UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Some aspects of iron limitation in a marine diatom Mueller, Bert 1985

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1985_A6_7 M82.pdf [ 4.65MB ]
Metadata
JSON: 831-1.0053144.json
JSON-LD: 831-1.0053144-ld.json
RDF/XML (Pretty): 831-1.0053144-rdf.xml
RDF/JSON: 831-1.0053144-rdf.json
Turtle: 831-1.0053144-turtle.txt
N-Triples: 831-1.0053144-rdf-ntriples.txt
Original Record: 831-1.0053144-source.json
Full Text
831-1.0053144-fulltext.txt
Citation
831-1.0053144.ris

Full Text

SOME ASPECTS OF IRON LIMITATION IN A MARINE DIATOM by BERT MUELLER B . S c . , U n i v e r s i t y o-f Winnipeg, Winnipeg, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Departments o-f Oceanography and Zoology) We accept t h i s t h e s i s as conforming t o the requ i r e d sta«da>-d THE UNIVERSITY OF BRITISH COLUMBIA May 1985 © BERT MUELLER, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of (^trip-o^ The University of B r i t i s h Columbia 1956 Main Mall ; ' • . Vancouver, Canada V6T 1Y3 Date (X^Ajii ; /9<H" DE-6 £3/81) ABSTRACT Batch c u l t u r e s o-f the marine diatom, Thalassiosir a pseudonana were grown i n the defined medium A q u i l without EDTA and with varying concentrations o-f added i r o n <Fe) . The response of c u l t u r e s t o Fe d e p l e t i o n was more r a p i d and dramatic when in vivo f l u o r e s c e n c e (a c o r r e l a t e of c e l l u l a r c h l o r o p h y l l a) was used as a measure of biomass i n s t e a d of c e l l d e n s i t y . In vivo f l u o r e s c e n c e and in vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y were found t o be more s e n s i t i v e and r e l i a b l e measures of Fe l i m i t a t i o n than c e l l d e n s i t y alone. Because the physiology of n u t r i e n t - l i m i t e d c e l l s changes r a p i d l y i n batch c u l t u r e , a chemostat was designed and constructed t o grow axenic c u l t u r e s of T. pseudonana under constant degrees of Fe l i m i t a t i o n . F e - l i m i t e d chemostat c u l t u r e s grown i n A q u i l without EDTA were a l s o p a r t l y l i m i t e d by s i l i c a t e . Despite growing t o higher c e l l d e n s i t i e s , F e - r e p l e t e batch c u l t u r e s were not s i l i c a t e l i m i t e d . I t thus appears that F e - s t r e s s c o n t r i b u t e s t o increased s i l i c a t e quotas i n t h i s organism. Doubling the s i l i c a t e c o ncentration of the medium a l l e v i a t e d s i l i c a t e l i m i t a t i o n and c e l l s responded t o Fe l i m i t a t i o n by co n t i n u i n g c e l l d i v i s i o n f o r a time while in vivo f l u o r e s c e n c e remained constant. Measured quotas of c e l l u l a r Fe were 370 a t t o m o l e s - c e l l - I (1 attomole i s 1 X lCr 8 moles) f o r an Fe - r e p l e t e c u l t u r e and ranged from 50 t o 100 attomol es-eel l - 1 f o r Fe-deplete batch and chemostat c u l t u r e s . Fe as s o c i a t e d with the c e l l s u r f a c e was not detected i n c e l l s grown i n a batch c u l t u r e which r e c e i v e d no added Fe or in an Fe-limited chemostat culture. As a comparison, the d i n o f l a g e l l a t e , Protogonyaulax tamarens is Clone D—255 grown in Fe-limited batch culture was -found to have an Fe quota of 116 f edit omoles-eel l " 1 which, however, was in close agreement with 7. pseudonana when Fe quotas were calculated on a per unit volume basis. A method for the measurement of Fe uptake by 3 3Fe .l a b e l l i n g was developed which estimated an uptake rate of 908 attomoles-cel l - 1-hr" 1 over a 10 min exposure to 450 nM Fe. Many of the methods developed and tested i n t h i s study should prove valuable i n future study of the iron requirement of phytoplankton and t h e i r adaptations to low-Fe stress. TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES vi LIST OF FIGURES v i i ACKNOWLEDGEMENTS ix Introduction. . - .......1 Chapter I: General Methods ....5 Test Organism 5 Isolation and Maintenance o-f Axenic Stock Cultures....5 Medium Preparation 6 Chapter II: Batch Culture Experiments 12 Introduction 12 Methods 13 Results and Discussion 14 Chapter I l l s Continuous Culture 39 Introduction 39 Materials and Methods 40 1. Construction of an Iron-limited Chemostat....40 2. Pumping Systems..... 43 3. Chemostat Out-flow 45 4. Chemostat Operation 46 5. Chemostat Monitoring 48 6. Confirmation of Fe-Li mi tat i on 48 7. Confirmation of the Axenic Condition of Chemostat Culture 49 8. Biomass Measurements 49 Results and Discussion 51 1. Achieving an Fe—Limited Chemostat-.. 51 1. 1 Chemostat Tr i al I.... 52 1.2 Chemostat T r i a l II 57 1.3 Chemostat T r i a l III 58 1.4 Chemostat T r i a l IV 64 Chapter IV: Determination of C e l l u l a r Fe 76 Introduction 76 Methods 76 Sampling and F i l t r a t i o n 78 Digestion 78 Reagents and Fe Determination 79 Fe Contamination 80 Ascorbate Washing Procedure 81 Results and Discussion 83 Chapter V; Determining Short Term Fe Uptake 88 Introduct i on 88 Methods 88 Results and Discussion 91 Summary. 93 Recommendations for Future Work 95 References. 99 v i L I S T OF TABLES TABLE I C o m p o s i t i o n o-f A q u i l - F e medium 10 TABLE I I C a l c u l a t e d Fe q u o t a s and -f l u o r e s c e n c e : F e r a t i o s . . 35 TABLE I I I P r o s and Cons o-f pumping s y s t e m s .....44 TABLE IV Summary o-f b a t c h c u l t u r e and c h e m o s t a t measurements 84 v i i LIST OF FIGURES FIGURE 1. Scheme f o r A q u i l - F e p r e p a r a t i o n 9 FIGURE 2. Growth as measured by c e l l c o n c e n t r a t i o n f o r Fe and no Fe t r e a t m e n t s 15 FIGURE 3. Growth as measured by in vivo f l u o r e s c e n c e f o r Fe and no Fe t r e a t m e n t s 17 FIGURE 4. In vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y f o r Fe and no Fe t r e a t m e n t s 18 FIGURE 5. R e l a t i o n between e x t r a c t e d c h l o r o p h y l l a and in vivo f l u o r e s c e n c e 20 FIGURE 6. In vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y f o r a range of added Fe c o n c e n t r a t i o n s 21 FIGURE 7. In vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y f o r a range of added Fe c o n c e n t r a t i o n s 24 FIGURE 8. R e l a t i o n s h i p between c e l l c o n c e n t r a t i o n and t h e c o n c e n t r a t i o n of added Fe. . ....28 FIGURE 9. R e l a t i o n s h i p between in vivo f l u o r e s c e n c e and t h e c o n c e n t r a t i o n of added Fe 29 FIGURE 10. R e l a t i o n s h i p between in vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y and t h e c o n c e n t r a t i o n of added Fe. 31 FIGURE 11. Schematic diagram of t h e chemostat a p p a r a t u s . . . 4 2 FIGURE 12. Chemostat Is C e l l d e n s i t y and in vivo f l u o r e s c e n c e over t i m e 54 FIGURE 13. Chemostat Is C o n f i r m a t i o n of F e - 1 i m i t a t i o n e x p e r i m e n t — in vivo f l u o r e s c e n c e 55 v i i i FIGURE 14. Chemostat Is Con-firmation of Fe-1 i m i t a t i o n experiment— c e l l d e n s i t y . . . . . . . 56 FIGURE 15. Chemostat I I I : C e l l d e n s i t y and i n vivo f l u o r e s c e n c e over time 59 FIGURE 16. Chemostat I I I : C o n f i r m a t i o n of F e - 1 i m i t a t i o n experiment- c e l l d e n s i t y 61 FIGURE 17. Chemostat I I I : C o n f i r m a t i o n of F e - 1 i m i t a t i o n experiment- i n vivo f l u o r e s c e n c e . . . . . 62 FIGURE IB. Chemostat IV: C e l l d e n s i t y and i n vivo f l u o r e s c e n c e over time...... 65 FIGURE 19. Chemostat IV: In vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y over time. 67 FIGURE 20. Chemostat IV: C o n f i r m a t i o n of F e - 1 i m i t a t i o n experiment— c e l l d e n s i t y 68 FIGURE 21. Chemostat IV: C o n f i r m a t i o n of F e - 1 i m i t a t i o n experiment- i n vivo f l u o r e s c e n c e 69 FIGURE 22. Chemostat IV: C o n f i r m a t i o n of F e - 1 i m i t a t i o n experiment- i n vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y 71 FIGURE 23. C a l i b r a t i o n of asc o r b a t e washing procedure..... 82 FIGURE 24. Test of quenching of "Fe by c e l l s 90 i x A C K N O W L E D G E M E N T S I express my s i n c e r e thanks t o my s u p e r v i s o r , Dr. A.G. Lewis f o r h i s concern, and guidance throughout t h i s study. Deep a p p r e c i a t i o n must a l s o be extended t o my research committee, Drs. R.J. Andersen, P.J. Harrison and T.F. Pedersen f o r t h e i r understanding and advice. Dr E . V . G r i l l s u p p l i e d the f e r r o z i n e reagent and c o n t r i b u t e d many us e f u l suggestions f o r the determination of i r o n . Frequent d i s c u s s i o n s with Dr. John Parslow, Peter Thompson, N e i l P r i c e and Greg Doucette were i n v a l u a b l e t o my understanding of chemostats and a l g a l c u l t u r i n g i n gener a l . Dr. Greg Boyer provided the c u l t u r e s of Protogonyaulax tamarBnsis. S p e c i a l thanks t o Junko Kazumi who often served as a sounding—board f o r my problems and ideas. The m u l t i d i s c i p i i n a r y atmosphere of the Oceanography Department provided me with a s t i m u l a t i n g environment f o r the p r a c t i c e and understanding of science. I g r a t e f u l l y acknowledge the Zoology Department f o r pr o v i d i n g me with Teaching A s s i s t a n t s h i p s and my su p e r v i s o r f o r summer f i n a n c i a l support. 1 INTRODUCTION Iron <Fe) i s an e s s e n t i a l m i c r o n u t r i e n t -for a l l l i v i n g c e l l s with the p o s s i b l e exception of the l a c t i c a c i d b a c t e r i a (Neilands, 1976). Although i t i s the f o u r t h most abundant element i n the Earth's c r u s t , i n o x i c aqueous s o l u t i o n s i t i s one of the l e a s t s o l u b l e of metals. In the presence of oxygen, F e ( I I ) i s r a p i d l y o x i d i z e d t o F e ( I I I ) which has an overwhelming propensity t o p r e c i p i t a t e as f e r r i c hydroxide (K B P lCr 3 9) at p h y s i o l o g i c a l pH's. F r e s h l y - p r e c i p i t a t e d c o l l o i d s of f e r r i c hydroxide can serve as a source of metabolic Fe but aging, heating and the r e s u l t a n t r e a c t i o n s of o l a t i o n and o x o l a t i o n convert them t o p r o g r e s s i v e l y poorer sources of b i o l o g i c a l l y a v a i l a b l e Fe (Lewin and Chen, 1971, 1973; Wells et a l . , 1983). The absolute requirement f o r Fe and i t s r e s t r i c t e d b i o a v a i l a b i l i t y are evidenced by the range of adaptations organisms have evolved t o sequester i t from the environment and withhold i t from t h e i r competitors. Siderophores, defined as h i g h — a f f i n i t y F e ( I I I ) t r a n s p o r t agents, have long been known t o be produced by t e r r e s t r i a l b a c t e r i a and fungi (Neilands, 1967). In a d d i t i o n t o making low environmental concentrations of F e d 11) a v a i l a b l e t o the siderophore producer, Fe can be excluded from microorganisms which do not possess the s p e c i f i c s i d e r o p h o r e - F e ( I I I ) receptor. Many i n f e c t i o u s b a c t e r i a o b t a i n Fe from w i t h i n t h e i r hosts by the production of siderophores. Hosts can counter by producing t h e i r own Fe binding and tr a n s p o r t compounds, such as t r a n s f e r r i n , l a c t o f e r r i n and conalbumin (Emery, 1980). In homeotherms, i t has been 2 suggested that -fever i s a mechanism of withholding Fe from b a c t e r i a s i n c e the discovery t h a t some i n f e c t i n g b a c t e r i a reduce or h a l t siderophore production at elevated temperatures ( G a r i b a l d i , 1972). Competition f o r Fe a l s o e x i s t s i n aquatic h a b i t a t s . Siderophores have been i s o l a t e d from some freshwater cyanobacteria (Simpson and Neilands, 1976; Murphy et a i . , 1976). Murphy et a i - (1976) found that blooms of cyanobacteria which a l s o corresponded t o periods of high Fe-uptake and hydroxamate-siderophore production suppressed the growth of euka r y o t i c algae. Laboratory s t u d i e s have shown that hydroxamate siderophores from cyanobacteria can i n h i b i t the growth of some chlorophytes (Murphy et a i - , 1976s Murphy, 1976; B a i l e y and Taub, 19B0). In oceans, the competition f o r Fe i s l e s s w e l l understood but s i m i l a r trends e x i s t . Using a bioassay technique f o r hydroxamic a c i d s , Goyne and Carpenter (1974) estimated t h a t 43Z of Sargasso Sea b a c t e r i a l i s o l a t e s produced i r o n — b i n d i n g compounds, p o s s i b l y siderophores. Armstrong and Van Baalen (1979) i s o l a t e d an hydroxamate siderophore from the marine cyanobacterium, Agmenellum quadruplicatum. An F e — s p e c i f i c organic c h e l a t o r was detected i n a c u l t u r e of the diatom, Chaetoceros social is but because the c u l t u r e was not axenic the source of the c h e l a t o r was ambiguous (Spencer et a i . , 1973). Further search f o r siderophores i n euk a r y o t i c phytoplankton produced negative r e s u l t s although no attempt was made to induce Fe l i m i t a t i o n i n the c u l t u r e s t e s t e d (Swallow et a i . , 1978; McKnight and Morel, 1979). The f i r s t d e f i n i t i v e example 3 o-F a siderophore produced by a e u k a r y o t i c phytoplankter was found by T r i c k et al - (1983a) who i s o l a t e d the compound, p r o r o c e n t r i n , from axenic F e - l i m i t e d c u l t u r e s of the d i n o f l a g e l l a t e , Prorocentrum minimum. Using s i m i l a r c u l t u r e techniques, T r i c k et al. (1983b) detected e x t r a c e l l u l a r hydroxamates i n F e - l i m i t e d axenic c u l t u r e s of Thalassiosira pseudonana and Dunaliella tertiolecta, a diatom and a green alg a r e s p e c t i v e l y . They c r e d i t the s u c c e s s f u l d e t e c t i o n of siderophores i n these organisms t o : 1) the use of axenic a l g a l c u l t u r e s t o e l i m i n a t e the p o s s i b i l i t y of a b a c t e r i a l o r i g i n of siderophores; 2) the c u l t u r i n g of phytoplankton under Fe— d e f i c i e n t c o n d i t i o n s ; 3) the choice of sampling times s i n c e the siderophores were found t o have extremely short residence times i n the medium; and 4) the c o n c e n t r a t i o n of siderophores from the c u l t u r e f i l t r a t e before d e t e c t i o n . The reasons given above f o r the s u c c e s s f u l d e t e c t i o n of siderophores u n d e r l i n e the importance of a c h i e v i n g and maintaining the appropriate c u l t u r e c o n d i t i o n s and p h y s i o l o g i c a l s t a t e . When the p h y s i o l o g i c a l s t a t e t o be achieved i s m i c r o n u t r i e n t l i m i t a t i o n , s p e c i a l care i s warranted s i n c e a small amount of contamination can lead t o m i c r o n u t r i e n t s u f f i c i e n c y . E q u a l l y important, one must ensure that the n u t r i e n t of i n t e r e s t i s the only n u t r i e n t which i s l i m i t i n g . If Fe l i m i t a t i o n or some aspect of i t i s t o be i n v e s t i g a t e d , i t i s e s s e n t i a l that the physiology of the organism i s c h a r a c t e r i z e d and c o n t r o l l e d . The o r i g i n a l purpose of the t h e s i s research was t o examine 4 the Fe metabolism o-f T. pseudonana with respect to the production of siderophores. However, because of the t e c h n i c a l problems encountered, the study was r e o r i e n t e d toward the development of c u l t u r e techniques which would permit the r e g u l a t i o n of the extent and t i m i n g of Fe l i m i t a t i o n . The developed techniques were then used t o study c e r t a i n aspects of Fe l i m i t a t i o n i n the diatom. C u l t u r e s of the t e s t organism, Thalassiosira pseudonana, were i s o l a t e d and maintained i n an axenic s t a t e . To reduce v a r i a t i o n i n c u l t u r e responses due t o seawater obtained from d i f f e r e n t seasons or sources, a m o d i f i c a t i o n of the defined c u l t u r e medium, A q u i l (Morel et a i . , 1979), was employed. I n i t i a l l y , J. pseudonana was grown i n batch c u l t u r e s t o determine the s p e c i f i c responses which are c h a r a c t e r i s t i c of Fe l i m i t a t i o n i n t h i s organism and the l e v e l s of added Fe which would e l i c i t symptoms of Fe l i m i t a t i o n . These batch c u l t u r e experiments formed the groundwork f o r growing T. pseudonana i n a chemostat designed f o r t r a c e metal l i m i t a t i o n . C e l l u l a r Fe was determined r a t h e r than medium Fe concentrations because: 1) previous s t u d i e s have shown that i n t r a c e l l u l a r l e v e l s of n u t r i e n t s c o n t r o l phytoplankton growth r a t e r a t h e r than e x t e r n a l n u t r i e n t l e v e l s (Davies, 1970; P r i c e and C a r e l 1 , 1964; Droop, 1968; Caperon, 1968; and Fuhs, 1969); and 2) ambient concentrations of Fe i n F e - l i m i t e d c u l t u r e s were expected t o be extremely low. To q u a n t i f y the organism's a b i l i t y t o take up Fe at v a r i o u s stages of Fe s t r e s s , a method was developed f o r the short-term exposure of c u l t u r e s t o r a d i o -l a b e l l e d Fe and i t s measurement i n c e l l s . 5 CHAPTER I G E N E R A L C U L T U R E M E T H O D S TEST ORGANISM The marine diatom Thalassiosira pseudonana (Hustedt) Hasle et Heimdal ( WHOI Clone 3-H, NEPCC# 58) was obtained from the North East P a c i f i c C u l t u r e C o l l e c t i o n ( U n i v e r s i t y of B r i t i s h Columbia). ISOLATION AND MAINTENANCE OF AXENIC STOCK CULTURES 7. pseudonana was acquired as a u n i a l g a l c u l t u r e . An axenic c u l t u r e was i s o l a t e d by exposing a s e r i e s of 50 ml stationary—phase c u l t u r e s grown i n A q u i l (Morel et al.,1979) t o a range of concentrations of an a n t i b i o t i c c o c k t a i l (Hoshaw and Rosowski, 1973). The c o c k t a i l contained 100 mg p e n i c i l l i n G and 50 mg streptomycin-SO, d i s s o l v e d i n 10 ml of d i s t i l l e d water t o which was added 10 mg of chloramphenicol d i s s o l v e d i n 1 ml of 95% ethanol. The c o c k t a i l was f i l t e r — s t e r i l i z e d (0.4 um Nuclepore polycarbonate f i l t e r ) before a d d i t i o n t o c u l t u r e s or agar p l a t e s . A f t e r 3 days of a n t i b i o t i c exposure, c u l t u r e s were streaked onto p l a t e s of A q u i l and 1.2% agar (Difco L a b o r a t o r i e s ) . The p l a t e s had been p r e v i o u s l y t r e a t e d by a s e p t i c a l l y spreading 2 drops of the a n t i b i o t i c c o c k t a i l onto the s u r f a c e and a l l o w i n g i t t o permeate the agar. A f t e r s t r e a k i n g , the p l a t e s were sealed with masking tape t o prevent water l o s s during the long i n c u b a t i o n times r e q u i r e d t o grow v i s i b l e T. pseudonana c o l o n i e s . V i s i b l e s i n g l e c o l o n i e s were examined with a stereomicroscope and a s e p t i c a l l y t r a n s f e r r e d t o s t e r i l e , a n t i b i o t i c - f r e e A q u i l . The axenic c o n d i t i o n of these c u l t u r e s was v e r i f i e d by acridine—orange d i r e c t s t a i n i n g (Hobbie et al. , 1977) and examination under an epif1uorescence microscope. B a c t e r i a l counts of c u l t u r e s were compared with counts of s t e r i l e (autoclaved) A q u i l . Samples of c u l t u r e were a l s o streaked onto STP b a c t e r i a l medium ( P r o v a s o l i et al., 1957) s o l i d i f i e d with 1.2.7. agar and onto a b a c t e r i a l medium c o n s i s t i n g of SOX stationary-phase T, pseudonana-culture f i l t r a t e and 502 f r e s h A q u i l s o l i d i f i e d with 1.2% agar. The b a c t e r i a — f r e e c u l t u r e of T. pseudonana which was used f o r a l l subsequent study was derived from the exposure of s t a t i o n a r y -phase c u l t u r e t o 17. a n t i b i o t i c c o c k t a i l . Axenic c u l t u r e s of T. pseudonana were r o u t i n e l y maintained by s t r e a k i n g l i q u i d c u l t u r e s onto A q u i l s o l i d i f i e d with 17. agar. I t was found t h a t T. pseudonana grew b e t t e r and produced l a r g e r c o l o n i e s on A q u i l p l a t e s i f l e s s agar was used t o s o l i d i f y the medium. Presumably i f the s o l i d medium contained more water the c o l o n i e s were l e s s prone t o d i f f u s i o n l i m i t a t i o n of n u t r i e n t s . When re q u i r e d f o r experimentation, s i n g l e c o l o n i e s were f i r s t t r a n s f e r r e d t o 200 ml of A q u i l c o n t a i n i n g f r e s h l y - p r e c i p i t a t e d f e r r i c hydroxide (4.5 X 10"7 M) but no EDTA (et h y l e n e d i a m i n e t e t r a a c e t i c acid) and l a t e r t r a n f e r r e d t o A q u i l c o n t a i n i n g n e i t h e r EDTA nor Fe. MEDIUM PREPARATION A l l experiments employed a m o d i f i c a t i o n of the marine algal-growth medium, A q u i l (Morel et a i . , 1979). Experiments by Wells (1982) have shown that EDTA i s not req u i r e d by T. pseudonana i f Fe i s s u p p l i e d as f r e s h l y - p r e c i p i t a t e d f e r r i c 7 hydroxide. The absence o-f EDTA would tend to increase the free io n i c a c t i v i t i e s of trace metals added to the medium and t h i s could r e s u l t i n toxic e f f e c t s to phytoplankton. However, Wells did not observe any decrease i n the growth rate of T. pseudonana when medium of t h i s formulation was used. As a metal chelator, EDTA could make portions of the unavailable Fe-f r a c t i o n available to the diatom and thus i n t e r f e r e with the goal of achieving an Fe-limited medium. The presence of EDTA in the medium might also complicate the search for e x t r a c e l l u l a r Fe—binding compounds which might be produced by Fe-limited cultures. For these reasons EDTA was excluded from a l l preparations of Aquil. Figure 1 shows the scheme for the medium preparation while Table I l i s t s the weights of i t s s a l t s , t h e i r stock solution and f i n a l concentrations. Standard ocean water (SOW) which contained the major Aquil s a l t s , was prepared in 20 l i t e r volumes by f i r s t adding the major s a l t s except MgCl 2H 20 to approximately 15 l i t e r s of g l a s s - d i s t i l l e d water (DW) and mixing u n t i l dissolved. Hygroscopic MgCl2-H20 was predried at 60° C, weighed, and then dissolved in the above solution. DW was added to make 20 l i t e r s . The SOW was then bubbled with acid cleaned (1 N H2S0«) and f i l t e r e d (0.4 um Nuclepore) a i r to eq u i l i b r a t e i t with a i r and consequently r a i s e the pH to i t s nominal value of 8.00. After e q u i l i b r a t i o n , the SOW was passed through a glass column containing the ion exchange r e s i n , Chelex 100, which had been previously acid cleaned and equilibrated to pH 8.00 and to the major cations found in SOW. This treatment served to reduce the level of transition-metal Figure 1. Scheme f o r the preparation of Aquil-Fe (modified from Morel et a i - , 1979). Refer t o Table f o r weights of s a l t s and vitamins. BIOTIN ( N H 4 ) 6 M o 7 0 2 4 4 H 2 0 C u S 0 4 - 5 H 2 0 MnCI 2 C o C I 2 6 H 2 0 4 H S Z n S 0 4 7 H 2 c 3 c 100 ml THIAMINE HCI r ~ i i liter 0.1 liter I ml I ml I ml 100 ml vitamin stock I liter trace metal stock 0.5 ml I ml N a H 2 P 0 4 N a C r N a N O , 3 c N a 2 S 1 0 3 NaC l NaCl N a 2 S 0 4 C a C I 2 2 H 2 0 KCI N a H C 0 3 KBr H 3 B 0 3 S r C I 2 6 H 2 0 NaF c M g C I 2 - 6 H 2 0 1 I 2 0 liters I ml I liter of A Q U I L - Fe Table I Composition o-f A q u i l - F e medium modified from Morel et al . (1979). I n i t i a l Stock F i n a l weight volume c o n c e n t r a t i o n c o n c e n t r a t i o n Substance (g) ( l i t e r s ) ( M ) ( M ) A q u i l s a l t s (SOW) NaCl 490.6 20 4.20 X 10"' 4.20 X 10" Na,S0. 