Marine Biology 34, 151-162 (1976) 9 by Springer-Verlag 1976
Comparison of Bacterial and Algal Utilization of Orthophosphate in an Estuarine Environment
M. A. Faust 1 and D. L. CorrelF
1 Chesapeake Bay Center for Environmental Studies, Smithsonian Institution; Edgewater, Maryland, USA, and 2 Radiation Biology Laboratory, Smithsonian Institution ; Rockville, Maryland, USA
Abstract
B a c t e r i a l u t i l i z a t i o n of o r t h o p h o s p h a t e in an e s t u a r i n e e n v i r o n m e n t has b e e n d i f f e r - e n t i a t e d f r o m a l g a l u t i l i z a t i o n by u s i n g f l o w - f i l t e r s of 5.0, 1.2 a n d 0 . 4 5 ~ m p o r e - size. E x a m i n a t i o n by l i g h t m i c r o s c o p y s h o w e d t h a t m o s t of the b a c t e r i a l p o p u l a t i o n p a s s e d t h r o u g h a 5.O-~um f i l t e r , w h e r e a s m o s t a l g a e w e r e r e t a i n e d . In all e x p e r i - m e n t s , b a c t e r i a l a n d a l g a l c e l l n u m b e r s a n d b i o m a s s w e r e e s t i m a t e d . P - u p t a k e by al- g a e a n d b a c t e r i a w a s c l o s e l y c o r r e l a t e d w i t h c e l l b i o m a s s . P - u p t a k e by a l g a e w a s h i g h o n l y in the s u m m e r m o n t h s , w h e r e a s P - u p t a k e by b a c t e r i a w a s h i g h t h r o u g h o u t the year. N e i t h e r a l g a l n o r b a c t e r i a l P - u p t a k e , h o w e v e r , w a s c o r r e l a t e d w i t h tem- p e r a t u r e or d i s s o l v e d o r t h o p h o s p h a t e , t o t a l o r g a n i c p h o s p h a t e or t o t a l p h o s p h a t e c o n c e n t r a t i o n s . C e l l b i o m a s s of a l g a e at a g i v e n time h a d a h i g h c o r r e l a t i o n w i t h d i s s o l v e d o r g a n i c p h o s p h a t e l e v e l s in 2 w e e k s p r i o r to s a m p l i n g (r = 0.830) and a low c o r r e l a t i o n in the 2 w e e k s f o l l o w i n g s a m p l i n g (r = 0 . 0 0 0 5 ) . A l g a l cell n u m b e r s h a d a h i g h c o r r e l a t i o n w i t h b a c t e r i a l c e l l n u m b e r s (r = 0 . 9 5 0 ) . T h e b i o m a s s of al- g a e a n d b a c t e r i a a l s o h a d a h i g h c o r r e l a t i o n (r = 0 . 9 0 2 ) . T h e r a t e of P - u p t a k e f r o m the w a t e r by a l g a e a n d b a c t e r i a v a r i e d w i t h s e a s o n a n d w i t h the s p e c i e s c o m p o s i t i o n of the n a t u r a l p o p u l a t i o n .
Introduction
O n e of the m a j o r d i f f i c u l t i e s in e s t a b - l i s h i n g t h e p a t h of p h o s p h o r u s c y c l i n g in the e s t u a r i n e e n v i r o n m e n t is the l a c k of i n f o r m a t i o n a b o u t the r o l e of v a r i o u s m i c r o o r g a n i s m s in this p r o c e s s . T h e r o l e of a l g a e in p h o s p h o r u s u p t a k e (P-uptake) w a s e m p h a s i z e d b y F u h s a n d C a n e l l i (1970) a n d T a f t (1974), w h o f o u n d m a r k e d d i f - f e r e n c e s in P - u p t a k e by a l g a l s p e c i e s . S o m e i n v e s t i g a t o r s t h o u g h t t h a t b o t h al- g a e a n d b a c t e r i a w e r e i m p o r t a n t (Harris, 1957; Lean, 1973), a n d o t h e r s l o o k e d u p o n b a c t e r i a as the m a j o r m i c r o o r g a n - isms r e s p o n s i b l e for p r i m a r y a s s i m i l a - t i o n of this e l e m e n t (Johannes, 1964, 1965; C o r r e l l e t al., 1975). T w o i n v e s t i - g a t o r s e x c l u d e d d i r e c t P - u p t a k e by zoo- p l a n k t o n (Harris, 1957), i n c l u d i n g fil- t e r - f e e d i n g p r o t o z o a n s (Hutner a n d P r o v a s o l i , 1955).
T h e lack of c o n c e n s u s s t e m s f r o m the f a c t t h a t m o s t i n v e s t i g a t o r s h a v e n o t a t t e m p t e d to d i s t i n g u i s h b e t w e e n a l g a l a n d b a c t e r i a l P - u p t a k e . T h e f r a c t i o n a - t i o n p r o c e d u r e e m p l o y e d w a s e i t h e r c o a r s e f i l t r a t i o n , w h i c h a l l o w e d b o t h
t y p e s of o r g a n i s m to f l o w t h r o u g h , or v e r y s m a l l p o r e - s i z e f i l t e r s , w h i c h re- t a i n e d b o t h o r g a n i s m s (Rigler, 1956;
H a r r i s , 1957; W a t t a n d Hayes, 1963; J o - h a n n e s , 1964, 1965; Lean, 1973; Taft, 1974).
In this study, our o b j e c t i v e was to u s e m o r e c o n t r o l l e d f i l t r a t i o n and m i c r o -
s c o p y to d i s t i n g u i s h b e t w e e n b a c t e r i a l a n d a l g a l u t i l i z a t i o n of i n o r g a n i c p h o s - p h a t e in the e s t u a r i n e h a b i t a t . S u c h a f i l t r a t i o n t e c h n i q u e has b e e n e m p l o y e d p r e v i o u s l y b y C o r r e l l et al. (1975) for p h o s p h o r u s and by D e r e n b a c h a n d Le B.
W i l l i a m s (1974) for c a r b o n u t i l i z a t i o n . In a d d i t i o n to s u c h size f r a c t i o n a - t i o n s of n a t u r a l p o p u l a t i o n s of m i c r o - o r g a n i s m s , t h e r e a r e o t h e r t y p e s of e v i - d e n c e t h a t b a c t e r i a m a y be t h e p r i n c i p a l a s s i m i l a t o r s of p h o s p h o r u s . P - u p t a k e w a s s i m i l a r in l i g h t a n d d a r k b o t t l e s (Taft,
1974; C o r r e l l et al., 1975), i n d i c a t i n g t h a t o r t h o p h o s p h a t e w a s t a k e n u p by h e t e r o t r o p h i c r a t h e r t h a n by a u t o t r o p h i c cells. T h i s f a c t s t r o n g l y i n d i c a t e s b a c - t e r i a l i n v o l v e m e n t , b u t c a n n o t be ac- c e p t e d as p o s i t i v e proof. A l t h o u g h a l g a l P - u p t a k e is g e n e r a l l y r e g a r d e d to be
152 M.A. Faust and D.L. Correll: Bacterial and Algal Use of Orthophosphate
p h o t o s y n t h e t i c in m a n y s p e c i e s , a l g a e a l s o c a n t a k e u p P in t h e d a r k u n d e r s o m e c o n d i t i o n s (Ketchum, 1939; K u e n z l e r a n d K e t c h u m , 1962). S e v e r a l a l g a e a r e e i t h e r o b l i g a t e h e t e r o t r o p h s (Faust a n d Gantt, 1973) or f a c u l t a t i v e h e t e r o t r o p h s if t h e n e c e s s a r y c a r b o n s o u r c e is a v a i l - a b l e ( H u t n e r a n d P r o v a s o l i , 1951, 1955), a n d i n o r g a n i c P - u p t a k e of a l g a e is g e n - e r a l l y s t i m u l a t e d by, b u t n o t d e p e n d e n t on, l i g h t (Healey, 1973).
