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Preliminary studies on the physiology of a psychrophilic Chlamydomonas (Chlorophyceae) Butler, Gary Lee 1970

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PRELIMINARY STUDIES ON THE PHYSIOLOGY OF A PSYCHROPHILIC CHLAMYDOMONAS (CHLOROPHYCEAE) by GARY LEE BUTLER B.A., U n i v e r s i t y o f M i s s o u r i , 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department o f BOTANY We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA June, 1970 In presenting th i s thes i s in pa r t i a l f u l f i lment o f the requirements fo r an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extensive copying of th i s thes i s for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th i s thes i s fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permission. Department of B o t a n v  The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date June 22, 1970 ABSTRACT An i n v e s t i g a t i o n o f an o b l i g a t e p s y c h r o p h i l i c g r een a l g a , Chlamydomonas s p . , was u n d e r t a k e n t o d e f i n e some o f i t s growth c h a r a c t e r i s t i c s . I t has a t e m p e r a t u r e optimum o f 5°C and a pH optimum o f 6.5. A v a r i e t y o f o r g a n i c supplements s t i m -u l a t e growth and b o t h NO^ and NH^+-N s e r v e as n i t r o g e n s o u r c e . Under optimum c o n d i t i o n s (5°C, pH 6.5, 0.1% g l u c o s e ) a maximum o f 1.37 d o u b l i n g s day 1 was o b t a i n e d . P h o t o s y n t h e s i s and r e s -p i r a t i o n have a t e m p e r a t u r e optimum o f 10°C, r e a c h i n g complete i n h i b i t i o n when t h e te m p e r a t u r e i s i n c r e a s e d t o 2 0°C. i i i TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES i v LIST OF FIGURES v ACKNOWLEDGEMENTS v i I . INTRODUCTION 1 I I . MATERIALS AND METHODS 3 I I I . RESULTS 8 A. E f f e c t o f t e m p e r a t u r e and g l u c o s e on growth. 8 B. Growth r a t e . 8 C. E f f e c t o f pH on growth. 11 D. E f f e c t o f n i t r o g e n s o u r c e s on growth. 11 E. E f f e c t o f o r g a n i c supplements on growth. 11 F. E f f e c t o f t e m p e r a t u r e on p h o t o s y n t h e s i s and 15 r e s p i r a t i o n . IV. DISCUSSION 17 V. SUMMARY 26 V I . LITERATURE CITED 27 IV LIST OF TABLES PAGE T a b l e 1. M o d i f i e d B e i j e r i n c k m i n e r a l medium 4 2. O r g a n i c supplements added t o b a s i c m i n e r a l medium a t t h e 0.1% l e v e l b e f o r e a u t o c l a v i n g 3. A l t e r a t i o n i n pH due t o growth 12 4. Rate o f p h o t o s y n t h e s i s and r e s p i r a t i o n as a 16 f u n c t i o n o f te m p e r a t u r e 5. Growth o f v a r i o u s C h l o r o p h y c e a e ( e x p r e s s e d as t h e 19 number o f d o u b l i n g s per day) V LIST OF FIGURES PAGE F i g u r e s 1-2. E f f e c t o f t e m p e r a t u r e on growth 3. Growth r a t e i n optimum c o n d i t i o n s (5°C, pH 6.5, 10 0 . 1 % g l u c o s e ) 4-5. E f f e c t o f pH on growth 10 6. E f f e c t o f n i t r o g e n s o u r c e s on growth 10 7-10. E f f e c t o f o r g a n i c supplements on growth 13-14 ACKNOWLEDGEMENTS The a u t h o r w i s h e s t o e x p r e s s h i s s i n c e r e thanks t o Dr. J a n e t R. S t e i n under whose gui d a n c e t h i s i n v e s t i g a t i o n was u n d e r t a k e n . T h i s s t u d y was s u p p o r t e d by funds from N.R.C g r a n t A-1035. S p e c i a l t h a n k s t o committee member, Dr. E.B. Tregunna, whose a s s i s t a n c e t h r o u g h o u t t h i s s t u d y was g r e a t l y a p p r e c i a t e d . I would l i k e t o thank my w i f e , J o y c e , and my p a r e n t s f o r t h e i r encouragement and u n d e r s t a n d i n g t h r o u g h o u t t h i s s t u d y . 1 INTRODUCTION Some members o f t h e group o f organisms known as a l g a e a r e a b l e t o e x i s t under extreme te m p e r a t u r e c o n d i t i o n s . A l g a e w h i c h l i v e i n snow a r e a b l e t o s u r v i v e and grow when t h e tem-p e r a t u r e i s near 0°C. T h i s i s m a n i f e s t e d i n t h e w i d e s p r e a d r e p o r t s o f c o l o r e d snow found i n s n o w f i e l d s o f a l p i n e , g l a c i a l and p o l a r r e g i o n s . V a r i o u s shades o f r e d , y e l l o w , g r e e n , orange and brown have been r e p o r t e d from t h e s e a r e a s o f semi-permanent and permanent snow (see K o l , 1968). The phenomenon o f c o l o r e d snow has been o f i n t e r e s t t o b o t h layman and s c i e n t i s t f o r many y e a r s . However, t h e i n v e s -t i g a t i o n s o f snow a l g a e have been o f a f l o r i s t i c n a t u r e and l i t t l e i s known o f t h e i r e c o l o g y and p h y s i o l o g y . The f l o r i s t i c s t u d i e s have been based on o b s e r v a t i o n s made from p r e s e r v e d ma-t e r i a l o r on f i e l d c o l l e c t i o n s i n t h e l a b o r a t o r y b e f o r e t h e o r -ganisms d e t e r i o r a t e d . As K o l (1968) n o t e d , because o f t h e l a c k o f f a c i l i t i e s f o