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Comparative qualitative analyses of hydrolysis products of extracellular polysaccharides Flodin, Patricia E. M. 1972

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COMPARATIVE QUALITATIVE ANALYSES OF HYDROLYSIS PRODUCTS OF EXTRACELLULAR POLYSACCHARIDES PRODUCED BY SOME YEASTS AND YEAST-LIKE FUNGI  by P a t r i c i a E . M. F l o d i n B.Sc,  U n i v e r s i t y o f B r i t i s h Columbia, 1969  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER  OF SCIENCE  i n t h e Department of Botany  We accept t h i s t h e s i s as conforming t o the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA August 1972  In p r e s e n t i n g t h i s t h e s i s  in p a r t i a l  f u l f i l m e n t o f the r e q u i r e m e n t s  an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, the L i b r a r y  s h a l l make i t  freely available for  I agree  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department  of  representatives.  It  this thesis for financial  written  Department  a  t  e  ftfy£-  /3  }  )9  Columbia  7^L  or  publication  g a i n s h a l l not be a l l o w e d w i t h o u t my  of  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  D  i s understood t h a t copying o r  permission.  that  r e f e r e n c e and s t u d y .  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s  by h i s  for  i  ABSTRACT  The o b j e c t i v e  of the experiments was t o compare  '  q u a l i t a t i v e l y the monosaccharides i n the h y d r o l y s i s products of the e x t r a c e l l u l a r p o l y s a c c h a r i d e s of s e v e r a l y e a s t s and y e a s t - l i k e f u n g i .  S p e c i f i c a l l y , t h e study was  aimed a t f i n d i n g s i m i l a r i t i e s and d i f f e r e n c e s  that  might  be u s e f u l i n suggesting and s u p p o r t i n g taxonomic r e l a t i o n ships.  Gas chromatography and paper chromatography were  used as methods o f a n a l y s e s i n an e f f o r t t o f i n d out what method i s s u f f i c i e n t a t the q u a l i t a t i v e l e v e l f o r distinguishing  some genera o f y e a s t s and y e a s t - l i k e  and what method would be b e s t at the q u a n t i t a t i v e  fungi;  level for  d i s t i n g u i s h i n g amongst some s p e c i e s o f the same genus. the  a n a l y t i c a l r e s u l t s i t was found t h a t  u s i n g the s o l v e n t s  paper  ethyl acetate: pyridine:  From  chromatography  water,  (8:2:2)  was s u f f i c i e n t f o r q u a l i t a t i v e d e t e r m i n a t i o n of the monosaccharides i n the e x t r a c e l l u l a r p o l y s a c c h a r i d e h y d r o l y s i s products.  However, i n d i c a t i o n s were t h a t  a n a l y s e s by gas chromatography, derivatives  quantitative  u s i n g the t r i m e t h y l s i l y l  of the monosaccharides would have been  in distinguishing  among s p e c i e s o f the same genus.  successful Two  groups were formed on the bases o f the q u a l i t a t i v e r e s u l t s .  Group I c o n t a i n e d two subgroups.  Subgroup I encompassed  those y e a s t s and y e a s t - l i k e f u n g i with the monosaccharides g a l a c t o s e , glucose, mannose, x y l o s e p r e s e n t i n the h y d r o l y s i s products  of t h e i r e x t r a c e l l u l a r p o l y s a c c h a r i d e s .  i n t h i s Subgroup I a r e : Cryptococcus mesenterica,  laurentii,  Included Tremella  B u l l e r a a l b a , Sporobolomyces odorus,  Sporobolomyces s i n g u l a r i s , and Rhodotorula  glutinis.  Sub-  group I I i s U s t i l a g o h o r d e i o n l y , with t h e monosaccharides g a l a c t o s e , glucose, mannose, and l a c k i n g x y l o s e . c o n t a i n s T a p h r i n a p o p u l i n a o n l y , with p r e s e n t and both g a l a c t o s e and x y l o s e The  two groups formed support  glucose and mannose absent. some o f t h e taxonomic  r e l a t i o n s h i p s t h a t have a l r e a d y been suggested. Tremella  - Cryptococcus  Group I I  The  taxonomic r e l a t i o n s h i p t h a t had  p r e v i o u s l y been p o s t u l a t e d on the b a s i s of s i m i l a r i t i e s i n e x t r a c e l l u l a r p o l y s a c c h a r i d e h y d r o l y s i s products,  morphology,  carbon a s s i m i l a t i o n p a t t e r n s , enzymatic x y l o s y l a t i o n r e a c t i o n , and s t a r c h f o r m a t i o n was supported.  Secondly,  the C r y p t o c o c c u s - B u l l e r a r e l a t i o n s h i p t h a t had been  suggested  on the b a s i s o f i n o s i t o l a s s i m i l a t i o n , l a c k o f pseudomycelium, and  s i m i l a r i t i e s i n s t a r c h s y n t h e s i s , was supported  by the  q u a l i t a t i v e a n a l y s i s o f the monosaccharides p r e s e n t i n the e x t r a c e l l u l a r polysaccharide h y d r o l y s i s products.  The  iii  monosaccharides and  Bullera  were t h e  found  i n both Cryptococcus  alba extracellular polysaccharides  same q u a l i t a t i v e l y .  Duality  species  of Sporobolomyces  further  work u s i n g q u a n t i t a t i v e  analyses. account and  This  of the d u a l i t y  p e r c e n t G+C  base  starch  Rho d o t o r u l a not  produce  gas  a n a l y s e s o f DNA.  chromatographic on  analyses  Taphrina  Cryptococcus  laurentii  inositol  but  inositol.  monosaccharides  extracellular  present i n the  polysaccharide hydrolysis laurentii  galactose  and  p r o d u c t s of b o t h  and R h o d o t o r u l a g l u t i n i s  Two  Cryptoare  x y l o s e whereas T a p h r i n a p o p u l i n a  l a c k s t h e s e two Results  does  Taphrina p o p u l i n a produces  s t a r c h but does not a s s i m i l a t e  coccus  glutinis  inositol:  assimilates  and  with  from R h o d o t o r u l a  assimilates  glutinis starch;  supported  shown i n a n t i g e n i c  laurentii. and  be  amongst  been p o s t u l a t e d  distinguished  Cryptococcus  produces  might  d u a l i t y had  p o p u l i n a c a n be and  laurentii  monosaccharides. o b t a i n e d from t h e q u a l i t a t i v e  the e x t r a c e l l u l a r  p o l y s a c c h a r i d e s produced  by  analyses of f u n g i may  be  important  taxonomically.  i n f o r m a t i o n may  be  along  with  morphology, c y t o l o g y , taxonomic  i s because the  u s e d when d e c i d i n g on  fungal relationships. considered  This  However, t h i s data and  relationship.  from o t h e r  qualitative  Perfect-Imperfect  information fields  such  should  be  as  genetics before hypothesizing  on  a  V  Contents  Page  Abstract  i  Contents  v  Tables  Figures  vi  v i i  Acknowledgements  x  Introduction  1  L i t e r a t u r e Review  3  M a t e r i a l s and Methods  12  Results  29  Discussion  48  References  58  Appendix  62  vi  Tables  Table  I  Page  The h o s t ,  location,  date,  c o l l e c t o r of  and  collection  fungus used i n the  each  experiments  (when k n o w n ) .  II  III  IV  V  Paper  Gas  13  Chromatography  Chromatography  results  results  Gas cflaxomatography-peak Areas  31  35  63  Gas chromatography-percent monosaccharide  67  vii  Figures  Figure  1  Page  Procedure f o r the production, and a n a l y s i s  isolation  of the e x t r a c e l l u l a r  polysaccharide  2  A p p a r a t u s f o r t h e IR-120 exchange  3  14  cation  resin  23  T r e m e l l a m e s e n t e r i c a and B u l l e r a  alba.  Separation of products using descending paper chromatography w i t h ethyl  4  acetate:  Sporobolomyces odorus  solvents  p y r i d i n e : water,  singularis,  laurentii .  32  Sporobolomyces  (#949) , S p o r o b o l o m y c e s  Cryptococcus  (8:2:2)  o d o r u s (#981) ,  Separation of  t h e p r o d u c t s u s i n g d e s c e n d i n g paper chromatography with acetate:  pyridine:  solvents water,  ethyl  (8:2:2).  33  Ustilago hordei, Rhodotorula Taphrina populina .  S e p a r a t i o n of  products  using descending  tography  with  pyridine:  water,  as  Bullera  acetate:  Separation of  laurentii.  derivatives  Separation of  as t r i m e t h y l s i l y l  alba.  derivatives.  Separation of  as t r i m e t h y l s i l y l  products  derivatives.  Sporobolomyces odorus  (#949).  ation  as  of the  chroma-  (8:2:2).  trimethylsilyl  Cryptococcus products  paper  solvents ethyl  Tremella mesenterica. products  glutinis,  products  Separ-  trimethylsilyl  derivatives.  Sporobolomyces odorus  (#981).  ation  as  of the  products  Separ-  trimethylsilyl  derivatives.  Sporobolomyces of  the  products  derivatives.  singularis. as  Separation  trimethylsilyl  IX  Figure  12  Page  Rhodotorula the products  glutinis. as  Separation of  trimethylsilyl  derivatives.  13  14  45  Ustilago  hordei.  Separation of  products  as t r i m e t h y l s i l y l  Taphrina populina. products  the  derivatives.  Separation of  as t r i m e t h y l s i l y l  46  the  derivatives.  47  X  ACKNOWLEDGEMENTS  First,  I would  like  t o t h a n k D r . R. J . B a n d o n i ,  Department  o f B o t a n y , U.B.C. f o r h i s c o n s t r u c t i v e  criticisms  on t h i s  thesis  concerning the d i r e c t i o n would  like  and f o r h e l p f u l  suggestions  o f my r e s e a r c h w o r k .  