81.8 20 2.88 X 10-2 2.88 X lO" 2 CaCl7-2H,0 30.8 20 1.05 X 10"s 1.05 X IO"2 KC1 14 20 9.39 X lO" 4 9.39 X 10'* NaHCO* 4 20 2.38 X lO" 3 2.38 X IO"3 KBr —> 20 8.40 X 10'* 8.40 X 10'4 H,B0, 0.6 20 4.85 X lO" 4 4.85 X 10"* SrCl,-6H,0 0.34 20 6.38 X 10"= 6.38 X i o - 3 NaF 0.06 20 7. 14 X lO" 3 7. 14 X 10"B MgCl,'6H,0 222 20 5.46 X lO" 2 5.46 X lO" 2 N u t r i ents NaH,P0.H,0 1.38 1 1.00 X IO"2 1.00 X IO"3 NaNQ, 8.50 1 1.00 X i o - ' 1.00 X i o - * Na,Si0,-9H?0 3.55 1 1.25 X IO"2 1.25 X i o - s Trace Metals CuSOa-SH^O 0.249 1 9.97 X IO"4 9.97 X IO"10 (NH. ) *MOTO j«-4HIO 0.265 1 1.50 X 10-3 1.50 X IO"' COC1,-6H,0 0.595 1 2.50 X i o - 3 2.50 X 10" MnCl2-4H?0 0.455 0. 1 2.30 X i o - 2 2.30 X 10"B ZnS0.-7H?a 0. 115 0. 1 4.00 X i o - 3 4.00 X i o - * Vi tamins B,? 0.011 0. 01 1. 10 X 10° g/1 5.5 X IO"7 g/1 B i a t i n 0.01 0. 1 1.00 X 10- g/1 5.0 X IO"7 g/1 Thiamine HC1 0.020 0. 1 2.00 X 10"' a/1 1.0 X 10"* g/1 11 contamination i n the SOW. The major n u t r i e n t s (NOV, P0,3_ and Si0 4" 4) were prepared according t o Morel et al. (1979) and were a l s o passed through a Chelex-100 column a f t e r a d j u s t i n g t h e i r pH t o 8.00. The metal (Cu, Mo, Co, Mn and Zn) and vitamin ( B i 2 , b i o t i n and thiamine) stock s o l u t i o n s were not passed through Chelex—100. Fe was e i t h e r excluded, t o y i e l d Aquil-Fe medium or added from 4.51 X 10-* M stocks of f r e s h l y prepared f e r r i c hydroxide p r e c i p i t a t e d from F e C l 3 (Wells et al., 1983) or added from Fe stock s o l u t i o n s ( a c i d i f i e d t o pH 2 with HC1) made from e l e c t r o l y t i c i r o n wire. A f t e r the a d d i t i o n of n u t r i e n t s , the medium was bubbled with f i l t e r e d (0.4 um Nuclepore) and a c i d cleaned (1 N H2S0») carbon d i o x i d e u n t i l the pH dropped t o approximately 5.5. Without t h i s pH r e d u c t i o n A q u i l s a l t s would p r e c i p i t a t e on a u t o c l a v i n g . Autoclaving and c o o l i n g the medium d i d not always r e t u r n the pH t o 8.00. Therefore i f axenic c u l t u r i n g was not e s s e n t i a l , the medium was bubbled with a c i d washed, but not s t e r i l e a i r t o r e e q u i 1 i b r a t e i t . If axenic c u l t u r e s were re q u i r e d the medium was a s e p t i c a l l y d i s t r i b u t e d among s t e r i l e c u l t u r e - f l a s k s , c losed and shaken sev e r a l times during the course of a day. The medium was r o u t i n e l y autoclaved i n a 12 l i t e r polycarbonate carboy. 1 2 CHAPTER II BATCH CULTURE EXPERIMENTS INTRODUCTION Due t o t h e i r s i m p l i c i t y , batch c u l t u r e s have d i s t i n c t advantages over more s o p h i s t i c a t e d c u l t u r i n g methods and are the obvious s t a r t i n g p o i n t -for research i n n u t r i e n t l i m i t a t i o n . Their s i m p l i c i t y - f a c i l i t a t e s the s t e r i l i z a t i o n of c u l t u r e v e s s e l s , media and the maintenance of axenic c u l t u r e s . S i m p l i c i t y , small s i z e and cost make batch c u l t u r e s amenable t o r e p l i c a t i o n and the subsequent e s t i m a t i o n of confidence i n measurements so t h a t the r e s u l t s of d i f f e r e n t treatments can be compared. In ge n e r a l , the observation of m i c r o b i a l growth i n batch c u l t u r e s a l l o w s r e l a t i v e l y easy a c q u i s t i o n of p r e l i m i n a r y data about the organism and the medium i n which i t grows. By t h e i r design, batch c u l t u r e s provide the mechanism whereby a microbe reaches n u t r i e n t l i m i t a t i o n . Since only a f i n i t e volume of medium i s contained i n a batch c u l t u r e the organism grows and consumes n u t r i e n t s u n t i l the n u t r i e n t i n l e a s t p r o p o r t i o n r e l a t i v e t o i t s needs i s exhausted. At t h i s p o i n t other n u t r i e n t s are s t i l l i n excess. Depending on the n u t r i e n t , the organism and i t s previous n u t r i t i o n a l s t a t e , such n u t r i e n t l i m i t a t i o n i n batch c u l t u r e s o f t e n occurs very r a p i d l y and consequently l i t t l e can be s a i d about how the organism approaches and adapts t o n u t r i e n t l i m i t a t i o n . Therefore, one u s u a l l y f i n d s batch c u l t u r e s i n one of two n u t r i t i o n a l s t a t e s : n u t r i e n t r e p l e t e or n u t r i e n t deplete. In order t o obta i n i n f o r m a t i o n on the physiology of a c u l t u r e i n an intermediate s t a t e of n u t r i e n t l i m i t a t i o n chemostat c u l t u r e s are re q u i r e d . 13 In t h i s study., the purpose of batch c u l t u r e experiments was t o determine the i n i t i a l e-f-fects of Fe l i m i t a t i o n on 7. pseudonana. This information would prove u s e f u l i n l a t e r i d e n t i f y i n g Fe l i m i t a t i o n i n chemostats. Batch c u l t u r e s were a l s o employed t o determine a c c u r a t e l y the medium concentrations of Fe tha t l i m i t the growth of 7- pseudonana i n A q u i l . This knowledge was needed t o formulate a chemostat medium that provided F e - l i m i t i n g c o n d i t i o n s but s t i l l allowed f o r the growth of a maximum number of c e l l s . High c e l l y i e l d s would f a c i l i t a t e the determination of Fe per c e l l and the d e t e c t i o n of e x t r a c e l l u l a r Fe—binding compounds. METHODS No attempt was made t o ensure that experimental batch c u l t u r e s remained axenic. Fe stocks used t o s p i k e media were not s t e r i l e s i n c e they were n e i t h e r autoclaved nor f i l t e r -s t e r i l i z e d . Wells et air (1983) showed that the process of au t o c l a v i n g Fe stock a c c e l e r a t e d the maturation of f r e s h l y -p r e c i p i t a t e d Fe t o l e s s b i o l o g i c a l l y a v a i l a b l e forms such as hematite and g o e t h i t e . F i l t e r s t e r i l i z a t i o n was not employed i n order t o avoid the adsorption of Fe t o membrane f i l t e r s or the f i l t e r apparatus (Gardner, 1982). Batch c u l t u r e s were i n o c u l a t e d with 7. pseudonana from Fe— st a r v e d , axenic stock c u l t u r e s t o y i e l d i n i t i a l c e l l c o n c e n t r a t i o n s of about 1000 c e l l s - m i - 1 . C u l t u r e s were contained i n 500 ml polycarbonate Erlenmeyer f l a s k s with polypropylene screwcap c l o s u r e s or i n 100 ml polycarbonate tubes covered with Saran Wrap. C u l t u r e s were 14 incubated at 17°C and i r r a d i a t e d with 120 uEin-m"2-s-1 o-f l i g h t c o ntinuously s u p p l i e d by " V i t a L i g h t " <Duro-Test) f l u o r e s c e n t lamps. C e l l c o n c e n t r a t i o n , determined by Coulter counter (Model Zf, Coulter E l e c t r o n i c s ) and i n vivo f l u o r e s c e n c e (Model 10, Turner Designs) were used t o monitor biomass d a i l y u n t i l s e v e r a l days i n t o s t a t i o n a r y phase. C h l o r o p h y l l a was e x t r a c t e d i n 90% acetone and measured by spectrophotometric absorption according t o S t r i c k l a n d and Parsons (1968). C h l o r o p h y l l a was computed according t o the formula given i n J e f f r e y and Humphrey (1975) f o r diatoms, chrysomonads and brown algae. RESULTS AND DISCUSSION T. pseudonana was f i r s t grown under low—Fe c o n d i t i o n s t o o b t a i n Fe—starved stock c u l t u r e s and t o observe the response of the organism as i t approached Fe l i m i t a t i o n . R e p l i c a t e f l a s k s c o n t a i n i n g 450 nM F e C l 3 and no added Fe were i n o c u l a t e d with F e - r e p l e t e T.pseudonana and growth was monitored by c e l l c o n c e n t r a t i o n . The two treatments showed no s i g n i f i c a n t d i f f e r e n c e i n biomass as measured by c e l l d e n s i t y although the "no added Fe" c u l t u r e s were v i s i b l y c h l o r o t i c , a pale green compared t o the darker, b r i g h t e r green of the F e — r e p l e t e c u l t u r e s . The experiment was repeated as above with the exception t h a t i n a d d i t i o n t o c e l l d e n s i t y , i n vivo f l u o r e s c e n c e was monitored t o q u a n t i f y the v i s u a l observations of the e a r l i e r experiment. The r e s u l t s ( F i g . 2) show that the c e l l d e n s i t y of the "no added Fe" c u l t u r e s c l o s e l y p a r a l l e l e d that of Fe-F i g u r e 2. Growth o-f Thalassiosira pseudonana as measured by c e l l c o n c e n t r a t i o n . F e - s u f - f i c i e n t T. pseudonana used as the inoculum. (•) denotes c u l t u r e s with 450 nM added Fe as F e C l s and (O) denotes no added Fe. P o i n t s are staggered t o the l e f l and r i g h t of the t r u e sampling time so that confidence i n t e r v a l s of the two treatments may be c l e a r l y d i s p l a y e d . E r r o r bars represent the 95% confidence i n t e r v a l f o r 3 c u l t u r e s . Symbol s i z e s may extend beyond the confidence i n t e r v a l s i n some cases. 16 r e p l e t e c u l t u r e s . However, when growth was measured by i n vivo f l u o r e s c e n c e the "no added Fe" c u l t u r e s reached much lower l e v e l s <Fig. 3 ) . IF i n vivo F l u o r e s c e n c e i s taken as a measure o-f c h l o r o p h y l l c o n c e n t r a t i o n these r e s u l t s may be i n t e r p r e t e d as a d e c o u p l i n g of growth when growth i s d e f i n e d as "an o r d e r l y i n c r e a s e of a l l chemical c o n s t i t u e n t s of an organism." At the l e v e l of Fe l i m i t a t i o n observed i n t h i s experiment, c e l l d i v i s i o n remained u n a f f e c t e d w h i l e c h l o r o p h y l l p r o d u c t i o n was reduced. T h i s suggests a decrease i n the c h l o r o p h y l l c e l l quota which may be i n d i c a t e d by a decrease i n the r a t i o of i n vivo f l u o r e s c e n c e t o u n i t c e l l c o n c e n t r a t i o n or u n i t c e l l d e n s i t y ( F i g . 4 ) . (For the convenience of o b t a i n i n g v a l u e s between 0 and 10, i n vivo f l u o r e s c e n c e was d i v i d e d by c e l l d e n s i t y i n u n i t s of 10 3 c e l l s - m l " 1 . ) S i n c e c h l o r o p h y l l s y n t h e s i s was a f f e c t e d b e f o r e c e l l d e n s i t y the c o n c l u s i o n must be t h a t c h l o r o p h y l l p r o d u c t i o n i s more s e n s i t i v e t o Fe d e p l e t i o n than i s c e l l d i v i s i o n . F e — d e f i c i e n c y c h l o r o s i s , a decrease i n c h l o r o p h y l l a due t o Fe s t a r v a t i o n , has long been observed i n t e r r e s t r i a l p l a n t s (Jacobson and O e r t l i , 1956; Young, 1967; P r i c e , 196S; and Subcommittee on Iron, 1979) and more r e c e n t l y i n a l g a e ( P r i c e and C a r e l l , 1964; Hayward, 1968; Davies, 1970; G l o v e r , 1977; and Anderson and Morel, 1982). In t h i s study, decreases i n i n vivo f l u o r e s c e n c e due t o low-Fe treatments c o r r e l a t e d well t o v i s u a l o b s e r v a t i o n s and t o the o p t i c a l d e n s i t i e s of c u l t u r e s a t the absorbance maximum of c h l o r o p h y l l a. However s i n c e decreases i n cytochrome f a c t i v i t y a l s o r e s u l t from Fe s t r e s s 1 7 FIGURE 3. Growth o-f Thalassiosira pseudonana as measured by in vivo f l u o r e s c e n c e . F e — s u f f i c i e n t T. pseudonana used as the inoculum . (•) denotes c u l t u r e s with 450 nM added Fe as F e C l s and CO) denotes no added Fe. P o i n t s are staggered t o the l e f t and r i g h t of the t r u e sampling time so that confidence i n t e r v a l s of the two treatments may be c l e a r l y d i s p l a y e d . E r r o r bars represent the 95% confidence i n t e r v a l f o r 3 c u l t u r e s . Symbol s i z e s may extend beyond the confidence i n t e r v a l s i n some cases. 0 1 2 3 4 5 6 7 TIME (days) FIGURE 4. In vivo fluorescence per 10 s c e l l s - m l * 1 versus time. F e - s u f f i c i e n t T. pseudonana used as the inoculum. (•) denotes c u l t u r e s with 450 nM added Fe as F e C l 3 and ( O ) denotes no added Fe. P o i n t s are staggered t o the l e f t and r i g h t of the t r u e sampling time so that confidence i n t e r v a l s of the two treatments may be c l e a r l y d i s p l a y e d . E r r o r bars represent the 957. confidence i n t e r v a l f o r 3 c u l t u r e s . Symbol s i z e s may extend beyond the confidence i n t e r v a l s i n some cases. ( i n a diatom and a chrysophyte; Glover, 1977), i n vivo f l u o r e s c e n c e may not be a r e l i a b l e measure of c h l o r o p h y l l a i n Fe-starved algae. Lesions i n the photosynthetic e l e c t r o n t r a n s p o r t system (such as an decrease i n the cytochrome f a c t i v i t y ) would be expected t o inc r e a s e i n vivo f l u o r e s c e n c e by d i v e r t i n g e n e r g e t i c e l e c t r o n s away from the photosynthetic e l e c t r o n t r a n s p o r t system and towards the reemission of that energy as l i g h t . To v e r i f y that i n vivo fluorescence was a r e l i a b l e i n d i c a t o r of c h l o r o p h y l l a, both were measured i n Fe-r e p l e t e and Fe-deplete batch c u l t u r e s and l a t e r i n F e - l i m i t e d chemostat c u l t u r e s . F i g u r e 5 shows th a t i n vivo fluorescence and c h l o r o p h y l l a are c l o s e l y c o r r e l a t e d (r 2= 0.97) i n c u l t u r e s from these sources. In the next batch c u l t u r e experiment (Experiment I I ) , Fe-starved T. pseudonana was used t o i n o c u l a t e a s e r i e s of f l a s k s which contained 4500, 450, 45, 4.5 and 0 nM added Fe. Fe was added as f r e s h l y - p r e c i p i t a t e d FeCl 3-6H 20 i n the manner of Wells et a l . , (1983). F i g u r e 6 shows that treatments of 4500 and 450 nM Fe r e s u l t e d i n values of i n vivo fluorescence per u n i t c e l l d e n s i t y which were con s i d e r a b l y above 2, i n d i c a t i n g F e - r e p l e t e c u l t u r e s . In treatments of 4.5 and 0 nM added Fe, i n vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y decreased t o below 1, which, from the r e s u l t s i n Figure 4 would i n d i c a t e Fe l i m i t a t i o n . C u l t u r e s c o n t a i n i n g 45 nM Fe grew t o s i m i l a r c e l l d e n s i t i e s as those with 10 and 100 times the added Fe, but achieved lower values of i n vivo fluorescence. This behavior i s evident i n Figu r e 6 which shows that between days 3 and 6 values of i n vivo fluorescence per u n i t c e l l d e n s i t y are below 2 0 125 0 5 10 15 20 in vivo FLUORESCENCE FIGURE 5. C o r r e l a t i o n between e x t r a c t e d c h l o r o p h y l 1 a and i n vivo -fluorescence i n Fe - r e p l e t e and Fe-deplete batch c u l t u r e s and i n F e - l i m i t e d chemostat c u l t u r e s o-f T. pseudonana. r 2=0.97. 21 3 - i o cn _i _i ui o ID o o z UJ o CO UJ cr o 3 o > 2 -added Fe (nM) • 450G A 450 • 45 A 4.5 O 0.0 0 1 r 3 4 TIME (days) T 5 T 6 FIGURE 6. In vivo -fluorescence per 10 s c e l l s - m l " 1 versus time -for c o n centrations o-f Fe which vary by an order magnitude. Fe -deplete T. pseudonana used as the inoculum. (Experiment II) 22 2 but never as low as 1. At t h i s intermediate degree o-f Fe l i m i t a t i o n c h l o r o p h y l l production was a f f e c t e d but not c e l l d i v i s i o n as was a l s o the case f o r the "no added Fe" treatment i n the previous experiment. This i n c i p i e n t Fe l i m i t a t i o n and the observation that c u l t u r e s t r e a t e d with 4.5 and 0 nli Fe appeared F e - l i m i t e d i n d i c a t e s that the upper t h r e s h o l d of added Fe concentrations which are l i m i t i n g to T, pseudonana are near 45 nM. A f t e r s t a t i o n a r y phase was reached, c u l t u r e s were spiked with 450 nM Fe while monitoring of c e l l d e n s i t y and i n vivo f l u o r e s c e n c e continued. No growth enhancement was observed f o r treatments of 4500, 450 or 45 nM Fe while the 4.5 or 0 added Fe treatments increased i n c e l l d e n s i t y and i n vivo f l u o r e s c e n c e . The growth enhancement i n the "no added Fe" treatment was e x h i b i t e d as a two—fold increase i n c e l l d e n s i t y and a greater than f i v e — f o l d i n c rease i n i n vivo fluorescence which gave an i n vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y r a t i o of 1.8 before the experiment was terminated. Another set of batch c u l t u r e s was te s t e d (Experiment I I I ) with the purpose of achieving a more accurate estimate of the c r i t i c a l Fe conc e n t r a t i o n s l e a d i n g t o Fe l i m i t a t i o n and the y i e l d s of c e l l d e n s i t y and i n vivo fluorescence t h a t they would allow. Fe—starved 7. pseudonana was i n o c u l a t e d i n t o f l a s k s c o n t a i n i n g a range of Fe concentrations from 1.79 t o 71.6 nM. The Fe stock s o l u t i o n s were made from e l e c t r o l y t i c Fe wire d i s s o l v e d i n concentrated HC1 and stored at pH 2. This ensured that a l l of the Fe remained i n s o l u t i o n and that stock c o n c e n t r a t i o n s would be as accurate as p o s s i b l e . The r e s u l t s of the 9-day c u l t u r i n g experiment ( F i g . 7) showed that Fe l i m i t a t i o n i s f i r s t evident at 17.9 nM Fe and not apparently evident at 35.8 nM. This c o n f l i c t s with r e s u l t s of Experiment II i n which symptoms of Fe l i m i t a t i o n were f i r s t observed at 45 nM Fe. More s p e c i f i c a l l y the treatment of 35.8 nM Fe y i e l d e d a fluo r e s c e n c e per u n i t c e l l d e n s i t y of about 2 . 6 at day 6 ( F i g . 7) whi l e i n Experiment II a treatment of 45 nM Fe at day 6 y i e l d e d a value of 1 . 6 ( F i g . 