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The regulation of blue-green algae by iron availability and calcite precipitation Murphy, Thomas P.D. 1987

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THE REGULATION OF BLUE-GREEN ALGAE BY IRON AVAILABILITY AND CALCITE PRECIPITATION THOMAS P. D. MURPHY B.Sc . Queens U n i v e r s i t y 1972 M.Sc. U n i v e r s i t y of Toronto 1976 A THESIS IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept th i s thes is as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1987 (c) Thomas P. D. Murphy, 1987 by in In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ~2-Qol~£> l> j The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) i i ABSTRACT The primary o b j e c t i v e of t h i s r e s e a r c h was t o determine i f changes i n i r o n a v a i l a b i l i t y i n f l u e n c e the p e r i o d i c i t y of blue-green a l g a l growth. A secondary goal was t o r e s o l v e how i r o n a v a i l a b i l i t y was r e l a t e d t o events such as c a l c i t e ( c a l c i u m carbonate) p r e c i p i t a t i o n and sediment n u t r i e n t r e l e a s e . The biogeochemical r e g u l a t i o n of blue-green a l g a l s u c c e s s i o n was s t u d i e d i n t h r e e e u t r o p h i c hardwater l a k e s l o c a t e d upon the Thompson P l a t e a u i n s o u t h - c e n t r a l B r i t i s h Columbia. The experimental approaches i n c l u d e d iri s i t u b o t t l e and l i m n o c o r r a l experiments, sediment core a n a l y s i s , monitoring of seasonal changes i n water chemistry, and whole-lake m a n i p u l a t i o n by hyp o l i m n e t i c a e r a t i o n , or c a l c i u m hydroxide a d d i t i o n . Growth and primary p r o d u c t i o n b i o a s s a y s were used t o e v a l u a t e i r o n a v a i l a b i l i t y . M i c r o b i a l c h e l a t o r s were i s o l a t e d from a l g a l c u l t u r e s and lake water, q u a n t i f i e d by a c h e l a t i o n assay, and used t o determine t h e i r i n s i t u e f f e c t s on a l g a l p r o d u c t i v i t y and b a c t e r i a l h e t e r o t r o p h y . Microbes were a b l e t o r e g u l a t e the b i o a v a i l a b i l i t y of i r o n . A l g a l s i d e r o p h o r e i s o l a t e s were r a p i d l y a s s i m i l a t e d i n l a k e water and they were h i g h l y s p e c i f i c f o r i r o n c h e l a t i o n . Moreover, c h e l a t o r c o n c e n t r a t i o n s i n Black Lake u s u a l l y exceeded the d i s s o l v e d i r o n c o n c e n t r a t i o n . Algae excreted c h e l a t o r s t h a t could suppress growth of some other s p e c i e s of algae by 90%, enhance the primary p r o d u c t i o n of some other a l g a l s p e c i e s by 30%, or suppress the h e t e r o t r o p h i c a c t i v i t y of b a c t e r i a by 14-98%. The degree of i r o n l i m i t a t i o n v a r i e d g r e a t l y d u r i n g the summer. In Black Lake, i r o n l i m i t a t i o n was more than t e n - f o l d i i i more i n t e n s e i n e a r l y summer than i n l a t e summer. Dense blooms of blue-green algae occurred i n Black Lake on l y a f t e r the i r o n content of the lake i n c r e a s e d from 20 to more than 100 ug/L. An i n c r e a s e i n i r o n c o n c e n t r a t i o n i n the water column of the thr e e l a k e s was caused by a midsummer sediment r e l e a s e of i r o n . Although sediment p y r i t e formation converted a v a i l a b l e i r o n i n t o r e f r a c t o r y i r o n i n both Chain and F r i s k e n l a k e s , the degree of i r o n l i m i t a t i o n v a r i e d g r e a t l y among the l a k e s . U n l i k e i n Black Lake, the algae i n Chain Lake were not l i m i t e d by i r o n a v a i l a b i l i t y . Phosphorus s o l u b i l i t y was a good index of i r o n a v a i l a b i l i t y . Black and F r i s k e n lakes had too l i t t l e i r o n f o r i r o n phosphate t o p r e c i p i t a t e , but the higher i r o n c o n c e n t r a t i o n i n Chain Lake r e g u l a t e d phosphorus s o l u b i l i t y . The d i f f e r e n c e s among lakes was p r i m a r i l y a f u n c t i o n of e x t e r n a l i r o n l o a d i n g , not sediment i r o n r e l e a s e . Chain Lake r e c e i v e d 10"^  more i r o n per 2 m than F r i s k e n or Black l a k e s . Carbonate e q u i l i b r i a i n t e g r a t e d the m i c r o b i a l responses t o i r o n enrichment. When i r o n a v a i l a b i l i t y was i n c r e a s e d i n the e p i l i m n i o n of Black Lake, a l g a l p r o d u c t i v i t y was enhanced which r e s u l t e d i n an i n c r e a s e i n pH and the c o p r e c i p i t a t i o n of more c a l c i t e and phosphorus than i n c o n t r o l treatments. The p r e c i p i t a t i o n of c a l c i t e c o u l d sediment as much as 90% of the algae and 97% of the phosphorus from the e p i l i m n i o n . The hypolimnia of the i r o n - e n r i c h e d l i m n o c o r r a l s had the lowest pH and h i g h e s t d i s s o l u t i o n of p r e c i p i t a t e d phosphorus. Three r e a c t i o n s , i r o n c h e l a t i o n , sediment i r o n r e l e a s e , and c a l c i t e p r e c i p i t a t i o n , can r e g u l a t e much of the p e r i o d i c i t y of blue-green a l g a l growth i n hardwater l a k e s . i v T a ble of Contents A b s t r a c t i i L i s t of Tables v i i i L i s t of F i g u r e s i x Acknowledgements x i i G l o s s a r y x i i i 1 I n t r o d u c t i o n 1 1.1 F a c t o r s R e g u l a t i n g the P e r i o d i c i t y of A l g a l Growth... 1 1.2 I r o n - R e q u i r i n g Reactions of Blue-Green Algae 4 1.2.1 Iron L i m i t a t i o n 10 2 Methods 15 2.1 Study Area 15 2.1.1 Black Lake 15 2.1.2 F r i s k e n Lake 19 2.1.3 Chain Lake 19 2.2 F i e l d Experiments 20 2.2.1 Sample C o l l e c t i o n 20 2.2.2 A e r a t i o n of Black Lake 21 2.2.3 Chemical Treatments of F r i s k e n Lake 21 2.2.4 L i m n o c o r r a l Experiments 22 2.2.5 Small I n - S i t u I ncubations 22 2.2.5.1 Calcium C h l o r i d e Experiment 22 2.2.5.2 Primary P r o d u c t i o n 23 2.2.5.3 Heterotrophy 24 , V 2.3 L a b o r a t o r y S t u d i e s and A n a l y s i s of F i e l d Samples.... 25 2.3.1 Chemical A n a l y s i s 25 2.3.2 3 2P-SRP A n a l y s i s 27 2.3.3 I r o n - B i n d i n g Assay 27 2.3.4 C h e l a t o r I s o l a t i o n 29 2.3.5 E l e c t r o n M i c r o s c o p i c A n a l y s i s 31 2.3.6 Sediment A n a l y s i s 31 3 R e s u l t s 32 3.1 B i o g e n i c Response t o Iron A v a i l a b i l i t y 32 3.1.1 I r o n - C h l o r o p h y l l a R e l a t i o n s h i p s 32 3.1.2 Seasonal A l g a l S u c c e s s i o n 36 3.1.3 Seasonal Changes i n I r o n C o n c e n t r a t i o n 37 3.2 C o n f i r m a t i o n of the Iron-Biomass R e l a t i o n s h i p 42 3.2.1 L a b o r a t o r y Bioassays with Black Lake Water 42 3.2.2 Fe-EDTA Limn o c o r r a l s i n Black Lake 47 3.2.3 F e - C i t r a t e L i m n o c o r r a l Experiment i n Black Lake 49 3.2.4 I r o n A v a i l a b i l i t y i n Chain Lake 55 3.3 Siderophore Ecology 57 3.3.1 C h e l a t o r Q u a n t i f i c a t i o n 57 3.3.2 Siderophore A s s o c i a t i o n with F i b r i l s 59 3.3.3 Siderophore S p e c i f i c i t y f o r Iron 62 3.3.4 Lake Siderophores 66 3.3.5 Enhanced Iron A v a i l a b i l i t y 69 3.3.6 A l l e l o p a t h i c P r o p e r t i e s of Siderophores 70 3.3.7 Siderophore I n f l u e n c e on Heterotrophy ... 72 3.4 E f f e c t of I r o n A v a i l a b i l i t y on Phosphorus Chemistry. 79 v i 3.4.1 Geographic V a r i a b i l i t y i n Fe-P Water Chemistry 79 3.4.2 Geographic V a r i a b i l i t y i n Sediment Iron R e a c t i v i t y . . 82 3.5 E f f e c t of Ir o n A v a i l a b i l i t y on C a l c i t e and Phosphorus P r e c i p i t a t i o n 86 3.5.1 Phosphorus Chemistry of Black Lake 86 3.5.2 C a l c i t e P r e c i p i t a t i o n i n Black Lake 89 32 3.5.2.1 P K i n e t i c s and P L i m i t a t i o n 91 3.5.3 C a l c i t e P r e c i p i t a t i o n i n Limn o c o r r a l s 91 3. 5.3.1 19 80 Fe-EDTA Li m n o c o r r a l s i n Black Lake 91 3.5.3.2 N i t r a t e I n d u c t i o n of C a l c i t e P r e c i p i t a t i o n 93 3.5.3.3 19 82 F e - C i t r a t e L i m n o c o r r a l s 94 3.5.3.4 C i t r a t e E f f e c t on P-CaC0 3 I n d i c a t e s I r o n L i m i t a t i o n 96 3.5.3.5 I n f l u e n c e of Weather on P-CaCO^ P r e c i p i t a t i o n 96 3.5.4 Cal c i u m C h l o r i d e I n d u c t i o n of C a l c i t e P r e c i p i t a t i o n . 99 3.5.4.1 C a l c i t e A n a l y s i s 101 3.6 C a l c i t e P r e c i p i t a t i o n - A Major Cause of A l g a l P e r i o d i c i t y 103 3.6.1 Pretreatment Water Chemistry 103 3.6.2 Lime-Induced C a l c i t e P r e c i p i t a t i o n 105 3.6.2.1 Suppression o f A l g a l Growth 107 3.6.3 Long-Term Enhancement of C a l c i t e P r e c i p i t a t i o n 107 4 D i s c u s s i o n 113 4.1 S p a t i a l and Temporal V a r i a t i o n i n Ir o n A v a i l a b i l i t y 113 4.2 C a l c i t e I n d u c t i o n of Iron L i m i t a t i o n 115 4.3 Sediment Iron Release 119 v i i 4.4 I n t e r r e l a t i o n s h i p of I r o n L i m i t a t i o n , C h e l a t i o n , and C a l c i t e P r e c i p i t a t i o n 121 4.5 M i c r o b i a l C o n t r o l of I r o n A v a i l a b i l i t y 122 4.6 Siderophores as Mediators of A l g a l S u c c e s s i o n 126 4.7 Siderophores as Regulators of B a c t e r i a l Heterotrophy 128 4.8 C a l c i t e P r e c i p i t a t i o n 130 4.8.1 V a r i a b i l i t y of C a l c i t e P r e c i p i t a t i o n 131 4.9 B i o l o g i c a l C o n t r o l of C a l c i t e P r e c i p i t a t i o n 133 4.10 M i c r o b i a l Responses t o C a l c i t e P r e c i p i t a t i o n 134 4.11 Phosphorus C o p r e c i p i t a t i o n with C a l c i t e 135 4.12 C o n c l u s i o n s 137 References 140 Appendix 1 D e t a i l s of L i m n o c o r r a l Experiments 151 Appendix 2 Iron A n a l y s i s i n Black. Lake 154 Appendix 3 Oxygen i n L i m n o c o r r a l s During C i t r a t e A d d i t i o n s . 157 Appendix 4 Improvements to the Iron B i n d i n g Assay 158 Appendix 5 Yellow Lake Sediment Chemistry 163 v i i i L i s t of T a b l e s 1 Some P h y s i c a l and Chemical Features of the Study Lakes 18 2 Timing and S i t e s of Experiments 20 3 T o t a l I r o n C o n c e n t r a t i o n s i n Chain Lake - 1984 38 4 T o t a l Iron C o n c e n t r a t i o n s i n F r i s k e n Lake - 1983, 1984.. 43 5 Primary P r o d u c t i o n i n Experimental E n c l o s u r e s i n Black Lake i n 1980 48 6 C i t r a t e C o n c e n t r a t i o n i n L i m n o c o r r a l s 52 7 L i m n o c o r r a l 1 4 C - C i t r a t e A s s i m i l a t i o n 53 8 E f f e c t of F r e e z i n g Anabaena f i l t r a t e on Iron C h e l a t i o n 58 9 E f f e c t of Metal A d d i t i o n on Fe C h e l a t i o n by the Anabaena f los-aquae s i d e r o p h o r e 62 10 E f f e c t of Metal A d d i t i o n of Fe C h e l a t i o n by the Aphanizomenon Siderophore 67 11 Black Lake Ir o n C o n c e n t r a t i o n and C h e l a t i o n C a p a c i t y J u l y 17, 1980 67 12 E f f e c t of E x t r a c t s Produced by Anabaena Under D i f f e r e n t Growth C o n d i t i o n s on the M i c r o b i a l Uptake of A c e t a t e . . . . 73 13 F l u x e s of I r o n i n Chain and F r i s k e n Lakes 83 14 C a l c i u m C o n c e n t r a t i o n s i n 1982 L i m n o c o r r a l s 98 15 Elemental A n a l y s i s of C r y s t a l 102 16 Calcium-Phosphate R a t i o s i n P r e c i p i t a t i o n Events, P r e c i p i t a t i o n Experiments, and Sediment 102 A l I r o n A n a l y s i s i n Black Lake 154 A2 Oxygen C o n c e n t r a t i o n s i n L i m n o c o r r a l s d u r i n g C i t r a t e A d d i t i o n s 157 A3 Yellow Lake Sediment Chemistry 163 i x L i s t of F i g u r e s 1 Key r e a c t i o n s i n t h i s study 5 2 Map of southern B r i t i s h Columbia and study s i t e s 16 3 Seasonal changes i n phytoplankton biomass ( c h l o r o p h y l l a) and p a r t i c u l a t e Fe i n Black Lake 33 4 Black Lake i r o n c o n c e n t r a t i o n s i n an a l g a l bloom 34 5 Iron and c h l o r o p h y l l a at 1.0 m i n F r i s k e n Lake 34 6 C h l o r o p h y l l a and i r o n c o n c e n t r a t i o n s i n Black, Chain, and F r i s k e n lakes 35 7 Seasonal changes i n oxygen c o n c e n t r a t i o n i n Black, Chain, and F r i s k e n l a k e s . 39 8 Iron geochemistry of Chain and F r i s k e n l a k e sediments 40 9 Seasonal changes i n temperature of the s u r f a c e sediments of Black, Chain, and F r i s k e n l a k e s 41 10 E f f e c t of i r o n enrichment on growth of algae from Black Lake 44 11 E f f e c t of a d d i t i o n s of Fe or the s i d e r o p h o r e i s o l a t e from Anabaena c y l i n d r i c a on primary p r o d u c t i o n i n Black Lake 46 12 Oxygen, and phosphorus c o n c e n t r a t i o n s i n the Fe-EDTA and Na-EDTA l i m n o c o r r a l s and the lake 48 13 Temporal v a r i a t i o n of heterotrophy i n the 1980 l i m n o c o r r a l s 50 14 C i t r a t e a s s i m i l a t i o n i n l i m n o c o r r a l s 52 15 Phosphorus, oxygen, and temperature i n the c i t r a t e l i m n o c o r r a l s i n Black Lake 54 X 16 Oxygen c o n c e n t r a t i o n s i n Chain Lake l i m n o c o r r a l s 56 17 I r o n t i t r a t i o n of EDTA, d e s f e r a l , and f i l t r a t e s from Anabaena f l o s - a q u a e c u l t u r e s 61 18 Metal displacement of i r o n from the Anabaena f los-aquae s i d e r o p h o r e 63 55 19 E l u t i o n of the F e - f i l t r a t e from an FeBC assay through a G-2 5 Sephadex column 65 20 A demonstration of s i d e r o p h o r e s p e c i f i c i t y - e f f e c t two s i d e r o p h o r e i s o l a t e s on primary p r o d u c t i o n of two a l g a e . . . 71 21 The e f f e c t of the Aphanizomenon siderophore i s o l a t e on the growth of Scenedesmus b a s i l i e n s i s 73 22 Suppression by s i d e r o p h o r e i s o l a t e s of b a c t e r i a l a s s i m i l a t i o n of a c e t a t e 75 23 E f f e c t of g l u c o s e on s u p p r e s s i o n of h e t e r o t r o p h y by an s i d e r o p h o r e i s o l a t e 76 24 E f f e c t of a c e t a t e on s u p p r e s s i o n of heterotrophy by an s i d e r o p h o r e i s o l a t e 76 25 Temporal v a r i a t i o n of heterotrophy i n Black Lake 78 26 Seasonal comparison of s o l u b l e r e a c t i v e phosphorus (SRP) c o n c e n t r a t i o n s i n Black, Chain, and F r i s k e n lakes 80 27 Chain Lake s o l u b l e r e a c t i v e phosphorus (SRP) 80 28 Chain Lake t o t a l phosphorus 81 29 Chain Lake sediment chemistry 85 30 Roche Lake sediment chemistry 87 31 F r i s k e n Lake sediment chemistry 87 x i 32 32 P-phosphate a n a l y s i s i n Black Lake Creek 88 33 Lake chemistry d u r i n g Aphanizomenon blooms 90 32 34 P a s s i m i l a t i o n d u r i n g c a l c i t e p r e c i p i t a t i o n 92 35 Hypolimnetic SRP and pH i n Black Lake c i t r a t e l i m n o c o r r a l s on Aug. 24, 1982 95 36 Depth p r o f i l e of c i t r a t e a s s i m i l a t i o n i n Black Lake c i t r a t e l i m n o c o r r a l s 95 37 C a C ^ induced c a l c i t e p r e c i p i t a t i o n 100 38 F r i s k e n Lake pretreatment SRP/TIC 104 39 A depth d i s t r i b u t i o n of SRP and TIC i n F r i s k e n Lake 104 40 F r i s k e n Lake e p i l i m n e t i c SRP 106 41 Suppression of a l g a l biomass by lime a p p l i c a t i o n . . . . 108 42 E f f e c t of c a l c i t e p r e c i p i t a t i o n of oxygen c o n c e n t r a t i o n 110 43 B i o t i c m o d i f i c a t i o n of lime t i t r a t i o n I l l 44 F r i s k e n Lake TIC 1983 I l l 45 Simulated mixing of F r i s k e n Lake 112 A l E f f e c t of i n c u b a t i o n time on c h e l a t i o n of i r o n by d e s f e r a l and a f i l t r a t e from a Anabaena f l o s - a q u a e c u l t u r e 159 A2 Iron b i n d i n g c a p a c i t y (FeBC) of a f i l t r a t e from a Anabaena f l o s - a q u a e c u l t u r e 160 A3 Ir o n b i n d i n g c a p a c i t y (FeBC) s t a n d a r d i z a t i o n w i t h d e s f e r a l 161 A4 Degradation of the si d e r o p h o r e from Anabaena f l o s - aquae by a c i d or base 162 ACKNOWLEDGEMENTS x i i I t h a n k my w i f e J u n e f o r h e r s u p p o r t t h r o u g h o u t t h i s s t u d y . I a p o l o g i z e t o my d a u g h t e r s M e l i s s a a n d R a c h e l f o r t h e h o u r s t h a t I was away. I t h a n k my f r i e n d s f o r t h e i r h e l p . D r . Ken H a l l p r o v i d e d v a l u a b l e g u i d a n c e t h r o u g h o u t t h i s s t u d y . D r . Tom N o r t h c o t e p r o v i d e d v a l u a b l e a d v i c e a n d d i r e c t i o n i n t h e o r g a n i z a t i o n a n d w r i t i n g o f t h e t h e s i s . S e v e r a l s t a f f f r o m C i v i l E n g i n e e r i n g a n d W e s t w a t e r p r o v i d e d o c c a s i o n a l a s s i s t a n c e . D r s . J a n B a r i c a , K e n t B u r n i s o n , D e n n i s D e l o r m e , a n d K e i t h R o d g e r s a r r a n g e d my e d u c a t i o n a l l e a v e f r o m t h e D e p a r t m e n t o f t h e E n v i r o n m e n t . D r . G a r y L e p p a r d p r o v i d e d d i r e c t i o n w i t h t h e e l e c t r o n m i c r o s c o p e a n a l y s i s . M r s . M u d r o c h h e l p e d g u i d e t h e g e o c h e m i c a l a n a l y s i s . D r . R a l p h D a l e y a n d Mr. C o l i n G r a y p r o v i d e d a d v i c e . Mr. F r e d M a h 1 s l a b o r a t o r y a n a l y z e d some o f t h e s a m p l e s f r o m F r i s k e n L a k e . D r . A r t T a u t z a l l o w e d t h e u s e o f e q u i p m e n t f r o m t h e B r i t i s h C o l u m b i a M i n i s t r y o f E n v i r o n m e n t a n d he p r o v i d e d f i n a n c i a l s u p p o r t . Mr. K e n A s h l e y a l l o w e d t h e u s e o f 1979-80 d a t a f r o m F r i s k e n L a k e . The B.C. E n v i r o n m e n t ' s l a b o r a t o r y a n a l y z e d many o f t h e s a m p l e s f r o m B l a c k L a k e . M r . C h r i s B u l l a l l o w e d t h e u s e o f h i s l a b o r a t o r y i n P e n t i c t o n a n d a f i e l d camp a t C h a i n L a k e . Mr. Don Holmes a l l o w e d t h e u s e o f h i s l a b o r a t o r y i n K a m l o o p s . Mr. J o h n C a r t w r i g h t s u p p l i e d b o a t s a n d h e l p f o r t h e F r i s k e n L a k e e x p e r i m e n t s . S e v e r a l s t a f f o f t h e M i n i s t r y o f E n v i r o n m e n t p r o v i d e d o c c a s i o n a l a s s i s t a n c e . x i i i G l o s s a r y A n d e s i t e , a v o l c a n i c rock composed of andesine and a v a r i e t y of magnesium s i l i c a t e s . A l l e l o p a t h y , the su p p r e s s i o n of the growth or occurrence of another p l a n t or microbe by excreted chemicals. A p a t i t e , a phosphorus c o n t a i n i n g m i n e r a l group c o n t a i n i n g f l u o r a p a t i t e , C a 5 ( P 0 4 ) 3 F , or h y d r o x y l a p a t i t e C a 5 ( P 0 4 ) 3 ( O H ) . The p r i n c i p a l m i n e r a l i s a carbonate c o n t a i n i n g v a r i e t y of f l u o r a p a t i t e . Axenic, without b a c t e r i a . BOD, b i o c h e m i c a l oxygen demand. C a l c i t e , a m i n e r a l , c a l c i u m carbonate, CaCO^. Hexagonal-rhombohedral c r y s t a l s . C h i a, c h l o r o p h y l l a, a p h o t o s y n t h e t i c pigment. C h l o r i t e , an i r o n c o n t a i n i n g c l a y m i n e r a l . EDAX, energy d i s p e r s i v e a n a l y s i s f o r x-rays. EDTA, ethylenediamine t e t r a a c e t i c a c i d , a c h e l a t i n g agent. E p i l i m n i o n , the s u r f a c e l a y e r of a s t r a t i f i e d l a k e . FeBC, i r o n b i n d i n g c a p a c i t y , the a b i l i t y of org a n i c matter t o maintain i r o n i n s o l u t i o n . F i b r i l , electron-opaque, c o l l o i d a l s i z e p a r t i c l e s t h a t o f t e n form a mucilaginous sheath around c e r t a i n a l gae. Heterotrophy, the m i c r o b i a l u t i l i z a t i o n of o r g a n i c matter f o r energy and growth. Hydroxamate, a f u n c t i o n a l group of a powerful group of si d e r o p h o r e s , c o n t a i n i n g two r e a c t i v e oxygen atoms t h a t are bound t o adjacent carbon and n i t r o g e n atoms. x i v Hypolimnion, the bottom l a y e r of a s t r a t i f i e d l a k e . L i m n o c o r r a l s , l a r g e e n c l o s u r e s t h a t i s o l a t e a p o r t i o n of a l a k e . PCS, a xylene based s c i n t i l l a t i o n f l u o r produced by Amersham. P i c o p l a n k t o n , m i c r o s c o p i c u n i c e l l u l a r algae t h a t are l e s s than 2.0 u i n s i z e . P y r i t e , a mi n e r a l FeS^, t h a t forms i n anoxic environments. Sephadex, a mo d i f i e d d e x t r a n . The dextran macromolecules are c r o s s l i n k e d t o g i v e a t h r e e - d i m e n s i o n a l network; the v a r i o u s s i z e pores r e t a r d the e l u t i o n of molecules as a f u n c t i o n of s i ze. SRP, s o l u b l e r e a c t i v e phosphorus. Siderophore, a low molecular weight or g a n i c compound (500-d a l t o n s ) t h a t has a high s p e c i f i c i t y f o r s t r o n g l y c h e l a t i n g f e r r i c i r o n and t h a t i s produced by microbes s u b j e c t e d t o i r o n d e f i c i e n c y t o a s s i s t the s o l u b i l i z a t i o n and a s s i m i l a t i o n of i r o n . 1 1 INTRODUCTION The primary g o a l of t h i s r e s e a r c h was t o determine i f changes i n i r o n a v a i l a b i l i t y i n f l u e n c e the p e r i o d i c i t y of b l u e -green a l g a l growth and r e g u l a t e the blue-green a l g a l dominance over other algae. A secondary o b j e c t i v e was t o r e s o l v e i f events such as c a l c i t e p r e c i p i t a t i o n , and sediment n u t r i e n t r e l e a s e , change i r o n a v a i l a b i l i t y . The p e r i o d i c i t y of a l g a l growth i n temperate l a k e s o f t e n f o l l o w s very s i m i l a r annual c y c l e s (Lund et a l . 1963). A t y p i c a l s p e c i e s p r o g r e s s i o n , i n temperate l a k e s t h a t s t r a t i f y i n summer, i s from s p r i n g diatoms t o summer blue-green a l g a e . The sea s o n a l p e r i o d i c i t y i n the growth of phytoplankton s p e c i e s i s d r i v e n by a mixture of b i o l o g i c a l processes and p h y s i c a l p e r t u r b a t i o n s (Trimbee and H a r r i s 1984) t h a t can l e a d t o p e r i o d i c changes i n n u t r i e n t a v a i l a b i l i t y (Lund et a l . 1975). However, the f a c t o r s t h a t r e g u l a t e a l g a l s u c c e s s i o n and the r e a c t i o n s t h a t determine the t i m i n g of the p e r i o d i c i t y a re i n c o m p l e t e l y understood (Hutchinson 1967, Reynolds 1982). 1.1 F a c t o r s R e g u l a t i n g the P e r i o d i c i t y of A l g a l Growth Water column s t a b i l i t y p l a y s a major r o l e i n the de t e r m i n a t i o n of phytoplankton p e r i o d i c i t y . Reynolds e t a l . (19 82) have shown t h a t sedimentation of diatoms i n s t r a t i f i e d water i s a major f a c t o r i n the su p p r e s s i o n of diatom growth i n summer. Blue-green algae possess gas vacuoles t h a t enable them t o r e g u l a t e t h e i r v e r t i c a l d i s t r i b u t i o n (Walsby 1977); thus, they may have a c o m p e t i t i v e advantage i n a s t r a t i f i e d l a k e . 2 Buoyancy c o n t r o l p r o v i d e s blue-green a l g a e some r e g u l a t i o n of exposure t o l i g h t , temperature, and n u t r i e n t s (Ganf and O l i v e r 1982). Although the response of s p e c i e s i s v a r i a b l e , blue-green a l g a e are g e n e r a l l y enhanced by high i l l u m i n a t i o n and warm water; whereas, diatoms are more t o l e r a n t of high t u r b i d i t y and c o o l water (Hutchinson 1967). Apparently, i n some l a k e s , the v e r t i c a l movement mediated by gas vacu o l e s can a l l o w blue-green a l g a e t o move i n t o n u t r i e n t r i c h deep water and r e t u r n t o the euphotic zone (Ganf and O l i v e r 1982). The s u p p r e s s i o n of diatom growth i s a l s o r e l a t e d t o changing n u t r i e n t a v a i l a b i l i t y . Diatoms r e q u i r e much more s i l i c o n than other algae, and s i l i c o n a v a i l a b i l i t y o f t e n l i m i t s diatom growth (Lund et a l . 1975, Hurley et a l . 1985). A f t e r the v e r n a l u t i l i z a t i o n of n i t r a t e by diatoms, n i t r o g e n f i x i n g blue-green algae o f t e n r e p l a c e diatoms which cannot u t i l i z e atmospheric n i t r o g e n (Wetzel 1975). D e c l i n i n g c o n c e n t r a t i o n s of other n u t r i e n t s such as phosphorus or carbon d i o x i d e can a l s o enhance blue-green a l g a l dominance (Shapiro 1973). Blue-green algae u s u a l l y s t a r t growing when the c o n c e n t r a t i o n of i n o r g a n i c n u t r i e n t s i s minimal (Hutchinson 1967). Blue-green algae are l e s s l i k e l y t o be eaten than are other a l g a e . The l a r g e s i z e of blue-green a l g a l trichomes l i m i t s zooplankton g r a z i n g (Ferguson e t a l . 1982); however, more complex f a c t o r s a r e i n v o l v e d . T r o p h i c l e v e l s t r u c t u r e such as the d e n s i t y of p r e d a t o r y f i s h can profoundly a l t e r the amount of zooplankton a l g a l g r a z i n g (Shapiro 1980). Lynch and Shapiro (1981) have shown t h a t the r e l a t i o n s h i p between Aphanizomenon and Daphnia i s a complex symbiotic r e l a t i o n s h i p . Furthermore, blue-green a l g a e can 3 e x c r e t e o r g a n i c c o m p o u n d s t h a t s u p p r e s s z o o p l a n k t o n g r a z i n g ( G e n t i l e a n d M a l o n e y 1 9 6 9 ; L a m p e r t 1 9 8 2 ; P o r t e r 1 9 7 3 , 1 9 7 7 ; P o r t e r a n d O r c u t t 1 9 8 0 ; S n e l l 1 9 8 0 ) . T h e a b i l i t y o f b l u e - g r e e n a l g a e t o s u p p r e s s o t h e r a l g a e b y t h e e x c r e t i o n o f t o x i c c o m p o u n d s i s a n o t h e r m e t h o d o f r e d u c i n g c o m p e t i t i o n { F o g g e t a l . 1 9 7 3 , H e l l e b u s t 1 9 7 4 , E l b r a c h t e r 1 9 7 6 , K e a t i n g 1 9 7 8 , K a y s e r 1 9 7 9 , W i l s o n e t a l . 1 9 7 9 , W o l f e a n d R i c e 1 9 7 9 , C h a n e t a l . 1 9 8 0 ) . N a t u r a l w a t e r s a r e s t r i k i n g l y i n h i b i t o r y w h e n c o l l e c t e d f r o m a r e a s r i c h i n a l g a e ( H u t c h i n s o n 1 9 6 7 ) . T h e a l l e l o p a t h i c r e a c t i o n s t h a t o c c u r i n b l u e - g r e e n a l g a l b l o o m s c o n t r i b u t e t o t h e l o w - s p e c i e s d i v e r s i t y o f t h e a l g a l b l o o m s ( H u t c h i n s o n 1 9 6 7 ) . B l u e - g r e e n a l g a e a r e a l s o a b l e t o s u p p r e s s b a c t e r i a l h e t e r o t r o p h y ( C h r o s t 1 9 7 3 , 1 9 7 5 ; D e l u c c a a n d M c C r a c k e n 1 9 7 7 , R e i c h a r d t 1 9 8 1 ) . C h r o s t ( 1 9 7 5 ) f o u n d t h a t t h e i n h i b i t o r y a g e n t w h i c h b l u e - g r e e n a l g a e e x c r e t e d w a s m o r e a c t i v e i n l i g h t . H e p r o p o s e d t h a t o n c e a l g a e s e t t l e f r o m t h e e u p h o t i c z o n e , b a c t e r i a w o u l d t h e n r a p i d l y d e c o m p o s e a l g a e a n d e n h a n c e o x y g e n d e p l e t i o n i n t h e d a r k h y p o l i m n i a . I n c o n t r a s t t o t h e e p i l i m n i o n , t h e h y p o l i m n i o n d o e s n o t h a v e a r e n e w a b l e s u p p l y o f o x y g e n . A t f i r s t , i r o n s o l u b i l i t y i s i n c r e a s e d a s t h e h y p o l i m n i o n b e c o m e s a n o x i c . H o w e v e r , a s t h e h y p o l i m n i o n b e c o m e s m o r e a n o x i c , s u l p h a t e r e d u c t i o n f o r m s s u l p h i d e w h i c h p r e c i p i t a t e s f e r r o u s s u l p h i d e s ( B a n o u b 1 9 7 7 ) . A n e n h a n c e d h y p o l i m n e t i c c o n s u m p t i o n o f o x y g e n c a n r e s u l t i n a n o x i a , a n d t h e f o r m a t i o n o f p y r i t e ( F e S 2 ) w h i c h i s a s t a b l e m i n e r a l i n a n o x i c e n v i r o n m e n t s ( B e r n e r 1 9 7 1 ) . I n e n v i r o n m e n t s w i t h l i t t l e a v a i l a b l e i r o n a n d r a p i d p y r i t e f o r m a t i o n , a r e a c t i o n t h a t d i s p l a c e s h e t e r o t r o p h y f r o m t h e 4 e p i l i m n i o n t o the hypolimnion and enhances the s e a s o n a l development of anoxia would reduce the a v a i l a b i l i t y of i r o n i n the hypolimnion. The t i m i n g of anoxia development w i l l d i r e c t l y i n f l u e n c e blue-green a l g a l s u c c e s s i o n . The p e r i o d i c i t y of blue-green a l g a l growth i s o f t e n l i n k e d t o the r e c r u i t m e n t of a l g a l c e l l s from l a k e sediments (Trimbee and H a r r i s 1984). In g e n e r a l , Aphanizomenon r e c r u i t m e n t i s favoured when the bottom water i s oxygenated (Lynch 19 80, Lynch and Shapiro 19 81, Trimbee and H a r r i s 1984). Other blue-green algae are u s u a l l y s t i m u l a t e d when the bottom water approaches anoxia (Reynolds et a l . 19 81, Trimbee and H a r r i s 19 84). Lund and Reynolds (19 82) suggest t h a t M i c r o c y s t i s propagules are not s t i m u l a t e d by an i n c r e a s e i n n u t r i e n t s , but respond t o changes i n lake dynamics which permit the formation of an anoxic hypolimnion. The a c c e l e r a t i o n of a l g a l s u c c e s s i o n when a l a k e becomes s t r a t i f i e d c o u l d be r e l a t e d t o changes i n the a v a i l a b i l i t y of i r o n . At f i r s t , i r o n becomes more s o l u b l e when hypolimnia become anoxic (Stumm and Morgan 1981). The f o l l o w i n g s e c t i o n d i s c u s s e s why blue-green a l g a l response t o a changing supply of i r o n should be c o n s i d e r a b l e . 1•2 I r o n - R e q u i r i n g R e a c t i o n s of Blue-Green Algae The ecology of blue-green a l g a l blooms i s complex and much of the complexity i s r e l a t e d t o r e a c t i o n s i n v o l v i n g i r o n ( F i g . 1). As e a r l y as 1937, Guseva (1937, 1939) s t a t e d t h a t i r o n was the c o n t r o l l i n g b i o g e n i c element f o r Anabaena and Aphani zomenon. More r e c e n t l y , C l a s e n and Berhardt (1974) observed t h a t b l u e -5 KEY REACTIONS IN THIS STUDY F i g u r e 1 Key r e a c t i o n s i n t h i s study. L i n e s denote a r e a c t i o n i n f l u e n c i n g another p r o c e s s . denotes a key r e a c t i o n . 6 g r e e n a l g a e r e s p o n d e d t o i r o n e n r i c h m e n t w i t h m o r e g r o w t h t h a n g r e e n a l g a e . A t l e a s t t w o l a b o r a t o r y s t u d i e s h a v e s h o w n t h a t b l u e - g r e e n a l g a e r e q u i r e m o r e i r o n t h a n g r e e n a l g a e ( S o e d e r e t a l . 1 9 7 1 , M o r t o n a n d L e e 1 9 7 4 ) . T h e s e o b s e r v a t i o n s a r e s u p p o r t e d b y t h e f o l l o w i n g s t u d i e s d e m o n s t r a t i n g t h a t i r o n - r e q u i r i n g r e a c t i o n s a r e i m p o r t a n t i n b l u e - g r e e n a l g a e . T h e e n z y m e s r e q u i r e d f o r n i t r o g e n f i x a t i o n c o n t a i n i r o n ( W h i t e e t a l . 1 9 6 8 ) ; t h e r e f o r e , w h e n b l u e - g r e e n a l g a e f i x n i t r o g e n t h e y r e q u i r e a d d i t i o n a l i r o n ( C a r n a h a m a n d C a s t l e 1 9 5 8 , S t e w a r t 1 9 6 9 , M u r p h y 1 9 7 6 ) . S i m p s o n a n d N e i l a n d s ( 1 9 7 6 ) f o u n d t h a t a n i r o n - d e f i c i e n t A n a b a e n a g r e w p o o r l y w h e n i t h a d t o f i x n i t r o g e n . W u r t s b a u g h a n d H o m e ( 1 9 8 3 ) f o u n d t h a t i r o n a d d i t i o n i n t o C l e a r L a k e s t i m u l a t e d n i t r o g e n f i x a t i o n b y a s m u c h a s 5 0 0 % . T h e a b i l i t y o f b l u e - g r e e n a l g a e t o r e c o v e r f r o m p e r i o d s o f n i t r o g e n l i m i t a t i o n i s o f t e n d e p e n d e n t u p o n n i t r o g e n f i x a t i o n ( M u r p h y a n d B r o w n l e e 19 8 1 , B r o w n l e e a n d M u r p h y 19 8 3 ) . H e a l e y a n d H e n d z e l ( 1 9 7 6 ) h a v e r e p o r t e d t h a t d e f i c i e n c y o f a n u n k n o w n n u t r i e n t , n o t p h o s p h o r u s , w a s r e s p o n s i b l e f o r t h e p e r i o d i c i n a b i l i t y o f A p h a n i z o m e n o n t o f i x n i t r o g e n , a n d t h e b l o o m c o l l a p s e . L e a n e t a l . ( 1 9 7 8 ) s u g g e s t e d t h a t a l a c k o f i r o n m a y h a v e b e e n t h e f a c t o r t h a t r e s t r i c t e d n i t r o g e n f i x a t i o n o f A n a b a e n a b l o o m s . B l u e - g r e e n a l g a e a r e t h e o n l y a l g a e w h i c h c a n f i x n i t r o g e n . T h u s , a r e q u i r e m e n t f o r i r o n m a y m a k e i r o n c h e l a t i o n m o r e o f a k e y r e a c t i o n i n b l u e - g r e e n a l g a l b l o o m s t h a n i n o t h e r t y p e s o f a l g a l b l o o m s . T h i s h y p o t h e s i s c a n b e s u p p o r t e d b y m a r i n e s t u d i e s . S t e w a r t ( 1 9 6 9 ) h a s p r o p o s e d t h a t t h e r e q u i r e m e n t f o r a d d i t i o n a l i r o n f o r n i t r o g e n f i x a t i o n a n d t h e g e n e r a l l a c k o f i r o n i n t h e o c e a n s 7 ( R y t h e r a n d K r a m e r 1 9 6 2 , S m a y d a 1 9 6 9 , M a t s u n a g a e t a l . 1 9 8 4 , R a o a n d Y e a t s 19 8 4 ) a r e r e s p o n s i b l e f o r t h e r e l a t i v e p a u c i t y o f p l a n k t o n i c h e t e r o c y s t c o n t a i n i n g b l u e - g r e e n a l g a e i n m a r i n e e n v i r o n m e n t s . T h e r e q u i r e m e n t f o r i r o n i n t h e r e p a i r o f p h o t o o x i d a t i v e d a m a g e a l s o s e e m s i m p o r t a n t . T h e i r o n - c o n t a i n i n g e n z y m e s ( c a t a l a s e , p e r o x i d a s e , a n d s u p e r o x i d a s e d i s m u t a s e ) a r e r e q u i r e d t o r e p a i r p h o t o o x i d a t i v e d a m a g e ( W h i t e e t a l . 1 9 6 8 , N e i l a n d s 1 9 8 1 b ) . C a t a l a s e s a r e k n o w n t o b e c o m e d r a m a t i c a l l y r e d u c e d i n i r o n - d e f i c i e n t b a c t e r i a b e f o r e g r o w t h i s a f f e c t e d i n a l o w l i g h t e n v i r o n m e n t ( P r i c e 1 9 6 8 ) ; t h e s e c e l l s w o u l d b e v u l n e r a b l e t o p h o t o o x i d a t i o n . L a n g e ( 1 9 7 4 ) o b s e r v e d t h a t i r o n - d e f i c i e n t b l u e - g r e e n a l g a e w e r e k i l l e d b y d i r e c t s u n l i g h t w i t h i n t h r e e h o u r s , b u t t h a t i r o n - e n r i c h e d a l g a e w e r e s t i l l h e a l t h y a f t e r 90 h o u r s o f e x p o s u r e t o d i r e c t s u n l i g h t . P h o t o o x i d a t i v e d e a t h o f b l u e - g r e e n a l g a e h a s o f t e n b e e n p r o p o s e d a s a m e c h a n i s m o f b l o o m c o l l a p s e ( A b e l i o v i c h a n d S h i l o 1 9 7 2 , E l o f f e t a l . 1 9 7 6 , C o u l o m b e a n d R o b i n s o n 1 9 8 1 , E l o f f e t a l . 