THE GREEN BACTERIA A PROBLEM IN HYDROBACTERIOLOGY By A lic e K a th ry n B ic k n e ll A THESIS S u b m i t t e d t o t h e S c h o o l o f G r a d u a t e S t u d i e s o f M ic h i g a n S t a t e C o l l e g e o f A g r i c u l t u r e and A p p l i e d S c i e n c e i n p a r t i a l f u l f i l l m e n t of th e re q u ire m e n ts f o r th e d eg ree of DOCTOR OF PHILOSOPHY D epartm ent o f B a c te r io lo g y 1950 ACKNOWLEDGMENT To D r. H. J . S t a f s e t h , H ead , D e p a rtm e n t o f B a c t e r i o l o g y , M ichigan. S t a t e C o l l e g e , t h e a u t h o r i s i n d e b t e d . F o r h i s u n d e r s t a n d i n g o f t h e p ro b le m s c o n f r o n t i n g t h e w o r k e r i n a new f i e l d o f b a c t e r i o l o g y a n d f o r h i s a i d i n e f f e c t i n g t h e m e c h a n ic s o f t h e f i e l d w o rk i n v o l v e d , I am m ost g r a t e f u l . To D r. W. L . M allm an u , P r o f e s s o r o f B a c t e r i o l o g y a t M i c h i ­ g a n S t a t e C o l l e g e , t o D r. S t a n l e y A . C a i n , D i r e c t o r o f R e s e a r c h , The C r a n b ro o k I n s t i t u t e o f S c i e n c e and t o D r. R o b e r t H a t t , D i r e c t o r , T h e C ra n b ro o k I n s t i t u t e o f S c i e n c e , t h e a u t h o r e x p r e s s e s h e r b e s t t h a n k s . W i t h o u t t h e a i d a nd e n c o u r a g e m e n t o f t h e s e s c i e n t i s t s , t h e w o rk r e p o r t e d u p o n h e r e c o u l d n e i t h e r h ave b e e n i n i t i a t e d n o r c o m p l e t e d . To a d m i r a t i o n a nd r e s p e c t f o r t h e s e w o r k e r s , t h e a u t h o r now ad d s a p p r e c i a t i o n . ^ jj, ^ * * * * * * * * ****** ** * i TABLE OF CONTENTS Page INTRODUCTION................................................................................................................................. 1 The P r o b l e m ................................................................................................................. S co pe o f t h e P r o b l e m ................................................................. 6 7 REVIEW OF THE LITERATURE.................................................................................................... DESCRIPTION OF ENVIRONMENT SELECTED FOR ECOLOGICAL STUDY..................... METHODS OF PROCEDURE.................................................... '.................................................... 8 11 12 1 . G e n e r a l ..................................................................................................................... 12 13 2 . F i e l d I n v e s t i g a t i o n s ......................................................................................... 3 . L a b o r a t o r y I n v e s t i g a t i o n s ............................................................................ 14 PRELIMINARY RECONNAISSANCE.............................................................................................. 15 METHODS OF PLANKTON ENUMERATION................................................................................. 16 THE VERTICAL AND HORIZONTAL DISTRIBUTION........................................... SEASONAL DISTRIBUTION......................................................................................................... 19 20 ENVIRONMENTAL FACTORS POSSIBLY AFFECTING THE ZONATION OF THE GREEN BACTERIA................................................................... A. P h y s i c a l 1. T e m p e r a t u r e ........................................................................................ 2 . H y d r o g e n - i o n c o n c e n t r a t i o n .................................................... 3 . L i g h t I n t e n s i t y .............................................................................. 24 28 29 B. C h e m ic a l 1 . D i s s o l v e d O x y gen .......................................................................... 2 . H y d ro g e n S u l p h i d e ................................................. 3 . O t h e r C h e m ic a l F a c t o r s . ......................................................... 36 36 37 C. A n a l y s i s o f t h e h y d r o s o l ............................................................................ 37 D. B i o l o g i c a l F a c t o r s 1 . The P h y t o p l a r i k t o n ........................................................................ 2 . The Z o o p l a n k t o n ............. 39 41 THE CULTURE OF THE GREEN BACTERIA.......................................................................... 52 A. C ru d e C u l t u r e s .................................................................................................... B. E n r i c h m e n t C u l t u r e s ............................. C. P u r e C u l t u r e s . .................................................................................................... 52 53 56 J ii TABLE OF CONTENTS ( C o n tin u e d ) Page GENERAL MORPHOLOGY...................................................................................................... 57 A . From E n r i c h m e n t C u l t u r e s ............................................................................ B. From P u r e C u l t u r e s ........................................................................................... 57 57 THE PIGMENTS OF THE GREEN BACTERIA....................................................................... 66 METABOLISM OF THEGREEN BACTERIA............................................................................... 71 DISCUSSION............................... 73 P r e l i m i n a r y D i s c u s s i o n ................................................ F a c t o r s P o s s i b l y I n f l u e n c i n g t h e D i s t r i b u t i o n o f G re e n B a c t e r i a ......................................................................... T e m p e r a t u r e a s a C o n t r o l l i n g F a c t o r ......................................................... D i s s o l v e d O x y g e n ....................................................................................................... The P r e s e n c e o f H y d ro g e n S u l p h i d e . ................................. L i g h t .................................................................................................................................. The C h e m ic a l C o n s t i t u e n t s o f t h e W a t e r , O t h e r t h a n D i s s o l v ­ ed Oxygen a n d H y d ro g e n S u l p h i d e .......................... B i o l o g i c a l F a c t o r s .............. T a x o n o m ic a l C o n s i d e r a t i o n s o f t h e G r e e n B a c t e r i a . . . 73 73 74 77 82 83 84 90 91 SUMMARY......................................................................................................................................... 96 CONCLUSIONS.................................................................................................................... 97 BIBLIOGRAPHY............................................................................................................................. 98 APPENDIX........................................................................................................................................ 104 iii i LIST OF TABLES T a b le I II III IV V VI V II V III IX X XI Page T e m p e r a t u r e R e c o r d s D ecem ber 1949 - J a n u a r y 1 9 5 0 ..................... App. T r a n s e c t o f Zone o f G re e n B a c t e r i a - S o d o n L a k e - 4 A ug. 1 9 4 9 ........................................................................................................................... 25 pH D e t e r m i n a t i o n s o f G re e n B a c t e r i a Zone T ak en on T r a n ­ sect 31 D i s s o l v e d Oxygen T re n d s - So do n L ake - J a n . 1949 - D ec. 1 9 4 9 ........................................................................................................................... 33 V e r t i c a l D i s t r i b u t i o n o f D i s s o l v e d Oxygen T T r a n s e c t So d o n L ak e - A u g u s t 1 9 4 9 .............................................................. ........... 34 D i s s o l v e d Oxygen i n R e l a t i o n t o Z o n a t i o n o f G re e n t e r i a - Sodon L a k e - A u g u s t 1 9 4 9 35 B ac­ C h e m ic a l A n a l y s e s - So do n Lake - 1 9 4 9 ................................................ 30 C h e m ic a l C o m p o s i t i o n o f t h e H y d r o s o l - S o d o n L ak e - 1 9 4 9 . 39 ( a ) Numbers p e r L i t e r o f D a p h n ia p u l e x , By S e a s o n and D e p th - S o d o n L ak e - 1 9 4 9 ............................................................. 42 ( b ) Numbers p e r L i t e r o f D ia p to m u s o r e g o n e n s i s , By S e a s o n a n d D e p th - So do n L a k e - 1 9 4 9 .................................................... 43 ( c ) Numbers p e r L i t e r o f C y c l o ps p r a s i n u s , By S e a s o n a n d D e p th - S o d o n L ake - 1 9 4 9 T . ......................................................... 44 A v a i l a b l e N u t r i e n t s i n Sodon L a k e 1 9 4 9 ....................... 46 Sodon L a k e - S p e c t r o g r a p h i c A n a l y s i s ................................................. 89 LIST OF TABLES T a b le I II III IV V VI V II V III IX T e m p e r a t u r e R e c o rd s D ecem ber 1949 - J a n u a r y 1 9 5 0 ........... ..... Page App. T r a n s e c t o f Zone o f Green. B a c t e r i a - S o d o n L a k e - 4 A ug. 1 9 4 9 ............................................................................................................................. pH D e t e r m i n a t i o n s o f G re e n B a c t e r i a Z one Taken o n ............................................................................ sect 25 T ran- D i s s o l v e d Oxygen T re n d s - S o d o n Lake - J a n . 1949 - D ec. 1 9 4 9 ............................................................................................................................. 33 V e r t i c a l D i s t r i b u t i o n o f D i s s o l v e d O xygen T T r a n s e c t So do n L a k e - A u g u s t 1 9 4 9 ............................................................................. 34 D i s s o l v e d Oxygen i n R e l a t i o n t o Z o n a t i o n o f G re e n t e r i a - So d on L a k e - A u g u s t 1 9 4 9 . . . . . 35 B ac­ C h e m ic a l A n a l y s e s - Sodon Lake - 1 9 4 9 . . .............................................. C h e m ic a l C o m p o s i t i o n o f t h e H y d r o s o l - S o d o n L a k e - 1 9 4 9 . 38 39 ( a ) Humbers p e r L i t e r o f D a p h n ia p u l e x , By S e a s o n and D e p th - S o d o n L a k e - 1 9 4 9 . . . ..................................... ( b ) Numbers p e r L i t e r o f D iaptornus o r e g o n e n s i s , By S e a s o n a n d D e p th - So do n L ak e - 1 9 4 9 . . . ............................................... 42 43 ( c ) Numbers p e r L i t e r o f C y c l o ps p r a s i n u s , By S e a s o n a n d D e p th - S o d o n L ak e - 1 9 4 9 . ............................................ X XI A v a ila b le N u trie n ts i n So do n L ak e 1 9 4 9 ................................................. Sod o n L a k e - S p e c t r o g r a p h i c A n a l y s i s ........................................... iv I 44 46 89 LIST OF FIGURES F ig u re . Page 1. The C o n to u r Map o f S o d o n L a k e ...................................................................... 10 2 ... P r o f i l e of S o d o n L a k e - L o c a t i o n o f S a m p lin g S t a t i o n s ........... 18 3. D i s t r i b u t i o n of G r e e n B a c t e r i a - S o d o n Lake - 1 9 4 9 . ................. 23 4. S e a s o n a l T e m p e r a t u r e T rends i n Sodon L a k e ....................... 26 5. pH D e t e r m i n a t i o n s - Sodon L a k e - 1 9 4 9 ................ 30 6. V a r i a t i o n o f M a tu r e E n t o m o s t r a c a n s - Sodon L a k e - F e b r u a r y - November 1 9 4 9 . . .................................................. 50 D e p th D i s t r i b u t i o n o f E n t o m o s t r a c a n s - S o d o n Lake F e b r u a r y - Novem ber 1 9 4 9 .............................................................................. 51 8. C e l l A g g r e g a t i o n o f C h lo r o b iu m l i m i c o l a . ............................................ 60 9. C. l i m i c o l a , sh ad o w c a s t e d ..................... 61 10. S i n g l e c e l l o f £ . l i m i c o l a ...................................... 62 11. M o r p h o l o g i c a l V a r i a n t s o f C. l i m i c o l a ................................... ... ............ 63 12. G re e n B a c t e r i a fr o m L a u t e r b o r n .................................................................... 65 13. A b s o r p t i o n S p e c tr u m o f G re e n B a c t e r i a ................................................... 67 14-. G r a p h i c a l P r e s e n t a t i o n o f E q u a t i o n s o f d e M a r t i n i ......................... 86 15. Die.gram o f Vacuum Tube P o t e n t i o m e t e r a nd A s s o c i a t e d S u b ­ m e rs io n . E l e c t r o d e A ssem b ly f o r M e a s u re m e n t o f Eh a n d p H .. 80 M o rp h o lo g y o f C. l i m i c o l a , From V a r i o u s A u t h o r s .......................... 94 7. 16. v ■ INTRODUCTION I n a tt e m p t i n g t o w r i t e a p a p e r on a q u a t i c b a c t e r i o l o g y , t h e w r i t e r f in d s h e r s e l f i n an alm o st u n e x p lo re d f i e l d . T here a r e b u t few s i g n p o s t s t o g u i d e t h e i n v e s t i g a t o r a n d t h o s e w h ic h do e x i s t a r e s u b je c t to d e b a ta b le i n t e r p r e t a t i o n s . I t sh o u ld be of v a lu e i n ex­ p l a i n i n g t h e ” r a i s o n - d ’ e t r e ” f o r t h i s p a r t i c u l a r p a p e r t o o f f e r some comments o f o t h e r w o r k e r s who h a v e f e l t t h e n e e d o f e x p l o r a t o r y a n d i n t e n s i v e w ork i n t h e f i e l d o f h y d r o b a c t e r i o l o g y a n d f o r c l o s e r c o ­ o p e r a t i o n w ith l i m n o l o g i s t s and e c o l o g i s t s in g e n e ra l. T h ien e m a n n ( 1 9 2 7 ) comments a s f o l l o w s : u The g r e a t e s t n e e d o f l i m n o l o g y , t h e s a t i s f a c t i o n o f w h ic h w o u ld b e o f b e n e f i t t o many a s s o ­ c ia te d d e p a rtm e n ts, is b a c t e r i o l o g i c a l in fo rm a tio n . ta rily I t may b e momen­ s u r p r is in g i f I i n s i s t t h a t h y d ro b a c te rio lo g y as such is v ir t u a lly n o n -e x iste n t. I t is t r u e t h a t t h e r e have been i n v e s t i g a ­ tio n s re g a rd in g b a c t e r i a l i v i n g i n w a te r, b u t f o r th e m ost p a r t th e y have had r e f e r e n c e p r i m a r i l y to p r a c t i c a l h y g ie n ic p ro b le m s . I t is w e l l known t h a t b a c t e r i a p l a y a n e x t r e m e l y i m p o r t a n t p a r t i n t h e c j^ c le o f l i f e - m a t e r i a l s ; i t may ev en b e t h e m o s t i m p o r t a n t p a r t , j u s t i f i e d i n a s s ig n in g d eg rees o f im p o rta n c e . may b e o u r m eth o d s i n w a t e r c h e m i s t r y , i f we a r e No m a t t e r how d e t a i l e d even th e m ost i n t e n s i v e d e lv in g i n t o p u r e ly ch em ical p r o c e s s e s i s n o t g o in g t o s o l v e f o r u s t h e m y ste ry o f t h e m e ta m o r p h o s is o f m a t t e r , i f we n e g l e c t b a c t e r i a l a c t i o n .......... What h a s b e e n s a i d h e r e a b o u t t h e i m p o r t a n c e o f b a c t e r i o l o g i c a l r e s e a r c h t o h y d r o b i o l o g y and l i m n o l o g y a p p l i e s a l s o t o f i s h e r i e s a n d b i o l o g y i n -1 - g e n e ra l. W h en ev er we e n c o u n t e r t h e d i f f i c u l t p r o b le m o f t h e c y c l e o f s u b s t a n c e s on t h i s e a r t h , we a l s o e n c o u n t e r t h e n e c e s s i t y o f c o n s i d e r ­ in g b a c t e r i a . ” W elch (193 5 ) s a y s : lim n o lo g y , i s ’’P u r e l i m n o l o g y , a s c o n t r a s t e d w i t h a p p l i e d p r i m a r i l y c o n c e r n e d w i t h t h e n o rm a l b a c t e r i a l p o p u l a t i o n s of u n c o n t a m i n a t e d ■•waters, a nd p a r t i c u l a r l y t h o s e w h ic h may b e r e g a r d e d a s o f m ore o r l e s s r e g u l a r o c c u r r e n c e i n t h e w a t e r s o f d i f f e r e n t k i n d s of la k e s . U n fo rtu n a te ly , th is i s a n a l m o s t unknow n f i e l d , s i n c e b a c t e r i o l o g i s t s a n d o t h e r s e m p lo y in g t h e m etho ds o f b a c t e r i o l o g y h a v e b e e n m a in ly c o n c e r n e d i n t h e p a s t w i t h p a t h o g e n i c b a c t e r i a . O nly a v e r y fe w l a k e s h a v e r e c e i v e d a n y s t u d y o f t h e b a c t e r i a n a t i v e t o th e m , and e v e n i n t h o s e few i n s t a n c e s t h e r e a r e many g a p s i n t h e a v a i l a b l e in fo rm a tio n . . . . The s t u d e n t who d e s i r e s a c o m p r e h e n s iv e l i s t o f s p e c i f i c a l l y i d e n t i f i e d b a c t e r i a ( t o g e n e r a a.ud t o s p e c i e s ) w h ic h com­ p o se t h e w a t e r b a c t e r i a i s doomed t o d i s a p p o i n t m e n t , s i n c e n o n e i s a v a i l a b l e f o r a n y u n m o d i f i e d l a k e i n N o r t h A m e ric a o r , i n s o f a r as t h e w r i t e r k n o w s , i n o t h e r c o n t i n e n t s . 11 H u b e r - P e s t a l o z z i ( 1 9 3 8 ) s t a t e s t h e p r o b le m i n t h i s w ay: " J e mehr d e r E i n b l o c k i n das L e b e n e i n e s S e e s v e r t i e f t w e rd e n s o i l , d e s t o mehr hi m u s se n a u c h d i e B a k t e r i e n i n d e n B e r e i c h d e r U n t e r s u c h u n g g e z o g e n w e rd e n ; j a man d a r f s o g a r b e h a u p t e n , d a s s f f i r d a s V e r s t a n d n i s d e r o f t s t a r k i n e i n a n d e r g r e i f e n d e n L e b e n s z y k l e n i n e in em S e e , b e s o n d e r s im e u t r o p h e n S e e , uberhaupfcfc f u r d e n g e s a m te n S t o f f h a u s h a l t i n e in e m s t e h e n d e n Gewa*sser, d i e K e n n tn is d e r T a t i g k e i t so w o h l d e r im f r e i e n W a s s e r a l s d e r im schlamme l e b e n d e n B a k t e r i e n u n e r l a s s l i c h i s t . " -2 - H o w ev er, t h e o p p o s i t e s i d e of t h e p r o b le m i s p r e s e n t e d w hen Jo rd an , in Ward a n d W h i p p l e 's F r e s h W a t e r B i o l o g y s t a t e s " T h e r e i s no s p e c i a l a n d c h a r a c t e r i s t i c c l a s s o f w a t e r b a c t e r i a b u t fo rm s f r o m t h e a i r , fr o m t h e s o i l , fro m d e co m p o sin g a n i m a l a n d p l a n t s u b s t a n c e s and fro m t h e h e a l t h y a n d d i s e a s e d t i s s u e s tim e s f i n d I t is o f a n i m a l s and p l a n t s may a t t h e i r way i n t o w a t e r " . a s a d co m m entary on t h e s t a t u s o f h y d r o b a c t e r i o l o g y i n th e U n i t e d S t a t e s t h a t t h e s t a t e m e n t by J o r d a n i s s t i l l p e r p e t r a t e d i n one o f t h e m o s t i m p o r t a n t s o u r c e b oo ks a v a i l a b l e t o t h e a q u a t i c b i o l o g i s t . I t is fo o lh a rd y t o s u g g e s t t h a t th e i n v e s t i g a t i o n r e p o r te d upon h e r e w i l l s o l v e many o f t h e p ro b le m s s u g g e s t e d by T hienem an n , W elch and H u b e r - P e s t a l o z z i . H ow ever, a f t e r f i v e y e a r s o f s t u d y on t h e n a t u r a l b a c t e r i o l o g y o f la k e s and r i v e r s , t h e a u t h o r f e e l s t h a t t h e r e i s some j u s t i f i c a t i o n in a tte m p tin g to p re s e n t th e r e s u lts of an e c o lo g ic a l s t u d y o f one t y p i c a l l y a q u a t i c g ro u p o f b a c t e r i a , t h e C h l o r o b a c t e r i a ceae. The p r e s e n t s t u d y r e p r e s e n t s , i n so f a r as t h e - a u t h o r i s aw are, a f i r s t a tte m p t t o stu d y a s p e c i f i c group of b a c t e r i a as i t i s iz e d in i t s organ­ n a t u r a l e n v iro n m en t. I n a n y a q u a t i c e n v ir o n m e n t w h e r e i n t h e r e e x i s t s a m e a s u r a b l e am ount o f h y d r o g e n s u l p h i d e g a s , s u f f i c i e n t l i g h t f o r t h e p r o c e s s o f p h o t o s y n t h e s i s t o t a k e p l a c e and a lo w ox y g en t h r e s h o l d , t h e r e may d e v e lo p o r g a n is m s w h ic h h a v e b e e n d e s i g n a t e d a s g r e e n b a c t e r i a . T hese b a c t e r i a , a c c o rd in g to van N ie l (1 9 3 2 ), a r e c a p a b le o f re d u c in g carbon d i o x i d e p h o t o c h e m i c a l l y w i t h o u t t h e l i b e r a t i o n o f o x y g e n u s i n g h y d ro g e n s u lp h id e a s th e hydrogen d o n o r. The g r e e n b a c t e r i a may b e f u r t h e r -3 - c h a r a c t e r i z e d a s b a c t e r i o - v i r i d i n - c o n t a i n i n g o r g a n is m s w h ic h d e v e l o p i n p u r e l y m i n e r a l m e d ia c o n t a i n i n g h y d r o g e n s u l p h i d e a n d w hose b i o ­ c h em ica l a c t i v i t i e s a r e a c t i v a t e d by a s u p p ly o f r a d i a n t e n e rg y . L a u te rb o rn (1915) e s t a b l i s h e d th e fa m ily C h lo r o b a o te r ia c e a e f o r t h o s e o r g a n is m s w h ic h s h o u l d b e i n c l u d e d i n t h e d e s c r i p t i o n g i v e n a b o v e . H o w ev e r, a s n o t h i n g was known o f t h e b i o c h e m i c a l a c t i v i t i e s g re e n b a c t e r i a u n t i l 1922, i t of th e i s o b v io u s t h a t t h e C h l o r o b a c t e r i a c e a e a s r e p r e s e n t e d by L a u t e r b o r n was a p u r e l y a r t i f i c i a l g r o u p . In th e f a m i l y C h l o r o b a c t e r i a c e a e L a u t e r b o r n c r e a t e d a new g e n u s C h l o r o b a c t e r iu m , w h ic h c o n s i s t e d o f one s p e c i e s C h i p r o b a c t e r i u x n s y m b i o t i c u m ; t h e g e n e r a S ch m id l e a , P e l o g l o e a , P e l o d i c t y o n a n d C h lo r o c h r o m a tiu m w e re a ls o in c lu d e d . 