6 ) i n d i c a t i n g mild Fe l i m i t a t i o n . The most obvious f a c t o r accounting f o r the d i f f e r e n t c o n c e n t r a t i o n s of Fe that induce symptoms of Fe l i m i t a t i o n i s that d i f f e r e n t sources of Fe were used i n the two experiments. In Experiment I I , where Fe was v a r i e d by powers of 1 0 , Fe was added as f r e s h l y - p r e c i p i t a t e d f e r r i c hydroxide made from F e C l 3 " 6 H 2 0 without pH adjustment. In Experiment I I I , Fe stock was made from elemental Fe wire d i s s o l v e d i n concentrated HC1, d i l u t e d and stored at pH 2 . Wells et a i . , (1983) have shown tha t aging, mild heating or au t o c l a v i n g a c c e l e r a t e the maturation of f e r r i c oxyhydroxide c o l l o i d s t o forms such as ge o t h i t e or hematite which are thermodynamical1y more s t a b l e and consequently are l e s s able t o provide a source of i o n i c Fe. The disagreement i n the concentrations of Fe tha t f i r s t e l i c i t Fe l i m i t a t i o n may suggest that f r e s h l y — p r e c i p i t a t e d f e r r i c hydroxide i s not as able t o provide a source of a v a i l a b l e Fe as the a c i d i f i e d Fe stock. This i s i n s p i t e of the f a c t t h a t the u n a c i d i f i e d Fe stock was added t o the medium w i t h i n one hour of prep a r a t i o n and was not t r e a t e d by heating or a u t o c l a v i n g . A d d i t i o n s of a c i d i f i e d Fe stock may provide a b e t t e r source of 24 FIGURE 7. In vivo fluorescence per 10= c e l l s - m l " 1 versus time f o r f i v e c o n centrations of Fe. Fe-deplete 7. pseudonana used as the inoculum. (Experiment I I I ) 25 b i o l o g i c a l l y a v a i l a b l e Fe because stock s o l u t i o n s do not form c o l l o i d a l f e r r i c oxyhydroxides at pH 2 which may l a t e r undergo maturation processes. When a c i d i f i e d Fe stock s o l u t i o n i s added to a r t i f i c i a l seawater 100% of the Fe i s i n i t i a l l y able t o provide a source of i o n i c Fe although i t may l a t e r undergo maturation processes i n the medium. D i l u t i o n of the Fe stock tends t o reduce the r a t e of c o l l o i d formation because of the reduced r a t e of f e r r i c i o n or f e r r i c hydroxide encounters (Wells et al.j 1983). On the other hand Fe stock which has not been pH adjusted has already undergone p r e c i p i t a t i o n , c o l l o i d formation and p o s s i b l y maturation before a d d i t i o n t o the medium. Therfore, when n o n — a c i d i f i e d Fe stock i s d i l u t e d i n t o a r t i f i c i a l seawater l e s s than 100% of the added Fe i s able t o provide a source of i o n i c Fe. Wells et a l . , (1983) d i d not observe Fe l i m i t a t i o n i n 7, pseudonana s u p p l i e d with 450 nM Fe aged f o r 1 week. However, they detected l i m i t a t i o n by monitoring c e l l d e n s i t y and comparing the growth of treatment c u l t u r e s t o c o n t r o l c u l t u r e s . The r e s u l t s of the present study, as we l l as work by T r i c k et al., (1983) demonstrate that c e l l d e n s i t y i s l e s s s e n s i t i v e t o Fe d e p l e t i o n than i s in vivo fluorescence. In a d d i t i o n , t h i s study shows th a t 7. pseudonana i s not Fe l i m i t e d , i n terms of c e l l d e n s i t y , by concentrations of f r e s h l y - p r e c i p i t a t e d Fe as low as 45 nM. Therefore, t o detect the e f f e c t of the small degree of Fe maturation which has occurred a f t e r one week of aging would r e q u i r e a more s e n s i t i v e i n d i c a t o r of Fe l i m i t a t i o n as w e l l as a lower q u a n t i t y of s u p p l i e d Fe. 26 Another -factor accounting f o r the disagreement i n the r e s u l t s of Experiments I I and I I I i s the d i f f e r e n t i n o c u l a used i n each experiment. Despite the f a c t that both b a t c h - c u l t u r e experiments used "Fe—starved" i n o c u l a the n u t r i t i o n a l h i s t o r i e s and previous s t a t e of growth of the two i n o c u l a may not have been i d e n t i c a l . This argues f o r a more c o n t r o l l e d method of batch c u l t u r i n g i n which the inoculum h i s t o r y has a l e s s s i g n i f i c a n t impact on the outcome of the batch c u l t u r i n g experiment. Such a method has been devised by Brand, G u i l l a r d and Murphy (1981) where batch c u l t u r e s are grown i n small tubes and t r a n s f e r r e d t o tubes of f r e s h medium before c e l l d e n s i t y reaches a concent r a t i o n where n u t r i e n t s l i m i t growth or waste products a f f e c t growth r a t e . The "acclimated growth r a t e " i s then c a l c u l a t e d only a f t e r the slopes of biomass versus time are s i m i l a r t o w i t h i n predetermined l i m i t s f o r s e v e r a l c u l t u r e t r a n s f e r s . A l t e r n a t i v e l y , c e l l s used f o r the i n o c u l a t i o n of c u l t u r e s could be made more uniform by employing a chemostat c u l t u r e growing at a constant growth r a t e as a source of inoculum. U l t i m a t e l y , the intended experiments could be done s o l e l y as chemostat c u l t u r e s . This would a l s o e l i m i n a t e the importance of the past h i s t o r y of the inoculum. Although the chemical form from which the Fe stock i s made (FeCls"6H20 or elemental Fe) should not a f f e c t i t as a n u t r i t i o n a l source, Fe stocks made from elemental Fe may be more accurate. The c h l o r i n e s a l t of Fe i s more d i f f i c u l t t o weigh a c c u r a t e l y due t o the v a r i a b l e number of waters of hydration a s s o c i a t e d with the molecule. Therefore, although i t i s l e s s l i k e l y than the above e x p l a n a t i o n s , the discrepancy i n 27 the r e s u l t s of Experiments I I and I I I could be due t o i n a c c u r a c i e s i n the prepar a t i o n of the Fe stock. A f i n a l e x p l a n a t i o n accounting f o r the disagreement i n the concentr a t i o n of Fe that i n i t i a l l y l i m i t s diatom growth i s that a p o r t i o n of the u n a c i d i f i e d Fe stock was l o s t due t o the adsorption of p r e c i p i t a t e d f e r r i c hydroxide t o the g l a s s w a l l s of the volumetric f l a s k i n which the stock was prepared. Such adsorption i s evidenced by a v i s i b l e c o a t i n g on the i n s i d e of g l a s s v o l u m e t r i c s a f t e r the f r e s h l y - p r e c i p i t a t e d f e r r i c hydroxide i s prepared and the unused p o r t i o n i s discarded. Davies (1968) documents the adhesion onto g l a s s s u r f a c e s of p r e c i p i t a t e d f e r r i c hydroxide i n a 0.7 M NaCl s o l u t i o n . Despite these d i f f i c u l t i e s the data from Experiments II and I I I were pooled. The best r e l a t i o n s h i p between c e l l c o n c e n t r a t i o n at s t a t i o n a r y phase and added Fe was obtained when c e l l c o n c e n t r a t i o n was normalized as a % of c o n t r o l c u l t u r e s ( F i g . 8 ) . The curve defined by the data p o i n t s becomes asymptotic at a concent r a t i o n of about 15 nM Fe suggesting that values below t h i s c o n c e n t r a t i o n l i m i t c e l l d e n s i t y . The best f i t r e l a t i o n s h i p between i n vivo f l u o r e s c e n c e at s t a t i o n a r y phase and concentrations of added Fe was obtained when i n vivo fluorescence was not normalized t o c o n t r o l c u l t u r e s ( F i g . 9). During exponential growth phase and f o r sev e r a l days i n t o s t a t i o n a r y phase iri vivo fluorescence i s a r e l i a b l e measure of c h l o r o p h y l l a ( F i g . 5 ) . Because Fe l i m i t a t i o n manifests i t s e l f as c h l o r o s i s , then over some Fe co n c e n t r a t i o n s , c h l o r o p h y l l production i s dependent upon 120 0 - 1 30 F e A D D E D I 60 (nM) n 90 FIGURE 8. The r e l a t i o n s h i p between the c e l l c o n c e n t r a t i o n of c u l t u r e s at s t a t i o n a r y phase (expressed as a percent of c o n t r o l c u l t u r e s ) and the concen t r a t i o n of added Fe. These are the combined r e s u l t s of Experiments I I and I I I . E r r o r bars represent the 957. confidence i n t e r v a l f o r three c u l t u r e s . I n s e r t shows the r e l a t i o n s h i p over a wider range of Fe concentrations. 1 0 FIGURE 9. The r e l a t i o n s h i p between in vivo fluorescence of c u l t u r e s at s t a t i o n a r y phase and the concen t r a t i o n of added Fe. These are the combined r e s u l t s of Experiments I I and I I I Er r o r bars represent the 95% confidence i n t e r v a l f o r three c u l t u r e s . Symbol s i z e s may extend beyond the confidence i n t e r v a l s i n some cases. Insert shows the r e l a t i o n s h i p over wider range of Fe concentrations. 30 a v a i l a b l e Fe. L i k e w i s e , i n vivo -fluorescence, a measure of c h l o r o p h y l l a, i s a l s o dependent upon a v a i l a b l e Fe. The r e l a t i o n s h i p becomes more apparent when the data are not normalized t o c o n t r o l c u l t u r e s which are not Fe l i m i t e d and hence c h l o r o p h y l l production i s independent of a v a i l a b l e Fe. The f a c t that t h i s i s not the case when c e l l d e n s i t y i s used as a measure of biomass may r e f l e c t the l e s s e r dependence of c e l l d i v i s i o n on a v a i l a b l e Fe. A comparison of Figure 9 and Figure 8 g r a p h i c a l l y demonstates that i n vivo f l u o r e s c e n c e i s a more s e n s i t i v e i n d i c a t o r of Fe s t r e s s than i s c e l l d e n s i t y . When fluo r e s c e n c e per u n i t c e l l d e n s i t y (an estimator of r e l a t i v e c e l l u l a r c h l o r o p h y l l quota) i s p l o t t e d against added Fe i t does not apparently decrease without l i m i t but l e v e l s o f f at a value above 1 ( F i g . 10). Several explanations may account f o r t h i s observation. Even i n batch c u l t u r e s where Fe was not added i t i s s t i l l present as a contaminant. I t i s i n excess of the heeds of.the organism, as evidenced by maximal growth r a t e s during exponential phase, u n t i l i t i s exhausted by d i v i d i n g c e l l s . G e n e r a l l y , i n n u t r i e n t — 1 i m i t e d batch c u l t u r e s i t i s found t h a t as c e l l d e n s i t y increases e x p o n e n t i a l l y the l i m i t i n g n u t r i e n t i s consumed at an exponential r a t e and c e l l s s h i f t from n u t r i e n t - s a t u r a t e d growth to n u t r i e n t - s t a r v e d s t a t i o n a r y phase, of t e n w i t h i n a s i n g l e c e l l generation (Darley, 1982; O'Brien, 1972). As a consequence, i n the case of F e — l i m i t e d media, c e l l s probably have l i t t l e time t o adapt to a low Fe environment and respond by simply h a l t i n g c e l l d i v i s i o n . Reduced c h l o r o p h y l l production i n conjunction with continued c e l l d i v i s i o n and perhaps decreasing c e l l s i z e may be 31 3 -i r 0 "i r 30 60 Fe ADDED (nM) "1 90 FIGURE 10. The r e l a t i o n s h i p between in vivo fluorescence per 10 s c e l l s - m l " 1 of c u l t u r e s at s t a t i o n a r y phase and the con c e n t r a t i o n of added Fe. These are the combined r e s u l t s of Experiments II and I I I . E r r o r bars represent the 95% confidence i n t e r v a l f o r three c u l t u r e s . 32 mechanisms whereby c e l l s adapt t o long term low—Fe s t r e s s . There-fore, by d i s c o n t i n u i n g c e l l d i v i s i o n a l i m i t may be placed on the redu c t i o n of c e l l u l a r c h l o r o p h y l l . In a d d i t i o n , there probably e x i s t s a lower l i m i t of c h l o r o p h y l l per c e l l which permits photosynthesis and growth, a l b e i t , at low r a t e s . That values of in vivo fluorescence per u n i t c e l l d e n s i t y below 1 can be a t t a i n e d by T. pseudonana i s demonstrated by two s e r i e s of batch c u l t u r e s i n which an F e - l i m i t e d chemostat c u l t u r e was used as the inoculum. The chemostat c u l t u r e was apportioned amoung polycarbonate f l a s k s and tubes and spiked with varying c o n c e n t r a t i o n s of Fe. I n i t i a l values of i n vivo fluorescence per c e l l were 0.68 while f i n a l values ranged from 0.37 f o r treatments of 0.36 nM Fe t o 0.93 f o r a d d i t i o n s of 179 nM Fe. Estimates of c e l l u l a r — F e quota can be c a l c u l a t e d from the ba t c h - c u l t u r e data by d i v i d i n g the d i f f e r e n c e i n added Fe conce n t r a t i o n s i n two treatments by the d i f f e r e n c e i n average c e l l d e n s i t i e s at s t a t i o n a r y phase f o r the same two treatments. This computation makes sever a l assumptions: 1) c u l t u r e s f o r which c a l c u l a t i o n s are made are a l l F e — l i m i t e d with respect t o c e l l d i v i s i o n ( therefore v i r t u a l l y a l l of the a v a i l a b l e Fe i s u t i l i z e d when s t a t i o n a r y phase i s reached); 2) the amount of Fe not u t i l i z e d by c e l l s i s n e g l i g i b l e or at l e a s t the same f o r a l l F e - l i m i t e d c u l t u r e s ; 3) a l l F e - l i m i t e d c u l t u r e s at s t a t i o n a r y phase have approached Fe d e p l e t i o n i n the same way and by the same p h y s i o l o g i c a l processes r e g a r d l e s s of the t o t a l amount of Fe i n the medium. Greater concentrations of Fe simply a l l o w the production of more c e l l s u n t i l Fe d e p l e t i o n of the medium occurs. I t should be noted that t h i s method of c a l c u l a t i n g c e l l u l a r — F e quota g i v e s an estimate of Fe per c e l l only f o r c e l l - d e n s i t y l i m i t e d c u l t u r e s . F e — s u f f i c i e n t c e l l s may cont a i n more Fe but cannot be r e l i a b l y estimated by t h i s means. Table II l i s t s the concentrations of added Fe, the average c e l l y i e l d at s t a t i o n a r y phase and the c a l c u l a t e d c e l l u l a r - F e quota f o r four experimental b a t c h - c u l t u r e runs. In most cases c e l l u l a r - F e quota (QFe) i s c a l c u l a t e d f o r c u l t u r e s t r e a t e d with two consecutive Fe concentrations w i t h i n the same s e r i e s of batch c u l t u r e s . Only one such estimate was made f o r Experiment II s i n c e only the 0 and the 4.5 nM Fe-spiked c u l t u r e s were Fe-l i m i t e d on the b a s i s of c e l l y i e l d . In Experiment I I I , s e v e r a l c a l c u l a t i o n s of QFo y i e l d e d values of 60 - 70 X10 - 1 8 moles F e - c e l l - 1 while f o r treatments of 17.9 nM Fe or greater the c a l c u l a t e d QFB's were e a s i l y over 200 attomoles F e - c e l l - 1 (1 attomole = IO - 1 8 moles). F i g u r e 8 shows that c u l t u r e s with greater than 15 nM Fe were not F e - l i m i t e d on the b a s i s of c e l l d e n s i t y . Therefore, c a l c u l a t i o n s of QFs f o r these c u l t u r e s may have over-estimated Fe per c e l l s i n c e i t i s not known whether a l l of the a v a i l a b l e Fe was u t i l i z e d . Likewise negative values of QFe do not meet the requirement of c e l l - d e n s i t y l i m i t e d c u l t u r e s . S i m i l a r estimates were made from Fe—spiked batch c u l t u r e s derived from an F e - l i m i t e d chemostat c u l t u r e (runs IVa and IVb). E s timating QFe using a d d i t i o n s of 1.79 and 7.16 nM Fe gi v e s 66.5 attomoles F e - c e l l - 1 f o r Experiment I I I and 27.7 attomoles F e c e l l - 1 f o r Experiment IVb. When c a l c u l a t i o n s are based on treatments of 7.16 and 17.9 nM Fe, the QF8 f o r Table I I . C a l c u l a t e d values o-F c e l l u l a r Fe quota and fluorescence:added Fe r a t i o s from data from 4 batch c u l t u r e experiments. Batch c u l t u r e s from experiments II and I I I used Fe-starved batch c u l t u r e s as t h e i r inoculum while batch c u l t u r e s from experiments IV a and b used an F e - l i m i t e d chemostat c u l t u r e . Experiment IVa batch c u l t u r e s were composed of 50 ml of chemostat c u l t u r e i n 100 ml polycarbonate tubes and experiment IVb c u l t u r e s were made up of 100 ml of chemostat c u l t u r e i n 500 ml polycarbonate f l a s k s . * Fe quota i s c a l c u l a t e d by d i v i d i n g the d i f f e r e n c e i n Fe conce n t r a t i o n of two treatments by the d i f f e r e n c e i n c e l l c o n c entrations of those c u l t u r e s at s t a t i o n a r y phase. ** Fluorescence:added Fe r a t i o i s c a l c u l a t e d by d i v i d i n g the d i f f e r e n c e i n in vivo f l u o r e s c e n c e at s t a t i o n a r y phase f o r two Fe treatments by the d i f f e r e n c e of t h e i r added Fe concentrations. *** These values are c a l c u l a t e d on the b a s i s of treatments with 1.79 and 7.16 nM added t o Fe i n c u l t u r e s . Fe CELL QUOTA * FLUORESCENCE: EXPERIMENTAL ADDED Fe CELL YIELD FLUORESCENCE (10" 1 8 moles Fe RATIO ** RUN (nM) (cells-l-») (arb. u n i t s ) c e l l - ' ) (uM"1) II 0 1.77 X 10" 1.6 4.4B 3.2 X 10s 2.6 31.3 223 I I I 1.79 1.56 X 10s 1.9 3.58 1.82 X 10° 2.6 68.9 391 7. 16 2.38 X 10° 3. 1 64.0 140 65.5*** 223** I I I 17.9 2.85 X 108 4. 1 229 93. 1 35.8 3.46 X 108 6.5 294 134 71.6 3.52 X 10° 7.0 5970 13.9 0. 358 3.9B X 10s 1.5 14.3 7. 38 X 10s 4. 17 41.1 192 26.9 7.21 X 10° 5.8 -7.4 130 179 6.79 X 10s 5.93 -3600 0.85 358 6.65 X 10s 5.83 -13000 -0.56 1.79 6. 11 X IO8 3.53 7. 16 8.05 X 108 4.43 27.7 168 17.9 9.31 X 10° 8.47 85.3 376 35. B 9.89 X 109 9.4 309 51.9 179 9.44 X 10° 9.4 -3200 0 36 Experiment I I I i s 229 attomoles F e - c e l l - 1 i n d i c a t i n g that c u l t u r e s with 17.9 nM Fe were probably not F e - l i m i t e d on the b a s i s of c e l l d e n s i t y . However, Experiment IVb y i e l d e d an estimated QFB o-f S5.3 attomoles F e - c e l l - 1 For the same treatment. These comparisons show that the chemostat-derived c u l t u r e s produce more c e l l s i n response t o a given amount o-f added Fe. They may do t h i s by reducing c e l l volume which i n t u r n would r e s u l t i n a reduced Fe per c e l l even though Fe per u n i t c e l l volume may remain constant. The lower estimates o-f QFe i n the chemostat-deri ved c u l t u r e s i s a l s o r e f l e c t e d i n a lower i n vivo fluorescence per u n i t c e l l d e n s i t y . Such a c o r r e l a t i o n between QF. and i n vivo fluorescence per u n i t c e l l d e n s i t y can be accounted f o r by the dependency on Fe f o r the production of c h l o r o p h y l l which i s measured by fluo r e s c e n c e . Again, the degree t o which c u l t u r e s such as those i n Experiment I I I can reduce QF8 or i n vivo fluorescence per u n i t c e l l d e n s i t y as adaptations t o F e - s t r e s s are probably r e s t r i c t e d by the speed with which batch c u l t u r e s approach n u t r i e n t l i m i t a t i o n . That some concentrations of added Fe s a t i s f i e d the Fe requirement of b a t c h - c u l t u r e - s t a r v e d c e l l s but not the Fe requirement of chemostat-culture c e l l s supports the con c l u s i o n t h a t the chemostat c e l l s were more Fe-stressed and that recovery from such s t r e s s can take s e v e r a l c e l l generations. The estimates of QF, which meet the c r i t e r i o n t h a t c u l t u r e s remain i n Fe l i m i t a t i o n with respect t o c e l l d i v i s i o n are i n the range of 30—100 attomoles F e - c e l l - 1 . This compares f a v o r a b l y with the estimate of 100 attomoles F e - c e l l - 1 f o r T. 37 pseudonana and T. oceanica by Sw i f t et al lin Murphy et a l , 1984). Anderson and Morel (1982) have estimated the c e l l u l a r Fe quota of F e - l i m i t e d Thalassiosira ueissflogii, a l a r g e r diatom, to be i n the range of 100-500 attomoles F e - c e l l " 1 . When QFa i s r e c a l c u l a t e d on a u n i t volume b a s i s the estimates of T. pseudonana are a f a c t o r of 2 times l a r g e r than those f o r T, Neissflogii. This d i f f e r e n c e might e a s i l y be accounted f o r by varying degrees of Fe l i m i t a t i o n , p oorly estimated c e l l s i z e s (Anderson and Morel do not repo r t c e l l volumes of t h e i r T. w e i s s f l o g i i c u l t u r e s ) , or by d i f f e r e n t r a t i o s of cytoplasmic volume (where most of the metabolic Fe i s presumed t o be located) t o c e l l volume. By assuming an average c e l l u l a r — F e quota of 70 attomoles F e - c e l l " 1 f o r batch c u l t u r e s of Fe-starved T. pseudonana one can estimate the amount of b i o l o g i c a l l y a v a i l a b l e Fe present i n Aquil-Fe as a contaminant. C u l t u r e s of Fe-starved T. pseudonana which received no added Fe u s u a l l y a t t a i n e d a c e l l d e n s i t y of 1.6 X 10 s c e l l s l " 1 . M u l t i p l y i n g the QF. by the c e l l d e n s i t y i n c e l l s ^ l - 1 g i v e s a t o t a l p a r t i c u l a t e Fe conc e n t r a t i o n of 11.2 nM Fe. This p a r t i c u l a t e Fe must have been derived from the medium s i n c e an Fe starved inoculum of IO 6 c e l l s - l " 1 can account f o r only 0.07 nM p a r t i c u l a t e Fe. The r a t i o of in vivo fluorescence:added Fe was c a l c u l a t e d by a method s i m i l a r t o that f o r QF. (Table I I ) . Here, the d i f f e r e n c e s i n in vivo f l u o r e s c e n c e of two consecutive Fe treatments was d i v i d e d by the d i f f e r e n c e i n the added Fe conce n t r a t i o n s . The r a t i o can be i n t e r p r e t e d as the e f f i c i e n c y of added Fe i n s t i m u l a t i n g an increase i n the c h l o r o p h y l l a. 38 The r e s u l t s i n Table II show tha t c u l t u r e s which do not appear to be F e - l i m i t e d from the c a l c u l a t i o n of Q F b nevertheless e x h i b i t i n c r e a s e s i n in vivo f l u o r e s c e n c e when Fe i s added. For example, c u l t u r e s spiked with 26.9 nM Fe i n Experiment IVa have a negative QF" but an in vivo f 1 uorescences added Fe r a t i o of 130. This i s simply a restatement of the observation that in vivo f l u o r e s c e n c e i s a more s e n s i t i v e measure of