 3 a) Physical processes 3 b) B i o l o g i c a l processes 5 3) Ki n e t i c s of nitrogen uptake 7 4) Pathways of nitrogen a s s i m i l a t i o n 9 5) Nutrient based competition 12 a) Competition i n a homogeneous environment 13 b) Competition i n a f l u c t u a t i n g environment 18 6) Purpose 19/ Chapter I. . EVIDENCE;. FOR THE GLUTAMINE SYNTHETASE PATHWAY" 01 AMMONIUM. ASSIMILATION.. 20 1) Summary 20 2) Introduction 21 -3) Mate r i a l s and Methods 22 4) Results 24 5) Discussion 30 i v Page II. LIMITING NUTRIENT PATCHINESS AS A FACTOR IN PHYTOPLANKTON ECOLOGY . 33~ 1) Summary 33 2) Introduction 34 3) Ma t e r i a l and Methods -35 3.1) Inoculum 36 3.2) Incubation 36 3.3) Inflow medium 36 3.4) Patchiness regimes ;.?7 a) System 1: Continual addition 37 b) System 2: 8 additions/day 37 c) System 3: 1 addition/day 37 3.5) Measurements 37 4) Results 39 4.1) Experiment 1 39 a) Ambient nutrients 39 b) Community structure 39 c) Nutrient uptake 42' 4.2) Experiment 2 45 5) Discussion 49 5.1) R e p l i c a b i l i t y 51 5.2) E c o l o g i c a l considerations 52 II I . CELL SIZE MANIPULATIONS IN PHYTOPLANKTON ASSEMBLAGES 57 1) Summary 57 2) Introduction -58 3) Methods 58 4) Results and Discussion 60, V Page IV. RESPONSE OF AMMONIUM LIMITED Skeletonema costatum AND Chaetooeros graoile TO LIMITING NUTRIENT PATCHINESS 65 1) Summary 65 2) Introduction 66 3) Materials and Methods 66 3.1) Measurements 67 4) Results 69 4.1) Fluorescence , 69 4.2) Carbon a s s i m i l a t i o n 69 4.3) Carbon/Nitrogen a s s i m i l a t i o n r a t i o s 69 4.4) C e l l numbers - 72 4.5) Nutrient uptake 72 a) I n t e r s p e c i f i c differences 72 b) I n t r a s p e c i f i c differences .72 5) Discussion 77 V. LIMITING NUTRIENT PATCHINESS AND PHYTOPLANKTON GROWTH: A CONCEPTUAL APPROACH 79 1) Summary 79 2) Introduction 80 3) Model 81 3.1) Nutrient uptake 82 3.2) Dependence of population growth rate on c e l l quota 82 3.3) Determination of quota 82 4) Insights into future experimental methodology 88 5) E c o l o g i c a l considerations 92 VI. A CONCEPTUAL APPROACH TO NUTRIENT BASED PHYTOPLANKTON ECOLOGY 95 SUMMARY 103 BIBLIOGRAPHY 105 APPENDIX I. CHEMOSTAT THEORY 114 v i Page APPENDIX I I . DETERMINATION OF NUTRIENT UPTAKE KINETIC PARAMETERS: A COMPARISON OF METHODS 115 1) Summary , 115 2) Introduction 115 3) Materials and Methods 118 3.1) Chemostat system and analyses 118 3.2) Uptake experiments 118 3.3) Uptake rate c a l c u l a t i o n 119 4) Results 120 4.1) Variable incubation time and v a r i a b l e substrate concentration 120 4.2) Constant incubation time and v a r i a b l e substrate concentration 120 4.3) Perturbation technique 120 5) Discussion 120 v i i LIST OF TABLES Table Page I Quantities of i n t r a c e l l u l a r free amino acids i n ammonium '." ' -li m i t e d Gymnodinium simplex at time zero and a f t e r ammonium perturbation: confidence l i m i t s of analysis are ± 3% . 25 II Quantities of i n t r a c e l l u l a r free amino acids, glutamate and glutamine, i n ammonium l i m i t e d Skeletonema aostatwn at time zero and a f t e r ammonium perturbation: confidence l i m i t s of analysis are ± 3% , , , , 27 III The composition of the i n i t i a l inocula for Experiments 1 and 2 41 IV Growth k i n e t i c parameters for the hypothetical species i n Figure 28. u i s the maximal growth rate, K i s the h a l f -max ' x saturation constant f o r growth l i m i t e d by resource X and K i s the h a l f - s a t u r a t i o n constant for growth l i m i t e d by resource Y , . , , 98 V K and V ( V ) values as determined f o r d i f f e r e n t incuba-s max max ti o n times using Method 2, i n which incubation time was ." constant at a l l substrate concentrations 124 VI K and V (V.) values as determined by the perturbation s max I J t-technique (Method 3) at d i f f e r e n t perturbation concentrations, 125 v i i i LIST OF FIGURES Figure Page 1 The nitrogen cycle i n the sea . ^ 2 Fluctuations i n l e v e l of free i n t r a c e l l u l a r amino acid pools i n response to addition of ammonium(perturbation at T=0) to ammoniumlimited Gymnodinium simplex: T=0 values are steady-state values immediately p r i o r to perturbation: 0 = glutamate, V. = glutamine 28 3 Fluctuations i n l e v e l of free i n t r a c e l l u l a r amino acid pools i n response to addition of ammonium(perturbation at T=0) to ammoniumlimited Gymnodinium simplex: T=0 values are steady-state values immediately p r i o r to perturbation: V = serine, 0 = glycine, A = alanine, T = i s o l e u c i n e , and Q = leucine 29 4 A schematic representation of the three culture systems. A l l systems were continuous flow (D = 0.3 d ^ ) . System 1 received NH^Cl c o n t i n u a l l y i n the inflow medium. System 2 received 8 additions/day, while system 3 received 1 addition/day of NH.C1. The f l u x of NH.C1 4 4 through a l l systems was 3 ug-at •£ "'"•day ^. The reactors were i n a water bath (13 ±. ,1°.C) as described i n the text 38 5 Inflow and outflow s i l i c a t e concentrations (Exp.l). System 1, ( 0 ) ; System 2, ( O ) ; System 3, (A) 4 0 i x Figure Page 6 Accumulative c e l l numbers (Exp.l) Thalassiosiva, (0) ; F l a g e l l a t e s , (V); Nitzschta, (.•); Chaetocevos, (?); Skeletonema, (*) ; T o t a l numbers, ( O ) : A, System 1: B, System 2 : C, S.ygtem 3 43 7 Relative c e l l numbers (Exp. 1). System 1, (0); ^ System 2 , (• ); System 3 , (A); Semicontinuous i culture CA) (see t e x t ) : A, percent Skeletonema: B, percent Chaetocevos 44 8 Disappearance of ammonium with time, following an ammonium perturbation at 0100 hr (Exp.l). System 1, (»); System 2 , (V); System 3 , (.0) , 4 6 9 Disappearance of ammonium with time, following an ammonium • perturbation at 1200 hr (Exp.l). System 1, ( • ); System 2 , (V); System 3 , (0) 47 10 Relative diatom numbers (Exp.2, System I ) : percent Skeletonema ( © ) and Chaetocevos (f) 4s 11 Surface l i g h t i n t e n s i t y ( 4 0 0 - 7 0 0 nm). A l i g h t f i l t e r (see text) was used to reduce i t by 50% during experiments. Experiment 1, (0); Experiment 2 , (•) 50 12 Possible changes i n competitive advantage between Chaetocevos and Skeletonema as a function of ammonium patchiness 53 13 A schematic representation of the possible r e l a t i o n s h i p between s p e c i f i c nutrient f l u x (nitrogen) and community structure, 55 Mean c e l l diameter as a function of the time between ammonium additions f o r Exp. 1 (0) and Exp. 2 (€>). The mean c e l l diameters i n each treatment were s i g n i f i c a n t l y d i f f e r e n t ( p = .01). Mean c e l l diameter was c a l c u -l a t e d from the mean c e l l volume assuming an equivalent sphere Possible changes i n competitive advantage between three marine phytoplankton as a function of ammonium patchiness Relative fluorescence over a 24 hr period f o r C. gvaoite (£j ; System 1, continual addition of ammonium and ft ; System 2 5 d a i l y addition of ammonium) and •S. oostatum (0; .System 1, •; System -2). A l l systems were continuous flow with a d i l u t i o n rate of 0.6 d ^ and grown under continuous l i g h t . The arrow represents the time of the ammonia addition to System 2. The point indicated f o r systems I represents the mean of four values taken throughout the day. .............................. . . . , Carbon f i x a t i o n rate for S. oostatum (0, System 1 , continual addition of ammonia and System 2, d a i l y addition of ammonia) and C. gvaoite ( L7 , System 1; continual addition of ammonia and fif , System 2;, d a i l y addition of ammonium). | indicates time of ammonium addit i o n to Systems 2. Bars represent 95% confidence x i Figure Page 18 The disappearance of ammonium as a function of time during a perturbation experiment for System 1 (continual addition of ammonium C. gracile, D , and S. costatum, 0 73 19 The disappearance of ammonium as a function of time during a perturbation experiment for System 2 ( d a i l y a ddition of ammonium C. graoile, ® and S. costatum, 74 20 The disappearance of ammonium as a function of time during a perturbation experiment for C. gvaeiZe " S,ystem 1 (continual addition of ammonium), Q , and System 2 (d a i l y addition of ammonium), M 75 21 The disappearance of ammoniujnas a function of time during a perturbation experiment for S. costatum System 1 (continual addition of ammonium ,~ 0^ and System 2 (d a i l y addition of ammonium), ® 76 22 A graphical representation of a temporally patchy, nutrient l i m i t e d environment. [S] i s the substrate concentration (yg-at • £ " ' ' ) . P i s the period of the patch (hr), the duration (hr) and T, the i n t e r v a l between successive pulses (hr). [S ] i s the substrate av concentration averaged over the patch period, P 86 x i i Figure Page 23 The growth rate, y, (hr "*") of two hypothetical species as a function of average substrate concentration, S , (yg-at • I and the patch period, P, (hr). The patch duration i s constant at 0.1 hr. Species A has the growth parameters: p /Q . =0.2 h r _ 1 ; K =0.2 yg-at • X,-1; and m mxn s y = 0.2 hr \ Species B has: -1 -1 p /Q . = 4.1 hr ; K =2.05 yg-at • I : and m mxn s y = 0.0512 h r " 1 . (see text for d e f i n i t i o n of symbols) 87 24 Growth rate of a patch-adapted population with growth parameters: p /Q . =2.5 h r - 1 : p max mxn K g = 0.8 yg-at • l'1; y = o . l h r " 1 ; Pn/ , Qmin = 0.1 hr 1 ; and T^ = 2.0 hr. (See text f o r d e f i n i t i o n of symbols) 89 25 Growth rate (y) as a function of average substrate concentration, ^ a v ^ f ° r t n e population i n Fig.3. The growth response at P = 0 i s represented as ( ) and at optimal patchiness C^ 0p t)» a s ( )• ^ 26 The growth rate (y) of a hypothetical species with growth parameters: [S 1= 0.1 yg-at • p /Q . = 0.325 h r " 1 ; o m mxn K = 0.1 yg-at • JT 1 and y = 0.155 h r " 1 9 1 x i i i Figure Page 27 The r e l a t i o n s h i p between s p e c i f i c nutrient f l u x D/S r a t i o and " r " and "K" competition strategy and the r e s u l t i n g phytoplankton community structure. The competition strategy i s represented as a continuum between " r " and "K" s t r a t e g i s t s 96 28 A dominance plane representing the outcome of competition, f o r the species i n Table IV, as a function of d i l u t i o n rate ( s p e c i f i c nutrient flux) and resource r a t i o . Representation of two species i n the same area i n d i c a t e stable coexistence, with both species l i m i t e d by a d i f f e r e n t resource 99 29 Ammonium uptake (hr "*") as a function of substrate concentration for P., pyvifoTmis grown i n an ammonium li m i t e d chemostat at 0.5 d \ The incubation time over which the uptake rate was calculated was the time at which the substrate concentration had dropped to hal f the o r i g i n a l concentration. Uptake rates were calculated using the true t = 0 substrate concentration, ©, and the f i r s t measured substrate concentration, 0 121 30 Determination of V ' , using method 2 (constant max incubation time at a l l substrate concentrations), as a function of incubation time. Bars represent one standard error 122 x i v Figure Page 31 The hal f saturation constant (K s) as determined by the perturbation technique f or d i f f e r e n t i n i t i a l substrate additions. Bars represent one standard error 123 32 Disappearance of ammonia with time showing the V • max and V. components 129 X V ACKNOWLEDGMENTS I wish to s i n c e r e l y thank my supervisor, Dr. Paul J . Harrison, f o r h i s advice, encouragement and the u n s e l f i s h g i f t of his time. In addition, I wish to thank Drs. T.R. Parson, F.J.R. Taylor and I.E.P. Taylor f o r the guidance afforded me as members of my research committee. John Parslow contributed s u b s t a n t i a l l y to the modelling e f f o r t s i n Chapter V, and i n so doing, broadened my understanding of phytoplankton growth dynamics. Lynne Quarmby provided t e c h n i c a l support i n the lab and ca r r i e d out many of the experiments i n Chapter IV. Douglas S. Scales maintained the autoanalyzer during the studies i n Chapter I I . Rosemay Waters graciously provided the cultures used i n t h i s study. I would also l i k e to thank A. Carruthers, Dr. G. Court, Dr. CO. Davis, Dr. R.E. DeWreede, B. Harrison, Dr. D. Tilman, J. Wehr and several anonymous reviewers f o r t h e i r comments, c r i t i c i s m and advice on various portions of t h i s work. A s p e c i a l thanks i s owed to my family whose patience and support f a c i l i t a t e d the completion of t h i s project. 1 INTRODUCTION 1) Marine phytoplankton and food chain ecology Marine phytoplankton are the f i r s t step i n many marine food chains. These microalgae require l i g h t , carbon dioxide, inorganic ions (NO^, NO^, NH*, PO^, SiO^) and sometimes trace amounts of organic compounds to be able to grow and produce the primary p a r t i c u l a t e material i n the sea. They are heavily grazed by herbivorous zooplankton which i n turn are prey to other organisms with higher trophic status. The morphology and s i z e of the phytoplankton determines to a large extent which organisms can consume them, and consequently they play an improtant r o l e i n mediating the r e s u l t i n g food chain. This idea was s u c c i n c t l y described by Ryther (1969) i n a c l a s s i c paper discussing three marine food chains: the oceanic, coastal and upwelling, and t h e i r p o t e n t i a l for f i s h production. The oceanic community i s characterized by low p r o d u c t i v i t y -2 -1 (^50 gC-m -yr ) and a standing stock of primary producers composed mainly of small f l a g e l l a t e s . The coastal food chain, termed the continental shelf food chain by Parsons and Takahashi (1973), has an average p r o d u c t i v i t y of 'vlOO gC -2 -1 •m -yr with an assemblage of primary producers co n s i s t i n g of large diatoms, d i n o f l a g e l l a t e s and nanoplankton. Upwelling ecosystems are characterized by Ryther's upwelling community. The primary p r o d u c t i v i t y of these regions -2 -1 averages^300 gC'm -yr to which the macrophytoplankton are the greatest contributors. The differences i n these three food chains, mediated i n a large part by the primary producers present, r e s u l t i n large differences i n the t o t a l f i s h production (harvestable resource) that can be supported by the system. Greve and Parsons (1977) proposed that the very nature of the phytoplankton i n a system may a f f e c t not only the production of higher trophic l e v e l s , as was argued by Ryther (1969), but also the species composition. Greve and Parsons suggested that there are two p r i n c i p a l pathways for transfer of energy i n a 2 marine food web. The f i r s t pathway proceeds from nanophytoplankton to ctenophores or medusae, while the second s t a r t s with large diatoms and term-inates with f i s h . The composition or community structure of the primary producers i n the world's ocean thus appears to be of paramount importance i n determining the pathways and ultimate y i e l d of marine food chains. The large economic import-ance a t t r i b u t e d to marine f i s h e r i e s makes i t important to understand the factors c o n t r o l l i n g the a b i l i t y of d i f f e r e n t phytoplankton groups to a t t a i n dominance i n the seas. The co n t r o l of dominance i n a phytoplankton community depends upon the net growth rates of the populations i n the community. The net growth rate i s the diffe r e n c e between the instantaneous rate of increase of the population and the loss terms due to grazing, sinking and advection. The population with the highest net growth w i l l eventually a t t a i n dominance i n the system. The instantaneous growth rate (y) of a population i s thus of great e c o l o g i c a l importance. The instantaneous growth rate i s affected by many factors including temperature, l i g h t and nutrient s . It i s generally accepted that temperature defines or l i m i t s the maximum 'poten t i a l ' growth rate of a population (Eppley, 1972) . Other factors such as nutrients and l i g h t w i l l determine the growth rate attained i f either of these i s l i m i t i n g . The major objective of the work reported i n t h i s thesis was to examine various aspects of nutrient l i m i t e d growth of marine phytoplankton, with p a r t i c u l a r reference to inorganic nitrogen. In the f i n a l chapter there i s an attempt made to integrate the nutrient e f f e c t s with other factors such as temperature and l i g h t i n a simple conceptual scheme. 3 2) Nitrogen i n the jiiarine ecosystem Nitrogen i n the sea i s found i n inorganic, organic and p a r t i c u l a t e pools. The dissolved inorganic nitrogen pool (DIN) i s composed mainly of molecular nitrogen, n i t r a t e , n i t r i t e and ammonium, whereas the dissolved organic n i t r o -gen pool (DON) p r i m a r i l y consists of urea, amino acids, creatine, peptides and nucleotides. Upon incorporation into phytoplankton or b a c t e r i a l biomass the nitrogen enters the p a r t i c u l a t e pool (PON). The fluxes between these pools are c o n t r o l l e d by b i o l o g i c a l , chemical and p h y s i c a l f a c t o r s . The amount of nitrogen i n a pool at any time i s a function of the a s s i m i l a t i o n , regeneration and trans-formation processes occurring i n the system. 2.1) Nitrogen c y c l i n g i n the sea: A schematic representation of nitrogen c y c l i n g i n the sea i s given i n Figure 1. a) Physical processes: Molecular nitrogen i s i n constant exchange between the sea surface and the atmosphere i n accordance with Henry's Law (Vaccaro, 1965). For the most part the surface of the oceans seems:to be saturated with molecular nitrogen (Fox, 1909; Rakestraw & Emmel, 1938; Benson & Parker, 1961). Molecular nitrogen i s of l i t t l e importance i n the b i o l o g i c a l c y c l i n g of nutrients except where i t i s used i n the processes of nitrogen f i x a t i o n by blue-green algae (Dugdale et a l . , 1964; Carpenter & McCarthy, 1975). Fixed nitrogen, i n the form of ammonium (NH^), n i t r a t e (NO^) and n i t r i t e (NO^) can also enter the sea from the atmosphere most often i n association with r a i n . The amount of t h i s f i x e d nitrogen varies considerably. Walsh et a l . (1978) measured concentrations (yg-at-£ - 1) of 0.03 N0~, 5.57 NH* and 14.18 N0~ i n the r a i n water from the New York Bight. This nitrogen f l u x would account for about 1% of the annual phytoplankton nitrogen budget i n the Bight. The concentration of nitrogenous nutrients i n rainwater may vary with the proximity to land and the extent of input into the a i r by the respective land mass. N 2 precipitation N I T R O G E N C Y C L E runoff Surface Euphotic Zone Nutricline advection Figure 1. The nitrogen cycle in the sea. 5 River input of f i x e d nitrogen can be s i g n i f i c a n t i n coastal and estuarine systems. Walsh et a l . (1978) using the data of Bowman (1977) and R i l e y (1959) estimated that the nitrogen f l u x i n the New York Bight, as a r e s u l t of r i v e r input, to be about 3 yg-at N-l "'"•yr \ or approximately 8% of yearly p r o d u c t i v i t y demand. Such allocthanous input would be i n s i g n i f i c a n t i n a system such as an o l i g o t r o p h i c oceanic gyre. A major proportion of nitrogenous inputs into the euphotic zone i s a r e s u l t of storm action. During these periods of disturbance, large quantities of deep n u t r i e n t - r i c h water are mixed up into the surface waters (Walsh et a l . , 1978). Upwelling of n u t r i e n t - r i c h , deep water i s a very important process i n many areas of the world. Such upwelling occurs at divergences which are most often found i n a s s o c i a t i o n with the eastern boundary currents (Dugdale, 1976; Wooster & Reid, 1963). The upwelling process o f f the coast of Peru i s of such magnitude that t h i s area represents one of the most productive areas i n the world oceans. Other factors such as turbulence across the n u t r i c l i n e also contribute to the p h y s i c a l l y c o n t r o l l e d fluxes. Losses of nitrogen from the system are p r i m a r i l y a r e s u l t of the sinking of p a r t i c u l a t e material including phytoplankton (Smayda, 1970), f e c a l material and d e t r i t u s . Other losses occur through organismal migration and advection. b) B i o l o g i c a l processes: Molecular nitrogen can enter the nitrogen cycle v i a the process of b i o l o g i c a l f i x a t i o n . The most commonly studied nitrogen f i x i n g organisms i n the seas are the blue-green algae and s p e c i f i c a l l y , members of the genus Osoi-llatori-a (Trichodesnrium) and some very small coccoid species (Watson, pers. comm.). I n i t i a l l y i t was suggested that ^ f i x a t i o n could be an important component of the nitrogen f l u x i n oceanic systems (Dugdale et a l . , 1964; Gpering et a l . , 1966). Other workers studying the Sargasso Sea and the c e n t r a l North P a c i f i c showed that N f i x a t i o n makes an i n s i g n i f i c a n t c ontribution to the nitrogen budget (Carpenter & McCarthy, 1975; Mague et a l . , 6 1974). However, Carpenter and Pr i c e (1977) have shown that i n the eastern Caribbean Sea, f i x a t i o n by Osci-llatoria sp. may be a very s i g n i f i c a n t component of nitrogenous fluxes. This apparently contradictory evidence probably r e s u l t s from the s p a c i a l and temporal patchiness of the f i x i n g organisms. The uptake of dissolved inorganic and organic nitrogen i s co n t r o l l e d by numerous fa c t o r s ; the concentration of the nutrient i n s o l u t i o n (Dugdale, 1969), the irradiance (Maclsaac & Dugdale, 1972), temperature (Goldman, 1977; Harrison, 1974) and the other nitrogenous nutrients present (Wheeler et a l . , 1974). The uptake process w i l l be described i n d e t a i l i n the next section. The nitrogenous compounds which are taken up are incorporated into primary p a r t i c u l a t e materials and then become susceptible to grazing pressure... The p a r t i c u l a t e nitrogen may be eit h e r assimilated and remain i n the p a r t i c u l a t e pool or eventually be excreted as ammonium, DON, or f e c a l material. Zooplankton excretion i s a very s i g n i f i c a n t component of nitrogen f l u x i n marine ecosystems. Ifalsh et a l . , (1978) showed that zooplankton excretion accounted for ^35% of the annual nitrogen .flux through the euphotic zone i n the New York Bight. Recent work by Goldman et a l . (1979) suggested that even though ambient nutrient concentrations i n ol i g o t r o p h i c areas of the sea may be undetectable, i t i s possible that phytoplankton growth rates are not nutrient l i m i t e d . This may be due to a dynamic balance between nutrient regeneration and a s s i m i l a t i o n r e s u l t i n g i n fluxes through the dissolved nutrient pool that are high enough to support high growth rates. Ammonium r e s u l t i n g from regeneration can be con-verted to NO^ and NO^ v i a b a c t e r i a l n i t r i f i c a t i o n (Vaccaro, 1965). The uptake and a s s i m i l a t i o n of dissolved inorganic nitrogen by phyto-plankton, with s p e c i f i c reference to ammonium are the major subjects of th i s t h e s i s . This i s an important component of the phytoplankton nitrogen budget, where a major portion of the nitrogen f l u x into a system may be due to ammonium regeneration (Dugdale & Goering, 1967; Dugdale, 1976; Harrison, 1978). 