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Physiological, ultrastructural and cytochemical studies on the utilization of various intermediates of… Huynh, Hanh Kim 1989

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PHYSIOLOGICAL, ULTRASTRUCTURAL AND CYTOCHEMICAL STUDIES ON THE UTILIZATION OF VARIOUS INTERMEDIATES OF THE PURINE CATABOLISM PATHWAY AS SOLE SOURCES OF NITROGEN BY MARINE PHYTOPLANKTERS By HANH KIM HUYNH B.Sc, The University of B r i t i s h Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1989 O Hanh Kim Huynh, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of SO I AH V The University of British Columbia Vancouver, Canada •ate TANUARi n ^ / m DE-6 (2/88) ABSTRACT Eleven s p e c i e s of marine m i c r o a l g a e belonging to s i x d i f f e r e n t taxonomic d i v i s i o n s were t e s t e d f o r t h e i r a b i l i t y to grow on a l l a n t o i n , a l l a n t o i c a c i d , hypoxanthine and urea as s o l e sources of n i t r o g e n . A l l s p e c i e s were ab l e to u t i l i z e the n i t r o g e n atoms of urea but only s i x of these were able to grow on a l l a n t o i c a c i d , while f i v e showed moderate to good growth i n hypoxanthine. None was a b l e to u t i l i z e a l l a n t o i n . The study of n i c k e l requirements f o r the growth of these microalgae on the d i f f e r e n t sources of n i t r o g e n , together with the r e s u l t s of i n h i b i t o r t e s t s suggest that those s p e c i e s capable of u t i l i z i n g both hypoxanthine and a l l a n t o i c a c i d c a t a b o l i z e p u r i n e s through the standard pathway of p u r i n e o x i d a t i o n d e s c r i b e d i n other microorganisms and higher p l a n t s . T h i s pathway leads to the p r o d u c t i o n of urea and i t s subsequent c o n v e r s i o n to u t i l i z a b l e ammonium. In the case of one s p e c i e s , Pavlova  l u t h e r i , growth on urea i s i n h i b i t e d by urease i n h i b i t o r s , while growth in a l l a n t o i c a c i d or hypoxanthine occurs i n the presence, of urease i n h i b i t o r s . The r e s u l t s suggest that i n t h i s case the c a t a b o l i c o x i d a t i o n of p u r i n e s and t h e i r d e r i v a t i v e s does not i n v o l v e urea p r o d u c t i o n and occurs through a pathway d i f f e r e n t from that observed i n the other s p e c i e s . C e l l s of Amphidinium c a r t e r a e grown on hypoxanthine undergo major u l t r a s t r u c t u r a l changes. These a f f e c t the p e r i c h r o m a t i n i c g r a n u l e s , the dictyosomes and dictyosome-d e r i v e d v e s i c l e s , the d i s t r i b u t i o n of the endoplasmic r e t i c u l u m , the number of mitochondria and mi c r o b o d i e s , and the s i z e and d i s t r i b u t i o n of the va c u o l a r compartment. Some of these u l t r a s t r u c t u r a l changes, such as i n c r e a s e i n endoplasmic r e t i c u l u m and the number of micr o b o d i e s , along w i t h the cy t o c h e m i c a l demonstration of both u r i c a s e and c a t a l a s e a c t i v i t i e s w i t h i n m i c r o b o d i e s , support the occurrence i n these microalgae of the standard pathway f o r the c a t a b o l i c d egradation of p u r i n e s . C e l l s of both D u n a l i e l l a t e r t i o l e c t a and Pavlova l u t h e r i grown on hypoxanthine a l s o undergo major u l t r a s t r u c t u r a l changes. These a f f e c t mainly the endoplasmic r e t i c u l u m , mitochondria and v a c u o l e s . The e f f e c t on mitochondria i s p a r t i c u l a r l y i n t e r e s t i n g s i n c e c y t o c h e m i c a l t e s t s r e v e a l the presence of both u r i c a s e and c a t a l a s e a c t i v i t i e s i n these o r g a n e l l e s . When one takes i n t o c o n s i d e r a t i o n that no microbodies are observed i n these microalgae and that u r i c a s e c o n t r o l s the key s t e p of the formation of a l l a n t o i n and H 2 O 2 through the o x i d a t i o n of u r a t e , i t becomes apparent that i n these m i c r o a l g a e mitochondria p a r t i c i p a t e i n the o x i d a t i v e d e g r a d a t i o n of p u r i n e s and t h e i r d e r i v a t i v e s and p l a y a major r o l e i n the or g a n i c N-budget of these microorganisms. iv TABLE OF CONTENTS: ABSTRACT i i ACKNOWLEDGEMENT V INTRODUCTION 1 MATERIALS & METHODS 5 RESULTS 10 DISCUSSION • 26 REFERENCES 49 APPENDIX 62 V ACKNOWLEDGEMENT I wish to express my s i n c e r e thanks and a p p r e c i a t i o n t o my resear c h s u p e r v i s o r , Dr. LUIS OLIVEIRA, who has supported my work i n the l a s t few ye a r s and made t h i s t h e s i s p o s s i b l e , to Dr. T. BISALPUTRA f o r l e t t i n g me use the f a c i l i t i e s i n h i s res e a r c h l a b o r a t o r y , to Mr. M. WEIS f o r . h i s t e c h n i c a l a s s i s t a n c e , to a l l my f r i e n d s i n the lab who always encouraged me during my s t a y i n the l a b o r a t o r y , t o Mr. W. HUNTER and a l l of the people i n the o f f i c e who have helped me i n the l a s t few y e a r s . Last but not l e a s t , to my Mom, Dad, and a l l of my s i s t e r s and brother who always encouraged me to proceed f u r t h e r with my e d u c a t i o n . 1 INTRODUCTION The availability of nitrogen for phytoplankton growth in coastal and open - ocean waters is often assessed by measuring the concentrations of inorganic nitrogen compounds. As pointed out by Antia et a l . (1975) such an approach overlooks the large pool of organic nitrogen present in . seawater. Apart from ammonium and nitrate, the two most common sources of inorganic nitrogen (Syrett, 1962; Morris, 1974), many algae can u t i l i z e various organic compounds as sole nitrogen sources for phototrophic growth. Although organic-N compounds are not always as effective as nitrate, many of these show the potential for supporting significant growth (see Antia et a l . , 1989, for a review). Urea is an important organic nitrogen source for phytoplankton growth (Oliveira and Antia, 1984) that can also be endogenously produced through metabolic pathways such as the aerobic degradation of purines (Naylor, 1970). Urea utilization by algae involves the production of two alternative urea - degrading enzyme - systems: Urease or ATP:urea amidolyase. The evidence shows that a given algal species may produce either urease or ATPrurea amidolyase but not both enzymes and that ATP:urea amidolyase is restricted in occurrence to some Chlorophycean algae (Bekheet and Syrett, 1977; Al-Houty and Syrett, 1984). Recent studies on the phototrophic growth of several marine phytoplanktonic algae with urea serving as sole source of nitrogen showed 2 that growth could only occur, in most cases, on addition of minute amounts of N i2 + to the growth media (Oliveira and Antia, 1984, 1986a). Of particular interest to these findings is the discovery that nickel is a constituent of jackbean (Canavalia ensiformis L.) urease (Dixon et a l . , 1975) and is required for urease activity in soybean tissue cultures (Polacco, 1977). A nickel requirement for urease activity was also reported for some microalgae (Syrett, 1981; Rees and Bekheet, 1982; Oliveira and Antia, 1986b). Antia et a l . (1989), in their review on the role of dissolved organic nitrogen in phytoplankton nutrition show widespread ab i l i t y on the part of microalgae to u t i l i z e purines and/or their derivatives as sole sources of nitrogen. The utilization of purines and purine derivatives such as hypoxanthine, xanthine, uric acid and allantoin as nitrogen sources for the phototrophic growth of marine microalgae has now been established for organisms from different taxonomic divisions (Antia et a l , , 1980a; Prasad, 1983; Shah and Syrett, 1984a). Some of these compounds are considered to be important organic N sources in certain marine environments, particularly inshore areas (van Baalen and Marler, 1963). The evidence suggests the existence in microalgae of a pathway of purine oxidation similar to that described for other microorganisms (Vogels and van der D r i f t , 1976) and higher plants (Reynolds et a l . , 1982). This pathway leads to the formation of urea and i t s subsequent conversion to utilizable ammonium (Antia et a l . , 1989). 3 However, as pointed out by Prasad (1983) and Antia et a l . (1989), the information available for the algae is at best fragmentary and restricted in most cases to tests dealing exclusively with growth studies (see also Antia e_t a l . , 1980b). The validity of the growth tests is further complicated by the fact that inappropriate concentrations of Ni2"1" in the culture medium may lead to misinterpretations regarding the ab i l i t y of the algae to u t i l i z e the intermediates of the pathway as sole sources of nitrogen. Consequently, the pathway of catabolism of purines and their derivatives in algae is far from understood. Equally unknown is the importance of the intermediates of this pathway on the organic N-budget of both coastal and. estuarine areas which are characterized by the seasonal occurrence of large pools of unidentified organic - N (Butler et a l . , 1979; Valiella and Teal,,1979). In this thesis, I report on the N i2 + requirement of 11 species of marine phytoplankters belonging to 6 different taxonomic divisions for growth on hypoxanthine, allantoin, allantoic acid and urea as sole sources of N. Since recent evidence suggests that in higher plants, purine N utilization may also proceed without the production of urea (Winkler et a l . , 1987, 1988), I have also conducted growth inhibition and enzymatic studies to help elucidate the mechanism(s) of oxidation of purines and their derivatives responsible for the growth of these 11 species of microalgae. 4 I have also come to realize that while some of the ultrastructural implications of the catabolic oxidation of purines have been well studied in higher plants, particularly in higher plants relying on nodular nitrogen fixation (Newcomb e_t a_l. , 1985; Kaneko and Newcomb, 1987; Webb and Newcomb, 1987), no such studies exist for microorganisms particularly the microalgae. Therefore, I also report on the ultrastructure and cytochemistry of the marine dinoflagellate Amphidin ium carterae, the Chlorophycean alga Dunaliella tertiolecta and the Prymnesiophyte Pavlova lutheri grown on nitrate, urea, allantoate and hypoxanthine in order to gain further insight into the mechanism(s) of purine- and purine derivatives - N utilization by these microalgae. 