Fe l i m i t a t i o n than i s c e l l d e n s i t y . The r a t i o s of F e / c e l l and in vivo fluorescences added Fe are only c a l c u l a t i o n s of the i n v e r s e of the slope of Figure 8 and the slope of Figure 9 r e s p e c t i v e l y . As higher values of added Fe are approached the p r o p e r t i e s of c e l l d e n s i t y and in vivo f l u o r e s c e n c e vary independently of added Fe and the r a t i o s r e f l e c t a l o s s of c o r r e l a t i o n of these p r o p e r t i e s t o added Fe. 39 CHAPTER I I I CONTINUOUS CULTURE INTRODUCTION Although batch c u l t u r e s have the advantage o-f t e c h n i c a l s i m p l i c i t y and. ease o-f r e p l i c a t i o n they a l s o have the disadvantage o-f p r e s e n t i n g the organism with a s e t of ever changing environmental c o n d i t i o n s (Jannasch, 1974). As a r e s u l t , i t i s d i f f i c u l t t o reproduce or maintain any p a r t i c u l a r p h y s i o l o g i c a l s t a t e of a p o p u l a t i o n of c e l l s . Because the l i m i t i n g n u t r i e n t i s being exhausted so r a p i d l y d u r i n g the d e c l i n i n g phase of ex p o n e n t i a l growth (Darley, 1982; O'Brien, 1972) i t i s d i f f i c u l t t o determine which p a r t of the growth curve best r e p r e s e n t s the c u l t u r e c o n d i t i o n s d u r i n g sampling. In a d d i t i o n , few i n d i v i d u a l s of a p o p u l a t i o n may a c t u a l l y be i n the same p h y s i o l o g i c a l s t a t e because the p h y s i o l o g y of the p o p u l a t i o n i s changing i n order t o adapt t o the most r e c e n t s e t of environmental c o n d i t i o n s and not a l l i n d i v i d u a l s change at the same time or a t the same r a t e . A chemostat or n u t r i s t a t circumvents these problems. The r e p r o d u c t i o n r a t e of the organism i s c o n t r o l l e d by d e l i v e r i n g new medium at a constant and predetermined r a t e and removing the chemostat c u l t u r e at the same r a t e so t h a t a constant c u l t u r e volume i s maintained. The c u l t u r e grows u n t i l the n u t r i e n t i n l e a s t abundance r e l a t i v e t o the organism's needs i s exhausted and t h e r e a f t e r , the r e p r o d u c t i o n r a t e of the organism i s determined by the r a t e of a d d i t i o n of new medium which c o n t a i n s the l i m i t i n g n u t r i e n t . A f t e r s e v e r a l g e n e r a t i o n s , c u l t u r e c e l l s adapt t o the degree of n u t r i e n t 40 s t r e s s brought on by the c o n t r o l l e d a d d i t i o n o-f new medium and an e q u i l i b r i u m i s reached between c e l l d i v i s i o n and c u l t u r e removal. At t h i s e q u i l i b r i u m or steady s t a t e , reproduction r a t e , c e l l p hysiology, c e l l c o n c e n t r a t i o n and n u t r i e n t and e x c r e t i o n product c o n c e n t r a t i o n a l l remain constant. If the l i m i t i n g n u t r i e n t i n the medium i s Fe, then the organism can be exposed to i n c r e a s i n g degrees of Fe l i m i t a t i o n by simply decreasing the r a t e of d e l i v e r y of new medium and consequently decreasing the growth r a t e of the organism. MATERIALS AND METHODS 1. CONSTRUCTION OF AN IRON-LIMITED CHEMOSTAT' The design and c o n s t r u c t i o n of the chemostat apparatus was constrained by s e v e r a l requirements. 1) To avoid the problems of metal contamination and adsorption l o s s of metals, p l a s t i c components were used throughout except f o r the pumphead assembly which was made of fused c r y s t a l l i n e alumina. Gardner (1982) has found that f o r d i l u t e s o l u t i o n s of l e a d , a d s o r p t i v e l o s s e s were greater when g l a s s f i l t e r apparatus was used versus polycarbonate equipment. Davies (1968) documents the adsorption of o x y f e r r i c hydroxides t o g l a s s surfaces i n an a r t i f i c i a l 0.7 M NaCl s o l u t i o n buffered with NaHC03 and a l s o observed that s t i r r i n g promoted the adsorption of p r e c i p i t a t e d Fe. 2) As w e l l as being "metal c l e a n " , p l a s t i c components must be a u t o c l a v a b l e t o a l l o w f o r the s t e r i l i z a t i o n of the apparatus. This requirement e l i m i n a t e d the use of polyethylene and p o l y v i n y l c h l o r i d e . 3) F i n a l l y , the components needed t o be mechanically 41 compatible t o produce s e a l s that were a i r t i g h t and closed t o b a c t e r i a l contamination. Figure 11 i s a schematic diagram o-f the chemostat apparatus. Both the medium r e s e r v o i r and the Fernbach c u l t u r e f l a s k were of polycarbonate (Nalgene). Teflon spaghetti tubing was used t o make connections between the medium r e s e r v o i r , pump, c u l t u r e vessel and the outflow b e l l . Small bore Teflon tubing (1.19 mm diameter) was employed t o achieve maximum f l u s h i n g of the medium i n f l o w and e f f l u e n t l i n e s . Large flow r a t e s w i t h i n the outflow l i n e were d e s i r a b l e t o minimize the chances of b a c t e r i a l contamination of the chemostat c u l t u r e by b a c t e r i a l growth w i t h i n the tubing. As w e l l , l a r g e flow r a t e s would ensure that the residence time of the c u l t u r e outflow was short so that c u l t u r e e f f l u e n t would be r e p r e s e n t a t i v e of the chemostat c u l t u r e . A i r t i g h t connections between the Teflon tubing and components were made by i n s e r t i n g the Teflon tubing i n t o polypropylene p i p e t t e t i p s cut t o s i z e and which were, i n t u r n , i n s e r t e d i n t o polypropylene tubes. The medium r e s e r v o i r was cl o s e d by a polypropylene screw cap bored t o accept a s i l i c o n e stopper which i n tu r n accepted polypropylene tubes. Three such tubes served t o bubble the medium with f i l t e r — s t e r i l i z e d a i r a f t e r a u t o c l a v i n g , draw medium from the r e s e r v o i r and t o introduce f i 1 t e r — s t e r i 1 i z e d a i r i n t o the r e s e r v o i r t o e q u i l i z e pressure as medium was removed. Because neoprene stoppers c o n t a i n high l e v e l s of t r a c e metals (A.G. Lewis, pers. comm.) they were not considered f o r use i n the chemostat. S i l i c o n e i s r e l a t i v e l y f r e e of t r a c e metal contamination, i s a c i d washable and aut o c l a v a b l e and f o r Figure 11. Schematic diagram of the chemostat apparatus. 1 C o l l e c t i o n v e s s e l , 2 Outflow b e l l (polypropylene), 3 Polypropylene t u b i n g , 4 Tefl o n spaghetti t u b i n g , 5 S i l i c o n e elastomer stopper, 6 T e l f o n coated s t i r bar, 7 Fernbach c u l t u r e f l a s k (polycarbonate), 8 Inflow t u b i n g , 9 R e c i p r o c a t i n g pump, 10 Medium r e s e r v o i r (polycarbonate), 11 Aquil-Fe medium, 12 Bubbling l i n e with 0.4 um Nuclepore f i l t e r , 13 A i r i n l e t with cotton plug K) these reasons would be the ma t e r i a l o-f choice. Unfort u n a t e l y , s i l i c o n e stoppers of the diameter needed t o stopper the 2.8 l i t e r Fernbach c u l t u r e f l a s k were not commercially a v a i l a b l e . Consequently, s i l i c o n e stoppers were custom made using the s i l i c o n e elastomer, Sylgard 184 (Dow—Corning). A number 8 s i l i c o n e stopper was embedded i n a mixture of Sylgard 184 and cu r i n g agent w i t h i n a 250 ml Tripour polypropylene beaker used as a mold. A f t e r c u r i n g , the enlarged stopper was rele a s e d from the mold and a c i d washed a f t e r holes were d r i l l e d t o accept polypropylene tubing. Although not r i g o r o u s l y t e s t e d , the use of t h i s stopper i n 2.8 l i t e r Fernbach f l a s k s d i d not apparently reduce the growth r a t e s of T.pseudonana batch c u l t u r e s or batch c u l t u r e s of Gymnodinium sanguineum (B. Doucette, pers. comm.). 2. PUMPING SYSTEMS A l l chemostats r e q u i r e a means of d e l i v e r i n g new medium t o the c u l t u r e v essel at p r e c i s e and constant flow r a t e s . In a d d i t i o n t o t h i s , a trace-metal-1imited chemostat must minimize trace—metal contamination from the pumping system. The components of the pumping system i n contact with the medium should be of i n e r t m a t e r i a l s and present a minimum of surface area i n order t o minimize adsorption-desorption phenomena. During the course of chemostat c o n s t r u c t i o n s e v e r a l means of d e l i v e r i n g medium t o the c u l t u r e vessel were considered and t e s t e d . A summary of the pros and cons of s e v e r a l pumping systems i s given i n Table I I I . A mechanical metering pump (Model RP-G6, FMI Metering, Inc., Oyster Bay, New York) with a V 4 inch p i s t o n , was chosen as i t s a t i s f i e d most of the Table I I I Advantages and disadvantages o-f various chemostat pumping systems. PUMPING SYSTEMS ADVANTAGES DISADVANTAGES Siphon pump - s i mpli c i ty -only tubing i s i n contact with medium -va r y i n g -flow r a t e s M a r i o t t e b o t t l e (Kubitschek, 1970) same as above -compensates -for changing l e v e l of medium i n r e s e r v o i r -long periods of time t o reach e q u i l i b r i u m -requires a i r t i g h t s e a l on medium r e s e r v o i r -bubbles or p r e c i p i t a t e lodge i n c a p i l l a r y tubing - d i f f i c u l t t o reproduce flow r a t e s E l e c t r o l y s i s Pump (Carpenter, 1968) -rate of pumping can be p r e c i s e l y c o n t r o l l e d -inexpensi ve -only gas and tubing contact medi um -pump i s autoclavable -due t o the c o m p r e s s i b i l i t y of the gas the response time i s long -sporadic pumping due t o r e s i s t a n c e i n medium l i n e s -requires a i r t i g h t s e a l on medium r e s e r v o i r P e r i s t a l t i c pump -only tubing i n contact with medium -wear and s t r e t c h of tubing causes v a r i a t i o n i n flow r a t e s Mechanical metering pump FMI Co. -does not re q u i r e a i r t i g h t seal on medium r e s e r v o i r - p i s t o n — c y l i n d e r assembly i s autoclavable —pumps medium i n p r e c i s e , d i s c r e t e pulses -a fused c r y s t a l l i n e alumina p i s t o n -c y l i n d e r assembly i s i n contact with the medium 45 requirements -for a trace-metal-1 i mi ted chemostat. The metering pump d i d however expose the medium t o n o n - p l a s t i c s u r f a c e s at the p i s t o n - c y l i n d e r assembly. The po r t s and connecting tubing were of polypropylene while the piston-pump assembly i t s e l f was made of fused c r y s t a l l i n e alumina (99.5% pure aluminum o x i d e ) . The pump d e l i v e r e d medium at small but c o n s i s t e n t flow r a t e s , by means of a r o t a t i n g , r e c i p r o c a t i n g p i s t o n which d i s p l a c e d the f l u i d i n d i s c r e t e pulses. Only the i n l e t - o u t l e t p o r t s and the p i s t o n — c y l i n d e r assembly came i n t o contact with the f l u i d being pumped. The p i s t o n - c y l i n d e r assembly was a l s o autoclavable. 3. CHEMOSTAT OUTFLOW Removal of c e l l s and spent medium from the c u l t u r e vessel i s commonly achieved by means of a siphon. The l e v e l of the outflow end of the siphon c o n t r o l s the l e v e l of c u l t u r e i n the vess e l and thus the c u l t u r e volume. However, the r a t e of outflow was found t o be r e l a t i v e l y i n s e n s i t i v e t o the l e v e l of c u l t u r e i n the vessel and adjustment t o the de s i r e d height was d i f f i c u l t and time consuming due t o the long time re q u i r e d f o r the r a t e of siphon outflow and c u l t u r e l e v e l t o come t o e q u i l i b r i u m . If the outflow tubing enters the c u l t u r e vessel from above (as i t must i n a Fernbach f l a s k ) r a t h e r than from below then c u l t u r e medium must be introduced i n t o the outflow tube by e i t h e r a b u i l d up of pressure w i t h i n the c u l t u r e vessel or by applying vacuum t o the outflow end of the siphon. C u l t u r e outflow was achieved by p r e s s u r i z a t i o n of the c u l t u r e vessel so that each drop of new medium pumped i n t o i t 46 was matched by a drop o-f c u l t u r e e x i t i n g the out-flow tube. This method allowed easy adjustment o-f c u l t u r e height as we l l as r a p i d r e p r e s s u r i z a t i o n o-f the c u l t u r e vessel i n order t o resume equal flow of medium i n t o and c u l t u r e out of the v e s s e l . This method d i d however r e q u i r e t h a t the c u l t u r e vessel be sealed a i r t i g h t , otherwise a l o s s of p r e s s u r i z a t i o n would r e s u l t i n an incr e a s e i n c u l t u r e volume with time. 4. CHEMOSTAT OPERATION In o p e r a t i o n , the medium was drawn from the r e s e r v o i r t o the p i s t o n pump and then d e l i v e r e d at a constant r a t e t o the c u l t u r e v e s s e l . As medium was drawn out, pressure w i t h i n the r e s e r v o i r was equa l i z e d by a l l o w i n g a i r t o enter through a gl a s s tube packed with cotton t o prevent b a c t e r i a l contami n a t i on. The chemostat was autoclaved i n two s e c t i o n s . One s e c t i o n included the medium r e s e r v o i r and the small bore Teflon tubing which would l a t e r be connected t o the pump i n f l o w p o r t . The other s e c t i o n c o n s i s t e d of the piston—pump assembly, the c u l t u r e - f l a s k i n f l o w l i n e , the stopper assembly and Fernbach f l a s k (which contained about 2 l i t e r s of medium), the c u l t u r e outflow l i n e and outflow b e l l . Connections between Teflon s p a g h e t t i tubing and chemostat components as well as components to be l a t e r connected were wrapped l o o s e l y i n Saran Wrap t o reduce the chance of b a c t e r i a l contamination of the chemostat a f t e r a u t o c l a v i n g . Connections which often loosened on aut o c l a v i n g could l a t e r be t i g h t e d by hand without f e a r of contamination. 47 The medium i t s e l f was autoclaved s e p a r a t e l y because the 22 l i t e r medium r e s e r v o i r d i d not - f i t u pright i n t o the a v a i l a b l e autoclaves. A f t e r a u t o c l a v i n g , medium was a s e p t i c a l l y t r a n s f e r r e d t o the s t e r i l e medium r e s e r v o i r v i a an autoclaved polypropylene tube and bubbled overnight with a c i d washed (1 N H2S04) and s t e r i l e f i l t e r e d (0.4 um Nuclepore) a i r t o hasten e q u i l i b r a t i o n with a i r . The medium i n s i d e the Fernbach f l a s k was e q u i l i b r a t e d with a i r by t i g h t l y stoppering the vessel and shaking i t seve r a l times during the course of a day. Previous t e s t i n g showed th a t the above two methods were adequate t o b r i n g the medium pH t o the nominal value of 8.00 f o r A q u i l . I n o c u l a t i o n was c a r r i e d out by a s e p t i c a l l y adding 10—40 mis of axenic, s t a t i o n a r y phase, Fe-deplete c u l t u r e of J. pseudonana d i r e c t l y i n t o the c u l t u r e vessel and r e p l a c i n g the stopper. The apparatus was then placed i n an environment chamber at 17°C and the c u l t u r e continuously i r r a d i a t e d by 115 uEin.m^-s-1 of l i g h t from a s i d e bank of " V i t a L i g h t " (Duro-Test) f l u o r e s c e n t lamps. Mixing of the c u l t u r e was achieved by means of a 7.5 cm Teflon coated s t i r bar r o t a t i n g w i t h i n the c u l t u r e vessel at 60 rpm. At t h i s time the c u l t u r e was brought up t o about 2.5 l i t e r s and p r e s s u r i z e d i n order t o obta i n samples of the c u l t u r e . The t e s t organism was grown e x p o n e n t i a l l y under these c o n d i t i o n s , e s s e n t i a l l y as a batch c u l t u r e , with the pump o f f except when c u l t u r e samples were re q u i r e d . C e l l c o n c e n t r a t i o n and in vivo fluorescence were monitored at l e a s t d a i l y u n t i l the c e l l d e n s i t y approached 1 X 10 3 c e l l s - m l — at which time pumping began at a predetermined 48 flow r a t e and continued u n t i l the end o-f the chemostat experiment. 5. CHEMOSTAT MONITORING C e l l c o n c e n t r a t i o n , in vivo -fluorescence and flow r a t e were monitored every 12 hours. The chemostat c u l t u r e was considered t o be at steady s t a t e when the c e l l c o ncentration and in vivo f l u o r e s c e n c e remained constant ( c o e f f i c i e n t of v a r i a t i o n l e s s than 5%) f o r f i v e consecutive generations. Flow r a t e i n t o the chemostat c u l t u r e vessel was determined by c o l l e c t i n g chemostat outflow i n t o a preweighed p a r t i c l e counting cuvette f o r a measured time i n t e r v a l . The cuvette was reweighed and the volume of outflow c a l c u l a t e d by d i v i d i n g the outflow weight by the d e n s i t y of seawater at a s a l i n i t y of 35 ppt and temperature of 17° C. Th i s volume was then d i v i d e d by the sampling time t o give flow r a t e . D i l u t i o n r a t e was c a l c u l a t e d by d i v i d i n g flow r a t e by the c u l t u r e volume which was determined at the time the e n t i r e c u l t u r e was harvested. 6. CONFIRMATION OF Fe LIMITATION When steady s t a t e was reached approximately 600 ml of chemostat e f f l u e n t was c o l l e c t e d i n a s t e r i l e polycarbonate f l a s k , mixed and 100 ml d i s t r i b u t e d i n t o each of s i x a c i d washed and s t e r i l i z e d 500 ml polycarbonate Erlenmeyer f l a s k s with polypropylene screw caps. Three f l a s k s were spiked with A q u i l c o ncentrations of the macronutrients: n i t r a t e , phosphate and s i l i c a t e and the trace—metal stock s o l u t i o n w h i l e the other three f l a s k s were spiked with 450 nM of f r e s h l y — p r e c i p i t a t e d 49 u n a c i d i f i e d FeCl 3-6H 20. These c u l t u r e s were incubated under the same c o n d i t i o n s as the chemostat c u l t u r e and monitored d a i l y -for c e l l c o n c e n t r a t i o n and in vivo -fluorescence u n t i l the c u l t u r e s reached s t a t i o n a r y phase. When Fe-treated c u l t u r e s had s i g n i f i c a n t l y (no overlap o-f 95% confidence i n t e r v a l s ) higher c e l l d e n s i t y and in vivo f l u o r e s c e n c e at s t a t i o n a r y phase compared t o the complement-nutrient—addition c u l t u r e s t h i s was taken as s u f f i c i e n t evidence t h a t the chemostat was F e - l i m i t e d . 7. CONFIRMATION OF THE AXENIC CONDITION OF CHEMOSTAT CULTURE Since some b a c t e r i a and fungi are known producers of siderophores (Neilands, 1967), the contamination of an a l g a l c u l t u r e by b a c t e r i a or fungi would lead t o u n c e r t a i n t i e s about the p o s s i b l e o r i g i n of e x t r a c e l l u l a r c h e l a t i n g agents which might be produced by the diatom i n response t o Fe— l i m i t a t i o n . Therefore, before h a r v e s t i n g the s t e a d y - s t a t e -chemostat c u l t u r e a sample of the e f f l u e n t was taken t o v e r i f y i t s axenic c o n d i t i o n . Two d i f f e r e n t enrichments of Aquil-agar p l a t e s (preparation described i n "GENERAL METHODS") were i n o c u l a t e d with chemostat outflow. A l i q u o t s of e f f l u e n t were a l s o s t a i n e d with a c r i d i n e orange (Hobbie et a i . , 1977) and examined f o r b a c t e r i a with an e p i f l u o r e s c e n c e microscope. (These 3 methods gave p o s i t i v e r e s u l t s f o r batch c u l t u r e s of Thalassiosira pseudonana which were grown f o r many months d i s r e g a r d i n g a s e p t i c techniques.) 