1906. The two l a t t e r g e n e r a w e r e o r i g i n a t e d by L a u t e r b o r n i n G e i t l e r a n d P a s c h e r ( 1 9 2 5 ) f o l l o w e d t h e same m a in o u t l i n e s o f c l a s s i f i c a t i o n as d id L a u te rb o rn b u t in c lu d e d s e v e r a l o th e r g en era and sp e c ie s. I n B e r g e y ’ s M anual o f D e t e r m i n a t i v e B a c t e r i o l o g y , s i x t h e d i ­ t i o n ( 1 9 4 8 ) i s i n c o r p o r a t e d t h e f o l l o i 'J i n g d e s c r i p t i o n o f t h e C h l o r o ­ b a c te ria c e a e . O rder E u b a c te r i a l e s Suborder P lio d o b ac te riin e ae F a m ily I I I . C h lo r o b a c te r ia c e a e G e i t l e r and P a s c h e r. ( C y a n o c h l o r i d i n a e - C h l o r o b a c t e r i a c e a e G e i t l e r a n d P a s c h e r , D ie S u s s w a s s e r f l o r a D e u ts c h la n d s , O s t e r r e i c h s und d e r S ch w eiz, J e n a , 12, 1925, 4 5 1 ; C h l o r o t h i o b a c t e r i a Bavendamm, E r g e b . B i o l . , 1 3 , 1 9 3 6 , 4 9 . G re e n b a c t e r i a , u s u a l l y of s m a l l s i z e , o c c u r r i n g s i n g l y o r i n c e l l m a s s e s o f v a r i o u s s h a p e s and s i z e s , d e v e l o p i n g i n e n v i r o n m e n t s c o n t a i n ­ in g r a t h e r h ig h c o n c e n tr a tio n s o f h y drog en s u lp h id e and exposed to lig h t. As a r u l e n o t c o n t a i n i n g s u l p h u r g l o b u l e s b u t f r e q u e n t l y d e ­ p o s i t i n g e lem en ta ry s u lp h u r o u ts id e th e c e l l s . C o n ta in g re e n p ig m e n t o f a c h l o r o p h y l l o u s n a t u r e , th o u g h n o t i d e n t i c a l w i t h t h e common g r e e n p la n t c h lo ro p h y lls nor w ith b a c te r io c h lo r o p h y ll. C ap ab le of p h o to s y n ­ t h e s i s i n t h e p r e s e n c e o f h y d r o g e n s u l p h i d e ; do n o t l i b e r a t e o x y g e n . -4 - Key I. F re e l i v i n g b a c t e r i a n o t i n tim a te ly a s s o c ia te d w ith o th e r m ic ro b e s. a. b a c te r ia n o t u n ite d in to w e ll d e fin e d c o lo n ie s G enus I C h lo r o b iu m a a . b a c te r ia u n ite d in to c h a r a c t e r i s t i c a g g re g a te s b. b a c t e r i a w ith o u t i n t r a c e l l u l a r su lp h u r g ra n u le s Genus I I P e l o d i c t y o n bb. b a c t e r i a w ith i n t r a c e l l u l a r s u lp h u r g ra n u le s Genus I I I C l a t h r o c h l o r i s II. G re e n b a c t e r i a f o u n d a s s y m b i o t i c a g g r e g a t e s w i t h o t h e r o r g a n is m s a. a g g re g ate s com posed o f g r e e n b a c t e r i a a n d p r o t o z o a Genus IV C h l o r o b a c t e r i u m a a . a g g r e g a t e s composed o f two d i f f e r e n t t y p e s o f b a c t e r i a b. a g g re g a te s s m a ll, b a r r e l sh a p e d , a c t i v e l y m o tile , and con­ s i s t i n g of a c e n t r a l , p o la r ly f la g e lla .te d , ro d -sh ap ed b a c te riu m w ith a c o v erin g o f g re e n s u lp h u r b a c t e r i a . Genus V C h lo r o c h r o m a tiu r a b b . a g g r e g a t e s l a r g e , c y l i n d r i c a l , n o n m o t i l e a n d com­ posed o f a c e n t r a l fila m e n to u s b a c te riu m w ith a more o r l e s s e x t e n s i v e c o v e r i n g o f g r e e n s u l p h u r b a c te ria . Genus VI C y l i n d r o g l o e a Some comment i s i n d i c a t e d as t o t h e sy stem of c l a s s i f i c a t i o n o f t h e g r e e n b a c t e r i a a s u s e d by S k u ja ( 1 9 4 B ) . S k u ja c o n sid e rs th e C h l o r o b a c t e r i a l e s a s one o f s i x o r d e r s u n d e r t h e phylum B a c t e r i o p h y t a . Slcuja f u r t h e r i n c o r p o r a t e s t h r e e f a m i l i e s , i . e . , P e lo s p h a e ra c e a e , C h lo r o b a c te r ia c e a e and C h lo ro c h ro m a tia c e a e u n d e r t h e o r d e r C h lo ro b a c ­ te ria le s. I t w i l l b e n o t e d t h a t S k u j a ' s c l a s s i f i c a t i o n i s som ew hat d i f f e r e n t t h a n t h a t w h ic h i s B e r g e y 's M a n u a l. in c o rp o ra te d in to th e s ix th e d itio n o f I n t h e M a n u a l, t h e g e n e r a P e l o s p h a e r a , T e t r a c h l o r i s a n d P e lo c h r o m a tiu m a r e n o t r e c o g n i z e d . 5- Of t h e s i x g e n e r a o f g r e e n b a c t e r i a i n c l u d e d i n B e r g e y 's T.Tanual l o c . c i t . , t h e r e i s f a c t u a l p r o o f o f t h e e x i s t e n c e o f b u t o ne l e g i t i ­ m a te genus i . e . , C h lo ro b iu m . A ll o th e r genera have been e re c te d s o l e l y on t h e b a s i s o f p r e p a r a t i o n s made fro m n a t u r a l s a m p le s a n d h a v e n o t t a k e n i n t o a c c o u n t a n y v a r i a t i o n p o s s i b l y c a u s e d by d i f f e r e n t e n v iro n m e n ta l c o n d i t i o n s . A member o f t h e g en u s C h lo ro b iu m h a s b e e n i s o l a t e d a nd grown i n p u r e c u l t u r e by v a n N i e l ( 1 9 3 1 ) . The P ro b le m T h ere e x i s t s t h e n f o r t h e o r g a n is m s d e s i g n a t e d a s ’’g r e e n b a c t e r i a " but l i t t l e i n f o r m a t i o n u p o n w h ic h t o b a s e a n a p p l i c a t i o n o f a c c e p t e d ta x o n o m i c a l p r i n c i p l e s . I t has been in f e r r e d t h a t such d a ta as a r e a v a i l a b l e h a v e b e e n o b t a i n e d fro m p r e p a r a t i o n s w h ic h , w h i l e d e s i g n a t e d a s " p u r e " c o u l d n o t h a v e b e e n so i n t h e t r u e s e n s e o f t h e w o rd . The t e r m " p u r e c u l t u r e " m u st b e r e s e r v e d f o r o n e w h ic h c o n s i s t s o f t h e p ro g e n y o f a s i n g l e c e l l . The e a r l y i n v e s t i g a t o r s o f t h e p u r p l e and g r e e n b a c t e r i a d i d n o t e f f e c t s i n g l e c e l l i s o l a t i o n s a nd c u l t u r e s w h ic h a p p e a r e d p u r e m i c r o s c o p i c a l l y w e re a c c e p t e d w i t h o u t e x a c t i n g c r i t e r i a . The e x p r e s s i o n "D am it w a r d i e R e i h k u l t u r e r r e i c h t " in th e e arly l i t e r a t u r e . appears fr e q u e n tly H o w ever, i t s h o u l d b e e m p h a s iz e d h e r e t h a t t h e p u r e c u l t u r e s r e f e r r e d t o i n t h e l i t e r a t u r e w e r e o b t a i n e d by t r a n s ­ f e r r i n g fro m one l i q u i d medium t o a n o t h e r o f t h e same c o n s i s t e n c y . I t seems o b v io u s t h a t t h e u s e o f l i q u i d m e d ia w o u ld , i n t u r n , t a t e th e u se of th e s in g le c e l l i s o l a t i o n te c h n iq u e . n e c e ssi­ I t f o l l o w s t h e n t h a t t h e -v a r io u s s p e c i e s o f g r e e n b a c t e r i a h ave b e e n e s t a b l i s h e d l a r g e l y on t h e b a s i s o f s i z e w i t h no p r o v i s i o n f o r v a r ia tio n w ith in th e s p e c ie s i t s e l f . In d e e d , even g e n era have been e r e c t e d on t h e same g e n e r a l b a s i s o f s i z e o r o f c e l l c o n g l o m e r a t e s c o n sid e re d as s t a t i c u n i t s . I f s i z e was n o t u s e d a s t h e d i s t i n g u i s h ­ in g c h a r a c t e r i s t i c to s e p a r a t e g e n e r a , th e n a n a s s o c i a t i o n w ith a n o t­ h e r b a c t e r i u m o r some l o w e r o r g a n is m was deem ed s u f f i c i e n t l y u n i q u e t o m e r i t a new d e s c r i b e d fo rm . S c o p e o f t h e P r o b le m I t i s t h e pu rp o se th e n of t h i s p aper to p re s e n t such in fo rm a tio n a s h a s b e e n o b t a i n e d fro m a n e c o l o g i c a l i n v e s t i g a t i o n o f t h e g r e e n b a c t e r i a a n d t o s u p p le m e n t s u c h i n f o r m a t i o n w i t h d a t a fr o m p u r e c u l ­ tu re s tu d ie s . I t seems n o t im p r o b a b l e t h a t t h e i n f o r m a t i o n o f f e r e d may b e o f v a l u e i n i n t e r p r e t i n g t h e b i o l o g i c a l r e l a t i o n s h i p s i n a n e c o s y s te m a n d t h a t some l i g h t may b e th ro w n u p o n t h e p r e s e n t ta x o n o m ical sta tu s of t h e g r o u p u n d e r i n v e s t i g a t i o n ; th e in flu e n c e of th e e n v ir o n m e n t on m o r p h o l o g i c a l v a r i a t i o n , w h i l e e x t r e m e l y d i f f i c u l t t o e v a lu a te , w i l l be none th e le s s ev id en t. -7 - REVIEW OF THE LITERATURE H i s t o r i c a l l y , th e i n v e s t ig a t i o n s on th e g re e n b a c t e r i a sh o u ld p a r a l l e l th o s e o f th e p u rp le s u lp h u r b a c t e r i a ; t h e l i t e r a t u r e on t h i s l a t t e r g r o u p i s v o lu m in o u s a n d a p p r o x i m a t e l y 100 y e a r s o l d . w h ile t h e e a rly i n v e s t i g a t o r s H o w ev e r, of th e p u rp le b a c te r ia n o te d th e o c c u r­ r e n c e o f t h e g r e e n fo rm s no e x t e n s i v e s t u d i e s o f t h e i r m o r p h o l o g y , p h y s i o l o g y a nd e c o l o g i c a l r e l a t i o n s h i p s w e re m a d e . W h ile P r i n g s h e i m ( 1 9 4 9 ) has i n d i c a t e d t h a t S z a f e r f i r s t d e s c r i b e d t h e g r e e n b a c t e r i a in 1911, a c tu a lly th e l i t e r a t u r e i s much o l d e r , v a n T ieg hem ( 1 8 8 0 ) m e n tio n e d a B a c i l l u s v i r e n s as b e in g g r e e n i n c o l o r . E n g e lm a n ( 1 8 8 2 ) s t a t e d t h a t B a c te riu m c h lo rin u m c o n ta in e d a c h l o r o p h y l l . D a n g e a rd (1B 90) d e s c r i b e d fo rm s w h ic h we now know m u st h a v e b e e n g r e e n b a c t e r i a . N ad so n (1 9 1 2 ) g a v e u s t h e f i r s t , m o rp h o lo g ic a l c h a r a c t e r i s t i c s . stu d ie s a c tu a l re c o g n iz a b le d e s c r ip tio n s Nads o n ’ s w ork was s u p p le m e n te d b y t h e o f B uder (1 9 1 3 ) , P e r f i l i e v ( 1 9 1 5 ) a n d Bavendamm ( 1 9 2 4 ) . of I t is (1 9 1 4 ), P a sc h e r (1 9 1 4 ), L a u te rb o rn ex tre m ely d i f f i c u l t t o e v a lu a te t h e w o rk o f t h e s e E u r o p e a n i n v e s t i g a t o r s a s i t a p p e a r s t h a t o n l y i n r a r e i n s t a n c e s d i d o n e b i o l o g i s t e v e r a c k n o w le d g e t h e c o n t r i b u t i o n s of h is c o lle a g u e s. For in s ta n c e , P e r f ilie v ( 1 9 1 4 ) d e s c r i b e d much more e x t e n s i v e l y t h e fo rm s c h a r a c t e r i z e d by L a u t e r b o r n e a r l y i n 1 9 1 4 . P e r f i l i e v lo c . c i t . , a lso p o in te d out t h a t th e g reen b a c te r ia d e sc rib e d by L a u t e r b o r n had b e e n p r e v i o u s l y d e p i c t e d by S z a f e r i n 1 9 1 1 . P e rfilie v l o c . c i t . , commented f u r t h e r t h a t t h e C h lo r o c h r o m a tiu m a g g r e g a t u m o f L a u t e r b o r n was i d e n t i c a l w i t h t h e C h lo ro n iu m m i r a b i l e o f B u d e r . B ut L a u t e r b o r n i n 1915 p u b l i s h e d a n e x t e n s i v e p a p e r ’’D ie s a p r o p e l i s c h e L e b e w e l t '1 i n w h ic h h e made no m e n t i o n o f t h e w ork o f e i t h e r N a d so n or P e rfilie v ! G e i t l e r and P a s c h e r (1 9 2 5 ) i n a n a p p en d ix t o P a s c h e r ’ s S u s s w a s s e r f l o r a , H e f t 1 2 , l i s t e d t h e known g r e e n b a c t e r i a a n d p r o p o s e d a c h a n g e f r o m t h e o l d e r C h l o r o b a c t e r i a c e a e t o G y a n o c h l o r i d i n a e , on t h e b a s i s t h a t d e f i n i t e p r o o f was l a c k i n g a s t o t h e b a c t e r i a l n a t u r e of th e g r e e n o rg a n ism s. H ow ever M e tz n e r ( 1 9 2 2 ) i n v e s t i g a t e d t h e p i g ­ m e n ts o f t h e ' g r e e n b a c t e r i a a n d o f f e r e d p r o o f t h a t t h e y d i d n o t c o n­ t a i n e i t h e r c h l o r o p h y l l a o r b o r b a c t e r i o c h l o r o p h y l l ; M e tz n e r l o c . c it., c a l l e d t h e p ig m e n t f o u n d i n t h e g r e e n b a c t e r i a , b a c t e r i o v i r i d i n . G e itle r and P ascher lo c . c i t . , e i t h e r w e re n o t aw are o f M e t z n e r ’ s w o rk o r e ls e d isre g a rd e d i t . The m o s t r e c e n t w o rk o n t h e g r e e n b a c t e r i a h as b e e n d o n e by v a n H i e l ( 1 9 3 1 ) . I n a d d itio n to a r e v is io n of G e itle r and P a s c h e r ’ s tax o n o m y of t h e g r e e n b a c t e r i a f o r t h e s i x t h e d i t i o n o f B e r g e y 's M a n u a l, v a n N i e l h a s made t h e g r e e n b a c t e r i a t h e s u b j e c t of e x t e n s i v e , t h e o r e t i c a l r e s e a r c h on t h e f u n d a m e n t a l n a t u r e o f p h o t o s y n ­ th e sis. -9 - SODON LAKE MIGHIGAN FIGURE I CONTOUR MAP N A IN L E T -) BOATHOUSE 100 -10- DESCRIPTION OF ENVIRONMENT SELECTED FOR ECOLOGICAL STUDY The a q u a t i c e n v iro n m e n t u n d e r i n v e s t i g a t i o n i s l o c a t e d i n O a k la n d C o u n t y , M i c h i g a n ( B l o o m f i e l d T o w n s h ip , S e c t i o n 2 0 , l a t i t u d e 4 2 ° 1 9 1 , l o n g i t u d e 83° 1 7 ’ ) , The l a k e i s a s m a l l g l a c i a l l a k e l o c a t e d i n t h e O u t e r D e f i a n c e M o r a in e a n d h a s a maximum d e p t h o f 56 f e e t ; im a te ly 5 .7 a c re s i t is app ro x ­ i n a r e a a t t h e s u r f a c e , 3 . 2 a c r e s w i t h i n t h e 20 f o o t d e p t h c o n t o u r a n d 1 . 0 a c r e w i t h i n t h e 40 f o o t i s o b a t h . The v o lu m e d e ­ v e lo p m e n t o f t h e l a k e i s 1 .2 1 , in d ic a tin g t h a t m o rp h o lo g ic a lly th e la k e a p p ears as a cone. The l a k e i s su rro u n d in g h i l l s sh o re. p r o t e c t e d fro m w in d a c t i o n by t h e a n d b y a r e d m a p l e - t a m a r a c k swamp w h i c h b o r d e r s t h e The l i t t o r a l a r e a , t o a d e p t h o f 12 f e e t , a l g a , C hara c o n t r a r i a . is covered w ith th e Beyond t h e 12 f o o t c o n t o u r t h e r e i s a n d a moss o f t h e g e n u s F o n t i n a l i s . -1 1 - some N i t e l l a METHODS OF PROCEDURE 1. G e n era l. D u r in g t h e p e r i o d o f f i e l d o b s e r v a t i o n s , J a n u a r y 2 2 , 1949 to J a n u a r y 1 , 1950, th r o u g h t h e c o o p e r a tio n of t h e C ranbrook I n s t i t u t e o f S c i e n c e a r r a n g e m e n t s w e re m ade f o r t h e r e g u l a r c o l l e c t i o n o f w a t e r sam p les fro m Sodon L a k e . 9 :3 0 i n t h e m o rn in g . S a m p lin g was d o n e b i - m o n t h l y , o r d i n a r i l y a t The d e e p e s t p a r t o f t h e l a k e (5 6 f e e t ) was s e ­ l e c t e d as a p erm anent s t a t i o n ; t h i s w as d e s i g n a t e d a s S t a t i o n A, t h e l o c a t i o n o f w h ic h may b e s e e n b y r e f e r e n c e t o F i g u r e 1 , T he C o n t o u r Map o f Sodon L ak e. S a m p l i n g was d o n e f r o m t h e s u r f a c e o f t h e l a k e t o t h e m u d - w a te r i n t e r f a c e , u s u a l l y a t i n t e r v a l s of fiv e f e e t. H o w e v e r, o t h e r d e p th i n t e r v a l s w ere sam p led f o r s p e c i a l s t u d i e s and w h e n ev e r any t h e r ­ m a l d i s t u r b a n c e was n o t e d . S a m p le s f o r b a c t e r i o l o g i c a l a n a l y s i s w e re c o l l e c t e d b o ttle s of th r e e l i t e r c a p a c ity . in s te r ile The s a m p l e s w e r e o b t a i n e d fr o m t h e d e s i r e d d e p t h s b y t h e u s e o f a K em m erer w a t e r s a m p l e r ( s e e a p p e n d i x fo r illu s tra tio n ); th is same d e v i c e s e r v e d t o s e c u r e m a t e r i a l f o r chem­ i c a l , p h y s ic a l and b i o l o g i c a l d e t e r m i n a t i o n s . Any d e p a r t u r e f r o m r o u ­ t i n e pro ced u res w i l l be in d ic a te d u n d er a p p r o p r ia te h e a d in g s . A w o rd s h o u l d b e s a i d h e r e c o n c e r n i n g t h e d i f f i c u l t i e s in h e re n t i n t h e o b t a i n i n g o f a w a te r sa m p le from a s p e c i f i c d e p th a n d , a t t h e sam e t i m e , i n s u r e t h a t t h e s a m p l e i s r e p r e s e n ta tiv e of t h a t p a r t i c u l a r d e p th and n o t c o n ta m in a te d by a n e x tra n e o u s f l o r a or fau n a. The f i r s t o b j e c t i o n , t h a t t h e sam p le s e c u re d is n o t r e p r e s e n t a t i v e o f a p a r t i c u ­ l a r d e p th , i s th is e a sily in v e stig a tio n , o v e rc o m e . I n t h e p r e l i m i n a r y w ork c o n c e r n e d w i t h t h e d e s ir e d sam p les w ere o b ta in e d i n s t e r i l e -1 2 - g la ss b o ttle s f i t t e d , w i t h a n i n l e t t u b e w h i c h was b r o k e n a t t h e d e s i r e d d e p t h by s e n d i n g down a m e s s e n g e r . tita tiv e ly H o w ev e r, n e i t h e r q u a l i t a t i v e l y nor quan­ d i d t h e p l a n k t o n d i f f e r fr o m t h a t o b t a i n e d b y u s i n g t h e K emmerer s a m p l e r . ap p aren t th a n r e a l . The c o n t a m i n a t i o n f r o m t h e s a m p l e r i t s e l f I t see m s t o t h e a u t h o r t h a t t h e r e i s i s m o re c o n sid e ra b le j u s t i f i c a t i o n i n u s i n g t h e K em m erer b o t t l e f o r s e c u r i n g s a m p l e s f o r b a c te rio lo g ic a l a n a ly sis. It i s re c o g n iz e d t h a t m e ta l b o t t l e s , in sta n c e s, are b a c te ric id a l o r b a c te r io s ta tic ; i n som e h ow ever, t h i s a c t i o n has b een o b s e rv e d o n ly on s t o r e d w a t e r s a m p le s Z o b e ll ( 1 9 4 1 ) . I t sh o u ld a l s o b e n o t e d t h a t t h e b a c t e r i c i d a l a c t i o n i s m o re p r o n o u n c e d w i t h b rig h tly p o lish e d s u rf a c e s . U nder t h e c o n d itio n s re p o rte d upon h e re , s a m p le s w e r e t r a n s f e r r e d f r o m t h e s a m p l e r t o s t e r i l e b o t t l e s In le s s th a n t h r e e m in u te s ; and t h e s a m p le r w a s, a t a l l t i m e s , h e a v i l y c o a te d w ith o x id e s . We s h o u l d l i k e t o make o n e f u r t h e r p o i n t i n c o n n e c t i o n w ith t h e u s e o f t h e m e ta l sa m p lin g d e v i c e . I f t h e m e ta l i s b a c t e r i c i d a l , t h e n t h e f l o r a w h i c h re m a in s a t t a c h e d t o t h e b o t t l e , w h i l e i t s t o r e d , s h o u l d b e k i l l e d a n d t h e b o t t l e w o u ld t h e r e f o r e b e , is b e in g in a sen se, ’’s t e r i l e ” when a g a i n p l a c e d i n o p e r a t i o n . 2. F ie ld In v e s tig a tio n s . th e f i e l d The f o l l o w i n g p r o c e d u r e s w e r e c a r r i e d o u t i n a t each b i-m o n th ly sam p lin g p e r io d : 1. C o l l e c t i o n o f sam p les f o r b a c t e r i o l o g i c a l a n a ly s e s 2 . C o l l e c t i o n o f sam p les f o r c h e m ic a l a n a ly s e s 3. C o l l e c t i o n o f sam ples f o r a n a ly s e s o f t h e h y d r o s o l 4 . C o l l e c t i o n of e u p la n k to n ( p h y to p la n k to n and z o o p la n k to n ) -1 3 - 5. V e r tic a l d i s t r i b u t i o n of te m p e ra tu re 6 . M e a s u re m e n t o f l i g h t i n t e n s i t i e s a t v a r io u s d e p th s 7 . S u s p e n s i o n o f s t a n d a r d 25 x 75 mm. g l a s s s l i d e s fo r a t t a c h m e n t s t u d i es 3 . L a b o ra to ry I n v e s t i g a t i o n s . The f o l l o w i n g p r o c e d u r e s w e r e c a r r i e d o u t i n t h e l a b o r a t o r y a t each b i-m o n th ly sam p lin g p e r io d : 1 . C h e m ic a l d e t e r m i n a t i o n s of d i s s o l v e d oxygen, h y d ro g e n s u lp h id e , c a rb o n a te , b ic a rb o n a te , n i t r a t e n itr o g e n , n i t r i t e n itro g e n , i r o n and p h o s p h o ro u s. 2. D e te rm in a tio n of h y d ro g e n -io n c o n c e n tra tio n 3 . E n u m e ra tio n and i d e n t i f i c a t i o n of p la n k to n 4 . A n a ly s is o f subm erged s l i d e s a s a c c e s s o r y i n f o r m a t i o n to p o p u la tio n s tu d ie s 5 . S p e c ia l b a c t e r i o l o g i c a l s t u d i e s , w h ic h w i l l be i n d i ­ c a te d u n d e r a p p r o p r ia te h e ad in g s. -1 4 - PRELIMINARY RECONNAISSANCE I n A u g u s t o f 1947 a g r e e n o r g a n i s m a p p e a r e d i n tr e m e n d o u s num­ b e r s i n t h e p h y t o p l a r i k t o n o f S o d o n L a k e ; t h e o r g a n i s m was p r e s e r v e d b u t no s t u d y was made o f i t . L a te r, i t was t e n t a t i v e l y id e n tifie d b y t h e a u t h o r a s C h lo r o b iu m l i m i c o l a Nads on a member o f t h e g r e e n s u l ­ fu r b a c te ria . From J a n u a r y 1949 t o J a n u a r y 1 9 5 0 , i n c o n n e c t i o n w i t h a b a c t e r i o l o g i c a l s u r v e y on t h e e c o l o g y , d i s t r i b u t i o n a n d n u t r i t i o n o f o t h e r a q u a t i c b a c t e r i a , we h a v e s t u d i e d t h e s e a s o n a l d i s t r i b u t i o n , t h e v e r t i c a l and h o r i z o n t a l d i s t r i b u t i o n , t h e c u l t u r e , c h em ica l, p h y s i­ c a l a nd b i o l o g i c a l e n v i r o n m e n t a n d m o r p h o l o g y o f t h e g r e e n b a c t e r i u m . METHODS OF PLANKTON EITOI-TERATIOl'T I n o r d e r to d e te r m in e t h e s e a s o n a l peaks and tr e n d s of th e g r e e n b a c t e r i a a n d o t h e r m em bers o f t h e p l a n k t o n , i t w as n e c e s s a r y t h a t an e n u m e ra tio n s ta n d a r d b e s o u g h t. W h i l e we a r e a w a r e t h a t many d i v e r s e q u a n t i t a t i v e p l a n k t o n m e th o d s e x i s t , Lackey (1 9 3 8 ) and L i t t l e f o r d m ent as t o et a l. ( 1 9 4 0 ) , t h e r e i s no g e n e r a l a g r e e ­ w hat c o n s t i t u t e s a c o u n tin g u n i t . h e r e to re v ie w o r t o (see p a rtic u la rly No a t t e m p t w i l l b e m ade e v a l u a t e t h e v a r i o u s q u a n t i t a t i v e m e th o d s w h i c h have been proposed f o r t r e a t i n g p la n k to n ic fo rm s; i t seem s o b v io u s t h a t th e p ro c e d u re a d o p ted m ust b e d e te rm in e d by th e c o n d itio n s th e f ie ld e x p e rim e n t, p a r t i c u l a r l y t h e r e l a t i v e ab u n d an ce and s i z e o f t h e o rg a n ism s u n d e r c o n s i d e r a t i o n . stric tly of H arvey (1 9 3 4 ) has s a i d n t h e o n ly q u a n t i t a t i v e m e th o d o f s a m p l i n g t h e p o p u l a t i o n i s t o f u g e a s m a l l sam p le o f w a t e r and c o u n t th e p l a n t s in i t ” . u n d e r th e c o n d itio n s o f t h e p re s e n t e x p e rim e n t, t r e a t e d c e n tri­ We h a v e , each o rg a n ism a s a u n i t an d h av e n o t a s s i g n e d c o lo n y f a c t o r s t o o rg a n ism s w h ic h e x is t as a g g re g a te s of s in g le c e l l s . By u s i n g s u c h a m e t h o d , w e h a v e e lim in a te d t h e p e r s o n a l f a c t o r a s f a r as ju d g m e n t o f t h e num ber o f c e lls in a c o lo ry is co n cern ed . The s p e c i f i c t e c h n i q u e w h i c h we h a v e u s e d f o r o u r e n u m e r a t i o n s is t h a t o f G. W. M a r t i n ( m s . ) a n d i s (m s . 