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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 by THOMAS P. D. MURPHY  B . S c . 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 in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to r e q u i r e d standard  THE UNIVERSITY  OF BRITISH COLUMBIA  September,  1987  (c) Thomas P. D. Murphy, 1987  the  In  presenting  degree  this  thesis in partial fulfilment of  requirements  for  an  of  department  this thesis for scholarly or  by  his  or  her  I further agree that permission for  purposes  permission.  Department of  ~2-Qol~£> l> j  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  extensive  may be granted by the head of  representatives.  It  is  understood  that  publication of this thesis for financial gain shall not be allowed without  DE-6(3/81)  advanced  at the University of British Columbia, I agree that the Library shall make it  freely available for reference and study. copying  the  copying  my or  my written  ii ABSTRACT The  primary  objective of t h i s  changes i n i r o n a v a i l a b i l i t y blue-green  algal  availability  was r e l a t e d  biogeochemical  was s t u d i e d Thompson  i n three  Plateau  t o events  r e g u l a t i o n of blue-green  eutrophic  experiments,  sediment core  hardwater l a k e s  included  algal  succession  l o c a t e d upon t h e  iri s i t u  bottle  of  seasonal  and w h o l e - l a k e m a n i p u l a t i o n  a e r a t i o n , or c a l c i u m hydroxide  production  and l i m n o c o r r a l  a n a l y s i s , monitoring  changes i n water c h e m i s t r y ,  availability.  (calcium  i n s o u t h - c e n t r a l B r i t i s h C o l u m b i a . The  approaches  primary  s u c h as c a l c i t e  bioassays  were u s e d  by  a d d i t i o n . G r o w t h and  to evaluate  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  iron  from  algal  c u l t u r e s and l a k e w a t e r , q u a n t i f i e d by a c h e l a t i o n a s s a y , used  t o determine t h e i r  bacterial  and  siderophore  they  chelator dissolved suppress  The  effects  on a l g a l  p r o d u c t i v i t y and  to regulate the b i o a v a i l a b i l i t y  i s o l a t e s were r a p i d l y  were h i g h l y s p e c i f i c concentrations iron  summer. I n B l a c k  Lake,  excreted  chelators that  species of algae  o f some o t h e r  the heterotrophic a c t i v i t y degree of i r o n  assimilated i n lake  water  Lake u s u a l l y exceeded t h e  c o n c e n t r a t i o n . Algae  production  of i r o n .  f o r i r o n c h e l a t i o n . Moreover,  i n Black  g r o w t h o f some o t h e r  the primary suppress  in situ  and  heterotrophy.  M i c r o b e s were a b l e Algal  iron  and s e d i m e n t n u t r i e n t r e l e a s e .  experimental  hypolimnetic  i n f l u e n c e the p e r i o d i c i t y of  g r o w t h . A s e c o n d a r y g o a l was t o r e s o l v e how  carbonate) p r e c i p i t a t i o n The  r e s e a r c h was t o d e t e r m i n e i f  algal  could  by 90%, e n h a n c e  s p e c i e s by 30%, o r  o f b a c t e r i a by 14-98%.  l i m i t a t i o n v a r i e d g r e a t l y during the iron  limitation  was more t h a n  ten-fold  iii more i n t e n s e  i n e a r l y summer  blue-green algae content increase lakes  occurred  of the lake  than  in late  i n B l a c k Lake o n l y  increased  after the iron  f r o m 20 t o more t h a n  in iron concentration  sediment p y r i t e formation  i n t o r e f r a c t o r y i r o n i n both Chain iron limitation  varied  B l a c k Lake, t h e algae  and F r i s k e n  Black  and F r i s k e n  lakes  the degree  Unlike in  C h a i n Lake r e g u l a t e d  iron  phosphorus s o l u b i l i t y .  was p r i m a r i l y a f u n c t i o n  index of i r o n  had t o o l i t t l e  i r o n phosphate t o p r e c i p i t a t e , but the higher  sediment  lakes,  i n C h a i n L a k e were n o t l i m i t e d b y i r o n  availability.  not  available iron  g r e a t l y among t h e l a k e s .  P h o s p h o r u s s o l u b i l i t y was a good  among l a k e s  of i r o n .  converted  availability.  in  100 u g / L . An  i n t h e w a t e r column o f t h e t h r e e  was c a u s e d b y a midsummer s e d i m e n t r e l e a s e Although  of  summer. Dense blooms o f  concentration  The d i f f e r e n c e s  of external  i r o n r e l e a s e . C h a i n Lake r e c e i v e d  iron for  iron  loading,  10"^ more i r o n p e r  2 m  than F r i s k e n  or Black  lakes.  Carbonate e q u i l i b r i a iron  integrated  responses t o  e n r i c h m e n t . 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  epilimnion resulted calcite  of B l a c k Lake, a l g a l  i n an i n c r e a s e  algae  i n the  p r o d u c t i v i t y was e n h a n c e d  which  i n pH and t h e c o p r e c i p i t a t i o n o f more  and p h o s p h o r u s t h a n  precipitation  i n c o n t r o l t r e a t m e n t s . The  of c a l c i t e could  s e d i m e n t as much as 90% o f t h e  and 97% o f t h e p h o s p h o r u s f r o m t h e e p i l i m n i o n . The  hypolimnia of the iron-enriched and  the microbial  highest  limnocorrals  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  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 blue-green  had t h e l o w e s t pH  algal  growth  i n hardwater  i r o n r e l e a s e , and  much o f t h e p e r i o d i c i t y o f lakes.  iv Table  of  Contents  Abstract  i i  List  of Tables  List  of F i g u r e s  viii ix  Acknowledgements  x i i  Glossary  xiii  1  Introduction  1  1.1  Factors Regulating the P e r i o d i c i t y  1.2  Iron-Requiring Reactions  1.2.1  Iron Limitation  10  2  Methods  15  2.1  Study Area  15  2.1.1  B l a c k Lake  15  2.1.2  F r i s k e n Lake  19  2.1.3  Chain  Lake  19  2.2  Field  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 B l a c k Lake  21  2.2.3  Chemical  21  2.2.4  Limnocorral Experiments  22  2.2.5  Small  22  2.2.5.1  Calcium C h l o r i d e Experiment  22  2.2.5.2  Primary  23  2.2.5.3  Heterotrophy  of Blue-Green  Treatments of F r i s k e n Lake  In-Situ  Incubations  Production  of A l g a l Growth... 1 Algae  4  24  ,  2.3  Laboratory  2.3.1  Chemical A n a l y s i s  2.3.2  3 2  Studies  V  and  A n a l y s i s of F i e l d  Samples....  25 25  P-SRP Analysis  27  2.3.3  Iron-Binding  2.3.4  Chelator  2.3.5  Electron Microscopic  2.3.6  Sediment A n a l y s i s  31  3  Results  32  3.1  Biogenic  3.1.1  Iron-Chlorophyll a Relationships  32  3.1.2  Seasonal  Algal  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  3.2  Confirmation  3.2.1  Laboratory  3.2.2  Fe-EDTA L i m n o c o r r a l s  3.2.3  Fe-Citrate Limnocorral  3.2.4  Iron A v a i l a b i l i t y  3.3  Siderophore  3.3.1  Chelator  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  Specificity  62  3.3.4  Lake S i d e r o p h o r e s  66  3.3.5  Enhanced  69  3.3.6  Allelopathic  3.3.7  Siderophore  3.4  Effect  of  Assay  27  Isolation  29 Analysis  31  Response t o I r o n A v a i l a b i l i t y  32  Succession  of the  37  Iron-Biomass R e l a t i o n s h i p  Bioassays  with  Black  in Black  Lake Water  42  Lake  Experiment  i n Chain  42  47 in Black  Lake  49  Lake  55  Ecology  57  Quantification  57  f o r Iron  Iron A v a i l a b i l i t y P r o p e r t i e s of Siderophores Influence  on  Heterotrophy  Iron A v a i l a b i l i t y  on  70 ...  72  Phosphorus Chemistry.  79  vi 3.4.1  Geographic V a r i a b i l i t y  i n Fe-P Water C h e m i s t r y  79  3.4.2  Geographic V a r i a b i l i t y  i n Sediment I r o n R e a c t i v i t y . .  82  3.5  Effect  of Iron 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 3.5.1  Phosphorus Chemistry  3.5.2  Calcite  Precipitation  86  of Black  Lake  i n Black  86  Lake  89  32 3.5.2.1  P Kinetics  and P L i m i t a t i o n  3.5.3  Calcite Precipitation  3. 5.3.1  19 80 Fe-EDTA L i m n o c o r r a l s  3.5.3.2  N i t r a t e Induction  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  3.5.3.4  Citrate  Effect  91  i n Limnocorrals i n Black  of C a l c i t e  on P - C a C 0  3  91  Lake  Precipitation  91 93 94  Indicates  Iron Limitation  96  3.5.3.5  I n f l u e n c e o f W e a t h e r on P-CaCO^ P r e c i p i t a t i o n  96  3.5.4  Calcium  Chloride Induction  99  3.5.4.1  Calcite  Analysis  3.6  Calcite  Precipitation  of C a l c i t e  Precipitation.  101 - A Major Cause o f A l g a l  Periodicity  103  3.6.1  Pretreatment  Water C h e m i s t r y  3.6.2  Lime-Induced C a l c i t e  Precipitation  105  3.6.2.1  Suppression  Growth  107  3.6.3  L o n g - T e r m Enhancement o f C a l c i t e  4  Discussion  4.1  Spatial  and T e m p o r a l V a r i a t i o n  4.2  Calcite  Induction  4.3  Sediment I r o n R e l e a s e  of Algal  103  Precipitation  107 113  i n Iron A v a i l a b i l i t y  of Iron L i m i t a t i o n  113 115 119  vii 4.4  Interrelationship and  Calcite  of  Iron Limitation,  Chelation,  Precipitation  Control  of  121  4.5  Microbial  Iron A v a i l a b i l i t y  4.6  S i d e r o p h o r e s as  M e d i a t o r s of  4.7  S i d e r o p h o r e s as  Regulators  Algal  of  Succession  122 126  Bacterial  Heterotrophy  128  4.8  Calcite  130  4.8.1  Variability  4.9  Biological  4.10  Microbial  4.11  Phosphorus C o p r e c i p i t a t i o n  4.12  Conclusions  Precipitation of  Calcite  C o n t r o l of  Precipitation Calcite  Responses t o C a l c i t e  Precipitation Precipitation  with C a l c i t e  131 133 134 135 137  References  140  Appendix  1  Details  of  Limnocorral Experiments  Appendix  2  Iron Analysis  Appendix  3  Oxygen  Appendix  4  Improvements t o  Appendix  5  Y e l l o w Lake Sediment Chemistry  i n Black. L a k e  in Limnocorrals During C i t r a t e A d d i t i o n s . the  Iron Binding Assay  151 154 157 158 163  viii List  of  Tables  1  Some P h y s i c a l and  Chemical  Features  of  the  Study Lakes and  18  2  Timing  Sites  of Experiments  3  Total  Iron Concentrations  i n Chain  4  Total  Iron Concentrations  i n F r i s k e n L a k e - 1983,  5  Primary  Production  20 L a k e - 1984  in Experimental  Enclosures  38 1984..  in  B l a c k L a k e i n 1980 6  Citrate Concentration  7  Limnocorral  8  Effect  1 4  48 in Limnocorrals  52  C-Citrate Assimilation  of F r e e z i n g Anabaena f i l t r a t e  53 on  Iron Chelation 9  Effect  58  of M e t a l  A d d i t i o n on Fe  t h e Anabaena f l o s - a q u a e 10  Effect  of Metal  Chelation  by  siderophore  A d d i t i o n o f Fe  62  C h e l a t i o n by  the  Aphanizomenon S i d e r o p h o r e 11  Black July  12  Effect  and  Chelation Capacity  1980  67  o f E x t r a c t s P r o d u c e d by  G r o w t h C o n d i t i o n s on  Anabaena Under  Different  the M i c r o b i a l Uptake of A c e t a t e . . . .  13  Fluxes of Iron  14  Calcium  15  Elemental  16  Calcium-Phosphate Ratios  i n Chain  Concentrations  and  F r i s k e n Lakes  i n 1982  Limnocorrals  Al  Iron Analysis in Black  A2  Oxygen C o n c e n t r a t i o n s  and  98 102  in Precipitation  Experiments,  73 83  A n a l y s i s of C r y s t a l  Precipitation  A3  67  Lake I r o n C o n c e n t r a t i o n 17,  43  Events,  Sediment  Lake in Limnocorrals  102 154  during  Citrate Additions  157  Yellow  163  Lake Sediment C h e m i s t r y  ix List  of  Figures  1  Key  reactions in this  2  Map  of  3  Seasonal  southern  study  British  5  Columbia  changes i n phytoplankton  ( c h l o r o p h y l l a)  and  4  Black  concentrations  5  I r o n and  6  C h l o r o p h y l l a and  Lake i r o n  Chain, 7  Seasonal  iron  changes  and  9  Seasonal  11  concentrations  iron  13  and  Chain,  and  e n r i c h m e n t on  in  34  Black,  in  Black, .  39  F r i s k e n lake sediments  40  surface  Frisken lakes  growth of a l g a e  41 from 44  o f a d d i t i o n s o f Fe  or on  the  siderophore  primary  isolate  production  in  Lake  Oxygen, and and  34  35  of Chain  f r o m Anabaena c y l i n d r i c a  12  bloom  Lake  Effect  Black  33  i n F r i s k e n Lake  i n oxygen c o n c e n t r a t i o n  sediments of B l a c k ,  Black  algal  changes i n temperature of the  of  Lake  Frisken lakes  Iron geochemistry  Effect  16  in Black  i n an m  sites  Frisken lakes  8  10  1.0  study  biomass  p a r t i c u l a t e Fe  c h l o r o p h y l l a at  and  Chain,  and  46 phosphorus c o n c e n t r a t i o n s  Na-EDTA l i m n o c o r r a l s and  Temporal v a r i a t i o n  of  the  i n the  Fe-EDTA  lake  heterotrophy  i n the  48 1980  limnocorrals  50  14  Citrate  52  15  P h o s p h o r u s , o x y g e n , and  assimilation in limnocorrals  limnocorrals  in Black  temperature  Lake  in the  citrate 54  X 16  Oxygen c o n c e n t r a t i o n s i n C h a i n  Lake l i m n o c o r r a l s  17  I r o n t i t r a t i o n o f EDTA, d e s f e r a l ,  and  filtrates  56 from  Anabaena f l o s - a q u a e c u l t u r e s 18  Metal displacement f los-aquae  of  iron  from  61 the  Anabaena  siderophore  63  55 19  20  Elution  of the  F e - f i l t r a t e from  through  a G-2 5 Sephadex column  an FeBC  assay 65  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 -  two  isolates  siderophore  on p r i m a r y  effect  p r o d u c t i o n of  two  algae.. . 21  The on  22  71  effect  o f t h e Aphanizomenon  Suppression  Effect an  24  by  siderophore  siderophore  of  bacterial 75  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  isolate  o f a c e t a t e on  siderophore  76  suppression of heterotrophy  by  isolate  25  Temporal v a r i a t i o n  26  Seasonal (SRP)  isolates  73  of a c e t a t e  o f g l u c o s e on  Effect an  isolate  t h e g r o w t h o f Scenedesmus b a s i l i e n s i s  assimilation 23  siderophore  comparison  of h e t e r o t r o p h y  76 i n B l a c k Lake  of s o l u b l e r e a c t i v e  concentrations in Black, Chain,  78  phosphorus  and  Frisken  27  lakes Chain Lake s o l u b l e  28  Chain  Lake t o t a l phosphorus  81  29  Chain  Lake sediment chemistry  85  30  Roche L a k e s e d i m e n t c h e m i s t r y  87  31  Frisken  87  r e a c t i v e phosphorus  Lake sediment c h e m i s t r y  (SRP)  80 80  xi 32 33  32  P-phosphate a n a l y s i s  Lake c h e m i s t r y d u r i n g  i n B l a c k Lake Creek Aphanizomenon  88  blooms  90  32 34 35  36  P a s s i m i l a t i o n during Hypolimnetic  SRP  limnocorrals  on Aug.  Depth p r o f i l e citrate  and pH  calcite precipitation i n B l a c k Lake  24,  92  citrate  1982  of c i t r a t e  95  a s s i m i l a t i o n i n Black  Lake  limnocorrals  95  37  CaC^  induced c a l c i t e p r e c i p i t a t i o n  38  F r i s k e n Lake p r e t r e a t m e n t  39  A d e p t h d i s t r i b u t i o n o f SRP  100  SRP/TIC  104  and T I C i n F r i s k e n  Lake  104  40  F r i s k e n L a k e e p i l i m n e t i c SRP  106  41  Suppression  42  E f f e c t of c a l c i t e  of a l g a l  b i o m a s s by l i m e  a p p l i c a t i o n . . . . 108  p r e c i p i t a t i o n o f oxygen  concentration  110  43  Biotic  modification  44  Frisken  45  Simulated  Al  E f f e c t of incubation desferal  Lake TIC mixing  of lime  titration  I l l  1983  I l l  of F r i s k e n  Lake  112  t i m e on c h e l a t i o n o f i r o n by  and a f i l t r a t e  f r o m a Anabaena  flos-aquae  culture A2  Iron  binding  Anabaena A3  Iron  159 capacity  flos-aquae  binding  (FeBC) o f a f i l t r a t e  from a  culture  capacity  160  (FeBC) s t a n d a r d i z a t i o n  with  desferal A4  Degradation of the siderophore aquae by a c i d o r b a s e  161 f r o m Anabaena  flos162  xii ACKNOWLEDGEMENTS  I  t h a n k my w i f e  June f o rh e r support  I  apologize  t o my d a u g h t e r s  I  was away.  I t h a n k my f r i e n d s  Dr. study. in  Ken H a l l  provided  D r . Tom N o r t h c o t e  M e l i s s a and Rachel f o r their  provided  Engineering Drs.  Rodgers arranged Environment. electron  geochemical advice.  Mr. F r e d  from F r i s k e n Dr.  analysis. Mah s 1  Frisken  Daley  A r tTautz  direction  and K e i t h  with the guide t h e  a n d Mr. C o l i n  Gray  provided  some o f t h e s a m p l e s  t h e u s e o f equipment  of Environment allowed  a n d he p r o v i d e d  L a k e . T h e B.C. E n v i r o n m e n t ' s  his  l a b o r a t o r y i n P e n t i c t o n and a f i e l d  John C a r t w r i g h t  financial data  from  laboratory analyzed  samples from B l a c k  Don H o l m e s a l l o w e d  from t h e B r i t i s h  t h e u s e o f 1979-80  the  provided  occasional assistance.  laboratory analyzed  allowed  Mr. Ken A s h l e y  experiments.  from  Lake.  Columbia M i n i s t r y support.  Several staff  Mrs. Mudroch helped  Dr. Ralph  this  from t h e Department o f t h e  Dr. Gary Leppard provided analysis.  that  and d i r e c t i o n  Dennis Delorme,  my e d u c a t i o n a l l e a v e  microscope  throughout  of the thesis.  Jan B a r i c a , Kent Burnison,  study.  f o r t h e hours  valuable advice  and Westwater p r o v i d e d  this  help.  valuable guidance  t h e o r g a n i z a t i o n and w r i t i n g  Civil  throughout  Lake. Mr. C h r i s B u l l  allowed  many o f  the use of  camp a t C h a i n  Lake. Mr.  t h e u s e o f h i s l a b o r a t o r y i n Kamloops. Mr.  supplied boats  Several  staff  and help  f o r t h e F r i s k e n Lake  of the Ministry  occasional assistance.  of Environment  xiii Glossary Andesite,  a v o l c a n i c rock  magnesium Allelopathy,  silicates.  the suppression  another Apatite,  composed o f a n d e s i n e and a v a r i e t y o f  of t h e growth or o c c u r r e n c e  p l a n t o r m i c r o b e by e x c r e t e d  a phosphorus c o n t a i n i n g m i n e r a l  fluorapatite, The  Ca (P04) F, 5  3  p r i n c i p a l mineral  of  chemicals. group  containing  or h y d r o x y l a p a t i t e  i s a carbonate  Ca (P0 ) (OH). 5  4  3  containing v a r i e t y of  fluorapatite. Axenic, BOD,  without  bacteria.  biochemical  Calcite,  o x y g e n demand.  a mineral,  rhombohedral  calcium carbonate,  EDAX,  an i r o n  containing clay  pigment.  mineral.  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, e t h y l e n e d i a m i n e Epilimnion, FeBC,  Hexagonal-  crystals.  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 Chlorite,  CaCO^.  iron  acid,  a c h e l a t i n g agent.  the surface l a y e r of a s t r a t i f i e d binding capacity, the a b i l i t y  maintain Fibril,  tetraacetic  iron  Heterotrophy,  of organic  matter t o  in solution.  electron-opaque,  a mucilaginous  lake.  colloidal  sheath  size  particles  around c e r t a i n  the microbial u t i l i z a t i o n  that often  form  algae.  of organic  matter f o r  e n e r g y and g r o w t h . Hydroxamate, a f u n c t i o n a l group o f a p o w e r f u l siderophores,  group o f  c o n t a i n i n g two r e a c t i v e o x y g e n atoms t h a t a r e  bound t o a d j a c e n t  c a r b o n and n i t r o g e n  atoms.  xiv Hypolimnion,  t h e bottom l a y e r of a s t r a t i f i e d  Limnocorrals,  large enclosures  PCS, a x y l e n e b a s e d Picoplankton,  that  scintillation  microscopic  isolate  fluor  unicellular  lake.  a portion  p r o d u c e d by  algae  that  of a  lake.  Amersham.  are less  than  2.0 u i n s i z e . Pyrite,  a mineral  FeS^,  Sephadex, a m o d i f i e d crosslinked size  that  forms  i n anoxic  environments.  d e x t r a n . The d e x t r a n m a c r o m o l e c u l e s a r e  to give  a three-dimensional  network; t h e v a r i o u s  p o r e s r e t a r d t h e e l u t i o n o f m o l e c u l e s as a f u n c t i o n o f  s i ze. SRP, s o l u b l e  reactive  Siderophore,  a low m o l e c u l a r w e i g h t o r g a n i c  daltons) ferric  that  phosphorus.  has a h i g h  i r o n and t h a t  of i r o n .  f o r strongly  chelating  i s p r o d u c e d by m i c r o b e s s u b j e c t e d  iron deficiency to assist assimilation  specificity  compound ( 5 0 0 -  t h e s o l u b i l i z a t i o n and  to  1 1  INTRODUCTION The  changes  primary goal in iron  green a l g a l over other  of t h i s  availability  r e s e a r c h was t o d e t e r m i n e i f influence the p e r i o d i c i t y  g r o w t h and r e g u l a t e t h e b l u e - g r e e n algae.  The  is  and sediment n u t r i e n t r e l e a s e ,  availability.  periodicity  follows very species  dominance  A s e c o n d a r y o b j e c t i v e was t o r e s o l v e i f e v e n t s  s u c h as c a l c i t e p r e c i p i t a t i o n , change i r o n  algal  of b l u e -  of a l g a l  similar  growth i n temperate l a k e s  annual c y c l e s  progression,  (Lund e t a l . 1 9 6 3 ) . A  i n temperate lakes  that  f r o m s p r i n g d i a t o m s t o summer b l u e - g r e e n  periodicity  i n t h e growth o f phytoplankton  mixture of b i o l o g i c a l  processes  often  stratify algae.  species  and p h y s i c a l  typical  i n summer,  The  seasonal  i s d r i v e n by a  perturbations  ( T r i m b e e a n d H a r r i s 1984) t h a t c a n l e a d t o p e r i o d i c c h a n g e s i n nutrient that the  availability  regulate algal timing  succession  of the p e r i o d i c i t y  (Hutchinson  1.1  (Lund e t a l . 1 9 7 5 ) . However, t h e f a c t o r s  1967, R e y n o l d s  Factors  Regulating  (19 82) water  are incompletely  plays  of phytoplankton  regulate their  periodicity.  Growth  in the  Reynolds e t a l .  of diatoms  in stratified  o f diatom growth i n  p o s s e s s gas v a c u o l e s  vertical distribution  may have a c o m p e t i t i v e  of Algal  i n the suppression  summer. B l u e - g r e e n a l g a e  understood  a major r o l e  have shown t h a t s e d i m e n t a t i o n i s a major f a c t o r  determine  1982).  the Periodicity  Water column s t a b i l i t y determination  and t h e r e a c t i o n s t h a t  t h a t e n a b l e them t o  (Walsby 1 9 7 7 ) ; t h u s ,  advantage i n a s t r a t i f i e d  lake.  they  2 Buoyancy c o n t r o l p r o v i d e s b l u e - g r e e n of  exposure  to light,  1982). Although  temperature,  t h e response  a l g a e some  and n u t r i e n t s  (Ganf  of species i s v a r i a b l e ,  a l g a e a r e g e n e r a l l y e n h a n c e d by h i g h i l l u m i n a t i o n whereas, diatoms water  a r e more t o l e r a n t  (Hutchinson  (Ganf The  and O l i v e r  availability.  nitrogen  availability  o f n i t r a t e by d i a t o m s ,  (Wetzel  nutrients  such  blue-green  often limits  usually  dominance  nutrients  i s minimal  Blue-green  zooplankton factors  size  (Hutchinson  likely  d i o x i d e can a l s o  enhance  algae  t o be e a t e n t h a n  of blue-green a l g a l  trichomes  are other  limits  e t a l . 1 9 8 2 ) ; however, more complex structure  can p r o f o u n d l y a l t e r  ( S h a p i r o 1980). Lynch  the r e l a t i o n s h i p  atmospheric  1967).  are involved. Trophic level  grazing  utilize  blue-green  when t h e c o n c e n t r a t i o n o f i n o r g a n i c  g r a z i n g (Ferguson  of p r e d a t o r y f i s h  growth  the vernal  ( S h a p i r o 1973). B l u e - g r e e n  algae are less  a l g a e . The l a r g e  than  concentrations of other  as p h o s p h o r u s o r c a r b o n  s t a r t growing  t o changing  diatom  nitrogen fixing  which cannot  1975). D e c l i n i n g  algal  algae to  D i a t o m s r e q u i r e much more s i l i c o n  a l g a e o f t e n r e p l a c e diatoms  that  growth i s a l s o r e l a t e d  e t a l . 1975, H u r l e y e t a l . 1 9 8 5 ) . A f t e r  utilization  algal  i n some l a k e s , t h e v e r t i c a l  1982).  o t h e r a l g a e , and s i l i c o n (Lund  and c o o l  r i c h d e e p w a t e r and r e t u r n t o t h e e u p h o t i c  suppression of diatom  nutrient  and warm w a t e r ;  by g a s v a c u o l e s c a n a l l o w b l u e - g r e e n  move i n t o n u t r i e n t zone  and O l i v e r  blue-green  of high t u r b i d i t y  1967). A p p a r e n t l y ,  movement m e d i a t e d  regulation  such  as t h e d e n s i t y  t h e amount o f  and S h a p i r o  zooplankton  (1981) have shown  between Aphanizomenon a n d D a p h n i a i s a  complex s y m b i o t i c r e l a t i o n s h i p . F u r t h e r m o r e ,  blue-green  a l g a e can  3 excrete  organic  (Gentile Porter  and  and  The the  Maloney  Orcutt  a b i l i t y  excretion  competition Keating 1979, when  compounds  of  Chan  a l .  collected  a l l e l o p a t h i c contribute  from  heterotrophy  which  would in  the  that  oxygen which In  does  is  in  algae  of  Elbrachter  1979,  Wolfe  of  the  1976,  and  s t r i k i n g l y  blue-green  Rice inhibitory  1967).  a l g a l algal  by  reducing  1974,  (Hutchinson  diversity  s e t t l e  In  forms 1977).  stable  mineral  reaction  from  as  the  The  blooms blooms  An  in  and  active  increased  the  anoxic  displaces  as  He bacteria  depletion the  oxygen. the  hypolimnion  becomes  precipitates  more  anoxic,  ferrous  hypolimnetic  consumption  formation  p y r i t e  (FeS2)  (Berner  1971).  of  environments  available  zone,  oxygen  of  agent  light.  epilimnion,  supply  which  in  euphotic  the  1977,  inhibitory  enhance  to  enhanced  anoxia,  that  and  b a c t e r i a l  McCracken  the  hypolimnion  sulphide  l i t t l e  the  renewable is  and  that more  contrast a  suppress  Delucca  algae  s o l u b i l i t y  in  a  1975;  to  was  have  with  able  excreted  result  environments  formation,  1977;  other  method  are  algae  found  However,  (Banoub  a  a l .  waters  occur  decompose  not  reduction  can  1973,  suppress  another  (1975)  algae  iron  anoxic.  sulphides  to  et  in  also  1973,  algae  once  f i r s t ,  sulphate  are  hypolimnia.  hypolimnion  becomes  Porter  Hellebust  rich  that  Chrost  rapidly  dark  At  is  Natural  low-species  (Chrost  1981).  then  algae  1973,  areas  1982;  grazing  1980).  1979, Wilson  algae  blue-green  proposed  a l .  zooplankton  1967).  Blue-green  Reichardt  Snell  1980).  the  Lampert  compounds  reactions  to  (Hutchinson  et  Kayser  et  suppress  blue-green  toxic  {Fogg  1978,  1969;  1980; of  that  iron  and  rapid  heterotrophy  p y r i t e  from  the  of  4 epilimnion  to the hypolimnion  and  enhances t h e  development of a n o x i a would reduce the  seasonal  the a v a i l a b i l i t y  of  iron  in  hypolimnion. The  t i m i n g of anoxia development w i l l d i r e c t l y  blue-green  algal  s u c c e s s i o n . The  growth i s o f t e n l i n k e d l a k e sediments  periodicity  of blue-green  t o the r e c r u i t m e n t of a l g a l  (Trimbee  and  Harris  influence  cells  algal  from  1 9 8 4 ) . In g e n e r a l ,  A p h a n i z o m e n o n r e c r u i t m e n t i s f a v o u r e d when t h e b o t t o m w a t e r i s oxygenated Harris  (Lynch  19 80,  1984). Other  Lynch  blue-green  the bottom water approaches and  Harris  and  19 8 4 ) . L u n d and  S h a p i r o 19 81, T r i m b e e  algae are u s u a l l y  anoxia Reynolds  (Reynolds (19 82)  s t i m u l a t e d when  e t a l . 19 81,  suggest  are not  nutrients,  t o changes i n l a k e dynamics which  respond  t h e f o r m a t i o n o f an The  acceleration  stratified iron.  why  c o u l d be  At f i r s t ,  anoxic  anoxic  (Stumm and  blue-green  of a l g a l  increase in permit  s u c c e s s i o n when a l a k e becomes  t o changes i n t h e a v a i l a b i l i t y  of  becomes more s o l u b l e when h y p o l i m n i a become  Morgan  algal  an  hypolimnion.  related  iron  s t i m u l a t e d by  Trimbee  that  M i c r o c y s t i s propagules but  and  1 9 8 1 ) . The  response  following  t o a changing  section discusses  supply of  iron  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 The  of 1).  ecology of blue-green  the complexity As  early  as  the c o n t r o l l i n g More r e c e n t l y ,  is related  1937,  Guseva  algal  Algae  blooms i s complex and  to reactions involving (1937, 1939)  stated  C l a s e n and  Berhardt  iron (Fig.  that  b i o g e n i c e l e m e n t f o r Anabaena and (1974) o b s e r v e d  much  iron  was  A p h a n i zomenon. that  blue-  5  KEY REACTIONS IN THIS STUDY  Figure  1  Key r e a c t i o n s influencing  in this  study.  another process.  Lines denote a r e a c t i o n denotes a key r e a c t i o n .  6 green  algae  green  algae.  blue-green a l . by  responded At  least  algae  1971, Morton the  following  reactions The (White  are  nitrogen  two  and  1974).  Lee  require  i r o n - d e f i c i e n t  The  Lake  l i m i t a t i o n  (Murphy  and  Hendzel  (1976) not  i n a b i l i t y  of  collapse.  Lean  have  the  been  Anabaena  in  19 8 1 ,  Brownlee  was  Aphanizomenon et  a l .  factor  a  and  deficiency  reaction  in  and  that  a  an  may  blue-green  as  500%.  periods  of  fixation Healey  and  periodic  the  bloom of  iron  fixation  only  fix  unknown  lack  nitrogen  iron  much  from  the  to  addition  19 8 3 ) .  of  for  nitrogen,  suggested  as  1958,  found  had  nitrogen  Murphy  for  algal  algae make a l g a l  may  of  which  can  iron blooms  than  blooms.  can that  and  upon  requirement  of  proposed  recover  the  types  hypothesis  iron  to  fix  (1976)  that by  iron  Castle  found  are  key  and  it  algae  a  et  supported  algae  when  responsible fix  are  contain  Neilands  fixation  restricted  Blue-green  Thus,  fixation  to  (19 78) that  that  that  (Soeder  poorly  dependent  reported  of  has  nitrogen  often  more  This (1969)  is  fixation  and  algae  than  iron-requiring  (Carnaham  (19 83)  blue-green  shown  algae  blue-green  grew  nitrogen  green  growth  algae.  