5 MATERIALS AND METHODS  A l g a l s p e c i e s Stock c u l t u r e s of the algae l i s t e d i n Table 1 were r o u t i n e l y maintained on n i t r a t e (as n i t r o g e n s o u r c e ) , under standard axenic c o n d i t i o n s , a c c o r d i n g to A n t i a and Cheng (1970). Growth t e s t s A l l t e s t s were c a r r i e d out i n c u l t u r e tubes at 18°C under continuous i l l u m i n a t i o n ( i r r a d i a n c e 95 - 100 uE.m~2.s- 1) from c o o l - white f l u o r e s c e n t lamps. The standard t e s t medium was that of 01 i v e i r a and A n t i a (1984) with the s a l i n i t y a d j u s t e d to 26%0 . In the case of O l i s t h o d i s c u s  l u t e u s and D u n a l i e l l a t e r t i o l e c t a , f i n a l s a l i n i t y of the t e s t medium was r e t a i n e d at 14%© . Unless otherwise s t a t e d , N i * , n i t r a t e , u r e a , a l l a n t o i c a c i d , a l l a n t o i n , hypoxanthine, c i t r a t e , a l l o p u r i n o l , 2 , 6 , 8 - t r i c h l o r o p u r i n e and hydroxyurea a d d i t i o n s were made to 4 mL of t e s t media before i n o c u l a t i o n with 0.2 mL a l i q u o t s of the a l g a l stock c u l t u r e was c a r r i e d o u t . The c o n c e n t r a t i o n s u t i l i z e d i n the experiments are given i n Tables 2 through 6. A l l t e s t media were f i l t e r - s t e r i l i z e d (0.2 jam pore s i z e p r e s t e r i l i z e d Nalgene f i l t e r s ) and a s e p t i c techniques were used throughout the t e s t s to ensure axenic growth c o n d i t i o n s . Growth was monitored d i r e c t l y i n c u l t u r e tubes by p e r i o d i c a l l y reading t h e i r o p t i c a l d e n s i t y at 600 nm (OB^QQ) 6 on a spectrophotometer a f t e r b r i e f vortex m i x i n g . Every growth t e s t i n c l u d e d a c o n t r o l without any added n i t r o g e n source. The growth measured on experimental n i t r o g e n sources was c o r r e c t e d f o r the r e s i d u a l growth, i f any, measured under those c i r c u m s t a n c e s . P l o t s of the c o r r e c t e d growth ag a i n s t the i n c u b a t i o n time p e r i o d were used to c a l c u l a t e the f o l l o w i n g three parameters: (1) ad a p t a t i o n p e r i o d = number of days from i n o c u l a t i o n to the f i r s t s i g n i f i c a n t i n c r e a s e i n OD600' (2) e x p o n e n t i a l growth r a t e = maximum incr e a s e i n O D600 Pe r ^a v d u r i n g e x p o n e n t i a l phase of growth, expressed as percentage of corresponding r a t e from n i t r a t e c o n t r o l , and (3) maximum y i e l d = maximum ODgQQ on the growth curve f o r a t e s t , expressed as percentage of s i m i l a r y i e l d from growth on n i t r a t e . The n i t r o g e n s t o i c h i o m e t r y f o r e q u i v a l e n t growth was d e f i n e d by the equation 0.5 mM hypoxanthine =0.5 mM a l l a n t o i n = 0.5 mM a l l a n t o i c a c i d = 1 mM urea = 2 mM n i t r a t e ( O l i v e i r a and A n t i a , 1984). C e l l f r e e e x t r a c t s P r e p a r a t i o n of c e l l - f r e e e x t r a c t s of the microalgae f o r dete r m i n a t i o n of enzyme a c t i v i t i e s was conducted a c c o r d i n g to the procedure d e s c r i b e d by Shah and S y r e t t (1984b). Enzyme assays were conducted f o r xanthine dehydrogenase ( S t i r p e and D e l l a C o r t e , 1969), u r i c a s e (Theimer and Beevers, 1971), a l l a n t o i n a s e and a l l a n t o i c a s e (Reinert and M a r z l u f , 1975). Whenever an enzyme a c t i v i t y was measured i t 7 was e s t a b l i s h e d that the r a t e was l i n e a r with time over the p e r i o d of assay and d i r e c t l y p r o p o r t i o n a l to the amount of c e l l - f r e e e x t r a c t added to the assay m i x t u r e . Further d e t a i l s are given i n the text (Table 7 ) . E l e c t r o n Microscopy F i x a t i o n s f o r e l e c t r o n , m i c r o s c o p i c o b s e r v a t i o n s were always c a r r i e d out at the same time of the day and stage of growth to minimize the impact of the c u l t u r e c o n d i t i o n s on the c e l l u l a r u l t r a s t r u c t u r e . Samples were co n c e n t r a t e d by g e n t l e c e n t r i f u g a t i o n and f i x e d f o r 3 hours at 4 °C with 2% to 2.5% (v/v) g l u t a r a l d e h y d e i n a s a l i n e phosphate (0.17 M) b u f f e r (PBS-pH 7.4) f o r the c e l l s of Amphidinium c a r t e r a e or i n a s a l i n e sodium c a c o d y l a t e b u f f e r s o l u t i o n (0.1M, pH 7.4) f o r D u n a l i e l l a t e r t i o l e c t a and Pavlova l u t h e r i . The c e l l s were then p o s t - f i x e d f o r 1.5 to 2 hours at 4°C with 1% (v/v) Os04 i n the same b u f f e r and r i n s e d thoroughly i n the a p p r o p r i a t e b u f f e r a f t e r each f i x a t i o n . F i n a l c o n c e n t r a t i o n of the samples, p r i o r to dehydration i n a graded methanol s e r i e s and f i n a l embedding i n Polybed (Epon) 812 ( L u f t , 1961), were conducted using the bovine serum albumin (BSA) technique ( O l i v e i r a et a l . , 1989). U l t r a t h i n s e c t i o n s were s t a i n e d with a s a t u r a t e d s o l u t i o n of u r a n y l a c e t a t e i n 50% methanol and l e a d c i t r a t e (Reynolds, 1963). 8 Cytochemistry Uricase cytochemistry. Cells were concentrated by centrifugation and fixed briefly for 5 minutes in 0.25% (v/v) formaldehyde. They were preincubated in the control medium for 5 minutes. This medium contained 3 mM CeCl3, 50 mM 3-amino-1,2,4- triazole and 0.001% Triton X-100 in 0.1M Pipes buffer (pH 9.4). The cells were then incubated for an extra 30 minutes at 37 °C in the experimental medium which was identical to the control one, except for the addition of 0.1 mM uric acid (Angermuller and Fahimi, 1986). After washing the cells thoroughly with Pipes buffer, they were fixed in 2% formaldehyde for 2 hours at 4°C with the same buffer, followed by post fixation with 1% osmium tetroxide for 1.5 hours at 4°C. The samples were rinsed in buffer after each fixation, dehydrated with a graded methanol series and embedded in Polybed 812. Control experiments were conducted in the presence of 2,6,8 - trichloropurine or oxypurine (2 mM), two competitive inhibitors of uricase. Catalase cytochemistry. For the cytochemical localization of catalase, the cells were fixed with 2.5% giutaraldehyde in the appropriate buffer, i.e. in a saline phosphate (0.17 M) buffer (PBS - pH 7.4) for Amphidinium carterae and in a saline sodium cacodylate buffer for Dunaliella  tertiolecta and Pavlova lutheri, for 2.5 hours and rinsed in the same buffer for 30 minutes at 4*C. The cells were then incubated in the standard 3,3' - diaminobenzidine (DAB) i n c u b a t i o n medium f o r 60 minutes at 37°C on a r o t a r y shaker ( F r e d e r i c k and Newcomb, 1969). T h i s medium co n t a i n e d 20 mg DAB (Sigma Chemical Co., S t . L o u i s , Mo.), 10 ml of 0.05 mM prop a n e d i o l b u f f e r (2-amino-2-methyl-1,3-propanediol) at pH 10.0, and 0.2 ml of 3% H202. The pH was a d j u s t e d to 9.0 p r i o r to i n c u b a t i o n of the c e l l s . F o l l o w i n g the i n c u b a t i o n , the c e l l s were r i n s e d i n b u f f e r f o r 30 minutes and post-f i x e d i n 1% osmium t e t r o x i d e i n the a p p r o p r i a t e b u f f e r f o r 0 1.5 hours at 4 C. A f t e r r i n s i n g , the samples were processed f o r e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n as d e s c r i b e d above. C o n t r o l experiments were conducted by e l i m i n a t i n g ^2^2 r r o m the i n c u b a t i o n medium or by adding 3-amino-1,2,4-triazole, a c a t a l a s e i n h i b i t o r . Morphometry C a l c u l a t i o n of the volume d e n s i t i e s ( i . e . the volume of i n d i v i d u a l components per t o t a l cytoplasm volume) of the endoplasmic r e t i c u l u m , m i t o c h o n d r i a , peroxisomes and vacuoles was c a r r i e d out i n e l e c t r o n micrographs enlarged to a f i n a l m a g n i f i c a t i o n of 36,000 X f o l l o w i n g the s t e r e o l o g i c procedures d e s c r i b e d by O l i v e i r a and F i t c h (1988). To compare means of parameters obtained from d i f f e r e n t n i t r o g e n regimes, Student's t t e s t was used (Snedecor and Cochran, 1967). Two means were co n s i d e r e d to d i f f e r s i g n i f i c a n t l y i f the p r o b a b i l i t y of e r r o r was p < 0.05. RESULTS I) Physiological studies Eleven species of microalgae representing different classes of phytoplankton were tested for their a b i l i t y to u t i l i z e different components of the purine catabolism pathway, i.e. hypoxanthine, allantoin, allantoic acid or urea, as the sole source of nitrogen (Table 1). Table 2 shows the results of the growth on urea of a l l species tested. Pavlova lutheri, Thalassiosi ra nordenskioldi i , Dunaliella tertiolecta, Aqmenellum quadruplicatum, Hymenomonas elongata, Amphidinium carterae, Isochrysis galbana and Nannochloropsis oculata showed good growth on urea. Both their growth rates and c e l l yields were approximately equal to those from equivalent nitrate. Thalassiosira pseudonana and 01isthodiscus luteus were also able to grow on urea but less efficiently than the other species, while Prymnesium parvum showed very poor growth rates and c e l l yields. Nickel - dependency for growth on urea was demonstrated in Amphidinium carterae and 01i sthodi scus luteus since no growth was observed in the absence or at low nickel concentrations (up to 0.01 uM). Growth on urea was improved by 63% for Thalassiosira nordenskioldi i by increasing the nickel supplementation. Growth was also improved (20 to 25%) in urea-grown cells of Hymenomonas elongata and Thalassiosira pseudonana when nickel was added into the culture medium-. In contrast, Pavlova lutheri, Dunaliella  tertiolecta, Aqmenellum quadruplicatum, Isochrysis galbana and Nannochiorops is oculata showed good growth on urea without nickel supplementation. Prymnesium parvum displayed very poor growth on urea with or without the addition of nickel to the culture medium (Table 2). Maximum growth rates and c e l l . yields were recorded at 1 uM N i2 + for Amphidinium carterae and these values started to decrease above this nickel concentration, especially at 10 uM Ni* . However, in 01Isthodiscus luteus, both maximum growth rates and c e l l yields were approximately equal at 1, 5 and 10 uM N i2 +. In Thalassiosira nordenskioldi i, . maximum values for both growth rates and c e l l yields were obtained at 1 uM Ni , with a very slight decrease in these parameters occurring at 5 and 10 uM nickel. Only a small improvement in c e l l yield values (20%) was observed in urea -grown cells of Thalassiosira pseudonana with 1 uM nickel compared with higher concentrations. In this case no major differences were noted in the values of the growth rate. In Hymenomonas elongata, maximum values for growth rate and c e l l yield were obtained at 1 uM N i2 + and they started to decrease at higher concentrations (Table 2). In a l l of the species tested, urea ut i l i z a t i o n requires only a short adaptation lag period (between 2 to 4 days), except for Prymnesium parvum where an adaptation period of 10 days was observed. Dunaliella tert iolecta, Aqmenellum  quadruplicatum and Nannochloropsis oculata showed a lag period at least twice as long in nickel-supplemented medium compared with cultures grown oh urea without nickel additions. The adaptation period decreased by 40 to 100% with increases in nickel supplementation in both Amphidinium carterae and Olisthodiscus luteus (Table 2). Table 3 shows the growth results of the same microalgae on allantoic acid. Hymenomonas elongata, Pavlova lutheri and Nannochloropsis oculata showed good growth on allantoic acid. Both maximum growth rates and c e l l yields of Hymenomonas elongata were approximately equal to those from equivalent urea. Although the maximum yields were similar for Pavlova lutheri and Nannochloropsis oculata when they were grown on urea or allantoic acid , their growth rates were different. The growth rates of allantoic acid-grown cultures of Pavlova lutheri were slightly higher (20%) than those from urea, while the opposite situation was detected in Nannochloropsis oculata, i.e. the growth rates of urea-grown cells were higher (25%) than those from allantoic acid. Dunaliella tert iolecta, Agmenellum quadruplicatum and Isochrysis galbana were also able to grow on allantoic acid but less e f f i c i e n t l y than in urea. A decrease of approximately 30% was detected in both maximum growth rates and c e l l yields of allantoic acid-grown cells of Dunaliella  tert iolecta, while a 15% decrease in the value of these parameters was recorded in Isochrysis galbana when compared with those from equivalent urea growth. Although the growth rates remained equal in urea- and allantoic acid-grown cells of Aqmenellum quadruplicatum, the maximum yields decreased by 50% in allantoic acid cultures. Growth was not detected in Prymnesium parvum, Thalassiosira nordenskioldii, Olisthodiscus luteus when allantoic acid was added into the culture medium as the sole source of nitrogen. Both growth rates and c e l l yields were slightly improved (15 to 20%) in allantoic acid-grown cel l s of Hymenomonas  elongata when nickel was added into the medium. The maximum values for these parameters were obtained at 1 uM Ni2"1", and they started to decrease at higher nickel concentrations (5 and 10 uM). At these higher concentrations, this microalga displayed an adaptation lag period of twice the duration of the one from lower nickel concentrations (1 i^M or lower). In contrast, Pavlova lutheri, Dunaliella  tertiolecta, Agmenellum quadruplicatum, Isochrysis galbana and Nannochloropsis oculata