Marine Biology 45, 39-52 (1978)
MARINE BIOLOGY
9 by Springer-Verlag 1978
Relationship between Phytoplankton Cell Size and the Rate of Orthophosphate Uptake: in situ Observations of an Estuarine
Population
E.S. Friebele, D.L. Correll and M.A. Faust
Smithsonian Institution, Chesapeake Bay Center for Environmental Studies; Edgewater, Maryland, USA
Abstract
O r t h o p h o s p h a t e u p t a k e by a n a t u r a l e s t u a r i n e p h y t o p l a n k t o n p o p u l a t i o n was e s t i m a t e d u s i n g two m e t h o d s : (I) 32p u p t a k e e x p e r i m e n t s in w h i c h filters of d i f f e r e n t p o r e sizes w e r e u s e d to s e p a r a t e p l a n k t o n s i z e - f r a c t i o n s ; (2) 33p a u t o r a d i o g r a p h y of phy- t o p l a n k t o n cells. R e s u l t s of the first m e t h o d s h o w e d t h a t p l a n k t o n cells larger than 5 ~um w e r e r e s p o n s i b l e for 2% of the t o t a l o r t h o p h o s p h a t e u p t a k e rate. 98% of the total u p t a k e rate o c c u r r e d in p l a n k t o n c o m p o s e d m o s t l y of b a c t e r i a , w h i c h
p a s s e d the 5 ~m s c r e e n and w e r e r e t a i n e d by the 0.45 Lum p o r e - s i z e filter. T h e r e was no o r t h o p h o s p h a t e a b s o r p t i o n by p a r t i c u l a t e s in a b i o l o g i c a l l y i n h i b i t e d c o n t r o l c o n t a i n i n g i o d o a c e t i c acid. O r t h o p h o s p h a t e u p t a k e r a t e s of i n d i v i d u a l p h y t o p l a n k t o n species w e r e o b t a i n e d u s i n g 33p a u t o r a d i o g r a p h y . The sum of t h e s e i n d i v i d u a l rates was very close to the e s t i m a t e d rate of u p t a k e by p a r t i c u l a t e s l a r g e r than 5 H/n in the 32p l a b e l l i n g e x p e r i m e n t . G e n e r a l l y , s m a l l e r cells w e r e f o u n d to h a v e a f a s t e r u p t a k e rate per bm3 b i o m a s s than larger cells. A l t h o u g h the n a n n o p l a n k t o n consti- tuted only about 21% of the t o t a l algal biomass, the rate of p h o s p h a t e u p t a k e by the n a n n o p l a n k t o n w a s 75% of the t o t a l p h y t o p l a n k t o n u p t a k e rate. R e s u l t s of the p l a n k t o n a u t o r a d i o g r a p h y s h o w e d that the p h o s p h a t e u p t a k e r a t e per u n i t b i o m a s s is a p o w e r f u n c t i o n of the s u r f a c e : v o l u m e r a t i o of a cell; the r e l a t i o n s h i p is ex- p r e s s e d by the e q u a t i o n Y = 2x70-11 xi.7, w h e r e Y is ~gP ~m-3 h-1 and x is the s u r f a c e : v o l u m e ratio. T h e s e r e s u l t s lend s u p p o r t to the h y p o t h e s i s that s m a l l e r cells h a v e a c o m p e t i t i v e a d v a n t a g e by h a v i n g faster n u t r i e n t u p t a k e rates.
Introdu~io. p l a n k t o n has v a r i e d b e t w e e n cells w i t h a
d i a m e t e r of 65 b m to those less than 3 b m . The p h y t o p l a n k t o n is n o r m a l l y an assem- However, it is c l e a r f r o m t h e s e studies b l a g e of o n e - c e l l e d or m u l t i - c e l l e d auto- and f r o m the w o r k of W a t t (1971), w h o trophs w i t h d i v e r s e shapes and sizes, used a u t o r a d i o g r a p h y to m e a s u r e p r i m a r y H u t c h i n s o n (1961) was p u z z l e d that such p r o d u c t i o n rates of i n d i v i d u a l p h y t o - d i v e r s i t y could exist in an i s o t r o p i c en- p l a n k t o n species, that the s m a l l e r cells v i r o n m e n t w h e r e c o m p e t i t i o n for re- p r o v i d e a large f r a c t i o n of the t o t a l sources is s u p p o s e d l y o c c u r r i n g .
P h y t o p l a n k t o n species not o n l y dis- play d i v e r s i t y in shapes and sizes, b u t they d i f f e r in t h e i r c o n t r i b u t i o n to the total a l g a l b i o m a s s and m e t a b o l i c a c t i v - ity. N a n n o p l a n k t o n (very s m a l l algal cells) which, in the past, w e r e over-
p r i m a r y p r o d u c t i o n in the e u p h o t i c zone.
A l t h o u g h n a n n o p l a n k t o n w e r e the m o s t im- p o r t a n t p r o d u c e r s in all e n v i r o n m e n t s s t u d i e d by M a l o n e (1971a), the n e t p l a n k - t o n : n a n n o p l a n k t o n p r o d u c t i v i t y and chlo- r o p h y l l r a t i o s w e r e h i g h e r in c o a s t a l wa- ters than in o c e a n i c waters. N a n n o p l a n k - looked w i t h net p h y t o p l a n k t o n sampling, ton p o p u l a t i o n s v a r i e d w i t h i n a n a r r o w h a v e r e c e n t l y b e e n r e c o g n i z e d as very im- margin, but g e n e r a l l y n e t p l a n k t o n stand- p o r t a n t a u t o t r o p h s in the p l a n k t o n com- ing crop i n c r e a s e d in areas of u p w e l l i n g
munity, along the C a l i f o r n i a C u r r e n t (Malone,
M a n y s t u d i e s h a v e d o c u m e n t e d the domi- 1971b). Y e n t s c h and R y t h e r (1959) also n a n c e of " n a n n o p l a n k t o n " in algal stand- r e p o r t e d a s t a b l e n a n n o p l a n k t o n p o p u l a - ing crop, c h l o r o p h y l l content, and pro- tion a c c o m p a n i e d by f l u c t u a t i o n s in net- d u c t i v i t y (Yentsch and Ryther, 1959; p l a n k t o n s t a n d i n g crop.
Malone, 1971a, b; M c C a r t h y et al., 1974; 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 per- Berman, 1975). The d e f i n i t i o n of n a n n o - haps the n a n n o p l a n k t o n are the b e s t
0025/3162/78/0045/0039/S02.8O
40 E.S. Friebele e t a l . : Phytoplankton Cell Size and Phosphorus Uptake
a d a p t e d s p e c i e s of t h e p h y t o p l a n k t o n c o m - h a p s c o m p e t i n g w i t h o t h e r c e l l s in a m u n i t y in m a n y a q u a t i c e n v i r o n m e n t s . P o s - n a t u r a l a q u a t i c e n v i r o n m e n t .
s i b l y t h e y r e p r e s e n t t h e m o r e o p p o r t u - n i s t i c s p e c i e s , s i n c e t h e y h a v e f a s t e r g r o w t h a n d m e t a b o l i c r a t e s t h a n l a r g e r s p e c i e s (Fogg, 1965). H o w e v e r , t h e i r a b i l i t y to m u l t i p l y r a p i d l y is d e p e n d e n t u p o n r a p i d a n d e f f i c i e n t u t i l i z a t i o n of a v a i l a b l e r e s o u r c e s . Do t h e n a n n o p l a n k - ton h a v e a c o m p e t i t i v e a d v a n t a g e in o b - t a i n i n g t h e r e q u i r e d n u t r i e n t s ?
M u n k a n d R i l e y (1952), w h o d e v e l o p e d a n u t r i e n t a b s o r p t i o n m o d e l b a s e d o n f l u i d d y n a m i c s , p r e d i c t e d t h a t s m a l l c e l l s h a v e a f a s t e r r e l a t i v e n u t r i e n t a b - s o r p t i o n r a t e t h a n l a r g e c e l l s . T h e y s t u d i e d m o r p h o l o g i c a l a n d s i z e v a r i a - t i o n s of p h y t o p l a n k t o n c e l l s a n d c o n - c l u d e d t h a t n e i t h e r t h e r a t e of s i n k i n g n o r n u t r i e n t a b s o r p t i o n i m p o s e s s e v e r e l i m i t s u p o n c e l l s of l e s s t h a n 20 H/n in size.
D u g d a l e (1967) p o i n t e d o u t t h e p o s s i - b i l i t y f o r c o m p e t i t i v e a d v a n t a g e of o n e p h y t o p l a n k t o n s p e c i e s o v e r a n o t h e r b a s e d u p o n M i c h a e l i s - M e n t e n n u t r i e n t u p t a k e k i n e t i c s . E p p l e y e t a l . (1969) o b t a i n e d k i n e t i c c o n s t a n t s for a n u m b e r of s p e - c i e s t a k i n g up a m m o n i a a n d n i t r a t e . T h e i r r e s u l t s s h o w e d s e v e r a l g e n e r a l
t r e n d s : (I) l a r g e s p e c i e s h a d h i g h K s
v a l u e s ; (2) s m a l l - c e l l e d s p e c i e s f r o m t h e o c e a n s h o w e d l o w K s v a l u e s ; (3) f a s t - g r o w i n g s p e c i e s s e e m e d to h a v e l o w e r K s v a l u e s t h a n s l o w g r o w e r s . H o w e v e r , t h e e f f e c t of n u t r i e n t u p t a k e r a t e s on s p e - c i f i c g r o w t h r a t e is m o d i f i e d b y e f f i -
Materials and Methods
T h i s s t u d y w a s c a r r i e d o u t on t h e R h o d e R i v e r , a s u b e s t u a r y on t h e w e s t e r n s h o r e of t h e C h e s a p e a k e B a y n e a r A n n a p o l i s , M a r y l a n d , USA. T h e R h o d e R i v e r is a s h a l -
l o w e s t u a r y w i t h a m e a n d e p t h of 2 m.
