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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 B.Sc,  KIM  HUYNH  The U n i v e r s i t y of B r i t i s h C o l u m b i a , 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 t h i s t h e s i s as conforming to the r e q u i r e d  THE  standard  UNIVERSITY OF BRITISH COLUMBIA February  O  Hanh  Kim  1989 Huynh,  1989  In presenting this thesis degree  at the  in partial fulfilment  of the  University of British Columbia, I agree that the  copying of this thesis for scholarly purposes or  by  his  for an advanced  Library shall make it  and study. I further agree that permission for extensive  freely available for reference department  requirements  or  her  representatives.  may be granted It  is  by the  understood  that  head of my 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  DE-6 (2/88)  TANUARi  n ^ / m  ABSTRACT  Eleven different to  grow  taxonomic on  as s o l e utilize  s p e c i e s of  the  t o grow  good g r o w t h The  that  hypoxanthine the s t a n d a r d  production  urea  l u t h e r i , growth  on  the  species  on  and In t h e  five  None was  of  to  these showed  able  sources  of  tests  utilizing  both  through  described in other  urea  not  one  is inhibited  The  oxidation  involve  pathway d i f f e r e n t  by  conversion  species, urease  or hypoxanthine  inhibitors.  the c a t a b o l i c  subsequent of  urea from  results of  to  f o r the  inhibitor  of  urea  able  catabolize purines  case  urease  does  and  s i x of  different  capable  its  presence, of  a  the  ability  requirements  results  in a l l a n t o i c acid  through  only while  study of n i c k e l  while growth  derivatives  but  were  six  h i g h e r p l a n t s . T h i s pathway l e a d s t o  ammonium.  t h i s case  species  acid,  allantoic acid  and  of  utilizable  hypoxanthine  pathway o f p u r i n e o x i d a t i o n  microorganisms  for their  in hypoxanthine.  with  those and  All  of urea  these microalgae  nitrogen, together suggest  acid,  on a l l a n t o i c  allantoin.  growth of  nitrogen.  n i t r o g e n atoms  belonging to  were t e s t e d  allantoin, allantoic  moderate t o utilize  divisions  s o u r c e s of  were a b l e  marine m i c r o a l g a e  inhibitors,  suggest  production  that observed  to  Pavlova  occurs  purines  the  i n the that  and and  in  their occurs  i n the  other  species. Cells  of  undergo major  Amphidinium  carterae  ultrastructural  grown  changes.  on These  hypoxanthine affect  the  perichromatinic derived  vesicles,  reticulum, the the  size  of  these  granules, the  the dictyosomes distribution  number o f  mitochondria  ultrastructural reticulum  changes,  and t h e  cytochemical demonstration  catalase  activities  occurrence  in  Dunaliella  also  These a f f e c t  interesting  key  in  step  oxidation  into  of  purines.  Pavlova  of  support  Cells  of  both  grown  on  changes.  i s particularly  in  that  these  organelles.  no m i c r o b o d i e s a r e  and t h a t u r i c a s e c o n t r o l s t h e  their  and H2O2  apparent  participate  p u r i n e s and  the  reveal the presence of  activities  i t becomes  degradation  and  reticulum, mitochondria  tests  consideration  mitochondria  in  along  uricase  lutheri  of the formation of a l l a n t o i n urate,  increase  both  on m i t o c h o n d r i a  these microalgae  of  as  major u l t r a s t r u c t u r a l  cytochemical  microalgae  role  and  and c a t a l a s e  takes  such  the endoplasmic  The e f f e c t  since  uricase  major  and m i c r o b o d i e s , and  o f t h e s t a n d a r d pathway f o r  of  undergo  mainly  vacuoles.  observed  endoplasmic  microbodies,  degradation  tertiolecta  hypoxanthine  When one  within  these microalgae  catabolic  both  the  number o f m i c r o b o d i e s ,  with the  and  dictyosome-  a n d d i s t r i b u t i o n o f t h e v a c u o l a r c o m p a r t m e n t . Some  endoplasmic  the  of  and  in  that  in  the  oxidative  derivatives  i n t h e o r g a n i c N-budget o f t h e s e  through the  and  these  play a  microorganisms.  iv TABLE  OF  CONTENTS:  ABSTRACT  ii  ACKNOWLEDGEMENT  V  INTRODUCTION  1  MATERIALS & METHODS  5  RESULTS  10  DISCUSSION  • 26  REFERENCES  49  APPENDIX  62  V  ACKNOWLEDGEMENT  I my  w i s h t o e x p r e s s my s i n c e r e  research  supervisor,  s u p p o r t e d my work possible, to facilities his  in  technical  i n the l a s t  Dr. T.  W.  HUNTER a n d  Last  but not l e a s t ,  and  brother  my  education.  for  a l l my my s t a y  a l l of the people  h e l p e d me i n t h e l a s t  OLIVEIRA,  who  letting  me  has  thesis  use the  l a b o r a t o r y , t o M r . M. WEIS f o r .  assistance, to me d u r i n g  LUIS  few y e a r s a n d made t h i s  BISALPUTRA  h i s research  always encouraged Mr.  Dr.  thanks and a p p r e c i a t i o n t o  friends  i n t h e l a b who  i n the l a b o r a t o r y , t o i n the o f f i c e  who have  few y e a r s . t o my Mom, D a d , a n d a l l o f my  who a l w a y s e n c o u r a g e d me t o p r o c e e d  sisters  further  with  1  INTRODUCTION  The a v a i l a b i l i t y of nitrogen for phytoplankton coastal and measuring  open  -  the  compounds. As approach  ocean  waters  concentrations  pointed out  overlooks  the  present in . seawater.  i s often of  by Antia  large  assessed  inorganic et a l .  pool  of  growth in by  nitrogen  (1975) such an  organic  nitrogen  Apart from ammonium and n i t r a t e , the  two most common sources of inorganic nitrogen (Syrett, 1962; Morris,  1974),  compounds as  many  algae  can  utilize  various  sole nitrogen sources for phototrophic growth.  Although organic-N n i t r a t e , many  compounds are not always as effective as  of these  show the  potential for supporting  s i g n i f i c a n t growth (see Antia et a l . , 1989, Urea  organic  i s an  phytoplankton  important  growth  organic  (Oliveira and  also be  endogenously produced  such as  the aerobic  nitrogen  source  A n t i a , 1984)  through  metabolic  that  for can  pathways  degradation of purines (Naylor, 1970).  Urea u t i l i z a t i o n  by algae  alternative urea  - degrading  ATP:urea amidolyase.  for a review).  involves the enzyme -  The evidence  production of  two  systems: Urease  or  shows that a given algal  species may produce either urease or ATPrurea amidolyase but not both  enzymes and that ATP:urea amidolyase i s r e s t r i c t e d  in occurrence  to  some  Chlorophycean  algae  Syrett, 1977; Al-Houty and Syrett, 1984). the phototrophic algae with  growth of  urea serving  several marine  as sole  (Bekheet  and  Recent studies on phytoplanktonic  source of nitrogen showed  2 that growth  could only occur, in most cases, on addition of 2+  minute amounts  of N i  Antia,  1986a).  1984,  findings i s  particular  interest  that nickel  1977). A  to  these  i s a constituent of  urease (Dixon  also reported  nickel requirement for  Rees and Bekheet, 1982;  Antia  et a l .  (1989), in  dissolved organic  et a l . ,  and/or  nitrogen.  on the their  some  for urease  microalgae  (Syrett,  O l i v e i r a and A n t i a , 1986b). their review  nitrogen in  widespread a b i l i t y purines  (Oliveira and  i s required for urease a c t i v i t y in soybean tissue  a c t i v i t y was  such as  growth media  ensiformis L.)  cultures (Polacco,  1981;  Of  the discovery  jackbean (Canavalia 1975) and  to the  on the  role of  phytoplankton n u t r i t i o n show  part of  microalgae  derivatives  as  to  sole  utilize  sources  of  The u t i l i z a t i o n of purines and purine derivatives hypoxanthine, xanthine,  nitrogen sources microalgae has  for now  d i f f e r e n t taxonomic 1983; Shah  the  been  phototrophic established  be important  marine environments, and Marler,  growth for  of  marine  organisms  from  divisions (Antia et a l , , 1980a; Prasad,  and Syrett, 1984a).  considered to  uric acid and a l l a n t o i n as  Some of these compounds are  organic N  sources  in  certain  p a r t i c u l a r l y inshore areas (van Baalen  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  conversion to  u t i l i z a b l e ammonium  urea and i t s subsequent (Antia  et  a l . , 1989).  3 However, as  pointed out  (1989), the  information available  fragmentary and  r e s t r i c t e d in  exclusively with 1980b).  The  by Prasad  for the algae i s at best  most cases  growth studies  validity  of  (1983) and Antia et a l .  to tests dealing  (see also  the  growth  Antia  tests  e_t a l . ,  is  further  complicated by the fact that inappropriate concentrations of 2 1  Ni " "  in the  regarding  culture medium may lead to misinterpretations  the  ability  intermediates of  of  the pathway  the  algae  as sole  to  utilize  the  sources of nitrogen.  Consequently, the pathway of catabolism of purines and their derivatives in algae i s far from understood. Equally unknown is the  importance of  the organic which are pools of  the intermediates  N-budget of  both coastal  of this pathway on and. estuarine  areas  characterized by the seasonal occurrence of large unidentified organic  - N  (Butler et  a l . , 1979;  V a l i e l l a and Teal,,1979). In  this thesis,  species of  marine phytoplankters  taxonomic d i v i s i o n s a l l a n t o i c acid evidence  I report on the N i  and urea  suggests  u t i l i z a t i o n may (Winkler et  belonging to on hypoxanthine,  of 11  6 different allantoin,  that  in  higher  plants,  purine  N  also proceed without the production of urea 1988), I have also conducted growth  enzymatic  mechanism(s) of  microalgae.  requirement  as sole sources of N. Since recent  a l . , 1987,  i n h i b i t i o n and  responsible  for growth  2 +  for  studies  oxidation of the  growth  to  help  elucidate  the  purines and their derivatives of  these  11  species  of  4 I  have also  come to  realize that  u l t r a s t r u c t u r a l implications purines  have  been  well  studied  Webb  and  e_t a_l. ,  Newcomb,  microorganisms p a r t i c u l a r l y also report marine  in  higher  plants,  relying on nodular nitrogen  1985; Kaneko  1987),  of the  of the catabolic oxidation of  p a r t i c u l a r l y in higher plants f i x a t i o n (Newcomb  while some  no  and Newcomb, 1987;  such  studies  the microalgae.  exist for  Therefore, I  on the ultrastructure and cytochemistry of the  dinoflagellate  Amphidin ium  Chlorophycean  alga  Prymnesiophyte  Pavlova  allantoate and  hypoxanthine  insight  the mechanism(s)  into  derivatives - N  Dunaliella lutheri  carterae,  tertiolecta grown  on  in order of  the and  nitrate,  to gain  purine-  and  u t i l i z a t i o n by these microalgae.  the urea,  further purine  5  MATERIALS AND  Algal  METHODS  species  Stock  cultures  routinely  of  maintained  standard axenic  the on  algae  nitrate  conditions,  listed (as  in  Table  nitrogen  according  1  were  source),  under  to A n t i a  and  Cheng  (1970).  Growth All  tests t e s t s were c a r r i e d  continuous  illumination  from c o o l  - white  medium was salinity luteus  that  and  Ni*  ,  was  (irradiance  to  26% 0  hydroxyurea  before culture  urea,  carried  experiments are were f i l t e r Nalgene the  tests  to  Growth periodically  out.  The  and  monitored  reading  uE.m~ .s standard  (1984) of  final  salinity  (0.2  acid,  the  of  the  stated,  allantoin,  2,6,8-trichloropurine 4 mL  aliquots  of  pore  of  the  test  media  algal  stock  utilized  2 t h r o u g h 6. size  A l l test  in  the  media  presterilized  t e c h n i q u e s were u s e d  growth  )  Olisthodiscus  made t o  jam  -1  test  with  case  concentrations  in Tables  ensure axenic was  mL  aseptic  2  100  The  18°C under  14%© . U n l e s s o t h e r w i s e  were  0.2  - sterilized  filters)  the  allopurinol,  with  given  -  allantoic  additions  inoculation was  at  tubes at  Antia  tertiolecta,  hypoxanthine, c i t r a t e , and  and  . In  retained  nitrate,  95  lamps.  