9 by Springer-Vedag 1977. Marine Diatoms Grown in Chemostats under Silicate or Ammonium. Limitation. III. Cellular Chemical Composition and Morphology of.
Marine Biology 43, 19-31 (1977)
MARINE BIOLOGY 9 by Springer-Vedag 1977
Marine Diatoms Grown in Chemostats under Silicate or Ammonium Limitation. III. Cellular Chemical Composition and Morphology of Chaetoceros debilis, Skeletonema costatum, and Thalassiosira gravida* P.J. I-larrbon, H.L. Conway**, R.W. Holmes*** and C.O. Davis**** Depertmem of Oceanography, Unlvertlty of WBzhington;Seattle, Wadzington, USA
Ab~
T h r e e m a r i n e diatoms, Skeletonema costatum, Chaetoceros debilis, and Thalassiosira g r a ~ d a w e r e g r o w n u n d e r no l i m i t a t i o n and a m m o n i u m or s i l i c a t e l i m i t a t i o n or starvation. C h a n g e s in cell m o r p h o l o g y w e r e d o c u m e n t e d w i t h p h o t o m i c r o g r a p h s of a m m o n i u m and s i l i c a t e - l i m i t e d and n o n - l i m i t e d cells, and c o r r e l a t e d w i t h o b s e r v e d changes in c h e m i c a l c o m p o s i t i o n . C u l t u r e s g r o w n u n d e r s i l i c a t e s t a r v a t i o n or l i m i t a t i o n s h o w e d an i n c r e a s e in p a r t i c u l a t e carbon, n i t r o g e n and p h o s p h o r u s and c h l o r o p h y l l a per unit cell v o l u m e c o m p a r e d to n o n - l i m i t e d cells; p a r t i c u l a t e s i l i c a per cell v o l u m e d e c r e a s e d . S i - s t a r v e d cells w e r e d i f f e r e n t from S i - l i m i t e d cells in that the former c o n t a i n e d m o r e p a r t i c u l a t e c a r b o n and s i l i c a per cell volume. The m o s t sens i t i v e i n d i c a t o r of s i l i c a t e l i m i t a t i o n or s t a r v a t i o n was the r a t i o C:Si, being 3 to 5 times h i g h e r than the values for n o n - l i m i t e d c e l l s . T h e r a t i o s S i : c h l o r o p h y l l a and S:P w e r e lower and N:Si was h i g h e r than n o n - l i m i t e d cells by a f a c t o r of 2 to 3. The o t h e r ratios, C:N, C:P, C : c h l o r o p h y l l a, N : c h l o r o p h y l l a, P : c h l o r o p h y l l a and N:P w e r e c o n s i d e r e d not to be s e n s i t i v e i n d i c a t o r s of s i l i c a t e l i m i t a t i o n or s t a r v a t i o n . C h l o r o p h y l l a, and p a r t i c u l a t e n i t r o g e n per unit cell v o l u m e d e c r e a s e d u n d e r a m m o n i u m l i m i t a t i o n and s t a r v a t i o n . N H 4 - s t a r v e d cells c o n t a i n e d m o r e chlorop h y l l a, carbon, nitrogen, silica, and p h o s p h o r u s per cell v o l u m e t h a n N H 4 - 1 i m i t e d cells. N : S i was the m o s t s e n s i t i v e ratio to a m m o n i u m l i m i t a t i o n or s t a r v a t i o n , b e i n g 2 to 3 times lower t h a n n o n - l i m i t e d cells. S i : c h l o r o p h y l l a, P : c h l o r o p h y l l a and N:P w e r e less s e n s i t i v e , w h i l e the ratios C:N, C : c h l o r o p h y l l a, N : c h l o r o p h y l l a, C:Si, C:P and Si:P w e r e the least sensitive. L i m i t e d cells had less of the l i m i t i n g n u t r i e n t per unit cell v o l u m e than s t a r v e d cells and m o r e of the n o n - l i m i t i n g nut r i e n t s (i.e., s i l i c a and p h o s p h o r u s for N H 4 - 1 i m i t e d cells). This s u g g e s t s that n u t r l e n t - l i m i t e d cells r a t h e r than n u t r i e n t - s t a r v e d cells s h o u l d be used along w i t h n o n - l i m i t e d cells to m e a s u r e the full r a n g e of p o t e n t i a l c h a n g e in c e l l u l a r chemical c o m p o s i t i o n for one s p e c i e s u n d e r n u t r i e n t limitation.