showed no signs of requirement for nickel supplementation since growth improvement was not recorded when nickel was added into the medium containing allantoic acid as the sole source of nitrogen. In fact, the adaptation lag period increased in duration when nickel was added to allantoic acid-cultures of Dunaliella tertiolecta, Agmenellum quadruplicatum, Isochrysis galbana and Nannochloropsis oculata, while no changes were detected in Pavlova lutheri. These observations are similar to those reported for urea-grown cultures (Table 3 compare with Thalassiosi ra pseudonana, Amphidinium carterae and Table 2). None of the species tested grew on allantoin with or without nickel supplementation. Table 4 shows the results of the growth of the microalgae on hypoxanthine. Prymnesium parvum displayed higher growth on hypoxanthine when compared with those from equivalent urea. An increase of more.than 50% in both maximum growth rates and c e l l yields was observed. An improvement of 10% and 30% in maximum yields and growth rates, respectively, was also detected in hypoxanthine-grown cells of Pavlova  lutheri when compared with urea-grown cultures. Although the maximum yields were approximately the same in urea- and hypoxanthine-growth in Dunaliella tertiolecta, Amphidinium  carterae and Nannochloropsis oculata, their growth rates were different. A 15% and '40% decrease in.the exponential growth rate values were observed in Nannochloropsis oculata and Dunaliella tert iolecta respectively, while an increase of 25% was noted in Amphidinium carterae. Growth was not observed in Hymenomonas elongata, Thalassiosira nordenskioldi i , Thalassiosi ra pseudonana, Aqmenellum  quadruplicatum, Olisthodiscus luteus and Isochrysis galbana when hypoxanthine was utilized as the sole source of nitrogen. Nickel-dependency for growth on hypoxanthine was only demonstrated in Amphidinium carterae, since no growth was observed in the absence or at low nickel concentrations (up to 0.01 uM) . Maximum growth rates and c e l l yields were recorded at 1 uM N i2 + and these values started to decrease above this concentration, especially at 10 uM N i2 +. The adaptation lag period of this hypoxanthine-grown culture decreased by approximately 75% in duration when nickel was added into the medium at 1 uM or above this optimal level (5 and 1 0 JJM N iz ). Prymnesium parvum, Pavlova luther i , Dunaliella tertiolecta and Nannochloropsis oculata showed no requirement for nickel supplementation since good growth was observed on hypoxanthine without addition of nickel into the culture media. No changes in the duration of the adaptation period were detected in hypoxanthine-grown cells of Pavlova lutheri, while the lag period was twice as long when nickel was added into the cultures of Dunaliella  tertiolecta and Nannochloropsis oculata• In Prymnesium  parvum, the lag period increased by 50% at 5 and 10 uM N i2 + when compared with lower nickel concentrations (Table 4). The optimal concentrations of urea, allantoic acid, hypoxanthine and nickel that support maximum growth of the microalgae are reported in Table 5. Hymenomonas elongata, Pavlova luther i , Dunaliella tert iolecta, Agmenellum  quadruplicatum and Isochrysis galbana displayed the highest values for growth rate and yield when grown with 1.0 mM of urea. In contrast, 2.0' mM of urea supported optimal growth for Amphidinium carterae and Nannochloropsi s oculata. Particularly noticeable in this respect was the growth response of Prymnesium parvum which showed the greatest improvement in growth rate and maximum yield values when supplemented with 4.0 mM urea. The optimal concentration of allantoic acid for those species able to u t i l i z e this organic compound as the sole source of nitrogen was determined to be 0.5 mM for Hymenomonas elongata, Pavlova  luther i , Dunaliella tert iolecta, Aqmenellum quadruplicatum, Isochrysis galbana, and 1.0.mM for Nannochloropsis oculata. In the case of hypoxanthine, 0.5 mM was the optimal concentration that supported growth of Pavlova lutheri, Dunaliella tert iolecta, 1.0 mM for Amphidinium carterae, Nannochloropsis oculata, and 2.0 mM for Prymnesium parvum. II) Chelation studies Nickel chelation, using citrate as the chelator, was also carried out to study microalgal dependency on this trace metal ion. In those organisms showing improvement or good growth on urea with nickel supplementation, growth was inhibited when citrate (5 mM) was added into the medium containing urea as the sole source of nitrogen. The growth inhibition could be reversed by the addition of excess nickel into the culture media (Figs. 1 and 2). In contrast, in the microalgae that showed no requirement for nickel supplementation , the addition of citrate into the medium did not affect either their growth rates or c e l l yields. Growth reduction or inhibition was not detected when citrate was added into allantoic acid-cultures of Pavlova lutheri, Dunaliella tert iolecta, Agmenellum quadruplicatum, Isochrysis galbana and Nannochloropsis oculata. However, growth inhibition was recorded in allantoic acid-grown ce l l s of Hymenomonas elongata. The inhibition was reversed by the Growth inhibition was detected in Amphidinium carterae when citrate was added into the culture medium containing hypoxanthine as the sole source of nitrogen. This inhibition was also reversed by the addition of excess nickel into the medium (Fig. 2). No changes in growth were observed when citrate was added into hypoxanthine cultures of Prymnesium  parvum, Pavlova luther i , Dunaliella tert iolecta and Nannochloropsis oculata. I l l ) Inhibition studies Growth inhibition studies were also carried out in this investigation. Allopurinol, 2,6,8-trichloropurine and hydroxyurea were added at various concentrations into the culture medium containing the appropriate organic nitrogen source (Table 6). The urea-growth of a l l species of microalgae, with the exception of Dunaliella tert iolecta, was inhibited by hydroxyurea. Hydroxyurea-dependent inhibition of growth was also observed among those species capable of growing with allantoic acid and/or hypoxanthine, with the exception of Dunaliella tertiolecta and Pavlova  luther i . Growth inhibition occurred when 1.0 uM of allopurinol was added to hypoxanthine (nickel was added where required) cultures of Prymnesium parvum, Amphidinium  carterae and Nannochloropsis oculata. Slightly higher concentrations of allopurinol were required for Pavlova addition of excess nickel into the medium (Fig. 1 ) . 18 lutheri (5 uM) and Dunaliella tertiolecta (2 uM) in order for growth to be inhibited. A similar pattern of growth inhibition responses was also detected in - these species when 2,6,8~trichloropurine was added to hypoxanthine-grown cultures. IV) Enzymatic studies Cell-free extracts of Amphidinium carterae, Dunaliella  tert iolecta and Pavlova lutheri were examined for xanthine dehydrogenase, uricase, allantoinase and allantoicase a c t i v i t i e s . No activity could be demonstrated in nitrogen-deprived or urea-grown c e l l s . However, with the exception of uricase a c t i v i t y , i t was possible to detect a l l other activ i t i e s in hypoxanthine-grown cel l s of these three species of microalgae (Table 7 ) . No inhibitors are known for allantoinase and allantoicase a c t i v i t i e s , but in the case of xanthine dehydrogenase, inhibition of activity could be demonstrated by u t i l i z i n g allopurinol (Fig. 3). V) Ultrastructural and Morphometric studies In nitrate-grown control cells of Amphidinium carterae, the nucleus always displays condensed chromosomes completely surrounded by perichromatinic granules which measure 300 + 30 nm in diameter (Figs. 4a, 5 and 8). In urea- (Fig. 5) and hypoxanthine-grown (Fig. 8) c e l l s , the perichromosomal granules are less abundan't and smaller in size, measuring 150 + 12 and 145 + 15 nm in diameter, respectively. These alterations are not an artifact of the plane of sectioning since they persist in serial sections. No obvious morphological differences can be observed in the nucleus and nucleolus of the cel l s 'of both Dunaliella tert iolecta and Pavlova lutheri grown in different nitrogen regimes (Figs. 16 to 23) . Dictyosomes are rather numerous in cells of Amphidinium  carterae grown on different nitrogen sources. The cis-face of the dictyosomes are closely associated with elements of the endoplasmic reticulum (E.R.) through transition vesicles (Figs. 11, 12 and 13, arrowheads). The trans-face of the dictyosomes is usually characterized by the presence of hypertrophied cisternae and smaller vesicular profiles measuring 70 + 5 nm in diameter. However, while these are moderately represented in nitrate and urea-grown cultures of Amphidinium carterae (Figs. 4a, 5, 11 and 12), they occur in large numbers in the hypoxanthine-grown cells (Figs. 6 and 13). . No significant ultrastructural alterations are detected in the dictyosomes of the cells of both Dunaliella  tert iolecta and Pavlova lutheri grown in a l l four different nitrogen regimes. In Amphidinium carterae, endoplasmic reticulum-like elements are often observed in close proximity to the trans-face of the dictyosomes (Figs. 12 and 13). In hypoxanthine -grown ce l l s of this microalga, E.R. elements frequently congregate in the immediate vicinity of chloroplasts and mitochondria without establishing direct contact with their envelopes (Fig. 7, arrows). Although not forming extensive congregations, E.R. elements are also observed in the vic i n i t y of chloroplasts and mitochondria in both urea-grown (Fig. 5, arrow), and nitrate-grown c e l l s of Amphidinium carterae (Fig. 4a, arrow). Morphometric measurements reveal that the volume density of the E.R. expressed per cytoplasm volume is 66% (p < 0.01) higher in hypoxanthine-grown cells compared to that in urea or nitrate. In Dunaliella tert iolecta, endoplasmic reticulum (ER) profiles are observed predominantly in the apical cytoplasm, some of these showing a consistent relationship to the cis-face of dictyosomes through the occurrence of transition vesicles. In hypoxanthine-grown c e l l s , congregation of E.R. elements is also observed in . the immediate vicinity of the trans-face of dictyosomes. Overall, the E.R. is more developed in hypoxanthine-growth than in ce l l s grown, in the three other sources of nitrogen (Fig. 19 c f . with Figs. 16, 17 and 18, arrowheads). Similar situation is detected in Pavlova lutheri, especially in hypoxanthine-grown c e l l s , where extensive development of the E.R. is observed -throughout the cytoplasm (Fig. 23 c f . with Figs. 20, 21 and 22, arrowheads). Morphometric measurements reveal that the volume density, of the E.R. expressed per cytoplasm volume . is 45 and 55% (p < 0.01) higher in hypoxanthine-grown cells of Dunaliella tertiolecta and Pavlova lutheri, respectively, compared with growth on the three other sources of nitrogen. 