Second, I  t o t h a n k D r . G. G. S . D u t t o n , D e p a r t m e n t o f  C h e m i s t r y , U.B.C. f o r t h e u s e o f t h e g a s c h r o m a t o g r a p h machine, for to  helpful  advice  paper, s o l v e n t s ,  concerning  and t a n k s .  Third,  h i s patience  I would  like  Also  c h r o m a t o g r a p h y I would  t h a n k D r . G. G. S . D u t t o n , M r . R. H. W a l k e r ,  S. K a b i r . for  chromatograph  like  and D r .  t o t h a n k my h u s b a n d  Bruce  and u n d e r s t a n d i n g w h i l e work f o r t h i s  d e g r e e was i n p r o g r e s s .  INTRODUCTION  In gather  general,  valuable  a classification  t h e p u r p o s e o f t h e e x p e r i m e n t s was t o  information scheme.  t h a t may a i d i n t h e s y n t h e s i s o f  The d a t a  obtained  tative  analyses  yeasts  a n d y e a s t - l i k e f u n g i may g i v e  already  postulated  T h i s process correlating part  of the e x t r a c e l l u l a r  the r e s u l t s  of previous  t o some o f t h e  fungal r e l a t i o n s h i p s .  re-examining,  adding  workers i s a  t o , and necessary  t h e o b j e c t i v e s o f t h e e x p e r i m e n t s were:  To determine q u a l i t a t i v e l y the  extracellular  yeasts 2.  support  o f some  o f any r e s e a r c h . Specifically,  1.  polysaccharides  Perfect-Imperfect  of c r i t i c a l l y  from t h e q u a l i -  the h y d r o l y s i s products  polysaccharides  of  produced by s e v e r a l  and y e a s t - l i k e f u n g i .  T o e m p l o y b o t h g a s c h r o m a t o g r a p h y and p a p e r  chromato-  g r a p h y a s methods o f a n a l y s i s a n d t o d e t e r m i n e : (a)  w h i c h method i s b e s t  at the q u a l i t a t i v e  (b)  w h i c h method i s b e s t  f o r separating  and 3.  species  of previous  genera  only,  o f t h e same g e n u s .  T o compare and c o n t r a s t t h e q u a l i t a t i v e those  level,  workers.  1  results  with  To compare and c o n t r a s t the yeasts and y e a s t - l i k e f u n g i i n v e s t i g a t e d with respect t o the monosaccharides i n t h e i r e x t r a c e l l u l a r polysaccharide h y d r o l y s i s products. To arrange the yeasts and y e a s t - l i k e f u n g i i n v e s t i g a t e d i n t o groups on the b a s i s of q u a l i t a t i v e s i m i l a r i t i e s or d i f f e r e n c e s i n the monosaccharides i n t h e i r e x t r a c e l l u l a r polysaccharide h y d r o l y s i s products. To suggest taxonomic r e l a t i o n s h i p s considering the above formed groups i n conjunction with data supplied from other f i e l d s such as morphology and g e n e t i c s . To examine the v a l i d i t y of using c e r t a i n biochemical analyses, i n p a r t i c u l a r , those of the e x t r a c e l l u l a r polysaccharides i n the synthesis of a c l a s s i f i c a t i o n scheme.  LITERATURE REVIEW  For used I  convenience,  i n the  yeasts  experiments w i l l  Subgroup I c o n t a i n s  laurentii  the  (Kufferath)  be  i n groups.  Tremella  mesenterica  Skinner,  Bullera alba e t Do  S p o r o b o l o m y c e s o d o r u s D e r x . , and  Lagerh.  yeast-like fungi  reviewed  Sporobolomyces s i n g u l a r i s P h a f f  Harrison.  and  Group I Subgroup I I Group I I i s T a p h r i n a  Fr.,  Cryptococcus  (Hanna) D e r x . ,  Carmo S o u s a ,  Rhodotorula  glutinis  i s Ustilago hordei populina  Group  (Fres.)  (Pers.)  Fr.  Group I Subgroup I The  extracellular  mesenterica  and  Cryptococcus  extensively.  The  c o n t a i n s both  an  polymer  S l o d k i , i n 1966  products  o f the  ratio  o f 0.5  Jennings, the  xylose,  : 4.4  i n 1971  Tremella  (43),  identified  mannose, and  : 3.8 (19),  the  Cryptococcus  contains 3  acid hydrolysis These  Fraser  products in  the  and  a neutral  glucan,  200«C-D-glucopyranose u n i t s  extracellular  laurentii  a neutral  n e u t r a l p o r t i o n of  p o l y s a c c h a r i d e . I t was  the  and  the  : 1 respectively. identified  mesenterica  glucuronic acid  and «Cl-4 i n t h e r a t i o o f 1:2  Similarly, by  p r o d u c e d by  i n s t r u c t u r e , composed o f  linkedoCl-6  Tremella  l a u r e n t i i have been i n v e s t i g a t e d  acidic heteropolysaccharide.  extracellular  linear  of  acidic heteropolysaccharide  glucan.  were O - a c e t y l ,  polysaccharides  respectively.  polysaccharide an  acidic  and  produced  a neutral  4 fraction. fraction  Abercrombie  t o be composed o f D-Mannose,  Glucuronic (24)  acid.  indicated  Glucuronic percent.  However,  studies  D-Xylose  f o u n d some t r a c e s  The n e u t r a l  portion linked  and  D-  seven  of galactose  t h e " b a c k b o n e " was  and t h e e n d g r o u p s were D - X y l o s e  and g l u c o s e . composed  and D - G l u c u r o n i c  of the e x t r a c e l l u l a r  polysaccharide  1-3, 1-4,  1-6  1-2  and/or  (24, 47) .  biosyn'theses o f the c a r b o h y d r a t e s found i n t h e a c i d i c  f r a c t i o n were a l s o (2).  a n d D-  s t u d i e s b y J e a n e s e t a l , i n 1964  showed t h a t  contained D-Glucose The  D-Xylose  O - A c e t y l was d e t e r m i n e d t o be  They a l s o  o f D-Mannose  ( 1 ) , found the a c i d i c  t h e p r e s e n c e o f D-Mannose,  acid.  Structural  acid.  e t a l , i n 1960  D-Mannose  i n v e s t i g a t e d by Abercrombie  and D - G l u c u r o n i c a c i d  e t a l , 1960  were f o r m e d  from t h e  h e x o s e s w i t h o u t any a p p r e c i a b l e breakdown o f t h e h e x o s e skeleton.  D - X y l o s e was  process i n v o l v i n g  formed  the loss  from t h e hexoses  o f c a r b o n s i x ; and D - X y l o s e a n d  L - A r a b i n o s e were b o t h c o n v e r t e d t o D-Mannose, D-Glucuronic  a c i d with rearrangement  t h a t may h a v e i n v o l v e d transketolases.  for 1962  industrial  the action  The c o n d i t i o n s  the e x t r a c e l l u l a r  m a i n l y by a  D-Xylose,  o f the pentose  skeleton  o f t r a n s a l d o l a s e s and  f o r maximum p r o d u c t i o n o f  polysaccharides  of Cryptococcus  p u r p o s e s were i n v e s t i g a t e d  laurentii  b y Cadmus e t a l ,  (12) . The  literature  and  suggests that  evidence e x i s t s f o r  5 postulating  first,  relationships  and  i n general,  Cryptococcus-Basidiomycetous  s e c o n d l y , more s p e c i f i c a l l y ,  Tremella relationships.  First,  the  Cryptococcus-Basidiomyce-  t o u s r e l a t i o n s h i p s were f o r m u l a t e d on  the b a s i s  analyses,  the  content  S t o r c k , i n 1956  a l s o determined fifty-five. that  (48), found  i n some B a s i d i o m y c e t e s Cryptococcus  t o be  f i v e Cryptococcus  y e a s t s t e s t e d had  i n 1968  content of  percent.  (33),  He be  found  hundred  forty-six  base  C+G  percent to  s p e c i e s o u t o f one  a G+C  o f DNA  percent  fifty  a l b i d u s G+C  N a k a s e and Komagata,  Cryptococcus-  to  and  forty  fifty-six  percent. Second,  the Cryptococcus-Tremella r e l a t i o n s h i p s  brought  to light  in  (41), proposed  1966  because  by  s e v e r a l workers a possible  I t was  that  similar  produced  Cryptococcus strains  xylose, Both of  polymers a l s o had  taxonomic  listed  by S l o d k i  D-Arabinose,  or The  lower ready  (32,  found  similar  that  t o those carbon  Slodki  et a l ,  relationship Cryptococcus  s p e c i e s of T r e m e l l a produced  by  assimilation patterns.  e t a l (41)  mannitol,  genera have the a b i l i t y  5.0  41).  o f s i m i l a r i t i e s b e t w e e n some members o f  and T r e m e l l a s p e c i e s .  All  (28,  were  adonitol,  assimilated and  to symthesize  glucose,  trehalose.  starch  at a  pH  41).  removal  by  acid hydrolysis  of  the x y l o s e  6 residues, relative has  perhaps  owing t o t h e i r  t o the mannose-glucuronic  acid  l e d t o the p r e p a r a t i o n of a u s e f u l  study of enzymatic is  apparent  xylosylation  donor n u c l e o t i d e , laurentii respect  to catalysis  ( 1 , 24)  Cryptococcus  t h e b i o s y n t h e s i s of from  transfer  from  d e x y l o s y l a t e d acceptor polymers  xylosyl  Cryptococcus with  UDP-xylose belonging t o  organism ( 4 1 ) . Under  certain  c o n d i t i o n s b a s i d i o s p o r e s o f some  T r e m e l l a l e s bud t o p r o d u c e that  (13).  (3). Extracts  of x y l o s y l  location  acceptor f o r the  and T r e m e l l a m e s e n t e r i c a a r e n o n s p e c i f i c  to p a r t i a l l y either  UDP-xylose  "backbone"  reactions  a s o u r c e o f enzymes c a t a l y z i n g  peripheral  are similar Fraser  yeast-like  colonies  i n culture  t o those o f Cryptococcus (28).  and J e n n i n g s p r e s e n t e v i d e n c e  Cryptococcus-Tremella relationships  against the  ( 1 9 ) . The  neutral  g l u c a n o f T r e m e l l a m e s e n t e r i c a h a s n o t b e e n shown t o contain  theOCl-3  laurentii  neutral  The resemble  linkages that glucan  species.  t h e d . 1 - 4 , oC 1-6 l i n k e d 11,  (1, 4 7 ) .  Tremella mesenterica neutral  structurally  Pullularia  are present i n the Cryptococcus  the neutral The r e a s o n s  glucan produced  by  f o r the resemblance  glucopyranose  19, 35, 45, 53, 54) .  