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THE  METABOLISM 
OF  ALGAE 


G.  E.  FOGG 


METHUEN'S  MONOGRAPHS  ON 
BIOLOGICAL  SUBJECTS 


Methueti's  Monographs  on  Biological  Subjects 
General  Editor:  Michael  Abercrombie 


THE  METABOLISM   OF  ALGAE 


4^/ 

THE  METABOLISM  OF/^^ 

ALGAE  C  ^ 


by 

G.  E.  FOGG 

B.Sc.(Lond.),  Ph.D. (Cantab.) 

READER   IN   BOTANY, 
UNIVERSITY   COLLEGE,    LONDON 


WITH    20   DIAGRAMS 


^  LONDON:   METHUEN  &  CO.   LTD. 

^     NEW  YORK:  JOHN  WILEY  &   SONS,   INC. 


First  published  in  igs3 


I.I 
CATALOGUE   NO.    4122/u    (mETHUEN) 

PRINTED   IN   GRE.'^T   BRITAIN 


To 

the  memory  of 

MY  FATHER 


PREFACE 

The  algae  are,  on  the  whole,  unobtrusive  organisms, 
and  although  their  beauty  of  form  and  variety  of  life 
history  have  not  failed  to  attract  the  attention  of  the 
morphologist,  the  student  of  metabolism  has  generally 
preferred  to  work  with  material  more  readily  available 
or  showing  more  obvious  chemical  activity.  How- 
ever, there  is  a  growing  recognition  that  algal  meta- 
bolism has  its  own  distinctive  features  and  that  its 
study  has  both  academic  and  economic  value.  In  this 
book  I  have  attempted  to  bring  together  information 
scattered  through  a  variety  of  scientific  publications 
into  a  general  account  of  the  subject  which  will  be 
of  interest  to  students  of  botany,  microbiology  and 
biochemistry. 

My  thanks  are  due  in  particular  to  Professors  F.  E. 
Fritsch,  F.R.S.,  and  W.  H.  Pearsall,  F.R.S.  There  is 
much  in  this  book  derived  from  their  teaching  for 
which  acknowledgement  cannot  be  made  by  citation 
of  published  works  and  without  their  encouragement 
and  help  I  could  not  have  persisted  in  this  field  of 
study.  I  am  also  grateful  to  my  colleague  P.  J.  Syrett 
for  his  helpful  criticism  of  the  manuscript;  such  errors 
as  still  occur  must  be  attributed  to  lack  of  thoroughness 
on  my  part,  not  his.  Finally  I  must  record  my  thanks 
to  those  persons,  named  in  the  list  of  references,  who 
have  allowed  me  to  quote  their  unpublished  results. 

G.  E.  F. 

UNIVERSITY   COLLEGE,    LONDON 

August  1952 


Vll 


CONTENTS 

CHAP.  PAGE 

PREFACE  Vii 

I  INTRODUCTION  I 

II  THE   PHOTOTROPHIC    ASSIMILATION    OF  CARBON            1 7 

III  THE   CHEMOTROPHIC   ASSIMILATION   OF  CARBON           48 

IV  AUTOTROPHIC  ASSIMILATION  WITH  SPECIAL  REFER- 

ENCE  TO   NITROGEN   METABOLISM  68 

V  HETEROTROPHIC   ASSIMILATION  84 

VI  THE   PRODUCTS   OF   METABOLISM  89 

VII  GROWTH   AND   METABOLISM  I06 

VIII  SUMMARY  AND  CONCLUSIONS  I30 
REFERENCES  1 34 
INDEX  145 


IX 


'I  should  rather  incline  to  believe,  that  that  wonderful  power  of 
nature,  of  changing  one  substance  into  another,  and  of  promoting 
perpetually  that  transmutation  of  substances,  which  we  may  observe 
every  where,  is  carried  on  in  this  green  vegetable  matter  in  a  more 
ample  and  conspicuous  way.' 


JOHN    INGENHOUSZ,    1779^^^ 


CHAPTER    I 
INTRODUCTION 

The  term  'alga'  is  a  difficult  one  to  define.  In  the  broad 
sense  in  which  it  is  used  by  modern  authorities^^^'  ^59  it 
includes  a  number  of  types  of  organism  (see  Table  i)  which 
differ  profoundly  one  from  another  in  cell  organization  and 
which  have  little  in  common  except  that  their  characteristic 
mode  of  nutrition  is  photosynthetic  and  that  they  cannot 
be  included  in  any  other  division  of  the  plant  kingdom. 
This  grouping  together  of  phylogenetically  remote  classes 
of  organisms,  while  artificial  from  some  points  of  view,  is 
nevertheless  convenient,  particularly  if  the  transformations 
of  matter  and  energy  which  comprise  their  metabolism  are 
to  be  considered.  Regarded  in  this  way,  these  organisms 
form  a  more  homogeneous  group  with  sufficiently  marked 
characteristics  to  justify  special  consideration. 

On  the  one  hand  algae  are  distinguished  from  the 
morphologically  more  complex  higher  plants  by  the  variety 
and  flexibility  of  chemical  activity  characteristic  of  the  more 
primitive  forms  of  life.  On  the  other  hand  they  differ  from 
organisms  such  as  bacteria,  fungi  and  protozoa,  in  having  a 
chemical  economy  based  upon  photosynthesis,  in  which  the 
accumulation  rather  than  the  breakdown  of  organic  matter 
predominates.  In  bulk  of  material  involved  this  algal  type 
of  metabolism  perhaps  exceeds  any  other.  The  total  yield 
of  photosynthesis  in  the  oceans,  in  which  algae  are  the  only 
photosynthetic  organisms,  has  been  estimated  to  be  from 
1-6  to  15-5x1010  tons  of  carbon  fixed  per  year,  and  is 
evidently  at  least  as  much  as  that  of  land  plants. ^47,  209  i^ 
soil  and  in  freshwater  algal  metabolism  is  generally  on  a 
lesser  scale  but  is  nevertheless  of  considerable  importance. 

It  is  not  altogether  surprising,  then,  that  the  metabolic 
activities  of  algae  should  have  attracted  attention  while  the 
description  and  classification  of  species  was  yet  in  a  chaotic 

I 


2  THE    METABOLISM    OF    ALGAE 

State.  In  books  published  in  1779  both  Priestley^^^  and 
Ingenhousz^^^  described  experiments  on  the  gas  exchanges 
accompanying  the  photosynthesis  of  the  simple  algae  which 
grew  by  chance  in  their  apparatus.  Priestley  was  sufficiently 
interested  in  this  'green  matter',  which  he  doubted  to  have 
'ever  been  properly  noticed  by  botanists',  to  make  further 
experiments  with  it,  observing,  among  other  things,  that 
its  growth  was  promoted  by  certain  organic  materials. 
Priestley  also  determined  the  composition  of  the  gas  en- 
closed in  the  bladders  of  marine  algae.  Ingenhousz  made 
fewer  experiments  with  algae  but  realized  more  of  their 
possible  role  in  the  economy  of  nature,  as  is  evident  from 
the  sentence  which  is  quoted  at  the  beginning  of  this  book. 

As  in  physiology  generally,  further  progress  in  the  study 
of  the  metabolism  of  algae  had  to  await  the  development 
of  the  physical  sciences  and  during  the  greater  part  of  the 
nineteenth  century  algae  were  studied  almost  exclusively 
from  morphological  and  taxonomic  points  of  view.  Thus 
Sachs-^®  in  his  text-book  of  plant  physiology,  published  in 
1882,  used  algae  as  examples  wherever  possible  but  was 
able  to  say  very  little  about  their  metabolism  beyond 
making  the  generalization  that  it  essentially  resembles  that 
of  higher  plants.  Experimental  investigation  of  algae  in- 
creased towards  the  close  of  the  century  but  then  followed 
three  principal  lines  which  remained  to  a  large  extent  inde- 
pendent for  a  considerable  period. 

Examination  of  the  chemical  constituents  of  algae  was 
until  recently  confined  to  the  larger  seaweeds  and  to  such 
smaller  forms  as  very  occasionally  occur  in  a  reasonably 
pure  state  in  sufficient  quantity  for  analysis.  Two  classes 
of  substance  have  received  most  attention.  Following  the 
description  of  alginic  acid  by  Stanford  in  1883,^^^  many 
studies  of  the  carbohydrates  and  related  compounds  present 
in  algae  have  been  made.  Other  workers  have  been  more 
interested  in  the  pigments  which  form  such  a  striking 
feature  of  these  organisms.  The  work  of  Willstatter  and 
Stoll  published  in  1913^^^  is  of  particular  importance  in 
forming  the  basis  of  our  present-day  knowledge  of  algal 
pigments.  Only  recently  has  interest  in  the  nitrogenous  and 


INTRODUCTION  3 

lipide  constituents  of  algae  developed.  Particularly  note- 
worthy series  of  papers  on  algal  chemistry  have  been  pro- 
duced by  Haas,  Colin,  Heilbron  and  Percival  and  their 
respective  collaborators  (see  references). 

A  second  type  of  investigation  has  been  concerned  with 
the  growth  of  algae  in  culture.  Microscopic  algae  were  not 
neglected  by  the  microbiologists  following  Pasteur  but  pro- 
gress in  their  investigation  w^as  slow  in  comparison  with 
that  in  other  branches  of  microbiology,  probably  because 
of  the  greater  technical  difficulties  in  culture  and  manipula- 
tion. Many  studies  were  made  with  cultures  which  were 
not  absolutely  pure  and  work  of  this  type,  for  example  that 
of  Molisch  published  in  1896,^^^  showed  the  mineral  re- 
quirements of  simple  algae  to  be  much  the  same  as  those  of 
higher  plants.  Further  reliable  work  on  nutrition  could  only 
be  carried  out  with  pure  cultures.  Beijerinck  in  1890  first 
described  the  isolation,  in  what  was  probably  pure  culture, 
of  Chlorella  vulgaris  and  similar  unicellular  green  algae  by 
means  of  the  methods  developed  by  Koch  for  bacteria. ^^ 
Subsequently  other  techniques  appropriate  to  particular 
kinds  of  algae  were  worked  out  but  these  need  not  be 
particularized  since  the  history  of  algal  culture  techniques 
has  been  summarized  by  Pringsheim,^^^  himself  a  dis- 
tinguished w^orker  in  this  field.  By  1920  many  species  had 
been  isolated  in  a  bacteria-free  state  and  investigations 
under  controlled  conditions  were  undertaken.  At  first  quali- 
tative studies  of  nutritional  requirements  (see  ref.  132,  for 
example)  predominated.  Precise  quantitative  methods  for 
the  investigation  of  the  growth  of  simple  algae  were  intro- 
duced by  Bristol  Roach  in  1926^^^  in  a  study  of  the  effects  of 
carbon  compounds  on  soil  algae.  Such  work  has  established 
the  main  features  of  the  growth  in  culture  and  the  major 
nutritional  requirements  of  those  algae  which  are  most 
easily  grown  under  laboratory  conditions.  More  direct 
studies  on  the  metabolism  of  algae  in  culture  were  begun 
by  Pearsall  and  Loose,  who  in  1937^^^  showed  that  the  main 
trends  of  the  chemical  changes  occurring  during  the  growth 
of  a  Chlorella  population  are  similar  to  those  occurring 
during  the  development  of  a  leaf  of  a  higher  plant. 


4  THE    METABOLISM    OF    ALGAE 

In  a  third  kind  of  investigation  algal  material  has  been 
used  in  short-term  biochemical  experiments.  Many  algae 
have  qualities  which  particularly  suit  them  for  precise 
physico-chemical  investigation  and  have  occasionally  been 
used  as  material  by  biochemists  interested  in  particular 
aspects  of  metabolism.  Engelmann  used  algae  extensively 
in  his  elegant  work,  published  in  1883,^^  on  the  light  factor 
in  photosynthesis.  Later,  in  1919,  Warburg^^o  described 
the  use  of  Chlorella  as  a  system  in  which  photosynthesis 
may  be  studied  under  the  least  complicated  conditions  and 
since  then  this  and  similar  species  have  played  an  increas- 
ingly important  part  in  the  elucidation  of  the  mechanism 
of  this  process.  The  investigations  of  oxidative  assimilation 
by  the  colourless  alga  Prototheca,  reported  by  Barker  in 
1935-6,22'  23  provide  an  example  of  work  with  algae  which 
is  of  fundamental  importance  in  another  field  of  bio- 
chemistry. 

The  distinction  between  these  various  lines  of  research, 
clear-cut  for  many  years,  is  now  disappearing  as  it  becomes 
more  generally  recognized  that  the  metabolism  of  the  algae 
is  a  distinct  field  of  study  in  itself  and  that  to  be  properly 
understood  its  various  aspects  must  be  related  one  to 
another.  The  comparatively  late  development  of  this  atti- 
tude is  shown  by  the  fact  that  no  comprehensive  reviews 
of  algal  physiology  or  biochemistry  appeared  until  those  of 
Myers  and'^Blinks  in  1951.200.  ^^  Part  of  the  present  increase 
in  interest  in  the  subject  is  a  reflexion  of  the  general  develop- 
ment in  microbiological  chemistry  that  has  occurred  in 
recent  years;  part  is  due  to  the  economic  necessity  of  obtain- 
ing information  about  organisms  which  appear  to  have 
considerable  potentialities  as  sources  of  materials  and  power. 
Seaweeds  have  always  been  utilized  by  man  to  some  extent 
and  are  now  finding  increasing  use  as  raw  materials  for  the 
production  of  a  variety  of  substances  valuable  in  industry.208 
The  capacity  for  synthesis  of  microscopic  algae  has  not  as 
yet  been  put  to  any  direct  economic  use  although  it  appears 
likely  that  eventually  their  value  in  this  respect  may  be 
greater  than  that  of  the  seaweeds.  The  harvesting  of  natur- 
ally occurring  planktonic  algae  does  not  appear  to  be  an 


INTRODUCTION 


economic  possibility  and  attention  has  so  far  been  concen- 
trated on  the  large-scale  culture  of  forms  such  as  Chlorella. 
The  use  of  mass  cultures  of  diatoms  for  the  production  of 
fat  was  first  suggested  by  Harder  and  von  Witsch^^^  during 
the  Second  World  War  and  the  suggestion  that  Chlorella 
might  be  used  for  the  same  purpose  was  made  indepen- 
dently in  1947  by  Spoehr  and  Milner.^^^  Since  high  photo- 
synthetic  efficiencies  can  be  more  easily  achieved  and  main- 
tained with  unicellular  algae  than  with  conventional  crop 
plants  the  idea  of  using  these  organisms  for  the  large-scale 
production  of  industrially  useful  organic  matter  is  an  attrac- 
tive one  and  has  been  the  subject  of  much  speculation, 
discussion  and  experiment.i^^,  262, 223.  204,  48 

In  the  present  state  of  our  knowledge  of  algal  metabolism 
the  facts  must  to  a  great  extent  be  interpreted  in  terms  of 
concepts  established  by  the  biochemical  investigation  of 
other  types  of  organisms.  There  can  be  little  doubt  that 
the  general  pattern  of  metabolism  in  algae  is  the  same  as 
that  in  other  forms  of  life.  Thus,  it  must  be  expected  that 
in  algae  life  involves  continual  synthesis  and  breakdown 
by  enzyme-catalysed  reactions  of  the  substances  of  high 
potential  chemical  energy,  such  as  proteins  and  nucleic 
acids,  which  make  up  the  fabric  of  their  protoplasm.  It  is 
less  certain  that  the  chemical  mechanisms  involved  in  these 
processes  in  algae  are  the  same  as  those  which  have  been 
found  in  other  organisms.  For  example,  although  the  re- 
markable similarity  of  reaction  sequences  found  to  take 
place  in  such  diverse  organisms  as  yeast  and  vertebrates 
suggests  that  the  mechanisms  by  which  carbohydrates  are 
broken  down  in  respiration  are  fundamentally  the  same  in 
all  organisms,  there  is  not  otherwise  much  justification  for 
assuming  that  the  respiratory  processes  of  algae  are  similar 
to  those  of  other  organisms.  The  facts  known  concerning 
algal  respiration  are  worth  considering  at  some  length  in 
this  connexion  since  this  will  give  the  best  idea  of  the  extent 
of  the  correspondence  between  the  chemical  mechanisms 
of  algae  and  of  other  organisms  and  will  also  provide  a 
useful  basis  for  the  discussion  in  subsequent  chapters  of 
processes  which  intermesh  with  respiration. 


6  THE    METABOLISM    OF    ALGAE 

Apart  from  determinations  of  the  rate  of  the  process  in 
various  species  (for  references  see  40)  general  studies  of 
algal  respiration  appear  to  have  been  made  only  by  Gene- 
voisii^  and  by  Watanabe.^^^.  295,  296 

The  respiration  of  a  hexose  sugar,  which  substance  may 
for  convenience  be  considered  to  be  the  immediate  sub- 
strate for  the  process,  takes  place  by  a  sequence  of  reactions 
which  may  in  the  first  place  be  separated  into  two  stages, 
one  of  breakdown  or  glycolysis,  and  one  of  oxidation  of  the 
products   of  glycolysis.   The   mechanism  of  glycolysis   is 
known  in  considerable  detail  from  studies  on  muscle  and 
yeast^®^'  '^^'  ^^  and  involves  the  phosphorylation  of  hexose 
followed  by  splitting  of  the  molecule  into  C3  compounds 
which  after  a  series  of  transformations  give  rise  to  pyruvic 
acid,  generally  regarded  as  the  end  product  of  the  process 
(Fig.  i).  Under  anaerobic  conditions,  pyruvic  acid  is  not 
removed  by  the  oxidative   mechanism  and  fermentation 
occurs,  the  intermediates  of  glycolysis  then  giving  rise  by 
mutual  oxidation-reduction  to  end  products  the  nature  of 
which  varies  according  to  the  organism  and  the  conditions 
to  which  it  is  exposed.  The  mechanism  of  glycolysis  in  algae 
does  not  appear  to  have  been  investigated  in  detail,  but  there 
is  indirect  evidence  that  it  follows  essentially  the  same 
course   as  that  just  outlined.   Thus,   substances  such  as 
pyruvic  acid,  phosphoglyceric  acid  and  triose  and  hexose 
phosphates,  which  are  known  to  be  intermediates  in  yeast 
and  muscle  glycolysis,  have  been  shown  to  be  present  in 
the  green  algae,  Chlorella  and  Scenedesmus.'''^  Pyruvic  acid 
has  been  found  to  be  a  suitable  substrate  for  respiration  in 
Chlorella,^"^  Prototheca}^  and   Ulvar^'^  among  the  Chloro- 
phyceae,  in  Myelophyciis,  a  brown  alga,^^^  and  in  Gelidium, 
a   red   alga,^^^   but   not,   however,   in   a   blue-green   alga, 
Cylindrospermum,  even  under  conditions  apparently  favour- 
able for  the  penetration  of  this  acid  into  the  cells. ^^®  Under 
anaerobic  conditions  various  species  of  Chlorophyceae  have 
been  found  capable  of  fermentations  of  a  mixed  acid  type^^^ 
and  glucose  is  fermented  by  Prototheca  to  give  lactic  acid 
as  the  only  product  as  in  muscle. ^^ 

In  the  aerobic  respiration  of  vertebrate  tissues  pyruvic 


INTRODUCTION 
CARBOHYDRATE 


® 


OHoC 


/CH,0@ 
h\h   HOy/^QH 


HO     H 
11 
2CH20(p).CHOH.CHO 

+4H-2H3PO4  %  -4H+2H3PO, 
2ADP+2CH20@.CH0H.C00(P) 

t  t 

2ATP+2CH20(P).CH0H.C00H 


FRUCTOFURANOSE-I   :  6- 
DIPHOSPHATE 


3-PHOSPHO- 

GLYCERALDEHYDE 


I  :  3-PHOSPHOGLYCERIC 

ACID 

3-PHOSPHOGLYCERIC    ACID 


1 


..^pN 


2CH20H.CHO(^.COOH      2-phosphoglyceric  acid 


+  2H,0  %  -2H2O 

2ADP+2CH2:C0(P).C00H 

t  t 

2ATP+2CH3.CO.COOH 


phospho-enolpyruyic 

ACID 
PYRUVIC    ACID 


FIG.  I.  Scheme  summarizing  the  reactions  of  glycolysis.  For  sim- 
plicity certain  reactions  have  been  omitted.  (p\  denotes  the 

phosphate  group,  ADP,  adenosine  diphosphate,  and  ATP, 
adenosine  triphosphate  (for  further  information  see  ref.  19). 

acid  is  completely  oxidized  to  give  carbon  dioxide  and 
water  by  means  of  the  system,  represented  rn  Fig.  2,  known 
as  the  Krebs  or  tricarboxylic  acid  cycle.^^  This,  or  some 
similar  system  involving  di-carboxylic  acids,  is  generally 
assumed  to  occur  in  all  aerobic  organisms.  Certain  of  the 
acids  concerned  have  been  shown  to  take  part  in  the  meta- 
bolism of  Chlorella  and  Scenedesmus^^  and  also  to  serve  as 
substrates  for  respiration  in  Chlorella,^^  Ulva,  Myelophycus 
and  Gelidium}^^  Cell-free  extracts  of  Chlorella  have  been 
found  to  contain  dehydrogenases  for  these  acids,  which  are 


8 

Pyruvic 

COOH 

I 

CO      - 

I 

CH3 

COOH 

I 
CH, 

I 
CO 

I 

COOH 


THE    :\IETABOLISM    OF    ALGAE 
Acetic  Citric 


•COa 

— >" 


COOH 

I 
CH. 


CH.COOH 

II 

C(OH) 

I 
COOH 


Oxaloacetic 


T2H 


COOH 

I 
CH2 

I 
CHOH 

COOH 
k 


Malic 


±u,o 


COOH 

I 
CH      '- 

II 
CH 

I 
COOH 

Fumaric 


T2H 


COOH 

I              -CO2 
CH2        < 

I  -aH 

CHo       +H20 

I 
COOH 

Succinic 


COOH 


TH,0 


CHo      ^^=- 


C(OH)COOH 

I 
CH2 

I 
COOH 


Iso-citric 


COOH 

I 
CO 

I 
CH2 

I 

CH2 

I 

COOH 


TCO. 


Aconitic 

COOH 

CH 

II 
C.COOH 

I 
CH, 

I 
COOH 


±HiO 


COOH 

I 
CHOH 

I 
CH.COOH 

i 

CH, 

I 
COOH 

A 

T2H 

COOH 

I 
CO 

I 

CH.COOH 

I 
CH2 

I 
COOH 


a-Ketoglutaric       Oxalosuccinic 


FIG.  2.  Scheme  summarizing  the  reactions  of  the  tricarboxylic  acid 
cycle  (for  further  information  see  ref.  19). 


INTRODUCTION  9 

known  to  be  concerned  in  the  cycle. ^^  The  respiration  of 
Chlorella,^^^  of  Myelophyciis  and  of  Gelidium'^^^  is  reduced 
in  the  presence  of  maionate,  which  is  a  specific  inhibitor 
for  succinic  dehydrogenase,  an  essential  for  the  tricarboxylic 
acid  cycle  represented  in  Fig.  2.  However,  maionate  has 
been  found  not  to  inhibit  the  respiration  of  Ulva^^^  or 
Cylindrospermum?^^  This  evidence  points  to  the  existence 
of  some  alternative  mechanism,  as  yet  unidentified,  for  the 
oxidation  of  succinic  acid  in  these  algae,  but  it  should 
be  noted  that,  as  has  happened  in  experiments  with 
Chlorella,^^'  ^^^  the  medium  used  for  Ulva  may  not  have 
been  sufficiently  acid  to  secure  adequate  penetration  of 
malonic  acid  into  the  cells  (see  p.  55).  There  is  also  some 
evidence  that,  in  addition  to  the  tricarboxylic  acid  system, 
Chlorella  contains  a  pyruvic  dehydrogenase  by  means  of 
which  the  direct  oxidation  of  this  substance  can  take  place.  ^* 
It  may  be  concluded  that  while  there  is  evidence  that  reac- 
tions concerned  in  the  tricarboxylic  acid  cycle  occur  in 
algae  there  is  as  yet  no  conclusive  proof  of  the  participation 
of  this  system  in  normal  algal  respiration. 

Pyruvic  acid  enters  the  tricarboxylic  acid  cycle  after 
oxidative  decarboxylation  to  yield  acetic  acid  which  then 
condenses  with  oxaloacetic  acid  to  give  citric  acid  (see  Fig.  2 
and  ref.  19).  The  presence  of  thiamine  (vitamin  B^),  in  the 
form  of  its  pyrophosphate,  co-carboxylase,  is  essential  if 
this  reaction  is  to  take  place.  Thus,  the  rate  of  oxidation  of 
pyruvic  acid  by  thiamine-deficient  Prototheca  is  greatly 
enhanced  by  the  addition  of  thiamine. ^^  The  presence  of 
thiamine  may  also  be  necessary  for  other  decarboxylations 
involved  in  the  tricarboxylic  acid  cycle.  Thiamine  has  been 
demonstrated  in  all  of  the  numerous  algae  in  which  it  has 
been  sought,  including  species  of  Chlorophyceae,  Phaeo- 
phyceae,  Rhodophyceae,^^^  Bacillariophyceae^^^  and  Myxo- 
phyceae,^'*^'  ^^^  and  those  forms  which  are  unable  to 
synthesize  it  or  one  of  its  constituent  parts  for  themselves 
must  be  supplied  with  the  appropriate  substance  if  they 
are  to  grow  (see  Chapter  V).  There  is  thus  no  reason  to 
doubt  that  thiamine  plays  an  essential  part  in  the  meta- 
bolism of  all  algae  and  this  suggests  a  similarity  of  their 


10  THE    METABOLISM    OF    ALGAE 

respiratory    mechanisms    to    those    of    other    forms    of 
Hfe. 

The  oxidations  which  occur  in  the  course  of  the  tri- 
carboxyHc  acid  cycle  are  accompUshed  by  means  of  dehy- 
drogenases, the  hydrogen  being  eventually  transferred  to 
free  oxygen.  This  terminal  process  often  occurs  through 
cytochromes,   hydrogen   carriers   which   appear  to   be  of 
universal  occurrence  in  aerobic  organisms. ^^  The  demon- 
stration of  cytochromes  in  algae  is  rendered  difficult  by  the 
presence  of  photosynthetic  pigments  but  it  has  been  accom- 
plished for  a  red  alga,  Porphyra,^^  for  VaucJuria}^^  which 
is  generally  classified  with  the  Chlorophyceae  but  which 
in  metabolism  more  resembles  a  member  of  the  Xantho- 
phyceae,  for  the  flagellate,  Euglena,^^^  and  for  the  brown 
seaweed,  Fuciis.^^^  It  must  be  noted,  however,  that  in  these 
last  three  algae  the  cytochrome  that  has  been  found  seems 
to  be  part  of  the  photosynthetic  rather  than  the  respiratory 
mechanism.^^^  The  respiration  of  Chlorella  supplied  with 
sugar  is  affected  by  carbon  monoxide  in  a  manner  char- 
acteristic of  oxidations  involving  cytochrome."^  The  pres- 
ence of  cytochrome  oxidase  in  Polytoniella  caeca,  a  colourless 
member  of  the  Chlorophyceae,  has  been  reported.^^^  These 
are  indications  that  the  cytochrome  system  is  of  general 
occurrence  in  algae  but  there  is  evidence  that  in  many 
species  alternative  oxidation  mechanisms  exist.  Thus  con- 
centrations of  cyanide  sufficient  to  inhibit  the  oxidation  of 
substrates  supplied  exogenously  (i.e.  in  the  external  medium) 
are  without  effect  on,  or  may  even  stimulate,  endogenous 
respiration  (i.e.  using  substrates  stored  within  the  cell)  in 
various    species    of    Chlorophyceae.'^^'  ^^^'  ^^*'  ^°^'  ^''*'  ^^® 
Cyanide  is  an  inhibitor  for  enzymes  containing  heavy  metals 
and  thus  prevents  the  operation  of  the  cytochrome  system. 
The  cyanide-stable  respiration  of  these  forms  may  possibly 
be  carried  on  by  enzymes  of  the  flavo-protein  type,  which 
are  cyanide-insensitive  and,  although  probably  chiefly  con- 
cerned with  intermediate  hydrogen  transfers,  are,  in  some 
cases,    capable    of    transferring    hydrogen    to    molecular 
oxygen. ^^  Riboflavin,  a  component  of  the  prosthetic  group 
of  the  flavo-proteins,  has  been  demonstrated  in  all  the  algae 


INTRODUCTION  II 

in  which  it  has  been  sought,  i.e.  representatives  of  the 
Chlorophyceae,  Euglenineae,  Phaeophyceae  and  Rhodo- 
phyceae.^'  ^^'  ^*^'  "^®  Peroxidase  and  polyphenol  oxidases 
have  been  shown  to  be  present  in  various  algae  ^^  but  the 
part  which  these  enzymes  play  in  respiration  has  not  yet 
been  established. 