21 No obvious morphological differences could be observed in the chloroplast and pyrenoid structures of Amphidinium  carterae. cells grown in the different nitrogen regimes (Figs. 4a, 5 and 6). In allantoic acid-grown cells of Dunaliella tert iolecta, most of the chloroplast volume is occupied by starch granules, while in nitrate-, urea- and hypoxanthine-grown cultures only a few starch granules are observed (Fig. 18, c f . with Figs. 16, 17 and 19). No obvious morphological differences are observed in the chloroplast of c e l l s of Pavlova luther i grown in the different nitrogen regimes (Figs. 20 to 23). Mitochondria exhibiting typical dinoflagellate tubular cristae can be seen throughout the c e l l cytoplasm of Amphidinium carterae. However, the number of mitochondria is strikingly different in nitrate-grown cells (19 + 3/section) compared with urea- (35 + 4/section) and hypoxanthine-grown (52 + 4/section) cultures of Amphidinium  carterae. This difference may be accounted for by the fact that mitochondria are more frequently observed undergoing division in urea and hypoxanthine-grown (Figs. 5 and 7) than in nitrate-grown cells (Fig. 4a). Morphometric measurements show that the volume density of the mitochondrial compartment increases by 78% and 47% (p < 0.01) in hypoxanthine and urea growth, respectively, compared to growth on nitrate. In Dunaliella tert iolecta and Pavlova lutheri, the mitochondria do not appear to differ morphologically when cells are grown in different nitrogen 22 sources. However, an increase in the number of mitochondrial profiles (Figs. 19 and 23) was detected in both species during hypoxanthine growth. Morphometric measurements show that the volume density of the mitochondrial compartment of hypoxanthine-grown cells of Dunaliella tert iolecta and Pavlova luther i increases by 40 and 45% (p < 0.01), respectively, in relation to growth on the other sources of nitrogen. In Amphidinium carterae, microbodies are rarely detected in nitrate-grown cultures, and although present in urea-grown c e l l s , they are far from abundant. Microbodies (415 + 15 nm in diameter) become readily visible when hypoxanthine is supplied to the growth medium along with nickel (Fig. 6, arrowheads). . Morphometric measurements show increases of 72% and 28% in the volume density of the peroxisomal compartment of cells grown on hypoxanthine and urea, respectively, compared to those grown.on nitrate. These are single membrane-bound organelles with a granular nucleoid-free matrix, frequently, seen in close association with membranous elements resembling endoplasmic reticulum profiles (Fig. 9). On occasion some microbody-like profiles appear as a terminal enlargement of endoplasmic reticulum cisternae (Fig. 6, arrow). Other micrographs show microbodies connected to each other by a narrow tubular system (Fig. 10, arrowhead). No microbodies are detected at any " time- in cells of Dunaliella tert iolecta and Pavlova  lutheri grown in a l l four nitrogen regimes. 2 3 The vacuolar apparatus of Amphidinium is subdivided into two major compartments. Both compartments are distinct and independent from the pusule (Fig. 4b). One of these compartments is represented by peripherally, located vacuoles, while the other is made up of several central vacuolar p r o f i l e s . In nitrate-grown cultures, the peripheral vacuoles are well developed and almost free of membranous inclusions (Fig. 4a). In urea-grown c e l l s , membranous inclusions can be seen in well-developed peripheral vacuoles (Fig. 5). Peripheral vacuoles are, however, less developed in hypoxanthine-grown cells (Fig. 6). In contrast, the central vacuoles in nitrate-grown cells are very small, and again there are no extensive accumulation of inclusions (Fig. 4a). In urea-grown cultures, the central vacuolar compartment is more developed, and contains a few membranous inclusions (Fig. 5). The central vacuoles are extremely well-developed, and contain large membranous inclusions in hypoxanthine-grown cells (Fig. 6). Morphometric studies reveal that the vacuolar compartment of hypoxanthine-grown cells of Dunaliella tertiolecta is 50 to 55% larger than that of ce l l s grown in the other sources of nitrogen (compare also F i g . 19 with Figs. 16 to 18). The vacuolar compartment of hypoxanthine- and allantoic acid-grown cells of Pavlova lutheri seems also consistently larger than that observed in ce l l s of the same organism supplied with nitrate or urea as sole sources of nitrogen (compare Figs. 22 and 23 with Figs. 20 and 21). This is confirmed by morphometric analysis that shows an increase of 67 to 72% (p < 0.01) in the volume density of this cellular compartment. Lipid granules are frequently observed in the cytoplasm of the cells of Dunaliella tert iolecta. These are particularly abundant in allantoic acid-grown cultures, compared to those grown in nitrate, urea or hypoxanthine (Fig. 18 cf. with Figs. 16, 17 and 19, "L"). Lipid granules are rarely detected in the cells of Pavlova lutheri (Figs. 20 to 23) or Amphidinium carterae (Figs. 4a, 5 and 6). VI) Cytochemical studies Cytochemical studies, using cerium chloride to demonstrate the occurrence of uricase a c t i v i t y , show deposition of reaction product within the microbody-like organelles of hypoxanthine-grown cells of Amphidinium carterae. Accumulation of reaction product is particularly intense in certain regions of the microbodies that could be the morphological equivalent of structureless nucleoids (Fig. 14, arrowheads). Control experiments, carried out in the presence of the inhibitors of uricase a c t i v i t y , 2,6,8-trichloropurine or oxypurine, eliminate the deposition of reaction product. In addition to uricase, these organelles also show intense deposition of reaction product indicative of catalase activity (Fig. 15). Deposition of reaction product is absent from samples incubated with aminotriazole, a catalase inhibitor, or without H2O2. In Dunaliella  tert iolecta and Pavlova luther i , the deposition of reaction 2 5 product, indicative of uricase activity, occurs within the cristae and in the outer compartment of the mitochondria (Figs. 24 and 28). Deposition of reaction product, however, occurs only after brief fixation (5 min), in very low concentrations of glutaraldehyde (0.25%) and in the presence of uric acid (Table 8). Control experiments show no deposition of reaction product (Figs. 25 and 29). In addition to uricase, mitochondria also show intense deposition of reaction product indicative of catalase activity in both Dunaliella tert iolecta and Pavlova luther i (Figs. 26 and 30). Deposition of reaction product occurs only at pH 9.0 in ce l l s incubated at 37°C and it is absent from samples incubated with aminotriazole or without H202 (Figs. 27 and 31, Table 8). A third type of reaction product deposition in mitochondria Of these microalgae is observed at pH 6.0 when the c e l l s are incubated at room temperature in the absence of H202. Under these circumstances, the deposition of reaction product is strongly inhibited by low concentrations of potassium cyanide which is indicative of cytochrome system-dependent oxidative activity. Furthermore, while the cytochrome system-dependent deposition of reaction product occurs in ce l l s grown in a l l four sources of nitrogen tested, uricase- and catalase-dependent deposition of reaction products are only observed in mitochondria of hypoxanthine grown cells (Table 8). . 26 DISCUSSION Growth Studies The results clearly demonstrate the widespread occurrence of urea - u t i l i z i n g capabilities on the part df a l l microalgae tested, with significantly greater growth exhibited by some species relative to the nitrate controls. The f a c i l i t y and efficiency of urea utilization by most of the species surveyed confirm the findings of previous authors that urea is one of the most important sources of organic-N available for growth of marine phytoplankton in the oceans (see Oliveira and Antia, 1984 for review). In a previous study, Prymnesium parvum showed excellent growth on urea, while Amphidinium carterae displayed a low nickel requirement for growth on this organic-N source. It was also shown that nickel supplementation was toxic for urea-grown cells of Olisthodiscus luteus (Oliveira and Antia, 1986a). In contrast, the present study shows, as previously observed by Droop (1955), poor growth on urea for Prymnesium parvum, while nickel is required at higher concentrations to support the urea-growth of Amph i d i n i um carterae and especially Olisthodiscus luteus (Table 2). I have utilized the same culture conditions and media as in the previous study. Therefore, I attribute the discrepancies to the use of different clones of these microalgae. The available evidence, on the distribution of urea-degrading enzymes, indicates that two alternative enzyme 27 systems (urease and ATPrurea amidolyase) occur i n a l g a e . ATP:urea amidolyase i s r e s t r i c t e d to some members of the Chlorophyceae, while urease occurs i n a l l other a l g a l c l a s s e s and some of the chlorophycean algae examined so f a r ( L e f t l e y and S y r e t t , 1973; Bekheet and S y r e t t , 1977; S y r e t t , 1981; A l - Houty and S y r e t t , 1984). More germane to t h i s problem was the i d e n t i f i c a t i o n of n i c k e l as a c o n s t i t u e n t of jackbean (Canavalia e n s i f o r m i s L.) urease (Dixon e_t a_l. , 1975) and soybean t i s s u e c u l t u r e s ( P o l a c c o , 1977). A n i c k e l requirement f o r urease a c t i v i t y was a l s o r e p o r t e d f o r some microalgae ( S y r e t t , 1981; Rees and Bekheet, 1982; O l i v e i r a and A n t i a , 1986b). T h e r e f o r e , the s t r i c t requirement f o r N i2 + e x h i b i t e d by Amphidinium  c a r t e r a e and O l i s t h o d i s c u s l u t e u s f o r growth on urea as s o l e source of organic-N suggests that urease i s the enzyme r e s p o n s i b l e f o r the co n v e r s i o n of urea i n t o u t i l i z a b l e ammonium ( P o l a c c o , 1977). T h i s i s confirmed by both the hydroxyurea- and c i t r a t e - d e p e n d e n t growth i n h i b i t i o n s observed when these microalgae are supplemented with urea as s o l e source of n i t r o g e n . The endogenous n i c k e l content of the sea water used i n the p r e p a r a t i o n of our t e s t media i s 3.42 nM and our estimate of endogenous n i c k e l i n the f i n a l media, e x c l u d i n g contamination from added s a l t s , i s i n the order of 2.7 and 1.4 nM f o r . t h e s a l i n i t i e s of 26 and 1 4% o u t i l i z e d i n the present study, r e s p e c t i v e l y ( O l i v e i r a and A n t i a , 1984). The growth enhancement observed i n urea-grown c e l l s of 28 Hymenomonas elongata, Thalassiosira nordenskioldi i and Thalassiosira pseudonana with nickel supplementation, therefore, implies that urease is also present in these microalgae. This is further confirmed by the strong growth inhibition observed when citrate, the natural Ni2 +-chelator found in nickel accumulating plants (Kersten et a l . , 1980), is added to the culture media. In contrast, the growth of Pavlova lutheri, Dunaliella tertiolecta, Aqmenellum  quadruplicatum, Isochrysis galbana and Nannochloropsis  oculata on urea displayed no signs of nickel-requirement. Furthermore, the addition of citrate did not affect the growth of these organisms. These data suggest that either these organisms have the capacity to readily concentrate Ni* from the nanomole levels normally occurring in seawater (see Oliveira and Antia, 1986a for review) or they use the ATP:urea amidolyase Ni2+-independent system for the conversion of urea directly to utilizable ammonium (Oliveira and Antia, 1986b). In the case of Pavlova lutheri, Agmenellum quadruplicatum, Isochrysi s galbana and Nannochloropsi s oculata, the f i r s t explanation is more likely to be true, since growth of these microalgae in urea as sole source of nitrogen is inhibited by hydroxyurea, a potent inhibitor of urease activity (Reithel, -197.1; Carvajal et a l . , 1982). This compound is without effect on the urea-supported growth of Dunaliella tertiolecta. Instead, this is inhibited by avidin, a known inhibitor of ATP:urea amidolyase a c t i v i t y . Furthermore, addition of 29 b i o t i n a b o l i s h e s the a v i d i n - i n d u c e d i n h i b i t i o n (unpublished r e s u l t s ) . These r e s u l t s are i n d i c a t i v e of the occurrence of ATP:urea amidolyase a c t i v i t y and e x p l a i n s the reason why a n i c k e l requirement i s absent from t h i s microalga ( L e f t l e y and S y r e t t , 1973; C a r v a j a l et a l . , 1982). . The long a d a p t a t i o n l a g p e r i o d observed i n urea-grown c e l l s of Prymnesium parvum together with i t s poor growth on t h i s n i t r o g e n source suggest that t h i s microalga may not be w e l l equipped to u t i l i z e the organic n i t r o g e n of u r e a . T h i s i s f u r t h e r confirmed by t h e i r i n a b i l i t y to grow e f f i c i e n t l y on t h i s organic n i t r o g e n source even at higher c o n c e n t r a t i o n s ( A n t i a e_t a l . , 1975). The problem i s not one of n i c k e l dependency s i n c e a d d i t i o n s of n i c k e l ions to the c u l t u r e media f a i l e d to improve growth. Since hydroxyurea completely i n h i b i t s