It a p p e a r s that, u n d e r c e r t a i n c o n d i - tions, b a c t e r i a m a y be m o r e i m p o r t a n t t h a n a l g a e in P - u p t a k e . B a c t e r i a - a c c e l e r a t e d p h o s p h a t e r e t u r n f r o m t h e s e d i m e n t to l a k e w a t e r (Hayes a n d
P h i l l i p s , 1958) t h u s a p p e a r e d to be r e -
by I m of line. B o t t l e s w e r e d i p p e d in b l a c k e p o x y p a i n t to d a r k e n them. T h e i n i t i a l s a m p l e of p l a n k t o n w a s t a k e n f r o m a d e p t h of I m w i t h a p e r i s t a l t i c p u m p ( C o r r e l l et al., 1 9 7 5 ) . S u b s a m p l e s of t h i s w a t e r w e r e a l s o t a k e n for c h e m - i c a l a n a l y s i s . 3 2 p - o r t h o p h o s p h a t e
( c a r r i e r - f r e e ) w a s t h e n a d d e d to t h e b o t - t l e of p l a n k t o n (2 U C / 2 5 0 ml) a n d t h e c o n t e n t s w e r e m i x e d t h o r o u g h l y , t h e t w o s y r i n g e n e e d l e s c a p p e d , a n d t h e b o t t l e i n c u b a t e d at a d e p t h of I m. A t a p p r o - p r i a t e t i m e i n t e r v a l s (2, 4, 6, 10, 15, 20, 25, 30, 40, 50, 60 min) t h e b o t t l e w a s r e c o v e r e d , a n d t h e s a m p l e w a s m i x e d b y s w i r l i n g . T h e n e e d l e c a p s w e r e re- m o v e d , a n d a I O - m l a l i q u o t w a s o b t a i n e d s p o n s i b l e f o r the r a p i d t u r n o v e r of p h o s - t h r o u g h t h e l o n g n e e d l e w i t h a 2 0 - m l p h o r u s (Rigler, 1956). In h i g h P m e d i a ,
t h r e e s t r a i n s of s o i l b a c t e r i a c o m p e t e d s u c c e s s f u l l y w i t h a l g a e b u t f a i l e d to do so at l o w P l e v e l s (I-3 ~ g - a t m/l; F u h s et al., 1972). E v e n t h o u g h m o s t l a b o r a t o - ry e x p e r i m e n t s w e r e c a r e f u l l y d e s i g n e d , J o h a n n e s (1964) w a r n e d t h a t l a b o r a t o r y e x p e r i m e n t s in w h i c h b a c t e r i a c o m p e t e d
g l a s s s y r i n g e .
A b o u t 10 m l of a i r w a s p u l l e d i n t o t h e s y r i n g e . T h e s a m p l e w a s t h e n p a s s e d t h r o u g h a s e r i e s of f i l t e r s in 2 5 - m m d i a m e t e r S w i n e x f i l t e r h o l d e r s (Milli- p o r e C o r p . , B e d f o r d , M a s s . ) . T h e f i r s t h o l d e r c o n t a i n e d N i t e x s c r e e n i n g w i t h a p o r e s i z e of 5 ~un (Tobler, E r n s t &
for p h o s p h o r u s w e r e u n n a t u r a l , s i n c e b a c - T r a b e r , Inc., E l m s f o r d , N . Y . ) , w h i c h w a s t e r i a l d e n s i t i e s w e r e s e v e r a l o r d e r s of c o a t e d a r o u n d t h e e d g e s w i t h s i l i c o n e m a g n i t u d e g r e a t e r t h a n t h o s e a t t a i n e d in
the sea.
It a p p e a r e d to us t h a t t h e r o l e of b a c t e r i a in P - u p t a k e in e s t u a r i e s w a s n o t clear, a n d t h a t f u r t h e r s t u d y o f t h i s p h e n o m e n o n w a s w a r r a n t e d .
Materials and Methods Sampling Site
M o n t h l y s a m p l i n g w a s c a r r i e d o u t in t h e m a i n b a s i n of t h e R h o d e R i v e r s u b e s t u a r y , an a r m of C h e s a p e a k e Bay, f r o m M a r c h 1973 t h r o u g h F e b r u a r y 1974. T h e s a m p l i n g s i t e in t h e s u b e s t u a r y ( L a t i t u d e 38 ~ 53' 02" N; L o n g i t u d e 76 ~ 32' 00" W) h a s a m e a n d e p t h of 4 m. T h e s a l i n i t y v a r i e d f r o m 4 . 7 ~ in t h e s p r i n g to 12~ in t h e f a l l of 1973. F r e s h w a t e r e n t e r s t h e s t u d y a r e a f r o m t h e M u d d y C r e e k w a t e r - shed, w h i c h has t h e f o l l o w i n g l a n d uses:
f o r e s t , b r u s h l a n d , m a r s h e s , n o n p a s t u r e g r a s s l a n d , p a s t u r e , c r o p l a n d . H u m a n p o p - u l a t i o n s a r e l o w ( O . 3 / h e c t a r e ) in t h i s w a t e r s h e d . S a l t w a t e r e n t e r s t h e s y s t e m f r o m the C h e s a p e a k e Bay.
Plankton Bottle Experiments
r u b b e r to f a c i l i t a t e s e a l i n g . T h e sec- o n d a n d t h i r d h o l d e r s c o n t a i n e d 1.2 a n d 0 . 4 5 ~ m p o r e - s i z e m e m b r a n e f i l t e r s (Mil- l i p o r e C o r p . , B e d f o r d , M a s s . ) , r e s p e c - t i v e l y . E n o u g h a i r w a s f o r c e d t h r o u g h a f t e r e a c h s a m p l e to r e m o v e a n y c h a n c e of e x c e s s m o i s t u r e o n t h e f i l t e r s . E a c h f i l t e r a n d 1-ml a l i q u o t of t h e f i n a l f i l t r a t e w e r e c o u n t e d . M e t a b o l i c a l l y - i n h i b i t e d c o n t r o l s w e r e r u n by d i s s o l v - ing i o d o a c e t i c a c i d (IAA), to a f i n a l c o n c e n t r a t i o n of 0 . 0 5 M, 5 m i n p r i o r to a d d i n g t h e 3 2 p - o r t h o p h o s p h a t e .
Radioisotope Counting and Uptake Calculations S a m p l e s w e r e c o u n t e d in a P a c k a r d , M o d e l 3320, l i q u i d s c i n t i l l a t i o n s p e c t r o m e t e r as d e s c r i b e d p r e v i o u s l y ( C o r r e ! l et al.,
1975). U p t a k e of c o u n t s / m i n (cpm) on v a r i o u s f i l t e r s w a s p l o t t e d a g a i n s t time.
A n i n t e r c e p t a n d s l o p e w e r e c a l c u l a t e d f r o m the u p t a k e d a t a p o i n t s in t h e t i m e r a n g e in w h i c h u p t a k e a p p e a r e d to be a p p r o x i m a t e l y l i n e a r , b y a l i n e a r l e a s t - s q u a r e s r e g r e s s i o n c a l c u l a t i o n . T h i s t i m e i n t e r v a l u s u a l l y v a r i e d f r o m 6 to 30 min. U p t a k e a f t e r t h i s p o i n t w a s u s u a l l y n o t l i n e a r , a n d the r e s u l t s w e r e c o n s i d e r e d at l e a s t p a r t i a l l y d u e to r e - c y c l i n g a n d w e r e n o t u s e d in t h e c a l c u - C l e a r g l a s s c y l i n d r i c a l b o t t l e s of 2 5 O m i l a t i o n s . T h e i n i t i a l s p e c i f i c a c t i v i t y c a p a c i t y w e r e e q u i p p e d w i t h r u b b e r s t o p - of P w a s c a l c u l a t e d f r o m t h e o r t h o p h o s - p e r s t h r o u g h w h i c h t w o 1 6 - g a u g e s y r i n g e p h a t e c o n c e n t r a t i o n o f t h e w a t e r a f t e r n e e d l e s h a d b e e n p l a c e d . O n e n e e d l e w a s f i l t r a t i o n t h r o u g h a O . 4 5 - ~ m p o r e - s i z e s h o r t a n d o n e e x t e n d e d w e l l i n t o t h e b o t - f i l t e r , a n d t h e c p m / m l of w h o l e w a t e r . tle. T h e b o t t l e w a s a t t a c h e d to a f l o a t T h e p a r t i c u l a t e u p t a k e , e x p r e s s e d as
M.A. Faust and D.L. Correll: Bacterial and Algal Use of Orthophosphate 153
~g P / h / l , w a s t h e n c a l c u l a t e d f r o m the s l o p e a n d the s p e c i f i c a c t i v i t y of t h e a v a i l a b l e P. C e l l s r e t a i n e d on t h e 0.5-~un p o r e - s i z e f i l t e r w e r e c o n s i d e r e d for u p t a k e c a l c u l a t i o n s to be a l g a l , a n d t h o s e r e t a i n e d o n t h e 1.2 a n d 0 . 4 5 - b m f i l t e r s to be b a c t e r i a l . R a t e s of u p t a k e i n t o s p e c i f i c s i z e c l a s s e s w e r e c o n v e r t e d to p e r c e n t a g e s . T o t a l P - u p t a k e r a t e s w e r e a l s o c a l c u l a t e d f r o m t h e r a t e o f d i s a p p e a r a n c e of r a d i o a c t i v - i t y f r o m t h e O . 4 5 - ~ m f i l t r a t e ( C o r r e l l et al., 1975). T o t a l u p t a k e r a t e s (from d i s a p p e a r a n c e of r a d i o a c t i v i t y in f i l - trate) w e r e t h e n m u l t i p l i e d b y t h e p e r - c e n t a g e s f o u n d in e a c h c l a s s of p a r t i c - u l a t e s to o b t a i n b e t t e r e s t i m a t e s of a b - s o l u t e r a t e s of u p t a k e b y t h a t c l a s s .