1 9 4 7 ) . d e s c r i b e d i n d e t a i l by V a n D e u s e n E s s e n t i a l l y , t h e m e th o d c o n s i s t s o f e n u m e ra tio n by d i r e c t c o u n t an d c a l c u l a t i o n o f t h e num ber o f o rg a n is m s p e r l i t e r o f l a k e w a te r. -1 6 - A S p e n c e r b i n o c u l a r m ic ro sc o p e eq u ip p ed w i t h a c o m b in a tio n o f 25x o c u l a r s a n d a 97x o b j e c t i v e was u s e d f o r e n u m e r a t i o n o f t h e b a c t e r i a ; f o r t h e p h y t o p l a n k t o n , 15x o c u l a r s a n d a 43 x o b j e c t i v e w e re u s e d ; f o r e n u m e r a t i o n o f t h e z o o p l a n k t o n , a c o m b i n a t i o n o f 15x o c u l a r s a n d a lOx o b j e c t i v e w as u s e d . The t h r e e l i t e r s a m p l e s f o r b a c t e r i a c o u n t s a n d f o r t h e p h y t o ­ p l a n k t o n w e r e p a s s e d t h r o u g h a F o e r s t C e n t r i f u g e ( 1 5 , 0 0 0 G ). The c o n­ c e n t r a t e was w a s h e d i n t o a s m a l l v i a l a n d t h e o r i g i n a l s a m p l e w a s r e ­ c e n trifu g e d . In a l l , t h i s p r o c e s s w as r e p e a t e d t h r e e t i m e s . The c on ­ c e n t r a t e was t h e n c o m p o s i t e d a n d c o u n t e d i m m e d i a t e l y o r p r e s e r v e d f o r l a t e r stu d y . One l i t e r s a m p le s f o r t h e z o o p l a n k t o n c o u n t w e r e s t r a i n e d t h r o u g h a nu m b e r 25 s i l k p l a n k t o n n e t a n d a t a l l y t h e c o n c e n t r a t e was m a d e. of e ac h o rg a n ism p r e s e n t i n The t o t a l was e x p r e s s e d a s t h e n u m b e r o f o rg an ism s p e r l i t e r o f l a k e w a t e r . -1 7 - THE VERTICAL AND HORIZONTAL DISTRIBUTION To d e t e r m i n e t h e v e r t i c a l a n d h o r i z o n t a l d i s t r i b u t i o n p r o f i l e o f t h e g r e e n b a c t e r i a , s e v e n s t a t i o n s w ere s e l e c t e d f o r sa m p lin g ; The l o c a t i o n s o f t h e s e s t a t i o n s a r e show n i n F i g u r e s 1 a n d 2 . S a m p le s w e r e s e c u r e d fr o m t h e f o l l o w i n g d e p t h s , m e a s u r e d fr o m t h e s u r f a c e : fo u r in c h e s, fo u r f e e t , te e n f e e t, e ig h t f e e t , tw e lv e f e e t , f o u r te e n f e e t , six ­ tw e n ty f e e t , t w e n t y - f o u r f e e t a n d t w e i i t y - e i g h t f e e t . th e tw e n ty -e ig h t fo o t le v e l, B elo w p h o to lo m e te r re a d in g s in d ic a t e d zero t r a n s m i s s i o n o f l i g h t and h e n c e a n u n f a v o r a b l e e n v iro n m e n t f o r t h e g r o w th o f t h e g r e e n b a c t e r i a e x i s t e d th e re . The s e v e n s t a t i o n s lo ­ c a t e d a l o n g t h e t r a n s e c t , h e r e a f t e r d e s i g n a t e d a s n T” a r e a s f o l l o w s : T1 a n d T7 ............ T2 a n d T6 ............. T3 a n d T5 ............ T4 M icro sc o p ic e x a m in a tio n o f t h e p h y to p la n k to n i n d i c a t e d w hat a p ­ p eared to b e a dense c o n c e n tr a tio n o f g re e n b a c t e r i a 5 fe e t. e x istin g a t 25t T h is o b s e r v a t i o n was f u r t h e r t e s t e d by d e t e r m i n i n g t h e a b s o r p ­ t i o n s p e c t r a o f t h e v a r i o u s s a m p l e s in ' a C o lem an J r . S p e c t r o p h o t o m e t e r . I n e v e r y c a s e , t h e z o n e o f c o n c e n t r a t i o n was s h a r p l y d e l i m i t e d a t 25?! 2 fe e t. The h o r i z o n t a l d i s t r i b u t i o n was d e t e r m i n e d o v e r t h e e n t i r e h o r i z o n t a l t r a n s e c t o f t h e l a k e a n d a g a i n sh ow ed t h a t t h e z o n e o f c o n ­ c e n t r a t i o n a p p e a re d t o be a t 2 5 l 2 f e e t and t o e x te n d s h o re w a rd t o m eet th e la k e b o tto m . -1 9 - SEASONAL DISTRIBUTION At th e s t a r t of th is i n v e s t i g a t i o n s t e r i l e 25 x 75 mm. g l a s s s l i d e s w ere p la c e d i n s p e c i a l c o n ta in e r s and su sp en d ed so t h a t t h e r e e x i s t e d a c o n tin u o u s a tta c h m e n t s u r f a c e fro m j u s t b e lo w t h e s u r f a c e o f t h e w a t e r t o t h e m u d - w a te r i n t e r f a c e . th e b a c te r ia d e p o site d upon th e s lid e s I t was p l a n n e d t o t r e a t i n a q u a n t i t a t i v e m a n n er, and t h u s p r o v i d e f o r s e a s o n a l d a t a on t h e b a c t e r i a l p o p u l a t i o n o f S o d o n Lake, S u c h a m e th o d h a d b e e n em p lo y e d b y H e n r i c i ( 1 9 3 3 ) i n h i s p i o n e e r w ork o n t h e a q u a t i c b a c t e r i a o f M i n n e s o t a l a k e s . F u r th e r m o re , Z o b e ll ( 1 9 4 1 ) a n d o t h e r s h a v e e m p h a s iz e d t h a t t h e b a c t e r i a l p o p u l a t i o n o f t h e open w a te r of l a k e s and oceans i s la r g e ly p e r ip h y tic j Z o b e ll l o c . c i t . , has p o s tu la te d t h a t th e r e l a t i v e l y sm a ll b a c t e r i a l p o p u la tio n r e p o r te d f o r l a k e s a nd r i v e r s i s d u e t o t h e f a c t t h a t a q u a t i c b a c t e r i a a r e r e ­ moved f r o m t h e p h y t o p l a r i k t o n by a t t a c h i n g t h e m s e l v e s t o s o l i d s u r f a c e s , th u s in c r e a s in g t h e i r s p e c i f i c g r a v ity and r a t e of s e d im e n ta tio n . The a s s u m p t i o n by H e n r i c i , Z o b e l l a n d o t h e r s t h a t t h e o r g a n i c m a t e r i a l d e p o s i t e d on s l i d e s r e p r e s e n t e d g ro w in g b a c t e r i a l c e l l s p roved t o be u t t e r l y f a l l a c i o u s . has A c h a n c e o b s e r v a t i o n , a r i s i n g fr o m i n a d v e r t e n t l y g ra m s t a i n i n g a s t e r i l e s l i d e w h ic h h a d b e e n s u s p e n d e d in s t e r i l e w a te r, r a is e d th e q u e s tio n as t o w h e th e r t h e m a t e r i a l b e in g d e p o s i t e d on t h e s u b m e rg e d s l i d e s a c c u m u la tio n of o rg a n ic m a t e r i a l . re p re se n te d b a c te r ia o r m e re ly an I t was fo u n d t h a t t h e a m o u n t o f ma­ t e r i a l w h ic h a c c u m u l a t e d on a g l a s s s l i d e i n a g i v e n p e r i o d o f t i m e c o u ld b e m easured f a i r l y a c c u r a t e l y . A w e t c o m b u s t i o n m e th o d w i t h p o t a s s i u m d i c h r o m a t e i n 50% s u l f u r i c a c i d was u s e d a s t h e o x i d i z i n g -2 0 - a g e n t;. The d i c h r o m a t e s o l u t i o n was 0 .0 1 5 N . The am ount o f m a t e r i a l a c c u m u l a t e d was d e t e r m i n e d by t h e d i f f e r e n c e i n b l a n k t i t r a t i o n s of e q u a l v o lu m e s o f t h e d i c h r o m a t e s o l u t i o n , a f t e r a c t i n g o n t h e s l i d e f o r 15 m i n u t e s a t 1 0 0 ° C. I t a p p eared d e f i n i t e l y t h a t a n a c c u m u la tio n o f o r g a n i c m a t e r i a l w o u ld t a k e p l a c e i n s t e r i l e w a t e r a s w e l l a s i n th e n a t u r a l la k e w a te r . m e rg e d s l i d e s C o n se q u e n tly , th e d a ta o b ta in e d from t h e su b ­ can n o t b e used in a q u a n tita tiv e sen se. O ur d i s c u s s i o n o f t h e s e a s o n a l d i s t r i b u t i o n o f t h e g r e e n b a c t e r i a i n Sodon Lake i s b a se d on th e p la n k to n c o u n ts . The s e a s o n a l a p p e a r a n c e o f t h e g r e e n b a c t e r i a h as b e e n e r r a t i c . U n d er t h e c o n d i t i o n s o f t h e p r e s e n t i n v e s t i g a t i o n , t h e y w e r e f i r s t n o t e d i n t h e p l a n k t o n on J a n u a r y 2 2 , 1949 a n d c o n t i n u e d t o b e p r e s e n t , a lth o u g h i n d im in is h in g n u m bers, u n t i l A p r i l . The g r e e n b a c t e r i a d i d not re a p p ea r in th e p h y to p la n k to n u n t i l J u ly ; in te n s iv e sa m p lin g , b o th d a ily and d iu r n a l f a i l e d t o rev eal t h e ir presence. From J u l y u n t i l N ovem ber, t h e y w e r e a g a i n m em bers o f t h e p l a n k t o n c o m m u n ity ; h o w e v e r , no t r a c e o f g r e e n b a c t e r i a h a s b e e n f o u n d i n t h e p l a n k t o n fr o m e a r l y N ovem ber t o t h e t e r m i n a t i o n o f t h i s stu d y . An e x a m i n a t i o n o f F i g u r e 3 w i l l sh o w t h e s e a s o n a l d i s t r i b u t i o n o f t h e g r e e n b a c t e r i a i n S o d o n Lake. From t h e a p p e a r a n c e o f t h e g r a p h , i t seem s p o s s i b l e t h a t a num­ e r i c a l p e a k o c c u r r e d i n J a n u a r y o f 1 9 4 9 ; a s e c o n d a n d o b v io u s p e a k o c ­ c u rre d i n A u g u st. We c a n n o t t h e o r i z e c o n c e r n i n g t h e f a t e of th e g reen b a c t e r i a a f t e r t h e e a r l y p a r t o f N ovem ber; i t i s t e m p t i n g t o p o s t u l a t e th a t, i f t h e o r g a n i s m s c o n t i n u e d t o b e members o f t h e p l a n k t o n -2 1 - c o m m u n ity , a n u m e r i c a l p e a k w o u ld h a v e b e e n r e a c h e d i n J a n u a r y o r F e b ru a ry o f 1950, We s h a l l d i s c u s s so m ew hat l a t e r , p o s s ib le reaso ns f o r th e d is a p ­ p e a ra n c e o f t h e g re e n b a c t e r i a from t h e p h y to p la n k to n of Sodon Lake, H ow ev er, we s h o u l d l i k e t o e m p h a s i z e h e r e t h a t , c o in c id e n t w ith th e o b s e r v a t i o n t h a t t h e g r e e n b a c t e r i a w e r e no l o n g e r r e s i d e n t s of th e p l a n k t o n c o m m u n ity , a c o m p l e t e r e i n v e s t i g a t i o n o f t h e l a k e was acc o m p ­ l i s h e d ; s e v e r a l a d d i t i o n a l s t a t i o n s w ere e s t a b l i s h e d and a l l in g d e p th s w e re sa m p le d . The r e s u l t s , in te rv e n ­ i n so f a r a s d e te r m in in g t h e p r e s e n c e o f t h e g r e e n b a c t e r i a was c o n c e r n e d , w e r e c o m p l e t e l y n e g a t i v e . -22- r u i N U O IN U ' S A M E D I M H CO. N O . 3 1 6 C El'TVIHOWTEKTAL FACTORS POSSIBLY AFFECTING THE 20NATI0N OF THE GREEN BACTERIA If each p h y s i c a l , ch em ical and b i o l o g i c a l f a c t o r u n d e r c o n s id e r ­ a t i o n i s a n a l y z e d i n d i v i d u a l l y b u t w i t h t h e r e a l i z a t i o n t h a t no o ne f a c to r o r s e t o f c o n d itio n s is e v e r re s p o n s ib le f o r a g iv e n s i t u a t i o n , t h e e n v i r o n m e n t a l h a b i t a t o f t h e g r e e n b a c t e r i a beco m es c l e a r . e v e r , we do n o t , a t t h i s p o in t, How­ o f f e r such in fo rm a tio n as p o s s ib le c l u e s t o c o n t r o l l i n g m e c h a n ism s b u t r a t h e r a s a g u i d e t o t h e o v e r a l l e c o l o g i c a l p i c t u r e o f Sodon Lake, The f o l l o w i n g e n v i r o n m e n t a l f a c t o r s h a v e b e e n i n v e s t i g a t e d t h r o u g h ­ out th e co u rse o f t h i s in v e s tig a tio n s A, P h y sic a l 1. T e m p e ra tu re 2 . H y d ro g e n -io n c o n c e n tr a tio n 3. L ig h t i n te n s ity , in c lu d in g r e la tio n s h ip to tu r b id ity B, C h e m ic a l 1, C h e m istry o f th e w a te r 2. S p e c tro c h e m istry o f th e w a te r C, B io lo g ic a l 1, P h y to p la n k to n 2. Z o o p lan k to n A, P h y s i c a l . 1. T e m p e ra tu re . of th is The t e m p e r a t u r e s ta k e n th ro u g h o u t th e e n t i r e p e rio d i n v e s t i g a t i o n a r e shown i n T a b l e I (A p p e n d ix ); te m p e r a tu r e r e a d i n g s t a k e n on a t r a n s e c t o f t h e z o n e o f g r e e n b a c t e r i a a r e i n d i ­ c a te d i n T a b le I I , These l a t t e r v a l u e s a r e re c o r d e d as o f A u g u st 4 , 194-9 a n d i n d i c a t e a p e r i o d when t h e g r e e n b a c t e r i a w e r e a t t h e i r -2 4 - TABLE I I TRANSECT OF ZONE OF GREEN BACTERIA, SODON LAKE, 4 ATTG. 4 9 . T e m p e ra tu re r e a d i n g s : D e p th 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 IB 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 D egrees F a h r e n h e i t T3 8 0 .6 8 0 .5 8 0 .2 7 8 .7 7 8 .1 7 7 .6 7 6 - 8 * 7a A 7 2 ,8 * 7 0 .4 6 6 .9 6 3 .3 5 9 .1 5 6 .7 5 4 .2 5 1 .7 4 9 .1 4 7 .3 4 5 .8 4 4 .6 4 3 .6 4 3 .0 4 2 .2 4 1 .7 4 1 .1 4 0 .9 4 0 .5 4 0 .2 4 0 .2 4 0 .1 4 0 .0 4 0 .1 4 0 .1 4 0 .1 4 0 .0 4 0 .1 4 0 .2 4 0 .5 T5 8 2 .3 8 1 .1 8 0 .5 7 9 .8 7 8 .8 7 7 .7 7 6 .6 7 3 .5 7 0 .7 6 7 .1 6 3 .2 6 0 .5 5 5 .9 5 3 .7 5 2 .1 4 9 .2 4 7 .2 4 5 .3 4 4 .3 4 3 .5 4 2 .8 4 2 .1 4 1 .5 4 1 .2 4 1 .0 4 0 .9 4 0 .7 4 0 .7 4 0 .5 4 0 .2 4 0 .2 4 0 .2 4 0 .2 4 0 .2 4 0 .3 4 0 .3 4 0 .4 4 0 .5 4 0 .5 4 0 .7 45 46 47 48 49 50 55 T4 7 9 .0 7 9 .0 7 8 .9 7 8 .7 7 8 .3 7 7 .4 7 5 .8 7 3 .2 6 9 .4 6 6 .3 6 3 .4 6 0 .3 5 6 .5 5 3 .2 5 1 .2 4 9 .1 4 7 .3 4 5 .6 4 4 .4 4 3 .8 4 3 .0 4 2 .4 4 1 .7 4 1 .2 4 1 .1 4 0 .8 4 0 .7 4 0 .7 4 0 .6 4 0 .5 4 0 .4 4 0 .9 4 0 .9 4 1 .0 4 1 .1 4 1 .2 * I n t e r m e d i a t e r e a d in g ta k e n a t 6 .5 f t . -2 5 - d e p th . i n S o d o n L ake k T hree Temperatare Trends xn ■ nnal Model Sdcwxng Temp Bim ensxonal 90° -to o -„h t 9 O0 "to L e f t V e r tic a l Zones ( S u r f fo e D e L Time (Jam* Terape r a 't u r e —2 6 - , „ _ 750 f .) n u m e ric al p e ak . A t h r e e d i m e n s i o n a l g r a p h s h o w in g s e a s o n a l t e m p e r a ­ t u r e t r e n d s o f Sod on L a k e i s p r e s e n t e d i n F i g u r e 4 . I t m u s t b e e m p h a s iz e d t h a t t h e t e m p e r a t u r e s d e l i m i t i n g t h e z o n e s of c o n c e n tra tio n vary b u t l i t t l e th ro u g h o u t th e y e a r . I t w ill be re ­ c a l l e d t h a t t h e zone o f c o n c e n tr a tio n f o r th e g re e n b a c t e r i a has b e en d e t e r m i n e d a t 25jf 2 f e e t . W h ile i t is re c o g n iz e d t h a t , i n a c c o r d a n c e w i t h V a n 't H o f f 's la w , a n d c o n s i d e r i n g t h e a c a d e m ic a p p r o a c h t o t h e e f f e c t o f t e m p e r a t u r e on p o p u l a t i o n s , p l a n k t o n p o p u l a t i o n s a r e d e t e r m i n e d l a r g e l y b y t h e te m ­ p e r a t u r e c h a r a c t e r i s t i c s of a body of w a t e r , i t m ust a l s o be n o te d t h a t t h e te m p e r a tu r e ra n g e from th e u p p e r l i m i t s zo n e t o t h e b o t t o m o f t h e l a k e i s o f th e green b a c te r ia r a r e l y m ore t h a n 1 ° F . A c t u a l l y v e r y l i t t l e fu n d a m e n ta l r e s e a r c h h a s b e e n done on t h e e f f e c t s o f t e m p e r a t u r e on t h e a c t i v i t i e s of in d iv id u a l p la n k to n ic o rg a n ism s; f o r t h e p e r io d o f o b s e r v a tio n o f t h e p r e s e n t i n v e s t i g a t i o n , t h e f a c t o r o f t e m p e r a t u r e d o e s n o t seem t o e x e r t a n a p p r e c i a b l e e f f e c t upon th e d i s t r i b u t i o n of g reen b a c t e r i a . In g e n e ra l, i n t h e l a t e s p r i n g a n d e a r l y sum m er, t h e t e m p e r a t u r e of s u r f a c e w a ters v a r ie s ow ing t o i n c r e a s e d s o l a r r a d i a t i o n ; t h e r e ­ s u l t i n g d i f f e r e n c e i n d e n s i t y o f t h e w a te r g iv e s r i s e t o t h e form a­ t i o n o f two d i s t i n c t l a y e r s o f w a te r, th e u p p er la y e r o r e p ilim n io n a n d t h e lo w e r l a y e r o r h y p o l i m n i o n . U n d e r some c o n d i t i o n s t h e te m ­ p e r a t u r e i s a p p ro x im a te ly t h e sam e a t a l l d e p th s I n t h e e p ilim n io n . B elow t h e e p i l i m n i o n i s t h e t r a n s i t i o n a l z o n e , known a s t h e therm o-r c lin e . H ere, th e te m p e ra tu re f a l l s -2 7 - ra p id ly w ith in c r e a s in g d e p th . I n t h e h y p o l i r r m i o n , t h e t e m p e r a t u r e , w h ic h o r d i n a r i l y is c o n sid e ra b ly l o w e r t h a n t h a t o f t h e e p i l i m n i o n d o e s n o t c h a n g e much w i t h i n c r e a s ­ in g d e p th . The f a c t o r o f t e m p e r a t u r e h e r e , s e e m s , i n t h i s in sta n c e , t o be m o re o f a n a c a d e m ic q u e s t i o n t h a n a p r a c t i c a l b i o l o g i c a l o n e . A ll te m p e ra tu re re c o rd s th ro u g h o u t t h i s w ith a F oxboro E l e c t r i c i n v e s t i g a t i o n w ere t a k e n T h erm o p h o n e ( S e e a p p e n d i x f o r i l l u s t r a t i o n ) . T h is i s a c t u a l l y a n e l e c t r i c t h e r m o m e t e r w h ic h a c t s u p o n t h e p r i n c i ­ p le t h a t th e r e s i s t a n c e of a c o n d u c to r t o t h e p a ssa g e of a n e l e c t r i c c u r r e n t changes w ith th e te m p e ra tu re , a ls o t h a t t h e r a t e r e s is ta n c e due to te m p e ra tu re d i f f e r s o f change in in d iff e r e n t m e ta ls. The th e r m o p h on e p o s s e s s e s a d v a n t a g e s o v e r m o s t o t h e r k i n d s o f s u b - s u r f a c e te m ­ p e r a t u r e m e asu rin g d e v ic e s i n t h a t i t c u ra te . is e x tre m ely s e n s i t i v e and a c ­ ( D e s c r i p t i o n l a r g e l y t a k e n f r o m W e lc h 1 9 4 8 ) . 2 . H y d ro g e n -io n c o n c e n t r a t i o n . th e r e s u l t s An e x a m i n a t i o n o f F i g u r e 5 w i l l sho w o f pH d e t e r m i n a t i o n s , t a k e n o v e r a p e r i o d o f t w e l v e m o n th s I n T a b le I I I a r e sh o w n t h e pH v a l u e s t a k e n on a t r a n s e c t o f t h e g r e e n b a c te ria peak. z o n e a t a p e r i o d w hen t h e g r e e n b a c t e r i a r e a c h e d a n u m e r i c a l A l l pH v a l u e s w e r e m e a s u r e d e l e c t r o m e t r i c a l l y , u s i n g a L e e d a n d N o r t h r u p pH m e t e r . O r d in a r ily , s u r f a c e w a te rs u n d erg o r e l a t i v e l y s m a ll changes i n pH v a l u e f r o m s e a s o n t o s e a s o n . G e n e r a l c h a n g e s i n pH o f a p r o g r e s ­ s i v e , a p p a r e n tly perm anent k in d a r e a b s e n t o r a r e so e x tre m e ly s l i g h t as to be in d is tin g u is h a b le . Each o rg a n ism h as i t s own t o l e r a t i o n r a n g e o f pH t e r m i n a t e d b y a minimum a n d a maximum a n d p o s s e s s e s a n optim um a t some i n t e r m e d i a t e p o sitio n * T hese t o l e r a t i o n ra n g e s a r e e x tre m e ly d i f f i c u l t , i f not im p o s s ib le , t o d e term in e u n d e r f i e l d c o n d itio n s . The l i t e r a t u r e c o n t a i n s n u m erou s p o s i t i v e c o n t e n t i o n s t h a t pH i s a n i m p o r t a n t l i m i t i n g f a c t o r f o r many o r g a n i s m s ; h o w e v e r , s o m e tim e s th e e v id en c e i s c o n f l i c t i n g f o r t h e same s p e c i e s . Many w o r k e r s r e g a r d t h e pH a s o f a t l e a s t s e c o n d a r y i m p o r t a n c e , a n d c e r t a i n i n v e s t i g a t o r s h a v e c l a i m e d t h a t no c o r r e l a t i o n o f a n y s o r t e x i s t s w i t h t h e pH r a n g e as i t occurs i n n a t u r a l w a te r s . A c c o r d i n g t o W e lc h * , i t h a s b e e n show n t h a t a c c l i m i t i z a t i o n e f f e c t s may b e m a n i f e s t e d n o t o n l y i n i n ­ d i v i d u a l s from w id e ly s e p a r a t e d p a r t s o f t h e g e o g r a p h i c a l ra n g e b u t, a l s o i n t h e i n s t a n c e o f i n d i v i d u a l s o f t h e sam e s p e c i e s w h i c h o c c u p y w a t e r s c l o s e l y a d j a c e n t b u t d i f f e r i n g w i d e l y i n t h e pH v a l u e . I t seem s p r o b a b l e t h a t pH, p e r s e , c a n b e o f l i t t l e s ig n ifi­ c a n c e ; b u t r a t h e r i s a n i n d i c a t i o n o f t h e c h e m i c a l c o m p le x e s i t i n g i n a n a q u a t i c e n v iro n m e n t. 3. L ig h t I n t e n s i t y . M e a s u re m e n ts o f t h e p e n e t r a t i o n o f l i g h t t h r o u g h t h e w a t e r a n d i c e w e r e m ade w i t h a s u b m e r s i b l e p h o t o m e t e r d e s i g n e d by D r. G e o rg e C l a r k o f H a r v a r d U n i v e r s i t y a n d e x e c u t e d b y F . S c h u e l e r o f W a lth a m , M a s s . , ( s e e a p p en d ix f o r i l l u s t r a t i o n ) . stru m e n t c o n s is ts o f tw o W e s to n p h o t r o n i c c e l l s , f o o t w a te rp ro o f c a b le to a g a lv a n o m e te r. 1 W e lc h , P . ' S . L im n o lo g y . E s s e n tia lly , th e in ­ c o n n e c t e d b y a 60 B e tw e e n t h e c e l l s th e re is a McGraw H i l l , Hew Y o r k , 1 9 3 5 , 471 p p . £IJ6£NE 0IETZ6EH tB . NO. 3 4 6 C ru iN T E P in u s . a . -30- TABLE I I I pH DETERMINATIONS OF GREEN BACTERIA ZONE TAKEN ON TRANSECT S ta tio n D e p th S u rface Two f e e t above u p p er l i m i t o f zone a s j u d g e d by c o lo r U pper l i m it o f zone T4 T2 T5 7 .3 7 .3 7 .3 6.6 6 .3 5 6 .1 5 6.6 6 .2 5 6 .4 6 .7 6 .3 6 .4 5 6.6 6.2 6 .4 M id d le o f zone L ow er l i m i t of v is ib le c o lo r -3 1 - fla sh e d opal d iffu s in g screen ; g la ss f i l t e r s was c a l i b r a t e d th e re is sp ace f o r in te rc h a n g e a b le and a l s o f o r l i g h t - r e d u c i n g f i l t e r s . The i n s t r u m e n t i n terras o f l i g h t i n t e n s i t y i n f o o t - c a n d l e s upon th e fa c e of th e d if f u s in g s c re e n . in c id e n t M e a s u r e m e n ts o f l i g h t i n t e n ­ s i t y w e r e m ade a t 12 o ' c l o c k n o o n o f e a c h r e g u l a r c o l l e c t i n g d a y . The t r a n s p a r e n c y o f t h e w a t e r a t v a r i o u s d e p t h s was m e a s u r e d w i t h a s p e c t r o p h o t o m e t e r ; a g r a p h was c o n s t r u c t e d s o t h a t t h e s p e c t r o ­ p h o to m e te r re a d in g s c o u ld b e t r a n s l a t e d in to p a rts p e r m illio n t u r ­ b id ity . A lm o s t n o t h i n g i s know n r e g a r d i n g t h e l i g h t r e q u i r e m e n t s o f t h e p la n k to n o f th e in la n d la k e s . d iv id u a l s p e c ie s has i t s I t is e n t i r e l y p ro b a b le t h a t each i n ­ own t o l e r a t i o n p o i n t . Comments a r e i n c l u d e d h e r e o n l y t o e m p h a s i z e t h e p o i n t t h a t b e l o w t h e 25 f o o t l e v e l , 0 . 