1 9 7 6 ) . I f b l u e - g r e e n a l g a e a r e m o r e s u s c e p t i b l e t o p h o t o o x i d a t i o n t h a n a r e o t h e r a l g a e , t h e s e n s i t i v i t y c o u l d b e a r e s u l t o f a n i m p a i r e d b u o y a n c y c o n t r o l . B l u e - g r e e n a l g a e a r e t h e o n l y a l g a e w i t h g a s v a c u o l e s . A r e d u c e d a b i l i t y t o r e g u l a t e g a s v a c u o l e s w o u l d l e a d t o t h e f o r m a t i o n o f s u r f a c e s c u m s a n d e x p o s u r e t o f u l l s u n l i g h t . A l a c k o f i r o n w o u l d s l o w r e s p i r a t o r y r e a c t i o n s r e q u i r i n g i r o n ( c y t o c h r o m e s , L a n k f o r d 1 9 7 3 ) a n d r e s p i r a t i o n i s r e q u i r e d t o r e g u l a t e g a s v a c u o l e s ( W a l s b y 1 9 7 7 ) . T h u s , i r o n -r e q u i r i n g e n z y m e s t h a t p r e v e n t p h o t o o x i d a t i o n ( c a t a l a s e s , p e r o x i d a s e s , a n d s u p e r o x i d e d i s m u t a s e ) m a y b e m o r e i m p o r t a n t i n 8 blue-green a l g a l blooms than i n other a l g a l blooms. Bacteriophages have a l s o been observed i n dying blue-green a l g a l blooms (Coulombe and Robinson 1981). Although other f a c t o r s i n f l u e n c e v i r a l a t t a c k s , they should occur more o f t e n d u r i n g p e r i o d s of i r o n l i m i t a t i o n . Bacteriophages enter b a c t e r i a v i a the same p r o t e i n s t h a t siderophores use t o enter the c e l l (Neilands 1981b). These p r o t e i n s are produced i n higher c o n c e n t r a t i o n s d u r i n g i r o n l i m i t a t i o n t o enhance the a s s i m i l a t i o n of siderophores (Neilands 1982). T h i s aspect of i r o n l i m i t a t i o n i s a p p a r e n t l y unresolved i n l a k e s . S e v e r a l o b s e r v a t i o n s t h a t blue-green algae produce siderophores (Smayda 1974, Murphy 1976, Murphy et a l . 1976, Simpson and N e i l a n d s 1976, Armstrong and Van Baalen 1979, B a i l e y and Taub 1980, McKnight and Morel 1980) and the r a r e p r o d u c t i o n of s i d e r ophores by other a l g a l d i v i s i o n s ( T r i c k e t a l . 1983a, 1983b) i n d i c a t e t h a t blue-green algae have a l a r g e i r o n requirement. Siderophores are e x c r e t e d by many prokaryotes when the supply of i r o n i s l i m i t e d (Neilands 1966, 1967, 1972, 1973; Lankford 19 73; Emery 19 82). Siderophores are low molecular weight o r g a n i c compounds t h a t s e l e c t i v e l y and s t r o n g l y c h e l a t e i r o n ; they e i t h e r r e a d i l y exchange the c h e l a t e d i r o n w i t h p r o t e i n r e c e p t o r s s i t e s on m i c r o b i a l c e l l s or they enter the c e l l b e f o r e r e l e a s i n g the i r o n . Only a few s t u d i e s have used r i g o r o u s chemical a n a l y s i s t o i d e n t i f y a l g a l s i d e r o p h o r e s . Simpson and N e i l a n d s (1976) were f o r t u n a t e i n t h a t the Anabaena s p e c i e s they s t u d i e d produced a compound (Schizokinen) t h a t e a r l i e r s t u d i e s i n t h e i r l a b o r a t o r y had i s o l a t e d from b a c t e r i a . T r i c k and coworkers (1983b) found 9 hydroxamate type siderophores i n i s o l a t e s from marine algae. Other s t u d i e s have r e l i e d upon b i o a s s a y s , r a d i o c h e m i c a l assays, or chemical a n a l y s i s of f u n c t i o n a l groups t o d e t e c t s i d e r o p h o r e s . In a l a b o r a t o r y , Murphy (1976) used the l a t t e r combination t o study s i d e r o p h o r e s . Sephadex chromatography w i t h 55Fe was able t o d e t e c t , p a r t i a l l y p u r i f y , and determine the approximate molecular weight of c h e l a t o r s . Compounds with no c h e l a t i o n such as g l y c i n e could not t r a n s m i t i r o n through a Sephadex column, but a c h e l a t o r such as c i t r a t e c o u l d . Chemical a n a l y s i s i n d i c a t e d t h a t the c h e l a t o r s e x c r e t e d by Scenedesmus b a s i l i e n s i s and Anabaena c y l i n d r i c a were c y c l i c p e p t i d e s ; these compounds had molecular weights around 30 0 d a l t o n s . Anabaena flos-aquae produced a c h e l a t o r with a molecular weight of about 900 d a l t o n s t h a t contained a hydroxamate group. The c h e l a t o r from Anabaena flo s - a q u a e formed a b r i c k red complex when s a t u r a t e d with i r o n . The c h e l a t o r s from Anabaena f l o s - a q u a e , Anabaena c y l i n d r i c a and Scenedesmus b a s i l i e n s i s were f u r t h e r s t u d i e d i n t h i s t h e s i s . The hydroxamate p r o d u c t i o n by Anabaena flos-aquae was r a d i c a l l y d i f f e r e n t from the hydroxamate p r o d u c t i o n i n T r i c k ' s study ( T r i c k et a l . 1983a). Murphy (1976) observed t h a t the c o n c e n t r a t i o n of e x c r e t e d hydroxamate i n c r e a s e d q u i c k l y up t o about ten days, s l o w l y i n c r e a s e d f o r another ten days and then remained co n s t a n t . T r i c k et a l . observed t h a t hydroxamate was e x c r e t e d i n a p u l s e and stayed i n s o l u t i o n f o r o n l y a few days. The s e l e c t i v e advantage t h a t blue-green algae have over green algae i n high-pH water (Shapiro 1984) may be r e l a t e d d i r e c t l y t o i r o n a s s i m i l a t i o n . Iron i s l e s s s o l u b l e i n high-pH water (Stumm and Morgan 19 81); thus, more complex i r o n -10 assimilation mechanisms may be required for growth in high pH water. 1.2.1 Iron Limitation Iron l i m i t a t i o n often has been observed in lakes without calcium carbonate p r e c i p i t a t i o n (Goldman 1966, Bernhardt et a l . 1971, Sakamoto 1971, A l l e n 1972, Clasen and Bernhardt 1974, Gerhold 1975, Lund et a l . 1975, Thurlow et a l . 1975, Happey-Wood and Pentecost 1981, Lin and Schelske 19 81, Paerl 19 82a). Complexation of iron with humic matter i s the best documented reaction mediating iron l i m i t a t i o n in softwater (Jackson and Hecky 1980). Although the r e a c t i v i t y and a v a i l a b i l i t y of humic iron varies greatly among lakes, in many lakes, most of the humic iron i s unreactive (Shapiro 1969). Recent studies have shown that iron i s rapidl y coated by humic acids (Picard and Felbeck 1976, Tipping 19 81, Baccini et a l . 19 82); presumably in some lakes, the coatings of humic acids reduce the r e a c t i v i t y and a v a i l a b i l i t y of iron. The a v a i l a b i l i t y of humic iron i s d i f f i c u l t to measure. Although a chemical assay, the ferrigram, can determine the r e a c t i v i t y and a v a i l a b i l i t y of humic iron in some lakes (Shapiro 1969), i t i s u n l i k e l y that any chemical f r a c t i o n a t i o n can resolve the complexities of a l l humic-iron chemistry. For example, Powell et a l . (1980) and Akers (1983) discovered a v a r i e t y of siderophores in s o i l extracts. Previously, these siderophore i s o l a t i o n s would have been c a l l e d f u l v i c or humic acids. The reactions of siderophores can be.complex. The a v a i l a b i l i t y of siderophore iron can be r e s t r i c t e d to only some species (O'Brien and Gibson 1970). Some siderophores have 11 a n t i b i o t i c properties; they mimic iron-sequestering siderophores (Neilands 1981a). The iron biochemistry of lakes should be as complex as s o i l iron biochemistry. Hardwater lakes that p r e c i p i t a t e calcium carbonate have often been c i t e d as being iron limited (Schelske 1961, 1962; Schelske et a l . 1963; Wetzel 1965, 1966, 1968; Lange 1971; Home 1974; Murphy and Lean 1975; Murphy et a l . 1976; Elder 1977; Elder and Home 1977; Wurtsbaugh and Home 1983). The rel a t i o n s h i p between calcium carbonate p r e c i p i t a t i o n and iron l i m i t a t i o n i s expected due to chemical and biochemical reasons. Calcium carbonate p r e c i p i t a t i o n can in t e r f e r e with iron uptake. Iron i s less soluble at the high pH found before and during calcium carbonate p r e c i p i t a t i o n (Stumm and Morgan 1981). Also, the cop r e c i p i t a t i o n of dissolved organic matter with calcium carbonate w i l l reduce the s o l u b i l i t y of iron in the euphotic zone (Wetzel 1975). Szaniszlo et a l . (1981) found that the addition of calcium carbonate to culture medium enhanced the production of siderophores, but that calcium concentration alone had no e f f e c t upon siderophore production. Other studies have indicated that some siderophores have an appreciable a f f i n i t y for chelation of calcium (Hider et a l . 1982). Perhaps calcium or calcium carbonate can suppress some limnetic iron-siderophore reactions. Calcium i s often concentrated on the surface of aquatic bacteria by excretion from microbial c e l l s (Morita 1980). The surface of blue-green algae i s often composed of microenvironments created by a covering of f i b r i l s (Leppard et a l . 1977; Leppard 1984a, 1984b). Thus, the concentration of calcium at the s i t e s of iron 12 uptake c o u l d be much higher than bulk water chemistry would suggest. A su p p r e s s i o n by c a l c i u m of i r o n a s s i m i l a t i o n would be much s t r o n g e r i n hardwater l a k e s than i n so f t w a t e r l a k e s . C alcium carbonate p r e c i p i t a t i o n i s c l o s e l y l i n k e d t o i r o n b i ogeochemistry v i a a l g a l p r o d u c t i v i t y . Any enhancement of a l g a l p r o d u c t i v i t y would i n c r e a s e the pH and carbonate c o n c e n t r a t i o n of the e p i l i m n i o n . Thus, i n c r e a s e d i r o n a v a i l a b i l i t y i n an i r o n - l i m i t e d l a k e should enhance a l g a l p r o d u c t i v i t y and c o u l d r e s u l t i n p r e c i p i t a t i o n of c a l c i u m carbonate. Small changes i n carbonate e q u i l i b r i a c o u l d i n f l u e n c e s e v e r a l l i m n e t i c p rocesses t h a t a re not d i r e c t l y r e l a t e d t o i r o n chemistry ( F i g . 1 ) . Cal c i u m carbonate p r e c i p i t a t i o n can p r e c i p i t a t e a l g a e (Rossknecht 19 80) and phosphorus (Murphy et a l . 1983) from the e p i l i m n i o n . These two processes can suppress the c o n c e n t r a t i o n of algae i n hardwater l a k e s i n summer ( S t a u f f e r 1985). The i n d u c t i o n of carbonate p r e c i p i t a t i o n may be the major l i m n e t i c r e a c t i o n t h a t m o d i f i e s the e f f e c t s of a change i n i r o n a v a i l a b i l i t y . S t a u f f e r (1985) has a l s o proposed t h a t a l a c k of mobile i r o n i n c a l c a r e o u s areas r e s u l t s i n too l i t t l e i r o n t o c o n t r o l phosphorus chem i s t r y . In other areas t h a t are r i c h i n i r o n , geochemical phosphorus r e a c t i o n s are c o n t r o l l e d by i r o n r e a c t i o n s (Bostrum 1984, Ryding 1985). As w e l l as t h i s s p a t i a l v a r i a t i o n i n the c o n t r o l of phosphorus s o l u b i l i t y and of i r o n a v a i l a b i l i t y , t h e r e may be a temporal v a r i a t i o n i n i r o n a v a i l a b i l i t y . Stumm and Morgan (19 81) proposed t h a t the degree of i r o n l i m i t a t i o n should v a r y among lak e s and t h a t i r o n a v a i l a b i l i t y should i n f l u e n c e the t i m i n g of an a l g a l bloom, not the biomass. The temporal v a r i a t i o n i n i r o n l i m i t a t i o n was demonstrated 13 i n a study by Murphy (19 76). In the Bay of Quinte, i r o n l i m i t a t i o n t h a t was d e t e c t e d by r a p i d i r o n uptake and the presence of s i d e r o p h o r e s , l a s t e d f o r l e s s than a week. However, the i r o n l i m i t a t i o n t h a t o c c u r r e d d u r i n g a blue-green a l g a l bloom was f o l l o w e d by a f o u r week p e r i o d of reduced a l g a l biomass. Seasonal changes i n i r o n geochemistry and the p e r i o d i c i t y of blue-green al g a e are both w e l l known, but the processes are u s u a l l y l i n k e d t o phosphorus a v a i l a b i l i t y . I r o n i s s t a b l e i n o x i d i z e d sediments (Williams et a l . 1971, Shukla et a l . 1971). However, i r o n and phosphorus are o f t e n r e l e a s e d from l a k e sediments when the oxygen c o n c e n t r a t i o n i s d e p l e t e d . Much more i s known about th e seasonal r e l e a s e of phosphorus from l a k e sediments than about i r o n r e l e a s e ; however, i n s e v e r a l s t u d i e s i r o n and phosphorus were r e l e a s e d i n synchrony from the sediments (Mortimer 1941, 1942; Banoub 1977; L i j k l e m a 1977). In some l a k e s , the s o l u b i l i t y of phosphorus has been shown t o be c o n t r o l l e d by the p r e c i p i t a t i o n of f e r r i c phosphate ( B i r c h 1976). In extreme anoxia, f e r r i c s u l p h i d e i s p r e c i p i t a t e d l e a v i n g phosphorus i n s o l u t i o n (Banoub 1977). Ryding (19 85) has proposed t h a t o r g a n i c i r o n c h e l a t o r s may enhance the s o l u b i l i t y of phosphorus by complexing i r o n and thus p r e v e n t i n g t h e p r e c i p i t a t i o n of f e r r i c phosphate. Thus, the geochemistry of phosphorus may be an e x c e l l e n t s i g n a l of i r o n a v a i l a b i l i t y . L i t t l e i s known about the c o u p l i n g of i r o n r e l e a s e from l a k e sediments and the a v a i l a b i l i t y of i r o n as a m i c r o n u t r i e n t . To r e s o l v e the impact of sediment i r o n r e l e a s e from seasonal changes i n phosphorus a v a i l a b i l i t y would r e q u i r e a l a k e w i t h high phosphorus c o n c e n t r a t i o n s t o s a t u r a t e b i o l o g i c a l 14 requirements. The goals of t h i s study were t o determine i f the seasonal changes i n blue-green a l g a l growth were r e l a t e d t o changes i n i r o n a v a i l a b i l i t y and t o r e s o l v e i f the blue-green a l g a l dominance over other microbes c o u l d be mediated by a si d e r o p h o r e r e g u l a t i o n of i r o n a v a i l a b i l i t y . To achieve t h i s o b j e c t i v e , f i e l d s t u d i e s were conducted i n t h r e e h y p e r t r o p h i c l a k e s t h a t had high phosphorus c o n c e n t r a t i o n s . The seasonal changes i n water chemistry and phytoplankton p e r i o d i c i t y were s t u d i e d . A s e r i e s of e n c l o s u r e experiments were conducted t o a s s i s t the i n t e r p r e t a t i o n of l i m n e t i c o b s e r v a t i o n s . Short-term b o t t l e i n c u b a t i o n s were used f o r primary p r o d u c t i o n and heterotrophy assays. For long-term i n c u b a t i o n s , l a r g e r e n c l o s u r e s ( l i m n o c o r r a l s ) were used t o avoid containment a r t i f a c t s . Long-term i n c u b a t i o n s i n s m a l l e n c l o s u r e s are prone t o s e v e r a l a r t i f a c t s . The high s u r f a c e area may s t i m u l a t e m i n e r a l p r e c i p i t a t i o n and growth of s p e c i e s t h a t are not common i n the open water. Moreover, the i s o l a t i o n of the water prevents gas exchange which i n t u r n can l e a d t o u n n a t u r a l changes i n pH. Li m n o c o r r a l s a l s o allowed o b s e r v a t i o n of a l g a l s u c c e s s i o n . Large e n c l o s u r e s can s t i l l have enhanced m i n e r a l p r e c i p i t a t i o n on the w a l l s . Thus, t o c o n f i r m the long-term responses, whole-lake ma n i p u l a t i o n s were a l s o conducted. The i n t e r p r e t a t i o n of the long-term responses t o i r o n enrichment r e q u i r e d an e v a l u a t i o n of c a l c i u m carbonate p r e c i p i t a t i o n . My s t u d i e s were i n t e g r a t e d with d e t a i l e d l a b o r a t o r y b i o a s s a y and chemical s t u d i e s where s p e c i f i c i n t e r a c t i o n s c o u l d be i n v e s t i g a t e d under more c o n t r o l l e d c o n d i t i o n s . 15 2 METHODS 2.1 Study Area The Thompson P l a t e a u i s l o c a t e d i n s o u t h - c e n t r a l B r i t i s h Columbia ( F i g . 2 ) . T h i s area has a temperate c o n t i n e n t a l c l i m a t e . The p l a t e a u i s i n the rainshadow of the Cascade Mountains which border the p l a t e a u on the west. R a i n f a l l v a r i e s from 75 cm on the highlands t o l e s s than 35 cm i n the s e m i a r i d v a l l e y s (MacMillan-B l o e d e l 1972). Two s t a t i o n s at Hedley, 48 km west of Black Lake, r e c e i v e a mean of 29 cm or 54 cm of r a i n a year a t e l e v a t i o n s of 425 m and 1,738 m, r e s p e c t i v e l y ( E n v i r o n . Can.). More r a i n and l e s s e v a p o r a t i o n a t higher e l e v a t i o n s r e s u l t s i n r u n o f f t h a t s u p p l i e s much of the water requirements of the v a l l e y s . B l a ck, Chain, and F r i s k e n lakes were the main study s i t e s ( F i g . 2, Tab l e 1). These l a k e s a l l had blue-green a l g a l blooms, high phosphorus c o n c e n t r a t i o n s , and f i s h k i l l s . The water flow i n midsummer ceased a t a l l s i t e s ; thus, the changes i n water chemistry d u r i n g summer were l a r g e l y c o n t r o l l e d by processes i n the l a k e s . Some samples were a l s o c o l l e c t e d from Yellow Lake (next t o Black Lake) and from Roche Lake (next t o F r i s k e n Lake). 2.1.1 Black Lake Black and Yellow l a k e s a r e s i t u a t e d upon a h i g h l a n d 16 km S.W. of P e n t i c t o n (Northcote and Halsey 1969, Northcote 1980). The n e i g h b o r i n g mountains r i s e 750 m above a steep g l a c i a l v a l l e y (Mathews 1944, L i t t l e 1961, Nasmith 1961). The s u r f a c e rock around Black Lake i s predominately an a p a t i t e r i c h Eocene a n d e s i t e (Bostock 1966, Church 1973, Parsons 1974). (Ti contour i n t e r v a l s i n meters 17 Figure 2 continued 18 E r o s i o n of a s u r f a c e l a y e r of a porous v o l c a n i c rock i s v e r y pronounced. Black Lake had s e v e r a l a spects t h a t made i t a p p r o p r i a t e f o r study: s m a l l s i z e , good road access , power supply, and i n t e n s e e u t r o p h i c a t i o n without anthropogenic wastes T a b l e 1 Some P h y s i c a l and Chemical F e a t u r e s of the Study Lakes Lake C h a r a c t e r i s t i c B l a c k Chain F r i s k e n L a t i t u d e 490°20' 490°42' 500°27' Longitude 1190°45' 1200 o16" 1200° 8' E l e v a t i o n (m) 750 1006 1138 Drainage Area (ha) 1532 4060 267 Lake Area (ha) 4 43.7 33.8 Max. Depth (m) 9 7.1 11 Mean Depth (m) 4.5 6.1 5.5 * R e t e n t i o n (yr) 1-5 0.3 20 C o n d u c t i v i t y 506 220 374 S p r i n g Fe (ug/L) 20 370 20 ** S p r i n g TP (ug/L) 300 20 300 ** Summer/Spring TP 0.2-0.5 10 0.5 IAP/Ksp C a C 0 3 & 7-23 2-10 5-20 * A l l v a l u e s a re e p i l i m n e t i c . R e t e n t i o n time i n Black and F r i s k e n l a k e s i s estimated from s e v e r a l measurements of stream flow. The hydrology of Chain Lake i s c o n t i n u o u s l y monitored by the B r i t i s h Columbia M i n i s t r y of Environment. uS/cm at 25°C. ** & TP = t o t a l phosphorus. Range of c a l c i u m carbonate s u p e r s a t u r a t i o n i n midsummer. 19 2.1.2 F r i s k e n Lake F r i s k e n Lake i s a h y p e r t r o p h i c l a k e 30 km S.S.E. from Kamloops. T h i s l a k e i s s i t u a t e d upon a p o o r l y d e f i n e d v o l c a n i c i n t r u s i o n c a l l e d the Wildhorse Mountain B a t h o l i t h t h a t formed d u r i n g the J u r a s s i c or post-lower Cretaceous p e r i o d ( C o c k f i e l d 1947, 1948). The rocks of t h i s v o l c a n i c i n t r u s i o n are r i c h i n a p a t i t e . F r i s k e n Lake was chosen f o r study because i t has a longer r e s i d e n c e time than B l a c k Lake; thus, the long-term e f f e c t s of a whole l a k e experiment c o u l d be monitored. 2.1.3 Chain Lake Chain Lake i s s i t u a t e d 45 km N.E. of P r i n c e t o n . The l a k e l i e s upon a J u r a s s i c v o l c a n i c i n t r u s i o n t h a t i s s i m i l a r i n age and composition t o the i n t r u s i v e s around F r i s k e n Lake (Rice 1946). The p l a t e a u around Chain Lake i s 300 m h i g h e r than the other s i t e s ; thus, more r a i n f a l l should f a l l i n the Chain Lake b a s i n . The o n l y data a v a i l a b l e i s f o r the v a l l e y which r e c e i v e s a mean r a i n f a l l of 52.7 cm a year ( E n v i r . Can. 26 years of d a t a ) . Two p r o p e r t i e s of Chain Lake support the hypothesis t h a t Chain Lake r e c e i v e s more r a i n f a l l than Black and F r i s k e n l a k e s ; the c o n d u c t i v i t y i s h a l f , and the r e s i d e n c e time of Chain Lake water i s o n l y a t e n t h t h a t of the other l a k e s (Table 1). Chain Lake i s s i m i l a r t o the other s i t e s i n t h a t i t r e c e i v e s a high phosphorus l o a d i n g from the n a t u r a l weathering of a p a t i t e (Murphy 1985). In s p i t e of having s i m i l a r geology, Chain Lake has a much higher i r o n c o n c e n t r a t i o n than the other two l a k e s (Table 1). The water e n t e r i n g Chain Lake i s r i c h i n i r o n (Water I n v e s t i g a t i o n s Branch of B r i t i s h Columbia, WIB, 1977). Chain Lake was chosen f o r study so t h a t the biogeochemistry of l a k e s w i t h low i r o n c o n c e n t r a t i o n s (Black and F r i s k e n l a k e s ) c o u l d be compared t o an i r o n - r i c h l a k e w i t h a s i m i l a r h a b i t a t . 2.2 F i e l d Experiments Whole-lake p e r t u r b a t i o n s , l a r g e e n c l o s u r e , and b o t t l e i n c u b a t i o n experiments were conducted i n Black, F r i s k e n and Chain l a k e s (Table 2). T a b l e 2 Timing and S i t e s of Experiments Year S i t e Black L. F r i s k e n L. Chain L. L a b o r a t o r y 1979 1980 m o n i t o r i n g a e r a t i o n m o n itoring a e r a t i o n Fe-EDTA l i m n o c o r r a l s c h e l a t o r i s o l a t i o n 1981 1982 F e - c i t r a t e l i m n o c o r r a l s i n i t i a l FeBC assay 1983 1984 Ca(OH) a d d i t i o n Ca(OH) a d d i t i o n l a k e monitoring lake m o n i t o r i n g improved FeBC assay FeBC I r o n - B i n d i n g C a p a c i t y , the a b i l i t y of o r g a n i c matter t o maintain i r o n i n s o l u t i o n 2.2.1 Sample C o l l e c t i o n Water samples were c o l l e c t e d with a t h r e e - l i t e r Van Dorn water sampler at s t a t i o n s l o c a t e d i n the deepest p a r t of the l a k e s . Samples were f i l t e r e d w i t h i n s i x hours. In 1983, samples 21 were c o l l e c t e d from two s t a t i o n s i n F r i s k e n Lake which were 100 m a p a r t . The two s i t e s r e p l i c a t e d very w e l l ; thus, i n 1984, samples were c o l l e c t e d from o n l y one s t a t i o n . Chain Lake water samples were c o l l e c t e d at one s t a t i o n i n the middle of the l a k e between the p r o v i n c i a l campground and the d i v e r s i o n . As WIB (1977) had found, t h i s s i t e appeared t o r e p r e s e n t a l l of Chain Lake w e l l . 2.2.2 A e r a t i o n of B l a c k Lake Black Lake was d i v i d e d i n t o two equal p a r t s by a Fabrene c u r t a i n (woven p o l y e t h y l e n e ) as p a r t of a h y p o l i m n e t i c a e r a t i o n study of the western s i d e of the l a k e (Ashley 1983). As h l e y aerated the l a k e t o a l l o w t r o u t t o s u r v i v e . The a e r a t i o n experiment was continued so t h a t samples c o u l d be c o l l e c t e d t o monitor the e f f e c t of a change i n oxygen c o n c e n t r a t i o n on i r o n chemistry, and m i c r o b i a l responses t o a change i n i r o n a v a i l a b i l i t y . 2.2.3 Chemical Treatments of F r i s k e n Lake Calcium hydroxide was added t o F r i s k e n Lake t o induce p r e c i p i t a t i o n of c a l c i u m carbonate; the n a t u r a l p r e c i p i t a t i o n of c a l c i u m carbonate i s too slow and u n p r e d i c t a b l e f o r study. The i n d u c t i o n of c a l c i u m carbonate p r e c i p i t a t i o n allowed more d e t a i l e d study of the e f f e c t s of carbonate p r e c i p i t a t i o n on n u t r i e n t ( i r o n and phosphorus) a v a i l a b i l i t y and a l g a l p r o d u c t i v i t y . A s l u r r y maker on a barge was used t o add the lime t o the l a k e (Murphy et a l . 1985). In 1983, lime was added t o the l a k e d u r i n g t h r e e r e l a t i v e l y dry p e r i o d s : June 16-17, 8.4 tonnes; 22 J u l y 26-27, 7.2 tonnes; and August 16-17, 7.2 tonnes ( t o t a l 22.8 t o n n e s ) . In 1984, lime was added i n one t r i p : May 26-28, 16 tonnes. The l a k e r e c e i v e d a t o t a l of 38.8 tonnes of C a f O H ^ over 2 the two year p e r i o d (114 g/m , 20.9 mg/L). The degree of c a l c i u m carbonate s a t u r a t i o n was determined w i t h the computer program PHREEQE (Parkhurst et a l . 1980). PHREEQE can perform complex t i t r a t i o n s w i t h d a t a from each l a y e r of a l a k e and then s i m u l a t e d e s t r a t i f i c a t i o n or c a l c i t e p r e c i p i t a t i o n . The program used the Debye-Huckle method t o c o r r e c t f o r i o n i c s t r e n g t h (Berner 1971). 2.2.4 L i m n o c o r r a l Experiments Large c o n t a i n e r s (2.0 m i n diameter, 5 or 7 m deep and 15,000 or 21,700 L) of t r a n s p a r e n t woven p o l y e t h y l e n e were used t o t e s t the long-term e f f e c t of n i t r a t e , EDTA, Fe-EDTA, c i t r a t e , or F e - c i t r a t e on a l g a l p r o d u c t i v i t y , c a l c i u m carbonate p r e c i p i t a t i o n , and phosphorus s o l u b i l i t y . See Appendix 1 f o r d e t a i l s . 2.2.5 Small I n - S i t u Incubations 2.2.5.1 Calcium C h l o r i d e Experiment To f a c i l i t a t e o b s e r v a t i o n s of c a l c i u m carbonate p r e c i p i t a t i o n , water samples were incubated with c a l c i u m c h l o r i d e . Water samples were c o l l e c t e d 1.0 meter from the s u r f a c e of Black Lake on Aug. 24 and Aug. 25, 1983, and e n r i c h e d with 10 and 20 mg/L of C a C l 2 r e s p e c t i v e l y . Samples with each treatment 2 were incubated e i t h e r i n f u l l s u n l i g h t i n the l a k e (1000 u E i m-23 1 - 2 - 1 s- ) or i n the shade (10 u.Ei m s ) f o r ten hours. A l l i n c u b a t i o n s were done i n open t e n - l i t e r P l e x i g l a s c y l i n d e r s . A f t e r the i n c u b a t i o n , the v e s s e l s were s t i r r e d and subsamples were immediately f i l t e r e d through 0.45 urn M i l l i p o r e c e l l u l o s e a c e t a t e f i l t e r s f o r a n a l y s i s of s o l u b l e r e a c t i v e phosphorus (SRP) and d i s s o l v e d c a l c i u m . Subsamples f o r SRP were analyzed w i t h i n t h r e e hours of c o l l e c t i o n . Both u n f i l t e r e d and f i l t e r e d samples were t r e a t e d w i t h 1.0 ml of 50% HC1 per 100 ml of sample and l a t e r analyzed f o r c a l c i u m . To f a c i l i t a t e c o l l e c t i o n of p a r t i c l e s i n the C a C ^ experiment, t h e v e s s e l s were l e f t u n d i s t u r b e d f o r 14 h. By deca n t i n g the s u r f a c e water, the s e t t l e d p a r t i c l e s c o u l d be con c e n t r a t e d 20 f o l d . F i f t y m i l l i l i t e r s of the c o n c e n t r a t e were f i l t e r e d through Whatman GF/C f i l t e r s . The f i l t e r s were immediately p l a c e d i n 50 ml s y r i n g e s with 20.0 ml of d i s t i l l e d water and 10.0 ml of e i t h e r CG^ or a i r . The s y r i n g e s were shaken f o r 20 s, and the gases were purged and r e p l a c e d with f r e s h C 0 2 or a i r . The s y r i n g e s were incubated at 40°C f o r one hour. The s y r i n g e samples were f i l t e r e d through Whatman GF/C f i l t e r s . SRP analyses were immediately done, and samples f o r d i s s o l v e d c a l c i u m were preserved f o r l a t e r a n a l y s i s . 2.2.5.2 Primary P r o d u c t i o n Primary p r o d u c t i v i t y response t o i r o n and c h e l a t o r s (EDTA, desferrioxamine-B, and t h r e e a l g a l c h e l a t o r s ) was measured by a l g a l uptake of 1.48 x 10 6 Bq of 1 4C-HC0 3 i n 300-ml BOD b o t t l e s t h a t were incubated i n the l a k e . R e l a t i v e l y long i n c u b a t i o n s (48 24 h) were used t o a v o i d m i s l e a d i n g s i g n a l s t h a t short-term i n c u b a t i o n s can produce (Lean and P i c k 1981). Dark b o t t l e s were used f o r c o n t r o l s . Samples were f i l t e r e d (0.45 urn M i l l i p o r e f i l t e r s ) , t r e a t e d with 1 ml of 0.5 N HC1 f o r 24 h, d r i e d (110°C), d i s s o l v e d i n PCS f l u o r (Amersham), and counted with a Beckman s c i n t i l l a t i o n counter. 2.2.5.3 Heterotrophy 14 M i c r o b i a l uptake of c i t r a t e was measured with C - c i t r a t e . Two a c t i v e and one c o n t r o l ( k i l l e d with 0.2 ml f o r m a l i n ) samples were e n r i c h e d with 7.11 ug C/L of 1,5 1 4 C - c i t r i c a c i d ( s p e c i f i c a c t i v i t y , 4.11 GBq/mM, Amersham). T e n - m i l l i l i t e r samples were incubated i n s i t u , i n 20.0 ml p l a s t i c s y r i n g e s f o r 2.0 h. A f t e r the i n c u b a t i o n , a f i l t e r assembly c o n t a i n i n g a 0.2 urn, 25 mm membrane f i l t e r was a t t a c h e d t o the s y r i n g e and the sample was f i l t e r e d i n t o the r e s e r v o i r tubes of a CC^ purging apparatus. The f i l t e r s were washed with 10.0 ml of d i s t i l l e d water, t r a n s f e r r e d t o s c i n t i l l a t i o n v i a l s and immediately i n a c t i v a t e d with 10.0 ml of s c i n t i l l a t i o n f l u i d . The f i l t r a t e i n the p u r g i n g apparatus was a c i d i f i e d by i n j e c t i n g 0.2 ml of 2 N ^SO^ through the stopper. The samples were purged w i t h C 0 2 ~ f r e e a i r from an aquarium pump. The 1 4C-C02 l i b e r a t e d from the s o l u t i o n was trapped i n 5.0 ml of s c i n t i l l a t i o n s o l u t i o n c o n t a i n i n g 0.3 ml of hyamine hydroxide i n a s c i n t i l l a t i o n v i a l connected t o a V i g r e a u x column. The hyamine hydroxide i s an e f f e c t i v e absorber of CO^; the Vigreaux column p r o v i d e s a l a r g e s u r f a c e area f o r good a b s o r p t i o n of carbon d i o x i d e . A f t e r purging f o r 20 min, the Vigreaux columns were washed with 5.0 ml of s c i n t i l l a t i o n s o l u t i o n . The samples were counted on a Beckman Isocap s c i n t i l l a t i o n counter and c o r r e c t e d f o r background and f o r quenching u s i n g an e x t e r n a l standard. The c o n t r o l samples were s u b t r a c t e d from the a c t i v e samples t o determine the a c t i v e uptake. The same procedure was 14 14 used f o r C-glucose and C-acetate uptake s t u d i e s . Recovery of 14 14 C from t e s t samples of C-bicarbonate i n the purging apparatus was 100.5% (n=10). 2.3 Laboratory S t u d i e s and A n a l y s i s of F i e l d Samples 2.3.1 Chemical A n a l y s i s Oxygen and temperature were measured with YSI meters t h a t were c a l i b r a t e d by Winkler t i t r a t i o n (APHA 1976). The pH was measured with a Corning pH meter. Inorganic carbon was measured by gas chromatography at the lake ( S t a i n t o n e t a l . 1977). Samples f o r c h l o r o p h y l l a a n a l y s i s were f i l t e r e d onto GF/C f i l t e r s w i t h i n two hours of c o l l e c t i o n , f r o z e n , and analyzed by DMSO e x t r a c t i o n (Burnison 1980). Although GF/C f i l t e r s are unable t o t r a p p i c o p l a n k t o n , t h i s l i m i t a t i o n was not important f o r a t l e a s t the summer p e r i o d . D i r e c t o b s e r v a t i o n with a f l u o r e s c e n t microscope of water samples t h a t were concentrated onto 0.2 jam Nuclepore f i l t e r s and s t a i n e d with a c r i d i n e orange (Daley 1979) i n d i c a t e d t h a t p i c o p l a n k t o n were on l y a minor c o n s t i t u e n t of the a l g a l biomass. For a l g a l enumeration and i d e n t i f i c a t i o n , samples were preserved with Lugol's s o l u t i o n (Vollenweider et a l . 1974) and s e t t l e d with the Ultermohl (19 31) technique. 26 I r o n content i n the samples was measured by a m o d i f i e d bathophenanthroline method ( S t r i c k l a n d and Parsons 1972). Samples f o r i r o n a n a l y s i s were f i l t e r e d at the l a k e through Whatman GF/C f i l t e r s . The f i l t r a t e was a c i d i f i e d with 2.0 ml of c o n c e n t r a t e d HNO^ per 500 ml of sample and r e f r i g e r a t e d u n t i l analyzed. P a r t i c u l a t e i r o n samples were kept f r o z e n u n t i l a n alyzed. Samples f o r c i t r a t e a n a l y s i s were p r e s e r v e d by f r e e z i n g . C i t r a t e was analyzed as the t r i m e t h y l s i l y l d e r i v a t i v e by a gas chromatography method (Stumpf and B u r r i s 1979). The method was changed i n the f o l l o w i n g ways; the samples were e l u t e d through an anion exchange r e s i n t o t r a p c i t r a t e (Dowex AG1-X8, formate form), c i t r a t e was e l u t e d from the column with 1.0 M formic a c i d , the e l u a n t was f r e e z e - d r i e d and resuspended i n 0.1 M NH^OH, d r i e d i n a d e r i v a t i z a t i o n v i a l at 50°C w i t h argon, and d e r i v a t i z e d with BSTFA [ N , 0 - b i s ( t r i m e t h y l s i l y l ) t r i f l u o r o a c e t a m i d e ] and p y r i d i n e . Some a l g a l s i d e r o p h o r e i s o l a t e s were hydrolyzed and analyzed f o r bound hydroxamate groups (Csaky 1948). A r s e n i c and s i l i c a were measured by the s i l v e r d i e t h y l d i t h i o c a r b a m a t e method and h e t e r o p o l y b l u e method r e s p e c t i v e l y (APHA 1976). Phosphorus was measured as t o t a l P and t o t a l d i s s o l v e d P a f t e r p e r c h l o r i c a c i d d i g e s t i o n . Samples f o r n i t r a t e , ammonium, n i t r i t e , d i s s o l v e d o r g a n i c n i t r o g e n , p a r t i c u l a t e n i t r o g e n , p a r t i c u l a t e carbon, d i s s o l v e d i n o r g a n i c carbon, t o t a l i n o r g a n i c carbon, c h l o r i d e , s u l p h a t e , c a l c i u m , magnesium, potassium and sodium were prepared i n a f i e l d l a b o r a t o r y , and shipped on i c e t o a l a b o r a t o r y f o r a n a l y s i s u s i n g Technicon a u t o a n a l y z e r methods (Environment Canada 1979). 2 7 2 . 3 . 2 3 2 P - S R P A n a l y s i s S t r e a m a n d e x t r a c t s o f s u r f a c e r o c k f r o m t h e B l a c k L a k e a r e a w e r e a n a l y z e d b y g e l c h r o m a t o g r a p h y t o s e p a r a t e o r t h o p h o s p h o r u s f r o m p o l y m e r i z e d - P o r o r g a n i c - P o f a d i f f e r e n t m o l e c u l a r s i z e ( L e a n 1 9 7 3 ) . R o c k s w e r e e x t r a c t e d b y f i r s t g r i n d i n g t h e m w i t h a m o r t a r a n d p e s t l e a n d t h e n e x t r a c t i n g 3 . 0 g o f p o w d e r w i t h 1 0 0 m l o f e i t h e r d i s t i l l e d w a t e r o r 1 . 0 N H C 1 i n a w r i s t a c t i o n s h a k e r . 3 2 P - P O ^ w a s u s e d a s a t r a c e r o f o r t h o p h o s p h a t e e l u t i o n . F i v e m l 3 2 s a m p l e s w e r e c o - i n j e c t e d w i t h P - P O ^ o n t o 2 . 5 b y 30 cm c o l u m n s p a c k e d w i t h G - 2 5 S e p h a d e x b e a d s ( P h a r m a c i a ) a n d e l u t e d w i t h 0 . 3 % s o d i u m c h l o r i d e a n d 0 . 0 2 % s o d i u m a z i d e . F i v e - m i l l i l i t e r a l i q u o t s 32 o f t h e e l u a n t w e r e a n a l y z e d f o r P a c t i v i t y b y C e r e n k o v c o u n t i n g a n d t h e n f o r S R P . 