8. BIOMASS MEASUREMENTS C e l l d e n s i t y , average cell-volume and cell-volume d i s t r i b u t i o n of steady-state-chemostat c u l t u r e s were measured 50 using a Model Zf Coulter Counter (Coulter E l e c t r o n i c s ) . By i n c r e a s i n g the lower t h r e s h o l d s e t t i n g by 10 u n i t s f o r consecutive counts of the same sample (while the s e n s i t i v i t y remained u n a l t e r e d ) , data were obtained which when p l o t t e d gave a frequency d i s t r i b u t i o n of cumulative c e l l number versus the lower t h r e s h o l d s e t t i n g . The lower t h r e s h o l d s e t t i n g s were p r e v i o u s l y c a l i b r a t e d with l a t e x spheres of known volumes s i z e d with a microscope and micrometer (Sheldon and Parsons, 1966). A cell—volume f r e q u e n c y - d i s t r i b u t i o n was then obtained by s u b t r a c t i n g a count taken at a s p e c i f i c t h r e s h o l d s e t t i n g from the count taken at the next higher t h r e s h o l d s e t t i n g . The c e l l frequency f o r a p a r t i c u l a r volume i n t e r v a l was m u l t i p l i e d by the mean volume of that i n t e r v a l and these volumes were summed up f o r each volume window t o g i v e the t o t a l c e l l volume per ml of sample. The average c e l l volume of the sample was obtained by d i v i d i n g the t o t a l c e l l volume per ml of sample by the number of c e l l s per ml. Because i n vivo fluorescence i s an i n d i r e c t measure of c h l o r o p h y l l and the i n vivo fluorescence:chlorophyl1 r a t i o can vary with p h y s i o l o g i c a l s t a t e , n u t r i e n t a v a i l a b i l i t y , l i g h t i n t e n s i t y and temperature ( K i e f e r 1973; Marra 1978; Setser et al. 1982) i t was employed i n chemostat experiments as an i n d i c a t o r t o confirm the onset of steady s t a t e and not as a measure of c u l t u r e biomass. Instead, c h l o r o p h y l l a was e x t r a c t e d i n 90% acetone, measured by spectrophotometry absorption according t o S t r i c k l a n d and Parsons (1968) and c h l o r o p h y l l a c a l c u l a t e d according t o the formula given i n J e f f r e y and Humphrey (1975) f o r diatoms, chrysomonads and brown algae. RESULTS AND DISCUSSION Since the flow r a t e of the chemostat was measured by ta k i n g samples from the outflow i n s t e a d of the i n f l o w any v a r i a t i o n or d r i f t i n the flow r a t e of the FMI metering pump could only be overestimated. Despite t h i s , the pump performed w e l l , flow r a t e s v a r i e d by 0.9"/. or l e s s , at the maximum s e t t i n g of the pump. The three t e s t s used t o check f o r the presence of b a c t e r i a or fungi (see GENERAL METHODS) i n batch c u l t u r e s were a l s o used t o confirm that the chemostat c u l t u r e s remained axenic. These t e s t s proved negative (no b a c t e r i a l or fungal contamination) f o r a l l chemostat experimental runs. 1. ACHIEVING AN Fe-LIMITED CHEMOSTAT The theory of chemostat k i n e t i c s makes three a priori assumptions (Rhee, 1980): 1) only one n u t r i e n t i s l i m i t i n g growth; 2) the c u l t u r e i s p e r f e c t l y mixed; and 3) a constant p r o p o r t i o n of c e l l s i n the chemostat i s v i a b l e . Assumption 2, that the c u l t u r e i s p e r f e c t l y mixed, i s not a b s o l u t e l y a t t a i n a b l e due t o the discontinuous method of i n t r o d u c i n g medium i n t o the c u l t u r e vessel (drops) and the f i n i t e time r e q u i r e d t o completely mix the new medium. Assumption 3 does not hold i n chemostats at extremely low d i l u t i o n r a t e s where, i n b a c t e r i a l chemostats i t . h a s been found that non—viable c e l l s have accounted f o r up t o 60-70% of the t o t a l c e l l count (Postgate and Hunter, 1962; Tempest et a i . , 1967). However, l i m i t a t i o n o-f growth by one n u t r i e n t can be a t t a i n e d by a d j u s t i n g n u t r i e n t r a t i o s so tha t a l l n u t r i e n t s are i n excess except -for the n u t r i e n t of i n t e r e s t . Thereafter, i n c r e a s i n g the c o n c e n t r a t i o n of the l i m i t i n g n u t r i e n t (while ensuring that i t remains the s o l e f a c t o r l i m i t i n g growth) only r e s u l t s i n i n c r e a s i n g the c e l l y i e l d of the steady-state-chemostat c u l t u r e . In t h i s p a r t i c u l a r study, the problem of ach i e v i n g an Fe l i m i t e d chemostat was compounded by the need t o produce high c e l l y i e l d s t o f a c i l i t a t e the assay f o r c e l l u l a r Fe. Because Fe i s a mi c r o n u t r i e n t i t i s expected t o be present i n c e l l s i n minute q u a n t i t i e s . High c e l l d e n s i t i e s would a l s o f a c i l i t a t e the d e t e c t i o n and q u a n t i f i c a t i o n of e x t r a c e l l u l a r metabolites such as siderophores which might be produced i n response t o a given Fe s t r e s s . 1.1 CHEMOSTAT TRIAL I I n i t i a l data on Fe l i m i t a t i o n i n batch c u l t u r e s suggested th a t a d d i t i o n s of 17.9 nM Fe t o Aquil—Fe would s t i l l produce F e - l i m i t e d c u l t u r e s . This was supported by the p l o t of i n vivo f l u o r e s c e n c e versus added Fe ( F i g . 9) as well as the p l o t of i n vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y versus added Fe ( F i g . 10). A c c o r d i n g l y , Chemostat I used Aquil-Fe enriched with 17.9 nM Fe and ran at a d i l u t i o n r a t e of 1.13 day 1. When grown i n batch c u l t u r e the maximum growth r a t e of T. pseudonana on t h i s medium was 1.4 day - 1 f o r four determinations. Therefore at 1.13 day - 1, the chemostat ran at about 80% of UM «x. Although the flow r a t e of the chemostat v a r i e d by l e s s than 0.9%, observed 53 values f o r i n vivo -fluorescence -fluctuated c o n s i d e r a b l y while c e l l d e n s i t y v a r i e d t o a l e s s e r degree ( F i g . 12). Values of in vivo fluorescence per u n i t c e l l d e n s i t y a l s o f l u c t u a t e d but were a l l above one. I t was expected t h a t a continuous F e - s t r e s s would cause c e l l s t o reach lower r a t i o s of i n vivo fluorescence per u n i t c e l l d e n s i t y . R e s u l t s of the experiment t o confirm Fe l i m i t a t i o n i n the chemostat were ambiguous. Up t o 72 hours ( F i g . 13), a l i q u o t s of "steady-state" chemostat c u l t u r e spiked with 450 nM f r e s h l y -p r e c i p i t a t e d F e C l 3 showed greater values of i n vivo f l u o r e s c e n c e than the complement—nutrient c u l t u r e s spiked with A q u i l c o ncentrations of n i t r a t e , phosphate, s i l i c a t e and metal mix. However, the same complement-nutrient t r e a t e d c u l t u r e s e x h i b i t e d a l a r g e r i n c r e a s e i n c e l l d e n s i t y than c u l t u r e s which re c e i v e d Fe alone ( F i g . 14). At t h i s time, i t was suspected that batch c u l t u r e s of 7. pseudonana grown i n A q u i l with s u f f i c i e n t Fe were next l i m i t e d by C0 2. Therefore, the t e s t c u l t u r e s may have reached a c o n d i t i o n where Fe and CQ2 were l i m i t i n g growth c o n c u r r e n t l y . To t e s t t h i s , a l l c u l t u r e s i n the con f i r m a t i o n of Fe l i m i t a t i o n experiment were spiked with 2.38 mM NaHCOs at 72 hours. This a d d i t i o n had no e f f e c t on the p r e v i o u s l y Fe-spiked c u l t u r e s but d i d s t i m u l a t e a considerable i n c r e a s e i n the i n vivo f l u o r e s c e n c e of the complement—nutrient t r e a t e d c u l t u r e s . Because those t e s t c u l t u r e s spiked with A q u i l n u t r i e n t s minus Fe should s t i l l be at l e a s t p a r t l y F e - l i m i t e d , i t i s most l i k e l y t h a t the NaHCQ3 a d d i t i o n provided the Fe-starved c u l t u r e s with Fe. From the amounts of NaHCD3 added t o Figure 12. Chemostat I grown i n Aquil-Fe plus 17.9 nM Fe at a d i l u t i o n r a t e of 1.13 day 1. A. C e l l d e n s i t y over time. B. In vivo f l u o r e s c e n c e over time. 55 8 - i UJ o z UJ (_> CO UJ cr o ZD O > 6 -4 --E 2 -0 —' Na H CO, - l 1 1 r 24 48 72 96 TIME (hours) 1 1 120 144 Figure 13. In vivo -fluorescence over time -for the conf i r m a t i o n of Fe l i m i t a t i o n experiment f o r Chemostat I. 100 ml a l i q u o t s of steady—state—chemostat c u l t u r e were d i s t r i b u t e d t o 500 ml polycarbonate Erlenmeyer f l a s k s , spiked with n u t r i e n t s and incubated under the same c o n d i t i o n s as the chemostat c u l t u r e . At time zero two batch c u l t u r e s were spiked with 450 nM Fe ( 0 ) and two with A q u i l c o n c e n t r a t i o n s of n i t r a t e , phosphate, s i l i c a t e and metal mix without Fe ( 0 ). At 72 hours a l l c u l t u r e s r e c e i v e d 2.38 mM NaHCOs- At 96 hours one of the Fe-treated c u l t u r e s (- - -) was spiked with 12.5 uM Na 2Si0 3. E r r o r bars represent the 95% confidence i n t e r v a l f o r two c u l t u r e s . Symbol s i z e s may extend beyond the confidence i n t e r v a l s i n some cases. 0 I 1 1 1 1 1 1 0 24 48 72 96 120 144 TIME (hours) Figure 14. C e l l d e n s i t y aver time -far the co n f i r m a t i o n of Fe l i m i t a t i o n experiment f o r Chemostat I. 100 ml a l i q u o t s of steady-state—chemostat c u l t u r e were d i s t r i b u t e d t o 5O0 ml polycarbonate Erlenmeyer f l a s k s , spiked with n u t r i e n t s and incubated under the same c o n d i t i o n s as the chemostat c u l t u r e . At time zero two batch c u l t u r e s were spiked with 450 nM Fe ( 0 ) and two with A q u i l concentrations of n i t r a t e , phosphate, s i l i c a t e and metal mix without Fe ( O At 72 hours a l l c u l t u r e s received 2.38 mM NaHC03. At 96 hours one of the Fe-treated c u l t u r e s (- — -) was spiked with 12.5 uM Na 2Si0 3. E r r o r bars represent the 95% confidence i n t e r v a l f o r 2 c u l t u r e s . Symbol s i z e s fliay extend beyond the confidence i n t e r v a l s i n some cases. 57 c u l t u r e s and the American Chemical S o c i e t y (A.C.S.) s p e c i f i c a t i o n s of i m p u r i t i e s f o r tha t l o t of NaHC03, i t was estimated t h a t as much as 23.8 nM Fe may have been added t o the t e s t c u l t u r e s . That the a d d i t i o n of NaHCQ3 d i d not immediately and s i g n i f i c a n t l y s t i m u l a t e an inc r e a s e i n the c e l l d e n s i t y of the Fe-spiked c u l t u r e s suggests that C0 2 was not l i m i t i n g growth i n c o n j u c t i o n with Fe. On the b a s i s t h a t the s i l i c a t e stock added t o the chemostat medium might not have been e n t i r e l y a v a i l a b l e due t o aging and pol y m e r i z a t i o n (McLachlan, 1979s Morel et a i - , 1979), 12.5 uM of f r e s h l y prepared s i l i c a t e stock was added t o one of the Fe spiked c u l t u r e s at 96 hours ( F i g . 13 and 14). This c u l t u r e responded by a s l i g h t i n c r e a s e i n i n vivo fluorescence but a l a r g e increase i n c e l l d e n s i t y t o a l e v e l equal t o tha t of c u l t u r e s spiked with A q u i l n u t r i e n t s minus Fe. 1.2 CHEMOSTAT TRIAL II The f a i l u r e of Chemostat I t o achieve a s o l e l y F e - l i m i t e d s t a t e was thought t o be caused by aging and po l y m e r i z a t i o n of the s i l i c a t e stock which reduced i t s a v a i l a b i l i t y f o r diatom growth. A two week delay between the time n u t r i e n t s were added t o the SOW and i n o c u l a t i o n of the medium may a l s o have played a part i n reducing s i l i c a t e a v a i l a b i l i t y . To avoid s i l i c a t e l i m i t a t i o n i n the next chemostat t r i a l no Fe was added t o the medium and s i l i c a t e was added from a stock s o l u t i o n prepared the same day. Chemostat II was run at a d i l u t i o n r a t e of 1.12 day 1. U n f o r t u n a t e l y , a f t e r a s i g n i f i c a n t p o r t i o n of medium i n the r e s e r v o i r was depleted, c u l t u r e growth r a t e s and c e l l d e n s i t y d e c l i n e d . Microscopic examination of c u l t u r e outflow 58 showed auxospores and male gametes suggesting that the culture was undergoing sexual reproduction. The pump was turned off to conserve medium u n t i l the culture returned to vegetative growth. Once vegetative growth and chemostat pumping resumed, the i n vivo fluorescence per unit c e l l density r a p i d l y dropped from about 5 to 0.66, where i t l e v e l l e d off for 3 days. In the confirmation of Fe-1imitation experiment for t h i s chemostat t r i a l those cultures spiked with Fe increased r a p i d l y in both c e l l density and i n vivo fluorescence while the controls did not respond. Because i n s u f f i c i e n t medium remained to r e l i a b l y e s t a b l i s h steady state and because the physiology may have been affected by the culture undergoing sexual phase i t was decided to end the chemostat experiment and use the culture as the inoculum for the batch culture experiments IVa and IVb discussed e a r l i e r . 1.3 CHEMOSTAT TRIAL III To avoid the dually l i m i t i n g condition suspected i n Chemostat I, medium was again prepared using f r e s h l y prepared s i l i c a t e stock and 7.16 nM Fe. Further batch culture r e s u l t s had shown that cultures with 7.16 nM added Fe limited growth of 7. pseudonaria on the basis of both c e l l density and i n vivo fluorescence. Chemostat III operated at a d i l u t i o n rate of 1.11 day 1 and achieved constant values of c e l l density and i n vivo fluorescence over time <Fig. 15). Steady-state values for i n vivo fluorescence per c e l l density l e v e l l e d off just below 1.5 indicating that the chemostat culture was probably not limited by Fe alone. •n a o H' c c ro rt H' •h 0 cn I—' 3 • c 0 T n T 0) zr ID rt ro in m 3 n 0 ro 0 tn 3 rt n m ID rt 0 r* »-i < t-l T a m •a ft«< T i 0 3 • £ IV fl 3> • H* 3 n ID I> XI *-« C M-a I - 1 m 1 • TI in ro rtTJ "< t-j c 0 in < ro ft Cr-3 ro 3 •n 00 n a> rt 00 CO ^ 0> O c CO ro in vivo FLUORESCENCE — ro oi J > _l I I I CELL DENSITY CD (cells ml" 1 X IO5 ) ro ot I I LI O 60 To i d e n t i f y p o s i t i v e l y the f a c t o r l i m i t i n g growth i n conjunction with Fe, the e n t i r e chemostat c u l t u r e was used as a source of inoculum f o r b a t c h — c u l t u r e a d d i t i o n experiments. On the b a s i s of c e l l d e n s i t y , an a d d i t i o n of 450 nM Fe e l i c i t e d the same response as c o n t r o l c u l t u r e s with no n u t r i e n t a d d i t i o n ( F i g . 16). The a d d i t i o n of 12.5 uM s i l i c a t e r e s u l t e d i n a l a r g e but not s t a t i s t i c a l l y s i g n i f i c a n t (95% confidence i n t e r v a l ) i n c r e a s e over the other two treatments. When in vivo f l u o r e s c e n c e i s considered ( F i g . 17), the no a d d i t i o n or 12.5 uM s i l i c a t e treatments d i d not s t i m u l a t e l a r g e i n c r e a s e s i n co n t r a s t t o c u l t u r e s spiked with 450 nM Fe. D i f f e r e n c e s i n in vivo fluorescence were s i g n i f i c a n t (95% confidence i n t e r v a l ) between the Fe and s i l i c a t e treatments but not s i g n i f i c a n t between the Fe and no a d d i t i o n treatments. Stationary—phase c e l l s from batch c u l t u r e s t r e a t e d with 450 nM Fe had an average cell-volume of 99.9 +/- 1.15 u s (95% confidence i n t e r v a l ) w hile the average volume of c e l l s of c u l t u r e s which received an a d d i t i o n a l 12.5 uM s i l i c a t e were 79.0 +/- 0.49 u 3. Untreated c u l t u r e obtained d i r e c t l y from the chemostat had an average c e l l volume of SI u 3. C u l t u r e s t r e a t e d with separate a d d i t i o n s of n i t r a t e , phosphate, ammonium, metal mix or vitam i n s d i d not d i s p l a y enhanced growth on the b a s i s of c e l l d e n s i t y or in vivo f1uorescence. G u i l l a r d et al. (1973) c a l c u l a t e d the y i e l d of T. pseudonana Clone 3—H t o range from 2.5—3.7 X 10* c e l l s - m l - 1 per uM s i l i c a t e removed from the c u l t u r e medium. On t h i s b a s i s , A q u i l , which co n t a i n s 12.5 uM s i l i c a t e , should y i e l d a maximum 61 o i 1 1 1 1 0 24 48 72 96 TIME (hours) F i g u r e 16. C e l l d e n s i t y over time -for the c o n f i r m a t i o n o-f Fe l i m i t a t i o n experiment For Chemostat I I I . 100 ml a l i q u o t s o-f steady-state-chemostat c u l t u r e were d i s t r i b u t e d t o 500 ml polycarbonate Erlenmeyer -flasks, spiked with n u t r i e n t s and incubated under the same c o n d i t i o n s as the chemostat c u l t u r e . At time zero, t r i p l i c a t e batch c u l t u r e s were spiked with 450 nM Fe, 12.5 uM Na 2Si0 3, or r e c e i v e d no a d d i t i o n . 62 9 -I U J o z U J 8 6 U J or o z> 5 3 0 -• • 450 nM Fe A 12.5 JIM N a 2 S i 0 5 O no addition r o n 1 r 24 48 72 TIME (hours) 1 96 Figure 17. In vivo con-f i r m a t i o n o-f Fe 100 ml a l i q u o t s o-f d i s t r i b u t e d t o 500 with n u t r i e n t s and chemostat c u l t u r e , were spiked with 450 nli Fe, 12.5 uM Na 2Si0 3, or rec e i v e d no addi t i on. fluorescence over time f o r the l i m i t a t i o n experiment f o r Chemostat steady-state-chemostat c u l t u r e were ml polycarbonate Erlenmeyer f l a s k s , incubated under the same c o n d i t i o n s I I I . s p i ked as the At time zero, t r i p l i c a t e batch c u l t u r e s 63 of 4.6 X 10 s c e l l s - m i - 1 . This i s about the usual maximum c e l l y i e l d i n batch c u l t u r e s of T. pseudonana grown i n 250 ml of Aq u i l i n 500 ml Erlenmeyer f l a s k s . Morel et al. (1979) a l s o s t a t e that the A q u i l f o r m u l a t i o n i s s i l i c a t e - l i m i t i n g t o diatoms. However, i n batch c u l t u r e s of T. pseudonana grown i n F e - s u f f i c i e n t A q u i l using F e — r e p l e t e i n o c u l a , 25 uM s i l i c a t e d i d not enhance growth over c o n t r o l c u l t u r e s which contained 12.5 uM s i l i c a t e . Doubling the A q u i l bicarbonate concentration s t i m u l a t e d f u r t h e r growth but d i d not double the f i n a l c e l l y i e l d . V i g o r o u s l y shaking c u l t u r e s each day r e s u l t e d i n enhanced growth as d i d growing c u l t u r e s i n 100 ml in s t e a d of 250 ml volumes i n the same 500 ml Erlenmeyer f l a s k s . Increased c e l l y i e l d upon, the a d d i t i o n of bicarbonate, vigorous shaking or growth i n higher s u r f a c e area t o volume r a t i o s suggests that the growth of T. pseudonana was l i m i t e d d i r e c t l y or i n d i r e c t l y by C0 2. Since doubling the bicarbonate c o n c e n t r a t i o n d i d not double the c e l l y i e l d , e i t h e r carbon a s s i m i l a t i o n was not l i m i t e d or another n u t r i e n t or f a c t o r became l i m i t i n g immediately a f t e r carbon, thus preventing a f u l l doubling of c e l l y i e l d . As we l l as being a s u b s t r a t e f o r growth, C0 Z a l s o serves t o b u f f e r pH. The growth of T. pseudonana t o higher c e l l d e n s i t i e s may be l i m i t e d by a b u i l d up of excreted products which i n c r e a s e the pH. Carbonic a c i d added e i t h e r v i a carbon d i o x i d e or bicarbonate may provide a source of hydrogen ions which r e s u l t s i n a pH drop, a l l o w i n g f u r t h e r growth. Recently, Doucette (pers. comm.) has observed t h a t c u l t u r e s of Gymnodinium sanguineum grown i n modified ESAW (Harrison et a i - , 1980) reach t h e i r maximum c e l l y i e l d at pH 9.0—9.1 and tha t 64 •further growth can be st i m u l a t e d by lowering the pH with HC1 or ascorbate. If growth at higher c e l l c o ncentrations i s l i m i t e d by pH t h i s would a l s o e x p l a i n why the f i n a l c e l l y i e l d of c u l t u r e s i s more v a r i a b l e when the pH of A q u i l i s not* adjusted a c c u r a t e l y t o 8.00 before i n o c u l a t i o n . Despite the f a c t that F e - r e p l e t e batch c u l t u r e s of T. pseudonana were not l i m i t e d by s i l i c a t e , the r e s u l t s of Chemostat I I I i n d i c a t e that s i l i c a t e was l i m i t i n g c e l l d i v i s i o n i n batch c u l t u r e s derived from the chemostat c u l t u r e . Although F e - i n s u f f i c i e n t batch c u l t u r e s (Batch c u l t u r e Experiment II) were not t e s t e d f o r s i l i c a t e l i m i t a t i o n , a d d i t i o n s of 450 nM FeCls t o F e — l i m i t e d c u l t u r e s s t i m u l a t e d i n c r e a s e s i n c e l l d e n s i t y and in vivo fluorescence t o the l e v e l s of Fe - r e p l e t e c o n t r o l c u l t u r e s . This suggests t h a t F e - l i m i t e d batch c u l t u r e s were not s i l i c a t e l i m i t e d , u n l i k e batch c u l t u r e s derived from the chemostat. 1.4 CHEMOSTAT TRIAL IV In order t o avoid the d u a l l y l i m i t i n g c o n d i t i o n encountered i n previous chemostat t r i a l s , the s i l i c a t e t o Fe r a t i o was increased by doubling the s i l i c a t e c o n c e n t r a t i o n from 12.5 t o 25 uM and by adding no Fe t o the medium. Chemostat IV ran at a d i l u t i o n r a t e of 1.11 days - 1. Although both c e l l d e n s i t y and in vivo fluorescence remained constant ( c o e f f i c i e n t of v a r i a t i o n < 5%) over at l e a s t 5 generations, Figure 18 shows tha t in vivo f l u o r e s c e n c e l e v e l l e d o f f as e a r l y as 96 hours while c e l l d e n s i t y increased u n t i l 144 hours before l e v e l l i n g o f f . This adaptation t o chemostat c o n d i t i o n s i s evident i n the a n ID 3 a io Ul c M "1 rt ID < c • 2 00 < • It) in 1 n 3-rt (D n 3 3 Oi O III rt Ul • ID ta CO 04 rt • rt 1-4 < a ID H. 0 c £ 0 rt M' 3 -h 0 H-M 3 3 c • 1 I> "1 0) ro rt c Ul ID n ID 0 1 3 •n n ID ID I z • H' < rt ID 3" T a Oi 3 • 3 • ID a • a • pa n ID >—' ID i—• m oo 0> 2 I tn (0 ro ro o in v i v o F L U O R E S C E N C E — ro oi I I L_ J C E L L D E N S I T Y ( c e l l s - m l - 1 X I O 5 ) o — ro w I I I CD O Ul 66 decreasing r a t i o of in vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y over time ( F i g . 19). At steady s t a t e , an a l i q u o t of the chemostat c u l t u r e was f i l t e r e d and the f i l t r a t e was assayed f o r r e a c t i v e s i l i c a t e according t o Parsons et al. (1984). D u p l i c a t e determinations of chemostat e f f l u e n t gave estimates of 28.45 +/- 0.11 uM r e a c t i v e s i l i c a t e . The average c e l l volume of chemostat c u l t u r e c e l l s was 40.45 +/- 0.19 u 3. In the experiment t o confirm Fe l i m i t a t i o n , 100 ml a l i q u o t s of chemostat c u l t u r e were d i s t r i b u t e d among f i f t e e n 500 ml Erlenmeyer f l a s k s and spiked with n u t r i e n t s . The a d d i t i o n s were: 1) 25 uM S i and 450 nM Fe; 2) 25 uM S i ; 3) 450 nM Fe; 4) 100 uM n i t r a t e p l u s 10 uM phosphate, A q u i l concentrations of metals excepting Fe, and A q u i l concentrations of v i t a m i n s . A no-addition c o n t r o l was a l s o included i n the treatments. On the b a s i s of c e l l d e n s i t y ( F i g . 