whatever growth i s d e t e c t e d i n the presence of u r e a , the more l i k e l y e x p l a n a t i o n f o r the poor r e s u l t s observed i s the impairment of the mechanism of urea uptake by these c e l l s . A c cording to A n t i a et. a l . , (1989), a l l a n t o i c a c i d has not been p r e v i o u s l y t e s t e d as an organic N-source f o r the growth of m i c r o a l g a e . Under our t e s t c o n d i t i o n s , only 6 of the 11 s p e c i e s s t u d i e d , showed moderate to good growth on t h i s n i t r o g e n s o u r c e . The growth observed i n 0.5 mM a l l a n t o i c acid-grown c u l t u r e s of Hymenomonas e l o n g a t a , Pavlova l u t h e r i and Nannochloropsis o c u l a t a i s e q u i v a l e n t to the one from 1 mM urea-supplemented c u l t u r e s . Since the N-atoms s t o i c h i o m e t r y of a l l a n t o i c a c i d versus urea i s 4:2, 30 the p r e v i o u s o b s e r v a t i o n suggests that these microalgae u t i l i z e the a l l a n t o i c a c i d - N as e f f i c i e n t l y as that from u r e a . These r e s u l t s may then be construed to imply that a l l a n t o i c a c i d might be c a t a b o l i z e d to urea and u r e i d o g l y c o l a t e by the enzyme a l l a n t o i c a s e ( A n t i a et al_. , 1989; Shah and S y r e t t , 1984b). T h i s i s supported by the deter m i n a t i o n of a l l a n t o i c a s e a c t i v i t y i n c e l l - f r e e e x t r a c t s of some of these microalgae (Table 7 ) . The i n h i b i t i o n of allantoate-supplemented c u l t u r e s of Hymenomonas elongata and Nannochloropsis o c u l a t a by hydroxyurea f u r t h e r supports t h i s i n t e r p r e t a t i o n . However, the absence of i n h i b i t i o n d i s p l a y e d by allantoate-grown c e l l s of Pavlova l u t h e r i t r e a t e d with hydroxyurea suggests that i n t h i s case urea may not be one of the products of the c a t a b o l i c a c t i o n of t h i s enzyme. The growth recorded i n 0.5 mM a l l a n t o i c acid-grown c e l l s of D u n a l i e l l a t e r t i o l e c t a , Agmenellum quadruplicatum and I s o c h r y s i s galbana was l e s s than that from 1.0 mM u r e a , e s p e c i a l l y f o r Agmenellum quadruplicatum (compare Table 3 with Table 2 ) . A n t i a et a l . , (1989) suggested t h a t c u l t u r e f a c t o r s , such as temperature, pH or i l l u m i n a t i o n c o u l d a f f e c t the c a p a b i l i t y of microalgae to decompose the e n t i r e s t r u c t u r e of a l l a n t o i c a c i d and u t i l i z e a l l 4 N atoms present i n the molecule; hence, the lower e f f i c i e n c y in the u t i l i z a t i o n of a l l a n t o i c a c i d - N . The i n h i b i t i o n of allantoate-grown c u l t u r e s of Agmenellum quadruplicatum and I s o c h r y s i s galbana by hydroxyurea suggests that c o n v e r s i o n of a l l a n t o i c a c i d to urea by the c a t a l y t i c a c t i o n of the 31 enzyme a l l a n t o i c a s e a l s o occurs i n these m i c r o a l g a e . Hydroxyurea showed no e f f e c t upon the growth of a l l a n t o a t e -supplemented c u l t u r e s of D u n a l i e l l a t e r t i o l e c t a . However, these r e s u l t s are not s u r p r i s i n g s i n c e ATP:urea amidolyase, not urease, was shown to be the enzyme r e s p o n s i b l e f o r the u t i l i z a t i o n of urea-N i n t h i s genus ( L e f t l e y and S y r e t t , 1973). The nickel-independence of ATPrurea. amidolyase e x p l a i n s the absence of N i2 +- r e q u i r e m e n t f o r growth on t h i s and other urea-producing sources of organic N ( O l i v e i r a and A n t i a , 1986b). Nickel-independence was a l s o demonstrated in a l l a n t o i c a c i d c u l t u r e s of Pavlova l u t h e r i , Agmenellum  quadruplicatum, I s o c h r y s i s galbana and Nannochloropsis  o c u l a t a . I n c o n t r a s t , the a d d i t i o n of c i t r a t e to a l l a n t o i c acid-supplemented c u l t u r e s of Hymenomonas elongata suppressed growth, suggesting that t h i s metal ion i s r e q u i r e d f o r growth on t h i s organic-N source. A l l a r t t o i n was a l s o t e s t e d f o r i t s a b i l i t y to support the growth of phytoplankton as the s o l e source of organic-N, but no growth was observed i n a l l s p e c i e s s t u d i e d . Except f o r c e r t a i n b e n t h i c - t y p e s p e c i e s which appear to be w e l l equipped f o r u t i l i z i n g a l l a n t o i n , A n t i a et a l . ,• . (1980b) suggested t h a t t h i s organic-N i s g e n e r a l l y a poor N-source for phytoplankton growth r e l a t i v e to the p u r i n e s . A s i m i l a r s i t u a t i o n was a l s o observed i n C h l o r e l l a . p y r e n o i d o s a (Ammann and Lynch, 1964). These i n v e s t i g a t o r s i n d i c a t e d that a c e l l u l a r p e r m e a b i l i t y b a r r i e r to a l l a n t o i n might p o s s i b l y be the cause of the absence of growth. 32 Hypoxanthine was chosen as the prime purine representative for this investigation not only on account of i t s established stability in natural sea water but also because unlike adenine or guanine, it does not possess any exocyclic nitrogenous group; hence any nitrogen utilization must depend on the ab i l i t y of a particular species to retrieve the purine-skeleton nitrogen (Antia et a l . , 1989). Out of the 11 species examined in this investigation, only 5 showed moderate to good growth on hypoxanthine. Prymnesium parvum proved to be rather interesting in the sense that it grows well on hypoxanthine, while poor growth was observed on urea. The situation is similar to the one previously reported by Antia et al.., ( 1975) for Chlamydomonas pa 11a. However, in the case of Prymnesium parvum, a higher substrate concentration (2 mM) in comparison with other hypoxanthine ut i l i z e r s (0.5 to 1 mM - Table 5) is required for growth to.occur. This suggests that this microalga may possess a less effective permease or uptake system for hypoxanthine (Antia et a_l. , 1980a). The growth observed in hypoxanthine-cultures of Pavlova lutheri, Nannochloropsis  oculata and Dunaliella tertiolecta was equivalent to the one from allantoic acid- and also urea-grown cells of these species (Table 5). Since both hypoxanthine and allantoic acid contain 4 N atoms versus 2 N atoms per molecule of urea, the evidence indicates that these 3 species of microalgae are efficient u t i l i z e r s of a l l the hypoxanthine -N. The hypoxanthine growth of these microalgae is not dependent upon N i2 supplementation and proceeds i n the presence of the N i2 + c h e l a t o r c i t r a t e . Amphidinium c a r t e r a e a l s o e x h i b i t e d good growth on hypoxanthine. However, t h i s s p e c i e s r e q u i r e s the a d d i t i o n of n i c k e l i n t o the c u l t u r e medium. The growth observed, under these circumstances', i s s i m i l a r to the one from e q u i v a l e n t urea p l u s N i2 + supplemented c u l t u r e s (compare Table 4.with Table 2)..The a d d i t i o n of c i t r a t e to h y p o x a n t h i n e - c u l t u r e s of Amphidinium  c a r t e r a e suppressed t h e i r growth. The growth suppression i s r e v e r s i b l e , s i n c e the a d d i t i o n of excess, n i c k e l i n t o the c u l t u r e medium r e s t o r e s i t . These data f u r t h e r support the nickel-dependency of t h i s process and confirms that urease i s the enzyme r e s p o n s i b l e f o r urea c a t a b o l i s m i n t h i s m i c r o a l g a . I n h i b i t o r S t u d i e s and Determination of Enzymatic a c t i v i t i e s As r e p o r t e d by F u j i h a r a and Yamaguchi (1978), a l l o p u r i n o l [4-hydroxypyrazolo (3,4-d) p y r i m i d i n e ] , an analogue of hypoxanthine, i s a potent i n h i b i t o r of the enzyme xanthine dehydrogenase. T h i s enzyme i s r e s p o n s i b l e for the o x i d a t i o n of hypoxanthine to x a n t h i n e , and t h i s to u r i c a c i d ( A n t i a et a l . , 1989). T h e r e f o r e , the growth i n h i b i t i o n observed i n hypoxanthine-grown c u l t u r e s of Prymnesium parvum, Pavlova  l u t h e r i , D u n a l i e l l a t e r t i o l e c t a , Amphidinium c a r t e r a e and Nannochloropsis o c u l a t a when a l l o p u r i n o l was added i n t o the c u l t u r e medium, i m p l i e s that the degradation of hypoxanthine -> xanthine -> u r i c a c i d i s dependent on the 34 a c t i v i t y of xanthine dehydrogenase. The demonstration of the occurrence of xanthine dehydrogenase a c t i v i t y i n some of these microalgae confirms t h i s i n t e r p r e t a t i o n (Table 7 ) . U r i c a s e (urate:oxygen oxidoreductase) i s a c u p r o p r o t e i n that c a t a l y z e s the o x i d a t i o n of urate i n the presence of oxygen, y i e l d i n g hydrogen peroxide ( H 2 O 2 ) , carbon d i o x i d e and a l l a n t o i n (Mahler et a l . , 1955). A number of substances are known to be e f f e c t i v e i n h i b i t o r s of u r i c a s e . These i n c l u d e o x y p u r i n e s , 2 , 6 , 8 - t r i c h l o r o p u r i n e and oxonate (Muller and M o l l e r , 1969 and r e f e r e n c e s c i t e d t h e r e i n ) . The growth i n h i b i t i o n of hypoxanthine-supplemented c u l t u r e s of these m i c r o a l g a e , t r e a t e d with t r i c h l o r o p u r i n e , i n d i c a t e s that u r i c a s e i s r e s p o n s i b l e f o r the u r i c a c i d -> a l l a n t o i n c a t a l y t i c c o n v e r s i o n . F a i l u r e to det e c t u r i c a s e a c t i v i t y i n crude c e l l - f r e e e x t r a c t s of the microalgae may be r e l a t e d to the h i g h s e n s i t i v i t y of t h i s enzyme to changes i n pH, among other f a c t o r s (Huynh and O l i v e i r a , 1989a, b ) . Indeed, Shah and S y r e t t (1984b) a l s o f a i l e d to det e c t u r i c a s e a c t i v i t y i n crude e x t r a c t s of the marine diatom Phaeodactylum  t r i c o r n u t u m , although a c t i v i t y was measurable i n p a r t i a l l y p u r i f i e d f r a c t i o n s of the same e x t r a c t . Urate o x i d a t i o n proceeds s i m u l t a n e o u s l y with the degradation by c a t a l a s e of the H 2 O 2 formed during the r e a c t i o n . I t i s then important to n o t i c e that cytochemical s t u d i e s of Amphidinium c a r t e r a e , D u n a l i e l l a t e r t i o l e c t a and Pavlova l u t h e r i r e v e a l the occurrence of both u r i c a s e and c a t a l a s e a c t i v i t i e s (Huynh and O l i v e i r a , 1989a, b ) . The determination i n crude c e l l 35 p r e p a r a t i o n s of some of the microalgae of a l l a n t o i n a s e and a l l a n t o i c a s e a c t i v i t i e s suggests then that the standard pathway of purine degradation a l s o occurs i n microalgae and i t i s r e s p o n s i b l e f o r the u t i l i z a t i o n of the n i t r o g e n of p u r i n e s and i t s d e r i v a t i v e s v i a urea p r o d u c t i o n (Vogels and van der D r i f t 1976; Reynolds et a l . , 1982). The f a c t that urease i n h i b i t o r s , with the e x c e p t i o n of D u n a l i e l l a  t e r t i o l e c t a and Pavlova l u t h e r i , block the growth of hypoxanthine-supplemented c u l t u r e s of the other three s p e c i e s able to grow on t h i s N source f u r t h e r supports t h i s i n t e r p r e t a t i o n . The lack of hypoxanthine-growth i n h i b i t i o n of D u n a l i e l l a t e r t i o l e c t a by urease i n h i b i t o r s r e f l e c t s the occurrence of ATPrurea amidolyase a c t i v i t y ( L e f t l e y and S y r e t t , 1973). The f a c t that u t i l i z a t i o n of hypoxanthine and a l l a n t o i c a c i d by Pavlova l u t h e r i remains u n a f f e c t e d i n the presence of urease i n h i b i t o r s suggests that the c a t a b o l i c o x i d a t i o n of p u r i n e s and t h e i r d e r i v a t i v e s does not i n v o l v e the p r o d u c t i o n of u r e a . It i s i n t e r e s t i n g r e g a r d i n g these f i n d i n g s that r e c e n t l y Winkler et a l . , (1987, 1988) showed that a l l a n t o i n c a t a b o l i s m i n soybean suspension c u l t u r e . c e l l s i s c a r r i e d