0 % t r a n s m i s s i o n was a l w a y s o b t a i n e d . T h is v a l u e i s t h e sam e r e g a r d l e s s of i c e c o v e r e d w i t h snow , i c e a l o n e o r c l e a r w a t e r . S i n c e no member o f t h e p h y t o p l a n k t o n w as s e c u r e d much b e l o w t h e 25 f o o t l e v e l , t h i s d e p th p ro b a b ly d e n o tes th e l i m i t o f t h e p h o to s y n th e tic th e e x c e p tio n of t h e b a c t e r i a l f l o r a , zone and, w ith p ro b a b ly c o in c id e s w ith t h e lo w e r l i m i t o f th e zone of b i o l o g i c a l a c t i v i t y . T r a n s p a r e n c y d e t e r m i n e s t h e d e p t h o f w a t e r w h ic h c a n s u p p o r t p h o t o s y n t h e s i s and t h e r e f o r e t h i s th e p e n e tra tio n of lig h t. q u a l i t y m u s t sho w som e r e l a t i o n t o O ur t u r b i d i t y v a l u e s a t t h e 25 f o o t l e v e l sho w a r a n g e o f 7 t o 20 p . p . m . B e lo w t h e 25 f o o t l e v e l , t u r b i d i t y v a l ­ u e s r a p i d l y beco m e h i g h e r b u t no d e t e c t a b l e p e n e t r a t i o n o f l i g h t o c c u r s . 2 We a r e i n d e b t e d t o J . H a r l e y a n d I . -3 2 - O 'l i t z k y f o r t h i s w ork. TABLE IV DISSOLVED OXYGEN TRENDS - SODON LAKE JAN. 1949 - DEC. 1949 P . P .m . D i s s o l v e d Oxygen D e p th D a te S u rface 15* 8' 16' Jan. No p o s i t i v e t e s t s Feb. n 24' 5 .0 0.0 25’ o b ta in e d tt it 20' tt March. 1 1 .4 A p ril 2 4 .5 1 5 .5 7 .8 0.0 May 3 1 .0 3 .0 1.8 0.0 0 .3 4 0.0 1.1 0.0 1 .7 0.0 0.1 0.0 1.1 0.0 0 .0 3 0.0 8.2 June 6 .3 1 8 .6 4 J u ly 4 .9 0 5 .2 0 Aug. 7 .7 8 .3 2 .8 0 1 .0 8 3 .9 S e p t. 1 3 .4 O c t. 5 .5 2 .5 N ov. 5 .5 5 .5 5 .5 D ec. 6.2 6.1 6.1 6 .0 4 1.8 2.1 0 .4 6 0.0 33 5 .6 TABLE V VERTICAL DISTRIBUTION OF DISSOLVED OXYGEN SODON LAKE L ocation Depth T TRANSECT AUGUST 1949 D.O.(ppm) D.O. (c c /l) % S at . 4" 11*04 7.85 142.0 4” 9.70 6.70 85.0 4* 7.30 5 .18 92.3 8« 6.25 4.32 74. 7 4” 6.88 4.81 86.8 4’ 6.67 4.66 81.6 e* 5.99 4.19 70.0 12 » 5.80 4.05 59.2 14* 1.80 1.26 16.9 16* 0.37 0.26 3 .3 20* 0.19 0.13 1.6 24* 0.14 0.10 1.1 28* 0.00 0.00 0 .0 4" 13.44 9.40 166.3 8* 6.65 4.66 81.3 12’ 2.16 4.23 71.1 14’ 2.69 1.42 22.2 16’ 1.89 1.83 16.7 20’ 0.46 1.32 3. 8 24’ 0.10 0.32 0.8 28’ 0.00 0.00 0.00 4" 7.10 4.97 92 .2 T5 4» 6.76 4.73 83.4 T5 8’ 7.32 5.12 85.5 16’ 1.57 1.10 14.2 24’ 0.00 0.00 0.00 T6 4” 12.83 8.99 8.99 T6 4’ 7.42 5.19 5.19 8* 6.79 4.75 4.75 4" 13.04 9.04 9.04 T1 T2 T2 T2 T3 T3 T3 T3 T3 T3 T3 T3 T3 T4 T4 T4 T4 T4 T4 T5 T5 Ts T6 T7 ' TABLE V I DISSOLVED OXYGEN IN RELATION TO 2 0 NATION OF GREEN BACTERIA SODON LAKE AUGUST 1949 D i s s o l v e d Oxygen D e p th ( F t . ) ppm 1 0 s 30 A.M. 2 : 0 0 P.M. 21 0 .2 4 0 .2 4 0 .2 4 22 0 .2 4 0 .2 4 0 .2 4 23 0.11 0 .1 3 0 .1 6 24 0.11 0 .1 3 0 .1 6 25 0 .0 8 0.12 0.12 -3 5 - 4 : 0 0 P .M . B . C h e m ic a l F a c t o r s 1 . D i s s o l v e d O xygen. The r a p i d W i n k l e r m e th o d o f o x y g e n d e t e r m i n a t i o n ■was u s e d t h r o u g h o u t t h i s in v e stig a tio n . The p r o c e d u r e u p t o a n d i n ­ c l u d i n g a c i d i f i c a t i o n was c a r r i e d o u t i n t h e f i e l d a n d t i t r a t i o n s w e r e e f f e c t e d im m e d ia te ly upon r e t u r n to t h e l a b o r a t o r y . s o l u t i o n s w ere such t h a t t h e f i n a l t i t r a t i o n p a rts C o n c e n tra tio n s of i n c . c . was e q u a l t o p e r m i l l i o n of d is s o lv e d oxygen, T h e r ia u lt (1 9 2 5 ); th e p e r cen t s a t u r a t i o n a n d c u b i c c e n t i m e t e r s p e r l i t e r w e r e s o m e tim e s f i g u r e d . I t is v i r t u a l l y b een o b ta in e d * little i m p o s s i b l e t o p r e s e n t a l l t h e d a t a w h ic h h a v e Many o f t h e d a t a a r e r e p e t i t i o u s and can o f f e r b u t a i d t o a n u n d e r s t a n d i n g o f t h e p r o b le m s i n v o l v e d i n t h i s stu d y . We a r e t h e r e f o r e p r e s e n t i n g i n T a b le IV w h a t we c o n s i d e r t o b e d i s ­ s o lv e d oxygen tr e n d s f o r t h e c o u r s e o f t h i s e x p erim en t. I n T a b le V a r e sho w n d i s s o l v e d o x y g e n v a l u e s f o r t h e e n t i r e T t r a n s e c t . In T a b l e V I , d i s s o l v e d o x y g e n v a l u e s w h ic h a r e i n d i r e c t r e l a t i o n t o t h e zone of g re e n b a c t e r i a a re t a b u la te d . 2 . H y d ro g e n S u l p h i d e . was d e t e r m i n e d . The v e r t i c a l d i s t r i b u t i o n o f h y d r o g e n s u l p h i d e The m e th o d u s e d was t h a t o f T h e r o u x , E l d r i d g e a n d M a llm a n n ( 1 9 3 6 ) . I t is c o n s is te n tly s t a t e d te ria in th e l i t e r a t u r e t h a t th e g re e n b a c ­ e x i s t i n an e n v iro n m e n t r i c h i n h y d ro g en s u lp h id e . W h eth e r p re v io u s i n v e s t i g a t o r s have a c t u a l l y d e term in e d th e p re s e n c e o f t h i s gas by a c t u a l ch em ical t e s t s o r have b een in f lu e n c e d i n t h e i r deduc­ t i o n s by t h e r e l a t i v e l y s t r o n g and o b v io u s o d o r, i s h a rd t o s a y . a c t u a l c h e m i c a l a n a l y s i s , t h e am ount o f h y d r o g e n s u l p h i d e i n S o d o n By L a k e -was, a t a l l t i m e s , v e r y s m a l l a n d f r e q u e n t l y p o s i t i v e t e s t s t h i s gas c o u ld n o t b e o b ta in e d . r a n g e f r o m 1 .0 6 5 ppm t o 0 . 6 ppm. fo r Our v a lu e s f o r h y d ro g e n s u l p h i d e It is re c o g n iz e d t h a t th e v a lu e s q u o te d a r e e x tre m e ly lo w b u t i t m u st a l s o b e o b v io u s t h a t w h e re t h e r e e x i s t s a tr e m e n d o u s c o n c e n t r a t i o n o f g r e e n b a c t e r i a , th e re m ust neces­ s a r i l y b e b u t l i t t l e m o re t h a n t r a c e a m o u n ts o f h y d r o g e n s u l p h i d e as th e g reen b a c te r ia a re c o n sta n tly c o n v e rtin g th is su b sta n c e to su lp h u r. 3 . O t h e r C h e m ic a l F a c t o r s . C h e m ic a l a n a l y s e s h a v e b e e n made r o u t i n e l y o f t h e f o l l o w i n g c h e m i c a l c o m p o n e n ts o f S o d o n Lakes b ic a rb o n a te , carbon d io x id e , c a r b o n a te , s u lp h a te , ir o n , n i t r a t e n itr o g e n and n i t r i t e n itro g e n . D e t e r m i n a t i o n s w e r e a l s o made o f t o t a l , so lv ed s o l i d s . suspended and d i s ­ U n le ss o th e r w is e s t a t e d , a l l p ro c e d u re s a r e i n a c c o rd ­ a n c e w i t h S t a n d a r d M e th o d s , n i n t h e d i t i o n , (1 9 4 6 ). I n g e n e ra l, d e te r­ m i n a t i o n s w e r e made d u r i n g t h e w i n t e r , s p r i n g , summer a n d f a l l when te m p e ra tu re re c o rd s in d ic a te d th e rm a l d is tu r b a n c e s . H o w e v e r, num erous o t h e r a n a l y s e s w e r e made i n a n e f f o r t t o d e t e r m i n e i n t e r m e d i a r y p r o ­ d u c ts i n s u lp h a t e m e ta b o lism ; a l s o , s e v e ra l d e te rm in a tio n s o f to ta l p h o s p h o r o u s w e re m ad e. of th e in v e s tig a tio n s a r e la rg e ly re p e titio u s The r e s u l t s and a r e n o t p re s e n te d i n t o t o . D a ta r e p r e s e n t a t i v e o f t h e v a r i o u s a n a l y s e s a r e shown i n T a b l e V I I . C. A n a l y s i s o f t h e h y d r o s o l S a m p le s o f t h e b o t t o m s o i l w e r e s e c u r e d by m e an s o f a n Ekman d r e d g e ( s i z e 2 2 . 6 by 2 2 . 8 c m . ) ; t h i s d re d g e is a s ta n d a rd p ie c e of lim n o lo g ic a l a p p a r a tu s a n d can be u s e d s u c c e s s f u l l y f o r sam p lin g a TABXjTS CHEM ICAL ANALYSES X vx 1949 SODON LAKE - (PARTS PER MILLION) J a m a iy 0.029 0.016 0.15 T .Solids 0 .0 S.S o lid s D. S o lid s 292.0 346.0 292.0 346.0 286.0 284.0 286.0 284.0 0 • 0 -W °2 0 » 0 39. 6 20.4 Fe293 00 • • 00 so4 0 • 0 1B5.0 295.0 C93 j C°2 H • 0 25 ft* bottom HC03 0 • 0 DOptyll 1 0 0 0 o• 0 0.013 0.03 ft 43. 6 39.5 !°i 1 190.0 250.0 0 0 25 f t . bottom • 0 February 0.03 0 0 i ! i 0 0 • 1 0 .0 2 0 .0 3 0 ° i i c o ; • • 42 . 7 i o ! H j 1 9 2 .0 302 .O 0 • 0 25 ft. b o t t o m ,0 0 9 * 0 0 March. 1 0 0 0 • • 00 • 0 H CO • 0 0 • 0 0 0 9 0 • 0 0 2 74.0 338.0 0 « 0 June 0 .0 0.02 • H 0 0 0 9 0 0 0.0 0.0 Ju ly 0 • 0 0 0 • • 0 0 0.0 0 .0 0.02 0.02 0 .0 OiOl 0.01 00 • 9 00 Trace 0 9 0 - . 25 f t . bottom Trace • 0 195.0 304.0 0 • 0 25 f t . bottom • 0 August 0 Trace • 0 0 • 0 0 25 f t . bottom 0 .0 0 .0 ■ 1 ! September 0 • 0 25 f t . bottom 0 .0 0 .0 0 .0 0.02 0.03 0.0 0 .0 0 .0 0 .0 0 .0 0.028 0.02 0 • 0 1 1 . 226.5 340.2 i? 0 October 25 f t . bottom 0 .0 0 H 0 .0 0.0 0 H 00 0 0.01 9 0 0 0.0 9 00 9 9 199.2 283. 8 00 Nov© m be r 25 f t . bottom 1 i 1 ■ • 1 1 00 9 0.01 I 0 9 0 9 0 .0 0 O.'O 0 85.6 1.79.9 0 December* 2S f t . bottom 2 78.0 284.0 - 2 82.0 350.0 CO 0 • 0 0.024 O. 008 t • 278.0 284.0 - 0 • 38.4 15.8 0 0 180.0 287.6 0 .0 M xy 0 25 f t . bottom 0 • 0 - ■- 0.016 0.012 0 • 42.0 40.8 0 • 0 178.0 190.0 0 25 f t . to tto m O • 0 A p ril s o f t b o tto m . The s a m p l e s w e r e t h o r o u g h l y m ix e d a n d a l i q u o t p a r t s s e l e c t e d f o r a n a l y s i s b y t h e S pu rw ay s o i l t e s t s . An e x a m i n a t i o n o f T a b l e V I I I w i l l show t h e c h e m i c a l c o m p o s i t i o n o f t h e h y d r o s o l . TABLE V I I I CHEMICAL COMPOSITION OF THE HYDROSOL - SODON LAKE - 1949 P a rts per M illio n S u lp h a te s 0.0 M agn esium 6 . 0 ppm N itra te s 0.0 P o ta ssiu m M a n g a n e se 0.0 P hosphorous tra c e 10.0 C a lc iu m 200.0 A lum i num tra c e F e rric iro n tra c e Ammonia n i t r o g e n tra c e D. B i o l o g i c a l F a c t o r s 1. The P h y t o p l a n k t o n . O u r p r i m a r y i n t e r e s t i n d e t e r m i n i n g t h e c o m p o si t i o n o f t h e p h y to p la n k to n o f Sodon Lake h a s b e e n t o s e e i f t h e r e e x i s t e d a m o re o r l e s s c o n s t a n t a s s o c i a t i o n o f some m em ber o r members o f t h e e u p la n k to n and th e g r e e n b a c t e r i a . su c h a n a s s o c i a t i o n does e x i s t . T h e r e i s some e v i d e n c e t h a t H o w e v e r, comments o f f e r e d h e r e m u s t be i n te r p r e te d as a p p ly in g to th e p a r t i c u l a r c o n d itio n s Sodon L ak e. re s u lts p resen t in P o s i t i v e c o n c lu s io n s m ust n e c e s s a r i l y be b a se d upon th e of in v e stig a tio n s o f o th e r la k es o f t h e sam e g e n e r a l t y p e a s Sodon. I n J a n u a ry , 1949, a t th e s t a r t of t h e i n v e s t i g a t i o n r e p o r te d upon h e r e , S c h ro e d e ria s e t i g e r a c o e x is te d w ith t h e g r e e n b a c t e r i a a t d e p th s r a n g i n g f r o m 22 t o 27 f e e t . Q u a n t i t a t i v e l y , t h e r e w ere a p p ro x im a te ly 5 9 , 9 4 1 S c h .ro a d e r i a p e r l i t e r of la k e w a te r. t e r i a was a b o u t t h e s a m e . The n u m b e r o f g r e e n b a c ­ A n u m e r i c a l p e a k o f 1 , 7 1 2 , 6 8 3 was r e a c h e d by S c h ro e d e ria on F ebruary 1 9 th . At t h i s b a c t e r i a was c o n s t a n t l y d e c l i n i n g . t i m e , t h e n u m b e r of g r e e n S c h r o e d e r i a was a b s e n t fr o m t h e p la n k to n d u rin g t h e i n t e r v a l A p r i l t o J u l y ; i t w i l l b e r e c a l l e d t h a t t h e g r e e n b a c t e r i a w e r e no l o n g e r members o f t h e p l a n k t o n a t t h i s tim e . From J u l y t o llo v e m b e r, S c h r o d e r i a a g a i n c o e x i s t e d i n t h e p l a n k ­ to n w ith th e g ree n b a c te r ia . P erh ap s no im p o rta n c e s h o u ld b e a t t a c h e d t o t h e a s s o c i a t i o n d e s ­ c rib e d ; how ever, s u c h a n a s s o c i a t i o n does seem s i g n i f i c a n t enough t o b e com m ented u p o n h e r e . P o s s i b l y som e a t t e n t i o n s h o u l d b e g i v e n b y th e p h y to lo g is t to th e p h y sio lo g y of S c h ro e d e ria . To c o m p l e t e t h e o v e r a l l p i c t u r e o f t h e p h y t o p l a n k t o n o f S o d o n L a k e a p r e l i m i n a r y l i s t o f t h e d o m in a n t f o r m s e n c o u n t e r e d fro m J a n u a r y , 1949 t o D e ce m b e r, 1 9 4 9 i s p r e s e n t e d h e r e . W h ile o u r d a t a p r o v id e f o r s e a s o n a l q u a n t i t a t i v e and q u a l i t a t i v e e v a l u a t i o n s , t h e i n f o r m a t i o n p r o v i d e d c o u l d b e o f no v a l u e i n d e t e r m i n i n g t h e r e l a t i o n s h i p s o f t h e g reen b a c te r ia . The f o l l o w i n g l i s t i s b y n o m eans c o m p l e t e , b u t i t s h o u ld s e r v e as a c l u e to t h e p re d o m in a tin g a l g a l f l o r a o f Sodon L ak e. The f o l l o w i n g members o f t h e e u p l a n k t o n h a v e b e e n o b s e r v e d : O s c i l l a t o r i a t e n u i s , C hroo co ccu s m in u tu s , A p h a n o th ec e n i d u l a n s , A p h a n iz o m e n o n f l o s - a q u a e , D a c t y l o c o c o o p s i s S m i t h i i , V o l v o x s p . , B u d o rjn a e le g a n s , O o c y s tis l a c u s t r i s , S c h r o e d e r ia s e t i g e r a , S e le n a s tr u m s p . , M icro sp o ra s p . , C ru c ig e n ia a l t e r n a r i a , C r u c ig e n ia r e c t a n g u l a r i s , -4 0 - D in o b r y o n s e r t u l a r i a , C l o s t e r i u m s p . , S t a u r a s t r u m s p . , C y c l o t e l l a s p . » S te p h a n o d is c u s s p . , T a b e l l a r i a s p . , F r a g i l a r i a s p . , Synedra s p . f A s t e r i o n e l l a s p . , N a v icu la s p . , 2. The Z o o p l a n k t o n . It is and P in n u l a r ia s p . th e p u rpose o f t h i s p h ase of our s tu d y to p r e s e n t q u a n t i t a t i v e d a t a s h o w in g t h e s e a s o n a l , v e r t i c a l d i s t r i b u t i o n o f c e r t a i n members o f t h e z o o p l a n k t o n a n d t o d i s c u s s b r i e f l y s i b l e r e l a t i o n s h i p of s e le c te d c h e m ic a l, to rs to th is p la n k to n . We a r e i n t e r e s t e d t h e pos­ p h y sic a l and b io lo g ic a l fa c ­ i n d e te r m in in g h e r e w h e th e r t h e r e i s a t y p i c a l z o o p l a n k t o n f a u n a a c c o m p a n y in g t h e g r e e n b a c t e r i a . I n g e n e r a l i t i s t r u e t h a t t h e z o o p l a n k t o n o f S o d o n L a k e i s dom­ i n a t e d b y t h r e e e n t o m o s t r a c a n s , D a p h n ia p u l e x , D ia p to m n s o r e g o n e n s i s a n d C y c lo p s p r a s i n u s ; o t h e r p l a n k t e r s , D a p h n ia l o n g i s p i n a a n d B osm ina c o r e g o n i , h a v e a p p e a r e d , b u t i n s u c h e x t r e m e l y s m a l l nu m bers t h a t t h e y may b e c o n s i d e r e d a s t r a n s i e n t members o f t h e p l a n k t o n c o m m u n ity . D a p h n ia l o n g i s p i n a , f o r i n s t a n c e , was f i r s t s e e n i n t h e p l a n k t o n May 1 s t ; its r e a p p e a r a n c e has n o t b e e n o b s e r v e d . W h e n e v e r t h e t r a n s i e n t fo rm s have b een n o te d in th e p la n k to n , t h e i r p re s e n c e has b een c o n fin e d to t h e 15 * 5 f o o t d e p t h . Members o f t h e z o o p l a n k t o n , o th e r th a n th o se i n d i c a t e d abo ve and n a u p l i i o f t h e c o p e p o d s , have b e e n v i r t u a l l y non­ e x iste n t. The t h r e e d o m in a n t m em bers o f t h e z o o p l a n k t o n i n d i c a t e d a b o v e h a v e b e e n a r r a n g e d a c c o r d i n g t o d e p t h a n d s e a s o n i n T a b l e s IX ( a ) , IX ( b ) a n d IX ( c ) . In tr a c in g th e s e a s o n a l p e r i o d i c i t y under th e c o n d itio n s of th is o f th e in d iv id u a l z o o p la n k te rs , e x p erim en t, w e a re a c t u a l l y t r a c i n g th e -4 1 - TABLE IX (a ) NUMBERS PER LITER OF DAPHNIA PULES, BY SEASON AND DEPTH, SODON LAKE, 1949 Samples tak en about 11:00 A.M. Depth in f t . 1 2/1 9 3 /5 3 /19 4 /2 n .s . n .s . n .s . Date 4 /1 6 4 /3 0 5 /1 4 5/28 6/11 6/25 7/9 T otals 7/23 8 /4 * 8 /2 0 9/3 4 21 3 n .s . 25 - - 3 s• 4 12 n .s . 9 /1 7 10/1 10/15 10 /2 9 6 1 5 1 89 3 37 21 14 10 1 37 21 16 1 3 3 1 14 1 4 12 11 ' 12 1 220 5 37 13 53 9 51 35 16 24 11 - 3 3 14 1 9 24 13 10 2 328 10 8 22 59 27 64 48 3 19 12 5 19 26 11 1 6 56 27 26 4 392 15 12 6 18 3 22 54 20 9 13 31 22 18 19 n .s . 8 68 2e 44 36 431 20 15 4 19 - 5 5 7 3 33 - 13 27 12 n .s. 10 19 22 13 24 245 25 - 1 1 - 6 37 - 2 - 5 - 9 5 n .s . - 7 5 8 3 3 82 30 - 2 1 - 2 - 2 5 2 1 2 2 1 n .s . 8 2 4 3 4 41 . 35 - - - 2 38 3 1 - - 1 3 2 - n. s , 1 - 1 4 1 57 40 - - 1 2 5 1 - - 1 3 - mm 2 n .s . 1 1 - 2 - 19 109 69 166 57 215 223 70 54 193 115 122 76 1899 * 103 73 49 68 88 82 Samples tak en a t 2 :0 0 P.M. TABLE IX (b ) LUMBERS PER LITER OF DIAPT0MD5 QRSGONENSIS, BY SEASON AND DEPTH, SODON LAKE, 1949 Samples tak en about 11:00 A.M. 2 /1 9 3 /5 3 /19 4 /2 n .s . n .s . n .s . Date 4 /1 6 4 /3 0 5 /1 4 5/28 6/11 6/25 7/9 1 3 3 n .s . 11 1 7 /2 3 B/4* B/20 9/3 - 5 n .s . 11 1 2 3 1 12 9 /1 7 1 0 /1 10/15 10/29 n .s . 13 2 8 - 59 - 2 18 7 12 5 94 2 3 1 3 1 4 2 16 2 1 2 2 3 8 2 13 2 6 4 - 9 3 7 15 4 17 13 113 3 15 18 4 4 11 3 26 - 5 8 8 2 6 3 5 6 8 8 143 3 8 2 1 1 16 IB 14 12 13 12 7 2 n .s . 2 4 2 7 2 126 2 4 - 1 - 3 9 3 43 15 9 6 2 n .s . 1 4 1 1 1 105 - 4 1 1 2 8 1 2 - 1 2 - - n .s . - 1 - 1 - 1 - - - - - - 2 2 - a * 1 1 n .s . 1 - - 2 M i 10 - - - 1 3 - 1 1 3 - 1 _ 1 n .s . - 2 - 1 - 14 - - - - - - - 1 2 - - 1 - n .s . - 1 - - - 5 13 35 24 17 53 36 63 22 14 * ▲ 89 65 42 44 24 40 Samples t a k e n a t 2:00 P.M. 16 57 29 24 693 TABLE IX (c ) NUMBERS PER LITER OF CYCLOPS PRASINTIS, BY SEASON AND DEPTH, SODON LAKE, 1949 Samples tak en about 11:00 A.M. Depth in f t , 1 2/1 9 3 /5 3 /19 4 /2 n , s • U.S. n . s . Date 4 /1 6 4 /3 0 5 /1 4 5/28 6/11 6/25 7/9 - - 2 n .s . 7 1 - 1 +. 7/23 8 /4 n .s . T otals 8/20 9/3 1 1 n .s . 9 /1 7 1 0 /1 10/15 10/29 9 14 5 4 45 2 - - ' 1 - - 7 10 12 4 1 - - 2 1 5 11 14 8 16 92 5 - - 2 - 2 9 13 16 8 3 1 - 1 - 5 18 4 16 12 110 10 - 1 10 4 2 4 10 15 20 5 4 2 4 - 1 12 8 5 14 121 15 - 1 28 2 3 11 15 15 19 11 6 M 10 n .s . 2 11 6 14 9 163 20 - 11 57 ,7 2 2 8 4 49 21 9 19 7 n .s . - 2 5 10 6 219 25 - 14 11 2 - 12 1 2 3 2 3 9 1 n .s . 2 - - 1 1 64 30 - 1 1 2 1 1 - 4 3 1 3 - 1 n .s . - - 3 - 2 23 35 - - 1 2 > - 2 1 2 2 1 - 1 n .s . 1 - - - - 13 40 - - - - - - - 2 1 1 - 1 - n .s . - - - - 1 6 0 28 111 19 10 48 16 63 54 59 65 856 59 78 110 47 28 31 28 * Samples taken a t 2 :0 0 P.M. s e a s o n a l h i s t o r y of th e t o t a l n e t p la n k to n as t h e n e t p la n k to n i s l i m i t e d t o t h e e n to m o stra ca n s l i s t e d ab o v e. The u n i v e r s a l c o n c e p t t h a t t h e a n n u a l t o t a l p l a n k t o n c u r v e i s one s h o w in g b u t tw o s h a r p m axim a a n d tw o w e l l d e f i n e d m in im a s h o u l d n o t be a c c e p te d w ith o u t r e s e r v a tio n ; u n d e r th e c o n d itio n s u p o n h e r e , t h e z o o p l a n k t o n show s p r o n o u n c e d i n c r e a s e s re p o rte d i n n u m b e rs d u r ­ i n g M a r c h , May, J u n e a n d S e p t e m b e r ; s e v e r a l m in im a c a n a l s o b e ob­ s e rv e d , F ig u re 6. The c l a s s i c c u r v e a l s o i n d i c a t e s t h a t t h e c u r v e o f t h e z o o p la n k to n c lo s e ly ap p ro x im a te s t h a t o f t h e p h y to p la n k to n ; d a t a a l s o show a d e p a r t u r e fro m t h i s our co n cep t in t h a t th e p h y to p la n k ­ t o n , e x c l u s i v e o f b a c t e r i a , was a l m o s t n o n - e x i s t e n t u n t i l l a t e M a rc h . T h is f a c t i s o f p a r t i c u l a r i n t e r e s t w hen a n e x p l a n a t i o n o f t h e n u t r i ­ t i o n o f th e en to m o stra ca n s is s o u g h t. I t is u s u a lly s ta te d or a t l e a s t in fe rr e d , i c a l and p h y sic a l fa c to rs t h a t t h e v a r i o u s chem­ o p e r a t i n g on a body o f w a t e r c o n t r o l th e q u a l i t y a n d q u a n t i t y o f t h e p l a n k t o n ; among o t h e r s , t h e f a c t o r s o f h y d ro g e n -io n c o n c e n tr a tio n , d is s o lv e d oxygen, te m p e ra tu re and in o r ­ g a n ic n u t r i e n t s a re in c lu d e d . H o w e v e r, H u t c h i n s o n ( 1 9 4 4 ) s t a t e s : " C le a r c u t c o r r e l a t i o n s b e tw ee n c h e m ic a l c o n d itio n s an d t h e q u a l i t a t i v e c o m p o sitio n o f th e p h y to p la n k to n (an d z o o p la n k to n ) a r e n o t t o b e ex­ p e c te d ." T h i s c o n c e p t may w e l l b e e x t e n d e d , i n t h e p r e s e n t i n v e s t i g a ­ t i o n , t o in c lu d e th e q u a n t i t a t i v e c o m p o sitio n o f th e p la n k to n a s w e ll. We h a v e p r e v i o u s l y i n d i c a t e d t h a t t h e t r a n s i e n t m embers o f t h e z o o ­ p l a n k t o n a p p e a r a t t h e 25 * 5 . f o o t d e p t h . The maximum n u m b e rs o f t h e r e s id e n t p o p u la tio n a ls o a p p e a r a t t h i s d e p th , F ig u re 7. -4 5 - F ro m in fo rm a tio n a v a ila b le in th e l i t e r a t u r e on p la n k to n d i s t r i b u t i o n , an e x p la n a tio n o f a p r e f e r e n t i a l d e p th i s n o t to o c l e a r . The pH a t a n y p a r t i c u l a r l e v e l o f S o d o n L a k e t e n d s t o c h a n g e b u t little th ro u g h o u t t h e se a so n ; a ls o t h e v a lu e s i n d i c a t e s l i g h t change in v e rtic a l d is trib u tio n . The f i g u r e s a r e l a r g e l y r e p e t i t i o u s , th e h y d r o g e n - i o n c o n c e n t r a t i o n a l w a y s b e i n g i n t h e a l k a l i n e r a n g e (pH 7 .3 5 - 7 .0 ) i n t h e e p ilim n io n and th e rm o c lin e r e g io n s . r a n g e i s a p p r o x i m a t e l y 1*1 pH u n i t s . The maximum v e r t i c a l A l s o a p p r e c i a b l e a m o u n ts o f f r e e CO w e r e r a r e l y f o u n d , t h e r a n g e b e i n g f r o m 3 . 