r m a i n t a i n i n g a low te m p e r a t u r e w h i l e t r a n s -p o r t i n g c o l l e c t i o n s o r i n t h e l a b o r a t o r y , most workers have not been a b l e t o c u l t u r e t h e s e a l g a e . P h y s i o l o g i c a l and e c o l o g i c a l s t u d i e s on p s y c h r o p h i l i c ( c o l d l o v i n g ) a l g a e have been l i m i t e d m a i n l y t o s t u d i e s on t h e p h y t o p l a n k t o n o f A r c t i c and a n t a r c t i c seas (Fogg, 1969; Horne, Fogg and E a g l e , 1969). A p r e l i m i n a r y e c o l o g i c a l s t u d y and p i g -ment a n a l y s i s o f t h e C h l o r o p h y c e a n a l g a Chlamydomonas n i v a l i s W i l l e c o l l e c t e d i n t h e Pyrenees has been completed (Lascombes and Rosch, 1957; V i a l a , 1966, 1967). T h i s a l g a i s o f t e n l i s t e d as t h e c a u s a t i v e agent o f r e d snow ( K o l , 1968). One st u d y o f 2 th e i n s i t u c a r b o n d i o x i d e f i x a t i o n by a random c o l l e c t i o n o f snow a l g a e o f t h e South Orkney I s l a n d s , A n t a r c t i c a , has been conducted (Fogg, 1967). S i n c e l i t t l e i s known about t h e e c o l o g y and p h y s i o l o g y of t h e snow a l g a e , t h i s s t u d y was u n d e r t a k e n t o d e t e r m i n e how t h i s a l g a has adapted p h y s i o l o g i c a l l y t o growth a t t e m p e r a t u r e s around 0°C. The s t u d y i n c l u d e d (1) e f f e c t o f t e m p e r a t u r e on growth, (2) maximum number o f d o u b l i n g s day x, (3) e f f e c t o f pH on growth, (4) e f f e c t o f n i t r o g e n s o u r c e s on grow t h , (5) whether m i x o t r o p h i c growth on o r g a n i c supplements i s g r e a t e r t h a n a u t o t r o p h i c growth and (6) t h e e f f e c t o f t e m p e r a t u r e on t h e r a t e o f p h o t o s y n t h e s i s and r e s p i r a t i o n . 3 MATERIALS AND METHODS A x e n i c c u l t u r e s o f a Chlamydomonas s p e c i e s were o r i g -i n a l l y i s o l a t e d by Dr. J a n e t R. S t e i n i n A p r i l , 1966. The c o l -l e c t i o n s were made from snow a t t h e 3 2 0 0 - f t l e v e l o f Mt. Seymour P r o v i n c i a l P a r k , near Vancouver, B r i t i s h C olumbia. The a l g a was p r e s e n t i n a d e p r e s s i o n i n a snow bank formed by r a i n w a t e r d r i p -p i n g from a l i m b o f a Chamaecyparis sp. t r e e . T h i s i s t h e same a l g a s t u d i e d by S t e i n and B i s a l p u t r a (1969). The b a s i c i n o r g a n i c medium was a m o d i f i e d B e i j e r i n c k m i n e r a l medium (Table 1 ) . NH^NO^ was r e p l a c e d w i t h NaNO^, (NH^^SO^ and u r e a t o v a r y t h e sou r c e o f n i t r o g e n ( F i g u r e 6 ) . O r g a n i c t e s t e r supplements (Table 2) were added t o t h e s t a n -d a r d B e i j e r i n c k medium. A l l c u l t u r e s o l u t i o n s were a u t o c l a v e d a t 15 l b f o r 5 min. The pH o f t h e c u l t u r e s o l u t i o n s was d e t e r m i n e d u s i n g a Beckman pH M e t e r , Model G. The pH f o r growth was a d j u s t e d t o 6.5^ .1 w i t h cone H^SO^ b e f o r e a u t o c l a v i n g . The pH v a l u e s i n T a b l e 3 were o b t a i n e d w i t h 2N H 2 S 0 4 f o r pH 7.0 o r lower and 2N KOH f o r l e v e l s above pH 7.0. The m a t e r i a l was grown under 150-250 f t - c c o o l w h i t e f l u o r e s c e n t l i g h t on a 16-hr l i g h t / 8 - h r d a r k c y c l e a t 0, 5, 10, 15 o r 20 - 2°C. C u l t u r e s were grown i n 250 o r 500 ml Erlenmeyer f l a s k s , e x c e p t f o r t h e oxygen e v o l u t i o n e x p e r i m e n t s w h i c h were grown i n 1000 ml f l a s k s , and s t o p p e r e d w i t h c h e e s e - c l o t h c o v e r e d c o t t o n p l u g s . The c u l t u r e s were n e i t h e r a e r a t e d nor a g i t a t e d . A l l c u l t u r e s were grown a t " s t a n d a r d c o n d i t i o n s " (5°C, pH 6.5, 16-hr l i g h t / 8 - h r dark) u n l e s s i n d i c a t e d o t h e r w i s e . T a b l e 1. M o d i f i e d B e i j e r i n c k M i n e r a l Medium* St o c k g / i Use ml/1 I . NH 4N0 3 15.00 10 I I . MgS0 4-7H 20 20.00 1 I I I . C a C l 2 - 2 H 2 0 10.00 1 IV. K H 2 P 0 4 36.28 10 V. K 2 H P 0 4 69.66 10 V I . T r a c e s t o c k * 1 *Trace s t o c k and b a s i c m i n e r a l medium from S t e i n , 1966 5 Table 2. Organic supplements added to basic mineral medium at the 0.1% l e v e l before autoclaving Acids Pentoses Acetate (Na) C i t r a t e (Na) Glycolate (Na) D(-) Arabinose D(-) Ribose D(+) Xylose Hexoses Disaccharides D(+) Glucose D(+) Galactose D(-) Fructose Maltose Sucrose Various Soluble starch (Fischer Cert) Peptone (Fischer Bio Cert) Tryptone (Fischer Bio Cert) Malt extract (Fischer Bacterological) Yeast extract (Difco Bacto) 6 The o p t i c a l density of the cultures was measured at 525 nm using a Beckman DB-G Grating Spectrophotometer. C e l l number used i n c a l c u l a t i n g the rates of photosynthesis and r e s p i r a t i o n was determined using a Coulter El e c t r o n i c s , Inc. Coulter Counter, Model