01 i v e i r a  Dunaliella  t e s t medium  in culture  fluorescent  of  adjusted  out  throughout  conditions.  directly  in  their optical density  culture at  600  tubes nm  by  (OB^QQ)  6  on  a  spectrophotometer a f t e r  growth t e s t source. was  included a  under t h o s e against  c o n t r o l without  f o r the  the  number o f  incubation three  days  increase  in  increase  in  600  nitrate  P  similar  e r  ^  a  v  curve  from  on  growth  equation  mM h y p o x a n t h i n e  =0.5  Cell  acid  = 1  mM u r e a  rate =  = 2  was  maximum  phase  of  rate  from  = maximum  ODgQQ on  percentage  nitrate.  The defined  mM a l l a n t o i n  mM n i t r a t e  =  significant  of corresponding  f o r equivalent  allantoic  the f i r s t  e x p r e s s e d as  growth  growth  period  exponential  stoichiometry 0.5  adaptation  maximum y i e l d  for a test,  measured  corrected  growth  during  sources  were u s e d t o c a l c u l a t e  (1)  exponential  and (3)  yield  period  as percentage  control,  growth  time  Every  nitrogen  i f any,  of the  inoculation to  OD600' (2) OD  Plots  parameters:  from  growth, expressed  Antia,  mixing.  any a d d e d  r e s i d u a l growth,  circumstances.  following  the  vortex  The g r o w t h m e a s u r e d on e x p e r i m e n t a l n i t r o g e n  corrected  the  brief  of  nitrogen by  the  = 0.5 mM  ( O l i v e i r a and  1984).  free  extracts  Preparation determination to the  of c e l l of  - free e x t r a c t s of the microalgae f o r  enzyme a c t i v i t i e s  procedure described  by  conducted  was c o n d u c t e d  Shah  and  Enzyme a s s a y s  were  (Stirpe  and  Della  Beevers,  1971),  a l l a n t o i n a s e and  Marzluf,  1975).  Whenever an enzyme a c t i v i t y  Corte,  Syrett  for  xanthine  1969),  uricase  according (1984b).  dehydrogenase (Theimer  and  a l l a n t o i c a s e ( R e i n e r t and was measured i t  7  was  established  period cell  of  -  the  and  directly  extract  added t o  assay  free  details  that  are  Electron  given  for electron,  always c a r r i e d growth to  to  for  2.5%  the  Further  7).  the  same t i m e of  impact of  and  appropriate  and  1 9 6 1 ) , were  1.5  Pavlova to  2  after  each  stained  with  et a l . ,  lutheri.  rinsed  (0.17  by 2% M)  in  1% the  methanol  812  (Luft, (BSA)  s e c t i o n s were  of u r a n y l a c e t a t e 1963).  cells  concentration  serum a l b u m i n  1989). U l t r a t h i n  7.4)  4°C w i t h  thoroughly  (Epon)  bovine  (Reynolds,  The  i n a graded  i n Polybed  a saturated solution  lead c i t r a t e  4 °C w i t h  (0.1M, pH  fixation. Final  the  of  c o n d i t i o n s on  hours at  to dehydration  conducted using  (Oliveira  m e t h a n o l and  and  embedding  stage  phosphate  buffer solution  buffer  technique  culture  and  were  of A m p h i d i n i u m c a r t e r a e or  same b u f f e r and  final  day  f o r 3 hours at  i n the  samples, p r i o r  the  in a saline  f o r the c e l l s  tertiolecta  the  observations  Samples were c o n c e n t r a t e d  fixed  post-fixed for  Os04  series  time over  mixture.  microscopic  sodium c a c o d y l a t e  Dunaliella  the  the assay (Table  glutaraldehyde  (PBS-pH 7.4)  were t h e n  of  with  p r o p o r t i o n a l t o t h e amount of  ultrastructure.  (v/v)  a saline  (v/v)  at the  centrifugation  buffer in  out  minimize  cellular  gentle  text  linear  Microscopy  Fixations  the  i n the  r a t e was  in  50%  8 Cytochemistry Uricase  cytochemistry.  centrifugation and  Cells  fixed b r i e f l y  (v/v) formaldehyde.  They were  were  for 5  concentrated minutes  preincubated in  medium for 5 minutes. This medium contained mM 3-amino-1,2,4Pipes buffer extra 30  t r i a z o l e and  in  by  0.25%  the control  3 mM CeCl3, 50  0.001% Triton X-100 in 0.1M  (pH 9.4). The c e l l s were then incubated for an  minutes at  37 °C  in the experimental medium which  was i d e n t i c a l to the control one, except for the addition of 0.1 mM  uric acid  washing the fixed in  (Angermuller  c e l l s thoroughly  2% formaldehyde  buffer, followed for 1.5  after each  1986).  After  buffer, they were  hours at 4°C with the same  fixation with 1% osmium tetroxide  4°C. The samples  fixation,  Fahimi,  with Pipes  for 2  by post  hours at  and  dehydrated  were rinsed  with  a  graded  in buffer methanol  series and embedded in Polybed 812. Control experiments were conducted in oxypurine  the  presence  (2 mM), two  Catalase cytochemistry. of catalase, in the (0.17 M) in  a  the c e l l s  appropriate  2,6,8 - trichloropurine or  competitive i n h i b i t o r s of uricase.  For the cytochemical l o c a l i z a t i o n were fixed with 2.5% giutaraldehyde  buffer,  i . e . in a  saline  phosphate  buffer (PBS - pH 7.4) for Amphidinium carterae and saline  t e r t i o l e c t a and in the  of  sodium  cacodylate  Pavlova l u t h e r i ,  same buffer  for 30  buffer  for Dunaliella  for 2.5 hours  minutes  and rinsed  at 4*C. The c e l l s were  then incubated in the standard 3,3' - diaminobenzidine  (DAB)  incubation  medium  (Frederick  and  DAB  (Sigma  and  prior  to  the  buffer 0.2 ml  o f 3%  medium c o n t a i n e d  1% osmium  20 mg  S t . L o u i s , M o . ) , 10 ml o f 0.05  H 2 0 2 . The  of the c e l l s .  were r i n s e d  in  This  shaker  (2-amino-2-methyl-1,3-propanediol)  incubation  cells  fixed  Newcomb, 1 9 6 9 ) .  Chemical Co.,  propanediol 10.0,  f o r 60 m i n u t e s a t 37°C on a r o t a r y  f o r 30  a t pH  adjusted  Following  in buffer  tetroxide  pH was  the  mM  t o 9.0  incubation,  minutes and p o s t -  i n the appropriate  buffer for  0  1.5 h o u r s for  electron  Control the  a t 4 C. A f t e r microscopic  observation  e x p e r i m e n t s were c o n d u c t e d  incubation  catalase  r i n s i n g , t h e s a m p l e s were as  described  by e l i m i n a t i n g  medium o r by a d d i n g  processed above.  ^2^2  r  r  o  m  3-amino-1,2,4-triazole, a  inhibitor.  Morphometry Calculation  o f t h e volume d e n s i t i e s  i n d i v i d u a l components endoplasmic  per t o t a l  reticulum,  c y t o p l a s m volume)  mitochondria,  v a c u o l e s was c a r r i e d o u t i n e l e c t r o n a  final  magnification  procedures  described  o f 36,000 X by  Oliveira  1967). the  t  t e s t was u s e d  Two means were c o n s i d e r e d  p r o b a b i l i t y of e r r o r  was  of  peroxisomes  the and  micrographs enlarged to  following  compare means o f p a r a m e t e r s o b t a i n e d regimes, Student's  ( i . e . t h e volume o f  and  the s t e r e o l o g i c  Fitch  (1988).  from d i f f e r e n t  To  nitrogen  ( S n e d e c o r and C o c h r a n ,  to d i f f e r  p < 0.05.  significantly i f  RESULTS  I)  Physiological studies Eleven  species  classes of utilize  of  microalgae  phytoplankton were  different  representing  different  tested for their a b i l i t y to  components  of  the purine  pathway, i . e . hypoxanthine, a l l a n t o i n ,  catabolism  allantoic  acid  or  urea, as the sole source of nitrogen (Table 1). Table  2 shows  species  the results of the growth on urea of a l l  tested.  nordenskioldi i ,  Pavlova  lutheri,  Dunaliella  quadruplicatum, Hymenomonas Isochrysis galbana  Thalassiosi ra  tertiolecta,  Aqmenellum  elongata, Amphidinium carterae,  and Nannochloropsis  oculata showed good  growth on urea. Both their growth rates and c e l l yields were approximately  equal  to  those  Thalassiosira pseudonana and able to  grow on  species, while  urea but  from  equivalent  01isthodiscus luteus were also  less e f f i c i e n t l y  Prymnesium  nitrate.  than the other  parvum showed very  poor growth  rates and c e l l y i e l d s . Nickel  - dependency for growth on urea was demonstrated  in Amphidinium growth  was  carterae and  observed  01i sthodi scus luteus  in the absence  or  at  since no  low  nickel  concentrations (up to 0.01 uM). Growth on urea was improved by 63%  for Thalassiosira  nickel supplementation. 25%)  in urea-grown  nordenskioldi i by Growth  cells  Thalassiosira pseudonana  of  was  also improved (20 to  Hymenomonas  when nickel  increasing the  was  elongata  added  into  and the  culture medium-. In contrast, t e r t i o l e c t a , Aqmenellum and Nannochiorops is without nickel very poor  1 uM  Ni  on  showed  good  growth  galbana on  urea  urea with or without the addition of  growth rates  (Table 2).  and c e l l . yields were recorded at  for Amphidinium carterae and these values started  to decrease 10 uM  Dunaliella  supplementation. Prymnesium parvum displayed  nickel to the culture medium  2 +  lutheri,  quadruplicatum, Isochrysis  oculata  growth  Maximum  Pavlova  above this  Ni* . However,  nickel concentration, e s p e c i a l l y at in 01Isthodiscus luteus, both maximum  growth rates  and c e l l yields were approximately equal at 1,  5 and  Ni  10 uM  values for at  1  2 +  . In Thalassiosira nordenskioldi i,. maximum  both growth  uM Ni  ,  with  a  parameters occurring improvement in -grown c e l l s  very at  slight  5 and  decrease  in  these  10 uM n i c k e l . Only a small  c e l l y i e l d values (20%) was observed in urea  of Thalassiosira  compared with  pseudonana with  higher concentrations.  differences were  were  1 uM nickel  In t h i s case no major  noted in the values of the growth rate. In  Hymenomonas elongata, c e l l yield  rates and c e l l y i e l d s were obtained  maximum  values for  obtained at 1 uM N i  2 +  growth rate and  and they started to  decrease at higher concentrations (Table 2). In only a  a l l of the species tested, urea u t i l i z a t i o n short adaptation  except for 10 days  lag period  Prymnesium parvum  was observed.  quadruplicatum and  requires  (between 2 to 4 days),  where an adaptation period of  Dunaliella  Nannochloropsis  tert i o l e c t a , oculata  Aqmenellum  showed  a  lag  period at compared  least twice as long in nickel-supplemented medium with  cultures  additions. The with  grown  adaptation  increases  oh  urea  without  period decreased  in nickel  by 40 to 100%  supplementation  in  Amphidinium carterae and Olisthodiscus luteus Table  nickel  both  (Table 2 ) .  3 shows the growth results of the same microalgae  on a l l a n t o i c a c i d . Hymenomonas elongata, Pavlova l u t h e r i and Nannochloropsis oculata acid.  Both  maximum  Hymenomonas elongata equivalent urea. for Pavlova  were d i f f e r e n t . cultures of  and  on  cell  allantoic yields  of  were similar  Nannochloropsis oculata  or a l l a n t o i c  when they  acid , their growth rates  rates of  allantoic  acid-grown  Pavlova lutheri were s l i g h t l y higher (20%) than urea, while  the opposite situation was detected  oculata, i . e . the growth rates of urea-  were higher  (25%) than  those  from  allantoic  D u n a l i e l l a tert i o l e c t a , Agmenellum quadruplicatum and  Isochrysis galbana but  rates  growth  were approximately equal to those from  The growth  in Nannochloropsis  acid.  good  Although the maximum yields  on urea  grown c e l l s  growth  l u t h e r i and  were grown  those from  showed  less  efficiently  approximately 30% and c e l l  than  in urea.  A  decrease  of  was detected in both maximum growth rates  y i e l d s of a l l a n t o i c acid-grown c e l l s of Dunaliella  tert i o l e c t a , while parameters was with those  were also able to grow on a l l a n t o i c acid  a 15%  decrease in the value  of  these  recorded in Isochrysis galbana when compared  from equivalent urea growth. Although the growth  rates remained  equal in urea-  and  allantoic  acid-grown  cells  of Aqmenellum  decreased by  quadruplicatum,  the maximum  yields  50% in a l l a n t o i c acid c u l t u r e s . Growth was not  detected in Prymnesium parvum, Thalassiosira n o r d e n s k i o l d i i , Thalassiosi ra  pseudonana,  Olisthodiscus luteus  Amphidinium  carterae  and  when a l l a n t o i c acid was added into the  culture medium as the sole source of nitrogen. Both growth rates and c e l l yields were s l i g h t l y improved (15 to  20%) in a l l a n t o i c acid-grown  c e l l s of  Hymenomonas  elongata when nickel was added into the medium. The maximum 2  1  values for these parameters were obtained at 1 uM Ni " ", and they started (5 and  10  to decrease uM).  