20), only the S i plu s Fe spiked c u l t u r e s s i g n i f i c a n t l y d i f f e r e d from . other treatments. The a d d i t i o n of s i l i c a t e alone d i d not increase c e l l d e n s i t y compared with other treatments. The greater c e l l d e n s i t y i n the S i pl u s Fe spi k e versus the Fe-only treatment can be accounted f o r i f the chemostat c u l t u r e was i n i t i a l l y l i m i t e d by Fe alone. Once Fe l i m i t a t i o n was overcome by the a d d i t i o n of Fe, s i l i c a t e was consumed and exhausted by successive c e l l d i v i s i o n s . On the b a s i s of in vivo fluorescence ( F i g . 21), only the treatments which included Fe enhanced growth. Again the s i l i c a t e p l u s Fe treatment s t i m u l a t e d greater in vivo fluorescence than the Fe—only a d d i t i o n and t h i s can be accounted f o r by the increased c e l l d e n s i t y of the former Figure 19. Chemostat IV grown i n Aquil-Fe with no added Fe at a d i l u t i o n r a t e o-f 1.11 day"1. In vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y over time. o E U >-CO UJ Q UJ O 12 -I 8 -4 -A + Si + Fe A + Si • + Fe O no addition • all nutrients - S i - F e n 1 1 1 1 1 24 48 72 96 120 144 TIME (hours) F i g u r e 20. C e l l d e n s i t y over time -for the con f i r m a t i o n of Fe l i m i t a t i o n experiment f o r Chemostat IV. 100 ml a l i q u o t s of steady-state—chemostat c u l t u r e were d i s t r i b u t e d t o 500 ml polycarbonate Erlenmeyer f l a s k s , spiked with n u t r i e n t s and incubated under the same c o n d i t i o n s as the chemostat c u l t u r e At time zero, t r i p l i c a t e c u l t u r e s were spiked with; 25 uM S i and 450 nM Fe, 25 uM S i , 450 nM Fe, a l l A q u i l n u t r i e n t s except S i and Fe, and 3 c u l t u r e s received no a d d i t i o n . 69 25 - i A + Si + Fe A + Si • + Fe O no addition • all nutrients - S i - F e 20 -5 -U J o z UJ o CO UJ or o 3 ^ io H o _> '> •- 5 H "I 1 1 1 1 1 24 48 72 96 120 144 TIME (hours) Figure 21. In vivo -fluorescence over time f o r the con f i r m a t i o n of Fe l i m i t a t i o n experiment f o r Chemostat IV. 100 ml a l i q u o t s of steady-state-chemostat c u l t u r e were d i s t r i b u t e d t o 500 ml polycarbonate Erlenmeyer f l a s k s , spiked with n u t r i e n t s and incubated under the same c o n d i t i o n s as the chemostat c u l t u r e . At time zero, t r i p l i c a t e c u l t u r e s were spiked with; 25 uM S i and 450 nM Fe, 25 uM S i , 450 nM Fe, a l l A q u i l n u t r i e n t s except S i and Fe, and 3 c u l t u r e s received no a d d i t i o n . 70 c u l t u r e s . When in vivo fluorescence per u n i t c e l l d e n s i t y was p l a t t e d against time ( F i g . 22), only those treatments which included Fe caused in vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y t o reach values beyond 1, i n d i c a t i n g that c e l l s had overcome Fe l i m i t a t i o n . The r i s e i n in vivo fluorescence per u n i t c e l l d e n s i t y was not s i g n i f i c a n t l y d i f f e r e n t between the s i l i c a t e p l u s Fe s p i k e and the Fe-only a d d i t i o n . A comparison of Figures 20, 21 and 22 demonstrates the u t i l i t y of using in vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y as an i n d i c a t o r of Fe-st r e s s e d c e l l s i n s t e a d of c e l l d e n s i t y or in vivo fluorescence alone. These r e s u l t s s t r o n g l y suggest that s i l i c a t e d i d not l i m i t the growth of the chemostat c u l t u r e and that Fe alone was the l i m i t i n g n u t r i e n t . The c e l l d e n s i t y of the stea d y - s t a t e chemostat was 1.4 X 10 s c e l l s m l - 1 with 28.45 uM s i l i c a t e remaining i n the medium ( s i l i c a t e contamination from the g l a s s c o n t a i n e r s used i n the preparation of A q u i l probably accounted f o r the e x t r a s i l i c a t e ) . S i l i c a t e was found t o l i m i t the c e l l d e n s i t y of Fe-spiked batch c u l t u r e s derived from the chemostat c u l t u r e t o about 7.9 X 10 s c e l l s - m l - 1 , 2.5 c e l l d i v i s i o n s away from the c e l l c o n c e n t r a t i o n of the chemostat c u l t u r e . Moreover, as Chemostat IV approached steady s t a t e , the c e l l d e n s i t y continued t o increase a f t e r in vivo fluorescence had l e v e l l e d o f f . In other chemostat t r i a l s in vivo fluorescence and c e l l d e n s i t y l e v e l l e d o f f simultaneously. The simplest i n t e r p r e t a t i o n i s that the Chemostat IV c u l t u r e had a s s i m i l a t e d a l l the a v a i l a b l e Fe, as evidenced by the constancy of in vivo f l u o r e s c e n c e , a f t e r which c e l l s responded by co n t i n u i n g 71 o - 2.5 A + Si + Fe A + Si • + Fe O no addition • all nutrients - S i - F e CO o — _l ° _J UJ o O I O UJ o CO UJ cr o 0.5 o > ~i r~ 1 r-24 48 72 96 TIME (hours) 120 ~1 144 Figure 22. In vivo -fluorescence per u n i t c e l l d e n s i t y over time -for the co n f i r m a t i o n o-f Fe l i m i t a t i o n experiment f o r Chemostat IV. 100 ml a l i q u o t s o-f steady-state-chemostat c u l t u r e were d i s t r i b u t e d t o 500 ml polycarbonate Erlenmeyer f l a s k s , spiked with n u t r i e n t s and incubated under the same c o n d i t i o n s as the chemostat c u l t u r e . At time z e r o , t r i p l i c a t e c u l t u r e s were spiked with; 25 uM S i and 450 nM Fe, 25 uM S i , 450 nM Fe, a l l A q u i l n u t r i e n t s except S i and Fe, and 3 c u l t u r e s r e c e ived no a d d i t i o n . 72 d i v i s i o n t o apportion the c h l o r o p h y l l and l i k e w i s e the Fe t o more c e l l s . This should not have occurred i f the chemostat c u l t u r e was s i l i c a t e l i m i t e d . A d d i t i o n a l l y , c e l l s from Chemostat IV e x h i b i t e d small c e l l volumes, 40.45 u 3, as compared to 81 u 3 i n Chemostat I I I and 100 u 3 i n p o r t i o n s of Chemostat I I I c u l t u r e s t r e a t e d with 450 nM Fe. The a d d i t i o n of s i l i c a t e alone to batch c u l t u r e s derived from Chemostat IV d i d not s t i m u l a t e i n c r e a s e s i n c e l l d e n s i t y beyond other treatments as would be expected i f the chemostat c u l t u r e was wholly or p a r t l y s i l i c a t e l i m i t e d . F i n a l l y , Chemostat IV reached values of in vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y below one. From previous batch c u l t u r e experiments, t h i s would i n d i c a t e Fe— s t r e s s e d c e l l s . Except f o r Chemostat I I , which went i n t o a sexual phase, other chemostat t r i a l s had r a t i o s above one. In the e a r l i e r experiments t o determine the l i m i t i n g n u t r i e n t i n A q u i l , F e — s u f f i c i e n t batch c u l t u r e s which received 25 uM s i l i c a t e d i d not show enhanced growth over F e - s u f f i c i e n t c u l t u r e s which contained 12.5 uM s i l i c a t e . I t was t h e r e f o r e concluded that 12.5 uM s i l i c a t e i n A q u i l was not l i m i t i n g the growth of T. pseudonana. F e — i n s u f f i c i e n t batch c u l t u r e s were not s p e c i f i c a l l y t e s t e d f o r s i l i c a t e l i m i t a t i o n , but the a d d i t i o n of Fe s t i m u l a t e d i n c r e a s e s i n in vivo fluorescence and c e l l d e n s i t y t o l e v e l s of F e - s u f f i c i e n t c o n t r o l s . The observation of s i l i c a t e l i m i t a t i o n concurrent with Fe l i m i t a t i o n i n an F e - l i m i t e d chemostat can be explained by s e v e r a l mechanisms. S i l i c a t e l i m i t a t i o n could r e s u l t from a form of metal 73 t o x i c i t y which i s magnified by Fe d e f i c i e n c y . Rueter et al. (1981) have shown that one e f f e c t of Cu t o x i c i t y was t o i n h i b i t the uptake of s i l i c i c a c i d and that t h i s e f f e c t could occur without reducing the growth r a t e . The degree of s i l i c a t e - u p t a k e i n h i b i t i o n could be increased by i n c r e a s i n g the Cu2* a c t i v i t y of the medium and a l s o r e s u l t e d i n decreasing the c e l l y i e l d at s t a t i o n a r y phase as well as i n c r e a s i n g the average c e l l -volume. Presumably i n h i b i t i o n of s i l i c a t e uptake a l s o i n h i b i t e d c e l l d i v i s i o n . The i n h i b i t i o n of s i l i c i c a c i d uptake was r e l i e v e d by i n c r e a s i n g the medium concentration of s i l i c a t e thus suggesting that the c u p r i c i o n and s i l i c a t e were competing f o r a common s i t e . The uptake of Cu i n t o the c e l l was a l s o increased by the Cu2* a c t i v i t y and decreased by an inc r e a s e i n s i l i c a t e c o n c e n t r a t i o n . Rueter and Morel (1981) have suggested that s i l i c a t e uptake i s mediated by a Zn—dependent system. They demonstrated t h a t the i n h i b i t i o n of s i l i c a t e uptake and increased Cu uptake can occur as the r e s u l t of Zn d e f i c i e n c y or low Zn2*:Cu2* r a t i o s and can be remedied by i n c r e a s i n g the a c t i v i t y of Zn2*. The f o r m u l a t i o n of A q u i l used i n t h i s study d i d not i n c l u d e the c h e l a t o r EDTA. Although the e x c l u s i o n of EDTA would tend t o increase the i o n i c a c t i v i t i e s of a l l t r a c e metals ( i n c l u d i n g Cu), no t o x i c e f f e c t s r e s u l t i n g i n growth r a t e depression have been observed when batch c u l t u r e s of 7. pseudonana were grown i n A q u i l without EDTA when Fe was su p p l i e d as 450 nM f r e s h l y - p r e c i p i t a t e d f e r r i c hydroxide (Wells, 1982? N. Z o r k i n , pers. comm.). In t h i s study no depression i n growth r a t e was observed when 7. pseudonana was 74 grown i n A q u i l without EDTA and with varying concentrations o-f Fe ( i n c l u d i n g no added Fe). Since Rueter et al. (1981) have observed t h a t the cupric—ion—dependent i n h i b i t i o n of s i l i c a t e uptake could occur without a f f e c t i n g growth r a t e t h i s mechanism cannot be e l i m i n a t e d on the b a s i s of growth r a t e depression. The demonstration of s i l i c a t e l i m i t a t i o n i n F e - l i m i t e d chemostat c u l t u r e s but not i n F e — s u f f i c i e n t batch c u l t u r e s can be accounted f o r by two mechanisms. In A q u i l without EDTA, Fe e x i s t s predominantly as c o l l o i d a l f e r r i c hydroxide (Wells et a i . , 1983) which c o n t r i b u t e s s i t e s f o r the e f f i c i e n t adsorption of Cu (Swallow et a l . , 1980). The adsorption of Cu would cause a decrease i n the c u p r i c — i o n a c t i v i t y and a r e s u l t a n t decrease i n any t o x i c e f f e c t s on phytoplankton due Cu. A l t e r n a t i v e l y , Fe may act as a b i o l o g i c a l antagonist of Cu t o x i c i t y , competing with Cu f o r the same uptake s i t e or t a r g e t of t o x i c i t y . The antagonism of some metals towards the t o x i c e f f e c t of others i s not an uncommon observation. Zn has been found t o counteract the i n h i b i t i o n of s i l i c a t e uptake caused by Cu t o x i c i t y or Zn d e f i c i e n c y (Rueter and Morel, 1981). Sunda et al . (1983) have shown that Mn can a l l e v i a t e Cu induced growth-rate r e d u c t i o n and Murphy et al. (1984) found that Fe a l s o a c t s as an antagonist of growth—rate l i m i t i n g C u — t o x i c i t y . A d d i t i o n s of Fe have been observed t o reverse the t o x i c e f f e c t s of Cd (Foster and Morel, 1982; Harrison and Morel, 1983). In higher p l a n t s Crooke (1954) found t h a t the c h l o r o s i s produced by an excess of t r a c e metals was reversed by the a p p l i c a t i o n of Fe d i r e c t l y t o the l e a f surface. Zn t o x i c i t y i n corn (Rosen et 75 a i . , 1977) was almost completely a l l e v i a t e d by the a d d i t i o n o-f Fe. These observations suggest t h a t a common mechanism o-f metal t o x i c i t y i n p l a n t s and phytoplankton may be a competition of t o x i c or excess metals f o r the uptake s i t e s of r e q u i r e d metals. By t h i s mechanism, t o x i c metals would act as analogues f o r r e q u i r e d metals at t h e i r uptake s i t e s , but would not be c h e m i c a l l y s i m i l a r enough to f u n c t i o n p r o p e r l y at the s i t e of metal usage. Consequently, the symptoms of metal t o x i c i t y would mimic the symptoms of required-metal d e f i c i e n c y . In higher p l a n t s , such as sugar beets, the symptoms of Co t o x i c i t y were s i m i l a r t o c h l o r o s i s brought about by Fe d e f i c i e n c y (Terry, 1981). In Thalassiosira weissfogii the symptoms of Cd t o x i c i t y were the same as those of Fe d e f i c i e n c y (Harrison et a i . , 1983). A f u r t h e r example i s found i n the siderophore—producing bacterium, Arthrobacter sp. (Rho, 1983). Exposure t o low l e v e l s of Cd (0.1-1.0 ppm) enhanced the production of hydroxylamine (an i n d i c a t o r of siderophore a c t i v i t y ) , suggesting t h a t Cd had produced an F e - d e f i c i e n t c o n d i t i o n t o which c e l l s responded by producing an F e — s p e c i f i c c h e l a t o r , t o bind Fe and f a c i l i t a t e i t s uptake i n t o the c e l l . 76 CHAPTER IV DETERMINATION OF CELLULAR Fe INTRODUCTION Evidence suggests t h a t the growth r a t e o-f phytoplankton i s f u n c t i o n a l l y dependent upon i n t r a c e l l u l a r — F e p o o l s r a t h e r than d i r e c t l y upon the medium c o n c e n t r a t i o n of Fe ( P r i c e and C a r e l l , 1964; Davies, 1970; Anderson and Morel, 1982). The ambient c o n c e n t r a t i o n s of Fe i n F e - l i m i t e d c u l t u r e s are a l s o expected t o be extremely low so t h a t the measurement of c e l l u l a r Fe, as well as being more p h y s i o l o g i c a l l y meaningful, i s l e s s d i f f i c u l t . If i n t r a c e l l u l a r Fe c o n t r o l s growth r a t e , then c e l l u l a r Fe quota (Q F o) c o u l d be used as a measure of Fe s t r e s s with which t o gauge other responses as the degree of Fe s t r e s s v a r i e s . The e s t i m a t i o n of c e l l u l a r - F e quota i s hampered by the extremely small Fe requirement of l i v i n g c e l l s and i t s u b i q u i t y as a trace—metal contaminant. Consequently, the e s t i m a t i o n of c e l l u l a r Fe r e q u i r e s t h a t ; 1) Fe a n a l y s i s be s e n s i t i v e , 2) sources of Fe contamination be kept t o a minimum, and 3) enough c e l l s are obtained so t h a t the Fe a s s o c i a t e d with them are above the d e t e c t i o n l i m i t and the " n o i s e " of background contamination. D i f f e r e n t i a t i o n of Fe on the c e l l from Fe i n the c e l l i s d e s i r a b l e i n order t o d i s t i n g u i s h between metabolic Fe and Fe t h a t has p h y s i c a l l y adsorbed t o the c e l l e x t e r i o r . METHODS I n i t i a l l y , c u l t u r e samples f o r Fe d e t e r m i n a t i o n were prepared by f i l t e r i n g an a l i q u o t of c u l t u r e onto a 0.4 um 77 polycarbonate Nuclepore - f i l t e r , digesting i t at room temperature i n concentrated n i t r i c acid -for several days and then d i l u t i n g to 105C n i t r i c acid with glass d i s t i l l e d water (DW). Fe i n the digested sample was measured using a Perkin-Elmer 560 graphite -furnace atomic absorption spectrophotometer with a Perkin-Elmer HGA—400 programmer. The r e s u l t s , however, were inconsistent and may have overestimated Fe. These problems were thought to stem from the incomplete digestion of c e l l s and the heterogeneous nature of the digestate (the f i l t e r was not digested and some c e l l s remained attached to i t ) . In addition, the presence of Si i n the diatom f r u s t u l e s may have in t e r f e r r e d with the Fe determination. Because Si i s not v o l a t i l e or r e a d i l y combustible i t does not come off during the charring steps used in graphite furnace atomic absorption spectrophotometry. During the atomization step Si does v o l a t i l i z e along with Fe and other metals and can absorb photons at wavelengths that are c h a r a c t e r i s t i c of Fe, thus leading to an overestimation of Fe. These problems were corrected by f i l t e r i n g algal culture samples onto f i l t e r s composed of a mixture of cellulose-acetate and c e l l u l o s e - n i t r a t e . These f i l t e r s were more e a s i l y and completely digested than polycarbonate f i l t e r s . A more rigorous digestion method also ensured the complete digestion of the f i l t e r and digestion of c e l l u l a r organics that might bind Fe. Interference of Fe determination due to Si was avoided by the use of a colorimetric technique i n which ferrozine ( 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine ) binds 78 f e r r o u s Fe and forms a magenta-colored complex w i t h i n the appropriate pH range. The procedure f o r determining Fe a s s o c i a t e d with T. pseudonana c e l l s i s now described i n d e t a i l . SAMFLING AND FILTRATION A l i q u o t volumes of batch or chemostat c u l t u r e s were measured i n acid-washed polymethylpentene graduated c y l i n d e r s . Known volumes were f i l t e r e d through M i l l i p o r e type AA f i l t e r s (0.8 um, 47 mm dia.) housed i n polycarbonate f i l t e r h olders and V i t o n 0-rings. Samples were f i l t e r e d at vacuum pressures l e s s than 20 mm of Hg i n a c l a s s 100 flow hood i n which a l l p o s s i b l e p a r t s were replaced with polypropylene. The f i l t e r holder and a n c i l l a r y apparati were a c i d washed (1 N HC1) and r i n s e d with g l a s s d i s t i l l e d water (DW) p r i o r t o use. A f t e r f i l t r a t i o n the f i l t e r s were placed i n p l a s t i c disposable p e t r i dishes and d r i e d at 60DC. The bottom of the dishes were l i n e d with Saran Wrap to prevent f i l t e r s from adhering t o them. DIGESTION B o r o s i l i c a t e g l a s s beakers and watch g l a s s e s were r i g o r o u s l y cleaned with Aqua-Regia (4 p a r t s cone. HC1 t o 1 part cone. HN0s) and r i n s e d with DW. F i l t e r s and Fe standards were placed i n the 100 ml beakers t o which was added 0.5 ml cone. HC104 and 4.5 ml of cone. HN03. Beakers were covered with watch g l a s s e s and heated on a h o t p l a t e u n t i l the HN03 b o i l e d away and the dense, white fumes of HC104 appeared. •9 79 REAGENTS AND Fe DETERMINATION Because o-f the r i g o r o u s d i g e s t i o n procedure used t o ensure the removal o-f a l l o r g a n i c s , the preparation o-f reagents was modified from Stookey (1970). The reducing agent (100 g-l~ l hydroxylamine hydrochloride) was prepared as a separate reagent as was the f e r r o z i n e s o l u t i o n (5.14 g - l _ l ) . Because the d i g e s t i o n was performed i n the absence of these reagents the h y d r o c h l o r i c a c i d was omitted from t h e i r p r e p a r a t i o n . The b u f f e r s o l u t i o n c o n s i s t e d of 400 g of ammonium acetate and 350 ml of concentrated ammonium hydroxide d i l u t e d t o 1 l i t e r with DW. These reagents were stored i n polypropylene b o t t l e s at 4°C. Fe standards were prepared from e l e c t r o l y t i c Fe wire, d i s s o l v e d i n concentrated HC1, d i l u t e d with g l a s s d i s t i l l e d water and stored i n high d e n s i t y polyethylene b o t t l e s at pH < 2. Digested samples were coaled t o room temperature and q u a n t i t a t i v e l y t r a n s f e r r e d t o 50 ml acid-washed (cone. HC1) g l a s s volumetric f l a s k s . To each volumetric f l a s k was added 1 ml hydroxylamine hydrochloride reagent, 1 ml f e r r o z i n e reagent and 1 ml b u f f e r s o l u t i o n . Testing showed that I ml of b u f f e r was s u f f i c i e n t t o r a i s e the pH of 0.5 ml of concentrated HC104 t o about 5, w i t h i n the range f o r c o l o r development. A f t e r d i l u t i n g t o 50 ml and a l l o w i n g at l e a s t 10 min f o r f u l l c o l o r development, the absorbance at 562 nm was measured i n a 10 cm path-length cuvette using a Bausch and Lomb Spectronic 2000 spectrophotometer with a DW blank. The absorbance of f i l t e r e d c u l t u r e samples was compared t o the absorbance of Fe standards prepared i n the same manner. 