B. Oxygen evolution was measured polarographically using a Yellow Springs Instrument Co. Oxygen Meter, Model 54. The oxy-gen electrode was mounted i n the side of a Plexiglas reaction chamber. The output of the oxygen meter was recorded using a Beckman Ten-Inch Linear-Log Potentiometer Recorder. The Plexiglas reaction chamber was a semi-cylinder 7.5 cm i n diameter and 2.5 cm i n depth. The chamber volume was 58 2 ml and the area of the face exposed to the l i g h t was 22 cm . Two 13 mm t e f l o n coated magnetic s t i r r i n g bars were used to agitate the reaction f l u i d . The chamber was immersed i n a Beckman Thermocirculator C i r c u l a t i n g Water Bath and Low Temper-ature Accessory to maintain 0, 5, 10, 15 or 20 - ,2°C. The u n i d i r e c t i o n a l l i g h t i n g was supplied by a 150 watt "Westinghouse spot l i t e " . The l i g h t i n t e n s i t y was maintained at 1000 f t - c as measured by a Gossen T r i l u x Foot Candle Meter. The oxygen electrode was calib r a t e d by taking the d i f -ference between the output of the system when the reaction medium was bubbled with nitrogen and when i t was a i r saturated. The l i n e a r i t y of response of the system was checked with a c t i v e l y growing yeast c e l l s . For the photosynthetic and respiratory experiments a sample of a c e l l culture i n exponential growth was d i l u t e d to 7 an o p t i c a l d e n s i t y o f 1.0 w i t h s t e r i l i z e d d i s t i l l e d w a t e r . The sample was bu b b l e d w i t h N 2 5 min t o remove most o f t h e d i s s o l v e d oxygen. The bu b b l e d c u l t u r e t h e n was a l l o w e d t o s t a n d 5 min f o r removal o f any bub b l e s formed d u r i n g t h e b u b b l i n g . F o r mea-surement o f p h o t o s y n t h e s i s and r e s p i r a t i o n a t t e m p e r a t u r e s o t h e r t h a n 5°C, t h e c e l l s were h e l d a t each t e m p e r a t u r e f o r 24 hr b e f o r e b e g i n n i n g t h e measurements. Subsequent b u b b l i n g w i t h CC^ had no e f f e c t on t h e r a t e o f e i t h e r p h o t o s y n t h e s i s o r r e s p i r a t i o n . 8 RESULTS E f f e c t o f Temperature and G l u c o s e on Growth. F i g u r e s 1 and 2 show t h e e f f e c t o f t e m p e r a t u r e and g l u c o s e on growth. The s h o r t e s t d i v i s i o n t i m e and t h e h i g h e s t -1 d e n s i t y i n c e l l s ml was o b t a i n e d a t 5°C. The c u l t u r e a t 0°C rea c h e d t h e same o p t i c a l d e n s i t y , b u t o n l y a t a s l o w e r growth r a t e . Growth d e c r e a s e s as t h e t e m p e r a t u r e i s i n c r e a s e d t o 10°C and 15°C, w i t h no growth a t 20°C. C e l l s m a i n t a i n e d a t 20°C f o r 24 hr d i e and w i l l n o t grow when t r a n s f e r r e d back t o 5°C. The a d d i t i o n o f 0.1% g l u c o s e t o t h e c u l t u r e medium does not i n c r e a s e t h e upper t e m p e r a t u r e l i m i t a t w h i c h growth i s p o s s i b l e ( F i g u r e 1 ) . However, t h e sugar does s t i m u l a t e growth -1 and i n c r e a s e t h e f i n a l d e n s i t y i n c e l l s ml a t each t e m p e r a t u r e where growth i s p o s s i b l e ( F i g u r e 1 ) . Growth Rate. F i g u r e 3 i s t h e growth r a t e under optimum c o n d i t i o n s (5°C, pH 6.5, 0.1% g l u c o s e ) . The r a t e was c a l c u l a t e d as l o g 2 -1 u n i t s and t h e v a l u e s used i n d i c a t e t h e d o u b l i n g s day f o r t h e p e r i o d o f growth from i n o c u l a t i o n i n t o f r e s h medium u n t i l t h e s t a t i o n a r y phase o f growth was o b t a i n e d . F o l l o w i n g i n o c u l a t i o n i n t o f r e s h medium, a l a g phase o f 6 days i s f o l l o w e d by 4 days of e x p o n e n t i a l growth b e f o r e a s t a t i o n a r y phase i s r e a c h e d . A maximum growth r a t e i s reached between day 9 and day 10 when -1 1.37 d o u b l i n g s day o c c u r . 9 F i g u r e s 1-2. E f f e c t of temperature on growth 9a 10 Figures 3. Growth r a t e i n optimum c o n d i t i o n s (5°C, pH 6.5, 0.1% glucose) 4-5. E f f e c t of pH on growth 6. E f f e c t of n i t r o g e n sources on growth 10a &! 5 8 & f> O 0> CO r-; 10 ••», q 3 3 f s J V O t J O S Q V a O N V Q t J O S O V ^ S O T 11 E f f e c t of pH on Growth. Figures 4 and 5 show the e f f e c t of pH on growth. A pH of 6.5 was found to be the optimum i n a pH range from 3.0 to 8.0. Increase i n c e l l number at pH 3.0, 4.0, 4.5 and 5.0 was accompanied by a metabolic a l t e r a t i o n of the pH by the c e l l s (see Table 3). E f f e c t of Nitrogen Sources on Growth. The e f f e c t of d i f f e r e n t nitrogen sources i s shown in Figure 6. This phase of the study was begun when a s h i f t i n pH of some of the cultures (Table 3) indicated that NO^ was being metabolized i n preference to NH^+. The r e s u l t s indicated that t h i s alga can metabolize either NH^+ or NO^ -N when each i s supplied separately. The f i n a l o p t i c a l density reached and the growth rate i n each case i s nearly equal, but the