iron  Simpson  Home  phosphorus,  nitrogen.  other  have  blooms.  chelation  and  Brownlee  nutrient,  fix  of  when  have  that  nitrogen  Anabaena  stimulated  a b i l i t y  nitrogen  for  more  observations  blue-green  1976).  Wurtsbaugh  Clear  in  additional  that  into  Murphy  than  These  therefore,  1969,  nitrogen.  iron  with  studies  demonstrating  required  Stewart an  laboratory more  1968);  they  enrichment  require  important  a l .  iron  studies  enzymes  et  to  the  be the  supported  by  requirement  general  lack  of  marine for  studies.  additional  iron  in  the  Stewart iron  oceans  for  7 (Ryther and  and  Yeats  Kramer  19 8 4 )  planktonic  1962,  are  Smayda  1969,  responsible  heterocyst  for  containing  Matsunaga  the  et  relative  a l .  1984,  paucity  blue-green  algae  the  of  in  Rao  of  marine  environments. The damage  requirement  also  (catalase, to  Catalases  environment  (Price  blue-green  algae  hours,  that  but  collapse  If than  impaired  buoyancy  with  vacuoles.  would  lead  to  the  sunlight.  A  requiring  iron  required requiring  to  lack  direct often and  Shilo  algae  are  the  a l .  formation of  iron  gas  that  healthy  to  three after  death  et  a l .  1976,  90 of  mechanism of  slow  to  could  be  algae  are  to  surface  vacuoles  superoxide  within  s t i l l  a  l i g h t  vulnerable  sunlight  susceptible  a  bloom  Coulombe  regulate scums  1973)  (Walsby  and  dismutase)  may  1977).  be  result  of  only  gas  an  algae  vacuoles  exposure  to  f u l l  reactions  and  photooxidation  photooxidation  the  respiratory  Lankford  prevent  i r o n - d e f i c i e n t  Eloff  a b i l i t y  would  low  that  as  in  1976).  more  of  a  be  proposed  Blue-green  reduced  in  Photooxidative  s e n s i t i v i t y  control.  Neilands reduced  affected  were  1972,  required  would  direct  algae  been  et  regulate  and  c e l l s  sunlight.  (cytochromes,  enzymes  peroxidases,  is  these  are  1968,  growth  Eloff  A  a l .  before  iron-enriched  algae,  et  dramatically  by  has  dismutase)  become  k i l l e d  to  enzymes  to  were  blue-green  gas  (White  observed  1981,  other  known  photooxidative  iron-containing  superoxidase  damage  1968);  (Abeliovich  are  The  repair  (1974)  algae  Robinson  in  Lange  exposure  blue-green  and  are  bacteria  photooxidation.  of  and  photooxidative  i r o n - d e f i c i e n t  hours  iron  important.  peroxidase,  repair  1981b).  seems  for  respiration Thus,  is  i r o n -  (catalases, more  important  in  8  blue-green  algal  blooms t h a n  Bacteriophages a l g a l blooms  in other  Robinson  influence v i r a l  a t t a c k s , they  p e r i o d s of  limitation.  1981b). These p r o t e i n s a r e produced iron  limitation  siderophores  Several  and of  Taub 1980,  requirement. supply  Lankford  1976,  by  other  Siderophores  of  iron  19 73;  either  Morel  algal  1980)  algae  releasing  the  Only identify fortunate compound had  algae  and  divisions  limitation  is  produce  Baalen  1979,  the r a r e  (Trick  1976, Bailey  production  e t a l . 1983a,  have a l a r g e  iron  is limited  (Neilands  1966,  and  are  low  1972,  1973;  molecular  strongly chelate  chelated  on m i c r o b i a l c e l l s  1967,  when  weight  iron;  iron with protein  or they  enter  the c e l l  before  iron.  a few algal  s t u d i e s have u s e d siderophores.  rigorous chemical  Simpson and  Neilands  i n t h a t t h e Anabaena s p e c i e s t h e y (Schizokinen)  isolated  of  a r e e x c r e t e d by many p r o k a r y o t e s  r e a d i l y exchange the  sites  concentrations  iron  Van  Emery 19 8 2 ) . S i d e r o p h o r e s  receptors  of  the  (Neilands  Murphy e t a l .  A r m s t r o n g and  o r g a n i c compounds t h a t s e l e c t i v e l y they  in higher  Murphy 1976,  1983b) i n d i c a t e t h a t b l u e - g r e e n  the  to enter the c e l l  that blue-green  M c K n i g h t and  siderophores  factors  in lakes.  (Smayda 1974, Neilands  other  more o f t e n d u r i n g  t o enhance the a s s i m i l a t i o n  observations  siderophores  blue-green  enter b a c t e r i a v i a  ( N e i l a n d s 1982). T h i s a s p e c t  apparently unresolved  Simpson and  occur  Bacteriophages use  in dying  1981). A l t h o u g h  should  same p r o t e i n s t h a t s i d e r o p h o r e s  during  blooms.  have a l s o been o b s e r v e d  (Coulombe and  iron  algal  that e a r l i e r  from b a c t e r i a . T r i c k  (1976) were  s t u d i e d produced  studies in their  and  analysis to  coworkers  a  laboratory  (1983b)  found  9  hydroxamate type Other  siderophores  s t u d i e s have r e l i e d  or chemical  study  purify,  and  a c h e l a t o r such  as  citrate  Anabaena c y l i n d r i c a molecular  that  combination  determine the  flos-aquae  no  analysis indicated  by  Scenedesmus b a s i l i e n s i s peptides;  a molecular  a brick  c h e l a t i o n such  c o u l d . Chemical  these  had  flos-aquae 900  daltons  c h e l a t o r f r o m Anabaena  r e d complex when s a t u r a t e d w i t h  iron.  c h e l a t o r s f r o m Anabaena f l o s - a q u a e , Anabaena c y l i n d r i c a  Scenedesmus b a s i l i e n s i s The  study  (Trick  from t h e hydroxamate p r o d u c t i o n  e t a l . 1 9 8 3 a ) . Murphy  concentration about  of e x c r e t e d  ten days,  constant.  excreted  i n a p u l s e and  green  directly water  selective  algae to  Trick  et a l . observed stayed  (Stumm and  was  in Trick's that  the  t e n d a y s and  t h a t hydroxamate  in solution  f o r o n l y a few  advantage t h a t blue-green ( S h a p i r o 1984)  may  be  assimilation.  Iron  Morgan  t h u s , more complex  19 8 1 ) ;  is less  algae  and  thesis.  h y d r o x a m a t e i n c r e a s e d q u i c k l y up  i n high-pH water iron  (1976) o b s e r v e d  s l o w l y i n c r e a s e d f o r another  remained  The  were f u r t h e r s t u d i e d i n t h i s  h y d r o x a m a t e p r o d u c t i o n by Anabaena f l o s - a q u a e  radically different  but  and  compounds  weight of about  a h y d r o x a m a t e g r o u p . The  formed  approximate  30 0 d a l t o n s . Anabaena  a chelator with  able  a Sephadex c o l u m n ,  were c y c l i c  weights around  contained  iron  55Fe was  through  the c h e l a t o r s excreted  produced  latter  w e i g h t o f c h e l a t o r s . Compounds w i t h  g l y c i n e c o u l d not t r a n s m i t  that  the  assays,  siderophores.  s i d e r o p h o r e s . Sephadex c h r o m a t o g r a p h y w i t h  molecular  The  (1976) u s e d  algae.  radiochemical  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  to detect, p a r t i a l l y  as  from marine  upon b i o a s s a y s ,  I n a l a b o r a t o r y , Murphy to  in isolates  have  to then was days.  over  related  s o l u b l e i n high-pH iron-  10 a s s i m i l a t i o n mechanisms may be r e q u i r e d f o r growth i n high pH water. 1.2.1  Iron L i m i t a t i o n Iron l i m i t a t i o n o f t e n has been observed  c a l c i u m carbonate p r e c i p i t a t i o n 1971,  i n l a k e s without  (Goldman 1966, Bernhardt  et a l .  Sakamoto 1971, A l l e n 1972, C l a s e n and Bernhardt 1974,  Gerhold 1975, Lund et a l . 1975, Thurlow et a l . 1975, Happey-Wood and Pentecost 1981, L i n and Schelske 19 81, P a e r l 19 82a). Complexation  of i r o n with humic matter  r e a c t i o n mediating  i s the b e s t documented  i r o n l i m i t a t i o n i n softwater  Hecky 1980). Although  (Jackson and  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  i r o n v a r i e s g r e a t l y among l a k e s , i n many l a k e s , most of t h e humic i r o n i s u n r e a c t i v e (Shapiro 1969). Recent s t u d i e s have shown t h a t i r o n i s r a p i d l y coated by humic a c i d s ( P i c a r d and F e l b e c k 1976, T i p p i n g 19 81, B a c c i n i e t a l . 19 82); presumably i n some l a k e s , the c o a t i n g s of humic a c i d s 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 i r o n i s d i f f i c u l t  t o measure.  Although a chemical assay, t h e f e r r i g r a m , 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 i r o n i n some l a k e s (Shapiro 1969), i t i s u n l i k e l y t h a t any chemical f r a c t i o n a t i o n can r e s o l v e the c o m p l e x i t i e s of a l l humic-iron chemistry. For example, Powell et  a l . (1980) and Akers  (1983) d i s c o v e r e d a v a r i e t y of  s i d e r o p h o r e s i n s o i l e x t r a c t s . P r e v i o u s l y , 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 a c i d s . The  r e a c t i o n s of s i d e r o p h o r e s can be.complex. The  a v a i l a b i l i t y of s i d e r o p h o r e i r o n can be r e s t r i c t e d t o o n l y some s p e c i e s (O'Brien and Gibson  1970). Some s i d e r o p h o r e s have  11 a n t i b i o t i c p r o p e r t i e s ; they mimic i r o n - s e q u e s t e r i n g  siderophores  (Neilands 1981a). The i r o n b i o c h e m i s t r y of l a k e s should be as complex as s o i l  iron  biochemistry.  Hardwater l a k e s t h a t p r e c i p i t a t e c a l c i u m carbonate o f t e n been c i t e d as being i r o n l i m i t e d Schelske e t a l . 1963; 1974;  Home  Murphy e t a l . 1976; E l d e r 1977;  Elder  Wurtsbaugh and Home 1983). The r e l a t i o n s h i p  between c a l c i u m carbonate expected  (Schelske 1961, 1962;  Wetzel 1965, 1966, 1968; Lange 1971;  Murphy and Lean 1975;  and Home 1977;  have  due t o chemical  Calcium carbonate  p r e c i p i t a t i o n and i r o n l i m i t a t i o n i s and b i o c h e m i c a l  reasons.  p r e c i p i t a t i o n can i n t e r f e r e with  iron  uptake. Iron i s l e s s s o l u b l e a t t h e high pH found b e f o r e and d u r i n g c a l c i u m carbonate  precipitation  (Stumm and Morgan 1981).  A l s o , t h e c o p r e c i p i t a t i o n of d i s s o l v e d o r g a n i c matter with c a l c i u m carbonate euphotic  w i l l reduce t h e s o l u b i l i t y o f i r o n i n t h e  zone (Wetzel  1975).  S z a n i s z l o e t a l . (1981) found carbonate  t h a t t h e a d d i t i o n of c a l c i u m  t o c u l t u r e medium enhanced the p r o d u c t i o n o f  s i d e r o p h o r e s , but t h a t c a l c i u m c o n c e n t r a t i o n alone had no e f f e c t upon s i d e r o p h o r e p r o d u c t i o n . Other s t u d i e s have i n d i c a t e d t h a t some siderophores calcium  have an a p p r e c i a b l e a f f i n i t y f o r c h e l a t i o n of  (Hider e t a l . 1982). Perhaps c a l c i u m or c a l c i u m  can suppress  some l i m n e t i c i r o n - s i d e r o p h o r e r e a c t i o n s . Calcium i s  o f t e n concentrated  on the s u r f a c e of a q u a t i c b a c t e r i a by  e x c r e t i o n from m i c r o b i a l c e l l s blue-green  carbonate  ( M o r i t a 1980). The s u r f a c e of  algae i s o f t e n composed o f microenvironments c r e a t e d  by a c o v e r i n g of f i b r i l s  (Leppard  e t a l . 1977; Leppard 1984a,  1984b). Thus, t h e c o n c e n t r a t i o n o f c a l c i u m a t t h e s i t e s of i r o n  12 uptake could  be much h i g h e r t h a n b u l k w a t e r c h e m i s t r y  s u g g e s t . A s u p p r e s s i o n by c a l c i u m o f i r o n much s t r o n g e r  i n hardwater  l a k e s than  Calcium carbonate p r e c i p i t a t i o n biogeochemistry v i a a l g a l productivity the  would  result  lake  should  in precipitation  carbonate p r e c i p i t a t i o n  iron  availability  influence  several  to iron  can p r e c i p i t a t e  algae  two  p r o c e s s e s can s u p p r e s s t h e c o n c e n t r a t i o n lakes  i n summer  the effects  calcareous  (Stauffer  may be t h e m a j o r  o f a change i n i r o n  (1985) has a l s o areas  results  ( B o s t r u m 1984, R y d i n g  1 9 8 5 ) . As w e l l  vary  proposed  that  among l a k e s and t h a t  timing  o f an a l g a l  The  of  reaction  a lack  iron  that  as t h i s  reactions  spatial variation in availability,  availability.  availability  iron  in iron,  by i r o n  and o f i r o n  in iron  of mobile  to control  are rich  the degree of i r o n  iron  These  of algae i n  are controlled  o f phosphorus s o l u b i l i t y  (19 81)  19 80)  availability.  in too l i t t l e  t h e r e may b e a t e m p o r a l v a r i a t i o n Morgan  1). Calcium  (Rossknecht  limnetic  proposed that  geochemical phosphorus r e a c t i o n s  control  (Fig.  processes  1985). The i n d u c t i o n  phosphorus c h e m i s t r y . In other areas t h a t  the  and c o u l d  (Murphy e t a l . 1983) f r o m t h e e p i l i m n i o n .  carbonate p r e c i p i t a t i o n  in  i n an  limnetic  chemistry  phosphorus  Stauffer  to iron  Any enhancement o f a l g a l  and  modifies  linked  o f c a l c i u m c a r b o n a t e . S m a l l changes i n  a r e not d i r e c t l y r e l a t e d  hardwater  lakes.  enhance a l g a l p r o d u c t i v i t y  carbonate e q u i l i b r i a could that  i s closely  w o u l d be  i n c r e a s e t h e pH and c a r b o n a t e c o n c e n t r a t i o n o f  e p i l i m n i o n . Thus, i n c r e a s e d  iron-limited  assimilation  i n softwater  productivity.  would  Stumm and  limitation  should  should  influence  the  bloom, n o t t h e biomass.  temporal v a r i a t i o n  in iron  l i m i t a t i o n was d e m o n s t r a t e d  13 in  a s t u d y by  limitation  Murphy  t h a t was  p r e s e n c e of  (19 7 6 ) .  In  the  detected  by  rapid  siderophores,  the  iron  limitation  was  followed  by  that occurred  S e a s o n a l changes i n i r o n  usually  are  sediments  However, i r o n  and  known a b o u t t h e  of reduced a l g a l  g e o c h e m i s t r y and  and  e t a l . 1971, often  the  1941,  iron  1942;  the  solubility  the  p r e c i p i t a t i o n of ferric  solution  (Banoub  Ryding enhance t h e preventing  o f p h o s p h o r u s has ferric  has  solubility  high  lake Much more i s lake  1977). In  b e e n shown t o be  studies sediments  some  lakes,  c o n t r o l l e d by  ( B i r c h 1976). In  organic  o f p h o s p h o r u s by  extreme  the  be  an  chelators  complexing  iron  e x c e l l e n t s i g n a l of  availability  r e s o l v e the  iron  f e r r i c phosphate. Thus,  i s known a b o u t t h e  s e d i m e n t s and  from seasonal lake with  Little  To  from  i s depleted.  Lijklema  proposed t h a t  t h e p r e c i p i t a t i o n of  micronutrient.  Shukla et a l . 1971).  i s p r e c i p i t a t e d l e a v i n g phosphorus  g e o c h e m i s t r y o f p h o s p h o r u s may  from lake  in  in  1977).  (19 85)  availability.  is stable  i n synchrony from the  phosphate  of  are  r e l e a s e ; however, i n s e v e r a l  Banoub 1977;  sulphide  periodicity  processes  Iron  bloom  biomass.  r e l e a s e of phosphorus from  p h o s p h o r u s were r e l e a s e d  (Mortimer  the  released  oxygen c o n c e n t r a t i o n  seasonal  the  a blue-green a l g a l  b o t h w e l l known, b u t  (Williams  sediments than about  anoxia,  during  phosphorus are  s e d i m e n t s when t h e  iron  i r o n u p t a k e and  l i n k e d t o phosphorus a v a i l a b i l i t y .  oxidized  iron  of Quinte,  l a s t e d f o r l e s s t h a n a week. However,  a f o u r week p e r i o d  blue-green algae  Bay  impact  of  coupling of  iron  sediment  changes i n phosphorus a v a i l a b i l i t y phosphorus c o n c e n t r a t i o n s  of  iron  as  a  iron  and  may thus  the iron release  release  would r e q u i r e  to saturate  biological  a  14 requirements. The  goals  of t h i s  changes i n b l u e - g r e e n iron  availability  dominance over regulation  study algal  were t o d e t e r m i n e i f t h e s e a s o n a l g r o w t h were r e l a t e d  and t o r e s o l v e i f t h e b l u e - g r e e n  of i r o n  availability.  To a c h i e v e  the  and p h y t o p l a n k t o n of e n c l o s u r e  interpretation  incubations, larger  production  precipitation  and  s u r f a c e a r e a may  a l s o allowed  enclosures  precipitation responses,  stimulate  o f t h e water p r e v e n t s  can s t i l l  have e n h a n c e d  whole-lake manipulations of t h e long-term  responses  s t u d i e s where s p e c i f i c  gas  i n pH.  succession. mineral long-term  were a l s o c o n d u c t e d . to iron  an e v a l u a t i o n o f c a l c i u m c a r b o n a t e  investigated  changes  observation of a l g a l  s t u d i e s were i n t e g r a t e d w i t h d e t a i l e d chemical  mineral  on t h e w a l l s . T h u s , t o c o n f i r m t h e  interpretation required  artifacts.  a r e prone t o s e v e r a l  exchange which i n t u r n can l e a d t o u n n a t u r a l  Large  heterotrophy  a n d g r o w t h o f s p e c i e s t h a t a r e n o t common i n t h e  open w a t e r . M o r e o v e r , t h e i s o l a t i o n  Limnocorrals  bottle  enclosures  t o avoid containment  incubations i n small enclosures The h i g h  high  to assist  observations. Short-term  assays.  artifacts.  l a k e s t h a t had  e x p e r i m e n t s were c o n d u c t e d  f o r primary  Long-term  objective, field  changes i n water  i n c u b a t i o n s were u s e d  ( l i m n o c o r r a l s ) were u s e d  siderophore  p e r i o d i c i t y were s t u d i e d .  of l i m n e t i c  For long-term  this  in three hypertrophic  p h o s p h o r u s c o n c e n t r a t i o n s . The s e a s o n a l  A series  algal  o t h e r m i c r o b e s c o u l d be m e d i a t e d by a  s t u d i e s were c o n d u c t e d  chemistry  t o changes i n  The  enrichment  precipitation.  My  l a b o r a t o r y b i o a s s a y and  interactions  c o u l d be  u n d e r 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 The  Columbia The  Area  Thompson P l a t e a u  ( F i g . 2 ) . T h i s a r e a has a t e m p e r a t e c o n t i n e n t a l c l i m a t e .  plateau  border  i s located i n south-central British  i s i n t h e rainshadow of t h e Cascade Mountains  t h e p l a t e a u on t h e w e s t . R a i n f a l l v a r i e s  highlands t o less Bloedel  than  35 cm i n t h e s e m i a r i d v a l l e y s  1 9 7 2 ) . Two s t a t i o n s  a t Hedley,  m and 1,738 m, r e s p e c t i v e l y  less  (Fig.  2, T a b l e  at a l l sites;  chemistry during  summer were l a r g e l y  2.1.1  t o B l a c k L a k e ) and f r o m  Black S.W. The  of the v a l l e y s .  thus,  kills.  of Penticton  controlled  Roche L a k e  lakes are situated  ( N o r t h c o t e and H a l s e y  n e i g h b o r i n g mountains r i s e  (Mathews 1944, L i t t l e  andesite  blooms,  The water f l o w i n  by p r o c e s s e s i n  from Y e l l o w  Lake  (next t o F r i s k e n L a k e ) .  Lake  and Y e l l o w  around B l a c k  algal  sites  t h e changes i n water  l a k e s . Some s a m p l e s were a l s o c o l l e c t e d  Black  i n runoff that  1 ) . T h e s e l a k e s a l l had b l u e - g r e e n  midsummer c e a s e d  Lake,  More r a i n and  and F r i s k e n l a k e s were t h e main s t u d y  h i g h p h o s p h o r u s c o n c e n t r a t i o n s , and f i s h  (next  (MacMillan-  a t e l e v a t i o n s of  (Environ. Can.).  s u p p l i e s much o f t h e w a t e r r e q u i r e m e n t s  the  a year  evaporation at higher elevations r e s u l t s  Black, Chain,  75 cm on t h e  48 km w e s t o f B l a c k  r e c e i v e a mean o f 29 cm o r 54 cm o f r a i n 425  from  which  1961, N a s m i t h  1966, C h u r c h  1969, N o r t h c o t e  1980).  750 m above a s t e e p g l a c i a l  Lake i s p r e d o m i n a t e l y  (Bostock  upon a h i g h l a n d 16 km  1961). The s u r f a c e an a p a t i t e r i c h  1973, P a r s o n s  1974).  rock  Eocene  valley  (Ti  contour  intervals  in  meters  17  F i g u r e 2 continued  18 Erosion  of a surface  layer  of a porous v o l c a n i c  rock i s very  p r o n o u n c e d . B l a c k L a k e had s e v e r a l a s p e c t s t h a t made i t appropriate  f o r study: small  s u p p l y , and i n t e n s e  Table 1  s i z e , good r o a d a c c e s s ,  e u t r o p h i c a t i o n without  power  a n t h r o p o g e n i c wastes  Some P h y s i c a l and C h e m i c a l F e a t u r e s o f  t h e Study  Lakes  Lake Characteristic  Black  Chain  Frisken  Latitude  490°20'  Longitude  1190°45'  1200 16"  1200°  750  1006  1138  Elevation  (m)  Drainage A r e a (ha)  1532  490°42' o  4060  Lake A r e a (ha)  4  43.7  Max. D e p t h  (m)  9  7.1  Mean D e p t h  (m)  4.5  6.1  *  1-5  Retention (yr) Conductivity Spring Fe  (ug/L)  ** S p r i n g TP  (ug/L)  **  Summer/Spring TP IAP/Ksp  CaC0  11 5.5 20 374  20  370  20  300  20  300  7-23  &  33.8  220  10 2-10  8'  267  506  0.2-0.5  3  0.3  500°27'  0.5 5-20  * A l l values are epilimnetic. R e t e n t i o n time i n B l a c k and F r i s k e n l a k e s i s e s t i m a t e d f r o m s e v e r a l measurements o f s t r e a m f l o w . The h y d r o l o g y o f C h a i n Lake i s c o n t i n u o u s l y m o n i t o r e d by the B r i t i s h Columbia M i n i s t r y o f Environment. uS/cm a t 25°C.  **  &  TP = t o t a l p h o s p h o r u s . Range o f c a l c i u m c a r b o n a t e s u p e r s a t u r a t i o n i n midsummer.  19 2.1.2  Frisken  Lake  F r i s k e n Lake i s a h y p e r t r o p h i c Kamloops. T h i s intrusion during 1947,  the  lake  called  J u r a s s i c or p o s t - l o w e r rocks  residence  2.1.3  of t h i s  time than Black  to the  plateau  sites;  b a s i n . The  thus,  the  only  more r a i n f a l l  of  52.7  is half,  a tenth  cm  a year  and  N.E.  a  monitored.  o f P r i n c e t o n . The  phosphorus l o a d i n g  a much h i g h e r 1 ) . The  i s 300  should  is similar  m higher  fall  ( E n v i r . Can.  s p i t e of  the  than Black  the  t h a t of the  (Murphy 1 9 8 5 ) . I n  (Table  in  lake in  age  (Rice than  the  i n the Chain  Lake  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  Chain Lake i s s i m i l a r a high  rich  long-term  i n t r u s i v e s around F r i s k e n Lake  L a k e r e c e i v e s more r a i n f a l l  is  km  p r o p e r t i e s of Chain Lake support  conductivity  formed  b e c a u s e i t has  Lake; thus,  around Chain Lake  only data  mean r a i n f a l l Two  chosen f o r study  i s s i t u a t e d 45  composition  other  volcanic  v o l c a n i c i n t r u s i o n are  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  1 9 4 6 ) . The  from  Lake  Chain Lake  and  S.S.E.  Cretaceous period ( C o c k f i e l d  o f a w h o l e l a k e e x p e r i m e n t c o u l d be  Chain  lies  km  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  a p a t i t e . F r i s k e n L a k e was  effects  30  the W i l d h o r s e Mountain B a t h o l i t h t h a t  1 9 4 8 ) . The  longer  lake  residence  26  hypothesis  and  (Table  to the  other  sites  iron concentration  that  data). Chain the  time of C h a i n Lake  lakes  having  of  Frisken lakes;  other  from the  years  than the  water  1). in that  i t receives  n a t u r a l weathering of similar  a  apatite  geology, Chain Lake other  water e n t e r i n g Chain Lake i s r i c h  two  has  lakes  in iron  (Water  Investigations  Branch of B r i t i s h  was  chosen f o r study  low  iron concentrations  compared  2.2  t o an  Field  so  iron-rich  that  the  (Black  1977). Chain  biogeochemistry of  and  Frisken  lakes)  lakes  could  Lake  with  be  lake with a s i m i l a r habitat.  Experiments  Whole-lake p e r t u r b a t i o n s , incubation  C o l u m b i a , WIB,  large enclosure,  e x p e r i m e n t s were c o n d u c t e d  lakes  (Table  Table  2  and  in Black,  bottle  Frisken  and  Chain  2).  T i m i n g and  S i t e s of  Experiments  Year  Site B l a c k L. monitoring aeration monitoring aeration Fe-EDTA limnocorrals  1979 1980  F r i s k e n L.  C h a i n L.  Laboratory chelator isolation  1981  initial FeBC assay  1982  Fe-citrate limnocorrals Ca(OH) addition Ca(OH) addition  1983 1984  FeBC I r o n - B i n d i n g Capacity, maintain i r o n i n s o l u t i o n  2.2.1  lake monitoring lake monitoring the  ability  of  improved FeBC a s s a y  organic  matter  Sample C o l l e c t i o n Water s a m p l e s were c o l l e c t e d w i t h a t h r e e - l i t e r Van  water sampler lakes.  at  stations located  Samples were f i l t e r e d  i n the  within  deepest part  s i x hours. In  1983,  of  Dorn the samples  to  21 were c o l l e c t e d  f r o m two  apart.  sites  The  two  s t a t i o n s i n F r i s k e n L a k e w h i c h were 100  replicated  were c o l l e c t e d  from only  were c o l l e c t e d  a t one  the p r o v i n c i a l  campground and  found, t h i s  2.2.2  site  Aeration Black  curtain study  one  station  appeared  of B l a c k  L a k e was  very  i n the middle of the t h e d i v e r s i o n . As  d i v i d e d i n t o two  lake to allow  e x p e r i m e n t was monitor the chemistry,  continued  effect and  of  samples  lake  WIB  samples  between  (1977)  had  a l l of Chain Lake w e l l .  Lake equal  p a r t s by  a  Fabrene  as p a r t o f a h y p o l i m n e t i c  of the w e s t e r n s i d e of the the  i n 1984,  s t a t i o n . C h a i n Lake water  to represent  (woven p o l y e t h y l e n e )  aerated  w e l l ; thus,  m  lake  (Ashley  1983).  t r o u t t o s u r v i v e . The so t h a t  samples c o u l d  aeration Ashley  aeration be  collected  a change i n oxygen c o n c e n t r a t i o n  m i c r o b i a l r e s p o n s e s t o a change i n  on  to  iron  iron  availability.  2.2.3  Chemical Treatments of F r i s k e n Calcium  hydroxide  precipitation calcium  of c a l c i u m  carbonate i s too  induction  of c a l c i u m  detailed  study  of the  nutrient  (iron  and  productivity. to the  was  lake  lake during  A  added t o F r i s k e n L a k e t o  carbonate; s l o w and  the  effects  of  unpredictable  f o r study.  allowed  a b a r g e was  (Murphy e t a l . 1 9 8 5 ) . I n  1983,  dry periods:  and used  of  The  more  carbonate p r e c i p i t a t i o n  phosphorus) a v a i l a b i l i t y  relatively  induce  natural precipitation  carbonate p r e c i p i t a t i o n  s l u r r y maker on  three  Lake  on  algal t o add  l i m e was  the  added t o  J u n e 16-17, 8.4  lime the  tonnes;  22 July  26-27, 7.2  tonnes;  t o n n e s ) . I n 1984, tonnes.  The  and  l i m e was  August added  16-17, 7.2  i n one  lake received a t o t a l  of  tonnes  trip:  38.8  May  (total  26-28,  22.8  16  tonnes of C a f O H ^  over  2 t h e two  year  The with  (114  g/m  , 20.9  t h e c o m p u t e r p r o g r a m PHREEQE perform  a l a k e and  precipitation. correct 2.2.4  The  program used  15,000 o r  precipitation,  to  1971).  (2.0  m  in diameter,  effect  of n i t r a t e ,  algal productivity,  and  layer  calcite  5 or  7 m deep  and  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 were  long-term on  each  Experiments  21,700 L )  or F e - c i t r a t e  or  from  t h e D e b y e - H u c k l e method  s t r e n g t h (Berner  containers  the  determined  ( P a r k h u r s t e t a l . 1980).  simulate d e s t r a t i f i c a t i o n  Limnocorral Large  s a t u r a t i o n was  complex t i t r a t i o n s w i t h d a t a  then  for ionic  to test  mg/L).  degree of c a l c i u m carbonate  PHREEQE can of  period  EDTA, Fe-EDTA,  calcium  phosphorus s o l u b i l i t y .  used  citrate,  carbonate  See  Appendix 1 f o r  details.  2.2.5  Small  2.2.5.1 To  In-Situ  Incubations  Calcium C h l o r i d e facilitate  precipitation,  Experiment  o b s e r v a t i o n s of c a l c i u m  w a t e r s a m p l e s were i n c u b a t e d w i t h  c h l o r i d e . Water s a m p l e s were c o l l e c t e d of B l a c k and  L a k e on Aug.  20 mg/L  of C a C l  were i n c u b a t e d  carbonate  2  either  24  and  Aug.  respectively. in full  25,  1.0 1983,  meter from and  Samples w i t h  sunlight  calcium  i n the  the  surface  enriched with each  lake  10  treatment  (1000  uEi  m-  2  23 s-  1  )  or i n t h e shade  - 2 - 1 (10 u.Ei m s ) f o r t e n 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 After  t h e i n c u b a t i o n , t h e v e s s e l s were s t i r r e d  were i m m e d i a t e l y acetate f i l t e r s and  Plexiglas  filtered  through  cylinders.  and s u b s a m p l e s  0.45 urn M i l l i p o r e  cellulose  f o r a n a l y s i s o f s o l u b l e r e a c t i v e p h o s p h o r u s (SRP)  d i s s o l v e d c a l c i u m . S u b s a m p l e s f o r SRP were a n a l y z e d  t h r e e hours of c o l l e c t i o n . were t r e a t e d w i t h later  analyzed To  Both u n f i l t e r e d  f o r calcium. collection  of p a r t i c l e s  e x p e r i m e n t , t h e v e s s e l s were l e f t  filtered  20 f o l d .  through  immediately  Fifty  milliliters  Whatman GF/C f i l t e r s .  placed  in the C a C ^  undisturbed  t h e s u r f a c e water, t h e s e t t l e d  concentrated  samples  1.0 ml o f 50% HC1 p e r 100 ml o f s a m p l e and  facilitate  decanting  and f i l t e r e d  within  f o r 14 h . By  particles  c o u l d be  of the concentrate The f i l t e r s  i n 50 ml s y r i n g e s w i t h  were  were  20.0 ml o f d i s t i l l e d  w a t e r and 10.0 ml o f e i t h e r CG^ o r a i r . T h e s y r i n g e s were s h a k e n for  20 s , and t h e g a s e s were p u r g e d  or a i r . The s y r i n g e s were i n c u b a t e d syringe analyses  s a m p l e s were f i l t e r e d were i m m e d i a t e l y  were p r e s e r v e d  2.2.5.2  for later  Primary  Primary  and r e p l a c e d w i t h  fresh  C0  2  a t 40°C f o r one h o u r . T h e  through  Whatman GF/C f i l t e r s .  