At  microalga displayed  at higher  these  nickel  higher  an adaptation  concentrations  concentrations,  lag period  of twice the  duration of  the one from lower nickel concentrations  or  In  lower).  contrast,  t e r t i o l e c t a , Agmenellum and Nannochloropsis for nickel  Pavlova  lutheri,  (1 ^iM  Dunaliella  quadruplicatum, Isochrysis  oculata showed  this  galbana  no signs of requirement  supplementation since growth improvement was not  recorded when  nickel was  a l l a n t o i c acid  added into  the medium containing  as the sole source of nitrogen. In f a c t , the  adaptation lag period increased in duration when nickel was added to a l l a n t o i c acid-cultures Agmenellum  quadruplicatum,  of D u n a l i e l l a t e r t i o l e c t a , Isochrysis  galbana  and  Nannochloropsis oculata,  while no  Pavlova l u t h e r i .  observations are similar to those  These  reported for urea-grown cultures  changes were detected in  (Table  3 compare  with  Table 2).  None of  the species  tested grew on a l l a n t o i n  with or without nickel supplementation. Table 4 shows the results of the growth of the microalgae on hypoxanthine.  Prymnesium parvum  on hypoxanthine urea. An  when compared  increase of  displayed higher growth  with those  more.than  from  equivalent  50% in both maximum growth  rates and  c e l l yields  and  in maximum yields and growth rates, r e s p e c t i v e l y ,  30%  was also  detected  was observed.  An improvement of 10%  in hypoxanthine-grown  c e l l s of Pavlova  l u t h e r i when compared with urea-grown c u l t u r e s . Although the maximum y i e l d s  were approximately  hypoxanthine-growth carterae and  A 15%  growth rate values and Dunaliella 25%  observed  in  nordenskioldi i ,  hypoxanthine  Amphidinium growth  rates  were observed in Nannochloropsis oculata respectively, while an increase  in Amphidinium Hymenomonas  was  carterae. Growth elongata,  Thalassiosi ra  quadruplicatum, Olisthodiscus when  their  and  and '40% decrease in.the exponential  tert iolecta  was noted  in urea-  in Dunaliella t e r t i o l e c t a ,  Nannochloropsis oculata,  were d i f f e r e n t .  of  the same  was not  Thalassiosira  pseudonana,  Aqmenellum  luteus and Isochrysis galbana  utilized  as  the sole  source  of  nitrogen. Nickel-dependency demonstrated  for growth  on hypoxanthine  in Amphidinium carterae,  was only  since no growth was  observed in the absence or at low nickel concentrations (up to 0.01  uM) . Maximum  recorded at  1 uM  Ni  growth rates 2 +  and  cell  yields  were  and these values started to decrease  above this  concentration, especially  adaptation lag period of decreased by added into (5 and  this  1 0 JJM  Ni  z  ).  uM  Ni  2 +  hypoxanthine-grown  approximately 75%  the medium at  at 10  .  The  culture  in duration when nickel was  1 uM  or above this optimal l e v e l  Prymnesium parvum,  Pavlova luther i ,  Dunaliella t e r t i o l e c t a 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  adaptation period of Pavlova  changes  was  t e r t i o l e c t a and  of the  were detected in hypoxanthine-grown c e l l s  l u t h e r i , while  when nickel  in the duration  added  the lag period was twice as long  into  the cultures  Nannochloropsis  oculata•  of  Dunaliella  In  Prymnesium  parvum, the lag period increased by 50% at 5 and 10 uM N i when compared with lower nickel concentrations The  optimal concentrations  hypoxanthine and  nickel that  of  urea,  luther i ,  quadruplicatum and  Dunaliella  for  allantoic  acid,  Hymenomonas elongata,  tert i o l e c t a ,  Agmenellum  Isochrysis galbana displayed the highest  values for growth rate urea.  (Table 4 ) .  support maximum growth of the  microalgae are reported in Table 5. Pavlova  2+  and y i e l d when grown with 1.0 mM of  In contrast, 2.0' mM of urea supported optimal growth Amphidinium  carterae  P a r t i c u l a r l y noticeable response of  and  in t h i s  Prymnesium parvum  improvement in growth rate supplemented with  Nannochloropsi s respect  which  was  showed  and maximum  oculata.  the growth the greatest  y i e l d values  when  4.0 mM urea. The optimal concentration of  a l l a n t o i c acid  for those  organic compound determined to  as  species  the sole  be 0.5  mM for  able  source  to of  utilize  this  nitrogen  was  Hymenomonas elongata, Pavlova  luther i , D u n a l i e l l a  tert i o l e c t a , Aqmenellum quadruplicatum,  Isochrysis galbana,  and 1.0.mM for Nannochloropsis oculata.  In  the case  of  hypoxanthine,  concentration that  supported  Dunaliella tert i o l e c t a ,  1.0 mM  0.5  mM  was  the optimal  growth  of  Pavlova  for Amphidinium  lutheri, carterae,  Nannochloropsis oculata, and 2.0 mM for Prymnesium parvum.  II)  Chelation studies Nickel c h e l a t i o n , using c i t r a t e as the chelator, was also  c a r r i e d out metal i o n . growth on  to study In those  organisms showing  urea with  inhibited when  microalgal dependency  as the  i n h i b i t i o n could  nickel into the culture media  supplementation  ,  by  added into  was  the medium  the addition  of  excess  (Figs. 1 and 2). In contrast,  showed no  the addition  did not affect either  growth  sole source of nitrogen. The growth  be reversed  in the microalgae that  trace  improvement or good  nickel supplementation,  c i t r a t e (5 mM) was  containing urea  on t h i s  requirement  for nickel  of c i t r a t e into the medium  their growth rates or  cell yields.  Growth reduction or i n h i b i t i o n was not detected when c i t r a t e was added Dunaliella  into a l l a n t o i c tert iolecta,  Isochrysis galbana  acid-cultures of Pavlova l u t h e r i , Agmenellum  and Nannochloropsis  quadruplicatum, oculata.  However,  growth i n h i b i t i o n was recorded in a l l a n t o i c acid-grown  cells  of Hymenomonas elongata. The i n h i b i t i o n addition of  excess  Growth i n h i b i t i o n citrate  was  nickel  into  was detected  added  into  was reversed by the (Fig.  the medium  1).  in Amphidinium carterae when  the culture  medium  containing  hypoxanthine as the sole source of nitrogen. This i n h i b i t i o n was also  reversed by the addition of excess nickel into the  medium  ( F i g . 2 ) . No changes  c i t r a t e was parvum,  added into  Pavlova  in growth were observed when  hypoxanthine cultures of Prymnesium  luther i ,  Dunaliella  tert i o l e c t a  and  Nannochloropsis oculata.  Ill)  Inhibition studies  Growth  i n h i b i t i o n studies were also c a r r i e d out in this  investigation.  Allopurinol,  hydroxyurea were culture medium source  added at  (Table  6 ) . The  capable of  by  growth was  into the  a l l species  of  tert i o l e c t a ,  Hydroxyurea-dependent  also observed among those species  growing with a l l a n t o i c acid and/or hypoxanthine,  Growth  Dunaliella tertiolecta  inhibition  a l l o p u r i n o l was  added to  where required)  cultures of  carterae  of  of Dunaliella  hydroxyurea.  with the exception of luther i .  urea-growth  the exception  inhibited  i n h i b i t i o n of  various concentrations  and  containing the appropriate organic nitrogen  microalgae, with was  2,6,8-trichloropurine  and  hypoxanthine  allopurinol  when  Pavlova  1.0 uM of  (nickel was  added  Prymnesium parvum, Amphidinium  Nannochloropsis  concentrations of  occurred  and  oculata. were  Slightly  required  higher  for Pavlova  18  lutheri (5  uM)  and  for growth  to be  Dunaliella t e r t i o l e c t a (2 uM) in order  inhibited. A  i n h i b i t i o n responses when  was also  similar pattern detected  of  in - these  growth species  2,6,8~trichloropurine was added to hypoxanthine-grown  cultures.  IV)  Enzymatic studies Cell-free  extracts of  tert i o l e c t a and  Amphidinium carterae, Dunaliella  Pavlova lutheri  dehydrogenase,  uricase,  a c t i v i t i e s . No  a c t i v i t y could  were examined for xanthine  allantoinase  and  allantoicase  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 , activities  in  i t was  possible  hypoxanthine-grown  to  detect  cells  of  a l l other these  three  species of microalgae (Table 7 ) . No i n h i b i t o r s 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 a l l o p u r i n o l ( F i g . 3).  V)  U l t r a s t r u c t u r a l and In  Morphometric  studies  nitrate-grown control c e l l s of Amphidinium carterae,  the nucleus always displays condensed chromosomes completely surrounded by 30 nm  perichromatinic granules  in diameter  (Figs. 4a,  and hypoxanthine-grown granules are 150 +  12  (Fig. 8)  less abundan't  which measure 300 +  5 and 8 ) .  In urea- ( F i g . 5)  c e l l s , the perichromosomal  and smaller  in s i z e , measuring  and 145 + 15 nm in diameter, r e s p e c t i v e l y . These  a l t e r a t i o n s are since  they  not an  persist  a r t i f a c t of the plane of sectioning in  serial  sections.  No  obvious  morphological differences can be observed in the nucleus and nucleolus of  the c e l l s  Pavlova lutheri  'of both  grown in  Dunaliella tert iolecta and  d i f f e r e n t nitrogen regimes (Figs.  16 to 23) . Dictyosomes  are rather numerous in c e l l s of Amphidinium  carterae grown of the  on d i f f e r e n t nitrogen sources. The cis-face  dictyosomes are c l o s e l y associated  with elements of  the endoplasmic reticulum (E.R.) through t r a n s i t i o n vesicles (Figs. 11,  12 and  13, arrowheads).  The trans-face of the  dictyosomes i s usually characterized hypertrophied measuring  cisternae  70 +  and  smaller  by  the  presence  vesicular  of  profiles  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  and 13). . No detected in  in the hypoxanthine-grown  significant ultrastructural  c e l l s (Figs. 6  alterations  are  the dictyosomes of the c e l l s of both Dunaliella  tert i o l e c t a and  Pavlova l u t h e r i grown in a l l four different  nitrogen regimes. In  Amphidinium  carterae,  endoplasmic  reticulum-like  elements are often observed in close proximity to the transface of  the dictyosomes (Figs. 12 and 13). In hypoxanthine  -grown c e l l s congregate in  of t h i s  microalga,  the immediate  E.R. elements frequently  v i c i n i t y of  mitochondria without establishing  chloroplasts  and  direct contact with their  envelopes  ( F i g . 7,  arrows).  extensive congregations,  Although  not  forming  E.R. elements are also observed in  the v i c i n i t y of chloroplasts and mitochondria in both grown  (Fig.  Amphidinium  5,  arrow),  carterae  measurements reveal expressed per  that the  cells  are  cytoplasm, some  transition  of these  vicinity  O v e r a l l , the  arrow).  volume density  to  Morphometric of  that  the  in  endoplasmic  predominantly  In  E.R.  in  urea  or  reticulum the  apical  the occurrence  hypoxanthine-grown  elements of  E.R. i s  of  i s 66% (p < 0.01) higher in  dictyosomes through  E.R.  cells  showing a consistent r e l a t i o n s h i p  vesicles.  congregation of  than in  4a,  compared  observed  cis-face of  immediate  (Fig.  Dunaliella tert i o l e c t a ,  (ER) p r o f i l e s  to the  nitrate-grown  cytoplasm volume  hypoxanthine-grown n i t r a t e . In  and  urea-  the  is  also  cells,  observed  trans-face  of  of  in . the  dictyosomes.  more developed in hypoxanthine-growth  c e l l s grown, in the three other sources of nitrogen  ( F i g . 19 c f . with F i g s . 16, 17 and 18, arrowheads). situation i s  detected in  Pavlova  lutheri,  Similar  especially  in  hypoxanthine-grown c e l l s , where extensive development of the E.R. i s observed -throughout the cytoplasm ( F i g . 23 c f . with F i g s . 20, reveal that  21 and 22, arrowheads). Morphometric measurements the volume  cytoplasm volume . i s hypoxanthine-grown Pavlova l u t h e r i ,  45  cells  density, of and of  55%  the E.R. (p  <  Dunaliella  respectively, compared  three other sources of nitrogen.  