lag phase i s extended for the NH^+ grown c e l l s . This extension i n the lag phase does not appear to be an adaptation to NH^+ as the r e s u l t s are the f i f t h i n a series of growth on (NH^^SO^. U t i l i z a t i o n of urea did not occur under the conditions present here (Figure 6) and growth after 2 weeks on urea was no more than growth i n nitrogen free medium. The c e l l s ml x had increased only s l i g h t l y a f t e r 3 months. Ef f e c t of Organic Supplements on Growth. Figures 7, 8, 9 and 10 show the e f f e c t of organic sup-12 T a b l e 3. A l t e r a t i o n i n pH due t o growth pH b e f o r e pH a f t e r 10 Absorbance a f t e r i n o c u l a t i o n days growth 10 days growth 3.0 5.2 0.456 3.5 3.8 0.319 4.0 5.1 0.413 4.5 5.2 0.456 5.0 5.5 0.481 5.5 5.6 0.458 6.0 6.0 0.638 6.5 6.5 1.288 7.0 7.0 0.497 7.5 7.5 0.381 8.0 7.8 0.056 13 Figures 7-8. E f f e c t of organic supplements on growth TIIV1E [DAYB1 14 F i g u r e s 9-10. E f f e c t o f o r g a n i c supplements on growth 14a 15 plements on growth. Numerous o r g a n i c supplements have been shown t o have a s t i m u l a t o r y e f f e c t on growth and m i x o t r o p h i c c u l t i v a t i o n may i n c r e a s e t h e growth s e v e r a l t i m e s o v e r c e l l s grown a u t o t r o p h i c a l l y . M i x o t r o p h i c growth by t h i s a l g a on a l l t h e o r g a n i c supplements e x c e p t s o l u b l e s t a r c h , was g r e a t e r t h a n a u t o t r o p h i c growth. P r e l i m i n a r y s t u d i e s i n d i c a t e t h a t s e v e r a l amino a c i d s a l s o have a s t i m u l a t o r y e f f e c t on growth. E f f e c t o f Temperature on P h o t o s y n t h e s i s and R e s p i r a t i o n . T a b l e 4 shows t h e r a t e o f p h o t o s y n t h e s i s and r e s p i r a t i o n as a f u n c t i o n o f t e m p e r a t u r e . B o t h t h e p h o t o s y n t h e t i c r a t e and th e r e s p i r a t o r y r a t e i n c r e a s e w i t h i n c r e a s i n g t e m p e r a t u r e up t o 10°C. As t h e t e m p e r a t u r e i s i n c r e a s e d t o 15°C, t h e p h o t o s y n -t h e t i c and r e s p i r a t o r y r a t e s b e g i n t o d e c l i n e and a t 20°C, p h o t o s y n t h e s i s i s i n o p e r a t i v e . There; i s a l s o a 77% d e c r e a s e i n r e s p i r a t i o n between 15°C and 20°C. T h i s d e c l i n e i n t h e r a t e s a f t e r t h e c e l l s a r e h e l d 24 h r a t 20°C, cannot be r e v e r s e d by r e t u r n i n g t h e c e l l s t o 5°C. 16 T a b l e 4. Rates o f p h o t o s y n t h e s i s and r e s p i r a t i o n as a f u n c t i o n o f t e m p e r a t u r e Temperature °0 P h o t o s y n t h e s i s * u mol 0 2 h r 1 1 0 8 c e l l s - 1 R e s p i r a t i o n u mol 0 2 h r 1 1 0 8 c e l l s - 1 0 5.61 2.58 5 9.65 3.12 10 14.00 3.54 15 10.05 2.79 20 0.00 0.69 * P h o t o s y n t h e s i s i s measured r a t e ; i t has n o t been c o r r e c t e d f o r r e s p i r a t i o n 17 DISCUSSION T h i s a l g a i s an o b l i g a t e p s y c h r o p h i l e w i t h an optimum growth t e m p e r a t u r e o f 5°C ( F i g u r e s 1 and 2) . As t h e temp e r a t u r e i s i n c r e a s e d , growth d e c r e a s e s , u n t i l a t 20°C d e a t h o c c u r s . The upper t e m p e r a t u r e l i m i t c o u l d n o t be i n c r e a s e d by t h e a d d i t i o n o f g l u c o s e ( F i g u r e 1 ) . G l u c o s e i n c r e a s e d t h e growth r a t e and the f i n a l c e l l s ml ^ o b t a i n e d , b u t o n l y a t t h o s e t e m p e r a t u r e s a t w h i c h t h e organism was c a p a b l e o f growth w i t h o u t g l u c o s e . Because i t i s an o b l i g a t e p s y c h r o p h i l e , t h e v e g e t a t i v e phase would be r e s t r i c t e d t o a l p i n e , g l a c i a l and p o l a r r e g i o n s where t h e t e m p e r a t u r e would remain below 10°C f o r an extended p e r i o d . A l g a e can s u r v i v e and grow a t t e m p e r a t u r e s a p p r o a c h i n g 0°C (Fogg, 1969) . Some workers have r e p o r t e d c u l t u r i n g *-at 20°C ( K o l and F l i n t , 1969) and a t c a . 5°C (Fukushima, 1963) o f a l g a e c o l l e c t e d from snow. However, few a l g a e y e t i s o l a t e d have an optimum t e m p e r a t u r e f o r growth o f 5°C o r lower (e.g. Bunt, Owens and Hoch, 1966; S t e i n and B i s a l p u t r a , 1969). Growth i n " s t a n d a r d c o n d i t i o n s " ( F i g u r e 3) f o l l o w s t h e c h a r a c t e r i s t i c p a t t e r n o f growth i n c u l t u r e s o f l i m i t e d volume (Fogg, 1966). A l a g phase (day 1-5) i s f o l l o w e d by an expo-n e n t i a l phase (day 6-10), but t h e r e d u c t i o n phase i s l i m i t e d and t h e s t a t i o n a r y phase ( b e g i n n i n g on day 11) f o l l o w s immedi-a t e l y a f t e r t h e e x p o n e n t i a l phase. The l a g phase i s l a r g e l y e l i m i n a t e d f o r c e l l s t r a n s f e r r e d d u r i n g t h e e x p o n e n t i a l phase. The maximum number o f d o u b l i n g s day x a r e compared t o t h o s e r e p o r t e d f o r s i m i l a r g r e e n a l g a e (Table 5) and as i t i s not known whether t h e l i g h t i n t e n s i t y used i n t h e p r e s e n t s t u d y 18 was s a t u r a t i n g , t h e maximum r e p o r t e d here may be lower t h a n would be o b t a i n e d f o r s a t u r a t i n g i n t e n s i t i e s . The maximum number o f d o u b l i n g s day 1 o b t a i n e d under t h e p r e s e n t c o n d i t i o n s i s lower t h a n t h e d o u b l i n g s day 1 r e p o r t e d f o r o t h e r Chlamydomonas s p e c i e s w i t h a growth optimum a t c a . 