l,~o4u~,
ents are w i t h d r a w n from the w a t e r in the p r o p o r t i o n s r e q u i r e d for g r o w t h (RedP h y t o p l a n k t o n g r o w t h r e q u i r e s the assimfield et al., 1963). F u r t h e r studies have i l a t i o n of i n o r g a n i c n u t r i e n t s from the s h o w n that the n u t r i e n t u p t a k e r a t i o or w a t e r in w h i c h they are growing. G e n e r a l - c e l l u l a r c h e m i c a l c o m p o s i t i o n can be ly, it is c o n s i d e r e d t h a t t h e s e n u t r i a l t e r e d by: b i o l o g i c a l factors such as cell size (Paasche, 1973b) and age of *Contribution No. 943 from the Department of c u l t u r e (Pugh, 1975); p h y s i c a l f a c t o r s Oceanography, University of Washington, such as light (intensity and p h o t o p e r i o d , Seattle, Washington 98195, USA. Collos and Lewin, 1976; Davis, 1976) and **Present address: Radiological and Environt e m p e r a t u r e (J~rgensen, 1968; Harrison, mental Research Division, Argonne National 1974); c h e m i c a l f a c t o r s such as s a l i n i t y Laboratory, Argonne, Illinois 60439, USA. (Pugh, 1975) s o u r c e of n i t r o g e n (nitrate ***Present address: Department of Marine Scior ammonium, S t r i c k l a n d et al., 1969) and ences, University of California, Santa Barn u t r i e n t l i m i t a t i o n . This study is conbara, California 93106, USA. c e r n e d e n t i r e l y w i t h the latter factor. ****Present address: Great Lakes Research DiviM u c h of the e a r l y w o r k on c h e m i c a l sion, The University of Michigan, Ann Arbor, c o m p o s i t i o n of m a r i n e p h y t o p l a n k t o n beMichigan 48109, USA. gan w i t h the e l e m e n t s n i t r o g e n and p h o s -
20
P.J. Harrison et al. : Chemical Composition and Morphology of Diatoms
p h o r u s b e c a u s e it was t h o u g h t that t h e s e two e l e m e n t s w e r e m o s t l i k e l y to limit p h y t o p l a n k t o n g r o w t h in the n a t u r a l env i r o n m e n t . E a r l y s t u d i e s by K e t c h u m and R e d f i e l d (1949) s h o w e d that d e f i c i e n c i e s of n i t r o g e n or p h o s p h o r u s in c u l t u r e m e d i u m c o u l d d e c r e a s e the N:P ratio in Chlorella pyrenoidosa by a factor of 2 for n i t r o g e n d e f i c i e n c y and i n c r e a s e the r a t i o by a f a c t o r of 5 for p h o s p h o r u s deficiency. B a t c h c u l t u r e s can be used to m e a s u r e the c h e m i c a l c o m p o s i t i o n of l o g a r i t h m i cally g r o w i n g (non-limited) cells and s t a r v e d cells, w h i c h are a s s u m e d to repr e s e n t the e x t r e m e s in the p o s s i b l e degrees of n u t r i e n t l i m i t a t i o n . H o w e v e r , at i n t e r m e d i a t e d e g r e e s of l i m i t a t i o n , and at lower c o n c e n t r a t i o n s of the limiting n u t r i e n t , the c h e m i c a l e n v i r o n m e n t c h a n g e s so q u i c k l y that the d e g r e e of l i m i t a t i o n can n e v e r be p r e c i s e l y known. Some w o r k e r s (Kilham, 1975; P a a s c h e , 1975) h a v e u s e d v e r y low cell c o n c e n t r a tions at low s u b s t r a t e c o n c e n t r a t i o n s so that the c h a n g e in the s u b s t r a t e c o n c e n t r a t i o n w o u l d be slower. H o w e v e r , it is d i f f i c u l t to c o n d u c t c h e m i c a l c o m p o s i t i o n m e a s u r e m e n t s at these low cell densities. This p r o b l e m in u s i n g b a t c h c u l t u r e s can be o v e r c o m e t h r o u g h the use of cont i n u o u s c u l t u r e t e c h n i q u e s , w h e r e the c u l t u r e can be m a i n t a i n e d at s t e a d y s t a t e at a s p e c i f i c g r o w t h rate and a s p e c i f i c d e g r e e of n u