expressed per  0.01)  higher  tertiolecta  in and  with growth on the  21 No obvious morphological differences could be observed in the  chloroplast  carterae.  and  pyrenoid  c e l l s grown  (Figs. 4a,  5 and  in the  6).  different  most of the  starch granules,  hypoxanthine-grown cultures observed  (Fig.  obvious  morphological  chloroplast  18, c f .  of  only a  with F i g s .  regimes  of  chloroplast volume  16, 17  Pavlova  seen throughout  are  and  19).  observed  luther i  exhibiting t y p i c a l  can be  nitrogen  is and  few starch granules are  d i f f e r e n t nitrogen regimes (Figs. 20  cristae  Amphidinium  while in n i t r a t e - , urea-  differences  cells  Mitochondria  of  In a l l a n t o i c acid-grown c e l l s of  Dunaliella tert i o l e c t a , occupied by  structures  grown  No  in  the  in  the  to 23). dinoflagellate tubular  the c e l l  cytoplasm  of  Amphidinium carterae. However, the number of mitochondria i s strikingly  different  in  nitrate-grown  hypoxanthine-grown (52  + 4/section) cultures of Amphidinium  that mitochondria d i v i s i o n in than  in  are more  urea and  show  be accounted  cells  that  the  volume  compartment  (p <  hypoxanthine and  in  ( F i g . 4a).  increases  and  for by the fact  frequently observed  mitochondrial 0.01)  4/section)  hypoxanthine-grown (Figs.  nitrate-grown  measurements  +  +  with  difference may  (35  (19  3/section) compared  carterae. This  urea-  cells  undergoing 5  and  7)  Morphometric  density  of  the  by  and  47%  78%  urea growth, respectively,  compared to growth on n i t r a t e . In Dunaliella tert iolecta and Pavlova l u t h e r i ,  the mitochondria  morphologically when  do not  appear to d i f f e r  c e l l s are grown in different nitrogen  22 sources.  However,  an  increase  mitochondrial  p r o f i l e s (Figs.  both  during  species  measurements  show  the  mitochondrial compartment  and 45%  (p <  the  19 and  hypoxanthine  that  Dunaliella tert iolecta  in  growth.  Morphometric  density  hypoxanthine-grown  and Pavlova  of  23) was detected in  volume  of  number  of  the  cells  of  luther i increases by 40  0.01), respectively, in relation to growth on  the other sources of nitrogen. In Amphidinium carterae, in nitrate-grown grown c e l l s ,  microbodies are rarely detected  cultures,  and although  they are far from abundant. Microbodies (415 +  15 nm in diameter) become readily v i s i b l e is supplied  when hypoxanthine  to the growth medium along with nickel ( F i g . 6,  arrowheads). . Morphometric measurements 72% and  present in urea-  28%  compartment  in of  the cells  respectively, compared single membrane-bound free matrix, membranous  volume grown  density on  of  the  of  peroxisomal  hypoxanthine  and  urea,  to those grown.on n i t r a t e . These are organelles with  frequently, seen in elements  show increases  resembling  a granular nucleoid-  close  association  endoplasmic  with  reticulum  p r o f i l e s ( F i g . 9). On occasion some microbody-like p r o f i l e s appear as  a  terminal enlargement of endoplasmic  cisternae  (Fig.  6,  arrow).  microbodies connected  to each  system  ( F i g . 10,  at any " time- in  arrowhead).  Other other by  micrographs a  narrow  reticulum show tubular  No microbodies are detected  c e l l s of Dunaliella tert i o l e c t a and Pavlova  l u t h e r i grown in a l l four nitrogen regimes.  23  The vacuolar apparatus of Amphidinium i s subdivided into two major  compartments. Both  independent  from  compartments  is  vacuoles, while  the  compartments are d i s t i n c t and  pusule  represented the other  ( F i g . 4b). One by  i s made  of  these  peripherally, located up of  several  central  vacuolar p r o f i l e s . In nitrate-grown cultures, the peripheral vacuoles are inclusions  well developed ( F i g . 4a). In  and almost free urea-grown  of membranous  cells,  membranous  inclusions can be seen in well-developed peripheral vacuoles ( F i g . 5 ) . Peripheral vacuoles are, however, less in hypoxanthine-grown central vacuoles again there  cells  ( F i g . 6). In  developed  contrast, the  in nitrate-grown c e l l s are very small, and  are no  extensive  ( F i g . 4a). In urea-grown  accumulation  cultures,  of  inclusions  the central  vacuolar  compartment i s more developed, and contains a few membranous inclusions ( F i g . 5 ) . well-developed, and hypoxanthine-grown reveal that c e l l s of that of  The central  contain large cells  extremely  membranous inclusions in  ( F i g . 6 ) . Morphometric  studies  the vacuolar compartment of hypoxanthine-grown Dunaliella  cells  (compare also compartment of  tertiolecta  grown in the other F i g . 19  with F i g s .  i s 50 to 55% larger than sources 16 to 18).  of  nitrogen  The vacuolar  hypoxanthine- and a l l a n t o i c acid-grown c e l l s  of Pavlova l u t h e r i seems also observed  vacuoles are  consistently larger than that  in c e l l s of the same organism supplied with n i t r a t e  or urea as sole sources of nitrogen (compare F i g s . 22 and 23 with F i g s .  20 and  2 1 ) . This  i s confirmed  by morphometric  analysis that  shows an  increase of 67 to 72% (p < 0.01) in  the volume density of this c e l l u l a r compartment. Lipid of  the  granules are frequently observed in the cytoplasm cells  of  Dunaliella  p a r t i c u l a r l y abundant compared to (Fig. 18  tert i o l e c t a .  in a l l a n t o i c  those grown  in n i t r a t e ,  These  acid-grown urea or  are  cultures,  hypoxanthine  c f . with F i g s . 16, 17 and 19, " L " ) . L i p i d granules  are rarely  detected  in  the c e l l s 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  reaction product  within the  hypoxanthine-grown Accumulation of certain  regions  cells  presence of  show  deposition of  microbody-like  organelles of  of  Amphidinium  carterae.  reaction product i s p a r t i c u l a r l y intense in of  the  morphological equivalent 14, arrowheads).  activity,  microbodies  that  of structureless  Control experiments,  the i n h i b i t o r s  of uricase  could  be  the  nucleoids ( F i g .  c a r r i e d out  in the  activity,  2,6,8-  trichloropurine or  oxypurine, eliminate the  reaction product.  In addition to uricase, these organelles  also show of catalase  deposition of  intense deposition of reaction product indicative activity  ( F i g . 15). Deposition  of  reaction  product i s absent from samples incubated with aminotriazole, a catalase  inhibitor,  tert iolecta and  or without  H2O2.  In Dunaliella  Pavlova luther i , the deposition of reaction  25 product, indicative cristae  and in  (Figs. 24  the outer  after  concentrations presence of  compartment of the mitochondria  brief fixation  of  addition  uric acid  to  deposition a c t i v i t y in (Figs. 26  min), in  (0.25%)  (Table 8). Control  reaction product  uricase, of  (5  glutaraldehyde  deposition of  only at  within the  and 28). Deposition of reaction product, however,  occurs only  no  of uricase a c t i v i t y , occurs  reaction  product  and  in  the  experiments show  (Figs. 25  mitochondria  very low  also  and 29). In  show  intense  of  catalase  indicative  both Dunaliella tert iolecta and Pavlova luther i  and 30). pH 9.0  from samples  Deposition of  reaction product  occurs  in c e l l s incubated at 37°C and i t i s absent  incubated with aminotriazole  or without  H202  (Figs. 27 and 31, Table 8 ) . A t h i r d type of reaction product deposition in at pH in the  mitochondria Of  6.0 when  the c e l l s are incubated at room temperature  absence of  deposition of  these microalgae i s observed  H202.  Under  these  circumstances,  the  reaction product i s strongly inhibited by low  concentrations of  potassium cyanide  which i s indicative of  cytochrome system-dependent oxidative a c t i v i t y .  Furthermore,  while the cytochrome system-dependent deposition of reaction product  occurs in  nitrogen tested,  c e l l s grown  in  a l l four  uricase- and catalase-dependent  of reaction  products are  only observed  hypoxanthine  grown c e l l s  (Table 8). .  sources  of  deposition  in mitochondria of  26  DISCUSSION  Growth  Studies  The results c l e a r l y demonstrate the widespread occurrence of  urea  -  utilizing  microalgae  tested,  exhibited  capabilities with  on  the  significantly  part  df a l l  greater  growth  by some species r e l a t i v e to the n i t r a t e c o n t r o l s .  The f a c i l i t y  and e f f i c i e n c y  the  surveyed  species  authors  that urea  organic-N available  of urea u t i l i z a t i o n by most of  confirm  the  findings  of  previous  i s one of the most important sources of for growth  of marine  the oceans (see O l i v e i r a and Antia,  phytoplankton in  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 c e l l s of  nickel supplementation  was toxic for urea-grown  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 i s required at higher concentrations to support the  urea-growth  of  Olisthodiscus luteus culture conditions Therefore, I  Amph i d i n i um (Table 2). and media  attribute the  d i f f e r e n t clones of these The  I have  as in  and  especially  utilized  the  the same  previous  discrepancies  to  the  study. use  of  microalgae.  available evidence,  degrading enzymes,  carterae  on the  indicates that  d i s t r i b u t i o n of two  alternative  ureaenzyme  27  systems  ( u r e a s e and  ATP:urea amidolyase Chlorophyceae, classes  occurs  e_t  1977). A  of  reported  a_l. ,  for  Bekheet, strict  the  jackbean  nickel  some  examined  so f a r  B e k h e e t and S y r e t t ,  1977;  organic-N  responsible ammonium  for  (Polacco,  hydroxyureaobserved  tissue  as  L.)  a  urease  activity  was  (Syrett,  1981;  2 +  1977). T h i s  by  urease  of  urea  is  as s o l e  the  into  i s confirmed  citrate-dependent  and  Amphidinium  f o r g r o w t h on u r e a  that  conversion  Rees  1986b). T h e r e f o r e , the  exhibited  luteus  also  enzyme  utilizable  by  growth  both  the  inhibitions  a r e supplemented with  urea as  of n i t r o g e n .  endogenous n i c k e l  the p r e p a r a t i o n estimate  nickel  f o r urease  when t h e s e m i c r o a l g a e  source The  and  Ni  of  germane  (Polacco,  suggests  the  1 9 8 4 ) . More  ensiformis  and A n t i a ,  and O l i s t h o d i s c u s  of  algae  cultures  microalgae  for  the algal  (Canavalia  1982; O l i v e i r a  members o f  in a l l  identification  requirement  in algae.  other  1975) and s o y b e a n  requirement  carterae  1973;  occur  t o some  A l - H o u t y and S y r e t t ,  p r o b l e m was  constituent  sole  urease  Syrett,  1981;  this  source  i s restricted  while  and  Syrett,  (Dixon  amidolyase)  and some o f t h e c h l o r o p h y c e a n  (Leftley  to  ATPrurea  of  contamination  of our  content  test  endogenous n i c k e l from  media  for.the  salinities  present  study, respectively  growth  enhancement  of  3.42  nM  26  in  used i n and  our  media, e x c l u d i n g  i s i n the order  (Oliveira  observed  is  i n the f i n a l  added s a l t s ,  1.4 nM  o f t h e sea water  o f 2.7 and  and 1 4 % o u t i l i z e d and A n t i a , urea-grown  i n the  1 9 8 4 ) . The cells  of  28  Hymenomonas  elongata,  Thalassiosira  pseudonana  therefore, implies microalgae.  Thalassiosira with  nordenskioldi i  nickel  that urease  supplementation,  i s also  present  in these  This i s further confirmed by the strong growth  i n h i b i t i o n observed  2+  when c i t r a t e , the natural N i - c h e l a t o r  found in nickel accumulating plants (Kersten et a l . , is added  to the culture media.  Pavlova  lutheri,  quadruplicatum, oculata on  Isochrysis  no signs  these organisms.  these organisms Ni*  tertiolecta,  galbana  Furthermore, the addition of  1980),  In contrast, the growth of  Dunaliella  urea displayed  growth of  and  and  Aqmenellum  Nannochloropsis  of nickel-requirement.  