glucan i s said to  units  i n both  are  ( 8 , 9,  7  Some s p e c i e s o f C r y p t o c o c c u s taxonomically  related  (32).  a n d B u l l e r a c o u l d be  T h e y a s s i m i l a t e i n o s i t o l and  b r e a k the e c - g l u c o s i d i c bonds o f m e l i b i o s e , methyl-D-glucoside. lactose, is  cellobiose,  present  Among  and p s e u d o m y c e l i u m  The  glutinis ninety  i s absent.  their  ability  Starch Some  and sucrose,  synthesis  strains  t o p r o d u c e and t h e genus  (32) .  extracellular Sporobolomyces  s p e c i e s have a l s o been described  assimilate  b a l l i s t o s p o r e s m i g h t be c l a s s e d w i t h  Cryptococcus  glutinis,  they  and s e v e r a l p e n t o s e s .  of B u l l e r a t h a t have l o s t discharge  others,  melezitose,  polysaccharides singularis,  as a s t r a i g h t  polysaccharide  polysaccharide  linked  o f two u n i d e n t i f i e d  t o be a p h o s p h o r y l a t e d  galactan  in  proportions.  equal  of  (20),  Rhodotorula  a hexose and a m e t h y l  products.  i n 1966 ( 4 3 ) , r e p o r t e d  approximately  et a l  1-3, {$ 1-4 D-mannopy-  They a l s o r e p o r t e d  p e n t o s e among h y d r o l y s i s Slodki,  Gorin  c h a i n mannan, composed o f a t l e a s t  units of alternately  ranose r e s i d u e s .  Rhodotorula  and Sporobolomyces  investigated.  the e x t r a c e l l u l a r  of  the e x t r a c e l l u l a r  Sporobolomyces  species  o f e C l - 3 , o< 1-6 l i n k e d D - G a l a c t o s e and  units  8 D - G a l a c t o s e - 6 - p h o s p h a t e were t h e o n l y  components  f o u n d on  hydrolysis. Gorin produced  e t a l (21) s t a t e d t h a t S p o r o b o l o m y c e s  a t r i s a c c h a r i d e , g a l a c t o s y l - l a c t o s e and a  saccaride,  g a l a c t o b i o s y l l a c t o s e from  Galactopyranosyl in  an e x t r a c e l l u l a r  and S p o r o b o l o m y c e s  to the e x t r a c e l l u l a r  of y e a s t s . that  species  antigen. give  Antigenic  analyses  five  composition  a high  giving 30,  by T s u c h i y a  of yeasts.  yet different  e t a l (51) i n d i c a t e d  chitin  content.  i n t h e group  that  The c e l l w a l l s o f  species  content,  seem t o be  similar  yeasts.  They  (or lack i t completely)  A mannan t h a t Fehling's  that d i d not  R h o d o t o r u l a and  f r o m most o t h e r  t o h a v e a low g l u c o s e  a p r e c i p i t a t e with  46) .  e t a l (20) t o  from t h e m a j o r i t y  each o t h e r .  S p o r o b o l o m y c e s and R h o d o t o r u l a  and  similar  o f R h o d o t o r u l a and  g r o u p s were s y n t h e s i z e d  of the m a j o r i t y  said  Sporobolo-  t h a t was  by G o r i n  s e t them a p a r t  cross reactions with  are  D-  and S p e n c e r ,  mannan f r o m  S p o r o b o l o m y c e s s p e c i e s were n o t c o n t a i n e d  in  The  o f R h o d o t o r u l a and S p o r o b o l o m y c e s h a d no common  Also  consisted  tetra-  glutinis.  characteristics  Sporobolomyces s p e c i e s  Phaff  singularis  mannan r e p o r t e d  produced by R h o d o t o r u l a Several  lactose.  u n i t s were/?l-4 l i n k e d .  1969 (36) r e p o r t e d  myces r o s e u s  be  singularis  i s capable  of  s o l u t i o n i s a b s e n t (15,  9 The  relationships  of Rhodotorula  and  Sporobolomyces  s p e c i e s t o the B a s i d i o m y c e t e s has  been put  s e v e r a l workers.  (48), working  S t o r c k , i n 1966  base a n a l y s e s found  a duality  i n both  and  species.  T h i s d u a l i t y was  in  the Rhodotorula  fifty  p e r c e n t and  Sporobolomyces  R h o d o t o r u l a m u c i l a g i n o s a both had percent. range in  A l l t h r e e o f t h e s e C+G  expressed  1968  that  a h i g h G+C  ratio  strong  implying a  sixty-five  Komagata,  s p e c i e s out  s p e c i e s o f y e a s t s t e s t e d had  t o be  i n f o r m a t i o n on t h e r e l a t i o n s h i p  life  cycles.  Rhodotorula  of  glutinis  found  that  Rhodosporidium  to  the h a p l o i d  (4). mating  Newell type  and  i s concerned demonstrated Rhodosporidium  Fell,  strains  s p h a e r o c a r p u m were i d e n t i c a l  Rhodotorula  was  the i m p e r f e c t stage of the U s t i l a g e n a c e o u s  t o r u l o i d e s by Banno, i n 1967 (34),  47.5  content.  Sporobolomyces s p e c i e s t o the Basidiomycetes  with t h e i r  the  Basidiomycetous  forty  and  ratio  urease  o f one  Further  of  N a k a s e and  (51).  and  readings are w i t h i n  In g e n e r a l , eleven Rhodotorula  65 p e r c e n t G+C  reflected  previously  relationship. hundred  DNA  t o h a v e a C+G  a C+G  species with content,  on  salmonicolor  for Basidiomycetes.  (33), found  a c t i v i t y had  found  by  Sporobolomyces  t h e a n t i g e n i c work o f T s u c h i y a m e n t i o n e d S p o r o b o l o m y c e s r o s e u s was  of  the  forward  with  in  1970,  of strains  of  10 Rhodotorula g l u t i n i s .  Lodder and Kreger van R i j  (31) and  Lodder e t a l (14) suggested t h a t the genus R h o d o t o r u l a i s an  i m p e r f e c t or degenerate B a s i d i o m y c e t e .  They s t a t e d  that  some of the s p e c i e s of the genus Rhodotorula, were t o be c o n s i d e r e d asporogenous Sporobolomyces s p e c i e s ,  since  Sporobolomyces without b a l l i s t o s p o r e p r o d u c t i o n would be indistinguishable van N i e l  from R h o d o t o r u l a s p e c i e s .  Kluyver and  (27) f i r s t r a i s e d the p o s s i b i l i t y t h a t the genus  Sporobolomyces might be o f Basidiomycetous o r i g i n .  This  was because the e j a c u l a t i o n mechanism of the b a l l i s t o s p o r e s of Sporobolomyces i s t h e same as the e j a c u l a t i o n mechanism of  basidiospores. van  der Walt and P i t o u t  on the DNA a n a l y s i s N  (52) submitted evidence based  f o r the e x i s t e n c e o f 2N ( d i p l o i d ) and  (haploid) g e n e r a t i o n s o f Sporobolomyces  salmonicolor.  However, because the b a l l i s t o s p o r e s o f d i p l o i d  colonies  produce o n l y a d i p l o p h a s e and the b a l l i s t o s p o r e s colonies  of h a p l o i d  produce o n l y a haplophase, the p o s s i b i l i t y  b a l l i s t o s p o r e s being b a s i d i o s p o r e s by S a i n c l i v i e r  o f the  (meiospores) as suggested  (39, 40) i s r u l e d o u t .  Banno, 1967 ( 4 ) ,  found no c o n j u g a t i o n between Rhodotorula and Sporobolomyces strains investigated, no  possible  and he t h e r e f o r e  stated  that  there was  r e l a t i o n s h i p between b a l l i s t o s p o r e p r o d u c t i o n by  11 S p o r o b o l o m y c e s and  the  sexual cycle  concerned  with  Rhodotorula.  Group I  Subgroup The  has  II  extracellular  not been  p o l y s a c c h a r i d e of U s t i l a g o h o r d e i  investigated.  Group I I Morphologically,  cultures  T a p h r i n a p o p u l i n a were s a i d Rhodotorula inositol,  (32).  i n 1952  b e t w e e n L i p o m y c e s and and m u l t i s p o r e d a s c i (29)  Cryptococcus  However, T a p h r i n a d o e s n o t  (55)  suggested  T a p h r i n a based found  in  1960  found t h a t  is  altogether different  the  i n both  and  assimilate  i t from C r y p t o c o c c u s ; i t  starch which d i s t i n g u i s h e s  Wickerham,  doubtful.  t o resemble  therefore distinguishing  does produce  make any  of the y e a s t stage of  on  i t from a  relationship  starch  genera.  production  However, K r a m e r ,  ascus development  from Lipomyces.  Rhodotorula.  i n Taphrina  This fact  r e l a t i o n s h i p b e t w e e n T a p h r i n a and  would  Lipomyces  MATERIALS AND  For in  three  cultures the  convenience,  sections.  M a t e r i a l s and M e t h o d s a r e d i s c u s s e d  Section A covers  for polysaccharide  isolation  C deals with  METHODS  maintenance o f  production,  of the e x t r a c e l l u l a r  Section B deals  polysaccharide  the a n a l y s i s of the crude  and S e c t i o n  extracellular  polysaccharide« Fungi Department Vancouver,  were o b t a i n e d  from t h e Mycology  of Botany, U n i v e r s i t y o f B r i t i s h B.C.  12  with  Laboratory, Columbia,  13  Table I  Fungus  Host  Location  Collection Date  Collector  Cryptococcus laurentii NRRLYB-4920 (U.B.C.#8114) Bullera alba (Hanna) D e r x . U.B.C. #983  Goldstream Provincial Park, B. C .  18/4/60  R.J.Bandoni  Sporobolomyces odorus U.B.C. #949  soil  Harrapa, West Pakistan  2/69  M.Rafiq  Sporobolomyces odorus. Derx. U.B.C. #981  Smut infected Citrus leaves  Nice, France  1930  H.G.Derx  Sporobolomyces singularis P h a f f e t Do Carmo-Sousa U.B.C. #8018  insect frass from dead Tsuqa.  Oregon, U.S.A.  1962  Tremella mesenterica R.J.B.#2259-6  on fallen Alnus  U.B .C. Endowment Lands  14/10/61  R.J.Bandoni  Ustilago hordei U.B.C. #570 (C . P e r s o n E3(-))  Hordeum  1964  P .L .Thomas  Taphrina populina U.B.C. #249  Poplus leaf  7/68  S.Reid  Rhodotorula glutinis U.B.C. #940 (I.F.O.#0559)  U.B.C. Endowment Lands  14  Figure  1  Procedure f o r the p r o d u c t i o n , and  a n a l y s i s o f the e x t r a c e l l u l a r  isolation, polysaccharide.  