The  transfer,  storage  and  utilization  of  the  free  energy 
released  in  respiration  appears  usually  to  be  effected  through 
phosphorylated  compounds.  The  best  known  example  of 
the  formation  of  such  compounds  is  one  occurring  in  the 
course  of  glycolysis  in  yeast,  that  in  which  the  energy 
released  by  the  oxidation  of  the  aldehyde  group  of  3-phos- 
phoglyceraldehyde  is  used  for  the  introduction  of  a  new 
phosphate  group  in  the  i-  position  with  a  relatively  small 
over-all  change  in  free  energy  (see  Fig.  i).  From  the 
I  :  3-phosphoglyceric  acid  which  is  produced,  the  phos- 
phate radical  is  transferred  to  adenosine  diphosphate  giving 
adenosine  triphosphate  in  which  form  it  may  be  used  for 
the  phosphorylation  of  other  substances  thus  raising  their 
potential  chemical  energy  content  to  the  level  necessary  for 
their  participation  in  synthetic  reactions.^^'  *^  Certain  of 
the  substances  involved  in  the  phosphorylation  cycle  of 
yeast,  e.g.  hexose  phosphates  and  phosphoglyceric  acid,  are 
known  to  be  present  in  Scenedesmus,  Chlorella^^'  ^^  and 
Euglena.^  Adenosine  triphosphate  itself  is  perhaps  absent 
from  Chlorella  but  phosphorylated  compounds  which  may  _^ 
have  a  similar  role  are  evidently  present  in  this  alga^^ 
together  with  an  enzyme  capable  of  hydrolysing  adenosine 
triphosphate.^*  Adenosine  di-  and  triphosphates  have  been 
identified  in  Eiiglena.^  The  relative  proportions  of  the 
different  phosphorus  containing  fractions  in  Chlorella  alter 
following  illumination  of  the  cells  in  a  manner  which  sug- 
gests that  phosphorylated  compounds  are  acting  as  energy 
carriers. ^^*  Evidence  that  the  manner  in  which  the  energy 
of  phosphate  bonds  is  utilized  in  synthesis  is  similar  in 
algae  and  in  other  organisms  is  available  in  the  case  of 
starch  or  glycogen  formation,  in  which  the  energy  necessary 
for  the  polymerization  of  glucose  is  provided  from  the  phos- 
phate bond   of  glucose- 1 -phosphate.^^'  *^  Phosphorylases 


12  THE    METABOLISM    OF    ALGAE 

catalysing  this  reaction,  similar  to  those  found  in  the 
tissues  of  higher  animals  and  plants,  have  been  reported  as 
present  in  Polytomella  caeca^^^'^^  and  in  the  blue-green 
alga  Oscillatoria  princeps}^^ 

The  available  evidence  thus  suggests  that  the  mechanism 
of  respiration  in  algae  is  of  the  same  general  pattern  as  that 
in  other  organisms,  but  that  certain  of  the  enzyme  systems 
involved  may,  at  least  in  some  species,  be  different  from 
those  found  in  yeast  or  vertebrates.  It  will,  however,  be 
noticed  that  much  of  the  evidence  which  has  been  quoted 
relates  to  species  of  Chlorophyceae  and  that  information 
about  the  mechanism  of  respiration  in  algae  belonging  to 
other  classes  is  meagre.  This  illustrates  a  point  which 
deserves  particular  emphasis.  Our  present  knowledge  of 
algal  metabolism  is  based  upon  the  examination  of  very  few 
types  out  of  the  great  variety  which  exists,  about  half  of 
the  investigations  referred  to  in  this  book  having,  in  fact, 
been  carried  out  with  species  of  Chlorococcales.  Species 
belonging  to  other  classes  have  received  a  little  attention 
but  it  cannot  yet  be  said  that  a  representative  sample  of 
the  various  types  has  been  examined  with  any  degree  of 
thoroughness.  Considerable  differences  in  metabolism  have 
been  found  to  exist  between  closely  related  species  of 
algae^^^  and  such  results  make  it  clear  that  generalizations 
based  upon  observations  on  one  form  only  should  be 
treated  with  reserve.  Table  i  shows  the  distribution  among 
the  several  classes  of  those  genera  of  algae  mentioned  in 
this  book. 

As  a  framework  for  the  consideration  of  the  variety  of 
types  of  metabolism  which  exists  among  the  algae  a  classi- 
fication appears  best  which  recognizes  that  the  manner  in 
which  an  organism  obtains  the  energy  necessary  for  the 
maintenance  of  its  life  and  the  organism's  powers  of  syn- 
thesis are  independent.  Organisms  have  for  a  long  time 
been  classified  broadly  into  autotrophs,  those  capable  of 
growth  on  inorganic  nutrients  only  (e.g.  photosynthetic 
organisms),  and  heterotrophs,  those  needing  organic  sub- 
stances for  growth.  The  inadequacy  of  this  classification 
was  made  apparent  by  the  discovery  that  certain  species, 


If- 

t 


INTRODUCTION 


13 


TABLE    I 

CLASSIFICATION    OF    THE   ALGAE   SHOWING    THE    GENERAL    FEATURES 

OF    THE    CLASSES    AND    THE    TAXONOMIC    POSITIONS    OF    THE 

GENERA    MENTIONED    IN    THE    TEXT 

Genera  marked  with  an  asterisk  include  colourless  species.  For 
further  information  on  classification  and  morphology  see  106,  107, 

259- 


CHLOROPHYTA 


Class 
Chlorophyceae 
(green  algae; 
flagellate,  coc- 
coid  and  fila- 
mentous forms; 
more  abundant 
in  fresh  than  in 
saltwater  and 
tending  to  be 
terrestrial) 


Order 

Volvocales 


Chlorococcales 


Ulotrichales 


Cladophorales 

Chaetophorales 

Oedogoniales 

Conjugales 

Siphonales 

Charales 


Genera 

Chlamydomonas,  Chloro- 
gonium,  Coccomyxa, 
Haematococcus,  Poly- 
toma*  Polytomella*, 
Tetrachloris 

A?ikistrodesmus,      Chlor- 
ella,  Prototheca* , 
ScenedesmiiSy  Trebouxia 

Enteromorpha,  Hor- 

ynidium,    Monostroma, 
Stichococcus,  Ulva 

Cladophora 

Oedogonhim 

Zygnema 

Valonia,  Vaucheria  (?) 

Nitella 


Xanthophyceae  . 
('yellow-green' 
algae;  flagellate, 
coccoid  and  fila- 
mentous forms; 
most  abundant 
in  freshwater) 

Bacillariophyceae 
(diatoms;  uni- 
cellular forms; 
widely  distri- 
buted in  fresh- 
water, marine 
and  terrestrial 
habitats) 


CHRYSOPHYTA 

Heterochloridales 
Heterococcales 
Heterotrichales 
Heterosiphonales 


Pennales 
Centrales 


Monodus 
Tribonema 


Asterionella,      Navicula, 
Nitzschia*,  Pinnularia 
Ditylium 


14 


THE    METABOLISM    OF    ALGAE 


Chr^'sophyceae     .      Chr^-somonadales       Synura,  Ochromonas 
(mainly     flagel-     Chrysosphaerales  — 

lates,  fresh-     Chr^'sotrichales  — 

water  and 

marine) 


PYRROPHYTA 


Cr^'ptophyceae     , 
(mainly     flagel- 
lates; freshwater    Cr>'ptococcales 
and  marine) 

Dinophyceae 
(mainly     flagel- 
lates; most 
abundant  in  the 
sea) 


Cr\'ptomonadales       Chilomonas*,  Crypto- 


(A)  Desmokontae 

(B)  Dinokontae: 
Dinoflagellata 


Dinococcales 
Dinotrichales 


monas 


Prorocentrum 

Gymnodinium, 
Peridinium 


Euglenineae    . 
(flagellates; 
most    abundant 
in  freshwater) 


EUGLENOPHYTA 


Astasia  *  Euglena 


Phaeophyceae 
(brown  sea- 
weeds; filament- 
ous and  more 
elaborate  forms; 
all  but  a  few- 
marine) 


Rhodophyceae 
(red  seaweeds; 
filamentous  and 
more  elaborate 
forms;  all  but  a 
few  marine) 


PHAEOPHYTA 

Ectocarpales 

Tilopteridales 

Cutleriales 

Sporochnales 

Desmarestiales 

Laminariales 


Sphacelariales 

Dictyotales 

Fucales 


RHODOPHYTA 

(A)  Bangioideae: 
Bangiales 

(B)  Florideae: 
Nemalionales 
Gelidiales 


Coilodesme,     Ectocarpus, 
Myelophycus 


Alaria,  Chorda,  Lamin- 
aria,  Macrocystis,  Sac- 
corhiza 


Ascophyllum,  Fucus, 
Pelvetia 


Porphyra,  Porphyridium 


Gelidiwn 


Cr>'ptonemiales    Corallina,  Harveyella* 


INTRODUCTION 

Gigartinales  Chondrus,  Gigartina, 

Iridaea 
Rhodymeniales    Rhodymenia 
Ceramiales  Bostrychia 


15 


Myxophyceae 
(blue-green 
algae;  unicellu- 
lar and  filament- 
ous forms  lack- 
ing a  nucleus  as 
found  in  other 
algae;  fresh- 
water, marine 
and  terrestrial) 


CYANOPHYTA 

Chroococcales 


Chamaesiphonales 

Pleurocapsales 

Nostocales 


Stigonematales 


Chroococcus,  Gloeocapsa 
Microcystis,  Synecho- 
coccus,  Synechocystis 


Anabaejia,  Anahaeniopsis, 
Aphanizomenon, 
Aulosira,     Beggiatoa*, 
Calothrix,      Cyli^idro- 
spermum,  Gloeotrichia, 
Nostoc,      Oscillatoria, 
Phorniidium, 
Tolypothrix 

Mastigocladus 


such  as  Euglena  pisciformis,  possess  chlorophyll  and  are 
photosynthetic  and  yet  require  organic  growth  factors. '^^  It 
is  thus  necessary  to  distinguish  between  the  use  of  organic 
substances  as  a  source  of  energy  and  their  use  as  growth 
factors  by  organisms  unable  to  synthesize  particular  meta- 
bolites for  themselves.  The  following  is  the  relevant  part 
of  a  classification  which  recognizes  this  distinction.^'^'  ^^* 

I.  Nomenclature  based  upon  energy  sources. 

A  Phototrophy:  energy  chiefly  provided  by  photo- 
chemical reaction. 

(i)  Photolithotrophy:  growth  dependent  upon  exo- 
genous inorganic  hydrogen  donors  (e.g.  most 
green  plants,  in  which  water  acts  as  the 
hydrogen  donor). 

(2)  Photo-organotrophy:  growth  dependent  upon 
exogenous  organic  hydrogen  donors  (e.g. 
photosynthetic  bacteria  belonging  to  the 
Athiorhodaceae,  in  which  organic  substances 
such  as  fatty  acids  act  as  hydrogen  donors  in 
photosynthesis^"*  ^) . 


l6  THE    METABOLISM    OF    ALGAE 

B  Chemotrophy:  energy  provided  entirely  by  dark 
chemical  reactions. 

(i)  Che?nolithotrophy:  growth  dependent  upon  oxi- 
dation of  exogenous  inorganic  substances  (e.g. 
the  'chemosynthetic'  bacterium  Nitrosomonas, 
which  oxidizes  ammonia  to  nitrite-^'^'  ^^'). 

(2)  Chemo-organotrophy:  growth  dependent  upon 
oxidation  or  fermentation  of  exogenous 
organic  substances  (e.g.  non-photosynthetic 
bacteria,  fungi  and  animals). 

II.  Nomenclature  based  upon  ability  to  synthesize  essential 
metabolites. 

A  Autotrophy:  all  essential  metabolites  are  synthesized 
(e.g.  most  green  plants), 
(i)  Autotrophy    seiisu    stricto:    ability    to    reduce 

oxidized  inorganic  nutrients  such  as  nitrate. 
(2)  Mesotrophy:  inability  to  reduce  one  or  more 
oxidized  inorganic  nutrients  (=need  for  one 
or  more  reduced  inorganic  nutrients  such  as 
ammonia). 
B  Heterotrophy:  not  all  essential  metabolites  are 
synthesized  ('=need  for  exogenous  supply  of 
one  or  more  essential  metabolites,  i.e.  growth 
factors  or  vitamins;  animals  and  many  fungi 
and  bacteria  are  of  this  type). 

Composite  names  may  be  used  for  the  concise,  if  un- 
couth, characterization  of  a  nutritional  type  with  respect 
to  the  chief  energy  source  as  well  as  to  the  capacity  for 
synthesis  of  essential  cell  constituents,  e.g.  Chlorella  vulgaris 
is  photolithoautotrophic,  whereas  Prototheca  zopfii  is 
chemo-organoheterotrophic.  To  avoid  confusion  it  should 
be  particularly  noted  that  the  terms  autotrophic  and  hetero- 
trophic as  used  in  this  book  are  as  re-defined  in  this 
classification. 


CHAPTER    II 

THE   PROTOTROPHIC   ASSIMILATION 
OF   CARBON 

Since  the  energy  transformations  occurring  in  the  meta- 
boUsm  of  Hving  organisms  are  chiefly  brought  about  by 
chemical  changes  in  carbon  compounds,  the  assimilation  of 
this  element  and  the  absorption  of  energy  cannot  be  con- 
sidered apart.  Photosynthesis,  the  characteristic  method  of 
carbon  assimilation  of  the  algae,  occupies  a  central  place  in 
the  metabolism  of  these  organisms  and  an  account  of  the 
process  is  an  indispensable  foundation  for  the  consideration 
of  their  other  chemical  activities. 

The  process  of  photosynthesis  may  be  represented  by  the 
following  over-all  equation: 

C02+2H20->(CH20)+H20+02     .         .      (i) 

i.e.  as  an  oxidation-reduction  in  which  water  acts  as  the 
hydrogen  donor  and  carbon  dioxide  as  the  hydrogen 
acceptor.  The  oxygen  evolved  in  photosynthesis  has  been 
shown  to  come  exclusively  from  the  water  used  and  there- 
fore two  water  molecules  are  needed  as  hydrogen  donors  for 
each  one  of  carbon  dioxide.  The  first  stable  product  of  the 
reduction  of  carbon  dioxide  may  for  present  purposes  be 
supposed  to  be  a  carbohydrate  and  is  accordingly  repre- 
sented as  (CHgO)  in  equation  (i).  The  photosynthetic 
quotient  (Op  =A02/-AC02)  is  then  unity.  Photosynthesis 
is  endergonic  and  proceeds  with  an  accumulation  of  at  least 
112  k-cal.  per  mole  of  carbon  dioxide  reduced.  This  energy 
is  obtained  as  light  of  wavelengths  between  about  400  and 
700  TnjLi,  absorbed  by  the  photosynthetic  pigments.  Chloro- 
phyll acts  as  the  photochemical  sensitizer  and  photosynthesis 
has  never  been  found  to  occur  in  its  absence.  The  minimum 
number  of  light  quanta  required  for  the  reduction  of  one 
molecule  of  carbon  dioxide  to  the  reduction  level  of  the 

17 


l8  THE    METABOLISM    OF    ALGAE 

carbon  in  carbohydrates  is  still  the  subject  of  debate  but  the 
weight  of  evidence  is  at  present  in  favour  of  this  number 
being  at  least  eight. 

Kinetic  studies  have  shown  that  photosynthesis  is  not  a 
simple  process  but  that  it  consists  of  both  a  photochemical 
reaction  and  others  able  to  proceed  in  the  dark.  Com- 
parative biochemistry  suggests  that  the  generation  of  reduc- 
ing power  and  the  assimilation  of  carbon  dioxide  are 
separate  processes  and  experimental  evidence  seems  to 
show  conclusively  that  in  isolated  chloroplasts  the  first  of 
these  processes  can  occur  independently  of  the  other. 
Reducing  power  is  generated  by  a  process  including  at  least 
one  'dark  reaction'  as  well  as  the  photochemical  reaction 
and  results  in  the  splitting  of  water  to  yield  an  oxidized 
portion,  which  is  ultimately  disposed  of  by  elimination  of 
elementary  oxygen,  and  hydrogen,  which  is  transferred  to 
some  as  yet  unidentified  intermediary  acceptor.  Hydrogen 
from  this  intermediary  substance  is  then  used  for  the 
reduction  of  the  assimilated  carbon  dioxide. 

This  concept  of  photosynthesis  is  based  on  results 
obtained  with  both  algae  and  higher  plants.  There  can  be 
little  doubt  that  in  all  important  respects  the  mechanism  of 
photosynthesis  is  the  same  in  algae  as  in  other  plants.  Good 
evidence  for  this  is  that  the  principal  photosynthetic  pig- 
ment in  all  classes  of  algae  and  in  the  higher  plants  is  the 
same,  i.e.  chlorophyll  aP'^  The  first  stable  product  of  the 
photosynthetic  fixation  of  carbon  dioxide  has  also  been 
found  to  be  the  same  in  higher  plants  as  in  the  algae 
Chlorella  and  Scenedesmiis.-^  It  may  be  noted  that  although 
the  principal  pigments  of  the  photosynthetic  bacteria  are 
chlorophylls  they  are  different  from  those  to  be  found  in  the 
algae  so  that  there  is  here  the  possibility  of  a  difference, 
although  it  seems  unlikely  that  it  can  be  a  very  profound 
one,  in  the  photochemical  mechanisms  of  the  two  types. 

In  the  following  sections  of  this  chapter  an  account 
will  be  given  of  such  features  of  photosynthesis  as  are 
especially  characteristic  of  the  algae  or  of  which  con- 
sideration is  necessary  for  the  understanding  of  other 
chemical    activities    of   these    organisms.    Detailed    treat- 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON      19 

ment  of  photosynthesis  is  to  be  found  in  a  number  of 
recent  pubUcations.^^^  248,  loo,  so,  99, 113, 261 

THE   ALGAL   PIGMENTS   AND   THE   UTILIZATION   OF   LIGHT 

IN   PHOTOSYNTHESIS 

Three  principal  types  of  pigment  are  to  be  found  in  algae. 
These  are  the  chlorophylls,  the  carotenoids  and  the  phyco- 
bilins.  The  chlorophylls  are  characterized  by  possessing  a 
cyclic  tetrapyrrolic  nucleus  containing  magnesium.  They 
are  soluble  in  fat  solvents  giving  fluorescent  solutions 
having  pronounced  absorption  bands  in  the  blue  or  blue- 
green  and  in  the  red  or  infra-red  (Fig.  3).  Carotenoid  pig- 
ments are  likewise  soluble  in  fat  solvents  but  have  a  yellow 
or  orange  colour  resulting  from  absorption  in  the  blue  and 
the  green  (Fig.  3).   Carotenoids  generally  contain  about 


0-S 


400       440       480        520        560       600       640        660 
mVE    LENGTH  IN  MjU 

FIG.  3.  Absorption  spectra  of  methanol  solutions  of  pigrnents 
extracted  quantitatively  from  cells  of  Navicula  minima  (after 
ref.  280). 


20  THE    METABOLISM    OF    ALGAE 

40  carbon  atoms,  most  of  which  are  united  into  a  long 
polyene  chain,  i.e.  one  having  alternating  double  and  single 
bonds.  Carotenoid  pigments  are  of  two  sorts,  the  carotenes, 
which  are  hydrocarbons,  and  the  xanthophylls,  which  are 
oxygen  derivatives  of  carotenes.  The  phycobilins  are  char- 
acterized by  having  a  metal-free  linear  tetrapyrrolic  chromo- 
phoric  group  linked  to  a  protein  of  the  globulin  type. 
Unlike  the  other  photosynthetic  pigments  they  are  soluble 
in  water  and  insoluble  in  fat  solvents.  The  differences 
between  the  two  types  of  phycobilin,  the  phycocyanins  and 
phycoerythrins,  are  not  clearly  marked,  but  the  former  are 
generally  blue  whereas  the  latter  are  generally  red  (for  an 
absorption  spectrum  of  a  phycobilin  see  Fig.  7).  Phyco- 
bilins are  strongly  fluorescent.  More  detailed  accounts  of 
the  chemistry  and  properties  of  these  three  classes  of  pig- 
ments are  to  be  found  elsewhere.^--  ^*'^»  ^*^'  ^'^^'  ^'^ 

The  principal  pigments  occurring  in  the  different  classes 
of  algae  are  shown  in  Table  2.  Chlorophyll  a  is  the  most 
abundant  chlorophyll  in  all  types  of  algae,  as  it  is  in  higher 
plants.  Chlorophyll  h,  which  is  also  to  be  found  in  higher 
plants,  occurs  in  the  Chlorophyceae  and  Euglenineae  but 
is  absent  from  the  other  classes,  in  which,  however,  chloro- 
phylls c,  d  or  e,  may  be  present,  ^-carotene  is  the  most 
abundant  carotene  in  all  classes,  but  a-carotene,  character- 
istic of  Chlorophyceae  and  higher  plants,  is  absent  from 
some  classes  and  is  replaced  in  the  Bacillariophyceae  by 
e-carotene.  The  xanthophylls  are  numerous  and  each  algal 
class  possesses  its  characteristic  sorts.  Those  of  the  Chloro- 
phyceae are  similar  to  those  to  be  found  in  higher  plants. 
It  is  to  be  noted  that  the  Siphonales  differ  somewhat  from 
other  orders  of  the  Chlorophyceae  in  their  carotenoids.  In 
the  Siphonales  a-  and  not  /5-carotene  is  more  abundant  and 

•  indicates  the  principal  pigment  of  its  group,  3  a  pigment 
comprising  less  than  half  the  total  pigments  of  its  group,  O  a 
pigment  comprising  only  a  small  fraction  of  the  total  pigments  of 
its  group,  —  absent,  •  incompletely  examined,  and  ?  uncertain. 
The  Cryptophyceae,  which  show  diverse  coloration  and  regarding 
the  pigments  of  which  nothing  is  known,  are  omitted  from  this 
table. 


TABLE   2 

THE    PRINCIPAL    PIGMENTS    OF    THE    DIFFERENT    CLASSES    OF    ALGAE 

(data  from  ref.  272) 


>:r 


Chlorophylls 
Chlorophyll  a 
Chlorophyll  h 
Chlorophyll  c 
Chlorophyll  d 
Chlorophyll  e 

Carotenes 
a-carotene 
^-carotene 
€-carotene 

Xanthophylls 
Lutein 
Zeaxanthin 
Violaxanthin 
Flavoxanthin 
Neoxanthin 
Siphonein 
Siphonoxanthin 
Fucoxanthin 
Neofucoxanthin 
Diatoxanthin 
Diadinoxanthin 
Dinoxanthin 
Neodinoxanthin 
Peridinin 
Myxoxanthin 
Myxoxanthophyll 
Un-named 

Phycohilins 

r-phycoerythrin 
r-phycocyanin 
c-phycoerythrin 
c-phycocyanin 


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22  THE    METABOLISM    OF    ALGAE 

although,  with  the  possible  exception  of  zeaxanthin,  the 
xanthophylls  typical  of  other  Chlorophyceae  are  present, 
two  xanthophylls  peculiar  to  this  order  are  more  abundant. 
Only  the  Rhodophyceae  and  Myxophyceae  possess  phyco- 
bilins,  phycoerythrin  being  more  characteristic  of  the 
former  and  phycocyanin  of  the  latter.  The  pigments  pro- 
duced by  these  two  classes  although  similar  are  not  identical, 
and  they  are  usually  termed  r-  or  c-  forms  according  to 
whether  they  occur  in  the  Rhodophyceae  or  Myxophyceae 
respectively. 

The  variation  to  be  found  in  the  chromatophore  pigments 
of  the  algae  stands  in  marked  contrast  to  the  conservatism 
in  this  respect  shown  by  higher  plants. 

Evidence  of  the  part  played  in  photosynthesis  by  these 
various  pigments  has  been  obtained  principally  by  com- 
parison of  the  absorption  spectra  of  algae  with  their  'action 
spectra'  for  photosynthesis.-^^  To  a  rough  approximation 
the  spectral  absorption  curve  of  an  alga,  when  effects  due 
to  the  scattering  of  light  inevitable  in  a  poly-phase  system 
are  reduced  to  a  minimum,  is  the  sum  of  those  of  its  indi- 
vidual pigments.  The  action  spectrum  curve  of  photosyn- 
thesis is  obtained  by  determining  the  photosynthetic 
efficiency  of  light,  usually  measured  in  terms  of  oxygen 
production,  at  different  wavelengths.  These  determinations 
must  be  made  at  low  light  intensity  since  with  light  satura- 
tion light  of  all  wavelengths  that  can  be  utilized  will  produce 
the  same  effect.  A  further  point  is  that  it  is  necessary  to 
make  the  comparisons  for  equal  numbers  of  quanta  not 
for  equal  energy  contents,  since  the  photochemical  effect 
of  a  light  quantum  remains  the  same  although  the  energy 
per  quantum  varies  with  wavelength.  If  all  the  energy 
absorbed  by  the  pigments  is  available  for  photosynthesis 
then  the  curves  for  the  absorption  and  action  spectra  should 
be  super-imposable  and  since  the  different  pigments  have 
absorption  maxima  at  different  wavelengths,  it  is  sometimes 
possible,  by  comparing  the  two  curves,  to  determine 
whether  or  not  light  absorbed  by  a  particular  pigment  is 
being  utilized  in  photosynthesis  or  not  (see  Figs.  6  and  7). 
Such  comparison,  however,  is  not  straightforward  be- 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON     23 

cause  it  is  only  possible  to  determine  the  absorption  spectra 
of  individual  pigments  after  extraction  from  the  plant 
whereas  it  is  known  that  the  absorption  properties  as  thus 
determined  may  differ  appreciably  from  those  which  a  pig- 
ment possesses  in  the  living  plant.  For  example,  the  main 
absorption  band  in  the  red  of  chlorophyll  has  a  peak  at 
about  660  mju  in  solvents  such  as  alcohol  and  acetone 
whereas  in  living  plants  this  peak  is  seen  to  be  shifted  by 
about  15  m/u  towards  longer  wavelengths.  The  striking 
change  in  colour,  from  brown  to  green,  which  brown  algae 
and  diatoms  undergo  upon  killing  is  due  to  a  displacement 
of  the  absorption  band  in  the  green  of  fucoxanthin  towards 
the  position  in  the  shorter  wavelengths  characteristic  of  the 
extracted  pigment. ^^^  Such  displacements  of  absorption 
bands  in  vivo  are  generally  ascribed  to  chemical  combina- 
tion of  the  pigments  with  each  other  and  with  lipide  and 
protein.  Allowance  can  be  made  for  such  alterations  in 
absorption  properties  in  interpreting  action  spectra  but 
where  the  absorption  bands  of  pigments  in  vivo  overlap, 
and  it  is  consequently  impossible  to  determine  the  positions 
of  peaks  accurately,  correction  must  be  arbitrary  and  there 
is  a  corresponding  uncertainty  in  the  conclusions  derived. 
A  further  assumption  that  must  be  made  in  the  interpreta- 
tion of  action  spectra  is  that  the  pigments  are  uniformly 
distributed  within  the  chromatophores.  If  there  is  unequal 
distribution  then  one  pigment  may  screen  another  and 
estimation  of  the  partition  of  light  absorption  among  the 
different  pigments  cannot  be  made  correctly  from  know- 
ledge of  absorption  properties  and  relative  concentrations 
only. 

Much  of  the  early  work  on  the  role  of  algal  pigments  in 
photosynthesis  was  made  using  colour  filters  which  were 
only  capable  of  isolating  broad  regions  of  the  spectrum. 
Such  work  sometimes  led  to  conclusions  which  have  been 
confirmed  by  more  critical  studies  but  it  is  unnecessary  to 
discuss  it  in  detail  here  (for  references  see  248,  107).  In 
the  more  modern  investigations  action  curves  for  photo- 
synthesis have  been  determined  using  narrow  spectral 
bands  isolated  by  means  of  monochromators. 

3 


24 


THE    METABOLISM    OF    ALGAE 


The  role  of  carotenoids  has  been  estabUshed  most  con- 
vincingly in  the  case  of  diatoms.'''^'  ^^^'  ^^^  In  an  investiga- 
tion with  Navicula  minima,  for  example,  the  quantum  yield 
of  photosynthesis  has  been  measured  at  different  wave- 
lengths  (Fig.   4)   and   compared  with  the   corresponding 


400      440 


480        520        560        600       640 
^AVE   LENGTH  IN  MjU 


680       720 


FIG.  4.  Quantum  yield  of  photosynthesis  by  Navicula  miniina  as  a 
function  of  wavelength  (after  ref.  280). 

estimates  of  the  distribution  of  light  among  chlorophylls, 
fucoxanthin  and  other  carotenoids  (Fig.  5).  The  corrections 
for  band  shifts  necessary  for  the  estimation  of  the  absorp- 
tion of  the  different  pigments  in  vivo  were  determined  using 
cells  extracted  wath  dilute  methanol  which  removed  chloro- 
phyll c  and  fucoxanthin  without  extracting  the  other  pig- 
ments. The  quantum  yield  of  photosynthesis  was  found  to 
be  nearly  constant  from  520  to  680  m//,  dropping  rapidly 
at  wavelengths  greater  than  680  m/<  and  showing  a  slight 
dip  in  the  blue  between  430  and  520  m^.  Comparisons 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON      2$ 

between  this  efficiency  curve  and  those  for  the  distribution 
of  absorption  among  the  pigments  /«  vivo  indicate  that  Hght 
absorbed  by  fucoxanthin  is  utiUzed  in  photosynthesis  with 
about  the  same  efficiency  as  that  absorbed  by  chlorophyll. 
Thus  at  550  m/f,  where  some  80  per  cent  of  the  light 
absorption  is  by  fucoxanthin,  there  is  no  decline  in  quan- 


/oo 


CHLOROPHYLLS 

FUCOXANTHIN 

OTHER 

CAROTENOIDS 


400 


450 


600  550  600 

^AVE  LENGTH  IN  M/A 


650 


70O 


FIG.  5.  Curves  showing  the  estimated  distribution  of  light  absorp- 
tion among  the  different  groups  of  pigments  in  live  cells  of 
Navicula  minima  (after  ref.  280). 

tum  yield  as  compared  with  that  at  650  m/«  where  100  per 
cent  of  the  light  is  absorbed  by  chlorophylls^  Light  absorbed 
by  other  carotenoids,  however,  does  not  appear  to  be  avail- 
able for  photosynthesis. 2^*^ 

Fucoxanthin  appears  to  be  effective  in  the  same  way  in 
Phaeophyceae,  comparison  of  the  action  and  absorption 
spectra  for  Coilodesme  (Fig.  6)  showing  a  high  photosyn- 
thetic  activity  in  the  region  500  to  560  mfx  where  absorption 
is   chiefly   by   this   pigment.^^^    In   green   algae   such   as 


26 


THE    METABOLISM    OF    ALGAE 


Chlorella/^  Uha  and  Monostroma^^^  light  absorbed  by 
carotenoids  appears  to  be  partly,  but  not  entirely,  utilized 
in  photosynthesis.  In  Chroococcus,  however,  light  absorbed 
by  carotenoids  seems  to  be  for  the  most  part  unavailable 
for  photosynthesis.  "^"^  Carotenoids  sometimes  occur  else- 
where in  the  protoplast  than  in  the  chromatophore  and  are 


/OO 


THALLUS  ABSORPTION 
ACTION  SPECTRUM 


400    440 


480      520     560      600     640 
WAVE    LENGTH  IN  Mju 


680      720      760 


FIG. 