0 t o 0 . 5 p . p . m . Inasm u ch 2 a s pH a n d f r e e COg a r e m e r e l y f a c t o r s i n a t o t a l c h e m i c a l c o m p l e x , i t i s im p r o b a b le th a t th e y a r e , i n th e m se lv e s , in d i c a t o r s o f e n v iro n m e n ta l p referen ce. The v e r t i c a l d i s t r i b u t i o n o f i n o r g a n i c n u t r i e n t s i n Sodon L ake i s a p p r o x im a t e l y t h e sam e a t a l l l e v e l s ; no a p p r e c i a b l e d i f f e r e n c e e x i s t s a t th e 5, 1 0 , 15 a n d 20 f e e t l e v e l s n o r i s s ig n ific a n t. th e seaso n al v a r ia tio n T h e r e a r e no e x t r e m e d e p a r t u r e s f r o m t h e t y p i c a l a n a l y ­ s i s a s show n i n T a b l e X. TABLE X AVAILABLE NUTRIENTS IN SODON LAKE, 1949 £S ro0 1 SsJ P a rts per M illio n NOg-N 0 .0 2 9 0 .0 P e 2 03 -P E 0 .1 S04 hco 3 C03 4 3 .6 1 9 2 .0 0 .0 -4 6 Ca 2 0 0 .0 K P 1 0 .0 T race Mn Mg 0 .0 6 .0 W h ile i t is re c o g n iz e d t h a t t h e in o r g a n ic c o n te n t of Sodon Lake i s s u r p r i s i n g l y s m a l l , t h e tw o i o n s f r e q u e n t l y o f f e r e d a s l i m i t i n g f a c t o r s f o r th e p h y to p la n k to n ( n i t r a t e - n i t r o g e n and p h o s p h a te -p h o s ­ p h o r o u s ) a r e p r e s e n t i n a m o u n ts e q u a l t o t h o s e r e p o r t e d f o r h i g h l y p r o ­ d u c tiv e la k e s . We a t t a c h s l i g h t , i f a n y , im p o rta n c e t o te m p e r a tu r e a s a g o v e rn ­ in g f a c t o r f o r th e d i s t r i b u t i o n s u n d e r i n v e s t i g a t i o n . L et us c o n sid e r a g a in th e f a c t t h a t th e m a jo r ity o f t h e e n to m o stra o an s a r e fo u n d a t t h e 15 + o r - 5 f o o t d e p t h . A t t h e s e d e p t h s , we h a v e t o c i t e j u s t tw o e x a m p l e s , a r a n g e o f 1 0 , 1 ° i n May a n d a r a n g e o f 2 7 , 5 ° i n A u g u s t , Tem­ p e r a tu r e o b v io u sly c a n n o t, i n i t s e l f , b e a c o n t r o l l i n g f a c t o r and th e t h e r m o c l i n e , w hen p r e s e n t i s no b a r r i e r t o t h e m i g r a t i o n s . W h ile t h e d i s s o l v e d o x y g e n c o n t e n t i s n e v e r c o n s i d e r e d t o b e a l i m i t i n g f a c t o r i n a n u n p o l l u t e d body o f w a t e r , Sodon Lake d o es e x h i b i t som e i n t e r e s t i n g o x y g e n r e l a t i o n s h i p s . From J a n u a r y 2 2 n d t o M arch 5 t h , a p o s i t i v e t e s t f o r d i s s o l v e d oxygen c o u ld n o t b e o b t a i n e d a t any d e p th f o r t h e s t a t i o n s e l e c t e d as t h e perm anent s a m p lin g p l a c e . th is As seem ed t o b e a r a t h e r u n u s u a l s i t u a t i o n , t h e t e s t s w e r e r e p e a t e d by c o -w o rk e rs u s i n g s e v e r a l d i f f e r e n t m eth o d s; t h e r e s u l t s w e r e n e g a ­ tiv e , From M arch 1 9 t h t o N ovem ber 1 5 t h o x y g e n i s p re sen t in th e e p ilim n io n an d th e rm o c lin e w h ile t h e h y p o lim n io n i s c o m p le te ly a n a e r o b ic . We h a v e b e e n u n a b l e t h r o u g h o u t t h e p e r i o d o f t h i s in v e s tig a tio n to ob­ t a i n a p o s i t i v e d i s s o l v e d o x y g e n t e s t b e l o w t h e 25 f o o t l e v e l . H o w e v e r, i t w i l l b e r e c a l l e d t h a t t h e b u lk o f th e p la n k to n p o p u la tio n i s found a t a b o u t t h e 20 f o o t l e v e l w h e r e t h e o x y g e n v a l u e s a r e a d e q u a t e f o r - 47- th e p la n k to n E n to m o stra c a . T h e s e o x y g e n v a l u e s a r e , h o w e v e r , a lw a y s l e s s t h a n 0 .2 o c . p e r l i t e r w h ic h , a c c o rd in g t o Pennak (1 9 4 6 ) i s c r i t i c a l p o in t f o r m ost p la n k to n E n to m o stra c a . a lo ss to th e We a r e c o m p l e t e l y a t e x p la in th e d i s t r i b u t i o n s d u rin g J a n u a r y , F e b ru a ry and M arch w h e n S o d o n L a k e was a n a e r o b i c . I t is p r o b a b l e t h a t a m o re d e l i ­ c a t e t e s t f o r o x y g e n , i . e . , t h e lu m in o u s b a c t e r i a m e th o d o f B e i j e r i n c k s h o u ld h av e b een u s e d t o d e t e c t m in u te t r a c e s o f oxygen a t t h e tim e s in d ic a te d . It is a ls o p o s s i b l e t h a t th e r e p o r te d a b se n c e o f oxygen i s m o re a p p a r e n t t h a n r e a l a n d d u e t o som e i n t e r f e r i n g s u b s t a n c e . H o w e v e r, t e s t s o x id a tio n s fo r th io su lfa te , an e x p ec te d p ro d u c t o f t h e b a c t e r i a l o f h y d ro g e n s u l p h i d e , w ere n e g a t i v e . A t any r a t e , i t m ust b e c o n c l u d e d t h a t t h e e n t o m o s t r a c a n s u n d e r i n v e s t i g a t i o n show a n e x ­ t r e m e l y h i g h t o l e r a n c e t o m a r g i n a l a m o u n ts o f o x y g e n a n d t h a t t h e f i g u r e c i t e d by Pennak m ust b e m o d ifie d a t l e a s t f o r t h i s In th is c o n n e c tio n , e x p e rim e n t. i t s h o u l d b e e m p h a s iz e d t h a t t h e e n t omo s t r a c a n s a l s o show c o n s i d e r a b l e t o l e r a n c e t o h y d r o g e n s u l f i d e a s m e a s u r a b l e a m o u n ts o f t h i s g a s a r e f o u n d j u s t b e lo v r t h e 25 f o o t l e v e l . A lm o s t n o t h i n g i s known r e g a r d i n g t h e l i g h t r e q u i r e m e n t s o f t h e p l a n k t o n o f in la n d la k e s . has i t s I t is e n tire ly own t o l e r a t i o n p o i n t . p o s s ib le t h a t each in d iv id u a l sp e c ie s A comment i s in c lu d e d h e r e o n ly t o em­ p h a s i z e t h e p o i n t t h a t b e l o w t h e 25 f o o t l e v e l 0.0/£ t r a n s m i s s i o n was o b ta in e d . In a s m u c h a s b u t f e w mem bers o f t h e p l a n k t o n w e r e o b t a i n e d much b e l o w t h e 25 f o o t d e p t h j T h is f i g u r e l i m i t s th e e x te n t o f th e p h o to s y n th e tic zone an d , w ith th e e x c e p tio n o f th e b a c t e r i a l f l o r a , p ro b a b ly c o in c id e s w ith th e lo w e r l i m i t o f th e zone of b i o l o g i c a l a c t i v ity . - 48- It is t o t h e b i o l o g i c a l f a c t o r s t h a t we t u r n f o r a p o s s i b l e e x ­ p la n a t io n of t h e d i s t r i b u t i o n o f t h e e n to m o stra c a n s. I t has been e m p h asiz ed t h a t t h e z o o p la n k to n a p p ro a c h e d a p e a k b e f o r e t h e p h y to ­ p la n k to n a p p e a re d i n m e a s u ra b le n um bers. I t w o u ld t h e n a p p e a r t h a t th e e n to m o s tra c a n s w ere in d e p e n d e n t o f th e a l g a l f l o r a f o r fo o d and t h a t t h e y m ust t u r n e i t h e r t o or to th e b a c te ria . c o l l o i d a l m a te r ia l p re s e n t i n t h e w a te r W h ile a n i n v e s t i g a t i o n o f t h e d i g e s t i v e t r a c t o f t h e c o p e p o d s s h o w s , i n som e i n s t a n c e s , o rg a n ism s t h e p re s e n c e o f m in u te g re e n (p ro b a b ly g re e n b a c t e r i a ) t h e s e d id n o t a p p e a r to b e i n a n a c tiv e s ta te of d ig e stio n . F ro m o u r b a c t e r i o l o g i c a l s t u d i e s , we h a v e c o n s id e ra b le e v id e n c e t h a t th e g r e a t e s t c o n c e n tr a tio n of b a c t e r i a e x i s t s j u s t b e l o w t h e 2 0 f o o t l e v e l a n d t h a t t h e i r n u m b e rs d i m i n i s h as t h e v e r t i c a l d i s t a n c e becom es l e s s . th e re Is I t w o u ld a p p e a r t h e n t h a t c ir c u m s ta n tia l e v id e n c e , a t l e a s t , t h a t t h e r e is a d e f i n i t e n u t r i t i o n a l r e s p o n s e by t h e copepods t o t h e b a c t e r i a l f l o r a i n t h e i r im m e d ia te e n v iro n m e n t. - 40- 70 EU G EN E DIETEGEN CO. N O. 316 C THE CULTURE OF THE GREEN BACTERIA A. C rude C u ltu r e s As h a s b e e n s t a t e d d e sc rip tio n s in t h e in tr o d u c tio n to t h i s of g en era and sp e c ie s based la rg e ly p aper, p u b lish e d of th e g reen b a c te r ia have been o n p r e p a r a t i o n s m ad e f r o m c r u d e c u l t u r e s o r fro m th e s tu d y o f t h e o rg an ism s a s th e y e x i s t e d i n t h e i r n a t u r a l e n v iro n m e n t. I t seem s o b v io u s s i t i o n ta k e; t h a t t h e d i v e r s i f i e d p r o c e s s e s o f m i c r o b i a l decom po­ p la c e i n a m anner t h a t c a n n o t be c o n t r o l l e d an d t h a t t h e r e s u l t i n g c ru d e c u ltu r e s m ust n e c e s s a r ily m e ta b o lic p ro d u c ts . c o n ta in a la rg e v a r ie ty T h e s e , i n t u r n , may i n f l u e n c e , of a s a n e n v iro n m e n ­ t a l r e s p o n s e , t h e b a c t e r i a l m o r p h o l o g y ; a n d may a c c o u n t f o r . I n som e m e a s u re , t h e num ber of g r e e n b a c t e r i a r e p o r t e d i n t h e e a r l y l i t e r a t u r e as d i s t i n c t g e n e ra and s p e c ie s . Our i n t e r e s t i n c r u d e c u l t u r e s o n l y . ' To t h i s end, g la s s has b e e n o f an e x p lo r a to r y n a tu r e c y lin d e r s w ere a b o u t one q u a r te r f i l l e d w ith t h e b l a c k mud o f t h e b o t t o m d e p o s i t s a n d u p o n t h e mud m s enough d e o x y g e n a te d w a t e r t o f i l l covered w ith g l a s s p la te s th e c y lin d e rs. T hese w ere th e n t o make h e r m e t i c a l l y s e a l e d u n i t s a n d s t o r e d in th e lig h t u n d er th e u su a l la b o ra to ry m ade t o d e p o site d c o n d itio n s. No a t t e m p t was c o n tr o l t h e e x p e rim e n ts. E x a m i n a t i o n o f o u r c r u d e c u l t u r e s h a s sh ow n a n u m b e r o f s e e m i n g l y d i f f e r e n t ty p e s s lig h tly o f g re e n b a c t e r i a w h ic h in c lu d e t h e s p h e r i c a l , rod , c u rv e d and s p i r a l s h a p e d o rg a n is m s r e p o r t e d up o n by o t h e r i n ­ v e stig a to rs. I n our o p in io n , c o n c l u s i o n s d raw n fro m t h e e x a m i n a t i o n o f c ru d e c u l t u r e s a r e w o r th le s s ; such c o n c lu s io n s a r e o n ly d e f i n i t i v e -5 2 - in t h a t th e p r e s e n c e o f t h e o rg a n ism is c o n firm e d . v a l i d in v e s t ig a t i o n of t h e g reen b a c t e r i a , F o r a c o n c lu s iv e and c r u d e c u l t u r e m eth o d s m u st b e s u p p le m e n te d b y t h o s e i n w h ic h t h e e n v ir o n m e n ta l c o n d i t i o n s c a n b e m o re a c c u r a t e l y c o n t r o l l e d a n d w h i c h c a n b e r e p r o d u c e d b y o t h e r i n v e s ­ tig a to rs. I t is h a r d t o c o n c e i v e t h a t t h e G erman S c h o o l o f B a c t e r i o l o g y , w h i c h owes much o f i t s p r e s t i g e t o t h e w o rk o f R o b e r t K o c h , in p a rt K och’ s p o s t u l a t e s and h is p u re c u l t u r e s t u d i e s , has p u b l i s h e d , as l a t e as 1913, d e s c r i p t i o n s of b a c t e r i a d e r iv e d w h o lly fro m o b s e r v a t i o n s on cru d e c u ltu r e s . B. E n r i c h m e n t C u l t u r e s I t m i g h t b e e x p e c t e d t h a t t h e d e v i s i n g o f a c u l t u r e m edium t o en­ h a n c e t h e g r o w t h o f t h e g r e e n b a c t e r i a w o u ld b e a c o m p a r a t i v e l y s i m p l e ta sk . H o w e v e r, s u c h was n o t t h e c a s e . The u s u a l p r o c e d u r e s , p r e p a r a ­ t o r y t o t h e f o r m u l a t i o n o f a n e x p e r i m e n t a l m ed iu m , w o u ld b e t h e c h e m i­ c a l a n a l y s i s o f t h e e n v i r o n m e n t i n w h ic h t h e o r g a n i s m i n q u e s t i o n i s g r o w in g a n d a n a n a l y s i s o f th e b a c t e r i a l a sh ; s h o u l d g i v e v a l u e s w h ic h a r e , a t l e a s t , g a n ic s a l t , re q u ire m e n ts M o st i n v e s t i g a t o r s t h e s e tw o d e t e r m i n a t i o n s in d ic a tiv e of th e g ro ss in o r ­ o f th e o rg a n ism u n d e r i n v e s t i g a t i o n . o f a q u a tic h a b i t a t s have fo llo w e d t h e s ta n d a r d p r o c e d u r e s f o r c h e m ic a l a n a l y s e s a s i n d i c a t e d by t h e A m e ric a n P u b l i c H e a lth A s s o c ia tio n ; b u t, i t is f e l t , t h a t such p ro ced u res a r e n o t a t a l l s u i t e d t o w a t e r s o f e x t r e m e l y lo w m i n e r a l c o n t e n t . tre m e n d o u s q u a n t i t i e s I n many c a s e s , , of w a te r m ust be e v a p o ra te d b e f o r e th e u s u a l a n a l y t i c a l m e th o d s c a n b e a p p l i e d . -5 3 - The t i m e e l e m e n t i s o f g r e a t im p o rta n c e h e r e i f on s i m u l t a n e o u s l y . it i s n e c e s s a r y t h a t many d e t e r m i n a t i o n s b e c a r r i e d A search of th e l i t e r a t u r e in d ic a te s th a t o th e r m e th o d s o f a n a l y s i s a r e a v a i l a b l e f o r w a t e r s o f lo w m i n e r a l c o n t e n t i n w h ic h t h e t o t a l s o l i d s a r e i n t h e r a n g e o f 250 p a r t s Iro n , per m illio n . f o r e x am p le, c a n be d e te r m in e d by t h e m e r c a p t o a c e t a t e m e th o d ; s o d i u m , b y t h e '’t r i p l e a c e t a t e m e t h o d s ” ; m a g n e s iu m , by t h e m e th o d o f K o l t h o f f a n d G i l l a n d c a l c i u m , b y t h e o x a l a t e m e th o d o f L i n g a n e . Some f u r t h e r com m ents on t h e s e m e th o d s w i l l b e e x p r e s s e d e l s e w h e r e i n th is paper. The r e s u l t s o f o u r a n a l y s e s b y r e f i n e d m e th o d s i n d i c a t e n o t h i n g m o re t h a n t h e m e re p r e s e n c e o f many i n o r g a n i c s u b s t a n c e s tio n s in c o n c e n tra ­ t o w h ic h t h e t e r m ’’t r a c e ” may c o n v e n i e n t l y b e a p p l i e d . q u e n tly , C onse­ o u r e n r i c h m e n t m edium h a s b e e n f o r m u l a t e d on t h e b a s i s th a t c a rb o n , h y d ro g en , oxygen, n i t r o g e n , phosp h o ru s and s u l f u r a r e i n d i s ­ p e n s a b l e c o m p o n e n ts o f a l l l i v i n g c e lls. n e c e s s a r y to t h e m e ta b o lism o f t h e c e l l , To t h e s e e l e m e n t s d eem ed m a g n e s iu m h a s b e e n a d d e d b e ­ cause o f th e f a c t t h a t i t i s th o u g h t to b e im p o rta n t i n m e ta b o lic p r o c e s s e s and a l s o t h a t i t is a com ponent o f b a c t e r i o c h l o r o p h y l l . It h a s b e e n a s s u m e d t h a t o t h e r s a l t r e q u i r e m e n t s w o u l d b e m e t by t h e p resen ce of tra c e e lem en ts i n o th e r w is e c h e m ic a lly p u re s u b s t a n c e s . W h ile B e i j e r i n c k e a r ly i n h is in v e stig a tio n s t e r i a u se d th e p r i n c i p l e of e n ric h m e n t c u l t u r e s , s u c h , h a s b e e n e m p lo y e d b u t l i t t l e o f th e p u rp le b a c ­ t h e te c h n iq u e , as by s u b s e q u e n t i n v e s t i g a t o r s . ^ W ith t h e a s s i s t a n c e o f D r. S t o n e , C h e m istry D e p a rtm e n t, M ic h ig a n S t a t e C o lle g e . B e i j e r i n c k p ro b a b ly added a s o u rc e o f hy d ro g en s u lp h id e t o d i t c h o r c a n a l w a t e r , t h u s m a k in g h i s c u l t u r e s , t o som e e x t e n t a t l e a s t , tia lly c o n tro lla b le . In ste a d p ar­ o f d i t c h o r c a n a l w a t e r , we h a v e u s e d a m edium o f t h e f o l l o w i n g c o m p o s i t i o n a s a n e n r i c h m e n t f o r o u r c r u d e c u ltu re s: s t e r i l e , a g e d l a k e w a t e r ........................ 1 0 0 0 m l . 0 .1 % HB4 C I ................................ K2 HP0 4 .................................................................... 0 . 1# M gC lg....................................................................... 0 .1 % 0 .1 % NaHCOg.................................................................... Na2 S .9 H 2 0 ............................................................ 0 .1 % The pH was a d j u s t e d t o 7 . 5 by t h e a d d i t i o n o f Na2 C0g a n d HgPO^.. The aqvieous s o l u t i o n s o f th e in o rg a n ic s a l t s , w ith t h e e x c e p tio n of t h e b i c a r b o n a t e a n d t h e s u l p h i d e , w e r e a u t o c l a v e d f o r 15 m i n u t e s a t 1 2 1 ° C . ; t h e s u l p h i d e s o l u t i o n was a u t o c l a v e d s e p a r a t e l y a n d a d d e d i n th e d e s ir e d c o n c e n tra tio n to th e o th e r in o rg a n ic s a l t s ; th e b ic a rb o n a te s o l u t i o n was s t e r i l i z e d by f i l t r a t i o n . The m edium was d i s p e n s e d i n t o screw capped b o t t l e s , v a r y i n g a m o u n ts o f t h e c r u d e c u l t u r e , seeded w ith s e a le d t o p ro v id e a n a e ro b ic con­ d i t i o n s a n d i n c u b a t e d a t ro o m t e m p e r a t u r e , u s i n g a s e v e n w a t t b u l b a s a so u rc e of illu m in a tio n . C u ltu r e s w ere in c u b a te d f o r s e v e n d a y s • F o l l o w i n g t h e i n c u b a t i o n p e r i o d , s e r i a l t r a n s f e r s w e r e m ade a n d t r e a t e d a s i n d i c a t e d a b o v e ; t h e t r a n s f e r p r o c e s s was c o n t i n u e d u n t i l t h e c u l ­ tu r e s ap p eared m ic ro s c o p ic a lly to c o n s is t o f b u t one ty p e o f c e l l . F rom t h e e n r i c h e d a n d p u r i f i e d c u l t u r e s , t r a n s f e r s w e r e m ade t o a m edium o f t h e sam e i n o r g a n i c c o n s ti tu te n ts as in d ic a te d b u t w ith th e a d d i t i o n o f O.Z% a g a r t o m ake a s e m i - s o l i d m edium ; t h i s m edium w as d i s ­ pensed in to screw cap tu b e s , in o c u la te d , u sin g th e shake c u ltu re -5 5 - te c h n i q u e , and s e a le d w ith s t e r i l e v a s p a r . T h e c u l t u r e s w e re i n c u b a t e d a s b e f o r e and k e p t f o r c o m p a ra tiv e p u rp o s e s . C. P u r e C u l t u r e s To i n s u r e c u l t u r e s w h i c h , i n t h e t r u e s e n s e o f t h e w o r d , c o u l d b e c o n s i d e r e d a s " p u r e ” , t h e s i n g l e c e l l i s o l a t i o n t e c h n i q u e was p r a c t i s ­ ed. U sin g a deF onbrune m ic ro m a n ip u la to r, s i n g l e c e l l s from th e n a t u r a l e n v i r o n m e n t w e r e i s o l a t e d , i n o c u l a t e d i n t o t h e d e f i n e d c u l t u r e m edium a n d t r e a t e d i n t h e sam e m a n n e r a s t h e e n r i c h e d c u l t u r e s . -5 6 - GENERAL MORPHOLOGY A. From E n r i c h m e n t C u l t u r e s The s t r i k i n g v a r i a t i o n i n m o r p h o l o g y o f t h e g r e e n b a c t e r i a a s p o r te d fro m th e l i t e r a t u r e m ust be re e m p h asiz ed h e r e . io n o f one s p e c ie s , b a c te ria re­ W ith t h e e x c e p t ­ C h lo ro b iu m l i m i c o l a , a l l d e s c r i p t i o n s of th e g reen re c o rd e d i n t h e l i t e r a t u r e have b e e n d e riv e d from o b s e r v a tio n s on cru d e' c u l t u r e s . As s o o n a s a c e l l f o r m was f o u n d w h i c h d i f f e r e d in b u t o n e m i n o r r e s p e c t fr o m o t h e r d e s c r i b e d f o r m s , a new s p e c i e s was c re a te d . V a ria b ility of t h e o rg a n ism u n d e r t h e i n f l u e n c e of d i f f e r e n t e n v i r o n m e n t a l c o n d i t i o n s was n o t c o n s i d e r e d ; a n d t h e r e f o r e a l l e x ist­ in g c l a s s i f i c a t i o n s a r e b a s e d u p o n m orph o lo g y a n d s i z e m e asu re m e n ts a s d e te rm in e d i n a n a t u r a l s t a t e ; s ta tic u n its. c e l l c o n g lo m e ra te s w ere c o n s id e r e d a s The p h e n o m en o n o f e n v i r o n m e n t a l v a r i a t i o n i s s o common t h a t i t m e r i t s no d i s c u s s i o n o t h e r t h a n t o p o i n t o u t t h a t by v a r y i n g th e en v iro n m e n t o f a n o rg a n ism m o rp h o lo g ic a l, v a r i a n t s can be p ro d u ced a lm o st a t w i l l . I n p r e p a r a t i o n s m ade f r o m o u r e n r i c h m e n t c u l t u r e s , t h e g r e e n b a c ­ t e r i a a p p e a re d as r o d s , a p p ro x im a te ly 2 .5 u by 1 .0 u , as c o c c i, as s p i r a l form s a n d a s c u rv e d v i b r i o l i k e o rg a n is m s . v a rie ty Ho o n e m o r p h o l o g i c a l c o u ld be s a i d t o b e d o m in a n t. B. From P u r e C u l t u r e s I n p r e p a r a t i o n s m ade f r o m c u l t u r e s s t a r t e d from s i n g l e c e l l s , g e n e r a l v a r i a t i o n i n m orph o lo g y i s a g a i n e v i d e n t . som e P erhaps a non- m o t i l e r o d a b o u t 1 . 0 u by 0 . 