80 Fe CONTAMINATION In p r e l i m i n a r y t e s t s of t h i s method o-f Fe determination, the measured absorbance of reagent blanks was much higher than expected. Because these high absorbance blanks were only observed when blanks were digested i t was suspected that Fe contamination occurred at some poin t i n the d i g e s t i o n procedure. A f t e r a l t e r i n g s e v e r a l aspects of the d i g e s t i o n procedure the reagent grade HN03 was found t o be a major source of Fe contamination. The l e v e l of Fe contamination i n A r i s t a r HNQ3 used was estimated at 42 ng-g_1. To reduce contamination from t h i s source, reagent grade HN03 was d i s t i l l e d once by g l a s s d i s t i l l a t i o n and twice by s u b — b o i l i n g d i s t i l l a t i o n from one Teflon b o t t l e t o another under cleanroom c o n d i t i o n s . These treatments brought the l e v e l of Fe contamination i n the HN03 down t o 19 ng-g-1. The c o n t r i b u t i o n of Fe contamination t o reagent blanks was f u r t h e r reduced by using 3 ml of HN03 i n s t e a d of 4.5 ml i n the d i g e s t i o n of f i l t e r s . Another source of contamination appeared t o be le a c h i n g of Fe from the w a l l s of the g l a s s beakers and watch g l a s s e s . To a c e r t a i n extent t h i s problem c o r r e c t e d i t s e l f s i n c e the amount of contamination from t h i s source decreased as the beakers were repeatedly used i n d i g e s t i o n s . Teflon beakers were used t o overcome the le a c h i n g problem but t h e i r use r e s u l t e d i n other drawbacks. Telfon beakers d i d not conduct heat w e l l which caused a doubling i n the d i g e s t i o n time. Because the Teflon beakers and watchglasses were white, i t was d i f f i c u l t t o see 81 the formation of the dense white fumes of HC104, which i n d i c a t e d that d i g e s t i o n was completed. The s e n s i t i v i t y of t h i s method of Fe determination was about 20 ng-g-1 with a p r e c i s i o n of +/— 35 ng-g_l (95% confidence i n t e r v a l ) f o r reagent blanks digested with l i i l l i p o r e AA f i l t e r s i n g l a s s beakers and watchglasses. The l i m i t of d e t e c t i o n and p r e c i s i o n of the purely chemical part of t h i s procedure i s , however, much greater than t h i s . The reduction i n s e n s i t i v i t y and the inc r e a s e i n v a r i a b i l i t y i s almost e n t i r e l y due t o Fe contamination which occurs during d i g e s t i o n . ASCORBATE WASHING PROCEDURE In order t o d i s t i n g u i s h between Fe i n the c e l l and Fe on the su r f a c e of the c e l l (associated with the c e l l membrane or the f r u s t u l e ) Fe was removed from the c e l l s u r f a c e by an ascorbate washing procedure (Anderson and Morel, 1982). The asorbate reduces f e r r i c Fe i n the medium and at c e l l s u r f a c e s to the more s o l u b l e f e r r o u s species which i s then separated from the c e l l s by f i l t r a t i o n . The washing procedure was c a l i b r a t e d f o r 7. pseudonana by exposing 200 ml a l i q u o t s of an Fe — r e p l e t e c u l t u r e t o 0.1 M ascorbate f o r i n c r e a s i n g lengths of time. Microscopic examination of c e l l s a f t e r the ascorbate treatment revealed shrinkage of c e l l s due t o p l a s m o l y s i s but no ruptured c e l l s were found. A p l o t of Fe versus time of exposure ( F i g . 23) supports t h i s f i n d i n g s i n c e Fe r e t a i n e d on the f i l t e r remained r e l a t i v e l y constant over time a f t e r an i n i t i a l dramatic decrease. H e r e a f t e r , metabolic Fe (Fe i n the c e l l ) was o p e r a t i o n a l l y defined as that Fe measured by the 82 5 n 4 -o> 3 -2 -I -0 - 1 r 0 T 10 — r 20 Time 1 30 (minutes) 40 " I 50 F i g u r e 23. C a l i b r a t i o n of ascorbate washing procedure. 200 ml a l i q u o t s of F e - r e p l e t e stationary-phase c u l t u r e were exposed t o 0.1 M ascorbate f o r i n c r e a s i n g lengths of time, f i l t e r e d and assayed f o r Fe. -ferrozine method a f t e r exposing a c u l t u r e t o 0. 1 M ascorbate f o r 15 min. RESULTS AND DISCUSSION An estimate of metabolic Fe per c e l l , or QFe, was obtained from the c a l i b r a t i o n experiment f o r the ascorbate washing procedure. QF. was estimated at 370 attomoles per c e l l f o r t h i s F e - r e p l e t e c u l t u r e (Table IV). The technique d i d not allow f o r the e s t i m a t i o n of Fe on the c e l l s u r f a c e because Fe as s o c i a t e d with non-ascorbate exposed and f i l t e r e d c e l l s could not be d i s t i n g u i s h e d from c o l l o i d a l Fe r e t a i n e d on the f i l t e r s . The estimate of DF, from an Fe- r e p l e t e batch c u l t u r e was three or more times greater than determinations f o r a batch c u l t u r e grown i n Aqui l - F e with no added Fe or f o r two chemostat c u l t u r e s which r e c e i v e d 17.9 or 0 nM added Fe. Chemostat I was shown t o be at l e a s t p a r t l y s i l i c a t e l i m i t e d (Chapter I I I : Continuous c u l t u r e ) . P a r t i a l s i l i c a t e l i m i t a t i o n would tend t o lessen the degree of Fe l i m i t a t i o n and would lead t o greater c e l l s i z e s by i n h i b i t i n g c e l l d i v i s i o n . These two f a c t o r s may have c o n t r i b u t e d t o the l a r g e r values of QF. f o r Chemostat I versus Chemostat IV which was shown t o be l i m i t e d by only Fe. The Fe-deplete batch c u l t u r e (# 37) e x h i b i t e d a l a r g e average cell-volume of 134 u 3. Such a c e l l volume i s w i t h i n the range of average cell-volumes observed i n s i l i c a t e - l i m i t e d c u l t u r e s i n t h i s study and i n Rueter et al., (1981) and may i n d i c a t e Cu-induced i n h i b i t i o n of s i l i c a t e l i m i t a t i o n (Chapter I I I ) . 0 F o was computed f o r Batch c u l t u r e #37 and f o r Chemostat I 84 ASCORBATE BATCH CHEMOSTAT CHEMOSTAT WASHING CULTURE I IV CALIBRATION #37 Fe ADDED TO MEDIUM (nM) 450 17.9 LIMITING FACTORS pH Fe Si ? Fe Si Fe CELL DENSITY (10 s c e l l s ! - 1 ) ,50 1 . 4 S 1.40 AVERAGE CELL VOLUME (u 3) 134 50 40 QFe (ascorbate washed) (attomoles-eel 1_1) 370 98 ** 90 110 ** 94 QFe (ascorbate unwashed) (attomoles-cel l" 1) 110 ** B8 94 ** 83 60 ** 46 CHOROPHYLL a (ugl- 1 ) 36 16 CHLOROPHYLL a PER CELL (f emtograms-cel 1 _ 1) 310 180 110 CHLOROPHYLL a PER UNIT CELL VOLUME (f emtograms-u-3) ;.6 !.9 Fe PER UNIT CELL VOLUME (attomoles-u - 3) 0.73 ** 2.2 ** 1.5 ** 0.67 1.9 1.2 CELLULAR CHLOROPHYLL a PER CELLULAR Fe (ug-nanomole-1) 3.3 2.: Table IV C h a r a c t e r i s t i c s 7. pseudonana c u l t u r e s grown under d i f f e r e n t c o n d i t i o n s of F e - s t r e s s . * Not done ** R e p l i c a t e determinations 85 on the b a s i s of c e l l u l a r Fe determined with and without ascorbate washing. Ascorbate washing d i d not lead t o c o n s i s t e n t l y lower values of QFe, thus suggesting that l i t t l e or no Fe was a s s o c i a t e d with the c e l l s u r f a c e i n these c u l t u r e s . This was expected i n chemostat c u l t u r e s where Fe introduced t o the c u l t u r e v i a f r e s h medium would be r a p i d l y absorbed and u t i l i z e d by F e - l i m i t e d , yet a c t i v e l y d i v i d i n g c e l l s . As a r e s u l t , l i t t l e Fe would remain t o p r e c i p i t a t e , form c o l l o i d s and p h y s i c a l l y adsorb t o c e l l s u r f a c e s . C e l l s from the inoculum which i n i t i a l l y may have possessed s u r f a c e -a s s o c i a t e d Fe would be d i l u t e d over time by newly d i v i d e d c e l l s and by the e x i t of c e l l s v i a the chemostat outflow. The f a c t that Fe a s s o c i a t e d with c e l l s u r f a c e s was not observed i n an Fe—deplete batch c u l t u r e probably i n d i c a t e s that the amount of adsorption was too small t o be detected by the present method and sample volume. The f l u c t u a t i o n i n QF« f o r r e p l i c a t e samples may have occurred because the amount of Fe being measured was near the l e v e l of background contamination. If c u l t u r e samples were ap p r e c i a b l y l a r g e r or i f c e l l s contained s i g n i f i c a n t l y more Fe, then background contamination would represent a smaller percentage of the r e a l s i g n a l , thus i n c r e a s i n g the p r e c i s i a n and confidence of measurement. Because no Fe a s s o c i a t e d with the c e l l s u r f a c e was observed i n batch c u l t u r e #37 and Chemostat I, and because Fe was not added to the medium f o r Chemostat IV, the e s t i m a t i o n of QFe a f t e r ascorbate washing was di s c o n t i n u e d and the sample volumes f o r Fe determination were 86 doubled t o 1 l i t e r . The values of QF. obtained f o r the F e — l i m i t e d batch and chemostat c u l t u r e s (Table I I I ) are w i t h i n the range of c a l c u l a t e d Fe quotas of batch c u l t u r e s estimated by s e r i a l Fe a d d i t i o n s (30-100 attomoles-cel l - ' , Table I I ) . As a comparison, the Fe content of the d i n o f l a g e l l a t e , Protogonyaulax tamarensis s t r a i n D—255 was determined without ascorbate washing. In two F e - l i m i t e d batch c u l t u r e s (0.1 uM added Fe) QFe was estimated t o be 22.7 and 34.8 femtomoles-cel 1 _ 1 (one femtomole i s equal to 1 X 10"1S moles) while i n two F e — r e p l e t e batch c u l t u r e s (6.56 uli added Fe) QF. was 105 and 128 femtomoles-cel 1 _ 1 . Assuming that a p r o l a t e spheroid approximates the shape of P. tamarensis, A. Cembella (pers. comm.) has c a l c u l a t e d i t s volume to be about 21,600 u 3. On the b a s i s of t h i s volume and using the Fe quotas determined f o r the Fe—deplete batch c u l t u r e s above, the c a l c u l a t e d Fe per u n i t c e l l volume i s 1.05 and 1.60 attomoles-u - 3 These values are i n very c l o s e accord with those c a l c u l a t e d f o r Thalassiosira pseudonana (Table I I I ) d e s p i t e the f a c t the two organisms belong t o d i f f e r e n t c l a s s e s . C h l o r o p h y l l a was measured i n Batch c u l t u r e #37 and i n Chemostats I and IV (Table I I I ) . Although the Fe-deplete batch c u l t u r e had a l a r g e c e l l u l a r c h l o r o p h y l l quota i n comparison t o the chemostat c u l t u r e s t h i s appears to have been due t o i t s l a r g e average c e l l volume. When c h l o r o p h y l l a was c a l c u l a t e d on a per u n i t volume b a s i s , c e l l s from the Fe-deplete batch c u l t u r e are c h l o r o p h y l l poor. A comparison of c h l o r o p h y l l per l i t e r , c h l o r o p h y l l per c e l l and c h l o r o p h y l l per u n i t c e l l volume between the two chemostat c u l t u r e s i s c o n s i s t e n t with a 87 greater degree of and more complete Fe l i m i t a t i o n i n Chemostat IV than Chemostat I. When Fe per c e l l i s c a l c u l a t e d on a per u n i t volume b a s i s , batch c u l t u r e c e l l s appear Fe poor due t o t h e i r l a r g e c e l l volumes. Considering t h i s same c h a r a c t e r i s t i c Chemostat IV appears more F e - l i m i t e d that Chemostat I. IF a c u l t u r e i s F e - l i m i t e d then Fe should c o n t r o l the amount of c h l o r o p h y l l produced and the r a t i o of c h l o r o p h y l l t o Fe might be expected t o remain constant. However i f Fe i s not l i m i t i n g , or only p a r t i a l l y l i m i t i n g then the production of c h l o r o p h y l l would not be expected t o be e n t i r e l y determined by the amount of Fe and the r a t i o of c h l o r o p h y l l t o Fe should increase over the F e - l i m i t e d r a t i o . I t f o l l o w s then, from the values of c h l o r o p h y l l a per c e l l u l a r Fe (Table I I I ) t h a t the Fe-depleted batch c u l t u r e was not as F e — l i m i t e d as were the chemostat c u l t u r e s . 88 CHAPTER V DETERMINING SHORT TERM Fe UPTAKE INTRODUCTION Given the small Fe requirement o-f the t e s t organism, the Fe-uptake r a t e measured by a decrease i n medium Fe or an i n c r e a s e i n c e l l u l a r Fe would be much too small t o be r o u t i n e l y measured by chemical means. Instead, a r a d i o i s o t o p e o-f Fe co u l d be used t o Follow uptake. s sFe i s an u n s t a b l e i s o t o p e of Fe which has a h a l f l i f e of 2.7 years and decays by e l e c t r o n capture t o "Mn (Brodsky, 1978). A f t e r decay t o ssMn, low energy X-rays are emitted which can be absorbed by a s c i n t i l l a n t and r e e m i t t e d a t v i s i b l e f r e q u e n c i e s t o be recorded by a s c i n t i l l a t i o n counter. METHODS 5 5Fe was obtained as carrier—free 5 3 F e C l 3 i n 0.1 N HC1 from Amersham, U.K. and was d i l u t e d t o a s p e c i f i c a c t i v i t y of 147 GBq-mole-' Fe and a c o n c e n t r a t i o n of 450 uM Fe. A l i q u o t s of c u l t u r e were exposed t o l a b e l l e d Fe i n the f o l l o w i n g manner. Ten u l of the s sFe l a b e l l e d Fe stock were de p o s i t e d a t the bottom of the b a r r e l of a 10 ml p l a s t i c d i s p o s a b l e s y r i n g e . The p i s t o n was r e p l a c e d and r e t u r n e d t o about the 0.5 ml mark. The s y r i n g e was then f i t t e d with an 18 gauge, 1.5 i n c h — l o n g d i s p o s a b l e needle i n which the metal needle b a r r e l was p u l l e d out and r e p l a c e d with a 3 cm l e n g t h of p o l y e t h y l e n e t u b i n g . Ten ml of c u l t u r e were drawn i n t o the s y r i n g e , i n the process mixing the c u l t u r e sample and the l a b e l l e d Fe. During i n c u b a t i o n s <10 min or 1 hr) the s y r i n g e s were o c c a s i o n a l l y i n v e r t e d t o ensure mixing. At the end of the 89 predetermined exposure time, the s y r i n g e was connected to a Swinnex - f i l t e r holder (polypropylene) v i a Luer lock - f i t t i n g s and the c u l t u r e was pushed through a 25 mm, 0.45 um M i l l i p o r e HA f i l t e r . Each f i l t e r was immediately r i n s e d with 5 ml of SOW, removed from the holder and placed i n a p e t r i d i s h l i n e d with Saran Wrap. F i l t e r s were d r i e d and stored i n a dryin g oven at 60°C. S 3Fe a c t i v i t y was determined by p l a c i n g the f i l t e r s i n 10 ml of Aquasol-2 (New England Nuclear) s c i n t i l l a t i o n f l u o r and counting f o r 10 min on a Unilux I I I l i q u i d s c i n t i l l a t i o n counter (Nuclear Chicago). In order t o d i s t i n g u i s h the p h y s i c a l adsorption of Fe onto c e l l s u rfaces and f i l t e r s from metabolic uptake of Fe i n t o c e l l s the a c t i v i t y of heat k i l l e d (50°C f o r 10 min) c o n t r o l c e l l s was subtracted from the a c t i v i t y of untreated c e l l s . To q u a n t i f y a c c u r a t e l y the amount of Fe i n c e l l s i t was necessary t o determine i f the counting of decay events was being quenched by c e l l s and then t o compensate f o r i t . A 70 ml a l i q u o t of F e — r e p l e t e c u l t u r e was exposed to 70 u l of the s sFe— l a b e l l e d Fe f o r 4 hours a f t e r which the c e l l s were k i l l e d with se v e r a l drops of Lugol's s o l u t i o n . Increasing volumes of t h i s c u l t u r e were then f i l t e r e d onto M i l l i p o r e HA 0.45 um f i l t e r s , d r i e d and counted as described above. The p l o t of counts per minute (cpm) versus the number of c e l l s f i l t e r e d ( F i g . 24) shows a l i n e a r r e l a t i o n s h i p up t o *" 15 X 10* c e l l s - m l - 1 where counting e f f i c i e n c y may have decreased. The same t e s t y i e l d e d l i t t l e d i f f e r e n c e or trend i n the AsB r a t i o (A i s a lower energy counting window while B i s the window normally used t o 90 16 -I 12 n o x 8 - \ E o. u 0 J r o i 5 — i r 10 15 Cells X IO6 1 20 F i g u r e 24. Measured a c t i v i t y of "Fe versus the number of c e l l s deposited on the f i l t e r . 91 count 3 SFe decay events) when the channels r a t i o method o-f est i m a t i n g quenching was employed. Since F e - l i m i t e d batch or chemostat c u l t u r e s normally had c e l l d e n s i t i e s of l e s s than 2 X 10 s c e l l s - m l " 1 , 10 mis of these c u l t u r e s would c o n t a i n no more than 2 X 10* c e l l s . This number of c e l l s i s we l l w i t h i n the l i n e a r p o r t i o n of the curve i n Fi g u r e 31 and t h e r e f o r e no quenching of S 3Fe should have occurred. RESULTS AND DISCUSSION The F e - l i m i t e d batch" c u l t u r e #37 e x h i b i t e d Fe uptake r a t e s of 910 attomoles-cel l-'-hr" 1 based on a 10 minute exposure t o l a b e l l e d Fe and 29 attomoles-cell-'-hr- 1 based on a 1 hour exposure. Such a l a r g e d i f f e r e n c e i n uptake r a t e s over the two exposure periods can be accounted f o r by the use of h e a t — k i l l e d c e l l s t o estimate p h y s i c a l l y adsorbed Fe. Fe which adsorbed onto h e a t - k i l l e d c e l l s and the f i l t e r were subtracted from the Fe a s s o c i a t e d with l i v e c e l l s and f i l t e r t o ob t a i n metabolic Fe. However, n e i t h e r p h y s i c a l adsorption of Fe nor metabolic uptake were l i n e a r over one hour exposures t o Fe. During the course of one hour, the r a t e of metabolic uptake d e c l i n e d f a s t e r than the uptake r a t e onto h e a t — k i l l e d c e l l s . Thus, metabolic Fe uptake was lower a f t e r 1 hour than a f t e r ten minutes. The best estimate of the maximum Fe-uptake r a t e was provided by the 10 minute exposure t o 450 nM Fe. This r a t e of 910 attomoles-cel l-'-hr - 1 i s l a r g e r than the maximum Fe uptake r a t e of 120 attomoles-cel l-'-hr-' observed i n Thalassiosira Meissflogii (Anderson and Morel, 1982). This 92 d i f f e r e n c e may be due t o the l a r g e r surface area per c e l l volume which T. pseudonana c e l l s present t o the environment i n comparison t o the much l a r g e r c e l l s of T„ tuexssflogii. Lower uptake r a t e s and l a r g e r c e l l volumes i n T. ueissflogii (T. w e i s f l o g i i are 8—25 times l a r g e r than T. pseudonana) would r e s u l t i n greater periods of Fe uptake before the Fe requirement i s s a t i a t e d . Anderson and Morel (1982) found that Fe uptake was l i n e a r over s e v e r a l hours, i n c o n t r a s t t o the non-linear uptake i n T. pseudonana over one hour. Since the DF» of c e l l s from F e - l i m i t e d batch c u l t u r e #37 was 100 attomoles-cel l* 1 (Table I I I ) , a 10 minute exposure t o 450 nM Fe would all o w these c e l l s t o ob t a i n at l e a s t another 150 attomoles-cel 1 _ 1. The Fe-uptake r a t e of Chemostat c u l t u r e s I and IV was determined but the a c t i v i t y of h e a t - k i l l e d c e l l s was found t o be greater than t h a t of l i v e c e l l s . Computed Fe uptake r a t e s would then be negative. The most reasonable explanation of these r e s u l t s i s th a t h e a t - k i l l i n g was so severe t h a t i t caused an increase i n c e l l s u r f a c e area and the number of s i t e s a v a i l a b l e f o r Fe adsorption. If a s i m i l a r phenomenon had occurred i n determining the Fe-uptake r a t e of the F e - l i m i t e d batch c u l t u r e #37 i t would have caused an underestimation of uptake r a t e . The f i l t e r s from which these determinations were made were stored f o r 4 months at 60 °C before t h e i r a c t i v i t y was determined on the s c i n t i l l a t i o n counter. I t i s , however, very u n l i k e l y that storage of the f i l t e r s over t h i s period of time would change t h e i r r e l a t i v e a c t i v i t y . SUMMARY Batch c u l t u r e experiments demonstrated t h a t i n vivo •fluorescence was a more appropriate measure of biomass f o r the study of Fe l i m i t a t i o n i n Thalass ios ir a pseudonana than was c e l l d e n s i t y . In vivo fluorescence was h i g h l y c o r r e l a t e d t o c h l o r o p h y l l a i n Fe — r e p l e t e and Fe—deplete batch c u l t u r e s and i n F e - l i m i t e d chemostat c u l t u r e s . In vivo f l u o r e s c e n c e per u n i t c e l l d e n s i t y proved t o be a s e n s i t i v e and r e l i a b l e i n d i c a t o r of Fe l i m i t a t i o n because i t e l i m i n a t e d the e f f e c t of c e l l d e n s i t y on the magnitude of i n vivo f l u o r e s c e n c e and was analogous t o c h l o r o p h y l l per c e l l . These r e s u l t s suggest that T. pseudonana responds t o Fe d e p l e t i o n by reducing c h l o r o p h y l l per c e l l before h a l t i n g c e l l d i v i s i o n . Because of the d i f f i c u l t y i n c o n t r o l l i n g the extent of Fe— s t r e s s i n batch c u l t u r e s , an F e — l i m i t e d chemostat was designed and constructed. A l l the chemostat components i n contact with medium or c u l t u r e were low i n t r a c e metals and metal adsoption s i t e s and were autoclavable. The chemostat, with a demonstrated a b i l i t y t o grow an a l g a l c u l t u r e at a constant Fe-l i m i t e d growth r a t e and t o maintain the c u l t u r e ' s axenic c o n d i t i o n should prove a va l u a b l e t o o l f o r the study of a l g a l siderophores and other adaptations t o F e - s t r e s s . Although s i l i c a t e was not l i m i t i n g i n F e — r e p l e t e batch c u l t u r e s grown i n A q u i l without EDTA i t became a l i m i t i n g n u t r i e n t , i n conjunction with Fe, i n F e - l i m i t e d chemostat c u l t u r e s . The l i m i t a t i o n occurred d e s p i t e the low c e l l d e n s i t i e s at which the chemostat operated and r e s u l t e d i n 94 