6.  Absorption  and  action  spectra  of  Coilodesme  californica. 
The  action  curve  for  photosynthesis,  corrected  to  relative 
rates  for  equal  numbers  of  incident  quanta,  has  been  made  to 
coincide  with  the  absorption  curve  at  675  m/ix.  The  relatively 
minor  divergence  between  the  two  curves  in  the  region  500 
to  560  m/x  suggests  that  light  absorbed  by  fucoxanthin  is 
effective  in  photosynthesis  (after  ref.  139). 

presumably  then  inactive  in  photosynthesis  and  it  may  be 
that  within  the  chromatophore  spatial  arrangements  exclude 
some  carotenoid  molecules  from  participation  in  active 
light  absorption.  Another  possibility  is  that  certain  types 
of  carotenoid  are  able  to  absorb  light  for  use  in  photo- 
synthesis but  that  others  are  quite  inactive  in  this  way. 

Because  their  main  absorption  bands  are  widely  separated 
from  those  of  the  chlorophylls,  the   phycobilins  provide 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON     27 

particularly  clear  examples  of  accessory  pigments  partici- 
pating in  photosynthesis.  In  Chroococcus  the  quantum  yield 
at  600  m/t  is  about  the  same  as  at  660  to  680  m^<,  where 
nearly  all  the  light  absorption  is  due  to  chlorophyll  a,  in 
spite  of  the  absorption  by  phycocyanin  at  this  wavelength 
being  about  six  times  that  of  chlorophyll  aJ'^  Thus  the 


/oo 


THALLUS  ABSORPTION 
ACTION  SPECTRUM 
EXTRACTED   PHYCOERYTHRIN 


400    440      480      620      560      600      640     680     720     760 
WAVE   LENGTH    IN  MjU 

FIG.  7.  Absorption  and  action  spectra  of  Porphyra  nereocystis.  The 
action  spectrum  for  photosynthesis  corresponds  more  closely 
to  the  absorption  spectrum  of  the  water  extract  of  the  alga, 
which  contains  phycoerythrin  as  the  principal  phycobilin 
pigment,  than  to  the  absorption  curves  of  chlorophylls  and 
carotenoids  (after  ref.  139). 

photosynthetic  efficiency  of  phycocyanin-  in  Chroococcus 
must  equal  that  of  chlorophyll.  The  phycobilins  in  Rhodo- 
phyceae  are  effective  in  the  same  way  and  in  certain  species, 
e.g.  Porphyra  nereocystis,  there  is  the  curious  situation  that 
light  absorbed  by  these  pigments  is  utilized  more  efficiently 
than  that  absorbed  by  the  chlorophylls  themselves. ^^^  This 
is  shown  in  Fig.  7,  in  which  the  photosynthetic  efficiency 
at  wavelengths  at  which  all  the  absorption  is  by  chlorophylls 


28  THE    METABOLISM    OF    ALGAE 

is  seen  to  be  much  less  than  that  at  wavelengths  where  the 
absorption  is  almost  entirely  due  to  phycobilins. 

Light  absorbed  by  chlorophyll  b  in  ChlorellaP^  and  by 
chlorophyll  c  in  Navicula^^^  appears  to  be  equally  available 
for  photosynthesis  with  that  absorbed  by  chlorophyll  a. 

Further  information  about  the  part  played  by  accessory 
pigments  in  photosynthesis  comes  from  studies  of  fluores- 
cence. Fluorescence  occurs  when  radiant  energy  absorbed 
by  a  substance  is  re-emitted  almost  instantaneously  as 
light.  This  light  is  necessarily  of  a  longer  wavelength  than 
that  which  excites  the  fluorescence  and  is  of  a  characteristic 
wavelength  for  a  given  pigment.  The  yield  of  fluorescence, 
i.e.  the  ratio  of  the  energy  emitted  to  that  absorbed,  gives 
information  regarding  the  fate  of  the  excitation  energy.  In 
living  Nitzschia  closterium  the  yield  of  chlorophyll  fluores- 
cence is  the  same  whether  it  is  excited  by  red  light  (600  m//.), 
absorbed  exclusively  by  chlorophyll,  or  by  blue-green  light 
(470  m/i),  three-quarters  of  which  is  probably  absorbed  by 
carotenoids."^  Similar  studies  with  Gigartina,  Iridaea,^^^ 
Porphyrtdium,''^'  ^^^  Porphyra  and  Oscillatoria^^  have  estab- 
lished that  light  absorbed  by  phycobilins  can  also  excite 
fluorescence  of  chlorophyll  a.  This  evidence  of  a  transfer- 
ence of  energy  from  accessory  pigments  to  chlorophyll 
suggests  that  light  absorbed  by  the  former  pigments  is  not 
utilized  directly  in  the  photochemical  reaction.  It  appears, 
in  fact,  that  all  light  energy  used  in  photosynthesis  must 
pass  through  chlorophyll  «.''*'  ^^^  Thus  in  Chlorella  only 
chlorophyll  a  fluoresces  and  its  fluorescence  is  excited  by 
light  absorbed  by  chlorophyll  h."^^  Since  chlorophyll  a  is 
the  principal  photosynthetic  pigment  in  all  the  algal  classes 
this  provides  evidence  that  the  photochemical  reaction  is 
of  the  same  nature  in  all  algae.  However,  it  has  been  found 
that  light  absorbed  by  phycobilins  is  sometimes  more 
effective  in  exciting  the  fluorescence  of  chlorophyll  a  than 
is  light  absorbed  by  this  chlorophyll  itself  and  that  light 
absorbed  by  chlorophyll  a  excites  the  fluorescence  of 
another  pigment  which  is  perhaps  chlorophyll  dJ^  These 
findings,  which  fit  in  with  the  observation  mentioned  above 
that  light  absorbed  by  phycobilins  is  more  efl"ective  in 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON     29 

photosynthesis  than  that  absorbed  by  chlorophyll,  can  be 
explained  if  it  is  assumed  that  chlorophyll  a  exists  in  two 
states  of  combination,  one  active  in  photosynthesis,  to 
which  energy  transference  from  phycobilins  is  possible,  and 
another,  inactive  in  photosynthesis,  from  which  energy  trans- 
ference to  the  unidentified  fluorescent  pigment  occurs.^^s 

After  it  was  first  postulated   by  Engelmann,^^'  ^^  the 
existence  of  chromatic  adaptation  among  algae  was  for  a 
long  time  the  subject  of  controversy  (see  107,  247,  248  for 
references).  This  problem,  of  w^hether  or  not  the  char- 
acteristic pigmentation  of  particular  forms  is  of  biological 
advantage  to  them,  is  now  largely  resolved  by  the  findings 
that   have    been   outlined    in   the   preceding   paragraphs. 
Chromatic  adaptation  is  most  clearly  exemplified  by  the 
algae  of  the  littoral  region  of  the  sea,  in  which,  as  a  general 
rule,  red  algae  occupy  the  lowest  zone  and  green  algae  the 
highest,  whilst  brown  algae  are  to  be  found  in  the  inter- 
mediate position.  Whereas  yellow  light  predominates  in  the 
sunlight  reaching  the  earth's  surface,  blue-green  light  is 
transmitted  to  the  greatest  extent  by  clear  seawater.  The 
pigmentation  of  the  littoral  algae  is  thus  complementary  to 
the  quality  of  light  characteristic  of  the  positions  which 
they  occupy  and  will  consequently  secure  maximum  light 
absorption.    Since    it   has    been    shown   that   the    energy 
absorbed  by  the  principal  accessory  pigments  is  utilized  in 
photosynthesis  it  is  clear  that  the  pigmentation  of  the  dif- 
ferent algae  is  such  as  to  enable  each  to  make  the  maximum 
use  of  the  light  energy  available  in  its  characteristic  habitat. 
Chromatic  adaptation  of  this  sort  is  genetically  determined, 
but  there  is  evidence  that,  in  some  species  at  least,  onto- 
genetic  adaptation   can   also   occur.^^^'  ^^^   However,    the 
zonation  of  algae  to  be  expected  from  this  simple  view  of 
chromatic  adaptation  may  be  blurred  by  several  circum- 
stances.  Thus  changes  in  the  proportions  of  individual 
photosynthetic  pigments  may  occur  in  response  to  variation 
in  intensity  as  well  as  in  quality  of  light  or  more  eflFective 
absorption    of   particular    wavelengths   may   be    obtained 
by  increase  in  total  concentration  of  pigments  or  by  in- 
crease in  thallus  thickness  rather  than  by  changes  in  the 


30  THE    METABOLISM    OF    ALGAE 

relative  amounts  of  the  individual  pigments.  It  is  also 
evident  that  a  species  in  which  a  particular  type  of  pig- 
mentation is  determined  genetically  may  become  adapted 
to  an  environment  in  which  its  pigmentation,  although  not 
a  disadvantage,  is  no  longer  an  advantage. 

THE   HYDROGEN   DONOR 

The  energy  made  available  through  absorption  by  the 
pigments  is  used  to  effect  reductions  by  means  of  hydrogen 
derived  from  some  specific  substance.  In  normal  photo- 
synthesis by  higher  plants  and  by  the  majority  of  algae  this 
hydrogen  donor  is  water.  In  certain  algae,  however,  other 
substances  can  be  utilized  at  least  as  the  ultimate  source  of 
hydrogen.  This  ability  to  make  use  of  a  wider  variety  of 
hydrogen  donors  in  photosynthesis  is  a  respect  in  which 
the  algae  resemble  the  bacteria  and  which  appears  to  be  a 
type  of  biochemical  variation  characteristic  of  the  more 
primitive  classes  of  organisms. 

The  process  of  'photoreduction'  by  means  of  elementary 
hydrogen,  which  can  occur  in  various  species  of  algae  after 
a  period  of  adaptation  under  anerobic  conditions,  was  dis- 
covered by  Gaffron  in  1939.^^^'  ^^"^  Adapted  algae  become 
able  to  carry  out  various  reactions  which  they  were  not 
able  to  effect  before.  In  the  dark  they  are  able  to  absorb 
hydrogen  from  an  atmosphere  containing  a  high  proportion 
of  this  gas,  providing  that  hydrogen  acceptors  such  as 
oxygen  are  available,  or  to  liberate  it  into  an  atmosphere 
of  pure  nitrogen  by  fermentation  of  a  substrate  such  as 
glucose.  Oxygen  and  hydrogen  can  thus  be  absorbed 
simultaneously  by  adapted  algae  and  the  reduction  of  carbon 
dioxide  may  be  coupled  with  this.  In  the  light,  exchanges 
involving  hydrogen  are  accelerated  and  photosynthesis 
from  carbon  dioxide  and  hydrogen  can  occur.  The  value  of 
the  quotient  AHg  'ACOg  is  in  accordance  with  the  over-all 
equation: 

CO2+2H2— >(CHoO)+H20  .         .      (2) 

i.e.  the  process  is  similar  to  the  photosynthesis  with  hydro- 
gen as  hydrogen  donor  which  occurs  in  the  purple  bac- 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON     3I 

teria.247  The  quantum  yield  of  this  process  is  of  the  same 
order  as  that  for  normal  photosynthesis.^*^ 

Hydrogenases,  enzymes  capable  of  introducing  hydrogen 
into  cellular  metabolism,  are  well  known  in  bacteria^^"?  and 
it  appears  that  in  certain  algae  there  are  similar  enzymes, 
which,  however,  only  become  active  after  reduction.  This 
reduction  occurs  during  adaptation  under  anaerobic  con- 
ditions and  is  reversed  when  de-adaptation  takes  place  in 
the  presence  of  oxygen  or  in  the  presence  of  carbon  dioxide 
if  the  intensity  of  illumination  exceeds  a  certain  limit.  It 
seems  likely  that  in  photosynthesis  by  adapted  algae  the 
photochemical  reaction  involves  the  decomposition  of  water 
as  in  normal  photosynthesis^*^  and  that  the  hydrogen  donors 
provided  by  the  hydrogenase  reaction  are  used  for  the 
reduction  of  the  oxidized  products  of  this  reaction,  which 
would  otherwise  be  disposed  of  by  processes  leading  eventu- 
ally to  the  liberation  of  oxygen.  If  the  light  intensity  is  high 
then  these  oxidation  products  accumulate  at  a  rate  faster 
than  that  at  which  they  can  be  reduced  by  the  hydrogenase 
system  and  this  leads  to  the  oxidation  and  inactivation  of 
the  hydrogenase.^*^  Certain  substances  such  as  hydroxyla- 
mine  and  o-phenanthroline  stabilize  adapted  algae  against 
de-adaptation  in  the  light,  evidently  by  inhibiting  the 
oxidation  of  the  hydrogenase  by  these  intermediates  of 
photosynthesis,  but  at  the  same  time  reduce  the  quantum 
efficiency  of  the  process  by  a  half.^^^'  ^^^ 

Not  all  algae  can  be  adapted  to  hydrogen  in  this  way. 
The  process  has  been  studied  principally  in  Scenedesmus  sp. 
and  has  been  found  to  occur  also  in  a  number  of  other 
algae,  but  many  species  have  been  found  not  to  possess  the 
property  (Table  3).  A  long  period  is  required  for  adaptation 
by  the  marine  algae  but  Chlamydomonas-moewusii  either 
does  not  require  adaptation  or  has  an  adaptation  period 
shorter  than  10  minutes.^^^  It  is  to  be  noted  that  the  distri- 
bution of  the  capacity  does  not  appear  to  depend  on  phylo- 
genetic  relationships.  The  capacity  is  present  in  members 
of  at  least  four  algal  classes  but  does  not  necessarily  occur 
in  all  members  of  a  taxonomic  group,  e.g.  it  is  absent  from 
Chlorella  whereas  it  occurs  in  Scenedesmus. 


32  THE  METABOLISM  OF  ALGAE 

TABLE  3 

ALGAE   WHICH    HAVE    BEEX    TESTED    FOR   ABILITY   TO    CARRY    OUT 

PHOTOREDUCTION  ^"^ 

Class  Species  successfully  Species  not  showing 

adapted  photoreduction 

Chlorophyceae     .    ScenedestJiiis  obliqiius  Chlorclla  pyrenoidosa 

Arikistrodesffius  sp. 

Chlamydomonas  moeuusii 

Ulva  lactuca 
Bacillariophyceae  —  Nitzschia  spp. 

Phaeophyceae       .    Ascophyllum  nodosum  — 

Rhodophyceae      .    Porphyra  lonbilicalis  — 

Porphyridiinn  criientiim 
M>'xophyceae       .    Synechococcus  elofigatus      Oscillatoria  sp. 

Synechocystis  sp.  Nostoc  ynuscorum 

Cylindrospermum  sp. 

This  type  of  photosynthesis  probably  does  not  occur  to 
any  great  extent  in  algae  under  natural  conditions.  After 
carrying  out  the  reaction  for  several  days  certain  algae  have 
been  found  to  have  shown  no  multiplication  or  increase  in 
chlorophyll  concentration  comparable  to  that  caused  by  a 
similar  period  of  normal  photosynthesis. ^^'^  It  is,  however, 
generally  found  that  green  algae  do  not  grow  under  anaero- 
bic conditions.  There  is  some  evidence  that  the  products  of 
photosynthesis  using  elementary  hydrogen  are  similar  to 
those  of  normal  photosynthesis^^'  ^^^  and  there  is  no  reason 
to  suppose  that  they  are  inherently  unsuitable  as  substrates 
for  growth.  Porphyridium,  a  red  alga,  has  been  found  to 
increase  in  dry  w^eight  and  pigmentation  while  carrying  on 
photosynthesis  using  elementary  hydrogen  under  anaerobic 
conditions. ^^^ 

A  blue-green  alga,  Oscillatoria,  and  a  diatom,  Pinnularia, 
have  been  reported  as  able  to  use  hydrogen  sulphide,  which 
is  actually  an  inhibitor  of  photosynthesis  in  higher  plants,  as 
hydrogen  donor. -^^'  ^^^  The  evolution  of  oxygen  is  here 
replaced  by  the  deposition  of  sulphur  within  the  cells  and 
the  prq^s  is  thus  apparently  similar  to  that  in  the  green 
sulphu^acteria,  represented  in  equation  3: 

COo+2HoS->(CHoO)+H20+2S     .         .      (3) 

Hydrogen-adapted    Synechococcus   and    Scenedesmus   have 
also  been  found  to  be  able  to  utilize  hydrogen  sulphide  as  a 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON     33 

hydrogen  donor  in  photosynthesis,  although  if  elementary 
hydrogen  is  present  this  is  used  preferentially.^^*  This  use 
of  hydrogen  sulphide  by  algae  has  not  yet  been  investigated 
in  detail,  but  there  are  indications  that  it  may  be  a  wide- 
spread phenomenon,  especially  among  Euglenineae  and 
other  flagellates. ^^° 

Photosynthetic  processes  utilizing  water,  elementary 
hydrogen  or  hydrogen  sulphide,  are  included  under  the 
heading  of  photolithotrophy  in  the  classification  of 
nutritional  types  given  in  the  first  chapter.  An  example  of 
photo-organotrophy,  in  which  an  organic  substance  acts 
as  hydrogen  donor,  is  provided  by  hydrogen-adapted 
Scenedesmus  which  will  utilize  substances  such  as  glucose 
in  preference  to  hydrogen  for  this  purpose. ^^^  No  case  of 
obligate  photo-organotrophy  in  an  alga  appears  to  have 
been  recorded  so  far. 

THE   ABSORPTION   OF   CARBON   DIOXIDE 

The  majority  of  algae  live  in  an  aquatic  environment  in 
which  carbon  dioxide  is  present  in  various  forms  in  equili- 
brium with  each  other: 

C02+H20^H2C03^HC03-+H+?=^C03--+2H+    .    (4) 

The  concentrations  of  these  several  forms  depend  on  a 
number  of  factors  including  hydrogen  ion  concentration, 
the  amount  of  base  in  excess  of  the  equivalent  of  the  strong 
acid  radicals  present,  the  partial  pressure  of  carbon  dioxide 
in  the  atmosphere  and  the  temperature.^*^'  ^^^  In  consider- 
ing the  activities  of  algae,  whether  in  culture  or  in  fresh- 
water or  seawater,  it  is  often  important  to  know  which  of 
these  forms  is  acting  as  the  immediate  source  of  carbon 
dioxide.  It  is  necessary  to  bear  in  mind,  of  course,  that  the 
form  in  which  carbon  dioxide  enters  the  cell  is  not  neces- 
sarily the  same  as  that  which  forms  the  immediate  substrate 
for  photosynthesis.  - 

It  is  generally  held  that  unionized  molecules  penetrate 
cell  membranes  more  rapidly  than  ions  and  in  agreement 
with  this  there  is  evidence  that  undissociated  carbon  dioxide 
is  utilized  by  certain  algae  whereas  bicarbonate  and  carbon- 
ate ions  are  not.  Thus  in  Chlorella  the  rate  of  photosynthesis 


34  THE    METABOLISM    OF    ALGAE 

has  been  found  to  be  dependent  on  the  concentration  of 
the  undissociated  form  even  though  both  ionic  forms  are 
present  in  considerable  excess^^^  and  direct  measurements 
have  shown  that  only  undissociated  carbon  dioxide  can 
penetrate  into  the  vacuole  of  Valonia  coenoc}tes.^^^  How- 
ever, the  results  with  Chlorella  cannot  be  considered  alto- 
gether reliable  since  the  buffer  solutions  used  not  only- 
varied  in  hydrogen  ion  concentration  but  were  very  alkaline 
for  physiological  media,  those  for  the  lower  concentrations 
of  undissociated  carbon  dioxide  having  pH  values  of  over 
10.^^^  In  Valonia  the  permeability  of  the  vacuole  membrane 
towards  carbon  dioxide  was  studied  whereas  for  photo- 
synthesis it  is  the  permeability  of  the  plasma  membrane, 
which  is  almost  certainly  different,  which  is  of  more 
importance. 

It  is  frequently  observed  that  algae  can  carry  on  rapid 
photosynthesis  in  alkaline  bicarbonate  solutions  in  which 
the  concentration  of  undissociated  carbon  dioxide  is  ex- 
tremely low\  This  may  be  explained  if  it  is  assumed  that 
the  bicarbonate  acts  indirectly  by  maintaining  the  supply 
of  undissociated  carbon  dioxide,  the  rate  of  diffusion  of 
which  would  otherwise  be  limiting,  in  the  vicinity  of  the 
cells,  but  the  effect  might  equally  well  be  due  to  the  absorp- 
tion of  the  bicarbonate  ions  themselves.  Active  absorption 
of  anions  is  a  property  common  to  all  growing  plant  cells 
and  there  seems  to  be  no  reason  for  supposing  that  bicar- 
bonate ions  are  exceptional  in  not  being  absorbed  by  this 
mechanism. 

Unequivocal  evidence  of  such  direct  utilization  of 
bicarbonate  ions  is  difficult  to  obtain  since  the  ratio 
[C02]/[HC03~]  in  solutions  cannot  be  altered  without 
changing  hydrogen  ion  concentration  too.  However,  over 
three  or  four  pH  units  around  neutrality  photosynthesis 
appears  not  to  be  directly  affected  by  hydrogen  ion  concen- 
tration."^®' 2^^'  ^^  Within  this  range  it  should  be  possible  to 
obtain  information  regarding  the  absorption  of  these  two 
forms  of  carbon  dioxide  by  study  of  the  performance  of 
algae  in  solutions  in  which  the  ratio  of  their  concentrations 
is  varied.  In  this  way  evidence  has  been  obtained  which 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON     35 

indicates  that  Scenedesmiis  quadricauda  is  able  to  utilize 
both  undissociated  carbon  dioxide  and  bicarbonate  ions, 
the  latter  being  more  readily  utilized,  only  10  to  20  /^  moles 
per  litre  being  required  for  maximum  growth  whereas  the 
corresponding  value  for  undissociated  carbon  dioxide  is 
80  1.1  moles  per  litre.^^^  Similar  experiments  with  Chlorella 
pyrenoidosa,  on  the  other  hand,  show  that  this  species  is 
not  able  to  utilize  bicarbonate  and  it  thus  appears  that 
there  may  be  two  types  of  algae  with  respect  to  carbon 
dioxide  absorption.^!*'  216  -Qqx^[i  the  Scenedesmus  and  the 
Chlorella  have  been  found  to  possess  carbonic  anhydrase 
so  that  the  difference  between  them  cannot  be  ascribed  to 
differences  in  ability  to  convert  carbon  dioxide  into  bicar- 
bonate.^!^  Experimental  evidence  confirms  the  expectation 
that  bicarbonate  absorption  is  an  active  process  of  anion 
absorption  rather  than  one  of  simple  diffusion  and  shows 
that  under  certain  circumstances  it  does  not  begin  until 
the  alga  has  been  activated  by  exposure  to  light  for  some 
time.217  The  mechanism  of  this  induction  effect  is  not 
known. 

Carbonate  ions  evidently  cannot  serve  directly  as  a  source 
of  carbon  dioxide  and  may  have  an  inhibitory  effect  upon 
gro^vth.^!^ 

By  absorption  of  carbon  dioxide  algae  may  increase  the 
alkalinity  of  the  medium  surrounding  them  to  the  point 
where  calcium  and  magnesium  carbonates  are  precipitated. 
Many  species,  particularly  those  belonging  to  the  Siphon- 
ales,  Rhodophyceae  and  Myxophyceae,  are  characterized 
by  the  production  of  calcareous  deposits  which  are  often 
incorporated  in  cell  membranes,  as  for  example  in  Coral- 
lina.^^^'  1^^  Such  calcification  is  generally  supposed  to  be 
brought  about  by  photosynthesis,  but  the  fact  that  calcareous 
and  non-calcareous  species  may  grow  side  by  side,  appar- 
ently in  similar  states  of  photosynthetic  activity,  suggests 
that  the  mechanism  may  not  be  so  simple.*^ 

In  general  the  total  concentration  of  all  forms  of  carbon 
dioxide  in  seawater  is  sufficient  not  to  limit  the  photo- 
synthesis of  unicellular  plants  under  conditions  likely  to  be 
met  with  in  the  open  sea.  This  is  shown  by  experiments 


36  THE    METABOLISM    OF    ALGAE 

with  Nitzschia  closterium,  for  example. ^^®  The  concentra- 
tions of  all  forms  of  carbon  dioxide  available  in  freshwater 
vary  much  more  than  those  in  seawater,  but  generally  they 
are  such  as  not  to  be  limiting  for  photosynthesis  by  phjto- 
plankton  except  in  the  surface  layers  of  waters  poor  in  dis- 
solved minerals  under  conditions  of  bright  sunlight  and 
calm.  With  bulkier  algae,  e.g.  Gelidium,  in  which  the  rate 
of  diffusion  through  the  tissues  up  to  the  site  of  fixation  is 
limiting,  the  saturating  concentration  of  carbon  dioxide  may 
be  considerably  higher  than  that  normally  to  be  found  in 
seawater.^^*  i 

THE   FIXATION   OF  CARBON   DIOXIDE 

The  transformations  undergone  by  intermediates  in  the 
metabolic  processes  of  plants  are  frequently  extremely 
rapid  and  until  recently  the  nature  of  those  involved  in  the 
photosynthetic  fixation  of  carbon  dioxide  remained  a  matter 
for  speculation,  the  available  analytical  techniques  being 
inadequate  for  the  separation  and  detection  of  the  minute 
amounts  of  the  substances  concerned.  Our  present  know- 
ledge of  the  intermediates  in  photosynthesis  is  based 
largely  upon  results  obtained  by  the  school  of  Calvin  and 
Benson  and  confirmed  and  extended  by  Gaff"ron  and  Fager, 
using  isotopic  tracer  techniques  in  conjunction  with  paper 
partition  chromatography  and  ion-exchange  methods.  In 
these  investigations  the  unicellular  algae  Scenedesmus  and 
Chlorella  have  been  the  principal  materials  for  study. 

If  Scenedesmus  or  Chlorella  is  supplied  with  radioactive 
carbon  dioxide  in  the  dark  it  is  found  that  malic,  succinic, 
fumaric,  citric,  glutamic  and  aspartic  acids  and  alanine, 
become  labelled  with  radioactive  carbon  and  together 
account  for  95  per  cent  of  the  total  radioactivity  of  the  algal 
products.  These  compounds  appear  to  be  labelled  by 
reversible  carboxylation  reactions  such  as  are  well  known 
in  other  organisms  and  which  occur  independently  of  the 
capacity  for  photosynthesis.  Under  anaerobic  conditions 
radioactive  carbon  fixed  in  this  manner  remains  in  the 
water-soluble  fraction,  only  when  aerobic  respiration  takes 
place  is  it  transferred  to  polysaccharide,  fat  or  protein.  If 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON      37 

the  alga  is  supplied  with  radioactive  carbon  dioxide  for  a 
short  period  in  the  light  then  killed  and  extracted  immedi- 
ately, it  is  found  that  not  only  is  the  rate  of  fixation  from 
ten  to  a  hundred  times  greater  than  that  in  the  dark,  but 
that  different  compounds  become  labelled,  phosphoglyceric 


SO  - 


o  ^o 

Q 

o 


O 

k 

ki 


20 


ORGANIC  PHOSPHATES 


O    PH0SPH06LYCER/C 
ACID 

O     MALIC  AND 

AS  PART  I C  AC  I DS^ 


MINUTES 

FIG.  8.  Distribution  of  radioactive  carbon  among  products  ex- 
tracted with  80  per  cent  ethanol  from  Scenedesrmis  sp.  after 
varying  periods  of  photosynthesis  in  the  presence  of  carbon 
dioxide  labelled  with  radioactive  carbon  (after  ref.  52). 


acid,  pyruvic  acid,  malic  acid,  alanine,  triose  and  hexose 
phosphates,  and  sucrose  now  being  the  principal  substances 
concerned. ^^'  ^^"^  As  the  period  of  exposure  of  the  actively 
photosynthesizing  alga  to  radioactive  carbon  dioxide  is 
shortened  a  greater  proportion  of  the  radioactive  carbon 
appears  in  phosphoglyceric  acid  (Fig.  8).  This  and  other 


38  THE    METABOLISM    OF    ALGAE  I 

evidence  has  established  that  this  substance  or  its  imme- 
diate precursor  is  an  intermediate  in  photosynthesis. ^2, 114 
Evidence  that  substances  such  as  pyruvic  acid  and  maHc 
acid  are  directly  involved  in  photosynthetic  carbon  dioxide 
fixation  is  less  satisfactorv. 