F r e s h w a t e r e n t e r s t h e r i v e r f r o m t h e M u d d y C r e e k w a t e r s h e d , a n d s a l i n e w a t e r e n t e r s f r o m t h e Bay. T h e e x p e r i m e n t s w e r e p e r f o r m e d in t h e fall, w h e n w a t e r t e m p e r a t u r e s w e r e d r o p p i n g , s o l u b l e r e - a c t i v e p h o s p h o r u s l e v e l s w e r e d e c l i n i n g a f t e r a l a t e s u m m e r p e a k , a n d p h y t o p l a n k - t o n and b a c t e r i a l n u m b e r s w e r e d e c r e a s - ing.
On O c t o b e r 31, 1974, a 24 1 w a t e r s a m - p l e w a s t a k e n w i t h a p e r i s t a l t i c p u m p f r o m I m d e p t h at a R h o d e R i v e r s t a t i o n
( L a t i t u d e 3 8 o 5 3 ' O O " N; L o n g i t u d e 7 6 0 3 2 ' 00" W), w h e r e t h e d e p t h is 4 m. T h e w a - ter w a s p l a c e d in g l a s s c a r b o y s a n d t r a n s p o r t e d to t h e S m i t h s o n i a n I n s t i t u - t i o n C h e s a p e a k e B a y C e n t e r d o c k w h e r e the e x p e r i m e n t s w e r e p e r f o r m e d . T h e w a - t e r w a s d i v i d e d i n t o e x p e r i m e n t a l b o t - t l e s w h i c h w e r e s u s p e n d e d f r o m t h e d o c k at I m w a t e r d e p t h .
T h e e x p e r i m e n t a l f l a s k for a u t o r a d i o g - r a p h y c o n s i s t e d of a 12 1 s p h e r i c a l
f l a s k w h i c h h a d b e e n a c i d - w a s h e d a n d e q u i p p e d w i t h a s t o p p e r a n d a w i r e h a r - n e s s , w i t h f l o a t a n d w e i g h t a t t a c h e d . T h e f l a s k w a s s u s p e n d e d in t h e w a t e r on c i e n c y of l i g h t u t i l i z a t i o n of t h e p h y t o - t h e s o u t h e r n s i d e of t h e d o c k at I m p l a n k t o n s p e c i e s . P a r s o n s a n d T a k a h a s h i d e p t h . T h e s t o p p e r w a s p i e r c e d b y 3
(1973) h a v e d e v e l o p e d a m o d e l w h i c h p r e - t u b e s : I f r o m a p e r i s t a l t i c p u m p , I f r o m d i c t s t h a t s m a l l c e l l s w i l l o u t g r o w l a r g - a s m a l l a i r pump, and I for t h e e x i t for er c e l l s in a l l e n v i r o n m e n t s e x c e p t air. F o r t h e d u r a t i o n of t h e e x p e r i m e n t , t h o s e w h e r e n u t r i e n t l e v e l s a n d l i g h t in- air was b u b b l e d g e n t l y t h r o u g h the flask t e n s i t i e s a r e h i g h . in o r d e r t o p r e v e n t t h e p l a n k t o n f r o m
T h e w o r k c i t e d a b o v e h a s i n c l u d e d s e t t l i n g . 2 0 0 ~ C i of 33p as H 3 P 0 4 w a s in- e i t h e r l a b o r a t o r y s t u d i e s of n u t r i e n t u p - j e c t e d i n t o t h e f l a s k t h r o u g h a l a r g e t a k e k i n e t i c s w i t h a l g a l c u l t u r e s u n d e r s y r i n g e n e e d l e . A t t i m e d i n t e r v a l s , 1 1 p r e s u m a b l y i d e a l c o n d i t i o n s , o r m o d e l s
u s i n g k i n e t i c d a t a t o p r e d i c t w h i c h c e l l s w i l l h a v e c o m p e t i t i v e a d v a n t a g e in c e r t a i n e n v i r o n m e n t s . T h e p u r p o s e of t h e f o l l o w i n g s t u d y w a s to o b s e r v e t o t a l a n d i n d i v i d u a l n u t r i e n t a b s o r p t i o n r a t e s of a d i v e r s e g r o u p of p h y t o p l a n k t o n c e l l s
s a m p l e s w e r e p u m p e d f r o m t h e f l a s k i n t o b o t t l e s c o n t a i n i n g b u f f e r e d f i x a t i v e
(final c o n c e n t r a t i o n : 0 . 5 % g l u t e r a l d e - h y d e a n d O . 0 1 M p o t a s s i u m p h o s p h a t e b u f - fer at p H 7.0). O n e m l of e a c h s a m p l e w a s a l s o p l a c e d in a s c i n t i l l a t i o n v i a l
for c o u n t i n g at t h e s a m e time. A f t e r u n d e r n a t u r a l c o n d i t i o n s . T h i s w a s a c c o m - e a c h s a m p l e w a s t a k e n , t h e p u m p w a s re- p l i s h e d b y m e a n s of 32p u p t a k e e x p e r i -
m e n t s e m p l o y i n g f i l t e r s to s e p a r a t e p l a n k t o n s i z e s a n d b y 33p a u t o r a d i o g r a - p h y of p l a n k t o n o r g a n i s m s . T h e l a t t e r t e c h n i q u e e n a b l e s t h e r e s e a r c h e r t o m e a - s u r e t h e r a t e of a m e t a b o l i c f u n c t i o n in an i n d i v i d u a l o r g a n i s m or s p e c i e s . T h u s , w e c a n d e t e r m i n e w h e t h e r s m a l l c e l l s d o h a v e a c o m p e t i t i v e a d v a n t a g e in n u t r i e n t u p t a k e w h e n t h e y a r e c o e x i s t i n g a n d p e r -
v e r s e d in o r d e r to c l e a r t h e p u m p l i n e s of w a t e r u n t i l the n e x t s a m p l e w a s t a k e n . O n e m l of w a t e r w a s t a k e n at t h e b e g i n - n i n g a n d at t h e e n d of t h e e x p e r i m e n t
for c o u n t i n g to c h e c k for leaks in t h e a p p a r a t u s .
T o t a l p h o s p h o r u s u p t a k e b y t h e p l a n k - t o n w a s m e a s u r e d a c c o r d i n g to t h e m e t h o d of F a u s t a n d C o r r e l l (1976), in t h r e e 2 5 0 m l b o t t l e s : light, d a r k , a n d o n e con-
E.S. Friebele e t al. : Phytoplankton Cell Size and Phosphorus Uptake 41
t a i n i n g t h e b i o l o g i c a l i n h i b i t o r , i o d o - a c e t i c acid. In e a c h e x p e r i m e n t , 2 ~Ci of c a r r i e r - f r e e 32p as o r t h o p h o s p h a t e w a s a d d e d at t i m e zero. T h e b o t t l e s w e r e
s u b m e r g e d at I m d e p t h and, at t i m e d in- t e r v a l s , the b o t t l e s w e r e r e t r i e v e d a n d a s u b s a m p l e w a s r e m o v e d . P a r t i c u l a t e s in t h i s s u b s a m p l e w e r e t h e n s e p a r a t e d ac- c o r d i n g to s i z e w i t h a 5 ~um p o r e - s i z e d
t h e s e w e r e c o u n t e d . T h i s p r o c e d u r e w a s r e p e a t e d u n t i l a l l g r a i n s a b o v e t h e c e l l w e r e o u t of f o c u s . S i n c e 33p is a m o d e r - a t e l y h i g h e n e r g y e m i t t e r w h i c h is l i k e - ly to p r o d u c e g r a i n s at p o i n t s w h i c h do n o t lie d i r e c t l y o v e r t h e p o i n t of e m i s - sion, s o m e i m a g e - s p r e a d i n g d u e to " c r o s s - fire" is to b e e x p e c t e d (Perry, 1964).
T h i s w a s t a k e n i n t o a c c o u n t v e r y r o u g h l y N i t e x s c r e e n a n d a 0 . 4 5 bun M i l l i p o r e m e m - by c o u n t i n g g r a i n s w i t h i n 5 ~ m of the b r a n e . T h e f i l t e r s a n d I m l of f i l t r a t e
w h i c h h a d p a s s e d t h e 0 . 4 5 Lum m e m b r a n e w e r e p l a c e d in s c i n t i l l a t i o n v i a l s for c o u n t i n g .