2.3.3 I r o n - B i n d i n g Assay . The i r o n - b i n d i n g c a p a c i t y (FeBC) of l a k e water was determined with a r a d i o i s o t o p e assay (Murphy et a l . 1983a). In t h i s assay, any unchelated i r o n was p r e c i p i t a t e d w i t h magnesium carbonate. The c h e l a t o r s were c a l i b r a t e d w i t h d e s f e r a l ( desferrioxamine-B, Ciba-Geigy) and the FeBC was expressed i n e q u i v a l e n t s of d e s f e r a l (uM). Some FeBC samples were f u r t h e r processed by gel-permeation chromatography (Murphy 1976). F i v e m i l l i l i t e r s of the t r e a t e d sample were i n j e c t e d onto 2.5 cm by 30 cm columns packed with G-25 Sephadex beads (Pharmacia) and e l u t e d w i t h 0.3% sodium c h l o r i d e and 0.02% sodium a z i d e . F i v e - m i l l i l i t e r a l i q u o t s of the 55 e l u a n t were analyzed f o r Fe. 28 For a comparative b i n d i n g assay of i r o n and other c a t i o n s , the FeBC assay was improved by s u b s t i t u t i n g 0.2 f o r 0.45 urn c e l l u l o s e a c e t a t e f i l t e r s , c a c o d y l a t e b u f f e r (pH 7.0) f o r TRIS b u f f e r , and i n c r e a s i n g the i n c u b a t i o n t o one hour. These assays used a l g a l f i l t r a t e s t h a t had been f r o z e n , and passed through a PM-30 u l t r a f i l t r a t i o n membrane (Amicon). ^ F e was measured by s c i n t i l l a t i o n c o u n t i n g . The e f f i c i e n c y of the PCS f l u o r (Amersham) was op t i m a l when 1.0 ml of 500 uM d e s f e r a l was added to the samples p r i o r t o a d d i t i o n of the f l u o r . Quench curves were needed f o r the c o l o u r e d metal s o l u t i o n s . Metals were added i n 0.001 N HC1. A t y p i c a l i r o n - b i n d i n g experiment c o n s i s t e d of 2.0 ml of a l g a l f i l t r a t e , 0.5 ml of 5 5 F e - F e C l 3 (7.4 x 10 4 Bq i n 0.01 N HC1), 0.5 ml of 0.01 N NaOH, 1.0 ml of b u f f e r , and enough double d i s t i l l e d water t o make a f i n a l volume of 10.0 ml. O n e - m i l l i l i t e r 55 samples were then taken f o r d e t e r m i n a t i o n of t o t a l Fe. A f t e r the i n i t i a l one hour i n c u b a t i o n w i t h the metals, MgCO^ (60 mg) was added t o each f l a s k . A f t e r another one hour i n c u b a t i o n , the s o l u t i o n s were f i l t e r e d and 1.0 ml of f i l t r a t e was c o l l e c t e d f o r de t e r m i n a t i o n of ^ F e remaining i n s o l u t i o n . Between experiments, the a l g a l f i l t r a t e s were f r o z e n . To determine t h e e f f e c t of pH changes d u r i n g c h e l a t o r i s o l a t i o n , a f i l t r a t e of Anabaena f l o s - a q u a e was p r e t r e a t e d with v a r i o u s amounts of HC1 or NaOH f o r one hour p r i o r t o the FeBC assay. L a t e r i n the study, the a d s o r p t i o n of si d e r o p h o r e i s o l a t e s t o the a l g a l c o l l o i d a l c o a t i n g s was s t u d i e d . The i r o n - b i n d i n g 29 assay was used on f i l t r a t e s from a l g a l c u l t u r e s t h a t had been r e c e n t l y f i l t e r e d ; f i l t e r e d , f r o z e n , thawed, and r e f i l t e r e d ; f i l t e r e d , f r e e z e - d r i e d , r e d i s s o l v e d , and r e f i l t e r e d ; or passed through an u l t r a f i l t r a t i o n membrane (PM-30, Amicon). The former treatments dehydrated and p r e c i p i t a t e d c o l l o i d s ; whereas, the l a t t e r treatment p h y s i c a l l y removed the c o l l o i d s . 2.3.4 C h e l a t o r I s o l a t i o n C h e l a t o r s were i s o l a t e d from Texas U n i v e r s i t y a l g a l c u l t u r e s , Scenedesmus b a s i l i e n s i s V i s c h e r 79, Anabaena f l o s - a q u a e [Lyngbye] 1444, and Anabaena c y l i n d r i c a Lemm 1447. These c u l t u r e s -2 -1 were grown wi t h a 16 h photoperiod, i n 100 uE m s of l i g h t from f l u o r e s c e n t lamps. T e s t s f o r b a c t e r i a l contamination of a l g a l c u l t u r e s were c a r r i e d out b e f o r e and a f t e r each experiment w i t h yeast e x t r a c t , beef e x t r a c t , and t h i o g y l c o l l a t e t e s t media ( D i f c o ) . C u l t u r e s were regarded as axenic i f no b a c t e r i a l or f u n g a l growth appeared i n the t e s t media f o r one week at 25°C. C o n f i r m a t i o n of the axenic s t a t u s of Anabaena c y l i n d r i c a , Anabaena f l o s - a q u a e , and Scenedesmus b a s i l i e n s i s was o b t a i n e d s e v e r a l times by t r a n s m i s s i o n e l e c t r o n microscopy. Stock c u l t u r e s were maintained i n Chu-10 medium ( N i c h o l s 19 73). To reduce the amount of i r o n i n the experimental medium, algae were s u b c u l t u r e d at l e a s t twice p r i o r t o the experiment i n medium without added i r o n . Although i r o n - r i c h algae s u b c u l t u r e d e a s i l y with a 1% inoculum, i r o n - d e f i c i e n t Anabaena f l o s - a q u a e needed a 10% inoculum to grow w e l l . Some i r o n - d e f i c i e n t c u l t u r e s of Anabaena f l o s - a q u a e would not grow. To minimize t h i s problem 30 i n e s t a b l i s h i n g new c u l t u r e s , s e v e r a l f l a s k s were i n o c u l a t e d and the b e s t growing c u l t u r e s were used. C h e l a t o r s were i s o l a t e d from axenic e x p o n e n t i a l phase batch c u l t u r e s (ten days o l d ) . For the l a r g e r i s o l a t i o n s , 100 ml c u l t u r e s were used to i n o c u l a t e bubbled 20 L c u l t u r e s . P r i o r t o c h e l a t o r e x t r a c t i o n , the a i r bubbler i n the c u l t u r e was turned o f f , the algae were allowed t o s e t t l e f o r four hours, and the medium was decanted and f i l t e r e d through c e l l u l o s e a c e t a t e membranes with 0.45 rim pores. Twenty l i t e r s of f i l t e r e d medium were f i r s t a c i d i f i e d t o pH 4.0, aerated f o r 30 min, a d j u s t e d t o pH 10, and s t i r r e d with 100 g of AG1-X8 anion exchange r e s i n (Bio-Rad, C l form) f o r 20 min. The r e s i n was packed i n t o 1.0 cm diameter columns and the c h e l a t o r was e l u t e d o f f the r e s i n with 0.01 N HC1. The f i r s t 14 i s o l a t i o n s used C-HCO^ t o l a b e l the o r g a n i c compounds (37 x 10 6 Bq per 100 ml c u l t u r e ) , and h e t e r o t r o p h i c bioassays w i t h l a k e water samples were used t o d e t e c t the most a c t i v e peak. T h i s peak was then p u r i f i e d f u r t h e r by u s i n g ^ F e - F e C l ^ and Sephadex chromatography (Murphy et a l . 1976). The c h e l a t o r s were d e s a l t e d by e l u t i o n through an i o n r e t a r d a t i o n r e s i n (Bio-Rad 14 55 . AG11A8). C and Fe were not necessary once the i s o l a t i o n procedure was developed. The pH of the e l u a n t from the i o n exchange column was used t o d e t e c t the important f r a c t i o n . Gel-permeation columns were r e p r o d u c i b l e as shown by p e r i o d i c 14 c a l i b r a t i o n with blue-dextran and C-glucose. A l l compounds t h a t were used i n the heterotrophy s t u d i e s , but not the i r o n - b i n d i n g assay, were passed through Sephadex columns. 31 2.3.5 E l e c t r o n M i c r o s c o p i c A n a l y s i s Samples from the C a C ^ experiment i n Black Lake and the lime treatment of F r i s k e n Lake were analyzed at the O n t a r i o Research Foundation or at McMaster U n i v e r s i t y on a Semco Nanolab 7 scanning e l e c t r o n microscope equipped with an EDAX microprobe f o r eleme n t a l a n a l y s i s . The beam was d i r e c t e d at the c e n t r e of the l a r g e r p a r t i c l e s . The s t a n d a r d l e s s a n a l y s i s used the Magic V computer program t o c a l c u l a t e the elemental composition (Yakowitz e t a l . 1973). 2.3.6 Sediment A n a l y s i s Sediment cores were taken from Yellow, Roche, F r i s k e n , and Chain lakes with a W i l l i a m s l i g h t w e i g h t c o r e r (Williams and Pashley 1978). The cores were d i v i d e d i n t o 2.0 cm s e c t i o n s w i t h i n an hour with the W i l l i a m s extruder (Williams and Pashley, u n p u b l i s h e d ) . The samples were then f r o z e n and l a t e r f r e e z e - d r i e d at the Canada Centre f o r Inland Waters. P r i o r t o a n a l y s i s , the samples were ground i n a sediment g r i n d e r , p e l l e t i z e d and analyzed f o r major elements ( S i , A l , Ca, Mg, Fe, Na, K, P, and Mn) by X-ray f l u o r e s c e n c e spectrometry (Mudroch and Duncan 1986). Determination of minerals p r e s e n t i n subsamples was c a r r i e d out q u a l i t a t i v e l y by a P h i l i p s X-ray d i f f r a c t i o n spectrometer. The p y r i t e content of the unground, f r e e z e - d r i e d sediment was determined by Mossbauer spectrometry (Manning et a l . 19 79). The r a t e s of sediment accumulation i n F r i s k e n and Chain l a k e s were determined by Pb-210 r a d i o c h e m i c a l d a t i n g (Robbins and Edgington 1975). 32 RESULTS 3.1 B i o g e n i c Response t o I r o n A v a i l a b i l i t y 3.1.1 I r o n - C h l o r o p h y l l a R e l a t i o n s h i p s The y e a r l y o s c i l l a t i o n s of a l g a l biomass i n Black Lake ( c h l o r o p h y l l a, F i g . 3) were c l o s e l y c o r r e l a t e d t o the p a r t i c u l a t e i r o n c o n c e n t r a t i o n ( F i g . 3) i n both the aer a t e d (r=0.93, n=12) and the c o n t r o l s i d e s of the l a k e (r=0.87, n=12). The higher i r o n c o n c e n t r a t i o n on the aerated s i d e of the l a k e was a s s o c i a t e d with a much higher a l g a l biomass ( c h i a, ug/L a e r a t e d / c o n t r o l : 97/45, 1979; 160/75, 1980, F i g . 3: v a l u e s are means of f i v e r e p l i c a t e s from 1.0 m). The higher a l g a l biomass on the aerated s i d e of the l a k e produced more oxygen i n the s u r f a c e of the l a k e , but the g r e a t e r oxygen demand i n the aer a t e d hypolimnion r e s u l t e d i n a shallower zone of o x i d i z e d s u r f a c e water ( F i g . 4). A f t e r the p r e c i p i t a t i o n of the a l g a l bloom by the second lime treatment of F r i s k e n Lake, the F r i s k e n Lake c h i a-Fe data showed a t r e n d s i m i l a r t o the Black Lake data ( F i g . 5). The t o t a l i r o n c o n c e n t r a t i o n s i n the e p i l i m n i o n were s t r o n g l y c o r r e l a t e d t o the e p i l i m n e t i c c h l o r o p h y l l a c o n c e n t r a t i o n (r= 0.82, n=23). P r i o r t o the lime treatment, no c o r r e l a t i o n e x i s t e d between i r o n c o n c e n t r a t i o n and c h l o r o p h y l l a ( F i g . 6). Although the i r o n c o n c e n t r a t i o n of Chain Lake a l s o i n c r e a s e d i n midsummer of 1983, the c o r r e l a t i o n of t o t a l i r o n t o c h l o r o p h y l l a c o n c e n t r a t i o n was not s i g n i f i c a n t ( F i g . 6). 33 200-, 1979 1980 •t F i g u r e 3 Seasonal changes i n phytoplankton biomass ( c h l o r o p h y l l a) and p a r t i c u l a t e Fe i n Black Lake. Values are means of two r e p l i c a t e s , except August, 1979, 1980 where n=5. E r r o r bars are one standard d e v i a t i o n . 34 OXYGEN mg. LITER*"1 50 WO 150 ZOO 250 8 0 1 0 0 1 5 0 200 250 300 Fe |ig. LITER-1 August 11 1079 F i g u r e 4 Black Lake i r o n c o n c e n t r a t i o n s i n an a l g a l bloom. August September F i g u r e 5 Iro n and c h l o r o p h y l l a i n F r i s k e n Lake a t 1.0 m. Values are means of fou r samples. E r r o r bars are one standard d e v i a t i o n . $ Denotes second lime a d d i t i o n . 35 150 100 H 50H 250 \ 100 01 3 O o 50 Frisken Lake • pre-treatment O post-treatment • • t • •• o o o •„ a 50 100 T 150 200 250 15CH 100H 100 200 300 Fe (ug-L-1) 400 500 F i g u r e 6 C h l o r o p h y l l and i r o n c o n c e n t r a t i o n s i n Bl a c k , Chain, and F r i s k e n l a k e s . 36 3.1.2 Seasonal A l g a l S u ccession The seasonal changes i n i r o n and c h l o r o p h y l l a c o n c e n t r a t i o n s were a s s o c i a t e d with changes i n the a l g a l s p e c i e s . During the s p r i n g a l g a l bloom i n A p r i l 1980 i n Black Lake (Mallomonas caudata, C y c l o t e l l a meneghiniana, Cocconeis  s c u t e l l u m , Diatoma elongatum, Cryptomonas e r o s a , M e r i d i o n  c i r c u l a r e , and Gomphonema s p . ) , the d i s s o l v e d i r o n decreased t o l e s s than 2 ug/L. During the same p e r i o d , the t o t a l i r o n i n the e p i l i m n i o n remained c o n s t a n t (20-25 ug/L, Appendix 2). The low d i s s o l v e d i r o n i n d i c a t e d the p o t e n t i a l f o r i r o n l i m i t a t i o n but r a p i d b i o l o g i c a l s u c c e s s i o n pre-empted a study of i r o n l i m i t a t i o n . The d i s t r i b u t i o n of the algae and zooplankton i n d i c a t e d t h a t the s p r i n g bloom i n Black Lake was terminated by i n t e n s e zooplankton g r a z i n g . On A p r i l 22, 1980, the c h l o r o p h y l l a d i s t r i b u t i o n i n d i f f e r e n t areas of the l a k e was q u i t e uniform (sample s i z e 200 ml, n=10, x=21.3+3.7 ug c h l a/L, c o e f f i c i e n t of v a r i a t i o n 17 . 4 % ) . On May 1, 1980, the c h l o r o p h y l l a d i s t r i b u t i o n i n d i c a t e d t h a t the phytoplankton d i s t r i b u t i o n was patchy (sample s i z e 200 ml, n=18, x=10.6+6.3 ng c h l a/L, c o e f f i c i e n t of v a r i a t i o n 59%). On May 1, Daphnia pulex was d i s t r i b u t e d i n dense swarms. At t h i s time, the f i l t r a t i o n of water samples produced f i l t e r s t h a t were e i t h e r green without Daphnia or they were almost c l e a r with 4-8 l a r g e Daphnia per sample. S i m i l a r e f f e c t s of zooplankton on blue-green algae were not observed. A l l l a k e s i n t h i s study developed Aphanizomenon blooms i n mid t o l a t e summer, but the t i m i n g of the blooms v a r i e d g r e a t l y . 37 The l a k e with the h i g h e s t i r o n c o n c e n t r a t i o n s , Chain Lake, developed Aphanizomenon blooms t h r e e weeks e a r l i e r than Black Lake. The blue-green a l g a l s u c c e s s i o n c o u l d occur very q u i c k l y . A f t e r the diatom bloom c o l l a p s e i n Black Lake i n 1979 and 1980, Anabaena and Aphanizomenon c o e x i s t e d at low c o n c e n t r a t i o n s i n Black Lake f o r a t l e a s t f o u r weeks. Much of the biomass of these algae was i n clumps; thus, they appeared t o have se p a r a t e n i c h e s . B e f o r e the August 1979 and August 1980 i n c r e a s e s i n i r o n , Anabaena d i e d r a p i d l y . On the morning of J u l y 3, 1979, Anabaena was observed c o n c e n t r a t e d i n the s u r f a c e meter of water i n green clumps with brown edges. Four hours l a t e r , Anabaena was found as brown clumps at 5.0 m ( c h i a 35 ug/L), w e l l below the o p t i m a l l i g h t l e v e l and at a depth where c h l o r o p h y l l a l e v e l s were u s u a l l y l e s s than 5 ug/L. I n i t i a l l y , the Aphani zomenon seemed u n a f f e c t e d by the death of the Anabaena; however, the death of the Anabaena c o i n c i d e d with enhanced h y p o l i m n e t i c oxygen d e p l e t i o n , an i n c r e a s e i n i r o n content of the water column, and the i n i t i a t i o n of the Aphani zomenon bloom. 3.1.3 Seasonal Changes i n I r o n C o n c e n t r a t i o n The i n c r e a s e of i r o n i n l a k e water, i n Black, Chain, and F r i s k e n l a k e s , o c c u r r e d when stream flows were i n s i g n i f i c a n t ; thus, i r o n must have been r e l e a s e d from the sediments. The i n f l u e n c e of oxygen c o n c e n t r a t i o n s on i r o n r e l e a s e was d i f f e r e n t i n the t h r e e l a k e s . In F r i s k e n Lake and the c o n t r o l s i d e of Black Lake, sediment i r o n r e l e a s e was not a s s o c i a t e d with a midsummer 38 r e d u c t i o n of oxygen content. The hypolimnia of Black and F r i s k e n l a k e s were r a r e l y o x i d i z e d ( F i g . 7 ) . The hypolimnion of Chain Lake was u s u a l l y o x i d i z e d but i t was anoxic when i r o n was r e l e a s e d i n August ( F i g . 7). In both F r i s k e n and Chain l a k e s , about 20% and 50% r e s p e c t i v e l y of the i r o n i s converted i n t o p y r i t e (FeS2, a m i n e r a l c o n t a i n i n g reduced i r o n , F i g . 8). Once p y r i t e was b u r i e d i n anoxic sediments, p y r i t e would be s t a b l e , and no r e f l u x i n g of t h i s i r o n i n t o the l a k e would occur. Some f e r r i c i r o n i s metastable i n these anoxic environments and a p u l s e of o r g a n i c matter decay c o u l d enhance the r a t e of i r o n r e d u c t i o n . Some of the f e r r o u s i r o n c o u l d enter the water column and some would form p y r i t e . Temperature was an important v a r i a b l e i n the sediment i r o n r e l e a s e at a l l s i t e s . Iron r e l e a s e o c c u r r e d e a r l i e s t i n Chain Lake, i n mid-July (Table 3). T h i s shallow l a k e mixes r e a d i l y ; thus, the sediments were warmer than the other s i t e s ( F i g . 9 ) . Ta b l e 3 T o t a l I r o n C o n c e n t r a t i o n s i n Chain Lake - 1984 I r o n C o n c e n t r a t i o n s (ug/L) Depth (m) J u l y 6 J u l y 16 Aug 10 Sept 1 426 377 236 252 2 322 633 3 264 379 812 1525 4 784 777 5 385 654 1967 741 I n l e t 893 219 39 ig u r e 7 Seasonal changes i n oxygen c o n c e n t r a t i o n i n Chain, B l a c k , and F r i s k e n l a k e s . A l l v a l u e s are mg/L. FRISKEN LAKE 70-60-50-IRCt TOTAL 40-% OF 30-• • 20- • • 10- • FeS 2 • Fe2* 0- i i 1 I T 1— 1 1— • F e 3 + 0 6 12 18 24 30 36 42 48 54 60 66 SEDIMENT DEPTH (cm) . F i g u r e 8 I r o n g e o c h e m i s t r y o f C h a i n a n d F r i s k e n l a k e s e d i m e n t s . MAY 1 JUNE 1 JUL? 1 AUGUST F i g u r e 9 Seasonal changes i n temperature of the s u r f a c e sediments of Black, Chain, and F r i s k e n l a k e s . 42 Iro n r e l e a s e i n Black Lake d i d not occur u n t i l the sediments were warmer than 10°C (Appendix A l , l a t e J u l y ) . A 2.6°C warming of the sediments by l a k e a e r a t i o n appeared t o enhance i r o n r e l e a s e on the a e r a t e d s i d e of Black Lake ( F i g . 9 ). Iro n r e l e a s e o c c u r r e d l a s t i n F r i s k e n Lake perhaps because the p r o f u n d a l sediments d i d not warm above 5°C u n t i l August (Table 4, F i g . 9 ). 3.2 C o n f i r m a t i o n of the Iron-Biomass R e l a t i o n s h i p Although the i r o n c o n c e n t r a t i o n i n Black and F r i s k e n l a k e s was s t r o n g l y c o r r e l a t e d with the a l g a l biomass ( F i g s . 3&5), these o b s e r v a t i o n s are not c o n c l u s i v e proof t h a t i r o n a v a i l a b i l i t y r e g u l a t e d a l g a l growth. The f o l l o w i n g microcosm s t u d i e s were used t o t e s t the i r o n - l i m i t a t i o n h y p o t h e s i s . 3.2.1 L a b o r a t o r y Bioassays with Black Lake Water When water samples, c o l l e c t e d on June 19, 19 79, were e n r i c h e d with 500 ug Fe/L, as F e C l ^ , i n an i n c u b a t o r , the a l g a l biomass i n c r e a s e d s i g n i f i c a n t l y ( F i g . 10). C o n t r o l samples had a l a g of s e v e r a l days b e f o r e growth was i n i t i a t e d and although the c o n t r o l samples then grew r a p i d l y , the f i n a l biomass produced was l e s s than the i r o n - e n r i c h e d samples. In a l l samples t h a t responded p o s i t i v e l y t o i r o n enrichment, Anabaena continued t o dominate the phytoplankton. Samples c o l l e c t e d l a t e i n the summer (Aug. 11, 1979), when the i r o n content of the water had i n c r e a s e d , d i d not show t h i s iron-enrichment response ( F i g . 10). The dominant a l g a l s p e c i e s i n l a t e summer, Aphani zomenon, would not grow i n the l a b o r a t o r y . 43 Tab l e 4 T o t a l I r o n C o n c e n t r a t i o n s i n F r i s k e n Lake 1983 * Depth June 16 J u l y 23 J u l y 27 Aug 18 Aug 25 Sept 20 1 27+4 18+1 26+5 24 36 83 2 22 31 137 3 18+2 34 30 134 4 32+2 35 129 5 68+3 39+10 45+12 131 125 43 6 559 536 80 7 791 923 39 8 94+11 226+102 219+245 1065+224 1253 444 1984 Depth June 15 J u l y 16 Aug 5 1 15 18 41 2 19 24 41 3 25 17 48 4 16 23 49 5 18 38 52 6 65 84 233 7 186 104 311 8 185 194 625 Fe ug/L depth i n meters 44 TIME (days) AUGUST 11, 1979 TIME (days) F i g u r e 10 E f f e c t of i r o n enrichment on growth of algae from Black Lake. Three samples were e n r i c h e d with 500 ug Fe/L (• ). ( A ) were c o n t r o l samples. O p t i c a l d e n s i t y a n a l y s i s has a c o e f f i c i e n t of v a r i a t i o n of 1.5%. Although the prime c o n t r o l l i n g v a r i a b l e i n the l a b o r a t o r y growth assays was probably i r o n c o n c e n t r a t i o n , the a b i l i t y of the dominant a l g a t o grow i n the l a b o r a t o r y appeared t o an important v a r i a b l e . In another assay on J u l y 3, 1979, some of the f l a s k s w i t h Anabaena dominance showed a p o s i t i v e response t o i r o n enrichment but o t h e r s , i n which diatoms r e p l a c e d Anabaena, d i d not show any response t o i r o n . Perhaps the high v a r i a n c e i n the i r o n - e n r i c h e d f l a s k s of June 19, 1979 ( F i g . 10) was caused by the v a r y i n g p r o p o r t i o n of diatoms prese n t with Anabaena. S i m i l a r v a r i a b l e r e s u l t s o c c u r r e d i n 1980 when a l a r g e diatom, Rhopaladia g i b b a , which was never observed i n the l a k e , q u i c k l y dominated Anabaena i n l a b o r a t o r y c u l t u r e s . Primary p r o d u c t i o n b i o a s s a y s i n the l a k e were b r i e f (48 h); thus, the problem w i t h a l g a l s u c c e s s i o n encountered i n 14 l a b o r a t o r y i n c u b a t i o n s was avoided. The a l g a l C - a s s i m i l a t i o n i n d i c a t e d a s i g n i f i c a n t s t i m u l a t i o n from i r o n enrichment, as F e C l ^ , o n l y i n e a r l y summer (compare C t o Fe i n F i g . 11). The 14 s i m i l a r s t i m u l a t i o n of a l g a l C - a s s i m i l a t i o n by an a l g a l s i d e r o p h o r e i s o l a t e (5 uM FeBC) on June 17 but not i n August ( F i g . 11) confirmed t h a t more i r o n was a v a i l a b l e i n August than i n June; i r o n c h e l a t e d by t h i s Anabaena c h e l a t e c o u l d be u t i l i z e d by a l l t e s t e d s p e c i e s ( t h r e e Anabaena s p e c i e s and two Scenedesmus s p e c i e s ) . The moderate s t i m u l a t i o n of primary p r o d u c t i o n observed from F e C l 3 a d d i t i o n i n the e a r l y summer of 1979, 1980 and 1981 was modest compared t o the responses observed with a l g a l c h e l a t o r s ( s e c t i o n 3.3.5). These s h o r t i n s i t u i n c u b a t i o n s were able t o assess the immediate e f f e c t of i r o n a d d i t i o n . An CONTROL APHANIZOMENON ANABAENA % 30 oc U J o 2 20 o Q O §F 10 oc < 5 oc O L CONTROL 4-AERATED 0J 4- 4-3-2-600 450 300 150 C A A+Fe C Fe A A+Fe C Fe A A+Fe C Fe A A+Fe 0 J C Fe A A+Fe JUNE 17 1979 JULY 19 1979 AUG. 30 1979 F i g u r e 11 E f f e c t of a d d i t i o n of Fe or the siderophore i s o l a t e from Anabaena c y l i n d r i c a on primary production i n Black Lake. C f A, Fe, and A+Fe are the c o n t r o l , s i d e r o p h o r e , i r o n alone, and siderophore plus i r o n treatments. C o n t r o l and aera t e d r e f e r t o the two si d e s of the lake. On J u l y 19 the two types of algae were separated p r i o r t o i n c u b a t i o n . Values are means of two r e p l i c a t e s . E r r o r bars a re one standard d e v i a t i o n . 47 e v a l u a t i o n of long-term responses t o i r o n enrichment r e q u i r e d the use of l i m n o c o r r a l i n c u b a t i o n s . 3.2.2 Fe-EDTA L i m n o c o r r a l s i n Black Lake In B l a c k Lake i n 1980, l i m n o c o r r a l s were e n r i c h e d w i t h Fe-EDTA or Na-EDTA. The i r o n c o n c e n t r a t i o n was maintained between 100 and 200 ug/L by monitoring t h e i r o n c o n c e n t r a t i o n and adding i r o n as needed (3 and 4 a d d i t i o n s i n f i r s t and second experiments r e s p e c t i v e l y ) . L i m n o c o r r a l b i o a s s a y s are more r e s t r i c t e d i n the type of i r o n source than are s m a l l b o t t l e s . Unchelated i r o n would p r e c i p i t a t e t o the bottom of the l i m n o c o r r a l . A c h e l a t o r must be used t o maintain i r o n i n s o l u t i o n ; thus, an a d d i t i o n a l c o n t r o l must be added t o determine i f the c h e l a t o r has an important e f f e c t . The i n i t i a l enrichment of l i m n o c o r r a l s on A p r i l 22, w i t h Fe-EDTA and Na-EDTA r e s u l t e d i n higher oxygen c o n c e n t r a t i o n s ( F i g . 12), presumably by i n c r e a s i n g a l g a l p r o d u c t i o n . By May 25, c a l c i u m carbonate p r e c i p i t a t i o n and perhaps zooplankton g r a z i n g had terminated the a l g a l bloom; thus, no d i f f e r e n c e s between c h l o r o p h y l l a c o n c e n t r a t i o n s i n the l i m n o c o r r a l s were apparent. The l a r g e r e d u c t i o n of phosphorus and c a l c i u m i n the s u r f a c e waters of the Fe-EDTA and Na-EDTA l i m n o c o r r a l s was p r i m a r i l y a r e s u l t of c o p r e c i p i t a t i o n of phosphorus with c a l c i t e (13 mg/L Ca and 140 ug/L P p r e c i p i t a t e d between May 25 and June 5; Ca/P molar r a t i o 77). A f t e r t h i s f i r s t s e t of l i m n o c o r r a l s had been emptied and r e f i l l e d on J u l y 30, 1980, t h e r e was no response t o Fe-EDTA enrichment and o n l y a s l i g h t response t o the Na-EDTA enrichment 48 F i g u r e 12 Oxygen and phosphorus i n Na-EDTA and Fe-EDTA l i m n o c o r r a l s and Black Lake. Table 5. Primary Production in Experimental Enclosures in Black Lake in 1980. 1 Limnocorral Date Fe-1 ED-1 C-2 Fe-2 ED-2 NO, LAKE May 22 28 40 J June 3 14 146 66 June 16 109 169 117 76 120 130 135 July 4 165 31 12 38 24 14 53 July 18 17 69 31 33 553 16 96 July 30 17 49 1030 28 175 Aug 12 77 251 192 115 976 56 730 Aug 28 46 166 38 156 589 13 124 Sept 10 50 350 52 33 310 20 83 A l l samples collected at 1 mf a l l units as (ug C L d . ) . 49 (greater c h i a, s i g n i f i c a n t at the 90% confidence l i m i t s ) . This seasonal decrease in e f fect of i ron enrichment was a l so observed in the growth and primary production bioassays and l a t e r l imnocorral experiments. The seasonal increase of i ron in the lake ( F i g . 3) was probabiy responsible for the seasonal reduction in the response of i ron enrichment in l imnocorra l s . Primary production in the l imnocorrals was h ighly v a r i a b l e ; however, the Na-EDTA l imnocorrals had the highest production measurements (Table 5). The heterotrophic a c t i v i t y was a l so var iab l e in the surface water and the Na-EDTA l imnocorrals had the highest heterotrophic ra tes . With only one exception, the heterotrophic a c t i v i t y was much higher in the Na-EDTA l imnocorrals in the ep i l imnia and hypolimnia during l a t e Ju ly and August ( F i g . 13). The heterotrophic assay indicates the capaci ty for heterotrophic metabolism; the assay can not measure b a c t e r i a l numbers or biomass. Doubts about the s u i t a b i l i t y of EDTA as a model chelator led to the r e p e t i t i o n of the i ron enrichment experiments with c i t r a t e as the i ron che la tor . C i t r a t e i s a weak chelator that can be u t i l i z e d by many microbes (Neilands 1981b) and plants ( T i f f i n 1966). 3.2.3 F e - C i t r a t e Limnocorral Experiment in Black Lake Limnocorrals were enriched with sodium c i t r a t e or f e r r i c c i t r a t e during the summer of 1982 in Black Lake. The c i t r a t e responses were qui te d i f f e r e n t from the EDTA responses. Part of the d i f ference was re la ted to the microb ia l u t i l i z a t i o n of c i t r a t e . Within f i v e days, c i t r a t e was undetectable in so lut ion 50 6LUC0SE UPTAKE F i g u r e 13 Temporal v a r i a t i o n of heterotrophy i n the 1980 l i m n o c o r r a l s . 51 ( F i g . 14, Tab l e 6). H e t e r o t r o p h i c uptake s t u d i e s with 1 4 C - c i t r a t e i n d i c a t e d t h a t 85+5.1% of the a s s i m i l a t e d 1 4 C l a b e l was converted i n t o carbon d i o x i d e w i t h i n two hours (Table 7). I n i t i a l l y , the oxygen c o n c e n t r a t i o n i n the sodium c i t r a t e - e n r i c h e d l i m n o c o r r a l s decreased. Within t h r e e days, the f e r r i c - c i t r a t e l i m n o c o r r a l s had s i g n i f i c a n t l y l e s s oxygen than the c o n t r o l l i m n o c o r r a l s . On June 26 and June 28 (the t h i r d and f i f t h day a f t e r n u t r i e n t enrichment), the f e r r i c - c i t r a t e l i m n o c o r r a l s had a mean of 0.90 mg 0 2/L l e s s 0^ than the c o n t r o l l i m n o c o r r a l s , and the s o d i u m - c i t r a t e l i m n o c o r r a l s had a mean of 0.49 mg C^/L l e s s 0^ than the c o n t r o l l i m n o c o r r a l s . Each mean va l u e was d e r i v e d from 24 v a l u e s ; thus, the d i f f e r e n c e i n treatments was h i g h l y s i g n i f i c a n t ( t t e s t , l e s s than 0.2% p r o b a b i l i t y of the treatments not having an e f f e c t , Appendix 3 ). A f t e r the c i t r a t e was u t i l i z e d , oxygen c o n c e n t r a t i o n s i n c r e a s e d i n a l l l i m n o c o r r a l s (Appendix 3, F i g . 15). Within t en days, the oxygen content of the s u r f a c e of the f e r r i c - c i t r a t e l i m n o c o r r a l s i n c r e a s e d more than the other l i m n o c o r r a l s ( F i g . 15). The treatments r e p l i c a t e d w e l l , and by a t t e s t , the f e r r i c - c i t r a t e l i m n o c o r r a l s had s i g n i f i c a n t l y more oxygen than the s o d i u m - c i t r a t e l i m n o c o r r a l s ( t t e s t , l e s s than a 5% p r o b a b i l i t y of the treatments not having an e f f e c t , Appendix 3). The u n u s u a l l y c o l d and cloudy weather d u r i n g t h i s experiment appeared t o suppress the i n t e n s i t y of the blue-green a l g a l growth ( F i g . 15). During t h i s l i m n o c o r r a l experiment, the c h l o r o p h y l l a content of the l i m n o c o r r a l s never exceeded 5 ug/L; the i r o n l i m n o c o r r a l s had the most c h l o r o p h y l l . F l a k e s of the blue-green a l g a Aphanizomenon f l o s - a q u a e appeared on l y i n the 52 1500 < b> a. O WE-F o 500-5.0 m C O R R A L S 0 10 20 TIME (DAYS) F i g u r e 14 C i t r a t e a s s i m i l a t i o n i n l i m n o c o r r a l s . A n a l y s i s i s on water 5.0 m from l a k e s u r f a c e . The mean c i t r a t e c o n c e n t r a t i o n s (•) of the two sodium c i t r a t e and two f e r r i c c i t r a t e l i m n o c o r r a l s i s shown with e r r o r bars of one standard d e v i a t i o n . Day zero was June 23, 19 82. Table 6 Citrate Concentration i n Limnocorrals Limnocorral June 24 June 26 June 28 June 30 Citrate 1 1 m 3 m 55.0+2.7 107.0 2.8+1.5 14.0+4.6 ND 0.8+0.01 4.0 Citrate 2 1 m 3 m 89.8+0.7 152.0+11.0 2.3+0.2 62.0+0.8 4.6+1.8 ND ND Iron 1 1 m 3 m 144.0+20.0 103.0+1.7 2.2+1.44 63.0+0.2 1.0+0.6 ND Iron 2 1 m 3 m 122.0+3.2 106.0 1.2+0.4 19.0+8.4 1.2+1.2 ND Samples analyzed twice are shown as the mean value + one standard deviation. After the additions of citrate on June 23, June 30, and Aug. 12, the calculated concentration would be 140 ug C/100 mL. Samples of June 30 were collected 4 h after the citrate enrichment. A l l values are ug C/100 mL. ND i s not detectable. Table 7 Limnocorral C-Citrate Assimilat io n Sample Depth Cont-1 Cont-2 NCL-1 MD3-2 C i t - 1 C i t --2 Fe-1 Fe-2 June 28 Cit-Net 1.0 m 7.89 6.65 4.37 5.93 32.4 83.8 18.6 36.8 Cit-Net 3.0 m 4.92 3.99 3.28 4.41 203.5 103.1 165.3 105.3 June 30 Cit-Net 1.0 m .693 .042 * 89.8 79.4 84.2 68.8 Cit-Gr 1.0 m 6.68 .205 * * * 582.0 * * Cit-Net 3.0 m 1.64 .478 * 320.0 248.0 286.0 53.8 Cit-Gr 3.0 m 9.55 .674 * * 1798.0 1702.0 1448.0 1270.0 Aug. 17 Cit-Net 1.0 m .73 .95 ND ND .22 ND ND .93 Cit-Net 3.0 m .82 .69 ND .439 .74 1.14 ND 3.72 ND=not detectable, *=no data, C i t = c i t r a t e , Gr=gross uptake ( t o t a l carbon a s s i m i l a t e d ) , Net=net uptake (gross C uptake-respired C). Cont-l=control-l limnocorral, Cont-2=control-2 limnocorral e t c . Gross C uptake = net C uptake + respired C. - 1 - 1 -2 A l l rates expressed as ng C L h xlO . 54 1(b) TIME (DAYS) JUL JUL AUG AUG 1 14 1 14 F i g u r e 15 Phosphorus, oxygen, and temperature i n the c i t r a t e l i m n o c o r r a l s i n Black Lake. (a) The mean SRP c o n c e n t r a t i o n ( ) and temperature ( ) of 1.0 m water from the 2 c o n t r o l , 2 n i t r a t e , 2 N a - c i t r a t e , and 2 F e - c i t r a t e l i m n o c o r r a l s . (b) The mean oxygen content of the N a - c i t r a t e (•) and Fe-c i t r a t e (•) l i m n o c o r r a l s at 1.0 m . The e r r o r bars are 1 standard d e v i a t i o n . Day 0 was June 23, 1982. f e r r i c - c i t r a t e l i m n o c o r r a l s . The f i n a l a d d i t i o n of sodium c i t r a t e and f e r r i c c i t r a t e (Aug. 12) was not as c l o s e l y s t u d i e d as the e a r l i e r a d d i t i o n s . I r o n enrichment d i d not appear t o s t i m u l a t e oxygen consumption or p r o d u c t i o n . Moreover, the oxygen content of a l l the l i m n o c o r r a l s decreased at t h i s time. Any d e t a i l e d i n t e r p r e t a t i o n of oxygen da t a was complicated by c a l c i u m carbonate p r e c i p i t a t i o n which oc c u r r e d i n a l l the l i m n o c o r r a l s and i n the l a k e . 3.2.4 I r o n A v a i l a b i l i t y i n Chain Lake Lim n o c o r r a l s were f i l l e d i n Chain Lake on J u l y 15, 1983 t o determine i f a l g a l oxygen p r o d u c t i o n was suppressed by a l a c k of a v a i l a b l e i r o n . The i r o n response was much d i f f e r e n t from i d e n t i c a l experiments conducted i n Black Lake. The l i m n o c o r r a l a d d i t i o n s of f e r r i c c i t r a t e d i d not r e s u l t i n a s i g n i f i c a n t s t i m u l a t i o n of a l g a l oxygen p r o d u c t i o n ( F i g . 16). A f t e r the t h i r d day, the oxygen content of the sodium c i t r a t e l i m n o c o r r a l s was higher than the f e r r i c c i t r a t e l i m n o c o r r a l s . In c o n t r a s t t o Black Lake, i n Chain Lake the m i c r o b i a l u t i l i z a t i o n of c i t r a t e consumed more oxygen than the algae produced. T h i s was t r u e i n both the sodium c i t r a t e and f e r r i c c i t r a t e l i m n o c o r r a l s . Iron d i d not s t i m u l a t e a l g a l oxygen p r o d u c t i o n ; thus, i t was concluded t h a t the algae i n Chain Lake were not l i m i t e d by a l a c k of i r o n . The Chain Lake l i m n o c o r r a l experiments i n d i c a t e t h a t i r o n i n Chain Lake was a v a i l a b l e t o algae, and was presumably r e a c t i v e . The r e l a t i o n s h i p between oxygen c o n c e n t r a t i o n and phosphorus s o l u b i l i t y supports t h i s hypothesis ( s e c t i o n 3 . 4.1). 56 CHAIN LAKE LIMNOCORRALS IS 25 31 F i g u r e 16 Oxygen i n Chain Lake l i m n o c o r r a l s . AC>2 i - s t n e mean change i n oxygen c o n c e n t r a t i o n s from mean i n i t i a l oxygen c o n c e n t r a t i o n of 10.0 mg/L at 1.0 m. Two l i m n o c o r r a l s were used f o r each treatment. C o e f f i c i e n t of v a r i a t i o n i s <0.5%. 