25°C (Table 5 ) . However, t h i s maximum v a l u e i s g r e a t e r t h a n t h a t r e p o r t e d f o r Chlamydomonas r e i n h a r d t i grown a t 18°C, C h l o r e l l a  p y r e n o i d o s a a t 10°C and Chlamydomonas r e i n h a r d t i a t 6°C. The Chlamydomonas s p e c i e s i n v e s t i g a t e d h e r e , a l t h o u g h growing a t te m p e r a t u r e s c o n s i d e r e d l i m i t i n g f o r m e t a b o l i c p r o c e s s e s , i s c a p a b l e o f v e g e t a t i v e l y r e p r o d u c i n g a t a r a t e a p p r o a c h i n g t h a t of a l g a e w h i c h n o r m a l l y grow a t 20°C o r h i g h e r . Optimum growth o f t h i s Chlamydomonas a t pH 6.5 i s w i t h -i n t h e range o f t h e pH measurements r e p o r t e d f o r snow. K o l (1968) l i s t s a range i n pH o f snow from 4.0 t o 7.0, w i t h t h e m a j o r i t y w i t h i n t h e range o f 5.0 t o 6.5. A l t h o u g h t h e optimum i s 6.5, t h i s o r g a n i s m i s c a p a b l e o f measurable growth a t pH 3.5 ( F i g u r e s 4 and 5 ) . K o l (1968) r e p o r t s t h a t t h e c o l o r and c h a r a c t e r i s t i c f l o r a found i n a snow bank a r e r e l a t e d t o t h e pH. She l i s t s 2 s p e c i e s o f Chlamydomonas w i t h g r e e n p i g m e n t a t i o n a t pH 6.0 t o 6.5 and 5 s p e c i e s w i t h r e d p i g m e n t a t i o n a t pH 4.5 t o 5.8. A l t h o u g h t h i s a l g a i s c a p a b l e o f measurable growth a t pH 3.5, a l t e r a t i o n i n pigment d i d not o c c u r . I f t h i s a l g a i s c a p a b l e of p r o d u c i n g a r e d pigment, t h e pH o f t h e environment i s not t h e c o n t r o l l i n g f a c t o r . Whether t h i s a l g a i s found t h r o u g h o u t a wide pH range i n t h e f i e l d i s not known. Table 5. Growth o f v a r i o u s Chlorophyceae ( e x p r e s s e d as the number of d o u b l i n g s per day) S p e c i e s Dbl d a y - 1 I l l u m i - * Temp °C R e f e r e n c e n a t i o n Chlamydomonas 4.2 25 B e r n s t e i n (1964) moewusii Chlamydomonas 3.8 + 25 S o r o k i n and r e i n h a r d t i K r a u ss (1958) Chlamydomonas 0.0 + ' 6 McCombie (1960) r e i n h a r d t i Chlamydomonas 1.35 ? 18 S t r o s s (1960) r e i n h a r d t i Chlamydomonas 0.5-1.37 ? 5 T h i s paper C h l o r e l l a 0.43 + 10 Fogg and p y r e n o i d o s a B e l c h e r (1961) * + means s a t u r a t i n g , ? means unknown 20 The i n c r e a s e i n pH a t 3.0, 4.0, 4.5 and 5.0 (Table 3) i n d i c a t e s a m e t a b l o i c a l t e r a t i o n o f the growth medium. Whether t h e a l g a i s s e c r e t i n g e x t r a c e l l u l a r p r o d u c t s o r i n c o r p o r a t i n g an i n o r g a n i c i o n p r e f e r e n t i a l l y i s not known. Other workers have shown t h a t under c e r t a i n c u l t u r e c o n d i t i o n s , a l g a e a r e c a p a b l e o f e x c r e t i n g c o n s i d e r a b l e q u a n t i t i e s o f e x t r a c e l l u l a r p r o d u c t s (Fogg, 1962). Fogg (1967) a l s o r e p o r t s t h a t up t o 7% of t h e car b o n f i x e d by snow a l g a e o f t h e South Orkney I s l a n d s appears as e x t r a c e l l u l a r p r o d u c t s a f t e r 2.5 h r . I f t h i s a l g a p r e f e r e n t i a l l y s e l e c t s o r i s c a p a b l e o f growth on o n l y NO-j -N, t h e r e would be an i n c r e a s e i n pH i n a medium c o n t a i n i n g NH^NO^ as t h e NO-j i s m e t a b o l i z e d . P r o c t o r (1957) o b s e r v e d w i t h a r e l a t e d a l g a (Haematococcus) t h a t a change i n pH i s i n d i c a t i v e o f t h e r a t i o i n which t h e two i o n s a r e b e i n g m e t a b o l i z e d . T h i s a l g a has t h e c a p a b i l i t y o f u s i n g e i t h e r NH^ "1" o r NO-j -N when each i s p r e s e n t e d s e p a r a t e l y ( F i g u r e 6 ) . Whether t h e organism s e l e c t s f o r NO^ when b o t h a r e p r e s e n t has n o t been s t u d i e d . The m e t a b o l i s m o f NO^ i n d i c a t e s t h e pr e s e n c e o f a NO^ r e d u c t a s e system, which i s c h a r a c t e r i s t i c o f many Ch l o r o p h y c e a e ( J a c o b i , 1962). When grown on NH^+-N t h e l a g phase i s extended, but t h e growth r a t e i s g r e a t e r d u r i n g t h e e x p o n e n t i a l phase. The NH^ "1" c u l t u r e w i l l a l s o r e a c h a c o n c e n t r a t i o n i n c e l l s ml 1 e q u a l t o the NO^ - c u l t u r e . Whether t h i s d i f f e r e n c e i n l a g t i m e i s due t o a p e r m e a b i l i t y d i f f e r e n c e between t h e NO^ and NH^ "1" o r due t o a d i f f e r e n t r a t e o f s y n t h e s i s o f an e s s e n t i a l growth f a c t o r i s not known. 