A u t o r a di o g r a p h y
F i x e d p l a n k t o n s a m p l e s w e r e c e n t r i f u g e d in the l a b o r a t o r y for 30 m i n at 5 0 0 0 x g at 5oc. T h e p e l l e t w a s w a s h e d t h r e e t i m e s w i t h 15 m l of 0 . 0 0 2 N H C I at 3 0 0 0 x g and r e s u s p e n d e d in I m l of d i s t i l l e d w a t e r . In o r d e r to s e p a r a t e c l u m p e d c e l l s , e a c h c e l l s u s p e n s i o n w a s s o n i - c a t e d for 10 s e c (Model W 140 C, H e a t S y s t e m Co., at 60 W o u t p u t ) a n d f i l t e r e d v i a g r a v i t y t h r o u g h 8 ~ m p o r e - s i z e
N u c l e p o r e f i l t e r s . T h e c e l l s w e r e r i n s e d
c e l l m a r g i n s . G r a i n c o u n t s w e r e m a d e f o r at l e a s t 20 c e l l s of e a c h s p e c i e s on e a c h a u t o r a d i o g r a m , i.e., at e a c h t i m e i n t e r v a l of t h e e x p e r i m e n t . B a c k g r o u n d g r a i n s w e r e c o u n t e d for a r e a s w h i c h h a d n o c e l l s , u n t i l t h e s t a n d a r d d e v i a t i o n of g r a i n s p e r u n i t a r e a w a s w i t h i n ~ 30%
of the m e a n for e a c h a u t o r a d i o g r a m . F o r e a c h t i m e p o i n t , t h e n u m b e r of b a c k - g r o u n d g r a i n s in t h e a r e a o c c u p i e d b y e a c h t y p e of p h y t o p l a n k t o n c e l l w a s c a l - c u l a t e d and s u b t r a c t e d f r o m t h e m e a n n u m - b e r of g r a i n s p e r cell.
A n a l y s e s for d i s s o l v e d p h o s p h o r u s w e r e p e r f o r m e d on w a t e r s a m p l e s w h i c h h a d b e e n f i l t e r e d t h r o u g h a 0 . 4 5 ~un p o r e - s i z e d M i l l i p o r e f i l t e r . I n o r g a n i c p h o s - p h o r u s w a s d e t e r m i n e d c o l o r i m e t r i c a l l y o f f t h e f i l t e r s a n d r e s u s p e n d e d in 2.2 m l on w h o l e a n d f i l t e r e d s a m p l e s b y r e a c - of d i s t i l l e d w a t e r , t i o n w i t h a m m o n i u m m o l y b d a t e a n d r e d u c -
t i o n w i t h s t a n n o u s c h l o r i d e ( A m e r i c a n L i q u i d - e m u l s i o n a u t o r a d i o g r a m s w e r e
p r e p a r e d a c c o r d i n g to t h e m e t h o d of P u b l i c H e a l t h A s s o c i a t i o n , 1971). T o t a l B o g o r o c h (1972), s o m e w h a t m o d i f i e d as d e - p h o s p h o r u s w a s a n a l y z e d b y t h e s a m e m e t h - s c r i b e d b y C o r r e l l e t a i . ( 1 9 7 5 ) . E a c h od a f t e r d i g e s t i o n w i t h p e r c h l o r i c a c i d c e l l s u s p e n s i o n w a s s o n i c a t e d for 15 s e c
a n d 3 d r o p s w e r e s p r e a d on a g e l a t i n - c o a t e d g l a s s s l i d e . S l i d e s of t h e 8 ~um f i l t r a t e w e r e a l s o m a d e . T h e s l i d e s w e r e a i r - d r i e d for 24 to 48 h.
a n d n e u t r a l i z a t i o n (King, 1932). M e t h o d s for r a d i o i s o t o p e c o u n t i n g w e r e as d e - s c r i b e d by F a u s t a n d C o r r e l l (1976).
P h y s i c a l p a r a m e t e r s w e r e m o n i t o r e d d u r - ing the e x p e r i m e n t s w i t h r e c o r d i n g in- s t r u m e n t s at t h e C h e s a p e a k e B a y C e n t e r In a d a r k r o o m , t h e p h o t o g r a p h i c N T B - 2 dock.
e m u l s i o n w a s d i l u t e d 1:1 w i t h d i s t i l l e d w a t e r a n d b r o u g h t to 4 4 o c in a w a t e r b a t h . T w o d r o p s of e m u l s i o n w e r e p l a c e d on e a c h s l i d e a n d t h e n s p r e a d to an e v e n t h i c k n e s s w i t h a s t a i n l e s s s t e e l r a c l e t t e
( B o g o r o c h , 1972). T h e s l i d e s w e r e al- l o w e d to d r y for I h at r o o m t e m p e r a t u r e
(ca. 2 3 o c ) , a n d w e r e t h e n p l a c e d in b l a c k l i g h t - t i g h t , d e s i c c a t e d s l i d e b o x - es a n d i n c u b a t e d at 4oc for a 3 - d a y ex- p o s u r e p e r i o d . T h e s l i d e s w e r e d e v e l o p e d in D e k t o l at 15oc for 2 m i n , r i n s e d for 10 sec, a n d f i x e d in a c i d f i x e r for 5 to 10 min, t h e n r i n s e d in r u n n i n g t a p w a t e r for 30 m i n . T h e s l i d e s w e r e c l e a r e d for I h in m e t h y l s a l i c y l a t e , a n d c o v e r s l i p s w e r e m o u n t e d w i t h P e r m o u n t .
G r a i n c o u n t s w e r e m a d e u s i n g a Z e i s s m i c r o s c o p e at 1 O O O X m a g n i f i c a t i o n u n d e r p h a s e - c o n t r a s t o p t i c s . T h e m i c r o s c o p e w a s f o c u s e d on t h e t o p of a c e l l a n d t h e g r a i n s in f o c u s w e r e c o u n t e d . T h e n t h e m i c r o s c o p e w a s f o c u s e d d o w n u n t i l t h e n e x t set of g r a i n s w a s in f o c u s a n d
T h e p o p u l a t i o n of a l g a l s p e c i e s w a s e s t i m a t e d in a I 1 s a m p l e of w a t e r b y the d i r e c t - c o u n t p r o c e d u r e of C a m p b e l l
(1973). T h e f i x e d c e l l s w e r e c o n c e n t r a t e d to I m l by c e n t r i f u g a t i o n at 5 0 0 0 x g for 30 min, s o n i c a t e d f o r 10 sec, a n d 0 . 0 3 m l w a s p l a c e d on a m i c r o s c o p e s l i d e . A
2.5 c m s q u a r e c o v e r s l i p w a s p l a c e d o v e r t h e l i q u i d a n d t h e n u m b e r of c e l l s of e a c h s p e c i e s in a n u m b e r of m i c r o s c o p e f i e l d s w a s c o u n t e d u n d e r 1 O O O X m a g n i f i - c a t i o n . E x c e p t f o r t h e r a r e r s p e c i e s , 1OO c e l l s of e a c h s p e c i e s w e r e c o u n t e d in o r d e r to e s t i m a t e c e l l n u m b e r s w i t h i n
20% w i t h a c o n f i d e n c e c o e f f i c i e n t of 95% (Lund e t a l . , 1958). V o l u m e a n d s u r - f a c e a r e a of p h y t o p l a n k t o n c e l l s w e r e c a l c u l a t e d f r o m l e n g t h a n d w i d t h m e a s u r e - m e n t s u s i n g s t a n d a r d f o r m u l a s f o r e i t h e r a rod, a s p h e r e , or o b l a t e s p h e r o i d . M o s t of t h e d i n o f l a g e l l a t e s w e r e a s s u m e d to be o b l a t e s p h e r o i d , w i t h an e c c e n t r i c - ity of 0.9.
42 E.S. Friebele et al.: Phytoplankton Cell Size and Phosphorus Uptake
Calculations
T h e t o t a l r a t e of p h o s p h a t e u p t a k e b y t h e p l a n k t o n w a s c a l c u l a t e d f r o m t h e r a t e of 32p d i s a p p e a r a n c e f r o m t h e f i l - t r a t e . A c c o r d i n g to t h e e q u a t i o n f o r a f i r s t - o r d e r r e a c t i o n , t h e r a t e c o n s t a n t , k, w a s o b t a i n e d f r o m t r a c e r m e a s u r e m e n t s :
- k t log C o / C - 2 . 3 0 3 '
w h e r e c is t h e d i s s o l v e d 32p r a d i o a c t i v - ity, c O is t h e a c t i v i t y at t i m e zero, a n d t is time. T h e s l o p e of a l i n e a r re- g r e s s i o n t h r o u g h t h e d a t a p l o t t e d as
log c v e r s u s t w a s u s e d to o b t a i n k. T h e s t e a d y s t a t e r a t e of d i s a p p e a r a n c e of o r t h o p h o s p h a t e f r o m s o l u t i o n w a s t h e n o b - t a i n e d as f o l l o w s :
P disappearance rate (~gPl -I h -l) k C o S w h e r e s is t h e s p e c i f i c a c t i v i t y of the p h o s p h a t e i o n (CPM ~g p-l) at t = O.
R a t e s of p h o s p h o r u s u p t a k e b y t h e t w o p a r t i c u l a t e s i z e s w e r e c a l c u l a t e d f r o m t h e s l o p e s of l i n e a r r e g r e s s i o n e q u a - t i o n s of t h e f i r s t p o r t i o n s of t h e t w o a p p e a r a n c e c u r v e s as f o l l o w s :
&c/At P uptake rate (#g P I-[ h -I) = S ' w h e r e & C / A t is t h e s l o p e r e p r e s e n t i n g
t h e c h a n g e in t h e 32p a c t i v i t y on e a c h of t h e f i l t e r s w i t h time.