C e l l s u s p e n s i o n s w e r e a p p l i e d to c l e a n m i c r o s c o p e s l i d e s . B a c t e r i a m a d e v i s i b l e b y t h e g r a m - s t a i n i n g t e c h n i q u e w e r e m e a - s u r e d a n d c o u n t e d o n s l i d e s . R o d - s h a p e d f o r m s a n d c o c c i w e r e c o u n t e d s e p a r a t e l y , a n d the n u m b e r of c e l l s in d i f f e r e n t s i z e c l a s s i f i c a t i o n s w a s r e g i s t e r e d . A l - gal c e l l s w e r e c o u n t e d s i m i l a r l y , b u t w i t h o u t s t a i n i n g .
D i f f e r e n t q u a n t i t i e s of c e l l s u s p e n - s i o n w e r e u s e d to d e t e r m i n e b a c t e r i a l a n d a l g a l c o u n t s . F o r t h e b a c t e r i a l c o u n t , 0.O1 m l of the f i x e d 3-ml s u s p e n - s i o n w a s used, a n d t h i s v o l u m e w a s
s p r e a d o n a 4 0 0 - m m 2 area. T h e n u m b e r of b a c t e r i a w e r e c o u n t e d on 100 m i c r o s c o p i c f i e l d s of e a c h s a m p l e at 1000 X m a g n i f i - c a t i o n . C e l l n u m b e r s of a l g a e w e r e e s t i - U p t a k e w a s a l s o e x p r e s s e d as 5 g P / m m 3 m a t e d u s i n g s e v e r a l O . 0 5 - m l a l i q u o t s h-] of a l g a l b i o m a s s or of b a c t e r i a l b i o - f r o m t h e 3 m l f i n a l s u s p e n s i o n s p r e a d
m a s s . u n d e r a c o v e r s l i p of k n o w n area. T h e
n u m b e r of a l g a e w e r e c o u n t e d o n 5 0 0 to 800 m i c r o s c o p i c f i e l d s of e a c h s a m p l e .
water Chemistry F r o m t h i s k n o w n a r e a a n d k n o w n v o l u m e ,
c a l c u l a t i o n s of t h e n u m b e r of c e l l s p e r W a t e r s a m p l e s for p h o s p h o r u s a n a l y s i s u n i t v o l u m e w e r e m a d e for e a c h s p e c i e s . w e r e t a k e n as d e s c r i b e d u n d e r p l a n k t o n A t t h e s a m e time, t h e s p e c i e s w e r e ex- b o t t l e e x p e r i m e n t s . A n a l y s i s for o r t h o - a m i n e d a n d i d e n t i f i e d . F r o m t h e m e a n p h o s p h a t e w a s c o n d u c t e d c o l o r i m e t r i c a l l y d i a m e t e r s a n d l e n g t h s of e a c h s i z e b y t e s t i n g t h e s a m p l e s ' r e a c 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 f t e r p o t a s s i u m p e r - s u l f a t e d i g e s t i o n ( A m e r i c a n P u b l i c H e a l t h A s s o c i a t i o n , 1971), s a m p l e s w e r e a n a l y z e d for t o t a l p h o s p h o r u s b y t h e s a m e p r o c e d u r e . A n a l y s i s w a s c o n d u c t e d b o t h b e f o r e a n d a f t e r f i l t r a t i o n t h r o u g h 0 . 4 5 - ~ m p o r e - s i z e f i l t e r s . T h e t o t a l o r - g a n i c p h o s p h a t e f r a c t i o n w a s c a l c u l a t e d as t h e t o t a l P m i n u s o r t h o p h o s p h a t e in u n f i l t e r e d w a t e r .
T h e r e s u l t s of w a t e r a n a l y s e s , m a d e at w e e k l y i n t e r v a l s b e t w e e n p l a n k t o n ex- p e r i m e n t s , w e r e a l s o u s e d in t h e a n a l - y s i s of c o n d i t i o n s p r i o r to a n d s u b s e - q u e n t to p l a n k t o n e x p e r i m e n t s .
g r o u p a n d t h e n u m b e r of m i c r o o r g a n i s m s p r e s e n t , t h e b i o m a s s w a s c a l c u l a t e d as d e s c r i b e d by R o d i n a (1972). T a k i n g t h e d i l u t i o n i n t o a c c o u n t , the b i o m a s s p e r u n i t of w a t e r w a s t h e n d e t e r m i n e d .
Electron M i c r o s c o p y
W a t e r s a m p l e s w e r e t a k e n as d e s c r i b e d u n d e r p l a n k t o n b o t t l e e x p e r i m e n t s . T h e s a m p l e s w e r e f i x e d w i t h 4% g l u t e r a l d e - h y d e in O . 1 M p h o s p h a t e b u f f e r , pH 7.0, f i n a l c o n c e n t r a t i o n . F i x e d c e l l s w e r e d e h y d r a t e d in a l c o h o l a n d e m b e d d e d in e p o n (Gantt et el., 1971). T h i n s e c t i o n s w e r e e x a m i n e d in a P h i l i p s , m o d e l 300, e l e c t r o n m i c r o s c o p e .
Biomass D e t e r m i n a t i o n s
C e l l n u m b e r s of b a c t e r i a (Rodina, 1972) a n d a l g a e ( C a m p b e l l , 1973) w e r e d e t e r - m i n e d b y d i r e c t - c o u n t p r o c e d u r e s on s a m - p l e s f i x e d to a f i n a l c o n c e n t r a t i o n of 0 . 4 % g l u t a r a l d e h y d e a n d O . 0 1 M s o d i u m p h o s p h a t e b u f f e r , pH 7.0. L i v e m a t e r i a l w a s a l s o c o l l e c t e d f o r t h e i d e n t i f i c a - t i o n of a l g a e . F i x e d s a m p l e s w e r e t h e n p r o c e s s e d in t h e l a b o r a t o r y . F i r s t t h e y w e r e c e n t r i f u g e d for 20 m i n at 2 0 0 0 x ~.
T h e p e l l e t w a s r e s u s p e n d e d a n d w a s h e d two m o r e t i m e s w i t h d i s t i l l e d w a t e r a n d r e s u s p e n d e d in 3 m l f i n a l v o l u m e . E a c h
Results
A s s o c i a t i o n of Microbial Cells with Particulates
T h e a s s o c i a t i o n of a l g a e a n d b a c t e r i a w i t h p a r t i c u l a t e s , as f o u n d in e s t u a r i n e - w a t e r s a m p l e s , is i l l u s t r a t e d in t r a n s - m i s s i o n e l e c t r o n m i c r o g r a p h s (Fig. I).
S o m e c e l l s w e r e f r e e of p a r t i c u l a t e s , b u t t h e m a j o r i t y w e r e s u r r o u n d e d b y p a r - t i c u l a t e s a d h e r i n g to s p e c i a l i z e d s t r u c - t u r e s of t h e m i c r o b i a l c e l l s u r f a c e
(Fig. la). T h e a d h e s i v e m a t e r i a l on t h e s e c e l l s v a r i e d in s t r u c t u r e . S o m e s a m p l e w a s t h e n s o n i c a t e d f o r 5 to 10 sec c o n s i s t e d of f i b r o u s s t r a n d s of v a r i o u s to b r e a k u p a n y c l u m p s of c e l l s (Model l e n g t h s f o r m i n g a t h i n l a y e r s i m i l a r to W 140C, H e a t S y s t e m s Co., at 60 W output), t h a t s e e n o n a b l u e - g r e e n a l g a l c e l l s u r -
154 M.A. F a u s t and D.L. Correll: B a c t e r i a l and A l g a l Use of O r t h o p h o s p h a t e
Fig. i. M i c r o o r g a n i s m s a s s o c i a t e d w i t h clay p a r t i c u l a t e s in w a t e r samples. S e c t i o n s t y p i c a l of cell- clay p a r t i c u l a t e a s s o c i a t i o n s , s h o w i n g 2 G r a m - n e g a t i v e b a c t e r i a (a) x 60100, 2 b l u e - g r e e n algae (b) x 34600, a n d a c y s t - l i k e o r g a n i s m (c) x 27200. The c e l l s are s u r r o u n d e d by clay p a r t i c l e s of v a r i o u s s h a p e s and sizes as d a r k - l a y e r e d or flaky a m o r p h o u s s t r u c t u r e s . Some c l a y p a r t i c l e s are in c l o s e as- s o c i a t i o n w i t h cell surface, c o n n e c t e d by f i b r o u s p o l y m e r i c strands. F i b r o u s s t r a n d s of v a r i o u s lengths form a thin l a y e r on the b l u e - g r e e n a l g a l cell s u r f a c e (b), s h o r t e r fibers are p r o t r u d i n g on the s u r f a c e of the c y s t - l i k e o r g a n i s m (c), a n d a m e s h of f i b r o u s s t r a n d s s u r r o u n d s the G r a m - n e g a t i v e b a c t e r i a (a). (Fibrous s t r a n d s and f i b e r s are i n d i c a t e d by arrows.)