SRP  done, and samples f o r d i s s o l v e d c a l c i u m analysis.  Production  productivity  desferrioxamine-B,  and t h r e e a l g a l  a l g a l u p t a k e o f 1.48 x 1 0 t h a t were i n c u b a t e d  response  6  Bq o f  1 4  t o i r o n a n d c h e l a t o r s (EDTA, c h e l a t o r s ) was m e a s u r e d by C-HC0  3  i n 300-ml BOD  i n the lake. Relatively  long  bottles  i n c u b a t i o n s (48  24 h) were u s e d  to avoid misleading signals  i n c u b a t i o n s can used  produce  for controls.  (Lean  and  filters),  treated with  1 ml  dissolved  i n PCS  (Amersham), and  scintillation  2.2.5.3  fluor  short-term  P i c k 1981). Dark b o t t l e s  Samples were f i l t e r e d of  that  0.5  N HC1  (0.45  urn M i l l i p o r e  f o r 24  counted  were  h, d r i e d with  (110°C),  a Beckman  counter.  Heterotrophy 14  M i c r o b i a l u p t a k e o f c i t r a t e was Two  active  and  one  control  were e n r i c h e d w i t h activity,  4.11  incubated  in situ,  After 25 mm  the  7.11  (killed  ug C/L  of  i n 20.0  ml  was  filters  were washed w i t h  10.0  transferred  to s c i n t i l l a t i o n  vials  the  pump. The  of s c i n t i l l a t i o n acidified  formalin)  C-citric  acid  by  samples  (specific  s a m p l e s were  s y r i n g e s f o r 2.0  h.  a s s e m b l y c o n t a i n i n g a 0.2  tubes ml  o f a CC^  and  the  purging  of d i s t i l l e d  urn,  sample  apparatus.  water,  immediately  inactivated  fluid.  The  f i l t r a t e in the  purging  injecting  0.2  ml  through  s a m p l e s were p u r g e d w i t h C 0 ~ f r e e 2  1 4  C-C02  liberated  of s c i n t i l l a t i o n  hydroxide The  ml  of  2 N ^SO^  stopper. The  ml  i n t o the r e s e r v o i r  was  0.2  C-citrate.  a t t a c h e d t o t h e s y r i n g e and  The  apparatus  1 4  plastic  filtered  ml  1,5  incubation, a f i l t e r  membrane f i l t e r  10.0  with  GBq/mM, Amersham). T e n - m i l l i l i t e r  was  with  measured w i t h  solution  in a s c i n t i l l a t i o n  hyamine h y d r o x i d e  Vigreaux  from  i s an  a i r from  t h e s o l u t i o n was  aquarium  trapped  c o n t a i n i n g 0.3  ml  vial  to a Vigreaux  connected  effective  absorber  of  an  in  5.0  hyamine  o f CO^;  column p r o v i d e s a l a r g e s u r f a c e a r e a f o r good  column.  the absorption  of  carbon  dioxide. After  were washed w i t h were c o u n t e d corrected  on a Beckman  Isocap  s o l u t i o n . The  scintillation  f o r b a c k g r o u n d and f o r q u e n c h i n g  samples t o determine for  f o r 20 min, t h e V i g r e a u x  5.0 ml o f s c i n t i l l a t i o n  s t a n d a r d . The c o n t r o l  used  purging  14  C-glucose  and  14  14  C-acetate  samples  counter  and  u s i n g an e x t e r n a l  s a m p l e s were s u b t r a c t e d f r o m the a c t i v e uptake.  columns  the active  The same p r o c e d u r e  was  uptake s t u d i e s . Recovery of  14 C from  was  test  100.5%  2.3  samples of  i n the purging  Chemical  S t u d i e s and A n a l y s i s o f F i e l d  were c a l i b r a t e d  Samples  Analysis  Oxygen and t e m p e r a t u r e by W i n k l e r  were m e a s u r e d w i t h Y S I m e t e r s t h a t titration  (APHA 1 9 7 6 ) . The pH  measured w i t h a C o r n i n g pH m e t e r . I n o r g a n i c c a r b o n by gas c h r o m a t o g r a p h y a t t h e l a k e Samples f o r c h l o r o p h y l l filters  (Burnison  least  of water  onto  GF/C  f r o z e n , and a n a l y z e d  l i m i t a t i o n was  t h e summer p e r i o d . D i r e c t  microscope  was m e a s u r e d  a a n a l y s i s were f i l t e r e d  1980). Although  trap picoplankton, this  was  ( S t a i n t o n e t a l . 1977).  w i t h i n two h o u r s o f c o l l e c t i o n ,  DMSO e x t r a c t i o n to  apparatus  (n=10).  Laboratory  2.3.1  C-bicarbonate  GF/C f i l t e r s not important  observation with  a r e unable for at  a fluorescent  s a m p l e s t h a t were c o n c e n t r a t e d  0.2 jam  filters  indicated  t h a t p i c o p l a n k t o n were o n l y a m i n o r c o n s t i t u e n t o f t h e  biomass.  were p r e s e r v e d and  settled  For a l g a l  orange  onto  Nuclepore  algal  and s t a i n e d w i t h a c r i d i n e  by  (Daley  e n u m e r a t i o n and i d e n t i f i c a t i o n ,  with Lugol's  solution  with the Ultermohl  (19 31)  1979)  samples  ( V o l l e n w e i d e r e t a l . 1974) technique.  26 Iron  content  i n the  s a m p l e s was  b a t h o p h e n a n t h r o l i n e method for  iron analysis The  filtrate  HNO^  500  ml  per  Particulate  ( S t r i c k l a n d and  were f i l t e r e d  filters.  of  was  at  s a m p l e and  analyzed  c h r o m a t o g r a p h y method in the  in  as  the  exchange r e s i n t o  eluant  was  BSTFA  freeze-dried  Burris  at  of  until  and  silica  diethyldithiocarbamate respectively  analyzed.  column w i t h  resuspended  50°C w i t h a r g o n ,  1.0  (Csaky  method and  p a r t i c u l a t e nitrogen,  inorganic  carbon, t o t a l  i n 0.1  gas  method  was  through  an  and  c a l c i u m , magnesium, p o t a s s i u m and  the  acid,  derivatized and  with  pyridine. and  analyzed  silver  heteropoly blue  acid  method  measured as  total P  and  digestion. dissolved  carbon, c h l o r i d e ,  organic  dissolved sulphate,  s o d i u m were p r e p a r e d  ice to a laboratory  T e c h n i c o n a u t o a n a l y z e r methods  formic  M NH^OH, d r i e d  p a r t i c u l a t e carbon,  inorganic  on  by  1948).  were measured by  P after perchloric  shipped  a  M  i s o l a t e s were h y d r o l y z e d  nitrogen,  and  freezing.  1 9 7 9 ) . The  Samples f o r n i t r a t e , ammonium, n i t r i t e ,  laboratory,  concentrated  s a m p l e s were e l u t e d  (APHA 1 9 7 6 ) . P h o s p h o r u s was  dissolved  GF/C  analyzed.  [N,0-bis(trimethylsilyl)trifluoroacetamide]  Arsenic  Samples  c i t r a t e (Dowex AG1-X8, f o r m a t e  and  bound h y d r o x a m a t e g r o u p s  total  and  from the  Some a l g a l s i d e r o p h o r e for  ml  trimethylsilyl derivative  trap  a derivatization vial  2.0  were p r e s e r v e d by  ways; t h e  eluted  modified  t h r o u g h Whatman  refrigerated until  (Stumpf  following  f o r m ) , c i t r a t e was the  with  a  Parsons 1972).  lake  i r o n s a m p l e s were k e p t f r o z e n  C i t r a t e was  anion  the  acidified  Samples f o r c i t r a t e a n a l y s i s  changed  measured by  in a  field  for analysis  using  ( E n v i r o n m e n t Canada  1979).  27 2.3.2  3  2  P-SRP  Stream  Analysis  and  extracts  were  analyzed  from  polymerized-P  (Lean  1973).  mortar of  and  either  by  gel  pestle  organic-P  were and  d i s t i l l e d  surface  rock  chromatography  or  Rocks  of  of  extracted  then  water  to a  1.0  by  N  the  separate  different f i r s t  extracting or  from  Lake  area  orthophosphorus molecular  grinding  3.0  g  of  in  a  wrist  HC1  Black  size  them  powder  with  with  action  a  100  ml  shaker.  32 P-PO^  was  used  as  a  tracer  of  orthophosphate  elution.  Five  ml  P-PO^  by  columns  32 samples  were  co-injected  packed  with  G-25  sodium  chloride  with  Sephadex and  beads  0.02%  sodium  onto  2.5  (Pharmacia) azide.  and  30  cm  eluted  with  F i v e - m i l l i l i t e r  0.3%  aliquots  32 of  the  and  eluant  then  2.3.3  for  analyzed  for  P  a c t i v i t y  by  counting  SRP.  iron-binding  determined assay,  c a p a c i t y (FeBC) o f l a k e w a t e r was  with a r a d i o i s o t o p e assay any u n c h e l a t e d  (Murphy e t a l . 1 9 8 3 a ) . I n  i r o n was p r e c i p i t a t e d  c a r b o n a t e . T h e c h e l a t o r s were c a l i b r a t e d (desferrioxamine-B, Ciba-Geigy) equivalents  with  desferal  o f d e s f e r a l (uM).  chromatography  by g e l - p e r m e a t i o n  (Murphy 1 9 7 6 ) . F i v e m i l l i l i t e r s  sample were i n j e c t e d Sephadex b e a d s  chloride  w i t h magnesium  and t h e FeBC was e x p r e s s e d i n  Some FeBC s a m p l e s were f u r t h e r p r o c e s s e d  G-25  Cerenkov  Iron-Binding Assay . The  this  were  onto  2.5 cm by 30 cm c o l u m n s p a c k e d  (Pharmacia)  and e l u t e d w i t h  a n d 0.02% 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  e l u a n t were a n a l y z e d  for  of the treated  55 Fe.  with  0.3% s o d i u m aliquots  of the  28 For  a comparative  binding assay of i r o n  t h e FeBC a s s a y was i m p r o v e d cellulose buffer, used  by s u b s t i t u t i n g  algal  filtrates  PM-30 u l t r a f i l t r a t i o n scintillation  (pH 7.0) f o r TRIS  t h e i n c u b a t i o n t o one h o u r .  that  had been f r o z e n ,  membrane ( A m i c o n ) .  c o u n t i n g . The e f f i c i e n c y  cations,  0.2 f o r 0.45 urn  acetate f i l t e r s , cacodylate buffer  and i n c r e a s i n g  and o t h e r  These  and p a s s e d  assays  through a  ^ F e was m e a s u r e d by  o f t h e PCS f l u o r  (Amersham) was o p t i m a l when 1.0 ml o f 500 uM d e s f e r a l was added to  t h e samples p r i o r  needed 0.001  to addition  f o r the coloured metal  of the f l u o r .  Quench c u r v e s were  s o l u t i o n s . M e t a l s were added i n  N HC1. A typical  iron-binding  filtrate,  HC1),  0.5 ml o f 0.01 N NaOH, 1.0 ml o f b u f f e r ,  5 5  Fe-FeCl  consisted  algal  distilled  0.5 ml o f  experiment  w a t e r t o make a f i n a l  3  (7.4 x 1 0  4  o f 2.0 ml o f  Bq i n 0.01 N a n d enough  double  volume o f 10.0 m l . O n e - m i l l i l i t e r 55  s a m p l e s were t h e n the i n i t i a l was  taken  one hour  added t o e a c h  f o r determination of t o t a l  Fe. After  i n c u b a t i o n w i t h t h e m e t a l s , MgCO^  (60 mg)  flask.  s o l u t i o n s were f i l t e r e d  After  another  filtrates  To d e t e r m i n e isolation, various  incubation,the  a n d 1.0 ml o f f i l t r a t e was c o l l e c t e d f o r  determination of ^ F e remaining the a l g a l  one hour  i n s o l u t i o n . Between  experiments,  were f r o z e n .  the effect  o f pH c h a n g e s d u r i n g  chelator  a f i l t r a t e o f A n a b a e n a f l o s - a q u a e was p r e t r e a t e d w i t h  amounts o f HC1 o r NaOH f o r one hour  prior  t o t h e FeBC  assay. Later to  i n t h e study, t h e adsorption of siderophore  the algal  colloidal  isolates  c o a t i n g s was s t u d i e d . T h e i r o n - b i n d i n g  29 a s s a y was recently  used  filtrates  filtered;  filtered, through  on  filtered,  freeze-dried,  dehydrated  frozen,  and  membrane  cultures that  thawed, and  redissolved,  an u l t r a f i l t r a t i o n  treatments  from a l g a l  and  latter  treatment p h y s i c a l l y  2.3.4  Chelator  refiltered;  colloids;  removed t h e  been  refiltered;  (PM-30, A m i c o n ) .  precipitated  had  or  passed  The  former  whereas,  the  colloids.  Isolation  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  algal  cultures,  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  -2 were grown w i t h a 16 from f l u o r e s c e n t algal  cultures  h p h o t o p e r i o d , i n 100  were c a r r i e d  with yeast e x t r a c t , beef  o u t b e f o r e and  extract,  ( D i f c o ) . C u l t u r e s were r e g a r d e d f u n g a l growth appeared Confirmation  and  of t h e a x e n i c s t a t u s  A n a b a e n a f l o s - a q u a e , and  19 7 3 ) . To  t h e amount o f i r o n  added  e a s i l y w i t h a 1% needed a 10%  experiment test  week a t  media or 25°C.  cylindrica, obtained  microscopy.  i n Chu-10 medium  (Nichols  i n t h e e x p e r i m e n t a l medium, t o the experiment  iron-rich  iron-deficient  t o grow w e l l .  light  i f no b a c t e r i a l  twice p r i o r  i r o n . Although  inoculum,  inoculum  each  Scenedesmus b a s i l i e n s i s was  a l g a e were s u b c u l t u r e d a t l e a s t medium w i t h o u t  after  o f Anabaena  c u l t u r e s were m a i n t a i n e d reduce  of  contamination of  m e d i a f o r one  s e v e r a l t i m e s by t r a n s m i s s i o n e l e c t r o n Stock  s  thiogylcollate  as a x e n i c  i n the t e s t  -1  uE m  lamps. T e s t s f o r b a c t e r i a l  cultures  in  algae subcultured  Anabaena f l o s - a q u a e  Some i r o n - d e f i c i e n t  o f A n a b a e n a f l o s - a q u a e w o u l d n o t grow. To m i n i m i z e  this  cultures problem  30 in  establishing  the  new  b e s t growing  cultures,  c u l t u r e s were  C h e l a t o r s were i s o l a t e d cultures  several  f l a s k s were i n o c u l a t e d  used.  from a x e n i c e x p o n e n t i a l phase b a t c h  ( t e n days  o l d ) . For the l a r g e r  isolations,  c u l t u r e s were u s e d  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  chelator off,  extraction,  decanted  and  membranes w i t h 0.45 Twenty l i t e r s 4.0,  filtered  f o r four  of f i l t e r e d  medium were f i r s t  a d j u s t e d t o pH  10,  and  (Bio-Rad, C l  The  diameter  c h e l a t o r was  packed eluted  hours,  to  turned  and  the  through c e l l u l o s e a c e t a t e  g o f AG1-X8 a n i o n e x c h a n g e r e s i n was  ml  rim p o r e s .  a e r a t e d f o r 30 min,  resin  100  t h e a i r b u b b l e r i n t h e c u l t u r e was  t h e a l g a e were a l l o w e d t o s e t t l e  medium was  and  into  1.0  cm  o f f the r e s i n  acidified stirred  with  f o r m ) f o r 20  columns and  w i t h 0.01  to  N HC1.  pH 100  min.  the  The  first  14 isolations  used  10  100  6  Bq p e r  water  samples  C-HCO^ t o l a b e l ml  culture),  were u s e d  T h i s peak was  and  (37  x  heterotrophic bioassays with  to detect  then p u r i f i e d  Sephadex c h r o m a t o g r a p h y  t h e o r g a n i c compounds  t h e most a c t i v e further  lake  peak.  by u s i n g ^ F e - F e C l ^ and  (Murphy e t a l . 1 9 7 6 ) .  The  chelators  were  desalted  by e l u t i o n t h r o u g h an i o n r e t a r d a t i o n r e s i n ( B i o - R a d 14 55 . AG11A8). C and Fe were n o t n e c e s s a r y o n c e t h e i s o l a t i o n p r o c e d u r e was  d e v e l o p e d . The  e x c h a n g e column was Gel-permeation  calibration were u s e d  used  pH  of the e l u a n t from the i o n  to detect  the important  c o l u m n s were r e p r o d u c i b l e 14  w i t h b l u e - d e x t r a n and  t h r o u g h Sephadex  as shown by  periodic  C - g l u c o s e . A l l compounds  i n the heterotrophy s t u d i e s ,  a s s a y , were p a s s e d  fraction.  but not t h e  columns.  that  iron-binding  31 2.3.5  Electron Microscopic Analysis Samples f r o m  treatment  i n B l a c k Lake and t h e lime  o f F r i s k e n L a k e were a n a l y z e d  Foundation scanning  the C a C ^ experiment  analysis.  larger particles.  equipped  w i t h an EDAX m i c r o p r o b e f o r  The beam was d i r e c t e d  at the centre of the  The s t a n d a r d l e s s a n a l y s i s u s e d  computer program t o c a l c u l a t e t h e e l e m e n t a l et  composition  (Yakowitz  Sediment A n a l y s i s S e d i m e n t c o r e s were t a k e n  Chain  lakes with a Williams  Pashley  from  Yellow,  1 9 7 8 ) . T h e c o r e s were d i v i d e d  hour w i t h  the Williams extruder  t h e Canada C e n t r e  s a m p l e s were g r o u n d analyzed  determined rates  of minerals  1975).  f r o z e n and l a t e r  freeze-dried  to analysis, the  ( S i , A l , C a , Mg, F e , Na, K, P, and  present X-ray  (Mudroch and Duncan  by M o s s b a u e r  diffraction  spectrometry  accumulation  1986).  i n s u b s a m p l e s was c a r r i e d o u t  of t h e unground, f r e e z e - d r i e d  o f sediment  determined  2.0 cm s e c t i o n s w i t h i n  i n a s e d i m e n t g r i n d e r , p e l l e t i z e d and  q u a l i t a t i v e l y by a P h i l i p s content  ( W i l l i a m s and  ( W i l l i a m s and P a s h l e y ,  fluorescence spectrometry  Determination  pyrite  into  f o r I n l a n d Waters. P r i o r  f o r major e l e m e n t s  Mn) by X - r a y  Roche, F r i s k e n , and  lightweight corer  u n p u b l i s h e d ) . The s a m p l e s were t h e n at  t h e Magic V  a l . 1973).  2.3.6  an  Research  o r a t M c M a s t e r U n i v e r s i t y on a Semco N a n o l a b 7  e l e c t r o n microscope  elemental  at the Ontario  spectrometer.  The  s e d i m e n t was  (Manning e t a l . 19 7 9 ) . The  i n F r i s k e n and C h a i n  b y Pb-210 r a d i o c h e m i c a l d a t i n g  (Robbins  l a k e s were and E d g i n g t o n  32 RESULTS 3.1  Biogenic  3.1.1  Response t o I r o n  Iron-Chlorophyll a Relationships The y e a r l y o s c i l l a t i o n s  (chlorophyll particulate  of a l g a l  a , F i g . 3) were c l o s e l y iron  (r=0.93, n=12) The  Availability  concentration  biomass i n Black c o r r e l a t e d t o the  ( F i g . 3) i n b o t h t h e  and t h e c o n t r o l s i d e s o f t h e l a k e  higher  iron  concentration  on t h e a e r a t e d  associated  with  a much h i g h e r  algal  aerated/control: means o f f i v e the of  aerated  1979;  lime  (r=0.87,  n=12).  s i d e of the lake ( c h i a,  o x y g e n demand i n t h e  algal  on  surface  aerated  zone o f o x i d i z e d  surface  4).  the p r e c i p i t a t i o n  showed a t r e n d  similar  concentrations  of the a l g a l  b l o o m by t h e s e c o n d  t o the Black  Lake d a t a  t o the lime  data  ( F i g . 5 ) . The  total  i n t h e 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  epilimnetic chlorophyll a concentration  Prior  are  biomass  t r e a t m e n t o f F r i s k e n L a k e , t h e F r i s k e n L a k e c h i a-Fe  iron  was  ug/L  1980, F i g . 3: v a l u e s  m). The h i g h e r  r e s u l t e d i n a shallower  (Fig. After  160/75,  r e p l i c a t e s f r o m 1.0  the l a k e , but the g r e a t e r  water  biomass  aerated  s i d e o f t h e l a k e p r o d u c e d more o x y g e n i n t h e  hypolimnion  the  97/45,  Lake  t r e a t m e n t , no c o r r e l a t i o n  concentration  and c h l o r o p h y l l a  concentration  of Chain  correlation  of t o t a l  not  significant  ( F i g .6).  iron  n=23).  e x i s t e d between  ( F i g . 6). Although the  Lake a l s o i n c r e a s e d  the  ( r = 0.82,  iron  iron  i n midsummer o f  1983,  to chlorophyll a concentration  was  33 200-,  1979  1980  •t  F i g u r e 3 S e a s o n a l changes i n p h y t o p l a n k t o n 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 F e i n B l a c k L a k e . V a l u e s a r e means o f two r e p l i c a t e s , e x c e p t A u g u s t , 1979, 1980 where n=5. E r r o r b a r s a r e one s t a n d a r d d e v i a t i o n .  34 OXYGEN mg. LITER*"  1  50  WO  150  ZOO  250  80100150  200  250 300  Fe |ig. LITER August 11 1079  -1  Figure  4  B l a c k Lake  iron concentrations  August  Figure  5  i n an a l g a l  bloom.  September  I r o n a n d c h l o r o p h y l l a i n F r i s k e n L a k e a t 1.0 m. V a l u e s a r e means o f f o u r s a m p l e s . E r r o r b a r s a r e 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  Frisken Lake  01 3  • pre-treatment O post-treatment  • 50 O  o  • t • •• o •„  o  o  a 50  100  100  200  T  150  200  250  300  400  500  15CH  100H  Fe  Figure  6  C h l o r o p h y l l and i r o n and F r i s k e n l a k e s .  (ug-L- ) 1  concentrations  i n Black,  Chain,  36 3.1.2  Seasonal A l g a l The  Succession  s e a s o n a l 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 w i t h c h a n g e s i n t h e a l g a l During  the spring  algal  bloom i n A p r i l  (Mallomonas c a u d a t a , C y c l o t e l l a  1980 i n B l a c k  meneghiniana,  species.  Lake  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 circulare, less  than  and Gomphonema  rapid  iron  decreased t o  2 ug/L. D u r i n g t h e same p e r i o d , t h e t o t a l  epilimnion dissolved  sp.), the dissolved  remained iron  constant  indicated  biological  (20-25 ug/L, A p p e n d i x  the potential  f o r iron  iron  i n the  2 ) . The low  l i m i t a t i o n but  s u c c e s s i o n pre-empted a study of i r o n  limitation. The  distribution  o f t h e a l g a e and z o o p l a n k t o n  indicated  that  t h e s p r i n g b l o o m i n B l a c k L a k e was t e r m i n a t e d by i n t e n s e z o o p l a n k t o n g r a z i n g . On A p r i l  22, 1980, t h e c h l o r o p h y l l  distribution  in different  (sample  200 m l , n=10, x=21.3+3.7  size  variation  17.4%).  indicated  that  size  ug c h l a / L , c o e f f i c i e n t o f  On May 1, 1980, t h e c h l o r o p h y l l  the phytoplankton d i s t r i b u t i o n  5 9 % ) . On May 1, D a p h n i a  swarms. A t t h i s filters almost  time,  a  distribution  was p a t c h y  clear  with  p u l e x was d i s t r i b u t e d  the f i l t r a t i o n  t h a t were e i t h e r  (sample  o f water samples  green without  4-8 l a r g e D a p h n i a  Daphnia  i n dense produced  o r t h e y were  p e r sample. S i m i l a r  effects  z o o p l a n k t o n on b l u e - g r e e n a l g a e were n o t o b s e r v e d . All  mid  uniform  200 m l , n=18, x=10.6+6.3 ng c h l a/L, c o e f f i c i e n t o f  variation  of  a r e a s o f t h e l a k e was q u i t e  a  lakes i n this  to late  study developed  Aphanizomenon blooms i n  summer, b u t t h e t i m i n g o f t h e blooms v a r i e d  greatly.  37 The  lake with  developed  the highest i r o n  c o n c e n t r a t i o n s , Chain  Aphanizomenon blooms t h r e e weeks e a r l i e r  Lake,  than  Black  Lake. The After  blue-green  the diatom  A n a b a e n a and  algal  Before  Aphanizomenon c o e x i s t e d a t low  the August  1979  observed  and On  concentrated  they  brown clumps a t  5.0  light  at a depth  level  usually  and  less  unaffected  than  by  the death  1980  the  an  3.1.3  Seasonal The  Frisken thus,  ug/L),  3,  Initially,  these niches.  1979,  Anabaena in  w e l l below the a levels  green  found  as  optimal were  t h e A p h a n i zomenon seemed  o f t h e A n a b a e n a ; however, t h e d e a t h hypolimnetic  content  of  oxygen  of t h e water column,  and  o f t h e A p h a n i zomenon bloom.  Changes i n I r o n  i n c r e a s e of  iron  Concentration  i n l a k e water,  l a k e s , o c c u r r e d when s t r e a m  r e l e a s e was  not  and  insignificant;  the sediments.  iron  t h e t h r e e l a k e s . I n F r i s k e n L a k e and iron  in Black, Chain,  f l o w s were  i r o n must have b e e n r e l e a s e d f r o m  Lake, sediment  1980,  iron,  Anabaena was  where c h l o r o p h y l l  i n f l u e n c e o f o x y g e n c o n c e n t r a t i o n s on in  increases in  hours l a t e r ,  increase in iron  initiation  t o have s e p a r a t e  i n t h e s u r f a c e meter of water  t h e Anabaena c o i n c i d e d w i t h enhanced depletion,  and  concentrations in  the morning of J u l y  ( c h i a 35  5 ug/L.  appeared  August  c l u m p s w i t h brown e d g e s . F o u r m  quickly.  f o u r weeks. Much o f t h e b i o m a s s o f  i n clumps; t h u s ,  Anabaena d i e d r a p i d l y . was  very  b l o o m c o l l a p s e i n B l a c k L a k e i n 1979  B l a c k Lake f o r a t l e a s t a l g a e was  succession could occur  r e l e a s e was  the c o n t r o l  associated with  The different  s i d e of  Black  a midsummer  38 reduction  o f o x y g e n c o n t e n t . The h y p o l i m n i a  l a k e s were r a r e l y L a k e was u s u a l l y released  o x i d i z e d ( F i g . 7 ) . The h y p o l i m n i o n o x i d i z e d b u t i t was a n o x i c when i r o n  i n August  of t h e i r o n  anoxic  this  sediments,  iron  matter  iron,  i n these anoxic  into pyrite  was  (FeS2,  a  F i g . 8 ) . Once p y r i t e was b u r i e d and no r e f l u x i n g o f  o c c u r . Some f e r r i c  iron i s  e n v i r o n m e n t s and a p u l s e o f o r g a n i c  decay c o u l d enhance t h e r a t e o f i r o n  the ferrous iron  of Chain  20% and 50%  p y r i t e would be s t a b l e ,  i n t o t h e l a k e would  metastable  l a k e s , about  i s converted  m i n e r a l c o n t a i n i n g reduced in  and F r i s k e n  (Fig. 7).  I n b o t h F r i s k e n and C h a i n respectively  of Black  r e d u c t i o n . Some o f  c o u l d e n t e r t h e w a t e r column and some w o u l d  form  pyrite. T e m p e r a t u r e was an i m p o r t a n t release at a l l s i t e s . i n mid-July  thus,  t h e s e d i m e n t s were warmer  Table  3  (Table 3). T h i s shallow than  Iron Concentrations  1  (m)  July 6 426  July 377  2 3  264  379  4 5  385  654  Inlet  893  219  16  l a k e mixes  the other  i n Chain  Iron Concentrations Depth  i n t h e sediment  Iron release occurred e a r l i e s t  Lake,  Total  variable  Aug  sites  10  i n Chain readily; (Fig. 9).  L a k e - 1984  (ug/L) Sept  236  252  322  633  812  1525  784  777  1967  741  iron  39  igure  7  S e a s o n a l c h a n g e s i n oxygen c o n c e n t r a t i o n i n C h a i n , 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 a r e mg/L.  70-  FRISKEN LAKE  60-  % OF TOTAL IRCt  50-  40-  3020-  •  •  •  •  1000  i  6  12  i  18  1  24  I  30  T  36  1—  1  42  48  • •  FeS Fe *  •  Fe  2  2  3 +  1—  54  60  66  SEDIMENT DEPTH (cm)  .Figure  8  Iron  geochemistry  of Chain  and F r i s k e n  lake  sediments.  MAY Figure 9  1  JUNE  1  JUL?  S e a s o n a l changes i n t e m p e r a t u r e o f t h e s u r f a c e C h a i n , and F r i s k e n l a k e s .  1  AUGUST  sediments o f Black,  42 Iron  release  i n B l a c k Lake d i d n o t occur u n t i l  warmer t h a n 10°C ( A p p e n d i x A l , l a t e s e d i m e n t s by l a k e a e r a t i o n the  aerated  last not  side  in Frisken warm above  3.2  July).  A 2.6°C warming o f t h e  appeared t o enhance  of B l a c k Lake  ( F i g . 9). Iron  iron  5°C u n t i l  August  (Table  r e l e a s e on  release  Lake perhaps because t h e p r o f u n d a l  occurred  sediments d i d  4, F i g . 9 ) .  C o n f i r m a t i o n of t h e Iron-Biomass R e l a t i o n s h i p Although the iron  was s t r o n g l y  correlated  concentration  i n B l a c k and F r i s k e n  with the a l g a l  biomass  observations are not conclusive proof that regulated to  t h e s e d i m e n t s were  test  3.2.1  algal  g r o w t h . The f o l l o w i n g  the iron-limitation  biomass  s t u d i e s were u s e d  with  on J u n e 19, 19 79, were  500 ug F e / L , as F e C l ^ ,  increased  significantly  i n an i n c u b a t o r ,  ( F i g . 10). Control  o f s e v e r a l d a y s b e f o r e g r o w t h was i n i t i a t e d  control  s a m p l e s t h e n grew r a p i d l y ,  than t h e i r o n - e n r i c h e d  responded p o s i t i v e l y  to iron  d i d n o t show t h i s  The  dominant  algal  species  not  grow i n t h e l a b o r a t o r y .  s a m p l e s had a  b i o m a s s p r o d u c e d was that  e n r i c h m e n t , Anabaena c o n t i n u e d t o  11, 1 9 7 9 ) , when t h e i r o n  increased,  the f i n a l  the a l g a l  and a l t h o u g h t h e  samples. In a l l samples  d o m i n a t e t h e p h y t o p l a n k t o n . Samples (Aug.  microcosm  availability  L a b o r a t o r y B i o a s s a y s w i t h B l a c k L a k e Water  enriched  less  iron  3&5), t h e s e  hypothesis.  When w a t e r s a m p l e s , c o l l e c t e d  lag  (Figs.  lakes  collected  late  i n t h e summer  c o n t e n t o f t h e w a t e r had  iron-enrichment  in late  response ( F i g .  10).  summer, A p h a n i zomenon, would  43 Table  4  Total  Iron Concentrations  in Frisken  Lake  1983  * Depth  J u n e 16  Aug 18  Aug 25  24  36  83  22  31  137  34  30  134  32+2  35  129  131  125  43  6  559  536  80  7  791  923  39  1253  444  1  July  23  18+1  27+4  July  27  26+5  2 18+2  3 4 5  39+10  68+3  8  94+11  226+102  45+12  219+245  1065+224  1984 Depth  J u n e 15  July  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  F e ug/L  depth i n meters  S e p t 20  44  TIME (days) AUGUST 11, 1979  TIME (days) Figure  10 E f f e c t o f i r o n e n r i c h m e n t on g r o w t h o f a l g a e f r o m B l a c k L a k e . T h r e e s a m p l e s were e n r i c h e d w i t h 500 ug F e / L ( • ). ( A ) were c o n t r o l s a m p l e s . Optical density a n a l y s i s h a s a c o e f f i c i e n t o f v a r i a t i o n o f 1.5%.  Although the  prime c o n t r o l l i n g v a r i a b l e  laboratory  g r o w t h a s s a y s was  probably  ability  the  t o grow i n t h e  of  dominant a l g a  to  an  important v a r i a b l e . In  of  the  to  i r o n enrichment but  variance  not  i n the  c a u s e d by  iron concentration, laboratory  a s s a y on  others,  July  show any  response to  iron-enriched  the  varying  i n which diatoms  3,  the  appeared 1979,  some  diatom, Rhopaladia gibba, quickly  f l a s k s o f J u n e 19,  proportion  w h i c h was  h);  thus,  the  1979  i n 1980  never observed  bioassays  problem with a l g a l  high  (Fig.  of diatoms p r e s e n t  dominated Anabaena i n l a b o r a t o r y Primary production  replaced  i r o n . Perhaps the  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  (48  the  f l a s k s w i t h Anabaena d o m i n a n c e showed a p o s i t i v e r e s p o n s e  Anabaena, d i d  was  another  in  10)  with  when a i n the  large lake,  cultures.  i n the  l a k e were  succession  brief  encountered  in  14 laboratory indicated FeCl^,  incubations a significant  only  similar  11)  a v o i d e d . The  stimulation  i n e a r l y summer  stimulation  siderophore (Fig.  was  isolate  confirmed  of  FeBC) on  J u n e 17  June; i r o n c h e l a t e d  by  a l l tested  species  by  this  as  i n F i g . 