O b t a i n i n g p h o s p h o r u s u p t a k e r a t e s of i n d i v i d u a l p h y t o p l a n k t o n s p e c i e s f r o m a u t o r a d i o g r a p h y r e q u i r e s a k n o w n r e l a - t i o n s h i p b e t w e e n g r a i n c o u n t s a n d a s p e - c i f i c a m o u n t of r a d i o a c t i v i t y . N o at- t e m p t w a s m a d e to q u a n t i f y the g r a i n y i e l d of 33p on K o d a k N T B - 2 e m u l s i o n . In- s t e a d , a y i e l d of 1.8 g r a i n s p e r e m i t t e d B p a r t i c l e w a s c h o s e n f r o m l i t e r a t u r e v a l u e s f o r i s o t o p e s w i t h s i m i l a r e n e r g y
l e v e l s a n d on f i l m s of s i m i l a r s e n s i t i v - i t y (Herz, 1959; P e r r y , 1964; Pelc, 1972;
P r e s c o t t , 1964). G r a i n c o u n t s w e r e f i r s t c o r r e c t e d for r a d i o a c t i v e d e c a y to t h e d a y of t h e e x p e r i m e n t . T h e m e a n n u m b e r of g r a i n s p e r c e l l w a s c o r r e c t e d for the l o s s of a c i d - s o l u b l e 33p d u r i n g a c i d w a s h i n g . A n a c i d - s o l u b l e f r a c t i o n of 17% of t o t a l c e l l P, as f o u n d in Chlorella pyrenoidosa b y B a k e r a n d S c h m i d t
(1964), w a s used. T h e ( c o r r e c t e d ) m e a n n u m b e r of g r a i n s p e r c e l l w a s t h e n c o n - v e r t e d t o c o u n t s p e r m i n u t e (cpm) p e r l i t e r for e a c h s p e c i e s as f o l l o w s :
Mean grains/cell x no. of cells/l cpm/l = 1.8 grains/count x 4320 rain exposure time"
S p e c i f i c a c t i v i t y of d i s s o l v e d o r t h o -
c o n c e n t r a t i o n . T h e s p e c i f i c a c t i v i t y of 33p04 d u r i n g the t i m e c o u r s e w a s c a l c u - l a t e d f r o m s p e c i f i c a c t i v i t y of 3 2 p O 4 u s i n g a r a t i o of t h e c o u n t s at t h e b e g i n - n i n g of the 33p a n d 32p e x p e r i m e n t s .
(The d i s s o l v e d o r t h o p h o s p h a t e c o n c e n t r a - t i o n r e m a i n e d c o n s t a n t . )
T h e r a t e of P u p t a k e d u r i n g e a c h t i m e i n t e r v a l w a s t h e n c a l c u l a t e d f o r e a c h a l g a l s p e c i e s as f o l l o w s :
A cpm
iAt - ~g P/iAt or ~g P i-i min-l- cpm/~g P
T h e P u p t a k e r a t e s w e r e t h e n a v e r a g e d o v e r a l l t i m e i n t e r v a l s for w h i c h t h e r e w a s a s l o p e , a n d a m e a n r a t e of p h o s p h o - r u s u p t a k e f o r e a c h s p e c i e s of p h y t o - p l a n k t o n w a s o b t a i n e d .
Results
V a l u e s for p h y s i c a l a n d c h e m i c a l p a r a m - e t e r s d u r i n g t h e e x p e r i m e n t s a r e p r e - s e n t e d in T a b l e I. T h e p l a n k t o n l a b e l - l i n g e x p e r i m e n t s w e r e c a r r i e d o u t f r o m a p p r o x i m a t e l y 1 0 . 0 0 to 1 6 . O O hrs, a n d the a u t o r a d i o g r a p h y e x p e r i m e n t w a s f r o m 0 9 . 3 0 t o 1 5 . 3 0 h r s . T h e l a r g e s t c h a n g e s in s o l a r r a d i a t i o n and t e m p e r a t u r e t o o k p l a c e in t h e l a t e r s t a g e s of t h e e x p e r i - m e n t s . T h e s o l u b l e r e a c t i v e p h o s p h o r u s c o n c e n t r a t i o n w a s l o w at 4 ~g 1-I, a n d a p p r o x i m a t e l y 50% of t h e t o t a l d i s s o l v e d P w a s o r g a n i c . M o s t p h o s p h o r u s f r a c t i o n s r e m a i n e d c o n s t a n t t h r o u g h o u t t h e e x p e r i - m e n t s , e x c e p t f o r an i n c r e a s e in t o t a l r e a c t i v e p h o s p h o r u s . T u r b i d i t y r e m a i n e d at 18 J a c k s o n u n i t s t h r o u g h o u t t h e day.
32p Tracer Experiments
32p u p t a k e w a s e s t i m a t e d b y m e a s u r i n g (I) t h e r a t e of 32p d i s a p p e a r a n c e f r o m t h e d i s s o l v e d o r t h o p h o s p h a t e p o o l in t h e f i l t r a t e , a n d (2) t h e r a t e of 32p a p p e a r - a n c e in t h e p a r t i c u l a t e f r a c t i o n . T h e re- s u l t s of t h e s e e x p e r i m e n t s a r e s h o w n in Figs. I a n d 2. V e r y l i t t l e u p t a k e oc- c u r r e d in t h e b o t t l e c o n t a i n i n g i o d o - a c e t i c a c i d (IAA), w h e r e b i o l o g i c a l u p - t a k e of p h o s p h o r u s w a s i n h i b i t e d . C h e c k s of t o t a l r a d i o a c t i v i t y at t h e b e g i n n i n g a n d at the e n d of e a c h b o t t l e e x p e r i m e n t c o n f i r m e d t h a t no 32p w a s l o s t to t h e s i d e s of t h e b o t t l e or b y l e a k a g e . 3 2 p o 4 d i s a p p e a r e d r a p i d l y f r o m t h e w a t e r in the d a r k b o t t l e . S i n c e t h i s u p t a k e w a s l o w w i t h I A A p r e s e n t , it w a s a s s u m e d t o b e d u e to b i o l o g i c a l a c t i v i t y . T h e l i g h t p h o s p h a t e at e a c h t i m e p o i n t in t h e b o t t l e w a s l o s t d u r i n g t h e e x p e r i m e n t . f i r s t 2 h w a s c a l c u l a t e d f r o m r a d i o a c t i v - H o w e v e r , t h e u p t a k e in l i g h t a n d d a r k i t y in t h e f i l t r a t e of t h e 32p u p t a k e ex- b o t t l e s h a s r a r e l y d i f f e r e d s i g n i f i c a n t - p e r i m e n t a n d t h e d i s s o l v e d o r t h o p h o s p h a t e ly in e x p e r i m e n t s of t h i s k i n d p e r f o r m e d
E.S. Friebele et al.: Phytoplankton Cell Size and Phosphorus Uptake 43
Table i. Physical and chemical parameters during 32p p l a n k t o n labelling and 33p autoradiography experiments, October 31, 1974
Time Solar Water Reactive phosphorus Total phosphorus
(hrs) radiation temper- (~g p i-i) (~g P 1-1 )
(g-cal ature Total Dissolved a Total Dissolved
cm-2 min-1) (oc)
8.OO 0.24 15.2
9.OO 0.50 15.2 6 4 49 10
iO.O0 0.70 15.3
ii.00 0.78 15.5
12.00 0.80 15.7
13.00 0.76 16.O
14.O0 0.64 16.4
15.00 0.40 17.0 16 4 45 7
16.00 0.20 17.2
aAlthough much has been written about other phosphorus compounds which might react w i t h molybdate if they are present in natural waters, we
assume for our purposes that dissolved reactive phosphorus and dis- solved orthophosphate are synonymous.
4.4 4.3
hl
< 4 . 2 ~~zk-'~ a
IK I..-
~ 4 . 1
U..
z__4.o
_ J
m 3 . 9 _ Ig
E 3 . a
n. o 3 . 7 _
3.6_
0
A
Q
@
i I I I i I
I O 0 2 0 0 3 0 0 T I M E (MIN)
Fig. i. Disappearance of 32p from the filtrate in dark bottle (circles) and bottle containing the m e t a b o l i c inhibitor iodoacetic acid, IAA
(triangles). The bottles were incubated in the Rhode River at I m depth for 4 h
O0 bJ
I - 7
--I
m 6
0
l -
n - O.
Z
- - 4
U) 0
n
. 3
--I
2
13.
r I
0
E) (9
0 . 4 5 . u m, d a r k
E)
5 @ m, dark
I I i , I , L
0 I O 0 2 0 0 3 0 0 T I M E ( M I N )
Fig. 2. Appearance of 32p in particulates. Cir- cles: 32p radioactivity on 5 and 0.45 ~m pore- sized filters in dark bottle; triangles: 32p radioactivity on 0.45 ~m p o r e - s i z e d filter in IAA bottle
i n t h e R h o d e R i v e r ( C o r r e l l et al., 1 9 7 5 ; F a u s t a n d C o r r e l l , 1 9 7 6 ) .
T h e a p p e a r a n c e o f 3 2 p i n s i z e f r a c - t i o n s o f p a r t i c u l a t e s i s s h o w n i n F i g . 2.
I n t h e I A A b o t t l e , t h e r e w a s n o P u p t a k e b y e i t h e r s i z e f r a c t i o n . T h e m a j o r i t y o f
d a r k - b o t t l e u p t a k e w a s b y o r g a n i s m s p a s s i n g t h e 5 ~ m s c r e e n a n d r e t a i n e d o n t h e 0 . 4 5 ~ m m e m b r a n e .