SLANTS MYP  20OC  PLATES MYP 25°C±2°C  **CENTRIFUCED l^.OOOxG., 30 MIN., 0°C.  FLASKS  CH5J& 125 ML., 250 ML.,  CELLS DISCARDED  7-14 DAYS, 250C±20C,  SHAKE  CELL-FREE MEDIUM RETAINED  ECONCENTRATED CELL-FREE MEDIUM ON FLASH EVAPORATOR.  EETHANOL-POTASSIUM ACETATE PRECIPITATION. K ETHANOL SOLUBLE IMPURITIES DISCARDED.  WHITE PRECIPITATE RETAINED.  EFREEZE-DRIED. EACID HYDROLYSIS, IN HS04, 2k HOURS, 2  90OC,  TUBES.  IN SEALED GLASS PYREX  BARIUM CARBONATE NEUTRALIZATION. Bag0u RESIDUE DISCARDED, IR-120 DEI0NI2ATI0N.  I  £NEUTRALIZED, DEIONIZED,  CONCENTRATED TO A SYRUP  I  PAPER CHROMATOGRAPHY ETHYL ACETATE:PYRIDINE:WATER (8:2:2)  HYDROLIZED SOLUTION CONCENTRATED ON THE FLASH EVAPORATOR.  E  CONCENTRATED TO DRYNESS  PREPARED TMS DERIVATIVES #10 MG. SAMPLE, 1 ML. PYRIDINE, .5 ML. HMDS, .25 ML. TMS.  EGAS CHROMATOGRAPHY  Section  A  Maintenance  of Cultures  for Polysaccharide  F u n g i were m a i n t a i n e d  on MYP  tubes a t 20°C.  The f u n g i  sterile  and c o o l e d ) w i r e  (flamed  cultures  t o MYP  plates.  medium  were t r a n s f e r r e d  MYP 1.  2.  i n culture with  a  l o o p f r o m t h e MYP  C o n t a m i n a n t s were r e v e a l e d  microscopic examination of the c u l t u r e s . was u s e d t o combat  Production  small tube by  Tetracycline  bacteria. Medium  used  f o r t u b e s and  plates.  Ingredients: Malt Extract  15 grams  Bacto Malt Extract, Difco Laboratories  Yeast  0.5  grams  Bacto Yeast Extract, Difco Laboratories  Soytone or Peptone  2.5  grams  Bacto Peptone, Bacto Soytone, D i f c o Laboratories  Distilled  1,000 millilitres  Extract  water  Agar  15 grams  Tetracycline  8 ml./litre o f medium as required.  Preparation  Plates:  Nutritional Biochemical  o f t h e Medium  The medium  was  autoclaved  f o r twenty minutes a t  15 p s i . , c o o l e d , p o u r e d , as d e s c r i b e d above,  solidified.  P l a t e s were i n o c u l a t e d  and u n u s e d p l a t e s were s t o r e d i n t h e  refrigerator. Tubes:  S l a n t s were p r e p a r e d  by f i l l i n g  required  amount o f m o l t e n a g a r .  solidify  with  the tube  resting  The agar  3.  Build  up o f  from  Hydrolysate increased  t h e agar, 5% G l u c o s e  agar  4.  were u s e d  and t r a n s f e r r e d  to build  Medium  to liquid  (CH5%G).  Liquid  g r a d u a l l y as the f u n g a l inoculum  fourteen  were i n o c u l a t e d  up a  C u l t u r e s were c u t l e n g t h w i s e o r  c u l t u r e s were s h a k e n r e c i p r o c a l l y to  The t u b e s  slants  inoculum  amount o f i n o c u l u m .  scraped  Unused  above.  P u r e c u l t u r e s on MYP large  with the  was a l l o w e d t o  a t an a n g l e .  were s t o r e d i n t h e r e f r i g e r a t o r . as d e s c r i b e d  tubes  Casein  CH5%G was  built  up.  Liquid  a t 25°C +_ 2°C f o r s e v e n  days.  C a s e i n H y d r o l y s a t e 5% G l u c o s e (for ten 2,800 ml. flasks)  Medium  ( 1 , 19)  Ingredients Medium  I  Distilled  water  Casein Hydrolysate  4  litres  15 grams - v i t a m i n F r e e ,  Salt  Free,  N u t r i t i o n a l Biochemical Corporation, Cleveland, O h i o , U.S.A.  17  KN0  5 grams  3  KH2PO4  20 grams  K2HPO4  15 grams  MgS0  4  Thiamine Trace  2.5  . 7H20  grams  5 milligrams  Hydrochloride  25  Elements S o l u t i o n  millilitres  Ingredients Trace  Elements S o l u t i o n  ZnSO^  . 7H2O  2.0  grams  CuS0  4  . 5H20  0.1  grams  MnSQ  4  . H 0  1.5  grams  2  FeSO,  2 .0 grams  7H 0 2  Distilled  1  Water  litre  Ingredients Medium I I Distilled  1  water  Glucose  250 grams  Tetracycline 5.  Preparation Medium  flasks.  8 ml./ l i t r e  as necessary  I was d i v i d e d among t e n c o n i c a l  were a u t o c l a v e d  ^  and I n o c u l a t i o n o f CH5%G Medium  F l a s k t o p s were c o v e r e d  conditions,  litre  with  foil.  2,800 m l . Media  I and I I  f o r t w e n t y m i n u t e s a t 15 p s i . Under  100 m l . a l i q u o t s o f Medium  each o f t h e t e n f l a s k s .  Each  sterile  I I were a d d e d t o  f l a s k was i n o c u l a t e d w i t h  a  s u s p e n s i o n o f y e a s t and s t e r i l e d i s t i l l e d w a t e r . cultures for  were s h a k e n on a r e c i p r o c a l  seven  to fourteen  shaker  a t 2 5°C + 2°C  days.  C a s e i n H y d r o l y s a t e 5% G l u c o s e Medium was because  i t was u s e d  Cryptococcus and  because  f o r polymer  laurentii  chosen  p r o d u c t i o n by b o t h  ( 1 ) , and T r e m e l l a m e s e n t e r i c a  i t was more c h e m i c a l l y d e f i n e d t h a n  MY B r o t h w i t h  Liquid  5% G l u c o s e  (19)  either  (22, 41, 44) o r t h e A u t o l y z e d  B r e w e r ' s Y e a s t Medium w i t h  5% G l u c o s e  (12, 24) .  19 Section B E x t r a c t i o n and I s o l a t i o n o f t h e E x t r a c e l l u l a r P o l y s a c c h a r i d e s 1.  C e n t r i f u g a t i o n o f t h e l i q u i d c u l t u r e and r e c o v e r y  of the  c e l l f r e e medium. A f t e r two weeks growth, t h e l i q u i d c u l t u r e was c e n t r i f u g e d a t 14,000 g., f o r t h i r t y m i n u t e s , a t z e r o centigrade.  degrees  The c e l l f r e e medium was r e t a i n e d and t h e  c e l l s were d i s c a r d e d .  Samples o f t h e c e l l f r e e medium  were checked m i c r o s c o p i c a l l y t o make s u r e t h a t a l l t h e c e l l s had been removed d u r i n g c e n t r i f u g a t i o n . 2.  E t h a n o l - P o t a s s i u m A c e t a t e p r e c i p i t a t i o n o f t h e crude . e x t r a c e l l u l a r polysaccharide  from t h e c e l l f r e e medium.  A s m a l l sample o f t h e c e l l f r e e medium was t o about o n e - h a l f  concentrated  i t s o r i g i n a l volume o f t h e f l a s h  A measured amount o f t h e c o n c e n t r a t e d slowly, with shaking,  evaporator.  medium was added  t o a known q u a n t i t y o f c o l d  ethanol.  S m a l l amounts o f p o t a s s i u m a c e t a t e were added t o f a c i l i t a t e the p r e c i p i t a t i o n o f t h e p o l y s a c c h a r i d e . p r e c e i p i t a t i o n appeared t h e q u a n t i t y o f  When a w h i t e concentrated  medium t h a t had been added t o t h e known q u a n t i t y o f e t h a n o l was r e c o r d e d . a large scale.  The p r e c i p i t a t i o n was t h e n c a r r i e d out on Dry i c e was added t o t h e e t h a n o l t o a g i t a t e  and  keep i t c o l d  during  the p r e c i p i t a t i o n .  precipitate  was r e t a i n e d  the  and e t h a n o l  ethanol  precipitate water as  soluble  was d i s s o l v e d  impurities.  i n a small  The w h i t e  amount o f  retained, water.  T h e w h i t e p r e c i p i t a t e was then d i s s o l v e d  i n a small  distilled  added  centrifuged  amount o f  to  ethanol  down,  distilled  The crude e x t r a c e l l u l a r p o l y s a c c h a r i d e  several  white  or decanting o f f  and t h e r e s u l t i n g a q u e o u s s o l u t i o n was  before.  into  by c e n t r i f u g i n g  The f i b r o u s  was  poured  round  bottom  f l a s k s and f r e e z e - d r i e d  overnight.  fluffy,  white,  freeze-dried  weighed  The  crude,  and  bottled.  Parafilm  was u s e d  p r o d u c t was  t o keep m o i s t u r e  out o f the  bottles.  3.  Hydrolysis  of the crude e x t r a c e l l u l a r  A portion polysaccharide  o f the f r e e z e - d r i e d  2  glass  Pyrex  (a)  A s l u r r y of barium  small  i n a beaker  f o r twenty-four  and d e i o n i z a t i o r f  exchange r e s i n .  N e u t r a l i z a t i o n with  prepared  4  tubes.  N e u t r a l i z a t i o n with Barium Carbonate w i t h IR-120 c a t i o n  a  crude e x t r a c e l l u l a r  was h y d r o l y z e d w i t h I N H S 0  h o u r s a t 100°C i n s e a l e d  4.  polysaccharide.  BaCO-3  c a r b o n a t e and d i s t i l l e d  i n t h e fume h o o d .  amount a t a t i m e ,  water  T h e s l u r r y was  to a flask containing  the  was added,  21 solution  of acid hydrolyzed material.  vigorously  T h e f l a s k was  and n e u t r a l i z a t i o n was d e t e r m i n e d  paper.  T h e r e s u l t i n g m i x t u r e was f i l t e r e d  funnel;  the c l e a r e d ,  the  Baso^ residue (b)  neutralized  was  the  Preparation  s o l u t i o n was r e t a i n e d a n d  o f IR-120 c a t i o n  volume o f t h e r e s i n .  i n a beaker  a l l t h e water  p a p e r was o b t a i n e d  been w e l l  stirred  r e s i n was washed c o m p l e t e l y  times  Enough 2N  a b a s i c r e a c t i o n on  o f NaOH.  The  2N HC1 was added  a v e r y p o s i t i v e a c i d pH was r e a c h e d .  r e s i n and t h e 2N HC1 were s t i r r e d  was  t h e n washed r e l a t i v e l y  was  repeated  very w e l l .  free of acid.  and t h e r e s i n was t h e n washed  The a c i d several  The washed r e s i n was t r a n s f e r r e d  c o l u m n and washed w i t h w a t e r u n t i l was  times  several  o f f each t i m e .  The  with water.  a t l e a s t four  f o r at l e a s t t e n minutes. free  a negative  The r e s i n treatment times  to a glass chloride  test  obtained (16). Preparation BaC0 A  3  o f t h e IR-120 c o l u m n f o r d e i o n i z a t i o n  neutralized glass  of t h e  polysaccharide  column o f s l u r r i e d  column  exchange r e s i n .  