3) K i n e t i c s of Nitrogen uptake The uptake rate of -.fixed' nitrogen (NH*, NO^, NO^, amino acids or urea) appears to be r e l a t e d to the concentration of the substrate i n s o l u t i o n by the Michaelis-Menten (1913) hyperbola which i s described by the following equation: v = v — max K + [S] s where V = nutrient uptake rate (hr ^) V = maximal nutrient uptake rate (hr 1 ) max S = concentration of the nutrient (ug-at'X, "*") K = concentration of the nutrient at which V = 1/2 V (ug«at«£ s max Numerous studies have shown the a p p l i c a b i l i t y of t h i s expression to nitrogen uptake by marine phytoplankton (Eppley et a l . , 1969; Eppley & Renger, 1974; Caperon & Meyer, 1972a & b). Nutrient uptake i s thought to be enzyme con t r o l l e d and therefore i t i s not s u r p r i s i n g that there i s a strong tempera-ture e f f e c t on the process. Eppley (1972) reported a Q^Q of 1.88 for phyto-plankton growth. In a nutrient saturated system or under conditions when uptake equals growth, t h i s value would also apply to nutrient uptake. Phytoplankton derive almost a l l t h e i r energy from l i g h t . It i s not su r p r i s i n g that uptake of many nutrients shows a strong dependence on l i g h t . Maclsaac and Dugdale (1972) showed the dependence of NO^ uptake on l i g h t . They described a r e l a t i o n s h i p between uptake and irradiance through a h a l f - s a t u r a -t i o n constant for l i g h t thus: V = V - [ T 1 -max where V = nutrient uptake rate (hr "*") V = maximal uptake rate of the l i m i t i n g nutrient (hr 1 ) max b 8 [I] = Irradiance (% surface radiation) K_ = Irradiance at which V = 1/2 V L t max Inorganic forms of nitrogen, e s p e c i a l l y NH*, are generally preferred to organic forms (Rees & Syrett, 1979; Wheeler et a l . , 1974; Wheeler, 1977). When ammonium i s added to a nitrate-grown phytoplankton population, n i t r a t e uptake i s suppressed (Eppley et a l . , 1969; Conway, 1977). The p o t e n t i a l uptake :rate of organic nitrogen increases r a p i d l y upon depletion of inorganic nitrogen and the strongly s e l e c t i v e uptake c h a r a c t e r i s t i c s disappear (wheeler et a l . , 1974). Numerous studies have been conducted over the l a s t decade on the k i n e t i c s of n u t r i t i o n a l ion uptake i n marine phytoplankton. The a b i l i t y of an organism to take up a l i m i t i n g nutrient r a p i d l y i s of great s u r v i v a l value. The h a l f -saturation constant f or nutrient uptake i s a measure of the a f f i n i t y of the organism for the l i m i t i n g nutrient. In environments where the ambient nutrient concentration i s low, a low h a l f - s a t u r a t i o n constant (high a f f i n i t y ) enables the organism to continue taking up nutrients at high rates and i s therefore considered a competitive advantage (Dugdale, 1976). Maclsaac and Dugdale (1969, 1972), Eppley et a l . (1973) and Carpenter and G u i l l a r d (1971) have shown that K g values were higher i n coastal (eutrophic) than i n oceanic (oligotrophic) phytoplankton assemblages. The a b i l i t y of an organism to take up a nutrient i s dependent upon both V and K and thus the determination of these values i s important to the max s understanding of the organism's f u n c t i o n a l r e l a t i o n s h i p to i t s environment. It i s now apparent that these uptake k i n e t i c parameters are more complicated than o r i g i n a l l y thought. These so-called "constants" are i n fact variables dependent on the n u t r i e n t - l i m i t e d growth rate of the population (Eppley & Renger, 1974; Conway & Harrison, 1977; McCarthy & Goldman, 1979). In addition 9 to t h i s complication many d i f f e r e n t methods of measuring nutrient uptake k i n e t i c s have been employed with no clear understanding as to how the r e s u l t s of various methods compare. Several methods of determining uptake k i n e t i c s on the same steady state population are presented i n Appendix I I . The r e s u l t s r e f l e c t a greater degree of complexity i n the determination and meaning of the uptake k i n e t i c parameters V and K than has hit h e r t o been expected. max s 4) Pathways of Nitrogen a s s i m i l a t i o n The f i r s t step i n the growth process occurs when a l i m i t i n g nutrient i s taken up and assimilated. The pathways by which a s s i m i l a t i o n occurs, t h e i r a b i l i t y to scavenge low l e v e l s of the l i m i t i n g nutrient, and the metabolic cost of operating the pathway are a l l important factors i n determining phyto-plankton a b i l i t y to compete f or a l i m i t i n g resource. Both n i t r a t e and n i t r i t e are reduced i n t r a c e l l u l a r l y to ammonium by the enzymes, n i t r a t e reductase and n i t r i t e reductase (Morris, 1974) before further a s s i m i l a t i o n can occur. Thus discussions of ammonium a s s i m i l a t i o n also include important steps i n the a s s i m i l a t i o n of n i t r a t e and n i t r i t e . Even some organic forms of nitrogen are converted both e x t r a c e l l u l a r l y (Belmont and M i l l e r , 1965; Saubert, 1957) and i n t r a c e l l u l a r l y (Stewart, 1977; Keys et a l . , 1978) to ammonium. It i s cl e a r that the pathway by which ammonium i s assimi-lated into organic plant constituents i s a major feature of nitrogenous bio-chemistry i n plants. A discussion of the regulation of n i t r a t e and n i t r i t e reduction i s not included i n t h i s thesis and the reader i s referred to Brown and Johnson (1977) f o r a comprehensive review of t h i s subject. The study of ammonium a s s i m i l a t i o n i n phytoplankton lags behind s i m i l a r studies i n higher plants ( M i f l i n & Lea, 1976) and ba c t e r i a (Stadman & Ginsburg, 1974). In these two groups of organisms i t i s thought that the enzyme respon-s i b l e f o r primary ammonium a s s i m i l a t i o n , under conditions of nitrogen l i m i t a -t i o n , i s glutamine synthetase (GS; E.C. 6.3.1.2) which catalyzes the conversion 10 j I j of the amino acid glutamate to glutamine i n the presence of NH^, ATP and Mg Glutamine can then p a r t i c i p a t e ' i n the synthesis of many c e l l u l a r nitrogen-ous compounds (Prusiner & Stadtman, 1973). The most common fate i s the production of two molecules of glutamate from one molecule of glutamine and a-ketoglutarate i n the presence of NAD(P)H. The enzyme catalyzing t h i s r eaction i s glutamine :2 oxoglutarate aminotransferase (GOGAT; E.C. 2.6.1.53) also known as glutamate synthase. The coupling of GS and GOGAT r e s u l t s i n a cycle using one molecule of NH*, a-ketoglutarate, ATP and NAD(P)H+H+ and y i e l d -ing one molecule of glutamate, ADP, P i and NAD(P) +. The glutamate which i s produced i n these reactions can be used to supply an amino group to a wide range of compounds. The most common reaction i s that of transamination i n which the glutamate amino group i s transferred to a a-keto acid ( M i f l i n & Lea, 1977). The discovery of GOGAT has been very recent ( M i f l i n & Lea, 1976). Early work on ammonium a s s i m i l a t i o n focused on the enzyme, glutamate dehydrogenase (GDH) which catalyzes the reductive amination of a-ketoglutarate to produce glutamate. Early evidence for t h i s pathway came from work on Candida utitus (Sims & Folkes, 1964; Folkes & Sims, 1974) which i s one of the few organisms that t r u l y lacks a GOGAT system ( M i f l i n & Lea,. 1976), and from reports that GDH can catalyze the in vitro a s s i m i l a t i o n of ammonium into amino acids ( M i f l i n & Lea, 1976). It may be k i n e t i c a l l y advantageous for an organism to possess glutamine synthetase when ammonium l e v e l s are low. The h a l f - s a t u r a t i o n constant (K ) of s GS for ammonium i s i n the micromole range, whereas the K g for GDH i s i n the mi l l i m o l e range (Falkowski & Rivkin, 1976; M i f l i n & Lea, 1976; Ahmed et a l . , 1977). Conversely, i t requires more energy to synthesize a molecule of glut a -mate v i a the GS/GOGAT pathway than v i a the GDH pathway ( i . e . , GS requires 1 ATP and GOGAT requires 1 NAD(P)H = 3 ATP). The GS/GOGAT system may be viewed 11 as a high energy/high a f f i n i t y system and GDH as a low energy/low a f f i n i t y system. Recent research with higher plants suggests that GDH i s used f o r a s s i m i l a t i o n when ammonium i s i n excess whereas the GS/GOGAT system operates when ammonium i s low (Stewart & Rhodes, 1977a). The two systems appear to be r e c i p r o c a l l y regulated through i n t r a c e l l u l a r glutamine concentration. When the glutamine concentration i s low (low ambient nitrogen), the GS/GOGAT system i s stimulated and GDH i s repressed. When glutamine i s high (high ambi-ent nitrogen) GDH i s derepressed and GS/GOGAT repressed. There have been few reports on the pathways of ammonia a s s i m i l a t i o n i n marine phytoplankton. In an early phytoplankton study '(Eppley & Rogers, 1970), and i n the early plant and b a c t e r i a l studies, glutamate dehydrogenase was the only system that was assayed. Further work on GDH i n marine phytoplankton was reported by Ahmed et a l . (1977). Falkowski and Rivkin (1976) drew attention to the fac t that GDH, with i t s low NH* a f f i n i t y , would be a poor ammonium scavenger under conditions of low ambient nitrogen concentrations and suggested that the large i n t r a c e l l u l a r ammonium pools (Eppley & Rogers, 1970) had been over-estimated due to nucleo-tide deamination during the extraction procedure. They also demonstrated that the GS/GOGAT system was operating i n marine phytoplankton. Its high a f f i n i t y f o r ammonium coupled with high in vitro a c t i v i t y indicated that t h i s pathway could be an important assimilatory pathway under nitrogen l i m i t a t i o n . Other recent enzymological studies have supported the contention that the GS/GOGAT system i s important i n the a s s i m i l a t i o n of ammonium (Edge & Ricketts, 1978). The evidence for use of the GS/GOGAT system under conditions of nitrogen l i m i t a t i o n i n marine phytoplankton i s r e s t r i c t e d to in vitvo enzyme k i n e t i c observations. Results i n Chapter I describe the pathway of ammonia a s s i m i l a t i o n i n two species of marine phytoplankton (Gymnodiniwn simplex (Dinophyceae) and Skeletonema costatwn (Bacillariophyceae)) determined using in vivo methods. 12 The f l u c t u a t i o n s i n the free i n t r a c e l l u l a r amino acid pools of the nitrogen-l i m i t e d , chemostat-grown phytoplankton were monitored following an ammonium perturbation. The r e s u l t s are then used to elucidate the apparent pathway of primary ammonium a s s i m i l a t i o n (Turpin & Harrison, 1978). 5) Nutrient-based competition Nutrient uptake i s only the f i r s t step i n the growth process. The nutrients which are taken up must be used e f f i c i e n t l y i f a population i s to grow. Monod (1942) proposed a simple model r e l a t i n g the growth rate of micro-organisms to the concentration of the l i m i t i n g nutrient according to the formula: [S] y ymax K + [S] s y = growth rate (hr 1 ) y = maximum growth (hr "*") max [S] = substrate concentration (yg-at•£ 1 ) ' K g = the substrate concentration promoting 1/2 maximal growth (yg-at - i T 1 ) This model has been most useful i n studying the processes of nutrient-based com-p e t i t i o n . A major weakness i s that i t assumes that the s p e c i f i c growth rate (time i s equal to the s p e c i f i c uptake rate of the l i m i t i n g nutrient. This assumption appears only to be true under conditions of steady state growth. Another problem i s that the model assumes a constant c e l l y i e l d per mole of l i m i t i n g nutrient. Recent studies on n u t r i e n t - l i m i t e d growth k i n e t i c s of marine phytoplankton have shown that t h i s assumption i s i n v a l i d because the amount of l i m i t i n g nutrient per c e l l v a r i e s over the growth range of the popu-l a t i o n (Caperon & Meyer, 1972a & b; Eppley & Renger, 1974; Paasche, 1973; Droop, 1970; Fuhs, 1969; Goldman & McCarthy, 1978). Many workers (Droop, 1968; Goldman & McCarthy, 1978) have shown that the amount of l i m i t i n g nutrient per c e l l (Q) v a r i e s i n response to the nutrient l i m i t e d growth rate according to 13 the r e l a t i o n s h i p proposed by Droop (1968) where: * = * ( 1 " y = growth rate (hr ~S y = growth rate when Q -> «> (hr "S Q = c e l l quota (y g - a t - c e l l "S = minimum quota needed f or growth to proceed ( y g - a f c e l l ^) a) Competition i n a homogeneous environment: Harder et a l . (1977) reviewed the processes involved during competition between microorganisms grown i n continuous c u l t u r e . Two general cases of s i n g l e nutrient-based competition are defined by the Monod model. In the f i r s t case, one of two species competing f o r a l i m i t i n g resource has both the higher y and the max lower f o r growth (as represented by species A below). Case 1 for Monod Competition / ior D (hr"1) Id max A Id max Q Species A - low K , hiqh y s max Species B - high K , low y s max [S] ( j jg-at-r 1) Regardless of the ambient substrate concentration, species A i s always able to outgrow species B. S i m i l a r i l y , i n a continuous culture, at any d i l u t i o n rate (D) (see Appendix I ) , species A w i l l maintain an ambient nutrient con-centration lower than that required f o r species B to maintain a growth rate equal or greater than the d i l u t i o n rate and hence B w i l l wash out. 14 In the second case, the growth vs. substrate curves of the two com-p e t i t o r s cross so that, at lower substrate concentrations or d i l u t i o n rates, species A w i l l win, but at higher concentrations or d i l u t i o n rates species B w i l l win. This condition i s attained when species A has a low U and a max low~ K f o r growth, whereas species B has a high y and a high K , Such a s max s s i t u a t i o n i s described below-: Case 2 f o r Monod Competition jU or D (hr-M [S] ' [S] (jjg-at-l-M Species A - low K , low y s max Species B - high K , high y s max In t h i s scheme, D' i s the d i l u t i o n rate at which species A and B maintain the same ambient substrate concentration while S' i s the substrate concentration where the growth rates of species A and B are equal. In t h i s system three r e s u l t s are t h e o r e t i c a l l y possible when the two species are competing f o r the same l i m i t i n g resource: (1) P A = WB when [S] = IS]' (2) UA < % when [S] > [S] • (3) \ > WB when [S] < [S]' And s i m i l a r i l y , i n a chemostat when: 15 (1) R A = y when D = D '.• (2) y. A y_ when D < D' (3) y„ = y„ when D > D' A B Simple models of t h i s type have been used to explain r e s u l t s obtained i n phytoplankton competition studies (Michelson et a l . , 1979; Titman, 1976; Tilman, 1977) and at the same time to provide a simple model f or i n t e r p r e t i n g nutrient-based competition i n the sea. The theory of competition f o r a sing l e l i m i t i n g nutrient was expanded by Taylor and Williams (1975) t o include many p o t e n t i a l l y l i m i t i n g resources. Subsequent experimental work by Tilman (1977; Titman, 1976) confirmed t h i s multiple resource-based competition theory. He established that when two r e -sources are p o t e n t i a l l y l i m i t i n g , the growth rate of the organism i s described by the concentration of the most l i m i t i n g n utrient, assuming the v a l i d i t y of the Monod expression. There thus e x i s t s a sharp switch from l i m i t a t i o n by one nutri e n t , to l i m i t a t i o n by another (Rhee, 1978). The r e l a t i v e supply rate of the two p o t e n t i a l l y l i m i t i n g nutrients at t h i s switch-over point can be deter-mined. At the point where l i m i t a t i o n switches from one nutri e n t to another, growth should be l i m i t e d equally by both nutrients as described below (Titman, 1976) : 2 or, [S,t] K s + [ S l ] = or, K. 1 K, '2 16 The r a t i o of nutrients needed t o obtain a growth rate which i s equally l i m i t e d by each nutrient i s therefore given by the r a t i o of the h a l f - s a t u r a t i o n con-stants f o r growth f o r the two nutrients i n question. I f the r a t i o of substrat-es i n the system i s greater than the r a t i o of h a l f - s a t u r a t i o n constants, the population i s l i m i t e d by the nutri e n t . I f the r a t i o of substrates i s less than the r a t i o of ha I f - s a t u r a t i o n constants, the population i s l i m i t e d by S^. As a r e s u l t , along a continuum of the r a t i o of two p o t e n t i a l l y l i m i t i n g n u t rients, there e x i s t s a region within which a population w i l l be S^-limited and another i n which i t i s S^-limited. Consequently when two species are cultured together there i s a range of substrate supply r a t i o over which species A would be l i m i t -ed by and species B by (providing K^/K^ ^ o r *" n e t w o s P e c i e s a r e n o t i d e n t i c a l ) . In t h i s region the populations are l i m i t e d by d i f f e r e n t resources and hence competitive exclusion does not take place. Outside t h i s region, when both species are l i m i t e d by the same resource, competition can be explained on the basis of Monod competition. Tilman--- (1977) used two species, Asterionella formosa and Cyclotella meneghiniana. Under phosphate l i m i t a t i o n , A. formosa out-competed C. meneghiniana at a l l substrate concentrations. Conversely, C. meneghiniana out competed A. formosa under a l l cases of s i l i c a t e l i m i t a t i o n . The respect-ive u vs. [S] curves are reproduced below: A. formosa C. meneghiniana C. meneghiniana A. formosa 17 The r a t i o s of K s i / K p 0 f o r ^- formosa and C. meneghiniana are 97 and 5.6, 1 4 re s p e c t i v e l y . Therefore, at Si/PO^ r a t i o s greater than 97, both species were PO^-limited and A. formosa won. At r a t i o s less than 5.6, both species were S i - l i m i t e d and C. meneghiniana won. In the zone between 97 and 5.6, coexist-ence occurred. The outcome of competition i s described diagrammatically below. -2 t o A. formosa C.meneghiniana rr coexistence c g dominates ' ' dominates Q | i i i 1000 100 10 i 1 Nutrient Ratio [Si ]/[ P0 A ] In Tilman's work, the d i l u t i o n r a t e of the continuous cultures e s s e n t i a l l y had no e f f e c t on the r e s u l t of competition. This can be interpreted as an example of "Case 1" Monod competition, i n other words the u vs, [S] curves f o r the two species do not cross. The examples of "Case 2" Monod competition discuss-ed e a r l i e r suggest that f u r t h e r i n s i g h t s i n t o nutrient based competition may be obtained by observing systems i n which both the d i l u t i o n r ate, or substrate concentration, and the r a t i o of p o t e n t i a l l i m i t i n g resources are important. This problem i s approached conceptually i n Chapter VI of t h i s t h e s i s . 18 b) Competition i n a f l u c t u a t i n g environment: It was assumed i n the preceding discussion of nutrient-based competition that there were both tempor-a l and s p a c i a l homogeneity i n the environment. There have been few attempts to understand the importance of f l u c t u a t i o n s i n n u t r i e n t supply that must e x i t i n nature. The f i r s t report of microalgal responses to f l u c t u a t i n g nutrient regimes was by Caperon (1969). The long-term growth response of Isochvysis galbana was followed i n response to numerous d i l u t i o n rate changes. Caperon (1969) showed that the r e s u l t s could be i n t e r p r e t e d i n a model that included a time lag response between the nutrient concentration and growth. The incorporat-ion of a time lag provided explanations of a number of the t r a n s i e n t responses observed by Caperon (1969). Grenney et a l . (1973) showed that a more complex growth model, which i n -cluded external nutrient concentration and three i n t r a c e l l u l a r nitrogen pools, also accommodated Caperon's (1969) data. Grenney et a l . (1973) used t h e i r model to explain the e f f e c t of f l u c t u a t i n g nutrient supply- rates ( d i l u t i o n rates) on the outcome of phytoplankton competition. They showed that with c e r t a i n low frequency (weeks) d i l u t i o n rate changes, co-existence between competing species could occur. Over the period of o s c i l l a t i o n , however, the population d e n s i t i e s of a l l species f l u c t u a t e d markedly. This study i n d i c a t e d the p o t e n t i a l importance of low frequency resource supply f l u c t u a t i o n s upon phytoplankton community structure. Three further questions a r i s e . 1) What i f the supply rate remains constant but the temporal or s p a c i a l d i s t r i -bution of the resource v a r i e s ? 2) How does t h i s a f f e c t the outcome of competition? 3) Is there an optimal degree of patchiness of the l i m i t i n g nutrient for a given species? 