D i s a p p e a r a n c e o f 3 2 p f r o m t h e w a t e r a p p e a r e d t o f o l l o w a p s e u d o f i r s t - o r d e r r e a c t i o n . I n F i g . I, i t c a n b e s e e n t h a t
44 E.S. Friebele et al.: Phytoplankton Cell Size and Phosphorus Uptake
Table 2. Estimates of population numbers and biomass of phytoplankton species from Rhode River station at i m depth on October 31, 1974
Species Size Volume Cells 1 -I Biomass % total
(~m) (~m 3) (x iO3) (mm 3 1-1) biomass Peridineum
trochoideum 25 x 28 10300 4.9 O.O5[ 2.5 Gymnodinium sp. 2 25 x 18 5890 [40.0 0.825 41.8 Gymnodinium
gracilentum 20 x iO 2090 72.3 O.151 7.6 Gymnodinium sp. 1 15 x 11 1300 68.3 0.0~9 4.5 Gyrodinium
mundulum 15 x iO 1180 22.3 0.026 1.3
Gymnodinium
galesianum ~5 x 15 i170 a 42.7 0.050 2.5 Gymnodinium
subroseum 11 x 8 546 33.4 O.018 0.9
Flagellate IO x IO 523 50.0 0.050 2.5
Euglena ascus 20 x 5 392 117 0.046 2.3
Katodinium
rotundatum 10-15 x 3-10 19oa ],300 0.247 12.5
Nannoplankton <5 34 12,400 0.422 21.4
Total 14,3OO 1.98
avalues from Campbell (1973).
t h e l o g 3 2 p i n s o l u t i o n d e c r e a s e s l i n e a r - d i n o f l a g e l l a t e s . P h y t o p l a n k t o n p o p u l a - ly f o r a p p r o x i m a t e l y t h e f i r s t 2 h. I t
t h e n l e v e l s o f f t o a n e q u i l i b r i u m v a l u e w h e n 3 2 p r e l e a s e i n t o t h e w a t e r b y p l a n k t o n i c o r g a n i s m s h a s b e g u n .
A s i n a n y f i r s t - o r d e r r e a c t i o n , t h e a p p e a r a n c e o f t h e " p r o d u c t " ( i . e . , 3 2 p i n p a r t i c u l a t e s ) is a c u r v e d l i n e . A l - t h o u g h t h e r a t e o f p h o s p h a t e u p t a k e i s c o n s t a n t , t h e r a t e o f a p p e a r a n c e o f 3 2 p i n p a r t i c u l a t e s d e c r e a s e s as t h e s p e c i f - ic a c t i v i t y o f o r t h o p h o s p h a t e d e c r e a s e s . T h e f i r s t p o r t i o n o f t h e c u r v e is f i t t e d t o a s t r a i g h t l i n e .
T h e t o t a l P u p t a k e r a t e c a l c u l a t e d f r o m t h e d i s a p p e a r a n c e o f 3 2 P 0 4 f r o m s o - l u t i o n is 1 . 0 7 b g P 1 -I h - 1 . U p t a k e r a t e s c a l c u l a t e d f r o m t h e r a t e o f 3 2 p
t i o n n u m b e r s a n d b i o m a s s a r e g i v e n i n T a b l e 2. O n l y o n e s p e c i e s , Katodinium rotun- datum, n u m b e r e d o v e r o n e m i l l i o n c e l l s p e r l i t e r . T h e n a n n o p l a n k t o n , w h i c h w a s c o m p o s e d o f m a n y s m a l l f l a g e l l a t e s , c h r y s o p h y t e s , a n d g r e e n a l g a e , w a s m o s t a b u n d a n t . S m a l l d i a t o m s w e r e q u i t e n u m e r - o u s , b u t m o s t o f t h e c e l l s a p p e a r e d t o b e s e n e s c e n t . I t c a n b e s e e n f r o m T a b l e 2
t h a t Gymnodinium s p e c i e s 2, K. rotundatum, a n d t h e n a n n o p l a n k t o n m a d e u p t h e l a r g - e s t p o r t i o n s o f t h e a l g a l b i o m a s s : 4 1 . 8 , 2 1 . 4 , a n d 1 2 . 5 % , r e s p e c t i v e l y .
33p Autoradiography
a p p e a r a n c e i n p a r t i c u l a t e s o n t h e 5 a n d F i g . 3 s h o w s t h e a p p e a r a n c e o f 3 3 p i n 0 . 4 5 Lum f i l t e r s a r e 2% ( 0 . 0 2 4 u g p 1-I h -I) f i x e d , a c i d - w a s h e d c e l l s ( c o n c e n t r a t e d a n d 9 8 % ( 1 . O 6 ~ g P 1 -I h - l ) , r e s p e c t i v e - f r o m I i o f w a t e r b y c e n t r i f u g a t i o n a n d ly, o f t h e t o t a l P u p t a k e r a t e .
Phytoplankton Population
T h e p h y t o p l a n k t o n i n t h e R h o d e R i v e r o n t h e d a y o f t h e e x p e r i m e n t s w a s n o t a s a b u n d a n t as d u r i n g t h e w a r m e r m o n t h s o f t h e y e a r , b u t t h e s p e c i e s c o m p o s i t i o n w a s q u i t e d i v e r s e . T e n m a j o r s p e c i e s o f
a l g a e w e r e o b s e r v e d , o f w h i c h m o s t w e r e
l a t e r u s e d f o r a u t o r a d i o g r a p h y ) w i t h t i m e . T h e r a t e o f p h o s p h o r u s i n c o r p o r a - t i o n i n t o a c i d - i n s o l u b l e c o m p o u n d s i n - s i d e p l a n k t o n c e l l s ( i n c l u d i n g b a c t e r i a ) w a s c a l c u l a t e d f r o m Fig. 3 as 0 . 7 6 8 ~ g P I-I h - l , w h i c h is a p p r o x i m a t e l y 7 5 % o f t h e t o t a l u p t a k e r a t e o b t a i n e d i n t h e
32p u p t a k e e x p e r i m e n t .
W e o b s e r v e d t h a t a u t o r a d i o g r a m s o f t h e R h o d e R i v e r s a m p l e s c o n t a i n e d a g r e a t e r n u m b e r o f b a c k g r o u n d g r a i n s t h a n
E.S. Friebele et al.: Phytoplankton Cell Size and Phosphorus Uptake 45
w a s f o u n d i n p l a n k t o n p r e p a r a t i o n s b y B r o c k a n d B r o c k ( 1 9 6 8 ) 9 T h i s p r o b l e m w a s a t t r i b u t e d t o t h e l a r g e n u m b e r of b a c - t e r i a p r e s e n t i n e s t u a r i n e w a t e r s 9 A u t o - r a d i o g r a m s o f p l a n k t o n a t t h e f i r s t t i m e - p o i n t s w e r e r e l a t i v e l y f r e e o f b a c k g r o u n d c o u n t s , b u t t h e n u m b e r o f b a c k g r o u n d g r a i n s i n c r e a s e d s t e a d i l y w i t h t i m e d u r - i n g t h e f i r s t 2 h; t h e n u m b e r t h e n r e - m a i n e d c o n s t a n t d u r i n g t h e r e m a i n d e r o f t h e e x p e r i m e n t . T h e r e f o r e , i t w a s n e c e s -
s a r y t o s u b t r a c t b a c k g r o u n d g r a i n s a t K e a c h t i m e - p o i n t as d e s c r i b e d i n " M a t e r i -
a l s a n d M e t h o d s "
F i g 9 4 i l l u s t r a t e s t h e i n c r e a s e i n
t h e m e a n n u m b e r o f g r a i n s p e r c e l l w i t h O t i m e f o r o n e d i n 9 s p e c i e s .
G e n e r a l l y , t h e s t a n d a r d d e v i a t i o n o f g r a i n c o u n t s f o r a s p e c i e s a t o n e t i m e p o i n t w a s w i t h i n ~ 30% o f t h e m e a n . T h e i n c r e a s e i n g r a i n s w a s r a p i d at f i r s t a n d t h e n l e v e l e d o f f w h e n t h e s p e c i f i c a c t i v i t y o f t h e o r t h o p h o s p h a t e p o o l i n t h e w a t e r h a d d e c r e a s e d 9 S i m i l a r c u r v e s w e r e o b t a i n e d f o r a l l 10 s p e c i e s of p h y - t o p l a n k t o n . A l l c u r v e s h a d s h a p e s s i m i - l a r t o t h e o n e s h o w n , w i t h t h e a b s o l u t e n u m b e r o f g r a i n s a n d t h e s l o p e d i f f e r i n g f o r e a c h s p e c i e s .