11).  C-assimilation  t h a t more i r o n was  in  C-assimilation  from i r o n enrichment,  (compare C t o F e 14  algal  (5 uM  algal  by  but  an  not  The  algal i n August  a v a i l a b l e i n August  Anabaena c h e l a t e  ( t h r e e Anabaena s p e c i e s  could  and  two  be  than  utilized  Scenedesmus  species). The from F e C l was  of primary production  addition  i n the  e a r l y summer o f  modest compared  to the  responses observed with  chelators able  moderate s t i m u l a t i o n  3  ( s e c t i o n 3.3.5). These s h o r t  t o assess the  immediate e f f e c t of  1979,  in situ  1980  observed  and  1981  algal  incubations  iron addition.  An  were  CONTROL  APHANIZOMENON  ANABAENA  600  AERATED  CONTROL  4-  % 30  4-  4-  450  oc UJ  3-  o  300  2 20 2-  o  Q O  150  §F 10 oc < 5 oc  OL  0  J  C  A  A+Fe  C  Fe  A A+Fe  C  Fe A A+Fe  JUNE 17 1979  C  Fe A A+Fe  JULY 19 1979  0  J  C  Fe  A A+Fe  AUG. 30 1979  F i g u r e 11 E f f e c t o f a d d i t i o n o f F e o r 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 c y l i n d r i c a on p r i m a r y p r o d u c t i o n i n B l a c k L a k e . C A, F e , and A+Fe a r e t h e c o n t r o l , s i d e r o p h o r e , i r o n a l o n e , and s i d e r o p h o r e p l u s i r o n t r e a t m e n t s . C o n t r o l a n d a e r a t e d r e f e r t o t h e two s i d e s o f t h e l a k e . On J u l y 19 t h e two t y p e s o f a l g a e were s e p a r a t e d p r i o r t o i n c u b a t i o n . V a l u e s a r e means o f two r e p l i c a t e s . E r r o r b a r s a r e one s t a n d a r d d e v i a t i o n . f  47 evaluation use  of  3.2.2  of  long-term  Fe-EDTA L i m n o c o r r a l s  and as  200  ug/L  needed  in Black  L a k e i n 1980,  Fe-EDTA o r Na-EDTA. The  iron  by  iron  of  iron  precipitate used  the  (3 and  source  must be  l i m n o c o r r a l s were e n r i c h e d  the  than  iron  iron  bioassays  are  and  second  with between adding  experiments  a r e more r e s t r i c t e d  small bottles.  Unchelated  in  iron  the would  l i m n o c o r r a l . A c h e l a t o r must  in solution;  added t o d e t e r m i n e  maintained  c o n c e n t r a t i o n and  4 additions in f i r s t  t o the bottom of the  to maintain  Lake  c o n c e n t r a t i o n was  monitoring  respectively). Limnocorral type  enrichment r e q u i r e d  limnocorral incubations.  In Black  100  responses to i r o n  i f the  thus,  an  be  additional control  c h e l a t o r has  an  important  effect. The Fe-EDTA and (Fig.  12),  initial  p r e s u m a b l y by  terminated  chlorophyll The  l i m n o c o r r a l s on A p r i l  Na-EDTA r e s u l t e d i n h i g h e r  calcium carbonate had  enrichment of  increasing algal  precipitation  the  algal  oxygen  and  bloom; t h u s ,  a concentrations  i n the  zooplankton  calcium  i n the  Na-EDTA l i m n o c o r r a l s was  result  of phosphorus with  140  ratio  ug/L  P precipitated  25,  grazing  between May  25  primarily a  calcite  and  surface  (13 mg/L  J u n e 5;  Ca/P  Ca molar  77). After  and  May  no d i f f e r e n c e s between  w a t e r s o f t h e Fe-EDTA and  and  By  l i m n o c o r r a l s were a p p a r e n t .  l a r g e r e d u c t i o n o f p h o s p h o r u s and  of c o p r e c i p i t a t i o n  with  concentrations  production.  perhaps  22,  refilled  on  e n r i c h m e n t and  this  first  J u l y 30,  s e t of 1980,  only a s l i g h t  l i m n o c o r r a l s had  t h e r e was  no  been  emptied  r e s p o n s e t o Fe-EDTA  r e s p o n s e t o t h e Na-EDTA  enrichment  48  Figure  12  Oxygen and p h o s p h o r u s limnocorrals  and B l a c k  i n Na-EDTA and Fe-EDTA Lake.  Table 5. Primary Production i n Experimental Enclosures i n Black Lake i n 1980.  1  Limnocorral Date May 22 June 3 June 16 July 4 July 18 J u l y 30 Aug 12 Aug 28 Sept 10  Fe-1 28 14 109 165 17  ED-1 40 146 169 31 69  77 46 50  251 166 350  C-2  Fe-2  117 12 31 17 192 38 52  A l l samples c o l l e c t e d a t 1 m  f  76 38 33 49 115 156 33  ED-2  NO,  LAKE  120 24 553 1030 976 589 310  130 14 16 28 56 13 20  66 135 53 96 175 730 124 83  J  a l l units as (ug C  L  d .).  49 (greater  c h i a,  significant  seasonal  decrease i n e f f e c t  at the 90% confidence  The seasonal  and l a t e r  i n c r e a s e of i r o n i n the  l a k e ( F i g . 3) was probabiy r e s p o n s i b l e i n the response  This  of i r o n enrichment was a l s o observed  i n the growth and primary p r o d u c t i o n bioassays l i m n o c o r r a l experiments.  limits).  f o r the seasonal  reduction  of i r o n enrichment i n l i m n o c o r r a l s .  Primary p r o d u c t i o n i n the l i m n o c o r r a l s was h i g h l y v a r i a b l e ; however,  the Na-EDTA l i m n o c o r r a l s had the  p r o d u c t i o n measurements  (Table 5 ) .  highest  The h e t e r o t r o p h i c a c t i v i t y was  a l s o v a r i a b l e i n the s u r f a c e water and the Na-EDTA l i m n o c o r r a l s had the h i g h e s t h e t e r o t r o p h i c r a t e s .  With o n l y one e x c e p t i o n ,  the  h e t e r o t r o p h i c a c t i v i t y was much higher i n the Na-EDTA l i m n o c o r r a l s i n the e p i l i m n i a and hypolimnia d u r i n g l a t e J u l y and August ( F i g . 13). for  The h e t e r o t r o p h i c assay i n d i c a t e s  the  capacity  h e t e r o t r o p h i c 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 led  to the r e p e t i t i o n of the i r o n enrichment experiments  c i t r a t e as the i r o n c h e l a t o r . be u t i l i z e d by many microbes  chelator with  C i t r a t e i s a weak c h e l a t o r t h a t can (Neilands 1981b) and p l a n t s  (Tiffin  1966).  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 B l a c k Lake L i m n o c o r r a l s were e n r i c h e d with sodium c i t r a t e or f e r r i c  c i t r a t e d u r i n g the summer of 1982 responses were q u i t e d i f f e r e n t  i n Black Lake. The c i t r a t e  from the EDTA responses. P a r t of  the d i f f e r e n c e was r e l a t e d to the m i c r o b i a l u t i l i z a t i o n of citrate.  Within f i v e days,  c i t r a t e was undetectable  in  solution  50  6LUC0SE UPTAKE  Figure  13 T e m p o r a l v a r i a t i o n limnocorrals.  of h e t e r o t r o p h y  i n t h e 1980  51 (Fig.  14, T a b l e  indicated  that  6). Heterotrophic  85+5.1% o f t h e a s s i m i l a t e d  i n t o carbon d i o x i d e  within  Initially, citrate-enriched ferric-citrate the  limnocorrals  limnocorrals  i n t h e sodium  decreased. Within  had s i g n i f i c a n t l y  three  days, the  l e s s oxygen  On J u n e 26 and J u n e 28  and t h e s o d i u m - c i t r a t e  than  ( t h e t h i r d and  was d e r i v e d  probability  f r o m 24 v a l u e s ; significant  thus,  had a mean o f  d a y s , t h e oxygen c o n t e n t increased  ( t t e s t , l e s s than  (Appendix  oxygen  0.2% Appendix 3 ) .  concentrations  3, F i g . 1 5 ) . W i t h i n  of the surface  more t h a n  E a c h mean  the difference i n  t h e c i t r a t e was u t i l i z e d ,  in a l l limnocorrals  limnocorrals  limnocorrals  o f t h e t r e a t m e n t s n o t h a v i n g an e f f e c t ,  After increased  than the c o n t r o l  than t h e c o n t r o l l i m n o c o r r a l s .  t r e a t m e n t s was h i g h l y  ten  of the f e r r i c - c i t r a t e  the other  limnocorrals ( F i g .  The t r e a t m e n t s r e p l i c a t e d w e l l , and by a t t e s t , t h e  ferric-citrate  limnocorrals  sodium-citrate  probability The  algal  growth  chlorophyll  had s i g n i f i c a n t l y more o x y g e n  limnocorrals  unusually  c o l d and c l o u d y  Appendix 3 ) .  weather d u r i n g  this  t o suppress the i n t e n s i t y of the blue-green  ( F i g . 15). During a content  iron limnocorrals  blue-green alga  than  ( t t e s t , l e s s t h a n a 5%  o f t h e t r e a t m e n t s n o t h a v i n g an e f f e c t ,  experiment appeared  the  converted  (Table 7 ) .  2  0.49 mg C^/L l e s s 0^  the  C-citrate  l a b e l was  had a mean o f 0.90 mg 0 / L l e s s 0^  limnocorrals,  15).  C  1 4  day a f t e r n u t r i e n t e n r i c h m e n t ) , t h e f e r r i c - c i t r a t e  limnocorrals  value  two h o u r s  1 4  t h e oxygen c o n c e n t r a t i o n  control limnocorrals.  fifth  uptake s t u d i e s with  this  limnocorral  of the l i m n o c o r r a l s  experiment, the  never exceeded  had t h e most c h l o r o p h y l l . F l a k e s  Aphanizomenon f l o s - a q u a e  appeared only  5 ug/L;  of the i n the  52  1500 <  5.0 m C O R R A L S  WE-  b> a. O F o  500-  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 w a t e r 5.0 m f r o m l a k e s u r f a c e . T h e mean c i t r a t e c o n c e n t r a t i o n s (•) o f t h e two s o d i u m c i t r a t e a n d 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 w i t h e r r o r b a r s o f one s t a n d a r d d e v i a t i o n . Day z e r o was J u n e 23, 19 82.  Table 6 C i t r a t e Concentration i n Limnocorrals Limnocorral Citrate 1 1 m 3 m  June 24  June 26  June 28  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  June 30  Samples analyzed twice are shown as the mean value + one standard deviation. A f t e r the additions of c i t r a t e on June 23, June 30, and Aug. 12, the calculated concentration would be 140 ug C/100 mL. Samples of June 30 were c o l l e c t e d 4 h a f t e r the c i t r a t e enrichment. A l l values are ug C/100 mL. ND i s not detectable.  Table 7  Sample June 28 Cit-Net Cit-Net June 30 Cit-Net Cit-Gr Cit-Net Cit-Gr Aug. 17 Cit-Net Cit-Net  Limnocorral  Depth  C-Citrate Assimilation  Cont-1  1.0 m 3.0 m  7.89 4.92  1.0 1.0 3.0 3.0  .693 6.68 1.64 9.55  m m m m  1.0 m 3.0 m  .73 .82  Cont-2 6.65 3.99  .042 .205 .478 .674 .95 .69  MD3-2  NCL-1 4.37 3.28  * * * * ND ND  5.93 4.41  *  Cit-1 32.4 203.5  83.8 103.1  89.8  79.4 582.0 248.0 1702.0  *  320.0 1798.0  * ND .439  C i t --2  .22 .74  ND 1.14  Fe-1 18.6 165.3  84.2  *  286.0 1448.0 ND ND  ND=not d e t e c t a b l e , *=no d a t a , C i t = c i t r a t e , Gr=gross u p t a k e ( t o t a l c a r b o n a s s i m i l a t e d ) , Net=net uptake (gross C u p t a k e - r e s p i r e d C ) . C o n t - l = c o n t r o l - l l i m n o c o r r a l , Cont-2=control-2 l i m n o c o r r a l e t c . G r o s s C u p t a k e = n e t C uptake + r e s p i r e d C. -1-1 -2 A l l r a t e s expressed as ng C L h xlO .  Fe-2 36.8 105.3  68.8  *  53.8 1270.0 .93 3.72  54  1(b)  TIME (DAYS) JUL 1  Figure  JUL 14  AUG 1  AUG 14  15 P h o s p h o r u s , o x y g e n , and t e m p e r a t u r e i n t h e 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 t e m p e r a t u r e ( ) o f 1.0 m w a t e r f r o m t h e 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 c o n t e n t o f t h e N a - c i t r a t e (•) and F e c i t r a t e (•) l i m n o c o r r a l s a t 1.0 m . T h e e r r o r b a r s a r e 1 s t a n d a r d d e v i a t i o n . Day 0 was J u n e 23, 1982.  ferric-citrate The (Aug. Iron  12)  limnocorrals.  final  was  not  a d d i t i o n of as  c l o s e l y studied  enrichment d i d not  production. decreased  complicated  occurred  i n a l l the  3.2.4  Iron  by  calcium  Availability  and  in Chain  identical  result (Fig.  limnocorral  in a significant 16).  citrate  A f t e r the  limnocorrals  limnocorrals.  In  algae produced. This ferric  citrate  production;  was  than  were n o t  iron  was  i t was  l i m i t e d by The  of  r e a c t i v e . The  citrate true  oxygen  J u l y 15,  c i t r a t e did  a l g a l oxygen  the  ferric  not  production  oxygen c o n t e n t o f  did  concluded  the  sodium  citrate  i n C h a i n Lake  not  that  lack  Lake.  i n both the  the  sodium c i t r a t e stimulate  the  algae  the  and  a l g a l oxygen  i n Chain  Lake  iron.  Chain Lake l i m n o c o r r a l  experiments  a v a i l a b l e to algae,  and  indicate was  supports t h i s  hypothesis  that  presumably  r e l a t i o n s h i p between o x y g e n c o n c e n t r a t i o n  phosphorus s o l u b i l i t y  a  consumed more o x y g e n t h a n  Iron  1983  much d i f f e r e n t f r o m  t o B l a c k Lake,  a l a c k of  i n C h a i n L a k e was  limnocorrals  s u p p r e s s e d by  ferric  of  the  limnocorrals.  thus,  of  stimulation  higher  or  lake.  was  in Black  t h i r d day,  microbial utilization  a l l the  i n C h a i n L a k e on  additions  contrast  additions.  oxygen consumption  i n the  i r o n r e s p o n s e was  experiments conducted The  earlier  citrate  Lake  were f i l l e d  a v a i l a b l e i r o n . The  ferric  carbonate p r e c i p i t a t i o n which  t o d e t e r m i n e i f a l g a l oxygen p r o d u c t i o n of  and  d e t a i l e d i n t e r p r e t a t i o n of  limnocorrals  Limnocorrals  the  oxygen c o n t e n t of  t i m e . Any  d a t a was  as  appear t o s t i m u l a t e  Moreover, the  at t h i s  sodium c i t r a t e  (section  and 3.4.1).  56  CHAIN LAKE  LIMNOCORRALS  IS  Figure  16  25  Oxygen AC>2 i -  s  t  31  i n Chain Lake l i m n o c o r r a l s . n  e  mean c h a n g e  f r o m mean i n i t i a l  i n oxygen  concentrations  oxygen c o n c e n t r a t i o n  o f 10.0  mg/L a t 1.0 m. Two l i m n o c o r r a l s were u s e d f o r e a c h 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 A  microbes  Ecology  s e r i e s of  regulated  a s s a y was  e x p e r i m e n t s was  the  developed  availability  to quantify  i n c u l t u r e s and  of  was  cation binding  complexing  availability algal  i n the  microdistribution  algal  and  and  An  e f f e c t s of  of  a s s a y was  iron-binding  hydroxamate s i d e r o p h o r e , coefficient  a  quantitative  insight into iron  d a t a on  chelators  siderophore  8 ) . The  compounds. T h i s  the  a s s a y does not  s o l u t i o n of  of  a well  chelators  in  the  were u s e d  to  i s o l a t e s on  measure t h e  have t h e  strong  to estimate a s s a y can  (Appendix was  precisely  4).  The  l e s s than  3.1%  molar c o n c e n t r a t i o n  concentration  compounds.  of  o f weak i r o n -  same i r o n - b i n d i n g  iron-binding  the  characterized  iron binding  example, a h i g h  compounds c o u l d  developed  namely, d e s f e r a l  of v a r i a t i o n of  c h e l a t o r s . For  dilute  thus,  of  Quantification  r a p i d l y measure e q u i v a l e n t s  binding  presence  provide  the  selectivity  i f these i r o n -  m i c r o d i s t r i b u t i o n of  c h e m i s t r y of  iron-binding  concentration  the  can  The  a l s o s t u d i e d . The  the  iron-complexing  bacterial productivity.  Chelator  (Table  to determine  how  iron-binding  i n l a k e w a t e r . The  lake  l a k e . The  e x p e r i m e n t s on  3.3.1  and  i n the  c u l t u r e medium was  design  amount o f  indicates iron limitation;  siderophores  to determine  i r o n . An  compounds were s i d e r o p h o r e s .  siderophores study of  evaluated  of  the  compounds p r e s e n t  conducted  capacity  as  a  58 Table  8  Effect  Treatment  o f F r e e z i n g A n a b a e n a F i l t r a t e s on I r o n C h e l a t i o n Filtrate #  DPMT  Totals C.V.  # #  #  DPM  C.V.  %Chelated  # #  * A. f . a. -1 Fresh Filtrate  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 Fresh Filtrate  10,151  .003  94,204  .007  10.7  FreezeDried  54,977  .007  69,802  .002  79.4  A. c y l . Fresh Filtrate  35,744  .022  102,486  .055  34.9  FreezeDried  19,353  .018  47,171  .014  41.0  *  **  *  A . f . a . - l and A . f . a . - 2 a r e two f i l t r a t e s Anabaena f l o s - a q u a e .  f r o mi c u l t u r e s o f  ** A. c y l . i s a f i l t r a t e f r o m a c u l t u r e o f A n a b a e n a  cylindrica.  DPM i s t h e mean number o f d i s i n t e g r a t i o n s p e r m i n u t e r a d i o a c t i v e i r o n i n two s a m p l e s . CV. iron.  i s the coefficient  of v a r i a t i o n  of the  o f DPM o f r a d i o a c t i v e  59 3.3.2  Siderophore Association with Fortunately,  cultures al.  I had s u p p l i e d  and t h e i r  material  extend from t h e c e l l  0.35  and Anabaena  phycosphere,  o f Anabaena  flos-aquae  to f i b r i l s  (Bell  microbes. Moreover, the m i c r o d i s t r i b u t i o n of  of a  and M i t c h e l l  w o u l d g r e a t l y change  species  of siderophore  by  The e s t a b l i s h m e n t  a microenvironment around a c e l l  the concentration  colloidal  colloidal  r e l a t i o n s h i p between t h e p r o d u c i n g  influences  (Leppard e t .  The  by 2.6 um.  adsorption  s a m p l e s o f my  15 t i m e s more  flos-aquae.  surface  cylindrica  1 9 7 2 ) , by s i d e r o p h o r e the  produces  t h a n Anabaena  fibrils um  Dr. Leppard w i t h  ultrastructure i s well defined  1 9 7 7 ) . Anabaena c y l i n d r i c a  fibrillar  Fibrils  and  competing  siderophores i s o l a t e s used i n  bioassays. Three d i f f e r e n t excreted  by Anabaena  experiments flos-aquae  1) When t h e f i l t r a t e s and  then r e f i l t e r e d ,  p e r c e n t a g e o f 2.3 uM increased was  from  frozen  (Table  Note t h a t Anabaena (Table  remaining  filtrate was  filtrate,  assay  t h a t was  frozen  that  and  the c h e l a t i o n  flos-aquae  increased  7.8  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  increased  w a t e r , and r e f i l t e r e d  only  slightly  8 ) . The f r e e z i n g and r e f i l t r a t i o n organic  The  t o 76% i n f i l t r a t e  t h e c h e l a t i o n c a p a c i t y o f an a l g a l  removes c o l l o i d a l  frozen  i n s o l u t i o n i n t h e FeBC  o f Anabaena  of d i s t i l l e d  cylindrica  colloid.  were  increased.  8 ) . In a s i m i l a r experiment,  when t h e f i l t r a t e volume  flos-aquae  the c h e l a t i o n capacity iron  the chelator  l o o s e l y bound t o a  f r o m Anabaena  51% i n u n f r o z e n  c a p a c i t y of another  equivalent  was  o n c e , and t o 101% i n f i l t r a t e  thawed t w i c e  fold  indicated that  after  (Table 8).  filtrate  freeze-drying  o f t h e thawed  m a t e r i a l . The c o l l o i d a l  from  filtrate  "fibrils"  that  60 coat many a l g a e ,  i n c l u d i n g Anabaena f l o s - a q u a e  1977), do not r e d i s s o l v e  from f r o z e n a l g a l  (Leppard et  al.  filtrates.  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 r e s p o n s i b l e increase  f o r the apparent  i n i r o n - b i n d i n g c a p a c i t y of an a l g a l f i l t r a t e  Anabaena f l o s - a q u a e  from 2 to 80 nM ( e q u i v a l e n t s  of  from  desferal).  3) Another i n d i c a t i o n t h a t c h e l a t o r s were not i n t r u e s o l u t i o n was found when the f i l t r a t e  from Anabaena f l o s - a q u a e  was  t i t r a t e d w i t h i r o n . When the c h e l a t o r was separated from the fibrils  by f r e e z e - d r y i n g and u l t r a f i l t r a t i o n and then used i n  FeBC assay, a s t r a i g h t replicate titrations chelators and  l i n e was produced ( F i g . 17).  of the p u r i f i e d Anabaena  the c o e f f i c i e n t s  the t i t r a t i o n s solutions  ( F i g . 17).  T h i s step response  each had one i n f l e c t i o n p o i n t i n the These experiments associated association  of the f r e s h  titration.  s u r f a c e of a l g a e . of s o l u t i o n s  are This of  s i d e r o p h o r e i s o l a t e s that are much more c o n c e n t r a t e d than filtrate.  algal  from d e s f e r a l or EDTA t h a t  supports the use i n bioassays  found i n the bulk  in  The p u r i f i e d  i n d i c a t e t h a t siderophores  weakly with the f i b r i l l a r  0.961  i s more r e a c t i v e when i n  i s adsorbed to the f i b r i l .  a l g a l c h e l a t o r response was d i f f e r e n t  (r ) were  had two i n f l e c t i o n p o i n t s  may i n d i c a t e that the c h e l a t o r  s o l u t i o n than when i t  In two  flos-aquae 2  of l i n e a r r e g r e s s i o n  0.968. The f r e s h a l g a l f i l t r a t e s  the  that  61  1  1  1  1  2  3 55  Figure  17  Iron  titration  Anabaena f l o s - a q u a e AFA-1  o f EDTA, d e s f e r a l ,  and f i l t r a t e s  cultures. filtrates  20 a n d 14 d a y s o l d  AFA-P i s a f i l t r a t e t h a t  ultrafiltration  1  5  Fe /ig/L  and AFA-2 a r e f r e s h  respectively.  1  4  and d i l u t e d  five  fold.  was p u r i f e d  by  from  62 3.3.3  Siderophore The  to  be  iron-binding  made  iron.  of  the  Relative  s p e c i f i c i t y  from  Anabaena  lanthanum 18).  orders  of  displace cobalt, five  or  9  copper  more  Effect  iron  of  Isolate  Cation  from  iron  to  iron  than  or  Mn  1.2  Na  3.75  Zn  6.5  a  isolate  radioactive  iron  to  be  about  of  zinc  were  that  than  three  iron  Solutions  concentrated  to  calcium, more  unlabelled  Fe  Chelation  of Cation into solution  by  the  * %Fe  88,  M mM  87  87  M M M M  in  assay  than  iron,  9).  concentration replicates  have  more  unlabelled  flos-aquae.  1.63  complexing  magnitude  had  Anabaena  K  of  the  on  650  solution,  orders  solutions  sodium,  evaluation  siderophore  Addition  Co  in  The  18).  for  an  siderophores  displace  (Fig.  more  3.75  attainable  two  concentrated  Concentration to d i s p l a c e Fe  highest  enabled  chelators  iron.  chromium  Ca  %Fe  the  required  (Table  Metal  also  compounds,  potassium,  magnitude  l i t t l e  Siderophore  and  radioactive  of  of  complexing  than  Iron  assay  organic  flos-aquae  manganese,  displaced  Table  for  magnitude  orders  capacity  most  Aluminum  the  for  s p e c i f i c i t y  to  high  (Fig.  S p e c i f i c i t y  94,  91  99,  99  82,  88  88,  91  63 C O L D - F e DISPLACEMENT O F 2.3 /iM  M  Fe  Cu DISPLACEMENT O F 2.3 /*M ^ F e  75J  c  26-  Cu (mM)  Figure  18  Metal displacement  of iron  flos-aquae siderophore. Values replicates.  Coefficient  from t h e Anabaena a r e means o f two  of v a r i a t i o n  i s less  t h a n 3%.  64 Al DISPLACEMENT OF 2.3 pM "Fe  Figure  18  Continued  65 La DISPLACEMENT OF 2.3  ui  • 1m 95% RECOVERY  s %  4  i  3  "Fe  3  Q7m 58% RECOVERY  >  i' c  u  a. 2-  *o— -o-— o — ~ o — o ~ - o - o  100 Figure  19  ELUTION VOLUME (ml)  E l u t i o n of the  5 5  Fe-filtrate  150  f r o m an FeBC  assay  t h r o u g h a G-25 Sephadex c o l u m n . T h e r e c o v e r y r e f e r s t o t h e proportion  of  5 5  Fe  that  eluted  through t h e column.  66 3.3.4  Lake Siderophores The  presence of low molecular weight c h e l a t o r s of i r o n i n  l a k e water c o u l d be observed e i t h e r by the FeBC assay or a 55 Sephadexpresent  Fe assay. These assays i n d i c a t e d t h a t c h e l a t o r s were  August 28,  1980  i n the n i t r a t e l i m n o c o r r a l , only i n the  s u r f a c e water ( F i g . 19). The  low molecular weight peak  (the  compounds e l u t i n g l a t e r on a Sephadex column) contained c h e l a t o r . The  high molecular weight peak c o u l d have been  c o l l o i d a l Fe-MgCO.j t h a t had on a f i l t e r .  The  a l g a l biomass and The in  a  not coagulated  enough t o be  water from seven meters had  retained  comparatively  little  no apparent c h e l a t i o n c a p a c i t y .  c h e l a t o r t h a t was  Black Lake i n 1982  was  i s o l a t e d from an Aphanizomenon bloom d i f f e r e n t from the Anabaena  flos-aquae  c h e l a t o r i n t h a t the Aphani zomenon c h e l a t o r d i d not have a hydroxamate group. The uM.  unconcentrated f i l t r a t e had  U n l i k e the Anabaena f l o s - a q u a e  s o l u t i o n s of sodium and Aphani zomenon c h e l a t o r flos-aquae  chelator,  a FeBC of  concentrated  c o b a l t suppressed i r o n c h e l a t i o n by (Table  1.5  the  10). In c o n t r a s t t o the Anabaena  c h e l a t o r , concentrated  s o l u t i o n s of aluminum  had  little  e f f e c t on c h e l a t i o n of i r o n by the Aphanizomenon c h e l a t o r  (Table  10). Although these c h e l a t o r s are v e r y d i f f e r e n t ,  the  c h e l a t i o n data i n d i c a t e t h a t both of these compounds are siderophores. The  iron binding capacity  observed t o be higher  than 2 uM  d i s s o l v e d i r o n on Aug. supersaturated  (FeBC) i n the l a k e was  11,  1979  i n 1980.  i r o n should  high l e v e l s  of  ( F i g . 4 ) were observed i n water  with oxygen, d u r i n g  p r e c i p i t a t i o n . Since  The  never  a p e r i o d of  calcite  be q u i t e i n s o l u b l e i n t h i s  Table  10  Effect  of  Metal  Addition  Cation  Concentration  Al  0.25  M  Co  0.25  M  Cu  on  Fe  Chelation  by  %Fe  in  the  Aphanizomenon  Siderophore  solution  69.9 0.6,  0.55  1 mM  2.4,  2.4  Cu  0.1  mM  5.1,  4.1  Na  1.25  M  7.6,  7.8  replicates  11  Table  Black  Lake  FeBC  Soluble  uM F e / L  ng  Sample  1  m  0.88  C - l  and  Iron Concentations  7 m  1  0.35  <1  Chelation Capacity  July  Particulate  Fe  1  7 m <1  m  40  80  350  230  150  200  80  100  EDTA-1  349  19 4  <10.*  <10.*  35  70  30  75  0.24  <1  0.12  <1  Fe-2  320  270  180  175  70  82  EDTA-2  204  331  <10*  <10*  70  80  0.79  0.20  <1  <1  20  78  LC  1  0.24  <1  8.00  75  100  LA  1.40  0.25  <1  1 8 . 00  94  123  N  0  3  *EDTA  interferes  samples  with  EDTA  side.  C - l ,  C-2  with had -  bathophenanthroline  reaction  to  lake  be  control  diluted.  LC  limnocorrals.  -  Fe  7 m  Fe-1  C-2  1 7 , 1980  Fe/L  Fe/L  m  -  & digestion  control  F e - 1 , Fe-2  -  side  increased  LA  iron-EDTA  -  lake  blank,  aerated  limnocorrals.  68 water, t h e high either of  a high  chelator The  in  iron concentration  f l u x o f i r o n f r o m t h e h y p o l i m n i o n o r by a b o u t  (assumes 1:1 c h e l a t o r  chelator  1980 was o n l y  hypolimnetic chelation  must have been m a i n t a i n e d by  concentration  to dissolved  capacity  than  the epilimnion  less  than  in algal  c u l t u r e s . Even d u r i n g  t h e FeBC o f t h e l a k e  than t h a t  observed  (Anabaena c y l i n d r i c a flos-aquae  b i o m a s s . However,  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  1 9 ) . T h e amount o f c h e l a t i o n c a p a c i t y  less  Lake  ( 4 0 % l e s s c h e l a t i o n by  Fig.  19 80,  of Black  s a m p l e s w i t h m i n i m a l a l g a l b i o m a s s had much l e s s  FeBC a s s a y , T a b l e  of  iron).  in the epilimnion  weakly r e l a t e d t o t h e a l g a l  4 uM  assay,  i n t h e l a k e was much  t h e A p h a n i zomenon b l o o m  (1.5 uM, c h i a 150 u g / L ) was much  i n 20 day o l d b l u e - g r e e n a l g a l  cultures  15 uM FeBC, 338 ug/L c h i a ; Anabaena  80 uM FeBC, 188 ug/L c h i a ) . I n s p i t e o f t h i s  large  d i f f e r e n c e between l a k e w a t e r a n d c u l t u r e f i l t r a t e s , t h e chelation capacity concentration could  f o r much o f t h e e a r l y summer. T h u s , a l g a l  control iron The  culture  siderophores  antibiotic  should  excretion  may be a r e f l e c t i o n  between l a k e w a t e r and  of the u t i l i z a t i o n of cultures  and f r e e o f b a c t e r i a . I n summer a l l l a k e s  siderophores or  in chelation capacity  i n t h e s e two h a b i t a t s . T h e a l g a l  dominant a l g a e ,  iron  bioavailability.  differences filtrates  unialgal  o f t h e l a k e water exceeded t h e d i s s o l v e d  several rarer algae,  had one o r two  and b a c t e r i a . I f  were i m p o r t a n t m e d i a t o r s o f s y m b i o t i c competitions,  the a s s i m i l a t i o n of  associations  siderophores  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 l a k e  B a c t e r i a may b o t h consume and p r o d u c e  were  siderophores.  water.  69 55 The axenic  r a t e of  a s s i m i l a t i o n of  Fe-siderophore  c u l t u r e of Anabaena f l o s - a q u a e ,  uM/d). However, r a p i d m i c r o b i a l siderophore from the  i s o l a t e s was  A n a b a e n a , t h a t was  back t o l a k e w a t e r on concentration c h e l a t o r was In  is relatively  a s s i m i l a t i o n of  observed  isolate, slow  two  4,  ^" C4  i n Black. L a k e . The  to the  culture  utilized  i n one  l a k e water samples  19 79,  a t an  (FeBC n o t  an  (<0.2  chelator  i s o l a t e d f r o m B l a c k L a k e , was  July  in  added  equivalent  measured);  20+  1%  of  the  day.  f r o m 1.0  m on  J u n e 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  utilized  i n one  cylindrica The  lake,  the  the  (7 uM  with the  the  limnocorral  summer  (Aug.30, A  3.3.5  Enhanced I r o n algal  August  (A  chelators  deformed  Scenedesmus s p e c i e s  day  f r o m J u n e 17  FeBC) of  was  this  (1.5  A.  uM/d). 4  was  i n the i r o n c o n t e n t of 14  the  and  to July  C-primary the  reduced  production e f f e c t of in  late  11).  algal  l e s s e f f e c t i n J u l y and  i n F i g . 11).  chelator  per  +1%  i s o l a t e d from a l a b o r a t o r y  Unlike  from Anabaena f l o s - a q u a e cylindrica  21  primary production  greatly stimulated  had  (7 uM  Availability  chelator  i n June but  on  1979,  in situ  bioassays,  chelator  in Fig.  Anabaena c y l i n d r i c a lake  increase  response of the  4,  utilized  utilization  seasonal  Anabaena c y l i n d r i c a  The  July  FeBC) was  in chelator  seasonal  bioassays,  (5 uM/d). On  chelator  reduction  consistent  day  chelator  and  d e s f e r a l and  at  of  low  two  algal  little  the  other  cells  chelators  isolated  the  t o l y s e . The  (10-50 uM  the  e f f e c t in  Anabaena s p e c i e s  concentrations  of  productivity in  Scenedesmus b a s i l i e n s i s ,  never caused cells  culture  and  FeBC).  