21 A l g a e must have a s u p p l y o f e s s e n t i a l n u t r i e n t s t o m a i n t a i n growth. The c h e m i c a l c o m p o s i t i o n o f snow has been s t u d i e d and measurable amounts o f C a + , M g + + , C I and NO^ have been found. A r e d d i s c o l o r a t i o n o f snow due t o f e r r u g i n o u s d e p o s i t s has been m i s t a k e n f o r c o l o r a t i o n due t o a l g a e (Fogg, 1967) . C o l l e c t o r s o f snow a l g a e r e p o r t t h e presence o f p l a n t and a n i m a l d e b r i s i n t h e i r c o l l e c t i o n s . Not o n l y a r e a l g a e found i n snow, but f u n g i , y e a s t s , p o l l e n and p r o t o z o a n s a r e a l s o p r e s e n t (see K o l , 1968). S u f f i c i e n t q u a n t i t i e s o f i n o r g a n i c and o r g a n i c m a t e r i a l a r e i n t r o d u c e d i n t o t h e snow by wind, r a i n , m e l t w a t e r and l a r g e r a n i m a l s t o s u p p o r t t h e growth o f a v a r i e t y o f o r g a n i s m s , t h e a l g a e i n c l u d e d . The d e c o m p o s i t i o n o f p l a n t and a n i m a l d e b r i s and t h e e x t r a c e l l u l a r p r o d u c t s from t h e m i c r o o r g a n i s m s p r e s e n t may p r o v i d e a s o u r c e o f o r g a n i c supplements (e.g. s u g a r s , v i t a m i n s , amino a c i d s ) , w h i c h t h i s Chlamydomonas c o u l d u t i l i z e d u r i n g m i x o t r o p h i c growth. A l l t h e o r g a n i c supplements i n v e s t i g a t e d , e x c e p t s o l u b l e s t a r c h , s t i m u l a t e d growth a t some ti m e d u r i n g t h e growth p e r i o d ( F i g u r e s 7-1,0) . The hexoses, y e a s t e x t r a c t and x y l o s e a r e m e t a b o l i z e d a f t e r a s h o r t l a g phase (4-6 days) and t h i s i s f o l l o w e d by a r a p i d i n c r e a s e i n c e l l number d u r i n g t h e exponen-t i a l phase. A c e t a t e , m a l t e x t r a c t , m a l t o s e , peptone and t r y p -tone have a l a g phase o f a p p r o x i m a t e l y t h e same l e n g t h (4-6 d a y s ) , b u t t h e f o l l o w i n g e x p o n e n t i a l phase i s c h a r a c t e r i z e d by a more g r a d u a l i n c r e a s e i n c e l l number. C i t r a t e , a r a b i n o s e and r i b o s e have an extended a d a p t i v e l a g phase, but d u r i n g 22 e x p o n e n t i a l growth t h e r a t e i s a p p r o x i m a t e l y e q u a l t o t h a t w i t h t h e hexoses. G l y c o l a t e and s u c r o s e i n h i b i t growth d u r i n g t h e p e r i o d o f a d a p t a t i o n . S u c r o s e e x h i b i t s a d o u b l e growth c u r v e ( d i a u x i e ) c h a r a c t e r i s t i c when two s u b s t r a t e s a r e p r e s e n t and one o f t h e c o n s t i t u e n t s i s b e i n g u t i l i z e d e x c l u s i v e o f t h e o t h e r (Monod, 1949). T h i s might be due t o t h e p a r t i a l breakdown o f t h e s u c r o s e i n t o i t s c o n s t i t u e n t s ( g l u c o s e and f r u c t o s e ) d u r i n g a u t o c l a v i n g o f t h e medium. The f i r s t growth phase may be due t o t h e u t i l i z a t i o n o f t h e two hexoses p r e s e n t b e f o r e t h e s u c r o s e i s m e t a b o l i z e d . I t i s known t h a t some a l g a e l i b e r a t e g l y c o l a t e i n t o t h e c u l t u r e medium ( T o l b e r t and Z i l l , 1956). I t has a l s o been shown t h a t t h e uptake of g l y c o l a t e by some a l g a e i s p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n up t o 50 mg 1 ^ (Nalewjko, Chowdhuri and Fogg, 1963). However, t h e i n h i b i t i o n and extended l a g phase p r e s e n t here may have r e s u l t e d from the c o m p a r a t i v e l y h i g h c o n c e n t r a t i o n o f g l y c o l a t e (1000 mg 1 x ) , which may even have been a s u b l e t h a l c o n c e n t r a t i o n under p r e s e n t c o n d i t i o n s . The r a t e o f p h o t o s y n t h e s i s o f t h i s Chlamydomonas (Table 4) i s s i m i l a r t o t h a t found by Fogg (1967) f o r snow a l g a e of t h e South Orkney I s l a n d s . Oxygen p r o d u c t i o n b e g i n s t o d e c l i n e as t h e t e m p e r a t u r e i s i n c r e a s e d above 10°C and i s c o m p l e t e l y i n h i b i t e d a t 20°C. The A n t a r c t i c marine d i a t o m , F r a g i l a r i a  s u b l i n e a r i s , has a s i m i l a r t e m p e r a t u r e optimum f o r growth (6°C) and has a d e c l i n i n g r a t e o f oxygen p r o d u c t i o n above 10°C (Bunt, Owens, and Hoch, 1966). They a l s o found t h a t t h e p h o t o s y n t h e t i c 23 pigment system o f t h e d i a t o m was d e s t r o y e d by p r o l o n g e d (24 hr) exposure t o t e m p e r a t u r e s above 2 0°C. However, i t i s not known whether t h e h i g h e r t e m p e r a t u r e s cause pigment d e c o m p o s i t i o n i n t h e Chlamydomonas o r n o t . Fogg (1967) e s t i m a t e d from t h e r a t e o f c a r b o