57 3.3 Siderophore Ecology A s e r i e s of experiments was conducted to determine how microbes r e g u l a t e d the a v a i l a b i l i t y of i r o n . An i r o n - b i n d i n g assay was developed to q u a n t i f y the amount of iron-complexing compounds pr e s e n t i n c u l t u r e s and i n l a k e water. The s e l e c t i v i t y of c a t i o n b i n d i n g was evaluated t o determine i f these i r o n -complexing compounds were s i d e r o p h o r e s . The presence of s i d e r o p h o r e s i n d i c a t e s i r o n l i m i t a t i o n ; thus, a q u a n t i t a t i v e study of s i d e r o p h o r e s i n the l a k e can provide i n s i g h t i n t o i r o n a v a i l a b i l i t y i n the l a k e . The m i c r o d i s t r i b u t i o n of c h e l a t o r s i n a l g a l c u l t u r e medium was a l s o s t u d i e d . The d a t a on the m i c r o d i s t r i b u t i o n and chemistry of the c h e l a t o r s were used t o design experiments on the e f f e c t s of s i d e r o p h o r e i s o l a t e s on a l g a l and b a c t e r i a l p r o d u c t i v i t y . 3.3.1 C h e l a t o r Q u a n t i f i c a t i o n An i r o n - b i n d i n g assay was developed t o estimate the c o n c e n t r a t i o n of i r o n - b i n d i n g compounds. T h i s assay can p r e c i s e l y and r a p i d l y measure e q u i v a l e n t s of a w e l l c h a r a c t e r i z e d hydroxamate si d e r o p h o r e , namely, d e s f e r a l (Appendix 4). The c o e f f i c i e n t of v a r i a t i o n of the i r o n b i n d i n g was l e s s than 3.1% (Table 8). The assay does not measure the molar c o n c e n t r a t i o n of the c h e l a t o r s . For example, a high c o n c e n t r a t i o n of weak i r o n -b i n d i n g compounds co u l d have the same i r o n - b i n d i n g c a p a c i t y as a d i l u t e s o l u t i o n of s t r o n g i r o n - b i n d i n g compounds. 58 Tabl e 8 E f f e c t of F r e e z i n g Anabaena F i l t r a t e s on Iro n C h e l a t i o n Treatment F i l t r a t e T o t a l s # DPMT C.V. # # # DPM C.V. # # %Chelated * A. f . a. -1 F r e s h F i l t r a t e 89,727 .031 174,810 .015 51.2 Frozen Once 136,918 .011 180,160 .002 75.0 Frozen Twice 125,350 .013 122,956 .029 101.9 * A.f.a.-2 F r e s h F i l t r a t e 10,151 .003 94,204 .007 10.7 F r e e z e -D r i e d 54,977 .007 69,802 .002 79.4 ** A. c y l . F r e s h F i l t r a t e 35,744 .022 102,486 .055 34.9 F r e e z e -D r i e d 19,353 .018 47,171 .014 41.0 * A . f . a . - l and A.f.a.-2 are two f i l t r a t e s from i c u l t u r e s of Anabaena f l o s - a q u a e . ** A. c y l . i s a f i l t r a t e from a c u l t u r e of Anabaena c y l i n d r i c a . DPM i s the mean number of d i s i n t e g r a t i o n s per minute of the r a d i o a c t i v e i r o n i n two samples. C V . i s the c o e f f i c i e n t of v a r i a t i o n of DPM of r a d i o a c t i v e i r o n . 59 3.3.2 Siderophore A s s o c i a t i o n with F i b r i l s F o r t u n a t e l y , I had s u p p l i e d Dr. Leppard wi t h samples of my c u l t u r e s and t h e i r u l t r a s t r u c t u r e i s w e l l d e f i n e d (Leppard e t . a l . 1977). Anabaena c y l i n d r i c a produces 15 times more c o l l o i d a l f i b r i l l a r m a t e r i a l than Anabaena f l o s - a q u a e . The c o l l o i d a l f i b r i l s extend from the c e l l s u r f a c e of Anabaena flos-aquae by 0.35 um and Anabaena c y l i n d r i c a by 2.6 um. The esta b l i s h m e n t of a phycosphere, a microenvironment around a c e l l ( B e l l and M i t c h e l l 1972), by si d e r o p h o r e a d s o r p t i o n t o f i b r i l s would g r e a t l y change the r e l a t i o n s h i p between the producing s p e c i e s and competing microbes. Moreover, the m i c r o d i s t r i b u t i o n of siderophores i n f l u e n c e s the c o n c e n t r a t i o n of sid e r o p h o r e i s o l a t e s used i n b i o a s s a y s . Three d i f f e r e n t experiments i n d i c a t e d t h a t the c h e l a t o r e x c r e t e d by Anabaena flos-aquae was l o o s e l y bound t o a c o l l o i d . 1) When the f i l t r a t e s from Anabaena fl o s - a q u a e were f r o z e n and then r e f i l t e r e d , the c h e l a t i o n c a p a c i t y i n c r e a s e d . The percentage of 2.3 uM i r o n remaining i n s o l u t i o n i n the FeBC assay i n c r e a s e d from 51% i n unfrozen f i l t r a t e , t o 76% i n f i l t r a t e t h a t was f r o z e n once, and t o 101% i n f i l t r a t e t h a t was f r o z e n and thawed twice (Table 8). In a s i m i l a r experiment, the c h e l a t i o n c a p a c i t y of another f i l t r a t e of Anabaena fl o s - a q u a e i n c r e a s e d 7.8 f o l d when the f i l t r a t e was f r e e z e - d r i e d , r e d i s s o l v e d i n an e q u i v a l e n t volume of d i s t i l l e d water, and r e f i l t e r e d (Table 8). Note t h a t the c h e l a t i o n c a p a c i t y of an a l g a l f i l t r a t e from Anabaena c y l i n d r i c a i n c r e a s e d o n l y s l i g h t l y a f t e r f r e e z e - d r y i n g (Table 8). The f r e e z i n g and r e f i l t r a t i o n of the thawed f i l t r a t e removes c o l l o i d a l o r g a n i c m a t e r i a l . The c o l l o i d a l " f i b r i l s " t h a t 60 coat many algae, inc luding Anabaena flos-aquae (Leppard et a l . 1977), do not red i sso lve from frozen a l g a l f i l t r a t e s . 2) The i n a b i l i t y of f i b r i l s to pass through an Amicon PM-30 u l t r a f i l t r a t i o n membrane seemed responsible for the apparent increase in i ron-b ind ing capaci ty of an a l g a l f i l t r a t e from Anabaena flos-aquae from 2 to 80 nM (equivalents of d e s f e r a l ) . 3) Another i n d i c a t i o n that chelators were not in true so lu t ion was found when the f i l t r a t e from Anabaena flos-aquae was t i t r a t e d with i r o n . When the chelator was separated from the f i b r i l s by freeze-drying and u l t r a f i l t r a t i o n and then used in the FeBC assay, a s t r a i g h t l i n e was produced ( F i g . 17). In two r e p l i c a t e t i t r a t i o n s of the p u r i f i e d Anabaena flos-aquae 2 chelators the c o e f f i c i e n t s of l i n e a r regression (r ) were 0.961 and 0.968. The fresh a l g a l f i l t r a t e s had two i n f l e c t i o n points in the t i t r a t i o n s ( F i g . 17). This step response of the fresh a l g a l so lut ions may ind icate that the chelator i s more react ive when in so lu t ion than when i t i s adsorbed to the f i b r i l . The p u r i f i e d a l g a l chelator response was d i f f e r e n t from des f era l or EDTA that each had one i n f l e c t i o n point in the t i t r a t i o n . These experiments ind icate that siderophores are associated weakly with the f i b r i l l a r surface of algae. This assoc ia t ion supports the use in bioassays of so lut ions of siderophore i so la tes that are much more concentrated than that found in the bulk f i l t r a t e . 61 1 1 1 1 1 1 2 3 4 5 5 5Fe /ig/L F i g u r e 17 Iro n t i t r a t i o n of EDTA, d e s f e r a l , and f i l t r a t e s from Anabaena f l o s - a q u a e c u l t u r e s . AFA-1 and AFA-2 are f r e s h f i l t r a t e s 20 and 14 days o l d r e s p e c t i v e l y . AFA-P i s a f i l t r a t e t h a t was p u r i f e d by u l t r a f i l t r a t i o n and d i l u t e d f i v e f o l d . 62 3 . 3 . 3 S i d e r o p h o r e S p e c i f i c i t y f o r I r o n T h e i r o n - b i n d i n g c a p a c i t y a s s a y a l s o e n a b l e d a n e v a l u a t i o n t o b e m a d e o f t h e s p e c i f i c i t y o f t h e c h e l a t o r s f o r c o m p l e x i n g i r o n . R e l a t i v e t o m o s t o r g a n i c c o m p o u n d s , s i d e r o p h o r e s h a v e a h i g h s p e c i f i c i t y f o r c o m p l e x i n g i r o n . T h e s i d e r o p h o r e i s o l a t e f r o m A n a b a e n a f l o s - a q u a e r e q u i r e d t w o o r d e r s o f m a g n i t u d e m o r e l a n t h a n u m o r c o p p e r t h a n i r o n t o d i s p l a c e t h e r a d i o a c t i v e i r o n ( F i g . 1 8 ) . A l u m i n u m a n d c h r o m i u m s o l u t i o n s h a d t o b e a b o u t t h r e e o r d e r s o f m a g n i t u d e m o r e c o n c e n t r a t e d t h a n u n l a b e l l e d i r o n t o d i s p l a c e t h e r a d i o a c t i v e i r o n ( F i g . 1 8 ) . S o l u t i o n s o f c a l c i u m , c o b a l t , m a n g a n e s e , p o t a s s i u m , s o d i u m , o r z i n c t h a t w e r e m o r e t h a n f i v e o r d e r s o f m a g n i t u d e m o r e c o n c e n t r a t e d t h a n u n l a b e l l e d i r o n , d i s p l a c e d l i t t l e i r o n ( T a b l e 9 ) . T a b l e 9 E f f e c t o f M e t a l A d d i t i o n o n F e C h e l a t i o n b y t h e S i d e r o p h o r e I s o l a t e f r o m A n a b a e n a f l o s - a q u a e . C a t i o n C o n c e n t r a t i o n o f C a t i o n t o d i s p l a c e F e i n t o s o l u t i o n * % F e C a 3 . 7 5 M 8 8 , 8 7 C o 6 5 0 mM 87 K 1 . 6 3 M 9 4 , 9 1 Mn 1 . 2 M 9 9 , 9 9 N a 3 . 7 5 M 8 2 , 8 8 Z n 6 . 5 M 8 8 , 9 1 h i g h e s t a t t a i n a b l e c o n c e n t r a t i o n i n a s s a y % F e i n s o l u t i o n , r e p l i c a t e s 63 C O L D - F e DISPLACEMENT OF 2.3 /iM M F e 75J c 26-Cu DISPLACEMENT OF 2.3 /*M ^Fe Cu (mM) F i g u r e 18 Metal displacement of i r o n from the Anabaena  f l o s - a q u a e s i d e r o p h o r e . Values are means of two r e p l i c a t e s . C o e f f i c i e n t of v a r i a t i o n i s l e s s than 3%. 64 Al DISPLACEMENT OF 2.3 pM "Fe F i g u r e 18 Continued 65 La DISPLACEMENT OF 2.3 "Fe % 4 i' i 3 c u a. 2-ui s 3 > • 1m 95% RECOVERY Q7m 58% RECOVERY *o— -o-— o—~o—o~-o-o 100 150 ELUTION VOLUME (ml) F i g u r e 19 E l u t i o n of the 5 5 F e - f i l t r a t e from an FeBC assay through a G-25 Sephadex column. The recover y r e f e r s t o the p r o p o r t i o n of 5 5 F e t h a t e l u t e d through the column. 66 3.3.4 Lake Siderophores The presence of low molecular weight chelators of iron in lake water could be observed either by the FeBC assay or a 55 Sephadex- Fe assay. These assays indicated that chelators were present August 28, 1980 in the n i t r a t e limnocorral, only in the surface water (Fig. 19). The low molecular weight peak (the compounds eluting l a t e r on a Sephadex column) contained a chelator. The high molecular weight peak could have been c o l l o i d a l Fe-MgCO.j that had not coagulated enough to be retained on a f i l t e r . The water from seven meters had comparatively l i t t l e a l g a l biomass and no apparent chelation capacity. The chelator that was isolated from an Aphanizomenon bloom in Black Lake in 1982 was d i f f e r e n t from the Anabaena flos-aquae chelator in that the Aphani zomenon chelator did not have a hydroxamate group. The unconcentrated f i l t r a t e had a FeBC of 1.5 uM. Unlike the Anabaena flos-aquae chelator, concentrated solutions of sodium and cobalt suppressed iron chelation by the Aphani zomenon chelator (Table 10). In contrast to the Anabaena  flos-aquae chelator, concentrated solutions of aluminum had l i t t l e e f f e c t on chelation of iron by the Aphanizomenon chelator (Table 10). Although these chelators are very d i f f e r e n t , the chelation data indicate that both of these compounds are siderophores. The iron binding capacity (FeBC) in the lake was never observed to be higher than 2 uM in 1980. The high levels of dissolved iron on Aug. 11, 1979 (Fig. 4 ) were observed in water supersaturated with oxygen, during a period of c a l c i t e p r e c i p i t a t i o n . Since iron should be quite insoluble in t h i s T a b l e 10 E f f e c t o f M e t a l A d d i t i o n o n F e C h e l a t i o n b y t h e A p h a n i z o m e n o n S i d e r o p h o r e %Fe i n s o l u t i o n C o n c e n t r a t i o n C a t i o n A l C o C u C u N a 0 . 2 5 M 0 . 2 5 M 1 mM 0 . 1 mM 1 . 2 5 M 6 9 . 9 0 . 6 , 0 . 5 5 2 . 4 , 2 . 4 5 . 1 , 4 . 1 7 . 6 , 7 . 8 r e p l i c a t e s T a b l e 11 B l a c k L a k e I r o n C o n c e n t a t i o n s a n d C h e l a t i o n C a p a c i t y - J u l y 1 7 , 1 9 8 0 S a m p l e F e B C S o l u b l e F e P a r t i c u l a t e F e uM F e / L ng F e / L F e / L 1 m 7 m 1 m 7 m 1 m 7 m C - l 0 . 8 8 0 . 3 5 <1 <1 4 0 80 F e - 1 3 5 0 2 3 0 1 5 0 2 0 0 8 0 1 0 0 E D T A - 1 3 4 9 19 4 < 1 0 . * < 1 0 . * 3 5 70 C - 2 0 . 2 4 0 . 1 2 <1 <1 3 0 7 5 F e - 2 3 2 0 2 7 0 1 8 0 1 7 5 70 82 E D T A - 2 2 0 4 3 3 1 < 1 0 * < 1 0 * 70 80 N 0 3 0 . 7 9 0 . 2 0 <1 <1 2 0 78 L C 1 0 . 2 4 <1 8 . 0 0 7 5 1 0 0 L A 1 . 4 0 0 . 2 5 <1 1 8 . 00 9 4 1 2 3 * E D T A i n t e r f e r e s w i t h b a t h o p h e n a n t h r o l i n e r e a c t i o n & d i g e s t i o n i n c r e a s e d b l a n k , s a m p l e s w i t h E D T A h a d t o b e d i l u t e d . L C - l a k e c o n t r o l s i d e L A - l a k e a e r a t e d s i d e . C - l , C - 2 - c o n t r o l l i m n o c o r r a l s . F e - 1 , F e - 2 - i r o n - E D T A l i m n o c o r r a l s . 68 water, the high i r o n c o n c e n t r a t i o n must have been maintained by e i t h e r a high f l u x of i r o n from the hypolimnion or by about 4 uM of c h e l a t o r (assumes 1:1 c h e l a t o r t o d i s s o l v e d i r o n ) . The c h e l a t o r c o n c e n t r a t i o n i n the e p i l i m n i o n of Black Lake i n 1980 was only weakly r e l a t e d t o the a l g a l biomass. However, hy p o l i m n e t i c samples w i t h minimal a l g a l biomass had much l e s s c h e l a t i o n c a p a c i t y than the e p i l i m n i o n (40% l e s s c h e l a t i o n by FeBC assay, Table 11; 98% l e s s c h e l a t i o n by g e l - f i l t r a t i o n assay, F i g . 19). The amount of c h e l a t i o n c a p a c i t y i n the l a k e was much l e s s than i n a l g a l c u l t u r e s . Even d u r i n g the Aphani zomenon bloom of 19 80, the FeBC of the la k e (1.5 uM, c h i a 150 ug/L) was much l e s s than t h a t observed i n 20 day o l d blue-green a l g a l c u l t u r e s (Anabaena c y l i n d r i c a 15 uM FeBC, 338 ug/L c h i a; Anabaena  fl o s - a q u a e 80 uM FeBC, 188 ug/L c h i a ) . In s p i t e of t h i s l a r g e d i f f e r e n c e between l a k e water and c u l t u r e f i l t r a t e s , the c h e l a t i o n c a p a c i t y of the l a k e water exceeded the d i s s o l v e d i r o n c o n c e n t r a t i o n f o r much of the e a r l y summer. Thus, a l g a l e x c r e t i o n c o u l d c o n t r o l i r o n b i o a v a i l a b i l i t y . The d i f f e r e n c e s i n c h e l a t i o n c a p a c i t y between l a k e water and c u l t u r e f i l t r a t e s may be a r e f l e c t i o n of the u t i l i z a t i o n of side r o p h o r e s i n these two h a b i t a t s . The a l g a l c u l t u r e s were u n i a l g a l and f r e e of b a c t e r i a . In summer a l l l a k e s had one or two dominant al g a e , s e v e r a l r a r e r algae, and b a c t e r i a . I f side r o p h o r e s were important mediators of symbiotic a s s o c i a t i o n s or a n t i b i o t i c c o m p e t i t i o n s , the a s s i m i l a t i o n of sid e r o p h o r e s should be q u i t e d i f f e r e n t i n c u l t u r e medium and la k e water. B a c t e r i a may both consume and produce s i d e r o p h o r e s . 69 55 The r a t e of a s s i m i l a t i o n of F e - s i d e r o p h o r e i s o l a t e , i n an axenic c u l t u r e of Anabaena f l o s - a q u a e , i s r e l a t i v e l y slow (<0.2 uM/d). However, r a p i d m i c r o b i a l a s s i m i l a t i o n of two ^"4C-s i d e r o p h o r e i s o l a t e s was observed i n Black. Lake. The c h e l a t o r from the Anabaena, t h a t was i s o l a t e d from Black Lake, was added back t o l a k e water on J u l y 4, 19 79, at an e q u i v a l e n t c o n c e n t r a t i o n t o the c u l t u r e (FeBC not measured); 20+ 1% of the c h e l a t o r was u t i l i z e d i n one day. In l a k e water samples from 1.0 m on June 17, 1979, 72 +10% 14 of the C - l a b e l l e d Anabaena c y l i n d r i c a c h e l a t o r (7 uM FeBC) was u t i l i z e d i n one day (5 uM/d). On J u l y 4, 1979, 21 +1% of t h i s A. c y l i n d r i c a c h e l a t o r (7 uM FeBC) was u t i l i z e d per day (1.5 uM/d). The r e d u c t i o n i n c h e l a t o r u t i l i z a t i o n from June 17 to J u l y 4 was c o n s i s t e n t with the seasonal i n c r e a s e i n the i r o n content of the 14 l a k e , the seasonal response of the i n s i t u C-primary p r o d u c t i o n b i o a s s a y s , the l i m n o c o r r a l b i o a s s a y s , and the reduced e f f e c t of the Anabaena c y l i n d r i c a c h e l a t o r on primary p r o d u c t i o n i n l a t e summer (Aug.30, A i n F i g . 11). 3.3.5 Enhanced I r o n A v a i l a b i l i t y The a l g a l c h e l a t o r i s o l a t e d from a l a b o r a t o r y c u l t u r e of Anabaena c y l i n d r i c a g r e a t l y s t i m u l a t e d a l g a l p r o d u c t i v i t y i n the l a k e i n June but had l e s s e f f e c t i n J u l y and l i t t l e e f f e c t i n August (A i n F i g . 11). U n l i k e d e s f e r a l and the c h e l a t o r s i s o l a t e d from Anabaena f l o s - a q u a e and Scenedesmus b a s i l i e n s i s , the A. c y l i n d r i c a c h e l a t o r never caused a l g a l c e l l s t o l y s e . The former c h e l a t o r s deformed c e l l s of two other Anabaena s p e c i e s and two Scenedesmus s p e c i e s a t low c o n c e n t r a t i o n s (10-50 uM FeBC). At 70 higher c o n c e n t r a t i o n s these c h e l a t o r s c o u l d r u p t u r e c e l l s of the above s p e c i e s . The l a c k of a n t i b i o t i c r e a c t i v i t y by the Anabaena c y l i n d r i c a c h e l a t o r makes i t an e x c e l l e n t c h e l a t o r t o make i r o n a v a i l a b l e to a l g a e . The s t i m u l a t e d a l g a l p r o d u c t i v i t y i n June ( F i g . 11) was p robably a r e s u l t of low l e v e l s of r e f r a c t o r y i r o n being made more a v a i l a b l e t o the a l g a e . Some of the p a r t i c u l a t e i r o n may have been weakly adsorbed t o humic matter. The c o n c e n t r a t i o n of c h e l a t o r used i n the assay i n F i g . 11 (5.0 riM FeBC) was two t o f i v e f o l d higher than any FeBC observed i n the l a k e i n summer of 19 80, but the c o n c e n t r a t i o n would be much more d i l u t e than i n the phycosphere around the a l g a l c e l l s . As noted e a r l i e r , siderophores appear t o be adsorbed weakly t o the f i b r i l s t h a t are found on the s u r f a c e of a l g a l c e l l s (3.3.2). 3.3.6 A l l e l o p a t h i c P r o p e r t i e s of Siderophores In Black Lake, Anabaena and Aphanizomenon had a s i g n i f i c a n t p o r t i o n of t h e i r biomass prese n t as almost u n i a l g a l clumps. The macro appearance of the clumps was d i s t i n c t enough t o a l l o w s e p a r a t i o n of two s p e c i e s of a l g a e . M i c r o s c o p i c examination showed t h a t the i s o l a t i o n s were at l e a s t 95% pure. A c h e l a t o r from the Anabaena clumps was i s o l a t e d and added t o f r e s h i s o l a t e s of Anabaena or Aphanizomenon i n the f i e l d . F i v e uM ( e q u i v a l e n t s of d e s f e r a l ) of the c h e l a t o r i s o l a t e d from the l a k e Anabaena s t i m u l a t e d the primary p r o d u c t i o n of Aphanizomenon but not Anabaena ( F i g . 20). Presumably, p r i o r t o my a d d i t i o n of c h e l a t o r , the Anabaena c h e l a t o r s a t i s f i e d the Anabaena i r o n requirement, but Aphani zomenon was not r e c e i v i n g an adequate supply of i r o n . i T ' S 6-1 5-0 J APHANIZOMENON ANABAENA Fe Sc Sc+Fe YL YL+Fe Fe Sc Sc+Fe YL YL+Fe JULY 19 1979 F i g u r e 20 A demonstration of siderophore s p e c i f i c i t y . The e f f e c t of the c o n t r o l (C), 10 uM Fe/L ( F e ) , 5 uM Scenedesmus b a s i l i e n s i s c h e l a t e ( S c ) , 5 uM Scenedesmus c h e l a t e + 10 uM Fe/L (Sc+Fe), 5 uM c h e l a t e i s o l a t e d from the Yellow Lake Anabaena (YL), and 5 uM YL c h e l a t e + 10 uM Fe/L (YL+Fe) on primary production of r e c e n t i s o l a t i o n s of Aphanizomenon and Anabaena. Values are means of two samples. E r r o r bars are one standard d e v i a t i o n . 72 The s p e c i f i c i t y of the c h e l a t o r a c t i v i t y was a l s o shown by the c h e l a t o r i s o l a t e d from a l a b o r a t o r y c u l t u r e of Scenedesmus  b a s i l i e n s i s (Sc., F i g . 20). F i v e uM of the Scenedesmus c h e l a t o r suppressed primary p r o d u c t i o n of Aphani zomenon, but not Anabaena. T h i s s u p p r e s s i o n was s t r o n g e r i f the c h e l a t o r was s a t u r a t e d with i r o n ; thus, the c h e l a t o r d i d not suppress Aphani zomenon by d e p r i v i n g i t of i r o n . The enhanced t o x i c i t y may i n d i c a t e t h a t Aphanizomenon t r e a t e d the Scenedesmus c h e l a t o r as a source of i r o n ; thus, the a d d i t i o n a l i r o n s t i m u l a t e d uptake of the t o x i c c h e l a t o r . The r e s u l t s from Black Lake i n d i c a t e d t h a t Aphanizomenon may r e q u i r e more i r o n than Anabaena. The Aphani zomenon c h e l a t o r was able t o suppress the growth of competing s p e c i e s . The c h e l a t o r was very t o x i c t o Scenedesmus  b a s i l i e n s i s when the s i d e r o p h o r e i s o l a t e was not s a t u r a t e d w i t h i r o n ( F i g . 21). The t o x i c i t y c o u l d have been r e l a t e d t o an enhancement of i r o n d e p r i v a t i o n . 3.3.7 Siderophore I n f l u e n c e on Heterotrophy Si n c e s i d e r o p h o r e i s o l a t e s i n f l u e n c e blue-green a l g a l growth, si d e r o p h o r e s probably i n f l u e n c e the growth of many other 14 b a c t e r i a . The a s s i m i l a t i o n of C - l a b e l l e d o r g a n i c compounds was used t o r e s o l v e the e f f e c t of s i d e r o p h o r e i s o l a t e s on b a c t e r i a . An a d d i t i o n a l c o n t r o l was used t o t e s t the p o s s i b i l i t y t h a t other low m olecular weight organic compounds were i n the a l g a l f i l t r a t e and were s u p p r e s s i n g the uptake of the ^ 4 C - l a b e l l e d s u b s t r a t e . In t h i s c o n t r o l , the same techniques t h a t were used f o r s i d e r o p h o r e i s o l a t i o n were used t o o b t a i n a f r a c t i o n from a c u l t u r e of Anabaena f l o s - a q u a e t h a t was grown wi t h i r o n . T h i s 73 TIME (days) F i g u r e 21 The e f f e c t of the Aphani zomenon s i d e r o p h o r e on the growth of Scenedesmus b a s i l i e n s i s . Values a re means of two c u l t u r e s . C o e f f i c i e n t of v a r i a t i o n i s l e s s than 10%. Table 12 E f f e c t of Iron A v a i l a b i l i t y on the T o x i c i t y of Anabaena F i l t r a t e s t o M i c r o b i a l A c e t a t e A s s i m i l a t i o n . A l g a l Growth Lake Water Gross Uptake Rate C o n d i t i o n s Treatment ug C L h xlO Iro n l i m i t a t i o n A l g a l e x t r a c t c o n t a i n s c h e l a t o r 2.39 Iron a d d i t i o n A l g a l e x t r a c t without c h e l a t o r 10.85 C o n t r o l no a l g a l e x t r a c t added 5.76 a c e t a t e concentration=2.75 ug C/L. Water c o l l e c t e d at 1 m from c o n t r o l s i d e of Black Lake on J u l y 31, 1980. c u l t u r e should not have produced any s i d e r o p h o r e (Murphy 1976). The i s o l a t e from the i r o n - s a t u r a t e d c u l t u r e s t i m u l a t e d h e t e r o t r o p h y w h i l e the i s o l a t e from an i r o n - d e f i c i e n t c u l t u r e depressed h e t e r o t r o p h y (Table 12). The s t i m u l a t i o n of h e t e r o t r o p h y c o u l d i n d i c a t e t h a t i r o n was being made a v a i l a b l e t o i r o n - l i m i t e d b a c t e r i a or t h a t a low molecular weight compound was s t i m u l a t i n g h e t e r o t r o p h i c a s s i m i l a t i o n of a c e t a t e . F o r t u n a t e l y , the s t i m u l a t i o n d i d not prevent the use of h e t e r o t r o p h i c b i o a s s a y s i n s t u d i e s of s i d e r o p h o r e - b a c t e r i a r e l a t i o n s h i p s ; the a l g a l s i d e r o p h o r e i s o l a t e s suppressed b a c t e r i a . The a b i l i t y of t h r e e a l g a l s i d e r o p h o r e i s o l a t e s t o suppress h e t e r o t r o p h i c a c t i v i t y was s t r o n g ( F i g . 22). B a c t e r i a l h e t e r o t r o p h y i n samples from a l l water depths was suppressed, and the g r e a t e s t s u p p r e s s i o n occurred i n the hypolimnion. The s u p p r e s s i o n o c c u r r e d e q u a l l y w e l l when these c h e l a t o r s were s a t u r a t e d with i r o n . Thus, the s u p p r e s s i o n was not r e l a t e d t o an enhancement of i r o n d e p r i v a t i o n . The i n h i b i t i o n by the s i d e r o p h o r e i s o l a t e s appeared t o be mediated by two r e a c t i o n s . The Anabaena siderophore i s o l a t e s (400 14 uM FeBC) enhanced r e s p i r a t i o n of C - l a b e l l e d a c e t a t e much more than e i t h e r the Scenedesmus sid e r o p h o r e i s o l a t e (10 uM FeBC) or c o n t r o l i n c u b a t i o n s (shaded area of F i g . 22). Another d i f f e r e n c e i n the p r o c e s s i n g of the s i d e r o p h o r e i s o l a t e s was r e v e a l e d i n the e f f e c t of g l u c o s e on siderophore i s o l a t e t o x i c i t y ( F i g . 23). The two s i d e r o p h o r e i s o l a t e s i n h i b i t e d g l u c o s e uptake when the c o n c e n t r a t i o n of g l u c o s e was low. At higher g l u c o s e c o n c e n t r a t i o n s , the i n h i b i t o r y e f f e c t s of the Anabaena (400 uM FeBC), but not the Scenedesmus (10 uM FeBC) siderophore i s o l a t e s , 75 ACETATE UPTAKE , ugCL"rh",x|0"* 0 2 4 6 8 10 12 14 16 1 1 1 1 1 j 1 1 2 5 CONTROL -Net CO, <u a> E CL Q 2 + 0 I 2 H—I 2 - t ^ ^ 5 9-6 H ANABAENA ( L A B ) Chelate ANABAENA  ( L A K E ) Chelate 2 -51 4 H Chelate • Fe I 2 H—I 9-Chelate + Fe H 1 2- IS 5-IB SCENEDESMUS Cheiote + 2-133 5-3 9 -B 2 H Cheiote • Fe F i g u r e 22 Suppression by side r o p h o r e i s o l a t e s of b a c t e r i a l a s s i m i l a t i o n of a c e t a t e . Samples from 2, 5 r and 9 meters were incubated at those depths with 3 side r o p h o r e i s o l a t e s and a c e t a t e . fcS^) r e p r e s e n t s a c e t a t e r e s p i r a t i o n . ACETATE, „gC I"1 i g u r e 24 E f f e c t of a c e t a t e on s i d e r o p h o r e s u p p r e s s i o n of heterotrophy. 77 were overcome. The t o x i c i t y of the Anabaena siderophore i s o l a t e could not be overcome by high concentrations of acetate ( F i g . 24) . The seasonal pattern of heterotrophic u t i l i z a t i o n of glucose in Black Lake formed a pattern that indicated suppression of b a c t e r i a l a c t i v i t y ( F i g . 25). A week af ter the co l lapse of the diatom bloom (May 11, 19 79), the greatest uptake of glucose was observed. A week af ter the co l lapse of a denser Anabaena bloom (July 18, 19 79), the lowest metabolic a c t i v i t y was observed ( F i g . 25) . The b a c t e r i a l a c t i v i t y increased af ter the co l lapse of the Aphani zomenon bloom (Aug. 31, 1979 ), but the a c t i v i t y was not as high as the spring value . Two factors explain the d i f ference between the two blue-green a l g a l blooms. 1) The Anabaena siderophore i s o l a t e suppressed bac ter ia heterotrophy ( F i g . 22) 2) The Aphani zomenon bloom was terminated by c a l c i t e p r e c i p i t a t i o n which lysed algae and appeared to enhance b a c t e r i a l a c t i v i t y . Siderophore ecology was not studied in a l l lakes . However, the phosphorus geochemistry indicated that Black Lake was very s i m i l a r to Fr i sken Lake and both of these lakes are qui te d i f f e r e n t from Chain Lake. Siderophore react ions are probably more important in Black and F r i s k e n lakes than in Chain Lake because the algae in Chain Lake would not need to produce siderophores. 78 GLUCOSE UPTAKE (pigC Lr'h^xK)- 2) CONTROL MAY 11 4 8 12 16 20 24 I l i I i i 2-3 9-JUNE 16 x t UJ Q -51 - . 4 JULY 19 J L 1 L 9 AUG 31 7 - 3 2 - B 5-9 1 2 5 • NET UPTAKE UJ MINERALIZATION g. ' ' t AERATED 4 8 12 16 20 24 _l I I i i i 1 2-5-9-2-6-J I - J •-a J L - ? - 1 2-5-9 J I 1 L F i g u r e 25 Temporal v a r i a t i o n of he t e r o t r o p h y i n Black Lake. 79 3.4 E f f e c t of Iron A v a i l a b i l i t y on Phosphorus Chemistry 3.4.1 Geographic V a r i a b i l i t y i n Fe-P Water Chemistry The most s t r i k i n g d i f f e r e n c e between Chain Lake and F r i s k e n and Black lakes was the seasonal change of phosphorus c o n c e n t r a t i o n s ( F i g . 26). In Chain Lake, the c o n c e n t r a t i o n of s o l u b l e r e a c t i v e phosphorus ( F i g . 27) or t o t a l phosphorus ( F i g . 28) was high o n l y i f the oxygen c o n c e n t r a t i o n was low ( F i g . 7). In Black or F r i s k e n l a k e , changes i n oxygen c o n c e n t r a t i o n was not c o r r e l a t e d with changes i n phosphorus c o n c e n t r a t i o n . In c o n t r a s t t o Chain Lake, Black Lake (Murphy e t a l . 1983a,b) and F r i s k e n Lake (Murphy et a l . 1985) had very l i t t l e r e a c t i v e i r o n . T h e r e f o r e , the best geochemical hypothesis f o r t h i s d i f f e r e n c e was t h a t higher r e a c t i v e i r o n c o n c e n t r a t i o n s r e s u l t e d i n much more f e r r i c phosphate p r e c i p i t a t i o n i n Chain Lake than i n Black or F r i s k e n l a k e . In p e r i o d s of high phosphorus c o n c e n t r a t i o n s i n Chain Lake i n 1973, 1976 ( F i g . 3 and 7 i n WIB 1977), and 1983 ( F i g . 27) the lower water column was anoxic ( F i g u r e 7). The i r o n data support the hypothesis t h a t i n anoxic water, i r o n i n the s u r f a c e sediments i s converted from i n s o l u b l e f e r r i c i r o n t o the more s o l u b l e f e r r o u s i r o n . The i r o n content of Chain Lake doubled from J u l y 6, 1984 t o September 14, 1984 (Table 3). The anoxic water contained a mean of 1.1 mg/L of i r o n . The changes i n water chemistry d u r i n g November 1983 i n Chain Lake were c o n s i s t e n t with the p r e c i p i t a t i o n of f e r r i c phosphate or another iron-phosphorus complex. The oxygen c o n c e n t r a t i o n had i n c r e a s e d from 4.4 to 8.0 mg/L. S i m i l a r changes occurred i n 1974 and 1975 ( F i g . 3 and p. 34 i n WIB 1977). 80 SOLUBLE REACTIVE PHOSPHORUS JLACK F i g u r e 26 Seasonal comparison of s o l u b l e r e a c t i v e phosphorus (SRP) c o n c e n t r a t i o n s i n Black, Chain, and F r i s k e n l a k e s . A l l Values are means of two samples. Black and F r i s k e n l a k e v a l u e s are means of two s t a t i o n s . c? CHAIN LAKE SOLUBLE REACTIVE PHOSPHORUS WO S R P (*q/L) F i g u r e 27 Chain Lake s o l u b l e r e a c t i v e phosphorus (SRP) A l l v a l u e s are means of two samples. CHAIN LAKE TOTAL PHOSPHORUS (HQ/L) F i g u r e 28 Chain Lake t o t a l phosphorus. I 50 NOV. 10,1983 82 3.4.2 Geographic V a r i a b i l i t y i n Sediment Iron R e a c t i v i t y The d i f f e r e n c e s between the water chemistry of Chain Lake and Black and F r i s k e n l a k e s are recorded i n the lake sediments. The f l u x e s of i r o n i n t o and out of the sediments p r o v i d e i n s i g h t i n t o i r o n a v a i l a b i l i t y . Complete i r o n budgets can only be c a l c u l a t e d f o r l a k e s t h a t have p y r i t e data and lead-210 r a d i o c h e m i c a l d a t i n g (Chain and F r i s k e n l a k e s , T a b l e 13). A comparison of the budgets has one l a r g e b i a s ; the sediments of Chain Lake are r e l a t i v e l y f l a t but the sediments of F r i s k e n Lake have much more s l o p e ( F i g . 2 ) . Resuspension from shallower sediments should r e s u l t i n enhanced sedimentation i n the r e l a t i v e l y s m a l l deep b a s i n of F r i s k e n Lake (sediment f o c u s s i n g ) . The d i s c r e p a n c y between the sedimentation of i r o n and h y d r a u l i c i r o n l o a d i n g (Table 13) supports the hypothesis t h a t the F r i s k e n Lake sediment core exaggerates the net i r o n sedimentation of the whole l a k e . Thus, the higher f l u x of i r o n i n t o Chain Lake r e l a t i v e t o F r i s k e n Lake (Table 13), i s g r e a t e r than the sediment core data i n d i c a t e . The e x t e r n a l i r o n l o a d i n g i s more than 200 f o l d higher and p y r i t e formation occurs more than 10 f o l d f a s t e r i n Chain Lake. 83 Table 13 Fluxes of Iron in Chain and Fr i sken Lakes Flux Fr i sken Lake Chain Lake Chain /Fr isken Hydraul ic „ Fe Loading 3 730 240 Sedimentation Rate Pb-210 1.25 7.8 6 Net Fe „ Sedimentation 120 470 4 Retention of Fe in lake (%) 4000 47 -Iron Reduction^ To P y r i t e 24 282 12 SedimenJ. Fe E f f l u x 35 238 7 Organic-C „„ Sedimentation 1400 4000 3 # -2 -1 * ** jig Fe cm yr . Sedimentation in mm per year. Increase in 2 ## —2 —1 water column iron from June to August, ug/cm . ug C cm yr The proport ion of iron found as p y r i t e , and the s t a b i l i t y of f e r r i c i r o n , are d i f f eren t in the sediments of Chain and Fr i sken lakes re spec t ive ly . In the Chain Lake sediment core, the concentration of p y r i t e increases u n t i l about 60% of the iron is present as p y r i t e ; i ron in the sediment core from Fr i sken Lake of the same age as the bottom of the Chain Lake core i s 20% p y r i t e . Re lat ive to Chain Lake, much more of the sediment iron in Fr i sken Lake appears to be stable as f e r r i c i r o n . Iron in sediments that had prec ip i ta ted f i f t y years ago (the bottom of the Chain Lake core) is 30% f e r r i c i r o n . Iron in the Fr i sken Lake 84 sediments of the same age as the bottom of the Chain Lake core is 60% f e r r i c iron. If differences in oxygen concentration produced t h i s difference then Chain Lake would have less oxygen. However, the water overlying the sediments of Frisken Lake has less oxygen than does Chain Lake (Fig. 7). X-ray d i f f r a c t i o n analysis indicated that in both lakes, some of the surface metastable iron i s in c h l o r i t e . Most of th i s metastable iron could not be characterized; t h i s technique can not measure iron bound to humic matter. In both Chain and Frisken lakes, the amount of iron released from the lake sediments was similar to the rate of p y r i t e formation; thus, p y r i t e formation i s an index of iron r e a c t i v i t y . Since most chemical variables are more favorable for p y r i t e formation in Frisken Lake than in Chain Lake, and Frisken Lake has proportionally less p y r i t e , these results indicate that the r e a c t i v i t y of iron i s much lower in Frisken Lake than in Chain Lake. The d i s t r i b u t i o n of iron, calcium, and phosphorus in Chain Lake indicates that geochemical reactions do not r e s t r i c t phosphorus mobility. The iron and calcium content is r e l a t i v e l y constant, whereas the phosphorus content i s enriched in the surface sediments. The phosphorus content was not correlated s i g n i f i c a n t l y to calcium (r=0.53) or iron content (r= -0.11) (Fig. 29). Carbonates are too d i l u t e to control phosphorus s o l u b i l i t y . The top two cm of sediment i s composed of only 2.1% calcium carbonate and the deeper sediments have no detectable calcium carbonate. With the probable exception of the surface sediments, iron i s also unable to influence phosphorus 300 Figure 29 Chain Lake sediment chemistry; ac id extractable iron and calc ium, b ioava i lab le phosphorus, water content, and age. 86 s o l u b i l i t y . In F r i s k e n , Roche (downstream from F r i s k e n Lake), and Yellow (downstream from Black Lake) l a k e s , the phosphorus content of the s u r f a c e sediments was i n v e r s e l y c o r r e l a t e d t o the i r o n content ( F r i s k e n Lake r=-0.81, n=6; Roche Lake r=-0.86, n=7; Yellow Lake r=-0.86, n=7). The i n v e r s e c o r r e l a t i o n was a r e f l e c t i o n of the s t r o n g c o n t r o l over phosphorus by c a l c i u m carbonate ( F r i s k e n Lake r=0.95, n=7; Roche Lake r=0.80, n=6; Yellow Lake r=0.95, n=7). The sediments c o n t a i n over 30% c a l c i u m carbonate ( F i g . 