A. former two At  70 higher above  concentrations  these chelators  chelator algae.  lack  of  antibiotic  makes i t an  The  probably  stimulated  a r e s u l t of  low  l e v e l s of  algae.  concentration  riM FeBC) was  the  lake  two  noted  the  earlier,  fibrils  3.3.6  of  of  the  that  used  i n the  fold  their  separation  of  showed t h a t from the  two  the  of  the  of  as  distinct  Anabaena  the  algae.  Microscopic l e a s t 95%  i s o l a t e d and  primary production  ( F i g . 20).  Anabaena c h e l a t o r  satisfied  but  A p h a n i zomenon was  not  be cells. to  (3.3.2).  a  significant  allow  examination  pure. A  chelator  field.  Five  uM  (equivalents  lake  Anabaena  of Aphanizomenon but  the  Anabaena  r e c e i v i n g an  The  added t o f r e s h i s o l a t e s  P r e s u m a b l y , p r i o r t o my  the  algal  enough t o  i s o l a t e d from the  stimulated  would  almost u n i a l g a l clumps.  of d e s f e r a l )  chelator  11  observed  cells  Aphanizomenon had  i n the  the  FeBC  algal  o f Anabaena o r Aphanizomenon of  may  Siderophores  i s o l a t i o n s were a t  A n a b a e n a clumps was  made  adsorbed weakly  of  to  was  in Fig.  concentration  surface  clumps was  species  t h a n any  a p p e a r t o be  f o u n d on  the  the  assay  phycosphere around the  biomass p r e s e n t  macro appearance of  i n the  higher  but  I n B l a c k L a k e , Anabaena and of  ( F i g . 11)  particulate iron  chelator  Allelopathic Properties  portion  the  matter.  siderophores are  t o make i r o n a v a i l a b l e  t o humic  19 80,  cylindrica  r e f r a c t o r y i r o n being  Some o f  to f i v e  i n summer o f  much more d i l u t e t h a n As  cells  t h e Anabaena  a l g a l p r o d u c t i v i t y i n June  have been w e a k l y a d s o r b e d The  r e a c t i v i t y by  excellent chelator  more a v a i l a b l e t o t h e  in  rupture  species. The  (5.0  could  a d d i t i o n of iron  not chelator,  requirement,  adequate supply  of  iron.  APHANIZOMENON  ANABAENA  6-1  i T '  S  5-  0  J  Fe  S c 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 o f t h e c o n t r o l ( C ) , 10 uM F e / 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 F e / L ( S c + F e ) , 5 uM c h e l a t e i s o l a t e d f r o m t h e Y e l l o w L a k e A n a b a e n a ( Y L ) , a n d 5 uM YL c h e l a t e + 10 uM F e / L (YL+Fe) on p r i m a r y p r o d u c t i o n o f r e c e n t i s o l a t i o n s o f Aphanizomenon and A n a b a e n a . V a l u e s a r e means o f two s a m p l e s . E r r o r b a r s a r e one s t a n d a r d d e v i a t i o n .  72 The the  specificity  chelator  of  the  chelator  a c t i v i t y was  i s o l a t e d from a l a b o r a t o r y  basiliensis  (Sc.,  F i g . 20).  Five  uM  of  a l s o shown  c u l t u r e of the  Scenedesmus  Scenedesmus  suppressed primary production  o f A p h a n i zomenon, b u t  This  i f the  suppression  iron;  thus,  depriving  the  was  stronger  chelator  i t of  i r o n . The  Aphanizomenon t r e a t e d iron;  thus,  the  c h e l a t o r . The  of  Scenedesmus c h e l a t o r  r e q u i r e more i r o n t h a n  species.  ( F i g . 21).  enhancement o f  The iron  The  was  chelator  siderophore toxicity  not  Anabaena.  saturated  a source  uptake of  the  of  toxic  that  t o suppress the  very  toxic to  i s o l a t e was  could  that  Anabaena.  able was  by  indicate as  with  not  growth  Scenedesmus  saturated  have b e e n r e l a t e d t o  with  an  deprivation.  Siderophore Influence Since  may  r e s u l t s from B l a c k Lake i n d i c a t e d  b a s i l i e n s i s when t h e  3.3.7  was  chelator  s u p p r e s s A p h a n i zomenon  enhanced t o x i c i t y  A p h a n i zomenon c h e l a t o r  competing  iron  not  additional iron stimulated  Aphanizomenon may The  the  did  chelator  by  siderophore  growth, s i d e r o p h o r e s  on  Heterotrophy  i s o l a t e s influence blue-green  probably  i n f l u e n c e the  algal  g r o w t h o f many  other  14 b a c t e r i a . The used An  a s s i m i l a t i o n of  to resolve  the  e f f e c t of  a d d i t i o n a l c o n t r o l was molecular weight  organic  and  were s u p p r e s s i n g  the  this  siderophore culture  c o n t r o l , the isolation  siderophore  possibility  compounds were i n t h e  uptake of  the  ^ C-labelled 4  same t e c h n i q u e s  were u s e d  o f Anabaena f l o s - a q u a e  to obtain t h a t was  compounds  i s o l a t e s on  used t o t e s t the  low  In  C-labelled organic  bacteria. that  algal  other  filtrate  substrate.  t h a t were u s e d a f r a c t i o n from  grown w i t h  was  iron.  for a This  73  TIME (days) F i g u r e 21 T h e e f f e c t o f t h e A p h a n i zomenon s i d e r o p h o r e on t h e g r o w t h o f Scenedesmus b a s i l i e n s i s . V a l u e s a r e means o f two c u l t u r e s . C o e f f i c i e n t o f v a r i a t i o n i s l e s s t h a n 10%.  Table  12 E f f e c t o f I r o n A v a i l a b i l i t y on t h e T o x i c i t y o f Anabaena F i l t r a t e s t o Microbial Acetate Assimilation.  A l g a l Growth  L a k e Water  Gross Uptake  Conditions  Treatment  ug  Iron  Algal extract contains chelator  Iron  limitation addition  Algal extract without chelator Control no a l g a l added  s i d e of  L  h xlO  2.39 10.85 5.76  extract  a c e t a t e c o n c e n t r a t i o n = 2 . 7 5 ug C/L. from c o n t r o l  C  Rate  Water c o l l e c t e d a t 1 m  B l a c k L a k e on J u l y 31, 1980.  culture The  should  isolate  from  heterotrophy depressed  not  have p r o d u c e d  the  while the  heterotrophy  could  iron-limited  bacteria  i s o l a t e from  algal  iron-deficient  ( T a b l e 1 2 ) . The  s t i m u l a t i o n of  i n d i c a t e t h a t i r o n was  1976).  heterotrophic assimilation  culture  b e i n g made a v a i l a b l e  or t h a t a low m o l e c u l a r  w e i g h t compound  The  of t h r e e a l g a l  ability  heterotrophic heterotrophy  activity  was  suppressed  i n samples from  suppression saturated  iron.  a l l w a t e r d e p t h s was  enhancement o f  Thus, the  to  suppress  Bacterial suppressed,  i n the hypolimnion.  o c c u r r e d e q u a l l y w e l l when t h e s e  with  and  The  c h e l a t o r s were  s u p p r e s s i o n was  not  related  to  an  iron deprivation.  inhibition  m e d i a t e d by  isolates  ( F i g . 22).  the g r e a t e s t suppression occurred  the  bacteria.  siderophore  strong  was  of h e t e r o t r o p h i c  in studies of siderophore-bacteria r e l a t i o n s h i p s ; isolates  to  of a c e t a t e . F o r t u n a t e l y ,  the use  siderophore  The  (Murphy  an  the s t i m u l a t i o n d i d not prevent bioassays  siderophore  iron-saturated culture stimulated  heterotrophy  stimulating  any  two  by  the siderophore  r e a c t i o n s . The  isolates  appeared  Anabaena s i d e r o p h o r e  to  be  isolates  (400  14 uM  FeBC) e n h a n c e d  than  either  control in  of  C-labelled  Scenedesmus s i d e r o p h o r e  i n c u b a t i o n s (shaded  o f g l u c o s e on  siderophore  concentration  not  the  isolate  low.  inhibitory  t h e Scenedesmus  i s o l a t e s was  isolate  inhibited  o f g l u c o s e was  concentrations, FeBC), but  siderophore  isolates  a c e t a t e much more (10 uM  area of F i g . 22). Another  the p r o c e s s i n g of the s i d e r o p h o r e  effect two  the  respiration  toxicity  difference  revealed in ( F i g . 23).  g l u c o s e u p t a k e when At  higher  effects (10 uM  FeBC) or  the The  the  glucose  o f t h e Anabaena FeBC) s i d e r o p h o r e  (400  uM  isolates,  75  ACETATE UPTAKE , ugCL"h"x|0"* r  0 2  2  4  6  8  10  1  1  1  1  1  ,  12 j  14  16  1  1  CONTROL  5  -Net  2  +  CO,  4 H  6  H  ANABAENA  2-  (LAB)  51 <u  Chelate • Fe  Chelate  a> E  0  I 2  I 2  H—I  H—I  ANABAENA  2-t^^  (LAKE)  CL 5 C h e l a t e + Fe  Chelate  Q 9-  9-  H 1  2-IS  SCENEDESMUS  + 2-133  5-3  5-IB Cheiote  Cheiote • Fe 9  Figure  2 H  -B  22 S u p p r e s s i o n b y s i d e r o p h o r e i s o l a t e s o f 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 f r o m 2, 5 a n d 9 m e t e r s were i n c u b a t e d a t t h o s e d e p t h s w i t h 3 s i d e r o p h o r e i s o l a t e s and a c e t a t e . fcS^) represents acetate respiration. r  ACETATE, „gC I" o f 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 1  i g u r e 24 Effect heterotrophy.  of  77  were overcome. The t o x i c i t y of the Anabaena s i d e r o p h o r e c o u l d not be overcome by high c o n c e n t r a t i o n s  isolate  of a c e t a t e ( F i g .  24) . The seasonal  p a t t e r n of h e t e r o t r o p h i c u t i l i z a t i o n of glucose  i n Black Lake formed a p a t t e r n t h a t bacterial activity  ( F i g . 25).  i n d i c a t e d s u p p r e s s i o n of  A week a f t e r  the c o l l a p s e  of  the  diatom bloom (May 11, 19 79), the g r e a t e s t uptake of g l u c o s e was observed.  A week a f t e r the c o l l a p s e  of a denser Anabaena bloom  ( J u l y 18, 19 79), the lowest metabolic a c t i v i t y was observed  (Fig.  25) . The b a c t e r i a l a c t i v i t y  the  increased after  the c o l l a p s e  of  Aphani zomenon bloom (Aug. 31, 1979 ), but the a c t i v i t y was not high as the s p r i n g v a l u e . Two f a c t o r s  e x p l a i n the  between the two b l u e - g r e e n a l g a l blooms.  as  difference  1) The Anabaena  s i d e r o p h o r e i s o l a t e suppressed b a c t e r i a heterotrophy 2) The Aphani zomenon bloom was terminated by  (Fig.  22)  calcite  p r e c i p i t a t i o n which l y s e d algae and appeared to enhance b a c t e r i a l activity. Siderophore ecology  was not s t u d i e d  the phosphorus geochemistry  in a l l lakes.  However,  i n d i c a t e d t h a t Black Lake was very  s i m i l a r to F r i s k e n Lake and both of these lakes are q u i t e different  from Chain Lake. Siderophore r e a c t i o n s are probably  more important i n Black and F r i s k e n lakes  than i n Chain Lake  because the algae i n Chain Lake would not need to produce siderophores.  78  GLUCOSE UPTAKE (pigC Lr'h^xK)- ) 2  AERATED 8 12 16 20 24  CONTROL MAY 11 4 I  l i  I  i  •  i  1  L  i  i  5 NET UPTAKE  J  L  -51  2-  -.  5-- J  4  JULY 19 1  J  I  J  L  J  I  9L  2-? 6-1  7 9- 3 AUG 31  Figure  I  UJ MINERALIZATION g.  9-  UJ Q  I  1  2  1  x t  _l  i  2-3  JUNE 16  4  8 12 16 20 24  25  •-a '  '  t  2-B  2-  5-  5-  9  9  Temporal v a r i a t i o n  of heterotrophy  i n Black  Lake.  79 3.4  Effect  3.4.1  Iron A v a i l a b i l i t y  Geographic V a r i a b i l i t y The  and  of  most s t r i k i n g  Black  l a k e s was  concentrations  the  28)  high  to Chain  with  Lake  1973,  low  ( F i g . 7).  of high  that  ferrous iron.  July  1984  from The  t o September  a mean o f  1.1  Frisken  for this difference r e s u l t e d i n much  i n Chain  Lake than  1 9 7 7 ) , and  in  Black  water, i r o n  14,  content 1984  of  (Table  f r o m 4.4  to  ( F i g . 3 and  8.0 p.  34  to the  more  Lake doubled  3 ) . The  anoxic  d u r i n g November  the p r e c i p i t a t i o n complex. The mg/L.  support  from  water  iron.  changes i n water c h e m i s t r y  iron-phosphorus  the  surface  iron  of Chain  Lake  ( F i g . 27)  iron data  i n the  insoluble ferric  i n Chain  1983  ( F i g u r e 7 ) . The  iron  mg/L  L a k e were c o n s i s t e n t w i t h  increased  7 i n WIB  anoxic  i s converted  or another  contrast  reactive iron.  concentrations  i n anoxic  soluble  The  little  phosphorus c o n c e n t r a t i o n s  ( F i g . 3 and  hypothesis  contained  very  not  lake.  1976  sediments  had  reactive iron  l o w e r w a t e r column was  1975  of  phosphorus ( F i g .  (Murphy e t a l . 1983a,b) and  phosphate p r e c i p i t a t i o n  In p e r i o d s  and  concentration  or t o t a l  the best geochemical hypothesis  or F r i s k e n  6,  Frisken  phosphorus  Lake, the  ( F i g . 27)  L a k e and  changes i n phosphorus c o n c e n t r a t i o n . In  that higher  the  Chemistry  oxygen c o n c e n t r a t i o n was  (Murphy e t a l . 1985)  more f e r r i c  in  i f the  Lake, Black  Therefore, was  Chemistry  o r F r i s k e n l a k e , c h a n g e s i n oxygen c o n c e n t r a t i o n was  correlated  Lake  Water  change o f  In Chain  r e a c t i v e phosphorus  In B l a c k  i n Fe-P  seasonal  ( F i g . 26).  only  Phosphorus  d i f f e r e n c e between C h a i n  soluble was  on  of  1983  ferric  1977).  Chain  phosphate  oxygen c o n c e n t r a t i o n  S i m i l a r changes o c c u r r e d  i n WIB  in  in  had 1974  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 B l a c k , C h a i n , and F r i s k e n l a k e s . A l l V a l u e s a r e means o f two s a m p l e s . B l a c k and F r i s k e n l a k e v a l u e s a r e means o f two s t a t i o n s .  CHAIN LAKE SOLUBLE REACTIVE PHOSPHORUS  c?  WO  S R P (*q/L)  F i g u r e 27 C h a i n L a k e s o l u b l e r e a c t i v e p h o s p h o r u s (SRP) A l l v a l u e s a r e means o f two s a m p l e s .  CHAIN LAKE  TOTAL PHOSPHORUS  (HQ/L)  I  50 NOV. 10,1983  Figure  28  C h a i n Lake t o t a l  phosphorus.  82 3.4.2  Geographic The  Variability  differences  and  Black  The  f l u x e s of i r o n  into  iron  calculated  i n Sediment I r o n  Reactivity  between t h e w a t e r c h e m i s t r y  and F r i s k e n l a k e s a r e r e c o r d e d  of Chain  i n the lake  sediments.  i n t o and o u t o f t h e s e d i m e n t s p r o v i d e  availability.  Complete  A comparison  i r o n b u d g e t s c a n o n l y be  ( C h a i n and F r i s k e n l a k e s , T a b l e 1 3 ) .  o f t h e b u d g e t s has one l a r g e b i a s ; t h e  sediments of Chain  Lake a r e r e l a t i v e l y  flat  but the sediments of  F r i s k e n L a k e have much more s l o p e  ( F i g . 2 ) . Resuspension  shallower  i n enhanced  sediments should  the r e l a t i v e l y  result  iron  s m a l l deep b a s i n o f F r i s k e n L a k e  l o a d i n g ( T a b l e 13) s u p p o r t s  t h e F r i s k e n Lake sediment c o r e exaggerates sedimentation  (sediment  than is  t h e sediment c o r e data  more t h a n  than  t o F r i s k e n Lake  10 f o l d  200 f o l d faster  indicate.  h i g h e r and p y r i t e  i n Chain  Lake.  of i r o n and  the hypothesis  that  the net iron  o f t h e whole l a k e . T h u s , t h e h i g h e r  Lake r e l a t i v e  from  sedimentation i n  f o c u s s i n g ) . The d i s c r e p a n c y between t h e s e d i m e n t a t i o n  i n t o Chain  insight  f o r l a k e s t h a t have p y r i t e d a t a and l e a d - 2 1 0  radiochemical dating  hydraulic  Lake  flux  of i r o n  (Table 13), i s g r e a t e r  The e x t e r n a l i r o n formation occurs  loading more  83 Table 13  Fluxes of Iron i n Chain and F r i s k e n Lakes  Flux  F r i s k e n Lake  Hydraulic „ Fe Loading  Chain Lake  3  Chain/Frisken  240  730  Sedimentation Rate Pb-210  1.25  Net Fe „ Sedimentation  120  470  4  R e t e n t i o n of Fe i n lake (%)  4000  47  -  Iron Reduction^ To P y r i t e  24  282  12  SedimenJ. Fe Efflux  35  238  7  1400  4000  3  Organic-C „„ Sedimentation #  7.8  6  -2 -1 jig Fe cm yr .  * ** Sedimentation i n mm per y e a r . Increase i n 2 ## —2 —1 water column i r o n from June to August, ug/cm . ug C cm yr  The p r o p o r t i o n of ferric  iron,  i r o n found as p y r i t e , and the s t a b i l i t y of  are d i f f e r e n t  lakes r e s p e c t i v e l y .  i n the sediments of Chain and F r i s k e n  In the Chain Lake sediment c o r e ,  c o n c e n t r a t i o n of p y r i t e i n c r e a s e s present  the  u n t i l about 60% of the i r o n  is  as p y r i t e ; i r o n i n the sediment core from F r i s k e n Lake of  the same age as the bottom of the Chain Lake core i s  20% p y r i t e .  R e l a t i v e to Chain Lake, much more of the sediment F r i s k e n Lake appears to be s t a b l e as f e r r i c i r o n .  iron in  Iron i n  sediments t h a t had p r e c i p i t a t e d 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 i n the F r i s k e n Lake  84 sediments 60% f e r r i c  of the same age as the bottom of the Chain Lake core i s i r o n . I f d i f f e r e n c e s i n oxygen c o n c e n t r a t i o n produced  t h i s d i f f e r e n c e then Chain Lake would have l e s s oxygen. However, the water o v e r l y i n g the sediments of F r i s k e n Lake has l e s s oxygen than does Chain Lake ( F i g . 7). X-ray d i f f r a c t i o n  analysis  i n d i c a t e d t h a t i n both l a k e s , some of the s u r f a c e metastable  iron  i s i n c h l o r i t e . Most of t h i s metastable i r o n c o u l d not be c h a r a c t e r i z e d ; t h i s technique can not measure i r o n bound t o humic matter. In both Chain and F r i s k e n l a k e s , the amount of i r o n from the l a k e sediments was  released  s i m i l a r t o the r a t e of p y r i t e  formation; thus, p y r i t e formation i s an index of i r o n  reactivity.  Since most chemical v a r i a b l e s are more f a v o r a b l e f o r p y r i t e formation i n F r i s k e n Lake than i n Chain Lake, and F r i s k e n Lake has p r o p o r t i o n a l l y l e s s p y r i t e , these r e s u l t s i n d i c a t e t h a t the r e a c t i v i t y of i r o n i s much lower i n F r i s k e n Lake than i n Chain Lake. The d i s t r i b u t i o n of i r o n , c a l c i u m , and phosphorus i n Chain Lake i n d i c a t e s t h a t geochemical r e a c t i o n s do not phosphorus m o b i l i t y . The  restrict  i r o n and c a l c i u m content i s r e l a t i v e l y  constant, whereas the phosphorus content i s e n r i c h e d i n the s u r f a c e sediments. The phosphorus content was  not c o r r e l a t e d  s i g n i f i c a n t l y t o c a l c i u m (r=0.53) or i r o n content (r= -0.11) ( F i g . 29). Carbonates  are too d i l u t e t o c o n t r o l phosphorus  s o l u b i l i t y . The top two cm of sediment c a l c i u m carbonate and the deeper  i s composed of o n l y  sediments  2.1%  have no d e t e c t a b l e  c a l c i u m carbonate. With the probable e x c e p t i o n of the s u r f a c e sediments,  i r o n i s a l s o unable t o i n f l u e n c e phosphorus  300  F i g u r e 29 Chain Lake sediment c h e m i s t r y ; a c i d e x t r a c t a b l e and c a l c i u m , b i o a v a i l a b l e phosphorus, water c o n t e n t , age.  iron and  86 solubility. In F r i s k e n ,  Roche  (downstream f r o m F r i s k e n L a k e ) , and  (downstream from B l a c k Lake) l a k e s , s u r f a c e sediments (Frisken  was  The  strong  o v e r p h o s p h o r u s by  r=0.95, n=7; The  X-ray  i n v e r s e c o r r e l a t i o n was  c o n t a i n over  diffraction  30%  analysis  indicated  m i n e r a l was  calcite.  3.5  of I r o n A v a i l a b i l i t y  Effect  Yellow of  that  and  the Lake  n=7).  ( F i g . 30,  the only  on C a l c i t e  Lake  (Frisken  Y e l l o w L a k e r=0.95,  calcium carbonate  the  content  a reflection  calcium carbonate  Roche L a k e r=0.80, n=6;  sediments  to the iron  Roche L a k e r=-0.86, n=7;  r=-0.86, n=7). control  the phosphorus c o n t e n t of  inversely correlated  L a k e r=-0.81, n=6;  Yellow  31).  carbonate  Phosphorus  Precipitation The  unusual  b i o g e o c h e m i s t r y o f t h e Thompson P l a t e a u  t h e r e a c t i o n s between p h o s p h o r u s and iron  Phosphorus Chemistry The  soluble  Lake Creek to that  that  r e a c t i v e phosphorus e n t e r e d B l a c k Lake  u g / L ) . The  volcanic  Sephadex r e s i n different  G-25  Lake (SRP)  content of the  (x=25 7 ug/L)  2 ug/L,  was  i n t h e Y e l l o w Lake Creek appeared  t o be  Sephadex b e a d s w i t h  size  (Lean  n=2)  and  1973).  volcanic  very  Yellow similar  concentration varied  s e p a r a t e s p h o s p h o r u s f r o m any  molecular  (25+  SRP  rock e x t r a c t  c o e l u t e d through  stream  of B l a c k  o f t h e l a k e (mean i n t e g r a t e d  t o 379  exposed SRP  to assess  availability.  3.5.1  228  c a l c i u m t o be u s e d  enabled  The  and  from  the  orthophosphate. 3 2  P0  4  The  ( F i g . 32).  o r g a n i c - P of a  arsenic  rock e x t r a c t  c o n t e n t of (30 ug/L)  the were  87  O  0.5  1  1.5  XFe  Figure 30 Roche Lake sediment chemistry; c a l c i u m , i r o n , and p h o s p h o r u s i n s a m p l e .  percent  of  total  Figure 30 F r i s k e n L a k e sediment chemistry; c a l c i u m , i r o n , and p h o s p h o r u s i n sample. ( • ) i s t h e age o f t h e s e d i m e n t .  percent  of  total  88  87  1  O  •  0.5  •  1  •  1.5  Figure 30 Roche Lake sediment chemistry; c a l c i u m , i r o n , and p h o s p h o r u s i n s a m p l e .  percent  of  total  Figure 30 F r i s k e n Lake sediment chemistry; c a l c i u m , i r o n , and p h o s p h o r u s i n s a m p l e . ( • ) i s t h e age o f t h e s e d i m e n t .  percent  of  total  88  89 less  than  arsenic the  10% o f t h e p h o s p h o r u s c o n t e n t  could  organic  n o t have  phosphorus content  u g / L ) . The s i l i c a was  i n t e r f e r e d with  too dilute  content  of four  of t h e samples;  thus,  my a n a l y s i s . S i m i l a r l y , s a m p l e s was  of the v o l c a n i c  rock  small  extract  t o i n t e r f e r e i n t h e phosphorus a n a l y s i s  (5-20  (20  mg/L)  (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 During  Lake  t h e A p h a n i zomenon b l o o m o f 1979, l a r g e q u a n t i t i e s o f  c a r b o n a t e were o b s e r v e d p r e c i p i t a t i n g on t h e i n c u b a t i o n on t h e i r o n - r i c h bottles  aerated  effervesced  v i g o r o u s l y when t r e a t e d w i t h  s a l t s were n o t o b s e r v e d time,  s i d e o f t h e l a k e . The s a l t s  t h e pH o f t h e l a k e  surface  increased  respect  during  prior  of the hypolimnion contrast  was c o n s t a n t ,  decreased  1980 s a m p l i n g  slightly.  l a k e . The a l g a l  similar  to that  increased. observations  ( F i g . 3 3 ) . C a l c i t e was n o t  and t h e p h o s p h o r u s I n t h e two weeks b e f o r e  o f t h e Aphanizomenon bloom,  P/L p r e c i p i t a t e d i n t h e s u r f a c e the  the a l k a l i n i t y  25 0 ug P/L i n t h e two  sharply with  t h e A p h a n i zomenon b l o o m o f 1979  observed, a l k a l i n i t y  August  while  t o A u g u s t 11, 1979. The a l k a l i n i t y and  19 80 o b s e r v a t i o n s  concentration  These  ( F i g . 3 3 ) . P h o s p h o r u s p r e c i p i t a t i o n i n t h e t o p 1.5  phosphorus content The  0.1 N HC1.  t o s a m p l e s c o l l e c t e d two  m o f t h e A p h a n i zomenon b l o o m was a t l e a s t week p e r i o d  on t h e  i n the c o n t r o l side of the lakes. At t h i s  and p h o s p h o r u s d e c r e a s e d w i t h weeks e a r l i e r  bottles  1.5 m o f w a t e r  l e s s than  10 ug  i n both sides of  b i o m a s s and w a t e r s t r a t i f i c a t i o n  o f 1979  t h e 12  i n 1980 was  ( F i g . 3 3 ) . T h e s e r e s u l t s were s u r p r i s i n g ,  b e c a u s e t h e l a k e w a t e r i n 1980 was  10-fold  and 1 9 - f o l d  90  2 4H  2-i 46 Alkalinity mg/L 8  6 Alkalinity mg/L 8 160  IE"  200  ^220  240  a4  6.8  9-2  9J6  AERATED  160  180  200  220  as  9L2  240~  —i—  0J6  CONTROL  F i g u r e 33 Lake chemistry d u r i n g Aphanizomenon phosphorus (PC< ), chlorophyll a (Chla), 4  blooms; oxygen  total (0 ), 2  temperature, a l k a l i n i t y , and pH on the aerated and c o n t r o l s i d e s of the l a k e , 12 August 1980 ( • ) , 11 August 1979 (•).  91 supersaturated sides  respect  to calcite  and  P-Limitation  on t h e c o n t r o l  and  aerated  of the lake.  3.5.2.1 In was  with  P-Kinetics July  1978,  so slow that  adsorption;  and June and J u l y  i t could  the high  phosphorus  1979,  assimilation  4  n o t be d i f f e r e n t i a t e d f r o m  SRP c o n c e n t r a t i o n s  assimilation.  P-P0  During  appeared  t h e 1979  phosphorus  to  saturate  bloom, p r e c i p i t a t i o n of  32 about  98%  o f t h e SRP r e s u l t e d  Phosphorus  turnover  the  control  two  sides  side  of the lake  3.5.3  h i n the aerated  This  large  (Fig.  a n d 46.0  difference  t o be r e l a t e d  34). h in  between t h e  directly to the  and 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 . after  this period  19 80 F e - E D T A L i m n o c o r r a l s limnocorrals  regulation Na-EDTA  of iron  treatments  and  alkalinity,  top  1.5  were used  availability. resulted  o f c a l c i u m and  1  Lake  i n a study  of  Early  microbial  i n t h e summer, Fe-EDTA a n d  i n a pH o f 9.5,  a decrease of  . Both values  productivity  a r e an o r d e r  from t h e a l g a l blooms  in the  i n other  than  of magnitude l e s s  than  limnocorrals increased  in  Relative  treatments.  was  less  less  (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 t h e Fe-EDTA a n d Na-EDTA  calcium  t w o w e e k s , t h e a l g a l b i o m a s s was  15 u g c h i a / L a n d t h e p r i m a r y _  i n Black  and complete p r e c i p i t a t i o n o f phosphorus  m of water. After  C L ^ d  those  uptake  4  precipitation.  Seven  ug  shortly  P-P0  Calcite Precipitation i n Limnocorrals  3.5.3.1  than  appeared  formation  bloom c o l l a p s e d  phosphorus  1.9  of the lake.  amount o f c a l c i t e The  t i m e was  i n rapid  or the by o n l y  170  lake 3  t o the lake  mg/L and  92  Figure  34  P uptake d u r i n g  Water f r o m 1.0 control  (  calcite  precipitation.  m from the s u r f a c e of aerated ) sides of  lake.  (- - -)  and  93 some l i m n o c o r r a l experiments, that either  these long-term responses  the a l g a l bloom and the bloom e f f e c t  concentrations  lasted  on oxygen  l e s s than a week, or t h a t another  such as c a l c i t e p r e c i p i t a t i o n ,  influenced  indicate  process  phosphorus  concentrations.  3.5.3.2 The  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 l i m n o c o r r a l treatment  induce c a l c i t e  t h a t had the g r e a t e s t a b i l i t y  and phosphorus p r e c i p i t a t i o n was  to  nitrate  enrichment. When n i t r a t e was used as a source of N, the pH i n c r e a s e d to 9.5 after  or 9.6  (Murphy et a l .  1983). Within two weeks  a n i t r a t e enrichment, the s u r f a c e water became d e p l e t e d  n i t r a t e and phosphorus, the pH i n c r e a s e d ,  and the a l k a l i n i t y  decreased much more than i n other l i m n o c o r r a l s . A l s o , c a l c i u m content  of the s u r f a c e water  any other l i m n o c o r r a l ( n i t r a t e l i m n o c o r r a l with c a l c i t e  (1.0  of  the minimum  m) was much lower than  l i m n o c o r r a l x=18.5 mg C a / L , other  formation x=25.7 mg C a / L , and  l i m n o c o r r a l s with no c a l c i t e formed a c r u s t on the w a l l s  formation x=35 mg C a / L ) .  Calcite  of the n i t r a t e l i m n o c o r r a 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 i m n o c o r r a l were l e s s than the c o n t r o l and the Fe-EDTA l i m n o c o r r a l s , and much l e s s than in  the Na-EDTA l i m n o c o r r a l s , or the l a k e (Murphy et  al.  1983a,b).  In the h y p o l i m n i o n , the phosphorus c o n c e n t r a t i o n was consistently  much higher i n the n i t r a t e l i m n o c o r r a l than i n  the  c o n t r o l l i m n o c o r r a l . Phosphorus i n c r e a s e d i n the hypolimnion by as much as 200 ug P / L with no i n c r e a s e  i n 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 i s s o l v e d much f a s t e r precipitated calcite.  than  T h i s p r e f e r e n t i a l phosphorus  the dissolution  indicates  t h a t p h o s p h o r u s p r e c i p i t a t i o n was a s u r f a c e  of phosphorus onto c a l c i t e  rather  adsorption  than a p r e c i p i t a t e o f c a l c i u m  phosphate.  3.5.3.3  1982 F e - C i t r a t e  The  1982 i r o n - c i t r a t e  additional calcium  Limnocorrals  information  limnocorral  on t h e r e g u l a t i o n o f a l g a l  provided p r o d u c t i v i t y by  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 calcium  t o o q u i c k l y t o have an e f f e c t on  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 calcium  Thus, c i t r a t e  could  precipitation  by m i c r o b i a l  and  treatments  only  by a s u b s e q u e n t  influence calcium oxidation  lowering  (section  carbonate  of c i t r a t e  t o carbon  Control  ( F e - c i t r a t e 8.5, 8.6; N a - c i t r a t e  t h e pH i n t h e h y p o l i m n i a  t o be  8.5, 8.5; NC> 8.7; 3  8.6, 8 . 5 ) . However, by Aug. 24, 1982, c i t r a t e  i n t o CC>2 r e d u c e d  dioxide  o f pH. T h e e f f e c t o f t h e c i t r a t e  a s s i m i l a t i o n upon t h e pH o f t h e e p i l i m n i a was t o o s m a l l detected  3.2.3).  conversion  significantly  (Fig.  35) . This  vertical  productivity.  zonation  o f pH was a r e s u l t o f m i c r o b i a l  A f t e r most o f t h e 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 ,  faster  a t 3.0 m t h a n  addition  of c i t r a t e  C - c i t r a t e uptake  ( J u n e 28, 1982) was much  a t 1.0 m ( F i g . 