t r i e n t l i m i t a t i o n . C h a n g e s in c h e m i c a l c o m p o s i t i o n of mar i n e p h y t o p l a n k t o n in c o n t i n u o u s c u l t u r e have b e e n s t u d i e d for v a r i o u s l i m i t i n g nutrients including nitrogen limitation (Caperon and Meyer, 1972; T h o m a s and Dodson, 1972; Conway, 1974; E p p l e y and Renger, 1974), p h o s p h o r u s l i m i t a t i o n (Fuhs, 1969; Fuhs et al., 1972; Droop, 1974; Perry, 1976), and s i l i c a t e l i m i t a tion (Paasche, 1973a; Davis, 1976; H a r r i s o n et al., 1976). In the a b o v e studies, l i t t l e a t t e m p t has b e e n m a d e to c o r r e l a t e the m o r p h o l o g i c a l c h a n g e s that a c c o m p a n y the c h a n g e s in c h e m i c a l c o m p o s i t i o n . Only P a a s c h e (1973a) has d o c u m e n t e d the m o r p h o l o g i c a l c h a n g e s that o c c u r in c o n t i n u ous c u l t u r e and a few s t u d i e s have b e e n c o n d u c t e d on s t a r v e d cells g r o w n in b a t c h c u l t u r e (Braruud, 1948; Holmes, 1966). P a a s c h e ' s p h o t o m i c r o g r a p h s show e x t e n s i v e m o d i f i c a t i o n s in the f r u s t u l e of Thalassiosira pseudonana w h e n it was grown under silicate limitation. This p a p e r p r e s e n t s d a t a on the cellular c h e m i c a l c o m p o s i t i o n and m o r p h o l o gy of t h r e e m a r i n e d i a t o m s that w e r e g r o w n in c h e m o s t a t s u n d e r s i l i c a t e or a m m o n i u m l i m i t a t i o n and s e m i - c o n t i n u o u s ly u n d e r u n l i m i t e d and n u t r i e n t - s t a r v e d
c o n d i t i o n s . The n u t r i e n t u p t a k e k i n e t i c s of t h e s e t h r e e species w i l l be p r e s e n t e d in a s u b s e q u e n t p a p e r (Conway and H a r r i s o n , 1977). Materials and Methods
T h r e e m a r i n e c e n t r i c d i a t o m s w e r e used in this study. The s o u r c e of Skeletonema costatum has b e e n p r e v i o u s l y d e s c r i b e d (Davis et al., 1973). Chaetoceros debilis Cleve was i s o l a t e d at F r i d a y Harbor, W a s h i n g t o n , in 1970 by Dr. J. L e w i n (Univ e r s i t y of W a s h i n g t o n ) . Thalassiosira gravida C l e v e was o b t a i n e d from the U n i v e r s i ty of C a l i f o r n i a c u l t u r e c o l l e c t i o n , S a n t a B a r b a r a (USA); it o r i g i n a t e d form e r l y f r o m the S c r i p p s I n s t i t u t e of Oceanography culture collection. The c h e m o s t a t s y s t e m was the same as that d e s c r i b e d p r e v i o u s l y (Davis et al., 1973; C o n w a y et al., 1976), except that the l i g h t i n g s y s t e m was changed. T h e n e w s y s t e m c o n s i s t e d of three, 110 W, daylight, f l u o r e s c e n t bulbs h e l d in a p a r a b o l i c r e f l e c t o r . A sheet of blue P l e x i glas (No. 2069, R o h m and Haas) 0.3 cm thick, was u s e d to m o d i f y the s p e c t r u m and m a k e it c o m p a r a b l e to 5 m u n d e r w a t e r light for c o a s t a l c o n d i t i o n s (described as J e r l o v t y p e 3 in Holmes, 1957). Cont i n u o u s light w a s u s e d and the i n t e n s i t y was 0.08 ly/min. The c u l t u r e s w e r e g r o w n at 18oc in a r t i f i c i a l s e a w a t e r (Davis e t a l . , 1973). N u t r i e n t s and v i t a m i n s w e r e a d d e d in the p r o p o r t i o n s of m e d i u m f (Guillard and Ryther, 1962). T h e 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 was about 8 to 10 ~gat/l -I in the i n f l o w m e d i u m , and the other n u t r i e n t s , t r a c e m e t a l s and vitamins w e r e c o n s i d e r e d to be s a t u r a t i n g at a p p r o x i m a t e l y f / 5 0 levels. The d i l u t i o n rates for the n u t