Examination  of  degradation  products  of  the  phospho- 
glyceric  acid  produced  in  short  periods  (5  to  10  seconds)  of 
photosynthesis  with  radioactive  carbon  dioxide  shows  radio- 
activity to  be  almost  entirely  confined  to  the  carboxyl  group. 
This  suggests  that  the  phosphoglyceric  acid  is  formed  by 
the  carboxylation  of  some  two  carbon  (C2)  compound.  As 
the  period  of  exposure  to  the  labelled  carbon  dioxide  is 
lengthened  radioactive  carbon  appears  to  increasing  ex- 
tents in  the  2  and  3  positions  in  phosphoglyceric  acid  and 
after  about  two  minutes'  exposure  the  molecule  is  uniformly 
labelled.  The  Cg  acceptor  thus  appears  to  be  itself  derived 
from  phosphoglyceric  acid.^^'  ^^'* 

These  well-established  facts  point  to  the  existence  of  a 
self-multiplying  fixation  cycle  which,  each  time  it  is  com- 
pleted, results  in  the  incorporation  of  three  molecules  of 
carbon  dioxide  and  the  formation  of  another  molecule  of 
phosphoglyceric  acid  for  each  one  initially  present.  One  of 
these  phosphoglyceric  acid  molecules  is  necessary  to  main- 
tain fixation  whilst  the  other  may  be  diverted  from  the 
cycle  and  used  for  other  purposes. 

The  nature  of  the  Cg  acceptor  and  the  manner  of  its 
regeneration  from  phosphoglyceric  acid  are  as  yet  unknown. 
There  is  no  evidence  that  the  acceptor  is  formed  by  con- 
densation of  two  Ci  molecules,  but  there  are  indications 
that  it  is  derived  from  C3  or  C4  compounds.  Thus,  if  plants 
are  illuminated  in  the  presence  of  radioactive  carbon  dioxide 
for  short  periods  so  that  labelled  C3  and  C4  substances  are 
formed,  a  further  period  of  illumination  in  the  absence  of 
carbon  dioxide  causes  the  disappearance  of  such  substances 
and  the  appearance  of  radioactive  glycollic  acid  and  glycine, 
both  C2  compounds.  This  suggests  a  close  relationship 
between  the  two  latter  compounds  and  the  C2  acceptor. ^^ 
As  an  example  of  the  type  of  fixation  cycle  which  may 
operate,  a  scheme  may  be  given  in  which  it  is  supposed 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON     39 

that  the  €3  acceptor  is  derived  by  spUtting  and  reduction 
of  oxaloacetic  acid.^^  It  will  be  seen  from  Fig.  9  that  in  this 
cycle,  which  involves  two  successive  carboxylations,  the 
three  substances  named  are  interconvertible  by  known  bio- 
chemical mechanisms.  There  is,  however,  no  direct  evidence 

C2  acceptor                  _^^.q^  phosphogly''^^  ^^^^ 

?  '- ^CH20H.CH0(P).C00H 


reduction 

+  CO2 

.CO.COOH  < CH3.CO.COOH 


splitting 

COOH.CH, 

oxaloacetic  acid  pyruvic  acid 

FIG.  9.  Scheme  showing  a  possible  manner  of  origin  of  the  C^ 
acceptor  in  photosynthesis  from  phosphoglyceric  acid  (after 
ref.  30). 

for  the  existence  of  this  cycle  and  it  is  open  to  criticism 
on  various  grounds. ^^^  The  evidence  for  the  various  other 
schemes  that  have  been  put  forward^^-  ^1^'  ^^^  is  equally 
unsatisfactory. 

A  fundamental  problem  which  is  yet  unsolved  is  that  of 
the  manner  in  which  the  reductions  involved  in  the  carbon 
dioxide  fixation  cycle  take  place.  Chloroplasts  separated  by 
suitable  techniques  from  living  cells,  or,  in  the  case  of  algae 
such  as  Chlorella,  intact  cells,  are  capable  of  carrying  out 
the  'Hill  reaction'  in  which,  under  the  influence  of  light, 
water  is  split  resulting  in  the  evolution  of  oxygen  and  the 
reduction  of  an  appropriate  hydrogen  acceptor.  A,  accord- 
ing to  the  equation: 

A+H2O— ^-HgA+iOa     .         .         .      (5> 

Substances  such  as  ferricyanide  and  quinone  can  act  as 
direct  hydrogen  acceptors  in  this  reaction,  but  carbon 
dioxide  cannot.^*'^'  ^^^'  ^^^  The  reduction  potential  devel- 
oped is,  in  fact,  insufficient  for  the  direct  reduction  of  the 
latter  substance  to  the  level  of  carbohydrate. ^^^  However, 
reduction  coupled  with  carbon  fixation  by  illuminated 
chloroplast  preparations  from  higher  plants  has  been  shown 
to  occur  in  the  presence  of  a  suitable  hydrogen  carrier. 
Triphosphopyridine  nucleotide  (co-enzyme  H)  is  such  a 

4 


40  THE    METABOLISM    OF    ALGAE 

substance  and  its  reduction  by  illuminated  chloroplasts  may- 
be coupled  with  a  reductive  carboxylation  such  as  that  of 
pyruvic  acid  to  malic  acid,  the  sequence  of  reactions  being: 

light 

HoO+coenzyme  II >•  coenzyme  II.H2+^02  .      (6) 

'malic'  enzyme 
CHa.CO.COOH  +  COa+coenzyme  II. H2 > 

COOH.CHa.CHOH.COOH+coenzyme  II      (7) 

The  so-called  'malic'  enzyme  which  catalyses  reaction 
(7)  has  been  shown  to  occur  in  a  number  of  plant 
species. ^^®'  "^^'  ^^  Although  an  extracellular  photosynthetic 
reaction  has  thus  been  brought  about,  it  is  doubtful  whether 
this  particular  reaction  is  concerned  in  normal  photo- 
synthesis.^®^' ^^  Malic  acid  does  indeed  appear  among  the 
products  of  short-term  photosynthesis  but  photosynthesis 
has  been  found  to  be  little  affected  by  the  presence  of  in- 
hibitors such  as  malonic  acid  which  completely  suppress 
the  formation  of  malic  acid.^^'  ^^ 

Further  information  regarding  the  link  between  the 
photochemical  reaction  and  the  reduction  of  carbon  dioxide 
comes  from  observations  on  the  enhanced  dark  fixation 
which  occurs  in  algae  and  in  other  plants  immediately 
following  a  period  of  normal  photosynthesis  or  a  period  of 
illumination  in  the  absence  of  carbon  dioxide  and  oxygen. 
In  both  cases  the  enhanced  dark  fixation  shows  character- 
istics of  photosynthetic  fixation. ^^'  ^^^>  ^^*  For  Chlorella^ 
studies  using  radioactive  carbon  dioxide  have  shown  that 
the  products  of  this  dark  fixation  following  illumination 
are  the  same  as  those  of  short-term  photosynthesis.  This 
has  been  used  as  evidence  in  favour  of  the  view  that  a 
reducing  product,  perhaps,  for  example,  reduced  coenzyme 
II,  survives  from  the  photochemical  reaction  and  is  capable 
of  complete  carbon  dioxide  reduction  in  the  absence  of 
light.  Such  a  substance  might  be  concerned  in  reductions 
at  more  than  one  point  in  metabolism,  for  example  in  the 
formation  of  hexose  sugar  from  phosphoglyceric  acid  or  in 
the  regeneration  of  the  Co  acceptor.^^  However,  it  has  been 
pointed  out  that  although  the  products  of  dark  fixation 
following  illumination  are  qualitatively  the  same  as  those 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON     4I 

of  short-term  photosynthesis  there  is  an  important  quantita- 
tive difference,  phosphoglyceric  acid  in  the  former  case 
constituting  a  much  greater  proportion  of  the  total.  In 
Scenedesmus  the  radioactive  carbon  fixed  in  the  dark  imme- 
diately following  illumination  is  found  almost  exclusively 
in  the  carboxyl  groups  of  phosphoglyceric  and  pyruvic 
acids.  Thus  it  appears  that  it  is  only  the  Cg  acceptor  that 
survives  after  the  cessation  of  illumination  and  that  the 
reductions  concerned  in  fixation  can  only  occur  in  the  light. 
This  view  is  supported  by  the  fact  that  reduction  in  the  Hill 
reaction  likewise  ceases  immediately  in  the  dark  and  by 
the  results  of  experiments  with  inhibitors. ^^^  This  implies 
an  intimate  association  between  the  reduction  required  in 
the  fixation  cycle  and  the  photochemical  reaction.  It  may 
be  that  there  is  only  one  specific  reduction  coupled  with 
the  photochemical  reaction  and  that  other  reductions  are 
accomplished  indirectly  at  the  expense  of  a  proportion  of 
the  products  of  photosynthesis  which  are  degraded  to  pro- 
vide the  necessary  energy.^^^  Our  knowledge  of  this  most 
important  step  in  photosynthesis  is  thus  still  in  an  elemen- 
tary stage. 

INTER-RELATIONS   BETWEEN   THE   CARBON   DIOXIDE 
FIXATION   CYCLE   AND    OTHER   METABOLIC    SYSTEMS 

The  close  connexion  existing  between  the  metabolic 
cycles  involving  sugars,  organic  acids  and  proteins  has  been 
recognized  for  some  time  and  the  concept  has  arisen  of  a 
'metabolic  pool'  of  intermediates  common  to  these  different 
processes  and  through  which  they  are  mutually  correlated. 
The  one  substance  now  known  with  a  degree  of  certainty 
to  be  an  intermediate  in  photosynthesis,  phosphoglyceric 
acid,  is  also  an  intermediate  in  glycolysis"  readily  trans- 
formable into  substances  involved  in  other  metabolic 
sequences  and  may  be  regarded  as  a  component  of  this 
'pool'.  This  and  other  considerations  (see  ref.  199)  make  it 
evident  that  the  photosynthetic  fixation  of  carbon  dioxide 
intermeshes  with  other  metabolic  processes  at  an  early 
stage.  This  idea,  which  is  contrary  to  the  concept  of  photo- 
synthesis which  prevailed  until  recently,  has  received  ample 


42  THE    METABOLISM    OF    ALGAE 

confirmation    from    studies    with    radioactive    carbon    as 
tracer. 

Since  the  first  recognizable  product  of  photosynthesis  is 
identical  with  an  intermediate  in  glycolysis,  a  process  which 
is  known  to  consist  of  fully  reversible  steps,  it  is  reasonable 
to  suppose  that  carbohydrate  formed  in  photosynthesis  is 
elaborated  by  simple  reversal  of  the  normal  glycolytic 
mechanism.  Among  the  products  found  to  be  labelled  in  algae 
after  periods  of  photosynthesis  in  the  presence  of  radio- 
active carbon  dioxide  of  15  to  60  seconds  duration  are  triose 
phosphates,  hexose  phosphates,  hexose  diphosphate  and 
sucrose.  This  rapid  appearance  of  radioactivity  in  the  sugars 
shows  that  there  is  a  close  connexion  between  the  photo- 
synthetic  mechanism  and  that  responsible  for  the  synthesis 
of  these  compounds  and  the  fact  that  several  of  the  labelled 
phosphate  esters  are  identical  with  those  involved  in  glyco- 
lysis suggests  that  the  pathway  of  synthesis  is  the  expected 
one.  Furthermore,  the  distribution  of  radioactivity  in  the 
3  :  4,  2  :  5  and  the  i  :  6  carbons  in  the  hexose  (see  formula 
on  p.  91)  corresponds  with  that  found  in  the  carboxyl  and 
in  the  2-  and  3-carbons  respectively  of  phosphoglyceric 
acid  as  would  be  expected  in  hexoses  formed  by  condensa- 
tion of  C3  substances  derived  from  this  compound.^-  In 
the  synthesis  of  sugars  from  phosphoglyceric  acid  a  reduc- 
tion of  the  carboxyl  to  an  aldehyde  group  occurs.  The 
source  of  the  hydrogen  necessary  for  this  reduction  may 
be  the  photochemical  reaction  itself,  but,  as  we  have  already 
seen,  it  is  more  probable  that  it  is  obtained  by  purely 
chemical  reactions  at  the  expense  of  a  proportion  of  the 
products  of  photosynthesis. 

The  accumulation  of  radioactivity  in  pyruvic  and  malic 
acids  during  short  periods  of  photosynthesis  by  Scenedesmus 
and  Chlorella  in  the  presence  of  radioactive  carbon  dioxide 
has  already  been  noted.  Radioactivity  appears  more  slowly 
in  succinic,  fumaric  and  citric  acids.^^'  ^^  These  acids  are 
all  concerned  in  the  tricarboxylic  acid  cycle  (Fig.  2)  and 
this  evidence  indicates  that  in  these  algae  carbon  fixed  in 
photosynthesis  may  enter  the  cycle  in  the  expected  way 
through  pyruvic  acid  (Fig.  10). 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON     43 


Labelled  amino-acids  appear  at  an  early  stage  when  algae 
photosynthesize  in  the  presence  of  radioactive  carbon 
dioxide.  Radioactivity  appears  first  in  aspartic  acid,  alanine, 
serine  and  glycine  among  amino-acids.^^  Aspartic  acid  and 


in 

<-^ 

J 
O 
ffl 
< 


ACID   METABOLISM 


r 


TCO5 


■^ 


±NH3 


CITRATE 


ra-KETOGLUTARATE  :^ 
-CO, 


•HIGHER   FATTY 

ACIDS 

N 


:iGLUTAMATE , 

NH3 


SUCCINATE 


MALATE 


GLUTAMINE 


ASPARAGINE 

k 


TNH3 

±NH3 
OXALO ACETATE  ^  *"  ASPARTATE- 


+  CO. 


GLYCEROL 


C0« 


±NH3 
PYRUVATE  ^,  ''ALANINE 

N 


TRIOSE 

phosphates' 


HEXOSE 
PHOSPHATES 


—  PHOSPHOGLYCERATE  GLYCINE 


\ 


+  CO2 


+  NH, 


C2   ACCEPTOR >-GLYCOLLATE 

L f 

PHOTOSYNTHESIS  ^ 


O 


PI 
> 

O 

r 


Fig.  10.  Scheme  showing  the  inter-relationships  between  photo- 
synthesis and  acid,  carbohydrate,  fat  and  protein  metaboHsm. 
For  the  sake  of  clarity  many  intermediate  reactions  have 
been  omitted. 

alanine  arise  by  the  addition  of  the  -NHg  group  to  oxalo- 
acetic and  pyruvic  acids  respectively.  Serine  may  perhaps 
be  derived  from  pyruvic  acid  and,  as  mentioned  above, 


44  THE    METABOLISM    OF    ALGAE 

glycine  seems  to  be  closely  related  to  the  Cg  acceptor. 
Radioactivity  accumulates  more  slowly  in  other  amino- 
acids,  e.g.  threonine,  phenylalanine,  glutamic  acid  and 
tyrosine^^  which  are  not  so  readily  derived  from  the  im- 
mediate products  of  photosynthesis. 

That  the  first  recognizable  product  of  photosynthesis  is 
a  phosphorylated  compound  is  evidence  of  a  connexion 
between  this  process  and  the  phosphorylation  cycles  in- 
volved in  respiration  and  cell  synthesis.  It  seems  unlikely 
on  theoretical  grounds  that  the  chemical  energy  produced 
by  the  photochemical  reaction  can  first  appear  in  the  form 
of  high  energy  phosphate  linkages^'*^  and  the  phosphoryla- 
tions involved  in  photosynthesis  are  more  probably  brought 
about  by  secondary  and  purely  chemical  reactions. 

All  the  substances  that  have  so  far  been  considered  occur 
in  the  water-soluble  fractions  of  the  algae.  Considerable 
proportions  of  tracer,  however,  appear  in  the  benzene- 
soluble  and  water-insoluble  fractions  in  the  course  of  quite 
short  periods  of  photosynthesis  (Fig.  ii).  This  demon- 
strates the  speed  with  which  the  products  of  photosynthesis 
are  incorporated  in  compounds  of  high  molecular  weight. 
The  tracer  appearing  in  protein  is  probably  that  in  the 
amino-acids  mentioned  above  as  being  closely  related  to 
the  immediate  products  of  photosynthesis. 

The  radioactivity  of  the  benzene-soluble  fraction  from 
cells  which  have  been  illuminated  for  only  40  seconds  in 
the  presence  of  radioactive  carbon  dioxide  has  been  shown 
not  to  be  due  to  contamination  with  water-soluble  sub- 
stances.^^ The  chlorophylls  remain  free  from  tracer  but 
tracer  carbon  is  distributed  uniformly  between  the  un- 
saponifiable  materials,  the  saturated  and  unsaturated  fatty 
acids,  and  the  water-soluble  saponification  products. ^^ 
Fatty  acids  are  probably  synthesized  from  Cg  units  derived 
from  acetic  acid,  or  a  derivative  of  this,  most  probably 
arising  by  oxidative  decarboxylation  of  pyruvic  acid. 
Evidence  for  this  comes  from  experiments  in  which  it  has 
been  shown  that  in  the  light,  whether  carbon  dioxide  is 
present  or  not,  acetate  is  converted  by  Scenedesmtis  to  fats 
as  well  as  to  tricarboxylic  acid  cycle  intermediates.^-  Algal 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON     45 

fatty  acids,  like  all  naturally  occurring  fatty  acids,  contain 
even  numbers  of  carbon  atoms  (see  Table  7)  thus  giving 
evidence  of  their  origin  from  Cg  fragments.  Again  there  is 
the  problem  of  w^hether  the  reductions  required  in  this 
synthesis  are  photochemical  or  only  indirectly  dependent 
on  photosynthesis. 


100 


5 


10 
MINUTES 


IS 


20 


FIG.  II.  Distribution  of  radioactive  carbon  in  Scenedesnius  obliquus 
after  varying  periods  of  photosynthesis  in  the  presence  of 
carbon  dioxide  labelled  with  radioactive  carbon.  A,  lipide 
and  pigment  fraction  ;  B,  water-soluble  fraction  ;  C,  residue, 
water-insoluble  fraction  (after  ref.  114). 

Demonstration  of  the  existence  of  common  intermediates 
in  photosynthesis  and  other  metabolic  processes  leads  to  the 
enquiry  as  to  how  these  systems  interact  under  the  dynamic 
conditions  of  the  living  organism.  Much  of  the  chapters  to 
follow  are  concerned  with  this  problem  but  certain  aspects 
may  best  be  considered  at  this  point.  The  nature  of  the 
interaction  of  photosynthesis  and  respiration  deserves  par- 


46  THE    METABOLISM    OF    ALGAE  , 

ticular  consideration  because  it  is  important  whenever  it  is 
necessary  to  measure  the  true  rate  of  photosynthesis.  There 
is  a  certain  amount  of  indirect  evidence  to  show  that  the 
view  that  is  commonly  held,  i.e.  that  respiration  continues 
at  the  same  rate  during  photosynthesis  as  under  otherwise 
comparable  conditions  in  the  dark,  is  correct.  Thus,  light 
itself,  in  the  absence  of  carbon  dioxide,  has  no  effect  on  the 
rate  of  respiration  of  Chlorella.^'^  More  direct  evidence  has 
been  obtained  recently  in  experiments  in  which  oxygen  up- 
take from  air  containing  the  heavy  isotope  of  oxygen  (O^^) 
has  been  followed  while  photosynthesis  using  normal  water 
(HgO^^)  was  taking  place.^^^  In  Chlorella  it  was  found  that 
respiration  continued  at  the  same  rate  in  the  dark  and  during 
photosynthesis,  the  labelled  oxygen  being  taken  up  at  the 
same  rate  regardless  of  whether  or  not  oxygen  was  being 
produced  by  photosynthesis  at  the  same  time.  However, 
with  other  algae  low  light  intensities  such  as  produce  rates 
of  photosynthesis  only  a  few  times  as  great  as  that  of 
respiration  were  found  sufficient  to  inhibit  uptake  of  oxygen 
from  the  air  to  a  considerable  extent.  In  Anabaena  sp. 
complete  inhibition  was  observed. ^^^  Such  inhibition  may, 
however,  be  only  apparent  since  the  oxygen  produced  in 
photosynthesis  may  be  used  preferentially  for  respiration 
and  whether  or  not  this  occurs  might  well  depend  on 
minor  differences  in  cell  or  protoplasmic  structure. 

The  results  of  studies  with  radioactive  carbon  dioxide 
have  also  been  interpreted  as  showing  that  respiration  is 
inhibited  by  photosynthesis.  In  Chlorella  exposed  in  the 
dark  to  radioactive  carbon  dioxide,  iso-citric  and  glutamic 
acids,  among  substances  participating  in  or  closely  con- 
nected with  the  tricarboxylic  acid  cycle,  acquire  high 
proportions  of  tracer  but  even  after  long  periods  of  photo- 
synthesis in  the  presence  of  radioactive  carbon  dioxide 
scarcely  any  radioactivity  can  be  detected  in  these  com- 
pounds.^^' ^-  If  it  is  assumed  that  respiration  in  Chlorella 
occurs  through  the  tricarboxylic  acid  cycle,  this  suggests 
that  respiration  is  suppressed  by  photosynthesis  in  this 
organism.  However,  it  must  be  remembered  that  other 
processes   than   respiration   and  photosynthesis   occur   in 


THE    PHOTOTROPHIC    ASSIMILATION    OF    CARBON     47 

green  cells  and  that,  principally  through  a-ketoglutaric  acid 
and  glutamine,  which  forms  a  reservoir  of  amino-  groups, 
the  tricarboxylic  acid  cycle  is  also  linked  with  protein 
synthesis  and  breakdown  (see  Fig.  10).  In  the  dark  the 
tricarboxylic  acid  cycle  is  maintained  with  oxaloacetic  acid 
derived  by  carboxylation  from  the  pyruvic  acid  produced 
in  glycolysis.  Under  these  conditions  its  intermediates 
rapidly  acquire  radioactive  carbon  supplied  in  carbon 
dioxide.  In  the  light  the  carbon  dioxide  concentration  in 
the  cell  is  reduced  by  photosynthetic  fixation  and  this  car- 
boxylation is  suppressed.  The  tricarboxylic  acid  cycle  is 
then  evidently  maintained  by  deamination  of  glutamic  acid 
to  yield  a-ketoglutaric  acid,  the  ammonia  liberated,  together 
with  carbon  skeletons  provided  by  photosynthesis,  being 
used  for  protein  synthesis.  According  to  this  view  the  tri- 
carboxylic acid  cycle  of  respiration  continues  unaffected 
during  photosynthesis,  but  is  then  maintained  from  different 
sources  to  those  which  maintain  it  in  the  dark.^^^  This 
explanation  is  consistent  with  the  observation  that  a  major 
portion  of  the  insoluble  products  formed  by  algae  in  short 
periods  of  photosynthesis  is  protein  and  that  glutamic  acid 
only  acquires  appreciable  amounts  of  tracer  after  longer 
times.  ^^ 

There  is  thus  no  conclusive  evidence  to  show  that  the 
rate  of  respiration  is  altered  to  any  great  extent  when 
photosynthesis  occurs. 


CHAPTER    III 

THE    CHEMOTROPHIC   ASSIMILATION 
OF   CARBON 

Many  algae  capable  of  photosynthesis  are  also  able  to 
assimilate  substances  of  high  potential  chemical  energy  and 
certain  forms,  not  possessing  photosynthetic  pigments,  are 
absolutely  dependent  upon  such  substances.  These  sub- 
stances evidently  do  not  contribute  any  chemical  groups 
that  are  'essential'  in  the  sense  that  vitamins  are  essential 
in  animal  nutrition;  their  function  is  that  of  furnishing  a 
readily  metabolized  source  of  energy.  In  considering  this 
chemotrophic  mode  of  nutrition  it  is  again  necessar\'  to  deal 
v^dth  the  assimilation  of  carbon  and  of  energy  together. 

CHEMOLITHOTROPHISM   IN   ALGAE 

When  the  substance  from  which  it  derives  its  energy  is 
inorganic  an  organism  is  said  to  be  chefnolithotrophic  or,  in 
the  older  terminology,  chemosytithetic.  Such  organisms  are 
independent  alike  of  light  and  of  organic  substances  as 
energy  sources  and  assimilate  carbon  dioxide  by  means  of 
energy  derived  by  oxidation  of  an  inorganic  substrate. 

Examples  of  chemolithotrophy  amongst  the  algae  are  less 
abundant  than  amongst  bacteria.  The  simultaneous  absorp- 
tion of  hydrogen  and  oxygen  coupled  with  the  reduction 
of  carbon  dioxide  by  hydrogen-adapted  Scenedesmus  has 
already  been  mentioned  on  p.  30.  The  metabolism  of 
Scenedesmus  is  here  similar  to  that  of  Bacillus  pantotrophiis 
or  B.  picnoticus,  which  utilize  the  energy  released  by  the 
'oxyhydrogen'  reaction: 

O2+2H2— >2H20  +  i37  k-cal.  .  .       (8) 

for  the  reduction  of  carbon  dioxide  to  bio-organic  sub- 
stances.2^'.  ^e?  -pj^g  value  of  the  ratio  AHg/AOg  for  Scene- 
desmus   carrying    out    the    oxyhydrogen    reaction    in    the 

48 


THE    CHEMOTROPHIC    ASSIMILATION    OF    CARBON     49 

presence  of  carbon  dioxide  shows  that  for  every  two  mole- 
cules of  hydrogen  transferred  to  oxygen,  up  to  one  molecule 
is  used  for  the  reduction  of  carbon  dioxide,^*^  i.e.  the 
over-all  equation  is: 

6H2+202+COo->(CHoO)+5H20     .  .       (9) 

However,  there  is  no  evidence  that  algae  such  as  Scenedes- 
mus  carry  out  this  reaction  to  any  considerable  extent  under 
natural  conditions  and  Chlamydomonas  moewusu,  another 
species  in  which  the  oxyhydrogen  reaction  occurs,  is  unable 
to  grow  in  the  dark  with  this  reaction  as  a  source  of 
energy.i^*  Thus,  although,  as  with  photoreduction,  this 
inability  to  grow  does  not  appear  to  be  due  to  an  inherent 
unsuitability  of  the  products  of  assimilation,  these  examples 
cannot  strictly  be  classed  as  chemolithotrophic  according 
to  the  definition  given  on  p.  16. 