out by two amidohydrolase r e a c t i o n s , a l l a n t o a t e amidohydrolase and u r e i d o g l y c o l a t e amidohydrolase. Under these circumstances the whole process proceeds without d e t e c t a b l e p r o d u c t i o n of u r e a . The a b i l i t y f o r urea u t i l i z a t i o n e x h i b i t e d by t h i s m i c r o a l g a may then represent a system independent from that of p u r i n e c a t a b o l i s m , such as that of the o r n i t h i n e c y c l e 36 i n v o l v i n g urea r e l e a s e from a r g i n i n e by a r g i n a s e ( N a y l o r , 1970). However, whether or not a two amidohydrolase system s i m i l a r to the one observed i n soybean e x i s t s i n Pavlova  l u t h e r i remains to be determined. U l t r a s t r u c t u r e and Cytochemical S t u d i e s on Amphidinium  c a r t e r a e Recent s t u d i e s have shown that high l e v e l s of the t r a n s i t i o n metals Fe, N i , Cu and Zn are found i n the condensed chromatin of a v a r i e t y of d i n o f l a g e l l a t e s ( f o r review see S p e c t o r , 1984). Studi e s on the uptake of 6 % i i n t o d i n o f l a g e l l a t e chromosomes r e v e a l that there i s a continuous i n c r e a s e i n the mean l e v e l of t h i s ion over a 24 hrs p e r i o d . T h i s was suggested e i t h e r to be i n d i c a t i v e of a continuous change in the balance of t r a n s i t i o n metals w i t h i n e x i s t i n g chromatin or to be r e l a t e d to the formation of new chromatin by continuous DNA s y n t h e s i s ( S i g e e , 1982). Although the f u n c t i o n of the p e r i c h r o m a t i n i c granules i s s t i l l unknown, the Spector et a l . (1981) model on d i n o f l a g e l l a t e chromosome o r g a n i z a t i o n suggests that they p l a y a r o l e i n the s t a b i l i z a t i o n of the chromosomes. The u l t r a s t r u c t u r a l changes i n the p e r i c h r o m a t i n i c granules may then r e f l e c t the response of the c e l l s to the presence of N i2 + i n the growth medium of both urea and hypoxanthine c u l t u r e s . In a recent review on the u l t r a s t r u c t u r e of d i n o f l a g e l l a t e s , Dodge and Greuet (1987) p o i n t out that i t i s u s u a l l y d i f f i c u l t to d i f f e r e n t i a t e between the forming ( c i s ) face and the maturing ( t r a n s ) face of dictyosomes i n these m i c r o a l g a e . In Amphidinium c a r t e r a e , the c i s - and t r a n s - f a c e s of the dictyosomes are d i s t i n c t i n a l l three n i t r o g e n n u t r i t i o n a l regimes t e s t e d . The c i s - f a c e shows a c l e a r r e l a t i o n s h i p with endoplasmic r e t i c u l u m elements which i n c l u d e s t r a n s i t i o n v e s i c l e s ( K r i s t e n , 1980). The c i s t e r n a e of the t r a n s - f a c e a r e , i n t u r n , always h y p e r t r o p h i e d . Another d i s t i n c t i v e f e a t u r e of the trans r e g i o n of the dictyosomes i s the presence of v e s i c u l a r - l i k e p r o f i l e s measuring 70 + 5 nm i n diameter. These are p a r t i c u l a r l y abundant i n hypoxanthine-grown c e l l s . S tudies on the e f f e c t s of heavy metals on the growth of microalgae have shown in c r e a s e d d ictyosomal a c t i v i t y (hypertrophy and v e s i c u l a t i o n ) at the t r a n s r e g i o n of the o r g a n e l l e s (Smith, 1983; Chan and Wong, 1987). In the case of Skeletonema  costatum, t h i s i n c r e a s e i n dictyosomal a c t i v i t y was suggested to be r e l a t e d to mechanisms of metal s e q u e s t r a t i o n (Smith, 1983). The presence of l a r g e membranous i n c l u s i o n s w i t h i n vacuoles i s i n d i c a t i v e of e x t e n s i v e autophagic a c t i v i t y (Marty et a l . , 1980). Why such widespread autophagic a c t i v i t y would develop i n hypoxanthine-grown c e l l s escapes our present understanding of the n u t r i t i o n a l e f f e c t s of d i f f e r e n t n i t r o g e n sources on c e l l u l a r u l t r a s t r u c t u r e . However, the e f f e c t i s not a d e l e t e r i o u s one, s i n c e growth in h y p o x a n t h i n e - n i c k e l supplemented medium proceeds as 38 e f f i c i e n t l y as i n n i t r a t e or urea p l u s n i c k e l c o n d i t i o n s . Recent s t u d i e s showed that i n a q u a t i c organisms s e v e r a l c e l l u l a r compartments ( i . e . G o l g i a p p a r a t u s , lysosomes) p l a y a major r o l e i n i n t r a c e l l u l a r metal homeostasis. The extent to which any one of these compartments i s i n v o l v e d i n metal bi n d i n g appears to depend on a number of f a c t o r s , i n c l u d i n g n u t r i t i o n a l regime and i n t e r a c t i o n s with competing metal ions (Fowler, 1987). The i n c r e a s e i n the vacuolar apparatus of Amphidinium c a r t e r a e c o u l d then, at l e a s t i n p a r t , be a r e f l e c t i o n of such p r o c e s s e s . The f a c t that an i n c r e a s e a l s o occurs i n the v a c u o l a r system of urea p l u s n i c k e l but not n i t r a t e / n i c k e l - f r e e supplemented c e l l s supports t h i s i n t e r p r e t a t i o n . Based on the d i s t r i b u t i o n of the enzymes of pu r i n e c a t a b o l i s m , the u r e i d e a l l a n t o i n i s h y d r o l y z e d to a l l a n t o i c a c i d by a l l a n t o i n a s e p o s s i b l y l o c a t e d i n the endoplasmic r e t i c u l u m (Hanks et a l , , 1981; 1983). The i n c r e a s e observed in the complexity and s i z e of the ER i n hypoxanthine-grown c e l l s seems then c o n s i s t e n t with the key r o l e t h i s o r g a n e l l e p l a y s i n the c a t a b o l i s m of p u r i n e s . The p r o l i f e r a t i o n i n endoplasmic r e t i c u l u m c o i n c i d e s a l s o i n these c e l l s with a l a r g e i n c r e a s e i n the number of m i c r o b o d i e s . Endoplasmic r e t i c u l u m elements are o f t e n c l o s e l y p o s i t i o n e d to the bounding- membrane of these o r g a n e l l e s and o c c a s i o n a l l y d i r e c t c o n t i n u i t y between ER and microbodies can a l s o be observed ( F i g . 4, a r r o w ) . The s i t u a t i o n resembles that of the root nodules of higher p l a n t s s p e c i a l i z e d f o r u r e i d e 39 p r o d u c t i o n and suggests that i n Amph i d i n i um c a r t e r a e microbodies may o r i g i n a t e from the ER (Newcomb et a l . , 1985; Kaneko and Newcomb, 1987; Webb and Newcomb, 1987). In some micrographs, microbodies are connected to one another by tu b u l a r p r o f i l e s ( F i g . 8, arrowhead). These are i n d i c a t i v e of the e x i s t e n c e of a peroxisomal r e t i c u l u m and suggest that microbodies may a l s o be formed by the f i s s i o n of p r e e x i s t i n g ones (Lazarow and F u j i k i , 1985). The f u n c t i o n a l s p e c i a l i z a t i o n of microbodies (e.g. peroxisomes) has been i n v e s t i g a t e d e x t e n s i v e l y i n higher p l a n t s (Beevers, 1979; T o l b e r t and Essner , 1981) but r a r e l y so with respect to m i c r o a l g a e . C o n s i d e r i n g the in f o r m a t i o n a v a i l a b l e , , i t seems that there are two groups of microbodies in the a l g a e . One group i s represented by u n s p e c i a l i z e d microbodies c h a r a c t e r i z e d mainly by the presence of c a t a l a s e and u r i c a s e a c t i v i t i e s . The other by o r g a n e l l e s f u n c t i o n a l l y resembling the glyoxysomes and peroxisomes of higher p l a n t s (Stabeneau, 1984). The demonstration of both u r i c a s e and c a t a l a s e a c t i v i t i e s i n Amphidinium c o u l d then be taken as i n d i c a t i v e of the occurrence of u n s p e c i a l i z e d microbodies i n the c e l l s of t h i s d i n o f l a g e l l a t e . However, the p r o l i f e r a t i o n of microbodies i s only observed i n hypoxanthine-growth which i s a s t r o n g i n d i c a t o r of some major a l t e r a t i o n ( s ) i n the endogenous metabolism of these c e l l s . In ur e i d e - p r o d u c i n g root nodules u r i c a c i d i s o x i d i z e d to a l l a n t o i n in the peroxisomes by u r i c a s e . T h i s r e a c t i o n produces ^2^2 which i s degraded by c a t a l a s e , a l s o l o c a t e d i n the peroxisomes (Hanks 40 et a l . , 1981; Schubert, 1986; Kaneko and Newcomb, 1987; Webb and Newcomb, 1987; Vaugh and S t e g i n k , 1987). These o b s e r v a t i o n s show t h a t hypoxanthine-growth of Amphidinium very l i k e l y occurs v i a i t s con v e r s i o n to urea through c a t a b o l i c o x i d a t i o n and that the l a r g e i n c r e a s e i n the microbody p o p u l a t i o n i s r e l a t e d to t h i s type of metabolism. The f a c t that no u r i c a s e and c a t a l a s e a c t i v i t i e s are de t e c t e d i n n i t r a t e or urea-grown c e l l s supports t h i s i n t e r p r e t a t i o n . In n i trate-grown c u l t u r e s microbodies are r a r e l y d e t e c t e d , a f a c t that confirms o b s e r v a t i o n s by other authors (Dodge and Crawford, 1968; K l u t et a l . , 1981). They are a l s o f a r from abundant i n urea-grown c e l l s compared with hypoxanthine-grown c e l l s . These f e a t u r e s i n c o n j u n c t i o n with the absence of enzymatic a c t i v i t y f o r u r i c a s e and c a t a l a s e suggest that these microbodies are f u n c t i o n a l l y d i f f e r e n t from those i n hypoxanthine-grown m a t e r i a l . In a recent study of the c a t a l a s e - n e g a t i v e microbodies of Amphidinium  c a r t e r a e , K l u t et a l . (1984) suggested that these o r g a n e l l e s might be regarded as a t y p i c a l glyoxysomes where the marker enzymes of the g l y o x y l a t e c y c l e may be absent or r e p r e s s e d . The evidence supports then the e x i s t e n c e i n t h i s m i c r o a l g a of two f u n c t i o n a l l y d i s t i n c t , n u t r i t i o n - r e l a t e d p o p u l a t i o n s of m i c r o b o d i e s . Such a s i t u a t i o n seems a l s o to occur i n Euglena where microbodies appear to perform l i k e peroxisomes d u r i n g a u t o t r o p h i c growth and as glyoxysomes 41 under heterotrophic conditions (Graves et a_l. , 1972; Collins and Merrett, 1975). Klut et a l . (1984) interpreted the lack of catalase in these microbodies as indicative of a corresponding lack of glycolate oxidase. Although l i t t l e is known of the overall mechanism of photorespiration in dinoflagellates, the available evidence supports the absence of glycolate oxidase from these organelles (Burris, 1977). It suggests instead that glycolate metabolism is initiated by glycolate dehydrogenase. This enzyme cannot transfer electrons directly to oxygen; hence, it does not form H202 during glycolate oxidation. In algae, glycolate dehydrogenase is usually located in mitochondria rather than microbodies (Stabenau, 1984). In Scenedesmus obiiquus, the increase in the number of mitochondria during adaptation to low C02 was suggested to reflect an increase in the activity of glycolate metabolism (Kramer and Findenegg, 1978). The occurrence of large numbers of mitochondria in cells of Amphidinium could then reflect, at least in part, their participation in photorespiration (Klut et a l . , 1981). Ultrastructural and Cytochemical Studies on Dunaliella tertiolecta and Pavlova lutheri 42 As p r e v i o u s l y d i s c u s s e d , the i n c r e a s e i n endoplasmic r e t i c u l u m r e p o r t e d to occur in c e l l s of the d i n o f l a g e l l a t e Amphidinium c a r t e r a e grown on hypoxanthine as s o l e source of n i t r o g e n , i s i n d i c a t i v e of the importance of the E.R. i n the u t i l i z a t i o n of hypoxanthine-N (Huynh and O l i v e i r a , 1989a). The l a r g e r development of the endoplasmic r e t i c u l u m (E.R.) i n hypoxanthine-grown c e l l s of D u n a l i e l l a t e r t i o l e c t a and Pavlova l u t h e r i seems then a l s o c o n s i s t e n t with the key r o l e t h i s o r g a n e l l e p l a y s i n the c a t a b o l i s m of p u r i n e s (Hanks et a l . , 1981; 1983). The o x i d a t i o n of u r a t