c i t r a t e did not affect  These data  the  suggest that either  have the capacity to  readily  concentrate  from the nanomole levels normally occurring in seawater  (see O l i v e i r a and Antia, ATP:urea  1986a  for review) or they use the  2+  amidolyase  Ni -independent  system  for  the  conversion of urea d i r e c t l y to u t i l i z a b l e ammonium (Oliveira and  Antia,  Agmenellum  1986b).  In  the case  quadruplicatum,  Nannochloropsi s  oculata,  of  Pavlova  Isochrysi s  the  first  lutheri,  galbana  explanation  and  i s more  l i k e l y to be true, since growth of these microalgae in urea as sole  source of  potent inhibitor Carvajal et a l . , the  of  ATP:urea  urease  1982). This  urea-supported  Instead, t h i s  nitrogen i s inhibited by hydroxyurea, a  growth  i s inhibited  amidolyase  activity  (Reithel,  -197.1;  compound i s without effect on of  Dunaliella  tertiolecta.  by a v i d i n , a known inhibitor of  activity.  Furthermore,  addition  of  29  biotin  abolishes  r e s u l t s ) . These  the  results  ATP:urea a m i d o l y a s e nickel and  . The cells this  1973;  long  of  source  this  suggest  by  organic  one  dependency  culture  media  the  poor  p r e s e n c e of  results  u r e a u p t a k e by  these  According not  been  this  11  one  studied, source.  acid-grown  lutheri from  1  and mM  atoms s t o i c h i o m e t r y  a  (Leftley  with  this  i n urea-grown  i t s poor growth  microalga  nitrogen  of  t o grow even  1975).  The  may  not  urea.  improve  whatever  at problem  is  is  not  ions  to  Since detected  explanation the  This  higher  growth.  i m p a i r m e n t of  be  efficiently  growth  more l i k e l y  on  for  the  mechanism  of  cells.  microalgae.  nitrogen  allantoic  the  i s the  to A n t i a  species  Pavlova  the  et. a l . , ( 1 9 8 9 ) ,  previously tested  g r o w t h of the  observed  source  to  of  r e a s o n why  s i n c e a d d i t i o n s of n i c k e l  inhibits  observed  occurrence  microalga  inability  failed  urea,  the  1982).  organic  e_t a l . ,  hydroxyurea completely in  the  nitrogen  (Antia  nickel  that  their  concentrations  the  from t h i s  lag period  to u t i l i z e  of  (unpublished  e x p l a i n s the  Prymnesium parvum t o g e t h e r  nitrogen  of  and  C a r v a j a l et a l . ,  further confirmed  on  inhibition  indicative  i s absent  adaptation  well equipped is  are  activity  requirement  Syrett,  avidin-induced  as  an  Under our  allantoic  organic test  growth  cultures  of  has  for  the  N-source  c o n d i t i o n s , only  showed m o d e r a t e The  acid  t o good g r o w t h  observed  in  Hymenomonas  Nannochloropsis oculata  0.5  allantoic  acid  i s equivalent  versus  on mM  elongata,  urea-supplemented c u l t u r e s . Since of  6 of  urea  the is  to N4:2,  30  the p r e v i o u s utilize  observation  the  allantoic  u r e a . These allantoic  acid  might by  Shah and  determination of  some  acid  r e s u l t s may  ureidoglycolate 1989;  suggests  then  be  be  catabolized  Nannochloropsis  oculata  This i s  activity (Table  allantoate-supplemented cultures  as t h a t  from  imply  that  urea  and  to  allantoicase  microalgae  microalgae  construed to  1984b).  of a l l a n t o i c a s e  of these  these  - N as e f f i c i e n t l y  t h e enzyme Syrett,  that  (Antia supported  in cell-free 7 ) . The  hydroxyurea of  further  cells  of  Dunaliella  Isochrysis  3 with  supports  entire  Antia such  i n the  allantoate-grown  of  acid  and  allantoic  acid-N.  o f Agmenellum  u r e a by  1.0 mM  urea,  (compare  Table  suggested  pH o r  that  illumination  t o decompose t h e utilize  hence, the lower  by h y d r o x y u r e a to  (1989)  acid-grown  quadruplicatum  t h a t from  of microalgae acid  with  enzyme.  allantoic  temperature,  molecule;  cultures  galbana  allantoic  as  al.,  of a l l a n t o i c  utilization  Isochrysis  et  displayed  treated  of t h i s  Agmenellum  than  this  u r e a may n o t be one  quadruplicatum  the c a p a b i l i t y  structure  atoms p r e s e n t the  2).  case  0.5 mM  was l e s s  Agmenellum  factors,  could affect  in  lutheri  action  tertiolecta,  galbana  for  Table  culture  of  that i n t h i s  growth r e c o r d e d  especially  in  Pavlova  the products of the c a t a b o l i c The  and  suggests  of  extracts  o f Hymenomonas e l o n g a t a and  by h y d r o x y u r e a  cells  by t h e  i n h i b i t i o n of  i n t e r p r e t a t i o n . However, t h e a b s e n c e o f i n h i b i t i o n by a l l a n t o a t e - g r o w n  et al_. ,  all 4  N  efficiency  The i n h i b i t i o n o f q u a d r u p l i c a t u m and  suggests  the c a t a l y t i c  that conversion action  of the  31  enzyme  allantoicase  Hydroxyurea  showed  also no e f f e c t  supplemented c u l t u r e s these not  results  urease,  1973).  The  explains and  Antia,  in  urea-producing  in a l l a n t o i c  this  genus  2+  of  acid  Isochrysis  galbana addition  cultures  of  suppressed  suggesting  that  f o r g r o w t h on t h i s was a l s o  growth o f p h y t o p l a n k t o n no  growth  was o b s e r v e d  tested  as the s o l e in a l l  equipped  for  allantoin,  for  phytoplankton  situation and  this  was a l s o  Lynch,  cellular the cause  1964).  amidolyase this  organic-N  These  Nannochloropsis  of c i t r a t e  to a l l a n t o i c  Hymenomonas this  elongata  metal  ion i s  source.  source  which  to support the  of o r g a n i c - N , but  appear  Antia  et  i s generally  growth r e l a t i v e observed  Agmenellum  s p e c i e s s t u d i e d . Except f o r  species  that  Syrett,  demonstrated  lutheri,  for i t s ability  benthic-type  suggested  f o r the  f o r g r o w t h on  and  organic-N  certain  utilizing  and  was a l s o  of P a v l o v a  c o n t r a s t , the  Allarttoin  amidolyase,  s o u r c e s o f o r g a n i c N ( O l i v e i r a and  cultures  growth,  However,  ATPrurea.  -requirement  acid-supplemented  required  (Leftley  Nickel-independence  quadruplicatum, oculata.In  tertiolecta.  t o be t h e enzyme r e s p o n s i b l e  absence of N i  1986b).  microalgae.  s i n c e ATP:urea  nickel-independence  the  other  urea-N  these  upon t h e g r o w t h o f a l l a n t o a t e -  a r e not s u r p r i s i n g  of  in  of D u n a l i e l l a  was shown  utilization  occurs  to  be  well  a l . ,• . (1980b)  a poor  to the p u r i n e s . A  N-source similar  i n C h l o r e l l a . p y r e n o i d o s a (Ammann investigators  permeability barrier  to allantoin  of the absence of growth.  indicated  that  a  might p o s s i b l y  be  32 Hypoxanthine was chosen as the prime purine representative for  this  investigation  established s t a b i l i t y  not  only  on  account  of i t s  in natural sea water but also because  unlike adenine or guanine, i t does not possess any exocyclic nitrogenous depend on  group;  hence  the a b i l i t y  any  of a  nitrogen  utilization  must  particular species to retrieve  the purine-skeleton nitrogen (Antia et a l . , 1989).  Out of  the 11 species examined in t h i s investigation, only 5 showed moderate to proved to well on urea.  good growth be rather  on hypoxanthine. Prymnesium parvum  interesting in the sense that i t grows  hypoxanthine, while The  situation  reported by However,  is  Antia et  in  the  case  of  possess a  (0.5  hypoxanthine-cultures of  one  on  previously  parvum,  comparison  a  higher  with  other  to 1 mM - Table 5) i s required  or  1980a).  Pavlova  uptake  system  for  The growth observed in  lutheri,  Nannochloropsis  was equivalent to the  a l l a n t o i c acid- and also urea-grown c e l l s of these  acid contain  5 ) . Since 4  N atoms  evidence  microalgae are -N. The  in  Dunaliella t e r t i o l e c t a  species (Table  urea, the  the  observed  for Chlamydomonas pa 11a.  permease  hypoxanthine (Antia et a_l. ,  one from  was  This suggests that t h i s microalga may  less e f f e c t i v e  oculata and  to  Prymnesium  (2 mM)  hypoxanthine u t i l i z e r s to.occur.  similar  al.., ( 1975)  substrate concentration  for growth  poor growth  both versus  indicates  efficient  hypoxanthine and a l l a n t o i c 2 N atoms per molecule of that these  3  species of  u t i l i z e r s of a l l the hypoxanthine  hypoxanthine growth  of  these  microalgae  is  not  d e p e n d e n t upon presence also  of  Ni  the N i  exhibited  species  2  similar  2 +  chelator  good g r o w t h  requires  medium. The  supplementation  the  the  addition  of  citrate  carterae  suppressed since  c u l t u r e medium  the  nickel  of  the  this  culture  of e x c e s s ,  process for  plus  Table of  Ni  2 +  2)..The  Amphidinium  growth s u p p r e s s i o n i s  i t . These d a t a  responsible  the  urea  4.with  g r o w t h . The  this  However,  into  to hypoxanthine-cultures  restores  enzyme  of  equivalent  the a d d i t i o n  nickel-dependency is  hypoxanthine.  (compare T a b l e  their  in  under t h e s e c i r c u m s t a n c e s ' , i s  from  supplemented c u l t u r e s  reversible,  on  addition  one  proceeds  c i t r a t e . Amphidinium c a r t e r a e  growth o b s e r v e d ,  to  and  and urea  nickel  further confirms  into  the  support that  catabolism  the  urease  in  this  microalga.  Inhibitor As  S t u d i e s and  Determination  r e p o r t e d by F u j i h a r a  [4-hydroxypyrazolo hypoxanthine,  is  (3,4-d) a potent  dehydrogenase. T h i s of h y p o x a n t h i n e al.,  and  Yamaguchi  pyrimidine], inhibitor  t o x a n t h i n e , and  cultures  lutheri, Dunaliella  tertiolecta,  oculata  medium,  hypoxanthine  ->  an  allopurinol analogue  of t h e enzyme  of  to u r i c  uric  of  xanthine oxidation (Antia  et  observed  in  Prymnesium p a r v u m , P a v l o v a Amphidinium  that ->  acid  inhibition  when a l l o p u r i n o l  implies xanthine  this  the growth  hypoxanthine-grown  culture  (1978),  activities  enzyme i s r e s p o n s i b l e f o r t h e  1989). T h e r e f o r e ,  Nannochloropsis  of E n z y m a t i c  the acid  was  carterae added  into  degradation i s d e p e n d e n t on  and the of the  34  activity  of  xanthine  the o c c u r r e n c e  of x a n t h i n e  these microalgae Uricase  confirms  and  allantoin  are  known  The of  t o be  and  growth  oxidoreductase) of urate  et a l . ,  i s a cuproprotein  i n the  inhibitors  1969  inhibition  presence of  carbon  ( H 2 O 2 ) ,  of  dioxide substances  uricase.  2,6,8-trichloropurine  Moller,  uricase  catalytic crude  and  and r e f e r e n c e s c i t e d  of hypoxanthine-supplemented  other  cell-free  factors  Failure  extracts  sensitivity  Syrett  crude  i s r e s p o n s i b l e f o r the u r i c  conversion.  the h i g h  (Huynh and  (1984b) a l s o  extracts  of  failed  was  proceeds  simultaneously  the  formed d u r i n g the r e a c t i o n .  that  Dunaliella occurrence  cytochemical tert iolecta of  Oliveira,  both  same  uricase  extract.  allantoin  i n p H , among  in partially  Urate  the degradation  studies and  indicates  Phaeodactylum  measurable  fractions  notice  cultures  may be r e l a t e d t o  diatom  purified  H2O2  therein).  1989a, b ) . I n d e e d , Shah  marine  with  oxonate  to detect uricase a c t i v i t y in  activity  of the  ->  enzyme t o c h a n g e s  Oliveira,  the  acid  These  to detect uricase a c t i v i t y in  of the microalgae  of t h i s  tricornutum, although  and  (Table 7 ) .  1 9 5 5 ) . A number o f  effective  of  i n some o f  these microalgae, t r e a t e d with t r i c h l o r o p u r i n e ,  that  and  (Mahler  demonstration  interpretation  hydrogen p e r o x i d e  oxypurines,  (Muller  this  the o x i d a t i o n  oxygen, y i e l d i n g  The  dehydrogenase a c t i v i t y  (urate:oxygen  that c a t a l y z e s  include  dehydrogenase.  