l a r g e r than average c e l l s . S i l i c a t e l i m i t a t i o n was overcome by doubling the s i l i c a t e c o n c e n t r a t i o n o-f the medium. These r e s u l t s are s i m i l a r t o the i n h i b i t i o n of s i l i c a t e uptake and incr e a s e i n s i l i c a t e quota i n 7„ pseudonana caused by high a c t i v i t i e s of Cu2* or low a c t i v i t e s of Zn 2 + (Rueter and Morel , 1981; and Rueter et a i . , 1981). This phenomenon may be important i n o l i g o t r o p h i c marine environments where low Fe a v a i l a b i l i t y might exacerbate l o w - s i l i c a t e c o n c e n t r a t i o n s . Such c o n d i t i o n s are expected t o c o n t r i b u t e t o a species s h i f t from diatoms t o organisms which do not r e q u i r e s i l i c a t e . Fe c e l l quotas were estimated by two methods. In one, c e l l quota was c a l c u l a t e d by the r a t i o of increased added Fe t o increased c e l l y i e l d . A l t e r n a t i v e l y , Fe was d i r e c t l y measured i n a sample of c e l l s and d i v i d e d by c e l l number. When the c e l l d e n s i t y of batch c u l t u r e s were l i m i t e d by Fe the former method provided c e l l quotas which agreed with c e l l quotas by d i r e c t determination. C e l l quotas ranged from 30—100 attomol es-cel 1 _ 1 f o r F e - l i m i t e d batch and chemostat c u l t u r e s and 370 attomoles-cel l " 1 f o r an Fe — r e p l e t e batch c u l t u r e . D i r e c t l y -determined c e l l quotas f o r two F e - l i m i t e d batch c u l t u r e s of the d i n o f l a g e l l a t e Protogonyaulax tamarensis (Clone D—255) were 22.7 and 34.8 femtomoles-cel 1 _ 1. However, when Fe per u n i t c e l l volume was computed the values f o r P. tamarensis c l o s e l y agreed with values c a l c u l a t e d f o r T. pseudonana. No de t e c t a b l e Fe was found on the su r f a c e s of T. pseudonana c e l l s when grown i n a batch c u l t u r e on A q u i l without added Fe or when grown i n an F e - l i m i t e d chemostat. A method f o r the determination of short-term Fe uptake was developed using 95 t h e r a d i o i s o t o p e , 3 SFe. An Fe-uptake r a t e of 9iO a t t o m o l e s - c e l l - 1 - h r - 1 was det e r m i n e d f o r an F e - l i m i t e d b a t c h c u l t u r e . R E C O M M E N D A T I O N S F O R F U T U R E WORK The f i n d i n g s of t h i s s t u d y suggest a number of improvements which would f a c i l i t a t e f u t u r e work. To e n s u r e t h a t Fe s t o c k s f o r a d d i t i o n t o c u l t u r e media a r e a c c u r a t e and remain b i o l o g i c a l l y a v a i l a b l e t h e y s h o u l d be made from a c c u r a t e l y weighed e l e m e n t a l Fe, d i s s o l v e d i n c o n c e n t r a t e d HC1, d i l u t e d and s t o r e d i n p l a s t i c b o t t l e s . A c i d i f i c a t i o n of t h e Fe s t o c k s h o u l d p r e v e n t p r e c i p i t a t i o n of Fe c o l l o i d s and a d s o r p t i o n t o t h e w a l l s of t h e c o n t a i n e r . I f d i a t o m s a r e t o be grown under Fe l i m i t a t i o n , p a r t i c u l a r l y i n a chemostat, s p e c i a l a t t e n t i o n i s w a r r a n t e d t o ens u r e t h a t s i l i c a t e l i m i t a t i o n does not o c c u r . T h i s may be a more i m p o r t a n t c o n s i d e r a t i o n when A q u i l i s used s i n c e i t i s low i n s i l i c a t e compared t o most o t h e r media. The consequences of t h e o m i s s i o n of EDTA from A q u i l s h o u l d be i n v e s t i g a t e d , e s p e c i a l l y w i t h r e s p e c t t o s i l i c a t e l i m i t a t i o n , p r i o r t o b e g i n n i n g a s e t of c u l t u r e e x p e r i m e n t s . When t r a c e metal c o n t a m i n a t i o n of c u l t u r e media i s a c o n s i d e r a t i o n , s t e r i l e f i l t r a t i o n has many advantages over a u t o c l a v i n g . S t e r i l e f i l t r a t i o n t h r o u g h p l a s t i c membrane f i l t e r s housed i n p l a s t i c h o l d e r s s h o u l d c o n t r i b u t e l e s s metal c o n t a m i n a t i o n t o media t h a n s t e r i l i z a t i o n i n a metal a u t o c l a v e . S t e r i l e f i l t r a t i o n i s a l s o l e s s l i k e l y t o a l t e r t h e s p e c i a t i o n of media by h e a t i n g , pH changes and p r e c i p i t a t i o n . 96 If axenic c u l t u r e s are r e q u i r e d , the p h y s i c a l removal of b a c t e r i a and other contaminating organisms from the media f a c i l i t a t e s t e r i l i t y t e s t s by e p i f l u o r e s c e n c e s t a i n i n g and microscopic examination. Autoclaved media s t i l l c o n t a in many b a c t e r i a v i s i b l e by e p i f l u o r e s c e n c e s t a i n i n g which are ne v e r t h e l e s s , non-viable. If these v i s i b l e , but non-viable c e l l s were removed, the r e l i a b i l i t y and confidence of s t e r i l i t y t e s t s would increase. The operation of the chemostat described i n t h i s study could be improved by a m o d i f i c a t i o n of the means of outflow. This m o d i f i c a t i o n would be made by running a tube i n t o the c u l t u r e v essel and submerging i t w i t h i n the c u l t u r e t o provide s t e r i l e f i l t e r e d a i r t o aerate and mix the c u l t u r e . This a i r would a l s o provide the a i r pressure t o f o r c e excess c u l t u r e through the outflow tube p o s i t i o n e d at the d e s i r e d c u l t u r e l e v e l . This method of outflow possesses c e r t a i n mechanical advantages; 1) the arrangement reduces the s t r i n g e n t requirement f o r an a i r t i g h t s e a l of the c u l t u r e v e s s e l , 2) i f the c u l t u r e vessel s e a l i s not a i r t i g h t i t provides a p o s i t i v e pressure t o prevent the entrance of contaminating organisms and metals, 3) the c u l t u r e l e v e l i s p o s i t i v e l y c o n t r o l l e d unless the flow of a i r i n t o the c u l t u r e vessel stops or a l a r g e leak i n the sea l develops. This arrangement a l s o provides a method f o r r a p i d l y mixing new medium i n t o the c u l t u r e . In t e s t s where food c o l o r i n g was dropped i n t o the center of a 2.8 l i t e r Fernbach f l a s k c o n t a i n i n g 2.5 l i t e r s of water, the r o t a t i o n of the s t i r — b a r at 60 rpm created a vortex which retarded 97 complete mixing. When dye was introduced away from the center of the vessel complete mixing was mar g i n a l l y f a s t e r . Removal of the s t i r - b a r and ass o c i a t e d magnetic s t i r r e r would allow the c u l t u r e t o be i l l u m i n a t e d more evenly by p l a c i n g the l i g h t source below the c u l t u r e v e s s e l . F i n a l l y , bubbling the c u l t u r e with a i r or an a i r - C 0 2 mixture would provide a mechanism f o r pH c o n t r o l of the c u l t u r e and thus prevent pH growth l i m i t a t i o n . When growing an organism i n a chemostat i t i s important t o ensure that only the intended l i m i t i n g n u t r i e n t a c t u a l l y l i m i t s growth. This might be done by growing a s e r i e s of chemostat c u l t u r e s at the same growth r a t e w h i l e varying only the medium concentration of the " l i m i t i n g " n u t r i e n t . If the n u t r i e n t t e s t e d i s the only l i m i t i n g n u t r i e n t then the c e l l y i e l d of the chemostat should increase l i n e a r l y with the con c e n t r a t i o n of the added n u t r i e n t . Operation of the chemostat at one of the lower or intermediate n u t r i e n t concentrations should provide a margin of e r r o r t o ensure t h a t no other n u t r i e n t becomes p a r t i a l l y l i m i t i n g . In measurements of Fe uptake, c e l l s from two chemostat c u l t u r e s showed higher Fe uptake i n the h e a t — k i l l e d c o n t r o l c e l l s than i n l i v e c e l l s . The most reasonable explanation of these f i n d i n g s i s th a t the heat treatment i n these cases was too harsh and the subsequent adsorption of Fe onto the c e l l s u r f a c e s d i d not a c c u r a t e l y d u p l i c a t e the adsorption onto l i v e c e l l s . The d i f f i c u l t y of k i l l i n g c e l l s without q u a n t a t i v e l y a l t e r i n g t h e i r c e l l s u r f a c e s can be be avoided by an ascorbate washing t o remove adsorbed Fe. This work leads t o a study of the trends of p h y s i o l o g i c a l 98 v a r i a b l e s such as c e l l volume, Fe c e l l — q u o t a , c h l o r o p h y l l a per c e l l , and Fe uptake over v a r y i n g degrees of Fe s t r e s s . Chemostat, r a t h e r than batch c u l t u r e s should be used t o c o n t r o l the degree of F e - s t r e s s experienced by the c u l t u r e . The demonstrated a b i l i t y of the chemostat t o maintain b a c t e r i a -f r e e c u l t u r e s a l s o a l l o w s the d e t e c t i o n and q u a n t i f i c a t i o n of s i d e r o p h o r e s without q u e s t i o n of t h e i r source. If a s i d e r o p h o r e producing organism i s grown i n the chemostat, l a r g e q u a n t i t i e s of s i d e r o p h o r e c o u l d be obtained f o r chemical and pharmacological c h a r a c t e r i z a t i o n by a d j u s t i n g the chemostat t o a growth r a t e which produces the maximum amount of s i d e r o p h o r e . 99 REFERENCES Anderson, M.A. and F.M.M. Morel. 1982. The i n f l u e n c e of aqueous i r o n c h e m i s t r y on the uptake o-f i r o n by the c o a s t a l diatom Thalassiosira weissflogii. Lzmnol, Oceanogr. 27(5):789-813. Armstrong, J.E. and C. Van Baalen. 1979. Iron t r a n s p o r t i n microalgae: the i s o l a t i o n and b i o l o g i c a l a c t i v i t y of a hydroxamate s i d e r o p h o r e from the blue-green a l g a fig men el 1 um qaadru.pl icatum. J. Ben. Microbiol . 111:253-262. B a i l e y , K.M. and F.B. Taub. 1980. E f f e c t s of hydroxamate s i d e r o p h o r e s (strong F e ( I I I ) c h e l a t o r s ) on the growth of algae. J. Phycol. 16:334-339. Brodsky, A.B. 1978. CRC Handbook of r a d i a t i o n measurement and p r o t e c t i o n . S e c t i o n A, Volume I: P h y s i c a l S c i e n c e Data. CRC Press, West Palm Beach. 693 p. Caperon, J . 1968. P o p u l a t i o n growth responses of Isochrysis galbana t o n i t r a t e v a r i a t i o n at l i m i t i n g c o n c e n t r a t i o n . Ecology 49:866-872. Carpenter, E . J . 1968. A simple inexpensive a l g a l chemostat. Lzmnol. Oceanogr. 13(4):720-721. Crooke, W.M., J.G. Hunter and 0. Vergnano. 1954. The r e l a t i o s h i p between n i c k e l t o x i c i t y and i r o n s upply. Ann. Appl. Biol. 41:311-324. Darley, W.M. 1982. A l g a l b i o l o g y : A p h y s i o l o g i c a l approach. B l a c k w e l l S c i e n t i f i c , Oxford. 168 p. Davies, A.G. 1970. Iron, c h e l a t i o n and the growth of marine phytoplankton: I. Growth k i n e t i c s and c h l o r o p h y l l p r o d u c t i o n i n c u l t u r e s of the e u r y h a l i n e f l a g e l l a t e Jlunalliela tertiolecta under i r o n l i m i t i n g c o n d i t i o n s . J. mar. Biol. Ass. U.K. 50:65-86. Davies. A.G. 1968. On the adhesion of c o l l o i d a l hydrous f e r r i c o x i d e t o g l a s s . J. Colloid Interface Sci. 28(1):48-53. Droop, M.R. 1968. Vitamin B 1 2 and marine ecology. IV. The k i n e t i c s of uptake, growth and i n h i b i t i o n i n Monochrysis lutheri. J. Mar. Biol. Ass. U.K. 48:689-733. Emery, T. 1980. Iron d e p r i v a t i o n as a b i o l o g i c a l defence mechanism. Nature. 287(30):776-777. 100 Emery, T. 1982. Iron metabolism i n humans and p l a n t s . f)m. Sci. 70:626-632. Foster, P.L. and F.M.M. Morel. 1982. Reversal of cadmium t o x i c i t y i n a diatom: An i n t e r a c t i o n between cadmium a c t i v i t y and i r o n . Limnol. Oceanogr. 27(4):745—752. Fuhs, 6.W. 1969. Phosphorus content and r a t e of growth i n the diatoms Cyclotella nana and Thaiassiosira f l u v i a t i l i s . J. Phycol. 5:312-321. Bardner, M.J. 1982. Adsorption of t r a c e metals from s o l u t i o n during f i l t r a t i o n of water samples. Mater Research Centre Technical Report TR-172. 40 p. G a r i b a l d i , J.A. 1972. Influence of temperature on the bi o s y n t h e s i s of i r o n t r a n s p o r t compounds by Salmanel1 a typhimurium. J. Bacterid. 110:262-265. Glover, H. 1977. E f f e c t s of i r o n d e f i c i e n c y on Isochrysis galbana (Chrysophyceae) and Phaeodactylum tricornutum ( B a c i l l a r i o p h y c e a e ) . J. Phycol. 13:208-212. Goyne, E.R. and E.J. Carpenter. 1974. Production of i r o n -binding compounds by marine microorganisms. Limnol. Oceanogr. 19(5):840-842. G u i l l a r d , R.R.L., P. Kilham and T.A Jackson. 1973. K i n e t i c s of s i l i c o n - l i m i t e d growth i n the marine diatom Thai assiosira pseudonana Hasle and Heimdal ^Cyclotella nana Hustedt). J. Phycol. 9:233-237. Harr i s o n , 6.1. and F.M.M. Morel. 1983. Antagonism between cadmium and i r o n i n the marine diatom Thaiassiosira weissflogii. J. Phycol. 19:495-507. Ha r r i s o n , P.J., R.E. Waters and F.J.R. Taylor. 1980. A broad spectrum a r t i f i c i a l seawater medium f o r c o a s t a l and open ocean phytoplanktan. J. Phycol. 16:28-53. Hobbie, J.E., R.J. Daley, and S. Jasper. 1977. Use of Nuclepore f i l t e r s f o r counting b a c t e r i a by fluorescence microscopy, fipp. Environ. Microbiol . 33 (5) : 1225—1228. Hoshaw, R.W. and J.R. Rosowski. 1973. Methods f o r Microscopic algae. In S t e i n , J.R. CEd.3 Handbook of Ph y c o l o g i c a l Methods: C u l t u r e Methods and Growth Measurements. P h y c o l o g i c a l S o c i e t y of America Inc., Cambridge U n i v e r s i t y Press, Cambridge. 448 p. Jacobson, L. and J . J . O e r t l i . 1956. The c o r r e l a t i o n between i r o n and c h l o r o p h y l l content i n c h l o r o t i c sunflower leaves. Plant Physiol. 31:199-204. 101 Jannasch, H.W. 1974. Steady s t a t e and the chemostat i n ecology. Lzmnol. Oceanogr. 19(4):716-720. J e f f r e y , S.W. and G.F Humphrey. 1975. New spectrophotometry equations f o r determining c h l o r o p h y l l s a, hT c% and c 2 i n higher p l a n t s , algae and n a t u r a l phytoplankton. Biochem. Physiol. Pflanzen. 167:191-194. K i e f e r , D.A. 1973. C h l o r o p h y l l a fluorescence i n marine c e n t r i c diatoms: Responses of c h l o r o p l a s t s t o l i g h t and n u t r i e n t s t r e s s . Mar. Biol. 23:39-46. Kubitschek, H.E. 1970. In t r o d u c t i o n t o research with continuous c u l t u r e . P r e n t i c e - H a l l , Englewood, N.J.. 195 p. Lewin, J . and C. H. Chen. 1971. A v a i l a b l e i r o n : A l i m i t i n g f a c t o r f o r marine phytoplankton. Limnol. Oceanogr. 16(4):670-675. Lewin, J . and C.H. Chen. 1973. Changes i n the concent r a t i o n of s o l u b l e and p a r t i c u l a t e i r o n i n seawater enclosed i n con t a i n e r s . Limnol. Oceanogr. 18(4):590-596. McKnight, D.M. and F.M.M. Morel. 1979. Release of weak and strong copper completing agents by algae. Limnol. Oceanogr. 24(5):823-837. McLachlan, J . 1979. Growth media-marine. In S t e i n , J.R. CEd.3, Handbook of P h y c o l o g i c a l Methods: C u l t u r e Methods and Growth Measurements. P h y c o l o g i c a l S o c i e t y of America Inc., Cambridge U n i v e r s i t y Press, Cambridge. 448 p. Harra, J . 1978. E f f e c t of short-term v a r i a t i o n s i n l i g h t i n t e n s i t y on photosynthesis of a marine phytoplankter: A la b o r a t o r y s i m u l a t i o n study. Mar. Biol. 46:191-202. Morel, F.M.M., Rueter, J.G., Anderson, D.M. and G u i l l a r d , R.R.L. 1979. A q u i l : A che m i c a l l y defined phytoplankton c u l t u r e medium f o r t r a c e metal s t u d i e s . J. Phycol. 15:135-141. Murphy, T.P. 1976. Some aspects of i r o n metabolism of algae. M.Sc. t h e s i s , U n i v e r s i t y of Toronto. 142 p. Murphy, L.S., R.R.L. G u i l l a r d and J.F. Brown. 1984. The e f f e c t s of i r o n and manganese on copper s e n s i t i v i t y i n diatoms: D i f f e r e n c e s i n the responses of c l o s e l y r e l a t e d n e r i t i c and oceanic s t r a i n s . Biol. Oceanogr. 3(2):187-201. Murphy, T.P., D.R.S. Lean and C. Nalewajko. 1976. Blue-green algae: Their e x c r e t i o n o-f i r o n — s e l e c t i v e c h e l a t o r s which enables them t o dominate other algae. Science. 192:900— 902. Neilands, J.B. 1967. Hydroxamic a c i d s i n nature. Science, 156:1443-1447. Neilands, J.B. 1976. Siderophores: d i v e r s e r o l e s i n m i c r o b i a l and human physiology. CInJ, Iron metabolism. Ciba Foundation Symposium 51. E l s e v i e r , Amsterdam. 391 p. Parsons, T.R., Y. Maita and CM. L a l l i . 1984. A Manual of Chemical and B i o l o g i c a l Methods f o r Seawater A n a l y s i s . Pergamon Press, Oxford. 173 p. Postgate, J.R. and J.R. Hunter. 1962. The s u r v i v a l of starved b a c t e r i a . J. Ben. Microbiol. 29:233-263. O'Brien, W.J. 1972. L i m i t i n g f a c t o r s i n phytoplankton algae: Their meaning and measurement. Science. 178:616—617. P r i c e , C A . 1968. Iron compounds and plan t n u t r i t i o n . Plant Physiol. 19:239-248. P r i c e , C A . and E.F. C a r e l l . 1964. Control by i r o n of c h l o r o p h y l l formation and growth i n Eaglena gracilis. Plant Physiol. 39:862-868. P r o v a s o l i , L., J.J.A. McLaughlin and M.R. Droop. 1957. The development of a r t i f i c i a l media f o r marine algae. Arch. Mikrobiol. 25:392-428. Rhee, G—Yul1. 1980. Continuous c u l t u r e i n phytoplankton ecology, fidv. Aquatic. Microbiol. 2:151-203. Rho, J . 1983. E f f e c t s of heavy metals on growth and hydroxylamine production by he t e r o t r o p h i c n i t r i f y i n g firthrobacter sp.. Ann. Meeting Amer. Soc. M i c r o b i o l . , New Orleans. 83:226. Rosen, J.A., C S . Pi k e and M.L. Golden. 1977. Zinc, i r o n , and c h l o r o p h y l l metabolism i n z i n c t o x i c corn. Plant Physiol. 59:1085-1087. Rueter, J.G., S.W. Chisholm and F.M.M. Morel. 1981. E f f e c t s of copper t o x i c i t y on s i l i c i c a c i d uptake and growth i n Thaiassiosira pseudonana. J. Phycol. 17:270-278. Rueter, J.6. and F.M.M. Morel. 1981. The i n t e r a c t i o n between z i n c d e f i c i e n c y and copper t o x i c i t y as i t a f f e c t s the s i l i c i c a c i d uptake mechanisms i n Thaiassiosira pseudonana. Limnol. Oceanogr. 26(1):67—73. 103 Setser, P.J., N.L. Buinasso, J r . , and D.R. Schink. 1982. D a i l y p a t t e r n s of fluorescence in vivo i n the c e n t r a l e q u a t o r i a l P a c i f i c . J. M a r . Res. 40: 453-471. Sheldon, R.W. and Parsons, T.R. 1966. A p r a c t i c a l manual on the use of the Coulter Counter i n marine scie n c e . Coulter E l e c t r o n i c Sales Company, Toronto. 66 p. Simpson, F.B. and J.B. Neilands. 1976. Siderochromes i n Cyanophyceae: I s o l a t i o n and c h a r a c t e r i z a t i o n of schi z o k i n e n from dnabaena sp.. J. Phycol. 12:44-48. Spencer, L.T., R.T. Barber and R.A. Palmer. 1973. The de t e c t i o n of f e r r i c s p e c i f i c organic c h e l a t o r s i n marine phytoplankton c u l t u r e s . CInJ P.C. Singer CEd3„ Food-drugs from the sea. Marine Technology S o c i e t y , Washington. Stookey, L.L. 1970. Ferrozine—A new spectrophotometry reagent f o r i r o n , final. Chem. 42 (7) : 779-781. Swallow, K.C., D.N.Hume and F.M.M. Morel. 1980. Sorption of copper and lead by hydrous f e r r i c oxide. Environ. Sci. Technol. 14:1326-1331. Swallow, K.C., J.C. W e s t a l l , D.M. McKnight, N.M.L. Morel and F.M.M. Morel. 1978. Po t e n t i o m e t r i c determination of copper complexation by phytoplankton exudates. Limnol. Oceanogr. 23(3):538-542. Subcommitee on Iron, National Academy of Sciences. 1970. Iron. Univ. Park Press, Baltimore. 248 p. Tempest, D.W., D. Herbert and P.J. Phipps. 1967. St u d i e s on the growth of fierobacter aeragenes at low d i l u t i o n r a t e s i n a chemostat. In E.O. Powel1, C.6.T. Evans, R.E. Strange and D.W. Tempest. EEds.3 M i c r o b i a l Physiology and Continuous C u l t u r e . Her Majesty's S t a t i o n a r y O f f i c e , London. 261 p. Terry, N. 1981. Physiology of t r a c e element t o x i c i t y i t s r e l a t i o n t o i r o n s t r e s s . J. Plant Nutri. 3 :561-578. T r i c k , C.G., R.J. Andersen, A. G i l l a m and P.J. H a r r i s o n . 1983. Prorocentrin". An e x t r a c e l l u l a r siderophore produced by the marine d i n o f l a g e l l a t e Prorocentrum minimum. Science. 219:306-308. T r i c k , C.G., R.J. Andersen, N.M. P r i c e , A. G i l l a m and P.J. Ha r r i s o n . 1983. Examination of hydroxamate—siderophore production by n e r i t i c e u k a r y o t i c marine phytoplankton. Mar. Biol. 75:9-17. 104 Young, P.A. 1967. Peanut c h l o r o s i s due t o i r o n d e f i c i e n c y . Plant Dis. Rep. 51:464-467. Wells, ti.L. 1982. The r o l e of c o l l o i d s i n p r o v i d i n g a source of i r o n t o phytoplankton. M.Sc. Thesis, U n i v e r s i t y of B r i t i s h Columbia. 116 p. Wells, M.L., Zorkin, N.G. and Lewis, A.6. 1983. The r o l e of c o l l o i d a l chemistry i n p r o v i d i n g a source of i r o n t o phytoplankton. J. M a r . Res. 41:731-746. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0053144/manifest

Comment

Related Items