M.A. Faust and D.L. Correll: Bacterial and Algal Use of Orthophosphate 155
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F M o n t h st i m e i n t e r c e p t o f a l i n e a r l e a s t - s q u a r e s r e g r e s s i o n i n d i c a t e s t h e a m o u n t of p h o s - p h a t e b o u n d to p a r t i c u l a t e s ( C o r r e l l et al., 1975). T h i s i n t e r c e p t w a s a b o u t e q u a l f o r light, dark, a n d i n h i b i t e d ex- p e r i m e n t s , i n d i c a t i n g t h a t t h i s d e v i a - t i o n f r o m a z e r o i n t e r c e p t w a s d u e to a d s o r p t i o n to p a r t i c u l a t e s r a t h e r t h a n b i o l o g i c a l u p t a k e .
A v a i l a b i l i t y o f P h o s p h o r u s in the W a t e r
T h e t o t a l p h o s p h o r u s r a p i d l y i n c r e a s e d d u r i n g t h e m o n t h s of May, J u n e a n d July, r e m a i n i n g h i g h u n t i l S e p t e m b e r , a n d r a p i d l y d e c r e a s e d to l o w l e v e l s t h e r e - a f t e r (Fig. 2a). T h e v a r i o u s i n o r g a n i c - P f r a c t i o n s a l l f o l l o w e d t h e s a m e p a t t e r n , b u t at l o w e r m a g n i t u d e s . T h e t o t a l o r - g a n i c p h o s p h o r u s p a t t e r n w a s s i g n i f i c a n t - ly d i f f e r e n t . It w a s h i g h d u r i n g two al- g a l b l o o m s in M a y a n d J u l y , a n d s l i g h t l y i n c r e a s e d in N o v e m b e r . C o m p a r i s o n of w a t e r t e m p e r a t u r e (Eig. 2b) a n d t h e v a r - i o u s f r a c t i o n s of i n o r g a n i c P i n d i c a t e d t h a t t h e r e w a s s o m e c o r r e l a t i o n b e t w e e n t h e i n o r g a n i c - P c o n c e n t r a t i o n s of t h e w a t e r a n d w a t e r t e m p e r a t u r e s (for the d i s s o l v e d o r t h o - P , r = 0 . 5 6 0 ; for p a r - t i c u l a t e o r t h o - P , r = 0 . 7 5 0 ) . In c o n - trast, t h e c o r r e l a t i o n b e t w e e n t e m p e r a - t u r e a n d t o t a l o r g a n i c - P in the w a t e r w a s l o w (r = 0 . 1 6 5 ) .
3 2 p - U p t a k e i n t o P a r t i c u l a t e s Fig. 2. Seasonal changes in phosphorus concentra-
tions (a) and water temperature (b) at Rhode 32p i n c o r p o r a t i o n i n t o p a r t i c u l a t e s re- River sampling station at 1-m depth. Open cir- r a i n e d o n 5 . O - ~ m p o r e - s i z e f i l t e r s w a s cles and solid line: total-P; crosses and solid I to 6% of t h e t o t a l , e x c e p t in J u n e a n d line: total organic-P; crosses and broken l i n e : J u l y (Table 1). 32p i n c o r p o r a t i o n i n t o total ortho-P; open circles and dashed line: dis- p a r t i c u l a t e s o v e r 5 ~ m s i z e w a s a l w a y s solved ortho-P; closed circles and solid line: l e s s t h a n f o r s m a l l e r - s i z e d p a r t i c u l a t e s . particulate orthophosphate D i r e c t m i c r o s c o p i c o b s e r v a t i o n s of the
1.2 a n d 0 . 4 5 - ~ m f i l t e r s i n d i c a t e d t h a t c h l o r o p h y l l - c o n t a i n i n g a l g a l c e l l s w e r e r e t a i n e d o n l y o c c a s i o n a l l y o n 1.2 a n d f a c e (arrows, Fig. Ib); s o m e w a s in t h e 0 . 4 5 - ~ m p o r e - s i z e f i l t e r s . T h i s l e d to f o r m of s h o r t d e n s e f i b e r s s u c h as t h o s e t h e c o n c l u s i o n t h a t b a c t e r i a w e r e t h e s h o w n on t h e s u r f a c e of the c y s t - l i k e o n l y s i g n i f i c a n t b i o l o g i c a l c o m p o n e n t re- o r g a n i s m i l l u s t r a t e d in Fig. Ib, c. P a r - t a i n e d on t h e s e t w o f i l t e r s . T h e p r o p o r - t i c u l a t e s a l s o v a r i e d in s h a p e a n d size, t i o n s of 32p r e t a i n e d o n v a r i o u s f i l t e r s a n d s o m e w e r e c l o s e l y a s s o c i a t e d w i t h c h a n g e d l i t t l e d u r i n g t h e year, i n d i c a t - t h e c e l l s u r f a c e , i n g t h a t w h e n c h a n g e s o c c u r r e d in t h e
W h e n t h e r e t e n t i o n of r a d i o a c t i v i t y b i o t a i n f l u e n c i n g t h e t o t a l u p t a k e , the b y f i l t e r s is u s e d to m e a s u r e p h o s p h o r u s n u m b e r s of b o t h a l g a e a n d b a c t e r i a a s s i m i l a t i o n , v a r i o u s n o n - l i v i n g p a r - c h a n g e d in a s i m i l a r m a n n e r .
t i c l e s , w h i c h m a y a d s o r b 3 2 p - c o n t a i n i n g T h e t o t a l a m o u n t of P i n c o r p o r a t e d c o m p o u n d s , a r e a l s o f i l t e r e d out, a n d i n t o p a r t i c u l a t e s (~g P / I h-l) v a r i e d t h e a d s o r p t i o n b y t h e n o n - l i v i n g p a r t i c u - w i t h s a m p l i n g t i m e (Table I). T o t a l f a t e s h a s to be e s t i m a t e d . T h e a d s o r p - P - u p t a k e w a s t h e h i g h e s t (480 to 500 t i o n of 32p to p a r t i c u l a t e s w a s m e a s u r e d ~ g P / I h-]) in M a y d u r i n g a P r o r o c e n t r u m
~ a d d i n g I A A to t h e s a m p l e 5 m i n b e f o r e m a r i a e - l e b o u r i a e b l o o m ; n e x t h i g h e s t P w a s a d d e d . T h i s c o m p o u n d e f f e c t i v e l y (39 to 42 ~ g P / I h -I) in N o v e m b e r , d u r - i n h i b i t s b i o l o g i c a l u p t a k e , a n d t h e z e r o ing a K a t o d i n i u m r o t u n d a t u m b l o o m , a n d t h e
156 M.A. Faust and D.L. Correll: Bacterial and Algal Use of Orthophosphate
Table i. Seasonal changes in P-uptake by plankton in the Rhode River at l-m depth
Date Treat- Apparent P-uptake into (]973- m e n t particles by accumula-
1974) tion into particles
>5 ~m <5 ~m
(~gP/i/h) % (~gP/1/h) %
(i) (2) (3) (4) (5) (6)
Total P-uptake into P-uptake into particles particles by disap- (Columns 4 & 6 times 7;
pearance from solu- MgP/i/h)
tion (~gP/i/h) >5 ~m <5 ~m
(7) (8) (9)
Mar. 28 L a 0.01 0.4 2.2
D b 0.01 0.3 3.1
May 16 L 2. iO 5.0 39.O
D 2.70 6.O 42 .O
June 14 L 0.73 8.8 7.6
D 0.46 15.4 2.~
July 9 L 1.50 41.9 2.1
D 0.28 7.0 3.7
Aug. 7 L 0.06 2.9 2.0
D O.O4 2.3 1.7
Sept. 6 L 0.06 3.2 1.8
D O.01 0.7 i .5
Oct. 3 L 0.08 3.8 2.0
D O.O2 O.7 2.7
Oct. 31 L 0.02 1.6 1.2
D 0.02 1.2 1.7
Nov. 26 L O.46 3.0 15.O
D 0.27 1.5 18.O
Dec. 18 L 0.03 3.2 0.9
D O.O2 2.8 O.7
Jan. 29 L O.O1 1.6 0.6
D 0.01 2.0 0.5
Feb. 28 L 0.01 1.2 0.8
D O.01 i .4 O.7
99.6 4.4 0.02 4.4
99.7 4.7 O.O1 4.7
95.0 500.0 25.00 470.0
94 .O 480.0 29.00 450.0
91.2 I0.0 0.80 9.1
84.6 10.O 1.50 8.4
58.1 8.5 3.60 4.9
93.0 3.2 0.22 3.0
97. i 15.O 0.43 15.O
97.7 22.0 0.50 21.O
96.8 7.9 0.25 7.6
99.3 26.0 0.17 26.0
96.2 5.9 0.22 5.7
99.3 5.3 0.03 5.3
98.4 2.5 0.04 2.4
98.8 2.1 0.02 2.1
97.0 42.0 i .30 41.O
98.5 39.0 0.58 38.0
96.8 i .6 0.05 I .5
97.2 0.6 0.02 0.6
98.4 0.6 O.01 0.6
98.O 0.6 0.01 0.5
98.8 i .2 0.01 I .2
98.6 2.0 0.02 2.0
a L ~ l i g h t bottle experiments.