The  colourless  organisms  belonging  to  the  genus  Beg- 
giatoa,  to  be  found  in  sewage-contaminated  water  and 
sulphur  springs,  have  long  been  known  to  be  chemolitho- 
trophic but  have  generally  been  classed  with  the  bacteria. 
However,  in  their  morphology  and  their  characteristic 
^method  of  movement  these  organisms  correspond  with 
blue-green  algae  of  the  genus  Oscillatoria  and  there  can  be 
little  doubt  that  they  should  be  classified  as  Myxophyceae.^^^ 
Species  of  Beggiatoa  oxidize  hydrogen  sulphide  to  elemen- 
tary sulphur  which  is  deposited  within  the  cells: 

O2+2H2S— >2H20+2S  +  i26  k-cals.    .  .     (10) 

When  the  hydrogen  sulphide  is  exhausted  the  sulphur  is 
further  oxidized  to  sulphate: 

02+§S+fH20  ->  f  SO4-  -  +  itH++98  k-cal.        .     (11) 

The  energy  released  is  used  for  the  reduction  of  carbon 
dioxide  and  Beggiatoa  spp.  are  able  by  this  means  to  grow 
in  the  complete  absence  of  organic  substrates.^*"^'  ^^'^ 

Chemolithotrophy  is  obviously  analogous  to  photo- 
trophy,  from  which  it  differs  apparently  only  in  the  source 
from  which  energy  is  derived.  Few  experimental  studies 
of  the  mechanism  of  the  process  have,  however,  been  made, 
and  it  is  not  known  whether  a  fixation  cycle  similar  to  that 


50  THE    METABOLISM    OF    ALGAE 

in  photosynthetic  organisms  operates  or  how  the  energy- 
yielding  reactions  are  linked  with  the  reduction  of  carbon 
dioxide.  ^'^^ 

FACULTATIVE   CHEMO-ORGANOTROPHY 

Many  algae  possessing  photosynthetic  pigments  are  able 
to  utilize  preformed  organic  substances  and  are  thus  enabled 
to  grow  in  the  dark  or  in  the  absence  of  carbon  dioxide. 
These  are  facultative  chemo-organotrophs.  Other  algae, 
having  no  photosynthetic  pigments,  are  obligate  chemo- 
organotrophs.  It  may  be  noted  that,  although  most  experi- 
mental work  has  been  carried  out  on  the  assimilation  of 
organic  substances  from  solution,  certain  forms  included 
in  the  algae  can  assimilate  particulate  food,  i.e.  show 
holozoic  nutrition. ^^^  Sometimes,  as  in  Ochromojias^  capaci- 
ties for  photosynthesis  and  holozoic  nutrition  are  present 
in  the  same  organism. ^*^" 

There  have  been  many  investigations  in  which  the  effects 
of  organic  substances  on  the  final  amount  of  growth 
achieved  by  algae  in  culture  have  been  recorded  in  qualita- 
tive or  semi-quantitative  terms  (a  historical  review  of  early 
work  is  given  in  ref.  258).  Such  work  has  established  that 
many  algae  possessing  photosynthetic  pigments  are  able 
to  grow  in  darkness  upon  substrates  such  as  sugars, 
alcohols,  organic  acids,  amino-acids,  peptones  and  pro- 
teins. These  same  substances  also  generally  stimulate  the 
growth  of  the  algae  in  the  light.  The  biological  advantage 
of  this  ability  to  make  use  of  organic  substances  to  algae 
inhabiting  soils  and  polluted  waters  and  to  symbiotic  forms 
need  not  be  emphasized.  So  far,  investigations  of  the 
assimilation  of  organic  compounds  by  algae  have  been  con- 
fined to  those  forms  which  are  easily  obtainable  in  pure 
culture  and  very  little  information  is  available  about  chemo- 
organotrophy  in  the  larger  marine  algae  although  it  has  been 
shown  that  numerous  organic  substances,  especially  fatty 
acids  and  amino-acids,  can  serve  as  substrates  for  the 
respiration  of  several  species  of  Chlorophyceae,  Phaeo- 
phyceae  and  Rhodophyceae.^^^  Ulva  lactuca,  however,  does 
not  appear  capable  of  chemotrophic  growth.^"^^ 


THE    CHEMOTROPHIC    ASSIMILATION    OF    CARBON     51 

Hexose  sugars  and  acetic  acid  are  the  substrates  most 
generally  utilized  by  algae,  but  individual  species  differ 
considerably  in  the  substances  which  support  growth  best. 
For  example,  fructose  supports  vigorous  growth  in  the  dark 
of  Cystococcus  (Trebouxia)  sp.  but  allows  only  poor  growth 
under  the  same  conditions  of  Scenedesmus  costulatus  var. 
chlorelloides?^^^  251  'pj^g  range   of  substrates   available  to 
Nostoc  punctiforme  is  very  similar  to  that  for  members  of 
the  Chlorophyceae  except  that  this  organism  is  able  to 
assimilate  polysaccharides  such  as  starch  and  inulin  whereas 
most  green  algae  do  not  appear  able  to  do  this.^^^  Only 
glucose  among  a  large  number  of  substances  tested  suffices 
to  support  growth  in  the  dark  of  Navicula  pelliculosa.^'^^ 
Flagellates  belonging  to  the  Euglenineae,  Cryptophyceae 
and  Volvocales  grow  best  when  provided  with  acetate, 
whereas  they  grow  poorly  or  not  at  all  upon  sugars,  and 
are  hence  often  known  as  'acetate  organisms'.^^^'  ^^^  Such 
organisms  are  abundant  in  situations  in  which  the  decom- 
position of  organic  matter  liberates  fatty  acids  and  alcohols 
in  relatively  high  concentrations,  e.g.  in  water  contaminated 
with  sewage.  An  extreme  example  of  this  type  is  afforded 
by   Chlorogonium  sp.,   which   cannot   utilize   any  organic 
energy   source   other   than   acetic   acid.^^*   Other   acetate 
organisms,  however,  are  able  to  make  use  of  a  wider  variety 
of  substrates.  Euglena  gracilis  can  grow  in  the  dark  if  pro- 
vided with  a  salt  of  a  lower  fatty  acid  such  as  acetic  or 
butyric.  Other  types  of  organic  acid,  tartaric,  lactic,  pyruvic, 
succinic  and  phosphoglyceric  for  example,  are  unsuitable. 
Among  the  lower  fatty  acids  only  those  with  an  even  number 
of  carbon  atoms  and  less  than  seven  carbon  atoms  in  all 
give  good  growth.18^  It  is  to  be  noted  that  not  all  acetate 
organisms  conform  to  this  pattern.   For  example,   other 
strains  of  Euglena  gracilis  are  able  to  utilize  succinic  acid.^^^ 
The  rule  regarding  the  utilization  of  fatty  acids  is  not  of 
general   application   among   algae   since  Prototheca  zopfii 
utilizes  fatty  acids  with  even  and  odd  numbers  of  carbon 
atoms  with  equal  readiness.^^ 

Determinations  of  the  final  populations  attained  in  cul- 
tures supplied  with  organic  substances  cannot  give  much 


52 


THE    METABOLISM    OF    ALGAE 


information  about  the  manner  in  which  these  substances 
are  utiUzed.  More  useful  comparisons  of  the  effects  of 
different  substrates  are  possible  if  growth  is  followed  quanti- 
tatively throughout  the  development  of  the  cultures.  A 
model  for  investigations  of  this  sort  is  that  of  Bristol 
Roach^^^'  ^^^  in  which  the  effect  of  a  number  of  organic 
substances  on  the  growth  of  Scenedesmus  costidatus  var. 
chlorelloides  was  studied.  Under  the  culture  conditions  used 
exponential  growth  lasted  for  several  days  and  the  relative 
growth  constant  (see  Fig.  12  and  page  107)  could  be  used 
for  the  quantitative  comparison  of  growth  in  the  presence 
of  different  substances.  The  results  obtained  by  Bristol 
Roach  are  summarized  in  Table  4.  It  is  to  be  noted  that 
in  a  light  intensity  which  was  not  saturating  for  photo- 

TABLE   4 

RELATIVE    GROWTH    CONSTANTS,    EXPRESSED    AS    PERCENTAGES 
OF    THE    MAXIMUM,    CHARACTERISTIC    OF    THE    GROWTH    OF 

Scenedesmus  costulatus  var.  chlorelloides  in  the  presence 

OF    VARIOUS    SUBSTRATES 

The  light  intensity  used  was  not  saturating  for  photosynthesis 2^° 


Glucose    (light)    . 

100 

Control    (light) 

.      60 

Maltose 

ICO 

Glycerol      ,, 

•      43 

Galactose     ,, 

94 

Glucose  (dark) 

.      40 

Sucrose         ,, 

.      .        84 

jVIannitol  (light) 

•      13 

Fructose       ,, 

.      .        73 

Xylose         „ 

0 

svnthesis  both  hexoses  and  disaccharides  accelerated  growth 
but  that  certain  substances,  particularly  the  pentose  sugar 
xylose,  had  an  inhibitor^'  effect. 

It  is  generally  accepted  that  the  reactions  in  which  the 
intermediates  of  metabolism  are  involved  are  reversible 
and  that  intermediates  and  enzymes  together  form  a  com- 
plex but  flexible  system  into  which  material  can  be  intro- 
duced at  many  points  and  through  which  material  flows  in 
a  direction  determined  rather  by  the  conditions  to  which 
the  organism  is  exposed  than  by  the  nature  of  the  mech- 
anism itself.  Because  the  respiratory  system  is  intermeshed 
with  other  metabolic  systems  then  it  would  seem  that  any 
substance  which  can  serve  as  a  substrate  for  respiration  and 
so  yield  energy  ought  at  the  same  time  to  be  capable  of 


THE    CHEMOTROPHIC    ASSIMILATION    OF    CARBON      53 


6-0 


GLUCOSE  SERIES 


GALACTOSE  SERIES 


--•--     GLYCEROL    SERIES 


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J^   4-0 

_ 

X-0 

u 

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j\''                              _  • 

Uj 

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Q. 

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W             ♦-'• 

J 

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CQ 

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0   3-0 

- 

P 

X 

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o.n 

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1 

1            r             1             1            1             1 

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^/4yS   O/^   GROWTH      . 


5 


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FIG, 


12.  Growth  of  Scenedesmus  costulatus  var.  chlorelloides  in  the 
hght  in  the  presence  of  various  organic  substrates.  The 
logarithms  of  the  bulk  of  alga  per  unit  volume  of  medium 
plotted  against  time  lie  approximately  along  straight  lines, 
i.e.  growth  was  exponential  during  the  period  of  the  experi- 
ment. In  each  case  the  slope  of  the  line  is  numerically  equal 
to  the  relative  growth  constant.  Further  explanation  is  given 
on  p.  107  (data  from  ref.  250). 


54  THE    METABOLISM    OF    ALGAE 

providing  the  carbon  skeletons  necessary  for  the  synthesis 
of  further  protoplasm  and  thus  support  chemotrophic 
growth.  There  is,  in  fact,  good  evidence  that  this  is  so  in 
certain  algae.  In  a  study  of  the  metabolism  of  ProtothecUy  in 
which  about  seventy  compounds,  including  fatty  acids, 
other  organic  acids,  carbohydrates,  alcohols,  ketones  and 
nitrogen-containing  compounds  were  used,  a  close  correla- 
tion was  found  between  the  growth  that  could  be  obtained 
with  a  particular  substrate  and  the  value  of  the  same  sub- 
stance as  a  substrate  for  respiration.^^  In  a  more  limited 
investigation  with  Chlorella  a  good  correspondence  has  like- 
wise been  found  between  the  values  as  substrates  for 
respiration  and  for  growth  of  a  number  of  organic  acids. ^"'  ^^ 
It  must  be  noted,  however,  that  such  a  correlation  has  not 
been  found  in  all  algae  that  have  been  examined.  Obligate 
phototrophs,  to  be  discussed  on  p.  59,  are  able  to  oxidize 
a  number  of  substances  which  they  are  not  able  to  utilize 
as  substrates  for  chemotrophic  growth,  and,  although 
Namcula  pelliculosa  is  apparently  only  able  to  use  glucose 
for  growth  in  the  dark,  nevertheless  citrate,  acetate,  pyru- 
vate, succinate  and  lactate  are  stimulatory  to  respiration.^"^ 

If  it  is  supposed  that  any  substance  which  is  an  inter- 
mediate in  metabolism  or  readily  convertible  into  one 
should  be  utilizable  as  a  substrate  for  chemotrophic  gro\\th 
provided  that  it  is  of  sufficient  potential  chemical  energy, 
then  it  is  necessary  to  explain  how  it  is  that  some  substances 
which  are  apparently  of  this  kind  cannot  be  assimilated  by 
certain  algae  and  how  it  is  that  different  species  vary  so  con- 
siderably in  their  requirements  for  chemotrophic  growth. 

One  possibility  is  that  a  substance  which  would  otherwise 
be  metabolized  may  not  be  able  to  enter  the  cell.  This  is 
obviously  so  in  the  case  of  bulky  molecules,  such  as  those 
of  proteins  and  polysaccharides,  which  are  unable  to  pene- 
trate the  plasma  membrane  unless  they  are  first  broken 
down  into  smaller  units.  Thus  most  algae  able  to  assimilate 
glucose  are  unable  to  utilize  glucose  polymers  such  as  starch 
or  glycogen  supplied  externally  although  they  may  store 
and  utilize  the  same  materials  within  their  cells.  The  pro- 
duction of  extracellular  enzymes  by  means  of  which  such 


THE    CHEMOTROPHIC    ASSIMILATION    OF    CARBON     55 

substrates  may  be  assimilated  appears  to  be  much  less 
common  in  algae  than  in  bacteria.  Certain  algae,  Scenedes- 
mus  costulatiis  var.  chlorelloides^'^^  and  Nitzschia  putrida^^^ 
for  example,  are  nevertheless  able  to  liquefy  gelatine,  a 
property  which  must  be  due  to  the  production  of  an  extra- 
cellular proteinase  (see  also  ref.  258). 

Since  ions  penetrate  into  cells  less  readily  than  undis- 
sociated  molecules  the  reaction  of  the  medium  is  of  great 
importance  in  determining  whether  or  not  ionizable  sub- 
stances can  be  assimilated.  Thus  all  but  the  weakest  organic 
acids  exist  almost  entirely  as  ions  in  alkaline  solution  and 
are  then  unable  to  penetrate  into  cells,  whereas  penetration 
of  the  same  acids  may  occur  readily  from  acid  solutions,  in 
which  the  undissociated  molecules  predominate.  If  this  is 
overlooked  then  it  may  appear  that  an  organic  acid  cannot 
be  utilized  for  chemotrophic  growth.  For  example,  from 
the  results  of  experiments  carried  out  at  pH  5-0  to  5-5  it 
was  concluded  that  pyruvic  acid  could  not  be  assimilated 
by  Prototheca  zopfii^^  but  further  work  using  media  adjusted 
to  pH  3-0  to  4-5  showed  that  this  substance  can  be  meta- 
bolized rapidly  by  this  organism. ^^  Similarly,  assimilation 
of  other  acids  known  to  be  intermediates  in  metabolism  can 
generally  be  demonstrated  if  conditions  are  adjusted  to  give 
a  sufficient  concentration  of  the  undissociated  form  in  the 
medium. 1^^  The  situation  may  be  further  complicated  if 
the  concentration  of  a  particular  acid  necessary  to  ensure 
adequate  penetration  reaches  the  toxic  level.  Ability  to 
resist  high  concentrations  of  free  fatty  acids  is  evidently  the 
important  characteristic  of  acetate  organisms.  Acetic  acid 
may  play  just  as  important  a  part  in  the  metabolism  of  other 
algae  as  it  does  in  that  of  acetate  organisms,  but  the  high 
concentrations  of  the  free  acid  which  the  latter  are  able  to 
tolerate  are  toxic  to  these  other  forms. ^^^ 

Another  possibility  is  that  an  enzyme,  necessary  for  the 
utilization  of  a  particular  substrate  and  regarded  as  of 
general  occurrence,  may  nevertheless  be  absent  from  a  par- 
ticular species.  The  clearest  example  of  this  is  the  inability 
of  certain  acetate  organisms  to  utilize  glucose  and  other 
sugars,  which  results  from  their  lack  of  hexokinase,  the 
5 


56  THE    METABOLISM    OF    ALGAE 

enzyme  responsible  for  the  phosphorylation  of  glucose  and 
fructose.^®®'  ^'  ^^^'  ^^  Polytornella  caeca,  for  instance,  stores 
a  starch-like  substance  which  it  is  able  to  synthesize  from 
glucose- 1 -phosphate  but  not  from  glucose,  maltose  or 
sucrose. ^^^'  ^^  Whereas  the  hexose  phosphates  are  inter- 
mediates in  metabolism,  the  sugars  themselves  are  not  and 
the  absence  of  the  enzymes  necessary  for  their  phosphoryla- 
tion does  not  impair  metabolic  activity  although  it  prevents 
direct  utilization  of  these  substrates. 

It  is  unlikely  that  all  the  vagaries  of  algae  in  the  assimila- 
tion of  organic  substrates  can  be  accounted  for  along  these 
lines.  The  metabolic  system  of  an  organism  is  not  infinitely 
adaptable  and  the  adjustment  of  the  proportions  of  the 
different  enzymes  necessary  to  cope  with  a  particular  sub- 
strate may  in  a  given  species  not  be  compatible  with  the 
general  economy  of  the  cell.  The  limits  within  which  adjust- 
ment is  possible  probably  vary  considerably  from  species  to 
species  and  may  be  expected  to  be  narrower  the  greater  the 
morphological  complexity  of  the  organism.  Adaptation  in 
bacteria  has  received  considerable  attention  and  it  has  been 
shown  that,  within  limits,  an  organism  may  be  trained  to 
attain  maximum  growth  rate  on  a  substrate  which  the 
original  material  was  unable  to  utilize.^*^  A  few  cases  of 
similar  adaptation  are  known  among  algae.  For  example, 
Scenedesmus  costulatus  var.  chlorelloides  exhibits  a  lag  period 
before  beginning  exponential  growth  with  maltose  as  a  sub- 
strate whereas  no  such  lag  is  shown  in  media  containing 
glucose,  sucrose  or  glycerol.  This  lag  period  may  be  inter- 
preted as  the  period  needed  for  the  development  of  maltase 
necessary  for  the  conversion  of  this  sugar  to  glucose. ^^*^  A 
further  example  is  afforded  by  a  strain  of  Chlorella  vulgaris 
which  has  been  found  to  grow  slowly  at  first  in  the  presence 
of  cellobiose  in  the  light  but,  after  several  transfers  in  the 
light  in  a  medium  containing  this  substance,  to  grow  almost 
as  rapidly  as  it  does  under  otherwise  comparable  conditions 
when  supplied  with  glucose. ^'^'^  Adaptation  evidently  is 
dependent  upon  suitable  environmental  conditions.  Thus 
Eugletia  gracilis  cannot  utilize  glucose  for  grovvth  under  the 
usual  conditions  of  culture,  but  in  an  atmosphere  containing 


THE    CHEMOTROPHIC    ASSIMILATION    OF    CARBON     57 

5  per  cent  of  carbon  dioxide  and  with  an  ammonium  salt 
as  the  nitrogen  source  it  attains  excellent  growth  upon  this 
substrate  after  a  lag  phase. ^^^  Certain  substances  appear 
sometimes  to  be  utilized  more  readily  in  the  dark  than  in 
the  light.  The  strain  of  Chlorella  vulgaris  mentioned  above, 
for  example,  grows  better  in  the  dark  than  in  the  light  when 
provided  with  lactose,  cellobiose  or  methyl-j5-D-glucoside, 
as  substrate.  These  three  compounds  are  all  /5-glucosides 
and  it  appears  that  adaptive  enzymes  are  formed  for  their 
utilization  and  that  the  rate  of  adaptation  is  slower  in  the 
light  when  the  photosynthetic  products  are  available  as  an 
alternative  source  of  carbon."^^' 

THE   RELATIONSHIP   BETWEEN   PHOTOSYNTHESIS   AND 
CHEMOTROPHIC   ASSIMILATION 

In  an  organism  which  is  capable  of  both,  phototrophic 
and  chemotrophic  assimilations  proceed  at  rates  which  are 
mutually  dependent.  For  Scenedesmus  costulatus  var.  Morel- 
hides  it  has  been  found  that  at  high  light  intensities  a 
maximum  rate  of  growth  is  attained  which  cannot  be  in- 
creased by  the  addition  of  glucose.-^^  With  reduced  light 
intensity  the  alga  absorbs  glucose  to  supply  the  deficiency 
due  to  retarded  photosynthesis  but  only  sufficiently  to  bring 
the  growth  rate  up  to  the  maximum.  At  still  lower  light 
intensities  the  growth  rate  falls,  approaching  the  value 
attained  in  complete  darkness  on  glucose  (see  Table  4). 
The  same  relationships  have  been  demonstrated  in  Chlorella 
pyrenoidosar^^  There  are  many  reports  of  the  growth  of 
algae  in  light  being  accelerated  by  the  addition  of  organic 
substrates  to  the  medium,  but  in  none  of  these  instances 
is  there  satisfactory  evidence  that  the  provision  of  carbon 
dioxide  was  sufficient  to  maintain  maximum  rates  of  photo- 
synthesis. No  case  has  yet  been  found  in  which  an  organic 
carbon  source  accelerated  growth  under  conditions  of  light- 
and  carbon  dioxide-saturation  of  photosynthesis. ^^^  It  thus 
appears  that  the  photosynthetic  mechanism  is  generally 
capable  of  saturating  with  its  products  the  synthetic  systems 
involved  in  growth  so  that  under  optimum  conditions  for 


58  THE    METABOLISM    OF    ALGAE 

photosynthesis  some  other  factor  than  carbon  assimilation 
limits  the  rate  of  growth. 

It  is  possible  that  light  may  have  effects  on  growth  other 
than  those  arising  from  photosynthesis.  If  Chlorella  vulgaris 
is  grown  in  the  presence  of  glucose  it  is  found  that  a  weak 
light  intensity  produces  a  considerable  increase  in  the  rate 
of  growth  in  the  exponential  phase  as  compared  with  that 
in  darkness,  but  that  further  increase  in  light  intensity  pro- 
duces comparatively  little  effect  even  though  carbon  dioxide 
is  not  in  short  supply.^^^  These  effects  may  be  indirectly 
due  to  photosynthesis  since  in  cultures  of  the  type  used  to 
obtain  these  results  oxygen  is  limiting  and  the  amount  of 
growth  that  can  take  place  in  older  cultures  is  dependent 
on  the  oxygen  evolved  in  photosynthesis.  In  the  exponential 
phase  the  medium  initially  contains  oxygen  and  it  may  be 
that  the  small  amounts  produced  by  photosynthesis  at  the 
lowest  light  intensities  are  sufficient  to  supplement  this  to 
a  level  at  which  the  maximum  growth  rate  can  be  main- 
tained.^^^  However,  the  effect  of  light  on  the  growth  rate  in 
the  exponential  phase  may  equally  well  be  explained  in 
other  ways.    For   example,   a   substance   which   in   small 
amounts  stimulates  growth  may  be  produced  by  a  photo- 
chemical reaction  other  than  photosynthesis.  Evidence  for 
such  an  effect  may  be  obtained  from  studies  of  the  effect 
of  light  upon  the  growth  of  cultures  in  the  absence  of 
carbon    dioxide.    For    Chlamydomonas  pseudococcum    and 
Hormidium  nitens  it  has  been  found  that  cultures  aerated 
with  carbon  dioxide-free  air  grow  better  in  the  light  than 
in  complete  darkness. ^^^  However,  really  vigorous  aeration 
is  needed  to  remove  from  a  culture  the  carbon  dioxide  pro- 
duced by  respiration  and  it  seems  probable  that  in  these 
experiments  a  small  amount   of  photosynthesis  occurred 
and  that  this  might  account  for  the  better  growth  of  the 
light  cultures. 2^^  Experiments  with  Chlorella  pyrenoidosa 
grown  in  cultures  vigorously  aerated  with  carbon  dioxide- 
free  air  have  failed  to  show  any  stimulatory  effect  of  light 
on  the  rate  of  growth  with  glucose  or  acetate  as  substrate. 
On  the  contrary,  light  retards  the  growth  of  Chlorella  on 
acetate  in  the  absence  of  carbon  dioxide.^^^  Thus  there 


THE    CHEMOTROPHIC    ASSIMILATION    OF    CARBON     59 

appears  to  be  no  conclusive  evidence  for  a  stimulatory  effect 
of  light  upon  the  growth  of  algae  apart  from  that  due  to 
photosynthesis.  However,  photochemical  reactions  other 
than  those  concerned  in  photosynthesis  are  known  to  occur 
in  plants,  e.g.  the  formation  of  chlorophyll  in  higher  plants 
involves  such  a  process,  and  it  is  possible  that  when  a  greater 
range  of  forms  has  been  examined  examples  of  these  will 
be  found  among  the  algae. 

OBLIGATE   PHOTOTROPHY 

From  the  account  that  has  been  given  above  it  would  be 
expected  that  all  photosynthetic  algae  would  also  be  capable 
of  chemo-organotrophic  nutrition.  However,  although 
many  algae  are  able  to  grow  in  the  dark  if  provided  with  a 
suitable  organic  substrate,  there  are  some  which  have  not 
so  far  been  grown  under  these  conditions  and  which  appear 
to  be  obligate  phototrophs.  A  strain  of  Chlorella  vulgaris, ^^ 
Chlamydomonas  spp.,^'^*  Prorocentrum  micans,  Peridinium 
sp.,^^  Anahaena  cylindrica,^^  and  perhaps  the  majority  of 
diatoms, 2*^'  ^'^  for  example,  are  apparently  of  this  type.  An 
analogous  situation  is  met  with  in  the  chemolithotrophic  1 
bacterium  Thiohacillus  thio-oxidans  in  which  sulphur  and 
carbon  dioxide  are  required  for  growth  and  can  be  replaced 
by  no  other  energy  or  carbon  source  respectively.^®^  The 
occurrence  of  obligate  phototrophism  is  evidently  sporadic 
as  far  as  systematic  position  is  concerned. 

Most  information  about  obligate  phototrophism  is  avail-  J 
able  for  Chlamydomonas  moewusii}'^^  Some  sixty-four  | 
organic  materials,  including  acids,  alcohols,  sugars,  phos- 
phate compounds  (including  phosphoglyceric  acid),  nitro- 
gen compounds  and  preparations  rich  in  growth  factors, 
have  been  tested  and  found  ineffective  as  substrates  for  the 
chemo-organotrophic  growth  of  this  flagellate.  Cell  extracts 
and  hydrolysates  of  the  organism  itself  and  filtrates  from 
light-grown  cultures  likewise  do  not  support  growth  in 
darkness.  A  most  curious  feature  is  that  whereas  control 
cultures  without  substrate  grow  on  being  returned  to  the 
light,  cultures  with  substrates  are  found  to  be  dead  although 
the  substrate  concentrations  used  are  not  toxic  in  light 


6o  THE    METABOLISM    OF    ALGAE 

cultures. ^^^  No  toxic  substance  inhibitory  to  the  organism 
when  growing  in  the  light  has  been  demonstrated  as  being 
produced  in  the  dark.^"^*  In  the  absence  of  carbon  dioxide 
no  growth  has  been  observed  on  any  substrates  either  in 
light  or  darkness;  this  seems  to  show  that  the  obligate 
phototrophy  does  not  in  this  case  depend  on  a  photo- 
chemical reaction  other  than  that  of  photosynthesis. i"^*  It 
has  already  been  noted  (p.  49)  that  a  chemolithotrophic 
mechanism  cannot  replace  photosynthesis  in  this  species. 

It  does  not  seem  that  substances  that  would  otherwise  be 
suitable  substrates  for  growth  are  unable  to  penetrate  into 
the  cells  of  C.  moewusii  since  acetate,  pyruvate  and  succinate 
are  readily  oxidized  by  the  organism  in  the  dark.^^*  Thus 
there  is  here  no  correlation  between  the  value  of  a  substance 
as  a  substrate  for  respiration  and  its  ability  to  support 
growth  such  as  has  been  noted  for  Prototheca  and  Chlorella 
(p.  54).  It  appears,  then,  that  energy  released  by  oxidation 
of  organic  substrates  is  not  available  for  at  least  one  syn- 
thetic mechanism  essential  for  the  growth  of  C.  moewusii. 
The  behaviour  of  an  ultra-violet  induced  mutant  of  C. 
moewusii,  in  which  the  photosynthetic  apparatus  is  im- 
paired,^ ^^  is  interesting  in  this  connexion.  This  mutant  is 
incapable  of  growth  in  purely  inorganic  media  unless  a  high 
concentration  of  carbon  dioxide  is  maintained,  e.g.  by 
aeration  with  5  per  cent  carbon  dioxide  in  air,  but  it  will 
grow  in  the  light  at  low  carbon  dioxide  tensions  in  media 
supplemented  with  substances  such  as  citrate,  fumarate, 
succinate,  pyruvate,  malate,  glucose  or  glycerol,  at  rates 
approaching  those  attained  by  the  wild  type.  The  mutant 
is  able  to  carry  out  the  Hill  reaction^^'  and  it  may  be  that 
the  part  of  the  photosynthetic  mechanism  which  is  impaired 
is  that  responsible  for  maintaining  the  concentration  of  the 
particular  form  of  carbon  dioxide  which  enters  into  the 
fixation  cycle,  e.g.  the  enzyme  concerned  might  be  carbonic 
anhydrase.  If  this  is  so,  then  the  supplementary  carbon 
sources  utilized  by  this  mutant  may  act  merely  by  being 
oxidized  to  give  carbon  dioxide  in  a  form  in  which  it  can 
be  fixed  photosynthetically  without  the  intervention  of  this 
system.  Another  possibility  is  that  oxidation  of  exogenous 


i 


THE    CHEMOTROPHIC    ASSIMILATION    OF    CARBON     6l 

substrates  can  be  used  as  a  supplementary  source  of  energy 
for  growth,  the  capacity  for  photosynthesis  of  the  mutant 
being  then  sufficient  for  an  essential  step  that  in  obligate 
phototrophs  is  specifically  associated  with  the  photochemical 
reaction  but  which  in  facultative  chemo-autotrophs  can 
be  by-passed  by  dark  reactions. 

It  may  be  noted  that  Euglena  gracilis,  although  a  faculta- 
tive chemo-organotroph,  responds  sluggishly  to  added 
organic  substrates  in  the  dark  after  having  been  cultured 
phototrophically.^^  Possibly  this  condition  represents  a 
stage  in  the  development  of  obligate  phototrophy. 

OBLIGATE   CHEMO-ORGANOTROPHY 

The  strains  of  Chlorella  generally  used  in  experimental 
work  are  facultative  chemo-organotrophs  which  during 
growth  in  darkness  produce  pigments  that  are  qualitatively 
the  same  as  those  produced  in  the  light  and  which  remain 
capable  of  beginning  photosynthesis  without  any  markedly 
prolonged  induction  period  on  exposure  to  light.^**'  ^^^  In 
these  cases  the  photosynthetic  mechanism  is  stable.  Chlor- 
ella variegata,  however,  if  cultured  in  the  presence  of 
organic  substrates  in  the  dark  becomes  yellow  and  only 
slowly  recovers  its  capacity  for  phototrophic  growth  when 
returned  to  the  light.  Some  cells  may  become  white  and 
in  them  the  capacity  for  producing  chlorophyll  is  irrevoc- 
ably lost.2'  Similarly,  Euglena  gracilis,  if  cultured  in  the 
dark  loses  chlorophyll  and  on  return  to  the  light  slowly 
becomes  photosynthetic  once  more.^^^  With  certain  strains 
of  this  species  completely  colourless  individuals  arise  during 
culture  in  the  dark  and  these  do  not  regain  their  green 
colour  in  the  light  and  are  thus  permanently  chemo- 
organotrophic.^*^ 

The  loss  of  photosynthetic  pigments  which  may  occur 
when  algae  are  cultured  in  the  dark  is  reversible  so  long  as 
leucoplasts,  i.e.  the  decolorized  chromatophores,  are  re- 
tained by  the  cells.  Irreversible  loss  of  pigments,  or  apo~ 
chlorosis,  takes  place  when  a  cell  containing  no  leucoplasts 
is  produced.  Production  of  such  cells  does  not  depend  on 
the  fortuitous  exclusion  of  leucoplasts  from  one  of  the 


62  THE    METABOLISM    OF    ALGAE 

products  in  cell  division  but  is  due  to  disorganization  of 
the  leucoplast  stroma  while  the  cell  remains  capable  of 
growth  and  division. -^^  This  disorganization  has  the  appear- 
ance of  resulting  from  a  gene-mutation.^'^'  ^*^ 

Spontaneous  apochlorosis  has  been  noted  only  rarely, 
but  chlorophyll-less  forms  have  been  induced  in  Chlorella 
vulgaris  by  irradiation  with  X-rays^^®  and  in  Euglena 
gracilis  by  treatment  with  streptomycin  or  by  exposure  to 
high  temperatures. 2'*^'  ^^®'  ^^^  Streptomycin  appears  to  be 
unique  among  antibiotic  substances  in  exerting  this  effect. ^^^ 
Streptomycin-induced  apochlorosis,  like  that  which  occurs 
spontaneously,  results  from  disintegration  of  the  chroma- 
tophores. 