19 Chapters I I , I I I , IV and V are concerned with these questions from experimental and modelling viewpoints, In Chapters II - IV, r e s u l t s are presented from experiments with phytoplankton grown i n chemostats either as u n i a l g a l or mixed species cultures, The average nutrient f l u x through each system was kept constant "but the temporal patchiness of the l i m i t i n g resource varied with respect to time, The e f f e c t s of competition and the physiology of the population are monitored to determine the importance of l i m i t i n g nutrient patchiness on the physiology, growth and competitive advantage of the various populations, Chapter VII reports an approach to the problem of competition for l i m i t i n g nutrients when the d i s t r i b u t i o n was patchy with respect to time. Growth curves for hypothetical species were generated that were dependent on the average substrate concentration i n the system and the degree of patchiness of that substrate, 6) Purpose This thesis contains r e s u l t s of several experimental approaches to phytoplankton p h y s i o l o g i c a l ecology, This makes i t , i n some respects, very general i n nature, An attempt has been made to synthesize the work into a cohesive study on the biochemical, p h y s i o l o g i c a l and e c o l o g i c a l l e v e l s of phytoplankton responses to resource f l u c t u a t i o n s , The study of marine phytoplankton at steady state has l e f t many unanswered questions about t h e i r f u n c t i o n a l r e l a t i o n s h i p to the environment. Since these organisms l i v e i n an environment which i s f l u c t u a t i n g , i t i s important to study the e f f e c t s of these f l u c t u a t i o n s on t h e i r physiology and growth. 20 Chapter I EVIDENCE FOR THE GLUTAMINE SYNTHETASE PATHWAY OF AMMONIUM ASSIMILATION 1) Summary An ammonium li m i t e d chemostat culture of Gymnodinium simplex (Lohm.) Kofoid et Swezy was perturbed with ammonium and fl u c t u a t i o n s i n the free i n t r a c e l l u l a r amino acid pools were followed 80 min. The steady-state value of glutamate was 2.07 x 10 ^ m o i - c e l l 1 and of glutamine was 0.31 x 10 ^ m o i - c e l l \ Five minutes a f t e r the perturbation, a su b s t a n t i a l r i s e i n glutamine was observed with corresponding decrease i n glutamate. A si m i l a r experiment was performed with an ammonium l i m i t e d culture of Skeletonema oostatvon (Grev.) Cleve. Two and one-half minutes a f t e r the perturbation, free i n t r a c e l l u l a r glutamate had decreased by 0.22 x 10 ^ moi - c e l l and glutamine had increased by 0.15 x 10 m o i - c e l l \ These observations are considered a r e s u l t of glutamine synthetase acting as the primary ammonium as s i m i l a t i n g enzyme. 21 2) Introduction The mechanisms of ammonium a s s i m i l a t i o n i n marine phytoplankton and higher plants have received increasing attention over the past few years. I n i t i a l l y i t was thought that glutamate dehydrogenase (GDH; EC. 1.4.1.3) was pr i m a r i l y responsible for ammonium a s s i m i l a t i o n (Basham & Kirk, 1964; Folkes & Sim, 1974; Sims & Folkes, 1964). Recent evidence indicates that the glutamine synthetase (GS; EC 6.3.1.2)/glutamate synthase (GOGAT; EC 2.6.1.53) system may be of primary importance i n ammonium incorporation, e s p e c i a l l y under ammonium l i m i t a t i o n (Arima & Kumazawa, 1977, Falkowski & Rivkin, 1976; Tempest et a l . , 1973). The pathways of nitrogen a s s i m i l a t i o n i n plants have been reviewed by M i f l i n and Lea (1976). Work by Sims and Folkes (1964) and Basham and Kirk (1964) coupled with the in vitro a b i l i t y of GDH to synthesize glutamate from a-ketoglutarate and ammonium were the main reasons f o r consider-ing GDH as the primary ammonium as s i m i l a t i n g enzyme. Further work on the k i n e t i c s of GDH i n marine phytoplankton revealed that i t had a low a f f i n i t y f o r ammonium. Eppley and Rogers (1970) showed that i n the marine diatom DityVum brightwellii (West) Grunow, GDH had a K for ammonium of 10 mM, m whereas the i n t r a c e l l u l a r ammonium pool concentration was between 5 and 10 mM. Falkowski and Rivkin (1976) found that the K of GDH for ammonium i n the m marine diatom Skeletonema oostatum (Grev.) Cleve was 28 mM, i n d i c a t i n g an unreasonably low a f f i n i t y for ammonium i f t h i s enzyme was i n fac t responsible for primary ammonium a s s i m i l a t i o n . They also suggested that the l e v e l s of i n t r a c e l l u l a r ammonium pools could e a s i l y be overestimated due to contamination and nucleotide deamination. Such an overestimation could have resulted' i n the erroneous conclusion that i n t r a c e l l u l a r ammonium l e v e l s were within the K m range of GDH. In other marine phytoplankton species, Ahmed et a l . (1977) found theammoniumK for GDH was between 4.5-10 mM. m 22 Tempest et a l . (1973) reported - that ammonium'limited bacteria- showed an i n i t i a l -increase ,in glutamine d i r e c t l y , following an ammonia perturbation -\They concluded" that the GS pathway of ammonium a s s i m i l a t i o n was operating but they did not show an i n i t i a l drop i n glutamate'and a corresponding r i s e i n glutamine. Such a covariance would be expected, over very short time i n t e r v a l s , i f the GS pathway was responsible f o r ammonium as s i m i l a t i o n (equation 1)'. " ' ' " + GS glutamate + NH. + ATP— > glutamine + ADP + P i (1) 4 divalent cation Other workers have shown that GS has a lower aiw.oniumK than GDH, thereby m suggesting that GS i s k i n e t i c a l l y more favorable for ammonium a s s i m i l a t i o n (Falkowski & Rivkin, 1976; Stewart & Rhodes, 1977). In vitro enzyme k i n e t i c r e s u l t s which provide evidence that the GS/GOGAT pathway operates but do not prove that this i s the-enzyme system functioning in vivo. Information regarding the products of ammonium a s s i m i l a t i o n i s needed. This chapter reports '"the r e s u l t s of studies on the response of ammonium l i m i t e d chemostat cultures of Gymnodinivm simplex and Skeletonema aostatvon "to an ammonium perturbation. The fl u c t u a t i o n s i n the free amino acid pools are used to elucidate the pathway of primary ammonium a s s i m i l a t i o n i n these organisms under ammonium l i m i t a t i o n . 3) Materials and Methods Gymnodinium simplex (NEPCC-119; Northeast P a c i f i c Culture C o l l e c t i o n , Department of Oceanography, The Un i v e r s i t y of B r i t i s h Columbia) and Skeleton-ema costatum (NEPCC-18b) were i s o l a t e d from P a c i f i c Ocean water samples taken on May 16, 1973 at 48°38'N, 126°00'W and June 20, 1977 from P a t r i c i a Bay, B.C., re s p e c t i v e l y . The two species were grown i n ammonium-limited chemostats i n a r t i f i c i a l seawater at 18°C as described by Davis et a l . (1973). G. "simplex was maintained i n a 6 - l i t e r , b o i l i n g f l a s k at a 23 d i l u t i o n rate of 0.25 d ^. S. costatum was maintained i n a 2 - l i t e r b o i l i n g f l a s k at a d i l u t i o n rate of 1.0 d \ The cultures were continuously s t i r r e d at 60 rpm with a magnetic s t i r r e r . Continuous i l l u m i n a t i o n was supplied by four fluorescent bulbs, three high-output V i t a l i t e (Durotest) bulbs and one day-l i g h t Powertube VHO Sylvania. This l i g h t was f i l t e r e d through a sheet of blue P l e x i g l a s (No. 2069, Rohm and Haas, P h i l a d e l p h i a ) , 0.3 cm thick to simu-l a t e the spectrum of 5 m underwater l i g h t f o r c o a s t a l areas. T o t a l irradiance -2 -1 -1 was 150 uEin«m «s . The inflow nutrient concentrations were 10 ug-at-A ammonium, 3.2 ug-at - i l ^ phosphate, 4.2 yg-at«£ ^ s i l i c a t e . In the S. costatum cultures, s i l i c a t e was added at 35 ug-at-£ Vitamins and trace metals were added as i n f medium ( G u i l l a r d & Ryther, 1962) but at a reduced concentration of f/20. Nutrient analysis was c a r r i e d out using a Technicon Autoanalyzer, and methods previously described by Davis et a l . (1973). C e l l numbers were determined by using an inverted microscope. The c e l l s were k i l l e d with a drop of Lugol's iodine s o l u t i o n before counting. Culture fluorescence was measured by a Turner (Palo A l t o , C a l i f . ) Model 111 fluorometer. Fluorescence, and c e l l and nutrient concentrations were monitored d a i l y and steady-state was obtained when there was no trend i n these parameters over a 5-day period. The ammonium perturbation consisted of shutting o f f the pump and then quickly i n j e c t i n g 3 ml of 10 mM ammonium chlo r i d e into the steady-state ammon-ium l i m i t e d chemostat culture which resulted i n a sudden increase i n ammonium concentration from 0.2 to ca. 5 yM. The disappearance of ammonium from the medium ( i . e . , the uptake) was followed during the experiment. Samples for amino acid analysis were c o l l e c t e d at 5, 10, 40 and 80 min a f t e r the perturba-t i o n . Values at time zero were the steady-state values immediately p r i o r to the perturbation. For amino acid extraction and ana l y s i s , one l i t e r of culture (500 ml i n 24 the case of S. oostatwn) was f i l t e r e d onto a 47 mm glass f i b e r f i l t e r (Reeve Angel) at 190 mm Hg negative pressure. A f t e r f i l t r a t i o n , the c e l l s and f i l t e r were washed with 10 ml 30% sodium formate s o l u t i o n to remove any seawater contaminants. At the predetermined sample time the f i l t e r was plunged into b o i l i n g 90% ethanol contained i n 15 ml screw top centrifuge tubes. The time required to f i l t e r , wash and k i l l the c e l l s was 1.5 min. The f i l t e r was broken up by vigorous shaking and vortex mixing (Vortex-Genie, Fischer, Bohemia, N.Y.). The f i l t e r paper-ethanol suspension was then centrifuged and the supernatant place i n 100 ml rotary evaporator f l a s k s . The extraction of the f i l t e r and c e l l s i n b o i l i n g 90% ethanol was repeated three times and the combined extracts were evaporated to dryness. A s p e c i a l r e f l u x tube (Buchi NS24/40) f o r amino acid analysis was used to avoid sample contamination which could occur from carryover of previous samples prepared on the same machine. The tube was rinsed with 90% ethanol before each analysis to minimize contamination. Amino acid analysis was c a r r i e d out using a Beckman 120C Amino Acid Analyzer. A c i d i c and neutral amino acids were separated using Li"*" form r e s i n which provided r e s o l u t i o n of asparagine and glutamine. The basic amino acids were separated using a 16 x 0.9 cm bed of Na + form r e s i n . Operating procedures were those outlined i n the Beckman Procedures Manual, A-TB-044, May 1967. When a standard s o l u t i o n containing 19 amino acids was extracted using the same procedures, the extraction e f f i c i e n c y ranged from 95-99%; glutamate and glutamine were >99%, whereas asparagine was ca. 95%. 4) Results Gymnodiniwn simplex: 6 — 1 The steady-state c e l l density was 25 x. 10 c e l l - 1 and the concentration of ammonium and phosphate was 0.2 and 1.5 yg-at•£ \ r e s p e c t i v e l y . The quantities of the free i n t r a c e l l u l a r amino acids following the ammonium perturbation are shown i n Table I. TABLE I . Quantities- of i n t r a c e l l u l a r free amino acids i n ammonium l i m i t e d SJymnodi.ni.ym; simp lex at time zero and a f t e r ammonium perturbation: confidence l i m i t s of analysis are ±3%. Amino acids Time (min) d o " 1 5 m o i ' c e l l 1) 0 5 10 40 80 alanine 0.7.5 0.72 1.21 ' 1.6 5 0.85 arginine ++a +++a ++ +a asparagirie ++ + +++ +4+ 4-aspartate ++ 44- 44- +4+ 44-cysteine b - - -glycine 0.39 0.45 1.18 0.65 0.55 glutamine 0.31 0.78 0.95 1.20 0.81 glutamate 2.07 1.31 1.72 1.97 1.87 h i s t i d i n e +++ 444- 0.26 44+ 44-i s o l e u c i n e + 44- 0.16 4- -leucine + 4+ 0.26 4- 4-l y s i n e 0.31 0.17 0.51 0.50 0.27 methionine + + + -phenylalanine + + 44+ 4- 4-p r o l i n e - - - -serine 0.74 0.50 1.51 0.56 0.52 threonine ++ 44- 4++ 44- 44-tryptophan - - - -tyrosine + 44- +++ 4- 4-v a l i n e 44 _ — 4-^ +, 44-, 4-44 = increasing l e v e l of d e t e c t a b i l i t y . - = not detectable. Glutamate was the most predominant free amino acid under steady-state conditions of ammonium l i m i t a t i o n with l e v e l s of 2.07 x 10 "'""'moi* c e l l "*". The other "major amino acids, at steady state, i n order of decreasing abundance were alanine, serine, glycine, glutamine and l y s i n e . Five minutes a f t e r the -15 -1 perturbation the l e v e l s of glutamate had dropped by 0.76 x 10 m o i - c e l l , whereas glutamate had increased by 0.47 x 10 "* " \ i o l - c e l l ^. The decreasing glutamate concentration recovered a f t e r 10 min coupled with a further increase i n glutamine (Table I, F i g . 2). The l e v e l s of both glutamate and glutamine increased up to 40 min and then decreased s l i g h t l y . The other major amino acids remained at more or less constant l e v e l s f o r the f i r s t 5 min following the perturbation. There was a marked increase by 10 min and then a continual decrease with time (Table I, F i g . 3). Ammonium concentration i n the culture medium remained above 4 uM throughout the perturb-ation experiment. Skeletonema costatum: The l e v e l s of two free amino acids, glutamate and glutamine, i n S. costatum before and 2.5 min following the ammoniumperturbation are given i n Table I I . During t h i s time period glutamate dropped by 0.22 x 10 "'""'moi-cell ^ . -15 -1 and glutamine rose by 0.15 x 10 m o i - c e l l 27 TABLE I I . Quantities;of. I n t r a c e l l u l a r free amino acids, glutamate and glutamine, i n ammonium l i m i t e d Skeletonema costatum at time zero and a f t e r ammonium perturbation:-confidence l i m i t s of analysis are ±3%. Amino acids Time (min) (I O " 1 5  m o i - c e l l " 1 0 .2.5 glutamate glutamine 1.28 0.79 1.06 0.94 28 0 V c 1 0 I 1 r - 1-< 0 5 10 40 80 Time (min.) Figure 2. Fluctuations i n l e v e l of free i n t r a c e l l u l a r amino acid pools i n response to addition of ammonium (perturbation at T=0) to ammonium l i m i t e d Gymnodinivm simplex: T=0 values are steady-state values immediately p r i o r to perturbation: 0 = glutamate, V = glutamine. 29 i Time (min.) Figure 3. Fluctuations i n l e v e l of free i n t r a c e l l u l a r amino acid pools i n response to addition of ammonium (perturbation at T=0) to ammonium l i m i t e d Gymnodinium simplex: T = 0 values are steady-state values immediately p r i o r to perturbation: V = serine, 0 = glycine, A = alanine, 7 = i s o l e u c i n e , and D = leucine. 30 5) Discussion If the GS pathway i s act i v e i n these two marine phytoplankton, we would expect (Equation 1) an equal molar decrease i n glutamate and increase i n glutamine immediately following the perturbation ( i . e . , a short enough time so that the GOGAT and transaminase systems would cause minimal i n t e r f e r e n c e ) . This i s what was observed. The decrease i n glutamate 5 min a f t e r perturbation for G. simplex was 0.76 x 1 0 - 1 5 m o l ' c e l l " 1 . This leaves 0.29 x 1 0 _ 1 5 m o l ' c e l l - 1 of glutamate unaccounted for i f an equal molar GS reaction occurs (Equation 1). Likewise i n S. costatum, 0.07 x 10 "'""'moi• c e l l of glutamate was unaccounted f o r . This discrepancy can be p a r t i a l l y explained by noting the s l i g h t increase i n the l e v e l s of other amino acids (glycine, leucine, tyrosine, isoleucine) i n G. simplex c e l l s , over the same period. The biosynthesis of these amino acids r e s u l t s i n loss of glutamate by the transaminase enzymes. Further interference could a r i s e from the production of glutamate by glutamate synthase but, as a r e s u l t of close agreement between the observed and expected r e s u l t s a f t e r 5 min, the contribution of GOGAT to the glutamate pool by that time was probably small. A f t e r 10 min the contribution of GOGAT to the gl u t a -mate pool appeared to be sub s t a n t i a l as indicated by the increased glutamate l e v e l s . Amino acids other than glutamate and glutamine were not quantitated i n the S. costatum experiment. If the GDH was responsible f or the primary a s s i m i l a t i o n of ammonium i n th i s organism we would expect to see an i n i t i a l r i s e i n the l e v e l s of gl u t a -mate rather than a drop. The reaction mediated by glutamate dehydrogenase i s : 31 a-ketoglutarate + NH^ + NAD(P)H + H + G D H > glutamate + NAD(P) + + H„0 (2) < 2. It would also be impossible to account f o r the rapid r i s e i n glutamine concentration i f the GDH system was s o l e l y responsible for ammonium as s i m i l a t i o n . Glutamate synthase, the enzyme responsible for the interconver-sion of glutamate and glutamine i s e s s e n t i a l l y i r r e v e r s i b l e , favoring the formation of glutamate from glutamine and a-ketoglutarate (Woolfolk et a l . , 1966). The reaction catalyzed by GOGAT i s : + GOGAT glutamine + a-ketoglutarate + NAD(P)H + H > 2 glutamate + NAD(P) + (3) In G. simplex, the responses of the other major amino acids to the perturbation, are markedly d i f f e r e n t from those exhibited by glutamate and glutamine. The r e l a t i v e l y constant l e v e l s of the other amino acids from 0 to 5 min are;consistent with the GS pathway being responsible f o r primary ammonium a s s i m i l a t i o n i n t h i s organism. The rapid increase i n the l e v e l s of these amino acids 10 min a f t e r the perturbation r e f l e c t s the rapid t r a n s f e r of the amino nitrogen throughout the amino acid pool as a r e s u l t of trans-aminase a c t i v i t y . I n t e r e s t i n g l y , glutamate never rose above i t s steady-state l e v e l . This implies that glutamate u t i l i z a t i o n increased r a p i d l y following the perturbation. The decrease i n the amino acid l e v e l s near the end of the time series was not due to ammonium l i m i t a t i o n as the ammonium l e v e l s never dropped below 4 yg-at • I 1 i n the external medium. This l e v e l i s w e l l above the for ammonium uptake f o r t h i s organism (Turpin, unpubl.). The decrease, best exhibited i n the amino acids other than glutamate and glutamine, i s undoubtedly due to some form of l i m i t a t i o n . I t i s possible, due to the low steady-state 32 growth rate (1/5 y ) of the organism that there was a reduction i n the max a c t i v i t y of many enzyme systems. This low sub-maximal growth rate could r e s u l t i n the i n a b i l i t y of the enzyme systems responsible f o r carbohydrate u t i l i z a t i o n and a4cetoglutarate production to provide an adequate source of carbon skeletons needed to allow the i n i t i a l rapid ammonium a s s i m i l a t i o n . The possible i n t r a c e l l u l a r competition for ATP-between ammonium uptake and a s s i m i l a t i o n and CO^ f i x a t i o n could also r e s u l t i n decrease of av a i l a b l e carbon skeletons (Falkowski & Stone, 1975). Ei t h e r of these p o s s i b i l i t i e s coupled with amino acid u t i l i z a t i o n , would account f o r the observed decrease i n the amino acid l e v e l s . .. The reason that a. drop i n glutamate, i n response to an ammonium addition, was not observed by other workers (Tempest et a l . , 1973) may be due to the high metabolic rates of the ba c t e r i a used and the i n a b i l i t y to obtain adequate short-term time seri e s data r e l a t i v e to the organism's metabolic rate. The experimental conditions were such that the organism's metabolic rate was slow enough to allow r e s o l u t i o n of the i n t r a c e l l u l a r glutamate drop. 33^ Chapter II LIMITING NUTRIENT PATCHINESS AS A FACTOR IN PHYTOPLANKTON ECOLOGY 1) Summary The e f f e c t of l i m i t i n g nutrient patchiness on community structure and species succession was examined i n natural phytoplankton communities held i n ammonium l i m i t e d continuous culture at a d i l u t i o n rate of 0,3 day \ Under a homogeneous d i s t r i b u t i o n of the l i m i t i n g nutrient members of genus Chaetoceros dominated but when ammonium was added d a i l y (patchy d i s t r i b u t i o n ) , SkeZetonema dominated. Intermediate patchiness gave r i s e to an assemblage dominated by both Chaetoceros and SkeZetonema. The nutrient uptake a b i l i t y of each assem-blage was determined three weeks a f t e r experiment i n i t i a t i o n . Each assemblage was best able to optimize uptake of ammonium under i t s p a r t i c u l a r patchy nutrient regime. Optimization of a patchy environment took place by an increased maximal uptake rate (V ) while optimization of a homogeneous max environment appeared to take place by increased substrate a f f i n i t y ( i . e . , low K g ) . It i s also shown that coexistence of two populations might be expected due to the patchiness of a single l i m i t i n g n u t r i e n t . The importance of pat c h i -ness i n r e l a t i o n to other factors which determine community structure i s discussed. 34 2) Introduction The importance of nutrients i n l i m i t i n g phytoplankton growth i n aquatic systems has long been r e a l i z e d . There are several mechanisms by which ambient nutrient concentrations may control phytoplankton growth and hence community structure. Species s p e c i f i c growth and nutrient uptake k i n e t i c s and a s s o c i -ated parameters (V , y and K ) have been proposed by Dugdale (1967) and TT13X TI13.X S shown by Eppley et a l . (1969) to be important i n explaining species succession. Titman (1976) and Tilman (1977) showed that ' community structure can be affected by d i f f e r e n t resource l i m i t a t i o n s ( s i l i -cate and phosphate) which act on d i f f e r e n t populations^ within the commun-i t y . This idea has also been developed i n simulation models of species comp-e t i t i o n (Taylor & Williams, 1975). Grenney et a l . (1973) suggested through t h e i r modelling e f f o r t s , that low frequency resource f l u c t u a t i o n s may r e s u l t i n an unstable coexistence of phytoplankton populations l i m i t e d by a si n g l e resource. Stross and Pemrick (1974) and Chisholm and Stross (1976) have provided a case for niche separation based on p e r i o d i c i t y i n nutrient uptake k i n e t i c s . Mickelson et a l . (1979) observed changes i n the outcome of competition between marine diatoms as a r e s u l t of changes i n continuous culture d i l u t i o n rate, or i n a more e c o l o g i c a l sense, the s p e c i f i c f l u x of the l i m i t i n g n utrient. In t h i s experimental system, species s e l e c t i o n occurs on the basis of growth k i n e t i c s . A l l the preceding studies considered only a homogeneous d i s t r i b u t i o n of the l i m i t i n g nutrient. There have been no studies on the e f f e c t of temporal patchiness of the l i m i t i n g n u t r i e n t on phytoplankton competition and growth. The a b i l i t y of various populations, or i n d i v i d u a l s of a popula-t i o n , to u t i l i z e a patchy resource could be instrumental i n mediating resource competition and hence species succession i n aquatic systems. This study was designed to answer two questions: 1) Can l i m i t i n g nutrient patchiness influence phytoplankton succession and community 35 structure? 