r i e n t l i m i t e d c u l t u r e s w e r e c h o s e n from the region w h e r e t h e r e was little or no c h a n g e in e f f l u e n t s u b s t r a t e c o n c e n t r a t i o n over a r a n g e of d i l u t i o n rates (defined as r e g i o n 2, H a r r i s o n et al., 1976). It was felt that d i l u t i o n rates from this region (generally D ~ I/3 to I/2 bmax) produced representative nutrient-limited cultures. Thalassiosira g r a ~ d a grows m o r e s l o w l y than the o t h e r two s p e c i e s and, t h e r e f o r e , it was g r o w n at a lower dilution rate in o r d e r to o b t a i n c h e m i c a l c o m p o s i t i o n d a t a from a c o m p a r a b l e reg i o n of the g r o w t h curve (region 2). Unl i m i t e d cells w e r e o b t a i n e d f r o m semic o n t i n u o u s c u l t u r e s (daily d i l u t i o n s of b a t c h cultures) in w h i c h cells w e r e g r o w ing l o g a r i t h m i c a l l y and n u t r i e n t s w e r e s a t u r a t i n g . S t a r v e d cells w e r e o b t a i n e d by a l l o w i n g t h e s e s e m i - c o n t i n u o u s cult u r e s of l o g a r i t h m i c a l l y g r o w i n g cells to d e p l e t e the l i m i t i n g n u t r i e n t (ammo-
P.J. Harrison et al. : Chemical Composition and Morphology of Diatoms
n i u m or silicate) in the m e d i u m w h i l e other n u t r i e n t s w e r e m a i n t a i n e d at satu r a t i n g levels. The s t a r v a t i o n p e r i o d lasted 72 h. At the end of this p e r i o d the cells w e r e still c a p a b l e of t a k i n g up the l i m i t i n g n u t r i e n t , i n d i c a t i n g that they w e r e still v i a b l e (Conway and H a r r i s o n , 1977). R e c o g n i t i o n of v i a b l e looking cells was f a c i l i t a t e d by counting live, r a t h e r than p r e s e r v e d samples. W h e n c u l t u r e s g r o w i n g in the chemostat r e a c h e d s t e a d y state, w h i c h was det e r m i n e d by no t r e n d in cell n u m b e r s or f l u o r e s c e n c e over s e v e r a l days, e f f l u e n t n u t r i e n t c o n c e n t r a t i o n s , cell n u m b e r s and v o l u m e s w e r e m e a s u r e d . S a m p l e s for p a r t i c u l a t e carbon, n i t r o g e n and s i l i c a w e r e o b t a i n e d by f i l t e r i n g about 1OO ml of the e f f l u e n t culture. N u t r i e n t analyses, f l u o r e s c e n c e meas u r e m e n t s and cell c o u n t i n g t e c h n i q u e s were the same as in p r e v i o u s studies (Davis et a l . , 1 9 7 3 ) . T h e c h l o r o p h y l l a det e r m i n a t i o n was s i m i l a r to the S C O R U N E S C O m e t h o d (Blasco, 1973); the m a i n m o d i f i c a t i o n was that the g l a s s - f i b e r f i l t e r s w e r e put d i r e c t l y into 90% acetone and a l l o w e d to e x t r a c t in the dark in a r e f r i g e r a t o r for 3 to 20 h. P a r t i c u l a t e s i l i c a (PSi) was a n a l y z e d by the s o d i u m f u s i o n p r o c e d u r e of B u s b y and Lewin (1967). P a r t i c u l a t e n i t r o g e n (PN) and c a r b o n (PC) w e r e a n a l y z e d w i t h a Carlo E r b a CHN a n a l y z e r (Model 1100). Cell v o l u m e s w e r e c a l c u l a t e d from m i c r o s c o p i c a l m e a s u r e m e n t s of cell diameter and length. T h e cells w e r e t a k e n to be c y l i n d r i c a l in shape, e x c e p t for Chaetoceros debilis w h i c h was a s s u m e d to be e l l i p t i c a l w i t h s e m i - a x e s of a and b = 3/4 a. T h e m e a n cell v o l u m e r e p o r t e d is from a m e a s u r e m e n t of 50 cells. P h o t o m i c r o g r a p h s w e r e t a k e n of live material under phase contrast using a Zeiss c o m p o u n d m i c r o s c o p e . E s t i m a t e s of p a r t i c u l a t e n i t r o g e n