R a t e s o f p h o s p h o r u s u p t a k e f o r a l l p h y t o p l a n k t o n s p e c i e s w e r e c o m p u t e d , as d e s c r i b e d i n t h e " M a t e r i a l s a n d M e t h o d s "
s e c t i o n , f r o m t h e i n c r e a s e i n g r a i n c o u n t s p e r c e l l o v e r t i m e . T h e s e u p t a k e r a t e s a r e p r e s e n t e d i n T a b l e 3, w i t h s e v e r a l p a r a m e t e r s o f c e l l d i m e n s i o n s 9 U p t a k e r a t e p e r c e l l is l a r g e s t f o r l a r g e c e l l s ; h o w e v e r , i t is o b v i o u s f r o m t h e d a t a i n t h e f i f t h c o l u m n o f T a b l e 3 t h a t t h e p h o s p h o r u s u p t a k e r a t e p e r ~ m 3 c e l l v o l u m e is h i g h e s t f o r s m a l l e r c e l l s . T h e r e l a t i o n s h i p b e t w e e n s i z e a n d u p t a k e r a t e is i l l u s t r a t e d m o r e c l e a r l y i n F i g . 5. A p l o t o f l o g P u p t a k e p e r c e l l
m a s s a g a i n s t l o g s u r f a c e : v o l u m e r a t i o ~150 f o r t h e v a r i o u s p h y t o p l a n k t o n s p e c i e s is l i n e a r . T h e l i n e r e p r e s e n t s t h e b e s t f i t
(r 2 = 0 . 9 3 ) o f t h e l i n e a r r e g r e s s i o n
e q u a t i o n , l o g Y = (-10 7) + (I 7 l o g x) ~I00 w h e r e Y is u p t a k e i n u g P p e r ~un 3 c e l l
v o l u m e p e r h o u r f o r a s p e c i e s a n d x is z t h e s u r f a c e : v o l u m e r a t i o 9 T a k i n g t h e ~ 50 a n t i l o g a r i t h m , t h e e q u a t i o n b e c o m e s Y =
(2 x 1 0 - 1 1 ) x 1.7, a n d w e c a n s e e t h a t P
u p t a k e o f t h e s e a l g a l c e l l s is a p o w e r 0 f u n c t i o n o f t h e i r s u r f a c e : v o l u m e r a t i o 9
T o t e s t w h e t h e r t h e o b s e r v e d r e l a t i o n - 0 s h i p b e t w e e n u p t a k e a n d s u r f a c e : v o l u m e
r a t i o is t r u e f o r o t h e r R h o d e R i v e r p h y - t o p l a n k t o n p o p u l a t i o n s r a t h e r t h a n j u s t f o r t h e o n e , w e a n a l y z e d t h e 3 3 p a u t o - r a d i o g r a p h y d a t a o f J u l y 9, 1 9 7 3 f r o m C o r r e l l et al. (1975) i n t h e s a m e w a y . T h e i r d a t a s h o w s a s i m i l a r c o r r e l a t i o n b e t w e e n g r a i n d e n s i t y p e r b m 3 c e l l v o l - u m e p e r h o u r a n d s u r f a c e : v o l u m e r a t i o
4
:3
2
0 (
0
9
I ~ - 9 9
f
O
I I I I I I
6 0 120 180 2 4 0 3 0 0 3 6 0 TIME (MIN)
Fig. 3. Autoradiography experiment. Appearance of 33p in acid-washed plankton cells and parti- culates with time. Rhode River water was in- cubated with 33p in 12 1 bottle in the light at i m depth. Each I 1 subsample was fixed, centri- fuged, and washed with dilute HCI. Solid line represents a least-squares regression equation giving best fit of first 7 data points; broken line is arbitrarily drawn
/ /
o _ _ _ _
@
/ / /
(D
I I I I t I
6 0 120 180 2 4 0 5 0 0 5 6 0
TIME (MIN)
Fig. 4. Gymnodinium species i. Mean number of grains per cell with time. Whole water samples containing this species were incubated with 33PO4 in the Rhode River at i m depth for 6 h.
Solid line gives best-fit linear regression of first 5 data points; broken line is arbitrarily drawn
Table 3. Surface areas, surface:volume ratios, and p h o s p h o r u s uptake rates for Rhode River phyto- plankton species, October 31, ~974. Uptake rates were computed from grain counts of autoradiograms.
For p h y t o p l a n k t o n cell counts and biomass estimates, see Table 2
Species S u r f a c e area Surface: uptake rate Uptake rate Uptake rate % Total phyto- (~m 2 cell-l) volume (~g P cell -I (~g P ~m -3 (~g P 1 -I p l a n k t o n uptake
ratio h-i x 10-9) h-i x 10 -12 ) h-i x 10 -3 ) rate per liter Peri dineum
trochoi deum 1927 Gymnodinium sp. 2 981 Gymnodinium
gracilentum 739 Gymnodinium sp. i 490 Gyrodinium
mundulum 464
Gymnodinium
galesianum 707 Gymnodinium
subroseum 261 F l a g e l l a t e 314 Euglena ascus 314 Katodinium
rotundatum 266 N a n n o p l a n k t o n 50 Total uptake rate
O. 188 1 i. 84 1 . 15 0.058 O. 15
O. 167 7.74 i . 31 1.084 2.84
O. 353 8.52 4.O7 0.616 1.62
O. 378 3.63 2 . 80 O.248 0.65
O. 394 2.60 2.21 0.058 O. 15
0.604 6.28 5 . 37 0.268 0.69
O.512 4.43 8.76 0.148 0.38
0.600 3.48 6.65 O. 174 O. 45
O. 801 5.92 15.1 0.692 I . 81
1 . 400 5.02 26.4 6.539 17.14
i . 480 2.30 67.7 28.52 74.75
38.41 = 0.038 ~ g P l - l h -I
- i 0
j - ICJ
E O.
, . - I i
U.I I-- Q .
Q.
L9 0 ..I
9
%
-t2
I I I I I
- 1.0 - 0 . 5
I J I I I
0 + Q 3
L O G ( S U R F / V O L )
Fig. 5. R e l a t i o n s h i p between p h o s p h o r u s uptake
( u p t a k e r a t e s w e r e n o t a v a i l a b l e ) . T h e r e g r e s s i o n e q u a t i o n i s Y = O . 1 7 x 2 . 4
(r 2 = 0 . 9 9 ) . T h e c o n v e r s i o n f r o m g r a i n s t o u p t a k e i n ~ g P i n v o l v e s s e v e r a l c o n - s t a n t s w h i c h a c c o u n t f o r t h e d i f f e r e n c e b e t w e e n t h e c o e f f i c i e n t s o f x i n t h e e q u a t i o n s f o r t h e 1 9 7 3 a n d 1 9 7 4 d a t a . H o w e v e r , t h e s i m i l a r i t y b e t w e e n e x p o - n e n t s o f x ( 1 . 7 f o r o u r d a t a , a n d 2 . 4 f o r t h e 1 9 7 3 d a t a ) is i n t e r e s t i n g .
T h e d a t a i n t h e s i x t h a n d s e v e n t h c o l - u m n s i n T a b l e 3 s h o w s t h a t n a n n o p l a n k t o n w e r e r e s p o n s i b l e f o r t h e l a r g e s t p a r t o f t h e p h o s p h o r u s u p t a k e b y t h e p h y t o p l a n k - t o n i n t h e R h o d e R i v e r a t t h i s t i m e o f y e a r . O t h e r s p e c i e s w h i c h s h o w e d s i g n i f i - c a n t P u p t a k e a r e Katodinium rotundatum, Gymnodinium graciientum, Gymnodinium s p e c i e s 2, a n d Euglena ascus. T h e s e s p e c i e s , w h i c h c o m p r i s e t h e l a r g e s t p o r t i o n s o f t h e p h y - t o p l a n k t o n b i o m a s s b e c a u s e o f e i t h e r l a r g e n u m b e r s o f c e l l s o r l a r g e c e l l s i z e , a r e a l s o r e s p o n s i b l e f o r t h e g r e a t - e s t p a r t o f t h e t o t a l p h o s p h o r u s u p t a k e p e r l i t e r . H o w e v e r , n o c l e a r - c u t r e l a - t i o n s h i p b e t w e e n t h e t o t a l b i o m a s s a n d t h e r a t e o f p h o s p h a t e u p t a k e b y e a c h s p e - c i e s is i n d i c a t e d .
W i t h t h e c o r r e l a t i o n i n F i g . 5 i n m i n d , a n e x a m i n a t i o n o f t h e r e l a t i o n s h i p b e t w e e n t o t a l c e l l s u r f a c e a r e a f o r a per ~m 3 cell volume p e r hour for various phyto- s p e c i e s a n d i t s P u p t a k e p e r l i t e r is plankton species and their surface:volume ratios, m o r e e n l i g h t e n i n g . N a n n o p l a n k t o n a n d Kato- Line drawn is linear regression of data: log Y = dinium rotundatum h a d t h e h i g h e s t s u r f a c e :
(-1o.7) + 1.7 log x (r 2 = 0.93) v o l u m e r a t i o s ( 1 . 4 8 a n d 1 . 4 ) a n d h i g h e s t
E.S. Friebele et al.: Phytoplankton Cell Size and Phosphorus Uptake 47
c e l l n u m b e r s ; t h e s e o r g a n i s m s w e r e re- f r a c t i o n a t i o n b y t h i s m e t h o d is n o t p e r - s p o n s i b l e f o r 75 a n d 17% r e s p e c t i v e l y , f e c t b y a n y m e a n s , h o w e v e r . S h e l d o n a n d of t h e t o t a l p h y t o p l a n k t o n P u p t a k e rate. S u t c l i f f e (1969) h a v e s t u d i e d the a c c u - In c o n t r a s t , s p e c i e s w i t h low s u r f a c e :
v o l u m e r a t i o s , s u c h as Gyrodinium mundulum (0.394) a n d P e r i d i n e u m t r o c h o i d e u m (O.188) a n d r e l a t i v e l y l o w c e l l n u m b e r s , h a d P u p t a k e r a t e s w h i c h w e r e O . 1 5 % of t h e t o - tal. Thus, w e f o u n d t h a t in the w a t e r c o l u m n , the P u p t a k e r a t e by a s p e c i e s is c o r r e l a t e d w i t h its t o t a l s u r f a c e area. T h e e q u a t i o n Y = (-1.4 x 10-3) +
(4.15 x 10-5 x), w h e r e x = s u r f a c e a r e a in m m -2 1-I for a s p e c i e s a n d Y = P up- t a k e in ~g P 1-I h-1 for t h a t s p e c i e s , is t h e l i n e a r r e g r e s s i o n of t h e data, w i t h r 2 = 0.90.