2) If so, are there p h y s i o l o g i c a l differences ( i . e . , nutrient uptake a b i l i t y ) among the r e s u l t i n g assemblages that would allow f or optimal use of the l i m i t i n g , nutrient i n a p a r t i c u l a r patchy regime? Continuous culture systems have been used extensively by microbiologists (Jannasch, 1967, 1968a & b; Meers, 1971, 1973; Veldkamp & Kuenen, 1973; Harder et a l . , 1977) to analyze factors i n f l u e n c i n g microbial s e l e c t i o n i n mixed species systems. This technique has been adapted f o r the study of competition i n phytoplankton communities (Dunstan & Menzel, 1971; Titman, 1976; Tilman, 1977; Mickelson et a l . , 1979; Harrison & Davis, 1979). Nitrogen i s the most frequent nutrient l r m i t i n g plant growth; i n ' the sea- Of i t s inorganic forms," ammonium i s the most r e a d i l y regenerated (Dugdale, 1976; Harrison, 1978). ..M chose to examine the .effects.of ammonium patchiness on ammonium l i m i t e d natural phytoplankton assemblages ' maintained i n continuous culture. 3) M a t e r i a l and Methods The experiments were conducted at the CEPEX (Controlled Ecosystem '.' Population Experiment) s i t e at Saanich I n l e t , Vancouver Island, B r i t i s h Columbia, Canada. Experiment 1 was conducted i n July 1978 and was duplicated (Experiment 2) i n August 1978. 3.1) Inoculum: A natural assemblage of marine phytoplankton was obtained from a large c o n t r o l l e d ecosystem enclosure (CEE) as described by Menzel and Case (1977). Assemblages for Experiment 1 and 2 were obtained from a sample integrated from 4-8 m i n CEE 78-2, on July 10 and August 9, 1978, re s p e c t i v e l y , and f i l t e r e d through 153 ym Nitex netting to remove any large zooplankton. The inoculum for Experiment 2 was allowed to grow for 4 days and then a small inoculum of Skeletonema costatum (Grev.) Cleve, Thalassiosira nordenskioldii Cleve, Chaetoceros socialis Lauder and C. constrictus Gran was added so that 36 these species, which were absent from the natural sample but present i n the inoculum for Experiment 1, could be observed i n the succession sequence. 3.2) Incubation: Cultures were maintained i n outdoor continuous cultures. Three l i t e r , b o r o s i l i c a t e , f l a t bottom, b o i l i n g f l a s k s were placed i n a w a t e r - f i l l e d P l e x i g l a s incubator system s i m i l a r to that described by Davis et a l . (1973). Temperature was maintained at 13 ± 1°C by a cooling unit (Haws HR4-20). Natural sunlight was attenuated and s p e c t r a l l y corrected to simulate J e r l o v Type 3 coastal water at 5 m (Holmes, 1957) by surrounding the incubator with 1/8" blue P l e x i g l a s (Rohm and Hass, #2069) (Davis et a l . , 1973). As the culture vessels were submerged i n flowing water, i n f r a - r e d wavelengths were also removed. 3.3) Inflow medium: Two hundred l i t e r s of Saanich Inlet surface water were c o l l e c t e d on July 11, 1978. The water was f i l t e r - s t e r i l i z e d using a 147 mm, 0.45 um M i l l i p o r e f i l t e r and stored i n a 2 0 0 - l i t e r Nalgene b a r r e l i n the dark i n a cold room for the duration of the experiment. Nutrient analyses indicated that the water had 0.5 yg-at-£ 1 t o t a l inorganic nitrogen (NO^, NOv;, NH^) -1 -«+ -1 -3-7.5 yg-at'£ SiO ^ . and 0.4 yg-at•£ PO.^- This water was used as a stock supply throughout the experiment. Aliquots were removed and enriched to the desired inflow concentrations as needed. Samples of the inflow medium were taken r e g u l a r l y to check nutrient concentrations, 3.4) Patchiness regimes: Three continuous cultures were set up as outlined below, with a constant d i l u t i o n rate of 0.3 day maintained by piston pumps (Fl u i d Metering, Inc.). Each culture received the same amount of ammonium each day and only the temporal d i s t r i b u t i o n of ammonium varied, from continual addition, 8 additions/day to 1 addition/day. 37 a) System 1: Continual addition (Fig. 4). Inflow medium for Experiment 1 -1 -1 was enriched to 10 ug-at.£ ammonium chloride, 3 yg-at.£ potassium phosphate (monobasic) and 20-45 ug-at-£ sodium s i l i c a t e . Vitamins and trace metals were added as f/25 ( G u i l l a r d & Ryther, 1962). The inflow medium during -3 -4 Experiment 2 was i d e n t i c a l to Experiment 1, except PO ^ and SiO were increased to 3.5 and 50 yg-at-£ \ .respectively. b) System 2: 8 additions/day (Fig. 4). D i l u t i o n rate and nutrient concentrations i n the inflow medium were i d e n t i c a l to System 1 except the l a t t e r contained no added NH^Cl. The NH^Cl additions were co n t r o l l e d by a separate pump which was turned on and off with a s p e c i a l l y modified timer (C i n c i n n a t i , Model 422). Additions were 1 min i n duration (0.94 ml of 1.2 mM NH^Cl) and occurred every 3 hr s t a r t i n g at 2400 hr. This resulted i n the d a i l y nitrogen f l u x being i d e n t i c a l to that of System 1 ( i . e . , 3 yg-at'£ day 1). This system, with i t s independent flow of seawater and non-limiting n u t r i e n t s , assured that the only v a r i a b l e between the two systems was the temporal d i s t r i b u t i o n of ammonium. c) System 3: 1 addition/day (Fig. 4). This system .was s i m i l a r to System 2, except the NH^Cl addition was at 0100 hr and consisted of 7.5 ml of 1.2 mM NH^Cl over a 1 min i n t e r v a l . The d a i l y ammonium f l u x through t h i s system was the same as the other systems (3 yg-at-£ "^day "*") . 3.5) Measurements: Culture e f f l u e n t s were c o l l e c t e d d a i l y and preserved for i d e n t i f i c a t i o n and enumeration i n Lugol's iodine. An inverted microscope was used for enumer-ation of samples. Fluorescence was monitored with a fluorometer (Turner Model 111) equipped with a high s e n s i t i v i t y door. Nutrients i n culture e f f l u e n t s were analyzed with a Technicon Autoanalyzer using methods previously described (Davis et a l . , 1973). Aft e r approximately 3 weeks of exposure to the nutrient regimes, the responses of the r e s u l t i n g assemblages to a nutrient pulse or perturbation 38 T i m e r I — J - — ; — S y s t e m 1 System 3 Sys tem 2 Figure 4. A schematic representation of the three culture systems. A l l systems were continuous flow (D = 0.3 d "*") . System 1 received NH^Cl continually i n the inflow medium; System 2 received 8 additions/day, while System .3 received 1 addition/day of NH^Cl. The f l u x of NH^Cl through a l l Systems was 3 yg-at•£ "*".day ''".-.The reactors' were, i n a water bath (13 + 1°C) as described i n the text. 39 (Caperon & Meyer, (1972b),was determined. Nutrient disappearance was followed with a Technicon Autoanalyzer. The a b i l i t y of each phytoplank-ton assemblage to respond to the various patchiness regimes was deter-mined i n t h i s manner. 4) Results 4.1) Experiment 1: a) Ambient nutrients: The continuous flow pumps were started a f t e r the inoculum had grown as a batch culture for 1 day. On day 2, " ^ ambient ammonia concentrations (measured each day between 1000-1200 hr) reached a maximum of 1.3, 0.8 and 0.9 yg-at•£ 1 for Systems 1, 2 and 3, re s p e c t i v e l y . For the rest of the experiment, ambient ammonium con-" centration never rose above 0.6 yg-at-£ ^. S i l i c a t e concentrations i n the inflow medium were raised p e r i o d i c a l l y throughout the experiment to' keep the"ambient l e v e l s w ell above s i l i c a t e l i m i t a t i o n s (Fig. 5). A f t e r day 7 there was a d i s t i n c t trend i n the ambient s i l i c a t e concentration with System 1 having;, the... highest concentration followed by 2 and 3, r e s p e c t i v e l y (Fig. 5). b) Community structure: The composition of the i n i t i a l community i s i l l u s t r a t e d i n Table I I I . Species present were Skeletonema costatwn} Chaetoaevds simplex' Ostenfeld, C. simile Cleve, C. eompvessus Lauder, C. eonstrictus} C. debilis Cleve, Thalassiosiro .votula Meunier, T. nordenskioldiiNitzsehia longissima (de Brebisson ex Kutzing) R a l f s , N. pungens Grunow, N. delicatissima Cleve, N. palea (Kutzing),W..Smith, Stephanopyxis tuvvis (Grev.) R a l f s , Cevataulina bevgonii (H. Peragallo) Schutt, small f l a g e l l a t e s and a few representatives of other diatom genera such as Rhizosolenia and Leptooylindvus. 4 0 . : i In f low Si l icate ( u g - a t - f 1 ) 2 1 2 3 2 0 3 0 35 4 0 4 5 r T I M E (DAYS) Figure 5. Inflow and outflow s i l i c a t e concentrations (Exp. 1). System 1, (0); System 2, ( O ) ; System 3, ( A ) . 41 Table I I I . The compostion of the i n i t i a l ' i n o c u l a for Experiments 1 and 2. Exp. 1 (xlO 6 c e l l - l " 1 ) Exp. 2 (xlO 6 c e l l - l - 1 ) SkeZetonema 0. 63 1.4 Chaetoceros 3.5 3.4 T h a l a s s i o s i r a 0.92 5.9 Stephanopyxis — 2.4 Cerataulina — 0.7 Nitzschia 0.96 1.4 F l a g e l l a t e s 1.6 8.6 Others 1.4 — TOTAL 9.01 23.5 42 The community structure i n a l l 3 systems remained constant f o r the f i r s t 3 days as evidenced by: 1) the domination by the genus Chaetoceros, and, 2) the constant r e l a t i v e abundances of SkeZetonema and Chaetoceros (Figs. 6A, B, C & 7A,B), f By day 5, $keJL&tonzma. w a s approximately 5 times more numerous i n the patchy systems (2 & 3) than i t was i n System 1 (Fig, 6A,B.,C). From day 9 to the termination of Experiment 1, SkeZetonema and Chaetoceros accounted for 65% to 85% of the t o t a l c e l l numbers i n a l l three treatments. The primary difference among the f i n a l assemblages was the r e l a t i v e proportions of Chaetoceros and SkeZetonema present, Chaetoceros continued to dominate System 1 u n t i l the end of the experiment (Figs, 6A & 7B). System 2 (8 additions/day) was dominated by Chaetoceros u n t i l day 16 a f t e r which SkeZetonema was the most abundant. Chaetoceros was dominant u n t i l day 9 i n System 3 (1 addition/day) with SkeZetonema dominating thereafter. An ammonium l i m i t e d semi-continuous culture was started at the same time as Experiment 1, It was d i l u t e d once a day and had the same turnover rate and ammonium f l u x as System 3, and thus, t h i s semi-continuous culture served as a r e p l i c a t e . After 3 weeks both Systems 3 and the semi-continuous culture r e p l i c a t e d very well (Figs. 7A & B) when considering the treatment differences, c) Nutrient uptake: Uptake rates were measured at the end of each experiment. The a b i l i t y of each culture to procure ammonium under the ammonium addition regimes of the other systems was determined. For example, System 3 received an addition of ammonium 'each.: day at. QiQQ-hr bringing the. reactor concentration to 3 ug-at'£ \ Therefore, t h i s addition was made to aliquots of each of the 3 cultures at 0100 hr and the disappearance of ammonium i n the dark was followed, 43 150 0 6 . 8 10 12 TIME-(DAYS) 14 16 18 20 Figure 6 . Accumulative c e l l numbers (Exp. 1) Thalassiosira, (0) ; F l a g e l l a t e s ( V ) ; Nitzsohia, ( 0 ) ; Chaetoceros, ( V ) ; Skeletonema, ( • ) ; Total numbers, ( a ) A, System 1 : B, System 2 : C, System 3 . 44 1004 fll £ c o -f-> tol 804 1004 in o L_ U| o Si ro SZ U Figure 7. Relative c e l l numbers (Exp. 1). System 1, (o); System 2, ( o ) ; System '3, ( A ) ; semi-continuous culture (A) (see t e x t ) : A, percent Skeletonema: B, percent Chaetocevos. 45 The decreases i n ammonium concentration with time f o r the 3 systems i s seen i n F i g , 8, The system previously exposed to t h i s once a day addition (System 3) had the highest uptake. System 2 was exposed to pulses of lower concentration but s t i l l exhibited f ar higher uptake than did System 1 which was subject to a r e l a t i v e l y homogeneous ammonium d i s t r i b u t i o n over the preceding 3 weeks, This uptake experiment was repeated during the day at 1200 hr, The pattern of uptake and magnitude of the rates during the day (Fig, 9) were e s s e n t i a l l y the same as those at night, with System 3>2>1, i n ranked order of a b i l i t y to take up the large pulse of the l i m i t i n g nutrient. The uptake c h a r a c t e r i s t i c s of a l l cultures when exposed to the addition regime of System 2 were examined by repeating the perturbation experiments •. with a smaller nutrient addition (0.38 yg-at•£~ 1) so that the ambient ammonium concentration was equivalent to the additions to System 2, At t h i s low NH^Cl concentration, the uptake of System 2 was so rapid that no ambient ammonium • was detected 2 min a f t e r the addition. It was, however, s t i l l detectable i n aliquots from Systems 1 and 3, In another experiment to further assess t h e i r rapid uptake a b i l i t i e s , a larger addition of NH^C1(1 yg-at • £ was added to another aliquot from each system. The ambient NH^ concentration was 0.36 yg-at•£ \ 2 min a f t e r the perturbation of System 2. Both Systems 1 and 3 had su b s t a n t i a l l y more ammonium remaining af t e r a s i m i l a r 2 min incubation. 4.2) Experiment 2: A dup l i c a t i o n experiment was i n i t i a t e d a few days a f t e r the termination of Experiment 1, Systems 2 and 3 were l o s t i n an accident. The differences i n the species composition between t h i s inoculum and that of Experiment 1 are shown i n Table I I I . The community structure of System 1, expressed as r e l a t i v e number of Skeletonema and Chaetocevos , i s shown i n Figure 10, The fact that Chaetocevos dominated over Skeletonema throughout the experiment 46 co i 3 . 4 -z O 250 TIME (MIN.) Figure 8. Disappearance of ammonium with time, following an ammonium perturba-t i o n at 0100 hr (Exp. 1). System 1, (•); System 2, ( V ) ; System 3, (0). ^ 5-i D) 250 TIME (M I N.) Figure. 9. Disappearance of ammonium with time, following an ammonium perturba-ti o n at 1200 hr (Exp. 1). System 1, (•); System 2, ( V ) ; System 3, (0). 48 100 ? 8 0 -0 2 4 6 8 10 12 14 16 18 20 Tl ME ( D A Y S ) Figure 10. Relative diatom numbers (Exp. 2, System 1): percent Skeletonema (• ) and Chaetocevos (•). .49 points to the high degree of consistency i n these data, even though both the I n i t i a l inofiulun(Table III) and the irradiance (Fig..11) d i f f e r e d markedly from that i n Experiment "1. 5) Discussion The primary difference i n community structure among the f i n a l assemblages was i n the proportion of SkeZetonema and Chaetoeevos present. D i f f e r e n c e s . i n f i n a l " composition must, therefore, have been mediated through differences i n competitive a b i l i t y f o r the l i m i t i n g nutrient over the range of patchiness presented. The reason that System 2 took Up low concentration pulses f a s t e r than System 3 (and yet the opposite holds true for high nutrient concentration pulses)' may'be due.to -the" complexity of the uptake -mechanism i t s e l f . Conway et a l . (1976) and Conway and Harrison (1977) showed that a f t e r addition of the l i m i t i n g nutrient to a phytoplankton culture there i s often an i n i t i a l , s h o r t - l i v e d , rapid uptake, followed by a more constant, possibly i n t e r n a l l y or feedback co n t r o l l e d uptake rate. Due to the dual . .. nature of the uptake mechanism, the preceding observations could be explained i f System 2 had the more rapid i n i t i a l uptake rate, but System 3 had; the more rapid i n t e r n a l l y c o n t r o l l e d or long term uptake rate (see also . ... . Appendix I I ) . The high uptake rate of the assemblages exposed to a patchy regime in d i c a t e d, s e l e c t i o n of populations best able to procure the l i m i t i n g nutrient i n i t s patchy d i s t r i b u t i o n . It can be i n f e r r e d that assemblage 1 with i t s low maximal uptake rate must therefore compensate by having a lower for ammonia than e i t h e r of the other assemblages. If t h i s were not the case, assemblage 1 could not have been selected for on the basis of nutrient uptake and growth a b i l i t y . Mickelson et a l . (1979) have used the same reasoning to rank growth k i n e t i c s of 3 species of diatoms based on t h e i r competitive a b i l i t i e s . 50 TIME (DAYS) Figure 11. Surface l i g h t i n t e n s i t y (400-700 nm). was used to reduce i t by 50% during experiments. Experiment 2, (•). A l i g h t f i l t e r (see text) Experiment 1, (0)-; 51 The r e s u l t s obtained here are consistent with other published work on phytoplankton physiology and competition. Conway and Harrison (1977) showed that when ammonium l i m i t e d Skeletonema oostatwn was perturbed with an addition i t was able to take up the pulse more r a p i d l y than a s i m i l a r culture of Chaetocevos debilis. Mickelson et a l . (1979) showed that i n ammonium li m i t e d continuous cultures (with a homogeneous ammonium d i s t r i b u t i o n ) Chaetocevos can, i n some cases, out-compete Skeletonema. 5.1) R e p l i c a b i l i t y : In any study i t i s important to draw attention to the reproducibility, and consistency of the r e s u l t s . In t h i s study, three treatments (3 v a r i a t i o n s i n the temporal d i s t r i b u t i o n of the l i m i t i n g nutrient) were employed. There are three sets of observations that i n d i c a t e consistency among treatments: F i r s t i s the trend i n community structure, with assemblage 1 being dominated by Chaetocevos; assemblage 2 codominated by Chaetocevos and Skeletonema and assemblage 3 by Skeletonema; second* the trend i n ambient s i l i c a t e concentrations, and t h i r d , the trend i n community physiology. R e p l i c a b i l i t y was demonstrated by a semi-continuous culture (d a i l y .. d i l u t i o n ) most c l o s e l y approximating System 3. The r e s u l t i n g trends i n community structure were very consistent (Fig. 7) i n s p i t e of the treatment differences (semi-continuous d i l u t i o n , daytime ammonia addition compared with continuous d i l u t i o n , nighttime ammonia add i t i o n ) . The d u p l i c a t i o n of System 1 i n Experiment 2 showed the same trend-in_community.structure: as'. System 1 i n Experiment 1, even though the inoculum and l i g h t conditions varied greatly. The i n t e r n a l consistency of the data and the r e p l i c a t i o n and d u p l i c a t i o n of predominant trends, indicates the consistency of the r e s u l t s as a function of the given treatments. 52 5.2) E c o l o g i c a l considerations: Limiting nutrient patchiness can occur by many mechanisms. The regeneration of nutrients i n the euphotic zone has been shown to be an important source of nutrients for phytoplankton growth (Eppley ,et: a l . , 1973; Dugdale, 1976). Since nutrient regeneration may occur at point sources, concentration gradients and patches may be maintained for some time due to the low contribution of turbulence to d i s s i p a t i o n at small s i z e scales. Upwelling, runoff, advection, d i e l zooplankton migration and other phenomena can cause large scale temporal and s p a c i a l nutrient patchiness. Nutrient patchiness would then appear to be a phenomenon that occurs on scales from micrometers (bacteria, zooplankton) to kilometers (upwelling and r u n o f f ) . These experiments showed that, under i d e n t i c a l d a i l y nutrient fluxes, the outcome,-of competition between the two dominant populations, SkeZetonema and Chaetoceros, was mediated by the patchiness of the l i m i t i n g nutrient.. Figure 12 shows a simple competition scheme where the competitive advantage of each group i s expressed as a f u n c t i o n o f patchiness. The two curves should.intersect.and the point of i n t e r s e c t i o n . represents the degree of patchiness mediating coexistence of the two groups. This could account for apparent long term coexistence seen i n natural systems where more than one organism i s l i m i t e d by a si n g l e resource. Optimization of a patchy l i m i t i n g nutrient environment, over the range tested, appears to occur by an enhanced maximal uptake rate (V ) while ' r r max adaptation to a homogeneous l i m i t i n g nutrient system appears to be more a function of substrate a f f i n i t y ( K g ) . Neither of these adaptive mechanisms should be considered mutually exclusive as uptake i s s t i l l a function of both V and K , at any nonsaturating substrate concentration. max s -53 Figure 12. SkeZetonema Possible changes in competitive advantage between Chaetoceros and as a function of ammonium patchiness. There are two. factors' of importance i n the outcome of competition for a patchy resource. The f i r s t are differences i n i n t e r s p e c i f i c n u t r i e n t uptake a b i l i t y . These inherent differences i n the genetic make-up of d i f f e r e n t phytoplankton species give r i s e to o v e r a l l differences i n compet-i t i v e a b i l i t y (Doyle, 1975). Superimposed on t h i s v a r i a b i l i t y i s i n t r a -s p e c i f i c v a r i a b i l i t y mediated by such factors as l i f e cycle stage (Davis et a l . , 1973), growth rate (Eppley & Renger, 1974; Turpin, unpubl.), past h i s t o r y (Chapter 4), l i g h t quantity and q u a l i t y and temperature. When a s h i f t i n communi structure i s observed, however, i t can be concluded that the i n t e r s p e c i f i c differences are of greater importance. The importance of temperature (Eppley, 1972) and l i g h t (Ryther, 1956) i n the establishment of upper l i m i t s f o r phytoplankton growth and a f f e c t i n g competition i s well documented (Goldman & Ryther, 1976). Under conditions of c o n t r o l l e d l i g h t and temperature i t has been shown that the s p e c i f i c f l u x of the l i m i t i n g resource can a f f e c t the outcome of competition (Meers, 1971; Harder et a l . , 1977; Mickelson, In Press; Mickelson et a l . , 1979; Harrison & Davis, 1979). A schematic representation of the e f f e c t s of the s p e c i f i c nutrient f l u x of the l i m i t i n g nutrient on community structure i s given i n F i g . 13. Once the general community structure has been set by the s p e c i f i c f l u x of the l i m i t i n g nutrient factors such as patchiness, fine-tune the system with respect to determining population dominance. The s p e c i f i c f l u x i n these systems was such that i t selected for fast-growing c e n t r i c diatoms. The patchiness imposed, determined which c e n t r i c s would dominate. At the same time that one nutrient i s l i m i t i n g f o r some species, other species may be l i m i t e d by other nutrients (Titman, 1976; Tilman, 1977; Rhee, 1978). The same argument f o r community fine-tuning by patchiness can be used for populations l i m i t e d by any resource. The importance of d i f f e r e n t i a l sinking and herbivore grazing (Steele & Frost, 1977) cannot be ignored as 5 5 ' Cent r i c Skeleton^ rha Chaetoceros .Thalassiosira Cyl inpir^hecai p e n n a t < 2 ..N.itzschia J Pyramirhonas Chrbbhibnas D O Q 9 X J +-> cs oo - M — 03 C9 C D) p ns b tl ° Q Specific N u t r i e n t Flux Figure 13. A schematic representation of the possible r e l a t i o n s h i p between s p e c i f i c nutrient f l u x (nitrogen) and community structure. 