3 6 ) . S h o r t l y (June 30),  citrate  a f t e r t h e second  a s s i m i l a t i o n was  much f a s t e r a t 3.0 m t h a n a t 1.0 m ( T a b l e  again  7 ) . The a s s i m i l a t i o n o f  14 C-citrate in  i n the control limnocorrals  was r e l a t i v e l y  samples c o l l e c t e d a t 1.0 and 3.0 m d e p t h s  thus,  t h e enhanced m i c r o b i a l  citrate  treated  limnocorrals  consistent  ( F i g . 36, T a b l e 7 ) ;  p r o d u c t i v i t y i n hypolimnia ofthe must have b e e n a r e s u l t o f t h e  5.0m CORRAL SRP &4  •C  &2•C  PH  • N0  &0-  3  Na Na  78-  • Fe *Fe 76-  50  — 1 — —  150  OO  SRP ,gLr Figure  35  1982.  1  SRP a n d pH a t 5.0 m i n l i m n o c o r r a l s Limnocorrals  were C, c o n t r o l ;  Na, s o d i u m c i t r a t e ; and F e , i r o n  on Aug. 24,  NO^, n i t r a t e ;  citrate.  500  400 LU  f  300-  Mil  |L  200H  5  100-  M  n  CONT-1 COMT-2 NO3-I MO3-2 LAKE CTT-1 C I T - 2 F«-1 Fe-2 1m 3m 1m 3 m 1m 3m 1m 3m 1m 3m im 3m 1m 3m i m 3m Im 3m  F i g u r e 36 14  Depth  C-citrate  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 . u p t a k e on June  (Cont-1, Cont-2), n i t r a t e sodium c i t r a t e  (Cit-1,  28, 1982 i n t h e (N0 ~1, 3  Cit-2),  (Fe-1, Fe-2) l i m n o c o r r a l s  The n e t control  NO.j-2),  and f e r r i c  i s shown.  citrate  96 citrate  e n r i c h m e n t . The  1 4  C-assimilation  data suggest that a  c h a n g e i n w a t e r c h e m i s t r y p r o d u c e d by c i t r a t e only  be o b s e r v e d  3.5.3.4  phosphorus c o n t e n t  reflected  the microbial  the  3 5 ) . The h y p o l i m n i a  highest  pH v a l u e s  Indicates  Iron  of the l i m n o c o r r a l  Limitation  hypolimnia  r e s p o n s e t o i r o n e n r i c h m e n t . The  phosphorus c o n c e n t r a t i o n (Fig.  was h i g h e r  i n t h e more a c i d i c  of t h e f e r r i c  phosphorus c o n c e n t r a t i o n  citrate  hypolimnia  limnocorrals  citrate  limnocorrals  were p r e s u m a b l y a r e s u l t o f t h e s t i m u l a t i o n  of m i c r o b i a l  productivity  phosphorus  by i r o n e n r i c h m e n t . The h i g h e r  either of  greater  The  of organic-P,  of  carbonate,  inhibition  or of  c a r b o n a t e as i t s e t t l e d i n t o  o f Weather on P-CaCO^ P r e c i p i t a t i o n  enigma o f why s e d i m e n t  iron release  resulted  in calcite  i n B l a c k L a k e 1979, b u t n o t i n 1980, was  t h e 1982 s t u d i e s . T h e a s y n c h r o n y changes i n l a k e  precipitation  be a r e s u l t o f  hypolimnia.  Influence  precipitation  and  regeneration  of phosphorus from c a l c i u m  l o w e r pH  3.5.3.5  in  microbial  hypolimnia could  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 u m  release the  i n t h e more a c i d i c  chemistry  in algal  indicate that  oxygen calcium  of phosphorus from t h e e p i l i m n i a o f  resolved  production carbonate  limnocorrals  was p a r t l y c o n t r o l l e d by w e a t h e r . The  had  and t h e l o w e s t pH. The l o w e r  i n the hypolimnia of the f e r r i c  concentration  would  i n the hypolimnia.  C i t r a t e E f f e c t on P-CaCO^  The  enrichment  seasonal  e p i l i m n i a was v e r y  pattern  of phosphorus d e p l e t i o n  similar in a l l limnocorrals  i n the  ( F i g . 1 5 ) . The  97 r a t e o f phosphorus  depletion varied  greatly  warm w a t e r o f J u n e , c o o l w a t e r o f J u l y , (Fig.  1 5 ) . The c o o l w a t e r o f J u l y ,  unusually  cloudy cold  Phosphorus epilimnion  supersaturated concentration of  u.g P L  several  cooling  1  d ^ ) . The w a t e r was warm, 20°C, and  fold  w i t h c a l c i u m c a r b o n a t e . The c a l c i u m 15 d a y s by an a v e r a g e  (Table 14).  15°C w a t e r o f J u l y ,  phosphorus u t i l i z a t i o n little.  was  The  of t h e water reduced t h e degree of c a l c i t e  and o x y g e n  f r o m 11.8 t o 5.5 f o l d .  production  occurred  Although t h e s u r f a c e water  rapid  with  i n June f r o m t h e  and t h e c a l c i u m c o n c e n t r a t i o n c h a n g e d  supersaturation  less  1982 was a s s o c i a t e d  precipitated  2.0 mg C a / L a t a d e p t h o f 1.0 m  undetectable  periods:  and warm w a t e r o f A u g u s t  appeared t o d e c r e a s e w i t h i n  In t h e c o o l e r  three  weather.  was r a p i d l y  O6.0  among  than f i v e - f o l d precipitation  2-3 weeks a f t e r  i n t h e c o o l water of J u l y .  i n the limnocorrals  of phosphorus  t h e peak oxygen  depletion  became v e r y r a p i d  biomass  i n J u l y was n e v e r  supersaturated with calcium carbonate, the  When t h e w a t e r warmed mean p h o s p h o r u s  The peak o f a l g a l  and c a l c i u m d i d n o t o c c u r  until  values.  b a c k up t o 17°C i n e a r l y A u g u s t , t h e a t a d e p t h o f 1.0 m i n t h e l i m n o c o r r a l s  (>9.4 ug P L ^ d  concentration decreased within  At t h i s  depth, the c a l c i u m  a month by a mean o f 9.5 mg C a / L .  I n t h e 1980 A p h a n i zomenon bloom, t h e c o l d e r w e a t h e r , m i x i n g of  t h e l a k e by a s t o r m , and t h e n a t u r a l d e l a y  formation probably prevented c a l c i t e  in crystal  precipitation.  Table  14  Calcium Concentrations  Treatment  Control 1 Control 2 Na-Citrate 1 Na-Citrate 2 Fe-Citrate 1 Fe-Citrate 2 Nitrate 1 Nitrate 2  All  June30  Julyl3  Limnocorrals  July23  Aug 2 4  lm  3m  lm  3m  lm  lm  3m  5m  49 48 49 49 49 49 48 48  50 49 49 48 49 49 49 50  48 48 46 48 46 46 47 47  49 47 45 48 48 48 48 49  46 49 48 48 45 44 48 46  41 40 38 37 33 34 38 47  41 39 40 39 36 36 42 42  41 38 40 40 38 38 41 42  v a l u e s a r e mg/L  to f i l l  i n t h e 1982  of Ca.  The  t h e l i m n o c o r r a l s was  51  calcium content mg/L.  of t h e water  used  99 3.5.4  Calcium In t h i s  was  induced  Chloride Induction  experiment, the p r e c i p i t a t i o n by  ( F i g . 37).  analysis  were u s e d  taken  with  during  Precipitation  of c a l c i u m  carbonate  i n c r e a s i n g the calcium c o n c e n t r a t i o n with  chloride  associated  of C a l c i t e  an  supersaturated  C h e m i c a l e x t r a c t i o n s and to determine  calcium.  with  i f precipitated  Calcium  a l g a l bloom calcite  p r o d u c t / s o l u b i l i t y product  direct  microscopic  phosphorus  s a l t s were added t o l a k e  ( c h i a 50 (pH  calcium  8.6,  ug/L) Ca  = IAP/K  t h a t was  40 mg/L,  = 7.63)  water  seven-fold  ion  on Aug  was  activity 24,  1982.  sp This  sample was  collected  calcium precipitation in  five  days).  constant oxygen  The  during  after  a p e r i o d of r a p i d phosphorus  (decreases  of  110+10 ug  phosphorus c o n c e n t r a t i o n  the  e x p e r i m e n t and  increase i n the  lake during  f o r the the  P/L;  10+1  in the next  lake  two  experiment  mg  and  Ca/L  was  days.  The  indicated that  t h e 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 vessels in  phosphorus c o n c e n t r a t i o n decreased (184  to  ug/L).  a phosphorus decrease  d a r k and  light  dark c o n t r o l , Another t h e Aug. 2°C  134  24  of t h e  and  CaCl /L 2  77  a t t a i n e d the  e x p e r i m e n t . The  P/L  treatments  Ca/L).  c h l o r i d e enrichment r e s u l t e d to  same f i n a l  and  83  pH  ug  P/L  (SRP)  done t h e  decreased 10  r e s p e c t i v e l y . The  mg  in  the  even  the  (8.8+0.05). next  day  to  maintained  seven hours  i n the c o n t r o l ,  decreased  123  t e m p e r a t u r e was  lake surface. Within  ug  in the c o n t r o l  respectively. A l l flasks,  s m a l l e x p e r i m e n t was  concentrations mg  f r o m 184  incubations  phosphorus c o n c e n t r a t i o n 75,  Calcium  least  replicate to within  (1100-1800)  f r o m 184 CaCl /L, 2  ug and  P/L 20  the t o 90  ,  mg  dissolved calcium  i n a l l of the C a C l  2  incubations  (4.0+3.0  INDUCED CALCITE PRECIPITATION Initial  INCUBATE WHOLE H 0 SAMPLE 10 h + CaCl  Ca(0H) CaCl SRP Ca  Concentration 10 20 184 53  2  2  2  -»• F i l t r a t e Treatment Control Ca C l Ca(0H)  Particles  2  2  Syringe + H 0 + C0  Syringe + H 0 + Air  Incubate lh 4°C  Incubate lh 4°C  2  1  -1  1  1  2  Filter  2  mg.L" mg.L ygP.L' mg.L'  SRP  pgP.L'  hv 130 83 29  DK 137 123 18  1  4  Filter  Filtrate SRP 149 gP.L - l Ca 75 mg.L' M  1  mg.L'  1  hv 52 56 47 o o  2  Filter  Ca  Filtrate SRP 25 ugP.L" Ca 37 mg.L"  1  1  F i g u r e 37 CaCl, induced c a l c i t e p r e c i p i t a t i o n . DK and hv represent dark and l i g h t i n c u b a t i o n s . Values are means of a n a l y s e s . 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 5%.  two  101 3.5.4.1  Calcite  A weak a c i d phosphorus that The  (Fig.  extraction  phosphorus c o u l d  3 7 ) . The c a r b o n d i o x i d e  19 7 4 ) .  i n the calcium chloride be q u i c k l y  incubations.  redissolved  by  t h e p a r t i c l e s and by e q u i l i b r a t i n g t h e sample w i t h CC^  enough f o r r a p i d  reduced  t h e pH t o 5.7 w h i c h  calcium carbonate d i s s o l u t i o n  concentration. algae;  precipitation  t h e pH f r o m  e f f e c t on t h e p h o s p h o r u s o r c a l c i u m  Carbon d i o x i d e  algae that  i s low  ( B e r n e r and Morse  E q u i l i b r a t i o n o f t h e sample w i t h a i r r e d u c e d  8.8 t o 8.2 and had l i t t l e  the  was u s e d t o c h a r a c t e r i z e t h e  precipitated  precipitated  cooling  Analysis  appeared  t o have l i t t l e  e f f e c t on  were c o l l e c t e d f r o m p e r i o d s w i t h o u t  d i d not release  p h o s p h o r u s when e q u i l i b r a t e d  calcite with  co . 2  Direct assist  microscopic  analysis  o f p r e c i p i t a t e s was u s e d t o  the i n t e r p r e t a t i o n of the C 0  2  extraction.  were c o l l e c t e d f r o m t h e Aug. 24 c a l c i u m c h l o r i d e the  end o f t h e i n c u b a t i o n  aggregated  into large  the  control  treatment  The  elemental analysis  period  by f i l t r a t i o n .  Precipitates experiment at C r y s t a l s had  " b a l l s " w i t h a mean d i a m e t e r o f 20 um i n but not i n t h e carbon d i o x i d e indicated  m a i n l y o f c a l c i u m and t h a t  that  silicon  the p a r t i c l e s  was an i m p o r t a n t  treatment. consisted constituent.  P o t a s s i u m , p h o s p h o r u s and z i n c were m i n o r c o n s t i t u e n t s calcite in  p a r t i c l e s (Table  1 5 ) . The r a t i o o f c a l c i u m t o p h o s p h o r u s  t h e s e p a r t i c l e s (Ca/P m o l a r r a t i o 72) c o r r e s p o n d e d  c h a n g e s i n c a l c i u m and p h o s p h o r u s o b s e r v e d rapid  of the  earlier  t o the  i n periods of  c a l c i u m and p h o s p h o r u s p r e c i p i t a t i o n i n t h e l a k e and  limnocorrals  (Table 16).  102  Table Oxide  15  Elemental A n a l y s i s of C a l c i t e Precision Oxide% 2  CaO  sio  2  K 0 2  P  2°5  ZnO A 1  2°3  Na 0 2  Crystal  sigma  51.78  0.59  17.11  0.24  2.33  0.14  0.92  0.07  0.85  0.13  0.81  0.06  .14  0.02  The computer p r o g r a m assumes a l l e l e m e n t s a r e p r e s e n t as simple o x i d e s ; t h e 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 t e c h n i q u e c o u l d be a c c u r a t e i f t h e a p p r o p r i a t e standards existed.  Table  16  Calcium/Phosphate Ratios i n P r e c i p i t a t i o n P r e c i p i t a t i o n E x p e r i m e n t s , and S e d i m e n t  Sample L-l May 25-June 5 L-2 J u l y 1 7 - J u l y 31 L-2 J u l y 31-Aug. 12 L-3 J u n e 5- J u l y 15 B l a c k Lake Aug. 80-79 Sed-1 0-2 cm Sed-2 10-12 cm CaCl Induced P r e c i p i t a t e 2  Ca mg/L 13 7 10 21.5 20 Ca mg/g 11.2 22.7 37.0  P ua/L 140 105 97 250 250 P mg/g .13 .22 .40  Events,  Ca/P 77 52 80 67 62 66 79 72  * molar r a t i o . L - l i s t h e Fe-EDTA l i m n o c o r r a l f r o m t h e f i r s t l i m n o c o r r a l experiment. L-2 and L-3 a r e t h e EDTA-2 and N i t r a t e l i m n o c o r r a l s f r o m t h e s e c o n d s e t o f 1980 l i m n o c o r r a l s i n B l a c k L a k e . The B l a c k L a k e Aug. 80-79 d a t a i s t h e d i f f e r e n c e o b s e r v e d i n w a t e r c h e m i s t r y f r o m an a l g a l b l o o m w i t h no c a r b o n a t e 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 c a r b o n a t e p r e c i p i t a t i o n (Aug.11, 1 9 7 9 ) . A l l w a t e r s a m p l e s were c o l l e c t e d 1.0 m e t e r , f r o m t h e s u r f a c e . A l l w a t e r c h e m i s t r y i s t h e c h a n g e i n mg L o f Ca and ug L of SRP between t h e two d a t e s . Sed-1 and Sed-2 a r e s e d i m e n t s a m p l e s f r o m t h e s u r f a c e and 10-12 cm h o r i z o n o f Y e l l o w L a k e .  103 These  additional  experiments  carbonate p r e c i p i t a t i o n importance  of c r y s t a l  to inhibitors  T h i s key  reaction  the iron  3.6  results  could  lime,  confirm that calcium solubility.  the s u s c e p t i b i l i t y  in highly variable  of  The the  precipitation.  the v a r i a b i l i t y  observed  experiments.  processes that  confirmed  and  e a s i l y produce  Calcite Precipitation The  2  c o n t r o l phosphorus  initiation  reaction  in  can  with C a C l  by  - A Major  Cause of A l g a l  Periodicity  occur during c a l c i t e p r e c i p i t a t i o n  inducing c a l c i t e precipitation  in Frisken  were  Lake w i t h  Ca(OH) . 2  3.6.1  P r e t r e a t m e n t Water  Chemistry  In p r e t r e a t m e n t samples correlations phosphorus (r=.735,  of t o t a l  (SRP)  n=21)  from F r i s k e n Lake,  inorganic  were o b s e r v e d  and  carbon  ( T I C ) and  soluble  reactive  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  i n the hypolimnion  (r=.973,  The  c o n c e n t r a t i o n s were i n t h e e p i l i m n i o n ,  SRP  t h e h i g h e s t were i n t h e h y p o l i m n i o n relationship phosphorus r=.955, The  existed  between t o t a l  (epilimnion  and  relationships  two  form  and  linear  n = l l ) . These  pretreatment data sets lowest TIC  two  significant  ( F i g . 3 9 ) . The inorganic  metalimnion  carbon  r=.753, n=21;  ( F i g . 38). and  same and  total  hypolimnion  n=ll). similarity  reflection  o f t h e SRP-TIC and T P - T I C r e l a t i o n s h i p s was  of the synchrony  b i o g e o c h e m i s t r y . The phosphorus  solubility  o f c a r b o n a t e and  carbonate e q u i l i b r i a and  presumably  algal  a  phosphorus  were c l o s e l y  related  p r o d u c t i o n . Most o f  to  104  SRP(pg/L)  Figure  38  F r i s k e n Lake pretreatment  SRP/TIC.  SRP (pg/L) 0  I  30  500  1000  1  1  35  40  1500  _ _ i  45  i  50  TIC (mg/L)  Figure  39  A depth d i s t r i b u t i o n  o f SRP and T I C i n F r i s k e n  Lake.  105 the t o t a l phosphorus was i n s o l u t i o n  ( e p i l i m n i o n and metalimnion  70.6%; hypolimnion 73.8%) and the s o l u b l e phosphorus was h i g h l y c o r r e l a t e d to the t o t a l phosphorus r=.912, n=21;  ( e p i l i m n i o n and metalimnion  hypolimnion r=.959, n = l l ) .  T h i s r e l a t i o n s h i p was  q u i t e s i m i l a r t o t h a t observed i n Black Lake where 85% of t o t a l phosphorus was i n  3.6.2  the  solution.  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 .  p r e d i c t e d the r e s u l t s  of the 1983 i n d u c t i o n of  38)  calcite  p r e c i p i t a t i o n by lime a p p l i c a t i o n to F r i s k e n Lake. The e p i l i m n e t i c TIC value SRP of  i n August 1983 of  30.5 mg/L would have a  zero i f the r e l a t i o n s h i p of F i g . 38 were e x t r a p o l a t e d ;  SRP was l e s s than 20 u g / L ( F i g . 40).  Furthermore, examinations  p a r t i c l e s w i t h an EDAX microprobe of an e l e c t r o n supported the hypothesis  of  microscope  t h a t c a l c i t e p r e c i p i t a t i o n removed  phosphorus from the e p i l i m n i o n (Table 15). the e p i l i m n i o n a f t e r  the  the 1984 t r i a l  Samples c o l l e c t e d  treatment contained  in  particles  r i c h i n c a l c i u m and phosphorus. Over a hundred n o n c e l l u l a r p a r t i c l e s were a n a l y z e d . The r a t i o of P/Ca i n ten  particles  v a r i e d between 0.64  to 0.04.  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  Only two p a r t i c l e s contained  iron,  t h a t these p a r t i c l e s were p y r i t e or  p y r i t e p r e c u r s o r s . These p a r t i c l e s were not w e 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  F i g u r e 40  EPILIMNETIC SRP  F r i s k e n Lake e p i l i m n e t i c 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 t o suppress  the  b l u e - g r e e n a l g a l blooms. The f i r s t a d d i t i o n of lime was too s m a l l to induce p r e c i p i t a t i o n . The second a d d i t i o n d i d not have an effect  after  content  48 h but a f t e r  had decreased  another two weeks, the c h l o r o p h y l l a  ( F i g . 41).  The 1984  i n d u c t i o n of  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 throughout the summer ( F i g . 41). exceeded four meters observations  calcite  of the  lake  The S e c c h i d i s k readings i n  and were deeper than the  1984  pretreatment  ( A s h l e y , B . C . Environment).  The lime treatment produced a long-term s u p p r e s s i o n of growth and enhancement  algal  of phosphorus p r e c i p i t a t i o n . Although no  lime was added to the l a k e i n 19 85, the c h l o r o p h y l l a content the lake was lower than pretreatment values B.C.  ( F i g . 41; A s h l e y ,  Environment). The phosphorus c o n c e n t r a t i o n s  in  the  e p i l i m n i o n of 19 85 were much lower than pretreatment values 40).  The S e c c h i d i s c readings  i n 1985  (5.5,  of  2.8  (Fig.  and 4.4 m) were  much deeper than pretreatment ones which were l e s s 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 r e d u c t i o n of a l g a l biomass,  concentrations,  the lower phosphorus  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 F r i s k e n  Lake i n 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 1984.  i n the hypolimnion i n 1983 and  The d i s s o l u t i o n of c a l c i t e  should enhance  p r e c i p i t a t i o n i n the e p i l i m n i o n when the l a k e  calcite  mixes.  108  FRISKEN LAKE  50- 1983 40302010-  Denotes L i m e Addition  50 1985  40302010-  JUN Figure  41  Suppression  Values one  JUL of a l g a l  AUG  b i o m a s s by l i m e  a r e means o f two s a m p l e s . The e r r o r  standard  deviation.  application. bars  represent  109 The  dissolution  composite  of m i c r o b i o l o g i c a l  decomposition induced oxygen  of c a l c i t e  i n t h e h y p o l i m n i o n was a  and g e o c h e m i c a l  of sedimenting  algae during either  calcite precipitation  results  by as much as 0.3 u n i t s w i t h i n  temperature  of the hypolimnion  of  thus, the t o t a l  calcite; The  effect  a model m i x i n g sequentially  inorganic  of c a l c i u m carbonate  of F r i s k e n  each  water  mixing. U n t i l  ( F i g . 4 3 ) . The pH  48 h. T h e low pH and  Lake  carbon  increased (Fig.  recycling  44).  i s i l l u s t r a t e d by  ( F i g . 4 5 ) . The model m i x e s  equilibria  t h e h y p o l i m n e t i c water mixes,  i n carbonate p r e c i p i t a t i o n  t h e w a t e r column d e s t r a t i f i e d that  the carbonate  supersaturated with c a l c i t e .  sequence of changes  (Fig.  decrease i n  t h e u n d e r l y i n g one m e t e r w a t e r l a y e r w i t h t h e  i s greatly  reaction  a n a t u r a l o r an  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  o v e r l y i n g w a t e r column and c a l c u l a t e s for  in a rapid  ( F i g . 42) and pH i n t h e h y p o l i m n i o n  decreased  r e a c t i o n s . The  in the f a l l  The p r e d i c t e d were o b s e r v e d  o f 1983. T h e  o c c u r r e d was an e n h a n c e d p r e c i p i t a t i o n  44). At t h i s  time,  half  the mixing  o f t h e phosphorus  when  first  of c a l c i t e  precipitated.  However, as t h e l a k e c o n t i n u e d t o mix a l l o f t h e p h o s p h o r u s redissolved An occur  (Fig.  analogous  enhancement o f c a l c i t e p r e c i p i t a t i o n  should  i n t h e s p r i n g when t h e l a k e warms and becomes  supersaturated that  40).  with c a l c i t e .  The 1985 w a t e r c h e m i s t r y  t h e 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.  indicated  FRISKEN LAKE  0  FRISKEN LAKE  mg/L  2  0  2  mg/L  BLACK LAKE  0  2  mg/L  F i g u r e 42 E f f e c t o f c a l c i t e p r e c i p i t a t i o n on o x y g e n . V a l u e s f r o m B l a c k and F r i s k e n l a k e s a r e means o f d a t a f r o m two s t a t i o n s . E r r o r b a r s r e p r e s e n t one s t a n d a r d d e v i a t i o n .  Ill  X  r-  Q. 8  60 pH JULY 27 1963  90  F i g u r e 43 The pH ( • ) and change i n pH ( o ) of F r i s k e n Lake 48 hours a f t e r lime a p p l i c a t i o n . Or  JULY 25  AUG  16  AUG 25  SEPT 20  a Ui O  5  30  40  50  I  ,  ,  I  ,  ,  30  40  50  30  40  50  40  50  TIC (mg/L)  F i g u r e 44 T o t a l i n o r g a n i c carbon (TIC) c o n c e n t r a t i o n water column of F r i s k e n Lake in 1983.  of  the  112  QJ  , -8  , -4  , , 0 +4  , +8  J  , , -8 -4  1 , , 0 +4 +8  J  1 i -8 -4  1 1 1 0 +4 +8  F i g u r e 45 S i m u l a t e d m i x i n g o f F r i s k e n L a k e . Numbers w i t h i n g r a p h r e f e r t o s e q u e n t i a l m i x i n g o f one m e t e r w a t e r l a y e r s . Numbers on x - a x i s a r e t h e d e g r e e o f s a t u r a t i o n o f c a l c i t e ( i . e . +4 i s f o u r f o l d supersaturated).  113 DISCUSSION The primary o b j e c t i v e a v a i l a b i l i t y can a f f e c t blooms was a c h i e v e d . v a r i a t i o n of  of demonstrating t h a t  the p e r i o d i c i t y of b l u e - g r e e n  i r o n l i m i t a t i o n agrees with other recent Ryding 1985,  where i r o n l i m i t a t i o n w i l l occur i s c o n c e n t r a t i o n of  Stauffer  Microbes excreted  of i r o n ,  chelators  that  the  sediments.  influenced  iron  The main l i m n e t i c  P y r i t e formation r e g u l a t e d the amount of i r o n  of a l g a l biomass.  event lake  release.  p r e c i p i t a t i o n , g r e a t l y a l t e r e d the amount  In Black and F r i s k e n lakes  of these four processes  from the sediments,  the  sequential  (iron chelation,  p y r i t e f o r m a t i o n , and c a l c i t e  iron  release  precipitation)  determined the types of s p e c i e s growing, the amount of biomass,  lake  i r o n a v a i l a b i l i t y was i r o n r e l e a s e from the  Another event, c a l c i t e  development  epilimnetic  c a l c i u m and phosphorus i n  a v a i l a b i l i t y and m i c r o b i a l s u c c e s s i o n .  sediments.  1985). When and  r e l a t e d to the  and the temperature of the l a k e  that regulated  studies  i r o n ; the phosphorus c o n c e n t r a t i o n i n the  the geochemistry  sediments;  algal  New i n f o r m a t i o n on the s p a t i a l and temporal  (Stumm and Morgan 1981,  water;  iron  algal  and the d u r a t i o n of a l g a l blooms. Both b i o c h e m i c a l and  geochemical  reactions  i n f l u e n c e d or c o n t r o l l e d the p e r i o d i c i t y of  b l u e - g r e e n 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 i n 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 i n i r o n a v a i l a b i l i t y i n my study  confirms the recent  hypotheses of Ryding  (1985); the supply of of  (1985) and S t a u f f e r  i r o n v a r i e s g r e a t l y among l a k e s .  The f l u x  i r o n i n t o F r i s k e n Lake was l e s s than 1% of t h a t e n t e r i n g Chain  114 Lake. The bedrock geology of the s i t e s i s 1947,1948; R i c e 1947) 1977)  similar  (Cockfield  but the water e n t e r i n g Chain Lake (WIB  had 50% l e s s a l k a l i n i t y than the water e n t e r i n g Black Lake  (Murphy et a l .  19 83b)  the Thompson P l a t e a u ,  and F r i s k e n Lake (Murphy et a l . a l k a l i n i t y may be a u s e f u l  19 85). On  guide to where  iron l i m i t a t i o n could occur. The c o n c e n t r a t i o n  of  i r o n in the water column was a rough  guide to when i r o n l i m i t a t i o n c o u l d o c c u r . In Black Lake, a d d i t i o n of  iron stimulated  iron concentration  a l g a l p r o d u c t i v i t y when the  was l e s s than 50 ug/L and b l u e - g r e e n  ambient algal  blooms only formed in lake water with high c o n c e n t r a t i o n s iron.  The Black Lake c h l o r o p h y l l a ( c h i a)  c o r r e l a t e d to the  iron concentration.  concentration  calcite  precipitation;  the  occurred d u r i n g blooms with no  precipitation.  I f my i n t e r p r e t a t i o n Lake i s  was  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 greatest chi a concentrations  of  of the c h i a-Fe c o r r e l a t i o n of B l a c k  c o r r e c t then the absence of a c h i a-Fe c o r r e l a t i o n i n  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  i r o n in Chain Lake. The lowest i r o n c o n c e n t r a t i o n  and i r o n d i d not s t i m u l a t e  of  in Chain Lake  (Table 3) was equal to 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 Lake (Appendix 2 ) ,  the  algal  in Black  oxygen  p r o d u c t i o n in Chain Lake. Furthermore, the Black Lake s t u d i e s i n d i c a t e d t h a t Aphanizomenon needed more i r o n than other therefore,  algae;  the dominance of Aphani zomenon i n Chain Lake confirmed  t h a t Chain Lake was not i r o n l i m i t e d . The i r o n data in F r i s k e n Lake was more complex than i n other  lakes.  Initially,  dense b l u e - g r e e n  a l g a l blooms  the  (70 ug c h i  115 a/L)  formed i n 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 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 to the concentrations.  iron  A f t e r 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 c o n c e n t r a t i o n s were c o r r e l a t e d to  iron  concentrations. The suppression of a l g a l growth i n 1984  i n F r i s k e n Lake was  s u r p r i s i n g i n t h a t the SRP 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 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 f a c t o r r e g u l a t i n g a l g a l p r o d u c t i v i t y ; however,  l i m i t e d i r o n a v a i l a b i l i t y may a l s o  been important. The t o t a l  have  i r o n 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  F r i s k e n Lake i n the e a r l y summer of 19 84 was l e s s than 20 u g / L . The s l i g h t 1984  i n c r e a s e of c h l o r o p h y l l a t h a t was observed i n August  i n F r i s k e n Lake was a s s o c i a t e d  i n the e p i l i m n i o n ( F i g .  4.2  with a d o u b l i n g of the  5).  C a l c i t e I n d u c t i o n of I r o n 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 i n F r i s k e n  Lake appeared to be a r e f l e c t i o n of enhanced  calcite  p r e c i p i t a t i o n . The enhancement of i r o n l i m i t a t i o n by p r e c i p i t a t i o n is consistent  mobility is  suppressed i n c a l c a r e o u s a r e a s .  consistent  s o i l s have l e s s a v a i l a b l e i r o n  Stauffer s 1  (1985). iron  hypothesis  calcareous  (Brown 1979). S t a u f f e r d i d not  propose a mechanism f o r the suppression of  Stauffer*s  i r o n because  with s o i l s t u d i e s t h a t have shown t h a t  My o b s e r v a t i o n s  calcite  with o b s e r v a t i o n s by S t a u f f e r  He proposed t h a t hardwater l a k e s have l i t t l e  is  iron  iron mobility.  i n F r i s k e n Lake were more s p e c i f i c  i n t h a t the carbonate suppression of  than  i r o n m o b i l i t y was  116 produced i n the l a k e , hypothesis  to e x p l a i n t h i s o b s e r v a t i o n ,  d i r e c t l y to c a l c i t e , was not a s s o c i a t e d (Table 14)  not i n the drainage b a s i n . The s i m p l e s t t h a t i r o n adsorbs  i s not supported by two o b s e r v a t i o n s .  with c a l c i t e c r y s t a l s  nor was i r o n p o s i t i v e l y  sediments (Table A3, F i g . 30,  Iron  in the water column  c o r r e l a t e d to c a l c i t e  i n the  31).  Without an assessment of i r o n a v a i l a b i l i t y , the l a c k of F e calcite  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 d u r i n g a l l  observations  of n a t u r a l c a l c i t e p r e c i p i t a t i o n i n t h i s  concentrations  hypothesis  i s t h a t most of  i r o n i n F r i s k e n and Black lakes was u n r e a c t i v e . i s c o n s i s t e n t with the e s t a b l i s h e d  a e r o b i c and a l k a l i n e lake water,  hypothesis  the m a j o r i t y of  p h y s i o l o g i c a l l y u n a v a i l a b l e forms  (Wetzel  The r e c i p r o c a l of S t a u f f e r ' s little  c o n s i s t e n t with the geochemistry  Ripl  is  (1986). He found that  sulphide; thus, must f i r s t  hypothesis  that  in as  t h a t lakes  with  is  of the lakes upon the Thompson should be  iron  a l s o c o n s i s t e n t with o b s e r v a t i o n s iron reacts  by  p r e f e r e n t i a l l y with  f o r f e r r i c phosphate r e a c t i o n s  saturate  This  1968).  P l a t e a u ; l a k e s with high phosphorus c o n c e n t r a t i o n s T h i s hypothesis  the  i r o n occurs  i r o n w i l l have high phosphorus c o n c e n t r a t i o n s  limited.  iron  i n the l a k e water were at a y e a r l y h i g h . The most  p l a u s i b l e e x p l a n a t i o n f o r these o b s e r v a t i o n s epilimnetic  study,  sulphide p r e c i p i t a t i o n  to o c c u r ,  iron  reactions.  F e r r i c r e a c t i o n s were able to r e g u l a t e the s o l u b i l i t y of phosphorus i n Chain Lake but not i n F r i s k e n or Black lakes 26).  