n f i x a t i o n t h a t t h e snow a l g a e growing a t 0°C would have a d o u b l i n g time o f about 23 days. C a l c u l a t i n g from t h e r a t e o f growth o f t h e c o n t r o l i n F i g u r e 7, t h i s Chlamydomonas growing a t 5°C and w i t h -out any o r g a n i c supplements, would have a d o u b l i n g t i m e o f a p p r o x i m a t e l y 20 days. The 1.37 d o u b l i n g s day 1 ( F i g u r e 3) o b t a i n e d when g l u c o s e was p r e s e n t demonstrates t h e a b i l i t y o f t h i s a l g a t o u t i l i z e e f f i c i e n t l y o r g a n i c supplements when t h e y a r e p r e s e n t . Other m e t a b o l i c p r o c e s s e s appear t o be l i m i t i n g above 10°C. A f t e r s e v e r a l days o f i n c u b a t i o n a t 10°C, t h e a l g a becomes n o n - m o t i l e and forms a m u c i l a g i n o u s s h e a t h . Whether p h o t o s y n -t h e s i s and r e s p i r a t i o n c o n t i n u e a t t h e same r a t e d u r i n g t h i s p e r i o d i s not known. However, i t does not seem l i k e l y t h a t t h e p h y s i o l o g i c a l p r o c e s s e s would r e m a i n t h e same i n t h e ensheathed form. Here i s a Chlamydomonas i s o l a t e d from snow wh i c h i s an o b l i g a t e p s y c h r o p h i l e and must grow a t t e m p e r a t u r e s lower t h a n 10°C. There a r e many s p e c i e s o f a l g a e r e p o r t e d from snow (see K o l , 1968), b u t i t i s n o t known how many a r e o b l i g a t e p s y c h r o p h i l e s s i m i l a r t o t h e one i n v e s t i g a t e d i n t h i s s t u d y . Other snow a l g a e must be i s o l a t e d and s t u d i e d b e f o r e any g e n e r a l c o n c l u s i o n s can be r e a c h e d about t h e p h y s i o l o g y o f 24 t h e s e o r g a n i s m s . A l t h o u g h t h i s a l g a i s c a p a b l e o f u s i n g a wide v a r i e t y o f o r g a n i c supplements, i t i s not known t o what e x t e n t t h e s e compounds a r e p r e s e n t i n t h e f i e l d . Snow a l g a e t e n d t o be c o n c e n t r a t e d i n snow-surface d r a i n a g e p a t t e r n s , d e p r e s s i o n s o r on t h e down-slope margins o f snow banks due t o t h e b u l k f l o w of m e l t w a t e r . P a r t i c l e s o f o r g a n i c d e b r i s w i l l a l s o accumulate i n t h e s e a r e a s and may p r o v i d e some o f t h e o r g a n i c supplements f o r m i x o t r o p h i c growth ( K o l , 1968). L i t t l e i s known about t h e e c o l o g y o f snow banks where a l g a e a r e found. Most c o l l e c t o r s o f snow a l g a e r e c o r d t h e temp-e r a t u r e and pH o f t h e snow and a g e n e r a l d e s c r i p t i o n o f t h e a r e a , but t h e r e a r e no p u b l i s h e d r e p o r t s o f snow samples a n a l y z e d f o r i n o r g a n i c i o n s , o r g a n i c c o n t e n t o r snow d e n s i t y . There appears t o be a c o r r e l a t i o n between t h e exposure t o t h e i n c i d e n t l i g h t and t h e organisms p r e s e n t i n t h e snow bank ( K o l , 1968; S t e i n and Amundsen, 1967) , but a d a i l y q u a n t i t a t i v e r e c o r d has not been o b t a i n e d f o r any c o l l e c t i n g s i t e . T h i s s t u d y has o u t l i n e d some o f t h e p h y s i o l o g i c a l c a p a b i l i t i e s o f t h i s snow a l g a . But o t h e r q u e s t i o n s remain unanswered. Does t h i s a l g a p o s s e s s t h e a b i l i t y t o w i t h s t a n d f r e e z i n g d u r i n g t h e n i g h t s when t h e te m p e r a t u r e drops below 0°C? I f growth and m e t a b o l i s m a r e handicapped by t h e slowness of c h e m i c a l r e a c t i o n s a t t e m p e r a t u r e s c l o s e t o 0°C, do t h e s e a l g a e p o s s e s s more e f f i c i e n t enzyme systems? Some o f t h e snow a l g a e appear t o go t h r o u g h a s e r i e s o f changes in__pigment k c o m p o s i t i o n d u r i n g t h e i r l i f e c y c l e . What change i n t h e 25 quantity and q u a l i t y of the l i g h t , temperature and nutrients i s responsible for t h i s a l t e r a t i o n i n pigments? I t i s hoped that future studies w i l l provide answers to some of these questions. SUMMARY An o b l i g a t e p s y c h r o p h i l e , Chlamydomonas sp., has a te m p e r a t u r e optimum f o r growth o f 5°C. When grown a u t o -t r o p h i c a l l y t h e p o p u l a t i o n w i l l d o u b l e once e v e r y 2 0 days. When grown m i x o t r o p h i c a l l y on g l u c o s e , 1.37 d o u b l i n g s day 1 were o b t a i n e d . The a l g a w i l l grow t h r o u g h o u t a pH range from 3.0 t o 7.5, but t h e optimum f o r growth i s pH 6.5. B o t h NO^ and NH^ + w i l l s e r v e as t h e n i t r o g e n s o u r c e , but no growth i s o b t a i n e d when u r e a i s t h e s o l e n i t r o g e n s o u r c e . V a r i o u s o r g a n i c supplements w i l l s t i m u l a t e g r o w t h , w i t h t h e hexoses c a u s i n g t h e l a r g e s t i n c r e a s e i n t h e growth r a t e . B oth p h o t o s y n t h e s i s and r e s p i r a t i o n have a t e m p e r a t u r e optimum o f 10°C, d e c r e a s i n g as t h e t e m p e r a t u r e i s i n c r e a s e d t o 20°C. A t 10°C t h e maximum r a t e o f p h o t o s y n t h e s i s i s 14.00 u mol hr 1 1 0 8 c e l l s ^. 