There  exists  in  nature  a  large  number  of  colourless  and 
therefore  obligatorily  chemotrophic  forms  which  can  readily 
be  recognized  as  related  to  pigmented  algae  because  of  simi- 
larities in  mdphology  and  in  storage  products. ^^^  These 
forms  have  apparently  been  derived  from  pigmented  species 
by  processes  similar  to  those  investigated  in  laboratory  ex- 
periments. Since  streptomycin-producing  actinomycetes  are 
common  in  soils  it  is  possible  that  this  substance  may  play 
an  important  part  in  inducing  apochlorosis  in  nature.  The 
pigmented  species  concerned  are  readily  able  to  utilize 
organic  substrates  so  that  colourless  forms  derived  from 
them  would  evidently  stand  a  good  chance  of  survival  under 
natural  conditions.  Only  a  few  of  the  colourless  algae  that 
have  been  described  can  be  mentioned  here.  Species  of  the 
genus  Astasia  are  similar,  except  for  the  absence  of  pig- 
ments, to  species  of  Euglena.  A.  longa  in  fact,  is  identical 
with  a  colourless  strain  of  E.  gracilis  obtained  in  culture 
experiments  and  has  been  renamed  E.  gracilis  forma 
hyalina.^^^  Polytoma  spp.  resemble  Chlamydomoiias  spp. 
except  in  the  matter  of  pigmentation  and  similarly  Poly- 
tomella  corresponds  to  Tetrachloris.'^^^  Prototheca  is  evi- 
dently a  colourless  Chlorella}'^ ^  ^^^  Colourless  species  have 
been  assigned  to  nearly  every  class  of  algae  with  unicellular 
representatives  (see  refs.  io6,  240,  243,  244).  Besides  those 
obviously  related  to  pigmented  species  there  is  a  consider- 
able number  of  forms  which  may  have  had  an  algal  origin 


THE    CHEMOTROPHIC    ASSIMILATION    OF    CARBON      63 

but  in  which  scarcely  any  indication  of  affinity  is  to  be 
found. 2*^  Among  the  morphologically  more  complex  algae 
certain  parasitic  species  of  Rhodophyceae,  e.g.  Harveyella, 
show  complete  absence  of  photosynthetic  pigments. ^°^ 

There  is  no  evidence  of  any  significant  alteration  in 
gro\\th  or  dark  metabolism  occurring  in  apochlorosis.  The 
requirements  for  chemotrophic  nutrition  and  the  storage 
products  are  the  same  in  corresponding  colourless  and  pig- 
mented forms. 240,  242 

OXIDATIVE   ASSIMILATION 

The  organic  substrate  used  in  chemo-organotrophic 
growth  must  serve  as  a  source  both  of  energy  and  of  carbon 
and  therefore  is  partly  oxidized  in  respiration  and  partly 
built  up  into  cell  material.  The  mechanism  of  such  oxidative 
assimilation  of  organic  substances  must  now  be  considered. 

Oxidative  assimilation  was  first  studied  in  ProtothecaP^  ^3 
Under  aerobic  conditions  the  supply  of  glucose  to  this 
organism  stimulates  respiration,  but  the  amount  of  oxygen 
consumed  for  a  given  amount  of  glucose  is  sufficient  to 
account  for  complete  oxidation  to  carbon  dioxide  and  water 
of  only  part  of  the  glucose,  the  rest,  about  30  per  cent, 
apparently  being  converted  to  cell  material.  Other  sub- 
stances, including  glycerol,  ethyl  alcohol,  acetic  acid  and 
propionic  acid,  can  be  partly  oxidized  and  partly  assimilated 
by  Prototheca  in  a  similar  manner.  Under  anaerobic  con- 
ditions glucose  cannot  be  used  for  building  cell  material 
but  is  fermented,  yielding  lactic  acid  in  almost  exactly  the 
amount  required  by  the  equation: 

CeHi206->2CH3.CH(OH).COOH    .  .     (12) 

Oxidative  assimilation  has  also  been  investigated  in  Chlor- 
ella  pyrenoidosa,^^^  C.  vulgaris^"^  and  Scmedesmus  quadri- 
cauda.^^^ 

In  investigations  with  Prototheca  the  amounts  of  sub- 
strate decomposed  and  oxygen  consumed  have  been  found 
to  be  in  agreement  with  the  hypothesis  that  a  carbohydrate 
is  the  principal  product  of  oxidative  assimilation,  being 
formed  according  to  a  simple  over-all  equation  such  as: 

CH3.COOH+02->(CH20)  +  C02+H20  .     (13) 


64  THE    METABOLISM    OF    ALGAE  j 

in  the  case  of  acetic  acid.^^  Similarly,  results  have  been 
obtained  in  experiments  on  the  oxidative  assimilation  of 
glucose  by  Chlorella  pyrenoidosa  in  accord  with  the  follow- 
ing equation:^^^ 

C6Hio06+02->5(CHoO)+C02+H20    .        .     (14) 

These  equations,  implying  a  definite  relationship  between 
oxidation  and  assimilation,  suggest  that  the  nature  of  the 
intermediate  products  of  the  decomposition  of  the  substrate 
may  be  more  important  in  determining  the  proportion 
which  is  oxidized  than  the  energy  relationships  involved.^* 

However,  the  proportion  of  substrate  which  is  oxidized 
is  not  as  fixed  as  these  results  suggest.  Not  only  does  the 
proportion  vary  from  species  to  species  but  it  may  alter  in 
the  same  organism  according  to  its  physiological  condition^* 
or  the  reaction  of  the  medium.^^i  If  it  is  supposed  that  a 
substrate  for  oxidative  assimilation  is  first  converted  into 
fragments  which  can  enter  the  metabolic  pool  then  in  most 
cases  these  fragments  will  be  of  one  sort  and  the  propor- 
tions oxidized  and  used  for  synthesis  will  depend  on  energy 
requirements  and  the  conditions  to  which  the  organism  is 
exposed.  For  example,  it  is  to  be  expected  that  in  the  oxida- 
tive assimilation  of  glucose  the  normal  glycolytic  pathway 
wall  be  followed  and  that  this  substance  after  phosphoryla- 
tion to  yield  hexose  phosphates  may  then  be  used  in  poly- 
saccharide synthesis  or  be  further  degraded  and  enter  the 
metabolic  pool  as  phosphoglycerate.  The  various  pathways 
by  which  phosphoglycerate  may  be  metabolized  are  indi- 
cated in  Fig.  10.  There  is  no  evidence  that  glucose  is 
assimilated  by  algae  in  any  other  way;  the  possibility  sug- 
gested by  equation  14,  for  example,  that  in  oxidative 
assimilation  it  is  converted  to  pentose  by  oxidation  and 
decarboxylation  receives  no  support  from  analytical  data.^^i 

A  further  example  that  may  usefully  be  discussed  is  that 
of  acetate,  a  substrate  which,  as  we  have  already  seen,  is 
the  most  generally  utilized  by  algae.  There  are  several  pos- 
sible ways  in  which  this  substance  might  enter  into  meta- 
bolism. Acetate,  by  reductive  carboxylation,  might  yield 
pyruvic  acid.  However,  the  oxidative  decarboxylation  of 


THE    CHEMOTROPHIC    ASSIMILATION    OF    CARBON     65 

pyruvic  acid  appeairs  to  be  an  irreversible  process  and  it  has 
been  found  that,  whereas  the  oxidation  of  glucose,  glycerol 
and  pyruvate,  by  thiamine-deficient  Prototheca  is  markedly 
stimulated  by  the  addition  of  thiamine  (which  is  necessary 
for  the  carboxylation  and  decarboxylation  of  pyruvic  acid, 
see  p.  9),  that  of  acetate  is  stimulated  to  a  much  smaller 
extent.  This  shows  that  pyruvate  is  not  an  intermediate  in 
the  oxidation  of  acetate  as  it  is  in  the  cases  of  glucose  and 
glycerol.^*  Another  possible  pathway,  involving  glycollic 
acid,  CHgOH.COOH,  as  an  intermediate,  seems  unlikely 
because  glycoUate  has  a  catalytic  effect  on  the  respiration 
of  Prototheca  whereas  acetate  does  not.^*  In  yeast,  acetate 
has  been  found  to  undergo  condensation  with  oxaloacetate 
to  form  citric  acid: 

CH3.COOH      CH2.COOH 

+  I 

CO.COOH  — >HO.C.COOH     .    .  (15) 

I  I 

CH2.COOH       CH2.COOH 

Acetic  acid  seems  not  to  take  part  directly  in  this  reaction 
but  must  first  be  converted  to  'activated  acetate',  probably 
an  acetylcoenzyme  A.^^  It  has  not  been  established  that 
acetate  is  oxidized  by  yeast  through  the  tricarboxylic  acid 
cycle  but  that  this  may  take  place  in  Scenedesmus  is  sug- 
gested by  the  appearance  of  radioactivity  in  citric  acid  and 
other  tricarboxylic  acid  cycle  intermediates  following  supply 
of  acetate  labelled  with  radioactive  carbon.  ^^  This  mech- 
anism can,  however,  only  account  for  the  oxidation  of 
acetate  since  the  operation  of  the  cycle  will  result  in  the 
liberation  as  carbon  dioxide  of  the  two  carbon  atoms  of 
acetate  and  the  regeneration  of  the  original  oxaloacetate 
(Fig.  2).  Assimilation  to  more  complex  substances  must 
follow  other  pathways.  In  certain  species  and  under  certain 
conditions  at  least,  e.g.  in  Scenedesmus  in  the  light,  acetate 
is  most  readily  assimilated  to  fats,^^  a  synthesis  that  most 
probably  takes  place  directly  from  'activated  acetate'  by 
successive  condensations  and  reductions. ^^'  ^^  Carbohydrate 
could  be  derived  from  acetate  if  this  were  first  transformed 


66  THE    METABOLISM    OF    ALGAE 

to  pyruvate.  The  condensation  of  two  molecules  of  acetic 
acid  to  give  succinic  acid: 

CH3.COOH    _,H     CHo.COOH 

+  .  I  .         .    (16) 

CH3.COOH      +-H      CH2.COOH 

has  been  demonstrated  in  certain  micro-organisms^®^'  ^^ 
and  if  a  similar  reaction  occurs  in  algae  pyruvic  acid  could 
be  formed  via  oxaloacetate  produced  by  the  operation  of 
the  tricarboxylic  acid  cycle  (Fig.  10).  It  is  to  be  noted  that 
the  effects  observed  in  thiamine-deficient  Prototheca  (see 
above)  do  not  exclude  the  possibility  that  assimilation  of 
acetate  occurs  in  this  way. 

Some  indication  of  the  manner  in  which  oxidation  and 
assimilation  are  related  is  given  by  the  effects  upon  them 
of  cyanide. ^^'*  In  Chlorella  vulgaris  low  concentrations  of 
cyanide  have  been  found  to  stimulate  endogenous  respira- 
tion. In  the  absence  of  cyanide  one-eighth  to  one-ninth  of 
added  glucose  may  be  completely  oxidized,  most  of  the 
remainder  being  assimilated  to  di-  or  polysaccharide.  Low 
concentrations  of  cyanide  reduce  the  rate  of  oxidation  of 
added  glucose  to  a  level  which  does  not,  however,  fall 
below  that  of  endogenous  respiration  under  the  same  con- 
ditions, but  at  the  same  time  produce  an  increase  in  the 
amount  of  oxygen  taken  up  for  a  given  amount  of  this 
substance.  That  is,  in  the  presence  of  low  cyanide  concen- 
trations more  of  the  added  glucose  is  oxidized  and  less  is 
synthesized  to  more  complex  substances.  The  facts  seem 
best  explained  on  the  assumption  that  glucose  may  be 
oxidized  by  either  of  two  mechanisms,  one  of  which  is  un- 
affected, the  other  blocked,  by  cyanide.  The  assimilation 
of  glucose  appears  to  be  coupled  with  the  cyanide-sensitive 
system  in  such  a  way  that  for  every  molecule  which  is  oxi- 
dized by  this  path  a  certain  fixed  number  of  molecules  are 
synthesized  to  more  complex  substances. ^^^  This  cyanide- 
sensitive  system  is  presumably  one  involving  a  cyto- 
chrome, evidence  for  the  presence  of  which  in  Chlorella 
has  already  been  mentioned  (p.  10),  with  a  phosphorylation 
cycle  as  a  means  of  energy  transfer  between  the  reactions 
of  oxidation  and  synthesis. 


A 


THE    CHEMOTROPHIC    ASSIMILATION    OF    CARBON     67 

Under  the  conditions  of  carbohydrate  deficiency  usually 
used  in  experimental  studies  of  oxidative  assimilation  the 
products  formed  by  Chlorella  and  Scenedesmus  are  chiefly 
polysaccharides,  the  exact  nature  of  which  has  not  been 
established. 2^^'  ^si  Xhese  products  may  subsequently  be 
broken  down  to  provide  for  the  synthesis  of  other  cell 
materials. 22  Under  other  circumstances  oxidative  assimila- 
tion presumably  results  directly  in  the  production  of 
lipides  or  proteins. 


CHAPTER    IV 

AUTOTROPHIC   ASSIMILATION   WITH 

SPECIAL   REFERENCE   TO   NITROGEN 

METABOLISM 

Given  a  suitable  source  of  energy  and  of  carbon,  auto- 
trophic organisms  are  able  to  synthesize  all  their  cell  con- 
stituents from  inorganic  materials.  Something  has  already 
been  said  of  the  synthesis  of  carbon  compounds  and  this 
chapter  must  be  chiefly  concerned  with  the  autotrophic 
assimilation  of  nitrogen.  This  emphasis  is  justified  since  the 
most  characteristic  components  of  living  matter  are  nitro- 
genous and  since  departure  from  the  autotrophic  mode  of 
life  is  most  frequently  manifest  as  an  inability  to  synthesize 
some  essential  nitrogenous  metabolite. 

Nitrogen  may  be  assimilated  by  autotrophic  algae  as  the 
element,  as  nitrate  or  as  ammonia. 

NITROGEN   FIXATION   BY   ALGAE 

The  ability  to  assimilate,  or  'fix',  the  elementary  nitrogen 
of  the  atmosphere  is  restricted  to  a  comparatively  few  organ- 
isms and  has  as  yet  been  demonstrated  with  certainty  only 
in  certain  bacteria,  i.e.  Azotobacter  spp.,  Clostridium  spp., 
Rhizohium  spp.  in  symbiotic  association  with  leguminous 
plants,  and  some  photosynthetic  bacteria,  and  in  certain 
Myxophyceae.-^^'  ^^^^ 

That  some  algae  are  able  to  fix  nitrogen  was  first  reported 
by  Frank  in  1889,^°^  four  years  before  the  first  isolation  of  a 
nitrogen-fixing  bacterium  was  announced  by  Winogradsky. 
Frank's  cultures  were,  however,  impure  and  must  have 
contained  numerous  organisms  other  than  algae  to  which 
the  observed  nitrogen  fi^xation  might  be  attributed.  The 
isolation  of  blue-green  algae  in  pure  culture  is  not  easy  and 
it  was  not  until  1928  that  satisfactory  evidence  of  nitrogen 

68 


AUTOTROPHIC    ASSIMILATION  69 

fixation  by  these  organisms  was  published."^  In  the  many 
subsequent  studies  in  which  a  capacity  for  nitrogen  fixation 
has  been  demonstrated  in  Myxophyceae  the  Kjeldahl 
method  has  generally  been  used  for  the  determination  of 
combined  nitrogen  but  nitrogen  fixation  has  also  been 
established  for  Nostoc  muscorum^^  and  Calothrix  parietina^^^ 
using  the  heavy  isotope  of  nitrogen  as  a  tracer. 

There  is  now  satisfactory  evidence  of  a  capacity  for  nitro- 
gen fixation  in  some  twenty-one  species  distributed  among 
the  following  genera  of  Myxophyceae:  Nostoc,  Anahaena, 
Cylindrospermum,  Aulosira  (for  references  see  89),  Calo- 
thrix,'^^''^  ^^^  Tolypothrix,  Anabaeniopsis,^^'^  Mastigocladus.^^ 
Further  research  will  no  doubt  reveal  many  more  nitrogen- 
fixing  species  in  this  class  but  it  is  certain  that  not  all  blue- 
green  algae  possess  the  property.  Evidence  that  species 
belonging  to  the  Chroococcales  and  to  the  Oscillatoriaceae 
are  nitrogen-fixing  is  unsatisfactory^^  and  certain  forms, 
e.g.  Phormidium  spp.,^®'  ^^^  Gloeocapsa  spp.,  Microcystis 
(Diplocystis)  aeruginosa  and  Aphanizomenon  flos-aquae,^^ 
have  been  found  to  be  incapable  of  growth  in  media  free 
from  combined  nitrogen. 

Work  with  pure  cultures  of  members  of  the  Chloro- 
phyceae  has  generally  shown  that  these  algae  cannot  fix 
nitrogen.  A  report  that  a  number  of  species  of  unicellular 
green  algae,  including  a  Chlorella,  possess  the  property^®^ 
has  been  shown  to  be  based  on  results  obtained  with  an 
unsuitable  analytical  technique.**  Nevertheless,  the  sugges- 
tion that  Chlorella  may,  under  certain  circumstances,  fix 
elementary  nitrogen  has  been  revived  as  a  result  of  an 
observation  that  old  cultures  of  this  alga  with  a  high  lipide 
content  contained  substantially  more  combined  nitrogen  as 
determined  by  the  absolute  method  of  Dumas  than  that 
originally  supplied.^^*  So  far  this  finding  has  not  been 
substantiated.^^^ 

While  there  is  no  evidence  to  suggest  that  algae  belong- 
ing to  classes  other  than  the  Myxophyceae  and  Chlorophy- 
ceae  include  species  capable  of  nitrogen  fixation  it  is  well 
to  remember  that  very  few  forms  have  as  yet  been  investi- 
gated critically  from  this  point  of  view. 


yo  THE    METABOLISM    OF    ALGAE 

It  is  evident  that  in  its  physiology  nitrogen  fixation  by 
blue-green  algae  resembles  that  in  other  organisms.  Thus 
nitrogen  fixation  can  only  occur  concurrently  with  growth 
and  is  suppressed  in  the  presence  of  readily  available  com- 
bined nitrogen  such  as  an  ammonium  salt.^'  Nitrate  in- 
hibits nitrogen  fixation  in  Anahaena  cylindrica  only  if  the 
alga  is  adapted  to  utilize  this  form  of  combined  nitrogen. ^^ 
Traces  of  molybdenum  are  essential  for  the  achievement 
of  maximum  rates  of  fixation  in  blue-green  algae  as  in  other 
nitrogen-fixing  organisms. '*^'  ^^'  ^^^  In  Nostoc  niuscorum  assi- 
milation of  free  nitrogen  does  not  occur  in  media  of  pH 
below  approximately  5-7.^^  Nitrogen  fixation  in  blue-green 
algae  may  occur  either  during  phototrophic  growth  or  in  the 
dark  if  a  suitable  carbon  source  is  provided. ^^'  ^®  In  Nostoc 
muscorum  growing  in  the  dark,  10  to  12  mg.  of  nitrogen 
have  been  found  to  be  fixed  per  gm.  of  glucose  utilized.^^ 

The  properties  of  the  nitrogen-fixing  enzyme  system  in 
Nostoc  muscorum  appear  to  be  very  similar  to  those  of 
Azotohacter  and  of  the  RhizobiumAegnme  system. ^^  The 
half  maximum  rate  of  fixation  occurs  at  a  partial  pressure 
of  nitrogen  of  about  0-02  atmosphere,  a  value  of  the  same 
order  as  those  for  Azotohacter  (0-02  atmosphere)  and  Tri- 
folium  pratense  (0-05  atmosphere).  Like  those  of  other 
nitrogen-fixing  organisms  the  enzyme  system  of  Nostoc  is 
specifically  inhibited  by  hydrogen  and  by  carbon  monoxide. 
In  bacteria  there  appears  to  be  some  correlation  between 
the  presence  of  hydrogenase  and  the  capacity  to  fix  nitro- 
gen^'^  but  this  correlation  does  not  hold  in  the  algae,  for 
Nostoc  muscorum  cannot  be  adapted  to  hydrogen  (Table  3) 
and  those  algae  possessing  a  hydrogenase  system  seem  to 
be  incapable  of  nitrogen  fixation.^^^ 

The  mechanism  by  which  nitrogen  fixation  is  accom- 
plished in  living  organisms  is  still  unknown,  but  it  is  gener- 
ally agreed  that  ammonia  is  the  first  recognizable  product 
although  in  some  organisms  in  certain  circumstances  this 
may  be  replaced  by  hydroxylamine.^^^  Nitrogen  fixed  in 
experiments  of  short  duration  accumulates  principally  in 
glutamic  and  aspartic  acids  in  Nostoc  muscorum,  as  it  does  in 
other  nitrogen-fixing  organisms. ^°^«  These  two  amino-acids 


AUTOTROPHIC    ASSIMILATION  71 

are  abundant  in  nitrogen-fixing  blue-green  algae^^-  ^^'^'  ^^^'  ^^ 
and  it  appears  that  through  them  the  nitrogen  fixed  enters 
into  general  metabolism. 

Nitrogen-fixing  blue-green  algae  appear  to  be  of  great 
importance  in  contributing  to  the  fertility  of  certain  habi- 
tats, tropical  soils  in  particular  and  possibly  some  types  of 
freshwater. ^^'  ^^'  ^oo  \  capacity  for  nitrogen  fixation  may 
be  important  to  certain  species  of  Myxophyceae  in  enabling 
them  to  colonize  unpromising  substrata.  Blue-green  algae 
are  often  found  in  symbiotic  association  with  other  types  of 
organisms  and,  in  certain  of  these  at  least,  nitrogen  fixation 
may  contribute  to  the  success  of  the  partnership.^^-  1^2 

THE   ASSIMILATION   OF  NITRATE 

The  majority  of  algae  are  able  to  utilize  nitrogen  supplied 
in  the  form  of  nitrate  for  the  synthesis  of  cell  material. 
Exceptions  are  to  be  found  among  colourless  flagellates,  in 
the  chemotrophic  growth  of  certain  pigmented  flagellates,  ^^^ 
and  in  Euglena  gracilis  var.  bacillaris,  which  will  not  grow 
phototrophically  with  nitrate  as  the  sole  nitrogen  source. ^'^^ 

The  nitrogen  of  nitrate  must  undergo  reduction  before  it 
can  be  incorporated  into  amino-acids,  nitrogenous  bases 
and  their  derivatives.  This  process  of  reduction  was  first 
investigated  by  Warburg  and  Negelein  using  Chlorella 
pyrenoidosa.^^^  In  order  to  obtain  a  reduction  of  nitrate  at  a 
rate  comparable  to  those  of  photosynthesis  and  respiration, 
these  workers  used  an  acid  medium,  of  pH  about  2-0,  from 
which  undissociated  nitric  acid  might  be  expected  to  pene- 
trate rapidly  into  the  cells.  Chlorella  in  this  medium  in  the 
dark  was  found  to  evolve  carbon  dioxide  extra  to  that  pro- 
duced by  respiration  under  comparable  conditions  in  the 
absence  of  nitrate  and  at  the  same  time  to  liberate  ammonia 
into  the  medium.  The  ratio  of  the  amounts  of  extra  carbon 
dioxide  and  ammonia  was  not  constant,  evidently  because 
some  of  the  ammonia  was  utilized  by  the  cells.  When  the 
cells  used  were  not  nitrogen  deficient  this  ratio  was  found 
to  approach  2  :  i ,  in  agreement  with  the  equation: 

HN03+2(CH20)->NH3+2C02+H20  .     (17) 

6 


72  THE    METABOLISM    OF    ALGAE 

where  (CHgO)  represents  a  carbohydrate  or  similar  sub- 
strate. 

Chlorella  placed  in  the  acid  nitrate  solution  in  the  light 
was  found  by  Warburg  and  Negelein  to  evolve,  not  extra 
carbon  dioxide,  but  oxygen  extra  to  that  produced  by 
photos3^nthesis  in  the  absence  of  nitrate  but  under  other- 
wise comparable  conditions.  This  was  explained  by  sup- 
posing that  the  carbon  dioxide  evolved  in  reaction  17  is  in 
illuminated  cells  used  for  the  production  of  more  (CH2O): 

2C02+2H20->2(CH20)+202       .         .    (18) 

However,  the  discrepancy  was  noticed  that  the  amount  of 
extra  oxygen  produced  in  the  light  was  greater  than  the 
amount  of  extra  carbon  dioxide  evolved  in  the  dark. 

The  possibility  must  be  borne  in  mind  that,  under  the 
acid  conditions  used  by  Warburg  and  Negelein,  the  meta- 
bolism of  Chlorella  may  have  been  abnormal.  However, 
subsequent  work  with  less  extreme  treatment  of  the  alga 
has  confirmed  their  principal  results.  It  appears  that  in 
media  of  low  pH  the  penetration  of  nitrate  into  the  cells 
is  not  increased  but  that  the  liberation  of  ammonia  is 
favoured. ^^  In  media  of  pH  4-5  no  ammonia  is  produced  by 
Chlorella  supplied  with  nitrate  but  the  gas  exchanges  taking 
place  are  similar  to  those  observed  by  the  earlier  workers. 
For  example,  the  photosynthetic  quotient  of  Chlorella  in 
the  presence  of  nitrate  has  been  found  to  be  1-47  as  com- 
pared with  the  value  of  i-o6  obtained  with  an  ammonium 
salt  as  the  nitrogen  source  but  under  otherwise  similar  con- 
ditions.^^ The  situation  is  complicated,  however,  since  the 
extent  of  nitrate  reduction  and  consequently  the  gas  ex- 
changes observed  are  dependent  on  the  previous  history  of 
the  cells  employed  in  the  experiment  and  on  the  conditions 
to  which  they  are  exposed  (Table  5).  At  high  light  inten- 
sities there  is  proportionately  less  nitrate  assimilation  and 
the  value  of  the  photosynthetic  quotient  approaches  unity 
indicating  that  the  chief  product  of  photosynthesis  is  carbo- 
hydrate. The  same  effect  is  achieved  if  the  cells  are  pre- 
viously depleted  of  carbohydrate  by  starvation.  If,  on  the 
other  hand,  the  concentration  of  carbohydrates  in  the  cells 


AUTOTROPHIC    ASSIMILATION  73 

is  already  high,  as  after  a  period  in  bright  light,  then  there 
is  no  restriction  on  the  use  of  the  products  of  photosynthesis 
for  the  synthesis  of  nitrogenous  substances  and  the  rate  of 
nitrate  assimilation  is  high  with  a  corresponding  value  of 
the  photosynthetic  quotient.  Since  no  nitrate  reduction 
occurs  when  ammonia  is  available  it  is  evident  that  nitrate 
is  only  reduced  in  a  manner  intimately  related  to  the  require- 
ments of  subsequent  nitrogenous  synthesis. ^^ 

TABLE  5 

PHOTOSYNTHETIC  QUOTIENTS  (Qp= AOg/- ACO2)  OF  Chlorella 
pyrenoidosa  in  the  presence  of  nitrate^^^ 


Previous  history  of  cells 

Observed  Qp 

Low  light 

High  light 

Grown  at  low  light  intensity 

Exposed  for  4  hr.  to  high  light  intensity 

Starved  for  3  days  in  darkness 

1-47 
2-50 

I-IO 

I-I4 

1-04 

Warburg  and  Negelein^^^  considered  nitrate  assimilation 
to  be  a  process  not  directly  connected  with  photosynthesis 
and  explained  the  increase  in  nitrate  reduction  on  illumina- 
tion as  due  to  the  effects  of  light  on  cell  permeability. 
Nevertheless  it  is  possible  that  nitrate  reduction  is  closely 
linked  with  the  photosynthetic  mechanism.  Thus,  nitrate 
might  be  reduced  by  a  substance  which  can  be  produced 
by  respiratory  processes  but  which  is  closely  related  to  a 
primary  product  of  photosynthesis  and  which  is  available 
in  greater  amounts  when  photosynthesis  is  taking  place. ^^^ 
It  is  also  possible  that  in  the  light  a  sensitized  photochemical 
reduction  of  nitrate  by  water  occurs,  i.e:  photosynthesis 
with  nitrate  substituted  for  carbon  dioxide:^^'' 

HN03+H20->NH3+202  .         .    (19) 

Evidence  that  this  takes  place  is  provided  by  the  observa- 
tion that  Chlorella  can  evolve  oxygen  in  the  light  in  the 
absence  of  carbon  dioxide  if  nitrate  is  present.^^*  Nitrate 
cannot,  however,  act  as  the  hydrogen  acceptor  in  the  Hill 


74  THE    METABOLISM    OF    ALGAE 

reaction.  Since  nitrate  reduction  can  also  occur  in  the  dark 
in  Chlorella  it  is  necessary,  if  it  is  accepted  that  photo- 
chemical reduction  occurs,  to  suppose  that  there  are  two 
distinct  mechanisms  for  the  reduction  of  nitrate.  Confirma- 
tion that  some  organisms  are  able  to  utilize  nitrate  under 
phototrophic  conditions  but  not  when  growing  chemo- 
trophically  would  give  strong  support  to  this  idea. 