The s t r o n g bonding of phosphorus to i n s o l u b l e f e r r i c  resulted  i n low phosphorus c o n c e n t r a t i o n s  L a k e ' s o x i d i z e d water  (<10  (Fig. iron  u g / L ) i n Chain  i n s p r i n g and e a r l y summer. F e r r i c  117 concentrations precipitation (Birch that and  1976,  The in  the  and  and  F r i s k e n l a k e s were t o o  adsorption  Stumm and  of phosphorus t o f e r r i c  Morgan  precipitation  iron  of  i r o n was  lake sediments. Observations  to react with  of  iron  correlated  sediments,  little  p h o s p h o r u s . The  to the  in this  correlated  calcite  study;  and  centimeter.  calcium  in Frisken  of the  an  in  the  be  a p u z z l e . The  13,  times higher  and  formation  r a t e of p y r i t e  (Berner  may  iron  was  of phosphorus and  able was  positively  from sediments i n  fold  faster  below  formation than  other  were  not  one  i n Chain  Lake  i n F r i s k e n Lake  F i g . 8).  proportion  iron  of  availability,  iron  concentrations should  (Berner  were o f t e n o x i d i z e d  sediment  undetectable  o f p y r i t e between C h a i n  availability  of  In F r i s k e n , Roche,  concentration  c a r b o n a t e was  i n Chain Lake than  undetectable  well  sedimentation  phosphorus c o n c e n t r a t i o n s  e v a l u a t i o n of  formation  13).  distribution iron  net  reflected  concentration.  more t h a n t e n  (Table  Without  formation  iron  Moreover, the  s e d i m e n t s was  pyrite  indicate  t h a t of C h a i n Lake  (Table  C h a i n L a k e s e d i m e n t s were d i f f e r e n t  sulphur  hydroxide  saturated  that the  limitation  n e g a t i v e l y c o r r e l a t e d t o the  had  not  i n F r i s k e n Lake i s o n l y a t h i r d  Yellow lake  sediments  for  1981). These o b s e r v a t i o n s  g e o c h e m i s t r y o f t h e w a t e r column was  a reflection  lakes  low  lakes.  organic-C be  or  sulphide Black  in Black  and  f o u n d as  the d i f f e r e n c e s  Frisken lakes p y r i t e was  have l i m i t e d  (Murphy 1 9 8 5 ) ;  sulphate  reduction  1971). Moreover, C h a i n Lake  ( F i g . 7)  1971). In  and  three  i n F r i s k e n Lake. C h a i n Lake of s u l p h a t e  oxygen s u p p r e s s e s  s p i t e of  would  often thus, and  sediments  pyrite  some a d v e r s e c o n d i t i o n s ,  the  118 r a t e of p y r i t e faster the  than  i n Chain  limiting  factors  s u l p h a t e was  that r e s t r i c t  of sulphate;  the lowest  i n hypolimnetic  F u r t h e r m o r e , t h e low o x y g e n  ferric  i n anoxic  Calcite precipitation  of p y r i t e ,  for pyrite  1 9 7 1 ) . The  a refractory  iron  mineral.  bloom i n F r i s k e n Lake site  for pyrite  reactive iron,  1 9 7 1 ) . The f o r m a t i o n  of p y r i t e  minimize the r e c y c l i n g  i n an a n o x i c  l a k e s would b l o c k suppression  (Berner  e n v i r o n m e n t would  sediment  of a l g a l  formation  iron  i n Chain  r e l e a s e (Table  decrease  or  1 3 ) . The  b i o m a s s by t h e l i m e t r e a t m e n t  F r i s k e n L a k e w o u l d be p a r t i a l l y m e d i a t e d by e n h a n c e d f o r m a t i o n . The r a p i d  matter  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  long-term  the  organic  b a c t e r i a t o p r o d u c e s u l p h i d e , would be h i g h e s t h e r e  Frisken  was  of iron m o b i l i t y i n a l k a l i n e  f o r m a t i o n . The o p t i m a l  formation,  iron  proceeds  i n F r i s k e n L a k e would be t h e s u r f a c e s e d i m e n t s ;  constituents and  an  of i r o n .  o f 90% o f t h e a l g a l  must have e n h a n c e d p y r i t e  of  i n F r i s k e n Lake  o f low a v a i l a b i l i t y  i s the formation  formation  (Berner  formation  mechanism o f s u p p r e s s i o n  formation  pyrite  in chlorite  slow r a t e of p y r i t e  a reflection  f o r m a t i o n . The p r e s e n c e  chlorite;  r e a d i l y by u s i n g t h e i r o n  was  ( F i g . 7) i n F r i s k e n  F r i s k e n Lake sediments i n d i c a t e d  complex more s t a b l e t h a n  lakes  c o n c e n t r a t i o n of  (Murphy e t a l . 1 9 8 5 ) .  concentrations  have e n h a n c e d p y r i t e  The  overcame  formation.  observed  samples  Lake s h o u l d  largely  of iron  500 ug/L and t h e odour o f h y d r o g e n s u l p h i d e  always obvious  iron  pyrite  t e n times  r e d u c t i o n i n F r i s k e n L a k e was n o t s t r o n g l y l i m i t e d  the supply  relatively  L a k e was more t h a n  i n F r i s k e n Lake; t h e a v a i l a b i l i t y  Sulphate by  formation  of  pyrite  o f pH by 0.1 t o 0.2 u n i t s i n 24 h  119 in  the anoxic  hypolimnion  after  lime treatment  d e c a y o f a l g a e . I f most o f t h e b a c t e r i a l associated could of  with  have g e n e r a t e d  s u l p h a t e was  could  s u l p h a t e r e d u c t i o n then  form The  as much as  about  5 uM;  100  thus,  uM  indicated  reductive activity the b a c t e r i a l  of H^S.  The  o n l y about h a l f  concentration limitation  formation  of s u l p h a t e  (Berner  is directly  activity  concentration of the  related  1971); t h u s ,  5-10  i n F r i s k e n L a k e . The  and  to  typical  r a t e of p y r i t e  predicts, the Lake  4.3  iron  r e l e a s e from  epilimnetic  formation  Sediment I r o n  times  higher  study,  iron  i r o n deeper  not  of a n o x i a .  reduced  mg/L. in  hypothesis  much l e s s  than  half  of F r i s k e n  iron  r e l e a s e of Chain  Lake  (1941, 1 9 4 2 ) , i . e .  Iron release in Black  and  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 i n anoxic water  i n s o l u b l e p r e v i o u s l y should  gradual decrease  i n the  this  t h e model o f M o r t i m e r  t h a t was  i n s o l u b l e . The  4  2).  c o n c e n t r a t i o n . P y r i t e would n o t d i s s o l v e ferrous  than  hypolimnetic  i n the hypolimnion  o n l y the sediment  the onset  l a k e s was  The  Release  consistent with  release with  As  o f B l a c k Lake had  c o n c e n t r a t i o n observed  ( T a b l e 4, A p p e n d i x  Frisken  iron  i n B l a c k L a k e would r e s u l t  the sediments.  the hypolimnion  In t h i s was  the  the degree of  s u l p h a t e c o n c e n t r a t i o n s i n B l a c k L a k e i n J u n e were 5 and  iron  iron  would be much g r e a t e r i n l a k e s w i t h more s u l p h a t e .  s u l p h a t e c o n c e n t r a t i o n i n B l a c k L a k e was  less  were  pyrite.  r a t e of p y r i t e  A faster  rapid  sediments  to ferrous iron,  remain  i n the c o n c e n t r a t i o n of  indicates that f e r r i c  apparently  i n mid  and  ferric  iron i s  summer, w h i c h  then  120 either  enters  eventually  the  w a t e r column or  Lake  an  at a l l s i t e s .  (mid-July).  important v a r i a b l e  Iron  This  release  (late  not  profundal  effect the  the  of  occurred  the  seasonal  earliest  sites.  Iron  not  the  aerated  in Frisken  the in  aeration  s i d e of  iron release  until  Black because  August.  seemed t o  change i n i r o n c o n c e n t r a t i o n  Black  10°C  Lake perhaps  warm above 5°C  sediment  lake  iron  in Chain  release  s e d i m e n t s by  on  last  sediments d i d  t e m p e r a t u r e on  strong  sediment  s e d i m e n t s were warmer t h a n  2.6°C warming o f  release  i n the  occurred  other  t o enhance i r o n r e l e a s e  Lake. Iron the  occur u n t i l  July). A  appeared  to  s h a l l o w l a k e mixes r e a d i l y ; t h u s ,  s e d i m e n t s were warmer t h a n t h e Lake d i d  sulphide  form p y r i t e .  T e m p e r a t u r e was release  reacts with  in the  The  produce water  columns. The  temperature of  p h y s i c a l mixing; i n f l u e n c e the related in  my  t h e s e two  sediment  to the  the  pattern  of  the  the  c o n t r o l s i d e of B l a c k The the  sediment  coincidence release  metabolic  of  iron release  activity  analogous t o the sediments  can  of  synchrony  to  more c l o s e l y  physical  mixing  a l l observations  in Frisken  by  except  Lake r e l a t i v e  to  Lake.  the  i s an  sedimentation  of t h e  iron indicates that  important  influence  on  aspect sediment  microbial mediation  (Ryding  in close  t h a n was  explain  iron release  sediment  release. A microbial  lake  v a r i a b l e s act  study; p h y s i c a l mixing slower  is largely controlled  i r o n r e l e a s e . T e m p e r a t u r e was  for  and  sediment  of  Anabaena b l o o m microbial  sediment  release  and  iron is  of phosphorus r e l e a s e  1 9 8 5 ) ; m i c r o b e s e n h a n c e or  sediment phosphorus r e l e a s e  of  iron  from  mediate  temperature enhances  microbial  121 metabolic a c t i v i t y .  4.4  Interrelationship  of I r o n L i m i t a t i o n ,  Chelation  and C a l c i t e  Precipitation To the  assess completely the iron  use of s e v e r a l  reactions,  l i m i t a t i o n of a lake  a s s a y s . Moreover, t h e impact  s u c h as c a l c i t e p r e c i p i t a t i o n ,  s y n c h r o n y o r many i r o n discovery that  of associated  must be r e s o l v e d i n  b i o a s s a y s c a n n o t be r e s o l v e d . The  calcite precipitation  response t o i r o n  requires  enrichment provided  could  strongly  insight  regulate the  i n t o many  o b s e r v a t i o n s . However, t h e e x p e r i m e n t a l u s e o f a c h e l a t o r t o maintain  iron  limitation  in solution  made r e s o l u 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  stimulate algal  and b a c t e r i a l  thus, a p r o c e s s o t h e r than  of the e f f e c t  difficult.  productivity  iron  of iron  EDTA a p p e a r e d t o  more t h a n Fe-EDTA;  a v a i l a b i l i t y may have b e e n  important. A  likely  explanation  primary production have s u p p r e s s e d e a s i l y blocked (Reynolds  and b a c t e r i a l  heterotrophy  calcite precipitation. by o r g a n i c  i s that  Calcite  EDTA  of may  precipitation i s  compounds, e s p e c i a l l y  chelators  1 9 7 8 ) . T h e s m a l l e r d e g r e e o f p h o s p h o r u s and c a l c i u m  precipitation  i n t h e EDTA l i m n o c o r r a l s  calcite precipitation calcite  f o r t h e a p p a r e n t EDTA s t i m u l a t i o n  precipitation,  would have a much which would r e s u l t i r o n would suppressing  indicated  occurred. Relative the algae  longer  less  to limnocorrals  i n t h e Na-EDTA  r e s i d e n c e time  that  limnocorrals  i n the euphotic  i n greater productivity.  with  The f i r m  zone  binding of  have p r e v e n t e d EDTA i n t h e Fe-EDTA l i m n o c o r r a l s calcite precipitation.  Iron  stimulated  from  microbes i n  122 the  Fe-EDTA l i m n o c o r r a l s ,  b u t t h e u s e o f EDTA as a c o n t r o l  t r e a t m e n t weakened t h e d e t e c t i o n  of t h e i r o n enrichment  response.  T h e s e d o u b t s a b o u t EDTA l e d t o t h e d e c i s i o n t o r e p e a t t h e iron  enrichment experiments with c i t r a t e  Citrate plants  i s a weak c h e l a t o r (Tiffin  in  that  and many m i c r o b e s  1966) c a n u t i l i z e  rate of microbial  citrate  was c o m p l e t e l y m e t a b o l i z e d provided  concentrations  iron chelated  an e x c e l l e n t o p p o r t u n i t y  oxygen c o n c e n t r a t i o n s  significant limiting  4.5  limnocorrals  evidence that  the algal  limnocorrals  A effects  study of a l g a l  precipitation.  limnocorrals  algal  oxygen  i r o n seemed t o was  had s i g n i f i c a n t l y limnocorrals  oxygen p r o d u c t i o n .  i n B l a c k Lake p r o v i d e d  an i n a d e q u a t e s u p p l y  of Iron  The  highly  o f i r o n was (Fig. 15).  Availability  siderophores  of iron limitation  had l o w e r  Once t h e c i t r a t e  p r o d u c t i v i t y o f B l a c k Lake  Microbial Control  rate of c i t r a t e  than t h e s o d i u m - c i t r a t e  3); iron stimulated  ferric-citrate  fortunate  t o observe  the control limnocorrals;  assimilated, the f e r r i c - c i t r a t e  (Appendix  was r e q u i r e d  t o separate the  from t h o s e produced by c a l c i t e  The s i d e 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 o f  siderophores  ( s e c t i o n 3 . 3 ) ; however, t h e p u r i t y o f t h e  siderophores  was n o t r i g o r o u s l y e s t a b l i s h e d . A l t h o u g h C o r b e t t  Chipko  and  enrichment.  enhance b a c t e r i a l oxygen u t i l i z a t i o n .  higher  19 81b)  by c i t r a t e . The  a f t e r a few d a y s . T h i s  the f e r r i c - c i t r a t e than  (Neilands  i n s o l u t i o n f o r days and t h e c h e l a t o r  long-term responses t o i r o n Initially,  chelator.  a s s i m i l a t i o n i n my s t u d y was  i r o n was m a i n t a i n e d  utilization  as t h e i r o n  and  (1978) f o u n d t h a t Sephadex c h r o m a t o g r a p h y o f h y d r o x a m i c  123 acids resulted e x c h a n g e and purified  my  i n compounds t h a t were more t h a n  Sephadex c h r o m a t o g r a p h y may siderophore  the b a c t e r i a l  This required  study  thus,  production, microbial  and  indicated  completely  no e f f e c t  of  on  the  had  t o be  heterotrophy bioassays of  and  iron  availability  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  small bottles  algal  ion  studies.  of l i m i t e d  regulation  over  t h i s u n c e r t a i n t y has  of the m i c r o b i a l a s p e c t s of  the use  chelators;  control  o f my  have  pure,  i s o l a t e s . With the p o s s i b l e exception  heterotrophy,  interpretation  not  99%  iron  used.  Growth,  indicated  availability  that  primary the  exerted a strong  b a c t e r i a l growth. A n a l y s i s of l a k e water  that c h e l a t o r s could 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  dissolved by  i r o n . The  e x c r e t i o n o f c h e l a t o r s was  Sephadex c h r o m a t o g r a p h y and  chelators  to control m i c r o b i a l metabolic  demonstrated The iron  in primary  specificity  ( F i g 18)  isolates  t h e FeBC a s s a y .  the hypothesis  The  activity  ability  isolates  Fe  of  was  heterotrophy  of the s i d e r o p h o r e  supports  is related  p r o d u c t i o n and  detected with  in  assays. chelating  that the a c t i v i t y  of  t o s i d e r o p h o r e s . Moreover, t h i s d a t a  these  disputes  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  algal  p r o d u c t i v i t y by  complexing  toxic  iron-  saturated  that et  i n my  assays,  s i d e r o p h o r e would not d e t o x i f y another  only a concentrated affinity  metals;  copper  treatment  metal.  c o u l d overcome a  f o r i r o n . T h i s h i g h degree of s p e c i f i c i t y  observed  a l . (1971),  siderophores.  by N e i l a n d s and  Emery  (1957),  an  In l a k e s ,  siderophore  is similar  Anderegg e t a l . (1963),  (1971) w i t h b a c t e r i a l and  fungal  to  Davis  124 The  specificity  indicate the  that  cell.  and  a strong  the  c o n t r o l of  Blue-green algae  maintaining  chelators  d i s t r i b u t i o n of a l g a l iron availability  a p p e a r t o have two  in close proximity  Anabaena c y l i n d r i c a  produces a c h e l a t o r  true  alga  and  solution. This  restrict  urn t h i c k l a y e r ) flos-aquae colloidal  excretes fibril  chelators  that  thaw. F r o z e n and  when f r o z e n ,  that  of  the  and  cell  thawed, and  refiltered refiltered  d e h y d r a t e d more e f f e c t i v e l y  freeze-thawing. Dehydrated  fibrils  (0.35 A.  to  isolating  Chelation freeze-  t h a t were thawed  deposit  of  fibrils  time. I s o l a t e s that  did  not  leave  a slime  a second time. F i b r i l s  by  freeze-drying  are  a  surface.  t h a n a f t e r one  a slimy  thawed a t h i r d  thawed, and  should  material  freeze-thawing.  f r o m Anabaena c u l t u r e s left  urn)  (Leppard e t a l . 1977).  after freeze-drying  twice s t i l l  in  (2.6  i s l o o s e l y adsorbed  extension  t h a n was  cell.  thick  a more e f f e c t i v e method o f  filtrates  when f r o z e n  freeze-dried,  t o be  was  higher  refiltered  filters  i s an  of  ( L e p p a r d e t a l . 19 7 7 ) .  that  near  a p p e a r s t o be  material  cell  cylindrica  a chelator  from f i b r i l s was  algal  p r o d u c e s much l e s s c o l l o i d a l  t h a n A.  Freeze-drying  capacity  fibrillar  w a t e r movement a r o u n d t h e  Anabaena f l o s - a q u a e  t o an that  occurs  strategies  i s covered with a very  dense l a y e r of c o l l o i d a l  chelators  relatively  than  on were layer appear  by  insoluble in  water. Resolution  that  bonded t o c o l l o i d a l concentration in  the  Anabaena f l o s - a q u a e  fibrils  of c h e l a t o r s .  FeBC f r o m 2 uM  between d i s s o l v e d  to and  80  uM  provides In  chelator  i n s i g h t i n t o the  a culture f i l t r a t e  after ultrafiltration,  adsorbed  c h e l a t o r s was  2/78  that  is loosely in  situ increased  the  equilibrium  uM.  The  125 f o l l o w i n g measurements were u s e d t o d e r i v e concentration  the  o f c h e l a t o r ; mean c e l l volume o f  in  situ  2 um  3  ,  cell  length  7 of  um,  1.8  cell  w i d t h of  and  fibril  length  the  p h y c o s p h e r e was  concentration  of  reactivity  the  the  of  The algae This  2.5%  total  (Leppard the  i n the  of  bioassays  that  filtered  (Plumb and The  Lee  medium. The  study of  the  use  criticism  seems l e s s  chelator  i n t o other  volume  of  the mM.  The  however, i f  in solution  with f i b r i l s  product  s i g n i f i c a n c e of now  4.3  i s unresolved;  a r e more c o n c e n t r a t e d  1973)  um,  a l . ) . The  was  of d i s s o l v e d c h e l a t o r  excretion  justifies  water exaggerates the  10  in  the  pM.  " l o s e " an also  et  c h e l a t o r was  concentration 110  1977  p h y c o s p h e r e was  chelator the  c e l l volume o f  c u l t u r e medium; t h u s ,  weak a s s o c i a t i o n o f c h e l a t o r s  discovery  insight  of  adsorbed  about  would not  freshly  1.8%  then the  p h y c o s p h e r e was  um  0.35  chelator  estimate that  accurate,  in  of  um,  1.2  3  of  i n the  explains bulk  s o l u t i o n s of  chelators in  that concentrating  the  lake  complexation  serious.  m i c r o d i s t r i b u t i o n provides  useful  (Murphy 1 9 7 6 ) . E a r l i e r 55 o b s e r v a t i o n s w i t h g e l chromatography of Fe-labelled algal f i l t r a t e s c o u l d n o t be c o m p l e t e l y r e s o l v e d . An u n c o n c e n t r a t e d filtrate  could  chelator  water.  than those found  organic-iron  why  carry very  studies  little  ^Fe  through the  column and  most  55 of  the  Fe  colloidal diluted  that passed  fraction.  to the  virtually w i t h a low  I f the  original  a l l of  the  through the  medium was  concentration  iron  could  indicate that  the  associated  freeze-dried, of the  culture  c h e l a t o r . The  enigma o f my  M.Sc.  with  i t could  be  and  p a s s t h r o u g h a Sephadex  molecular weight organic  U.B.C. s t u d i e s  column was  column  r e s u l t s of study  was  my  a  126 p r o d u c e d by medium and  the  adsorption  isolation  of  of  the  the  chelator  chelator  to f i b r i l s  from f i b r i l s  by  in  fresh  freeze-  drying. One in  the  problem r e l a t e d t o the  B l a c k Lake s t u d y ,  concentrated,  frozen,  membrane. C h e l a t i o n  or  i s that passed  capacity  underestimated. Fortunately, simple.  The  greater  than the  chelators  chelator  could  t h r o u g h an  i n t e r p r e t a t i o n of  iron  the  4.6  S i d e r o p h o r e s as  M e d i a t o r s of A l g a l  The  that  inhibit  or  algal  which microbes could  stimulate  coexisting  species  succession.  siderophore increased  The  culture  and  decreasing  Thus,  1974,  Elbrachter  isolates  W o l f e and  these studies,  the  the  could  species  the  access  either  but  can  not  iron  1979,  be  1967,  of  concentration microbial  availability. found  i n many  Fogg e t a l .  1978,  a  influence  m e d i a t i o n of  Keating  Rice  was  microbial  r a t e of u t i l i z a t i o n  (Hutchinson 1976,  and  still  grow.  siderophores  a siderophore  is  Succession  algal  i n B l a c k L a k e as  lake bioassays  data  l a k e water  iron  c h a n g e s i n a l l e l o p a t h y can  W i l s o n e t a l . 1979, of  the  would d e c r e a s e w i t h a change i n i r o n  seasonal  all  one  indicates that  indicates that  Similar  Hellebust  siderophore  ( F i g . 20)  isolates  succession  of  soluble  could  Kayser  1973,  1979,  Chan e t a l . 1 9 8 0 ) .  f i l t r a t e from e i t h e r a c u l t u r e or  l a k e w a t e r sample s u p p r e s s e d  the  highly probable that  these f i l t r a t e s  some o f  growth of  not  have b e e n  the  concentration.  iron  observation  found  ultrafiltration  i n t h e s e samples may  complex a l l o f  regulate  association  some l a k e w a t e r s a m p l e s were  measured c h e l a t i o n c a p a c i t y dissolved  fibril  another a l g a . contained  In a  It is  to  127 siderophores. Laboratory  s t u d i e s have d e m o n s t r a t e d  compounds, w i t h many p r o p e r t i e s o f t h e growth of a competing The  a n t a g o n i s m was  that  iron  primarily  not  and  after  the parent  The indicates algal  The  concentrated  (Paerl  specific  toxicity  to l y s i s  clumps. S y m b i o t i c  (19 8 2 a ) .  and  algal  suppress  iron  1976).  availability  inhibition  siderophore  from Anabaena  the c e l l s had  flos-  basiliensis  became r o u n d e d  ruptured.  siderophore  isolates  could regulate symbiotic a s s o c i a t i o n s a s s o c i a t i o n s between b a c t e r i a and  blue-green  I r o n c o m p l e x e d by was  cells  algal  Murphy  l y s e Scenedesmus  of t h e s e  that siderophores  algae  the c h e l a t o r produced  preferentially  near the  have b e e n documented  a s s i m i l a t e d by  h e t e r o c y s t s o f A.  by both  oscillaroides  1982b).  Other aspects s u c c e s s i o n . The iron-limited indicates  of  comparatively  iron  assimilation  restricted  w a t e r and  that during  low-affinity  1980).  of  algal  s e v e r a l h o u r s most o f t h e c e l l s  Anabaena o s c i l l a r o i d e s bacteria  could  alga. Prior  clumps o f n i t r o g e n f i x i n g Paerl  could  ( F i g . 21;  (lOx mature c u l t u r e c o n c e n t r a t i o n )  but  by  species  a suppression  aquae or Anabaena c y l i n d r i c a  in  siderophores,  a d d i t i o n c o u l d r e v e r s e . However, o t h e r  r e a c t i o n s were p r e s e n t . isolates  algal  that excreted  algal  inefficient  also influence algal  g r o w t h o f A p h a n i zomenon i n  i t s rapid  iron-uptake  may  growth  blooms t h i s  in iron-rich alga u t i l i z e s  system. These a s s i m i l a t i o n and  water a  systems  n o n - s p e c i f i c (Neilands et a l .  are  128 4.7  Siderophores Three a l g a l  suppress  siderophore  bacterial  respiration by  as R e g u l a t o r s  i s o l a t e s were a b l e t o  assimilation  isolates  indicated  s i d e r o p h o r e s . The Black  Lake a l s o  of simple  f r o m two  Delucca  idea that iron bacteria  is  The  Siuda  t h a t Anabaena i n h i b i t e d  McCracken  availability  1977,  algae  (1978).  mediates a l g a l  Chrost  bacteria.  suppression  in The  (Chrost the  of  They hypothesized  b a c t e r i a more i n t h e  excretory  light  i n the dark.  than  s i d e r o p h o r e or a n t i b i o t i c  production  Lake, the  iron-binding data  chelators  are  4-10  fold  heterotrophy  limitation  is  Chrost  that algal  of g r e a t e r i r o n  a reflection  isolates  (1973,1975) and  c o u l d be  and  and  products Chrost's  and  chi a  higher  i n the e p i l i m n i o n . In  ( T a b l e 11,  F i g u r e 19)  more c o n c e n t r a t e d  Black  indicated  that  i n the e p i l i m n i o n than  hypolimnion.  The  p r o d u c t i o n of s i d e r o p h o r e s  iron-rich  environments l i k e  s h o u l d be  the hypolimnion  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 reflect (i.e.  algal  1 9 8 1 ) ; however,  siderophore  between b a c t e r i a l  the  the  i s w e l l known  Reichardt  negative correlations  in  enhanced  new.  t o o b s e r v a t i o n s by  suppressed  greatly  The  s e a s o n a l 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  s u p p r e s s i o n o f b a c t e r i a by  similar  o r g a n i c compounds.  a s u p p r e s s i o n o f b a c t e r i a by  indicated  and  greatly  Anabaena s p e c i e s . T h e s e  s u p p r e s s i o n o f b a c t e r i a by b l u e - g r e e n 1973,  Heterotrophy  o f a s s i m i l a t e d o r g a n i c compounds was  siderophore  disruptions  of B a c t e r i a l  a lower  iron  iron-limited  capacity  than  requirement.  t h e e p i l i m n i a . The  of a l a k e  in hypolimnia  Hypolimnia  l i m n o c o r r a l s ) had  minimal i n  with  (Neilands may  little  also iron  much l e s s i r o n - b i n d i n g  l a c k of c h e l a t o r s i n the  129 h y p o l i m n i a was Bacteria  can  g r e a t e r e x c r e t i o n of  iron  than  (section  rapid. in  r e q u i r e more  enhancement by  light  of  limitation  The  hypolimnion  the hypolimnion  could  i t settles  does not can  of  have a  become  a residual  o x y g e n demand can  renewable  would  i n f l u e n c e the  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  (White  accumulate i n in  e p i l i m n i o n d u r i n g t h e Anabaena b l o o m i n B l a c k L a k e as of h e t e r o t r o p h y  the  anoxic  A s u p p r e s s i o n of oxygen consumption  inhibition  result  into  d e c a y o f o r g a n i c compounds i s i n e f f i c i e n t  a l . 1968); t h u s , hypolimnion.  iron  being oxidized a f t e r  s u p p l y of o x y g e n ; t h u s , Anoxic  the  1.2).  i n t h e e p i l i m n i o n by  hypolimnion.  iron  as  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 d e g r a d a t i o n  the organic matter  iron-rich  the  the microbes  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 s u c h  organic matter  the  and  subsequent siderophore e x c r e t i o n c o u l d r e f l e c t  In the  et  c h e l a t o r s by  An  activity.  l i m n o c o r r a l s was  i n d i c a t e t h a t a u t o t r o p h i c microbes  photooxidation  rapidly.  iron  ( N e i l a n d s 1967)  of these  h e t e r o t r o p h i c microbes.  demand and need o f  t o low m i c r o b i a l m e t a b o l i c  i n the hypolimnia  e p i l i m n i a may  in  related  produce siderophores  heterotrophy The  not  the  a result  of  seasonal  which enhances  pyrite  formation. 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  experiments  were i n c o m p l e t e l y r e s o l v e d . I n c o n t r a s t t o  experiments, not  the a d d i t i o n  overcome t h e t o x i c i t y  unexpected another  response  of  iron  isolates  supports  and  in heterotrophy  of the s i d e r o p h o r e the a l t e r n a t i v e  compound w i t h a n t i b i o t i c  siderophore  inhibition  was  also  induced  by  experiments  isolates. hypothesis  p r o p e r t i e s was  also  iron  algal  This that  i n the  limitation.  did  130 Many m i c r o b e s e x c r e t e a n t i b i o t i c s t h a t siderophores  (Prelog  can  with  interact  microbial  antibiotics  microbes soils  species  complicates  competition;  availability  The  1968, Winkelmann 1 9 7 4 ) . T h e s e  siderophores t o result  antagonism of other uncertainty  control  that  however, t h e s i g n i f i c a n c e by t h e p o t e n t i a l  siderophore  general  regulator  changes  iron  effect of  control  of p o p u l a t i o n  availability  presence of  had o v e r  aquatic  of microbes i n relationships  i n humans  ( W e i n b e r g 1974,  over  availability  iron  structure  can s h i f t  and any f o r c e  community  is a  that  structure.  Precipitation  Although t h i s availability  of iron  limitation.  isolates  bacteria  1978). The m i c r o b i a l  Calcite  This  1981b, Emery 1 9 8 2 ) , p l a n t - p a t h o g e n  (Emery 1 9 8 2 ) , and e n t e r i c  4.8  antibiotics  o f t h e mechanisms o f  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  (Neilands  1975,  (Musher e t a l . 1 9 7 4 ) .  a r e i n d u c e d by i r o n  homologs o f  in synergistic  an i n t e r p r e t a t i o n  i s unaltered that  are structural  thesis  on a l g a l  of i r o n  began as a s t u d y o f t h e e f f e c t  periodicity,  availability  the long-term  limnetic  other  on a l g a l  factors  modified the  productivity.  response t o i r o n  of iron  Interpretation  enrichment  required  a  study  o f c a l c i u m c a r b o n a t e g e o c h e m i s t r y . The c r a s h o f b l u e - g r e e n  algal  blooms was sometimes r e l a t e d  precipitation, the  precipitation  suppression key  and o n l y  reaction  indirectly to iron  chemistry.  o f c a l c i t e seemed t o overcome t h e  of b a c t e r i a l that  directly to calcite  heterotrophy. C a l c i t e  i s related  to iron  chemistry  Moreover, microbial  precipitation i n several  is a  ways.  131 C a r b o n a t e c h e m i s t r y has been w e l l importance of c a l c i t e p r e c i p i t a t i o n g r o w t h has o n l y r e c e n t l y precipitation  been w e l l  studies  al.  o f phosphorus  Calcite  and W e t z e l 1972,  in lakes  phosphorus.  