b D:dark bottle experiments.
l o w e s t d u r i n g t h e w i n t e r m o n t h s . O b - s e r v e d t o t a l P - u p t a k e r a t e s c a l c u l a t e d b y 3 2 p d i s a p p e a r a n c e f r o m t h e w a t e r v a r -
i e d b y t h r e e o r d e r s o f m a g n i t u d e . P - u p t a k e w a s a l s o e x p r e s s e d p e r b i o m a s s o f a l g a e a n d o f b a c t e r i a (~g P / m m 3 h -I) f o r s a m p l e s a t v a r i o u s t i m e s o f t h e y e a r
( F i g s . 3, 4). H i g h c o r r e l a t i o n w a s f o u n d b e t w e e n P - u p t a k e b y p a r t i c u l a t e s l a r g e r t h a n 5 fun i n s i z e a n d a l g a l b i o m a s s
( F i g . 3). A l i n e a r l e a s t - s q u a r e s r e g r e s - s i o n c a l c u l a t i o n g a v e t h e e q u a t i o n :
~ g P - u p t a k e h -I = 0 . 3 4 + ( O . O 3 3 ) ( m m 3 o f a l g a l b i o m a s s / l ) . T h e c o r r e l a t i o n c o e f - f i c i e n t w a s 0 . 9 9 . T h e m e a n u p t a k e
(ng P / m m 3 h -I) i n t o t h i s s i z e c l a s s w a s O . 1 4 • 0 . 2 5 i n t h e d a r k o r 0 . 1 7 • 0 . 1 8 i n t h e l i g h t . A h i g h c o r r e l a t i o n w a s a l s o f o u n d b e t w e e n P - u p t a k e b y s m a l l e r p a r t i c u l a t e s a n d b a c t e r i a l b i o m a s s ( F i g .
4). A l i n e a r l e a s t - s q u a r e s r e g r e s s i o n c a l c u l a t i o n g a v e t h e e q u a t i o n : ~ g P -
u p t a k e / l h -I = 4 9 + ( 2 7 ) ( m m 3 o f b a c t e r i a l b i o m a s s / l ) . T h e c o r r e l a t i o n c o e f f i c i e n t w a s 0 . 8 8 0 . T h e m e a n P - u p t a k e (ug P / m m 3 h - l ) i n t o t h i s s i z e c l a s s w a s 6 . 4 • 8 . 2 i n t h e l i g h t a n d 6 . 8 • 7 . 9 i n t h e d a r k . A l t h o u g h h i g h c o r r e l a t i o n e x i s t e d b e ~ t w e e n t o t a l P - u p t a k e p e r c e l l b i o m a s s
( F i g s . 3, 4), t h e r o l e o f a l g a e a n d b a c - t e r i a i n P - u p t a k e v a r i e d w i t h t h e n a t u - r a l p o p u l a t i o n . A l g a l a n d b a c t e r i a l P - u p t a k e w a s h i g h i n M a y , J u n e a n d J u l y w h e n P. m a r i a e - l e b o u r i a e , n a n n o p l a n k t o n a n d G y m n o d i n i u m s p e c i e s w e r e d o m i n a n t . I n c o m p a r i s o n , K a t o d i n i u m s p e c i e s w a s d o m i n a n t i n N o v e m b e r , w h e n h i g h P - u p t a k e w a s d u e t o t h e b a c t e r i a .
P - u p t a k e o r P l e v e l s i n t h e w a t e r s h o w e d v e r y l i t t l e c o r r e l a t i o n w i t h w a - t e r t e m p e r a t u r e . F o r P - u p t a k e i n t o c e l l s l a r g e r t h a n 5 ~ m , t h e f o l l o w i n g c o r r e l a - t i o n c o e f f i c i e n t s w e r e f o u n d : t o t a l P, 0 . 0 4 8 ; d i s s o l v e d o r t h o - P , 0 . 0 2 9 ; t o t a l
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log10 Biomass of aigae m m 3 / t
Fig. 3. C o r r e l a t i o n b e t w e e n P - u p t a k e i n t o p a r - t i c l e s >5 p m a n d a l g a l b i o m a s s . C r o s s e s s h o w l i g h t - a n d c l o s e d c i r c l e s d a r k - b o t t l e e x p e r i - m e n t s . P: M a y P r o r o c e n t r u m m a r i a e - l e b o u r i a e b l o o m ; G: J u l y G y m n o d i n i u m n e l s o n i i bloom; N:
J u n e n a n n o p l a n k t o n b l o o m ; K: N o v e m b e r K a t o d i n i u m r o t u n d a t u m b l o o m . A l i n e a r l e a s t - s q u a r e s r e g r e s - s i o n c a l c u l a t i o n g a v e f o r t h e l i g h t (L) d a t a y = 0 . 5 0 9 + 0 . 0 3 0 6 x (r = 0.984) a n d f o r t h e d a r k d a t a y = O . 1 8 4 + 0 . 0 3 5 7 x (r = 0 . 9 9 7 ) . L o g - a r i t h m s o f t h e v a l u e s w e r e u s e d f o r c o n v e n i e n c e in p r e p a r i n g t h e f i g u r e , b u t n o t in t h e s t a t i s - t i c a l a n a l y s e s
x P - L 9
X
• x x
x K - L
" K - D
X
x N - L x o N - D
x G - L
" G - D
.0
I I I
0 .1.0 + 2 0
Loglo Biomass of b a c t e r i a m m 3 / l
Fig. 4. C o r r e l a t i o n b e t w e e n P - u p t a k e i n t o p a r - t i c l e s <5 p m a n d b a c t e r i a l b i o m a s s . S y m b o l s as in Fig. 3. A l i n e a r l e a s t - s q u a r e s r e g r e s s i o n c a l c u l a t i o n g a v e Y = 4 9 . 1 + 2 7 . 2 2 X (r = 0 . 8 7 9 ) . L o g a r i t h m s o f t h e v a l u e s w e r e u s e d f o r c o n v e - n i e n c e in p r e p a r i n g t h e f i g u r e , b u t n o t in t h e s t a t i s t i c a l a n a l y s e s
T a b l e 2. T o t a l c e l l n u m b e r s (cells x 104/ml) of v a r i o u s b a c t e r i a l s i z e c l a s s e s a n d t h e i r s e a s o n a l o c c u r r e n c e a t s a m p l i n g s t a t i o n in R h o d e R i v e r at l-m d e p t h
B a c t e r i a l D a t e (1973-1974),
s i z e M a r . M a y J u n e J u l y Aug. Sept. Oct. Oct. Nov. Dec. Jan. "Feb.
c l a s s e s 28 16 14 9 7 6 3 31 26 18 29 28
(~m)
0 . 6 x 0 . 6 9 . 7 O O IOO 0 IiO 6 0 . 7 O O 0 5 . 8 8 . 9
1.O x 1.O 8.1 9 8 0 12.8 7 1 0 4 0 0 112 2 1 . 7 120 5 1 . 3 11.3 4 . 4 2.2
1.7 x 1.7 0 . 8 O 3 9 . 4 iO O O 13.3 0 0 O 4 . 5 18.9
0 . 5 x 1.5 8.5 110 O 0 245 0 2 9 . 8 150 9 8 . 4 4 9 . 1 191 215 0 . 5 x 5 . 0 2 6 . 7 O o O O 3.6 6 . 8 O 103 8 0 . 6 16.4 11.9
0 . 5 x 8 . 5 O 6 7 0 O 6 100 15.3 5.3 0 47 19.1 2.6 2.7
1.0 x 5 . 0 0 O 46 2 26 7.8 7.6 22 10 O 8.7 2.7
i.O x i00 O O o 0 O 1.4 0 . 2 0 O O 0 O
2 . 0 x 4 . 0 O O O 0 5 O O 0 0 O O O
T o t a l 5 3 . 8 1,760 9 8 . 2 8 2 8 871 2 5 0 145 292 3 1 0 160 234 263 c e l l s / m l
158 M.A. F a u s t and D.L. Correll: Bacterial and Algal Use of Orthophosphate
Table 3. Total cell numbers (cells/ml) of various algal classes and their seasonal occurrence at sam- pling station in Rhode River at a l-m depth. -: None present
Algal classes Date (1973-1974)
Mar. May June July Aug. Sept. Oct. Oct. Nov. Dec. Jan. Feb.