30, 31). X-ray d i f f r a c t i o n a n a l y s i s i n d i c a t e d t h a t the o n l y carbonate m i n e r a l was c a l c i t e . 3.5 E f f e c t of I r o n A v a i l a b i l i t y on C a l c i t e and Phosphorus  P r e c i p i t a t i o n The unusual biogeochemistry of the Thompson P l a t e a u enabled the r e a c t i o n s between phosphorus and c a l c i u m t o be used t o assess i r o n a v a i l a b i l i t y . 3.5.1 Phosphorus Chemistry of Black Lake The s o l u b l e r e a c t i v e phosphorus (SRP) content of the Yellow Lake Creek t h a t entered Black Lake (x=25 7 ug/L) was v e r y s i m i l a r t o t h a t of the l a k e (mean i n t e g r a t e d c o n c e n t r a t i o n v a r i e d from 228 t o 379 ug/L). The SRP i n the Yellow Lake Creek and the exposed v o l c a n i c rock e x t r a c t appeared t o be orthophosphate. The SRP c o e l u t e d through G-25 Sephadex beads w i t h 3 2 P 0 4 ( F i g . 32). Sephadex r e s i n separates phosphorus from any organic-P of a d i f f e r e n t molecular s i z e (Lean 1973). The a r s e n i c content of the stream (25+ 2 ug/L, n=2) and v o l c a n i c rock e x t r a c t (30 ug/L) were 87 O 0.5 1 1.5 XFe F i g u r e 30 Roche Lake sediment chemistry; percent of t o t a l c a l c i u m , i r o n , and phosphorus i n sample. F i g u r e 30 F r i s k e n Lake sediment chemistry; percent of t o t a l c a l c i u m , i r o n , and phosphorus i n sample. ( • ) i s the age of the sediment. 88 87 1 • • • O 0.5 1 1.5 F i g u r e 30 Roche Lake sediment chemistry; percent of t o t a l c a l c i u m , i r o n , and phosphorus i n sample. F i g u r e 30 F r i s k e n Lake sediment chemistry; percent of t o t a l c a l c i u m , i r o n , and phosphorus i n sample. ( • ) i s the age of the sediment. 88 89 l e s s than 10% of the phosphorus content of the samples; thus, a r s e n i c c o u l d not have i n t e r f e r e d with my a n a l y s i s . S i m i l a r l y , the o r g a n i c phosphorus content of fo u r samples was s m a l l (5-20 ug/L). The s i l i c a content of the v o l c a n i c rock e x t r a c t (20 mg/L) was too d i l u t e t o i n t e r f e r e i n the phosphorus a n a l y s i s (APHA 1976). 3.5.2 C a l c i t e P r e c i p i t a t i o n i n Black Lake During the Aphani zomenon bloom of 1979, l a r g e q u a n t i t i e s of carbonate were observed p r e c i p i t a t i n g on the i n c u b a t i o n b o t t l e s on the i r o n - r i c h a e r a t e d s i d e of the l a k e . The s a l t s on the b o t t l e s e f f e r v e s c e d v i g o r o u s l y when t r e a t e d with 0.1 N HC1. These s a l t s were not observed i n the c o n t r o l s i d e of the l a k e s . At t h i s time, the pH of the l a k e s u r f a c e i n c r e a s e d w h i l e the a l k a l i n i t y and phosphorus decreased with r e s p e c t t o samples c o l l e c t e d two weeks e a r l i e r ( F i g . 33). Phosphorus p r e c i p i t a t i o n i n the top 1.5 m of the Aphani zomenon bloom was at l e a s t 25 0 ug P/L i n the two week p e r i o d p r i o r t o August 11, 1979. The a l k a l i n i t y and phosphorus content of the hypolimnion i n c r e a s e d . The 19 80 o b s e r v a t i o n s c o n t r a s t s h a r p l y with o b s e r v a t i o n s d u r i n g the Aphani zomenon bloom of 1979 ( F i g . 33). C a l c i t e was not observed, a l k a l i n i t y was constant, and the phosphorus c o n c e n t r a t i o n decreased s l i g h t l y . In the two weeks b e f o r e the 12 August 1980 sampling of the Aphanizomenon bloom, l e s s than 10 ug P/L p r e c i p i t a t e d i n the s u r f a c e 1.5 m of water i n both s i d e s of the l a k e . The a l g a l biomass and water s t r a t i f i c a t i o n i n 1980 was s i m i l a r t o t h a t of 1979 ( F i g . 33). These r e s u l t s were s u r p r i s i n g , because the l a k e water i n 1980 was 1 0 - f o l d and 1 9 - f o l d 90 2 4H 6 8 Alkalinity mg/L 2-i 4-6 8 160 IE" 200 ^220 240 Alkalinity mg/L 160 180 200 220 240~ a4 6.8 9-2 9J6 AERATED as 9L2 CONTROL —i— 0J6 Figure 33 Lake chemistry during Aphanizomenon blooms; t o t a l phosphorus (PC<4), ch lorophy l l a (Chla) , oxygen ( 0 2 ) , temperature, a l k a l i n i t y , and pH on the aerated and contro l sides of the lake , 12 August 1980 ( • ) , 11 August 1979 ( • ) . 91 s u p e r s a t u r a t e d w i t h r e s p e c t t o c a l c i t e on t h e c o n t r o l a n d a e r a t e d s i d e s o f t h e l a k e . 3.5.2.1 P - K i n e t i c s a n d P - L i m i t a t i o n I n J u l y 1978, and J u n e and J u l y 1979, P-P0 4 a s s i m i l a t i o n was s o s l o w t h a t i t c o u l d n o t be d i f f e r e n t i a t e d f r o m p h o s p h o r u s a d s o r p t i o n ; t h e h i g h SRP c o n c e n t r a t i o n s a p p e a r e d t o s a t u r a t e p h o s p h o r u s a s s i m i l a t i o n . D u r i n g t h e 1979 b l o o m , p r e c i p i t a t i o n o f 32 a b o u t 98% o f t h e SRP r e s u l t e d i n r a p i d P-P0 4 u p t a k e ( F i g . 34). P h o s p h o r u s t u r n o v e r t i m e was 1.9 h i n t h e a e r a t e d a n d 46.0 h i n t h e c o n t r o l s i d e o f t h e l a k e . T h i s l a r g e d i f f e r e n c e b e t w e e n t h e two s i d e s o f t h e l a k e a p p e a r e d t o be r e l a t e d d i r e c t l y t o t h e amount o f c a l c i t e f o r m a t i o n a n d i n d i r e c t l y t o t h e p r o d u c t i v i t y . The b l o o m c o l l a p s e d s h o r t l y a f t e r t h i s p e r i o d o f c a l c i u m a n d p h o s p h o r u s p r e c i p i t a t i o n . 3.5.3 C a l c i t e P r e c i p i t a t i o n i n L i m n o c o r r a l s 3.5.3.1 19 80 Fe-EDTA L i m n o c o r r a l s i n B l a c k L a k e S e v e n l i m n o c o r r a l s w e r e u s e d i n a s t u d y o f m i c r o b i a l r e g u l a t i o n o f i r o n a v a i l a b i l i t y . E a r l y i n t h e summer, Fe-EDTA a n d Na-EDTA t r e a t m e n t s r e s u l t e d i n a pH o f 9.5, a d e c r e a s e o f c a l c i u m a n d a l k a l i n i t y , a n d c o m p l e t e p r e c i p i t a t i o n o f p h o s p h o r u s i n t h e t o p 1.5 m o f w a t e r . A f t e r two w e e k s , t h e a l g a l b i o m a s s was l e s s t h a n 15 ug c h i a/L and t h e p r i m a r y p r o d u c t i v i t y was l e s s t h a n 170 ug C L ^ d _ 1 . B o t h v a l u e s a r e an o r d e r o f m a g n i t u d e l e s s t h a n t h o s e f r o m t h e a l g a l b l o o m s i n o t h e r l i m n o c o r r a l s o r t h e l a k e (n=8). The o x y g e n c o n t e n t o f t h e w a t e r i n c r e a s e d b y o n l y 3 mg/L i n t h e Fe-EDTA and Na-EDTA t r e a t m e n t s . R e l a t i v e t o t h e l a k e a n d 9 2 F i g u r e 34 P uptake d u r i n g c a l c i t e p r e c i p i t a t i o n . Water from 1.0 m from the s u r f a c e of ae r a t e d (- - -) and c o n t r o l ( ) s i d e s of l a k e . 93 some l imnocorra l experiments, these long-term responses indicate that e i ther the a l g a l bloom and the bloom ef fect on oxygen concentrations las ted less than a week, or that another process such as c a l c i t e p r e c i p i t a t i o n , influenced phosphorus concentrat ions . 3 .5.3.2 N i t r a t e Induction of C a l c i t e P r e c i p i t a t i o n The l imnocorra l treatment that had the greatest a b i l i t y to induce c a l c i t e and phosphorus p r e c i p i t a t i o n was n i t r a t e enrichment. When n i t r a t e was used as a source of N, the pH increased to 9.5 or 9.6 (Murphy et a l . 1983). Within two weeks af ter a n i t r a t e enrichment, the surface water became depleted of n i t r a t e and phosphorus, the pH increased, and the a l k a l i n i t y decreased much more than in other l imnocorra l s . A l s o , the minimum calcium content of the surface water (1.0 m) was much lower than any other l imnocorra l (n i t ra te l imnocorral x=18.5 mg C a / L , other l imnocorral with c a l c i t e formation x=25.7 mg C a / L , and l imnocorrals with no c a l c i t e formation x=35 mg C a / L ) . C a l c i t e formed a crust on the walls of the n i t r a t e l imnocorra l . The biomass and p r o d u c t i v i t y of the n i t r a t e l imnocorra l were less than the contro l and the Fe-EDTA l imnocorra l s , and much less than in the Na-EDTA l imnocorra l s , or the lake (Murphy et a l . 1983a,b). In the hypolimnion, the phosphorus concentration was cons i s t en t ly much higher in the n i t r a t e l imnocorral than in the contro l l i m n o c o r r a l . Phosphorus increased in the hypolimnion by as much as 200 ug P /L with no increase in a l k a l i n i t y . The p r e c i p i t a t e d phosphorus d isso lved much faster than the p r e c i p i t a t e d c a l c i t e . This p r e f e r e n t i a l phosphorus d i s s o l u t i o n i n d i c a t e s t h a t phosphorus p r e c i p i t a t i o n was a s u r f a c e a d s o r p t i o n of phosphorus onto c a l c i t e r a t h e r than a p r e c i p i t a t e of c a l c i u m phosphate. 3.5.3.3 1982 F e - C i t r a t e L i m n o c o r r a l s The 1982 i r o n - c i t r a t e l i m n o c o r r a l treatments p r o v i d e d a d d i t i o n a l i n f o r m a t i o n on the r e g u l a t i o n of a l g a l p r o d u c t i v i t y by c a l c i u m carbonate p r e c i p i t a t i o n . C i t r a t e was a s s i m i l a t e d too q u i c k l y t o have an e f f e c t on c a l c i u m p r e c i p i t a t i o n v i a c h e l a t i o n of c a l c i u m ( s e c t i o n 3.2.3). Thus, c i t r a t e c o u l d only i n f l u e n c e c a l c i u m carbonate p r e c i p i t a t i o n by m i c r o b i a l o x i d a t i o n of c i t r a t e t o carbon d i o x i d e and by a subsequent lowering of pH. The e f f e c t of the c i t r a t e a s s i m i l a t i o n upon the pH of the e p i l i m n i a was too s m a l l t o be det e c t e d ( F e - c i t r a t e 8.5, 8.6; N a - c i t r a t e 8.5, 8.5; NC>3 8.7; C o n t r o l 8.6, 8.5). However, by Aug. 24, 1982, c i t r a t e c o n v e r s i o n i n t o CC>2 reduced the pH i n the hypolimnia s i g n i f i c a n t l y ( F i g . 35) . T h i s v e r t i c a l zonation of pH was a r e s u l t of m i c r o b i a l p r o d u c t i v i t y . A f t e r most of the c i t r a t e of the f i r s t enrichment 14 had been a s s i m i l a t e d , C - c i t r a t e uptake (June 28, 1982) was much f a s t e r at 3.0 m than at 1.0 m ( F i g . 36). S h o r t l y a f t e r the second a d d i t i o n of c i t r a t e (June 30), c i t r a t e a s s i m i l a t i o n was again much f a s t e r at 3.0 m than at 1.0 m (Table 7). The a s s i m i l a t i o n of 14 C - c i t r a t e i n the c o n t r o l l i m n o c o r r a l s was r e l a t i v e l y c o n s i s t e n t i n samples c o l l e c t e d at 1.0 and 3.0 m depths ( F i g . 36, Table 7); thus, the enhanced m i c r o b i a l p r o d u c t i v i t y i n hypolimnia of the c i t r a t e t r e a t e d l i m n o c o r r a l s must have been a r e s u l t of the 5.0m CORRAL SRP &4 • C &2-PH • C &0- • N03 Na 7 8 - Na • Fe *Fe 76- — 1 — — 150 50 OO SRP ,gLr1 F i g u r e 35 SRP and pH at 5.0 m i n l i m n o c o r r a l s on Aug. 24, 1982. L i m n o c o r r a l s were C, c o n t r o l ; NO^, n i t r a t e ; Na, sodium c i t r a t e ; and Fe, i r o n c i t r a t e . 500 400 300-LU f Mil | L 200H 5 100-M n CONT-1 COMT-2 NO3-I MO3-2 LAKE CTT-1 CIT-2 F « - 1 F e - 2 1m 3m 1m 3m 1m 3m 1m 3m 1m 3m im 3m 1m 3m im 3m Im 3m F i g u r e 36 Depth p r o f i l e of c i t r a t e a s s i m i l a t i o n . The net 14 C - c i t r a t e uptake on June 28, 1982 i n the c o n t r o l (Cont-1, Cont-2), n i t r a t e (N0 3~1, NO.j-2), sodium c i t r a t e ( C i t - 1 , C i t - 2 ) , and f e r r i c c i t r a t e (Fe-1, Fe-2) l i m n o c o r r a l s i s shown. 96 c i t r a t e enrichment. The 1 4 C - a s s i m i l a t i o n data suggest t h a t a change i n water chemistry produced by c i t r a t e enrichment would o n l y be observed i n the hypolimnia. 3.5.3.4 C i t r a t e E f f e c t on P-CaCO^ I n d i c a t e s Iron L i m i t a t i o n The phosphorus content of the l i m n o c o r r a l h ypolimnia r e f l e c t e d the m i c r o b i a l response t o i r o n enrichment. The phosphorus c o n c e n t r a t i o n was higher i n the more a c i d i c hypolimnia ( F i g . 35). The hypolimnia of the f e r r i c c i t r a t e l i m n o c o r r a l s had the h i g h e s t phosphorus c o n c e n t r a t i o n and the lowest pH. The lower pH values i n the hypolimnia of the f e r r i c c i t r a t e l i m n o c o r r a l s were presumably a r e s u l t of the s t i m u l a t i o n of m i c r o b i a l p r o d u c t i v i t y by i r o n enrichment. The higher phosphorus c o n c e n t r a t i o n i n the more a c i d i c hypolimnia c o u l d be a r e s u l t of e i t h e r g r e a t e r m i c r o b i a l r e g e n e r a t i o n of organic-P, of i n h i b i t i o n of phosphorus c o p r e c i p i t a t i o n with c a l c i u m carbonate, or of r e l e a s e of phosphorus from c a l c i u m carbonate as i t s e t t l e d i n t o the lower pH hypolimnia. 3.5.3.5 I n f l u e n c e of Weather on P-CaCO^ P r e c i p i t a t i o n The enigma of why sediment i r o n r e l e a s e r e s u l t e d i n c a l c i t e p r e c i p i t a t i o n i n Black Lake 1979, but not i n 1980, was r e s o l v e d i n the 1982 s t u d i e s . The asynchrony i n a l g a l oxygen p r o d u c t i o n and changes i n l a k e chemistry i n d i c a t e t h a t c a l c i u m carbonate p r e c i p i t a t i o n of phosphorus from the e p i l i m n i a of l i m n o c o r r a l s was p a r t l y c o n t r o l l e d by weather. The seasonal p a t t e r n of phosphorus d e p l e t i o n i n the e p i l i m n i a was very s i m i l a r i n a l l l i m n o c o r r a l s ( F i g . 15). The 97 r a t e of phosphorus d e p l e t i o n v a r i e d g r e a t l y among t h r e e p e r i o d s : warm water of June, c o o l water of J u l y , and warm water of August ( F i g . 15). The c o o l water of J u l y , 1982 was a s s o c i a t e d with u n u s u a l l y cloudy c o l d weather. Phosphorus was r a p i d l y p r e c i p i t a t e d i n June from the e p i l i m n i o n O6.0 u.g P L 1 d ^ ) . The water was warm, 20°C, and s u p e r s a t u r a t e d s e v e r a l f o l d with c a l c i u m carbonate. The c a l c i u m c o n c e n t r a t i o n appeared t o decrease w i t h i n 15 days by an average of 2.0 mg Ca/L a t a depth of 1.0 m (Table 14). In the c o o l e r 15°C water of J u l y , phosphorus u t i l i z a t i o n was und e t e c t a b l e and the c a l c i u m c o n c e n t r a t i o n changed l i t t l e . The c o o l i n g of the water reduced the degree of c a l c i t e s u p e r s a t u r a t i o n from 11.8 t o 5.5 f o l d . The peak of a l g a l biomass and oxygen p r o d u c t i o n o c c u r r e d i n the c o o l water of J u l y . Although the s u r f a c e water i n the l i m n o c o r r a l s i n J u l y was never l e s s than f i v e - f o l d s u p e r s a t u r a t e d with c a l c i u m carbonate, the r a p i d p r e c i p i t a t i o n of phosphorus and c a l c i u m d i d not occur u n t i l 2-3 weeks a f t e r the peak oxygen v a l u e s . When the water warmed back up t o 17°C i n e a r l y August, the mean phosphorus d e p l e t i o n at a depth of 1.0 m i n the l i m n o c o r r a l s became very r a p i d (>9.4 ug P L ^ d At t h i s depth, the c a l c i u m c o n c e n t r a t i o n decreased w i t h i n a month by a mean of 9.5 mg Ca/L. In the 1980 Aphani zomenon bloom, the c o l d e r weather, mixing of the l a k e by a storm, and the n a t u r a l d e l a y i n c r y s t a l f o rmation probably prevented c a l c i t e p r e c i p i t a t i o n . Table 14 Calcium Concentrations i n the 1982 L i m n o c o r r a l s Treatment June30 J u l y l 3 July23 Aug 2 4 lm 3m lm 3m lm lm 3m 5m C o n t r o l 1 49 50 48 49 46 41 41 41 C o n t r o l 2 48 49 48 47 49 40 39 38 N a - C i t r a t e 1 49 49 46 45 48 38 40 40 N a - C i t r a t e 2 49 48 48 48 48 37 39 40 F e - C i t r a t e 1 49 49 46 48 45 33 36 38 F e - C i t r a t e 2 49 49 46 48 44 34 36 38 N i t r a t e 1 48 49 47 48 48 38 42 41 N i t r a t e 2 48 50 47 49 46 47 42 42 A l l v alues are mg/L of Ca. The c a l c i u m content of the water used to f i l l the l i m n o c o r r a l s was 51 mg/L. 99 3.5.4 Calcium C h l o r i d e I n d u c t i o n of C a l c i t e P r e c i p i t a t i o n In t h i s experiment, the p r e c i p i t a t i o n of c a l c i u m carbonate was induced by i n c r e a s i n g the c a l c i u m c o n c e n t r a t i o n w i t h c a l c i u m c h l o r i d e ( F i g . 37). Chemical e x t r a c t i o n s and d i r e c t m i c r o s c o p i c a n a l y s i s were used t o determine i f p r e c i p i t a t e d phosphorus was a s s o c i a t e d with c a l c i u m . Calcium s a l t s were added t o l a k e water taken d u r i n g an a l g a l bloom ( c h i a 50 ug/L) t h a t was s e v e n - f o l d s u p e r s a t u r a t e d w i t h c a l c i t e (pH 8.6, Ca 40 mg/L, i o n a c t i v i t y p r o d u c t / s o l u b i l i t y product = IAP/K = 7.63) on Aug 24, 1982. sp T h i s sample was c o l l e c t e d a f t e r a p e r i o d of r a p i d phosphorus and c a l c i u m p r e c i p i t a t i o n (decreases of 110+10 ug P/L; 10+1 mg Ca/L i n f i v e d a y s ) . The phosphorus c o n c e n t r a t i o n i n the l a k e was constant d u r i n g the experiment and f o r the next two days. The oxygen i n c r e a s e i n the l a k e d u r i n g the experiment i n d i c a t e d t h a t the a l g a e were p h o t o s y n t h e t i c a l l y a c t i v e . The phosphorus c o n c e n t r a t i o n decreased l e a s t i n the c o n t r o l v e s s e l s (184 t o 134 ug/L). C a l c i u m c h l o r i d e enrichment r e s u l t e d i n a phosphorus decrease from 184 to 123 and 83 ug P/L i n the dark and l i g h t i n c u b a t i o n s r e s p e c t i v e l y . A l l f l a s k s , even the dark c o n t r o l , a t t a i n e d the same f i n a l pH (8.8+0.05). Another s m a l l experiment was done the next day t o r e p l i c a t e the Aug. 24 experiment. The temperature was maintained to w i t h i n 2°C of the l a k e s u r f a c e . Within seven hours (1100-1800) the phosphorus c o n c e n t r a t i o n (SRP) decreased from 184 ug P/L to 90 , 75, and 77 ug P/L i n the c o n t r o l , 10 mg C a C l 2 / L , and 20 mg C a C l 2 / L treatments r e s p e c t i v e l y . The d i s s o l v e d c a l c i u m c o n c e n t r a t i o n s decreased i n a l l of the C a C l 2 i n c u b a t i o n s (4.0+3.0 mg Ca/L). INDUCED CALCITE PRECIPITATION INCUBATE WHOLE H20 SAMPLE 10 h + CaCl2 Fi l ter Particles Syringe + H20 + C02 Incubate lh 4°C F i l ter F i l t rate SRP 149 MgP.L Ca 75 mg.L' 1 Figure 37 - l Syringe + H20 + Air Incubate lh 4°C 4 F i l te r F i l t rate SRP Ca In i t ia l Concentration Ca(0H)2 CaCl2 SRP Ca 10 mg.L"1 20 mg.L - 1 184 ygP.L' 1 53 mg.L' 1 -»• F i l t rate SRP pgP.L' 1 Ca mg.L' 1 Treatment hv DK hv Control Ca C l 2 Ca(0H)2 130 137 83 123 29 18 52 56 47 CaCl, ugP.L"1 mg.L"1 induced c a l c i t e p r e c i p i t a t i o n . 25 37 o o DK and hv analyses. represent dark Coef f i c i ent of and l i g h t incubations. Values v a r i a t i o n was less than 5%. are means of two 101 3.5.4.1 C a l c i t e A n a l y s i s A weak a c i d e x t r a c t i o n was used t o c h a r a c t e r i z e the phosphorus t h a t p r e c i p i t a t e d i n the c a l c i u m c h l o r i d e i n c u b a t i o n s . The p r e c i p i t a t e d phosphorus c o u l d be q u i c k l y r e d i s s o l v e d by c o o l i n g the p a r t i c l e s and by e q u i l i b r a t i n g the sample with CC^ ( F i g . 37). The carbon d i o x i d e reduced the pH to 5.7 which i s low enough f o r r a p i d c a l c i u m carbonate d i s s o l u t i o n (Berner and Morse 19 74). E q u i l i b r a t i o n of the sample with a i r reduced the pH from 8.8 to 8.2 and had l i t t l e e f f e c t on the phosphorus or c a l c i u m c o n c e n t r a t i o n . Carbon d i o x i d e appeared to have l i t t l e e f f e c t on the a l g a e ; algae t h a t were c o l l e c t e d from p e r i o d s without c a l c i t e p r e c i p i t a t i o n d i d not r e l e a s e phosphorus when e q u i l i b r a t e d w i t h c o 2 . D i r e c t m i c r o s c o p i c a n a l y s i s of p r e c i p i t a t e s was used t o a s s i s t the i n t e r p r e t a t i o n of the C 0 2 e x t r a c t i o n . P r e c i p i t a t e s were c o l l e c t e d from the Aug. 24 c a l c i u m c h l o r i d e experiment at the end of the i n c u b a t i o n p e r i o d by f i l t r a t i o n . C r y s t a l s had aggregated i n t o l a r g e " b a l l s " w i t h a mean diameter of 20 um i n the c o n t r o l treatment but not i n the carbon d i o x i d e treatment. The elemental a n a l y s i s i n d i c a t e d t h a t the p a r t i c l e s c o n s i s t e d mainly of c a l c i u m and t h a t s i l i c o n was an important c o n s t i t u e n t . Potassium, phosphorus and zin c were minor c o n s t i t u e n t s of the c a l c i t e p a r t i c l e s (Table 15). The r a t i o of c a l c i u m t o phosphorus i n these p a r t i c l e s (Ca/P molar r a t i o 72) corresponded t o the changes i n c a l c i u m and phosphorus observed e a r l i e r i n p e r i o d s of r a p i d c a l c i u m and phosphorus p r e c i p i t a t i o n i n the l a k e and l i m n o c o r r a l s (Table 16). 102 T a b l e 15 Elemental A n a l y s i s of C a l c i t e C r y s t a l Oxide Oxide% P r e c i s i o n 2 sigma CaO 51.78 0.59 s i o 2 17.11 0.24 K 2 0 2.33 0.14 P 2 ° 5 0.92 0.07 ZnO 0.85 0.13 A 1 2 ° 3 0.81 0.06 Na 20 .14 0.02 The computer program assumes a l l elements are present as simple oxides; the a n a l y s i s i s p r e c i s e but not n e c e s s a r i l y a c c u r a t e . The technique c o u l d be a c c u r a t e i f the a p p r o p r i a t e standards e x i s t e d . Table 16 Calcium/Phosphate R a t i o s i n P r e c i p i t a t i o n Events, P r e c i p i t a t i o n Experiments, and Sediment Sample Ca mg/L P ua/L Ca/P L - l May 25-June 5 13 140 77 L-2 J u l y 17-July 31 7 105 52 L-2 J u l y 31-Aug. 12 10 97 80 L-3 June 5- J u l y 15 21.5 250 67 Black Lake Aug. 80-79 20 250 62 Ca mg/g P mg/g Sed-1 0-2 cm 11.2 .13 66 Sed-2 10-12 cm 22.7 .22 79 C a C l 2 Induced P r e c i p i t a t e 37.0 .40 72 * molar r a t i o . L - l i s the Fe-EDTA l i m n o c o r r a l from the f i r s t l i m n o c o r r a l experiment. L-2 and L-3 are the EDTA-2 and N i t r a t e l i m n o c o r r a l s from the second s e t of 1980 l i m n o c o r r a l s i n Black Lake. The Black Lake Aug. 80-79 data i s the d i f f e r e n c e observed i n water chemistry from an a l g a l bloom with no carbonate p r e c i p i t a t i o n (Aug.12, 1980) t o one c o n t a i n i n g r a p i d carbonate p r e c i p i t a t i o n (Aug.11, 1979). A l l water samples were c o l l e c t e d 1.0 meter,from the s u r f a c e . A l l water chemistry i s the change i n mg L of Ca and ug L of SRP between the two dates. Sed-1 and Sed-2 are sediment samples from the s u r f a c e and 10-12 cm h o r i z o n of Yellow Lake. 103 These a d d i t i o n a l experiments with C a C l 2 c o n f i r m t h a t c a l c i u m carbonate p r e c i p i t a t i o n can c o n t r o l phosphorus s o l u b i l i t y . The importance of c r y s t a l i n i t i a t i o n and the s u s c e p t i b i l i t y of the r e a c t i o n t o i n h i b i t o r s r e s u l t s i n h i g h l y v a r i a b l e p r e c i p i t a t i o n . T h i s key r e a c t i o n c o u l d e a s i l y produce the v a r i a b i l i t y observed i n the i r o n experiments. 3.6 C a l c i t e P r e c i p i t a t i o n - A Major Cause of A l g a l P e r i o d i c i t y The processes t h a t occur d u r i n g c a l c i t e p r e c i p i t a t i o n were confirmed by i n d u c i n g c a l c i t e p r e c i p i t a t i o n i n F r i s k e n Lake w i t h lime, Ca(OH) 2. 3.6.1 Pretreatment Water Chemistry In pretreatment samples from F r i s k e n Lake, s i g n i f i c a n t c o r r e l a t i o n s of t o t a l i n o r g a n i c carbon (TIC) and s o l u b l e r e a c t i v e phosphorus (SRP) were observed i n both the e p i l i m n i o n / m e t a l i m n i o n (r=.735, n=21) and i n the hypolimnion (r=.973, n = l l ) . These two pretreatment data s e t s form two l i n e a r r e l a t i o n s h i p s ( F i g . 38). The lowest TIC and SRP c o n c e n t r a t i o n s were i n the e p i l i m n i o n , and the h i g h e s t were i n the hypolimnion ( F i g . 39). The same r e l a t i o n s h i p e x i s t e d between t o t a l i n o r g a n i c carbon and t o t a l phosphorus ( e p i l i m n i o n and metalimnion r=.753, n=21; hypolimnion r=.955, n = l l ) . The s i m i l a r i t y of the SRP-TIC and TP-TIC r e l a t i o n s h i p s was a r e f l e c t i o n of the synchrony of carbonate and phosphorus biogeochemistry. The carbonate e q u i l i b r i a were c l o s e l y r e l a t e d to phosphorus s o l u b i l i t y and presumably a l g a l p r o d u c t i o n . Most of 104 SRP(pg/L) F i g u r e 38 F r i s k e n Lake pretreatment SRP/TIC. SRP (pg/L) 0 500 1000 1500 I 1 1 _ _ i i 30 35 40 45 50 TIC (mg/L) F i g u r e 39 A depth d i s t r i b u t i o n of SRP and TIC i n F r i s k e n Lake. 105 the t o t a l phosphorus was in so lut ion (epil imnion and metalimnion 70.6%; hypolimnion 73.8%) and the soluble phosphorus was highly corre la ted to the t o t a l phosphorus (epil imnion and metalimnion r=.912, n=21; hypolimnion r=.959, n = l l ) . This r e l a t i o n s h i p was qui te s imi lar to that observed in Black Lake where 85% of the t o t a l phosphorus was in s o l u t i o n . 3.6.2 Lime-Induced C a l c i t e P r e c i p i t a t i o n The simple r e l a t i o n s h i p between TIC and SRP ( F i g . 38) predicted the re su l t s of the 1983 induct ion of c a l c i t e p r e c i p i t a t i o n by lime app l i ca t ion to Fr i sken Lake. The ep i l imnet ic TIC value in August 1983 of 30.5 mg/L would have a SRP of zero i f the r e l a t i o n s h i p of F i g . 38 were extrapolated; the SRP was less than 20 ug/L ( F i g . 40). Furthermore, examinations of p a r t i c l e s with an EDAX microprobe of an e lectron microscope supported the hypothesis that c a l c i t e p r e c i p i t a t i o n removed phosphorus from the epi l imnion (Table 15). Samples co l l ec ted in the epi l imnion af ter the 1984 t r i a l treatment contained p a r t i c l e s r i c h in calcium and phosphorus. Over a hundred nonce l lu lar p a r t i c l e s were analyzed. The r a t i o of P/Ca in ten p a r t i c l e s var ied between 0.64 to 0.04. Only two p a r t i c l e s contained i r o n , and these p a r t i c l e s contained no phosphorus; the high Fe/S r a t i o (0.35 to 0.56) indicates that these p a r t i c l e s were p y r i t e or p y r i t e precursors . These p a r t i c l e s were not we l l c r y s t a l l i z e d but they were not c e l l u l a r . FRISKEN LAKE EPILIMNETIC SRP Figure 40 Fr isken Lake epi l imnet ic SRP. 107 3 .6 .2 .1 Suppression of A l g a l Growth The induced c a l c i t e p r e c i p i t a t i o n was able to suppress the blue-green a l g a l blooms. The f i r s t addi t ion of lime was too small to induce p r e c i p i t a t i o n . The second addi t ion did not have an e f fec t a f ter 48 h but af ter another two weeks, the c h l o r o p h y l l a content had decreased ( F i g . 41). The 1984 induct ion of c a l c i t e p r e c i p i t a t i o n suppressed the c h l o r o p h y l l a content of the lake throughout the summer ( F i g . 41). The Secchi disk readings in 1984 exceeded four meters and were deeper than the pretreatment observations (Ashley, B . C . Environment). The lime treatment produced a long-term suppression of a l g a l growth and enhancement of phosphorus p r e c i p i t a t i o n . Although no lime was added to the lake in 19 85, the c h l o r o p h y l l a content of the lake was lower than pretreatment values ( F i g . 41; Ashley, B . C . Environment). The phosphorus concentrations in the epi l imnion of 19 85 were much lower than pretreatment values ( F i g . 40). The Secchi d i sc readings in 1985 (5.5, 2.8 and 4.4 m) were much deeper than pretreatment ones which were less than 2.0 m. 3.6.3 Long-Term Enhancement of C a l c i t e P r e c i p i t a t i o n The reduction of a l g a l biomass, the lower phosphorus concentrat ions , and improved c l a r i t y of 19 85 appeared to be produced by the 19 84 lime treatment. No lime was added to Fr i sken Lake in 19 85. The improved water q u a l i t y may have been produced by the d i s s o l u t i o n of c a l c i t e in the hypolimnion in 1983 and 1984. The d i s s o l u t i o n of c a l c i t e should enhance c a l c i t e p r e c i p i t a t i o n in the epi l imnion when the lake mixes. 108 FRISKEN LAKE 50-40-30-20-10-1983 50 1985 40-30-20-10-Denotes Lime Addition JUN JUL AUG F i g u r e 41 Suppression of a l g a l biomass by lime a p p l i c a t i o n . Values are means of two samples. The e r r o r bars r e p r e s e n t one standard d e v i a t i o n . 109 The d i s s o l u t i o n of c a l c i t e i n the hypolimnion was a composite of m i c r o b i o l o g i c a l and geochemical r e a c t i o n s . The decomposition of sedimenting algae d u r i n g e i t h e r a n a t u r a l or an induced c a l c i t e p r e c i p i t a t i o n r e s u l t s i n a r a p i d decrease i n oxygen ( F i g . 42) and pH i n the hypolimnion ( F i g . 43). The pH decreased by as much as 0.3 u n i t s w i t h i n 48 h. The low pH and temperature of the hypolimnion both c o n t r i b u t e t o the d i s s o l u t i o n of c a l c i t e ; thus, the t o t a l i n o r g a n i c carbon i n c r e a s e d ( F i g . 44). The e f f e c t of c a l c i u m carbonate r e c y c l i n g i s i l l u s t r a t e d by a model mixing of F r i s k e n Lake ( F i g . 45). The model mixes s e q u e n t i a l l y the u n d e r l y i n g one meter water l a y e r w i t h the o v e r l y i n g water column and c a l c u l a t e s the carbonate e q u i l i b r i a f o r each mixing. U n t i l the hy p o l i m n e t i c water mixes, the mixing water i s g r e a t l y s u p e r s a t u r a t e d with c a l c i t e . The p r e d i c t e d sequence of changes i n carbonate p r e c i p i t a t i o n were observed when the water column d e s t r a t i f i e d i n the f a l l of 1983. The f i r s t r e a c t i o n t h a t o c c u r r e d was an enhanced p r e c i p i t a t i o n of c a l c i t e ( F i g . 44). At t h i s time, h a l f of the phosphorus p r e c i p i t a t e d . However, as the l a k e continued t o mix a l l of the phosphorus r e d i s s o l v e d ( F i g . 40). An analogous enhancement of c a l c i t e p r e c i p i t a t i o n should occur i n the s p r i n g when the l a k e warms and becomes su p e r s a t u r a t e d with c a l c i t e . The 1985 water chemistry i n d i c a t e d t h a t the enhanced p r e c i p i t a t i o n of c a l c i t e d i d occur. FRISKEN LAKE 0 2 mg/L FRISKEN LAKE 0 2 mg/L BLACK LAKE 0 2 mg/L F i g u r e 42 E f f e c t of c a l c i t e p r e c i p i t a t i o n on oxygen. Values from Black and F r i s k e n lakes are means of data from two s t a t i o n s . E r r o r bars r e p r e s e n t one standard d e v i a t i o n . I l l X r -Q. 8 6 0 9 0 pH JULY 27 1963 Figure 43 The pH ( • ) and change in pH ( o ) of Fr i sken Lake 48 hours after lime a p p l i c a t i o n . Or a Ui O 5 JULY 25 AUG 16 30 40 50 AUG 25 SEPT 20 I , , I , , 30 40 50 30 40 50 T I C (mg/L) 40 50 Figure 44 T o t a l inorganic carbon (TIC) concentration of the water column of Fr i sken Lake in 1983. 112 Q J , , , , , J , , 1 , , J 1 i 1 1 1 - 8 - 4 0 +4 +8 - 8 - 4 0 +4 +8 - 8 - 4 0 +4 +8 F i g u r e 45 Simulated mixing of F r i s k e n Lake. Numbers w i t h i n graph r e f e r t o s e q u e n t i a l mixing of one meter water l a y e r s . Numbers on x- a x i s are the degree of s a t u r a t i o n of c a l c i t e ( i . e . +4 i s four f o l d s u p e r s a t u r a t e d ) . 113 DISCUSSION The primary object ive of demonstrating that i ron a v a i l a b i l i t y can af fec t the p e r i o d i c i t y of blue-green a l g a l blooms was achieved. New information on the s p a t i a l and temporal v a r i a t i o n of iron l i m i t a t i o n agrees with other recent studies (Stumm and Morgan 1981, Ryding 1985, Stauffer 1985). When and where iron l i m i t a t i o n w i l l occur is re lated to the ep i l imnet ic concentration of i r o n ; the phosphorus concentration in the lake water; the geochemistry of i r o n , calcium and phosphorus in the sediments; and the temperature of the lake sediments. Microbes excreted chelators that influenced iron a v a i l a b i l i t y and microb ia l succession. The main l imnet ic event that regulated iron a v a i l a b i l i t y was iron release from the lake sediments. P y r i t e formation regulated the amount of i ron re lease . Another event, c a l c i t e p r e c i p i t a t i o n , great ly a l t ered the amount of a l g a l biomass. In Black and Fr i sken lakes the sequential development of these four processes ( iron che la t ion , iron re lease from the sediments, p y r i t e formation, and c a l c i t e p r e c i p i t a t i o n ) determined the types of species growing, the amount of a l g a l biomass, and the duration of a l g a l blooms. Both biochemical and geochemical react ions influenced or contro l l ed the p e r i o d i c i t y of blue-green a l g a l growth. 4 .1 S p a t i a l and Temporal V a r i a t i o n in Iron A v a i l a b i l i t y The s p a t i a l v a r i a t i o n in iron a v a i l a b i l i t y in my study confirms the recent hypotheses of Ryding (1985) and Stauffer (1985); the supply of iron var ies great ly among lakes . The f lux of iron into Fr i sken Lake was less than 1% of that entering Chain 114 Lake. The bedrock geology of the s i t e s i s s i m i l a r (Cockf ie ld 1947,1948; Rice 1947) but the water entering Chain Lake (WIB 1977) had 50% less a l k a l i n i t y than the water entering Black Lake (Murphy et a l . 19 83b) and Fr i sken Lake (Murphy et a l . 