5 u a p p e a r s m o re o f t e n t h a n o t h e r m o r p h o l o g ­ ic a l v a ria n ts. H o w e v e r, n e i t h e r s h a p e n o r s i z e i s a c o n s t a n t -5 7 - m o rp h o lo g ic a l f e a t u r e . C e ll c o n g lo m e ra te s a r e p r e s e n t , t h e s e b e in g s u rro u n d e d b y c a p s u l a r m a t e r i a l w h ic h c an be d i s s o l v e d o f f by s u i t ­ a b le b a c te r io l o g ic a l te c h n iq u e s . E le c tr o n m ic ro sco p e p ic tu r e s sh o w tw o t o t h r e e a r e a s o f heavy c o n d e n s a tio n , a p p ro x im a te ly 0 .2 5 u i n d ia m e te r as d e te rm in e d by r e f e r ­ ence to p a r tic le s o f p o ly sty re n e la te x . The c o n d e n s e d a r e a s d is p e rs e d by e x tr a c tio n w ith e th y l a lc o h o l; can b e from a s tu d y o f t h e a b ­ s o r p t i o n s p e c t r a o f t h e g r e e n b a c t e r i a , t h e r e i s som e e v i d e n c e t h a t t h e z o n e s o f c o n c e n t r a t i o n may r e p r e s e n t a n u c l e i c a c i d c o m p o n e n t . I n F ig u re s 8 , 9 , 10 a n d 1 1 , t h e m o r p h o l o g y o f t h e g r e e n b a c t e r i a a s d e ­ te r m in e d b y t h e e l e c t r o n m ic ro s c o p e i s shown. I n F ig u re 8 is m a te ria l; sh ow n a n a g g r e g a t i o n o f c e l l s t h e r o d a n d c o c c u s fo r m s p r e d o m i n a t e h e r e . sam e t y p e o f g r o u p i n g i s shadow c a s t t h e o b j e c t s . s u r f a c e to p o g rap h y i s in c o rp o ra te d in to t h i s b a sis of s iz e . te riu m . s u r r o u n d e d by m ucous In F ig u re 9, th e i n d i c a t e d b u t a n a t t e m p t h a s b e e n m ade t o The r e s u l t i s n o t e n t i r e l y s u c c e s s f u l b u t t h e e v id e n t; p a r tic le s of p o ly s ty re n e l a te x have been p r e p a r a t io n in o rd e r t o o f f e r a c o m p a ra tiv e I n F i g u r e 10 i s sh ow n a s i n g l e c e l l o f t h e g r e e n b a c ­ I n c o r p o r a t e d i n t o F i g u r e 11 i s a r o d s h a p e d c e l l a n d a c o c c u s shaped c e l l ; t h e s e a r e t h e tw o m o r p h o l o g i c a l v a r i a n t s o f C. l i m i c o l a w h ic h a p p e a r i n o u r c u l t u r e s . We h a v e i d e n t i f i e d t h e o r g a n i s m s t u d i e d i n p u r e c u l t u r e a s C h l o r o b iu m l i m i c o l a N a d s o n . I t seem s p o s s i b l e t h a t t h e C h l o r o c h r o m a t i u m a g g re g a tu m o f L a u te r b o r n , h is P e lo d ic ty o n c l a t h r a t i f o r m e and h is genus P e l o g l o e a p r o b a b l y r e p r e s e n t g r o w t h fo r m s o r e n v i r o n m e n t a l v a r i a n t s -5 6 - of C h lo ro b iu m l i m i c o l a . Some c o n c l u s i o n s i n re g a r d t o t h e m o rp h o lo g y o f t h e s e l a t t e r o r g a n i s m s c a n b e d ra w n b y r e f e r e n c e t o F i g u r e 1 2 . I t ap­ pears of r a t h e r o b v io u s t h a t v a r i a t i o n i s w h e th e r p u re c u ltu r e s o r cru d e c u ltu r e s to b e e x p e c te d re g a r d le s s a r e a n a ly z e d . H o w e v e r, w e do n o t h a v e s u f f i c i e n t d a t a t o j u s t i f y m o re c o n c l u s i v e s t a t e m e n t s . se v e ra l c u ltu re s o f C h l o r o b i u m l i m i c o l a w h ic h we h a v e s t u d i e d d o s h o w d i f f e r e n t m o rp h o lo g y and c e l l a g g r e g a t i o n s , sta n tia l The e v id e n c e , a t l e a s t , in d ic a tin g t h a t such v a r i a t i o n s c lo s e c irc u m ­ re sem b le th o s e d e s ­ c rib e d f o r o th e r d i s t i n c t sp e c ie s o f g reen b a c te r ia . F i g u r e B. Cel] a r r r e r a t i o n o f C b l o r o M n . l i m i c o l a F ig u re 9. ^ »strv ■*> s rf' " v^ irji./»r c r^ v . ? ^ w V-v-\ V £-* - i ^ i c o i s - » s h a d o w c a s t e d F ig u re 10. l.OVk S in gle c e l l - 62- o? _C. 1 i m i c o l a . F ig u re 11. M o rp h o lo g ic a l v a r i a n t s o f C. lim ic o la F ig u re 12. m m t\ui />\,i ,!Nt^ >.’■/, / / " I 's>\v.'», / r ' Vf '.* -'>=*' ' 'f *♦ »' i ' *i / JV .vif .iw u 1^ « '; v.'i'.V ^ V y ’,’ '> 1 V . r ' - t >:•>.i'i .V / v Key t o F i g u r e 12 T a k e n f r o m D ie s a p r o p e l i s c h L e b e x v elt by L a u t e r b o r n 3 4-36 C h lo ro b a c te riu m sy m b io tic u m n o v . s p e c . 29 S c h m id le a l u t a o l a S c h m id le 32 P e lo g lo e a c h lo rin a L a u te rb o rn 30-31 P e lo g lo e a b a c i l l i f e r a nov. s p e c . 32 P e lo d ic ty o n c la th ra tifo rm e S z a fe r 37 C h lo ro c h ro m a tiu m a g g re g a tu m L a u te r b o r n -6 4 - THE PIGMENTS OF THE GREEN BACTERIA V e r y f e w d a t a e x i s t on t h e s p e c t r a l p r o p e r t i e s b a c te ria . of t h e green M e t z n e r ( 1 9 2 2 ) h a s i n d i c a t e d t h a t t h e g r e e n b a c t e r i a do n o t c o n t a i n p h y c o c y a n i n , t h e p r e s e n c e o f w h i c h w o u ld p l a c e t h e s e o r g a n ­ ism s c l o s e t o t h e b l u e - g r e e n a l g a e , t h e C y a n o p h y c e a e . M etz n e r, l o c . c i t . , has a ls o in tim a te d t h a t t h e c h lo ro p h y ll o f th e g re e n b a c t e r i a , b a c te rio v irid in , d iffe rs from t h e c h l o r o p h y l l o f t h e h i g h e r p l a n t s and from t h a t o f t h e g re e n a l g a e . v a n N ie l (1 9 3 1 ) has a c c e p te d th e id e a s ex p re ssed above i n re g a rd to th e p ig m e n ts o f t h e g r e e n b a c t e r i a b u t h i s c o n c l u s i o n s a r e b a s e d u p o n t h e w o rk o f M e t z n e r a n d do n o t r e p r e ­ s e n t e i t h e r a r e i n v e s t i g a t i o n o f M e t z n e r * s w o rk o r e x p l o r a t o r y w o r k o f h is own. te ria is T h e r e fo r e a r e e x a m in a tio n o f t h e p ig m e n ts of th e green b a c ­ in d ic a te d h ere. A b s o r p tio n s p e c t r a w ere d e te r m in e d f o r c e l l s u s p e n s i o n s and f o r c e ll e x tra c ts of m a te r ia l. th e c e ll o f th e g reen b a c t e r i a , u s in g p u re c u ltu r e s a s th e s o u rc e A Beckman S p e c t r o p h o t o m e t e r was u s e d f o r t h i s w o r k . For e x t r a c t s , t h e c e l l u l a r m a t e r i a l w as e x t r a c t e d i n t h e d a r k w ith e th y l a lc o h o l; t h e r e s i d u e from t h i s a l c o h o l i c w ith v a r io u s s o lv e n ts i.e ., e x t r a c t was t r e a t e d carb o n d is u lp h id e , benzene, carbon t e t r a ­ c h l o r i d e , c h lo ro fo rm , d io x a n e and p e tro le u m e t h e r . H o w e v e r, a b s o r p t i o n s p e c t r a o f t h e e x t r a c t i o n s w ith t h e v a r i o u s s o l v e n t s m e n tio n e d p ro v ed t o b e d e p e n d e n t on t h e r e f r a c t i v e i n d e x o f t n e s o l v e n t . p e t r o l e u m e t h e r was a d o p t e d a s t h e s e c o n d a r y s o l v e n t . o f F i g u r e 13 w i l l show t h e a b s o r p t i o n s p e c t r u m f r o m ( l ) s io n s of g re e n b a c t e r i a and (2 ) th e a l c o h o l i c -6 6 - e x tra c t. For a s ta n d a rd , An e x a m i n a t i o n c e ll suspen­ 0586078576 EUCENED IEIZGEIICO .N O, 316c In o r d e r to i n t e r p r e t th e a b s o r p tio n sp e c tru m of t h e g re e n b a c t e r i a , i t seems n e c e s s a r y t h a t a n a n a l y t i c a l c o m p a r i s o n b e made o f t h e a b s o r p ­ t i o n s p e c tr a of o th e r u n i c e l l u l a r c h lo r o p h y ll- c o n ta in in g o rg a n ism s. S p e c ia l a t t e n t i o n s h o u ld be p a id to t h e a b s o r p tio n sp e c tru m o f th e b l u e g reen a lg a e because i t g reen b a c te r ia . in sta tu s is to th is g r o u p t h a t G e i t l e r w o u ld a s s i g n t h e I t w i l l b e re m e m b e re d t h a t G e i t l e r p r o p o s e d a c h a n g e o f t h e g r e e n b a c t e r i a from t h e C h l o r o b a c t e r i a c e a e o f L a u t e r b o r n t o t h e C y an o c h lo ro d in ea e ( G e i t l e r and P a s c h e r ) , T h is c h a n g e was r e c o m ­ m ended l a r g e l y on t h e b a s i s t h a t d e f i n i t e p r o o f was l a c k i n g a s t o t h e e x iste n c e , from in th e g re e n b a c t e r i a , o f a c h lo r o p h y llo u s p ig m e n t d i f f e r e n t t h a t w h ic h e x i s t e d i n t h e b l u e - g r e e n a l g a e . Many d a t a a r e a t h a n d f o r t h e c o m p a r i s o n o f o f c h lo r o p h y ll- c o n ta in in g o rg a n ism s. th e a b so rp tio n sp e c tra An e x t e n s i v e l i t e r a t u r e i s a v a i l ­ a b l e fr o m i n v e s t i g a t i o n s i n t h e f i e l d o f p h o t o s y n t h e s i s . H o w e v e r, i t m u s t b e e m p h a s iz e d t h a t t h e s p e c i f i c t e c h n i q u e s u s e d t o s t u d y p h o t o s y n ­ t h e s i s i n - v i t ro a r e n o t g e n e r a l l y a v a i l a b l e t o t h e b a c t e r i o l o g i s t . We r e f e r h e re , p r im a r ily , to th e d i s r u p tio n of th e c e l l by u l t r a so n ic v ib ra tio n s; t h i s t e c h n i q u e p e r m i t s t h e u s e o f a n e x t r a c t w h ic h g i v e s t h e same a b s o r p t i o n b a n d s a s t h a t o f t h e p i g m e n t w hen p r e s e n t i n t h e p la stid s. As we h a v e n o t b e e n a b l e t o p r e p a r e ’’c e l l j u i c e " b a c te ria , e x tra c ts of g reen o u r d a ta a n d th e i n t e r p r e t a t i o n t h e r e o f m ust b e b a s e d on c e l l s u s p e n s i o n s a n d e x t r a c t i o n s m ade w i t h o r g a n i c s o l v e n t s . H o w e v e r, s u c h c o m p a r i s o n s a s w i l l b e made b e t w e e n t h e a b s o r p t i o n s p e c t r a o f t h e g r e e n -6 8 - b a c t e r i a and t h o s e o f o t h e r one c e l l e d c h l o r o p h y l l - c o n t a i n i n g o r g a n ­ is m s w i l l b e b a s e d o n s i m i l a r e x t r a c t i o n s o f t h e s e p l a n t s w i t h t h e same o r g a n i c s o l v e n t . I n F i g u r e 13 i s show n t h e a b s o r p t i o n s p e c t r u m o f ( 1 ) a c e l l s u s ­ p e n s io n o f g re e n b a c t e r i a and ( 2 ) th e a l c o h o l i c maximum a t a b o u t 740 mu. i s i t is e x tra c t, A stro n g i n d i c a t e d f o r t h e c e l l s u s p e n s io n ; how ever, o b v i o u s t h a t t h e r e i s a s h i f t t o t h e s h o r t e r w ave l e n g t h s f o r t h e a lc o h o lic e x t r a c t , w h ic h p l a c e s t h e a b s o r p t i o n maximum a t a b o u t 670 mu. In a s e rie s of b e a u tifu l e x p e r i m e n t s H u b e r t ( 1 9 3 5 ) w as a b l e t o show t h a t t h e s p e c t r a o f c h l o r o p h y l l ( t h a t i s , t h e p o s i t i o n o f t h e m a x i­ ma) i n o r g a n i c s o l v e n t s d e p e n d e d o n t h e r e f r a c t i v e i n d e x o f t h e s o l v e n t used. A c c o r d i n g l y , t h e c o m p a r i s o n s now t o b e n o t e d a r e b a s e d on c e l l s u s p e n s io n s and e x t r a c t s w ith e t h y l a l c o h o l ; th u s d u p l i c a t i n g th e c o n ­ d i t i o n s u n d e r w h ic h t h e a b s o r p t i o n c u r v e s o f t h e g r e e n b a c t e r i a w e r e draw n. The d a t a t o b e s u m m a riz e d r e g a r d i n g t h e a b s o r p t i o n s p e c t r a o f t h e b l u e - g r e e n a l g a e a n d t h e g r e e n a l g a e a r e so w e l l known t h a t i t i s n o t th o u g h t n e c e s s a r y to docum ent th e m . w ith r e l a t i v e l y cru d e in s tru m e n ts . The d a t a a r e r e p r o d u c i b l e e v e n For th e b lu e -g re e n a lg a e , u sin g O s c i l l a t o r i a s p . as a n ex am p le, and u s i n g a c e l l s u s p e n s io n , e x ist; tw o maxima o n e o f t h e s e maxima i s a t 680 m u . , t h e o t h e r , a t 620 mu. 68 0 i s t h e p o s i t i o n o n t h e a b s o r p t i o n b a n d o f c h l o r o p h y l l a n d 620 r e p r e s e n t s th e p h y c o cy a n in . Now i n a n a l c o h o l i c e x tr a c t o f O s c i l l a t o r i a , th e c h l o r o p h y l l maximum i s s h i f t e d t o a b o u t 6 7 0 . 5 mu. U sin g C h l o r e l l a s p . a s a n e x a m p le o f t h e g r e e n a l g a e , t h e a b s o r p t i o n maximum i s a t 680 mu; -6 9 - in an a lc o h o lic e x t r a c t , t h i s maximum i s s h i f t e d t o 664 mu. a p p a r e n t t h a t a s h i f t t o t h e s h o r t e r •wave l e n g t h s a lc o h o lic e x tra c ts th e green a lg a e . I t is ta k es p la c e i n th e of t h e g re e n b a c t e r i a , th e b lu e - g r e e n a lg a e and To c l a r i f y t h e s e d a t a f o r f u r t h e r d i s c u s s i o n , we may l o o k a t th e m i n t a b u l a r f o r m . A b s o r p t i o n Maxima ( m u . ) of G reen B a c t e r i a G re e n A l g a e B lu e -g re e n A lg a e C e ll S u sp e n sio n 740 6B0 680 a n d 620 A lc o h o lic E x tra c t 670 664 6 7 0 .5 W hat a r e we t o c o n c l u d e f r o m a c o m p a r i s o n o f t h e a b s o r p t i o n s p e c ­ tra o f th e g re e n a lg a e , th e b lu e -g re e n a lg a e and th e g reen b a c te r ia ? v a n l T i e l ( 1 9 4 4 ) h a s s a i d HThe g r e e n s u l p h u r b a c t e r i a c o n t a i n a c h l o r o ­ p h y l l w h ic h i s m o s t c e r t a i n l y n o t i d e n t i c a l w i t h e i t h e r b a c t e r i o c h l o r o p h y ll or c h lo ro p h y ll a o r b . ” But t h i s v e r y s tr o n g s ta te m e n t i s b a se d u p o n o n e d e t e r m i n a t i o n made by M e t z n e r i n 1 9 2 2 . O b v i o u s l y , w hen u s i n g c e l l s u s p e n s i o n s , t h e r e i s a s m a l l d i f f e r e n c e ( 6 0 m u .) b e t w e e n t h e a b ­ s o r p tio n s p e c tr a of th e g reen a lg a e and th e g reen b a c t e r i a ; fo r th e a lc o h o lic t i o n maxima i s e x tra c ts how ever, o f t h e s e o rg a n ism s t h e d i f f e r e n c e i n a b s o r p ­ r e d u c e d t o 6 mu. Such a d i f f e r e n c e i s r e a s o n f o r t h e s ta t e m e n t q u o te d fro m v a riN ie l. h a rd ly a v a l i d C o n tra ry to th e o p in io n e x p r e s s e d by P r i n g s h e i n (194-9) t h a t t h e p i g m e n t s o f t h e g r e e n b a c t e r i a w i l l p ro b a b ly b e t h e m ost s u i t a b l e f e a t u r e s upon w h ic h t o b a s e a c l a s ­ sific a tio n , i t a p p e a r s t h a t a m o re v a l i d r a t i o n a l e m u s t b e f o u n d i f a c l e a r d e m a rk a tio n o f t h e lo w e r a l g a e and t h e g re e n b a c t e r i a i s so u g h t. METABOLISM OF THE GREER BACTERIA The t e r m ’' a u t o t r o p h i c - p h o t o s y n t h e t i c 1’ h a s b e e n c o i n e d t o d e s c r i b e t h e m e ta b o lism of t h e g re e n b a c t e r i a . The e x p r e s s i o n i s s i n g u l a r l y d e s c r i p t i v e in a s m u c h a s t h e g r e e n b a c t e r i a d e v e l o p i n e n t i r e l y m e d ia , c o n ta i n in g h y d ro g en s u l p h i d e . in o rg a n ic L ig h t o f s u f f i c i e n t i n t e n s i t y and q u a l i t y to a c t i v a t e a p h o to c h em ica l r e a c t i o n m ust a ls o b e p ro v id e d . The c l a s s i c e q u a t i o n f o r p h o t o s y n t h e s i s o f t h e g r e e n p l a n t s re p re se n te d a s : C02 + H2 0 L i Sh t CHgO + Og. is F o rm ald eh y d e, i n t h i s e q u a t i o n r e p r e s e n t s a compound f o r m e d b y t h e r e d u c t i o n o f c a r b o n d i o x i d e . F o r p h o t o s y n t h e s i s a s c a r r i e d on b y t h e g r e e n b a c t e r i a , a g e n e r a l i z e d e q u a t i o n w o u ld b e r e p r e s e n t e d a s : CC>2 t 2 H2 S ^ i g h t In th e p h o to sy n th e sis of t h e g reen p l a n t , th e r e a c t i o n of w a te r w ith carb o n d io x id e g iv e s r i s e to t h e p ro d u c tio n o f oxygen. is s o w e l l known t h a t i t m e r i t s no d i s c u s s i o n . as e f f e c te d by th e g reen b a c t e r i a d i f f e r s + HgO +■ 2 S . T h is phenom enon H o w e v e r, p h o t o s y n t h e s i s i n a t l e a s t two m a j o r a s p e c t s f r o m t h a t c a r r i e d on b y t h e h i g h e r p l a n t s . - W ith t h e g r e e n b a c t e r i a , th e c o n v e rsio n o f carb o n d io x id e i n t o a r e d u c tio n p ro d u c t is e n t i r e l y upon th e p re s e n c e o f s u lp h id e i n th e e n v iro n m e n t. dependent A second m a j o r d i f f e r e n c e i n t h e m e t a b o l i s m o f t h e two g r o u p s o f p l a n t s is th e o b s e r v a b l e f a c t t h a t no o x y g e n i s p r o d u c e d a s t h e r e s u l t o f t h e p h o t o ­ sy n th e tic a c tiv itie s of t h e green b a c t e r i a . The m o s t s e n s i t i v e t e s t know n t o d e m o n s t r a t e t h e p r e s e n c e o f o x y g e n i s m e th o d d e v e l o p e d b y B e i j e r i n c k . o f oxygen i n q u a n t i t i e s t h e lu m in o u s b a c t e r i a T h is m e th o d w i l l d e t e c t t h e p r e s e n c e o f t h e o r d e r o f m a g n itu d e o f a m i l l i o n t h o f a m icro g ram . E v e n t h e u s e o f B e i j e r i n c k ’ s m e th o d f a i l s t o sh o w t h e p r e s e n c e o f oxygen a s a b y -p r o d u c t o f b a c t e r i a l p h o t o s y n t h e s i s . H y p o th e tic a lly , th e p ro c e ss of b a c t e r i a l p h o to s y n th e sis can be r e c o n s tr u c te d as in d ic a te d i n th e fo llo w in g e q u a tio n s : C02 * 2H2S 1,1 Sh t CHgO + H2 0 * 2S or 2C02 * H2S + 2H2 0 JA ? .A 2(CI-I2 0 ) + H2S 0 4 The two e q u a t i o n s g i v e n a b o v e c a n b e g e n e r a l i z e d a s C02 + 2 Hr, A A jiA CH2 0 + 2A + HgQ H e r e , H2A i s u s e d t o d e s i g n a t e a n y r e d u c i n g a g e n t o r h y d r o g e n d o n o r w h ile A is th e o x id a tio n o r d e h y d ro g e n a tio n p ro d u c t. -7 2 - DISCUSSION P re lim in a ry D isc u ssio n We h a v e , t h u s f a r , in th is p a p e r p r e s e n t e d d a t a on c e r t a i n e c o ­ l o g i c a l f a c t o r s w h ic h o p e r a t e w i t h i n a b o d y o f w a t e r a n d w h ic h h a v e b e e n i n t e r p r e t e d by s t u d e n t s o f a q u a ti c b io lo g y as e x e r tin g p o s i t i v e or n e g a tiv e e f f e c ts upon a q u a tic p o p u la tio n s . I t m ust b e e m p h asized h e re t h a t e c o lo g i c a l e le m e n ts c an n e v e r o p e r a te in d e p e n d e n tly o f each o t h e r b u t t h a t t h e y m u s t b e c o n s i d e r e d a s members o f a n i n f i n i t e l y c o m p le x e c o s y s t e m , e a c h c o m p o n e n t o f w h i c h i s n e c e s s a r y a n d v i t a l t o t h e dyn am ism o f t h e e n v i r o n m e n t . I n a tte m p tin g to a n a ly z e o r to e v a lu a te th e d a ta o b ta in e d d u rin g th e c o u rse of t h i s i n v e s t i g a t i o n , one is f a c t o r s a l l o f w h ic h w o u ld a p p e a r t o c o n fro n te d by a m u ltitu d e of in flu e n c e a d is tr ib u tio n . g e n e r a l l y c o n ce d e d t h a t f o r m ost p l a n t s and an im als I t is such a d i s t r i b u ­ t i o n d o e s e x i s t a n d t h a t a s u i t a b l e c r i t e r i o n s u c h a s C h a u v e n e t s may be a p p lie d . It re m a in s t o b e s e e n w h e th e r t h e b a c t e r i a u n d e r i n v e s t i ­ g a t i o n c a n b e t r e a t e d as a n e c o l o g i c a l u n i t i n a m anner co m p arab le t o o t h e r members o f t h e f l o r a a n d f a u n a . We s h a l l , in th e fo llo w in g p ag es, a tte m p t to a n a ly z e in d iv id u a lly each p o s s ib le c o n t r o l l i n g f a c t o r u n d e r c o n s i d e r a t i o n b u t a lw a y s w i t h t h e r e a l i z a t i o n t h a t no o n e f a c t o r o r s e t o f c o n d i t i o n s i s ever re ­ s p o n s i b le f o r a g iv e n d i s t r i b u t i o n . F a c t o r s P o s s ib ly I n f l u e n c i n g t h e D i s t r i b u t i o n o f G reen B a c t e r i a I t seem s j u s t i f i a b l e to u s e as a m a jo r p re m ise th e s ta te m e n t t h a t t h e num ber o f g r e e n b a c t e r i a fo u n d i n a p a r t i c u l a r e n v iro n m e n t a t a -7 3 s t a t e d t i m e i s a f u n c t i o n o f two c o m p le x v a r i a b l e s ; u n d o u b te d ly a r e (1 ) th e r e s u l t a n t of fo rces t h e s p e c i e s and ( 2 ) t h e r e s u l t a n t o f f o r c e s tio n of th e sp e c ie s. th e se v a ria b le s fa v o rin g re p ro d u c tio n of fa v o rin g d e a th o r e x tin c ­ E v e n th o u g h o p t i m a l e n v i r o n m e n t a l c o n d i t i o n s f o r r e p r o d u c t i o n may e x i s t , s u r v i v a l c o n d i t i o n s may b e u n f a v o r a b l e a n d t h e r e s u ltin g b a c t e r i a l p o p u la tio n w i l l b e lim it e d ; i f s u rv iv a l c o n d itio n s a r e f a v o r a b l e , a l a r g e p o p u l a t i o n may r e s u l t i n s p i t e o f t h e f a c t t h a t t h e e l e m e n t s f o r g r o w th a n d r e p r o d u c t i o n a r e n o t o p t i m a l . T he r e m a r k s e x p r e s s e d a b o v e may b e s u m m a riz e d i n t h e f o r m o f a d i f f e r e n t i a l e q u a ­ t i o n w h e re in t h e tim e is e x p r e s s e d a s t h e c o n s t a n t K; F a n d F 1 r e p r e ­ s e n t th e r e s u lta n t of fa v o ra b le and u n fa v o ra b le fo rc e s d N ^ EdF dF or re sp e c tiv e ly . d H ^ KdFf dF T em p eratu re as a C o n t r o l l i n g F a c to r We s h a l l f i r s t d i s c u s s t e m p e r a t u r e a s a p o s s i b l e c o n t r o l l i n g f a c ­ t o r a f f e c t i n g o r r e g u l a t i n g th e d i s t r i b u t i o n o f C h lo ro b iu m l i m i c o l a . We h a v e p r e v i o u s l y i n d i c a t e d , i n a p r e l i m i n a r y d i s c u s s i o n o f t h e f a c t o r o f te m p e r a tu r e , t h a t m ost a q u a ti c e n v i r o n m e n t s show a t l e a s t tw o m a j o r t h e r m a l c h a r a c t e r i s t i c s w h i c h c a n b e i n t e r p r e t e d a s h a v i n g some e c o l o g i cal sig n ific a n c e . T h e s e two m a in t h e r m a l c h a r a c t e r i s t i c s a r e ( 1 ) some fo rm o f s e a s o n a l change a n d (2 ) t h e e x i s t e n c e of a n a r e a c a l l e d th e th e rm o c lin e . T h i s l a t t e r t e r m was c o i n e d b y B i r g e i n 1897 t o a c c o u n t f o r a th e rm a l l a y e r w h e re in th e f a l l F. per fo o t of v e r t i c a l d is ta n c e . i n t e m p e r a t u r e w as a t l e a s t 0 . 5 4 8 ° The a r e a s a b o v e a n d b e lo w t h e th e r m o c l i n e a r e known r e s p e c t i v e ^ a s t h e e p i l i m n i o n and t h e h y p o l i m n i o n ; -7 4 - h ere, th e te m p e ratu re f a l l is l e s s th a n 0 .5 4 8 ° F. p e r f o o t o f v e r t i c a l d ista n c e . T he w a t e r s o f S o d o n L a k e d e p a r t fr o m t h e c l a s s i c in te rp re ta tio n o f t e m p e r a t u r e e x p r e s s e d a b o v e i n a t l e a s t tw o m a j o r a s p e c t s . are ( l) These a n i n c r e a s e i n te m p e r a tu r e from t h e to p t o th e b o tto m o f t h e h y p o lim n io n , and (2 ) a p erm an en t s t a g n a t i o n o f th e h y p o lim n io n th r o u g h ­ ou t th e y e a r. "While t h e f i r s t o f t h e s e o b s e r v a b l e ph eno m ena h a s b e e n r e p o r t e d by Newcombe ( 1 9 4 8 ) a n d s u b s t a n t i a t e d by t h e a u t h o r , p e r a tu r e in v o lv e d is th irty foot le v e l, r a r e l y m o re t h a n 0 . 