a f t e r t h e r e s i n a n d 2N NaOH h a d  and l e f t  t o the r e s i n u n t i l  exchange r e s i n  T h e r e s i n was washed  NaOH was a d d e d t o t h e r e s i n s o t h a t litmus  through a  o f t h e IR-120 c a t i o n  r e s i n was p l a c e d  with water, d e c a n t i n g  litmus  discarded.  Regeneration The  with  swirled  IR-120 was p r e p a r e d  as i n  Figure rod  2.  A plug  at the  of  g l a s s w o o l was  bottom of  the  column.  IR-120 c a t i o n e x c h a n g e r e s i n was water rod  and  was  small of  poured  removed and  attached  A piece  to  the  washed s e v e r a l solution  Molisch  The  sulphuric  rubber tubing  times with  glass  distilled  p r o d u c t s was  f l a s k was  used t o  general, a c i d on  the M o l i s c h sugars  and  phenolic  sulphuric  side of  the  column  i n t o the  was  the  (18,  26,  substances.  a c i d was  was  water  till  negative.  deionized  products.  grams o f  down t h e  The  56) a c t i o n of  subsequent r e a c t i o n of  percent  column  column.  0.05  Concentrated  the  poured  t e s t , i f performed i n a t e s t tube:  fifteen  Another  neutralized  t e s t i s the  i n 1 ml.  The  water, then the  this  carbohydrate  of  a s c r e w c l a m p were  column.  collect  t e s t for Carbohydrates  drops of  of  washed t h r o u g h w i t h d i s t i l l e d  p r o d u c t s formed w i t h  t o two  rod.  settle.  and  glass  distilled  a glass  top  a  amount  with  i n s e r t e d a t the  of neutralized h y d r o l y s i s  Molisch In  slurried  r e s i n allowed to  bottom of the  was  required  t e s t for carbohydrate material  Erlenmeyer  solution  of  The  column a l o n g  was  of h y d r o l y s i s  This material  An  the  g l a s s wool p l u g  IR-120.  the  i n t o the  i n s e r t e d using  a d d e d by  the  Specifically, to  in  approximately  o f w a t e r was  alcohol  strong  added  s o l u t i o n of pouring  it  one  naphthol. slowly  t e s t tube t o form a l a y e r under t h e  sugar  23  Figure 2  Apparatus  for  t h e IR-120 c a t i o n  exchange  resin.  glass column ring stand with screw clamp  glass wool plug •IR-120 cation exchange resin  glass wool plug  Erlenmeyer flask  —deionized solution  24 solution. violet  In the presence  color  appeared  of carbohydrate  a t the  because o f the formation of their that  r e a c t i o n with  furfural  naphthol  (18).  t h e mechanism o f t h e r e a c t i o n  influence t o the  o f 15M  straight  dehydration This  and  sulphuric acid c h a i n aldehyde ring  i s f o l l o w e d by  hydroxylmethyl and  interface  furfural,  material a  o f t h e two  derivatives  and  I t i s believed proceeds  from form  liquids  under  (26)  the  the glucopyranose with  form  subsequent  closure to 5-hydroxymethylfurfural. the h y d r o l y t i c  scission  of the  group w i t h the p r o d u c t i o n of furfural  degradation products,  5-  formaldehyde or  furfural  polymers.  Important  P r o p e r t i e s of Amberlite  The  cation  sulfonated, apparent is  exchange r e s i n  polystyrene type  d e n s i t y (average)  20-50 mesh; t h e v o i d  ation  i s 98%  49-55% and  the t o t a l  meq./ml. m i n .  c  (minimum).  and  IR-120. i s strongly  acidic,  o f medium p o r o s i t y .  The  i s 0.77g/ml.; t h e mesh s i z e  volume,  35-40%; d e g r e e  of  (wet)  regener-  Moisture holding capacity i s  exchange c a p a c i t y i s , by  by weight  (dry),  5.0  volume,  meq./g.min.  1.75  25 Section  C  Q u a l i t a t i v e A n a l y s i s of the Crude E x t r a c e l l u l a r Hydrolysis 1.  Gas  2 .  Paper  1.  Gas  Products  Chromatography Chromatography  Chromatography  Preparation  of the  neutralized,  trimethylsilyl  extracellular s m a l l round paration  neutralized,  p o l y s a c c h a r i d e was  bottom  flask  o f t h e TMS  To  on  the  approximately  suspension  flash  10 mg.  of KOH  HMDS ( h e x a m e t h y l d i s i l a z a n e ) , and  o f TMS,  The  The  out  about t h i r t y  or  a covered  seconds,  then  m i n u t e s a t room t e m p e r a t u r e .  The  pre-  i n the  fume  added 1 m l .  pellets).  To  0.25  stoppered  in a  sample was  ml.  s o l u t i o n became c l o u d y  f l a s k was  ground g l a s s stopper for  carried  o f TMS on  allowed Formation  and to  of  solution ml.  (trimethyl-  the  addition  ammonium  immediately cork  the  a d d e d 0.5  presumably because o f the p r e c i p i t a t e  chloride.  the  to dryness  evaporator.  o f m a t e r i a l i n p y r i d i n e was  chlorosilane).  of  deionized, hydrolyzed  evaporated  d e r i v a t i v e s was  anhydrous p y r i d i n e (kept over or  derivatives  deionized hydrolysis products.  A p o r t i o n of the  hood.  Polysaccharide  with  a  shaken v i g o r o u s l y stand  for 5  o f t h e TMS  derivatives  occurred  rapidly  a t room t e m p e r a t u r e .  g r o u p s were s i l y l a t e d was  quantitative  as p r e s e n t e d v  - S i - CI The  »  HO-C-  of  and  *  carried  |  i n j e c t e d with  then  programmed  f l o w was  for  10 ml.) .  2.  Paper  o u t on an F and M  equal weights  *  approximately  HC1  machine.  720 d u a l  column  i n . coiled  ( t o w i t h i n 20  mg.)  The c o l u m n s were three  a t 2° per minute t o h o l d a t 88 m l . p e r m i n u t e  minutes  220°. (6.8 seconds  Chromatography  A p o r t i o n of the hydrolyzed, p o l y s a c c h a r i d e was  flash  amounts o f t h e d i l u t e d flamed,  Base  a glass syringe  i s o t h e r m a l l y a t 190° f o r approximately  Helium  cooled, wire  Chromatography Paper.  evaporated  (H2O)  carried  neutralized  and d e i o n i z e d  to a syrup.  Small  s y r u p were a p p l i e d w i t h  l o o p t o 24 i n . b y 7.4 A t t h e same t i m e ,  made o f known m o n o s a c c h a r i d e s . was  +  The two c o l u m n s were 8 f t . x 0.25  columns packed w i t h  reaction  (23) i s :  20% SF 96 on 60-80 mesh D i a t o p o r t S.  held  derivatives  p o r t o f t h e gas chromatography  C h r o m a t o g r a p h y was  copper  and S c h e i n o s t  m a t e r i a l was  into the injector  instrument.  o f t h e TMS  v I Base—» - Si-O-C-  +  T  silylated  hydroxyl  (49). In general, the s i l y l a t i o n  by H e n g l e i n  +  /  and t h e y i e l d  A l l free  Whatman  s p o t s were  Descending  out using the s o l v e n t s e t h y l  in.  a  paper  acetate:  #1  also  chromatography pyridine:  water  i n the r a t i o s  o f 8:2:2, i n an e q u i l i b r a t e d  chromatography tank  f o r a p p r o x i m a t e l y 48 h o u r s .  c h r o m a t o g r a m s were r e m o v e d f r o m developed  The  then  o f t h e AgNC-3 d i p principle  on t h e T o l l e n ' s  behind  silver  i s reduced  aldehyde  dried,  The  w i t h AgNC>3 d i p (50) .  Preparation  silver  t h e tank,  glass  t h e AgN03 d i p p r o c e s s i s b a s e d  mirror test  by aldehydes  or ketone  f o r carbohydrates.  The  and s u g a r s c o n t a i n i n g f r e e  groups ( 8 ) .  I n g r e d i e n t s (50) 1.  Acetone-AgN03  Solution  This reagent ml. with  s o l u t i o n was p r e p a r e d b y d i l u t i n g  o f s a t u r a t e d aqueous s i l v e r acetone,  the s i l v e r  n i t r a t e which  with  t o 20 m l .  shaking,  until  s e p a r a t e s on t h e a d d i t i o n o f Spreading  of the sparing s o l u b i l i t y  (0.014% a t 23°C f o r c r y s t a l l i n e  2.  solution  and a d d i n g water d r o p w i s e ,  acetone has r e d i s s o l v e d . because  nitrate  0.1  of the spots i s l i m i t e d  o f sugars  i n acetone.  glucose.)  E t h a n o l - NaOH S o l u t i o n The  by d i l u t i n g  0.5N s o l u t i o n  o f NaOH i n a q u e o u s e t h a n o l was made  s a t u r a t e d aqueous NaOH s o l u t i o n  with ethanol.  28 3 .  6N Ammonium Procedure The  through after  hydroxide  (50)  dried  paper  chromatogram s t r i p  the AgN03-acetone r e a g e n t s o l u t i o n  each  run through.  once t h r o u g h o x i d e was  The  immediately produced.  r e d u c t i o n was dissolved  by  judged  The  the  standards. the  R  for  48 h o u r s  it  was  which  to  drying  passed silver  sugars  formed  excess  silver  i n 6N  i t was  When  oxide  was  ammonium h y d r o x i d e  washed w i t h w a t e r  and  H2S.  i d e n t i t y of the  positions  F  then  Brown  Reducing  of the s t r i p  after  twice,  rapidly  s p o t s c o u l d have been r e n d e r e d j e t b l a c k by  momentary e x p o s u r e The  was  passed  a t room t e m p e r a t u r e .  complete,  immersion  a few m i n u t e s ,  dried.  d r y paper  the e t h a n o l - N a O H s o l u t i o n .  dense b l a c k spots of s i l v e r  for  was  s p o t s was  determined  by  o f t h e unknown s p o t s w i t h t h o s e o f  T h i s method was  values. and  more a c c u r a t e t h a n  comparing  the  calculating  S i n c e some o f t h e c h r o m a t o g r a m s were r u n the solvent  considered unreliable  front  had  run o f f the  to calculate  paper,  the r a t i o  movement o f a s p o t t o t h e movement o f t h e s o l v e n t  of  the  front.  