19 85). On the Thompson Plateau, a l k a l i n i t y may be a usefu l guide to where i ron l i m i t a t i o n could occur. The concentration of i ron in the water column was a rough guide to when iron l i m i t a t i o n could occur. In Black Lake, addi t ion of iron stimulated a l g a l p r o d u c t i v i t y when the ambient i ron concentration was less than 50 ug/L and blue-green a l g a l blooms only formed in lake water with high concentrations of i r o n . The Black Lake c h l o r o p h y l l a (chi a) concentration was corre la ted to the iron concentrat ion. Some of the v a r i a b i l i t y of the c h i a-Fe c o r r e l a t i o n was caused by c a l c i t e p r e c i p i t a t i o n ; the greatest ch i a concentrations occurred during blooms with no c a l c i t e p r e c i p i t a t i o n . I f my in terpre ta t ion of the c h i a-Fe c o r r e l a t i o n of Black Lake is correct then the absence of a c h i a-Fe c o r r e l a t i o n in the Chain Lake data ( F i g . 6) should r e f l e c t the high a v a i l a b i l i t y of i ron in Chain Lake. The lowest i ron concentration in Chain Lake (Table 3) was equal to the highest i ron concentration in Black Lake (Appendix 2), and iron d id not st imulate a l g a l oxygen production in Chain Lake. Furthermore, the Black Lake studies indicated that Aphanizomenon needed more iron than other algae; therefore , the dominance of Aphani zomenon in Chain Lake confirmed that Chain Lake was not iron l i m i t e d . The i ron data in Fr i sken Lake was more complex than in the other lakes . I n i t i a l l y , dense blue-green a l g a l blooms (70 ug c h i 115 a/L) formed in water with r e l a t i v e l y l i t t l e iron (28 ug F e / L ) , and the c h l a concentrations were not corre la ted to the iron concentrat ions . Af ter the lime a p p l i c a t i o n , a l g a l growth was r e s t r i c t e d and c h l a concentrations were corre la ted to i ron concentrat ions . The suppression of a l g a l growth in 1984 in Fr i sken Lake was s u r p r i s i n g in that the SRP concentration of the epi l imnion was above 50 ug/L throughout the summer. The enhancement of carbonate p r e c i p i t a t i o n may have been a factor regulat ing a l g a l p r o d u c t i v i t y ; however, l imi ted iron a v a i l a b i l i t y may a lso have been important. The t o t a l i ron concentration in the epi l imnion of Fr i sken Lake in the ear ly summer of 19 84 was less than 20 ug /L . The s l i g h t increase of c h l o r o p h y l l a that was observed in August 1984 in Fr i sken Lake was associated with a doubling of the i ron in the epi l imnion ( F i g . 5). 4.2 C a l c i t e Induction of Iron L i m i t a t i o n The establishment of the c h l a-Fe r e l a t i o n s h i p in Fr i sken Lake appeared to be a r e f l e c t i o n of enhanced c a l c i t e p r e c i p i t a t i o n . The enhancement of i ron l i m i t a t i o n by c a l c i t e p r e c i p i t a t i o n is consistent with observations by Stauffer (1985). He proposed that hardwater lakes have l i t t l e iron because i ron mobi l i ty i s suppressed in calcareous areas. S t a u f f e r 1 s hypothesis i s consistent with s o i l studies that have shown that calcareous s o i l s have less ava i lab le i ron (Brown 1979). Stauffer d id not propose a mechanism for the suppression of iron m o b i l i t y . My observations in Fr i sken Lake were more s p e c i f i c than Stauffer*s in that the carbonate suppression of iron mobi l i ty was 116 produced in the lake , not in the drainage bas in . The simplest hypothesis to explain th i s observation, that i ron adsorbs d i r e c t l y to c a l c i t e , i s not supported by two observations. Iron was not associated with c a l c i t e c r y s t a l s in the water column (Table 14) nor was iron p o s i t i v e l y corre la ted to c a l c i t e in the sediments (Table A3, F i g . 30, 31). Without an assessment of i ron a v a i l a b i l i t y , the lack of Fe -c a l c i t e p r e c i p i t a t i o n would be s u r p r i s i n g because during a l l observations of natural c a l c i t e p r e c i p i t a t i o n in t h i s study, iron concentrations in the lake water were at a year ly high. The most p laus ib l e explanation for these observations i s that most of the ep i l imnet ic iron in Fr i sken and Black lakes was unreact ive . This hypothesis is consistent with the establ ished hypothesis that in aerobic and a l k a l i n e lake water, the majority of iron occurs as p h y s i o l o g i c a l l y unavai lable forms (Wetzel 1968). The r e c i p r o c a l of S tauf f er ' s hypothesis that lakes with l i t t l e iron w i l l have high phosphorus concentrations i s consistent with the geochemistry of the lakes upon the Thompson Plateau; lakes with high phosphorus concentrations should be i ron l i m i t e d . This hypothesis is a lso consistent with observations by R i p l (1986). He found that iron reacts p r e f e r e n t i a l l y with sulphide; thus, for f e r r i c phosphate react ions to occur, i ron must f i r s t saturate sulphide p r e c i p i t a t i o n react ions . F e r r i c react ions were able to regulate the s o l u b i l i t y of phosphorus in Chain Lake but not in Fr i sken or Black lakes ( F i g . 26). The strong bonding of phosphorus to inso luble f e r r i c i ron resulted in low phosphorus concentrations (<10 ug/L) in Chain Lake's oxidized water in spring and ear ly summer. F e r r i c 117 c o n c e n t r a t i o n s i n Black and F r i s k e n lakes were too low f o r p r e c i p i t a t i o n or a d s o r p t i o n of phosphorus to f e r r i c hydroxide ( B i r c h 1976, Stumm and Morgan 1981). These o b s e r v a t i o n s i n d i c a t e t h a t s u l p h i d e p r e c i p i t a t i o n of i r o n was not s a t u r a t e d i n F r i s k e n and Black l a k e s . The i r o n geochemistry of the water column was r e f l e c t e d w e l l i n the l a k e sediments. Observations t h a t the net sedimentation of organic-C i n F r i s k e n Lake i s o n l y a t h i r d t h a t of Chain Lake may be a r e f l e c t i o n of i r o n l i m i t a t i o n (Table 13). In F r i s k e n , Roche, and Yellow lake sediments, l i t t l e of the sediment i r o n was able to r e a c t with phosphorus. The d i s t r i b u t i o n of phosphorus was n e g a t i v e l y c o r r e l a t e d t o the i r o n c o n c e n t r a t i o n and p o s i t i v e l y c o r r e l a t e d t o the c a l c i t e c o n c e n t r a t i o n . Chain Lake sediments were d i f f e r e n t from sediments i n other lakes i n t h i s study; i r o n and phosphorus c o n c e n t r a t i o n s were not c o r r e l a t e d and c a l c i u m carbonate was un d e t e c t a b l e below one centimeter. Moreover, the r a t e of p y r i t e formation i n Chain Lake sediments was more than ten f o l d f a s t e r than i n F r i s k e n Lake sediments (Table 13, F i g . 8). Without an e v a l u a t i o n of i r o n a v a i l a b i l i t y , the d i f f e r e n c e s i n the formation of p y r i t e between Chain and F r i s k e n lakes would be a p u z z l e . The p r o p o r t i o n of i r o n found as p y r i t e was t h r e e times higher i n Chain Lake than i n F r i s k e n Lake. Chain Lake o f t e n had u n d e t e c t a b l e c o n c e n t r a t i o n s of sulphate (Murphy 1985); thus, sulphur a v a i l a b i l i t y should have l i m i t e d s u lphate r e d u c t i o n and p y r i t e formation (Berner 1971). Moreover, Chain Lake sediments were o f t e n o x i d i z e d ( F i g . 7) and oxygen suppresses p y r i t e formation (Berner 1971). In s p i t e of some adverse c o n d i t i o n s , the 118 r a t e of p y r i t e formation i n Chain Lake was more than t en times f a s t e r than i n F r i s k e n Lake; the a v a i l a b i l i t y of i r o n overcame the l i m i t i n g f a c t o r s t h a t r e s t r i c t p y r i t e f ormation. Sulphate r e d u c t i o n i n F r i s k e n Lake was not s t r o n g l y l i m i t e d by the supply of s u l p h a t e ; the lowest observed c o n c e n t r a t i o n of sul p h a t e was 500 ug/L and the odour of hydrogen s u l p h i d e was always obvious i n h y p o l i m n e t i c samples (Murphy e t a l . 1985). Furthermore, the low oxygen c o n c e n t r a t i o n s ( F i g . 7) i n F r i s k e n Lake should have enhanced p y r i t e formation. The presence of f e r r i c i r o n i n anoxic F r i s k e n Lake sediments i n d i c a t e d an i r o n complex more s t a b l e than c h l o r i t e ; p y r i t e formation proceeds r e a d i l y by u s i n g the i r o n i n c h l o r i t e (Berner 1971). The r e l a t i v e l y slow r a t e of p y r i t e formation i n F r i s k e n Lake was l a r g e l y a r e f l e c t i o n of low a v a i l a b i l i t y of i r o n . The mechanism of suppression of i r o n m o b i l i t y i n a l k a l i n e l akes i s the formation of p y r i t e , a r e f r a c t o r y i r o n m i n e r a l . C a l c i t e p r e c i p i t a t i o n of 90% of the a l g a l bloom i n F r i s k e n Lake must have enhanced p y r i t e f ormation. The optimal s i t e f o r p y r i t e formation i n F r i s k e n Lake would be the s u r f a c e sediments; the c o n s t i t u e n t s f o r p y r i t e formation, r e a c t i v e i r o n , o r g a n i c matter and b a c t e r i a t o produce s u l p h i d e , would be h i g h e s t here (Berner 1971). The formation of p y r i t e i n an anoxic environment would minimize the r e c y c l i n g of i r o n . A d o u b l i n g of the r a t e of p y r i t e formation i n Chain or F r i s k e n l a k e s would block sediment i r o n r e l e a s e (Table 13). The long-term s u p p r e s s i o n of a l g a l biomass by the lime treatment of F r i s k e n Lake would be p a r t i a l l y mediated by enhanced p y r i t e f ormation. The r a p i d decrease of pH by 0.1 to 0.2 u n i t s i n 24 h 119 i n the anoxic hypolimnion a f t e r lime treatment i n d i c a t e d r a p i d decay of a l g a e . I f most of the b a c t e r i a l r e d u c t i v e a c t i v i t y were a s s o c i a t e d with sulphate r e d u c t i o n then the b a c t e r i a l a c t i v i t y c o u l d have generated as much as 100 uM of H^S. The c o n c e n t r a t i o n of s u l p h a t e was about 5 uM; thus, only about h a l f of the i r o n c o u l d form p y r i t e . The r a t e of p y r i t e formation i s d i r e c t l y r e l a t e d t o the c o n c e n t r a t i o n of s u l p h a t e (Berner 1971); thus, the degree of i r o n l i m i t a t i o n would be much g r e a t e r i n lakes with more s u l p h a t e . The s u l p h a t e c o n c e n t r a t i o n i n Black Lake was 5-10 times h i g h e r than i n F r i s k e n Lake. The t y p i c a l e p i l i m n e t i c and h y p o l i m n e t i c s u l p h a t e c o n c e n t r a t i o n s i n Black Lake i n June were 5 and 4 mg/L. A f a s t e r r a t e of p y r i t e formation i n Black Lake would r e s u l t i n l e s s i r o n r e l e a s e from the sediments. As t h i s h y p o thesis p r e d i c t s , the hypolimnion of Black Lake had much l e s s than h a l f the i r o n c o n c e n t r a t i o n observed i n the hypolimnion of F r i s k e n Lake (Table 4, Appendix 2). 4.3 Sediment Iron Release In t h i s study, o n l y the sediment i r o n r e l e a s e of Chain Lake was c o n s i s t e n t with the model of Mortimer (1941, 1942), i . e . r e l e a s e with the onset of anoxia. Iron r e l e a s e i n Black and F r i s k e n l a k e s was not a s s o c i a t e d w i t h a r e d u c t i o n i n oxygen c o n c e n t r a t i o n . P y r i t e would not d i s s o l v e i n anoxic water and f e r r o u s i r o n t h a t was i n s o l u b l e p r e v i o u s l y should remain i n s o l u b l e . The g r a d u a l decrease i n the c o n c e n t r a t i o n of f e r r i c i r o n deeper i n the sediments i n d i c a t e s t h a t f e r r i c i r o n i s reduced to f e r r o u s i r o n , a p p a r e n t l y i n mid summer, which then 120 e i t h e r e n t e r s the water column or r e a c t s with s u l p h i d e t o e v e n t u a l l y form p y r i t e . Temperature was an important v a r i a b l e i n the sediment i r o n r e l e a s e at a l l s i t e s . Iron r e l e a s e occurred e a r l i e s t i n Chain Lake ( m i d - J u l y ) . T h i s shallow l a k e mixes r e a d i l y ; thus, the sediments were warmer than the other s i t e s . Iron r e l e a s e i n Black Lake d i d not occur u n t i l the sediments were warmer than 10°C ( l a t e J u l y ) . A 2.6°C warming of the sediments by l a k e a e r a t i o n appeared to enhance i r o n r e l e a s e on the aerated s i d e of Black Lake. Iron r e l e a s e occurred l a s t i n F r i s k e n Lake perhaps because the p r o f u n d a l sediments d i d not warm above 5°C u n t i l August. The e f f e c t of temperature on sediment i r o n r e l e a s e seemed t o produce the s t r o n g seasonal change i n i r o n c o n c e n t r a t i o n i n the water columns. The temperature of the sediment i s l a r g e l y c o n t r o l l e d by p h y s i c a l mixing; these two v a r i a b l e s a c t i n c l o s e synchrony t o i n f l u e n c e the sediment i r o n r e l e a s e . Temperature was more c l o s e l y r e l a t e d t o the p a t t e r n of i r o n r e l e a s e than was p h y s i c a l mixing i n my study; p h y s i c a l mixing can e x p l a i n a l l o b s e r v a t i o n s except f o r the slower sediment i r o n r e l e a s e i n F r i s k e n Lake r e l a t i v e t o the c o n t r o l s i d e of Black Lake. The c o i n c i d e n c e of the sedimentation of the Anabaena bloom and the r e l e a s e of sediment i r o n i n d i c a t e s t h a t m i c r o b i a l metabolic a c t i v i t y i s an important aspect of sediment i r o n r e l e a s e . A m i c r o b i a l i n f l u e n c e on sediment r e l e a s e of i r o n i s analogous t o the m i c r o b i a l mediation of phosphorus r e l e a s e from l a k e sediments (Ryding 1985); microbes enhance or mediate sediment phosphorus r e l e a s e and temperature enhances m i c r o b i a l 121 metabolic a c t i v i t y . 4.4 I n t e r r e l a t i o n s h i p of Iron L i m i t a t i o n , C h e l a t i o n and C a l c i t e P r e c i p i t a t i o n To assess completely the i r o n l i m i t a t i o n of a l a k e r e q u i r e s the use of s e v e r a l assays. Moreover, the impact of a s s o c i a t e d r e a c t i o n s , such as c a l c i t e p r e c i p i t a t i o n , must be r e s o l v e d i n synchrony or many i r o n b i o a s s a y s cannot be r e s o l v e d . The d i s c o v e r y t h a t c a l c i t e p r e c i p i t a t i o n could s t r o n g l y r e g u l a t e the response t o i r o n enrichment p r o v i d e d i n s i g h t i n t o many o b s e r v a t i o n s . However, the experimental use of a c h e l a t o r t o maintain i r o n i n s o l u t i o n made r e s o l u t i o n of the e f f e c t of i r o n l i m i t a t i o n and c a l c i t e p r e c i p i t a t i o n d i f f i c u l t . EDTA appeared t o s t i m u l a t e a l g a l and b a c t e r i a l p r o d u c t i v i t y more than Fe-EDTA; thus, a process other than i r o n a v a i l a b i l i t y may have been important. A l i k e l y e x p l a n a t i o n f o r the apparent EDTA s t i m u l a t i o n of primary p r o d u c t i o n and b a c t e r i a l heterotrophy i s t h a t EDTA may have suppressed c a l c i t e p r e c i p i t a t i o n . C a l c i t e p r e c i p i t a t i o n i s e a s i l y blocked by org a n i c compounds, e s p e c i a l l y c h e l a t o r s (Reynolds 1978). The s m a l l e r degree of phosphorus and c a l c i u m p r e c i p i t a t i o n i n the EDTA l i m n o c o r r a l s i n d i c a t e d t h a t l e s s c a l c i t e p r e c i p i t a t i o n o c c u r r e d . R e l a t i v e t o l i m n o c o r r a l s with c a l c i t e p r e c i p i t a t i o n , the algae i n the Na-EDTA l i m n o c o r r a l s would have a much longer r e s i d e n c e time i n the euphotic zone which would r e s u l t i n g r e a t e r p r o d u c t i v i t y . The f i r m b i n d i n g of i r o n would have prevented EDTA i n the Fe-EDTA l i m n o c o r r a l s from suppressing c a l c i t e p r e c i p i t a t i o n . Iron s t i m u l a t e d microbes i n 122 the Fe-EDTA l i m n o c o r r a l s , but the use of EDTA as a c o n t r o l treatment weakened the d e t e c t i o n of the i r o n enrichment response. These doubts about EDTA l e d t o the d e c i s i o n t o repeat the i r o n enrichment experiments w i t h c i t r a t e as the i r o n c h e l a t o r . C i t r a t e i s a weak c h e l a t o r and many microbes (Neilands 19 81b) and p l a n t s ( T i f f i n 1966) can u t i l i z e i r o n c h e l a t e d by c i t r a t e . The r a t e of m i c r o b i a l c i t r a t e a s s i m i l a t i o n i n my study was f o r t u n a t e i n t h a t i r o n was maintained i n s o l u t i o n f o r days and the c h e l a t o r was completely metabolized a f t e r a few days. T h i s r a t e of c i t r a t e u t i l i z a t i o n p r o v i d e d an e x c e l l e n t o p p o r t u n i t y t o observe long-term responses t o i r o n enrichment. I n i t i a l l y , the f e r r i c - c i t r a t e l i m n o c o r r a l s had lower oxygen c o n c e n t r a t i o n s than the c o n t r o l l i m n o c o r r a l s ; i r o n seemed t o enhance b a c t e r i a l oxygen u t i l i z a t i o n . Once the c i t r a t e was a s s i m i l a t e d , the f e r r i c - c i t r a t e l i m n o c o r r a l s had s i g n i f i c a n t l y higher oxygen c o n c e n t r a t i o n s than the s o d i u m - c i t r a t e l i m n o c o r r a l s (Appendix 3); i r o n s t i m u l a t e d a l g a l oxygen p r o d u c t i o n . The f e r r i c - c i t r a t e l i m n o c o r r a l s i n Black Lake pr o v i d e d h i g h l y s i g n i f i c a n t evidence t h a t an inadequate supply of i r o n was l i m i t i n g the a l g a l p r o d u c t i v i t y of Black Lake ( F i g . 15). 4.5 M i c r o b i a l C o n t r o l of Iron A v a i l a b i l i t y A study of a l g a l siderophores was r e q u i r e d t o separate the e f f e c t s of i r o n l i m i t a t i o n from those produced by c a l c i t e p r e c i p i t a t i o n . The side r o p h o r e i s o l a t e s had many p r o p e r t i e s of siderophores ( s e c t i o n 3.3); however, the p u r i t y of the siderophores was not r i g o r o u s l y e s t a b l i s h e d . Although C o r b e t t and Chipko (1978) found t h a t Sephadex chromatography of hydroxamic 123 a c i d s r e s u l t e d i n compounds t h a t were more than 99% pure, i o n exchange and Sephadex chromatography may not have completely p u r i f i e d my s i d e r o p h o r e i s o l a t e s . With the p o s s i b l e e x c e p t i o n of the b a c t e r i a l heterotrophy, t h i s u n c e r t a i n t y has no e f f e c t on the i n t e r p r e t a t i o n of my s t u d i e s . T h i s study of the m i c r o b i a l aspects of i r o n a v a i l a b i l i t y r e q u i r e d the use of l i m i t e d s u p p l i e s of n a t u r a l l y o c c u r r i n g c h e l a t o r s ; thus, s m a l l b o t t l e s had t o be used. Growth, primary p r o d u c t i o n , and h e t e r o t r o p h y bioassays i n d i c a t e d t h a t the m i c r o b i a l r e g u l a t i o n of i r o n a v a i l a b i l i t y e xerted a s t r o n g c o n t r o l over a l g a l and b a c t e r i a l growth. A n a l y s i s of l a k e water i n d i c a t e d t h a t c h e l a t o r s c o u l d c o n t r o l the a v a i l a b i l i t y of a l l 55 d i s s o l v e d i r o n . The e x c r e t i o n of c h e l a t o r s was d e t e c t e d with Fe by Sephadex chromatography and the FeBC assay. The a b i l i t y of c h e l a t o r s t o c o n t r o l m i c r o b i a l metabolic a c t i v i t y was demonstrated i n primary p r o d u c t i o n and heterotrophy assays. The s p e c i f i c i t y of the siderophore i s o l a t e s i n c h e l a t i n g i r o n ( F i g 18) supports the hypothesis t h a t the a c t i v i t y of these i s o l a t e s i s r e l a t e d t o s i d e r o p h o r e s . Moreover, t h i s d a t a d i s p u t e s the a l t e r n a t i v e hypothesis t h a t o r g a n i c c h e l a t o r s enhance a l g a l p r o d u c t i v i t y by complexing t o x i c metals; i n my assays, an i r o n -s a t u r a t e d s i d e r o p h o r e would not d e t o x i f y another metal. In l a k e s , o n l y a concentrated copper treatment c o u l d overcome a s i d e r o p h o r e a f f i n i t y f o r i r o n . T h i s high degree of s p e c i f i c i t y i s s i m i l a r t o t h a t observed by N e i l a n d s (1957), Anderegg et a l . (1963), Davis et a l . (1971), and Emery (1971) with b a c t e r i a l and f u n g a l s i d e r o p h o r e s . 124 The s p e c i f i c i t y and the d i s t r i b u t i o n of a l g a l c h e l a t o r s i n d i c a t e t h a t a s t r o n g c o n t r o l of i r o n a v a i l a b i l i t y occurs near the c e l l . Blue-green algae appear t o have two s t r a t e g i e s of m a i n t a i n i n g c h e l a t o r s i n c l o s e p r o x i m i t y to an a l g a l c e l l . Anabaena c y l i n d r i c a produces a c h e l a t o r t h a t appears t o be i n t r u e s o l u t i o n . T h i s a l g a i s covered with a very t h i c k (2.6 urn) and dense l a y e r of c o l l o i d a l f i b r i l l a r m a t e r i a l t h a t should r e s t r i c t water movement around the c e l l (Leppard et a l . 1977). Anabaena f l o s - a q u a e produces much l e s s c o l l o i d a l m a t e r i a l (0.35 urn t h i c k l a y e r ) than A. c y l i n d r i c a (Leppard et a l . 19 77). A. f l o s - a q u a e e x c r e t e s a c h e l a t o r t h a t i s l o o s e l y adsorbed t o a c o l l o i d a l f i b r i l t h a t i s an e x t e n s i o n of the c e l l s u r f a c e . F r e e z e - d r y i n g was a more e f f e c t i v e method of i s o l a t i n g c h e l a t o r s from f i b r i l s than was freeze-thawing. C h e l a t i o n c a p a c i t y was higher a f t e r f r e e z e - d r y i n g than a f t e r one f r e e z e -thaw. Frozen f i l t r a t e s from Anabaena c u l t u r e s t h a t were thawed and r e f i l t e r e d twice s t i l l l e f t a s l i m y d e p o s i t of f i b r i l s on f i l t e r s when f r o z e n and thawed a t h i r d time. I s o l a t e s t h a t were f r e e z e - d r i e d , thawed, and r e f i l t e r e d d i d not l e a v e a slime l a y e r when f r o z e n , thawed, and r e f i l t e r e d a second time. F i b r i l s appear t o be dehydrated more e f f e c t i v e l y by f r e e z e - d r y i n g than by f reeze-thawing. Dehydrated f i b r i l s are r e l a t i v e l y i n s o l u b l e i n water. R e s o l u t i o n t h a t the Anabaena f l o s - a q u a e c h e l a t o r i s l o o s e l y bonded to c o l l o i d a l f i b r i l s p r o v i d e s i n s i g h t i n t o the in s i t u c o n c e n t r a t i o n of c h e l a t o r s . In a c u l t u r e f i l t r a t e t h a t i n c r e a s e d i n FeBC from 2 uM to 80 uM a f t e r u l t r a f i l t r a t i o n , the e q u i l i b r i u m between d i s s o l v e d and adsorbed c h e l a t o r s was 2/78 uM. The 125 f o l l o w i n g measurements were used t o d e r i v e the i n s i t u 3 c o n c e n t r a t i o n of c h e l a t o r ; mean c e l l volume of 2 um , c e l l l e n g t h 7 3 of 1.8 u m , c e l l width of 1.2 u m , t o t a l c e l l volume of 10 um, and f i b r i l l e n g t h of 0.35 u m (Leppard 1977 et a l . ) . The volume of the phycosphere was 1.8% of the c u l t u r e medium; thus, the c o n c e n t r a t i o n of c h e l a t o r i n the phycosphere was 4.3 mM. The r e a c t i v i t y of the adsorbed c h e l a t o r i s u n r e s o l v e d ; however, i f the estimate t h a t 2.5% of the c h e l a t o r was i n s o l u t i o n was a c c u r a t e , then the c o n c e n t r a t i o n of d i s s o l v e d c h e l a t o r i n the phycosphere was about 110 pM. The weak a s s o c i a t i o n of c h e l a t o r s with f i b r i l s e x p l a i n s why algae would not " l o s e " an e x c r e t i o n product i n the bulk water. T h i s d i s c o v e r y a l s o j u s t i f i e s the use of s o l u t i o n s of c h e l a t o r s i n b i o a s s a y s t h a t are more concentrated than those found i n the f r e s h l y f i l t e r e d medium. The c r i t i c i s m t h a t c o n c e n t r a t i n g l a k e water exaggerates the s i g n i f i c a n c e of o r g a n i c - i r o n complexation (Plumb and Lee 1973) now seems l e s s s e r i o u s . The study of c h e l a t o r m i c r o d i s t r i b u t i o n p r o v i d e s u s e f u l i n s i g h t i n t o other c h e l a t o r s t u d i e s (Murphy 1976). E a r l i e r 55 o b s e r v a t i o n s with g e l chromatography of F e - l a b e l l e d a l g a l f i l t r a t e s c o u l d not be completely r e s o l v e d . An unconcentrated f i l t r a t e c o u l d c a r r y very l i t t l e ^ F e through the column and most 55 of the Fe t h a t passed through the column was a s s o c i a t e d w i t h a c o l l o i d a l f r a c t i o n . I f the medium was f r e e z e - d r i e d , i t c o u l d be d i l u t e d t o the o r i g i n a l c o n c e n t r a t i o n of the c u l t u r e and v i r t u a l l y a l l of the i r o n c o u l d pass through a Sephadex column wit h a low molecular weight or g a n i c c h e l a t o r . The r e s u l t s of my U.B.C. s t u d i e s i n d i c a t e t h a t the enigma of my M.Sc. study was 126 produced by the a d s o r p t i o n of the c h e l a t o r t o f i b r i l s i n f r e s h medium and i s o l a t i o n of the c h e l a t o r from f i b r i l s by f r e e z e -d r y i n g . One problem r e l a t e d t o the c h e l a t o r f i b r i l a s s o c i a t i o n found i n the Black Lake study, i s t h a t some lake water samples were not c o n c e n t r a t e d , f r o z e n , or passed through an u l t r a f i l t r a t i o n membrane. C h e l a t i o n c a p a c i t y i n these samples may have been underestimated. F o r t u n a t e l y , i n t e r p r e t a t i o n of the d a t a i s s t i l l s imple. The measured c h e l a t i o n c a p a c i t y of the l a k e water was g r e a t e r than the d i s s o l v e d i r o n c o n c e n t r a t i o n . Thus, m i c r o b i a l c h e l a t o r s c o u l d complex a l l of the s o l u b l e i r o n and the access t o i r o n c o u l d r e g u l a t e which microbes could grow. 4.6 Siderophores as Mediators of A l g a l Succession The o b s e r v a t i o n t h a t s i d e r o p h o r e i s o l a t e s could e i t h e r i n h i b i t or s t i m u l a t e ( F i g . 20) one a l g a l s p e c i e s but not a c o e x i s t i n g s p e c i e s i n d i c a t e s t h a t siderophores can i n f l u e n c e a l g a l s u c c e s s i o n . The d e c r e a s i n g r a t e of u t i l i z a t i o n of siderophore i s o l a t e s i n Black Lake as the i r o n c o n c e n t r a t i o n i n c r e a s e d i n d i c a t e s t h a t a siderophore mediation of m i c r o b i a l s u c c e s s i o n would decrease with a change i n i r o n a v a i l a b i l i t y . S i m i l a r seasonal changes i n a l l e l o p a t h y can be found i n many c u l t u r e and l a k e bioassays (Hutchinson 1967, Fogg et a l . 1973, H e l l e b u s t 1974, E l b r a c h t e r 1976, K e a t i n g 1978, Kayser 1979, Wilson et a l . 1979, Wolfe and R i c e 1979, Chan e t a l . 1980). In a l l of these s t u d i e s , the f i l t r a t e from e i t h e r a c u l t u r e or a l a k e water sample suppressed the growth of another a l g a . I t i s h i g h l y probable t h a t some of these f i l t r a t e s contained 127 s i d e r o p h o r e s . L a b o r a t o r y s t u d i e s have demonstrated t h a t excreted a l g a l compounds, wit h many p r o p e r t i e s of s i d e r o p h o r e s , c o u l d suppress the growth of a competing a l g a l s p e c i e s ( F i g . 21; Murphy 1976). The antagonism was p r i m a r i l y a s u p p r e s s i o n of i r o n a v a i l a b i l i t y t h a t i r o n a d d i t i o n c o u l d r e v e r s e . However, other i n h i b i t i o n r e a c t i o n s were p r e s e n t . The concentrated a l g a l s i d e r o p h o r e i s o l a t e s (lOx mature c u l t u r e c o n c e n t r a t i o n ) from Anabaena f l o s -aquae or Anabaena c y l i n d r i c a c ould l y s e Scenedesmus b a s i l i e n s i s but not the parent a l g a . P r i o r t o l y s i s the c e l l s became rounded and a f t e r s e v e r a l hours most of the c e l l s had r u p t u r e d . The s p e c i f i c t o x i c i t y of these s i d e r o p h o r e i s o l a t e s i n d i c a t e s t h a t siderophores c o u l d r e g u l a t e symbiotic a s s o c i a t i o n s i n a l g a l clumps. Symbiotic a s s o c i a t i o n s between b a c t e r i a and clumps of n i t r o g e n f i x i n g blue-green algae have been documented by P a e r l (19 82a). I r o n complexed by the c h e l a t o r produced by Anabaena o s c i l l a r o i d e s was p r e f e r e n t i a l l y a s s i m i l a t e d by both b a c t e r i a and a l g a l c e l l s near the h e t e r o c y s t s of A. o s c i l l a r o i d e s ( P a e r l 1982b). Other aspects of i r o n a s s i m i l a t i o n may a l s o i n f l u e n c e a l g a l s u c c e s s i o n . The r e s t r i c t e d growth of Aphani zomenon i n i r o n - l i m i t e d water and i t s r a p i d growth i n i r o n - r i c h water i n d i c a t e s t h a t d u r i n g a l g a l blooms t h i s a l g a u t i l i z e s a l o w - a f f i n i t y iron-uptake system. These a s s i m i l a t i o n systems are c o m p a r a t i v e l y i n e f f i c i e n t and n o n - s p e c i f i c (Neilands e t a l . 1980). 128 4.7 Siderophores as Regulators of B a c t e r i a l Heterotrophy Three a l g a l s i d e r o p h o r e i s o l a t e s were able t o g r e a t l y suppress b a c t e r i a l a s s i m i l a t i o n of simple organic compounds. The r e s p i r a t i o n of a s s i m i l a t e d o r g a n i c compounds was g r e a t l y enhanced by s i d e r o p h o r e i s o l a t e s from two Anabaena s p e c i e s . These d i s r u p t i o n s i n d i c a t e d a s u p p r e s s i o n of b a c t e r i a by the a l g a l s i d e r o p h o r e s . The seasonal p a t t e r n of h e t e r o t r o p h i c a c t i v i t y i n Black Lake a l s o i n d i c a t e d t h a t Anabaena i n h i b i t e d b a c t e r i a . The s u p p r e s s i o n of b a c t e r i a by blue-green algae i s w e l l known (Chrost 1973, Delucca and McCracken 1977, R e i c h a r d t 1981); however, the i d e a t h a t i r o n a v a i l a b i l i t y mediates a l g a l s u p p r e s s i o n of b a c t e r i a i s new. The s u p p r e s s i o n of b a c t e r i a by s i d e r o p h o r e i s o l a t e s i s s i m i l a r t o o b s e r v a t i o n s by Chrost (1973,1975) and Chrost and Siuda (1978). They hypothesized t h a t a l g a l e x c r e t o r y products suppressed b a c t e r i a more i n the l i g h t than i n the dark. C h r o s t ' s negative c o r r e l a t i o n s between b a c t e r i a l h e t e r o t r o p h y and c h i a c o u l d be a r e f l e c t i o n of g r e a t e r i r o n l i m i t a t i o n and higher s i d e r o p h o r e or a n t i b i o t i c p r o d u c t i o n i n the e p i l i m n i o n . In Black Lake, the i r o n - b i n d i n g data (Table 11, F i g u r e 19) i n d i c a t e d t h a t c h e l a t o r s are 4-10 f o l d more conc e n t r a t e d i n the e p i l i m n i o n than i n the hypolimnion. The p r o d u c t i o n of siderophores should be minimal i n i r o n - r i c h environments l i k e the hypolimnion of a l a k e (Neilands 1967). A l a c k of s i d e r o p h o r e p r o d u c t i o n i n hypolimnia may a l s o r e f l e c t a lower i r o n requirement. Hypolimnia with l i t t l e i r o n ( i . e . i r o n - l i m i t e d l i m n o c o r r a l s ) had much l e s s i r o n - b i n d i n g c a p a c i t y than the e p i l i m n i a . The l a c k of c h e l a t o r s i n the 129 hypolimnia was not r e l a t e d t o low m i c r o b i a l metabolic a c t i v i t y . B a c t e r i a can produce siderophores (Neilands 1967) and the h e t e r o t r o p h y i n the hypolimnia of these l i m n o c o r r a l s was r a p i d . The g r e a t e r e x c r e t i o n of i r o n c h e l a t o r s by the microbes i n e p i l i m n i a may i n d i c a t e t h a t a u t o t r o p h i c microbes r e q u i r e more i r o n than h e t e r o t r o p h i c microbes. An enhancement by l i g h t of i r o n demand and subsequent siderophore e x c r e t i o n c o u l d r e f l e c t the need of i r o n - c o n t a i n i n g enzymes i n r e a c t i o n s such as p h o t o o x i d a t i o n ( s e c t i o n 1.2). In the l a k e , a s u p p r e s s i o n of m i c r o b i a l degradation of o r g a n i c matter i n the e p i l i m n i o n by i r o n l i m i t a t i o n c o u l d r e s u l t i n the organic matter being o x i d i z e d a f t e r i t s e t t l e s i n t o the i r o n - r i c h hypolimnion. The hypolimnion does not have a renewable supply of oxygen; thus, the hypolimnion can become anoxic r a p i d l y . Anoxic decay of o r g a n i c compounds i s i n e f f i c i e n t (White et a l . 1968); thus, a r e s i d u a l oxygen demand can accumulate i n the hypolimnion. A suppression of oxygen consumption i n the e p i l i m n i o n d u r i n g the Anabaena bloom i n Black Lake as a r e s u l t of i n h i b i t i o n of heterotrophy would i n f l u e n c e the seasonal asynchrony i n oxygen p r o d u c t i o n / u t i l i z a t i o n which enhances p y r i t e f o r m a t i o n . The b i o c h e m i c a l r e a c t i o n s i n the heterotrophy i n h i b i t i o n experiments were i n c o m p l e t e l y r e s o l v e d . In c o n t r a s t t o a l g a l experiments, the a d d i t i o n of i r o n i n heterotrophy experiments d i d not overcome the t o x i c i t y of the siderophore i s o l a t e s . T h i s unexpected response supports the a l t e r n a t i v e hypothesis t h a t another compound with a n t i b i o t i c p r o p e r t i e s was a l s o i n the siderophore i s o l a t e s and was a l s o induced by i r o n l i m i t a t i o n . 130 Many microbes e x c r e t e a n t i b i o t i c s t h a t are s t r u c t u r a l homologs of side r o p h o r e s ( P r e l o g 1968, Winkelmann 1974). These a n t i b i o t i c s can i n t e r a c t with siderophores t o r e s u l t i n s y n e r g i s t i c antagonism of other s p e c i e s (Musher et a l . 1974). T h i s u n c e r t a i n t y complicates an i n t e r p r e t a t i o n of the mechanisms of m i c r o b i a l c o m p e t i t i o n ; however, the s i g n i f i c a n c e of i r o n a v a i l a b i l i t y i s u n a l t e r e d by the p o t e n t i a l presence of a n t i b i o t i c s t h a t are induced by i r o n l i m i t a t i o n . The c o n t r o l t h a t s i d e r o p h o r e i s o l a t e s had over a q u a t i c microbes i s analogous t o s i d e r o p h o r e r e g u l a t i o n of microbes i n s o i l s (Neilands 1981b, Emery 1982), plant-pathogen r e l a t i o n s h i p s (Emery 1982), and e n t e r i c b a c t e r i a i n humans (Weinberg 1974, 1975, 1978). The m i c r o b i a l c o n t r o l over i r o n a v a i l a b i l i t y i s a g e n e r a l r e g u l a t o r of p o p u l a t i o n s t r u c t u r e and any f o r c e t h a t changes i r o n a v a i l a b i l i t y can s h i f t community s t r u c t u r e . 