56 as factors that would have to be included i n any model accounting for phytoplankton d i v e r s i t y . Based on e a r l i e r arguments that coexistence could be expected on a s i n g l e resource due to patchiness, one would expect the maximum t h e o r e t i c a l number of coexisting species to be equal to two times the number of l i m i t i n g resources. When considering a l l these previously mentioned factors i n addition to d i v e r s i t y maintenance by allelopathy (DeFreitas & Frederickson, 1978) and the contemporaneous d i s e q u i l i b r i u m hypothesis ,of Richerson et a l . (1970), there i s seemingly l i t t l e reason to invoke a "paradox of the plankton" (Hutchison, 1961). There are several p o t e n t i a l p r a c t i c a l a p plications of l i m i t i n g n u t r i e n t patchiness i n manipulating phytoplankton communities. By using the correct frequency of nutrient additions coupled with correct nutrient fluxes and r a t i o s , favorable species may be selected f or use i n aquaculture systems. The Great Central Lake f e r t i l i z a t i o n project (Takahashi & Nash, 1973) has resulted i n enhanced f i s h y i e l d s following a r e a l bombing of the lake with nutrients (Stockner, pers. comm.). A l t e r a t i o n of the frequency of bombing could lead to s e l e c t i o n of more favorable primary producers which i n turn might r e s u l t i n enhanced herbivore production and increases i n f i s h y i e l d s . The recent work by Marra (1978a; 1978b) indic a t e d the p o t e n t i a l importance of f l u c t u a t i n g l i g h t regimes i n terms of primary p r o d u c t i v i t y . Both Marra's work and t h i s study seem to show that i n t e r p r e t a t i o n of growth' only as a function of constant environmental c o n d i t i o n s " w i l l not lead to the accurate understanding and p r e d i c t i o n of phytoplankton dynamics i n the sea. The environment of a phytoplankton c e l l i s continually f l u c t u a t i n g and-, therefore, work should begin to focus on understanding the growth dynamics of these organisms under f l u c t u a t i n g environmental conditions. 57 Chapter I II CELL SIZE MANIPULATION IN PHYTOPLANKTON ASSEMBLAGES 1) Summary In cultures of natural phytoplankton and a mixed culture of diatoms, the mean c e l l diameter that was selected f o r i n species competition experiments was rela t e d to the time between l i m i t i n g nutrient (ammonium) additions ( i . e . , temporal patchiness). The mean c e l l s i z e increased as the frequency of the nutrient addition decreased. The p o s s i b i l i t y that l i m i t i n g nutrient p a t c h i -ness may be of some importance i n c e l l s i z e s e l e c t i o n i n nature i s also discussed. 58 2) Introduction Factors regulating the c e l l s i z e of phytoplankton i n the sea have been discussed (Malone, 1971; Semina, 1971, 1972; Parsons & Takahashi, 1973b; Laws 1975) and there has been considerable controversy as to t h e i r importance (Parsons & Takahashi, 1973b; Hecky & Kilham, 1974; Parsons & Takahashi, 1974; Malone, 1975; Parsons & Takahashi, 1975). Phytoplankton c e l l s i z e has been suggested to be important i n determining trophic l e v e l structure and the e f f i c i e n c y of food chain energy transfer (Parsons et a l . , 1967; Ryther, 1969; Parsons & LeBrasseur, 1970; Greve & Parsons, 1977). I f t h i s hypothesis i s to be tested i t w i l l require the manipulation of c e l l s i z e i n natural phytoplankton assemblages. This i s not possible at present. This chapter reports how c e l l s i z e was manipulated i n diatom-dominated laboratory cultures and i n cultures of natural phytoplankton populations, by changing the temporal patchiness of the l i m i t i n g nutrient (ammonium). 3) Methods A natural phytoplankton sample taken from 4-8 m i n a c o n t r o l l e d ecosystem enclosure (CEE) at the CEPEX s i t e (Menzel & Case, 1977) was f i l t e r e d through a 150 ym mesh to remove any large zooplankton. Diatoms predominated i n the sample and the most numerous species were ThcLtaiA^OA-iAJX. noK.&QM&\lLoL<iLL, ChaCLtodQAOi) spp. and SkoZoXonma. COStotum. U n i d e n t i f i e d small f l a g e l l a t e s made up < 40% of the sample c e l l numbers. Further d e t a i l s of the composition of t h i s i n i t i a l sample are given i n Experiment 2 i n Chapter 2, p. 41. Cultures were grown: i n 3 - l i t e r f l a s k s and incubated at 13 it 1°C i n a w a t e r - f i l l e d P l e x i g l a s incubator system (Ch. 3). Natural sunlight was attenuated by 50% and s p e c t r a l l y corrected by surrounding the incubator with 1/8" blue P l e x i g l a s . D e t a i l s of the incident r a d i a t i o n during the experimental period are given i n Experiment 2 i n Chapter 2, p. 50. 5 9 The culture medium consisted of a 2 0 0 - l i t e r sample c o l l e c t e d from 0-4 m i n the CEE and f i l t e r - s t e r i l i z e d using a membrane f i l t e r (0.45 ym) . This water was found to be nitrogen depleted (<0.5 yg-at.«£ \)' and was' enriched to give f i n a l concentrations of ammonium chlo r i d e , 10 yg-at-£ potassium phosphate -1 -1 (monobasic), 3 yg-at•£ ; s i l i c a t e , 50 yg-at•£ and vitamins and trace metals to f/25 ( G u i l l a r d &. Ryther, 1962). The natural phytoplankton sample described above was used to inoculate three outdoor cultures (Exp. 1) i n which the l i m i t i n g n u t r i e n t , ammonium, was added continuously i n one culture and semi-continuously at d i f f e r e n t time i n t e r v a l s i n the other two cultures. Culture 1 was a continuous flow culture with a d i l u t i o n rate of 0.3 day Culture 2 was a semi-continuous culture,, d i l u t e d every day ( d i l u t i o n rate = 0.3 day "*") with inflow medium i n which the ammonium enrichment of 10 yg-at•£ 1 was omitted. This culture received i t s ammoniumsupply as a discre t e pulse of 9 yg-at•£ 1 every 3 days. Culture 3 was i d e n t i c a l to culture 2 except that 21 yg-at•£ 1 ammoniumwas added once every 7 days. A comparison of the treatments used i n a l l three cultures indicates that both the d i l u t i o n rate and the l i m i t i n g nutrient f l u x (21 yg-at NH*-week ^) wore i d e n t i c a l i n a l l three cultures and only the frequency of additions (temporal d i s t r i b u t i o n ) of the l i m i t i n g nutrient varied. A s i m i l a r experiment (Exp. 2) was conducted i n the laboratory as a test of d u p l i c a t i o n . Cultures were grown i n a l i g h t regime of 16L:8D and an i r r a d --2 -1 iance of 150 yEin'm -sec . U n i a l g a l cultures of Chaetocevos sp. (#277), Skeletonema costatvm (#18b) and Thalassiosiva novdenskioldii (#252) .(North-east P a c i f i c Culture C o l l e c t i o n , Department of Oceanography, U.B.C.) were mixed together and maintained i n three cultures as described i n Experiment 1, with the exception of culture 1 which was a semi-continuous cul t u r e , d i l u t e d d a i l y . Since the temperature was higher (18°C) i n t h i s experiment, the d i l u t i o n rate was increased to 0.5 day 1 i n order to achieve a s i m i l a r degree of nitrogen l i m i t a t i o n as used i n Experiment 1. Nitrogen fluxes were 6 0 adjusted so the weekly ammonium f l u x (35 yg-at-£ """) through a l l cultures was i d e n t i c a l . Samples of the inflow medium were taken r e g u l a r l y to check expected nutrient concentrations and samples from the cultures were frequently analyzed to determine ambient nutrient concentrations. A f t e r three weeks of treatment, the .organisms 'present i n both Experiments 1 and 2 were i d e n t i f i e d and counted. C e l l volumes of the most dominant species were calculated from measurements of c e l l dimensions of 50 c e l l s , using an eyepiece micrometer and an inverted microscrope. 4) Results and Discussion Nutrient analyses of the culture e f f l u e n t indicated that the ammonium was undetectable i n the continuous flow culture. In culture 2, the 9 y g - a f £ _ 1 addition of ammonium f e l l below detectable l e v e l s by the end of the f i r s t day and as a r e s u l t i t was N-starved for the following 2 days u n t i l another pulse was given. Culture 3 depleted the 21 yg-at•£ 1 ammonium addition in•two days, r e s u l t i n g i n a -5-day period of N^ataxyation before the next weekly ammonium pulse. Figure 14 shows the mean c e l l diameter of the dominant species (>85% of culture biomass) from Experiments 1 and 2 as a function of the time between ammonium additions. There i s a s i g n i f i c a n t increase ( t - t e s t , p=.01) i n c e l l s i z e over the range of treatments. Experiment 1, System 1 (continual addition) was dominated by Chaetocevos sp., System 2 by Skeletonema costatwn and System 3 by Thalassiosiva novdenskiold-ii. I t i s also of i n t e r e s t to note that i n addition to the trend of increasing c e l l s i z e with low frequency patchiness, the large c e l l s , such as Thalassiosiva and Skeletonema > formed chains, whereas the small. Chaetocevos remained s i n g l e - c e l l e d . Experiment 2 demonstrated the same trend as Experiment 1 with the mean c e l l diameter increasing with the time between ammonium additions (Fig. 14). 61 2 3 4 T I M E (DAYS) Figure 14. Mean c e l l diameter as a function of the time between ammonium additions for Exp. 1 (0) and Exp. 2 (•). The mean c e l l diameters between each treatment "in each experiment were s i g n i f i c a n t l y , d i f f e r e n t (p = .01). Mean c e l l diameter was calculated from the mean c e l l volume assuming an equivalent sphere. Skeletonema, however, washed out of a l l cultures soon a f t e r the experiment was i n i t i a t e d . This was probably due to the poor condition of the inoculum 1. Culture 1 was dominated by Chaetocevos sp.;, i n culture 2, Chaetocevos sp. and Thalassiosiva novdenskiold-ii codominated, while culture 3 was dominated by T. novdenskoldii. These r e s u l t s are consistent with other recent studies. Nitrogen additions were made on a weekly basis to the nitrogen-limited CEE community from which the i n i t i a l inoculum for Experiment 1 was taken. The dominant phytoplankters over a 60-day period were generally l a r g e - c e l l e d diatoms, such as Stephanopyxis tuvvis, Cevataulina bergonii and Thalassionema nitzschiodes (Parsley & Davis, pers. comm.). Mickelson (in press) found that over a wide range of d i l u t i o n rates (continual l i m i t i n g nutrient addition) i n nitrogen-l i m i t e d continuous cultures, small c e l l s dominated. He suggested that c e l l s i z e must be determined, therefore, by factors other than nutrient supply rates. Data from Eppley et a l . (1969) suggest that small c e l l s have a d i s t i n c t advantage i n si t u a t i o n s i n which there i s a constant supply of the l i m i t i n g nutrient because of t h e i r lower values compared to large c e l l s . When the nutrient supply becomes patchy, large c e l l s apparently gain the advantage. Even though r e s p i r a t i o n rates were not determined i n t h i s study, i t i s tempt-ing to suggest that i n a low-frequency patchy environment, small c e l l s , with a higher s p e c i f i c r e s p i r a t i o n rate (Laws, 1975; Banse, 1976) tend to "burn themselves up" before the appearance of the next nutrient pulse. If r e s p i r a -t i o n i s a s i g n i f i c a n t f actor i n species s e l e c t i o n , the competition scheme that has been proposed for high-frequency l i m i t i n g nutrient patchiness (Turpin & Harrison, 1979; Ch. 3) could be expanded to include the possible e f f e c t of size-dependent r e s p i r a t i o n losses at lower frequency 1 The Skeletonema inoculum appeared to be i n poor health. C e l l s were very t h i n , chains were short and often clumped. This was i n contrast to the large vigorous c e l l s seen i n the natural sample. 63 patchiness (Fig. 15). The r e s u l t s of these indoor and outdoor culture studies i n d i c a t e that by varying the frequency of addition of the l i m i t i n g n u t r i e n t , natural phyto-plankton populations can be manipulated to produce communities dominated by e i t h e r large or small c e l l s . Since nutrient f l u x i s a coarse tuning v a r i a b l e tending to regulate s e l e c t i o n between phytoplankton groups (Turpin & Harrison, 1979; Ch. 3), i t may be possible to study c e l l s i z e s e l e c t i o n within f l a g e l -l a tes by using a much lower d i l u t i o n rate than was used i n t h i s study. The r o l e of pulsed nutrient supplies i n determining c e l l s i z e i n natural systems i s not known. The importance of grazing ( M c A l l i s t e r et a l . , 1960; Parsons et a l . , 1967; Malone, 1971; Steele & Frost, 1977) and sinking (Semina, 1972) i n c o n t r o l l i n g c e l l s i z e is.not to be denied, Nevertheless, i t does seem possible, that a wide range i n temporal patchiness of the nutrient supply may control s e l e c t i v e ce'll s i z e growth i n the sea. Increasing Cell Size D e c r e a s i n g S p e c i f i c — " • • —1 ••-R e s p i r a t i o n R a t e ? N H 4 Patchiness D) co +-> c ro I n c r e a s i n g m a x i m a l = 1>-U p t a k e r a t e ( V m a x ) I n c r e a s i n g S u b s t r a t e A f f i n i t y ( l o w K s ) Figure 15. Possible changes i n competitive advantage between three marine phytoplankton as a function of ammonium patchiness. 65 Chapter IV RESPONSE OF AMMONIUM LIMITED SkeZetonema costatum AND Chaetoceros graciZe TO LIMITING NUTRIENT PATCHINESS 1 ) Summary The e f f e c t s of pulsed ammonium additions on the ammonia l i m i t e d marine diatoms, SkeZetonema costatum and Chaetoceros graciZe were examined. It was found that ammonium patchiness produced p e r i o d i c i t i e s i n carbon a s s i m i l a t i o n and in vivo fluorescence. Changes i n nutrient uptake a b i l i t y under varying l i m i t i n g nutrient patchiness regimes ind i c a t e that a given population may be able to adapt i t s nutrient uptake c h a r a c t e r i s t i c s , thereby optimzing the temporal d i s t r i b u t i o n of the l i m i t i n g resource. S. costatum shows a greater a b i l i t y to u t i l i z e a pulse of ammonium than C. graciZe. This evidence i s consistent with the outcome of the competition studies i n Chapter 2. 66 2) Introduction In Chapters II and I I I , l i m i t i n g nutrient patchiness was examined as a fac t o r a f f e c t i n g the outcome of competition i n phytoplankton assemblages. It was shown that the temporal d i s t r i b u t i o n of the l i m i t i n g nutrient affected the outcome of competition and the r e s u l t i n g p h y s i o l o g i c a l c h a r a c t e r i s t i c s of the community. The observation that the assemblage dominating under patchy conditions had a higher V than the one dominating under homogeneous condi-max tions, suggested that i n t e r s p e c i f i c v a r i a b i l i t y i n the nutrient uptake char-a c t e r i s t i c s was of great importance i n raffecting competition. The remaining questions were: to what extent can a given species adapt to a given patchy environment, and to what degree does i n t r a s p e c i f i c v a r i a b i l i t y allow for optimization of a patchy environment? In attempts to answer these questions, two species of marine phytoplankton were grown i n u n i a l g a l continuous cultures under a range of ammonium patchiness conditions. Their p h y s i o l o g i c a l response to these t r e a t -ments was monitored through nutrient uptake and photosynthesis experiments, i n addition to the monitoring of culture fluorescence and c e l l numbers. The marine diatoms, SkeZetonema *eo,stectum and Chae.toceros gvaciZe> were, studied, to gain insight into;the p h y s i o l o g i c a l mechanisms contributing to the out^ come of competition-observed'in studies i n Chapter 2. In those studies S. costatum dominated under the patchy conditions and a small- Chaetoceros sp. , s i m i l a r to C. graciZe, dominated under the homogenous nutrient-conditions. 3) Materials and Methods The inocula were obtained from the Northeast P a c i f i c Culture C o l l e c t i o n (NEPCC) at The Uni v e r s i t y of B r i t i s h Columbia (SkeZetonema costatum, NEPCC 18b; Chaetoceros graciZe, NEPCC 294) and were grown i n 6 - l i t e r continuous cultures at a d i l u t i o n rate of 0.6 d 1 under conditions described i n Chapter 1. Inflow medium was a r t i f i c i a l seawater (Davis et a l . , 1973) enriched to f/2 ( G u i l l a r d 67 & Ryther, 1962) with phosphate and s i l i c a t e and to f/25 with vitamin and trace metals. The l i m i t i n g n u t r i e n t , ammonium was added as ammonium chloride to a concentration of 30 ug-at-£ \ This ••established a d a i l y ammonium f l u x through the cultures of 18 pg-atN«£ "'"•day "*". The cultures were grown at 18°C -2 -1 and the irradiance was 150 uEin-m «s , continuous l i g h t (Ch. 1). Once steady-state had been attained,the continuous cultures described above were divided into two x 2 - l i t e r . f l a s k s with a d i l u t i o n rate of 0.6 d 1 . System 1 for both species had the same inflow medium as the parent culture, whereas System 2 had no added ammonia i n the inflow medium. System 2, for both species, received a s i n g l e d a i l y ammonia addition at 1300 hr, con s i s t i n g of 12.7 ml of 2.83 mM ammonium chloride s o l u t i o n . This resulted i n an i d e n t i c a l ammonia f l u x through both Systems 1 and 2 of 18 ug-atN.£ "'"• day "*". The addition was c o n t r o l l e d by a s p e c i a l l y modified timer ( C i n c i n n a t i , Model 422) and a c a l i b r a t e d metering pump.(Fluid Metering Inc., New Jersey). A diagrammatic representation of these systems appears in-.Fig. 4 along with an assessment of the experimental design. 3.1) Measurements: Culture e f f l u e n t s were c o l l e c t e d d a i l y and preserved i n Lugol's iodine. An inverted microscope was used for enumeration of samples. In vivo f l u o r -escence was monitored with a fluorometer (Turner, Model H I ; equipped with a high s e n s i t i v i t y door. Nutrients i n culture e f f l u e n t s were analyzed with a Technicon Autoanalyzer using methods previously described (Davis et a l . , 1973). Aft e r two weeks of exposure to the nutrient regimes, the response of the populations to an ammonia pulse or perturbation (Caperon & Meyer,1972b) was determined. A 250 ml . ; sample was removed from a chemostat and perturbed with 1.59 ml of 2.83 mM ammonium ch l o r i d e . This r a i s e d the ambient concentra-t i o n of ammonia to 18 ug-at.£ \ the same concentration that was obtained 68 immediately a f t e r the once per day pulse i n System 2. Since System 2 cultures were acclimated to the pulse occurring at 1300 hr, the uptake experiments for these cultures were c a r r i e d out at t h i s same time. Ammonium •disappearance was then followed with an Autoanalyzer. The a b i l i t y of the various phyto-plankton populations to respond to the d i f f e r e n t patchiness regimes was determined.. Carbon a s s i m i l a t i o n was measured over a 24 hr period to observe the influence of the d a i l y ammonia addition on photosynthetic a c t i v i t y . This was accomplished by placing 50 ml of the culture i n a 50 ml screw top test -1 14 tube and adding 0.4 ml of 2.5 uCi-ml NaH CO^. The samples from System 1 were incubated for 1 hr, whereas the samples from System 2 were incubated for 8, 3 hr i n t e r v a l s extending over the 24 hr period. The shorter incubation time f o r System 1 was chosen to minimize the e f f e c t s of nitrogen starvation that occurs upon removing the sample from t h i s continual ammonium addition system. A longer incubation time could be used for the samples from System 2 because the l i m i t i n g nutrient Ammonium ) was added at only one time each day. Following the incubations, samples were f i l t e r e d onto 0.45 um M i l l i p o r e f i l t e r s and suspended i n 15 ml of Scinti-Verse s c i n t i l -l a t i o n f l u i d . The r a d i o a c t i v i t y i n the samples was determined using a.Unilux III, l i q u i d s c i n t i l l a t i o n counter. Average d a i l y C/N a s s i m i l a t i o n r a t i o s were calculated f or a l l systems a f t determining the t o t a l d a i l y carbon a s s i m i l a t i o n and the calculated d a i l y nitrogen a s s i m i l a t i o n . Daily carbon a s s i m i l a t i o n for System 1 was calculated by mult i p l y i n g the mean hourly carbon a s s i m i l a t i o n rate by 24. The d a i l y carbon a s s i m i l a t i o n for System 2 was calculated by summing the 8, 3 hr incuba-t i o n values. Average d a i l y nitrogen a s s i m i l a t i o n was equated to the d a i l y ammonia fl u x through the culture. Correction was made for the washout of a portion of the anmionlum i n System 2 (as ammonia was added i n a d i s c r e t e pulse 69 and, as the uptake of t h i s pulse was not instantaneous some washout occurred). This correction did not have to be applied to System 1 as there was e s s e n t i a l l y no d e t e c t i b l e ammonia i n the outflow. 4) Results 4.1) Fluorescence: Culture fluorescence i n the homogeneous cultures remained constant throughout the experiment, whereas i t fluctuated markedly i n the once a day ammonia addition culture, System 2 (Fig. 16). This f l u c t u a t i o n ' .was.most noticeable i n -S. aostatwn. The minimum culture fluorescence occurred at ^ 1900 hr, 4 hr a f t e r the addition of the ammonium pulse, while the fluorescence maximum occurred at ^ 0300 hr, 14 hr a f t e r the pulse. C. gracile (System 2) showed some f l u c t u a t i o n i n culture fluorescence with a minimum at ^ 1700 hr, 6 hr a f t e r the ammonium addition, and a maximum at ^ 0300 hr, 14 hr a f t e r the addition. 4.2) Carbon a s s i m i l a t i o n : System 1, for both species, showed a constant carbon a s s i m i l a t i o n rate over the 24 hr period (Fig. 17). In the once a day ammonium addition cultures, carbon a s s i m i l a t i o n fluctuated i n response to the d a i l y ammonium addition. For both species, a maximum was observed at ^ 0300 hr, 14 hr a f t e r the ammonium addition and the minimum occurred d i r e c t l y following the ammonium addition. 