5 ° F . ( 3 9 . 2 ° - 3 9 . 7 ° F . ) a t t h e r e p r e s e n t i n g t h e m i d d l e p o r t i o n of- t h e h y p o l i m n i o n ; an d a p o o r l y d e f i n e d r a n g e o f 0 . 1 ° F . in te rfa c e . t h e r a n g e o f te m ­ "While i t ( 4 1 . 0 ° - 4 0 . 9 ° F . ) a t t h e m u d - w a te r c a n b e shovm t h a t som e .m e t a b o l i c p r o p e r t i e s la k e b e a r a d i r e c t r e l a t i o n s h i p to i t s th e rm a l c o n d itio n s , of a i t can, in no w a y , b e d e m o n s t r a t e d t h a t d i c o t h e r m y o r t e m p e r a t u r e i n v e r s i o n o f t h e h y p o lim n io n i s a c o n t r o l l i n g f a c to r f o r a q u a tic p o p u la tio n s. The s e c o n d t h e r m a l c h a r a c t e r i s t i c o f S o d o n L a k e t o b e d i s c u s s e d i.e ., t h e p erm an en t s t a g n a t i o n o f t h e h y p o lim n io n th ro u g h o u t t h e y e a r , has, i t seem s t o u s , a n i m p o r t a n t b e a r i n g on t h e b a c t e r i a l p o p u l a t i o n s and a l l o th e r p o p u la tio n s in v e s tig a te d d u rin g th e c o u rse of t h i s stu d y . I t h a s r e p e a t e d l y b e e n e m p h a s iz e d t h a t t h e d e p t h i n t e r v a l o f 25 t 5 f e e t d e l i m i t s t h e z o n e of b i o l o g i c a l p r o d u c t i v i t y o f S o d o n L a k e . s t a t e m e n t m u st n o t b e c o n s t r u e d a s i n c l u d i n g t h e a c t i v i t i e s T h is of th e a n a e r o b i c b a c t e r i a , f o r t h e s e o r g a n i s m s r e a c h t h e i r maximum n u m b e rs a t th e m u d -w ater i n t e r f a c e . H o w e v e r, i n r e s p e c t t o t h e p o s s i b l e i n f l u e n c e -7 5 - o f t h e h y p o l i m n i o n , i f we c o n s i d e r t h a t t h e 3 0 f o o t d e p t h i n t e r v a l r e p r e s e n ts t h e m id d le p o r t i o n o f t h e h y p o lim n io n , th e n t h e r e i s con­ c l u s i v e e v i d e n c e t h a t t h e g r e e n b a c t e r i a , o t h e r members o f t h e p h y t o ­ p l a n k t o n a n d t h e z o o p l a h k t o n r e a c h t h e i r maxima n e a r t h i s zone o f d e m a rk a tio n . h y p o lim n e tic R e f e r e n c e t o T a b l e I w i l l sho w t h a t a n a l m o s t c o n s t a n t t e m p e r a t u r e e x i s t e d t h r o u g h o u t t h e y e a r a t t h e 30 f o o t d e p t h . H o w e v e r, c o n s i d e r a b l e c a u t i o n m u st b e u s e d i n a s s i g n i n g a d e g r e e o f im p o rta n c e to te m p e r a tu r e a s a r e g u l a t o r y f a c t o r f o r any d i s t r i b u t i o n . C e r t a i n l y , t e m p e r a t u r e may b e e x p e c t e d t o i n f l u e n c e t h e m e t a b o l i c a c tiv itie s of b a c t e r i a a n d may c a u s e r e s t r i c t i o n a s t o t h e k i n d s p r e s e n t i n a g iv e n e n v iro n m e n t; b u t te m p e r a tu r e , p e r s e , c an in f l u e n c e a b a c t e r i a l d i s t r i b u t i o n o n l y i n t h a t i t may r e g u l a t e t h e a v a i l a b i l i t y of m a te ria ls fo r s y n th e s is . I t is d i s c o n c e r t i n g t o r e a d , i n much o f th e a v a il a b le lim n o lo g ic a l l i t e r a t u r e , p e ra tu re upon p o p u la tio n s . of th e p o s itiv e e ffe c ts A ll such sta te m e n ts o f te m ­ s h o u l d b e m ade d e f i n i ­ t i v e by s t a t i n g t h e d e f i n i t e r o l e t h a t t e m p e r a t u r e i s t h o u g h t t o p l a y . I f food m a t e r i a l i s p r e s e n t f o r t h e g r o w th o f b a c t e r i a , a r i s e i n te m ­ p e r a t u r e may a c c e l e r a t e m e t a b o l i s m b u t o n ly w h i l e s u f f i c i e n t m a t e r i a l is p r e s e n t f o r s y n th e s is . Once t h i s p o in t is re a ch ed , th e te m p e ra tu re may a c t u a l l y c a u s e a d i m i n i s h i n g o f t h e b a c t e r i a l p o p u l a t i o n . e x c e p tio n s in d ic a te d , la tio n is it is W ith t h e e n t i r e l y p ro b a b le t h a t t h e b a c t e r i a l popu­ in d e p e n d e n t o f t h e f a c t o r o f te m p e r a tu r e . H o w e v e r, we l o o k u p o n t h e d e m a r k a t i o n z o n e o f t h e h y p o l i m n i o n a s a c o n v e n i e n t d e p t h w h ic h r e p r e s e n t s a n e c o t e n e l e v e l ; -7 6 - it is h e re , p ro b a b ly , t h a t th e th e rm a l in flu e n c e s of th e s h a llo w w a te r and deep w a t e r m e rg e a nd p o s s i b l y o f f e r a b a r r i e r t o b i o l o g i c a l m i g r a t i o n s . D i s s o l v e d O xygen We h a v e i n d i c a t e d , per s e , in th e p re c ed in g p arag rap h t h a t te m p e ra tu re , p ro b a b ly has l i t t l e , th e g reen b a c te r ia . i f any, e f f e c t upon th e d i s t r i b u t i o n of H ow ever we m u s t now c o n s i d e r t h e f a c t o r o f o x y g e n d e p le t io n as a f u n c tio n of te m p e ra tu re and b i o l o g i c a l a c t i v i t y . e x tre m e ly d i f f i c u l t , i n l i g h t o f p r e s e n t d ay k n o w le d g e, It t o do m o re th a n th e o r iz e c o n c e rn in g th e a b s o lu te h y p o lim n ia l oxygen d e f i c i t . a r e a w a r e o f t h e w o rk o f A l s t e r b e r g is We (1 9 3 0 ), H u tc h in so n (1 9 3 8 ) and o f K u s n e tz o w a n d K a r s i n k i n ( 1 9 3 1 ) ; t h e s e i n v e s t i g a t o r s have a tte m p te d to e x p la i n th e d e p l e t i o n o f oxygen i n th e h y p o lim n io n by v a r i o u s m eans. A l s t e r b e r g has p ro p o s e d t h e m i c r o s t r a t i f i c a t i o n th e o r y w h ic h i n f e r s t h a t t h e c h i e f c o n su m p tio n o f oxygen i s i n t h e b o t t o m d e p o s i t s ; w hen th e s e d e p o s its a r e e x h au ste d of oxygen, th e d e p l e t i o n c o n tin u e s i n t o th e h y p o lim n io n . H u t c h i n s o n c o m p u te s t h e " r e a l ” o x y g e n d e f i c i t by a d d ­ i n g t o t h e a p p a r e n t d e f i c i t t h e am o u n t o f o x y g e n n e c e s s a r y t o o x i d i z e s u c h s u b s t a n c e s a s m e t h a n e a n d h y d r o g e n s u l p h i d e w h ic h e m a n a te f r o m t h e b o tto m d e p o s it s and b u b b le th r o u g h th e h y p o lim n io n . K u s n e tz o w a n d K a r s in k in s u p p o rt t h e th e o r y t h a t t h e oxygen d e p l e t i o n i n t h e h y p o lim ­ n io n i s due to t h e o x y g en c o n su m p tio n by b a c t e r i a . It is p ro b a b le t h a t no o ne t h e o r y c a n a c c o u n t f o r t h e a n a e r o b i c c o n d i t i o n f o u n d i n t h e h y p o l i m n i o n ; o u r d a t a w o u ld o f f e r some s u p p o r t t o t h e t h e o r y of K u s n e tz o w a n d K a r s i n k i n i n t h a t e n o rm o u s n u m b e rs o f b a c t e r i a a r e p r e s e n t -7 7 - i n t h e h y p o lim n io n o f Sodon L ak e. H o w e v e r, t o t h e e f f e c t o f b a c t e r i a l a c t i o n m ust b e added t h a t o f t h e hy d ro g en s u lp h id e gas b u b b lin g th ro u g h t h e h y p o l i m n i o n a n d t h u s r e m o v in g q u a n t i t i e s It is of oxygen. n o t d i f f i c u l t t o c o r r e l a t e o u r d a t a on t h e d i s t r i b u t i o n o f th e g ree n b a c t e r i a w ith th e o b serv ed h y p o lim n ia l d e f i c i t . b a c te ria are The g r e e n e n t i r e l y a n a e r o b i c a n d we h a v e n o t b e e n a b l e t o s t i m u ­ l a t e th e g ro w th o f t h e s e o rg a n is m s w ith e v en m in u te t r a c e s of oxygen. M any o f t h e o t h e r g r o u p s o f b a c t e r i a w h i c h we h a v e f o u n d i n t h e h y p o l i m ­ n i o n w h i l e c l a s s i f i e d a s ’’a e r o b i c ” may p r o p e r l y b e d e s i g n a t e d a s m i c r o a e ro p h ilic o r f a c u l t a t i v e a n a e r o b i c a n d w o u ld t h e r e f o r e b e a b l e t o v iv e f o r a tim e u n d e r s t r i c t l y a n a e ro b ic c o n d itio n s . These g ro u p s h i b i t t o l e r a t i o n rang es e x te n d in g a t l e a s t t o t h e f o r t y su r­ ex­ f o o t d e p th in te rv a l. W h i l e we h a v e d e m o n s t r a t e d t h a t , th ro u g h o u t th e p e rio d of t h i s v e s t i g a t i o n , an a n a e ro b ic c o n d itio n has d e p th i n t e r v a l , a n a e ro b io sis in ­ p r e v a i l e d b e l o w t h e 25 "t 2 f e e t i t w as t h o u g h t t h a t a d d i t i o n a l d a t a i n r e g a r d t o c o u ld b e o b ta in e d i f v a r i o u s d e p t h s w e r e know n. It is th e o x id a tio n - r e d u c tio n p o te n tia ls a t g e n e r a lly conceded t h a t th e p o t e n tia ls e x i s t i n g i n l a k e w a t e r a n d muds a r e o x i d a t i o n - r e d u c t i o n p o t e n t i a l s . To t h i s e n d , a n a p p a r a t u s f o r m e a s u r i n g Eh a n d pH, a t a n y d e s i r e d d e p t h , h a s b e e n d e s i g n e d b y R. J . H a r l e y , m ent o f M ich ig an S t a t e C o lle g e . c o n sists of th e B a c te rio lo g y D e p a rt­ E sse n tia lly , th e m e a su rin g a p p a r a tu s o f a L eeds and N o r th r u p vacuum tu b e p o t e n t i o m e t e r ; t h e r e q u i r e d e l e c t r o d e s a r e em bedded i n W ards B i o - P l a s t i c . 78- The g l a s s e le c tro d e f a r pH r e a d i n g s , t h e p l a t i n u m e l e c t r o d e f o r Eh d e t e r m i n a t i o n s a n d t h e c a lo m e l h a l f - c e l l a r e a r r a n g e d a s i n d i c a t e d i n F ig u r e 15 . Each e l e c ­ t r o d e -w ire i s a t t a c h e d t o o n e w i r e o f a t h r e e c o n d u c t o r s h i e l d e d w a t e r ­ p r o o f c a b l e 60 f e e t i n l e n g t h ; t h e w i r e s f r o m t h e c a b l e a r e c o n n e c t e d t o a L e e d s a n d U o r t h r u p A s s e m b ly U n i t , n u m b e r 7 6 6 3 -A 1 . e te r is re c tio n s eq u ip p ed w i t h a s c a l e , T h is p o t e n t i o m ­ c a l i b r a t e d i n pH u n i t s ; t e m p e r a t u r e c o r ­ f o r t h e pH r e a d i n g s a r e m ade b y a c o m p e n s a t i n g d i a l o n t h e panel board of th e in s tru m e n t. By m eans o f a t o g g l e s w i t c h , Eh m e a s u r e ­ m e n ts a r e made on t h e sam e s c a l e a s f o r pH m e a s u r e m e n t s . A ll m easure­ m e n ts o f p o t e n t i a l s a r e r e f e r r e d t o t h e s t a n d a r d h y d r o g e n e l e c t r o d e . D e te rm in a tio n s o f Eh a n d pH w e r e made i n s i t u o n M arch 1 1 , 1 9 5 0 ; a t t h i s tim e , a n e i g h t in c h i c e c o v e r e x is t e d to g e t h e r w ith a p p ro x i­ m a t e l y s e v e n i n c h e s o f sn ow . A t t h e s u r f a c e 2 .0 p .p .m . o f d is s o lv e d o x y g e n w e r e d e t e c t e d w h i l e t h e c o r r e s p o n d i n g v a l u e a t 20 f e e t was 1 . 0 p .p .m . A p o s i t i v e t e s t f o r d i s s o l v e d oxygen c o u ld n o t b e o b ta in e d b e ­ lo w t h e 25 f o o t d e p t h i n t e r v a l . H y d ro g e n s u l p h i d e c o u l d n o t b e d e ­ t e c t e d i n t h e u p p e r l a y e r s o f w a t e r b u t was p r e s e n t i n v a l u e s r a n g i n g f r o m 2 . 0 p . p . m . a t t h e 40 f o o t l e v e l t o 7 . 5 p . p . m . a t t h e 50 f o o t l e v e l . F e r r o u s i r o n was p r e s e n t i n t r a c e a m o u n t s . The pH r a n g e was f r o m B .5 a t t h e s u r f a c e t o 6 .5 a t t h e m u d -w a te r i n t e r f a c e . The p r e s e n c e o f t h e g r e e n b a c t e r i a T h erefo re, c o u ld n o t be d e m o n stra te d . in a p p ro a c h in g a d is c u s s io n o f t h e r e s u l t s o f o u r Eh m e a s u r e ­ m e n t s , we a r e c o n s i d e r i n g a s i t u a t i o n w h e r e i n t h e g r e e n b a c t e r i a e ith e r do n o t e x i s t o r t h e i r num bers a r e s o s m a l l a s t o r e n d e r th e m u n d e c t e c t a b l e by c o n v e n tio n a l c o l l e c t i n g m e th o d s. -7 9 - FIGURE DI AGRAM AND OF 15 VACUUM ASSOCIATED T UB E P OT E NT I 0 ME TE R SUBMERSION ELECTRODE a s s e m b l y FOR MEASUREMENT Of EH AND p H P O T E NTI O M E T E R n o u j Pc. GLass i i -8 0 - f t RcF A t t h e b e g i n n i n g o f t h e a n a e r o b i c z o n e , we o b t a i n e d a n Eh o f +0,2748 v o l t s ; t h i s f i g u r e was r e l a t i v e l y f e e t was r e a c h e d . c o n s t a n t u n t i l a d e p t h o f 50 A t t h e 50 f o o t d e p t h i n t e r v a l , a n Eh o f + 0 . 2 5 5 8 v o l t s was o b t a i n e d . The i m p o r t a n t q u e s t i o n t o b e d i s c u s s e d i s : "How i s p o t e n t i a l of a p a r t i c u l a r m a g n itu d e to b e i n t e r p r e t e d ? " t h a t th e r e have been s e v e r a l in v e s t i g a t i o n s d iffe re n t lakes. I t is tr u e involving th e d eterm in a tio n of p o t e n t i a l s as th e y e x i s t i n n a t u r a l w a t e r s . c l u s i o n t o b e drawn fro m t h e s t u d i e s an e le c tro d e But t h e o n ly v a l i d c o n ­ is t h a t the p o te n tia ls vary i n W h ile q u a n t i t a t i v e methods s h o u ld g i v e r e s u l t s w h ic h may b e i n t e r p r e t e d d i r e c t l y , it se e m s t h a t a s t a t i c tem m ust b e p r e s e n t i n o r d e r f o r t h e r e s u l t s or co n tro lled sy s­ to be m eaningful. Ifiuch o f t h e d a t a w h i c h a r e a v a i l a b l e h a v e come f r o m s t u d i e s o n t h e l a b o r a t o r y c u ltiv a tio n of anaerobes; here, conditions po ssib le under laboratory c u ltiv a tio n . a re c o n tro lle d in s o fa r as is H o w e v e r , we m u s t r e a l i z e t h a t even i n a s o - c a l l e d " c o n t r o l l e d s t a t e " n o t only i s t h e o x i d a t i o n - r e d u c t i o n p o t e n t i a l changing b u t a l s o t h e h y d ro g e n -io n c o n c e n tr a tio n and th e c h e m i c a l c o m p o s i t i o n o f t h e m e diu m o r , m o r e p r o p e r l y s p e a k i n g , t h e m ilie u is in a s ta t e of flu x . d itio n s fu rth e r as, I f a t t e m p t s a r e made t o s t a n d a r d i z e c o n ­ f o r i n s t a n c e , b y c o n t r o l l i n g pH, we w i l l no l o n g e r be d e alin g w ith a n a tu r a l system . Now i f our a tte n tio n is d ire c te d to a n a t u r a l b o d y o f w a t e r a s a c u l t u r e me diu m f o r u n t o l d t y p e s and n u m b e rs of o rg an ism s, th e problem and t h e i n t e r p r e t a t i o n o f e x p e rim e n ta l d a ta a r e r a i s ed t o e x p o n e n t i a l h e i g h t s . -8 1 - I n t h e l a s t a n a l y s i s , we c a n o n l y r a i s e q u e s t i o n s Eh o f n a t u r a l w a t e r s . Perhaps f u r t h e r i n v e s t i g a t i o n s concerning th e along t h i s lin e w i l l be p r o f i t a b l e . Some o f t h e q u e s t i o n s t o b e a n s w e r e d b e f o r e p o s i ­ tiv e in terp retatio n s of e le c tr o d e p o t e n t i a l s b e made a r e : i n a n a t u r a l sy stem can d o e s t h e e l e c t r o d e p o t e n t i a l r e p r e s e n t t h e s t a t e o f some p articu lar r e v e r s i b l e system o r s y ste m s ; does t h e p o t e n t i a l r e p r e s e n t a positiv e c o r r e l a t i o n w ith oxygen t e n s i o n ; a n d , does t h e . p o t e n t i a l r e p r e s e n t a n a v e r a g e o f s e v e r a l s y s t e m s , t o some e x t e n t , in equilibrium w ith each o th e r? If i t can be shown t h a t t h e r e i s a s i g n i f i c a n t d i f f e r e n c e i n e l e c ­ t r o d e p o t e n t i a l s w he n t h e g r e e n b a c t e r i a a r e p r e s e n t i n d e t e c t a b l e q u an tities, some l i g h t w i l l b e s h e d u p o n t h e c o n d i t i o n s of not only th e g reen b a c te r ia but a ls o of e x iste n c e of t h e f a u n a a n d f l o r a w h i c h c o - e x i s t w ith th e g re e n b a c t e r i a i n t h e a n a e ro b ic zone. The P r e s e n c e o f H y d r o g e n S u l p h i d e A t no t i m e d u r i n g t h e c o u r s e o f t h i s i n v e s t i g a t i o n has t h e h y d ro ­ g e n s u l p h i d e c o n t e n t of Sodon Lake b e e n l a r g e and f r e q u e n t l y a p o s i ­ tiv e te s t f o r hydrogen s u lp h id e could n ot be o b ta in e d . has b e en fro m 0 t o 1.065 p .p .m . ra th e r sm all q u a n titie s . The a n n u a l r a n g e Some a t t e n t i o n s h o u l d b e g i v e n t o t h e s e C om paratively l a r g e v a lu e s f o r hydrogen s u l ­ p h i d e i n t h e i m m e d i a t e e n v i r o n m e n t .of t h e g r e e n b a c t e r i a , 0 . 1 g ra m s p e r l i t e r a t a d e p t h o f 1 8 . 7 m e t e r s , 0 .2 B 2 grams p e r l i t e r , a n d 0 . 3 grams p e r l i t e r a t a d e p t h o f 43 m e t e r s h a v e b e e n r e p o r t e d b y S z a f e r ( 1 9 1 0 ) , Nads on ( 1 9 1 2 ) a nd Bavendamm ( 1 9 2 4 ) . -8 2 - It is r a t h e r hard t o a c c e p t th e values c i t e d a b o v e when c o m p a r e d w i t h a d e m o n s t r a b l e f a c t ; t h a t i n t h e zone o f c o n c e n t r a t i o n of t h e g r e e n b a c t e r i a , s u lp h id e is b eing converted i n t o elem en tal s u lp h u r. nam ely, th e hydrogen Thus t h e r e m u s t n e c e s s a r i l y b e m i n u t e a m o u n ts o f t h e g a s i n t h e i m m e d i a t e e n v i r o n m e n t of th e green b a c te r ia , "While o u r v a l u e s f o r h y d r o g e n s u l p h i d e i n c r e a s e w ith th e v e r t i c a l d is ta n c e (an expected r e s u l t ) t h e c o n c e n tr a tio n of t h e g r e e n b a c t e r i a does n o t i n c r e a s e w i t h t h e v e r t i c a l d i s t a n c e ; fact, t h e zone o f c o n c e n t r a t i o n of t h e g r e e n b a c t e r i a lim ite d a t th e tw e n ty -fiv e fo o t depth. in is sharply de­ We a r e p r o p o s i n g h e r e t h a t t h e s m a l l amount o f h y d ro g e n s u l p h i d e found i n t h e zone of c o n c e n t r a t i o n i s t h e c o n d i t i o s i n e qua. n o n f o r t h e e x i s t e n c e o f t h e g r e e n b a c t e r i a . The d e h y d r o g e n a t i o n o f h y d r o g e n s u l p h i d e a n d t h e r e d u c t i o n o f c a r b o n d io x id e w ith th e r e s u ltin g lo s s of oxygen c r e a t e s a n a n a e r o b ic c o n d i­ t i o n w ith o u t which t h e g re e n b a c t e r i a c a n n o t e x i s t . L ight There must b e , u n d e r a g i v e n s e t of c o n d i t i o n s , a c r i t i c a l value f o r l i g h t i n t e n s i t y which marks t h e p o i n t a t which l i g h t becomes t h e l i m i t i n g f a c t o r f o r growth of t h e g re e n b a c t e r i a . in tricacies is A d isc u ssio n of the of l i g h t e v a l u a t i o n i s beyond th e sco p e o f t h i s paper; it c e r t a i n t h a t l i g h t m easurem ents and i n t e r p r e t a t i o n of t h e r e s u l t i n g d a t a b e l o n g i n t h e ha nd s o f a c o m p e t e n t p h y s i c i s t . We c a n o n l y i n d i c a t e h e r e t h a t a s u f f i c i e n t a m ou nt o f l i g h t t o a c t i v a t e a p h o t o c h e m i c a l p r o ­ c e s s does p e n e t r a t e t h r o u g h t h e w a t e r o f Sodon Lake t o t h e u p p e r l i m i t s o f th e a n a e ro b ic zone. alw ays o b ta in e d ; B e l o w t h e 25 f o o t l e v e l 0,0%, t r a n s m i s s i o n was t h i s v a l u e i s t h e same f o r i c e w i t h s no w , i c e a l o n e o r w ater. -8 3 - I t may b e o f i n t e r e s t t o c i t e some e x a m p l e s o f l i g h t v a l u e s o b ­ t a i n e d by v a r io u s workers in aquatic in v e stig a tio n s. i n d i c a t e s t h a t th e com pensation i n t e n s i t y i . e . , C la r k (1939) t h a t i n t e n s i t y of l i g h t which w i l l a c t i v a t e a p h o t o s y n t h e t i c p r o c e s s and b e lo w w hich photosynthesis i s o v e r b a l a n c e d by t h e p r o c e s s p r o x i m a t e l y 30 t o 40 f o o t c a n d l e s . of re s p ira tio n , is ap­ W ilson (1935) c i t e s f i g u r e s of 4 00 t o 600 f o o t c a n d l e s f o r t h e sam e s e t o f c o n d i t i o n s ; a n d G r e e n b a n k (1945) s t a t e s t h a t t h e amount o f l i g h t w h ich w i l l p e n e t r a t e o n e and o n e -h a lf f e e t of c le a r ic e is s u f f ic ie n t fo r photosynthesis to take p lace. tity It is obvious t h a t s e r i o u s d is a g r e e m e n ts e x i s t as t o t h e quan­ o f l i g h t n e c e ssa ry t o a c t i v a t e a photochem ical p ro c e s s . d a t a show e x t r e m e l y l o w v a l u e s f o r t h e p h o t o s y n t h e t i c z o n e , Our own ranging from 1 .5 t o 1 .0 f o o t c a n d le s . The C h e m i c a l C o n s t i t u e n t s o f t he W a t e r , O t h e r t h a n D i s s o l v e d Oxy g e n and Hydrogen S u lp h id e L i t e r a t u r e d e a l i n g w i t h t h e b i o l o g y of la k e s abounds w i t h t a b l e s of chem ical d e te r m in a tio n s p u rp o r tin g t o b e a c c u r a t e a n a ly s e s o f th e chem ical n a tu r e o f a q u a tic environm ents. is examined c a r e f u l l y , If th is chem ical l i t e r a t u r e one u n d e b a ta b le c o n c lu s io n m ust be drawn. This c o n c l u s i o n , w h i c h we s h a l l l a t e r q u a l i f y b y c i t i n g p e r t i n e n t e x a m p l e s , i s t h a t many c h e m i c a l d a t a r e p o r t e d i n t h e l i t e r a t u r e a r e u n t e n a b l e w i t h w h a t i s known a b o u t c h e m i c a l b e h a v i o r . A b a s ic assum ption i n w ater a n a l y s i s , p h th a le in -m e th y l orange a l k a l i n i t y , c o n s i d e r i n g now p h e n o l - i s t h a t t h e b i c a r b o n a t e a nd h y d r o x i d e -8 4 - i o n s may n o t e x i s t i n t h e s a m e w a t e r ; correct. th is assum ption is certain ly not D©Martini (1 9 3 8 ) has c a l l e d a t t e n t i o n t o t h i s v e r y o b v io u s f a c t and a l s o to t h e in a d e q u a c ie s o f f r e e c a rb o n d i o x i d e d e t e r m i n a t i o n s . A g a i n , t h e v a l u e s o b t a i n e d f o r h y d r o x i d e a l k a l i n i t y c o m p u t e d by t i t r a ­ t i o n a r e no t i n agreem ent w ith t h e hydroxyl io n c o n te n t as determ ined from th e h ydrogen-ion c o n c e n tra tio n of w a te r . The s u g g e s t i o n i s t h e r e ­ f o r e o f f e r e d t h a t t h e c a rb o n d i o x i d e , b i c a r b o n a t e , normal c a r b o n a t e and h y d r o x id e c o n t e n t of n a t u r a l w a t e r s s h o u ld b e c a l c u l a t e d from t h e pH a n d t o t a l a l k a l i n i t y p h y sica l chem istry. o f t h e s e w a t e r s by means o f e q u a t i o n s b a s e d on By t h i s m e t h o d t h e d i f f e r e n c e s in in d iv id u a l te c h ­ n i q u e s w i l l h,e m i n i m i z e d a n d a m o r e s u i t a b l e r a t i o n a l e w i l l b e a v a i l a b l e f o r com paring th e c h e m is tr y o f d i f f e r e n t ty p e s o f w a te r . An e x a m i n a t i o n o f F i g u r e 14 w i l l s h o w a p h y s i c a l c h e m i s t r y a p p r o a c h t o t h e d e t e r m i n a ­ t i o n o f t h e r e l a t i o n s h i p b e t w e e n pH, t o t a l a l k a l a n i t y a n d c a r b o n d i o x ­ ide; th is lo c. cit. g r a p h was c o n s t r u c t e d by u s i n g t h e e q u a t i o n s o f D e M a r t i n i , S im ilar fig u res c a n b e c o n s t r u c t e d t o show t h e r e l a t i o n s h i p f o r b i c a r b o n a t e a l k a l a n i t y and normal a l k a l a n i t y ; and t h e t o t a l a l k a l a n i t y , a n d , k n o w i n g t h e pH t h e d e s i r e d component c a n be r e a d d i r e c t l y from t h e c h a r t . I n v ie w of t h e a t t e n t i o n c u r r e n t l y b e in g d i r e c t e d t o a c o n s i d e r a ­ tio n of tra c e elem ents as l i m i t i n g f a c t o r s i n a q u a ti c environm ents, i t s h o u l d b e em phasized t h a t t h e s t a n d a r d methods o f a n a l y s e s as s u g g e s t e d by t h e A m e ric a n P u b l i c H e a l t h A s s o c i a t i o n a r e n o t s u i t a b l e f o r d e t e c t ­ in g s m a l l amounts o f i r o n , sodium , magnesium , p o t a s s i u m a n d o t h e r e l e ­ m e n t s ; u n f o r t u n a t e l y , many o f t h e r e p o r t s -8 5 - in th e l i t e r a t u r e a re based #$()+('#+*+'&)%)%)*#''&)#%*&)%&()*#+')*)$()($#'#+#*(&&$+#$)#&&++$('')*$()#$&(&)%)()%#('&+$$&&)$& EUGENE D IETZGEN CO, N O . 