T h e s u m of i n d i v i d u a l p h y t o p l a n k t o n P u p t a k e r a t e s as g i v e n in T a b l e 3 is 0 . 0 3 8 ~g P 1-I h-1. T h e r a t e s w e r e c a l - c u l a t e d a s s u m i n g t h a t a c i d - s o l u b l e P w a s 17% of t o t a l c e l l P. If t h e a c i d - s o l u b l e P f r a c t i o n w e r e a s s u m e d to be 50% as it is in s o m e d i a t o m s , t h e t o t a l u p t a k e r a t e w o u l d be 0 . 0 6 3 ~g P 1-I h-1. T h e s u m of t h e r a t e s of i n d i v i d u a l s p e c i e s o b t a i n e d f r o m a u t o r a d i o g r a p h y is in v e r y g o o d a g r e e m e n t w i t h t h e r a t e of 0 . 0 2 4 ~g P 1 -I h -I for p l a n k t o n c e l l s " l a r g e r t h a n 5 %~m" o b t a i n e d in the 32p u p t a k e e x p e r i - m e n t s .
Discussionand ~nclusions
T h e e x p e r i m e n t s p r e s e n t e d h e r e w e r e de-
r a c y of s i z e s e p a r a t i o n of p l a s t i c p a r - t i c l e s w i t h d i f f e r e n t t y p e s a n d p o r e s i z e s of f i l t e r s . T h e i r r e s u l t s s h o w t h a t m a n y p a r t i c l e s w i t h a d i a m e t e r l e s s t h a n the s t a t e d p o r e s i z e of t h e f i l t e r are r e t a i n e d d u e to c l o g g i n g of p o r e s a n d o b s t r u c t i o n of p a r t i c l e p a t h t h r o u g h t h e f i l t e r s . T h e t y p e of f i l t e r used, as w e l l as its p o r e size, d e t e r m i n e d t h e q u a l i t y of s e p a r a t i o n o b t a i n e d ; s c r e e n s r a t h e r t h a n m e m b r a n e f i l t e r s g a v e t h e b e s t s e p a r a t i o n . In o u r e x p e r i m e n t , p a r - t i c l e s s m a l l e r t h a n 5 ~m ( i n c l u d i n g the n a n n o p l a n k t o n ) w e r e p r o b a b l y r e t a i n e d b y the 5 ~ m s c r e e n . B a c t e r i a a r e n o t o r i o u s for a t t a c h i n g to s u r f a c e s , i n c l u d i n g s u s p e n d e d s e d i m e n t s a n d o t h e r p l a n k t o n cells. T h i s p h e n o m e n o n m i g h t l e a d t o o v e r e s t i m a t i o n of p h o s p h a t e u p t a k e b y p l a n k t o n " l a r g e r t h a n 5 H/n" in size.
A n d e r s o n (1965) r e p o r t e d e x p e r i m e n t s w i t h s i z e f r a c t i o n a t i o n of p l a n k t o n off t h e O r e g o n C o a s t . H e f o u n d t h a t l a r g e f r a c t i o n s of p h y t o p l a n k t o n p a s s e d t h r o u g h a 5 Lum f i l t e r , w h i l e v e r y l i t t l e p a s s e d t h r o u g h a 0 . 8 ~ m f i l t e r . B e r m a n (1975), w o r k i n g in t h e G u l f of C a l i f o r n i a , f o u n d
13 to 83% of t o t a l c a r b o n f i x a t i o n of n a t u r a l p h y t o p l a n k t o n p o p u l a t i o n s w a s a s s o c i a t e d w i t h o r g a n i s m s p a s s i n g a 3 ~ m N u c l e p o r e f i l t e r . T h e r e s u l t s of t h e s e
s i z e f r a c t i o n a t i o n e x p e r i m e n t s d e p e n d , of c o u r s e , u p o n t h e s i z e d i s t r i b u t i o n of s i g n e d t o m e a s u r e t h e p h o s p h o r u s u p t a k e n a t u r a l p h y t o p l a n k t o n p o p u l a t i o n s . W h i l e of a n a t u r a l e s t u a r i n e p h y t o p l a n k t o n c o m - t h e s e t w o s t u d i e s w e r e c a r r i e d o u t in m u n i t y u n d e r n a t u r a l c o n d i t i o n s . E n v i r o n - o p e n m a r i n e w a t e r s , s i z e f r a c t i o n a t i o n m e n t a l v a r i a b l e s in t h e s e e x p e r i m e n t s
s u c h as s o l a r r a d i a t i o n , w a t e r t e m p e r a - ture, s a l i n i t y , a n d n u t r i e n t l e v e l s , w e r e t h o s e o c c u r r i n g n a t u r a l l y in t h e R h o d e R i v e r . T h e r e f o r e , t h e n u t r i e n t up- t a k e r a t e s o b s e r v e d in t h i s s t u d y a r e l i k e l y to b e t h o s e w h i c h o c c u r w h e n a
e x p e r i m e n t s w i t h p l a n k t o n f r o m a s h a l l o w e a s t - c o a s t e s t u a r y s h o w e d m o s t a l g a l c e l l s to be r e t a i n e d b y a 5 ~ m N i t e x s c r e e n (Faust a n d C o r r e l l , 1976). A l s o , a u t o r a d i o g r a m s of t h e f i l t r a t e of o u r c e l l s u s p e n s i o n s f i l t e r e d v i a g r a v i t y t h r o u g h an 8 ~ m N u c l e p o r e f i l t e r c o n - d i v e r s e g r o u p of a l g a e is p r e s e n t a n d ex- t a i n e d f e w p h y t o p l a n k t o n c e l l s . A l t h o u g h cess n u t r i e n t s a r e n o t a v a i l a b l e .
T h e r a t e of t r a n s f e r of p h o s p h o r u s f r o m t h e d i s s o l v e d o r t h o p h o s p h a t e p o o l i n t o p l a n k t o n o r g a n i s m s w a s m e a s u r e d b y
(I) t r a c i n g t h e m o v e m e n t of 3 2 p 0 4 i n t o s i z e f r a c t i o n s of p a r t i c u l a t e s , a n d (2) b y f o l l o w i n g t h e m o v e m e n t of 3 3 p o 4 i n t o i n d i v i d u a l s p e c i e s of p h y t o p l a n k t o n . U s i n g t h e f i r s t m e t h o d , t h e t o t a l p h o s - p h o r u s u p t a k e r a t e of t h e p l a n k t o n w a s d e t e r m i n e d to b e a p p r o x i m a t e l y I ~g P 1-I h-1. A c o n t r o l e x p e r i m e n t w i t h b i o - l o g i c a l i n h i b i t o r s h o w e d v e r y l i t t l e ad- s o r p t i o n of the r a d i o a c t i v e t r a c e r to n o n - l i v i n g p a r t i c l e s . P l a n k t o n c e l l s r e - t a i n e d on t h e 5 ~ m s c r e e n w e r e r e s p o n s i - b l e for a b o u t 2% of t h e u p t a k e (0.024 ~g P 1-I h - l ) , w h i l e t h o s e p a s s i n g t h e 5 ~um s c r e e n d i s p l a y e d 98% of the u p t a k e . S i z e
m i c r o s c o p i c e x a m i n a t i o n of t h e f i l t e r s w a s n o t m a d e in t h i s s t u d y , it is as-
s u m e d t h a t the m a j o r i t y of p h y t o p l a n k t o n w a s r e t a i n e d b y t h e 5 ~ m s c r e e n as s h o w n by p r e v i o u s s t u d i e s in t h e s a m e e s t u a r y
(Faust a n d C o r r e l l , 1976).
A l t h o u g h t h e r e a r e n o r e s u l t s f r o m t h e l i g h t b o t t l e , w e h a v e f o u n d t h a t t o - t a l P u p t a k e a n d u p t a k e b y d i f f e r e n t p a r - t i c u l a t e s i z e s g e n e r a l l y d o n o t v a r y s i g - n i f i c a n t l y b e t w e e n l i g h t a n d d a r k b o t - t l e s in 32p t r a c e r e x p e r i m e n t s on t h e R h o d e R i v e r ( C o r r e l l et al., 1975; F a u s t a n d C o r r e l l , 1976). T a f t et al. (1975) a l s o o b t a i n e d i d e n t i c a l r e s u l t s w i t h l i g h t a n d d a r k b o t t l e s in 32p t r a c e r ex- p e r i m e n t s w i t h p l a n k t o n of C h e s a p e a k e Bay. Thus, it a p p e a r s t h a t t h e o b s e r v e d P u p t a k e d o e s n o t r e q u i r e l i g h t a n d is
48 E.S. Friebele e t al.: Phytoplankton Cell Size and Phosphorus Uptake
n o t d i r e c t l y d r i v e n b y p h o t o s y n t h e s i s ~ T h e s e c o n d c o n s i d e r a t i o n w a s the ef- t h i s u p t a k e a l s o is n o t i n h i b i t e d n o t i c e - f e c t of c e l l s i z e a n d s h a p e u p o n g r a i n a b l y by light.