and s i l i c a w e r e m a d e by d i r e c t a n a l y s i s and by mass b a l a n c e (inflow m i n u s o u t f l o w n u t r i e n t c o n c e n t r a t i o n s ) . D i r e c t analyses w e r e f o u n d to be m o r e v a r i a b l e (Harrison et al., 1976) and t h e r e f o r e part i c u l a t e silica, n i t r o g e n and P h O s p h o r u s w e r e m e a s u r e d by m a s s b a l a n c e in this study. H i g h e r v a r i a b i l i t y in the d i r e c t a n a l y s i s was also o b s e r v e d by C a p e r o n and M e y e r (1972). The mass b a l a n c e m e t h od a s s u m e s that a n e g l i g i b l e a m o u n t of the i n o r g a n i c n u t r i e n t in the i n f l o w m e d i u m is c o n v e r t e d into d i s s o l v e d organic compounds. R ~
Cell Morphology
D i s t i n c t d i f f e r e n c e s in cell m o r p h o l o g y of a s p e c i e s r e s u l t e d from g r o w t h u n d e r
21
s i l i c a t e - l i m i t e d (Si-limited), a m m o n i u m l i m i t e d (NH4-1imited), and n o n - l i m i t e d nutrient conditions. These differences for the t h r e e s p e c i e s are shown in the p h o t o m i c r o g r a p h s (Fig. 1). T h e m o r p h o logical c h a n g e s in s t a r v e d cells w e r e not as s t r i k i n g as in limited cells, but o t h e r w i s e they w e r e similar. P h o t o m i c r o g r a p h s of s t a r v e d cells have not been included. A c o m p a r i s o n of S i - l i m i t e d , nonlimited, and N H 4 - 1 i m i t e d cells of Skeletonema costatum is m a d e in Fig. IA, B and C, r e s p e c t i v e l y . The n u m b e r of cells/ chain was g e n e r a l l y g r e a t e r than 10 for n o n - l i m i t e d cells, g r e a t e r than 6 for N H 4 - 1 i m i t e d cells and less than 4 for S i - l i m i t e d cells. The length of the conn e c t i n g rods b e t w e e n cells was n o r m a l (3 ~um) for n o n - l i m i t e d and N H 4 - 1 i m i t e d cells and almost zero for S i - l i m i t e d cells. M a n y S i - l i m i t e d cells a p p e a r e d to have more or larger c h r o m a t o p h o r e s than n o n - l i m i t e d or N H 4 - 1 i m i t e d cells. The largest cell v a c u o l e was o b s e r v e d for N H 4 - 1 i m i t e d cells. Si- and N H 4 - 1 i m i t e d cells w e r e u s u a l l y v e r y long and narrow, t y p i c a l l y about 1 2 ~m x 3 ~m. The c h a n g e s
in m o r p h o l o g y of Chaetoces h o w n in Fig. ID, E and F. A g a i n the n~nlber of c e l l s / c h a i n was less (-4) u n d e r s i l i c a t e l i m i t a t i o n than under no l i m i t a t i o n (-10) or a m m o n i u m limi t a t i o n (-10). The l e n g t h of the p r o j e c t ing setae was n o r m a l u n d e r no l i m i t a t i o n and a m m o n i u m limitation, but setae w e r e s h o r t e r or a b s e n t u n d e r s i l i c a t e limitation. U s u a l l y t h e r e w e r e two c h r o m a t o phores r a t h e r t h a n one, u n d e r s i l i c a t e limitation. The size of the v a c u o l e in r e l a t i o n to the rest of the cell was d i f f i c u l t to estimate. ros debilis are
Changes
in the m o r p h o l o g y of Thalassiow e r e s i m i l a r to the other two species (Fig. IG, H and I). The n u m b e r of c e l l s / c h a i n was lowest u n d e r s i l i c a t e l i m i t a t i o n (10) g e n e r a t i o n s b e f o r e a s t e a d y s t a t e is o b t a i n e d and m o d i f i c a t i o n in c h e m i c a l c o m p o s i t i o n is complete. T h e r e f o r e , in l a b o r a t o r y s t u d i e s to det e r m i n e the full r a n g e of c h e m i c a l comp o s i t i o n for o n e s p e c i e s u n d e r one nutrient limitation, nutrient-limited r a t h e r than n u t r i e n t - s t a r v e d cells s h o u l d be used, a l o n g w i t h n o n - l i m i t e d cells.