316.C P R I N T E D IN U .S .A . -86- upon S t a n d a r d Methods. Some means w h i c h a r e s u i t a b l e f o r t h e d e t e r m i n a ­ t i o n of t h e f o u r elem ents l i s t e d above w i l l be o f f e r e d ; t h e s e m e th o d s a r e a d e q u a t e f o r w a te r s w hich c o n t a i n 150-250 p a r t s p e r m i l l i o n o f to ta l so lid s. Iron: The t h i o c y a n a t e m e t h o d a s i n d i c a t e d i n S t a n d a r d M et hods i s not s a t i s f a c t o r y h e re because such a procedure is not p a r t i c u l a r l y sen sitiv e. Among t h e m o r e s e n s i t i v e m e t h o d s a v a i l a b l e f r o m t h e l i t e r a ­ tu r e a r e th e m e rc a p to a c e ta te , th e o rth o p h e n a n th ro lin e and th e s a l i c y l a doxim e m e t h o d s . Of t h e s e , only t h e m e r c a p t o a c e t a t e method g iv e s a c o l o r which i s s u i t a b l e f o r u s e i n N e s s l e r t u b e s . In addition, t h i s m e th o d doe s n o t r e q u i r e c o n c e n t r a t i o n o f t h e s a m p l e e v e n a t a c o n c e n t r a t i o n o f 0.1 p.p .m . of i r o n . A n i o n s do n o t a f f e c t t h e c o l o r a n d l o w c o n c e n t r a ­ t i o n s o f t h e h e a v y m e t a l s do n o t i n t e r f e r e . Sodium; The c h l o r o p l a t i n a t e m e t h o d f o r p o t a s s i u m a n d t h e c a l c u l a ­ t i o n o f sodium from t h e w e i g h t o f t h e m ix e d c h l o r i d e s i s a t l e a s t o n l y a c ru d e a p p r o x im a tio n when t h e the a lk a lie s . condition; s a m p le c o n t a i n s o n ly afe w p . p . m . o f The ' ' t r i p l e a c e t a t e " m e th o d s a r e p r a c t i c a l f o r s u c h a t h e te c h n i q u e of B a r b e r and K o l t h o f f (1928) i s M ag ne s iu m : re co mm end ed . K o l t h o f f and G i l l a m ( 1 9 4 1 ) u s e a c o l o r i m e t r i c d e t e r ­ m i n a t i o n o f m a gnes iu m by a d s o r b i n g t i t a n y e l l o w o n p r e c i p i t a t e d mag­ nesium h y d ro x id e . Potassium ; 7/hen t h e p i c r i c a c i d t e s t f o r p o t a s s i u m i s n e g a t i v e , t h e m e th o d o f C a l e y I t was d e c i d e d ( 1 9 3 1 ) may b e u s e d . to subject a v e r t i c a l s e rie s Sod on La ke t o s p e c t r o g r a p h i c a n a l y s i s . -8 7 - ofw a te r sam ples from T he s e a n a l y s e s w e r e t h o u g h t t o b e n e c e s s a r y i n a s m u c h a s Newcomb ( 1 9 5 0 ) h a s r e p o r t e d f o r Sod on Lake a biochem ical s t r a t i f i c a t i o n of s o lu b le phosphorus, s i l i c a , c a l c i u m a nd iro n . N e ith e r ou r chemical a n a ly se s by r o u ti n e p ro ced u res n o r t h e r e ­ su lts o f s p e c t r o g r a p h i c a n a l y s e s s u p p o r t Newcomb’ s c o n t e n t i o n o f chem­ i c a l s t r a t i f i c a t i o n i n Sodon Lake. The p r o c e d u r e i n v o l v e d i n a s p e c t r o g r a p h i c a n a l y s i s c a n b e sum­ m arized as fo llo w s : t h e s a m p l e i s b u r n e d i n a n a r c a n d s p a r k s t a n d by a high v o lta g e sp ark . The l i g h t e m i t t e d b y t h e s a m p l e i s d i s p e r s e d i n t o a s p e c t r u m a n d p h o t o g r a p h e d by a s p e c t r o g r a p h . The l i n e s on t h e p l a t e a r e i d e n t i f i e d by a c o m p a r a t o r a n d t h e i r b l a c k n e s s m e a s u r e d by a m icrophotom eter. J a r r e l l - A s h e q u i p m e n t was u s e d f o r t h e s p e c t r o - c h em ical w ork, and t h e a s s i s t a n c e o f Dr. J . B e d e l l , P r e s i d e n t o f th e N atio n al Spectrographic L a b o ra to rie s, i s ' g r a t e f u l l y acknow ledged. Much o f t h e d a t a o b t a i n e d a r e l a r g e l y r e p e t i t i o u s ; this i s a n ex­ p e c t e d r e s u l t i n a s m u c h a s S o d o n La ke i s known t o e x h i b i t a n i n c o m p l e t e overturn. The r e s u l t s o f t h e s p e c t r o g r a p h i c a n a l y s e s a r e i n d i c a t e d i n T able XI. The s p e c i f i c c h e m i c a l c o n d i t i o n s f o u n d i n S o d o n L a k e a r e n o t p a r ti c u l a r l y unique. A l k a l i n i t y i s d u e e n t i r e l y t o b i c a r b o n a t e s w h ic h a r e p r e s e n t i n l a r g e am ounts. The q u a n t i t i e s o f f r e e c a r b o n d i o x i d e a r e s m a l l as would b e e x p e c te d i n a h a rd w a t e r l a k e . N itra te nitrogen a n d p h o s p h a t e p h o s p h o r o u s , t h e two i o n s g i v e n c o n s i d e r a b l e a t t e n t i o n a s l i m i t i n g f a c t o r s f o r p l a n k t o n p r o d u c t i o n , a r e p r e s e n t i n a m o u n ts equal to th o se re p o rte d f o r p ro d u c tiv e la k e s . The e l e m e n t s r e p o r t e d a s b e i n g p r e s e n t i n t r a c e a m o u n ts i n S o d o n L a k e may b e o f g r e a t -8 B - TABLE X I SODON LAKE - SPECTROGRAPHIC ANALYSIS* Maj o r M in o r 0.0141 g / l over 10/ 1-10/ Mg Low V e r y Low 0.0 1 / 0 .001/ 0.0041 g / l 0 .1 -1 .0 / 0.0004 g / l 0.1 / 0.000041 g / l 0.0000041 g / l B Pe P Ni Si A1 Bi Cu Cr Ti Mo Mn Co V Pb Ca Sn N a(trace) Ag T r a c e Based upon t h r e e l i t e r samples t a k e n a t v a r i o u s d e p t h s . -8 9 - i m p o r t a n c e t o t h e o r g a n i s m s p r e s e n t o r t h e y may b e o f no i m p o r t a n c e . It is th e contention of th is au th o r t h a t , u n t i l inform ation is a v a i l ­ a b l e show ing t h e amount o f each a v a i l a b l e n u t r i e n t o r elem ent p r e s e n t i n a l a k e a n d a c t u a l l y u s e d by i n d i v i d u a l o r g a n i s m s , r o u ti n e chem ical d eterm in atio n s a r e of l i t t l e v a lu e in e x p la in in g th e com positional d i f f e r e n c e s and d i s t r i b u t i o n s . The i d e a s u g g e s t e d may b e i m p o s s i b l e o f e x e c u t i o n and p u r e l y i n t h e r e a l m o f t h e o r y j b u t i t scien tific i s hoped t h a t t h e o r i z i n g r e g a r d i n g some o f t h e p r o b l e m s o f l a k e b i o l o g y may l e a d t o new m e t h o d s o f a p p r o a c h . I t is not th e purpose of t h i s Sodon Lake and i t s paper to d is c u s s t h e chem istry of p ossible effects on p r o d u c t i v i t y . R a t h e r , we a r e i n t e r e s t e d i n k n o w i n g i f t h e c h e m i c a l c o m p o s i t i o n o f t h e w a t e r ha s some o b s e r v a b l e c o n s e q u e n c e u p o n t h e d i s t r i b u t i o n o f t h e g r e e n b a c t e r i a . M ortim er (1939) has i n d i c a t e d t h a t t h e chem ical co m p o sitio n of w a t e r i s a p p r o x i m a t e l y t h e same a t a l l d e p t h s a n d v a r i e s b u t s l i g h t l y w ith th e seaso n s. Our i n v e s t i g a t i o n s , in v o lv in g th e chem istry of th e w a t e r o f S o d o n L a k e o v e r a p e r i o d o f one y e a r s ee m t o s u p p o r t M o r t i m e r ’ s co n tention. The c o n c l u s i o n i s t h e r e f o r e r e a c h e d t h a t t h e f l u c t u a t i o n s i n t h e a m o u n t s o f d i s s o l v e d s u b s t a n c e s i n t h e w a t e r g i v e no c l u e a s t o th e d is tr ib u tio n of a q u atic b a c te ria . B io lo g ic al Factors I t w o u l d b e i n t e r e s t i n g i f we c o u l d c o n c l u d e f r o m a n a n a l y s i s o f o u r d a ta t h a t a s i g n i f i c a n t l y u n iq u e p h y to p la n k to n and zo o p lan k to n accom panied t h e d i s t r i b u t i o n of t h e g r e e n b a c t e r i a , . is n o t the case. Such, a p p a re n tly , D aphnia p u l e x , Diaptomus o r e g o n e n s i s and Cyclops -9 0 - p rasinus ( t h e t h r e e m o s t common members o f t h e z o o p l a n k t o n o f S o d o n Lake) a r e c o s m o p o lita n i n t h e i r d i s t r i b u t i o n ; ev er, u nder t h e environm ental co n d itio n s t h e s e o r g a n i s m s d o , how­ p r e s e n t i n Sodon Lake, exhibit a n a d a p t i v e t o l e r a n c e t o m i n u t e amounts o f oxygen and t o m e a s u r a b le quan tities of hydrogen s u lp h id e . A t o t a l o f 117 D a p h n i a p u l e x , 29 D ia p t o m u s o r e g o n e n s i s a n d 45 C y c l o p s p r a s i n u s w e r e c o l l e c t e d a t d e p t h s where a p o s i t i v e t e s t f o r d i s s o l v e d oxygen co u ld n o t be o b ta in e d . I t is planned, i n t h e near f u t u r e , to a tte m p t d u p lic a tio n of t h e d ata on t h e copepod d i s t r i b u t i o n u n d e r l a b o r a t o r y c o n d i t i o n s . The p h y t o p l a n k t o n , w h i l e q u a l i t a t i v e l y r e s t r i c t e d , d o e s n o t see m to be p e c u li a r l y d i f f e r e n t from t h a t o f o th e r hard w a ter la k e s . W hile a more d e t a i l e d s t u d y of t h e p h y t o p l a n k t o n w i l l f o r m t h e b a s i s o f a n o t ­ her study, it is s u f f i c i e n t t o s t a t e h e r e t h a t t h e r e a r e no i n d i c a t i o n s o f c o m p o s i t i o n a l d i f f e r e n c e s b e tw ee n Sodon Lake and o t h e r s m a l l g l a c i a l lakes. The B a c i l l a r i a e w e r e p e r h a p s t h e m o s t n u m e r o u s o f t n e p l a n k t o n i c f o r m s , b o t h a s t o t h e number o f s p e c i e s a n d t h e n um be r o f i n d i v i d u a l s . The t e r m ” s e l e c t i o n b i o t a ” , a n e x p r e s s i o n d e n o t i n g a g r o u p o f o r g a n i s m s o r d i n a r i l y w i d e l y d i s t r i b u t e d a nd a b l e t o w i t h s t a n d a v a r i e t y o f e n ­ vironm ental c o n d itio n s, is perhaps d e s c r i p t i v e of b o th t h e phytoplarik- t o n and t h e z o o p l a n k t o n o f Sodon Lake. Taxonomical C o n s i d e r a t i o n s o f t h e G reen B a c t e r i a W hile i t t i n c t species i s p o s s i b l e t h a t f u t u r e r e s e a r c h may d i s c l o s e many d i s ­ of green b a c te r ia , it is c e r ta in th a t, a t p re se n t, the C h lo ro b a cteriac e ae re p re s e n t a very a r t i f i c i a l -9 1 - grouping. Only a s b acterio lo g ical is i n f o r m a t i o n f r o m a l a r g e n um be r o f a q u a t i c h a b i t a t s o b t a i n e d a n d a n a l y z e d , c a n m e a n i n g f u l a d v a n c e s b e m ad e. It is c e r­ t a i n t h a t p u r e c u l t u r e s t u d i e s m u s t b e c a r r i e d on i n c h e m i c a l l y d e f i n e d and r e p r o d u c i b l e m e d ia , Y Jh il e i t i s recognized t h a t th e la b o ra to ry c u l t i v a t i o n of b a c t e r i a c r e a t e s an u n n a t u r a l environm ent ( u n n a tu r a l i n t h e s e n s e t h a t s u c h a method c a n n o t r e c r e a t e a n e c o l o g i c a l s y ste m ) i t is t h e o n l y means by w n i c h c o n t r o l o f t h e e n v i r o n m e n t c a n b e a s s u r e d . The t y p e s p e c i e s o f t h e g r e e n b a c t e r i a , Chlorobium l i m i c o l a N a d s o n , i s t h e o n l y member o f t h e C h l o r o b a c t e r i a c e a e w h i c h we h a v e i s o ­ l a t e d and grown i n p u r e c u l t u r e , A d e s c r i p t i o n o f C, l i m i c o l a f o l l o w s : C e l l s : v a r i o u s shapes and s i z e s , markedly dep en d en t upon e n v iro n m en tal conditions; s p h e r i c a l to ovoid, ab o u t 0,5 u to 1 ,0 u i n d iam eter; ro d s , g e n e r a l l y 0 , 7 u by 1 , 0 t o 2 . 5 u ; r e g u l a r l y p r o d u c e mucus, c a u s i n g t h e f o r m a t i o n o f c e l l c o n g lo m e ra te s o f d i f f e r e n t s i z e and s h a p e , b u t n o t , as a r u l e , m otile; of c h a r a c t e r i s t i c appearance; c o lo r, y ello w ish -g reen ; non- i n v o l u t i o n fo r m s common; s t r i c t l y a n a e r o b i c ; a p p a r e n t l y d e p e n d ­ e n t upon hydrogen s u l p h i d e and l i g h t , No d e v e l o p m e n t i n o r g a n i c m e d i a . As we h a v e o b s e r v e d no g r e e n b a c t e r i a i n o u r c o l l e c t i o n s w h i c h can n o t be p la c e d i n t h e genus C hlorobium , a d i s c u s s i o n o f o t h e r r e p o r t e d genera must b e b a se d upon d e s c r i p t i o n s e x is tin g in the l i t e r a t u r e . The g e n u s P e l o d i c t y o n L a u t e r b o r n a n d t h e g e n u s C l a t h r o c h l o r i s G e i t l e r a r e s u p p o s e d l y d i s t i n g u i s h a b l e by t h e f a c t t h a t t h e f o r m e r i n ­ cludes green b a c t e r ia w ith o u t i n t r a c e l l u l a r su lp h u r g lo b u le s; the genus C l a t h r o c h l o r i s i n c l u d e s t h o s e g r e e n b a c t e r i a which p o s s e s s i n t r a ­ c e ll u la r s u lf u r globules. Both g e n e ra a r e d e s c r i b e d as o c c u r r i n g a s f c e ll aggregates. R epresentatives ed i n p u r e c u l t u r e . globules of n e i t h e r genus have b e e n o b t a i n ­ The r e p o r t e d p r e s e n c e o f i n t r a c e l l u l a r s u l f u r i n C l a t h r o c h l o r i s s u l f u r i c a is published d e s c rip tio n s i n c o n s i s t e n t ’w i t h a l l o t h e r o f t h e C h l o r o b a c t e r i a c e a e ; t o d a t e , C_. s u l ­ f u r i c a i s t h e only genus d e s c r i b e d as c o n t a i n i n g i n t r a c e l l u l a r s u lfu r granules. The g e n e r a C h l o r o b a c t e r i u m L a u t e r b o r n , C h l o r o c h r o i n a t i u m L a u t e r b o r n a n d C y l i n d r o g l o e a P e r f i l i e v s ee m t o h a v e no t a x o n o m i c a l l e g i t ­ imacy j t h e s e g e n e r a c o m p r i s e t h o s e g r e e n b a c t e r i a f o u n d a s s y m b i o n t s w ith o th e r organism s. The g e n e r a h a v e b e e n e r e c t e d p r o v i s i o n a l l y o n t h e a s s u m p t i o n t h a t g e n e r i c r a n k may b e u s e d t o d e s i g n a t e a s t a b l e com­ plex. H o w ev e r, t h e r e i s som e e v i d e n c e t h a t t h e c o m p l e x e s m e n t i o n e d above a r e n o t s t a b l e and hence t h e g re e n c o n s t i t u t e n t s s h o u ld be a s ­ s i g n e d t o e i t h e r new o r e x i s t i n g g e n e r a . I t i s o n c e more t e m p t i n g t o s u g g e s t t h a t C hlorobium r e p r e s e n t s , i n s o f a r as p r e s e n t know ledge i s c o n c e rn e d , t h e only l e g i t i m a t e genus of green b a c t e r i a . P e l o d i c t y o n may p r o p e r l y b e a n e n v i r o n m e n t a l v a r i ­ a n t o f Chlorobium l i m i c o l a . P r i n g s h e i m ( 1 9 4 9 ) , c o m m e n ti n g on o r g a n i s m s w h o se r e l a t i o n s h i p w i t h t h e b a c t e r i a and t h e b l u e - g r e e n a l g a e i s d i s p u t e d , s t a t e s : v ariab ility of v a n l l i e l ’s Chlorobium i s r e m a r k a b l e , . . . . . ’’ The This p o ly ­ morphism s u r p a s s e s t h a t o f e v e ry o t h e r m ic r o o r g a n is m and s h o u ld b e c o n f i r m e d by t h o r o u g h s c r u t i n y " . £. I n F i g u r e 16 t h e m o r p h o l o g y o f l i m i c o l a a s d e t e r m i n e d by v a n N i e l , Slcuja a n d t h e a u t h o r i s p r e s e n t e d . -9 3 - F ig u re 16. C hlorobium l i m i c o l a (various au th o rs) f C hlorobium l i m i c o l a a f t e r Skuja Chlorobium l i m i c o l a a f te r van M el (from pure c u l t u r e ) C hlorobium l i m i c o l a c a m e r a l u c i d a x 1900 (from pure c u l t u r e ) B icknell -9 4 - x 2000 I t mu st b e f o r c i b l y e m p h a s i z e d t h a t t h e p l e o m o r p h i s m a s shown by v a n N iel cannot be d em o n strated from o u r p u re c u l t u r e s t u d i e s ; it is a l s o o b v i o u s t h a t C_. l i m i c o l a , a s f i g u r e d by S k u j a shows no ne o f t h e p l e o m o r p h i s m s t r e s s e d by v a n K i e l . -9 5 - SUMMARY 1. The b a c t e r i o l o g i c a l s t u d y r e p o r t e d u p o n h e r e r e p r e s e n t s one phase of an i n t e n s i v e lim n o lo g ic a l i n v e s t i g a t i o n o f a s m a ll, g la c ia l lake. 2. V arious e c o l o g i c a l f a c t o r s were s t u d i e d i n a n e f f o r t t o d e t e r ­ m i n e t h e e f f e c t of a n e c o l o g i c a l c o m p le x u p o n t h e d i s t r i b u t i o n o f a s p e c i f i c member o f t h e g r e e n b a c t e r i a i . e . , C hlorobium lim ico la. 3. The f o l l o w i n g p o s s i b l e c o n t r o l l i n g f a c t o r s t h o u g h t t o i n f l u e n c e th e d i s t r i b u t i o n of green b a c t e r i a w ere i n v e s t i g a t e d : tu re, 4. tem pera­ pH, l i g h t a nd c h e m i c a l c o m p o n e n t s o f t h e w a t e r . The c h a r a c t e r of t h e e u p l a n k t o n was d e t e r m i n e d i n a n a t t e m p t t o a s c e r t a i n i f a p e c u l i a r phyto and z o o p la n k to n accom panied t h e d i s t r i b u t i o n of th e green b a c t e r i a . 5. The a b s o r p t i o n s p e c t r a o f C. l i m i c o l a w e r e d e t e r m i n e d a n d com­ p a re d w i t h t h o s e of a g r e e n a l g a and a b l u e - g r e e n a l g a . 6. E l e c t r o n m i c r o s c o p e s t u d i e s o f t h e m o r p h o l o g y o f C_. l i m i c o l a were p r e s e n te d . 7. P u r e c u l t u r e s t u d i e s o f C. l i m i c o l a w e r e d e s c r i b e d . -9 6 - CONCLUSIONS The f o l l o w i n g t e n t a t i v e c o n c l u s i o n s a r e o f f e r e d w i t h t h e r e a l i ­ z a t i o n t h a t f u t u r e r e s e a r c h may c a u s e some 1. re v e rs a l of opinion: Changes i n t e m p e r a t u r e c a n n o t b e shown t o c o r r e l a t e w i t h t h e ob­ served d i s t r i b u t i o n of green b a c te r ia . 2. The h y p o l i m n e t i c o x y g e n d e f i c i t i s t h o u g h t n o t t o b e t h e p r i m a r y re a so n f o r r e s t r i c t i n g t h e g re e n b a c t e r i a t o a n oxygenless zone. 3. Changes i n c o n c e n t r a t i o n s of d i s s o l v e d and t o t a l s o l i d s p r o b a b l y h a v e no e f f e c t u p o n t h e d i s t r i b u t i o n u n d e r c o n s i d e r a t i o n ; th is s ta te m e n t a l s o a p p ea rs t o be t r u e i n re g a rd t o t h e p o s s i b l e e f ­ fects of s l i g h t changes i n t h e c h e m ic a l c o m p o s i tio n of t h e w a te r on t h e d i s t r i b u t i o n . 4. Light i n t e n s i t y , in t h a t i t r e s t r i c t s th e depth o f th e photosyn­ t h e t i c zone, is a p o s i t i v e f a c t o r in th e d i s t r i b u t i o n of t h e green b a c te r ia . 5. The p r e s e n c e o f h y d r o g e n s u l p h i d e , t h e s u b s e q u e n t d e h y d r o g e n a t i o n of t h i s s u b s t a n c e and t h e r e d u c t i o n o f c a r b o n d i o x i d e w i t h t h e c r e a t i o n of an a n a e r o b i c en v iro n m en t a r e p r o b a b ly t h e most im por­ tan t facto rs i n d e te r m in in g t h e environm ent of t h e green b a c t e r i a . -9 7 - BIBLIOGRAPHY A l l g e i e r , R . , B. H a f f o r d and C. J u d a y 1 9 4 1 . O xidation-R eduction P o t e n t i a l s a nd pH o f La ke W a t e r s a n d o f L a k e S e d i m e n t s . Trans. W i s . A c a d . S c i . , A r t s and L e t . , 3 3 : 1 1 5 - 1 3 3 . A l s t e r b e r g , G. 1930. D ie t h e r m i s c h e n u n d c h e m i s c h e n A u s g l e i c h e i n d e n S e e n z w i s c h e n Boden a n d 7 f a s s e r k o n t a k t S o w i e i h r e b i o l o g i s c h e Bedeutung. I n t . Rev. H y d r o b . , 2 4 . B a r b e r , H. H . , a n d I . M. 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W inogradsky, S. 1888. B e i t r a g e z u r M o rp h o lo g ie und P h y s i o l o g i c d e r B akterien. H eft I : Z u r M o r p h o l o g i e un d P h y s i o l o g i e d e r S c h w e f e l ­ bak terien . L eipzig. Z o b e l l , C. E . 1941. A p p aratu s f o r C o l l e c t i n g w a t e r sam ples from d i f ­ f e r e n t depths f o r b a c t e r i o l o g i c a l a n a l y s i s . J o u r . Mar. 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