4.3) Carbon/Nitrogen a s s i m i l a t i o n r a t i o s : The average d a i l y C/N a s s i m i l a t i o n r a t i o s (by atoms) for S. costatum were 14.3±1.2 and 12.8±1.7 for Systems 1 and 2, r e s p e c t i v e l y . This d i f f e r e n c e was not s i g n i f i c a n t (p = 0.3). The average d a i l y C/N a s s i m i l a t i o n r a t i o s for C. gvacile were 11.6 ± 1.2 and 10.0 ± 1.8 for Systems a and 2, r e s p e c t i v e l y . These differences were again not s i g n i f i c a n t (p = 0.07). There was, however, a s i g n i f i c a n t difference between the two species grown i n both the homogeneous and patchy conditions (p = 0.0001 and p = 0.03, r e s p e c t i v e l y ) . 70 Figure 16. Relative fluorescence over a 24 hr period f o r C. gvacile (Q , System 1, continuous addition of ammonia; and fl| , System 2, d a i l y addition of ammonia) and S. costatum (0, System 1; System 2). A l l systems were continuous flow with a d i l u t i o n rate of 0.6 d 1 and grown under continuous l i g h t . The arrow represents the time of the ammonia addition to System 2. The point indicated f o r System 1 represents the mean of four values taken throughout the day. 71 3.<H 0 -) 1 1 1 1 1 1 1 — — i 1 1 1 1 1 1 1 1 1 1 1 1 i i 1400 1600 2000 2400 0400 0800 1200 TIME Figure 17. Carbon f i x a t i o n rate f o r S. costatum, (0, System 1, continual addition of ammonia; and • , System 2, d a i l y addition of ammonia) and C. grac-LXe,,' — ( 8 f , System .continual' : addition -of-'ammonia; and Cf , System 2, d a i l y addition of ammonia) j y indicates time of ammonia addition to System 2. Bars represent 95% confidence l i m i t s . 72 4.4) C e l l numbers; No trends were apparent i n the c e l l d e nsities i n any of the systems, 8 —1 System 1 f o r 5, costatum had an average density of 7,3 ± 0.4 x 10 cells•£ 8 —1 whereas System 2 exhibited an average density of 7,4 ± , 08 x 10 cells1'£ 8 —1 C. gracile (System 1) had an average c e l l density of 4,7 ± 0,3 x 10 cell-£~ , 8 -1 whereas the average density of System 2 was 4.6 ± 1,2 x 10 cell-£ 4.5) Nutrient uptake: a) I n t e r s p e c i f i c d i f f e r e n c e s : The nutrient uptake response of System 1 (homogeneous d i s t r i b u t i o n of the l i m i t i n g nutrient) to a perturbation equiva-lent to the d a i l y addition of System 2 i s represented i n F i g , 18, S, costatum exhibited a very rapid i n i t i a l uptake (Y^ ; see Appendix II) over the f i r s t 3 min following the perturbation, when compared to C, gracile. Subsequent uptake (V : see Appendix II) i s also more rapid for S, costatum, The nutrient uptake response of System 2 (da i l y addition) to i t s d a i l y ammonium pulse i s shown i n Figure 19, \ The general features of the r e s u l t s are s i m i l a r to System 1 with S, costatum having a higher V (3 min) and a continued higher, longer-term uptake, V_^ , than C, gracile, b) I n t r a s p e c i f i c differences; C. gracile: The only diff e r e n c e between the nutrient uptake a b i l i t y of the homogeneous population and the one addition per day population was i n the i n i t i a l rapid uptake (V' ), In the patchy-grown Til cLX population, V' was greater than the homogeneous culture (Fig, 20), TT13.X S. costatum: No major difference i n nutrient uptake rates was apparent between the two culture conditions (Fig. 21), There appeared to be some smaller differences, however, between the two treatments, System 2 exhibited a higher and, as a consequence, the patch-adapted population could take up a d a i l y pulse of ammonium fa s t e r than the homogeneous population (System 1), 73 Figure 18. The disappearance of ammonium as a function of time during a perturbation experiment for System 1 (continual addition of ammonium) C. graoile, Q / and S. costatum , 0 . o I cn 5 184 TIME (minutes) Figure 19. The disappearance of ammonium as a function of time during a perturbation experiment for System. 2 (da i l y addition of ammonium) Q. gracile, ffif and S. costatum, 9 . 75 Figure 20. The disappearance of ammonium as a function of time during a perturbation experiment for C. gracile, System 1 (continual addition of ammonium Q , and System 2 (daily addition of ammonium) @ . 76 Figure 21. The disappearance of ammonium as a function of time during a perturbation experiment for S. oostatvm, System 1 (continual addition of ammonium) „ 0 '•> and System 2 (daily addition of ammonium), • . 77 5) Discussion L i m i t i n g nutrient patchiness (ammonium) i n i t i a t e s a number of p h y s i o l o g i c a l p e r i o d i c i t i e s i n phytoplankton populations. The p e r i o d i c i t y i n carbon f i x a t i o n did not, however, s i g n i f i c a n t l y a l t e r the average d a i l y C/N a s s i m i l a t i o n r a t i o s i n the two species tested. The p e r i o d i c i t y i n carbon f i x a t i o n could be explained i n terms of an i n t r a c e l l u l a r energy a l l o c a t i o n mechanism. When the l i m i t i n g nutrient i s a v a i l a b l e , energy i s shunted to the uptake process at the expense of carbon f i x a t i o n (Falkowski & Stone, 1975). A f t e r the a v a i l a b l e nitrogen has been assimilated, enhanced carbon f i x a t i o n occurs. Ohmori & •Hattori (1978) obtained some biochemical*evidence to support t h i s type of energy a l l o c a t i o n . They showed that the addition of ammonium to an ammoniumlimited phytoplankton culture resulted i n a rapid drop i n i n t r a c e l l u l a r ATP. This ATP drop was shown to be a r e s u l t of glutamine synthetase a c t i v i t y . P e r i o d i c i t i e s i n chlorophyll a fluorescence could be due to e i t h e r a change i n c h l o r o p h y l l a l e v e l s or a change i n photosynthetic e f f i c i e n c y . As c h l o r o p h y l l a was not d i r e c t l y measured, i t was. not possible to determine the underlying p h y s i o l o g i c a l mechanism mediating the fluorescence f l u c t u a t i o n s . Under a l l treatments S. costatum had a higher nutrient uptake rate than did C. gracile. This i s i n agreement with the work of Conway and Harrison (1977) who worked on S. costatum and C. debilis. The two C. gracile popula-tions (1 and 2) showed some i n t e r e s t i n g differences i n uptake a b i l i t y depending on the nutrient past h i s t o r y under which they were grown. The patch-adapted T population (System 2) showed a much greater V than the homogeneous popula-max t i o n , while the subsequent uptake rate, V , was the same for both populations. As a r e s u l t the population grown on a patchy nutrient source was best.able to take up a pulse of the l i m i t i n g n u t r i e n t . S. costatum also showed s l i g h t differences i n nutrient uptake a b i l i t y i n response to l i m i t i n g nutrient patchiness. Over the f i r s t 3 min the 78 i homogeneous culture had a s l i g h t l y higher V but the V. was greater i n ° max 1 the patch-adapted system. Consequently, the population grown under conditions of once a day ammonia addition was best able to optimize uptake of the nutrient with that patchy temporal d i s t r i b u t i o n . Other workers (Chisholm & Stross, 1976) have induced p e r i o d i c i t i e s i n phytoplankton c e l l u l a r metabolism by use of light:dark cycles. When EugZena gvaoiZis was grown, under l i g h t : dark cycles,. d i u r n a l f l u c t u a t i o n s i n ' 14 C a s s i m i l a t i o n and V for phosphate uptake were observed. max The major differences between the physiology of the populations grown on homogeneous or patchy nutrient d i s t r i b u t i o n s was that the pulsed populations established; a p e r i o d i c i t y - i n a number of parameters such as chlorophyll-a fluorescence and carbon a s s i m i l a t i o n . The observation that c e l l numbers remained r e l a t i v e l y constant throughout the 24 hr cycle i n System 2 supported the work of Caperon (1969). He suggested that phytoplankton growth was an i n t e g r a t i v e r e s u l t of the n u t r i t i o n a l past h i s t o r y of the population over the preceding 24 hr period. Changes i n nutrient uptake a b i l i t y suggested that populations grown on a pulsed nutrient system tended to exhibit higher nutrient uptake rates than those grown on homogeneous d i s t r i b u t i o n of the l i m i t i n g nutrient. This phenomenon w i l l be explored further i n Chapter V and i t s p o t e n t i a l e f f e c t on phytoplankton growth and competition w i l l be discussed. In summary, the r e s u l t s of competition observed i n Chapters II and III appear to be due almost e n t i r e l y to i n t e r s p e c i f i c n u t r i e n t uptake dif f e r e n c e s . The r o l e of . i n t r a s p e c i f i c v a r i a b i l i t y i n the competition for the l i m i t i n g nutrient i s minor when compared to the i n t e r s p e c i f i c differences. Neverthe-l e s s , the a b i l i t y of a species to a l t e r i t s physiology i n response to a patchy environment may be important i n the.optimization, of a p a r t i c u l a r nutrient regime. -79 Chapter V LIMITING NUTRIENT PATCHINESS AND PHYTOPLANKTON GROWTH: A CONCEPTUAL APPROACH 1) Summary A t h e o r e t i c a l framework i s developed to explore the e f f e c t s of l i m i t i n g nutrient patchiness on phytoplankton growth. Growth rate i s represented as a function of the average ambient substrate concentration i n the medium, the degree of patchiness and the patch duration. Phytoplankton growth, i n r e l a t i o n to the external substrate concentration, i s mediated by the c e l l quota f o r the l i m i t i n g n utrient. Two general conclusions can be drawn from t h i s study. F i r s t , the degree of patchiness i n the environment can a f f e c t i n d i v i d u a l growth rates and thus a l t e r community structure even though there i s no change i n the average ambient nutrient concentration. Second, f o r patch-adapted populations, the apparent K g for growth can be lowered s i g n i f i c a n t l y by making the d i s t r i -bution of the l i m i t i n g nutrient patchy with respect to time. The i n s i g h t s which t h i s model provides into future experimental'methodologies.-are also discussed. 6 80 2) Introduction The a p p l i c a t i o n of the Monod equation (1) (Monod, 1942) vi = ,y. max ' K + [ S ] [ S ] U) growth rate, (hr ) max maximum growth rate (hr ) K [ S ] s substrate concentration (yg-at- I ) h a l f - s a t u r a t i o n constant (yg-at-£ 1 to nutrient l i m i t e d phytoplankton growth has generally been unsuccessful i n describing growth on nutrients other than carbon (Droop, 1968; Caperon & *.' Meyer, 1972a;Fuhs et . a l . , 1972; Goldman et al., 1974; Harrison et a l . , 1976; Goldman &^McCarthy, 1978). In many of these l a t t e r cases nutrient l i m i t e d growth rates were described better as a function of the c e l l quota (Droop, 1968; Caperon & Meyer, 1972a;Goldman & McCarthy, 1978). The preceding studies were a l l conducted under steady state conditions with a r e l a t i v e l y homogeneous d i s t r i b u t i o n of the l i m i t i n g n u t r i e n t . However, i t has been shown recently that following an addition of the l i m i t i n g nutrient to a nutrient l i m i t e d culture, uptake rate f a r exceeds growth rate (Conway et al.,1976; Conway & Harrison, 1977; McCarthy & Goldman, 1979). Davis, Breitner and Harrison (1978) provided a model that simulated s i l i c a t e l i m i t e d diatom growth at a steady state as well as the transient uptake response to a s i l i c a t e addition. The degree to which the transient uptake rate exceeded growth was dependent upon the species, the nutrient i n question (Conway & Harrison, 1977) and the degree of nutrient l i m i t a t i o n (McCarthy & Goldman, 1979; Eppley & Renger, 1974). The a b i l i t y of some species to respond more r a p i d l y than other species to a patch of the l i m i t i n g nutrient could provide a. basis for explaining resource 81 competition and niche separation. Further work (Turpin & Harrison, 1979; Chapters I I I , IV & V) has shown that t h i s i s the case with the temporal d i s -t r i b u t i o n of the l i m i t i n g nutrient causing modification of community structure and physiology. Nutrient l i m i t a t i o n i s w e l l documented i n many freshwater and marine ecosystems. The c y c l i n g of nutrients for u t i l i z a t i o n by the primary producers occurs by d i f f u s i o n from.the n u t r i e n t - r i c h .water below the thermo-c l i n e , advection (runoff, mixing and. upwelling) and regeneration, e i t h e r by zooplankton. or ba c t e r i a . These mechanisms of nutrient supply are_not evenly d i s t r i b u t e d over either time or space (Shanks & Trent, 1979). As a r e s u l t , the supply of'nutrients to a. system:. i s r n o t homogenous with respect to time or space. Spacial patchiness, i n r e l a t i o n to a phytoplankton c e l l , could be modelled i d e n t i c a l l y to temporal patchiness since i n both cases there would' be a f i n i t e time between patch encounters. It i s known that d i f f e r e n t species of nutrient l i m i t e d phytoplankton respond to the addition of the l i m i t i n g nutrient with d i f f e r e n t uptake rates and that nutrient patchiness can be expected i n aquatic eco-systems . This chapter attempts to demonstrate how patchiness, average substrate concentration and growth rate of a'phytoplankter .could be r e l a t e d . . The e c o l o g i c a l implications of such i n t e r r e l a t i o n s h i p s and the i n s i g h t s into future experimental methodology w i l l be discussed. 3) Model This model was developed to predict the growth of nutrient l i m i t e d phytoplankton under conditions of l i m i t i n g nutrient patchiness. The model was designed to account f o r steady state growth k i n e t i c s as w e l l as to provide i n s i g h t into the e f f e c t s of nutrient patchiness on phytoplankton growth. The model components are outlined below. 82 3.1) Nutrient uptake: The amount of nutrient taken up by a c e l l per unit time, p , ( y g - a t - c e l l 1 •hr ^) i s given by the following equation: ... P M [ s ] P = m K G + [S] (2) p = maximum uptake rate per c e l l (yg-at• cell;- -hr "*") m [S] = substrate concentration (yg-at•£ "*") KV. = h a l f saturation constant (yg-at* £ "*") In simulations, p^ - i s assumed to be constant (Dugdale, 1977), or i t i s varied i n response to the c e l l quota using the data of McCarthy and Goldman (1979). 3.2) Dependence of population growth rate on c e l l quota: The growth rate of the population i s determined as a function of the c e l l quota using the equation of Droop (1968) ; M = y <i - Q I N I N / Q ) ' ( 3 ) y = growth rate (hr "*") y = growth rate when (Q -> °° (hr 1 ) Q = c e l l quota ( y g - a t " c e l l "*") Q.. = minimum quota needed for growth to proceed ( y g - a t * c e l l 1 ) mxn o r vr-o 3.3) Determination of quota: The change i n quota per unit time (Q) ( g - a f c e l l "^-hr ^) i s the net r e s u l t of an increase i n Q due to uptake and a d i l u t i o n of Q as a r e s u l t of growth as described by the following equation: * " K V [ S ] " P ( 1 ^ m i n / ^ ( 4 ) s At steady state growth, with a constant substrate concentration [S] , Q w i l l approach equilibrium when Q = 0 such that equation (4) reduces to: 83 Wm [S] K s + [S] y ( Q " W or Q = Q m i n + p m [ S ] / ( K s + [ S ]>^ (5) The steady state growth rate of the population i s obtained by s u b s t i t u t i n g equation (5) into equation (3) as follows: V = P < 1 - min Q +_S m l n y {K + [S]} or rearranging the above equation y i e l d s : yp y = m :s] (6) uQ • + P "Q . K » mm m^ V- min s yQ . +P M mm m + [S] Therefore at steady state, the dependence of growth (y) on substrate [S] i s described by a rectangular hyperbola. The population's maximal growth rate, (y )_ under any set of conditions (see equation 6) i s H13.X described by: yp m max yQ . + P M mm m (7) and the hal f saturation constant for growth -(see equation 6) by: • 1 The equation f o r steady state growth as a function of external nutrient; concentration was derived by Droop (1968). My terminology follows that of Dugdale (1977). 84 K' = Vmin Qmin (8) S uQ • + P" v mm m I have chosen to c a l l the h a l f saturation constant f o r growth, K/ , to avoid confusion with the h a l f - s a t u r a t i o n constant for uptake (K ). This s expression f o r K' indicates that i t i s a v a r i a b l e , dependent on both K s s and p , providing y and Q . are constant under a l l conditions, m mm Since y and K' depend on three c e l l parameters, y , ^  m , and K max s ^ r s , min two organisms can have the same dependence of y on [S] at steady state ( i . e . , same y and K' ) and s t i l l have d i f f e r e n t growth and uptake para-TTlciX s meters (y , ^ m and K ) . As an extreme example, a population with Q s m y = 0.2 hr 1 , Pm = 0.2 hr 1 and K =0.2 yg-at• 1 1 w i l l show_-the same steady Q • s mxn growth k i n e t i c s as a population with y = 0.105 hr \ Pm = 2.1 hr 1 and Q . _ l mxn K =2.1 yg-at•1 . s ° The s i m i l a r i t y i n growth k i n e t i c s ends at steady state. As the nutrient a v a i l a b l e to the c e l l fluctuates over time, differences a r i s e i n the a b i l i t i e s of the two species to procure the nutrients and grow. To i l l u s t r a t e t h i s point, the average growth rate of c e l l s exposed to an environment i n which the nutrients come i n short pulses, separated by periods of nu t r i e n t s t a r v a t i o n , has been calculated. The nutrient concentration during the pulse i s adjusted i n order to maintain some fixed concentration t ^ a v ] when averaged over the period of the patch, P (Fig. 22). For the simulations, the duration of the pulse, D , was held constant at 0.1 hr and the i n t e r v a l between pulses, T varied. Holding D^constant allowed us to use the compartment model e f f e c t i v e l y . In a more complex s i t u a t i o n with a varying pulse duration , the model would have :to be modified to include i n t e r n a l pool(s) and feedback control of the uptake rates (Davis et a l . , 1978; DeManche et a l . , 1979). 85 When a population i s exposed to a p e r i o d i c nutrient supply (Fig. 22) the c e l l quota responds according to equation 5, and approaches an equilibrium with Q increasing during the pulse and decreasing during the absence of the nu t r i e n t . An average population growth rate i s calculated over t h i s cycle. At equilibrium, for a given patchy regime, the absence of a delay between Q and u (Cunningham &. Maas, 1978) does not a f f e c t the r e s u l t . As might be expected, steady state populations having the same u and K' but d i f f e r -max s ent values of u , _pm and K , show quite d i f f e r e n t growth rates under the Q S same patchy nutrient regimes. To emphasize t h i s behaviour a second set of parameters can be chosen such that i n a steady state system ( i . e . , p = 0 , when the l i m i t i n g nutrient i s homogeneously dis t r i b u t e d ) species A may out-grow species B but as the system becomes patchy, B would outgrow A (Fig.23). Simulations were run using a v a r i a b l e p as a function of c e l l quota m which was calculated from the data of McCarthy and Goldman (1979). This v a r i -able, , combined with the growth k i n e t i c data from the same organism (Gold-man & McCarthy, 197 8) produced r e s u l t s s i m i l a r i n general appearance to F i g . 23A with growth rate decreasing with lower frequencies of patchiness. This demonstrates that a high V alone w i l l not allow an organism to grow max better under patchy conditions at a given average substrate concentration. This i s contrary to the assertion made by McCarthy and Goldman (1979). Some preliminary work with n a t u r a l marine phytoplankton communities (Turpin & Harrison, 1979) and u n i a l g a l cultures (Ch. IV) suggest that the maximum uptake rate of a population increases with the time between nutrient pulses. To determine the possible e f f e c t s of such a condition, a r e l a t i o n s h i p between p and T i s presented (equation 9) which allows enhancement of l i m i t i n g nutrient uptake when the d i s t r i b u t i o n of that nutrient i s patchy. Although t h i s r e l a t i o n s h i p can not be v a l i d a t e d with e x i s t i n g data, i t allows a representation of enhanced uptake i n a patchy system. The equation i s : 86 Figure 22. A graphical representation of a temporally patchy, nutrient l i m i t e d environment. [S] i s the substrate concentration (ug-at*£ ^ ) . P i s the p e r i o d i c i t y of the patch (hr), the duration (hr) and T , the i n t e r v a l between successive pulses (hr). [S ] i s the substrate concentra-•3.V t i o n averaged over the patch period, P . 87 102 102 8-2 Figure 23. The growth rate, y , (hr ) of two hypothetical species as a x r function of average substrate concentration,S , (jg-at • Ji, - 1) and the patch period 3.V P , (hr). The patch duration'is constant at 0.1 hr. Species A has the growth -1 - -1 - -1 parameters: p /Q ...= 0^2 hr K: = -0.2 yg-at • £ - •: and, y. •= 0.2 -hr .: m mm s '. * Species B-'has:: p /Q . = 4.1 h r " 1 ; K = 2.05 yg-at-£ _ 1; and, y = 0.0512 h r " 1 . m mm s 88 P max. m P P' Q~7 m m mxn ( . Q . Q . 1\ + Tmm mxn h T, = the i n t e r v a l , T, at which p i s 1/2 p h ' P ' P max _^ _^ p • = maximum increase i n uptake under patchy conditions ( y g - a t - c e l l «hr ) p TT13.X p ' = maximum uptake under homogeneous nutrient d i s t r i b u t i o n s m ( y g - a t - c e l l "'"-hr 1 ) T = i n t e r v a l between pulses (hr) The r e s u l t i n g community then grows at an enhanced rate when the l i m i t i n g nutrient i s patchy with respect to time even though [S ] i s constant (Fig. clV 24). In other words, the e f f e c t i v e K' of the community i s lowered under ' s conditions of optimal patchiness (Fig. 25) i f a population can demonstrate an enhancement i n uptake a b i l i t y i n response to patchiness. A s i m i l a r enhancement of y as a function of patchiness can be obtained i f one uses an [S ] value (a substrate concentration below which uptake i s o zero). An example of t h i s simulation i s given i n F i g . 26. 4) Insights Into Future Experimental Methodology The use of the chemostat i s precluded i n e l u c i d a t i n g the r e l a t i o n s h i p between growth rate, (y) , average substrate concentration, [S ] , and patchiness, (P) . When a patch i s added to a chemostat population, the nutrient patch remains i n the medium u n t i l i t i s assimilated or washed out. This would not allow the i n v e s t i g a t o r to predetermine [S ] or the patch av duration as these parameters would depend on the uptake rate of the culture. Also the average growth rate would be predetermined or set by the d i l u t i o n rate and i t would be independent of both t ^ a v ] a n C i ^ providing they are within the l i m i t s of growth for the organism i n question. The only method by which a y vs. [S ] and P p l o t could be generated using a chemostat 89 90 - i r 0-1 0-2 Ks'at POPT 0-41 , 05 l<s at P.0 [ S a v ] Figure 25. Growth rate (y) as a function of average substrate concentration [S ] for the population i n F i g . 