29  RESULTS  The those  results  a r e p r e s e n t e d i n two  o b t a i n e d from paper  sections.  chromatography.  First,  Second,  those  obtained  from gas c h r o m a t o g r a p h y .  The  obtained  from paper  a r e shown i n T a b l e I I .  Figures  3,  4,and 5 a r e r e p r e s e n t a t i o n s  chromatograms. chromatography representations the results for  The  tentative  a r e shown i n T a b l e I I I . of the a c t u a l  gas  results  Figures  f o r gas  6-14  chromatograms.  chromatography  paper  are  Because  were  sufficient  determinations of the monosaccharides  the extracellular was  results  of the a c t u a l  qualitative  o b t a i n e d from paper  qualitative  emphasis  chromatography  qualitative  polysaccharide hydrolysates,  placed  on t h e gas  in  n o t much  chromatography.  Only pre-  l i m i n a r y gas chromatography  e x p e r i m e n t s were c a r r i e d  The  were d e t e r m i n e d t e n t a t i v e l y  identities  first,  assuming  o f the peaks that  t h e peaks  represented the  t h a t were shown t o be p r e s e n t b y t h e p a p e r examinations of the h y d r o l y s a t e s .  out.  monosaccharides  chromatographic  Secondly, the  individual  p e a k r e t e n t i o n t i m e s were compared w i t h t h e r e t e n t i o n o f known m o n o s a c c h a r i d e s conditions cation  (17, 37,  49).  c o u l d have been  chromatographed  by  under  times  similar  However, more p o s i t i v e  identifi-  o b t a i n e d by e x p e r i m e n t s u s i n g  the  30 peak enhancement  technique.  This  i n v o l v e s a d d i n g a known  monosaccharide t o t h e h y d r o l y s a t e peaks a r e e n h a n c e d  on t h e g a s c h r o m a t o g r a m s .  e n h a n c e d w o u l d most saccharide  and t h e n o b s e r v i n g  likely  be t h o s e  t h a t was a d d e d .  This  which  The peaks  o f t h e known mono-  process  w o u l d be  carried  out  f o r a l l t h e m o n o s a c c h a r i d e s t h o u g h t t o be p r e s e n t i n  the  hydrolysate.  appear  The g a s and p a p e r c h r o m a t o g r a p h y  t o be t h e same.  identification  But, u n t i l  gas chromatography r e s u l t s  the  g a s chromatograms doing  extracellular yeast-like  systematic  i n v o l v i n g p e a k enhancement  the  searchers  this  should  results kind of  i s c a r r i e d out  are inconclusive.  However,  remain i n the t h e s i s .  Re-  q u a n t i t a t i v e g a s c h r o m a t o g r a p h y on t h e  polysaccharides  p r o d u c e d b y some y e a s t s and  f u n g i may want t o r e f e r  some o f t h e g a s c h r o m a t o g r a m s .  t o and i n t e r p r e t  ( S e e A p p e n d i x page  further 62.)  31 Table II PAPER CHROMATOGRAPHY  Gr oup  RESULTS Monosaccharides i n the Extracellular Polysaccharide Hydrolysis Products  Fungus  G a l a c t o s e G l u c o s e Mannose X y l o s e Group I Subgroup I  Cryptococcus laurentii NRRL YB-4920 (UBC #8114) Tremella mesenterica RJB #2259-6  Group I Sub group I I Group I I  Bullera a l b a (Hanna) Derx UBC #983  +  +  +  +  Sporobolomyces odorus UBC #949  +  +  +  +  Sporobolomyces odorus UBC #981  +  +  +  +  Rhodotorula glutinis UBC #940  +  +  +  +  S p o r ob o 1 omy c e s singularis UBC #8018  +  +  +  +  Ustilago hordei UBC #570 (C.Person E3(-))  +  +  +  +  +  Taphrina populina UBC #249  Figure  Tremella  mesenterica  Separation  3  and  of products  using  p a p e r c h r o m a t o g r a p h y and acetate:  Bullera alba.  p y r i d i n e : water,  descending  solvents (8:2:2).  ethyl  Tremella mesenterica  OO  CO  Xylose Arabinose  X  in  a a: Mannose s z Glucose Galactose  Bullera alba Xylose Arabinose Q  a: < a z  Mannose Glucose Galactose  *  CO  cO  Figure  Sporobolomyces  4  singularis,  Sporobolomyces  odorus  (#949), S p o r o b o l o m y c e s  odorus  laurentii.  of products using descending  Separation  paper chromatography pyridine:  water,  and  (8:2:2).  (#981) C r y p t o c o c c u s  solvents ethyl  acetate:  Figure  5  Ustilago hordei, Rhodotorula g l u t i n i s , singularis, products  Taphrina  using  populina.  Sporobolomyces  Separation  of  descending paper chromatography  solvents ethyl acetate:  p y r i d i n e : water,  and  (8:2:2) .  Mannose Glucose Xylose Galactose Arabinose Taphrina populina 249 Ustilaqo hordei 570  o o  Sporobolomyces singuloris  Xylose Arabinose Mannose Glucose Galactose Rhodotorula glutinis  CD  35 Table I I I  Results  Gas C h r o m a t o g r a p h y  Fungus  Tremella mesenterica  Cryptococcus laurentii  Peak  Retention Time (Minutes)  Tentative Identification  1  2.9  Solvent  2  13.8  Xylose  3  17.5  Xylose  4  19.8  Xylose  5  22.6  G a l a c t o s e , Mannose  6  25.7  Galactose  7  27.4  Galactose, Mannose  8  30.5  Glucose  1  19.7  Xylose  2  23.0  Xylose  3  26.6  G a l a c t o s e , Mannose  4  31.8  Galactose, Mannose  37.0  Glucose  Glucose,  Glucose,  36 Table  III  (Continued)  Gas C h r o m a t o g r a p h y R e s u l t s  Peak  Fungus  Bullera  alba  Sporobolomyces o d o r u s (#949)  Retention Time (Minutes)  Tentative Identification  1  2.9  solvent  2  4.5  solvent  3  6.2  solvent  4  18.0  Xylose  5  20.4  Xylose  6  23.2  Galactose,  7  25.9  Galactose  8  28.7  Galactose, Mannose  Mannose.  Glucose,  33.4  Glucose  1  17.4  Xylose  2  19.1  Xylose  3  21.2  Xylose  4  24.0  Galactose,  Mannose  5  26.6  Galactose,  Mannose  6  29.3  Galactose  7  31.9  Galactose, Mannose  8  37.0  Glucose  Glucose,  Table  37  I I I (Continued)  Gas C h r o m a t o g r a p h y R e s u l t s  Fungus  Sporobolomyces o d o r u s (#981)  Sporobolomyces singularis  Rhodotorula glutinis  Peak  Retention Time (Minutes)  Tentative Identification  1  17.8  Xylose  2  19.6  Xylose  3  22.0  Xylose  4  28.1  Galactose,  5  31.0  Galactose  6  33.8  Galactose, Mannose  38.7  Glucose  1  18.8  Xylose  2  28.2  Galactose,  Mannose  3  30.2  Galactose, Mannose  Glucose,  34.2  Glucose  1  3.0  Solvent  2  15.3  Xylose  3  17.3  Xylose  4  22.2  Galactose,  5  24.8  Galactose  6  26.4  Galactose, Mannose  30.4  Glucose  Mannose  Glucose,  Mannose  Glucose,  38 T a b l e I I I (Continued) Gas Chromatography R e s u l t s  Fungus  Ustilago hordei  Taphrina populina  Peak  Retention Time (Minutes)  Tentative Identification  1  23.5  G a l a c t o s e , Mannose  2  25.0  Galactose  30.2  Galactose, Mannose  36.2  Glucose  1  14.7  Mannose  2  23.5  Mannose  3  24.3  Glucose  4  29.0  Glucose  5  50.3  unidentified  Glucose,  Figure  6  Tremella mesenterica. of  products  derivatives.  as  Separation  trimethylsilyl  i  DETECTOR RESPONSE  Figure  Cryptococcus of  laurentii.  p r o d u c t s as  derivatives„  7  Separation  trimethylsilyl  DETECTOR RESPONSE  Figure  Bullera as  alba.  8  Separation  trimethylsilyl  of  products  derivatives„  DETECTOR  RESPONSE  Figure  Sporobolomyces of  odorus  9  (#949).  products as t r i m e t h y l s i l y l  Separation derivatives«  DETECTOR If  RESPONSE  Figure  Sporobolomyces  10  odorus  (#981).  o f p r o d u c t s as t r i m e t h y l s i l y l  Separation derivatives.  DETECTOR RESPONSE  0)  CD  00  Figure  Sporobolomyces of products  11  singularis.  Separation  as t r i m e t h y l s i l y l  derivatives.  Sporobolomyces singularis  T^JTQ CVJ  ftp $>  1  '  RETENTION TIME (MINUTES)  Figure  Rhodotorula g l u t i n i s . products  12  Separation of  as t r i m e t h y l s i l y l  derivatives  DETECTOR RESPONSE  Figure  Ustilago as  hordei.  trimethylsilyl  13  Separation  of  derivatives.  products  Figure  Taphrina  populina.  14  Separation  p r o d u c t s as t r i m e t h y l s i l y l  of  derivatives  1  . Ul St  1  1  i n ro ro KJ cu  1  1  1  1  1  ro Q in  Q CD  co  RETENTION TIME (MINUTES)  48  DISCUSSION  The previous For  d i s c u s s i o n r e l a t e s my  workers.  convenience,  were a s s i g n e d subgroups. Tremella  the yeasts  t o two  Fungi  and  groups.  p r o d u c e d by  xylose  absent.  contain  divided into  the  absent.  glucose  Rhodotorula  mannose and  of the  extracellular  glucose  Group I I c o n s i s t e d of  and  mannose p r e s e n t ;  I n t e r e s t i n g l y , the  two  odorus, The  xylose  Subgroup and  Taphrina  galactose  some f u n g i t h a t h a v e a l r e a d y b e e n s u g g e s t e d A Cryptococcus-Tremella  s h i p was  forward  first  put  Members o f t h e two saccharide  by  genera are  production,  They a l s o have s i m i l a r  K o b a y a s h i and similar  isolation  and  II  mannose  and  groups formed  omically related.  were  poly-  f u n g i i n Subgroup I . galactose,  two  laurentii  glutinis.  glucose,  included Ustilago hordei with  populina with  yeast-like fungi investigated  G r o u p I was  and  i n the hydrolysates  xylose  deductions.  B u l l e r a a l b a , Sporobolomyces  monosaccharides galactose,  present;  of  i n Subgroup I were: C r y p t o c o c c u s  mesenterica,  saccharides  t o those  I t a l s o makes some l o g i c a l  Sporobolomyces s i n g u l a r i s  present  results  above as  taxonomic Tubaki  taxon-  relation-  (28).  in extracellular a n a l y s i s ( 1 , 19,  carbon a s s i m i l a t i o n patterns  poly24,  and  44).  