4.8 C a l c i t e P r e c i p i t a t i o n Although t h i s t h e s i s began as a study of the e f f e c t of i r o n a v a i l a b i l i t y on a l g a l p e r i o d i c i t y , other f a c t o r s m o d i f i e d the e f f e c t of i r o n a v a i l a b i l i t y on a l g a l p r o d u c t i v i t y . I n t e r p r e t a t i o n of the long-term l i m n e t i c response t o i r o n enrichment r e q u i r e d a study of c a l c i u m carbonate geochemistry. The c r a s h of blue-green a l g a l blooms was sometimes r e l a t e d d i r e c t l y t o c a l c i t e p r e c i p i t a t i o n , and onl y i n d i r e c t l y t o i r o n chemistry. Moreover, the p r e c i p i t a t i o n of c a l c i t e seemed t o overcome the m i c r o b i a l s u p p r e s s i o n of b a c t e r i a l heterotrophy. C a l c i t e p r e c i p i t a t i o n i s a key r e a c t i o n t h a t i s r e l a t e d t o i r o n chemistry i n s e v e r a l ways. 131 Carbonate chemistry has been w e l l documented; however, the importance of c a l c i t e p r e c i p i t a t i o n as a r e g u l a t o r of a l g a l growth has only r e c e n t l y been w e l l e s t a b l i s h e d . C a l c i t e p r e c i p i t a t i o n suppresses a l g a l growth by enhancing a l g a l sedimentation and by p r e c i p i t a t i n g phosphorus. C a l c i t e p r e c i p i t a t i o n of phosphorus has been demonstrated i n l a b o r a t o r y s t u d i e s (Otsuki and Wetzel 1972, Stumm and Morgan 1981) and r e c e n t l y i n l a k e s (Rossknecht 1980, Avnimelach 1983, Murphy e t a l . 1983a). 4.8.1 V a r i a b i l i t y of C a l c i t e P r e c i p i t a t i o n Although the thermodynamics of carbonate r e a c t i o n s are w e l l c h a r a c t e r i z e d (Stumm and Morgan 1981), the i n i t i a t i o n of p r e c i p i t a t i o n i s d i f f i c u l t t o p r e d i c t . C a l c i t e p r e c i p i t a t i o n i s r e s t r i c t e d by k i n e t i c l i m i t a t i o n s (Berner and Morse 1974), org a n i c compounds (Chave and Suess 1970, Otsuki and Wetzel 1973, Reddy 1979) and phosphate (Berner and Morse 1974, Walter and Hanor 1979). In my s t u d i e s , phosphate and d i s s o l v e d o r g a n i c matter were about 0.9% and 10% r e s p e c t i v e l y of the p r e c i p i t a t i n g c a l c i t e f l o e . Phosphate and d i s s o l v e d o rganic carbon s u p p r e s s i o n of c a l c i t e p r e c i p i t a t i o n must c o n t r i b u t e t o the high degree of s u p e r s a t u r a t i o n of Black and F r i s k e n lakes with c a l c i t e . In the J u l y of 1982, c o l d weather r e s t r i c t e d c a l c i t e p r e c i p i t a t i o n . The c o o l i n g of the e p i l i m n e t i c waters from 20°C t o 14.6°C reduced the degree of c a l c i t e s u p e r s a t u r a t i o n from 11.8 t o 5.5 f o l d . The c o l d weather was unusual; moreover, c a l c i t e p r e c i p i t a t i o n was blocked when the water was s t i l l g r e a t l y s u p e r s a t u r a t e d . Other f a c t o r s must c o n t r i b u t e t o the v a r i a b i l i t y 132 of c a l c i t e p r e c i p i t a t i o n . Geochemists believe that c a l c i t e c r y s t a l formation i s prone to spontaneous nucleation (Reddy and Nancollas 19 71, Reynolds 19 78). Geochemists overcome the random length of time for nucleation to occur by saturating their laboratory solutions with calcium carbonate dust (Reddy et a l . 1981). This technique would change too many variables ( l i g h t i n t e n s i t y , dissolved organic carbon and phosphorus concentrations, and a l g a l buoyancy) for most b i o l o g i c a l studies. A pH stat, such as Shapiro (1984) has used in limnocorrals, may be a very useful to prevent c a l c i t e p r e c i p i t a t i o n in bioassay experiments. Considerable v a r i a b i l i t y in the i n i t i a t i o n of c a l c i t e p r e c i p i t a t i o n in lakes has been observed. Lehman (19 80) found that "Lake water e q u i l i b r i a were so close to the saturation l i m i t for CaCC>3 at the pH of ca. 8.2 that the increased productivity caused by the nutrients led quickly to c a l c i t e p r e c i p i t a t i o n " . Rossknecht (1977) proposed that a lack of "seed" crystals to i n i t i a t e p r e c i p i t a t i o n , resulted in the observed one week delay from the peak of a l g a l productivity to p r e c i p i t a t i o n of c a l c i t e . S i m i l a r l y , Koschel et a l . (1983) have observed delays of two to four weeks in the p r e c i p i t a t i o n of c a l c i t e a f t e r the peak of oxygen production. A stimulation of c a l c i t e p r e c i p i t a t i o n could occur in any enrichment of hardwater with a l i m i t i n g nutrient. The addition of the l i m i t i n g nutrient causes an i n i t i a l stimulation of primary production which raises the pH and causes c a l c i t e p r e c i p i t a t i o n . This reaction was most pronounced in the Black Lake n i t r a t e limnocorral. Nitrate assimilation r e s u l t s in hydroxide excretion 133 (Goldman and Brewer 19 80). Moreover, n i t r a t e has been used t o s t i m u l a t e the p r e c i p i t a t i o n of c a l c i u m carbonate (Morita 1980, No v i t s k y 1981, Brownlee and Murphy 1983). Ir o n enrichment of the Black Lake l i m n o c o r r a l s a l s o e f f e c t i v e l y s t i m u l a t e d c a l c i t e p r e c i p i t a t i o n . The densest a l g a l blooms i n e i t h e r the l i m n o c o r r a l s or i n Black or F r i s k e n l a k e s o c c u r r e d when c a l c i t e p r e c i p i t a t i o n was e i t h e r not observed or was delayed by more than t h r e e weeks. At times, the p e r i o d i c i t y of blue-green a l g a l blooms was r e g u l a t e d by c a l c i t e p r e c i p i t a t i o n . 4.9 B i o l o g i c a l I n d u c t i o n of C a l c i t e P r e c i p i t a t i o n Another cause of the v a r i a b l e i n d u c t i o n of c a l c i t e p r e c i p i t a t i o n i s b i o l o g i c a l r e g u l a t i o n of c a l c i u m carbonate p r e c i p i t a t i o n . C e r t a i n l y , algae mediate c r y s t a l n u c l e a t i o n ; the type of c r y s t a l p r e c i p i t a t e formed i s dependent upon the type of algae present (Darley 1974). However, whether microbes gain energy r e l e a s e d i n c a l c i u m carbonate p r e c i p i t a t i o n i s an o l d co n t r o v e r s y i n both geology and b i o l o g y (Kuznetsov 1970). Ca l c i u m carbonate p r e c i p i t a t i o n i s so c l o s e l y r e l a t e d t o m i c r o b i a l metabolic a c t i v i t y t h a t b i o c h e m i c a l r e a c t i o n s cannot be r e a d i l y d i s c r i m i n a t e d from geochemical r e a c t i o n s (Megard 1968, K e l t s and Hsu 1978). However, some microbes are much b e t t e r c a t a l y s t s of c a l c i u m carbonate p r e c i p i t a t i o n than others ( M o r i t a 1980). S t a b e l (1986) found t h a t c a l c i t e p r e c i p i t a t i o n i n Lake Constance, S w i t z e r l a n d , was c a t a l y z e d by only some of the many s p e c i e s of algae observed growing i n water s u p e r s a t u r a t e d with c a l c i u m carbonate. S t a b e l (19 86) a l s o found t h a t p o p u l a t i o n s of algae able t o i n i t i a t e 134 c a l c i t e p r e c i p i t a t i o n a r i s e r e g u l a r l y a t d e f i n e d p e r i o d s . T h u s , t h e c o m p o s i t i o n o f t h e b i o l o g i c a l c o m m u n i t y c a n i n f l u e n c e c a l c i u m c a r b o n a t e p r e c i p i t a t i o n ; t h e r e i s an i m p o r t a n t b i o l o g i c a l m e d i a t i o n o f t h e a l g a l p e r i o d i c i t y i m p o s e d by c a l c i t e p r e c i p i t a t i o n . 4.10 M i c r o b i a l R e s p o n s e s t o C a l c i t e P r e c i p i t a t i o n O t h e r b i o l o g i c a l r e a c t i o n s d u r i n g c a l c i u m c a r b o n a t e p r e c i p i t a t i o n a r e u n r e s o l v e d . The i n i t i a t i o n o f c a l c i t e p r e c i p i t a t i o n by t h e a d d i t i o n s o f c a l c i u m c h l o r i d e r e s u l t e d i n t h e l y s i s o f a b o u t a t h i r d o f t h e A p h a n i zomenon. None o f t h e a l g a e i n t h e c o n t r o l f l a s k s l y s e d . L y s i s o f a l g a e a l s o o c c u r r e d d u r i n g t h e c a l c i t e p r e c i p i t a t i o n t h a t was o b s e r v e d i n B l a c k L a k e i n 19 79 a n d i n t h e l i m e - m e d i a t e d i n d u c t i o n o f c a l c i t e p r e c i p i t a t i o n o f F r i s k e n L a k e . R a p i d c h a n g e s i n m i c r o b i a l p o p u l a t i o n s t r u c t u r e w o u l d o c c u r i f c a l c i t e s u p p r e s s e d s i d e r o p h o r e a c t i v i t y . S z a n i s z l o e t a l . (19 81) f o u n d t h a t c a l c i u m c a r b o n a t e e n h a n c e d s i d e r o p h o r e p r o d u c t i o n . The m i c r o b i a l n e c e s s i t y t o e n h a n c e s i d e r o p h o r e p r o d u c t i o n c o u l d i n d i c a t e a s u p p r e s s i o n o f s i d e r o p h o r e r e a c t i v i t y . T h r e e m e chanisms c o u l d p r o d u c e t h i s s u p p r e s s i o n . 1) C a l c i t e c o u l d r e a c t w i t h t h e f i b r i l l a r m a t e r i a l t h a t s i d e r o p h o r e s a r e a s s o c i a t e d w i t h . T h i s r e a c t i o n c o u l d p h y s i c a l l y p r e v e n t s i d e r o p h o r e r e a c t i o n s . I n t h e i r o n - b i n d i n g s t u d y , f a i l u r e t o i s o l a t e t h e c h e l a t o r s f r o m t h e f i b r i l l a r c o l l o i d s r e s u l t e d i n c a r b o n a t e c o p r e c i p i t a t i o n o f t h e c h e l a t o r s . 2) C a l c i u m c o u l d c o m p e t e d i r e c t l y w i t h i r o n f o r s i d e r o p h o r e c o m p l e x a t i o n . The r e a c t i o n s o f c a l c i u m ( H i d e r e t a l . 1982) and 135 c a l c i t e (Reynolds 1978) with some c h e l a t o r s are w e l l e s t a b l i s h e d ; however, the s p e c i f i c i t y of the side r o p h o r e c h e l a t i o n suggests t h a t other r e a c t i o n s e x i s t . 3) C a l c i t e c o u l d r e a c t w i t h the s i t e s of side r o p h o r e a s s i m i l a t i o n . T h i s type of i n t e r f e r e n c e has been observed with polymyxin (Newton 1953). Newton found t h a t d i v a l e n t ions i n t e r f e r e d with the a n t i b a c t e r i a l a c t i v i t y of polymyxin by b l o c k i n g the s i t e s of polymyxin a s s i m i l a t i o n . The i n h i b i t i o n of side r o p h o r e a c t i v i t y by c a l c i t e would lead t o a c o l l a p s e of symbiotic a s s o c i a t i o n s mediated by s i d e r o p h o r e s . For microbes with weak i r o n a s s i m i l a t i o n systems, such as Aphanizomenon, i r o n l i m i t a t i o n would be r a p i d l y enhanced. The i n i t i a t i o n of an i r o n - c h l o r o p h y l l c o r r e l a t i o n a f t e r the two lime treatments of F r i s k e n Lake ( F i g . 5) may have been e s t a b l i s h e d by p r e c i p i t a t i o n of i r o n c h e l a t o r s . H a l f of the d i s s o l v e d o r g a n i c carbon (30 — > 15 mg/L) was p r e c i p i t a t e d from F r i s k e n Lake by the i n d u c t i o n of c a l c i u m carbonate p r e c i p i t a t i o n . 4.11 Phosphorus C o p r e c i p i t a t i o n w i t h C a l c i t e Phosphorus l i m i t a t i o n c o u l d be another mechanism producing the c o l l a p s e of a l g a l blooms d u r i n g c a l c i t e p r e c i p i t a t i o n . In Black Lake, the temporal s e p a r a t i o n of a l g a l oxygen p r o d u c t i o n and phosphorus p r e c i p i t a t i o n ( F i g . 15), and the c o i n c i d e n c e of phosphorus and c a l c i u m p r e c i p i t a t i o n showed t h a t c a l c i u m carbonate p r e c i p i t a t i o n c o u l d r e g u l a t e phosphorus s o l u b i l i t y . The 32 . • . . . r a p i d a s s i m i l a t i o n of P-PO^ du r i n g c a l c i t e p r e c i p i t a t i o n i n d i c a t e d t h a t phosphorus l i m i t a t i o n c o u l d be induced by c a l c i t e p r e c i p i t a t i o n . 136 The i n d u c t i o n of P - c a l c i t e p r e c i p i t a t i o n c o u l d remove most of the phosphorus from the e p i l i m n i o n of a hardwater l a k e . The maximum p r e c i p i t a t i o n induced i n the C a C ^ i n c u b a t i o n of Black Lake was 10 mg CaCO^/L and 100 ug P/L. L i n e a r e x t r a p o l a t i o n of t h i s response t o the l a r g e s t amount of c a l c i t e p r e c i p i t a t i o n observed i n Black Lake (50 mg CaC0 3/L) i n d i c a t e s a p o t e n t i a l p r e c i p i t a t i o n of 500 ug P/L. Berner and Morse (1974) produced t h i s degree of phosphorus p r e c i p i t a t i o n i n pseudo-seawater. The r e l a t i v e importance of geochemical and b i o c h e m i c a l phosphorus p r e c i p i t a t i o n was not r e s o l v e d . However, phosphorus p r e c i p i t a t i o n d u r i n g c a l c i t e p r e c i p i t a t i o n events p r o v i d e d i n s i g h t i n t o the e f f e c t of i r o n a v a i l a b i l i t y on carbonate e q u i l i b r i u m and i n t u r n h e t e r o t r o p h i c p r o d u c t i o n . The carbonate e q u i l i b r i a are a good i n t e g r a t i o n of primary p r o d u c t i o n and heterotrophy. The pH i n the hypolimnia of the c i t r a t e l i m n o c o r r a l experiments was lowest i n the f e r r i c c i t r a t e l i m n o c o r r a l s . A lower pH i s the expected r e s u l t of g r e a t e r h e t e r o t r o p h i c p r o d u c t i o n of CG^ i n the hypolimnia which i n t u r n , i s the a n t i c i p a t e d e f f e c t of g r e a t e r primary p r o d u c t i o n i n the e p i l i m n i a ; these F e - l i m n o c o r r a l s had the g r e a t e s t oxygen p r o d u c t i o n i n the e p i l i m n i a . The higher phosphorus c o n c e n t r a t i o n i n the hypolimnia of the F e - l i m n o c o r r a l s i n d i c a t e s t h a t e i t h e r more P - c a l c i t e d i s s o l v e d as a r e s u l t of g r e a t e r CG^ p r o d u c t i o n or t h a t more org a n i c phosphorus was metabolized. E i t h e r r e a c t i o n confirms t h a t more h e t e r o t r o p h i c a c t i v i t y o c c u r r e d i n the hypolimnia of the f e r r i c c i t r a t e l i m n o c o r r a l s and t h a t i n t u r n , more primary p r o d u c t i o n o c c u r r e d i n the e p i l i m n i a . 137 The s t r o n g c o r r e l a t i o n of phosphorus t o c a l c i u m i n the sediments of Yellow, F r i s k e n , and Roche lakes i n d i c a t e s t h a t not a l l of the p r e c i p i t a t e d P - c a l c i t e r e d i s s o l v e d i n the hypolimnion. Although the i n i t i a l P - p r e c i p i t a t e cannot be a p a t i t e (Koutsoukos e t a l . 1980), the s t a b l e component of P - c a l c i t e p r e c i p i t a t i o n may be a p a t i t e . I n i t i a l l y , phosphorus adsorbs t o k i n k s i n the c a l c i t e c r y s t a l (Berner and Morse 1974) and t h i s complex u l t i m a t e l y forms h y d r o x y l a p a t i t e (Stumm and L e c k i e 19 70, G r i f f i n and Ju r i n a k 1974). Brown (1980) suggested t h a t h y d r o x y l a p a t i t e c o u l d form i n aqueous c a l c i t i c limestone suspensions. Ryding (19 85) proposed t h a t lime a p p l i c a t i o n t o a lake would enhance a p a t i t e f o r m a t i o n . In F r i s k e n Lake, lime a p p l i c a t i o n d i d not appear t o enhance a p a t i t e formation; about 95% of the p r e c i p i t a t e d phosphorus r e d i s s o l v e d . The i n v e r s e c o r r e l a t i o n between i r o n and p h o s p h o r u s / c a l c i t e i n d i c a t e s t h a t d i f f e r e n t v a r i a b l e s c o n t r o l the s o l u b i l i t y of these two geochemical subsets. P y r i t e i s very s t a b l e i n these anoxic sediments; P - c a l c i t e can be d i s s o l v e d by the h e t e r o t r o p h i c p r o d u c t i o n of C 0 2. T h i s d i f f e r e n c e i n s t a b i l i t y of these two minerals must c o n t r i b u t e t o the establishment of low i r o n and high phosphorus c o n c e n t r a t i o n s i n many hardwater l a k e s . 4.11 CONCLUSIONS Iron a v a i l a b i l i t y i n f l u e n c e s the p e r i o d i c i t y of blue-green a l g a l growth. Blue-green a l g a l blooms occur i n Black Lake o n l y a f t e r i r o n i s r e l e a s e d from the lake sediments. P r i o r t o the mid summer i n c r e a s e i n i r o n , microbes i n Black Lake produce low molecular weight c h e l a t o r s t h a t s e l e c t i v e l y complex i r o n . The 138 b i o a v a i l a b i l i t y of chelated iron can be r e s t r i c t e d to p a r t i c u l a r species. Furthermore, the chelator concentration in lake water can exceed the dissolved iron concentration; thus, microbial chelator production can control the a v a i l a b i l i t y of iron. The competition for iron i s associated with the suppression of competing algae and bacteria by either the d i r e c t t o x i c i t y of siderophores or an associated excretion of an a n t i b i o t i c . These reactions enable some algae to produce th e i r own microenvironment. Iron l i m i t a t i o n in lakes varies greatly within geological formations mainly because iron loading varies greatly between lakes in the same area. On the Thompson Plateau, the po t e n t i a l for iron a v a i l a b i l i t y i s indicated by a high concentration of phosphorus in oxidized water in either the f a l l or spring. Iron-limited lakes can have too l i t t l e iron for f e r r i c phosphate reactions to regulate the s o l u b i l i t y of phosphorus. The lack of iron to p r e c i p i t a t e phosphorus and the high loading of phosphorus from the weathering of apatite r e s u l t s in very high concentrations of phosphorus O 2 0 0 ug/L) in Black and Frisken lakes. The high iron and phosphorus loading into Chain Lake re s u l t s in high soluble phosphorus concentrations only for b r i e f periods when oxygen concentrations are less than 4 mg/L. Iron biogeochemsitry i s c l o s e l y coupled to c a l c i t e p r e c i p i t a t i o n . Iron enrichment of iron-limited hardwater lakes can lead to rapid p r e c i p i t a t i o n of c a l c i t e . However, c a l c i t e p r e c i p i t a t i o n can be delayed for at least three weeks after the onset of c a l c i t e supersaturation. The delay period and the amount of p r e c i p i t a t i o n varies greatly. In many bioassays in hardwater 139 l a k e s , the e f f e c t of i r o n enrichment can be i n t e r p r e t e d more e a s i l y by an a n a l y s i s of the s h i f t s i n the carbonate e q u i l i b r i a than by a measurement of a l g a l biomass. Phosphorus adsorbs d i r e c t l y to c a l c i t e . Over 90% of the p r e c i p i t a t e d phosphorus r e d i s s o l v e s i n the hypolimnion; thus, u n l i k e i n i r o n - r i c h l a k e s , the c o n c e n t r a t i o n of s o l u b l e phosphorus i s v ery high i n s p r i n g and f a l l when the h y p o l i m n e t i c water mixes and becomes o x i d i z e d . However, the sedimented phosphorus t h a t remains a s s o c i a t e d with c a l c i t e i n the l a k e sediments i s s t a b l e . In l a k e sediments with more than 10% c a l c i u m carbonate, the d i s t r i b u t i o n of phosphorus i s s t r o n g l y c o r r e l a t e d t o the d i s t r i b u t i o n of c a l c i u m . The p r e c i p i t a t i o n of c a l c i t e enhances the sedimentation of a lgae and many algae l y s e d u r i n g c a l c i t e p r e c i p i t a t i o n . The decay of algae i n the hypolimnion enhances the p o t e n t i a l f o r p y r i t e f o rmation which i n t u r n minimizes the r e c y c l i n g of i r o n . Thus, c a l c i t e p r e c i p i t a t i o n i n e u t r o p h i c s t r a t i f i e d l akes w i l l enhance p y r i t e f ormation, and i f the supply of i r o n t o the l a k e i s low, p y r i t e formation w i l l r e s u l t i n i r o n l i m i t a t i o n . In g e n e r a l , the enhancement of i r o n l i m i t a t i o n by p y r i t e formation w i l l be more important i n hardwater lakes than i n s oftwater lakes where s u l p h a t e r e d u c t i o n can be l i m i t e d by an inadequate supply of s u l p h a t e . Four r e a c t i o n s , i r o n c h e l a t i o n , sediment i r o n r e l e a s e , p y r i t e f o r m a t i o n , and c a l c i t e p r e c i p i t a t i o n , can r e g u l a t e much of the a l g a l s u c c e s s i o n and v a r i a b i l i t y of m i c r o b i a l p r o d u c t i v i t y i n hardwater l a k e s . 140 REFERENCES A b e l i o v i c h , A . , and M. S h i l o . 1972. Photooxidative death of blue-green algae. J . B a c t e r i o l . 111:682-689. Akers , H.A. 1983. M u l t i p l e hydroxamic ac id microb ia l iron chelators (siderophores) in s o i l s . S o i l Sc. 135:156-160. A l l e n , H . L . 1972. Phytoplankton, photosynthesis , micronutrient i n t e r a c t i o n s , and inorganic carbon a v a i l a b i l i t y in a soft-water Vermont lake , p.63-83. In G . E . Likens (ed. ) Nutr ients and Eutrophica t ion . A l l e n Press I n c . , Lawrence Kansas. American Publ ic Health Assoc ia t ion . 19 76. Standard methods for the examination of water, sewage, and wastewater, 14th ed. American Publ ic Health Assoc ia t ion , Washington, D . C . Anderegg, G.W. , F . L ' E p l a t t e n i e r , and F . M . Cal lahan . 1963. Hydroxamate complex 111. Iron (111) exchange between sideramins and complexones. Helv. Chim. Acta . 46:1409-1422. Armstrong, J . E . and C. Van Baalen. 1979. Iron transport in microalgae: the i s o l a t i o n and b i o l o g i c a l a c t i v i t y of a hydroxamate siderophore from the blue-green alga Agmenellum quadruplicatum. J . Gen. M i c r o b i o l . 111:253-262. Ashley, K . I . 1983. Hypolimnetic aerat ion of a n a t u r a l l y eutrophic lake: phys i ca l and chemical e f f ec t s . Can. J . F i s h . Aquat. S c i . 40:1343-1359. Avnimelech, Y. 1983. Phosphorus and calcium s o l u b i l i t i e s in Lake Kinneret . Limnol . Oceanogr. 28:640-646. B a c c i n i , P . , E . Gr ieder , R. S t i e r l i , and S. Goldberg. 1982. The inf luence of natural organic matter on the adsorption propert ies of mineral p a r t i c l e s in lake water. Schweiz. Z. Hydrol . 44:99-116. B a i l e y , K . M . , and F . B . Taub. 1980. Ef fec t s of hydroxamate siderophores (strong Fe (III) chelators) on the growth of algae. J . Phycol . 16:334-339. Banoub, M.W. 19 77. Experimental inves t iga t ion on the release of phosphorus in r e l a t i o n to i ron in freshwater/mud system, p. 324-330. In H . L . Golterman (ed.) Interact ions Between Sediments and Freshwater. Dr . W. Junk, The Hague. B e l l , W., and R. M i t c h e l l . 1972. Chemotactic and growth responses of marine bac ter ia by a l g a l e x t r a c e l l u l a r products. Limnol . Oceanogr. 19:833-839. Berner, R.A. 1971. P r i n c i p l e s of Chemical Sedimentology. McGraw-Hi l l . New York. Berner, R . A . , and J.W. Morse. 1974. D i s so lu t ion k i n e t i c s of calc ium carbonate in sea water IV. Theory of c a l c i t e d i s s o l u t i o n . Am. Jour. S c i . 274:108-134. Bernhardt, H . , J . Clasen, and H. S c h e l l . 1971. Phosphate and t u r b i d i t y contro l by f l o c c u l a t i o n and f i l t r a t i o n . Amer. Water Works Assoc. 63:355-368. B i r c h , P . B . 1976. The r e l a t i o n s h i p of sedimentation and nutr ient c y c l i n g to the trophic status of four lakes in the Lake Washington drainage. Ph.D. D i s s e r t a t i o n . Univ . Washington, Sea t t l e . 141 Bostock, H.H. 1966. F e l d s p a r and quartz phenocrysts i n the S h i n g l e Creek Porphyry, B r i t i s h Columbia. Ge o l . Surv. Can. B u l l . 126. 70p. Bostrum, B. 1977. P o t e n t i a l m o b i l i t y of phosphorus i n d i f f e r e n t types of l a k e sediment. I n t . Revue ges. H y d r o b i o l . 69:457-474. Brown, J.C. 1979. Iron and p l a n t s , p. 57-78. In I r o n . M e d i c a l and B i o l o g i c a l E f f e c t s of Environmental P o l l u t a n t s S e r i e s , U n i v e r s i t y Park Press, B a l t i m o r e . Brown, J.L. 1980. Calc i u m phosphate p r e c i p i t a t i o n i n aqueous c a l c i f i c limestone suspensions. J . E n v i r o n . Qual. 9:641-644. Brownlee, B.G., and T.P. Murphy, 1983. Ni t r o g e n f i x a t i o n and phosphorus turnover i n a h y p e r t r o p h i c p r a i r i e l a k e . Can. J . F i s h . Aq. S c i . 40:1853-1860. Burnison, B.K. 1980. M o d i f i e d dimethyl s u l f o x i d e (DMSO) e x t r a c t i o n f o r c h l o r o p h y l l a n a l y s i s of phytoplankton. Can. J . F i s h . Aquat. S c i . 37:729-733. Carnaham, J.E., and J.E. C a s t l e . 1958. Some requirements of b i o l o g i c a l n i t r o g e n f i x a t i o n . J . B a c t e r i o l . 75:121-124. Chan, A.T., R.J. Andersen, M.J. LeBlanc, and P.J. H a r r i s o n . 1980. A l g a l p l a t i n g as a t o o l f o r i n v e s t i g a t i n g a l l e l o p a t h y among marine microalgae. Mar. B i o l . 59:7-13. Chave, K.E., and E. Suess, 1970. Calcium carbonate s a t u r a t i o n i n seawater: e f f e c t s of d i s s o l v e d o r g a n i c matter. Limnol. Oceanogr. 15:633-637. Chrost, R.J. 1973. I n h i b i t o r s produced by algae as an e c o l o g i c a l f a c t o r a f f e c t i n g b a c t e r i a i n water ecosystems. A c t a M i c r o b i o l o g i c a P o l o n i c a 7:125-133. Chrost, R.J. 1975. I n h i b i t o r s produced by algae as an e c o l o g i c a l f a c t o r a f f e c t i n g b a c t e r i a i n water. I I . A n t i b a c t e r i a l a c t i v i t y of algae d u r i n g blooms. Acta M i c r o b i o l o g i c a P o l o n i c a 7:167-176. Chr o s t , R.J. and W. Siuda. 1978. Some f a c t o r s a f f e c t i n g the h e t e r o t r o p h i c a c t i v i t y of b a c t e r i a . Acta M i c r o b i o l o g i c a P o l o n i c a 27: 129-138. Church, B.N. 1973. Geology of the White Lake B a s i n . B.C. Dep. Mines Pet. Resour. B u l l . 61. 120p. Clase n , J . , and H. Bernhardt. 1974. The use of a l g a l assays f o r determining the e f f e c t of i r o n and phosphorus compounds on the growth of v a r i o u s a l g a l s p e c i e s . Water Res. 8:31-44. C o c k f i e l d , W.E. 1947. N i c o l a , Kamloops, and Yal e D i s t r i c t Map. Geol Surv. Can. Sheet 921 (East H a l f ) . C o c k f i e l d , W.E. 1948. Geology and m i n e r a l d e p o s i t s of N i c o l a map-area, B r i t i s h Columbia. Can. Dept. Mines Resources Memoir 249. 42p. Co r b e t t , M.D. and B.P. Chipko. 1978. P u r i f i c a t i o n of hydroxamic a c i d s by the use of SP-Sephadex. J . Chromatogr. 151:379-383. Coulombe, A.M., and G.G.C. Robinson. 1981. C o l l a p s i n g Aphani zomenon f l o s - a q u a e blooms: p o s s i b l e c o n t r i b u t i o n s of p h o t o o x i d a t i o n , 0 o t o x i c i t y , and cyanophages. Can. J . Bot. 59:1277-1284. Csaky, T.Z. 1948. On the e s t i m a t i o n of bound hydroxylamine i n b i o l o g i c a l m a t e r i a l s . Acta Chem. Scand. 2:450-454. Daley, R.J. 1979. D i r e c t e p i f l u o r e s e n c e enumeration of n a t i v e a q u a t i c b a c t e r i a ; uses, l i m i t a t i o n s , and comparative accuracy. Sp. Tech. Pub. 695. Am. Soc. T e s t . Mat. 142 D a r l e y , W.M. 1974. S i l i f i c a t i o n and c a l c i f i c a t i o n , p. 655-675. In W.D.P. Stewart (ed) A l g a l P h y s i o l o g y and Bi o c h e m i s t r y . Univ. C a l i f o r n i a P r e s s . B e r k e l e y . D a v i s , W.B., M.J. McCauley, and B.R. Byers. 1971. Iron requirements and aluminum s e n s i t i v i t y of an hydroxamic a c i d - r e q u i r i n g s t r a i n of B a c i l l u s meqaterium. J . B a c t e r i o l . 105:589-594. Delucca, R., and M.D. McCracken. 1977. Observations on i n t e r a c t i o n s between n a t u r a l l y - c o l l e c t e d b a c t e r i a and s e v e r a l s p e c i e s of algae. H y d r o b i o l o g i a 55:71-75. E l b r a c h t e r , M. 1976. P o p u l a t i o n dynamic s t u d i e s on phytoplankton c u l t u r e s . Mar. B i o l . 35:201-209. E l d e r , J.F. 1977. I r o n uptake by freshwater algae and i t s d i e l v a r i a t i o n , p. 346-357. In H. Drucker and R.E. Wildrung (eds.) B i o l o g i c a l I m p l i c a t i o n s of Metals i n the Environment. T e c h n i c a l Information Center, Energy Research and Development A d m i n i s t r a t i o n . E l d e r , J.F., and A.J. Home. 1977. B i o s t i m u l a t o r y c a p a c i t y of d i s s o l v e d i r o n f o r cyanophycean blooms i n a n i t r o g e n - r i c h r e s e r v o i r . Chemosphere 9:525-530. E l o f f , J . N., Y. S t e i n i t z , and M. S h i l o . 1976. P h o t o o x i d a t i o n of cy a n o b a c t e r i a i n n a t u r a l c o n d i t i o n s . Appl. M i c r o b i o l . 31:119-126. Emery, T. 1971. Role of f e r r i c h r o m e as a f e r r i c ionophore i n U s t i l a g o sphaerogena. B i o c h e m i s t r y 10:1483-1488. Emery, T. 19 82. Iron metabolism i n humans and p l a n t s . Am. S c i . 70:626-633. Environment Canada. 19 79. A n a l y t i c a l Methods Manual. Inland Water D i r e c t o r a t e . Water Q u a l i t y Branch, Ottawa, Can. Ferguson, A.J.D., J.M. Thompson, and C.S. Reynolds. 1982. S t r u c t u r e and dynamics of zooplankton communities maintained i n c l o s e d systems, with r e s p e c t t o the a l g a l food supply. J . Plankton Research 4:523-543. Fogg, G.E., W.D.P. Stewart, P. Fay, and A.E. Walsby. 1973. The blue-green a l g a e . Academic P r e s s , New York. Ganf, G.G. and R.L. O l i v e r . 1982. V e r t i c a l s e p a r a t i o n of l i g h t and a v a i l a b l e n u t r i e n t s as a f a c t o r c a using replacement of green algae by blue-green a l g a e i n the plankton of a s t r a t i f i e d l a k e . J . Ecology 70:829-844. G e n t i l e , J.H., and T.E. Maloney. 1969. T o x i c i t y and environmental requirements of a s t r a i n of Aphani zomenon f l o s - a q u a e (L.) R a l f s . Can. J . M i c r o b i o l . 15:165-173. Gerhold, R.N. 1975. A l g a l n u t r i t i o n a l b i o a s s a y s of Lake Wylie, North C a r o l i n a . In E . J . Middlebrooks, D.H. Falkenborg, and T.E. Maloney (eds) B i o s t i m u l a t i o n and n u t r i e n t assessment. E.P.A., C o r v a l l i s , Oregon. Goldman, C R . 1966. M i c r o n u t r i e n t l i m i t i n g f a c t o r s and t h e i r d e t e c t i o n i n n a t u r a l phytoplankton p o p u l a t i o n . 122-136. In: Primary p r o d u c t i o n i n Aqu a t i c Environments, ed. C.R. Goldman. Mem 1st. I t a l . I d r o b i o l . 18 Suppl. U n i v e r s i t y of C a l i f o r n i a P r e s s . Goldman, J . C , and P.G. Brewer. 1980. E f f e c t of n i t r o g e n source and growth r a t e on phytoplankton-mediated changes i n a l k a l i n i t y . Limnol. Oceanogr. 25:352-357. 143 Green, D . B . , T . J . Logan, and N . E . Smeck. 1978. Phosphate adsorpt ion-desorpt ion c h a r a c t e r i s t i c s of suspended sediments in the Maumee River basin of Ohio. J . Environ . Qual . 7:208-212. G r i f f i n , R . A . , and J . J . Jur inak . 1974. K i n e t i c s of phosphate i n t e r a c t i o n with c a l c i t e . S o i l . S c i . Amer. Proc. 38:75-79. Guseva, K . A . 1937. The hydrobiology and microbiology of the Ucha reservo ir of the Moscow-Volga canal 11. Microb io log ia 6:449-464. Guseva, K . A . 19 39. Bloom on the Ucha r e s e r v o i r . B i u l . Moskov. O b s l i c h . I spytat . Pr i rody: Otdel B i o l . 48:30-32. Happey-Wood, C . M . , and A. Pentecost. 1981. A l g a l bioassay of the water from l inked but contrast ing Welsh Lakes. Freshwater B i o l . 11:473-491. Healey, F . P . , and L . Hendzel. 1976. P h y s i o l o g i c a l changes during the course of blooms of Aphani zomenon f los-aquae. J . F i s h . Res. Board Can. 33:36-41. Hel lebust , J . A . 1974. E x t r a c e l l u l a r products, p . 838-863. In W.D.P. Stewart (ed.) A l g a l Physiology and Biochemistry. Berkeley, U n i v e r s i t y of C a l i f o r n i a Press . Hider , R . C . , A. Rahim Mohd-Nor, J . S i l v e r , and J . B . Nei lands . 1982. Coordination of calcium by iron enterobact in . J . Inorganic Chem. 17:205-213. Home, A . J . 1974. The importance of i ron as a factor c o n t r o l l i n g blue-green blooms in some eutrophic lakes . 38th Annual Meeting Amer. Soc. of Limnol . Oceanogr. Abstract . Hurley , J . P . , G . J . Kenoyer, and C . J . Bowser. 1985. Ground water as a s i l i c a source for diatom production in a precipi tat ion-dominated lake . Science:227:1576-1578. Hutchinson, G . E . 1967. A T r e a t i s e on Limnology. Wiley. New York. Jackson, T . A . , and R . E . Hecky. 1980. Depression of primary p r o d u c t i v i t y by humic matter in lake and reservo ir waters of the boreal forest zone. Can. J . F i s h . Aquat. S c i . 37:2300-2317. Kayser, H. 1979. Growth in terac t ions between marine d i n o f l a g e l l a t e s in mult ispecies cu l ture experiments. Mar. B i o l . 52:357-369. Keat ing, K . I . 1978. Blue-green a l g a l i n h i b i t i o n of diatom growth: t r a n s i t i o n from mesotrophic to eutrophic community s t ruc ture . Science 199:971-973. K e l t s , K . , and K . J . Hsu. 1978. Freshwater carbonate sedimentation, p . 295-323. In A. Lerman (ed.) Lakes: Chemistry, Geology, Physics . Spr inger -Ver lag , New York. Koschel R . , J . Benndorf, G. P r o f t , and F . Recknagel. 1983. C a l c i t e p r e c i p i t a t i o n as a natural contro l mechanism of eutrophicat ion . Arch . Hydrobio l . 98:380-408. Koutsoukos, P. Z. Amjad, M.B. Tomson, and G. H. Nancol las . 1980. C r y s t a l l i z a t i o n of calcium phosphates. A constant composition study. Am. Chem. Soc. 102:1553-1557. Kuznetzov, S . J . 1970. The M i c r o f l o r a of Lakes and i t s Geochemical A c t i v i t y . Univ . Texas Press . Lampert, W. 1982. Further studies on the i n h i b i t o r y e f fect of the tox ic blue-green Microcys t i s aeruginosa on the f i l t e r i n g rate of zooplankton. Arch . Hydrobio l . 95:207-220. Lange, W. 1971. L i m i t i n g nutr ient elements in f i l t e r e d Lake E r i e water. Water Res. 5:1031-1048. 144 Lange, W. 1974. C h e l a t i n g agents and blue-green a l g a e . Can. J . M i c r o b i o l . 20:1311-1321. Lankford, C E . 1973. B a c t e r i a l a s s i m i l a t i o n of i r o n . Chemical Rubber Co. C r i t . Rev. M i c r o b i o l . 2:273-331. Lean, D.R.S. 1973. Phosphorus dynamics i n la k e water. S c i e n c e 179:678-680. Lean, D.R.S., C F . H . L i a o , T.P. Murphy, and D.S. P a i n t e r . 1978. The importance of n i t r o g e n f i x a t i o n i n l a k e s . E c o l . B u l l . (Stockholm) 26:41-51. Lean, D.R.S., and F.R. P i c k . 1981. P h o t o s y n t h e t i c response of la k e plankton t o n u t r i e n t enrichment: a t e s t f o r n u t r i e n t l i m i t a t i o n . Limnol. Oceanogr. 26:1001-1019. Lehman, J.T. 1980. N i t r o g e n l o a d i n g : I n f l u e n c e on d i s s o l v e d i n o r g a n i c carbon i n n a t u r a l waters. Prepared f o r O f f i c e of Water Research and Technology, Washington, D.C # PB81-142226.b Leppard, G.G., A. M a s s a l s k i , and D.R.S. Lean. 1977. Electron-opaque m i c r o s c o p i c f i b r i l s i n l a k e s : t h e i r demonstration, t h e i r b i o l o g i c a l d e r i v a t i o n and t h e i r p o t e n t i a l s i g n i f i c a n c e i n the r e d i s t r i b u t i o n of c a t i o n s . Protoplasma. 92:289-309. Leppard, G.G. 1984a. R e l a t i o n s h i p s between f i b r i l s , c o l l o i d s , c hemical s p e c i a t i o n , and the b i o a v a i l a b i l i t y of t r a c e heavy metals i n s u r f a c e waters - A review. NWRI, C o n t r i b u t i o n No. 84-45. B u r l i n g t o n , O n t a r i o . Leppard, G.G. 1984b. The u l t r a s t r u c t u r e of l a c u s t r i n e sedimenting m a t e r i a l s i n the c o l l o i d a l s i z e range. Arch. H y d r o b i o l . 