28 16 14 9 7 6 3 31 26 18 29 28
Dinophyceae - 200,000 702 8,O61 950 599 262
Chrysophyceae - - 384 - 160 36 238
Parasinophyceae . . . 82
Chlorophyceae 250 - 74 - 80 58 468
Bacillariophyceae IOO - 292 - 82 22 174
Cryptophyceae 60 300 - 230 ii0 14 96
Euglenophyceae 110 - - 1,2OO 942 18 -
Haptophyeeae . . . . 92 - -
Flagellates 140 75 - 230 41 6 82
Nannoplankton 800 50 48 404 160 56 69
Total cells/ml 1,460 200,425 1,5OO 10,125 2,617 809 1,471
408 597 682 59 14
250 14 50 80 240
94 4O 64O II 6O
434 - 174 59
1,475 66 186 25 241
382 26 226 115 180
22 - 580
252 58 778 98
4 - - 8 130
4 24 384 16 113
3,325 825 3,700 314 1,135
p a r t i c u l a t e - P , 0 . O 9 1 ; t o t a l o r t h o - P , 10 B 0 . 0 3 4 ; p a r t i c u l a t e o r t h o - P , 0 . 0 6 2 ; t e m -
p e r a t u r e , O . O 1 2 . F o r P - u p t a k e i n t o c e l l s
S m a l l e r t h a n 5 b m , t h e f o l l o w i n g c o r r e l a - ]07 t i o n c o e f f i c i e n t s (r) w e r e f o u n d : t o t a l - P ,
O . O 2 1 ; d i s s o l v e d o r t h o - P , 0 . 0 2 9 ; t o t a l 105 p a r t i c u l a t e - P , 0 . 1 7 7 ; t o t a l o r t h o - P ,
0 . 0 4 6 ; p a r t i c u l a t e o r t h o - P , 0 . 0 6 4 ; t e m - p e r a t u r e , 0 . 0 0 8 5 . T h e r a t i o o f P - u p t a k e
i n t o c e l l s l a r g e r t h a n 5 ~um t o P - u p t a k e _ ~ 1 0 5 i n t o c e l l s s m a l l e r t h a n 5 ~un h a d a l o w
c o r r e l a t i o n w i t h t h e l e v e l o f a v a i l a b l e
d i s s o l v e d o r t h o - P (r = 0 . 0 0 3 3 ) . ]0&
Changes in Algal and Bacterial Population and Biomass D u r i n g the Year
,0
10 2 T h e n u m b e r o f b a c t e r i a , e s t i m a t e d b y __(
d i r e c t c o u n t s , I m b e l o w t h e w a t e r s u r -
face, ranged between 5xi05 and 107 80
c e l l s / m l t h r o u g h o u t t h e y e a r ( F i g . 5 a ) .
T h e h i g h e s t b a c t e r i a l n u m b e r s w e r e o b - ~ s e r v e d d u r i n g t w o d i f f e r e n t a l g a l b l o o m s , ~ 2 0P
a Brorocentrum mariae - lebouriae b l o o m i n E M a y a n d a Gymnodinium nelsonii b l o o m i n E J u l y . A f t e r t h e J u l y b l o o m , b a c t e r i a l ~ ~ 15 n u m b e r s d e c l i n e d a n d r e m a i n e d a l m o s t c o n -
s t a n t f r o m S e p t e m b e r t o F e b r u a r y a t l e v - E o e l s s l i g h t l y a b o v e 1 0 6 c e l l s / m l . I n g e n - ~ I0 e r a l , s m a l l G r a m - n e g a t i v e o r g a n i s m s (I b m
i n d i a m e t e r ) w e r e p r e d o m i n a n t i n s p r i n g a n d s u m m e r , w h i l e r o d s ( 0 . 5 x 1 . 5 t o 8 . 5 ~ m i n s i z e ) i n c l u d i n g G r a m - p o s i t i v e o r g a n i s m s ( T a b l e 2) w e r e p r o m i n e n t d u r - i n g t h e f a l l a n d w i n t e r . T h e n u m b e r o f a l g a l c e l l s ( d e t e r m i n e d a l s o b y d i r e c t c o u n t s ) w a s b e t w e e n 1 0 2 a n d 1 0 c e l l s / m l , w i t h t h e e x c e p t i o n o f t w o a l g a l b l o o m s
( a )
\ ,---.\
~/
X ~ X " " X ' " " X ~ ' X - - X!
Z
%s ~ A
I l I \
I 1 1 %
/ l,
-o" ~o"
I I I I I I I I
V
I I I l
\i I I
11 i \ I I i l
x x
"~'~
I i l , , , ,
M A M 3 3 A 5 0 N D 3 F Months
Fig. 5. Seasonal distribution of cell numbers w h i c h o c c u r r e d i n M a y a n d J u l y w h e n t h e and biomass at Rhode River sampling station at a a l g a l c o u n t r e a c h e d 1 0 5 a n d 1 0 4 c e l l s / m l , i-m depth. Solid lines: bacteria; dashed lines:
r e s p e c t i v e l y ( F i g . 5 b ) . T h e r e w a s n o algae
M.A. Faust and D.L. Correll: Bacterial and Algal Use of Orthophosphate 159
a p p a r e n t i n c r e a s e in t o t a l n u m b e r of e i t h e r b a c t e r i a or a l g a e in N o v e m b e r , b u t K a t o d i n i u m r o t u n d a t u m w a s t h e d o m i n a n t a l g a l s p e c i e s . T h e t o t a l c e l l n u m b e r s p e r m l of v a r i o u s a l g a l c l a s s e s a n d t h e i r s e a s o n a l o c c u r r e n c e a r e s h o w n in T a b l e 3.
T h e h i g h e s t b a c t e r i a l b i o m a s s w a s 16.9 m m 3 / l d u r i n g t h e p r o r o c e n t r u m m a r i a e - l e b o u r i a e b l o o m . B a c t e r i a l b i o m a s s v a r - ied b e t w e e n 1.1 a n d 6.3 m m 3 / l t h e r e s t of t h e y e a r (Fig. 5B). T h e a l g a l b i o m a s s f o l l o w e d a l g a l c e l l n u m b e r s a n d w a s 150 t i m e s h i g h e r in M a y a n d 10 t i m e s h i g h e r in July, d u r i n g t h e t w o a l g a l b l o o m s , t h a n d u r i n g t h e r e s t of t h e y e a r . L a r g e d i n o f l a g e l l a t e s w e r e t h e d o m i n a n t a l g a l s p e c i e s w i t h r e s p e c t to b i o m a s s d u r i n g the s u m m e r , a n d n a n n o p l a n k t o n of l e s s t h a n 10 ~m s i z e w e r e d o m i n a n t d u r i n g t h e r e s t of t h e year.
On t h r e e o c c a s i o n s , t h e d o m i n a n t al- g a l s p e c i e s w e r e P r o r o c e n t r u m m a r i a e - l e -
a m e a s u r e of t h e m e t a b o l i c a c t i v i t y of t h e o r g a n i s m s w i t h r e g a r d to P - u p t a k e .