oxidation  by c a t a l a s e o f  I t i s then  important to  of Amphidinium  Pavlova  lutheri  carterae, reveal  and c a t a l a s e a c t i v i t i e s  1989a, b ) . The d e t e r m i n a t i o n  the  (Huynh  i n crude  cell  35  preparations  of  allantoicase  activities  pathway of it  is  purine  der  urease  the m i c r o a l g a e suggests  degradation  responsible for  p u r i n e s and van  some of  the  1976;  and  also  that  occurs  v i a urea  with  the  Pavlova  the  the  exception block  the  growth  species able  N source  further  l a c k of  of D u n a l i e l l a  tertiolecta  occurrence  ATPrurea  Syrett,  of  1 9 7 3 ) . The  and  allantoic  the  presence  catabolic not  by  of  the  these  ( 1 9 8 7 , 1988) suspension  that  Pavlova  urease  oxidation  involve  regarding  acid  urease  amidolyase  fact  lutheri  showed t h a t culture  of  . cells  is  reactions,  ureidoglycolate  amidohydrolase.  inhibition  reflects  the  (Leftley of  and  hypoxanthine  suggests  their  that  the  derivatives  does  It  recently  allantoin  this  remains u n a f f e c t e d i n  urea.  that  amidohydrolase  the  and  of  three  supports  activity  utilization  of p u r i n e s  findings  other  inhibitors  inhibitors  production  the  hypoxanthine-growth  by  that  Dunaliella  of  i n t e r p r e t a t i o n . The  fact  and  of  cultures  this  and  (Vogels  1 9 8 2 ) . The  hypoxanthine-supplemented t o grow on  standard  n i t r o g e n of  production  lutheri,  and  in microalgae  of  Reynolds et a l . ,  inhibitors,  tertiolecta  then  utilization  i t s derivatives  Drift  of a l l a n t o i n a s e  is  interesting  Winkler  catabolism carried  allantoate  in  out  et  al.,  soybean by  two  amidohydrolase  Under  these  and  circumstances  whole  process  proceeds without d e t e c t a b l e production  u r e a . The  ability  for  microalga of p u r i n e  may  then  urea  represent  c a t a b o l i s m , such  utilization a system as  that  e x h i b i t e d by  independent of  the  from  ornithine  of  this that cycle  36 involving  urea  release  1 9 7 0 ) . However,  whether  similar  to  t h e one  lutheri  r e m a i n s t o be  Ultrastructure  from  arginine  or  not  observed  a two  in  by  arginase  (Naylor,  amidohydrolase  soybean e x i s t s  system  in Pavlova  determined.  and  Cytochemical  Studies  on  Amphidinium  carterae Recent transition  s t u d i e s have metals  Fe, N i ,  condensed chromatin review  see  shown  of a  Cu  that and  variety  S p e c t o r , 1984).  high Zn  of  Studies  into dinoflagellate  chromosomes  continuous  i n t h e mean l e v e l  increase  hrs period. continuous existing  change  Although  by the  suggested  i n the b a l a n c e  chromatin  chromatin  still  T h i s was  or t o be  continuous function  unknown,  the  of  chromosome  play  i n the  a role  ultrastructural then Ni  2 +  reflect in  the  found  in  the  dinoflagellates  (for  on  6 % i  t h e u p t a k e of that  of t h i s t o be  t o the  there  al.  organization  a  a  24  indicative metals  of a  within  f o r m a t i o n of  synthesis  et  is  ion over  (Sigee,  the p e r i c h r o m a t i n i c  1982).  granules  (1981)  suggests  new  is  model that  on they  of t h e chromosomes.  The  i n the p e r i c h r o m a t i n i c g r a n u l e s  may  response  the growth  DNA  stabilization  changes  the  of t r a n s i t i o n  related  Spector  dinoflagellate  of  are  reveal  either  levels  of the  medium of  cells  both  urea  to the presence and  of  hypoxanthine  cultures. In  a  recent  review  d i n o f l a g e l l a t e s , Dodge  and  on Greuet  the  ultrastructure  (1987) p o i n t out  of  that i t  is  usually  (cis)  difficult  face  and  the  these m i c r o a l g a e . trans-faces nitrogen clear  of  to  differentiate  maturing  In A m p h i d i n i u m  the dictyosomes  nutritional  regimes  transition  vesicles  of  the  trans-face  are,  Another  distinctive  dictyosomes  is  measuring  70  abundant  +  of  dictyosomal  vesiculation) Chan  costatum,  at  and Wong, this  s u g g e s t e d t o be (Smith,  cells. of  region  1 9 8 7 ) . In  increase related  in  cisternae  region  are  of  the  profiles  particularly  on  microalgae  the  effects  have  shown  (hypertrophy  and  of the o r g a n e l l e s the  which  hypertrophied.  Studies  activity  the t r a n s  shows a  vesicular-like These  and  three  elements  1 9 8 0 ) . The  trans  in diameter.  the growth  cis-  cis-face  always  the of  forming  in a l l  reticulum  turn,  presence  5 nm  m e t a l s on  increased  case  of  dictyosomal  (Smith,  Skeletonema  activity  t o mechanisms o f m e t a l  was  sequestration  1983).  The  presence  vacuoles  is et  activity  different  of  large  indicative al.,  would  our p r e s e n t  membranous  of  1980).  extensive Why  develop in  nitrogen effect  sources is  hypoxanthine-nickel  such  inclusions autophagic  widespread  hypoxanthine-grown  u n d e r s t a n d i n g of  However, t h e in  the  The  (Kristen,  i n hypoxanthine-grown  of h e a v y  (Marty  feature  the  are d i s t i n c t  tested.  in  the  face of d i c t y o s o m e s i n  carterae,  r e l a t i o n s h i p with endoplasmic  includes  1983;  (trans)  between  the on  not a d e l e t e r i o u s supplemented  activity autophagic  cells  nutritional cellular  within  escapes  effects  of  ultrastructure. one,  medium  since  growth  proceeds  as  38  efficiently  as  in n i t r a t e  Recent s t u d i e s  showed t h a t  cellular  compartments  a major  role  to which binding  any  ions  one  (Fowler,  of  occurs  the  in  plus  carterae  vacuolar  nitrate/nickel-free  i s involved  a number of  increase could  such p r o c e s s e s .  several  m e t a l h o m e o s t a s i s . The  interactions  1 9 8 7 ) . The  organisms  a p p a r a t u s , lysosomes)  these compartments  and  nickel conditions.  aquatic  ( i . e . Golgi  t o depend on  of A m p h i d i n i u m  in  of  regime  reflection  urea  in i n t r a c e l l u l a r  appears  nutritional  or  The  i n the  vacuolar  metal  apparatus  l e a s t i n p a r t , be an  urea plus  supplemented  in metal  competing  fact that  s y s t e m of  extent  factors, including  with  then, at  play  cells  increase  a  also  n i c k e l but  not  supports  this  enzymes of  purine  interpretation. B a s e d on catabolism, acid  by  distribution  ureide  allantoin  allantoinase possibly  reticulum in  the  the  the  (Hanks  size  cells  seems t h e n c o n s i s t e n t  plays  in  the  endoplasmic large  catabolism  reticulum  increase  elements  are  bounding-  membrane  of  continuity  observed the  root  (Fig.  of  ER  w i t h the  often these  4, a r r o w ) . higher  in  observed  organelle  proliferation  and  in  with  a  Endoplasmic  positioned  microbodies  situation  plants  this  microbodies.  organelles  The  endoplasmic  in these c e l l s  closely  and  allantoic  increase  role  The  also  the  to  in hypoxanthine-grown  key  of p u r i n e s .  between ER  n o d u l e s of  the  number of  reticulum  direct  i s hydrolyzed  1 9 8 3 ) . The  coincides  i n the  the  located  e t a l , , 1981;  c o m p l e x i t y and  of  to  the  occasionally can  also  resembles that  specialized for  be of  ureide  39  production  and  microbodies  may o r i g i n a t e  Kaneko and  Newcomb, 1987;  micrographs, tubular of  suggests  from  microbodies  profiles  that  in  Webb and Newcomb, 1 9 8 7 ) . In some  are connected  ( F i g . 8,  ones  may a l s o  The  functional  arrowhead).  been  plants  1979; T o l b e r t  respect to  by  These a r e i n d i c a t i v e that  of p r e e x i s t i n g  1985).  peroxisomes) has  so w i t h  another  r e t i c u l u m and s u g g e s t  specialization  (Beevers,  t o one  be formed by t h e f i s s i o n  (Lazarow a n d F u j i k i ,  carterae  t h e ER (Newcomb e t a l . , 1985;  t h e e x i s t e n c e of a p e r o x i s o m a l  microbodies  Amph i d i n i um  of  investigated  microbodies  extensively  and E s s n e r ,  in  (e.g. higher  1981) b u t r a r e l y  microalgae. Considering the information  a v a i l a b l e , , i t seems t h a t t h e r e a r e two g r o u p s o f m i c r o b o d i e s in  the  a l g a e . One  group  is  represented  microbodies  c h a r a c t e r i z e d mainly  and  activities.  uricase  resembling  the  (Stabeneau, catalase  the c e l l s  1984).  is a  The d e m o n s t r a t i o n i n Amphidinium  of t h i s  functionally  uric  of both c o u l d then  plants  uricase  and  be t a k e n a s  of u n s p e c i a l i z e d m i c r o b o d i e s i n  i s only observed  strong indicator  nodules  of c a t a l a s e  d i n o f l a g e l l a t e . However, t h e p r o l i f e r a t i o n  endogenous m e t a b o l i s m root  by t h e p r e s e n c e  The o t h e r by o r g a n e l l e s  of the o c c u r r e n c e  of m i c r o b o d i e s  unspecialized  glyoxysomes and peroxisomes of h i g h e r  activities  indicative  by  acid  o f some of these  i n hypoxanthine-growth  which  major a l t e r a t i o n ( s )  i n the  cells.  i s oxidized  p e r o x i s o m e s by u r i c a s e . T h i s r e a c t i o n d e g r a d e d by c a t a l a s e , a l s o  located  In  to  ureide-producing  allantoin  produces  i n the  ^2^2 w h i c h i s  i n the peroxisomes  (Hanks  40  et  al.,  and  1981; S c h u b e r t ,  Newcomb,  1987;  observations  show  very  occurs  likely  catabolic  via  oxidation  fact  that  detected  in  Vaugh  and  Stegink,  t h a t hypoxanthine-growth  the  i s related  no  uricase  nitrate  or  and  to  large  to t h i s  These  Amphidinium  urea  through  increase  i n the  type of  catalase  urea-grown  1987). of  i t s conversion  and t h a t  microbody p o p u l a t i o n The  1986; Kaneko a n d Newcomb, 1987; Webb  metabolism.  activities  are  supports  this  cells  interpretation. In  nitrate-grown  detected, a fact  cultures  microbodies  t h a t c o n f i r m s o b s e r v a t i o n s by o t h e r  (Dodge a n d C r a w f o r d ,  1968; K l u t e t a l . ,  far  in  from  abundant  urea-grown  hypoxanthine-grown c e l l s . These the absence suggest from  those  study  of enzymatic  that  of  features  activity  these microbodies  the catalase-negative  regarded as  enzymes o f The  the glyoxylate  evidence  supports  of  two f u n c t i o n a l l y  of  microbodies.  Euglena  atypical  where  distinct,  Such a  f o r u r i c a s e and c a t a l a s e  material. microbodies  peroxisomes during  different  In of  a  recent  Amphidinium  that these  organelles  g l y o x y s o m e s where t h e marker or r e p r e s s e d .  the existence i n t h i s nutrition-related  situation  microbodies  with  i n conjunction with  c y c l e may be a b s e n t  then  authors  compared  are functionally  i n hypoxanthine-grown  rarely  1 9 8 1 ) . They a r e a l s o  cells  c a r t e r a e , K l u t e t a l . (1984) s u g g e s t e d m i g h t be  are  seems  appear  a u t o t r o p h i c growth  also to and  microalga  populations to  occur  perform as  in like  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  glycolate oxidase. mechanism  of  of a corresponding lack of  Although l i t t l e  photorespiration  i s known of the overall  in  dinoflagellates,  the  available evidence supports the absence of glycolate oxidase from these that  organelles  glycolate  dehydrogenase. d i r e c t l y to  metabolism This  usually located  in  (Stabenau, 1984).  suggests instead  initiated  cannot i t does  In algae,  by  glycolate  transfer  electrons  not form  rather  In Scenedesmus  than  reflect  glycolate  metabolism  occurrence of  an  increase  (Kramer  large numbers could then  and  and  r e f l e c t , at  microbodies  in  was  activity  of  1978).  The  the  Findenegg, in  least in  cells  of  part, their  (Klut et a l . , 1981).  Cytochemical  t e r t i o l e c t a and Pavlova lutheri  is  to low C0 2  of mitochondria  p a r t i c i p a t i o n in photorespiration  Ultrastructural  during  obiiquus, the increase in  of mitochondria during adaptation to  H202  glycolate dehydrogenase  mitochondria  suggested  Amphidinium  1977). It  is  enzyme  oxygen; hence,  glycolate oxidation.  the number  (Burris,  Studies  on  Dunaliella  42  As  previously  reticulum  discussed,  reported  to occur  Amphidinium c a r t e r a e nitrogen, the  is  1989a).  