Reduction  of  nitrate  to  ammonia  takes  place  in  plants 
in  several  stages.  Under  certain  conditions  nitrite  is  liber- 
ated instead  of  ammonia  when  Chlorella^^^  or  Ankistrodes- 
mus^^^  reduce  nitrate  in  an  acid  medium.  Both  nitrite  and 
hydroxylamine  appear  to  be  intermediates  in  the  reduction, 
the  sequence  being: 

NO3-— >N02-— >NH3(OH)+— >NH4+        .     (20) 

It  may  be  noted  here  that  nitrite  can  serve  as  a  source  of 
nitrogen  for  some  algae,  although  it  is  toxic  except  in  low 
concentrations, ^^^'  ^^°  and  that,  in  low  concentrations, 
hydroxylamine  is  available  as  a  nitrogen  source  for  certain 
organisms  and  may,  indeed,  be  the  form  in  which  the 
nitrogen  of  nitrate  enters  into  the  general  metabolism  of 
the  organism. 2^^'  ^^^ 

Nitrate  assimilation  by  green  algae  is  inhibited  by  low 
concentrations  of  cyanide,  an  indication  that  heavy  metals 
are  concerned  in  the  process. ^^^'  ^^^'  ^^®  As  in  higher  plants, 
manganese  appears  to  play  a  specific  part  in  the  reduction 
of  nitrate  by  algae^^"  and  traces  of  molybdenum  have  been 
found  to  be  essential  for  the  achievement  of  maximum 
rates  of  nitrate  assimilation  by  Anabaena.^^^ 

THE   ASSIMILATION   OF   AMMONIUM   NITROGEN 

A  mmonium  salts  can  be  utilized  as  sole  source  of  nitrogen 
by  most  algae  and  are  generally  more  readily  assimilated 
than  is  nitrate.  If  the  two  forms  are  supplied  together,  the 
ammonium  nitrogen  is  utilized  preferentially  and  the  nitrate 
is  onlv  consumed  when  the  ammonia  is  exhausted.^"*'  ^^°'  ^^^ 
If  Chlorella  growing  in  a  medium  containing  nitrate  is 
supplied  with  an  ammonium  salt  the  rate  of  gas  exchange 
alters  immediately,  showing  no  adaptation  to  this  latter 
source  of  nitrogen  to  be  necessary. ^^  The  obvious  explana- 


AUTOTROPHIC    ASSIMILATION  75 

tion  of  these  facts  is  that  nitrate  must  be  reduced  before  its 
nitrogen  can  be  utiHzed  in  cell  synthesis  whereas  the  nitro- 
gen in  ammonia,  being  already  at  the  same  level  of  reduc- 
tion as  that  in  bio-organic  compounds,  can  participate 
immediately  in  metabolism.  The  fact  that  an  organism  able 
to  utilize  nitrate  must  possess  some  mechanism  extra  to 
that  required  for  ammonia  assimilation  is  recognized  in  the 
distinction  drawn  between  autotrophs  sensu  stricto,  which 
are  able  to  reduce  oxidized  inorganic  nutrients,  and  meso- 
trophs,  which  require  one  or  more  reduced  inorganic 
nutrients  such  as  ammonia  (see  p.  16). 

Secondary  effects  of  ammonia  absorption  make  it  a  less 
favourable  source  of  nitrogen  than  nitrate.  Ammonium 
nitrogen  enters  living  cells  most  readily  as  undissociated 
ammonium  hydroxide,  with  the  result  that  the  medium 
surrounding  the  cells  becomes  more  acid  and  may  eventu- 
ally reach  a  pH  sufficiently  low  to  inhibit  growth.^^s  While 
absorption  of  nitrate  causes  a  medium  to  become  more 
alkaline,  the  change  is  relatively  less  and  this  form  of  nitro- 
gen is  generally  used  in  preference  to  ammonium  salts  in 
making  up  media  for  algae.  Only  if  pH  changes  are  reduced 
by  buffering  or  by  some  other  means,  is  growth  with 
ammonium  salts  as  good  as  that  with  nitrate.  Ammonium 
nitrogen,  even  in  slight  excess,  may  have  a  profound  influ- 
ence on  the  course  of  metabolism  and  can  exert  effects, 
which  may  or  may  not  be  deleterious,  apart  from  those 
attendant  upon  changes  in  hydrogen  ion  concentration. 
Even  concentrations  of  the  order  of  0-5x10-4  M  of  an 
ammonium  salt  suppress  the  formation  of  heterocysts  by 
Anahaena  cylindrical^  and  although  growth  of  Prorocentrum 
spp.  and  Peridinium  spp.  is  possible  in  the  presence  of 
0-2x10-5  M  ammonium  chloride,  it  is  inhibited  by  a  con- 
centration of  0-2  X  iQ-^  M.^^  ' 

As  with  nitrate,  the  uptake  of  ammonium  nitrogen  by 
algal  cells  depends  on  the  previous  history  of  the  cells  and 
on  the  conditions  to  which  they  are  exposed.^^s  The  addi- 
tion of  an  ammonium  salt  to  a  suspension  of  nitrogen- 
starved  Chlorella  vulgaris  is  followed  immediately  by  a  rapid 
assimilation  of  ammonia  which  continues  until  either  the 


76  THE    METABOLISM    OF    ALGAE 

ammonia  in  the  medium  or  some  carbon  reserve  within  the 
cells  becomes  exhausted.  Ammonia  uptake  by  cells  not 
initially  deficient  in  nitrogen  is  much  less  marked  but  is 
increased,  although  to  a  level  lower  than  that  of  nitrogen- 
deficient  cells,  if  glucose  is  supplied  in  the  medium.  The 
supply  of  glucose  to  nitrogen-deficient  cells  does  not  in- 
crease their  rate  of  ammonia  assimilation.  These  observa- 
tions were  made  with  cell  suspensions  kept  in  the  dark; 
light  has  little  effect  on  the  rate  of  ammonia  assimilation  by 
nitrogen-deficient  cells  but  doubles  the  assimilation  rate  of 
normal  cells. 

TABLE   6 

RATES    OF    OXYGEN    ABSORPTION    (mM.^/mG.    DRY   WT./hR.)    BY 

Chlorella  vulgaris^' ^ 


Nitrogen-starved  fno  glucose 
cells  l  +  i?o  glucose 

no  glucose 


Normal  cells 


+  1 


glucose 


Before  addition 
of  (NH4)oSO« 


2-74 

7-75 

3-05 

12-48 


After  addition 
of  (NH4)2SO« 


i6-o8 

i6-66 

8-52*  2-32 

17-53 


*  This  rate  was  maintained  for  10  minutes  only. 

The  assimilation  of  ammonia  by  nitrogen-starved  Chlor- 
ella is  accompanied  by  a  marked  increase  in  respiration  rate. 
When  the  addition  of  an  ammonium  salt  to  a  cell  suspension 
is  not  followed  by  very  rapid  ammonia  assimilation  respira- 
tion is  not  stimulated  in  this  manner  (Table  6).  The  assimi- 
lation of  ammonia  and  the  respiration  accompanying  it  are 
both  cyanide-sensitive.  Fermentation,  in  cell  suspensions 
in  an  atmosphere  of  nitrogen,  is  not  aflPected  by  addition  of 
ammonia.  There  is  thus  a  close  correlation  between  the 
rates  of  ammonia  assimilation  and  of  aerobic  respiration. ^^^ 

The  ammonia  assimilated  by  nitrogen-starved  Chlorella 
is  converted  largely  into  soluble  organic  compounds.  At 
first,  most  of  the  ammonia  which  disappears  from  the 
medium  can  be  accounted  for  as  free  or  combined  amino- 
nitrogen  and  amide  nitrogen  within  the  algal  cells  (Fig.  13). 


AUTOTROPHIC    ASSIMILATION 


77 


100O 


90O 


1  2 

TIME    IN    HOURS 

FIG.  13.  Changes  in  the  amounts  of  various  chemical  fractions  in 
Chlorella  vulgaris  following  the  supply  of  ammonia  to  nitrogen- 
deficient  cells  (after  ref.  276). 


78  THE    METABOLISM    OF    ALGAE 

As  assimilation  continues,  these  fractions  account  for  less 
of  the  ammonia  taken  up,  basic  amino-acids,  particularly 
arginine,  lysine  and  ornithine,  being  formed  instead.^'®'  ^^^ 
Corresponding  with  the  assimilation  of  ammonia  there  is  a 
disappearance  of  non-reducing  sugar  and  of  polysaccharide 
from  the  cells  (Fig.  13)  and  it  is  evident  that  the  carbon 
skeletons  for  the  nitrogenous  compounds  formed  are  derived 
from  these  substances. ^"^  When  glucose  is  supplied,  all  the 
ammonia  nitrogen  added  to  the  cells  is  eventually  converted 
to  insoluble  nitrogen  and  the  cells  return  to  the  nitrogen- 
starved  condition.  When  no  glucose  is  supplied  a  large  pro- 
portion of  the  ammonia-nitrogen  assimilated  remains  in 
the  cells  in  a  soluble  organic  form.^'''^ 

The  mechanisms  whereby  ammonia  is  incorporated  into 
organic  substances  in  algae  have  not  yet  been  investigated 
in  detail.  In  higher  plants  supplied  with  ammonia  labelled 
with  heavy  nitrogen,  heavy  nitrogen  appears  more  rapidly 
and  extensively  in  glutamic  acid  than  in  any  other  amino- 
acid,  suggesting  that  there  is  ready  exchange  between 
ammonia  and  its  amino-group  and  that  this  is  the  principal 
route  of  entry  of  ammonia  into  metabolism. ^^  Glutamic 
acid,  which  evidently  occurs  in  substantial  proportions  in 
most  algae  (see  Table  8)  and  which  is  more  readily  assimil- 
ated than  other  amino-acids  by  algae  such  as  Scenedesmus^ 
and  Eiiglena  spp.,^^^  appears* to  occupy  a  similar  special 
position  in  the  nitrogen  metabolism  of  these  organisms  and 
that  this  is  so  for  Nostoc  muscoriun  has  been  confirmed 
using  heavy  nitrogen  as  a  tracer.  ^°^«  Glutamic  acid  is  formed 
in  plant  tissues  from  oc-ketoglutaric  acid  by  a  reversible  pro- 
cess involving  a  reduction  catalysed  by  glumatic  dehydro- 
genase: 


.41 


COOH  COOH  COOH 

C:0        1h5)      C:NH      +2H     CH.NH2 


(CHo)o  (CH2)2  (CHo)2 

I  I  I      ' 

COOH  COOH  COOH 

a-Ketoglutaric  a-Iminoglutaric  Glutamic 
acid                       acid  acid 


(21) 


AUTOTROPHIC    ASSIMILATION  79 

a-Ketoglutaric  acid  may  be  derived  through  the  tricar- 
boxylic acid  cycle  from  carbohydrate  or  from  the  primary 
products  of  photosynthesis  by  the  pathways  indicated  in 
Fig.  10.  A  corresponding  mechanism  for  the  formation  of 
the  related  aspartic  acid  from  oxaloacetic  acid  is  unknown'*^ 
and  aspartic  acid  does  not  appear  to  occupy  a  similar  central 
position  in  the  metabolism  of  Chlorophyceae  to  that  of 
glutamic  acid.^'  ^  There  is  evidence  that  in  algae  other 
amino-acids  are  formed  from  glutamic  and  aspartic  acids 
by  transamination  processes  such  as  have  been  found  to 
occur  in  other  organisms, ^^'  ^^^"  e.g.: 

COOH  COOH  COOH  COOH 


CH.NH2  +  C:0 

I  I 

C^H2  Cri3 


COOH 

Aspartic 
acid 


Pyruvic 
acid 


C:0 

CH2 

I 
COOH 

Oxaloacetic 
acid 


+       CH.NH2        (22) 
CH3 

Alanine 


The  necessary  keto-acid  in  this  particular  example  may  be 
provided  directly  either  by  glycolysis  or  by  photosynthesis. 
Transaminase  systems  catalysing  the  transfer  of  the  amino- 
group  from  aspartic  acid,  alanine  and  leucine  to  a-keto- 
glutaric  acid  and  from  aspartic  acid,  glutamic  acid  and 
leucine  to  pyruvic  acid  have  been  demonstrated  in  Chlorella 
vulgaris}^^^  The  synthesis  of  the  basic  amino-acids,  arginine, 
lysine  and  ornithine,  which  appear  to  be  of  particular  im- 
portance in  C  vulgaris,"''^  has  been  studied  in  detail  in 
other  organisms^^'  ^^  but  there  is  nothing  to  indicate 
whether  or  not  the  mechanisms  that  have  been  found  occur 
in  algae  also. 

Amide  formation  is  of  general  occurrence  in  plants^^  and 
the  observed  increase  in  amide  in  Chlorelta  supplied  with 
ammonia^'^  is  in  agreement  with  expectation.  Glutamine 
formation  in  animal  tissues,  higher  plants  and  bacteria,  has 
been  found  to  be  dependent  on  aerobic  respiration  and 
evidently  involves  a  phosphorolytic  reaction,  the  energy 
necessary  for  the  formation  of  the  amide  linkage  being 
derived  from  adenosine  triphosphate:^^'  *^ 


8o  THE    METABOLISM    OF    ALGAE 

COOH  CO.NH2 

I  ! 

(CH,)2  (CHa)^ 

ATP  +   I  +  NH3— >ADP  +  H3PO4  +   I  .     (23) 

CH.NH2  CH.NH2 


COOH 
Glutamic  acid 


COOH 

Glutamine 


The  mechanism  of  amide  formation  in  algae  must  be  sup- 
posed to  be  essentially  similar. 


A' 


Polysaccharide 


^  Organic  acids  ^ 
fi 


CO., 


Amino-acids  -  r^Amide 


A' 


A' 


Glucose 


Protein 


FIG. 


14.  Scheme  showing  the  probable  inter-relationships  between 
the  processes  involved  in  ammonia  assimilation  in  Chlorella 
vulgaris.  A  represents  a  substance  such  as  adenosine  diphos- 
phate and  A',  a  substance  such  as  adenosine  triphosphate 
(after  ref.  277). 


It  will  be  evident  from  the  preceding  paragraphs  that 
the  mechanisms  of  ammonia  assimilation  and  respiration  are 
interdependent.  The  correlation  existing  between  the  rates 
of  the  two  processes  may  be  explained  if  it  is  assumed  that 
the  rate  of  respiration  is  normally  limited  by  the  amount 
available  of  some  substance,  such  as  adenosine  diphosphate, 
which  can  act  as  an  acceptor  for  the  high  energy  phosphate 
groups  produced  in  this  process.^"^  It  has  been  pointed  out 


AUTOTROPHIC    ASSIMILATION  8l 

above  that  the  synthesis  of  amides  requires  energy  which 
can  be  supplied  from  high  energy  phosphate  groups  and 
it  seems  Ukely  that  the  synthesis  of  amino-acids  and  proteins 
must  involve  the  consumption  of  energy  from  a  similar 
source. ^^'  *^  Upon  addition  of  ammonia  to  nitrogen-starved 
cells  the  synthesis  of  nitrogenous  substances  presumably 
resuhs  in  the  disappearance  of  high  energy  phosphate 
groups  and  makes  more  acceptor  available  so  that  respiration 
may  proceed  more  rapidly.  In  normal  cells,  in  which  syn- 
thesis of  organic  nitrogenous  substances  from  ammonia 
does  not  occur,  no  high  energy  phosphate  groups  are 
utilized  and  so  the  respiration  rate  is  not  increased.  This 
mechanism  is  represented  in  Fig.  14,  in  which  A  stands  for 
a  substance  such  as  adenosine  diphosphate  and  A'  for  a 
substance  such  as  adenosine  triphosphate.  In  agreement 
with  this  hypothesis  it  has  been  found  that  addition  of 
adenylic  acid  or  of  dinitrophenol,  which  promotes  break- 
down of  adenosine  triphosphate,  increases  the  respiration 
rate  of  nitrogen-starved  Chlorella,  and  that  the  soluble 
organic  phosphorus  fraction  in  the  cells  decreases  after 
addition  of  ammonia.^'*^ 

THE   ASSIMILATION   OF   NITROGEN   IN   ORGANIC 

COMBINATION 

The  reactions  by  which  nitrogen  is  transferred  from  one 
carbon  residue  to  another  during  nitrogen  metabolism  are 
evidently  reversible  in  the  majority  of  cases  and,  just  as  in 
the  case  of  carbon  metabolism,  we  are  justified  in  imagining 
a  flexible  system  into  which  material  may  be  introduced 
at  several  points.  The  expectation  that  any  substance 
which  is  an  intermediate  in  nitrogen  metabolism,  or  readily 
convertible  into  such,  can  serve  as  a  nitrogen  source  if  it 
is  able  to  penetrate  the  plasma  membrane,  appears  to  be 
borne  out  by  the  observed  facts.  Thus  there  are  many 
records  of  autotrophic  algae  being  able  to  utilize  various 
organic  nitrogenous  substances  as  their  only  source  of 
nitrogen  (e.g.  ^^^' ^^''  3'*'^'  ^'  '^'  ^'i^).  Since  this  form  of  nitro- 
gen assimilation  is  facultative  and  does  not  involve  any  loss 


82  THE    METABOLISM    OF    ALGAE 

in  synthetic  ability  it  is  more  appropriately  considered  here 
rather  than  under  the  heading  of  heterotrophism. 

The  first  step  in  the  assimilation  of  a-amino-acids,  such 
as  glycine  and  alanine,  is  evidently  deamination,  so  that 
their  nitrogen  enters  into  metabolism  as  ammonia.^'  ^  Thus 
during  the  growth  of  Scenedesmus  ohliquus  in  the  light  in  a 
medium  containing  glycine,  which  forms  a  good  source  of 
nitrogen  for  this  alga,  considerable  amounts  of  ammonia 
are  liberated.^  Since  S.  obliquus  is  able  to  grow  slowly  in 
the  dark  with  glycine  as  its  sole  source  of  carbon,  it  might 
be  that  this  excess  ammonia  is  produced  because  the  rate 
of  deamination  of  the  glycine  is  high  as  a  result  of  the 
utilization  of  the  carbon  residue  under  conditions  of  carbon 
deficiency.  Evidently  this  cannot  be  so,  since  if  glucose  is 
supplied  together  with  glycine  to  illuminated  cultures  the 
rate  of  deamination  per  cell  remains  the  same,  although  less 
ammonia  appears  in  the  medium  because  of  the  more  rapid 
growth  of  the  alga.  It  appears,  therefore,  that  deamination  of 
glycine  by  S.  ohliquus  is  independent  of  carbon  metabolism 
and  is  in  excess  of  requirements.^  In  Chlorella  vulgaris  the 
rate  of  deamination  of  this  amino-acid  is  relatively  slower 
than  in  Scenedesmus  and  limits  the  rate  of  growth  of  the  alga 
with  the  result  that  no  ammonia  is  liberated  provided  that 
the  medium  is  neutral.'*  Similarly  Chlorella,  Haematococcus 
and  Zygnema  spp.  deaminate  alanine  relatively  slowly  so 
that  no  ammonia  is  liberated  from  the  cells,  whereas 
Ankistrodesmus,  Stichococcus  and  Hormidium,  are  of  the 
same  type  as  Scenedesmus  and  give  off  an  excess  of  ammonia 
to  the  medium  when  supplied  with  these  amino-acids.^ 
Bacterial  deaminases  have  a  maximum  rate  of  reaction  at 
about  pH  8-0  but  the  optimum  for  these  algal  deaminases 
is  on  the  acid  side  of  neutrality,  between  pH  5  and  6.^-  * 
The  algae  in  which  deamination  has  been  demonstrated 
frequently  occur  in  waters  contaminated  with  organic 
matter  and  must  play  a  considerable  part  in  the  degradation 
of  nitrogenous  substances  in  such  situations. 

Aspartic  acid,  glutamic  acid,  asparagine,  succinamide 
and  glutamine,  have  also  been  found  to  be  suitable  as  sole 
nitrogen  sources  for  various  green  algae.®'  ^'  ^  Aspartic  acid 


AUTOTROPHIC    ASSIMILATION  83 

does  not  appear  to  be  deaminated  during  assimilation,  its 
amino-group  evidently  being  utilized  by  transamination® 
as  in  reaction  22.  In  media  containing  amides,  ammonia 
may  be  liberated  in  small  quantities  during  the  growth  of 
Chlorophyceae,  evidently  as  a  result  of  deamidation  taking 
place  independently  of  deamination  and  transamination.' 


CHAPTER    V 
HETEROTROPHIC   ASSIMILATION 

Heterotrophic  organisms  are  unable  to  synthesize  the  full 
range  of  organic  substances  necessary  for  life  and  are  conse- 
quently dependent  on  exogenous  sources  for  one  or  more 
essential  metabolites.  In  the  algae  autotrophism  is  evidently 
the  primitive  condition,  the  heterotrophic  habit  being 
derived  from  it  by  specialization  leading  to  loss  of  particular 
synthetic  mechanisms. 

There  are  many  indications  that  a  requirement  for  specific 
organic  growth  factors  is  of  frequent  occurrence  among 
algae  but  few  definite  instances  have  been  recorded.  Partly, 
this  is  because  the  isolation  of  algae  has  usually  involved 
'enrichment  culture'*  in  a  mineral  medium,  a  procedure 
which  tends  to  select  species  without  organic  growth  sub- 
stance requirements  and  which  results  in  our  present  know- 
ledge of  algal  metabolism  being  biased  in  favour  of  com- 
pletely autotrophic  forms.^'®  There  is  also  the  technical 
difficulty  of  establishing  requirements  for  substances  which 
may  be  active  at  exceptionally  high  dilutions. ^^^  Many 
reports  of  growth  factor  requirements  among  flagellates 
cannot  be  confirmed^^^  and  these  will  not  be  discussed  here. 

THE   AMINO-ACID   REQUIREMENTS   OF   ALGAE 

Twenty  or  more  different  a-amino-acids  appear  to  be 
essential  in  living  matter  and,  if  any  part  of  the  mechanism 
for  the  synthesis  of  these  from  ammonia  and  appropriate 
carbon  residues  is  absent,  the  organism  concerned  must 
obtain  the  particular  amino-acids  from  exogenous  sources. 

Eiigletia  deses  can  only  grow  if  provided  with  a  suitable 
amino-acid  such  as  aspartic  acid.^^^  Here  the  requirement 

*  An  enrichment  culture  is  one,  inoculated  with  a  mixture  of 
species,  in  which  particular  conditions  favour  the  development  of 
certain  organisms  whilst  hindering  that  of  others  (see  ref.  241). 

84 


HETEROTROPHIC    ASSIMILATION  85 

is  evidently  not  for  a  specific  amino-acid  and  it  appears  that 
the  mechanism  which  is  lacking  is  one  which  forms  amino- 
groups  from  ammonia,  for  example  that  represented  in 
equation  21.  Given  an  amino-acid  which  can  participate  in 
transamination  reactions  (equation  22)  then  synthesis  of 
other  amino-acids  is  possible.  Glycine  and  phenylalanine  are 
not  suitable  nitrogen  sources  for  Euglena  deses^^^  and  it  may 
be  significant  that  of  these  the  former  apparently  does  not 
participate  in  transamination  reactions  in  other  organisms. 
Chlamydomonas  chlamydogama,  an  obligate  phototroph, 
provides  an  example  of  an  alga  with  specific  amino-acid 
requirements,  viz.  for  histidine  and  aspartic  acid.^^^  Here 
the  missing  mechanisms  lie  towards  the  end  of  synthetic 
sequences  rather  than  on  the  main  track  of  synthesis  as  in 
Euglena  deses. 

THE   VITAMIN   REQUIREMENTS   OF  ALGAE 

If  an  organism  is  unable  to  synthesize  for  itself  a  com- 
ponent of  an  enzyme  system,  only  minute  amounts  of  the 
metabolite  concerned  need  to  be  supplied  from  exogenous 
sources  to  permit  growth  to  take  place.  Growth  factors  of 
this  type  are  generally  known  as  vitamins. 

Most  algae  are  able  to  synthesize  for  themselves  vitamin 
Bi  or  thiamine,  the  importance  of  which  in  metabolism  has 
already  been  discussed  (p.  9),  in  quantities  sufficient  for 
their  needs. ^  However,  a  requirement  for  this  substance  has 
been  demonstrated  in  a  number  of  algae.  It  may  be  noted 
that  the  amounts  of  dissolved  thiamine  present  in  natural 
waters  are  evidently  enough  to  support  the  growth  of  these 
forms.i*^'  177  'Yht  thiamine  molecule  consists  of  thiazole 
and  pyrimidine  portions  and  synthetic  disabilities  may  result 
in  an  organism  being  unable  to  synthesize,  either  or  both 
of  these  portions  or  to  unite  them  to  give  thiamine.  A 
requirement  for  thiamine  as  such  has  not  yet  been  demon- 
strated in  an  alga.  The  thiazole  portion  only  is  required  by 
Polytoma  ocellatum  whereas  Euglena  gracilis  can  manufac- 
ture this  for  itself  but  requires  an  exogenous  supply  of  the 
pyrimidine  portion.^^^  Both  the  pyrimidine  and  thiazole 
portions  must  be  supplied  for  the  growth  of  Polytomella 


86  THE    METABOLISM    OF    ALGAE 

caeca,  Chilotnonas  paramoecium'^^^  and  Prototheca  zopfii}^ 
A  mutant  requiring  thiamine  of  Chlamydomofias  moewusii 
has  been  induced  by  ultra-violet  irradiation  and  responds  to 
as  little  as  io~^'^  gm./ml.  of  the  substance.  Here  it  is  the 
pyrimidine  portion  alone  which  is  required,  the  ability  to 
synthesize  thiazole  remaining  unimpaired. ^'^'^ 

The  requirement  of  Euglena  gracilis  for  thiamine  is  of 
particular  interest  in  that  this  substance  has  been  found  to 
be  necessary  for  phototrophic  growth  but  not  when  gluta- 
mate  is  supplied  either  in  the  light  or  the  dark.^^  The  sig- 
nificance of  these  facts  is  not  clear  since  although  it  is 
possible  for  the  decarboxylation  of  pyruvic  acid,  for  which 
thiamine  is  essential,  to  be  by-passed  if  glutamate  is  the 
carbon  source  (see  Fig.  lo),  thiamine  appears  to  be  just  as 
necessary  for  the  oxidative  decarboxylations  occurring  in 
the  tricarboxylic  acid  cycle,  which  presumably  operates  in 
Euglena.  Possibly  the  inability  of  Euglena  gracilis  to  synthe- 
size thiamine  is  not  absolute  and  glutamate  exerts  sufficient 
thiamine-sparing  action  to  enable  growth  to  take  place. 

Traces  of  vitamin  Bjg,  the  anti-pernicious  anaemia 
factor  of  animals,  are  necessary  for  the  growth  of  Euglena 
gracilis,  E.  stellata  and  Astasia  spp.  It  has  been  suggested 
that  the  requirement  is  characteristic  of  species  of  Eugle- 
nineae  in  general,  but  it  can  also  occur  in  representatives  of 
other  classes,  e.g.  Chlamydomonas  chlamydogama}^^  Vita- 
min Bi2,  like  folic  acid,  may  be  concerned  in  the  synthesis 
of  desoxyribosenucleic  acid.^^*'  The  vitamin  B12  require- 
ment of  Euglena  gracilis  is  the  same  whether  grov^th  takes 
place  in  the  light  or  in  the  dark.*^ 

A  constituent  of  folic  acid,  /)-amino-benzoic  acid,  has 
been  recognized  as  an  essential  metabolite  as  a  result  of  the 
observation  that  it  antagonizes  the  inhibition  of  the  growth 
of  micro-organisms  by  its  analogue,  sulphanilamide.^®^ 
p-Amino-benzoic  acid  evidently  plays  as  important  a  role 
in  the  metabolism  of  some  algae  as  it  does  in  that  of  other 
organisms  since  it  has  been  found  to  neutralize  the  inhibi- 
tory effects  of  sulphanilamide  on  the  growth  of  Nitzschia 
spp.^^*^  and  of  Chlamydomonas  ffioewusii.^'^''  While  p-amino- 
benzoic  acid  has  been  found  to  be  a  growth  factor  for  several 


HETEROTROPHIC    ASSIMILATION  87 

bacterial  species, -^"^  a  requirement  for  it  in  an  alga  appears 
so  far  only  to  have  been  found  in  an  artificially  induced 
mutant  of  Chlamydomonas  moewusUy'^^  ^'^  In  the  nutrition 
of  this  mutant  ^-amino-benzoic  acid  may  be  replaced  by 
aniline  at  an  efficiency  of  about  i  per  cent,  but  it  is  remark- 
able that  aniline  does  not  have  a  corresponding  neutralizing 
effect  on  the  inhibition  of  the  wild-type  cells  by  sulphan- 

ilamide.1^6,  177 

UNIDENTIFIED    GROWTH    FACTORS 

Besides  these  few  instances  in  which  growth  factors  have 
been  identified,  it  may  be  as  well  to  mention  briefly  examples 
in  which  a  requirement  for  less  completely  characterized 
growth  factors  has  been  found,  as  an  indication  of  the 
extent  to  which  heterotrophism  occurs  among  the  algae. 