24. The growth response at P = 0 i s represented as ( ) and at optimal patchiness (P ) , as (— ). 91 0.08 -f Figure 26. The growth rate (y) of a hypothetical species with growth parameters [S ] = 0.1 y g - a t - A - 1 ; /Q . = 0.325 h r " 1 ; K =0.1 yg-at-£ _ 1; and r o m min s a y = 0.155 h r " 1 . 92 would be to follow [S] over the period of the patch, to determine [S ]. av There are two manor problems with t h i s method. The f i r s t - i s that [S 1 J ^ av would be a function of the culture uptake rate and the concentration of the pulse. The second problem i s the complicating e f f e c t of d e c l i n i n g substrate concentration following the creation of the patch. This varying nutrient l e v e l and varying patch duration would make the i n t e r p r e t a t i o n of r e s u l t s f a r more d i f f i c u l t . The construction of u. vs [S] and P p l o t s , under conditions of constant patch duration (Dp) , as i n Figures 23 - 26, would be impossible. What i s needed i s a system where the experimenter can set t^ a v-I » P and D , while measuring the r e s u l t i n g growth rate. At present such a P system does not e x i s t f or phytoplankton cultures. 5) E c o l o g i c a l Considerations Two general conclusions are drawn from t h i s study. F i r s t , the degree of patchiness i n the environment can a f f e c t growth rates of d i f f e r e n t species and thus a l t e r community structure even though there i s no change i n the average ambient nutrient concentration. Second, for patch-adapted populations, the apparent for growth can be lowered s i g n i f i c a n t l y by making the d i s -t r i b u t i o n of the l i m i t i n g nutrient patchy with respect to time. Figure 25 shows how growth may vary with respect to average substrate concentration under homogeneous, (P = 0), and optimal patchiness, t ) • l n s o m e simula-tions the K/ was over an order of magnitude lower than the f o r growth under homogeneous conditions assuming that there i s an enhanced as a function of patchiness. The presence of an ] value, a substrate concentration below which growth does not occur, also r e s u l t s i n an enhancement i n growth as a function of patchiness at l i m i t i n g substrate concentrations. Figure 26 shows the growth response of a hypothetical species with an [S Q] value. D i f f e r e n t species with d i f f e r e n t uptake a b i l i t i e s could then be expected 93 to dominate under various patch regimes even though [S ] does not change. Hence, not only i s the ambient nutrient concentration i n the environment im-portant i n determining species composition, but i t s temporal and s p a c i a l d i s t r i -bution may be equally important. Nutrient patchiness i n nature could range over many orders of magnitude, from millimeters (Shank's & Trent, 1979) to kilometers. Response to t h i s wide range of patchiness would be expected to be very d i f f e r e n t . Small scale-patchiness would tend to generate a regime with pulses of short duration (as seen by the c e l l ) . Growth r e s u l t i n g from nutrients procured i n these patches could occur outside the patch at some l a t e r time as a r e s u l t of uncoupling of uptake and growth (Caperon, 1969; Cunningham & Maas, 1978). Large scale patchiness would tend to r e s u l t i n pulses of long duration with growth and an increase i n biomass taking place i n the patch. Random f l u c t u a t i o n s i n the d i s t r i b u t i o n of the l i m i t i n g n u t r i e n t could then give r i s e to a form of coexistence, with species l i m i t e d by a common nutri e n t . In a simulat ion model, Grenney, et a l . - (1973) showed, that fl u c t u a t i o n s i n chemostat d i l u t i o n rates and inflow concentrations could r e s u l t i n the coexistence of several species i n a s i n g l e reactor. There are other factors that have not been considered i n the model but they are l i k e l y to be associated with communities that are adapted to a patchy environment. Of great importance f o r a patch-adapted species i s the a b i l i t y to store excess nutrients which would be used when exogenous n u t r i -ent supplies are lacking. This a b i l i t y could be manifested i n the form of large i n t r a c e l l u l a r inorganic pools. Species with large pools would take up large amounts of nutrient before the pool f i l l e d and uptake decreased to an i n t e r n a l l y c o n t r o l l e d rate, termed , by Davis, et al . (.1.978). Organisms with small i n t e r n a l pools would not be.able to sustain the rapid i n i t i a l uptake due to-more rapid pool f i l l i n g (DeManehe 94 et a l . , 1979) C e l l s i z e may also be r e l a t e d to an organism's a b i l i t y to survive i n a patchy nutrient l i m i t e d environment. A moving c e l l would increase i t s chances of encountering a patch. If i t possessed 'chemosensory'-motility coupling (Spero, pers. comm.), i t might further increase i t s chances of being able to stay i n the patch. Such a s i t u a t i o n would increase the duration of the nutrient exposure, hence further optimize uptake and growth. Other considerations such as phased c e l l d i v i s i o n i n a population and the r e s u l t i n g phased nutrient uptake would need to be considered i n a complete simulation model. The e f f e c t s of l i g h t and the r e s u l t i n g d i e l p e r i o d i c i t y would also a f f e c t uptake of various nutrients and p o s s i b l y modify trends i n the community structure controlled p r i m a r i l y by n u t r i e n t s . This work has provided a conceptual framework f o r the evaluation of the importance of nutrient patchiness i n determining phytoplankton growth and community structure. The problem at t h i s time i s the lack of information on the time and space scales of the patches being discussed and the nutrient concentrations within them. Future research should approach t h i s problem from the b i o l o g i c a l , chemical and p h y s i c a l viewpoints i n an attempt to further understand t h i s p o t e n t i a l l y important phenomenon. 95 Chapter VI A CONCEPTUAL APPROACH TO NUTRIENTVBASED PHYTOPLANKTON ECOLOGY Since Dugdale (1967) postulated the importance of nutrient concentration i n determining phytoplankton community structure, many models have been proposed to account f o r the e f f e c t of nutrients on phytoplankton growth, competition and succession. Few models deal with an in t e g r a t i o n of factors such as d i l u t i o n r a t e s 1 , nutrient r a t i o s , l i g h t and temperature. Those that do (Kremmer & Nixon, 1978), follow a highly mechanical approach. In . thi s chapter, I attempt to combine a number of simple nutrient based growth models, to. integrate the e f f e c t s of nutrient r a t i o s ? nutrient fluxes j'.temperature and light,, and to present a simple conceptual approach to nutrient based phytoplankton ecology. The importance of d i l u t i o n rates i n determining the outcome of chemostat competition experiments i s we l l documented for both b a c t e r i a (Harder et a l . , 1977) and phytoplankton (Michelson et a l . , 1979; Harrison & Davis, 1979). A schematic representation of the importance of the d i l u t i o n rate, or the s p e c i f i c f l u x of nitrogen through a nitrogen l i m i t e d system i n determining the general phytoplankton community structure i s presented in- F i g . 27 (Turpin & Harrison, 1979). At high s p e c i f i c nutrient fluxes fast growing c e n t r i c diatoms dominate while at low fluxes, u - f l a g e l l a t e s dominate. This scheme agrees with laboratory experiments (Michelson et a l . , 1979; Harrison & Davis, 1979), and f i e l d observations In which the outcome of 1 This i s equivalent to the s p e c i f i c f l u x of the l i m i t i n g nutrient ( i . e . ds/dt . -1. — j r ^ : — = time ) at steady-state. 96 1.0, to o t -< Q z O h-rr 0 o_ O QC 0_ ( Skeletonema Chaetoceros Thalassiosira Cylmdrothecan „ .... . . IPennate Nitzschia J Pyramimonas Chroomonas Specific Nu t r ien t F lux — Demand /Supp ly TJ fc +J o JS Q O Q C9 TJ r r r o O £= 03 E 17 o o <> p »i Figure 27. The r e l a t i o n s h i p between s p e c i f i c nutrient f l u x , D/S r a t i o , and " r " and "K" competition strategy and" the r e s u l t i n g phyt-oplanktpn community structure.The competition, strategy i s represented as a continuum between " r " and "K" s t r a t e g i s t s . 97 competition was related to d i l u t i o n rate or s p e c i f i c nutrient f l u x . Recent work showed the importance of nutrient r a t i o s , . • independent of the absolute magnitude of a given f l u x (Titman, 1976; Tilman, 1977). Such work has confirmed resource-based competition theory, s t i p u l a t i n g that under i d e a l conditions ( i d e n t i c a l m o r tality rates or temporal and s p a c i a l homogeneity) coexistence of two competing species can take place only when both/ species are l i m i t e d by d i f f e r e n t resources. The only reason that the outcome of competition was f l u x independent i n the preceding work was because the growth-rate (.y), vs, substrate, .[S],, curves for the chosen species did not i n t e r s e c t (Titman, 1976;. Tilman, 1977). Using species whose p vs. [S] curves i n t e r s e c t , i t i s possible to demonstrate the i n t e r a c t i o n of both the s p e c i f i c f l u x or d i l u t i o n rate and the nutrient r a t i o . This was accomplished by estimating Monod (1942) growth parameters for four hypothetical species competing for two d i f f e r e n t poten-" t i a l l y l i m i t i n g n u t r i e n t s , X and Y (Table IV). The outcome of competition between the four hypothetical species i s given as a function of both the d i l u t i o n rate and the nutrient r a t i o s i n F i g . 28. The boundaries mediated by changes i n d i l u t i o n rate were determined by the i n t e r s e c t i o n of y vs. [S] curves (Harder et a l . , 1977) (see Introduction). The boundaries mediated by resource r a t i o s were determined as described by Titman (1976) (see Introduc-t i o n ) . . The r e s u l t i s that both the d i l u t i o n rate and the r a t i o of l i m i t i n g nutrients i n t e r a c t i n such a way that s u b s t a n t i a l changes to the community occur, ranging from complete dominance by one species to coexistence of a number of combinations of two species. If the y vs. [S] curves of the species i n question did not cross, then the resource r a t i o would be the only factor determining the outcome of competition. In the natural environment the s p e c i f i c nutrient f l u x and the r a t i o of the nutrient fluxes may i n t e r a c t also. In a system where nutri e n t fluxes were 98 Table IV. Growth k i n e t i c parameters f o r the hypothetical species i n Figure 28. u i s the maximal growth rate, K Is the h a l f - s a t u r a t i o n constant f o r growth max x 6 l i m i t e d by resource X and K i s the h a l f - s a t u r a t i o n constant f o r growth l i m i t e d y by resource Y. •\ -PARAMETERS y m a x K X K y K /K x y A 1.0 4.0 0.1 40 B 0.85 3.0 0.05 60 C 0.88 2.0 0.5 4 D 1.0 2.5 0.25 10 99 Dominance Plane 1.0 ns ~o CJ A +-> co on c o -M Z3 5 0.54 B O -A & D B & D D D 100 60 40 10 4 X / Y X l i m i t e d — > • Y l i m i t e d Figure 28. A dominance plane representing the outcome of competition, f or the species i n Table IV ,as a function of d i l u t i o n rate ( s p e c i f i c nutrient flux) and resource r a t i o . Representation ' of two species i n the same area indicate stable coexistence with both species l i m i t e d by a d i f f e r e n t resource. 100 determined mainly by p h y s i c a l processes such as transport across a thermocline, increases i n mixing rates would tend to increase a l l nutrient fluxes simultan-eously, r e s u l t i n g i n l i t t l e change i n r a t i o s . Conversely, when nutrient fluxes are c o n t r o l l e d predominantly by b i o l o g i c a l phenomena, the n u t r i e n t r a t i o s may change due to d i f f e r e n t i a l regeneration. Since both p h y s i c a l and b i o l o g i c a l factors play an important r o l e i n the nutrient dynamics of natural systems, both the magnitude and r a t i o of fluxes can be expected to vary quite dramat-i c a l l y with space or time. In an attempt to integrate the possible e f f e c t s of temperature and l i g h t on the structure of a nutrient-based system, a demand/supply (D/S) continuum was imposed, such that at high f l u x rates, S was large and D/S approached zero. When l i t t l e nutrient was added to the system (low f l u x ) , S was small and D/S approached i n f i n i t y (Fig. 27). A s i m i l a r approach has been taken by Kilham and Kilham .(in prep;').. The r e s u l t of changing temperature and l i g h t can then be viewed through i t s e f f e c t on the D/S continuum as a r e s u l t of changes i n demand (D). An increase i n water temperature within a given range would increase the growth p o t e n t i a l of the community (Eppley, 1972), and therefore increase the demand (D) for the l i m i t i n g resource and hence increase D/S.. This increase i n temperature should r e s u l t i n a change i n community structure s i m i l a r to that implemented through decreasing the s p e c i f i c nutrient f l u x , which also increases D/S by decreasing S. Consequently, a low temperature c e n t r i c diatom community would be expected to s h i f t through a pennate community to a u - f l a g e l -l a t e dominated assemblage i n response to a temperature increase and a concomi-tant increase i n D/S. Such a r e s u l t has been demonstrated by Goldman &Ryther (B76 ) where an increase i n temperature drove nitrogen-limited phytoplankton assem-blages from c e n t r i c through pennate diatoms to f l a g e l l a t e s . An increase i n l i g h t that i s below the saturating l e v e l would also tend 101 to increase the growth p o t e n t i a l of the community r e s u l t i n g i n an increase i n the p o t e n t i a l demand (D) for the l i m i t i n g resource and consequently, community structure should tend to s h i f t to that governed by higher D/S r a t i o s . Some evidence also supports t h i s s i m p l i f i e d approach (Harrison & Davis, 1979). They found that when the l i g h t i n t e n s i t y was decreased for a culture growing at a low d i l u t i o n rate, i t resulted i n natural .assemblages of phytoplankton that were s i m i l a r to those i n cultures that were growing at a..higher l i g h t and d i l u t i o n rate. Temperature and l i g h t e f f e c t s can be conceptualized through t h e i r i n t e r a c t i o n with the D/S continuum and to some extent may mimic s p e c i f i c f l u x changes. Changes i n temperature and l i g h t are obviously more complex than indicated by our D/S continuum, e s p e c i a l l y at the extremes of the temperature and l i g h t ranges for a given species. The example of changes i n l i g h t and temperature mimicing s p e c i f i c f l u x changes may be r e s t r i c t e d to small changes of l i g h t and temperature occurring at intermediate values within a species range. There i s a continuum of " r " and "K" competition strategy (Pianka, 1970) corresponding to the D/S continuum. An " r " selected, organism i s selected on the basis of high growth rates, whereas a "K" selected organism i s selected on i t s a b i l i t y to compete for the l i m i t i n g resource. When D/S i s low, s e l e c -t i o n occurs for fast growing " r " selected organisms, such as the c e n t r i c diatoms. When D/S i s high, competition for the l i m i t i n g resource i s high and the "K" s t r a t e g i s t , the f l a g e l l a t e s , succeed. In conclusion, t h i s simple approach allows for the i n t e g r a t i o n of some of the major f a c t o r s , such as nutrient f l u x , nutrient r a t i o , l i g h t and tempera-ture, that a f f e c t nutrient-based phytoplankton ecology. Nutrient f l u x governs the supply, (S), of the l i m i t i n g nutrient while changes i n sub-optimum temperature and l i g h t a f f e c t the demand, (D), for the nutrient. Large changes i n these:, parameters appear to a f f e c t between group (e.g. diatoms vs. f l a g e l l a t e s ) dominance, which i s also the case for large changes i n nutrient r a t i o s . On the other hand, patchiness of the l i m i t i n g nutrient or frequency of addition of the l i m i t i n g nutrient has been termed a fine-tuning v a r i a b l e since t h i s parameter appears to se l e c t for c e r t a i n species within the phyto-^ plankton group that was selected by the major s e l e c t i o n f a c t o r s . 103 SUMMARY The f l u c t u a t i o n s i n the free i n t r a c e l l u l a r amino acid pools of ammonia li m i t e d Gymnodinium simplex and Skeletonema costatum, i n response to an ammonia perturbation, are best explained i f the enzyme glutamine synthetase (EC. 6.3.1.2) acts as the primary ammonium a s s i m i l a t i n g enzyme. Changes i n the l e v e l s of a l l measurable amino acids i n G. simplex following the per-turbation indicated that the amino group was r a p i d l y shunted between the constituents of the amino acid pool. The temporal patchiness of a l i m i t i n g n u t r i e n t Ammonium )affected the outcome of phytoplankton competition. When the l i m i t i n g nutrient was homo-geneously d i s t r i b u t e d with time, members of the genus Chaetoceros dominated, while under patchy conditions (d a i l y ammonia ad d i t i o n ) , Skeletonema dominated. It was shown that each r e s u l t i n g assemblage was best able to optimize uptake under i t s p a r t i c u l a r patchy regime. Optimization of a patchy environment took place by an increase i n the maximal uptake rate (V ), while optimiza-max t i o n of a homogeneous environment appeared to take place by an increased substrate a f f i n i t y ( i . e . , low K ). Lim i t i n g nutrient patchiness (ammonia) was shown to a f f e c t the mean.cell diameter that was selected i n phytoplankton competition, experiments.. .Low frequency patchiness selected large, c e l l s while high' frequency-patchiness and homogeneous d i s t r i b u t i o n of the l i m i t i n g nutrient selected small.,.cells. Limiting nutrient patchiness induced p e r i o d i c i t i e s - i n : c a r b o n ! a s s i m i l a t i o n and in vivo fluorescence i n u n i a l g a l cultures of S.. costatum and C. gracile. Observed changes.in nutrient uptake a b i l i t y under varying ^ limiting, nutrient patchiness-regimes suggested that, a given population-may. adapt-its nutrient uptake c h a r a c t e r i s t i c s to optimize the' temporal d i s t r i b u t i o n of the l i m i t i n g resource. This i n t r a s p e c i f i c v a r i a b i l i t y i n nutrient uptake i s minor when compared to i n t e r s p e c i f i c d ifferences. 104 A t h e o r e t i c a l framework i s developed to explore the e f f e c t s of l i m i t i n g n u t r i e n t patchiness on phytoplankton growth. Two general conclusions can be drawn from t h i s study. F i r s t , the degree of patchiness of the l i m i t i n g n u t r i e n t i n the environment can a f f e c t i n d i v i d u a l growth rates and thus a l t e r community structure even though there was no change i n the average ambient nutrient concentration. Second, for patch-adapted populations, the apparent K' for growth may possibly be lowered by making the l i m i t i n g nutrient patchy with respect to time. Gross changes i n nutrient-based phytoplankton community structure were mediated by the s p e c i f i c f l u x of the l i m i t i n g n u t r i e n t . Low s p e c i f i c fluxes re-sulted i n f l a g e l l a t e dominated assemblages, while high s p e c i f i c fluxes resulted i n diatom dominated assemblages. The e f f e c t s of temperature and l i g h t on community structure can be conceptualized through t h e i r e f f e c t on a Demand/ Supply continuum and hence mimic s p e c i f i c f l u x changes. Phytoplankton respond to f l u c t u a t i o n s i n nutrient supply at a l l l e v e l s of b i o l o g i c a l organization (biochemical, p h y s i o l o g i c a l and e c o l o g i c a l ) . Nutrient pulses are r a p i d l y taken up and assimilated. Differences i n the a b i l i t y of species to procure nutrients i n t h i s manner r e s u l t i n changes i n competitive advantage as a function of the patchiness of the resource. 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Hence the d i l u t i o n rate, D , has units of time \ The change i n c e l l numbers i n a chemostat i s a function of the growth rate of the population, u (hr "*"), defined as — • -j^ - , and the d i l u t i o n rate, D . The equation for net growth i s : dx dt = (y - D)x (1) where x = cell'£ 1 at time t. dx When the culture i s at steady state, such that -g^ r = 0 , then from equation 1 y = D. By i n t e g r a t i n g and rearranging equation 1, we obtain the so l u t i o n f o r the s p e c i f i c growth rate of a population i n a chemostat as: y = D + — £n x/x (2) t o where x = cell«£ 1 at time o o x = cell«£ 1 at time t If the culture i s at steady state, equation 2 reduces to y = D . For a more det a i l e d d e s c r i p t i o n of chemostat theory see Herbert et a l . (1956). 115 Appendix II DETERMINATION OF NUTRIENT UPTAKE KINETIC PARAMETERS: A COMPARISON OF METHODS 1) Summary The marine chrysophyte, Pseudoped-inella pyviform-is N. Carter, was grown i n ammonium l i m i t e d continuous culture. This steady state population was used to carry out a comparison of three methods presently used to determine the nutrient uptake k i n e t i c parameters, V and K . The f i r s t two methods max s involved a multiple f l a s k incubation where d i f f e r e n t concentrations of sub-s t r a t e were added to each f l a s k and therefore the culture past h i s t o r y was constant f o r each uptake determination. These two methods were s i m i l a r except the incubation time was v a r i a b l e (method 1), or short and constant (method 2). The t h i r d method, the perturbation method, involved monitoring the uptake of one large addition of the substrate to a culture. Hence, i n t h i s method, the past h i s t o r y varied during the experiment. Results i n d i c a t e for n u t r i e n t l i m i t e d cultures that the_parameters, V max and K^ , are best estimated by employing a short, constant incubation time at varying substrate concentrations (method 2). It appears that t h i s method determines the i n i t i a l maximum uptake rate, r e l a t i v e l y free of feedback regula-t i o n , when incubation time i s very short. The short incubation time i s neces-sary because the measured V decreases with increasing incubation time. max Method 3 provides valuable information on a t h i r d uptake parameter, , the approximate rate of a s s i m i l a t i o n of the l i m i t i n g nutrient, that i s not obtained using e i t h e r of the other methods. 