27 LITERATURE CITED B e r n s t e i n , E. 1964. P h y s i o l o g y o f an o b l i g a t e p h o t o a u t o t r o p h (Chlamydomonas m o e w u s i i ) . I . C h a r a c t e r i s t i c s o f s y n c h r o n o u s l y and randomly r e p r o d u c i n g c e l l s and a h y p o t h e s i s t o e x p l a i n t h e i r p o p u l a t i o n c u r v e s . J . P r o t o z o o l . 11:56-74. Bunt, J . S., Owens, 0. van H. and Hoch, G. 1966. E x p l o r a t o r y s t u d i e s on t h e p h y s i o l o g y and e c o l o g y o f a p s y c h r o -p h i l i c m arine d i a t o m . J . P h y c o l . 2:96-100. Fogg, G. E. 1962. E x t r a c e l l u l a r p r o d u c t s . I n , L e w i n , R. A., ed. P h y s i o l o g y and B i o c h e m i s t r y o f a l g a e . Academic P r e s s . New Y ork. p 475-489. Fogg, G. E. 1966. A l g a l C u l t u r e s and P h y t o p l a n k t o n E c o l o g y . U n i v e r s i t y o f W i s c o n s i n P r e s s . Madison. 126p. Fogg, G. E. 1967. O b s e r v a t i o n s on t h e snow a l g a e o f t h e South Orkney I s l a n d s . P h i l . T r a n s . R. Soc. (London), s e r . B. 252:279-287. Fogg, G. E. 1969. S u r v i v a l o f a l g a e under a d v e r s e c o n d i t i o n s . Symp. Soc. Exp. B i o l . 23:123-142. Fogg, G. E. and B e l c h e r , J . H. 1961. P h y s i o l o g i c a l s t u d i e s on a p l a n k t o n i c ^u-alga. V e r h . I n t . V e r . L i m n o l . 14:893-896. Fukushima, H. 1963. S t u d i e s on c r y o p h y t e s i n Japan. J . Yokohama Munic. U n i v . , s e r . C. 43:1-146. Home, A . J . , Fogg, G.E. and E a g l e , D.J. 1969. S t u d i e s i n s i t u o f t h e p r i m a r y p r o d u c t i o n o f an a r e a o f i n s h o r e A n t a r c t i c sea. J . Mar. B i o l . A s s . U.K. 49:393-405. J a c o b i , G. 1962. Enzyme systems. I n , Lewin, R.A., ed. P h y s i o l o g y and B i o c h e m i s t r y o f A l g a e . Academic P r e s s . New Y o r k , p 125-140. K o l , E. 1968. K r y o b i o l o g i e B i o l o g i e . I n , Thienemann, A., ed. L i m n o l o g i e des Schnees und E i s e s " I . K r y o v e g e t a t i o n . E. S c h w e i z e r b a r t s c h e V e r l a g s b u c h h a n d l u n g . S t u t t g a r t . 216p. K o l , E. and F l i n t , E.A. 1969. A l g a e i n g r e e n i c e from t h e B a l l e n y I s l a n d s , A n t a r c t i c a . New Zealand J . B o t . 6:249-261. Lascombes, G. and Rosch, J . 1957. C o n d i t i o n s de v i e du c r y o p l a n c t o n e t c o n d i t i o n s p h y s i q u e s s u r Mars. E x t r . 28 Mem. Soc. Roy. S c i . ( L i e g e ) , s e r . 4. 18:202-208. McCombie, A.M. 1960. A c t i o n s and i n t e r a c t i o n s o f t e m p e r a t u r e , l i g h t i n t e n s i t y and n u t r i e n t c o n c e n t r a t i o n on t h e growth o f t h e green a l g a Chlamydomonas r e i n h a r d i Dangeard. J . F i s . Res. Bd. Can. 17:871-894. Monod, J . 1949. The growth o f b a c t e r i a l c u l t u r e s . Ann. Rev. M i c r o b i o l . 3:371-394. Na l e w a j k o , C , Chowdhuri, N. and Fogg, G. E. 1963. E x c r e t i o n o f g l y c o l l i c a c i d and t h e growth o f a p l a n k t o n i c C h l o r e l l a . I n , Japanese S o c i e t y o f P l a n t P h y s i o l o g i s t s , S t u d i e s on M i c r o a l g a e and P h o t o s y n t h e t i c B a c t e r i a , p 171-183. P r o c t o r , V.W. 1957. P r e f e r e n t i a l a s s i m i l a t i o n o f n i t r a t e i o n by Haematococcus p l u v i a l i s . A m e r . J . B o t . 44:141-143. S o r o k i n , C. and K r a u s s , R.W. 1958. The e f f e c t s o f l i g h t i n t e n s i t y on t h e growth r a t e s o f g r e e n a l g a e . P l a n t P h y s i o l . 33:109-113. S t e i n , J.R. 1966. Growth and m a t i n g o f Gonium p e c t o r a l e ( V o l v o c a l e s ) i n d e f i n e d media. J . P h y c o l . 2:23-28. S t e i n , J.R. and Amundsen, C C . 1967. S t u d i e s on snow a l g a e and f u n g i from t h e F r o n t Range o f C o l o r a d o . Can. J . B o t . 45:2033-2045. S t e i n , J.R. and B i s a l p u t r a , T. 1969. C r y s t a l l i n e b o d i e s i n an a l g a l c h l o r o p l a s t . Can. J . B o t . 47:233-236. S t r o s s , R.G. 1960. Growth r e s p o n s e o f Chlamydomonas and Haematococcus t o t h e v o l a t i l e f a t t y a c i d s . Can. J . M i c r o b i o l . 6:611-617. T o l b e r t , N.E. and Z i l l , L.P. 1956. E x c r e t i o n o f g l y c o l i c a c i d by a l g a e d u r i n g p h o t o s y n t h e s i s . J . B i o l . Chem. 222:895-906. V i a l a , G. 1966. L ' a s t a x a n t h i n e chez l e Chlamydomonas n i v a l i s W i l l e . C R . Acad. S c i . , s e r D. 263:1383-1386. V i a l a , G. 1967. Recherches s u r l e Chiamydomonas n i v a l i s W i l l e dans l e s P y r e n e e s . Soc. B o t . F r a n c e , B u l l . 114:75-79. 

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