e and the subsequent degradation by c a t a l a s e of the H202 formed in the u r i c a s e r e a c t i o n are u s u a l l y compartmentalized in m i c r o b o d y - l i k e o r g a n e l l e s , the peroxisomes (Schubert, 1981; Huynh and O l i v e i r a , 1989a). The i n t r i g u i n g f a c t i n both D u n a l i e l l a t e r t i o l e c t a and Pavlova l u t h e r i i s the l o c a t i o n of these r e a c t i o n s , s i n c e no m i c r o b o d y - l i k e o r g a n e l l e s are d e t e c t e d i n c e l l s grown on a l l four sources of n i t r o g e n t e s t e d . I t i s then s i g n i f i c a n t that c y t o chemical d e p o s i t i o n of r e a c t i o n products i n d i c a t i v e of the occurrence of urate oxidase and c a t a l a s e a c t i v i t i e s i s observed in m i t o c h o n d r i a . It i s i n t e r e s t i n g a l s o to n o t i c e that these c o i n c i d e with an i n c r e a s e i n the volume d e n s i t y of the m i t o c h o n d r i a l compartment. The c y t ochemical l o c a l i z a t i o n of u r i c a s e and c a t a l a s e a c t i v i t i e s i n m itochondria i s an unusual f i n d i n g , although u r i c a s e a c t i v i t y was reported before to be a s s o c i a t e d with the m i t o c h o n d r i a l f r a c t i o n of soybean seeds (Glyc ine max A62-1: 43 nodulating variety). It is interesting to notice that in this case uricase activity was only detected during certain periods of plant development (Tajima and Yamamoto, 1975). The situation resembles that observed in both Dunaliella  tert iolecta and Pavlova lutheri, since uricase activity is only detected in hypoxanthine but not allantoic acid, urea or nitrate supported growth. The question of the specificity of the cytochemical reactions must be carefully considered. The pH optimum for urate oxidase activity was shown to be above 9, although in soybean radicles an optimum pH of 7.0 was reported (Muller and Moller, 1969; Tajima and Yamamoto, 1975). In this last instance, however, the enzymatic activity proved to be due not to uricase but to two other enzymes, diamine oxidase and peroxidase (Tajima et a l , 1985). It is important to notice then that the deposition of reaction product indicative of uricase activity in mitochondria of both Dunaliella  tertiolecta and Pavlova lutheri occurs at high pH values and rapidly declines with decreasing pH (Table 8). Angermuller and Fahimi (1986 and references cited therein) showed that urate oxidase activity is extremely sensitive to aldehyde fixation. Even short fixation periods of 20 min with 1% aldehyde produces a 90% loss in activity (Yokota and Nagata, 1974). Deposition of reaction product can only be obtained in our material by lowering the concentration of glutaraldehyde to 0.25% (v/v) and using a (very short) 5 min fixation period. It is also important to emphasize that 44 no deposition of reaction product occurred in mitochondria of these two microalgae when uric acid was omitted from the incubation medium. Trichloropurine has been extensively used in cytochemical studies as a control test for the identification of uricase-dependent reaction product deposition in organelles of a variety of plant c e l l s . Under these conditions staining is almost completely or even totally abolished (Huynh and Oliveira, 1989a, and references cited therein). In the case of both Dunaliella tert iolecta and Pavlova lutheri, no deposition of reaction product occurs in mitochondria when the cel l s are incubated in the presence of trichloropurine. The results are then consistent with those used by other authors for the cytochemical demonstration of urate oxidase activity in both plant and animal cells (see Angermuller and Fahimi, 1986; Huynh and Oliveira, 1989a for reviews). 3,3'-Diaminobenzidine (DAB) is a widely utilized substrate in the cytochemical localization of peroxidatic or oxidative activities of peroxidase, catalase and the mitochondrial cytochrome system, respectively (Frederick, 1987). Deposition of cytochrome dependent reaction products are known to be optimized at pH 6.0, in the absence or the presence of very low levels of H2O2 and at room temperature. The reaction is usually abolished or strongly inhibited by low concentrations of potassium cyanide. Reaction product deposition with these characteristics is also detected in mitochondria of Dunaliella tertiolecta and Pavlova lutheri 45 grown i n a l l four sources of n i t r o g e n (Table 8 ) . T h i s evidence suggests r e a c t i o n product to be formed as a consequence of the cytochrome system, i n c l u d i n g cytochrome c oxidase a c t i v i t y ( S i l v e r b e r g and Sawa, 1974; T a y l o r and H a l l , 1978; Olah and M u e l l e r , 1981). These r e a c t i o n s are d i s t i n c t from the r e a c t i o n observed i n mitochondria of both microalgae at pH 9.0 and a f t e r longer f i x a t i o n p e r i o d s . Under these c o n d i t i o n s , d e p o s i t i o n of r e a c t i o n products occurs only i n the presence of higher l e v e l s of H202 when the c e l l s are incubated at 37°C. The d e p o s i t i o n of r e a t i o n products i s completely a b o l i s h e d by a m i n o t r i a z o l e , a non-competitive i n h i b i t o r of c a t a l a s e , but i t i s not a f f e c t e d by potassium c y a n i d e , a non-competitive i n h i b i t o r of the m i t o c h o n d r i a l cytochrome system or by t r i c h l o r o p u r i n e , an i n h i b i t o r of u r i c a s e . These c h a r a c t e r i s t i c s are c o n s i d e r e d i n d i c a t i v e of the occurrence of c a t a l a s e a c t i v i t y i n mitochondria of both microalgae (Novikoff and G o l d f i s h e r , 1969; S i l v e r b e r g and Sawa, 1.974; van der Rhee et al., 1977; Olah and M u e l l e r , 1981). Furthermore,.as i n the case of u r i c a s e a c t i v i t y , c a t a l a s e -dependent d e p o s i t i o n of r e a c t i o n product i s only observed i n mitochondria of hypoxanthine-grown c e l l s (Table 8 ) . These f i n d i n g s c o n t r a s t with the cytochrome-dependent d e p o s i t i o n of r e a c t i o n products that i s observed i n mitochondria of both microalgae grown on a l l four sources of n i t r o g e n ; hence, they suggest that s y n t h e s i s of u r i c a s e and c a t a l a s e 46 and t h e i r l o c a l i z a t i o n i n mitochondria i s s u b s t r a t e dependent. An i n c r e a s e i n s t a r c h g r a n u l e s , c y t o p l a s m i c and c h l o r o p l a s t l i p i d s has been shown to occur i n a s s o c i a t i o n with aging in c e l l s of D u n a l i e l l a p r i m o l e c t a (Eyden, 1975) and D u n a l i e l l a t e r t i o l e c t a (Hoshaw and Maluf, 1981) grown p h o t o a u t o t r o p h i c a l l y . S i m i l a r o b s e r v a t i o n s have a l s o been r e p o r t e d i n aging c e l l s of both photoautotrophic and p h o t o h e t e r o t r o p h i c a l l y grown c u l t u r e s of Chroomonas s a l i n a ( A n t i a et a l . , 1973). However, the i n c r e a s e i n s t a r c h and l i p i d s observed i n a l l a n t o i c acid-grown c e l l s of D u n a l i e l l a  t e r t i o l e c t a can not be a t t r i b u t e d to aging s i n c e the c e l l s used i n t h i s study were obtained from the e a r l y e x p o n e n t i a l growth phase. Why such an i n c r e a s e i n s t a r c h and l i p i d s would only develop in a l l a n t o i c acid-grown c e l l s escapes our present understanding of the n u t r i t i o n a l e f f e c t s of d i f f e r e n t n i t r o g e n sources on c e l l u l a r u l t r a s t r u c t u r e . N e v e r t h e l e s s , the e f f e c t i s not a d e l e t e r i o u s one, s i n c e growth i n a l l a n t o i c a c i d c u l t u r e s proceeds as e f f i c i e n t l y as in n i t r a t e , urea or hypoxanthine-supplemented c e l l s . An i n t e r a c t i o n between c h l o r o p l a s t s and vacuoles with respect to the r e g u l a t i o n of n i t r o g e n metabolism was r e p o r t e d i n C h l o r e l l a ( T i s c h n e r , 1984). However, whether a s i m i l a r i n t e r a c t i o n can account f o r the i n c r e a s e s observed i n the volume d e n s i t y of the v acuolar compartments of both D u n a l i e l l a t e r t i o l e c t a and Pavlova l u t h e r i remains to be determined. 47 Conclusions In c o n c l u s i o n , i t can be s a i d that some microalgae possess the a b i l i t y to u t i l i z e p u r i n e s and/or t h e i r d e r i v a t i v e s as s o l e sources of N. The c a t a b o l i c o x i d a t i o n of these compounds seem to occur i n most of these microalgae by the standard pathway of pu r i n e o x i d a t i o n d e s c r i b e d f o r other microorganisms (Vogels and van der D r i f t , 1976) and higher p l a n t s (Reynolds et a_l. , 1982).. However, in the case of the Prymnesiophyte Pavlova l u t h e r i , o x i d a t i o n of p u r i n e s seems to take p l a c e without f i n a l c o n v e r s i o n to u r e a . T h i s i n d i c a t e s the occurrence i n t h i s microalga of a m o d i f i c a t i o n of the pathway of p u r i n e - d e r i v e d N u t i l i z a t i o n ( A n t i a et a l . , 1989) and resembles the s i t u a t i o n r e p o r t e d f o r soybean suspension c e l l c u l t u r e s (Winkler et a l . , 1987, 1988). The growth of Amphidinium in organic N-sources (urea and hypoxanthine) p l u s N i2 + produces major u l t r a s t r u c t u r a l changes. Some of the changes ( i . e . s i z e of p e r i c h r o m a t i n i c g r a n u l e s , number of dictyosome-derived v e s i c l e s , s i z e and d i s t r i b u t i o n of the v a c u o l a r apparatus) can be a t t r i b u t e d to mechanisms of metal homeostasis. Other changes ( i . e . i n c r e a s e s i n ER and microbodies) i n d i c a t e the occurrence of a mechanism f o r the c a t a b o l i c degradation of p u r i n e s s i m i l a r to that observed i n higher p l a n t s . The demonstration of both u r i c a s e and c a t a l a s e a c t i v i t i e s i n microbodies of hypoxanthine-grown c e l l s supports t h i s i n t e r p r e t a t i o n . 48 The growth of D u n a l i e l l a t e r t i o l e c t a and Pavlova l u t h e r i i n hypoxanthine a l s o produces u l t r a s t r u c t u r a l changes ( i . e . i n c r e a s e s i n E.R. and mitochondria) d i r e c t l y r e l a t e d to the occurrence of a mechanism f o r n i t r o g e n u t i l i z a t i o n through the c a t a b o l i c d egradation of p u r i n e s and t h e i r d e r i v a t i v e s . 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Van Nostrand Rei n h o l d Co., New York, 72 - 79. 6 2 .APPENDIX  FIGURE EXPLANATION Symbols: CH (or Ch)= chloroplast, ER = endoplasmic reticulum L = lipid inclusion, M = mitochondrion, m = microbodies, N = nucleus, n = nucleolus, Pu = pusule, Py = pyrenoid, V = vacuole Figure 1 - Growth curves of Hymenomonas elongata on urea (1 mM) without citrate addition (A), with 5 mM citrate (A) followed by the addition of 2 5 uM N i2 + at day 1 6 ( i ) , and also on allantoic acid ( 0 . 5 mM) without citrate addition (•), with 5 mM citrate (•) followed by the addition of 2 5 ^jM Ni2 + at day 16 ( 1 ) . Corresponding control growth on nitrate ( 2 mM) with or without citrate addition is shown (X) . Figure 2 - Growth curves of Amphidinium carterae on urea (1 mM) without citrate addition ( A ) , with 5 mM citrate (A).followed by the addition of 2 5 uM Ni2 + at day 14 ( T ) , and also on hypoxanthine ( 0 . 5 mM) without citrate addition (•), with 5 mM citrate ( O ) followed by the addition of 2 5 JJM N i2 + at day 10 (• t ) • Corresponding control growth on nitrate ( 2 mM) with or without citrate addition is shown ( X ) . 