2) Introduction Nutrient uptake by marine phytoplankton can be r e l a t e d to the ambient concentration of the nutrient by a rectangular hyperbola, s i m i l a r to the Michaelis-Menten equation f o r enzyme k i n e t i c s where, V = V . [S]/(K + [ S ] ) max s 116 and V i s the uptake v e l o c i t y (hr 1). 1, V the maximal v e l o c i t y , [S] the max concentration of l i m i t i n g nutrient and K the h a l f - s a t u r a t i o n constant s representing the value of [S] where V = V 12 . The determination of the max nutrient uptake k i n e t i c parameters, V and K , have been useful i n explain-max s ing competition for the l i m i t i n g nutrients i n the marine system (Dugdale, 1967; Eppley et a l . , 1969; Tilman and Kilham, 1976; also see Ch. 1). Frequently nutrient uptake i s determined i n d i r e c t l y by measuring the decreasing concentration of the l i m i t i n g nutrient i n the culture medium. Direct measurements of nutrient uptake rates are also made by using isotopes X5 32 30 such as N, P, and S i . Nutrient incorporation into the c e l l i s then determined a f t e r a sui t a b l e incubation time. In the methods which i n d i r e c t l y measure uptake rate by the disappearance of the nutrient from the medium, there are several possible approaches. The f i r s t determinations of V and K were conducted on batch cultures which max s had j u s t run out of nu t r i e n t s , however, i f the culture was without nutrients f o r too long a period, the subsequently determined uptake was "non-linear" (Eppley et a l . , 1969; Eppley &-Thomas, 1969). The general protocol for t h i s method was to set up a serie s of 5 to 10 f l a s k s to which d i f f e r e n t concentra-tions of the l i m i t i n g nutrient were added. The experiment was i n i t i a t e d by adding a sub-sample of a few hundred m i l l i l i t e r s , of the culture to the f l a s k s . The cultures were incubated f o r d i f f e r e n t times. The c r i t e r i o n f o r termin-ating the experiment was when the l i m i t i n g nutrient concentration was thought to be reduced to approximately h a l f the concentration of the o r i g i n a l addition or a f t e r a depletion of 2 yg-at•£ 1 at higher substrate l e v e l s . The uptake rate f o r the incubation period was then associated with the mean substrate concentration i n the f l a s k s . Therefore, i n t h i s method, past h i s t o r y was constant ( i . e . , each f l a s k had the same inoculum) and time of incubation and substrate addition varied. 117 The second approach was i d e n t i c a l to the f i r s t , except that the time of incubation of the cultures exposed to d i f f e r e n t substrate concentrations was constant and r e l a t i v e l y short. Rhee (1978) used a constant incubation time of 20 min for Soenedesmus i n an attempt to a l l e v i a t e past h i s t o r y e f f e c t s of n i t r a t e uptake during the experiment. Another approach to measuring nutrient uptake rates was developed by Caperon and Meyer (1972b). This i s termed the perturbation method. These workers grew the experimental culture i n a chemostat and upon reaching steady state, the culture was perturbed by adding a r e l a t i v e l y large addition of the l i m i t i n g nutrient (e.g., 10 ug-at«£ ^ ) . Continual sampling and analyses with an a.utoanalyzer provided a time seri e s of disappearance of the l i m i t i n g nutrient from the culture u n t i l steady state was regained or the l i m i t i n g nutrient was completely taken up. The changing uptake rate was then re l a t e d to the average nutrient concentration over the sample period. This approach was also used by Conway et a l . (1976). In t h i s l a t t e r work, s i l i c a t e or ammonium-limited cultures exhibited a surge i n the uptake rate, termed V , or V by Goldman and McCarthy (1978), immediately a f t e r the addition of max J J ' J the l i m i t i n g n u t r i e n t . This method thus incorporated a v a r i a b l e past-history e f f e c t into the parameter determination. The population at the end of the experiment w i l l have been exposed to high nutrient concentrations for a longer time than i t was at the beginning of the experiment. The i n t e r v a l over which the uptake rate i s calculated i s constant and a function of the autoanzlyzer-sampling speed. Since i t i s unclear how the choice of e i t h e r of these three methods af f e c t s the values of V and K determination, a systematic comparison of max s these methods was undertaken i n t h i s study. Each method i s then discussed i n r e l a t i o n to the other two and recommendations are made as to the s u i t a b i l i t y of the various methods. 118 3) Methods and Materials 3.1) Chemostat system and analyses: Fseudoped-inella pyvifovmis N. Carter was obtained from the Northeast P a c i f i c Culture C o l l e c t i o n , Department of Oceanography, The U n i v e r s i t y of B r i t i s h Columbia, Vancouver, Canada. The culture was grown at 18°C i n a 6 - l i t e r b o r o s i l i c a t e flat-bottomed b o i l i n g f l a s k and under continuous l i g h t -2 -1 -1 with an irradiance of 150 yEin-m -s . The d i l u t i o n rate was 0.5 d . The ammonia-limited inflow medium was a r t i f i c i a l seawater (Davis et a l . , 1973) enriched with f/20 vitamins and trace metals (Guillard'& Ryther, 1962). The concentrations of the macronutrients, ammonia, s i l i c a t e and phosphate were 10, 45 and 5.5 ug-at-£ \ r e s p e c t i v e l y . The methods for nutrient a n a l y s i s , c e l l counts and fluorescence were described previously (Davis et a l . , 1973). When no trend was observed i n the e f f l u e n t nutrient concentrations, c e l l numbers of fluorescence f o r several days, the culture was assumed to be at steady state and the following experi-ments were i n i t i a t e d . 3.2) Uptake experiments: Since a large 6 - l i t e r chemostat was used, 200 ml could be removed from the chemostat without appreciably changing (4%) the d i l u t i o n rate. The 200 ml subsample that was removed was replaced by pumping i n new medium by the time the next experiment was performed. A small amount of the l i m i t i n g nutrient (ammonia) was added to the subsample and the culture was immediately incubated under previous growth conditions. Samples were taken every 3 min s t a r t i n g 2 min a f t e r the nutrient addition. Ammonia disappearance was followed u n t i l depletion occurred. A f t e r that time the f l a s k was rinsed and f i l l e d with another 200,-ml zsub^sample .from'.the:" chemostat to which another concentration of the., l i m i t ing nutrient was added and the same continuous sampling repeated. These s e r i e s of uptake experiments were performed over a l i m i t i n g substrate concentration range from 0.5 to 20 yg-at•£ \ These time ser i e s data allow 119 the c a l c u l a t i o n of uptake rates using three methods. Method 1 determined the uptake rate over the time i t took f o r the substrate to reach a concentration of ^1/2 i t s o r i g i n a l concentration. Method 2 used constant time i n t e r v a l s of eith e r 2, 5, 17 or 30 min f o r a l l uptake c a l c u l a t i o n s . Method 3 used the instantaneous rate of disappearance of the nutrient and re l a t e d i t to the average substrate concentration over that i n t e r v a l . The i n i t i a l substrate concentration ( i . e . , at T = 0) could not be accurately determined by sampling immediately a f t e r the substrate was added to the culture and thoroughly mixed. Therefore, the same substrate additions were made to f i l t e r e d chemostat e f f l u e n t and measurement of the concentration of the l i m i t i n g nutrient was made and taken to represent the concentration at T = 0. The uptake rates that were calculated were rel a t e d to the nutrient concentration at the middle of the time i n t e r v a l over which the uptake rate was calculated. Uptake rates for Method 1 were also calculated by taking the i n i t i a l substrate concentration as the f i r s t measurement taken a f t e r the substrate addition rather than the true i n i t i a l substrate concentrations at T = 0. 3.3) Uptake rate c a l c u l a t i o n s : Uptake rates were calculated from measurements made during the v a r i a b l e substrate addition procedure described above. Uptake rates were calculated as described by Conway et a l . (1976). The p a r t i c u l a t e values of nitrogen used i n the ca l c u l a t i o n s was determined at steady state from a mass balance where the disappearance of the l i m i t i n g nutrient was assumed to be equal to the increase i n p a r t i c u l a t e nitrogen i n the culture. The nutri e n t uptake k i n e t i c paramenters, V and K , were determined by a d i r e c t hyperbola f i t and s t a t -nicix s i s t i c a l analysis was made using the program of Cleland (1967). 120 4) Results 4.1) Variable incubation time and v a r i a b l e substrate concentration, Method 1; The r e s u l t s from t h i s method are given i n Figure 29. When the uptake rate was calculated using the true T = 0 concentration (see Methods) the V vs. [S] curve generated was not i n the form of a rectangular hyperbola. If the f i r s t measured concentration was used as the i n i t i a l substrate concentra-t i o n a rectangular hyperbola could f i t the data. In t h i s case V was max 0.27 ± 0.04 (s.e.) h r " 1 and -K was 0.14 +0.12 (s.e.) pg-at/fc 4.2) Constant incubation time and v a r i a b l e substrate concentration, Method 2: In Table V, the estimates of the uptake parameters V and K are max s given as a function of the incubation time used to determine them. These r e s u l t s indicated that the. shorter the incubation time, the larger the estimate of the maximum uptake rate obtained. Figure 30 shows the r e l a t i o n s h i p of V max with the incubation time used f o r i t s determination. No observable trend was exhibited i n the h a l f - s a t u r a t i o n constant for uptake (K g) as a function of incubation time. 4.3) Perturbation technique, Method 3: The uptake parameter estimates are given i n Table VI as a function of the concentration of the perturbation. The V from these experiments were a l l max remarkably s i m i l a r , ranging from 0.24 + 0.02 to 0.27 ± .0.04 h r " 1 . The h a l f -saturation constants, however, showed a great deal of v a r i a b i l i t y depending on the magnitude of - the i n i t i a l perturbation. The larger the perturbation used, the greater the K g estimate. A graphic representation of t h i s r e l a t i o n s h i p i s given i n F i g . 31. 5) Discussion It i s apparent from the r e s u l t s of t h i s study that the method chosen to determine uptake k i n e t i c parameters greatly a f f e c t s the value of the estimate obtained. Therefore, comparison of k i n e t i c estimates f o r various phytoplankton 121 V A R I A B L E I N C U B A T I O N T I M E 0.8 0.6 f 0.4 0.2 0 0 2 4 6 S(ug-at-r 1 ) Figure 29. Ammonia uptake ,(hr 1 ) as a function of substrate concentration . - . -1 for P. pyvifovmts grown i n an ammonia l i m i t e d chemostat at 0.5 d . The incubation time over which the uptake rate was calculated was the time at which the substrate concentration had dropped to ha l f .of the o r i g i n a l concentra-t i o n . Uptake rates were calculated using the true t = 0 substrate concentra-t i o n , #, and the f i r s t measured substrate concentration, 0. 122 T I M E ( m i n ) T Figure 30. Determination of V , using Method 2 (constant incubation time nicix at a l l substrate concentrations) as a function of incubation time. Bars represent one standard error. 123 t Figure 31. The h a l f - s a t u r a t i o n constant (K ) as determined by the s J perturbation technique f o r d i f f e r e n t I n i t i a l 1 substrate -additions. Bars represent-one standard, error. Table V. K and V (V ') values as determined f or d i f f e r e n t s max max incubation times using method 2 in-which incubation, time was constant a t - a l l substrate concentrations. Incubation Time K ± s.e. V (V ') ± s max max (min) (yg-at • I "*") (hr" 1) 2 0.32 ± .09 1.72 ± .07 5 0.05 '+• .07 0.76 ± .03 11 0.04 ± .02 0.47 ± .01 17 0.18 ± .04 0.42 ± .01 30 0.45 ± .13 0.37 ± .02 Table. VI. K and V (V.)'values as determined by the perturbation s max 1 • • • J " -technique (method 3) at d i f f e r e n t perturbation concentrations. Perturbation K ±s.e. V (V.) ±s.e. s max 1 (jig-at • a'1) 5.0 0.97 ±.26 0.24 ±.02 2.5 0.21 ±.09 0.27 ±.02 1.0 0.15 ±.05 0.24 ±.04 0.5 0.08 ±.03 0.27 ±.04 126 species i n the l i t e r a t u r e should be jnade with caution since the actual method used to determine the parameters may bias the r e s u l t s . Recent work by Burmaster and Chisholm- (1979) -have compared the estimate of parameters obtained through the d i r e c t incorporation method (isotope uptake) and the disappearance method. Providing a true t = 0 substrate value was not obtained i n the disappearance method and absorption was co n t r o l l e d f o r i n the incorporation -method, l i t t l e d i f f e r e n c e i n the two methods was apparent. Consequently, the conclusion of the study outlined i n t h i s appendix should be applicable to studies employing the isotope incorporation method. Of the three methods used, Method 2, the constant time, v a r i a b l e substrate method, gave the highest estimate of V e s p e c i a l l y over short-incubation max ' .••.:.;>,. periods. This i s a r e s u l t of the n o n - l i n e a r i t y i n nutrient uptake rate with time exhibited by t h i s and other organisms (Conway et a l . , 1976; Conway & Harrison, 1977) i n response to a perturbation. Figure 32 shows the disappear-ance of ammonia with time for P. piriformis for a ^3 y g - a t 1 perturbation. The i n i t i a l rapid disappearance followed by a slower, long-term uptake response i s the phenomenon responsible f or much of the v a r i a b i l i t y i n V estimates, max depending on the incubation time used. If short incubation times are employed, the i n i t i a l rapid uptake phenomenon i s weighted more heavily i n the estimate, and consequently high maximal uptake rates are determined. If longer incuba-t i o n times are used, the slower long-term uptake response (V^) (Conway et a l . , 1976) i s weighted more heavily i n the estimate, r e s u l t i n g i n a lower estimate of V . This r e s u l t i s not unique for ammonia uptake. Similar decreases i n max 14 uptake rate with increasing incubation time have been observed for C uptake (Marra, 1978a). The decreased uptake rate following a perturbation has been r a t i o n a l i z e d previously (Conway et a l . , 1976; Conway & Harrison, 1977). These authors f e l t that the i n i t i a l rapid uptake (hereafter r e f e r r e d to as V ' a f t e r McCarthy max 127 & Goldman, 1979) represented the true uptake p o t e n t i a l of the steady state population, whereas represented an a s s i m i l a t i o n phase. An a d d i t i o n a l explanation i s that the i n i t i a l rapid uptake, v m ^ x , may be a c e l l surface adsorption phenomenon and V may be the membrane transport component. This study was not extensive enough to resolve t h i s p o s s i b i l i t y . Consequently, t h i s study w i l l r e f e r to disappearance of the nutrient from the medium as uptake. In s p i t e of the fact that knowledge i s lacking about the exact process observed when nutrient uptake parameters are determined, the population's a b i l i t y to remove ammonium from the environment i s s t i l l being measured. There-fore, determining the maximum nutrient procurement (uptake a b i l i t y ) of a popu-l a t i o n requires use of an extremely short incubation time i n order to minimize the e f f e c t s of feedback regulation of the uptake rate. The shortest uptake i n t e r v a l used i n t h i s study was 2 min. This was the f a s t e s t time i n which the nutrient could be added, mixed and the c e l l s f i l t e r e d . If a shorter time i n t e r v a l were used the measured uptake response may have been even greater, as i i t has not yet been possible to show the l i n e a r i t y of V over that i n i t i a l J max T 2 min. As a r e s u l t the 2-min V estimate i s probably an under-estimate of max the true maximum uptake rate. In order to acknowledge the dependence of the i V estimate on the time of incubation we suggest that future designations max & & 6 include the time over which the uptake response was measured ( i . e . , V max (2 min)). Use of the perturbation technique, as o r i g i n a l l y used by Caperon and Meyer (1972b), measures the component, as defined by Conway et a l . (1976) and t i s a great under-estimate of the i n i t i a l rapid uptake phenomenon, V . The max V component, however, i s of great e c o l o g i c a l s i g n i f i c a n c e i f the population i s exposed to f l u c t u a t i o n s i n nutrient l e v e l s of long duration (low frequency patchiness). 128 The estimates from the perturbation technique show a great deal of v a r i a b i l i t y depending on the i n i t i a l concentration of the addition (Fig.30). The curve through these data represents a v i s u a l extrapolation to a IC value at a 0 yg-at«£ 1 perturbation, i n other words, the 1C of the unperturbed culture. This f i g u r e shows that the a f f i n i t y f o r the l i m i t i n g nutrient r a p i d l y decreases .after the population has been exposed to a nutrient pulse. The low l e v e l perturbations are taken up r a p i d l y and do not a f f e c t the popula-tion's n u t r i e n t - l i m i t e d state. On the other hand, large perturbations which are taken up over a period of hours by a n u t r i e n t - l i m i t e d population, represents a s u b s t a n t i a l modification i n the population's nutrient physiology. For example, uptake of a 5 yg-at'£ 1 of ammonium.represents a 50% increase i n population nitrogen quota; (the populations used i n t h i s study had a p a r t i c u -l a t e nitrogen value of 10 yg-at«£ "*"). The v a r i a b l e incubation time r e s u l t s were calculated i n two ways. The f i r s t method used the true T = 0 nutrient value. The uptake v e l o c i t y was then calculated over the time i n t e r v a l required for the ambient nutrient concentration to drop to half the o r i g i n a l concentration. This method resulted i n data that did not f i t a rectangular hyperbola (Fig. 29). The reason for t h i s d i f f e r e n t curve i s due to varying contributions of the rapid uptake phenomenon. For the low concentration uptake determinations, the i n i t i a l r apid uptake phenomenon accounts for most of the nutrient disappearance because the uptake i n t e r v a l i s short. Therefore, at low concentrations we measure high uptake v e l o c i t i e s . At high concentrations the i n i t i a l rapid uptake accounts for only a small portion of the t o t a l uptake. This i s because the.' incubation time i s long and there i s a large contribution to t o t a l uptake by the V. component. This r e s u l t s i n the lower uptake estimates at high concentrations. If the f i r s t measured nutrient concentration i s used as the i n i t i a l sub-strate concentration and uptake v e l o c i t i e s are calculated, then the 129 TIME (hr) t Figure 32. Disappearance of ammonium-with time, showing the V and V max 1 components. 130 data f i t a rectangular hyperbola (Fig. 29). The V ' , as obtained rn 3.x by t h i s method, was i d e n t i c a l to the of the perturbation method. The h a l f -saturation constant as determined by t h i s method was 0.14 ±0.12 yg-at•£ 1 ammonium. The major problem with t h i s method i s that each uptake determination represents a population with a d i f f e r e n t past h i s t o r y or nu t r i e n t time expo-sure. This could r e s u l t i n d i f f e r e n t contributions of feedback e f f e c t s , by the nutrient taken up, on the subsequent uptake rate. It appears that the best method for determining the maximum uptake rate i s to use a constant incubation time at varying substrate concentrations (method 2). The reporting of data should include a notation as to the incubation time used. The perturbation technique (method 3) and the v a r i a b l e time incubation method (method 1) both give an estimate of the of the population. The determination of the h a l f - s a t u r a t i o n constant i s not straightforward. Using the perturbation technique at varying i n i t i a l n utrient l e v e l s and extrapolating to a 0 yg-at•£ 1 perturbation appears to be one method (Fig.31). Unpublished work (Harrison, pers. comm.) suggests that t h i s may not be univ e r s a l for a l l phytoplankton species. In f a c t , he found that the size of the perturbation had no e f f e c t on the K value determined by t h i s method. s Using a constant incubation time gives K g estimates that overlap at the 95% confidence l i m i t s , regardless of the incubation times used. The v a r i a b l e incubation time also gives an estimate encompassed by the 95% confidence i n t e r -v a l s of the constant incubation time method. Therefore, the K data are not s of s u f f i c i e n t r e s o l u t i o n to resolve any trends that may e x i s t . I t i s apparent from the r e s u l t s of t h i s study that caution must be exercised when comparing uptake k i n e t i c values which were determined using d i f f e r e n t methods. The point must be made that data i n t h i s study pertain 131 only to a nitrogen l i m i t e d population. When l i m i t a t i o n i s not severe, uptake appears to be l i n e a r over time (Eppley & Thomas, 1969; Eppley et a l . , 1969). The degree of uptake complexity would then appear to be a function of the degree of nitrogen l i m i t a t i o n , with the contribution of the V ' component max increasing with increasing nutrient deficiency (McCarthy & Goldman, 1979) and decreasing incubation time. 

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