The  increase  in c e l l s  grown on  indicative  utilization  the  of  the  of h y p o x a n t h i n e - N  role  this  lutheri organelle  (Hanks e t a l . , 1981; The  oxidation  catalase usually  of  (Huynh the  Pavlova  lutheri  four  that  in  of  urate  H 2 02  and  formed  is  observed  notice  of  nitrogen  of  uricase  with the of  key  purines  urate  these  It  is  mitochondrial  is  an  activity  reported  before  fraction  of  on  grown  significant  catalase  indicative activities  interesting increase  be  soybean seeds  and  in c e l l s  finding, to  the  no  catalase  unusual  are  reactions, since  compartment.  and  by  1989a).  tertiolecta  reaction products  w i t h an  mitochondria  organelles,  t e s t e d . I t i s then  o x i d a s e and  uricase  reaction  Oliveira,  detected  of  of  mitochondrial  reticulum  subsequent d e g r a d a t i o n  Dunaliella  are  localization  was  in  tertiolecta  catabolism  the  l o c a t i o n of  these c o i n c i d e the  Dunaliella  Huynh and  in mitochondria.  that  density  in  both  organelles  occurrence  E.R.  Oliveira,  endoplasmic  in microbody-like  cytochemical deposition  the  the  1981; in  i s the  s o u r c e s of  of  the  the  of  1983).  fact  microbody-like all  plays  (Schubert,  intriguing  of  source  and  seems t h e n a l s o c o n s i s t e n t  compartmentalized  peroxisomes The  the  sole  i m p o r t a n c e of  l a r g e r d e v e l o p m e n t of  Pavlova  endoplasmic  the d i n o f l a g e l l a t e  h y p o x a n t h i n e as  (E.R.) i n h y p o x a n t h i n e - g r o w n c e l l s and  of  in  The  also  i n the  to  volume  cytochemical  activities although  associated  in  uricase with  ( G l y c i n e max  the  A62-1:  43 nodulating v a r i e t y ) . this case  It i s  interesting to  notice that  in  uricase a c t i v i t y was only detected during certain  periods of  plant development  The situation  resembles that  tert iolecta and only detected  (Tajima and  Yamamoto, 1975).  observed in  both  Pavlova l u t h e r i , in hypoxanthine  Dunaliella  since uricase a c t i v i t y is  but not a l l a n t o i c a c i d , urea  or nitrate supported growth. The  question of  reactions must  the s p e c i f i c i t y  of  the  cytochemical  be c a r e f u l l y considered. The pH optimum for  urate oxidase a c t i v i t y was soybean radicles  shown to be above 9,  an optimum  and Moller, 1969;  pH of 7.0 was  reported (Muller  Tajima and Yamamoto, 1975).  instance, however,  the enzymatic  although in  In this last  a c t i v i t y proved to be due  not to uricase but to two other enzymes, diamine oxidase peroxidase  (Tajima  then that  the deposition  uricase  activity  t e r t i o l e c t a and and  rapidly  et a l ,  in  1985). It i s important to notice of reaction product indicative of  mitochondria  Pavlova l u t h e r i declines  Angermuller and  and  with  Fahimi (1986  of  occurs  both  Dunaliella  at high pH values  decreasing  pH  (Table  8).  and references c i t e d therein)  showed that urate oxidase a c t i v i t y i s extremely sensitive to aldehyde f i x a t i o n .  Even short  fixation periods  of 20  min  with 1% aldehyde produces a 90% loss in a c t i v i t y (Yokota and Nagata, 1974). obtained in  Deposition of  our material  glutaraldehyde to min fixation  0.25%  reaction product  by lowering (v/v)  can only be  the concentration of  and using  a (very  short) 5  period. It i s also important to emphasize that  44  no deposition  of reaction  product occurred in mitochondria  of these two microalgae when incubation medium.  uric acid was omitted from the  Trichloropurine  used in cytochemical studies identification  of  has  as a  been  control  uricase-dependent  extensively  test  for the  reaction  product  deposition in organelles of a variety of plant c e l l s . Under these conditions  staining  i s almost  completely  or  even  t o t a l l y abolished (Huynh and O l i v e i r a , 1989a, and references cited therein). and Pavlova  In the case of both Dunaliella tert iolecta  lutheri,  no  deposition  occurs in mitochondria when presence  of  consistent  those  reaction  product  the c e l l s are incubated in the  trichloropurine.  with  of  The  used  by  results  other  are  authors  then  for the  cytochemical demonstration of urate oxidase a c t i v i t y in both plant and  animal c e l l s  (see Angermuller  Huynh and O l i v e i r a , 1989a 3,3'-Diaminobenzidine in  the  oxidative  mitochondrial cytochrome 1987). Deposition are known  for reviews).  (DAB) i s a widely u t i l i z e d substrate  cytochemical activities  localization of  peroxidase,  system,  of  peroxidatic  catalase  respectively  or  and the  (Frederick,  of cytochrome dependent reaction products  to be optimized at pH 6.0,  presence of very low levels of The reaction  and Fahimi, 1986;  i s usually  low concentrations  H2O2  in the absence or the  and at room temperature.  abolished or strongly inhibited by  of potassium  cyanide. Reaction  deposition with  these c h a r a c t e r i s t i c s  mitochondria of  Dunaliella t e r t i o l e c t a  i s also  product  detected in  and Pavlova lutheri  45  grown  in  a l l four  evidence  sources  suggests  of  reaction  c o n s e q u e n c e of t h e c y t o c h r o m e oxidase Hall,  activity  1978;  These  nitrogen product  and M u e l l e r ,  of both  longer  p e r i o d s . Under  reaction  levels  products  of  H 2 02  deposition  of  aminotriazole, a it  is  inhibitor  of  the  trichloropurine, characteristics of  catalase  (Novikoff van  der  et  a l . , 1977; i n the  dependent d e p o s i t i o n mitochondria  of  findings contrast reaction  hence, they  case  of  with  the  that  grown on  suggest  that  of h i g h e r  a b o l i s h e d by  system  uricase.  indicative  both  Silverberg and  a l l four  These  and Sawa, 1.974; Mueller,  1981).  catalase-  i s only observed i n (Table 8 ) .  cytochrome-dependent i s observed  by  microalgae  of u r i c a s e a c t i v i t y , product  or  of the o c c u r r e n c e  of  Olah  of r e a c t i o n  deposition  non-competitive  cytochrome  hypoxanthine-grown c e l l s  products  both m i c r o a l g a e  1969;  after  of c a t a l a s e , but  cyanide, a  mitochondria  and G o l d f i s h e r ,  Furthermore,.as  of  in  and  conditions,  inhibitor  inhibitor considered  9.0  i s completely  mitochondrial  are  pH  observed  a r e i n c u b a t e d a t 3 7 ° C . The  by p o t a s s i u m  activity  Rhee  cells  products  an  a  T a y l o r and  i n the presence  non-competitive  not a f f e c t e d  at  these  occurs only  reation  as  cytochrome c  from t h e r e a c t i o n  microalgae  when t h e  This  1981).  in mitochondria  of  8).  formed  and Sawa, 1974;  reactions are d i s t i n c t  fixation  t o be  system, i n c l u d i n g  (Silverberg  Olah  (Table  These  deposition  i n mitochondria  sources  s y n t h e s i s of u r i c a s e  of  of  nitrogen;  and c a t a l a s e  46  and  their  localization  in  mitochondria  is  substrate  dependent. An  increase  chloroplast  lipids  with aging and  in  in  has  cells  Dunaliella  starch been  Similar  reported  cells  aging  photoheterotrophically et  lipids  observed  used  in  this  present  not  Why  nitrogen  nitrate,  the  between  can  density  Dunaliella determined.  and  of  Chroomonas s a l i n a  increase  in starch of  since  early  in starch  not  cellular a  chloroplasts  the  cells  exponential and  lipids  escapes  of  nitrogen  p r o c e e d s as  effects  one,  of  since  efficiently  1984).  account of  tert iolecta  for  the and  vacuoles with  metabolism However,  the  vacuolar Pavlova  was  lutheri  respect  a  remains  in  similar  observed  compartments  as An  reported  whether  increases  our  ultrastructure.  deleterious  and  and  Dunaliella  hypoxanthine-supplemented c e l l s .  (Tischner,  interaction  is  on  grown  photoautotrophic  nutritional  acid cultures  u r e a or  regulation  Chlorella  volume  effect  in allantoic  interaction to  sources  1981)  acid-grown c e l l s  the  1975)  been  from the  increase  of  (Eyden,  acid-grown c e l l s  in a l l a n t o i c  N e v e r t h e l e s s , the  in  an  association  Maluf,  a t t r i b u t e d to aging  understanding  different  growth  be  and  have a l s o  cultures  However, t h e  such  develop  and  both  s t u d y were o b t a i n e d  growth p h a s e . would o n l y  of  in a l l a n t o i c  can  in  observations  grown  a l . , 1973).  tert iolecta  shown t o o c c u r  (Hoshaw  photoautotrophically.  (Antia  cytoplasmic  of D u n a l i e l l a p r i m o l e c t a  tert iolecta  in  granules,  in  of  the both  to  be  47  Conclusions In  conclusion, i t  possess  the  derivatives these the  can  ability as  sole  be  to  said  utilize  sources  of N. The  compounds seem t o o c c u r  that  purines  plants  (Vogels  (Reynolds  and  e t a_l. ,  of  i n most of t h e s e m i c r o a l g a e  by  van  d e s c r i b e d for other  der D r i f t ,  1976)  lutheri,  oxidation  to  without  final  conversion  indicates of  the  al.,  the o c c u r r e n c e  pathway o f 1989)  suspension The  and  in this  cultures  the  hypoxanthine)  plus  c h a n g e s . Some  of the  distribution mechanisms increases  of  2 +  metal ER  and  that observed  uricase  and  major  catalase  hypoxanthine-grown c e l l s  (Antia  1988).  supports  this  (urea  can  be  Other  size  changes  the  and  attributed  of p u r i n e s  to  (i.e.  occurrence  demonstration in  and  ultrastructural  vesicles,  degradation  activities  et  of p e r i c h r o m a t i n i c  indicate  i n h i g h e r p l a n t s . The  This  modification  1987,  ( i . e . size  microbodies)  the  seems  urea.  in organic N-sources  homeostasis.  of  r e p o r t e d f o r soybean  of d i c t y o s o m e - d e r i v e d  a mechanism f o r t h e c a t a b o l i c to  situation  produces  changes  to  utilization  the v a c u o l a r a p p a r a t u s )  of in  Ni  higher  of p u r i n e s  of a  (Winkler et a l . ,  g r o w t h of A m p h i d i n i u m  g r a n u l e s , number  microalga  purine-derived N  resembles  cell  and  1982).. However, i n t h e c a s e  Pavlova  place  their  oxidation  Prymnesiophyte take  microalgae  and/or  catabolic  s t a n d a r d pathway of p u r i n e o x i d a t i o n  microorganisms  some  of  similar of  both  microbodies  of  interpretation.  48 The in (i.e. to  growth of D u n a l i e l l a  hypoxanthine  also  increases  i n E.R.  the  through  occurrence  u r i c a s e and  The catalase  of m i c r o b o d i e s , allantoin reaction  and was  the  produces and  mitochondria)  activities  the d i s p o s a l transferred  to  of  of the  lutheri  that  and  i n the  oxidation  ^2^2  mitochondria.  related  utilization  demonstration  the  changes  directly  purines  suggests  s t e p of  Pavlova  for nitrogen  degradation  cytochemical  key  and  ultrastructural  of a mechanism  the c a t a b o l i c  derivatives.  tertiolecta  their  of  both  absence  of u r a t e  produced  in  to  this  49  REFERENCES  AL  -  HOUTY  F.A.A. and  of  SYRETT  urease/urea  P . J . , 1984. 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In E l e c t r o n  and  Methods,  Van  Nostrand  62  .APPENDIX FIGURE EXPLANATION Symbols: CH (or Ch)= chloroplast, ER = endoplasmic reticulum L = l i p i d inclusion, M = mitochondrion, m = microbodies, Pu = pusule,  Figure 1 - Growth (1 mM)  N = nucleus,  n = nucleolus,  Py = pyrenoid, V = vacuole  curves of Hymenomonas elongata without citrate  addition  on urea  (A), with 5 mM  citrate (A) followed by the addition of 2 5 uM N i  2+  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 N i  16 ( 1 ) . ( 2 mM)  2+  at day  Corresponding control growth on nitrate 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 at day  1 4 ( T ) , and also on hypoxanthine  without citrate (O)  addition  ( 2 mM)  with  shown ( X ) .  2+  ( 0 . 