101:521-530. L i j k l e m a , L. 1977. The r o l e of i r o n i n the exchange of phosphate between water and sediments, p. 313-317. In H.L. Golterman (ed.) I n t e r a c t i o n s Between Sediments and Freshwater. Dr. W. Junk. The Hague. L i n , C.K., and C L . Schelske. 1981. Seasonal v a r i a t i o n of p o t e n t i a l n u t r i e n t l i m i t a t i o n t o c h l o r o p h y l l p r o d u c t i o n i n southern Lake Huron. Can. J . F i s h . Aquat. S c i . 38:1-9. L i t t l e , H.W. 1961. K e t t l e R i v e r map area (west h a l f ) . B.C. Geol. Surv. Can. P r e l i m . Ser. 15-1961. Lund, J.W.G., F.J.H. Mackereth, and C.H. Mortimer. 1963. Changes i n depth and time of c e r t a i n chemical and p h y s i c a l c o n d i t i o n s and of the standing crop of A s t e r i o n e l l a formosa Hass. i n the no r t h b a s i n of Windermere i n 1947. Lund, J.W.G., G.H.M. Jaworski, and C. Butterwick. 1975. A l g a l b i o a s s a y of water from Bleham Tarn, E n g l i s h Lake d i s t r i c t and the growth of p l a n k t o n i c diatoms. Arch. H y d r o b i o l . Suppl. 49:49-69. Lund, J.W.G., and C.S. Reynolds. 1982. The development and o p e r a t i o n of l a r g e l i m n e t i c e n c l o s u r e s i n Blelham t a r n , E n g l i s h D i s t r i c t , and t h e i r c o n t r i b u t i o n t o phytoplankton e c o l o g y . Prog, p h y c o l . Res. 1:1-65. Lynch, M. 1980. Aphanizomenon blooms: a l t e r n a t e c o n t r o l and c u l t i v a t i o n by Daphnia pulex. In W.C. K e r f o o t (ed.) E v o l u t i o n and ecology of zooplankton communities. S p e c i a l Symposium Volume 3. Amer. Soc. Limnol. Oceanogr. Lynch, M. and J . Shap i r o . 1981. P r e d a t i o n , enrichment, and phytoplankton community s t r u c t u r e . Limnol. Oceanogr. 26:86-102. 145 M a c M i l l a n - B l o e d e l Co. 1972. The Okanagan f o r e s t . F o r e s t a l k 1:13-15. Manning, P.G., J.D.H. W i l l i a m s , and M.N. C h a r l t o n . 1979. Mossbauer s p e c t r a l s t u d i e s of the d i a g e n e s i s of i r o n i n a s u l p h i d e - r i c h sediment c o r e . Nature 280:134-136. Mathews, W.H. 1944. G l a c i a l l a k e s and i c e r e t r e a t i n s o u t h - c e n t r a l B r i t i s h Columbia. Trans. Roy. Soc. Can. Ser. 3, sec. 4, 38:39-58. Matsunaga, K., K. I g a r a s h i , S. Fukase, and H. Tsubota. 1984. Behaviour of o r g a n i c a l l y - b o u n d i r o n i n seawater e s t u a r i e s . E s t . Coast. S h e l f S c i . 18:615-622. McKnight, D.M., and F.M.M. Morel. 1980. Copper complexation by side r o p h o r e s from filamentous blue-green a l g a e . Limnol. Oceanogr. 25:62-71. Megard, R.O. 1968. P l a n k t o n i c p h o t o s y n t h e s i s and the environment of c a l c i t e carbonate d e p o s i t i o n i n l a k e s . I n t e r i m Rept. 2 Limnol. Res. Center. U. Minn. M o r i t a , R.Y. 1980. C a l c i t e p r e c i p i t a t i o n by marine b a c t e r i a . G e o m i c r o b i o l . J . 2:63-82. Mortimer, C.H. 1941. The exchange of d i s s o l v e d substances between mud and water i n l a k e s : I and I I . J . E c o l . 29:280-329. Mortimer, C.H. 1942. The exchange of d i s s o l v e d substances between mud and water i n l a k e s : I I I and IV. J . E c o l . 30:147-201. Morton, S.D., and T.H. Lee. 1974. A l g a l blooms: p o s s i b l e e f f e c t s of i r o n . E n v i r o n . S c i . Technol. 80:673-674. Mudroch, A. and G.A. Duncan. 1986. D i s t r i b u t i o n of metals i n d i f f e r e n t s i z e f r a c t i o n s of sediment from the Niagara R i v e r . J . Great Lakes Res. 12:117-126. Murphy, T.P., and D.R.S. Lean. 1975. The d i s t r i b u t i o n of i r o n i n a c l o s e d ecosystem. Verh. I n t e r n a t . V e r e i n . Limnol. 19:258-266. Murphy, T.P. 1976. Some aspects of i r o n metabolism of algae. M.Sc. T h e s i s , Univ. of Toronto. Murphy, T.P., D.R.S. Lean, and C. Nalewajko. 1976. Blue-green a l g a e : T h e i r e x c r e t i o n of i r o n s e l e c t i v e c h e l a t o r s enables them t o dominate other algae. Science 192:900-902. Murphy, T.P., and B.G. Brownlee. 1981. Ammonia v o l a t i l i z a t i o n i n a h y p e r t r o p h i c p r a i r i e l a k e . Can. J . F i s h . Aquat. S c i . 38:1035-1039. Murphy, T.P., K.J. H a l l , and I. Y e s a k i . 1983a. B i o g e n i c r e g u l a t i o n of i r o n a v a i l a b i l i t y i n a e u t r o p h i c hardwater l a k e . S c i . T o t a l E n v i r o n . 28:37-50. Murphy, T.P., K.J. H a l l , and I. Y e s a k i . 1983b. C o p r e c i p i t a t i o n of phosphate wi t h c a l c i t e i n a n a t u r a l l y e u t r o p h i c l a k e . Limnol. Oceanogr. 28:58-69. Murphy, T.P., K.J. H a l l , K.I. Ashley, A. Mudroch, M. Mawhinney, H j . F r i c k e r . 1985. In - l a k e p r e c i p i t a t i o n of phosphorus by lime treatment. NWRI C o n t r i b u t i o n S e r i e s 85-167, B u r l i n g t o n , O n t a r i o . Murphy, T.P. 1985. The e f f e c t of a water d i v e r s i o n on a e u t r o p h i c l a k e . NWRI C o n t r i b u t i o n S e r i e s 85-168, B u r l i n g t o n , O n t a r i o . Musher, D.M., C. Saenz, and D.P. G r i f f i t h . 1974. I n t e r a c t i o n between acetohydroxamic a c i d and 12 a n t i b i o t i c s a g a i n s t gram-negative pathogenic b a c t e r i a . A n t i m i c r o b . Ag. Chemother. 5:106-110. 146 Nasmith, H..1961. Late g l a c i a l h i s tory and s u r f i c i a l deposits of the Okanagan V a l l e y , B r i t i s h Columbia, Dep. Mines Pet. Resour. B u l l . 46. Nei lands , J . B . 1957. Some aspects of microb ia l i ron metabolism. B a c t e r i o l . Rev. 21:101-111. Nei lands , J . B . 1966. Natura l ly occurring non-porphyrin iron compounds. S t r u c t . Bonding 1:59-108. Nei lands , J . B . 1967. Hydroxamic acids in nature. Science 156:1443-1447. Nei lands , J . B . 1972. M i c r o b i a l i ron transport compounds (siderochromes), p. 167-202. In G. Eichhorn (ed. ) Inorganic Biochemistry. E l s e v i e r Publ i shing C o . , Amsterdam. Nei lands , J . B . 1973. Chemistry of i ron in b i o l o g i c a l systems, p. 13-42. In S .K. Dhar (ed.) Metal Ions in B i o l o g i c a l Systems. Plenum Press , New York. Nei lands, J . B . , T . Peterson, and S .A. Leong. 1980. High a f f i n i t y i ron transport in microorganisms, p. 263-278. In A . E . M a r t e l l (ed.) Inorganic Chemistry in Biology and Medicine, Am. Chem. Soc. Nei lands, J . B . 1981a. M i c r o b i a l i ron compounds. Ann. Rev. Biochem. 50:715-731. Nei lands, J . B . 1981b. Iron absorption and transport in microorganisms. Ann. Rev. N u t r i . 1:27-46. Nei lands , J . B . 1982. M i c r o b i a l envelope proteins re lated to i r o n . Ann. Rev. M i c r o b i o l . 36:285-309. Newton, B . A . 1953. Reversal of a n t i b a c t e r i a l a c t i v i t y of polymyxin by d iva lent ions . Nature. 172:160-161. Northcote, T . G . , and T . G . Halsey. 1969. Seasonal changes in the limnology of some meromictic lakes in southern B r i t i s h Columbia. J . F i s h . Res. Bd. Can. 26:1763-1787. Northcote, T . G . 1980. Morphometrically conditioned eutrophy and i t s amel iorat ion in some B r i t i s h Columbia lakes , p.305-316. In J . Bar ica and L . R . Mur (eds.) Hypertrophic Ecosystems. Dr . W. Junk, The Hague. Novitsky, J . A . 1981. Calcium carbonate p r e c i p i t a t i o n by marine b a c t e r i a . Geomicrobiol . J . 2:375-388. O'Br ien , E . G . , and F . Gibson. 1970. Biochim. Biophys. Acta 215:393-402. Otsuk i , A . , and R . G . Wetzel. 1972. Coprec ip i ta t ion of phosphate with carbonates in a marl lake . Limnol . Oceanogr. 17:763-767. Otsuk i , A. and R . G . Wetzel. 1973. Interact ion of yellow organic acids with calcium carbonate in freshwater. Limnol . Oceanogr. 18:490-493. P a e r l , H.W. 1982a. Factors l i m i t i n g p r o d u c t i v i t y of freshwater ecosystems, p. 75-110. In K . C . Marshal l (ed.) Advances in M i c r o b i a l Ecology. Plenum Pres^, New York. P a e r l , H.W. 1982b. F e a s i b i l i t y of Fe autoradiography as performed on N - f i x i n g Anabaena spp. populations and associated b a c t e r i a . App l . Environ . M i c r o b i o l . 43:210-217. Parkhurst , D . L . , D . C . Thorstenton, and L . N . Plummer. 1980. PHREEQE a computer program for geochemical c a l c u l a t i o n s . USGS Report WRI-80-96. Parsons, D . C . 1974. S o i l - p l a n t re la t ionsh ips around an in land , sa l ine slough. M.Sc. Thes i s , S o i l Science, U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B . C . 147 P i c a r d , G.L., and G.T. Felbeck J r . 1976. The complexation of i r o n by marine humic a c i d . Geochim. Cosmochim. A c t a 40:1347-1350. Plumb, R.H.Jr., and G.F. Lee. 1973. A note on the i r o n - o r g a n i c r e l a t i o n s h i p i n n a t u r a l water. Water Res. 7:581-585. P o r t e r , K.G. 1973. S e l e c t i v e g r a z i n g and d i f f e r e n t i a l d i g e s t i o n of algae by zooplankton. Nature 244:179-180. P o r t e r , K.G. 1977. The p l a n t - a n i m a l i n t e r f a c e i n freshwater ecosystems. American S c i e n t i s t 65:159-170. P o r t e r , K.G., and J.D. O r c u t t . 1980. N u t r i t i o n a l adequacy, m a n a g e a b i l i t y , and t o x i c i t y as f a c t o r s t h a t determine the food q u a l i t y of green and blue-green algae f o r Daphnia. p. 268-281. In W.C. K e r f o o t (ed.) E v o l u t i o n and Ecology of Zooplankton Communities. New England P r e s s . Hanover, N.H. Powell, P.E., G.R. C l i n e , C P . P . R e i d , and P.J. S z a n i s z l o . 1980. Occurrence of hydroxamate siderophore i r o n c h e l a t o r s i n s o i l s . Nature 287:833-835. P r e l o g , V. 1968. Iron c o n t a i n i n g a n t i b i o t i c s and m i c r o b i c growth f a c t o r s . Pure. Appl. Chem. 6:327-338. P r i c e , C A . 1968. Iron compounds and p l a n t n u t r i t i o n . Ann. Rev. P l a n t P h y s i o l . 19:239. Rao, D.V.S., and P.A. Yeats. 1984. E f f e c t of i r o n on phytoplankton p r o d u c t i o n i n the Sargasso Sea. J . Exp. Mar. B i o l . E c o l . 81:281-289. Reddy, M.M., and G.H. N a n c o l l a s . 1971. The c r y s t a l l i z a t i o n of c a l c i u m carbonate 1. Isotope exchange and k i n e t i c s . J . C o l l o i d I n t e r f a c e S c i . 36:166-172. Reddy, M.M. 1979. K i n e t i c i n h i b i t i o n of c a l c i u m carbonate formation by wastewater c o n s t i t u e n t s , p. 32-58. In A.J. Rubin (ed.) Chemistry of Wastewater Technology. Ann Arbor S c i e n c e , Michigan. Reddy, M.M., L.N. Plummer, and E. Busenburg. 1981. C r y s t a l growth of c a l c i t e from c a l c i u m carbonate s o l u t i o n s at constant PC0 ? and 25 eC - a t e s t of a c a l c i t e d i s s o l u t i o n model. Geochim. Cosmochim. A c t a 45:1281-1289. R e i c h a r d t , W. 1981. I n f l u e n c e of methlyheptone and r e l a t e d phytoplankton n o r o c a r t e n o i d s on h e t e r o t r o p h i c a q u a t i c b a c t e r i a . Can. J . M i c r o b i o l . 27:144-147. Reynolds, C S . 1982. Phytoplankton p e r i o d i c i t y : i t s m o t i v a t i o n , mechanisms and ma n i p u l a t i o n . Freshwater B i o l . Assoc. Ann. Rep. 50:60-74 Reynolds, C.S., J . . Thompson, A.J.D. Ferguson, and S.W. Wiseman. 19 82. Loss processes i n the p o p u l a t i o n dynamics of phytoplankton maintained i n c l o s e d ecosytems. J . Plankton Res. 4:561-600. Reynolds, C S , G.H.M. Jaworski, H.A. Cmiech, and G.F. Leedale. 1981. On the annual c y c l e of the blue-green a l g a M i c r o c y s t i s  aeruginosa Kutz emendin. P h i l . Trans. R. soc. LoncH B. ,293:419-477. Reynolds, R.C 1978. Polyphenol i n h i b i t i o n of c a l c i t e p r e c i p i t a t i o n i n Lake Powell. Limnol. Oceanogr. 23:585-597. R i c e , H.M.A. 1946. P r i n c e t o n map area, Sheet 92H (east h a l f ) , G e o l . Surv. Can. R i p l , W. 1986. I n t e r n a l phosphorus r e c y c l i n g mechanisms i n shallow l a k e s , p. 138-142. In G. R e d f i e l d , J.F. Taggart, and L.M. Moore (eds. ) Lake and R e s e r v o i r Management V o l . I I Proc. 5th Annual Conf. I n t . Symp. N. Arm. Lake Manage. Soc. 148 Nov. 19 85. Ryding, S. 1985. Chemical and m i c r o b i o l o g i c a l processes as r e g u l a t o r s of the exchange of substances between sediments and water i n shallow e u t r o p h i c l a k e s . I n t . Revue ges. H y d r o b i o l . 70:657-702. Robbins, J.A., and D.N. Edgington. 1975. Determination of r e c e n t sedimentation r a t e s i n Lake Michigan u s i n g Pb-210 and Cs-137. Geochim. Cosmochim. A c t a 39:285-304. Rossknecht, V.H. 1977. On the autochtonous p r e c i p i t a t i o n of c a l c i t e i n the Lake of Constance ( i n German). Arch. H y d r o b i o l . 81:35-54. Rossknecht, V.H. 1980. Phosphate removal w i t h c a l c i u m carbonate p r e c i p i t a t i o n i n the Lake of Constance (Obersee) ( i n German). Arch. H y d r o b i o l . 88:328-344. Ryther, J . , and D.D. Kramer. 1962. R e l a t i v e i r o n requirements some c o a s t a l and o f f s h o r e plankton algae. Ecology 42:444-446. Sakamoto, M. 1971. Chemical f a c t o r s i n v o l v e d i n the c o n t r o l of phytoplankton p r o d u c t i o n i n the Experimental Lakes Area, Northwestern O n t a r i o . J . F i s h . Res. Board Can. 28:203-213. Schelske, C L . 1961. The a v a i l a b i l i t y of i r o n as a f a c t o r l i m i t i n g primary p r o d u c t i v i t y i n a marl l a k e . Ph.D. T h e s i s , U n i v e r s i t y of Michigan. Schelske, C L . 1962. I r o n , o r g a n i c matter, and other f a c t o r s l i m i t i n g primary p r o d u c t i v i t y i n a marl l a k e . S c i e n c e 136:45-46. Schelske, C.L., F.F. Hooper, and E . J . H a e r t l . 1963. Responses of a marl lake t o c h e l a t e d i r o n and f e r t i l i z e r . E cology 43:646-653. Shapiro, J . 1969. Iron i n n a t u r a l waters - i t s c h a r a c t e r i s t i c s and b i o l o g i c a l a v a i l a b i l i t y as determined with the f e r r i g r a m . Verh. I n t e r n a t . V e r e i n . Limnol. 17:456-466. Shapi r o , J . 1973. Blue-green a l g a e : why they become dominant. Science 179:382-384. Shapiro, J . 1980. The importance of t r o p h i c l e v e l i n t e r a c t i o n s t o the abundance and s p e c i e s composition of algae i n l a k e s . In J . B a r i c a and L.R. Mur (eds.) Hypertrophic Ecosystems. Dr. W. Junk, The Hague. Shapiro, J . 1984. Blue-green algae dominance i n l a k e s : the r o l e and management s i g n i f i c a n c e of pH and C0 o. I n t . Revue, ges. H y d r o b i o l . 69:765-780. Z Shukla, S.S., J.K. Syers, J.D.H. W i l l i a m s , D.E. Armstrong, and R.F. H a r r i s . 1971. S o r p t i o n of i n o r g a n i c phosphate by l a k e sediments. S o i l S c i . Soc. Amer. Proc. 35:244-249. Simpson, F.B., and J.B. N e i l a n d s . 1976. Siderochromes i n cyanophyceae: i s o l a t i o n and c h a r a c t e r i z a t i o n of s c h i z o k i n e n from Anabaena sp. J . P h y c o l . 12:44-48. Smayda, T . J . 1969. F u r t h e r enrichment experiments u s i n g the marine c e n t r i c diatom C y c l o t e l l a nana (clone 13-1) as an assay organism, p.493-512. In J.D. Costlow (ed.) F e r t i l i t y of the Sea. Gordon and Breach Science P u b l i s h e r s , New York. Smayda, T . J . 1974. Bioassay of the growth p o t e n t i a l of the lower N a r r a g a n s e t t Bay over an annual c y c l e u s i n g the diatom T h a l a s s i o s i r a pseudonana. Limnol. Oceanogr. 19:889-901. S n e l l , T.W., 1980. Blue-green algae and s e l e c t i o n i n r o t i f e r p o p u l a t i o n s . O e c o l o g i a 46:343-346. 149 Soeder, C.J., H. M u l l e r , H.D. Payer, and H. S c h u l l e . 1971. M i n e r a l n u t r i t i o n of p l a n k t o n i c algae: some c o n s i d e r a t i o n s , some experiments. M i t t . I n t . Ver. Limnol. 19:39-58. S p i r o , T.G. and P. Saltman. 1969. P o l y n u c l e a r complexes of i r o n and t h e i r b i o l o g i c a l i m p l i c a t i o n s . S t r u c t . Bond. 6:116-156. S t a b e l , H.H. 1986. C a l c i t e p r e c i t a t i o n i n Lake Constance: Chemical e q u i l i b r i u m , sedimentation, and n u c l e a t i o n by alg a e . Limnol. Oceanog. 31:1081-1093. S t a i n t o n , M.P., M.J. Capel, and F.A.J. Armstrong. 1977. Chemical a n a l y s i s of f r e s h water. Can. Dept. E n v i r o n . M i s c e l l a n e o u s S p e c i a l P u b l i c a t i o n No. 25. S t a u f f e r , R.E. 1985. R e l a t i o n s h i p s between phosphorus l o a d i n g and t r o p h i c s t a t e i n c a l c a r e o u s lakes of southeast Wisconsin. Limnol. Oceanogr. 30:123-145. Stewart, W.D.P. 1969. N i t r o g e n f i x a t i o n i n the sea, p. 537-564. In J.D. Costlow (ed.) F e r t i l i t y of the Sea. Gordon and Breach S c i e n c e P u b l i s h e r s , New York. S t r i c k l a n d , J.D.H., and T.R. Parsons. 1972. A p r a c t i c a l manual of seawater a n a l y s i s . B u l l . F i s h . Res. Board Can. Stumm, W., and J.O. L e c k i e . 1970. Phosphate exchange with sediments; i t s r o l e i n the p r o d u c t i v i t y of s u r f a c e waters. Proc. F i f t h I n t e r n a t . Water P o l l u t i o n Res. Conf. Pergamon P r e s s . Stumm, W., and J . J . Morgan. 1981. Aquatic Chemistry. W i l e y - I n t e r s c i e n c e , New York. Stumpf, D.K., and R.H. B u r r i s . 1979. A micromethod f o r the p u r i f i c a t i o n and q u a n t i f i c a t i o n of org a n i c a c i d s of the t r i c a r b o x y l i c a c i d c y c l e i n p l a n t t i s s u e s . A n a l . Biochem. 95:311-315. S z a n i s z l o , P.J., P.E. Powell, C P . P . Reid , and G.R. C l i n e . 1981. Pr o d u c t i o n of hydroxamate siderophore i r o n c h e l a t o r s by e c t o m y c o r r h i z a l f u n g i . Mycologia 73:1158-1174. Thurlow, D.L., R.B. Davis, and D.R. S a s s e v i l k . 1975. Primary p r o d u c t i v i t y , phytoplankton p o p u l a t i o n s and n u t r i e n t b i o a s s a y s i n China Lake, Maine, U.S.A. Verh. I n t . V e r e i n . Limnol. 19:1029-1036. T i f f i n , L.O. 1966. Iron t r a n s l o c a t i o n I I . C i t r a t e / i r o n r a t i o s i n p l a n t stem exudates. P l a n t . P h y s i o l . 41:515-518. T i p p i n g , E. 19 81. The a d s o r p t i o n of a q u a t i c humic substances by i r o n o x i d e s . Geochim. Cosmochim. Acta. 45:191-199. T r i c k , C.G., R.J. Andersen, A. G i l l a m , and P.J. H a r r i s o n . 1983a. P r o r o c e n t r i n : An e x t r a c e l l u l a r s i d e r o p h o r e produced by the marine d i n o f l a g e l l a t e Prorocentrum minimum. Sc i e n c e 219:306-308. T r i c k , C.G., R.J. Andersen, N.M. P r i c e , A. G i l l a m , and P.J. H a r r i s o n . 1983b. Examination of hydroxamate sid e r o p h o r e p r o d u c t i o n by n e r i t i c e u k a r o y t i c marine phytoplankton. Mar. B i o l . ( B e r l ) 75:9-18. Trimbee, A.M., and G.P. H a r r i s . 1984. Phytoplankton p o p u l a t i o n dynamics of a smal l r e s e r v o i r : use of sedimentation t r a p s t o q u a n t i f y the l o s s of diatoms and r e c r u i t m e n t of summer bloom-forming blue-green algae. J . Plankton Res. 6:897-918. Utermohl, H. 1931. Neuewege i n der q u a n t i t a t i v e n E r f a s s u n g des plan k t o n s . Verh. i n t . V e r e i n t h e o r . angew. Limnol. 5:567-596. 150 V o l l e n w e i d e r , R.A. 1974. A manual on methods f o r measuring primary p r o d u c t i o n i n a q u a t i c environments. IBP Handbook No 12. B l a c k w e l l S c i . , Oxford. Walsby, A.E. 1977. The gas vacuoles of blue-green a l g a e . S c i e n t i f i c American. 237:90-97. Walter, L.M., and J . S. Hanor. 1979. E f f e c t of orthophosphate on the d i s s o l u t i o n k i n e t i c s of b i o g e n i c magnesium c a l c i t e s . Geochim. Cosmochim. A c t a 43:1377-1385. Water I n v e s t i g a t i o n s Branch. 1977. Observations of water q u a l i t y i n the Chain-Link-Osprey system. P a r t 1. B r i t i s h Columbia Min. E n v i r . F i l e 0290862. Weinberg, E.D. 1974. I r o n and s u s c e p t i b i l i t y t o i n f e c t i o u s d i s e a s e . S c i e n c e 184:952-956. Weinberg, E.D. 1975. Metal s t a r v a t i o n of pathogens by h o s t s . B i o s c i e n c e 25:314-318. Weinberg, E.D. 1978. Iron and i n f e c t i o n . M i c r o b i o l . Rev. 42:45-66. Wetzel, R.G. 1965. P r o d u c t i v i t y and n u t r i e n t r e l a t i o n s h i p s i n marl lakes of n o r t h e r n Indiana. I n t . Ver. Theor. Angew. Limnol. Verh. 16:321-332. Wetzel, R.G. 1966. P r o d u c t i v i t y and n u t r i e n t r e l a t i o n s h i p s i n marl lakes of Northern Indiana. Verh. I n t . Ver. Limnol. 16:331-332. Wetzel, R.G. 1968. D i s s o l v e d o r g a n i c matter and phytoplankton p r o d u c t i v i t y i n marl l a k e s . M i t t . I n t . Ver. Limnol. 14:261-270. Wetzel, R.G. 1975. Limnology. W.B. Saunders Co., P h i l a d e l p h i a . White, A., P. Handler, and E.L. Smith. 1968. P r i n c i p l e s of B i o c h e m i s t r y . McGraw-Hill Co., New York. White, A., P. Handler, and E.L. Smith. 1968. P r i n c i p l e s of B i o c h e m i s t r y . McGraw-Hill. Toronto. W i l l i a m s , J.D.H., J.K. Syers, R.F. H a r r i s , and D.E. Armstrong. 1971. F r a c t i o n a t i o n of i n o r g a n i c phosphate i n c a l c a r e o u s l a k e sediments. S o i l . S c i . Amer. Proc. 35:250-255. W i l l i a m s , J.D.H., and A.E. Pashley. 1978. Lightweight c o r e r designed f o r sampling v e r y s o f t sediments. J . F i s h . Res. Board Can. 36:241-246. Wilson, J.T., S. Greene, and M. Alexander. 1979. E f f e c t of i n t e r a c t i o n s among algae on n i t r o g e n f i x a t i o n by blue-green algae (Cyanobacteria) i n f l o o d e d s o i l s . Appl. E n v i r o n . M i c r o b i o l . 38:916-921. Winkelmann, G. 1974. M e t a b o l i c products of microorganisms. Arch. M i c r o b i o l . 98:39-50. Wolfe, J.M., and E.L. R i c e . 1979. A l l e l o p a t h i c i n t e r a c t i o n s among al g a e . J . Chem. E c o l . 5:533-542. Wurstbaugh, W.A., and A.J. Home. 1983. Iron i n e u t r o p h i c C l e a r Lake, C a l i f o r n i a . Can. J . F i s h . Aquat. S c i . 40:1419-1429. Yakowitz, H., R.L. Myklebust, and K.F.J. H e i n r i c h . 1973. FRAME: an on l i n e c o r r e c t i o n procedure f o r q u a n t i t a t i v e e l e c t r o n probe m i c r o a n a l y s i s . U.S. N a t i o n a l Bureau of Standards Tech. Note 796. 151 A p p e n d i x 1 D e t a i l s o f L i m n o c o r r a l E x p e r i m e n t s 19 80 B l a c k L a k e L i m n o c o r r a l E x p e r i m e n t s S e v e n l i m n o c o r r a l s o f t r a n s p a r e n t woven p o l y e t h y l e n e w e r e u s e d t o t e s t t h e e f f e c t o f i r o n a n d n i t r a t e e n r i c h m e n t on a l g a l p r o d u c t i v i t y . The l i m n o c o r r a l s w e r e 2 .0 m e t e r s i n d i a m e t e r a n d 7.0 m e t e r s d e e p w i t h a s e a l e d b o t t o m . A f l o a t - c o l l a r o f s t y r o f o a m a n d p l y w o o d p r e v e n t e d any w a t e r e x c h a n g e w i t h t h e l a k e . S t a r t i n g on 20 May 1980, one l i m n o c o r r a l was e n r i c h e d e v e r y two weeks w i t h 1.82 g o f r e a g e n t g r a d e KNO^ ( F i s h e r ) . A p a d d l e was u s e d t o d i s t r i b u t e t h e n i t r a t e t h r o u g h o u t t h e l i m n o c o r r a l . I d e a l l y t h e t o p f o u r m e t e r s w o u l d h a v e b e e n i n c r e a s e d t o 200 ug N/L. The s u r f a c e n i t r a t e c o n c e n t r a t i o n i m m e d i a t e l y a f t e r n i t r a t e e n r i c h m e n t was 350 ug N/L. I r o n was a d d e d i n t h e f o r m o f an EDTA-Fe c o m p l e x ( 3 : 1 m o l a r r a t i o E D T A : F e ) . The c o n c e n t r a t i o n o f i r o n was m o n i t o r e d b i w e e k l y by g r a p h i t e f u r n a c e a t o m i c a b s o r p t i o n and t h e s o l u b l e i r o n l e v e l s w e r e m a i n t a i n e d b e t w e e n 100 and 200 ug F e / L . E D T A - l i m n o c o r r a l s w e r e e n r i c h e d w i t h e q u i v a l e n t c o n c e n t r a t i o n s o f EDTA as w e r e t h e Fe-EDTA t r e a t m e n t s . C o n t r o l l i m n o c o r r a l s w e r e m o n i t o r e d a l o n g w i t h t h e t r e a t m e n t s . 152 19 82 Black Lake L i m n o c o r r a l Experiment Li m n o c o r r a l s of t r a n s p a r e n t woven p o l y e t h y l e n e , 2.0 meters i n diameter and 5.0 meters deep, were made by F a l s e Creek P l a s t i c s (Vancouver,B.C.). A c o l l a r t h a t was r e s i s t a n t t o u l t r a v i o l e t l i g h t was sewn t o the top and extruded styrofoam was i n s e r t e d i n t o the hollow c o l l a r f o r f l o t a t i o n . The bottom of the l i m n o c o r r a l s were s e a l e d and anchored with ropes and co n c r e t e b l o c k s v i a attached e x t e r n a l l o o p s . The improvements i n des i g n r e l a t i v e t o 1980, f a c i l i t a t e d f i l l i n g of the l i m n o c o r r a l s and r e p l i c a t i o n of the treatments. E i g h t l i m n o c o r r a l s were f i l l e d June 13-14, 1982 wit h lakewater 2.0 meters from the lake s u r f a c e u s i n g a l a r g e water pump. Two l i m n o c o r r a l s r e c e i v e d t h r e e a d d i t i o n s of 18.4 g of commercial grade CafNO^^* Two l i m n o c o r r a l s r e c e i v e d t h r e e a d d i t i o n s of 18.4 g of CaCNO^) 2» and 78.76 g of sodium c i t r a t e . Two l i m n o c o r r a l s r e c e i v e d 18.4 g of CafNO^^f a n d a n i r o n s o l u t i o n . The i r o n s o l u t i o n was made by f i r s t d i s s o l v i n g 4.55 g of F e C l j and 192.12 g of c i t r i c a c i d and then a d j u s t i n g the pH to 7.0 with NaOH. Two l i m n o c o r r a l s were l e f t u n t r e a t e d . The n u t r i e n t s were added June 23, June 30, and Aug. 12. In the August 12 treatment, n i t r a t e was not added t o the c i t r a t e l i m n o c o r r a l s . On June 23, the n u t r i e n t s were d i s s o l v e d i n 50 l i t e r s of lakewater and pumped the s o l u t i o n t o f i v e depths. For the other two treatments the n u t r i e n t s were d i s s o l v e d i n 1.0 l i t e r of lakewater and added t o the s u r f a c e of the l i m n o c o r r a l with a paddle s t i r r i n g the water. With i d e a l mixing, the i n i t i a l 153 n u t r i e n t c o n c e n t r a t i o n s would have been: 200 U9" N/L, 1.4 mg C - c i t r a t e / L , or 100 ug Fe/L. Chain Lake L i m n o c o r r a l Experiments Li m n o c o r r a l s with the 1982 design were used. E i g h t l i m n o c o r r a l s were f i l l e d J u l y 15 and en r i c h e d on J u l y 18. The n i t r o g e n l i m n o c o r r a l s were e n r i c h e d with 8.3 g of NH^Cl which should have produced a c o n c e n t r a t i o n of 200 ug N/L. Iro n (3.42 g F e C l j per l i m n o c o r r a l ) was mixed with c i t r i c a c i d (96 g per l i m n o c o r r a l ) , the pH was adj u s t e d t o 7.0 wit h NaOH, and q u i c k l y the s o l u t i o n s were added t o the l i m n o c o r r a l s . Two l i m n o c o r r a l s r e c e i v e d 96 g of c i t r i c a c i d t h a t was a l s o n e u t r a l i z e d w i t h NaOH. A l l l i m n o c o r r a l s , i n c l u d i n g what i s r e f e r r e d t o i n the t e x t as "untreated l i m n o c o r r a l s " r e c e i v e d 1.98 g of KH 2P0 4. A l l n u t r i e n t a d d i t i o n s were made by mixing the n u t r i e n t s i n 50 L of water and then pumping the s o l u t i o n i n t o the l i m n o c o r r a l s at f i v e depths. 154 Appendix 2 Table A l . Iron analysis in Black Lake - May 9, 1978. Depth Dissolved Particulate Aerated Control Aerated Control Surface 6 5 4 3 2M 8 4 19 12 4M 20 17 44 26 6M 22 26 182 57 8M 25 13 202 45 9M 28 88 119 69 A l l values are in ng Fe/L. 155 Table Al continued 1979 Data: Dissolved Particulate Date Depth Aerated Control Aerated Contrc June 15 2M 13 - 29 25 5M 22 - 30 11 9M 19 - 30 11 July 4 Surf 17 11 31 28 3M 10 10 29 20 5M 20 20 84 27 9M 22 24 97 57 July 17 2M 3 11 40 15 5M 51 7 60 48 9M 8 18 123 100 Aug 11 0.5M <1 5 94 50 1M 2 250 90 27 2M 119 184 111 60 3M 4 2 92 29 5M 8 203 260 69 9M 600 - 632 273 Aug 30 1M <1 2 43 37 5M 101 90 86 52 9M 102 85 100 32 Oct 26 2M 10 12 30 28 8M 14 18 44 62 Dec 13 2M 9 10 20 21 8M 11 12 18 15 A l l values are in ug Fe/L. 156 Table Al continued 1980 Data: Dissolved Particulate Date Depth Aerated Control Aerated Control Jan 22 2M 18 17 13 -8M 29 - 13 -Apri l 23 2M 2 2 35 27 5M 3 4 39 45 8M 10 13 42 41 May 12 2M - 13 42 41 8M - 13 - -May 21 Surf - - 59 -2M - - 46 -5M - - 34 -7M - - 38 -June 4 Surf 25 18 6 6 IM 20 22 7 7 2M 20 25 6 8 3M 18 20 27 7 5M 20 30 30 11 8M 11 35 30 11 July 17 IM 1 1 10 12 3M 4 2 20 17 Aug 11 IM 4 0 93 59 3M 5 3 184 72 5M 7 8 194 98 7M - - 453 326 9M 18 17 - 422 A l l values are in ug Fe/L. Appendix 3 Table A2 Oxygen Concentrations in Liimccorrals During Citrate Additions ( m g / L ) Fe -C1t ra te L imnocorrals Depth June J u l y August 24th 26th 28th 13th 22nd 10th 17 th 24th 0 8 . 5 8 . 8 8 .4 8 . 7 9.2 11.2 1 3 . 3 12.2 18.0 2 0 . 0 17 .9 18.2 18.2 16.6 17.4 16.8 1 8 . 8 9.2 8 .35 8.75 9 . 8 11.2 13.8 11.5 20 .0 17.0 17 .9 1 9 . 0 15 .3 16.6 16.8 16.2 2 9 .4 10.2 9.4 10.4 9 .5 .10.3 11 .3 8 . 6 20 .0 16.6 10.2 1 7 . 0 12 .8 13.1 14 .0 14.4 3 9 .2 10.2 9.55 10.2 8 . 5 ' 9 . 4 9.2 8 . 3 12.0 11 .0 8 . 2 8 . 8 10.8 12.6 9.2 9 .2 4 9.1 9 . 9 9 .25 10.0 8 . 0 8 . 5 7 .9 7 .0 3 .0 6 . 0 8 . 1 7 . 8 10.1 10.6 6 . 7 2 . 0 5 9 . 0 9 .9 8 . 0 9 .0 6 .1 6 . 5 5 . 0 4 . 2 2 .4 4 . 3 8 . 0 7.7 5 .5 7 .0 1 .0 0.7 Na -C1t rate L imnocorra ls Depth June J u l y August 24th 26th 28th 13th 22nd 10th 17th 24th 0 8 . 7 8 . 8 9.1 9 . 8 10.6 12 .0 11.0 13 .0 16.5 16 .0 14.6 16 .2 14.4 13.4 14.2 15.4 1 9 . 0 9 . 5 9.1 9 .8 10.5 12 .0 12.2 15.0 13.5 14.6 15.2 16 .2 - 1 3 . 6 12 .9 14 .3 13.1 2 9.7 10.1 10.0 10.2 10 .0 11 .0 10 .8 13.6 13.5 14.6 1 6 . 3 16.1 11 .2 11.6 13 .0 12 .2 3 9 .6 9 .9 9 .9 10.2 9.2 9 . 0 9.1 9 . 0 12.6 11 .8 9 .4 8 . 8 11.2 10.6 10 .0 9.1 4 9 .4 9.7 9 .6 9.7 7 .5 7 .9 6 .7 7.4 5.6 5 .6 8 . 2 4 . 2 9 . 8 9 . 0 8 . 6 6 . 8 5 9 . 0 9.4 8.1 8 .4 6 . 7 7 .0 6 . 5 4.1 4 . 0 1.8 7.6 3 .6 8 . 0 7 .0 3 .6 1 .3 Contro l L imnocorrals Depth June J u l y August 24 th 26th 28th 13th 22nd 10th 17th 24th 0 8 .4 8 .6 8 . 8 9.2 10.4 9 .4 12.8 17.4 15.9 1 5 . 8 18.4 15 .0 16.1 14.4 16 .0 14.2 1 8 . 5 8 . 8 9 . 0 9 .2 10.5 9.4 13.0 2 0 . 0 15.9 16.1 18.4 14.4 16.0 12.0 16.4 14.4 2 8 . 9 9 .4 9.4 9 .6 9 . 8 9 . 8 12.0 20 .0 16.1 16.6 16 .8 11.8 12.5 9 . 8 14.8 14 .0 3 9 . 0 9.5 9 .4 9 .6 9 . 3 9 .4 9 .5 12.4 9 . 8 14.4 10.4 4 . 2 12.4 9 .6 11.4 8 . 0 4 9 . 0 9 .5 9 .0 9 . 5 8 . 7 9 . 3 8 . 8 9 . 0 8 .2 5 .4 9 . 8 2 . 6 12.2 8 . 4 1 1 . 0 6 . 7 5 8 .7 8 .4 8 . 3 7 .8 7 .8 7 .3 6 .4 6 . 0 7.7 3 .8 9.4 2 . 5 3 .0 6 .4 4 . 2 3.2 158 Appendix 4 Improvements t o the I r o n - B i n d i n g Assay U n f o r t u n a t e l y , the assay was i n i t i a l l y used b e f o r e the opt i m a l i n c u b a t i o n time was determined. The c h e l a t i o n i s not complete w i t h i n the h a l f hour i n c u b a t i o n t h a t was i n i t i a l l y used ( F i g . A l ) . T h i s procedure r e s u l t e d i n a s m a l l e r range of l i n e a r responses and a sampling e r r o r of about 10% ( c o e f f i c i e n t of v a r i a t i o n ) . H a l f hour i n c u b a t i o n s were used f o r the f o l l o w i n g work: the i r o n b i n d i n g c a p a c i t y of the c h e l a t o r s used i n the heterotrophy, primary p r o d u c t i o n i n c u b a t i o n s , and measurements of l a k e s i d e r o p h o r e s . In one hour i n c u b a t i o n s , the standard curve was l i n e a r throughout the c o n c e n t r a t i o n range t h a t was encountered ( F i g . A2). Blanks were very low and the s e n s i t i v i t y of the r a d i o i s o t o p e method was good ( F i g . A3). In one hour i n c u b a t i o n s , the c o e f f i c i e n t of v a r i a t i o n was l e s s than 2%. Another e r r o r may have a r i s e n i n the use of a c i d s or bases with i o n exchange r e s i n s . Some of the side r o p h o r e r e a d i l y broke down i n the presence of weak a c i d or base ( F i g . A4). Presumably l i t t l e of t h a t break down product was i n the b i o a s s a y s . F r e e z e - d r y i n g i s a b e t t e r method of c o n c e n t r a t i n g s i d e r o p h o r e s than the ion-exchange method. The u n c e r t a i n t y a s s o c i a t e d with knowing the c o n c e n t r a t i o n of the s i d e r o p h o r e i n the microenvironment of the c e l l s was a much bigge r problem than the a n a l y t i c a l problems. Strong q u a l i t a t i v e statements are s t i l l p o s s i b l e . 159 TIME COURSE DES 2CXXH I o. o 1500H A.f.a. I 100CH 500H 1 2 3 4 5 6 TIME (hours) F i g u r e A l E f f e c t of i n c u b a t i o n time on c h e l a t i o n of i r o n by d e s f e r a l (DES) and a f i l t r a t e from a Anabaena f l o s - a q u a e c u l t u r e ( A . f . a . ) . 160 - 1 1 1 1 1— 0 2 0 4 0 6 Q B t o ALIQUOT A.f.a. F i g u r e A2 Iron b i n d i n g c a p a c i t y (FeBC) of a f i l t r a t e from Anabaena f l o s - a q u a e c u l t u r e . A l i q u o t s of A . f . a . r e f e r s the degree of d i l u t i o n of the c u l t u r e f i l t r a t e . 161 75,000 n Fe B C Calibration 60,000 45,000 A CL o Jg 30,000 15,000 A 1,000 Desferal \ig/L 2,000 F i g u r e A3 I r o n b i n d i n g c a p a c i t y (FeBC) s t a n d a r d i z a t i o n with d e s f e r a l . 0.15H 0.10H I O CO Z 005H NaOH SIDEROPHORE DEGRADATION BY ACID OR BASE 0.05 004-I 0 . 0 5 H O X 0.02H 0D1H Fe BC (uM-L"1) F i g u r e A4 Degradation of the siderophore from Anabaena f l o s - aquae by a c i d or base A p p e n d i x 5 T a b l e A3 Y e l l o w L a k e S e d i m e n t C h e m i s t r y D e p t h s i o 2 A 1 2 0 3 F e 2 ° 3 MgO C a O N a 2 0 K 2 0 T i 0 2 MnO P 2 ° 5 c m 0 - 2 5 4 . 8 6 6 . 1 1 3 . 5 5 2 . 1 6 1 5 . 7 2 0 . 8 2 1 . 8 5 0 . 4 1 0 . 1 4 0 . 3 0 4 - 6 4 3 . 7 5 8 . 7 0 3 . 9 9 2 . 22 1 9 . 3 6 0 . 8 7 2 . 2 5 0 . 4 2 0 . 0 6 0 . 3 2 6 - 8 3 8 . 0 7 6 . 1 7 2 . 3 9 1 . 3 6 2 6 . 8 9 0 . 8 1 1 . 6 4 0 . 2 8 0 . 1 0 0 . 3 8 8 - 1 0 3 3 . 9 6 4 . 0 6 1 . 6 5 1 . 0 2 3 1 . 6 5 0 . 7 6 1 . 1 1 0 . 1 9 0 . 0 5 0 . 4 4 1 0 - 1 2 3 3 . 7 0 3 . 9 4 1 . 7 3 1 . 0 4 3 1 . 8 3 0 . 5 4 1 . 0 4 0 . 1 9 0 . 0 5 0 . 5 1 1 2 - 1 4 3 6 . 77 5 . 4 5 2 . 5 8 1 . 5 0 2 8 . 5 2 0 . 6 4 1 . 4 2 0 . 24 0 . 0 5 0 . 4 3 1 4 - 1 6 3 1 . 9 6 4 . 8 3 2 . 2 3 1 . 5 6 3 1 . 8 2 0 . 8 0 1 . 1 0 0 . 2 0 0 . 0 4 0 . 4 8 1 8 - 2 0 4 2 . 6 6 4 . 2 7 2 . 6 5 0 . 9 6 2 0 . 3 5 0 . 3 5 0 . 8 8 0 . 2 9 0 . 0 6 0 . 4 8 2 8 - 3 0 4 7 . 3 3 1 . 2 2 1 . 5 0 0 . 5 2 2 1 . 8 2 0 . 2 0 0 . 1 0 0 . 0 9 0 . 0 5 0 . 6 2 3 8 - 4 0 4 4 . 8 5 2 . 5 5 1 . 7 0 0 . 6 0 2 3 . 3 6 0 . 3 6 0 . 6 4 0 . 1 8 0 . 0 4 0 . 5 8 4 8 - 5 0 4 9 . 0 9 1 . 6 3 1 . 5 7 0 . 5 3 2 0 . 42 0 . 3 4 0 . 2 8 0 . 1 3 0 . 0 5 0 . 5 2 A l l v a l u e s a r e e x p r e s s e d a s % d r y w e i g h t . T h e X - r a y f l u o r e s e n c e a n a l y s i s r e p o r t s t h e t o t a l c o n c e n t r a t i o n a s i f t h e e l e m e n t w a s p r e s e n t a s a . s i m p l e o x i d e . T h e p r e s e n c e o f c a l c i t e ( c a l c i u m c a r b o n a t e ) w a s c o n f i r m e d b y X - r a y d i f f r a c t i o n a n a l y s i s . 

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