Ecological
Significance
In a d d i t i o n to c o n f o u n d i n g t a x o n o m i c studies, c h a n g e s in m o r p h o l o g y i n d u c e d by s i l i c a t e or a m m o n i u m l i m i t a t i o n m a y also i n c r e a s e s i n k i n g rates. The r e d u c t i o n in the l e n g t h of the setae in Chaetoceros debilis and in the l e n g t h of the rods j o i n i n g the cells of Skeletonema costatum that o c c u r r e d u n d e r s i l i c a t e limitat i o n have p r e v i o u s l y b e e n s h o w n to inc r e a s e s i n k i n g r a t e s (Smayda and Boleyn, 1966; Smayda, 1970). T h e s e o b s e r v a t i o n s s u g g e s t that the s i n k i n g rate of cells u n d e r s i l i c a t e l i m i t a t i o n m a y be h i g h e r t h a n u n d e r a m m o n i u m l i m i t a t i o n , due to the s u b s t a n t i a l m o d i f i c a t i o n and r e d u c t i o n in e x t e n s i o n s of the f r u s t u l e that do n o t o c c u r u n d e r a m m o n i u m limitation. F u t u r e i n v e s t i g a t i o n s in this d i r e c t i o n are needed. The p h o t o m i c r o g r a p h s of the cells (Fig. I) s u g g e s t that it is p o s s i b l e to v i s u a l l y a s s e s s the n u t r i t i o n a l status of m a r i n e d i a t o m s by c o r r e l a t i n g v i s u a l s y m p t o m s i n d u c e d by l i m i t a t i o n or s t a r v a t i o n w i t h the p a r t i c u l a r e l e m e n t in s h o r t s u p p l y such as n i t r o g e n or silicon. The u s e f u l n e s s of this a p p r o a c h in f i e l d s t u d i e s w i l l d e p e n d p a r t i a l l y on the m a g n i t u d e of the s i n k i n g rates and the d e p t h of the t h e r m o c l i n e . T i t m a n and K i l h a m (1976) h a v e s h o w n that s i n k i n g rate increases substantially, shortly after the o n - s e t of p h o s p h a t e s t a r v a t i o n , and it b e g i n s to d e c r e a s e a b o u t 12 h a f t e r p h o s p h a t e e n r i c h m e n t . Due to this r e a s o n a b l y r a p i d a d j u s t m e n t of s i n k i n g r a t e by cells, it is p o s s i b l e that cells c o u l d q u i c k l y sink into h i g h - n u t r i e n t , d e e p w a t e r (where r e c o v e r y f r o m l i m i t a tion w o u l d begin) b e f o r e n u t r i e n t d e f i c i e n t s y m p t o m s are w e l l d e v e l o p e d . The c h e m i c a l c o m p o s i t i o n of Thalassiosira p s e u d o n a n a g r o w n in a c h e m o s t a t u n d e r p h o s p h o r u s or n i t r o g e n l i m i t a t i o n has been c o m p a r e d and the e f f e c t on r a t i o s e v a l u a t e d (Perry, 1976). P e r r y s u g g e s t e d
29
that r a t i o s w h i c h d i f f e r e d by a f a c t o r of 5 or m o r e could be c o n s i d e r e d u s e f u l d i a g n o s t i c p a r a m e t e r s in d e t e r m i n i n g nut r i t i o n a l status of p h y t o p l a n k t o n . Based on this c r i t e r i o n , o n l y one ratio, N:Si, w o u l d be u s e f u l in d i s t i n g u i s h i n g bet w e e n Si- and N - l i m i t e d p h y t o p l a n k t o n . This r a t i o was s i m i l a r for all t h r e e species, w i t h an a v e r a g e v a l u e of 5.1 for S i - s t a r v e d or limited, 2.0 for nonlimited, and 0.8 for N H 