5 mM)  (•), with 5 mM citrate  followed by the addition of 2 5 JJM N i  10 (• t ) •  uM N i  25  2+  at day  Corresponding control growth on nitrate or  without  citrate addition  is  63  Figure  3  - Detection cell  -  of x a n t h i n e dehydrogenase  free  previously  grown  presence of 340 nm  f o r the  Figure  4a  of  Figure  + hypoxanthine  vicinity  5 - Longitudinal  section  vicinity  Figure  6  1 pM  7  4b  through a c e l l  t o ER i n  shows  grown  part  i n 1 mM i n the  s e c t i o n through a c e l l  grown  i n 0.5  of the c h l o r o p l a s t .  and 1 pM n i c k e l . A r r o w h e a d s w h i l e t h e arrow  developing  from ER - l i k e  - Section  through a  shows  division.  8 - Nucleus  Figure  9 - Microbody  shows a  numerous  elements.  mitochondria t o ER  - ER a s s o c i a t i o n cell.  grown  undergoing  elements.  o f a h y p o x a n t h i n e / n i c k e l - grown  - grown  point  microbody  hypoxanthine/nickel -  Arrows p o i n t  Figure  nickel  points  in 2  t o ER  hypoxanthine  cell  grown  points  to m i c r o b o d i e s ,  Figure  (+) , and  + a l l o p u r i n o l (•).  n i c k e l . Arrow  - Longitudinal mM  the  enzyme +  + hypoxanthine  of c h l o r o p l a s t . F i g u r e region.  u r e a and  (O),  extract  n i c k e l . Arrow  the pusule  in  measurements, a t  s e c t i o n through a c e l l  and no  carterae  hypoxanthine  . Absorbance  ( A ) , enzyme  nitrate  the  2 +  Amph i d i n i um  mM  enzyme o r  - Longitudinal mM  of  on 0.5  1 pM N i  allopurinol enzyme  extracts  activity in  cell.  i n a hypoxanthine/  64  Figure  10  -  Microbodies  structure nickel  Figure  Figure  11  connected  (arrowhead)  - grown  grown  indicated  by  12 -  a  in  narrow  an  tubular  hypoxanthine/  cell.  - Associations nitrate-  by  between cell.  dictyosome  -  Transition  ER  in  vesicles  a  are  arrowheads.  ER - d i c t y o s o m e  grown c e l l .  associations  in a urea/nickel  Transition vesicles  -  a r e i n d i c a t e d by  arrowheads.  Figure  13 -  ER - d i c t y o s o m e  nickel  -  grown  transition  Figure  14  cell.  of  15  16  activity  t o 19  -  Dunaliella (Figure  - grown  of r e a c t i o n  hypoxanthine/nickel  Figures  to  in - grown  cell.  product a  grown  acid  (Figure  urea  of  of a  cell.  tertiolecta mM  indicative  microbody  sections  1  (arrowheads)  i n m i c r o b o d i e s of a  Longitudinal  16),  allantoic  points  product  of u r i c a s e a c t i v i t y  - Deposition catalase  hypoxanthine/  Arrowhead  reaction  hypoxanthine/nickel  Figure  in a  vesicles.  - Deposition indicative  associations  t h r o u g h c e l l s of  in  2  (Figure  18) and 0.5 mM  mM 17),  nitrate 0.5  mM  hypoxanthine  65  (Figure  19).  endoplasmic  Figures  20  t o 23 Pavlova 1 mM  to elements  of the  reticulum.  -  Longitudinal  lutheri  urea  (Figure  Arrowheads p o i n t  grown  (Figure  22)  i n 2 mM  21),  and 0.5  Arrowheads p o i n t  sections  mM  through  nitrate  0.5  mM  (Figure 20),  allantoic  hypoxanthine  to.elements  c e l l s of  acid  (Figure 23).  of the  endoplasmic  reticulum.  Figures  24  and 28  -  activity (Figure on  Cytochemical  localization  i n m i t o c h o n d r i a of D u n a l i e l l a 24)  and P a v l o v a  hypoxanthine  lutheri  (0.5  mM)  as  of  uricase  tertiolecta  (Figure sole  28) grown source  of  nitrogen.  Figures  25  and 29  -  dependent  reaction  mitochondria and  Cytochemical  Pavlova  26  and 30 activity (Figure on  •  -  product  lutheri  ( F i gure  nitrogen.  with  25) 2  mM  .  Cytochemical  and P a v l o v a  hypoxanthine  in  (Figure  29) t r e a t e d  localization  i n m i t o c h o n d r i a of D u n a l i e l l a 26)  of u r i c a s e -  deposition  of D u n a l i e l l a t e r t i o l e c t a  trichloropurine.  Figures  inhibition  (0.5  lutheri mM)  as  of  tertiolecta  (Figure sole  catalase  30) grown source  of  66  Figures  27  and  31  -  dependent  Cytochemical reaction  inhibition  product  of D u n a l i e l l a t e r t i o l e c t a  and  lutheri  (Figure  amino-1,2,4,-triazole.  catalase-  deposition  mitochondria Pavlova  of  31)  (Figure  treated  with  in 27) 3-3'  Figure 1 - Hymenomonas elongata Nickel chelation with Urea/Allantoic acid as N-source O.6-1  X~"""* A  A — A ^ - X A  Legend A Urco-cH jk Ureq+cIJ _ i>  •  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 Growth Period [days]  24  26  28  30  32  34  Figure 3 - Amphidinium carterae Xanthine Dehydrogenase Activity 1-1  ...+  +  ..+  0.8-  .+• E c  O 0.6•<*  £2,  d) o c o •8 O  V)  0.4-  Si  < 0.2  I,-'--"  — o — o — o — 0 - 7 - 0 — 0 —— 0 — 0 O'—o—o—o—o—o——o—  1  .  .  .  Legend  O |nr A Enz+AM  A  + Enz+HyR 1  0  1  1  1  1  2  1  1  3  1  1  4  1  1  5  1  1  1  1  1  1  6 7 8 Time [minutes]  1  1  9  1——I  10  1  1  11  1  1  12  1  1  1 1  13  14  •  Enz+Hyp+AII  •  72  73  7L>  75  76  78  Table 1 - Microalgae used i n the present study STRAIN/CLONE (isolator/supplier/source)  ALGA PRYMNESIOPHYCEAE J-SQchrysiLs galbana Hymenomonas elongata Prymnesium parvum Carter Pavlova l u t h e r i  Leftley, Leftley, Droop, Leftley,  SMBA SMBA SMBA SMBA  No. 58 No. 62 No. 65 No. 261  BACILLARIOPHYCEAE Thalassiosira nordenskioldii Thalassiosira pseudonana  Cleve  Rao, Bedford I n s t i t u t e , Darmouth , N.S. Neil P r i c e , U.B.C.  CHL OROPHYCEAE Dunaliella t e r t i o l e c t a  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. C a t t o l i c o ,  Seattle  EUSTIGMATOPHYCEAE Nannochloropsis oculata  L e f t l e y , 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 y i e l d (%) Growth parameters are expressed as percentage of those i n media containing n i t r a t e (2 mM) without n i c k e l . (—) = no growth  NICKEL 0  4  ALGA  nuun  a  b  Isochrysis galbana  4  Hvmenomonas elonaata  3  c  s  10"-3  1  c  a  b  x  SUPPLEMENTATION  10"•2  1  c  a  b  a  b  109 91  5  110 91  5  106 89  5  53  83  2  55  80  2  60  82  2  Prymnesium parvum  10 23  34  10 21  25  10 20  Pavlova l u t h e r i  4  96  103  4  96  104  4  Thalassiosira nordenskioldi 12  73  79  2  80  85  Thalassiosira pseudonana  2  76  46  2  72  Dunaliella t e r t i o l e c t a  2  122 110  4  92  Aamanellum quadruplicatum  2  102 103  5  x  10" 1 c  (uM)  1 a  b  105 90  5  66  85  3  24  10 20  92  97  4  89  2  84  86  2  50  2  69  48  110  4  88  112 110  5  5 c  a  b  110 91  5  80  98  4  22  10 16  90  4  96  105 103  2  70  106  4  89  107 103  10 c  a  b  c  103 88  5  102 89  76  90  6  70  82  18  10 16  18  10 17  19  103  4  90  93  4  91  92  2  133 117  2  120 110  2  118 110  52  2  76  69  2  70  68  2  65  60  108  4  77  110  4  80  103  4  81  105  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  4  90  89  4  99  90  4  97  89  4  91  90  Nannochloropsis oculata  2  107 94  4  98  91  4  95  88  -SI  Table 3 - Growth of microalgae on a l l a n t o i c acid (0.5 mM) with nickel supplementation. (a) adaptation period (days); (b) exponential growth rate (%) ; (c) maximum y i e l d (%) Growth parameters are exoressed as percentage of those in media containing n i t r a t e (2 mM) without nickel. (—) = no growth NICKEL 4 x 10"-3  0  SUPPLEMENTATION  1 x 10"-2  1 x 10"-1  (uM)  1  10  5  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 l u t h e r i  —  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 t e r t i o l e c t a  2  78  83  4  85  84  4  82  80  4  81  83  4  85  Aamanellum quadruplicatum  2  98  55  5  110 55  6  102 52  6  101 51  5  109 55  84  79  78  4  80  81  10 98  51  10 99  52  4  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 m i c r o a l g a e on hypoxanthine (0.5 mM) w i t h n i c k e l s u p p l e m e n t a t i o n . (a) a d a p t a t i o n period (days); (b) e x p o n e n t i a l , growth r a t e (%) ; (c) maximum y i e l d (%) Growth parameters a r e expressed as percentage of those i n media c o n t a i n i n g nitrate (2 mM) w i t h o u t nickel (—) = no growth NICKEL 4 x 10"•3  0 ALGA a  b  c  I s o c h r y s i s qalbana  __  Hymenomonas e l o n a a t a  —  4  84  Pavlova l u t h e r i  6  135 113  nordenskioldil  Thalassiosira  pseudonana  Dunaliella tertiolecta  c  b  1 x 10*-2  1 X 10"  a  a  —  Prymnesium parvum  Thalassiosira  a  83  b  c  —  4  86  85  6  124 113  b  1  c  87  6  121 108  81  (uM)  1 a  —  4  b  5 c  a  b  —  4  83  6  123 112  85  10 c  a  —  4  84  83  6  129 112  b  c  —  6  81  83  6  115 10 5  6  80  6  116 108  4  83  83  »  —  2  79  .  110  4  —  79  —  112  4  82  Aamanellum q u a d r u o l i c a t u m  —  —  —  Amnhidinium c a r t e r a e  —  —  —  Olisthodiscus luteus  —  —  —  Nannochloropsis o c u l a t a  SUPPLEMENTATION  2  91  86  4  89  88  4  85  110  4  76  105  4  —  7  71  4  81  112  4  78  —  58  4  —  82  79  —  95  —  90  4  4  87  82  4  72  4  —  80  104  —  89 .  —  84  109  72  54  —  76  4  81  79  82  Safrle 5 - Optimal c o n c e n t r a t i o n s of n i c k e l ( i f r e q u i r e d ) , u r e a , a l l a n t o i c a c i d and hypoxanthine (where a p p l i c a b l e ) t h a t s u p p o r t maximum y i e l d s of the m i c r o a l g a e (—) = no growth  ALGA  NICKEL (uM)  UREA (mM)  ALLANTOIC ACID (mM)  HYPOXANTHINE (mM)  I s o c h r y s i s aalbana  0  1  0.5  Hymenomonas  1  1  0.5  —  Prymnesium parvum  0  4  —  2  Pavlova  0  1  0.5  0.5  Dunaliella tertiolecta  0  1  0.5  0.5  Aqmanellum quadruDlicatum  0  1  0.5  —  Amphidinium c a r t e r a e  1  2  —  1  Nannochloropsis  0  2  1  1  elonqata  lutheri  oculata  e x t e r n a l a d d i t i o n of n i c k e l was not r e q u i r e d  83  Table 6 - C o n c e n t r a t i o n s (uM) o f a l l o p u r i n o l ( A l ) , 2,6,8 - t r i c h l o r o p u r i n e (Tc) and h y d r o x y u r e a '(Hu) r e q u i r e d f o r 50% i n h i b i t i o n o f m i c r o a l g a l growth i n media supplemented w i t h hypoxanthine, a l l a n t o i c a c i d or urea as o r g a n i c N-sources  Organic N - Source / I n h i b i t o r s  Alga Urea Hu  Allantoic  acid  Hypoxanthine  Hu  Al  Tc  Hu  1  1  -  Hymenomonas e l o n a a t a  10  10  -  Prymnesium parvum  10  -  -  -  1  5  10  Pavlova  5  500  5  5  500  5  -  -  -  1  -  -  -  Dunaliella tertiolecta  50 0  500  2  5  500  Aamanellum  10  10  -  -  Amohidinium c a r t e r a e  1  -  1  10  1  Olisthodiscus  5  -  -  -  -  10  10  1  10  10  Isochrysis  qalbana  lutheri  Thalassiosira  nordenskioldii  Thalassiosira  Dseudonana  quadruplicatum  luteus •  Nannochloropsis  oculata  symbol (-) i n d i c a t e s  t h a t the m i c r o a l g a does not grow on t h a t p a r t i c u l a r N-source  8 4  Table  7  -  Xanthine  dehydrogenase  allantoicase microalgae  (ALC)  grown  (XD),  a c t i v i t i e s  i n  allantoinase i n c e l l  -  free  (ALN)  and  e x t r a c t s  of  hypoxanthine  Crude  Extract  A c t i v i t i e s  1  Alga XD  ALN  Amphidinium  carterae  3 . 1 + 0 . 2  D u n a l i e l l a  t e r t i o l e c t a  2.2  ±  2.9  ±  Pavlova  1  l u t h e r i  Enzyme and  a c t i v i t i e s  represent  a r e  averages  1.9  ±  0.3  2.3  ±  0 .1  0.3  . 3.0  +  0.2  1.5  ±  0 .3  0.1  2.6  ±  0.2  1.7  ±  0 .2  expressed of  ALC  three  as  nmol  separate  product assays  m i n  - 1  mg  p r o t e i n  Table 8 - Summary of the occurrence and characteristics of the cytochemical reactions observed in mitochondria of Dunaliella t e r t i o l e c t a and Pavlova l u t h e r i grown on four different sources of nitrogen  Cytochemical Nitrogen  Requirements  Source  Incubation  H2O2  pH  Nitrate  None  6.0  Urea  None  Allantoate Hypoxanthine  Substrate  Hypoxanthine  Uric acid  Hypoxanthine  Symbols:  H  Parameters  Temperature  Pre - Fixation  Inhibitor  C(%)  T(mim)  22-27*C  1% Ga  30-60  KCN  Cytochrome system  6.0  22-27°C  1% Ga  30-60  KCN  Cytochrome system  None  6.0  22-27*C  1% Ga  30-60  KCN  Cytochrome Bystem  None  6.0  22-27 *C  1% Ga  30-60  KCN  Cytochrome system  Yes  9.0  37 °C  0.25% Ga  5  TC/OX  Uricase  Yes  9.0  37 °C  1-2% Ga  60-90  activity  AT  2°2 hydrogen peroxide, C = concentration of prefixative, T = time (duration) of prefixation, KCN = potassium cyanide, OX = oxypurines, AT = aminotr iazole =  Enzyme  Catalase  Ga » glutaraldehyde TC « tr ichloropur ine  

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