T h e m a j o r i t y of u p t a k e is b y s m a l l o r g a n i s m s w h i c h i n c l u d e f e w a l g a e ; l i g h t or t h e a b s e n c e of l i g h t h a s l i t t l e e f - f e c t u p o n t h e i r P u p t a k e . T h e r e f o r e , m o s t of the p h o s p h a t e u p t a k e m u s t b e at- t r i b u t e d to b a c t e r i a . M a n y l a b o r a t o r y s t u d i e s h a v e e s t a b l i s h e d t h e r a p i d u p - t a k e of i n o r g a n i c p h o s p h e r u s b y b a c t e r i a
(Rigler, 1956; H a r r i s , 1957; J o h a n n e s , 1964; Rhee, 1972; B e u c h l e r a n d D i l l o n , 1974). W e h a v e l e s s k n o w l e d g e of t h i s
d e n s i t y . F o r a p o i n t s o u r c e of r a d i o a c - t i v i t y l o c a t e d at t h e c e n t e r of a disc, t h e n u m b e r of g r a i n s r e s u l t i n g f r o m t h e e m i t t e d ~ p a r t i c l e w h i c h a r e c o u n t e d o v e r a l a r g e d i s c w i l l b e h i g h e r t h a n t h o s e c o u n t e d o v e r a s m a l l disc. H o w e v e r , t h i s e f f e c t is g r e a t l y r e d u c e d b y c o u n t - ing g r a i n s at t h e m a r g i n s a n d s e v e r a l m i c r o m e t e r s b e y o n d . T h e d a t a p r e s e n t e d b y K n o e c h e l a n d K a l f f (1976) s h o w s t h a t t h e g r a i n c o u n t i n g e f f i c i e n c y o v e r a c e l l 10 Lum in d i a m e t e r , w h e n c o u n t i n g is p r o c e s s o c c u r r i n g in n a t u r e . R i g l e r ' s e x - e x t e n d e d 5 Lum b e y o n d c e l l m a r g i n s , is p e r i m e n t s (1956) w i t h l a k e p l a n k t o n i n -
d i c a t e d a h i g h p h o s p h a t e u p t a k e b y b a c - t e r i a . T a f t (1974) a n d T a f t e t el. (1975) a r g u e t h a t b a c t e r i a a r e n o t i m p o r t a n t c o n s u m e r s of i n o r g a n i c p h o s p h a t e in es- t u a r i n e w a t e r s . T h e y n o t e t h a t p a r t i c u - l a t e s a b s o r b i n g p h o s p h a t e in t h e i r s t u d - ies h a d N a n d P to c h l o r o p h y l l a r a t i o s w h i c h a r e c l o s e to t h o s e of m o s t a l g a l c e l l s . In c o n t r a s t , F a u s t a n d C o r r e l l
(1976) h a v e s h o w n a h i g h c o r r e l a t i o n b e - t w e e n p h o s p h a t e u p t a k e r a t e s by p a r t i c u - l a t e s s m a l l e r t h a n 5 Lun a n d b a c t e r i a l b i o m a s s in e s t u a r i n e w a t e r s at v a r i o u s t i m e s of t h e y e a r .
O n l y 2% of t h e p h o s p h a t e u p t a k e w a s b y o r g a n i s m s r e t a i n e d o n t h e 5 %um s c r e e n , p r e s u m a b l y a l g a e . A l t h o u g h t h e a l g a l a b - s o r p t i o n r a t e s e e m s a r e m a r k a b l y s m a l l p a r t of t h e t o t a l u p t a k e , it is c o n - f i r m e d b y t h e s u m of i n d i v i d u a l s p e c i e s r a t e s o b t a i n e d f r o m a u t o r a d i o g r a p h y . T h e p h y t o p l a n k t o n r a t e f r o m t h e f i l t r a t i o n t e c h n i q u e is 0 . 0 2 4 ~g P 1-I h-l, as c o m - p a r e d w i t h 0 . 0 3 8 to 0 . 0 6 3 ~g P 1-I h-1 f r o m a u t o r a d i o g r a p h y , d e p e n d i n g u p o n t h e v a l u e u s e d for t h e a c i d - s o l u b l e P c o n - t e n t of t h e p h y t o p l a n k t o n c e l l s .
T h e t e c h n i q u e of a u t o r a d i o g r a p h y w a s u s e d to c o m p a r e t h e p h o s p h o r u s u p t a k e r a t e s of i n d i v i d u a l s p e c i e s of p h y t o - p l a n k t o n . K n o e c h e l a n d K a l f f (1976) h a v e c r i t i c a l l y r e v i e w e d t h e u s e of g r a i n - d e n s i t y a u t o r a d i o g r a p h y for c o m p a r i s o n of m e t a b o l i c a c t i v i t y in p l a n k t o n s p e - cies. T h e y s u b m i t t h a t e r r o r s r e s u l t f r o m s e v e r a l s o u r c e s : (I) i n a d e q u a t e c e l l p r e p a r a t i o n ; (2) f a i l u r e to c o n - s i d e r t h e e f f e c t of c e l l s i z e a n d g e o m - e t r y on g r a i n d e n s i t y ; (3) i n a d e q u a t e m e t h o d s of d e t e r m i n i n g g r a i n y i e l d .
In t h i s s t u d y , t h e m e t h o d of c e l l p r e p a r a t i o n w a s n o t d e t r i m e n t a l to t h e c e l l s , as n o a c e t o n e , h a r s h f i x a t i v e s , h e a t , or h i g h c e n t r i f u g e s p e e d s w e r e used. F i x a t i o n w a s c o m p l e t e : F r o t a r g o l s t a i n i n g of t h e g l u t e r a l d e h y d e - f i x e d c e l l s s e v e r a l m o n t h s l a t e r s h o w e d e x c e l - l e n t f i x a t i o n a n d n o d e t e r i o r a t i o n of c e l l o r g a n e l l e s .
75%. T h u s , s i n c e g r a i n c o u n t i n g w a s ex- t e n d e d t h a t far in t h i s s t u d y , t h e e f f i - c i e n c y w a s r e d u c e d v e r y l i t t l e . A l s o , s i n c e o u r c e l l s w e r e w i t h i n a n a r r o w s i z e r a n g e , a n y r e d u c t i o n in e f f i c i e n c y w o u l d b e n e a r l y c o n s t a n t f o r a l l t h e
cells. T h e a u t h o r s a l s o n o t e t h a t s e l f - a b s o r p t i o n of r a d i a t i o n b y the c e l l s m a y c a u s e e r r o r . H o w e v e r , s e l f - a b s o r p t i o n is g r e a t e r in l a r g e c e l l s ; s i n c e m o s t of o u r c e l l s w e r e less t h a n 20 Lum in d i a m - eter, t h i s e f f e c t w o u l d r e d u c e a c t u a l g r a i n c o u n t s b y 15% at m o s t , a c c o r d i n g to e s t i m a t e s b y K n o e c h e l a n d K a l f f (1976).
F o r t h e s m a l l e s t c e l l s , s e l f - a b s o r p t i o n w o u l d r e s u l t in a r e d u c t i o n in g r a i n c o u n t s of o n l y a f e w p e r c e n t .
F i n a l l y , e r r o r is i n t r o d u c e d in e s t i - m a t i n g t h e g r a i n y i e l d of a r a d i o a c t i v e i s o t o p e on a p a r t i c u l a r e m u l s i o n or film.
I d e a l l y , an i n t e r n a l s t a n d a r d i z a t i o n b e - t w e e n g r a i n s a n d d i s i n t e g r a t i o n s h o u l d b e m a d e u s i n g c e l l s w i t h a k n o w n r a d i o - a c t i v i t y . As s t a t e d b e f o r e , no a t t e m p t at t h i s w a s m a d e in t h i s study. H o w e v e r , t h e c o m p a r i s o n b e t w e e n a l g a l s p e c i e s is n o t a f f e c t e d by a n y e r r o r in t h e g r a i n y i e l d , as it is m e r e l y a c o n s t a n t u s e d in c a l c u l a t i o n of a l l u p t a k e r a t e s . T h e s u m of i n d i v i d u a l p h o s p h a t e u p t a k e r a t e s , i.e., t h e t o t a l p h y t o p l a n k t o n u p t a k e rate, m i g h t r e f l e c t an e r r o r in t h e g r a i n y i e l d , b u t the g r a i n y i e l d u s e d
(1.8) w o u l d h a v e to b e u n d e r e s t i m a t e d b y an o r d e r of m a g n i t u d e b e f o r e a s i g n i f i - c a n t d i f f e r e n c e in t h e t o t a l p h y t o p l a n k - t o n u p t a k e r a t e w o u l d b e o b s e r v e d . T h e s o u r c e s of e r r o r w h i c h K n o e c h e l a n d K a l f f l i s t a r e s i g n i f i c a n t o n l y if a u t o - r a d i o g r a p h y is u s e d f o r s t u d y i n g l a r g e p h y t o p l a n k t o n c e l l s w i t h a h i g h e n e r g y i s o t o p e , or if o n e is c o m p a r i n g t h e r e - s u l t s of s e v e r a l d i f f e r e n t a u t o r a d i o g - r a p h y e x p e r i m e n t s in w h i c h t h e r e a r e d i f - f e r e n c e s in t o t a l r a d i o a c t i v i t y .
As n o t e d in t h e r e s u l t s , t h e a u t o - r a d i o g r a m s c o n t a i n e d a n u m b e r of b a c k - g r o u n d g r a i n s w h i c h i n c r e a s e d w i t h time.
A u t o r a d i o g r a m s of t h e f i l t r a t e o b t a i n e d by f i l t e r i n g 3 3 p - l a b e l l e d c e l l s u s p e n - s i o n s t h r o u g h an 8 H/n p o r e - s i z e f i l t e r