T h i s t e c h n i q u e w a s b a s e d o n t h e o b - s e r v a t i o n t h a t m o s t of the b a c t e r i a l p o p - u l a t i o n p a s s e d t h r o u g h a 5-~un p o r e - s i z e f i l t e r , w h e r e a s p h y t o p l a n k t o n w e r e re- t a i n e d o n it. E v e n t h o u g h w e s e p a r a t e d a l g a e f r o m b a c t e r i a as w e l l as o u r c o n - d i t i o n s p e r m i t t e d , w e r e c o g n i z e d t h e d i f f i c u l t y in s e p a r a t i n g m i c r o o r g a n i s m s f r o m n a t u r a l w a t e r s i n t o c l e a r l y - d e f i n e d s i z e g r o u p s . S o m e b a c t e r i a l r o d s w e r e l o n g e r t h a n 5 ~m; o t h e r s a d h e r e d to
l a r g e n o n - l i v i n g p a r t i c u l a t e s , or to v a r - i o u s a l g a e as r e p o r t e d b y o t h e r i n v e s t i - g a t o r s (Bell a n d M i t c h e l l , 1972; J o n e s ,
1972). In c o n t r a s t , s o m e b r o k e n c e l l s w e r e r e t a i n e d o n t h e s m a l l e r p o r e - s i z e f i l t e r s in s o m e s a m p l e s . W i t h all t h e s e d i f f i c u l t i e s , the 5 - b m p o r e - s i z e f i l t e r s t i l l a p p e a r s u s e f u l in s e p a r a t i n g
e s t u a r i n e p l a n k t o n i c a l g a e f r o m b a c t e r i a . A n o t h e r p r o b l e m w a s h o w to d i s c r i m i -
b o u r i a e , G y m n o d i n i u m n e l s o n i i a n d K a t o d i n i u m n a t e b e t w e e n 3 2 p - o r t h o p h o s p h a t e b o u n d to
r o t u n d a t u m . It w a s m o r e d i f f i c u l t to d e t e r - n o n - l i v i n g p a r t i c u l a t e s a n d 32p i n c o r p o - m i n e the s p e c i e s of b a c t e r i a , a n d t h e y
w e r e m e r e l y c l a s s i f i e d a c c o r d i n g to s i z e (Table 2). T h e m o r p h o l o g y of b a c t e r i a v a r i e d a c c o r d i n g to s e a s o n . T h e n u m b e r of c e l l s in c e r t a i n c l a s s i f i c a t i o n s w a s h i g h at o n e s a m p l i n g a n d z e r o at s u b s e - q u e n t s a m p l i n g s . A l i n e a r l e a s t - s q u a r e s r e g r e s s i o n c a l c u l a t i o n g a v e t h e r e s u l t : n u m b e r of b a c t e r i a l c e l l s / m l = 2 3 . 6 +
(O.69) (algal c e l l s / m l ) , (r = 0 . 9 5 0 ) . T h e c o r r e l a t i o n b e t w e e n a l g a l a n d b a c t e r i a l b i o m a s s w a s a l s o high, (r = O . 9 0 1 ) , in- d i c a t i n g t h a t c o n d i t i o n s f a v o r i n g b a c - t e r i a l g r o w t h a l s o f a v o r e d a l g a l g r o w t h .
T h e b i o m a s s o f p h y t o p l a n k t o n h a d a h i g h c o r r e l a t i o n (r = 0.803) w i t h t h e l e v e l of d i s s o l v e d o r g a n i c - P p r e s e n t in t h e p r e c e e d i n g 2 w e e k s a n d a l o w c o r r e - l a t i o n (r = 0 . 0 0 0 5 ) w i t h t h e l e v e l of d i s s o l v e d o r g a n i c - P p r e s e n t in t h e s u c - c e e d i n g 2 w e e k s . T h e s a m e c o r r e l a t i o n s e x i s t e d b e t w e e n d i n o f l a g e l l a t e b i o m a s s a n d d i s s o l v e d o r g a n i c - P .
Discussion
W e h a v e a t t e m p t e d to d i s t i n g u i s h P- u p t a k e b y a l g a e f r o m t h a t b y b a c t e r i a in an e s t u a r i n e p l a n k t o n i c c o m m u n i t y b y u s i n g a d i f f e r e n t i a l f i l t r a t i o n t e c h - n i q u e . A l l e x p e r i m e n t s w e r e c o u p l e d w i t h t h e e s t i m a t i o n of e x i s t i n g b i o m a s s
t h r o u g h o u t a y e a r . T h i s a l l o w e d us to c a l c u l a t e P - u p t a k e p e r b i o m a s s a n d a l s o p e r v o l u m e of w a t e r , w h i c h g a v e us n o t o n l y a m e a s u r e of the c h a n g e in b i o m a s s w i t h e n v i r o n m e n t a l c o n d i t i o n s , b u t a l s o
r a t i o n i n t o the l i v i n g p a r t i c u l a t e s . C o n - t r o l e x p e r i m e n t s on 3 z p - u p t a k e in t h e p r e s e n c e of IAA, w h i c h i n h i b i t s b i o l o g i - cal u p t a k e , g a v e an e s t i m a t e of t h e ad- s o r p t i o n of t h e t r a c e r on s u r f a c e s w h i c h a r e l a r g e l y c o m p o s e d o f n o n - l i v i n g p a r - t i c u l a t e s . O u r m e t h o d of c a l c u l a t i o n of P - u p t a k e c o r r e c t s f o r t h i s n o n b i o l o g i c a l P - b i n d i n g . T h e o r t h o p h o s p h a t e p o o l s i z e a n d its s p e c i f i c a c t i v i t y , as w e l l as f r e q u e n t d a t a p o i n t s in t h e e a r l y p h a s e of t h e e x p e r i m e n t , w e r e a l s o n e e d e d for t h e k i n e t i c c a l c u l a t i o n s ( C o r r e l l e t al., 1975). O n l y t h e u p t a k e w h i c h o c c u r r e d a f t e r t h e v e r y r a p i d a d s o r p t i o n of p h o s - p h a t e to t h e s u r f a c e s of p a r t i c u l a t e s c o u l d be u s e d in t h e c a l c u l a t i o n of b i o - l o g i c a l P - u p t a k e r a t e s . It is p o s s i b l e t h a t w e u n d e r e s t i m a t e d t h e r e l a t i v e m a g - n i t u d e of b a c t e r i a l u p t a k e c o m p a r e d to a l g a l u p t a k e , s i n c e w e d e t e r m i n e d t h i s r a t i o by m e a s u r i n g t h e s l o p e s of u p t a k e i n t o p a r t i c u l a t e s r e t a i n e d on f i l t e r s . S i n c e b a c t e r i a l u p t a k e is v e r y f a s t c o m - p a r e d w i t h a l g a l u p t a k e , t h e r e w o u l d be a t e n d e n c y to u n d e r e s t i m a t e b a c t e r i a l u p t a k e . S i n c e a l g a l u p t a k e w a s v e r y small, the m a g n i t u d e o f t h i s e r r o r c o u l d n o t be v e r y great.
T h e d i f f i c u l t i e s in e s t i m a t i n g s t a n d - i n g c r o p u s i n g t o t a l p l a s m a v o l u m e or c a r b o n p e r u n i t c e l l v o l u m e of a n a t u r a l s a m p l e h a v e b e e n d e s c r i b e d in d e t a i l
( C a m p b e l l , 1973). In t h i s s t u d y w e m e a - s u r e d s t a n d i n g c r o p of a l g a e a n d b a c - t e r i a b y c e l l c o u n t s . T h i s m a y p l a c e a d i s p r o p o r t i o n a t e e m p h a s i s o n t h e s m a l l e r - c e l l e d s p e c i m e n s , w h e r e t h e t o t a l b i o - m a s s of a n u m b e r of s m a l l c e l l s m a y ac-
I@0 M.A. Faust and D.L. Correll: Bacterial and Algal Use of Orthophosphate
t u a l l y b e l e s s t h a n a f e w c e l l s of a l a r g e r s p e c i e s . W i t h t h i s m e t h o d w e c a n - n o t d i f f e r e n t i a t e b e t w e e n d e a d or l i v e c e l l s . S m a l l e r c e l l s i z e p r o v i d e s a g r e a t e r p r o p o r t i o n of s u r f a c e - t o - v o l u m e r a t i o , w h i c h c a n e n a b l e m o r e r a p i d u p - t a k e of n u t r i e n t s a n d p e r m i t a m o r e r a p - id m e t a b o l i s m of t h e s e s m a l l e r s p e c i e s . Thus, u s e of p l a s m a v o l u m e in c a l c u l a t - ing s t a n d i n g c r o p m a y o v e r e m p h a s i z e t h e i m p o r t a n c e of l a r g e r s p e c i e s ( C a m p b e l l , 1973). H o w e v e r , w e w i s h e d to be a b l e to e x p r e s s P - u p t a k e p e r b i o m a s s of c e l l s . To e s t i m a t e the v o l u m e of c e l l s , w e n e e d e d to k n o w t h e i r d i m e n s i o n s , w h i c h c a n b e d e t e r m i n e d o n l y b y d i r e c t m i c r o - s c o p i c e x a m i n a t i o n . J o n e s (1972) h a s d i s - c u s s e d t h e v a l i d i t y of d i r e c t m i c r o -
and b a c t e r i a 20 t i m e s h i g h e r t h a n t h e r e s t of the year.