produce  49 starch  (32,  41).  S i m i l a r m o r p h o l o g y and  a t i o n r e a c t i o n s are My support  results  present  the r e l a t i o n s h i p  Cryptococcus found  a l s o important  laurentii  biochemical  present  Tremella mesenterica. xylose  and  found  be  present  of  both 19,  both  The  the  i n the e x t r a c e l l u l a r  24,  41,  44) .  laurentii Galactose  amounts f r o m C r y p t o c o c c u s  or  may  have been p r e s e n t  possibly, than has  the not  g a l a c t o s e i s more t i g h t l y  been p r e v i o u s l y i s o l a t e d  e l e c t r o n m i c r o s c o p i s t w o r k i n g on  hydrolysate  mesenterica  (24)  and h a d  a contaminant  or  o t h e r m o n o s a c c h a r i d e s and  to  cells  from  to the  under c e r t a i n analyzed.  fungal c e l l  cell  t h a t might  centrifugation. bound  and  not  mesenterica.  e n d o c e l l u l a r m a t e r i a l from y e a s t  h a v e b r o k e n down d u r i n g c u l t u r i n g  workers  a neutral glucan  laurentii  as  and  been r e p o r t e d o n l y i n  been r e p o r t e d p r e v i o u s l y from T r e m e l l a Galactose  and  mannose,  of previous  Tremella  had  and  laurentii  polysaccharide and  I  extracellular  of glucose,  results  may  (41).  mannose  Cryptococcus  presence  41) .  between  Tremella mesenterica  both  xylosyl-  information that  an a c i d i c x y l o m a n n a n and  Cryptococcus  32,  i n the h y d r o l y s a t e s o f the  i s i n agreement w i t h  who  wall  28,  already suggested  p o l y s a c c h a r i d e s p r o d u c e d by  trace  (3, 13,  the monosaccharides g a l a c t o s e , glucose,  x y l o s e t o be  (1,  enzymatic  cell  Or, wall  conditions Perhaps  w a l l s and  an using  50 radioactive  tracers  w o u l d be  T r e m e l l a m e s e n t e r i c a has linkages  support  laurentii  the  b e e n s u g g e s t e d as similar  starch  being  melibiose,  melezitose,  both produce s t a r c h had  not  and  and  and  and  Cryptococcus  (32).  Bullera  are  alba  Cryptococcus  My  produce  findings  laurentii  may  Cryptococcus r e l a t i o n s h i p s more d i r e c t l y  Bullera  alba  and  that  q u a l i t a t i v e l y the  pertain  to  give  giving  saccharide with the  evidence  on  the  bases  pseudomycelium; Both  genera  bonds  of  They  If  i n the the  also  Bullera  genus  monosaccharides  same a s  those  added s u p p o r t t o  laurentii  of  of the  Bullera-  However, t h e  biochemical  results  support to  a  relationship.  r e s u l t s , b o t h Sporobo1omyces  S p o r o b o l o m y c e s o d o r u s p r o d u c e d an  has  ballistospores,  in general.  Cryptococcus  shown i n my  of  pseudomycelium.  b e e n known t o  as  Cryptococcus have  methyl«C-D-glucoside .  lack  1-3  species.  break the o C - g l u c o s i d i c  them m i g h t h a v e b e e n i n c l u d e d  As  (19),  taxonomically related lack  Though  contain  substantial  carbon a s s i m i l a t i o n .  inositol  and  is still  g e n e r a l m o r p h o l o g y and  p r o d u c t i o n and  cultures  there  genera B u l l e r a  assimilate  some o f  b e e n shown t o  a r e l a t i o n s h i p b e t w e e n t h e s e two  Members of  of  yet  this.  i n i t s extracellular polysaccharide  Cryptococcus to  not  able to resolve  extracellular  monosaccharides galactose,  singularis poly-  glucose,  mannose  51 and  xylose  and  glucose  i n the h y d r o l y s a t e .  i n the Sporobolomyces  polysaccharide hydrolysate of  Gorin  cellular  be  i s i n agreement w i t h  (36). Xylose  odorus had n o t been p r e v i o u s l y The  of Sporobolomyces  the results  had n o t been r e p o r t e d  polysaccharide  of the e x t r a c e l l u l a r  Rhodotorula  g l u t i n i s were g a l a c t o s e ,  to  t h e mannan, R h o d o t o r u l a  methyl pentose. I  mannose and  similar  g l u t i n i s produced  t o those Sporobolo-  galactose  a h e x o s e and a  or g l u c o s e  i n the Rhodotorula  hexose o f G o r i n  t h e pentose t h a t I found  Rhodotorula  produced by  g l u t i n i s and  polysaccharide hydrolysate  the u n i d e n t i f i e d  xylose,  glucose,  i n the  Gorin et a l stated that i n addition  Perhaps e i t h e r  d e t e r m i n e d t o be p r e s e n t  extracellular as  polysaccharide  Rhodotorula  (20, 3 6 ) .  hydrolysate.  t o be p r e s e n t  Mannose may be p a r t o f a mannan  myces s i n g u l a r i s  previously to  analyzed.  monosaccharides t h a t I found  mannans p r o d u c e d b y b o t h  o f P h a f f and  produced by Sporobolomyces  hydrolysate  xylose.  the r e s u l t s  o f mannose i n t h e e x t r a -  i n the Sporobolomyces s i n g u l a r i s  extracellular  extracellular  i s i n agreement w i t h  polysaccharide hydrolysate  present  The  singularis  e t a l ( 2 1 ) . The f i n d i n g  singularis Spencer  The p r e s e n c e o f g a l a c t o s e  glutinis  c o u l d be t h e same  et a l (20). Also,  t o be p r e s e n t  glutinis extracellular  that  hydrolysate  i n the c o u l d be a  52 derivative al  of the u n i d e n t i f i e d  methyl  pentose  of Gorin e t  qualitative  f o r the U s t i l a g o h o r d e i  (20) . The  extracellular  results  p o l y s a c c h a r i d e a r e new.  The  monosaccharides  p r e s e n t w e r e : g a l a c t o s e , g l u c o s e and mannose.  Xylose  was  absent. I n d i c a t i o n s were t h a t qualitative important  quantitative,  a n a l y s e s u s i n g gas chromatography might  a r e most c l o s e l y r e l a t e d .  some members o f b o t h R h o d o t o r u l a suggested  (32,  by a n t i g e n i c  33, 48, 5 1 ) , may  chromatographic The  and S p o r o b o l o m y c e s  a n d p e r c e n t G+C  results  Rhodotorula  a n a l y s e s may  prove  gas  can o n l y d i s t i n g u i s h glutinis  However, more  for distinguishing  moment a r e c a r b o n  analyses of  whether t h i s  laurentii.  these  useful  those of  Taphrina populina  and C r y p t o c o c c u s  i n this  t o be a n o t h e r  T a p h r i n a s p e c i e s from  and C r y p t o c o c c u s .  t h e known t e s t s  that  analyses  f o r T a p h r i n a p o p u l i n a a r e new.  s p e c i e s are necessary t o determine  My r e s u l t s  amongst  be s u p p o r t e d b y q u a n t i t a t i v e  means o f d i s t i n g u i s h i n g Rhodotorula  A duality  analyses.  case the q u a l i t a t i v e  other  be more  i n d e t e r m i n i n g what s p e c i e s o f S p o r o b o l o m y c e s a n d  Rhodotorula  was  instead of  genera  i s so.  from Some o f  at the  a s s i m i l a t i o n and o b s e r v a t i o n o f s t a r c h  53 production.  Taphrina  starch; Rhodotorula inositol  as  production,  and  However, t h e b y Kramer Lipomyces.  related  My  But  and  cellular The  asci  t o be  Cryptococcus  is  results  found asci  i n both  polysaccharide with  genera  the  W i c k e r h a m showed t h a t t h e  was  in  populina  produced  monosaccharides product.  Lipomyces  extra-  composed o f mannose o n l y  Lipomyces-Taphrina r e l a t i o n s h i p  (55). shown  from t h o s e  i n the h y d r o l y s i s  was  i n starch  i n Taphrina  showed t h a t T a p h r i n a  polysaccharide  postulated  similarities  altogether different  mannose p r e s e n t  S l o d k i and  and  produces  s p e c i e s have been  because o f  multispores  an e x t r a c e l l u l a r glucose  Lipomyces  development o f the  (29)  and  (32).  and  taxonomically  negative  i s starch negative;  positive  Taphrina  is inositol  (42).  seems d o u b t f u l a t  this  point. Barnett, major unsolved that:  (1)  biochemical  i n 1957  problems of yeast  into  tests  are  for d i f f e r e n t  four  T h e s e were  different  sets  groups of y e a s t s .  of  (2)  t o have a q u a n t i t a t i v e e v a l u a t i o n of  necessary  to separate  a l r e a d y named g r o u p s ,  activity  taxonomy.  T h e r e i s a need t o d e v i s e  w o u l d be h e l p f u l many t e s t s  ( 5 ) , s t a t e d t h a t t h e r e were  described  should  say be  different  species.  considered  (3)  It how  strains The  biochemical  quantitatively.  54 (4)  The c o n d i t i o n s f o r each o f t h e s e  assessed  c r i t i c a l l y and l a b i l e  In  1961, B a r n e t t  be  developed  i n which t h e r e  morphological biochemical  (6) s u g g e s t e d  criteria  tests  t e s t s n e e d s t o be  c h a r a c t e r s should  are designed  supreme i m p o r t a n c e , for (2)  spores  g r e a t e r p r e c i s i o n , and  t o g i v e more i n f o r m a t i o n .  Morphological  (1)  Sporulation tests  Negative  criteria  to  specify precisely.  In  1966, B a r n e t t  (3)  Biochemical  be  of the r e s u l t s  t o u n d e r l i e those  and a s e a r c h  tests  are crude.  (1) t h e p r e s e n c e  of  can lead t o  o f many o t h e r  tests.  o f l a r g e numbers o f t e s t s  u s e d t o make e v i d e n t t h e b i o c h e m i c a l  likely  are given  are equivocable.  and a b s e n c e i n o t h e r s  c o r r e l a t i o n s between t h e r e s u l t s analyses  tests  major  a r e i l l d e f i n e d and d i f f i c u l t  (7) s t a t e d t h a t :  enzymes i n some y e a s t s  (2)  has three  a r r a y s o f media a r e used,  i s necessary.  