4 - s t a r v e d or limited. O t h e r r a t i o s i n c l u d i n g C:Si, N:P, Si:P and S i : c h l o r o p h y l l a w e r e d i f f e r e n t by a factor of at least 2. T h e 5 r a t i o s that w o u l d be least u s e f u l in d i s t i n g u i s h i n g b e t w e e n s i l i c a t e or n i t r o g e n l i m i t a t i o n w e r e C:N, C:P, C : c h l o r o p h y l l a, N : c h l o r o p h y l l a and P : c h l o r o p h y l l a. G e n e r a l l y , t h e s e r a t i o s i n c r e a s e d for b o t h s i l i c a t e and a m m o n i u m l i m i t a t i o n , but for d i f f e r e n t reasons. (The N : c h l o rophyll a ratio under ammonium limitat i o n was an exception.) In all cases, the c h a n g e in t h e s e 5 ratios was small and could not be e a s i l y d e t e c t e d u s i n g natural phytoplankton populations. Due to the p r o b l e m s of m e a s u r i n g the c h e m i c a l c o m p o s i t i o n of living p h y t o p l a n k t o n in the ocean, the n u t r i t i o n a l status of p h y t o p l a n k t o n in the f i e l d is b e s t s t u d i e d by c o m b i n i n g a n u m b e r of a p p r o a c h e s . T h e s e are: m e a s u r i n g one or two of the m o r e s e n s i t i v e c h e m i c a l comp o s i t i o n ratios, v i s u a l l y e x a m i n i n g the cells for s y m p t o m s of c e r t a i n n u t r i e n t d e f i c i e n c i e s , and m e a s u r i n g u p t a k e kin e t i c s on a t i m e - s e r i e s basis.
Acknowledgements. This research was supported by National Science Foundation Grants GB 6394, GA 31093 and GB 20182. The AutoAnalyzer~ was maintained by G. Friederich, and D. Harmon assisted in data analysis and sampling. The CHN analyzer was maintained by R. Feller and J. Vidal.
t ~ m
C~sd
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30
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Date of final manuscript
acceptance:
April
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26-77. Ed. by M.N. Bill. New York: Wiley Interscience 1963 Smayda, T.J.: The suspension and sinking of phytoplankton in the sea. Oceanogr. mar. biol. A. Rev. 8, 353-414 (1970) and B.J. Boleyn: Experimental observations on the flotation of marine diatoms. II. Skeletonema costatum and Rhizosolenia setigera. Limnol. Oceanogr. ii, 18-34 (1966) Strickland, J.D.H., O. Holm-Hansen, R.W. Eppley and R.J.Linn: The use of a deep tank in plankton ecology. I. Studies on the growth and composition of phytoplankton crops at low nutrient levels. Limnol. Oceanogr. 14, 23-34 (1969) Thomas, W.H. and A.N. Dodson: On nitrogen deficiency in tropical Pacific oceanic plankton. II. Photosynthetic and cellular characteristics of a chemostat grown diatom. Limnol. Oceanogr. 17, 515-523 (1972) Titman, D. and P. Kilham: Sinking in freshwater phytoplankton: some ecological implications of cell nutrient status and physical mixing processes. Limnol. Oceanogr. 21, 409-417 (1976) Werner, D.: Die Kiesels~ure im Stoffwechsel yon Cyclotella cryptica, Reiman, Lewin and Guillard. Arch. Mikrobiol. 55, 278-308 (1966) -
Dr. Paul J. Harrison Institute of Oceanography and Department of Botany University of British Columbia Vancouver V6T IW5, British Columbia Canada
15, 1977. Communicated by M.R. Tripp, Newark