Calcite  has been d e m o n s t r a t e d i n l a b o r a t o r y Stumm and Morgan 1981)  ( R o s s k n e c h t 1980,  V a r i a b i l i t y of C a l c i t e  A v n i m e l a c h 1983,  and  Murphy e t  Precipitation  A l t h o u g h t h e thermodynamics characterized  (Stumm and Morgan  precipitation  is difficult  restricted organic  Hanor  by k i n e t i c  compounds  Reddy 1979)  limitations  0.9%  precipitation. 14.6°C  and W e t z e l  1974,  of the  organic  precipitation  The c o o l i n g  was  1973,  organic precipitating  carbon  suppression  must c o n t r i b u t e t o t h e h i g h d e g r e e o f  o f 1982,  cold  is  W a l t e r and  cold  lakes with c a l c i t e .  weather  restricted  of the e p i l i m n e t i c  calcite  w a t e r s f r o m 20°C t o  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  f o l d . The  of  1974),  p h o s p h a t e and d i s s o l v e d  o f B l a c k and F r i s k e n  the July  Otsuki  and 10% r e s p e c t i v e l y  well  precipitation  ( B e r n e r and Morse  f l o e . P h o s p h a t e and d i s s o l v e d  supersaturation  Calcite  ( B e r n e r and Morse  studies,  calcite precipitation  In  1981), t h e i n i t i a t i o n  to predict.  and p h o s p h a t e  1 9 7 9 ) . I n my  calcite  of carbonate r e a c t i o n s a r e  (Chave and S u e s s 1970,  m a t t e r were a b o u t  5.5  algal  1983a).  4.8.1  of  of  s u p p r e s s e s a l g a l g r o w t h by e n h a n c i n g a l g a l  (Otsuki  recently  however, t h e  as a r e g u l a t o r established.  s e d i m e n t a t i o n and by p r e c i p i t a t i n g precipitation  documented;  w e a t h e r was  u n u s u a l ; moreover,  b l o c k e d when t h e w a t e r was  s u p e r s a t u r a t e d . Other f a c t o r s  still  f r o m 11.8  to  calcite greatly  must c o n t r i b u t e t o t h e 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 b e l i e v e t h a t c a l c i t e c r y s t a l formation i s prone t o spontaneous n u c l e a t i o n  (Reddy and  N a n c o l l a s 19 71,  19 78). Geochemists overcome the random length nucleation calcium  t o occur by  carbonate dust  of time f o r  saturating their laboratory  s o l u t i o n s with  (Reddy et a l . 1981). T h i s technique would  change too many v a r i a b l e s carbon and  Reynolds  (light intensity, dissolved  phosphorus c o n c e n t r a t i o n s ,  most b i o l o g i c a l s t u d i e s . A pH used i n l i m n o c o r r a l s , may  and  organic  a l g a l buoyancy) f o r  s t a t , such as Shapiro (1984) has  be a very u s e f u l t o prevent  calcite  p r e c i p i t a t i o n i n b i o a s s a y experiments. C o n s i d e r a b l e v a r i a b i l i t y i n the p r e c i p i t a t i o n i n lakes has that  i n i t i a t i o n of  calcite  been observed. Lehman (19 80)  found  "Lake water e q u i l i b r i a were so c l o s e to the s a t u r a t i o n  f o r CaCC> at the pH 3  of ca. 8.2  t h a t the  increased  limit  productivity  caused by the n u t r i e n t s l e d q u i c k l y to c a l c i t e p r e c i p i t a t i o n " . Rossknecht  (1977) proposed t h a t a lack of "seed" c r y s t a l s t o  i n i t i a t e p r e c i p i t a t i o n , r e s u l t e d i n the observed one  week delay  from the peak of a l g a l p r o d u c t i v i t y to p r e c i p i t a t i o n of  calcite.  S i m i l a r l y , Koschel et a l . (1983) have observed delays of two  to  f o u r weeks i n 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 s t i m u l 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 c o u l d  occur i n  enrichment of hardwater with a l i m i t i n g n u t r i e n t . The the l i m i t i n g n u t r i e n t causes an production  initial  which r a i s e s the pH and  T h i s r e a c t i o n was  any  a d d i t i o n of  s t i m u l a t i o n of primary  causes c a l c i t e p r e c i p i t a t i o n .  most pronounced i n the Black Lake n i t r a t e  l i m n o c o r r a l . N i t r a t e a s s i m i l a t i o n r e s u l t s i n hydroxide  excretion  133 (Goldman and Brewer stimulate Novitsky Black  19 8 0 ) .  the precipitation  has been u s e d t o  of calcium carbonate  ( M o r i t a 1980,  1981, B r o w n l e e and Murphy 1 9 8 3 ) . I r o n e n r i c h m e n t o f t h e  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  precipitation.  The d e n s e s t  limnocorrals  or i n Black  precipitation  was e i t h e r  t h r e e weeks. A t t i m e s ,  Biological  algal  calcite  blooms i n e i t h e r t h e  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  not observed  o r was d e l a y e d  the p e r i o d i c i t y  of blue-green  was r e g u l a t e d by c a l c i t e  4.9  Moreover, n i t r a t e  by more algal  than  blooms  precipitation.  Induction  of C a l c i t e  Precipitation  Another cause of t h e 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 precipitation  i s biological  precipitation. type  Certainly,  algae  of c r y s t a l p r e c i p i t a t e  algae  present  energy  mediate c r y s t a l n u c l e a t i o n ; t h e  formed  geology  precipitation  and b i o l o g y  carbonate  precipitation  metabolic  a c t i v i t y that biochemical  Hsu  i s d e p e n d e n t upon t h e t y p e o f  i n calcium carbonate  i n both  discriminated  carbonate  ( D a r l e y 1 9 7 4 ) . However, w h e t h e r m i c r o b e s  released  controversy  r e g u l a t i o n of calcium  i s so c l o s e l y  from geochemical  (Kuznetsov  related  gain  i s an o l d 1970).  to microbial  r e a c t i o n s c a n n o t be  reactions  Calcium  (Megard  readily  1968, K e l t s and  1978). However, some m i c r o b e s a r e much b e t t e r c a t a l y s t s  carbonate found  precipitation  than  others  that c a l c i t e precipitation  of c a l c i u m  ( M o r i t a 1980). S t a b e l  i n Lake C o n s t a n c e ,  Switzerland,  was c a t a l y z e d by o n l y some o f t h e many s p e c i e s o f a l g a e growing  i n water  (19 86)  a l s o found  supersaturated  with  that populations  calcium carbonate.  of algae  able  (1986)  observed Stabel  to initiate  134 calcite the  precipitation  composition  of  arise  the  biological  carbonate  precipitation;  mediation  of  the  algal  regularly  there  at defined periods.  community can  i s an  periodicity  Thus,  influence calcium  important  biological  i m p o s e d by  calcite  precipitation.  4.10  Microbial Other  Responses t o C a l c i t e  biological  precipitation  are  precipitation  by  the  lysis  algae  about  i n the  during in  of  19 79  the  control  i n the  precipitation Rapid if  production. production  1)  siderophores prevent to  carbonate 2)  lysed. Lysis  the  could  observed  activity.  occurred  in Black  calcite  siderophore  siderophore  could produce t h i s  react with  of  of  the  fibrillar  the  suppression. material that  reaction could  physically  iron-binding study,  fibrillar  occur  siderophore  enhance  a suppression  r e a c t i o n s . In  could  Lake  S z a n i s z l o et a l .  enhanced  associated with. This  The  also  Lake.  siderophore  coprecipitation  complexation.  algae  i n d u c t i o n of  c h e l a t o r s from the  Calcium  of  in  the  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 would  siderophore  isolate  calcite  t h e A p h a n i zomenon. None o f  lime-mediated  indicate  are  of  t h a t was  Three mechanisms  Calcite  carbonate  calcium chloride resulted  microbial necessity to  could  reactivity.  initiation  that calcium carbonate The  calcium  precipitation  suppressed  found  of  flasks  Frisken  changes  calcite  (19 81)  of  The  a d d i t i o n s of  a third  calcite  and  reactions during  unresolved. the  Precipitation  colloids  failure  resulted  in  the c h e l a t o r s .  compete d i r e c t l y  r e a c t i o n s of  calcium  with  iron  (Hider et  for  siderophore  a l . 1982)  and  135 calcite  (Reynolds  1978) w i t h  some c h e l a t o r s  are well  established;  however, t h e s p e c i f i c i t y o f t h e s i d e r o p h o r e c h e l a t i o n that  other 3)  reactions  Calcite  assimilation. polymyxin  The to  with the s i t e s of siderophore  type of i n t e r f e r e n c e  with the a n t i b a c t e r i a l  the s i t e s of polymyxin  of symbiotic  Aphanizomenon, initiation  iron  o f an i r o n - c h l o r o p h y l l  precipitation  chelators.  of i r o n  (30 — >  induction  (Fig.  and  with  separation  phosphorus p r e c i p i t a t i o n  rapid  assimilation  indicated  that  precipitation.  of the d i s s o l v e d  of  32  could  from F r i s k e n  by  organic L a k e by t h e  Calcite producing  c a l c i t e p r e c i p i t a t i o n . In of a l g a l  oxygen  production  ( F i g . 1 5 ) , and t h e c o i n c i d e n c e o f  p h o s p h o r u s and c a l c i u m p r e c i p i t a t i o n carbonate p r e c i p i t a t i o n  t h e two l i m e  be a n o t h e r mechanism  blooms d u r i n g  Black Lake, t h e temporal  after  precipitation.  Phosphorus l i m i t a t i o n c o u l d of a l g a l  enhanced. The  5) may have been e s t a b l i s h e d Half  Phosphorus C o p r e c i p i t a t i o n  collapse  s y s t e m s , s u c h as  correlation  15 mg/L) was p r e c i p i t a t e d  of c a l c i u m carbonate  lead  m e d i a t e d by s i d e r o p h o r e s .  l i m i t a t i o n w o u l d be r a p i d l y  Lake  the  ions  assimilation.  assimilation  treatments of Frisken  4.11  divalent  with  a c t i v i t y o f p o l y m y x i n by  associations  m i c r o b e s w i t h weak i r o n  carbon  has been o b s e r v e d  i n h i b i t i o n o f s i d e r o p h o r e a c t i v i t y by c a l c i t e w o u l d  a collapse  For  react  (Newton 1 9 5 3 ) . Newton f o u n d t h a t  interfered blocking  exist.  could  This  suggests  showed t h a t  calcium  r e g u l a t e p h o s p h o r u s s o l u b i l i t y . The  . P-PO^ d u r i n g  • calcite  phosphorus l i m i t a t i o n c o u l d  . . . precipitation  be i n d u c e d by c a l c i t e  136 The of  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  could  remove most  the phosphorus from the e p i l i m n i o n of a hardwater l a k e .  maximum p r e c i p i t a t i o n L a k e was this  10  mg  in Black  precipitation this  CaCO^/L and  response to the  observed  induced  of  500  100  largest  Lake  ug  P/L.  P/L.  relative  precipitation insight  during  CaC0 /L) i n d i c a t e s a  equilibria  and  was  of  Morse  (1974) p r o d u c e d  i n pseudo-seawater.  not  r e s o l v e d . However,  precipitation  iron  biochemical  events  availability  on  i n t e g r a t i o n of primary  phosphorus  provided carbonate  in turn heterotrophic production.  a r e a good  potential  3  B e r n e r and  calcite  i n t o the e f f e c t  equilibrium  precipitation  i m p o r t a n c e o f g e o c h e m i c a l and  phosphorus p r e c i p i t a t i o n  Black  L i n e a r e x t r a p o l a t i o n of  degree of phosphorus p r e c i p i t a t i o n The  i n c u b a t i o n of  amount o f c a l c i t e  (50 mg  ug  in the C a C ^  The  The  carbonate  production  and  heterotrophy. The  pH  i n the  hypolimnia  e x p e r i m e n t s was  lowest  l o w e r pH  expected  i s the  production  o f CG^  anticipated  these  production  i n the  in  the  i n the  effect  epilimnia;  hypolimnia  in the  confirms  citrate  limnocorral  ferric  citrate  limnocorrals. A  result  of g r e a t e r  hypolimnia  which  of g r e a t e r primary  Fe-limnocorrals e p i l i m n i a . The  had  p h o s p h o r u s was  of t h e  more p r i m a r y  ferric  production  citrate occurred  in  the  the  oxygen  phosphorus  concentration  indicates that  o f g r e a t e r CG^  metabolized.  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  hypolimnia  in turn, i s  the g r e a t e s t  higher  a result  heterotrophic  production  of the F e - l i m n o c o r r a l s  more P - c a l c i t e d i s s o l v e d as t h a t more o r g a n i c  of the  either  production  Either reaction  occurred  l i m n o c o r r a l s and in the e p i l i m n i a .  in that  the in turn,  or  137 The  strong  c o r r e l a t i o n o f phosphorus t o c a l c i u m  sediments of Yellow, F r i s k e n , all  and Roche l a k e s  indicates that not  of the p r e c i p i t a t e d P - c a l c i t e redissolved  Although  the i n i t i a l  P-precipitate  et  a l . 1980), t h e s t a b l e  be  apatite. I n i t i a l l y ,  crystal  i n the hypolimnion.  c a n n o t be a p a t i t e  (Koutsoukos  component o f P - c a l c i t e p r e c i p i t a t i o n may  phosphorus adsorbs t o k i n k s  i n the c a l c i t e  ( B e r n e r and M o r s e 1974) and t h i s complex u l t i m a t e l y  hydroxylapatite 1 9 7 4 ) . Brown  (Stumm and L e c k i e  (1980) s u g g e s t e d  aqueous c a l c i t i c that  i nthe  that  apatite  Griffin  Lake,  and  hydroxylapatite  limestone suspensions. Ryding  lime a p p l i c a t i o n t o a lake  In F r i s k e n  19 70,  forms  Jurinak  could  (19 85)  would e n h a n c e a p a t i t e  form i n  proposed formation.  lime a p p l i c a t i o n d i d n o t appear t o enhance  formation;  about  95% o f t h e p r e c i p i t a t e d  phosphorus  redissolved. The  inverse  indicates that  c o r r e l a t i o n between i r o n and  different variables  phosphorus/calcite  c o n t r o l the s o l u b i l i t y of  t h e s e two g e o c h e m i c a l s u b s e t s . P y r i t e i s v e r y s t a b l e anoxic  s e d i m e n t s ; P - c a l c i t e c a n be d i s s o l v e d  production  of C 0 . This  difference  2  m i n e r a l s must c o n t r i b u t e high  4.11  i n these  by t h e h e t e r o t r o p h i c  in stability  o f t h e s e two  t o t h e e s t a b l i s h m e n t o f low i r o n and  phosphorus c o n c e n t r a t i o n s  i n many h a r d w a t e r  lakes.  CONCLUSIONS Iron  availability  influences  the p e r i o d i c i t y of blue-green  algal  g r o w t h . B l u e - g r e e n a l g a l blooms o c c u r  after  iron i s released  summer i n c r e a s e  from t h e lake  i n i r o n , microbes  molecular weight c h e l a t o r s  that  i n B l a c k Lake  only  s e d i m e n t s . P r i o r t o t h e mid  i n B l a c k L a k e p r o d u c e low  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 c h e l a t e d i r o n can be r e s t r i c t e d t o p a r t i c u l a r s p e c i e s . Furthermore, the c h e l a t o r c o n c e n t r a t i o n i n l a k e water can exceed 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 p r o d u c t i o n can c o n t r o l t h e a v a i l a b i l i t y of i r o n . The of  competition  f o r i r o n i s a s s o c i a t e d with t h e s u p p r e s s i o n  competing algae and b a c t e r i a by e i t h e r the d i r e c t t o x i c i t y of  siderophores  or an a s s o c i a t e d e x c r e t i o n of an a n t i b i o t i c . These  r e a c t i o n s enable some algae t o produce t h e i r  own  microenvironment. Iron l i m i t a t i o n i n l a k e s v a r i e s g r e a t l y w i t h i n g e o l o g i c a l formations  mainly because i r o n l o a d i n g v a r i e s g r e a t l y between  l a k e s i n the same area. On the Thompson P l a t e a u , t h e p o t e n t i a l for  iron a v a i l a b i l i t y  i s i n d i c a t e d by a high c o n c e n t r a t i o n of  phosphorus i n o x i d i z e d water i n e i t h e r the f a l l I r o n - l i m i t e d lakes can have t o o l i t t l e  or s p r i n g .  iron for f e r r i c  phosphate  r e a c t i o n s t o r e g u l a t e the s o l u b i l i t y of phosphorus. The l a c k of i r o n t o p r e c i p i t a t e phosphorus and t h e high l o a d i n g of phosphorus from the weathering of a p a t i t e r e s u l t s i n very c o n c e n t r a t i o n s of phosphorus O 2 0 0  high  ug/L) i n Black and F r i s k e n  l a k e s . The high i r o n and phosphorus l o a d i n g i n t o Chain Lake r e s u l t s i n high s o l u b l e phosphorus c o n c e n t r a t i o n s only f o r b r i e f p e r i o d s when oxygen c o n c e n t r a t i o n s a r e l e s s 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 i r o n - l i m i t e d hardwater l a k e s can l e a d t o r a p i d 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  f o r a t l e a s t t h r e e weeks a f t e r the  onset of c a l c i t e s u p e r s a t u r a t i o n . The d e l a y p e r i o d and the amount of  p r e c i p i t a t i o n v a r i e s g r e a t l y . In many b i o a s s a y s  i n hardwater  139 lakes,  the  easily  by  t h a n by  effect an  of  iron  analysis  of  e n r i c h m e n t can the  a measurement of  shifts  algal  i n the  unlike  in  phosphorus  lakes,  i s very high  the  in  spring  and  becomes o x i d i z e d .  phosphorus t h a t  remains a s s o c i a t e d  is stable.  carbonate, to  the  precipitation  a l g a e and algae  i n the  of  during  the  in eutrophic i f the  supply  pyrite  formation w i l l  result  in  sulphate reduction  limitation  can  sedimented i n the  lake 10%  is strongly  calcium  correlated  lakes be  sedimentation  iron by  potential  recycling  stratified  and  i n hardwater  hypolimnetic  calcite precipitation.  formation,  important  when t h e  c a l c i t e enhances the  in turn minimizes  iron  thus,  soluble  with c a l c i t e  pyrite  enhancement of  of  However, t h e  phosphorus  the  hypolimnion;  fall  h y p o l i m n i o n enhances the  precipitation  of  calcium.  many a l g a e l y s e  formation which calcite  of  of  more  equilibria  l a k e s e d i m e n t s w i t h more t h a n  distribution  distribution  The  of  the  In  in the  concentration  w a t e r m i x e s and  sediments  carbonate  c a l c i t e . 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N o t e 796.  151  1  Appendix  19 80  used  D e t a i l s of  Black  Lake L i m n o c o r r a l  Seven  l i m n o c o r r a l s of  to  test  the  productivity.  effect  meters deep w i t h  and  plywood  two  weeks w i t h  was  used  Ideally  the  20  on  350  Iron  added  was  g  were e n r i c h e d  nitrate  reagent  the  meters  float-collar  g r a d e KNO^  nitrate  have been  concentration  of  and  styrofoam  lake. enriched A  every  paddle  the l i m n o c o r r a l .  increased  immediately  200  to  after  ug  nitrate  N/L.  atomic  form  of  100  an of  EDTA-Fe c o m p l e x  200  Control  molar  was  monitored  and  the  soluble iron  absorption and  (3:1  iron  ug  Fe/L.  equivalent concentrations  treatments.  the  (Fisher).  throughout  algal  i n diameter  l i m n o c o r r a l was  meters would  between  with  one  were  e n r i c h m e n t on  exchange w i t h  concentration  Fe-EDTA t r e a t m e n t s . the  and  woven p o l y e t h y l e n e  bottom. A  water  in the  furnace  were m a i n t a i n e d  with  of  ug  E D T A : F e ) . The  graphite  iron  1980,  nitrate  e n r i c h m e n t was  by  any  four  surface  transparent  of  May  1.82  top  Experiments  sealed  to distribute  The  ratio  a  prevented  Starting  Experiments  l i m n o c o r r a l s w e r e 2.0  The  7.0  N/L.  Limnocorral  biweekly levels  EDTA-limnocorrals of  EDTA a s  l i m n o c o r r a l s were monitored  were along  the  152  19 82  B l a c k Lake L i m n o c o r r a l Limnocorrals  in  Experiment  of transparent  woven p o l y e t h y l e n e ,  d i a m e t e r and 5.0 m e t e r s d e e p , were made by F a l s e  Plastics  (Vancouver,B.C.). A c o l l a r  ultraviolet inserted  light  limnocorrals blocks  were s e a l e d  v i a attached  relative  Eight lakewater  forflotation.  external  loops.  concrete i n design  of t h e l i m n o c o r r a l s and  of the treatments. limnocorrals  pump. Two l i m n o c o r r a l s  were f i l l e d  received  commercial grade CafNO^^*  2  Two l i m n o c o r r a l s  received  The i r o n  J u n e 13-14, 1982 w i t h surface  three  using  a large  additions  Two l i m n o c o r r a l s  o f 18.4 g o f C a C N O ^ ) »  solution.  The b o t t o m o f t h e  T h e improvements  filling  2.0 m e t e r s f r o m t h e l a k e  additions  s t y r o f o a m was  and a n c h o r e d w i t h r o p e s a n d  t o 1980, f a c i l i t a t e d  replication  Creek  t h a t was r e s i s t a n t t o  was sewn t o t h e t o p and e x t r u d e d  i n t o t h e hollow c o l l a r  2.0 m e t e r s  o f 18.4 g o f  received  three  and 78.76 g o f s o d i u m  18.4 g o f C a f N O ^ ^ f  s o l u t i o n was made by f i r s t  a  n  d  a  n  citrate.  iron  d i s s o l v i n g 4.55 g  o f F e C l j and 192.12 g o f c i t r i c  a c i d and t h e n a d j u s t i n g  7.0 w i t h NaOH. Two l i m n o c o r r a l s  were l e f t  The the  12 t r e a t m e n t ,  limnocorrals.  the  liter  untreated.  n i t r a t e was n o t added t o t h e c i t r a t e  On J u n e 23, t h e n u t r i e n t s were d i s s o l v e d  o f l a k e w a t e r and pumped t h e s o l u t i o n t o f i v e  other  t h e pH t o  n u t r i e n t s were added J u n e 23, J u n e 30, a n d Aug. 12. I n  August  liters  water  depths. For  two t r e a t m e n t s t h e n u t r i e n t s were d i s s o l v e d  o f l a k e w a t e r and added t o t h e s u r f a c e  with a paddle s t i r r i n g  t h e water. With  i n 50  i n 1.0  of the limnocorral  i d e a l mixing,  the i n i t i a l  153  nutrient  concentrations  C-citrate/L,  Limnocorrals  nitrogen should  July  15 and e n r i c h e d  were e n r i c h e d a concentration  per limnocorral)  were u s e d .  with  limnocorrals,  "untreated additions  on J u l y  18. The  o f 200 ug N/L. I r o n  was mixed w i t h c i t r i c  t h e pH was a d j u s t e d  96 g o f c i t r i c  Eight  8.3 g o f NH^Cl w h i c h  acid  (3.42 g  (96 g p e r  t o 7.0 w i t h NaOH, a n d q u i c k l y  s o l u t i o n s were added t o t h e l i m n o c o r r a l s .  received All  were f i l l e d  have p r o d u c e d  limnocorral), the  Experiments  w i t h t h e 1982 d e s i g n  limnocorrals  FeClj  have b e e n : 200 U9" N/L, 1.4 mg  o r 100 ug F e / L .  Chain Lake L i m n o c o r r a l  limnocorrals  would  Two  limnocorrals  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.  i n c l u d i n g what i s r e f e r r e d t o i n t h e t e x t as  limnocorrals"  received  1.98 g o f K H P 0 . A l l n u t r i e n t  were made by m i x i n g t h e n u t r i e n t s  2  4  i n 50 L o f w a t e r and  t h e n pumping t h e s o l u t i o n i n t o t h e l i m n o c o r r a l s  at five  depths.  154  Appendix 2 Table A l . Iron analysis in Black Lake - May 9, 1978.  Depth  Dissolved  Particulate  Aerated  Control  Aerated  6  5  4  3  4  4M 6M 8M  8 20 22 25  17 26  19 44 182  12 26 57  13  202  45  9M  28  88  119  69  Surface 2M  A l l values are in ng Fe/L.  Control  155  Table Al continued 1979 Data:  Date June 15  Depth 2M  Surf 3M  13 22 19 17 10  5M 9M 2M  5M 9M July 4  July 17  Aug 11  Aug 30  Oct 26 Dec 13  Dissolved Aerated Control  5M 9M 0.5M 1M 2M 3M 5M 9M 1M 5M 9M 2M 8M 2M 8M  -  Particulate Aerated Contrc 29 30  25 11 11  11 10  30 31 29  28  20  20  84  27  22 3 51 8 <1 2 119 4 8 600 <1 101 102 10 14  24 11  97 40  57 15  7 18  60 123  48 100  5 250 184 2 203  94 90 111 92 260 632 43 86 100 30 44 20 18  50 27 60 29 69 273 37  9 11  A l l values are in ug Fe/L.  -  -  -  2 90 85 12 18 10 12  20  52 32 28 62 21 15  156  Table Al continued 1980 Data:  Date Jan 22 A p r i l 23  May 12  Depth 2M 8M 2M  18 29 2  5M 8M  3 10  2M  -  8M May 21  June 4  July 17 Aug 11  Dissolved Aerated Control  Surf 2M 5M 7M Surf IM 2M 3M 5M 8M IM 3M IM 3M 5M 7M 9M  -  -  -  25 20 20 18 20 11 1 4 4 5 7  -  18  A l l values are in ug Fe/L.  17  -  2 4  13 13 13  -  -  18 22 25 20 30 35 1 2 0 3 8  -  17  Particulate Aerated Control  -  13 13 35  27  39 42 42  45 41 41  -  -  46 34  -  38 6 7 6 27 30 30 10  -  59  20 93 184 194 453  -  -  -  -  6 7 8 7 11 11 12 17 59 72 98 326 422  Appendix 3 Table A2 Oxygen Concentrations i n Liimccorrals During C i t r a t e A d d i t i o n s Fe-C1trate Depth 0 1 2 3 4 5  Na-C1trate Depth  (mg/L)  Limnocorrals 24th 8.5 8.8 8.8 9.2 9 . 4 10.2 9 . 2 10.2 9.9 9.1 9.0 9.9  June 26th 8.4 8.7 8 . 3 5 8.75 9.4 10.4 9.55 10.2 9.25 10.0 8.0 9.0  28th 9.2 11.2 9 . 8 11.2 9 . 5 .10.3 8.5 ' 9 . 4 8.0 8.5 6.5 6.1  July 13th 1 3 . 3 12.2 13.8 11.5 8.6 11.3 9.2 8.3 7.9 7.0 5.0 4.2  22nd 18.0 2 0 . 0 20.0 17.0 20.0 16.6 12.0 11.0 3.0 6.0 4.3 2.4  10th 17.9 18.2 17.9 19.0 10.2 1 7 . 0 8.8 8.2 8.1 7.8 8.0 7.7  August 17 th 18.2 16.6 1 5 . 3 16.6 1 2 . 8 13.1 1 0 . 8 12.6 10.1 10.6 5.5 7.0  24th 1 7 . 4 16.8 1 6 . 8 16.2 1 4 . 0 14.4 9.2 9.2 6.7 2.0 1.0 0.7  Limnocorrals 24th 8.7 8.8 9.0 9.5 9.7 10.1 9.6 9.9 9.4 9.7 9.0 9.4  June 26th 9.1 9.8 9.1 9.8 10.0 10.2 9 . 9 10.2 9.6 9.7 8.1 8.4  28th 10.6 1 2 . 0 10.5 12.0 10.0 11.0 9.2 9.0 7.5 7.9 6.7 7.0  July 13th 11.0 13.0 12.2 1 5 . 0 10.8 13.6 9.1 9.0 6.7 7.4 6.5 4.1  22nd 16.5 16.0 13.5 1 4 . 6 13.5 14.6 12.6 1 1 . 8 5.6 5.6 4.0 1.8  10th 14.6 16.2 15.2 1 6 . 2 1 6 . 3 16.1 9.4 8.8 8.2 4.2 7.6 3.6  August 17th 14.4 1 3 . 4 -13.6 12.9 11.2 11.6 11.2 1 0 . 6 9.8 9.0 8.0 7.0  24th 14.2 15.4 1 4 . 3 13.1 13.0 12.2 10.0 9.1 8.6 6.8 3.6 1.3  C o n t r o l Limnocorrals Depth 24 th 8.4 8.6 0 8.5 1 8.8 9.4 8.9 2 9.5 3 9.0 4 9.0 9.5 8.4 8.7 5  June 26th 9.2 8.8 9.0 9.2 9.4 9.6 9.4 9.6 9.5 9.0 7.8 8.3  28th 10.4 9.4 9.4 10.5 9.8 9.8 9.4 9.3 9.3 8.7 7.8 7.3  July 13th 22nd 12.8 17.4 15.9 15.8 13.0 2 0 . 0 15.9 16.1 12.0 20.0 16.1 1 6 . 6 9 . 5 12.4 9 . 8 14.4 8.2 5.4 8.8 9.0 6.4 6.0 7.7 3.8  10th 18.4 1 5 . 0 18.4 1 4 . 4 16.8 11.8 10.4 4.2 9.8 2.6 9.4 2.5  August 17th 16.1 14.4 16.0 12.0 12.5 9.8 12.4 9.6 12.2 8.4 3.0 6.4  24th 16.0 14.2 16.4 14.4 14.8 14.0 11.4 8.0 11.0 6.7 4.2 3.2  0 1 2 3 4 5  158  Appendix  4  Improvements t o t h e I r o n - B i n d i n g  Unfortunately, optimal  t h e assay  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  A l ) . This procedure r e s u l t e d i n a smaller  r e s p o n s e s and a s a m p l i n g variation).  Half  work: t h e i r o n heterotrophy, lake  hour  primary  e r r o r o f about  range of l i n e a r  f o r the following  c a p a c i t y o f t h e c h e l a t o r s used production  used  10% ( c o e f f i c i e n t o f  i n c u b a t i o n s were u s e d  binding  i n the  i n c u b a t i o n s , and measurements o f  siderophores. I n one hour  throughout A2).  used b e f o r e the  i n c u b a t i o n t i m e was d e t e r m i n e d . The c h e l a t i o n i s n o t  complete w i t h i n the h a l f (Fig.  was i n i t i a l l y  Assay  Blanks  the concentration were v e r y  method was good coefficient  incubations, the standard  curve  linear  r a n g e t h a t was e n c o u n t e r e d  low and t h e s e n s i t i v i t y  ( F i g . A 3 ) . I n one hour  of v a r i a t i o n  was  was l e s s  than  (Fig.  of the r a d i o i s o t o p e  incubations, the 2%.  A n o t h e r e r r o r may have a r i s e n i n t h e u s e o f a c i d s o r b a s e s with  i o n exchange r e s i n s .  Some o f t h e s i d e r o p h o r e  readily  down i n t h e p r e s e n c e o f weak a c i d  or base  little  was i n t h e b i o a s s a y s .  o f t h a t b r e a k down p r o d u c t  Freeze-drying than  ( F i g . A 4 ) . Presumably  i s a b e t t e r method o f c o n c e n t r a t i n g  siderophores  t h e i o n - e x c h a n g e method. The  the  broke  u n c e r t a i n t y associated with  siderophore  bigger  knowing t h e c o n c e n t r a t i o n o f  i n the microenvironment  problem than  statements a r e s t i l l  the a n a l y t i c a l possible.  of the c e l l s  problems. Strong  was a much qualitative  159  TIME COURSE DES  2CXXH  A.f.a.  1500H  I  I  o. o 100CH  500H  1  2  3  TIME (hours) Figure A l  E f f e c t of incubation  desferal culture  t i m e on c h e l a t i o n  (DES) and a f i l t r a t e (A.f.a.).  4  from a Anabaena  5  6  o f i r o n by flos-aquae  160  -1  02  1  04  1  1  1—  06  QB  to  ALIQUOT  A.f.a.  F i g u r e A2 I r o n b i n d i n g c a p a c i t y (FeBC) o f a f i l t r a t e f r o m 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 o f 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  2,000  Desferal \ig/L F i g u r e A3  Iron binding  desferal.  capacity  (FeBC) s t a n d a r d i z a t i o n w i t h  SIDEROPHORE DEGRADATION BY ACID OR BASE NaOH  0.05  0.15H  004-I  0.10H 0.05H  I O  O X  CO  Z  0.02H 005H  0D1H  Fe BC (uM-L" ) 1  F i g u r e A4  Degradation  o f t h e s i d e r o p h o r e f r o m Anabaena  flos-  aquae by a c i d  o r base  Appendix T a b l e A3  Depth  5 Yellow  sio  2  Lake Sediment Chemistry  A1  2  0  3 F  e  2 ° 3  MgO  CaO  Na 0  K 0  T i 0  1.85 2. 25  0.41  0.14  0.30  0.06 0.10  0.32  2  2  2  MnO  P  2°5  cm 0-2  54.86  6.11  3.55  2.16  15.72  0.82  4-6 6-8  43.75 38.07  8.70 6.17  2 . 22  19.36  4.06 3.94  26.89 31.65  10-12 12-14  33.96 33.70 3 6 . 77  5.45  1.73 2.58  1.36 1.02 1.04 1.50  0.87 0.81  8-10  3.99 2.39 1.65  14-16 18-20  31.96 42.66  4.83 4.27  2.23 2.65  28-30  47.33  1.22  38-40  44.85  2.55  48-50  49.09  1.63  A l l The was conf  1.64  0.42 0.28  31.83 28.52  0.76 0.54 0.64  1.11 1.04 1.42  0.19 0.19 0 . 24  0.05 0.05  0.44 0.51  0.05  1.56  31.82  0.80  1.10  0.20  0.04  0.43 0.48  0.96 0.52  20.35  0.35  1.50  21.82  0.20  0.88 0.10  1.70 1.57  0.60  23.36  0.62 0.58  2 0 . 42  0.64 0.28  0.06 0.05 0.04  0.53  0.36 0.34  0.29 0.09 0.18 0.13  0.05  0.52  0.38  0.48  v a l u e s a r e e x p r e s s e d as % d r y weight. X-ray fluoresence analysis reports the t o t a l concentration as i f the element p r e s e n t as a . s i m p l e o x i d e . The p r e s e n c e of c a l c i t e (calcium carbonate) was i r m e d by X - r a y d i f f r a c t i o n analysis.  

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