should  a r e no s p o r u l a t i o n t e s t s ,  are given  These a r e :  avoided.  that a c l a s s i f i c a t i o n  He s t a t e d t h a t t h e D u t c h c l a s s i f i c a t i o n weaknesses.  be  could  features that are  correlations.  W i t h r e f e r e n c e t o my e x p e r i m e n t s ,  i t i s evident  that  a q u a n t i t a t i v e a n a l y s i s u s i n g GLC would h a v e b e e n b e t t e r f o r distinguishing  b e t w e e n s p e c i e s o f t h e same g e n u s ,  Sporobolomyces s i n g u l a r i s is  a l s o apparent  e.g.  and S p o r o b o l o m y c e s o d o r u s .  t h a t t o be s t a t i s t i c a l l y  It  correct i n implying  55 taxonomic r e l a t i o n s h i p s , methods and t e c h n i q u e s isolating from those  researchers  for culturing  should  t h e f u n g i and f o r  and a n a l y z i n g t h e e x t r a c e l l u l a r fungi that  u s e t h e same  polysaccharide  a r e t o be c o m p a r e d .  V a r i a t i o n s i n the  p r o p e r t i e s o f t h e p o l y s a c c h a r i d e may be d e p e n d e n t o n t h e fermentative the A  conditions .  constitution  These v a r i a t i o n s a r e r e l a t e d t o  and m o l e c u l a r  size  short discussion o f fermentative  importance the  i n this respect  growth p e r i o d .  glucan, both  at four  i s relevant here.  a t seven days  (44), f i v e  was p r o d u c e d o n l y  Cryptococcus  conditions that are of First,  (24) and s i x d a y s  after  laurentii  ( 1 9 ) , and b y  and T r e m e l l a  at twelve  days  ( 4 3 ) . Secondly,  important  factor  was s u i t a b l e f o r p o l y m e r  (1, 1 9 ) .  galactans  t h e pH o f t h e medium i s a n An i n i t i a l  production  pH o f 6.4 t o 6.8  by C r y p t o c o c c u s  laurentii  12) and a pH o f 6.0 was r e q u i r e d f o r p h o s p h o r y l a t e d  galactan production workers found at  (12). Neutral  mesenterica  s p e c i e s produced phosphorylated  to consider.  Cryptococcus  f o u r t e e n days growth by  Sporobolomyces  (1,  consider  A c i d i c x y l o m a n n a n was p r o d u c e d b y  Tremella mesenterica laurentii  of the polymer ( 2 4 ) .  by Sporobolomyces s p e c i e s  t h a t Sporobolomyces  pH 6.0 b u t no p o l y s a c c h a r i d e  trisaccharide  (43) .  Other  s i n g u l a r i s h a d good  production.  growth  Yields of  and t e t r a s a c c h a r i d e i n c r e a s e d a t pH 3.75.  Above pH is  4.0  a third  saccharide  no  o l i g o s a c c h a r i d e was  production.  Maximum p o l y m e r p r o d u c t i o n  laurentii  occurred  a t 22°C  at  Sporobo1omyces  ( 1 2 ) ; by  by R h o d o t o r u l a and  glutinis  other  the p o l y s a c c h a r i d e I n most c a s e s  (21);  at  production  of  the  extracellular  g o v e r n e d by  the  cultural  study be  of the best  select  those  be  24°C  This  polysaccharides.  of the  the  aeration,  that analytical Because the  biochemical  results  are  so  closely  that not  of the  and  the  be  Perhaps  a  parameters would  researcher  the  (12).  and  w o u l d be  v a r i a b l e and qualitative  able use  or  c e l l w a l l amino a c i d s m i g h t  analyses  and  structure  taxonomically.  p a r a m e t e r s t h a t were l e a s t  more u s e f u l t h a n  pH,  laurentii  p o l y s a c c h a r i d e s may  instance,  (21);  Fourthly,  c o n d i t i o n s i t i s apparent  i s because the  glutinis  22°C  (20).  polysaccharides  q u i t e a few  analyses  Sporobolo-  t r a c e s o f MnS04 i n c r e a s e d  so  comparable.  For  at  Cryptococcus  p a r a m e t e r s t o use  them t a x o n o m i c a l l y . quantitative  as  by  extracellular  encompassing  valuable.  singularis  constant  contents  ( 1 2 ) ; by  polyby  by R h o d o t o r u l a  t h e medium, t e m p e r a t u r e ,  statistically  of the  and  20°C and  m i g h t be  one  in extracellular  a t 25°C  f a c t o r s such  g r o w t h p e r i o d were k e p t  analyses  Temperature  importance  myces s i n g u l a r i s  aeration  (21) .  c o n d i t i o n of  Cryptococcus  20°C  formed  extracellular  to  With regard should  the fact  to the Cryptococcus-Tremella  that Tremella  t o h a v e 1-3 l i n k a g e s  mesenterica  i n i t s extracellular  has  Cryptococcus  laurentii  the  proposed taxonomic r e l a t i o n s h i p ?  many t e s t s a r e n e c e s s a r y Cryptococcus  laurentii  and T r e m e l l a  statement optimal possible  that I think  classification  I n other  as  against  w o r d s , how  mesenterica?  that Tremella  as h a s C r y p t o c o c c u s  weighted h e a v i l y against  shown  polysaccharide  (19) be w e i g h t e d h e a v i l y  b e e n shown t o h a v e 1-3 l i n k a g e s  polysaccharide  h a s not been  t o prove t h e r e l a t i o n s h i p between  I don't t h i n k t h a t t h e f a c t not  relationships  mesenterica  has  i n i t s extracellular laurentii  this relationship.  i s quite  Personally  fitting.  c a n be d e f i n e d ,  up t o t h e p o i n t o f i n h e r e n t  should  be  J o h n s o n made a  He s a i d  t h a t no  b u t improvement i s  instability  (25) .  58  REFERENCES  1.  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Table V shows the percent monosaccharide sugar.  of the t o t a l  These percentages were c a l c u l a t e d u s i n g c o r r e c t i o n  f a c t o r s f o r the monosaccharides (17»37»^9)»  i n equilibrium solutions  Second, some mention of q u a n t i t a t i v e y i e l d s  of crude e x t r a c e l l u l a r polysaccharides should be made. Tremella mesenterica, cryptococcus l a u r e n t i i , B u l l e r a a l b a , sporobolomyces  odorus, sporobolomyces  and Rhodotorula g l u t i n i s  singularis,  a l l produced approximately  one t o two grams of crude polysaccharide i n approximately s i x l i t r e s of c u l t u r e medium.  Taphrina p o p u l i n a and  U s t i l a g o horde1 both produced l e s s than one gram of crude polysaccharide i n s i x l i t r e s of c u l t u r e medium.  62  Table I V  63 Peak Number  Fungus  Tremella mesenterica  Total  Tentative Identification  Solvent 2  Xylose  235  3  Xylose  7, 040  4  Xylose  7, 847  5  G a l a c t o s e , Mannose  6  Galactose  7  G a l a c t o s e , Glucose,  8  Glucose  Mannose 10,594 1, 414 39,981  1  Xylose  376  2  Xylose  7,943  G a l a c t o s e , Mannose Galactose, Mannose Glucose Area  11,610 1,241  Area  Cryptococcus laurentii  Total  Peak Area  40,763  Glucose, 251,730 266,866 567,678  Table  IV  (continued)  Fungus  Bullera alba  Total  Peak Number  Peak Area  Tentative Identification  1  Solvent  2  Solvent  3  Solvent  4  Xylose  2, 701  5  Xylose  3, 548  6  G a l a c t o s e , Mannose  7  Galactose  8  G a l a c t o s e , Glucose,  9  Glucose  13,568 495 Mannose  5,564 1, 412  Area  Sporobolomyces odorus (#949) ]  Total  6k  27,288  Xylose  389  Xylose  2,439  3  Xylose  3,953  4  G a l a c t o s e , Mannose  71  5  G a l a c t o s e , Mannose  22,724  6  Galactose  12,306  7  G a l a c t o s e , Glucose,  8  Glucose  Area  Mannose  36,460 15,072 93,414  Table  IV  (continued) *5  Peak Number  Fungus  Sporobolomyces odorus 1 (#981)  Total  Tentative Identification  Peak Area  Xylose  1, 007  Xylose  4, 691  Xylose  8,074  4  G a l a c t o s e , Mannose  27,010  5  Galactose  19,782  6  G a l a c t o s e , Glucose,  7  Glucose  Mannose  6, 766  Area  Sporobolomyces singularis 1  50,024  117,354  Xylose  287  2  G a l a c t o s e , Mannose  6, 357  3  G a l a c t o s e , Glucose, Mannose  1,095  4  Glucose  1,471  Total  Area  Rhodotorula glutinis  1  Solvent  2  Xylose  688  3  Xylose  1,378  4  G a l a c t o s e , Mannose  9, 724  5  Galactose  1, 537  6  G a l a c t o s e , Glucose,  9,210  Glucose Total  Area  Mannose  14,368 11,709 1Q.ADA  Table  IV (continued)  Fungus  Peak Number  66  Tentative Identification  Peak Area  Ustilago hordei  1  G a l a c t o s e , Mannose  527  2  Galactose  190  3  G a l a c t o s e , G l u c o s e , Mannose  4  Glucose  T o t a l Area  1,233 653 2,603  Taphrina populina  1  Mannose  3,469  2  Mannose  2 55  3  Glucose  1,374  4  Glucose  1,567  T o t a l Area  6, 665  Table V  67  Fungus  Monosaccharide  Tremella mesenterica  xylose Galactose  Cryptococcus  Bullera  laurentii  alba  —  Sporobolomyces  odorus  mm  odorus GFWT)  of T o t a l Sugar  38.00  9*50  Mannose  46.90  Glucose  5«60  Xylose  1.40  Galactose,Kannose  22.40  Glucose  76.20  Xylose  23.00  Galactose  Sp o r ob o l oinyc e s  percent  6.16  Mannose  62.4-0  Glucose  8.44  Xylose  7.24  Galactose  45.10  Mannose  21.36  Glucose  26.30  Xylose  11.70  Galactose  57.50  Mannose  21.39  Glucose  9.41  Table v (continued) Fungus  68 Monosaccharide  Sporobolomyces s i n g u l a r l s  Rhodotorula g l u t i n l s  Tjstllago horde!  Taphrina p o p u l i n a  Xylose  Percent of T o t a l Sugar 3»30  Galactose, Mannose  70.76  Glucose  25.94  Xylose  5.20  Galactose  13*34  Mannose  33.16  Glucose  48.30  Galactose  25.00  Mannose  34.30  Glucose  40.70  Mannose  56.00  Glucose  44.00  

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