63 F i g u r e 3 - D e t e c t i o n of xanthine dehydrogenase a c t i v i t y i n c e l l - f r e e e x t r a c t s of Amph i d i n i um c a r t e r a e p r e v i o u s l y grown on 0.5 mM hypoxanthine i n the presence of 1 pM N i2 +. Absorbance measurements, at 340 nm f o r the enzyme or e x t r a c t ( O ) , enzyme + a l l o p u r i n o l ( A ) , enzyme + hypoxanthine (+) , and enzyme + hypoxanthine + a l l o p u r i n o l (•). Fi g u r e 4a - L o n g i t u d i n a l s e c t i o n through a c e l l grown i n 2 mM n i t r a t e and no n i c k e l . Arrow p o i n t s to ER i n the v i c i n i t y of c h l o r o p l a s t . F i g u r e 4b shows part of the pusule r e g i o n . F i g u r e 5 - L o n g i t u d i n a l s e c t i o n through a c e l l grown i n 1 mM urea and 1 pM n i c k e l . Arrow p o i n t s to ER i n the v i c i n i t y of the c h l o r o p l a s t . F i g u r e 6 - L o n g i t u d i n a l s e c t i o n through a c e l l grown i n 0.5 mM hypoxanthine and 1 pM n i c k e l . Arrowheads poi n t to m i c r o b o d i e s , while the arrow shows a microbody developing from ER - l i k e elements. F i g u r e 7 - S e c t i o n through a hy p o x a n t h i n e / n i c k e l - grown c e l l shows numerous mitochondria undergoing d i v i s i o n . Arrows p o i n t to ER elements. Fig u r e 8 - Nucleus of a h y p o x a n t h i n e / n i c k e l - grown c e l l . F i g u r e 9 - Microbody - ER a s s o c i a t i o n i n a hypoxanthine/ n i c k e l - grown c e l l . 64 F i g u r e 10 - Microbodies connected by a narrow t u b u l a r s t r u c t u r e (arrowhead) i n an hypoxanthine/ n i c k e l - grown c e l l . F i g u r e 11 - A s s o c i a t i o n s between dictyosome - ER i n a n i t r a t e - grown c e l l . T r a n s i t i o n v e s i c l e s are i n d i c a t e d by arrowheads. F i g u r e 12 - ER - dictyosome a s s o c i a t i o n s i n a u r e a / n i c k e l -grown c e l l . T r a n s i t i o n v e s i c l e s are i n d i c a t e d by arrowheads. F i g u r e 13 - ER - dictyosome a s s o c i a t i o n s i n a hypoxanthine/ n i c k e l - grown c e l l . Arrowhead p o i n t s to t r a n s i t i o n v e s i c l e s . F i g u r e 14 - D e p o s i t i o n of r e a c t i o n product (arrowheads) i n d i c a t i v e of u r i c a s e a c t i v i t y i n microbodies of a h y p o x a n t h i n e / n i c k e l - grown c e l l . F i g u r e 15 - D e p o s i t i o n of r e a c t i o n product i n d i c a t i v e of c a t a l a s e a c t i v i t y i n a microbody of a hy p o x a n t h i n e / n i c k e l - grown c e l l . F i g u r e s 16 to 19 - L o n g i t u d i n a l s e c t i o n s through c e l l s of D u n a l i e l l a t e r t i o l e c t a grown i n 2 mM n i t r a t e ( F i g u r e 16), 1 mM urea (Figure 17), 0.5 mM a l l a n t o i c a c i d (Figure 18) and 0.5 mM hypoxanthine 65 (Figure 19). Arrowheads p o i n t to elements of the endoplasmic r e t i c u l u m . F i g u r e s 20 to 23 - L o n g i t u d i n a l s e c t i o n s through c e l l s of Pavlova l u t h e r i grown i n 2 mM n i t r a t e ( F i g u r e 20), 1 mM urea ( F i g u r e 21), 0.5 mM a l l a n t o i c a c i d (Figure 22) and 0.5 mM hypoxanthine ( F i g u r e 23). Arrowheads p o i n t to.elements of the endoplasmic r e t i c u l u m . F i g u r e s 24 and 28 - Cytochemical l o c a l i z a t i o n of u r i c a s e a c t i v i t y i n mitochondria of D u n a l i e l l a t e r t i o l e c t a ( F i g u r e 24) and Pavlova l u t h e r i ( Figure 28) grown on hypoxanthine (0.5 mM) as s o l e source of n i t r o g e n . F i g u r e s 25 and 29 - Cytochemical i n h i b i t i o n of u r i c a s e -dependent r e a c t i o n product d e p o s i t i o n i n mitochondria of D u n a l i e l l a t e r t i o l e c t a ( F i g u r e 25) and Pavlova l u t h e r i ( F i gure 29) t r e a t e d with 2 mM t r i c h l o r o p u r i n e . . F i g u r e s 26 and 30 - Cytochemical l o c a l i z a t i o n of c a t a l a s e a c t i v i t y i n mitochondria of D u n a l i e l l a t e r t i o l e c t a ( F igure 26) and Pavlova l u t h e r i (Figure 30) grown on hypoxanthine (0.5 mM) as s o l e source of • n i t r o g e n . 66 F i g u r e s 27 and 31 - Cytochemical i n h i b i t i o n of c a t a l a s e -dependent r e a c t i o n product d e p o s i t i o n i n mitochondria of D u n a l i e l l a t e r t i o l e c t a ( F i g u r e 27) and Pavlova l u t h e r i ( F i g u r e 31) t r e a t e d with 3-3' a m i n o - 1 , 2 , 4 , - t r i a z o l e . Figure 1 - Hymenomonas elongata Nickel chelation with Urea/Allantoic acid as N-source O.6-1 X~"""* A A — A ^ - X A L egend A Urco-cH jk Ureq+cIJi>_ • AlJ.aeld-elt • All.ocld+clt X Nlt±clt , Growth Period [days] Figure 2 - Amphidinium carterae Nickel chelation with Urea/Hypoxanthine as N-sources 0 2 A 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Growth Period [days] Figure 3 - Amphidinium carterae Xanthine Dehydrogenase Activity 1-1 0.8-E c O 0.6-•<* £2, d) o c o •8 O 0.4-V) Si < ...+ + ..+ .+• 0.2 I,-'-" O'—o—o—o—o—o——o— 1 . . . A —o—o—o—0-7 -0—0— — 0 — 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1——I 1 1 1 1 1 1 1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Legend O |nr A Enz+AM + Enz+HyR • Enz+Hyp+AII Time [minutes] • 72 73 7L> 75 76 78 Table 1 - Microalgae used in the present study ALGA STRAIN/CLONE (isolator/supplier/source) PRYMNESIOPHYCEAE J-SQchrysiLs galbana  Hymenomonas elongata Prymnesium parvum Carter Pavlova lutheri Leftley, SMBA No. 58 Leftley, SMBA No. 62 Droop, SMBA No. 65 Leftley, SMBA No. 261 BACILLARIOPHYCEAE Thalassiosira nordenskioldii Cleve Rao, Bedford Institute, Darmouth , N.S. Thalassiosira pseudonana Neil Price, U.B.C. CHL OROPHYCEAE Dunaliella tertiolecta Butcher Wood Hole Oceanographic Institution CYANOPHYCEAE Agmflriellum quadruplicatum Van Baalen, ATCC No. 2726 4 DINOPHYCEAE Amphidinium carterae Hulburt J . McLachlan (Halifax NRC) CHRYSOPHYCEAE Olisthodiscus luteus Carter R.A. Cattolico, Seattle EUSTIGMATOPHYCEAE Nannochloropsis oculata Leftley, SMBA No. 66 Table 2 - Growth of microalgae on urea (1 mM) with nickel supplementation. (a) adaptation period (days); (b) exponential growth rate (%); (c) maximum yield (%) Growth parameters are expressed as percentage of those in media containing nitrate (2 mM) without nickel. (—) = no growth NICKEL SUPPLEMENTATION (uM) ALGA 0 4 s 10" -3 1 x 10" •2 1 x 10" 1 1 5 10 n u u n a b c a b c a b c a b c a b c a b c a b c Isochrysis galbana 4 109 91 5 110 91 5 106 89 5 105 90 5 110 91 5 103 88 5 102 89 Hvmenomonas elonaata 3 53 83 2 55 80 2 60 82 2 66 85 3 80 98 4 76 90 6 70 82 Prymnesium parvum 10 23 34 10 21 25 10 20 24 10 20 22 10 16 18 10 16 18 10 17 19 Pavlova lutheri 4 96 103 4 96 104 4 92 97 4 89 90 4 96 103 4 90 93 4 91 92 Thalassiosira nordenskioldi 12 73 79 2 80 85 2 84 86 2 105 103 2 133 117 2 120 110 2 118 110 Thalassiosira pseudonana 2 76 46 2 72 50 2 69 48 2 70 52 2 76 69 2 70 68 2 65 60 Dunaliella tertiolecta 2 122 110 4 92 110 4 88 106 4 89 108 4 77 110 4 80 103 4 81 105 Aamanellum quadruplicatum 2 102 103 5 112 110 5 107 103 5 109 107 5 112 110 7 106 101 7 108 103 AmDhidinium carterae — — — 9 48 65 6 69 95 6 68 80 6 50 68 Olisthodiscus luteus — — — 4 42 40 2 63 66 2 72 76 2 65 64 Nannochloropsis oculata 2 107 94 4 98 91 4 95 88 4 90 89 4 99 90 4 97 89 4 91 90 -SI Table 3 - Growth of microalgae on allantoic acid (0.5 mM) with nickel supplementation. (a) adaptation period (days); (b) exponential growth rate (%) ; (c) maximum yield (%) Growth parameters are exoressed as percentage of those in media containing nitrate (2 mM) without nickel. (—) = no growth NICKEL SUPPLEMENTATION (uM) 0 4 x 10" -3 1 x 10" -2 1 x 10" -1 1 5 10 a b c a b c a b c a b c a b c a b c a b c Isochrysis aalbana 4 99 74 5 99 74 5 96 71 5 95 73 5 99 74 5 91 69 5 90 70 Hymenomonas elonaata 4 65 76 4 70 78 4 80 89 4 79 88 4 83 93 8 77 86 8 70 72 Prymnesium parvum — — — — — — — Pavlova lutheri 6 115 109 6 115 113 6 101 102 6 99 101 6 115 110 6 97 100 6 95 101 Thalassiosira nordenskioldi A Thalassiosira pseudonana — — — — — — Dunaliella tertiolecta 2 78 83 4 85 84 4 82 80 4 81 83 4 85 84 4 79 78 4 80 81 Aamanellum quadruplicatum 2 98 55 5 110 55 6 102 52 6 101 51 5 109 55 10 98 51 10 99 52 AmDhidinium carterae — — — — — — — Olisthodiscus luteus — — — — — — — Nannochloropsis oculata 2 81 89 4 79 88 4 77 86 4 75 82 4 76 85 4 75 81 4 70 76 Table 4 - Growth of microalgae on hypoxanthine (0.5 mM) with n i c k e l supplementation. (a) adaptation period (days); (b) e x p o n e n t i a l , growth ra t e (%) ; (c) maximum y i e l d (%) Growth parameters are expressed as percentage of those i n media c o n t a i n i n g n i t r a t e (2 mM) without n i c k e l (—) = no growth NICKEL SUPPLEMENTATION (uM) ALGA 0 4 x 10" •3 1 x 10* -2 1 X 10"1 1 5 10 a b c a b c a b c a b c a b c a b c a b c Is o c h r y s i s qalbana __ Hymenomonas elonaata — — — — — — — Prymnesium parvum 4 84 83 4 86 85 4 87 81 4 83 85 4 84 83 6 81 83 6 80 83 Pavlova l u t h e r i 6 135 113 6 124 113 6 121 108 6 123 112 6 129 112 6 115 10 5 6 116 108 T h a l a s s i o s i r a n o r d e n s k i o l d i l » T h a l a s s i o s i r a pseudonana — . — — — D u n a l i e l l a t e r t i o l e c t a 2 79 110 4 79 112 4 82 110 4 76 105 4 79 112 4 78 109 4 83 104 Aamanellum quadruolicatum — — — — — — — Amnhidinium carterae — — — 7 71 58 4 95 90 4 89 72 4 72 54 Ol i s t h o d i s c u s luteus — — — — — . — — Nannochloropsis o c u l a t a 2 91 86 4 89 88 4 85 82 4 81 84 4 87 82 4 80 76 4 81 7 9 82 Safrle 5 - Optimal concentrations of n i c k e l ( i f r e q u i r e d ) , urea, a l l a n t o i c acid and hypoxanthine (where applicable) that support maximum yields of the microalgae (—) = no growth ALGA NICKEL (uM) UREA (mM) ALLANTOIC ACID (mM) HYPOXANTHINE (mM) Isochrysis aalbana 0 1 0.5 Hymenomonas elonqata 1 1 0.5 — Prymnesium parvum 0 4 — 2 Pavlova l u t h e r i 0 1 0.5 0.5 Dunaliella tertiolecta 0 1 0.5 0.5 Aqmanellum quadruDlicatum 0 1 0.5 — Amphidinium carterae 1 2 — 1 Nannochloropsis oculata 0 2 1 1 external addition of n i c k e l was not required 83 Table 6 - Concentrations (uM) of a l l o p u r i n o l ( A l ) , 2,6,8 - tr i c h l o r o p u r i n e (Tc) and hydroxyurea '(Hu) required for 50% i n h i b i t i o n of microalgal growth in media supplemented with hypoxanthine, a l l a n t o i c acid or urea as organic N-sources Alga Organic N - Source / In h i b i t o r s Urea A l l a n t o i c acid Hypoxanthine Hu Hu Al Tc Hu Isochrysis qalbana 1 1 - - -Hymenomonas elonaata 10 10 - - -Prymnesium parvum 10 - 1 5 10 Pavlova l u t h e r i 5 500 5 5 500 T h a l a s s i o s i r a n o r d e n s k i o l d i i 5 - - - -T h a l a s s i o s i r a Dseudonana 1 - - - -D u n a l i e l l a t e r t i o l e c t a 50 0 500 2 5 500 Aamanellum quadruplicatum 10 10 - - -Amohidinium carterae 1 - 1 10 1 Olisthodiscus luteus • 5 - - - -Nannochloropsis oculata 10 10 1 10 10 symbol (-) indicates that the microalga does not grow on that p a r t i c u l a r N-source 8 4 T a b l e 7 - X a n t h i n e d e h y d r o g e n a s e ( X D ) , a l l a n t o i n a s e ( A L N ) a n d a l l a n t o i c a s e ( A L C ) a c t i v i t i e s i n c e l l - f r e e e x t r a c t s o f m i c r o a l g a e g r o w n i n h y p o x a n t h i n e A l g a C r u d e E x t r a c t A c t i v i t i e s 1 XD A L N A L C A m p h i d i n i u m c a r t e r a e 3 . 1 + 0 . 2 1.9 ± 0.3 2.3 ± 0 .1 D u n a l i e l l a t e r t i o l e c t a 2 .2 ± 0.3 . 3.0 + 0.2 1.5 ± 0 .3 P a v l o v a l u t h e r i 2 . 9 ± 0.1 2.6 ± 0.2 1.7 ± 0 .2 1 E n z y m e a c t i v i t i e s a r e e x p r e s s e d a s n m o l p r o d u c t m i n - 1 mg p r o t e i n a n d r e p r e s e n t a v e r a g e s o f t h r e e s e p a r a t e a s s a y s Table 8 - Summary of the occurrence and characteristics of the cytochemical reactions observed in mitochondria of Dunaliella tertiolecta and Pavlova lutheri grown on four different sources of nitrogen Cytochemical Parameters Nitrogen Requirements Incubation Pre - Fixation Inhibitor Enzyme Source Substrate H2O2 pH Temperature C(%) T(mim) activity Nitrate None 6.0 22-27*C 1% Ga 30-60 KCN Cytochrome system Urea None 6.0 22-27°C 1% Ga 30-60 KCN Cytochrome system Allantoate None 6.0 22-27*C 1% Ga 30-60 KCN Cytochrome Bystem Hypoxanthine None 6.0 22-27 *C 1% Ga 30-60 KCN Cytochrome system Hypoxanthine Uric acid Yes 9.0 37 °C 0.25% Ga 5 TC/OX Uricase Hypoxanthine Yes 9.0 37 °C 1-2% Ga 60-90 AT Catalase Symbols: H 2 ° 2 = hydrogen peroxide, C = concentration of prefixative, Ga » glutaraldehyde T = time (duration) of prefixation, KCN = potassium cyanide, TC « tr ichloropur ine OX = oxypurines, AT = aminotr iazole 

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