T h e r e l a t i v e l y l o w c o n c e n t r a t i o n s of o r t h o p h o s p h a t e in the w a t e r , w h i c h p r e - v a i l e d t h r o u g h o u t the s e a s o n w i t h the e x c e p t i o n of the s u m m e r , d i d n o t in- f l u e n c e the P a s s i m i l a t i n g - a b i l i t y of b a c t e r i a a n d a l g a e , w h i c h h a d h i g h c o r - r e l a t i o n s o n l y w i t h b i o m a s s . T h u s , o u r r e s u l t s i n d i c a t e t h a t P - u p t a k e p e r u n i t b i o m a s s w a s i n d e p e n d e n t of P c o n c e n t r a - t i o n s a n d of w a t e r t e m p e r a t u r e . T h i s d i f - f e r s f r o m t h e r e s u l t s of K r a m e r et al.
(1972), w h o c o r r e l a t e d P l e v e l s and to- tal p r o d u c t i v i t y .
C o m p e t i t i o n b e t w e e n a l g a e a n d b a c - s c o p i c c o u n t s a n d o t h e r m e t h o d s to e s t a b - t e r i a has b e e n d e s c r i b e d in l a b o r a t o r y l i s h the d e g r e e of a s s o c i a t i o n of a l g a e
a n d b a c t e r i a in a f r e s h - w a t e r e n v i r o n - m e n t . He f o u n d a h i g h c o r r e l a t i o n b e -
t w e e n a l g a l c e l l n u m b e r s a n d v i a b l e b a c - t e r i a l c o u n t s , b u t n o t w i t h d i r e c t
c o u n t s of b a c t e r i a . H o w e v e r , w e o b t a i n e d a h i g h c o r r e l a t i o n b e t w e e n a l g a l a n d b a c t e r i a l c e l l n u m b e r s u s i n g d i r e c t c o u n t s . It a p p e a r e d t h a t b a c t e r i a l n u m - b e r s f o l l o w a l g a l c e l l n u m b e r s . T h i s t r e n d g a v e a h i g h p o s i t i v e c o r r e l a t i o n n o t o n l y w i t h c e l l n u m b e r s b u t w i t h b i o - m a s s e s t i m a t i o n s ; r = 0 . 9 5 0 a n d 0 . 9 0 2 , r e s p e c t i v e l y .
T h e d i f f e r e n t i a l f i l t e r t e c h n i q u e c l e a r l y d e m o n s t r a t e d t h a t the r e l a t i v e c o n t r i b u t i o n of a l g a e a n d b a c t e r i a to P - u p t a k e v a r i e d w i t h s e a s o n . D u r i n g the p e r i o d f r o m A u g u s t to M a y , P w a s a s s i m i - l a t e d m o s t l y b y b a c t e r i a , a n d t h e a l g a l c o n t r i b u t i o n to P - u p t a k e w a s less t h a n
6% of t o t a l P - u p t a k e . D u r i n g J u n e a n d July, P - u p t a k e by a l g a e in t h e l i g h t in- c r e a s e d to 9 a n d 42% of the t o t a l P-
c u l t u r e s , w h e r e g r o w t h of a l g a e w a s s e v e r e l y l i m i t e d b y the p r e s e n c e of b a c - t e r i a a f t e r e x t e r n a l P h a d b e e n ex- h a u s t e d , b u t g r o w t h of b a c t e r i a w a s u n - a f f e c t e d by t h e p r e s e n c e of a l g a e (Rhee, 1972). In o u r study, no s t r o n g c o m p e t i - t i v e a d v a n t a g e in P - u p t a k e by b a c t e r i a o v e r a l g a e at l o w a v a i l a b l e p h o s p h a t e l e v e l s w a s i n d i c a t e d . T h u s , t h e r a t i o of a l g a l to b a c t e r i a l u p t a k e h a d a l o w c o r - r e l a t i o n w i t h d i s s o l v e d o r t h o - P (r = 0 . 0 0 3 3 ) .
A l t h o u g h a l g a l a n d b a c t e r i a l P - u p t a k e , as m e a s u r e d in t h i s study, w e r e b o t h h i g h l y c o r r e l a t e d w i t h t h e i r r e s p e c t i v e b i o m a s s e s , h i g h s t a n d a r d d e v i a t i o n s f r o m the m e a n s w e r e f o u n d . F o r e x a m p l e , t h e m e a n a l g a l u p t a k e p e r b i o m a s s in t h e l i g h t w a s O . 1 7 ng P / m m 3 / h , a n d the s t a n - d a r d d e v i a t i o n w a s O . 1 8 . O n e r e a s o n for t h i s h i g h d e v i a t i o n w a s u n d o u b t e d l y the v a r i a t i o n in c e l l s i z e d i s t r i b u t i o n f r o m e x p e r i m e n t to e x p e r i m e n t . H o w e v e r , the h e a l t h or g r o w t h r a t e of t h e c e l l p o p u l a - u p t a k e , r e s p e c t i v e l y . T h e h i g h e r P - u p t a k e t i o n w a s p r o b a b l y t h e d o m i n a n t f a c t o r . t h r o u g h o u t the y e a r by b a c t e r i a , c o m -
p a r e d w i t h a l g a e , c l e a r l y i n d i c a t e s the i m p o r t a n c e of b a c t e r i a in P a s s i m i l a t i o n in e s t u a r i n e e n v i r o n m e n t s , as h a s a l s o b e e n s h o w n for the m a r i n e e n v i r o n m e n t
(Johannes, 1964). H i g h P - u p t a k e d u e to a l g a e is p o s s i b l e o n l y in t h e s u m m e r . T h i s is d u e to h i g h e r n u m b e r s of a l g a e at t h a t t i m e r a t h e r t h a n to h i g h e r P a s - s i m i l a t i n g - a b i l i t y p e r cell. A n i n c r e a s e in p o p u l a t i o n d e n s i t y a n d a d e c r e a s e in p r i m a r y p r o d u c t i v i t y p e r u n i t of s t a n d - ing c r o p in the s u m m e r h a s b e e n r e p o r t e d
( F i n d e n e g g , 1965). P o s s i b l e c a u s e s a r e e x t i n c t i o n of light, n u t r i e n t c o m p e t i - tion, a n d i n c r e a s e d c o n c e n t r a t i o n of e x - c r e t e d a l g a l p r o d u c t s . D o u b t l e s s , t h e s e
A n o t h e r i n t e r e s t i n g r e s u l t of t h i s s t u d y w a s t h a t c e l l s l a r g e r t h a n 5 ~ml
(Fig. 3; T a b l e I) h a d e s s e n t i a l l y i d e n - t i c a l P - u p t a k e r a t e s in b o t h l i g h t a n d d a r k n e s s . T h i s c o u l d be d u e in p a r t to s o m e b a c t e r i a l c e l l s c o n t a m i n a t i n g t h e 5-~um s i z e class, s i n c e t h e a v e r a g e P- u p t a k e by b a c t e r i a p e r b i o m a s s w a s 40 t i m e s the a v e r a g e for a l g a e . S i n c e v e r y l i t t l e b a c t e r i a l b i o m a s s w a s r e t a i n e d on the 5-H/n f i l t e r , t h i s is p r o b a b l y n o t a v e r y m a j o r f a c t o r in t h e i n t e r p r e t a t i o n of t h e s e r e s u l t s . In s o m e e x p e r i m e n t s
(i.e., J u l y 9 a n d O c t o b e r 3), P - u p t a k e in t h e d a r k w a s m u c h l e s s t h a n in t h e light. In o t h e r s (i.e., M a r c h 28, O c t o - f a c t o r s m a y h a v e o c c u r r e d in the R h o d e h e r 31, N o v e m b e r 26, a n d D e c e m b e r 18), R i v e r d u r i n g t i m e s w h e n t h e a l g a l p o p u l a - u p t a k e in the d a r k w a s a b o u t h a l f t h a t t i o n w a s h i g h a n d w h e n t h e h i g h e s t s u p - in the light. In t h e rest, it w a s a b o u t p o r t e d b i o m a s s of a l g a e w a s 150 t i m e s t h e s a m e or e v e n h i g h e r . T h e s e d i f f e r -