There  are  several  reports  indicating  that  the  growth  of 
marine  algae  may  in  some  cases  depend  on  organic  sub- 
stances present  in  seawater.^^^  Thus,  Ditylium  brightwelli 
has  been  found  to  require  for  vigorous  growth  in  artificial 
seawater  two  organic  substances,  or  groups  of  substances, 
in  addition  to  mineral  salts.  One  of  these  factors  may  be 
replaced  by  organic  compounds  containing  sulphur  in  the 
grouping,  — S— CH2.CH(NH2)COOH.  The  other  sub- 
stance, or  group  of  substances,  is  present  in  extracts  of 
algae  and  of  yeast. ^^^  Seawater  from  the  surface,  particularly 
that  from  the  littoral  zone,  has  been  found  to  contain  sub- 
stances necessary  for  the  germination  of  sporelings  of 
Enteromorpha  and  Ulva  spp.,  which  do  not  appear  to  be  of 
an  inorganic  nature  and  which  are  absent  from  water  taken 
from  a  depth  of  35  to  40  metres. ^^^  A  marine  dinoflagellate, 
Gymnodinium  sp.,  cannot  be  subcultured  indefinitely  in  the 
absence  of  an  organic  factor  or  factors  occurring  in  soil 
extract.^'^^  The  addition  of  soil  extract  is  frequently  neces- 
sary to  obtain  growth  of  soil  and  freshwater  algae  in  cul- 
ture.^'^^  Thus,  Cryptomonas  ovata  and  Synura  uvella  will 
not  grow  in  a  medium  found  suitable  for  other  planktonic 
algae,  until  an  extract  of  soil  or  lake  sediment  is  added. 
Ashing  destroys  the  activity  of  these  extracts.^^*  Soil  ex- 
tract presumably  contains  a  wide  variety  of  substances 
7 


88  THE    METABOLISM    OF    ALGAE 

which  may  act  as  growth  factors  and  different  factors  may  be 
concerned  in  different  cases.^^^  There  is  some  evidence  that 
Gloeotrichia  echinulata  requires  a  thermolabile  growth  factor 
present  in  garden  soil.^^*  It  is  commonly  observed  that  algae 
which  have  grown  well  in  impure  culture  cease  to  thrive 
after  isolation  in  bacteria-free  culture.  Gloeotrichia  nutans, 
for  example,  is  of  this  type.^^  This  is  an  indication  that 
such  species  require  organic  growth  factors. 


CHAPTER    VI 
THE   PRODUCTS   OF   METABOLISM 

In  the  previous  chapters  the  pathways  by  which  materials 
assimilated  by  algae  enter  into  metabolism  have  been  traced 
as  far  as  possible.  Now,  the  nature  of  the  final  products  of 
metabolism  must  be  considered.  Something  of  the  nature  of 
the  transformations  which  the  intermediates  that  have  so 
far  been  identified  must  undergo  before  these  final  products 
appear  can  be  inferred  by  analogy  with  processes  that  have 
been  discovered  in  other  organisms,  but  information  ob- 
tained by  experiment  with  algae  themselves  is  for  the  most 
part  lacking  as  yet.  For  present  purposes  it  seems  best,  after 
noting  the  existence  of  this  gap  in  our  knowledge,  to  review 
briefly  that  which  is  known  of  the  chemical  nature  of  the 
major  products  of  algal  metabolism  without  speculation  as 
to  the  pathways  by  which  they  are  synthesized. 

SOLUBLE   EXTRACELLULAR  PRODUCTS 

Algae,  in  general,  inhabit  aquatic  environments.  As  we 
have  seen,  it  is  possible  for  organic  substances  to  enter  algal 
cells  from  the  environment  and  so  the  possibility  must  be 
considered  that  the  passage  of  similar  substances  can  take 
place  equally  readily  in  the  opposite  direction  with  the  result 
that  appreciable  proportions  of  the  products  of  metabolism 
are  liberated  in  extracellular  form  from  healthy  algal  cells. 

In  pure  cultures  of  species  of  Chlorophyceae,  such 
as  Scenedesmus,  Chlorella  and  Coccomyxa  spp.,  from  2 
to  12-5  per  cent  of  the  total  carbon  assimilated  has 
been  found  to  appear  in  a  soluble  form  in  the  external 
medium.^®®'  ^^^'  ^®*'  ^^^'  ^^^  The  amount  of  such  extra- 
cellular carbon  increases  as  cultures  age  and  proportions  as 
high  as  12*5  per  cent  have  only  been  observed  in  old  cul- 
tures^^^  in  which,  it  may  be  suspected,  autolysis  may  have 
occurred  to  some  extent.  The  nature  of  the  extracellular 

89 


90  THE    METABOLISM    OF    ALGAE 

compounds  of  the  Chlorophyceae  has  not  been  determined 
but  Anabaena  cylindrica  has  been  found  to  Uberate  extra- 
cellular pentose  in  amounts  of  up  to  1-4  per  cent  of  its  dry 
weight  as  well  as  nitrogenous  substances  which  may  repre- 
sent a  considerably  greater  proportion  of  the  total  carbon 
fixed  by  the  alga.^^  It  is  to  be  noted  that  the  dissolved 
organic  matter  to  be  found  both  in  freshwater  and  seawater 
consists  principally  of  substances  of  a  nitrogenous  nature 
and  of  pentosans. ^•'^  Organic  acids,  alcohols  and  other 
extracellular  products  commonly  found  in  cultures  of  bac- 
teria and  fungi  have  never  been  found  in  appreciable  quan- 
tities in  filtrates  from  algal  cultures. 

Extracellular  nitrogenous  products  in  cultures  of  nitrogen 
fixing  algae  have  been  noted  frequently^^'  ®^'  ®'^'  ^^'  ^*^and  it 
appears  that  the  liberation  of  such  substances  may  be  char- 
acteristic of  most  members  of  the  Myxophyceae,  whether 
they  fix  nitrogen  or  not.^^  For  Anabaena  cylindrica  the  total 
amount  of  extracellular  substances  is  relatively  greatest  in 
young  cultures,  in  which  they  may  account  for  over  50  per 
cent  of  the  nitrogen  assimilated,  and  the  substances  con- 
cerned are  evidently  not  produced  by  autolysis  of  dead  cells. 
The  liberation  of  extracellular  combined  nitrogen  appears 
invariably  to  accompany  growth  although  its  relative  extent 
varies  according  to  culture  conditions.  The  substances 
concerned  are  principally  polypeptides  but  in  young  cul- 
tures a  considerable  proportion  of  the  nitrogen  may  be 
present  as  amide. ^'^  It  is  not  known  whether  similar  sub- 
stances are  liberated  by  algae  belonging  to  other  groups 
but  such  excretion  easily  escapes  notice  in  the  usual  culture 
media  made  up  with  ample  amounts  of  nitrate  or  ammonium 
salts. 

It  may  be  agreed  that  'conservation  of  carbon  and  a  mini- 
mal level  of  excretion  is  probably  generally  characteristic 
of  the  green  algae'  and  that  in  addition  to  their  role  in 
raising  the  energy  level  of  carbon  'they  also  provide  the 
mechanism  of  carbon  accumulation  and  can  ill  afford  waste- 
ful luxuries  of  excretion'. ^^^  This  generalization,  however, 
does  not  necessarily  apply  to  species  belonging  to  other 
groups  of  algae  and  even  the  low  levels  of  excretion  reported 


THE  PRODUCTS  OF  METABOLISM        9I 

for  members  of  the  Chlorophyceae  may  become  important 
where  growth  of  these  algae  takes  place  on  a  large  scale. 
The  direct  nutritional  value  to  most  other  organisms  of 
algal  excretion  products  is  probably  slight^^^'  ^^  but  their 
indirect  effects  on  organic  production  are  perhaps  sometimes 
considerable.^^ 

CARBOHYDRATES   AND   RELATED    SUBSTANCES 

Carbohydrates  may  occur  in  considerable  proportions  in 
algae  either  as  intracellular  reserve  materials  or  as  cell  wall 
constituents  and  more  is  known  of  this  class  of  algal  product 
than  of  any  other.  Most  of  the  carbohydrates  that  will  be 
mentioned  are  built  up  from  units  containing  a  heterocyclic 
six-membered,  or  pyranose,  skeleton.  It  may  be  helpful  to 
the  reader  of  this  section  to  note  that,  in  order  that  the 
spatial  relationships  of  the  atoms  may  be  shown,  this  ring 
is  conventionally  represented  thus: 


the  plane  of  the  ring  being  supposed  to  be  at  right  angles 
to  that  of  the  paper,  with  the  thickened  edge  foremost. 
The  formation  of  this  ring  from  a  simple  chain  results  in  a 
further  carbon  atom,  that  numbered  i,  becoming  asym- 
metric. Thus  there  are  two  optical  isomers  of  the  pyranose 
form  of  D-glucose: 


HO  X^^      'V  OH  HO 


a-D-glucopyranose  j3-D-gIucopyranose 


92  THE    METABOLISM    OF    ALGAE 

Further  information  on  the  structure  and  nomenclature  of 
carbohydrates  may  be  found  in  a  number  of  books,  e.g.^-^ 

Glucose  or  other  reducing  sugars  occur  in  low  concen- 
trations in  species  of  Chlorophyceae^^"'''  ^'^^'  ^^^  but  are 
present  only  in  traces  or  are  absent  in  demonstrable  quantity 
in  representatives  of  the  Bacillariophyceae,^*^  Phaeophy- 
ceae,i2i,  127,  173  Rhodophyceae^^'  i^s,  i69  ^nd  Myxophy- 
ceae.^-^'  ^'^  Free  pentoses,  like  hexoses,  are  evidently  of 
little  quantitative  importance  in  algae.  Mannitol,  a  hexa- 
hydric  alcohol  which  may  be  derived  by  reduction  from 
mannose  or  fructose,  occurs  in  considerable  quantity  in 
species  of  Phaeophyceae,  in  which  it  may  sometimes  form 
more  than  30  per  cent  of  the  dry  weight. ^^  Mannitol  is  to 
be  found  in  only  a  few  higher  plants  but  appears  to  be  of 
general  occurrence  in  the  Phaeophyceae. ^^^'  ^^^'  ^'^  It  is 
absent  from  those  representatives  of  the  Rhodophyceae  in 
which  it  has  been  sought^^^  and  does  not  appear  to  have 
been  recorded  as  occurring  in  any  other  algal  class. 

CHoOH  CH2OH 

I  '  I 

HOCH  HOCH 

I  I 

CH 


O 


HCOH 

I 
HCOH 

I 
— CH2 


HOCH 
I 
HCOH 

I 
HCOH 

CH2OH 

D-mannitol  D-mannitan 

An  anhydride  of  mannitol,  mannitan,  has  been  reported  as 
occurring  in  Pelvetia  canaltculata.^^^  Dulcitol  and  sorbitol, 
which  are  isomeric  with  mannitol,  have  been  found  in  Bos- 
trychia  scorpioides  and  Iridaea  laminarioides  but  not  in  a 
numberof  other  species  of  Rhodophyceae.^^^' ^^^'  ^^^  Flori- 
doside,  which  is  evidently  glycerol  a-D-galactoside,  is 
abundant  and  generally  distributed  in  the  Rhodophy- 
ceae.^^' ^^"'  ^^^  It  is  remarkable  among  naturally  occurring 
glycosides  in  belonging  to  the  a-series.  In  many  species  of 


THE    PRODUCTS    OF    METABOLISM  93 

Ceramiales,     Gigartinales    and    Cryptonemiales,    sodium 
mannoglycerate  is  more  abundant  than  floridoside.^"'' 


HO 


Floridoside 

Among  oligosaccharides,  sucrose  occurs  in  appreciable 
quantities  in  Chlorella  and  Scenedesmus^^  but  does  not 
appear  to  have  been  found  in  more  than  traces  in  represen- 
tatives of  classes  of  algae  other  than  the  Chlorophyceae. 
Trehalose,  i -[a-D-glucopyranosido]-a-D-glycopyranoside, 
which  is  commonly  to  be  found  in  fungi  but  not  in  higher 
green  plants,  is  present  in  various  species  of  Myxophy- 
ceae^^^'  ^'^  and  in  freshwater  species  of  Rhodophyceae.^® 

Glucose  polymers  form  the  principal  intracellular  reserve 
products  in  several  classes  of  algae.  The  starch  of  the 
Chlorophyceae  stains  blue  or  purple  with  iodine  and  appears 
to  be  essentially  similar  to  that  of  higher  plants.  Starch 
contains  two  principal  constituents,  i.e.  amylose,  which 
consists  of  long  unbranched  chains  of  a-D-glucopyranose 
units  united  by  i  :  4  linkages,  and  amylopectin,  also  con- 
sisting of  a-D-glucopyranose  units  but  having  branched 
chains.  The  starch  of  Polytomella  caeca  contains  84  to  87 
per  cent  of  amylopectin. ^^  Floridean  starch,  which  occurs 
in  the  form  of  small  grains  staining  brown  with  iodine  or, 
on  swelling,  violet,  is  the  characteristic  reserve  product  of 
the  Rhodophyceae.^^^  It  consists  entirely  of  glucose  residues 
but  is  structurally  different  from  normal  starch  in  contain- 
ing a  large  proportion  of  i  :  3  linkages  and  is  resistant  to 
attack  by  /5-amylase.^*  Paramylum  (paramylon),  the  charac- 
teristic reserve  product  of  the  Euglenineae,  does  not  stain 
with  iodine  but  yields  mostly  glucose  upon  hydrolysis^  ^^ 
and  is  evidently  related  to  starch.  A  starch-like  product 
giving  a  blue  colour  with  iodine  either  directly^^^  or   on 


94 


THE    METABOLISM    OF    ALGAE 


boiling^*®  occurs  in  the  Cryptophyceae  and  in  Chilomonas 
paramoecium  appears  to  consist  of  about  equal  parts  of 
amylose  and  amylopectin.^^^  A  polysaccharide,  the  presence 
of  which  causes  the  chromatoplasm  to  stain  reddish  brown 
with  iodine,  generally  occurs  in  the  Myxophyceae^^' 
although  it  is  not  always  demonstrable  in  these  algae. 221 
This  polysaccharide,  which  has  been  generally  considered 
to  be  glycogen  but  which  is  perhaps  better  designated  as 
Myxophycean  starch,  evidently  occurs  in  the  cells  in  the 
form  of  submicroscopic  granules.^ '^  Oscillatoria princeps  has 
been  found  to  contain  an  enzyme  synthesizing  a  glycogen- 
like  polysaccharide  having  14  to  16  glucose  residues,  which 
may  or  may  not  be  identical  with  Myxophycean  starch, 
from  glucose- 1 -phosphate. ^•^^  Starch  and  similar  sub- 
stances appear  to  be  absent  from  the  Phaeophyceae. 

Another  type  of  glucose  polymer  is  represented  by 
laminarin,  a  soluble  reserve  product  giving  no  colour  with 
iodine  and  occurring  in  the  Phaeophyceae,  e.g.  in  Asco- 
phyllum,  Fucus  and  Laminaria  spp.  to  the  extent  of  up  to 
25  per  cent  on  a  dry  weight  basis. ^^  Laminarin  has  been 
reported  as  being  absent  from  certain  members  of  the 
Phaeophyceae,  e.g.  Pelvetia  canaliculata  and  Chorda  and 
Ectocarpiis  spp.^'^  but  this  has  been  shown  to  be  incorrect 
in  the  case  of  the  Pelvetia.^''  Normal  laminarin  is  built  up  of 
/5-D-glucopyranose  units  linked  through  C^  and  C3  Upon 
hydrolysis  with  mineral  acid  it  yields  a  disaccharide,  lamin- 
aribiose  or  3-/5-D-glucosyl  D-glucose.^®  Laminarin  prob- 
ably has  the  following  constitution:  ^^ 


CHPH 


HOH 


Laminarin  may  occur  in  another  modification,  the  relation 
of  which  to  the  normal  form  is  not  known.^^^^ 


THE  PRODUCTS  OF  METABOLISM        95 

Leucosin,  the  characteristic  reserve  product  of  the 
Chrysophyceae  and  perhaps  also  of  the  Baciilariophyceae, 
occurs  as  rounded  gUstening  granules,  soluble  in  water, 
which  do  not  stain  with  iodine. i^^'  ^"^  Nothing  is  known 
of  its  chemical   structure    but    it   is  evidently  a  polvsac- 

Charide.260.  270,  244a 

Cellulose  is  composed  of /?-D-glucopyranose  units  linked 
into  long  chains  through  the  Cj  and  C4  positions.  As  in  the 
higher  plants  it  is  the  characteristic  cell  wall  component  in 
the  Chlorophyceae  and  many  studies,  made  by  X-ray 
analysis  and  other  physical  methods,  of  cell  wall  structure 
have  utilized  members  of  this  class,  particularly  Valonia 
spp.,  as  material  e.g.,^^^  Cellulose  is  not,  however,  invari- 
ably present  in  members  of  the  Chlorophyceae. ^^^^  Cellu- 
lose, as  demonstrated  by  microchemical  tests,  is  prominent 
in  the  envelopes  of  Dinophyceae.^'^^  It  occurs  generally  in  the 
Phaeophyceae  and  Rhodophyceae,  amounting  to  2  to  15  per 
cent  of  the  dry  weight  in  these  algae^^^  and  its  fundamental 
similarity  in  the  Phaeophyceae  and  in  the  higher  plants  has 
been  confirmed  by  chemical  and  X-ray  examination.--^ 
Cellulose  occurs  sporadically  in  the  Myxophyceae  and  ap- 
pears to  be  generally  lacking  in  the  Xanthophyceae,  Chryso- 
ph^^ceae  and  Baciilariophyceae,^^^'  ^^'^  although  X-ray 
evidence  shows  its  presence  in  Tribonema.^^^^ 

Algin,  the  characteristic  intercellular  substance  of  the 
Phaeophyceae,  of  which  it  generally  forms  about  25  per 
cent  on  a  dry  weight  basis, ^^  is  a  calcium-magnesium  salt  of 
alginic  acid,  a  polymer  of  /?-D-mannuronic  acid  having  the 
following  probable  constitution:^® 


COOH 


n 


96  THE    METABOLISM    OF    ALGAE 

Because  of  its  special  colloidal  properties  alginic  acid  finds 
considerable  use  in  industry.-®^ 

A  polysaccharide  yielding  only  xylose  upon  hydrolysis 
has  been  reported  as  present  in  Rhodymenia  palmata.  This 
xylan  contains  both  i  :  3  and  i  :  4  linkages. ^-'^ 

Other  polysaccharides  constituting  the  intercellular  ma- 
terial of  marine  algae  are  composed  of  residues  esterified  by 
sulphuric  acid.  Because  of  this,  a  considerable  proportion  of 
the  ash  constituents  of  marine  algae  are  present  in  the  living 
plants  in  chemical  combination  with  the  intercellular  ma- 
terial.^^^  Such  sulphuric  esters  have  so  far  only  been  found 
in  the  Rhodophyceae  and  Phaeophyceae  and  in  animals  and 
have  not  been  reported  as  occurring  in  the  bacteria  or 
M}^ophyceae.  It  has  been  suggested  that  the  sulphate  group 
plays  a  similar  part  in  the  synthesis  of  these  polysaccharides 
as  does  the  phosphate  group  in  the  synthesis  of  starch^^® 
but  there  is  as  yet  no  evidence  to  show  whether  this  is 
actually  so. 

Agar,  or,  more  properly,  agar-agar,  is  the  most  familiar 
polysaccharide  of  this  type,  being  extensively  used  as  a 
substratum  for  the  culture  of  micro-organisms  and  for  a 
variety  of  medical  and  industrial  purposes.-"^  Its  structure 
has  not  yet  been  fully  elucidated.  Many,  but  not  all,  Rhodo- 
phyceae yield  extracts  which  gel^^^  and  the  tendency  is 
to  apply  the  term  agar  to  all  the  substances  concerned 
although  they  certainly  differ  in  chemical  constitution.  A 
commercial  agar,  probably  derived  from  Gelidium  sp.,  has 
been  found  to  contain  D-galactopyranose  units  linked  by 
I  :  3  linkages  together  with  L-galactose  residues  linked  with 
the  others  through  Cj  and  C4  and  esterified  by  sulphuric 
acid  at  Cg.^^^  These  two  components  are  respectively: 


HO 


/ 


CH2OH 


O     H 


HO 


H-     and 


THE  PRODUCTS  OF  METABOLISM 


97 


The  ratio  of  D-  to  L-galactose  residues  has  been  variously- 
reported  as  9  :  i^^^  and  15  :  2^^^  and  the  exact  constitution 
of  the  polysaccharide  is  not  yet  agreed  upon.^^^ 

Chondrus  crispus,  carragheen,  which  is  now  used  as  a 
source  of  agar,^^^  yields  two  principal  colloidal  substances, 
one  more  soluble  in  cold  water  than  the  other  and  giving  a 
viscous  solution,  and  another  forming  a  gel  more  readily.^^^ 
The  difference  in  physical  properties  between  these  two 
appears  to  be  due  to  the  former  being  a  mixture  of  sodium 
and  potassium  salts,  whereas  the  latter  is  chiefly  a  calcium 
salt,  the  polysaccharide  concerned,  carragheenin,  being 
evidently  the  same  in  both.*^  The  principal  constituents 
of  carragheenin  are  D-galactose  residues  linked  through 
Cj  and  Cg  and  each  having  a  sulphate  residue  on  C4.  Car- 
ragheenin also  contains  L-galactose  and  other  residues. ^^^ 
Gigartina  stellata  ^^  and  Iridaea  laminarioides^^'^  evidently 
contain  polysaccharides  of  a  similar  nature. 

Fucoidin^^''''  ^'^^  is  a  mucilaginous  material  extractable 


-O3S.O 


-O3S.O 


H 

OH 

H 


H  L/CH5  \ 
H  |S^  WO/ 
J{ O    O  ^j ^H 


-038.0 


OH         H 


-O3S.O 


-O3S.O 


98  THE    METABOLISM    OF    ALGAE 

from  many  Phaeophyceae,  e.g.  Fiicus  spp.  and  Laminaria 
cloustoni.  The  principal  constituents  of  this  polysaccharide 
are  L-fucose  (a  methyl  pentose)  residues  esterified  by  sul- 
phuric acid.  These  residues  are  evidently  united  chiefly  by 
I  :  2-a  links;  for  a  possible  constitution  see  p.  97.^-^'  ®^ 

There  is  some  evidence  that  chitin,  which  is  composed 
of  N-acetylglucosamine  residues  and  which  is  a  charac- 
teristic component  of  the  cell  wall  of  fungi  and  of  the 
exoskeleton  of  insects,  occurs  in  the  cell  walls  of  certain 
species  of  Xanthophyceae.^^^" 

Many  other  substances  of  a  carbohydrate  nature,  variously 
described  as  pectins,  mucilages  and  hemicelluloses,  are  to 
be  found  in  algae  but  have  not  as  yet  been  adequately 
characterized  (see  refs.  106,  107,  221,  117,  169,  170). 


LIPIDES 


Table  7  summarizes  results  of  fatty  acid  analyses  of  vari- 
ous algae.  These  results  are  in  general  agreement  with  those 
of  less  extensive  analyses  made  on  other  algae  representative 
of  the  same  classes. ^^^'  ^^'  ^^" 

The  Cjg  acid,  palmitic  acid,  appears  to  be  the  most  gener- 
ally abundant  of  the  saturated  acids.  In  algae  of  all  classes, 
unsaturated  acids  predominate,  principally  those  having 
16,  18  and  20  carbon  atoms.  An  exception  to  this  is  found 
in  the  fructifications  of  higher  marine  algae  such  as  Alaria 
crassifolia,  of  which  over  50  per  cent  of  the  fatty  acids  are 
saturated. 2' 9  Having  regard  to  the  considerable  variations 
in  the  composition  of  the  fatty  acid  fraction  that  may  occur 
in  a  single  species  (see  p.  118)  it  does  not  appear  that  there 
are  any  consistent  differences  between  the  fatty  acids  pro- 
duced by  algae  of  different  classes  and  higher  plants  growing 
in  similar  habitats. 

The  fatty  acids  of  Chlorella  sp.,  Scenedesmus  sp.  and 
Nitzschia  palea  are  present  as  triglycerides^^'  but  it  has 
been  reported  that  freshly  collected  marine  diatoms,  e.g. 
N.  closterium,  may  contain  considerable  proportions  of  free 
fatty  acids,  apparently  irrespective  of  species. ^^  An  explana- 
tion of  this  difference  has  not  yet  been  put  forward  and 


THE  PRODUCTS  OF  METABOLISM 
TABLE  7 


99 


FATTY   ACIDS,    EXPRESSED    AS    PERCENTAGES    BY   WEIGHT    OF    TOTAL 
FATTY    ACIDS,    OF    ALGAE    REPRESENTATIVE    OF    VARIOUS    CLASSES    AND 

OF   A    HIGHER    PLANT 

Degrees  of  unsaturation  are  given  in  parenthesis 


Saturated 

Unsaturated 

Class  and  species 

Cx4 

C^, 

Cx8 

Cx« 

Ci, 

C,8 

C20 

C22 

Chlorophyceae 

Chlorella  pyrenoidosa^^* 



8 

4 

27 

(-4-4H) 

61 

(-3-4H) 

"" 

~ 

Oedogonium  sp.^" 

2 

20 

I 

" 

32 

(-3-iH) 

35 
(-4-6H) 

9 

I 

Cladophora  sauteri^''^ 

12 

lO 

2 

trace 

19 
(-4-7H) 

49 
(-3-8H) 

8 
(-7-iH) 

Nitella  opaca"-'' 

6 

i8 

3 

3 
(-2H) 

34 
(-2SH) 

23 
(-4-5H) 

13 
(-5-8H) 

Bacillariophyceae 

Nitzschia  closterium^'" 

8 

17 

2 

I 

36 
(-3-4H) 

20 
(-5-3H) 

16 
(-7-oH) 

Phaeophyceae 

Fucus  vesiculosus^''^ 

9 

7 

2 

I 

(-2-Ih) 

63 
(-3-oH) 

13 

(-7-3H) 

~" 

Laminaria  digitata^'' 

6 

14 

I 

2 

II 

(-2-oH) 

42 
(-4-2H) 

24 
(-8-iH) 

Alaria  crassifolia'^''^ 

5 

19 

O 

4 

(-2H) 
12 

(-4H) 

II 
(-4H) 

(-6H) 

19 

(-8H) 

Rhodophyceae 

Rhodymenia  palniata^'^ 

4 

19 

I 

trace 

6 
(-2-9H) 

20 
(-4-5H) 

36 
(-9-2H) 

13 

Higher  Plant 

Elodea  (Anacharis) 

I 

IS 

5 

2 

25 

.      39   ^ 

12 

canadensis^'^ 

1 

(-30H) 

(-4-9H) 

(-6-oH) 

there  would  otherwise  appear  to  be  no  difference  between 
the  fats  of  marine  and  freshw^ater  algae. ^'^^ 

In  addition  to  fats,  a  fat  solvent  extract  of  any  organism 
contains  lesser  amounts  of  other  materials,  such  as  photo- 
synthetic  pigments,  sterols  and  hydrocarbons,  which  are 
unrelated  chemically  to  the  true  lipides  and  which  are 
generally  classed  as  lipoids.  Of  these,  the  photosynthetic 
pigments  have  already  been  discussed  in  an  earlier  chapter. 


100 


THE    METABOLISM    OF    ALGAE 


The  unsaponifiable  lipoid  fractions  of  all  algae  appear  to 
contain  small  amounts  of  the  paraffin  hydrocarbon  hentria- 
contane,  C3iH64.^*^'  ^^  Sterols,  similar  to  those  of  higher 
plants  which  are  grouped  under  the  generic  name  of  sito- 
sterol, have  been  found  in  the  Chlorophyceae,  Xantho- 
phyceae  and  Rhodophyceae.^*^'  ^^  A  more  well-defined 
compound,  fucosterol,  occurring  in  representatives  of  most 
algal  classes  but  not  in  higher  plants,  is  the  only  sterol 
produced  by  the  Phaeophyceae.^^  The  Chlorophyceae  are 
somewhat  variable  in  the  sterols  which  they  contain;  thus 
Oedogoniiim  sp.  contains  only  sitosterol ;^^^  Cladophora 
saiiteri  and  Nitella  opaca  contain  both  sitosterol  and  fuco- 
sterol;^^^  the  principal  sterol  of  Chlorella  pyrenoidosa  is  ergo- 
sterol,  a  sterol  which  is  otherwise  known  only  from  fungi 
and  lichens;^^^  and  Scenedesmus  ohliquiis  contains  chon- 
drillasterol  amounting  to  about  23  per  cent  of  the  unsaponi- 
fiable fraction,  apparently  to  the  exclusion  of  other  sterols. ^^ 


CH3 

I 
CgHj  3Crl2CH2^^ 


CHCH3      CH3 

II  I 

(CH  3)  2CHCCH  oCH  2CH 


HO\/\/ 
j3-sitosterol 


/\y\/~ 


HOX/X/ 
fucosterol 

C2H5 


CH, 


(CH3)2CHCH(CH3)CH:CHCH(CH3)    (CH3)2CHCHCH:CHCH 


'\/\/' 


HO\/\/ 
ergosterol 


HO\/\/ 
chondrillasterol 


THE    PRODUCTS    OF    METABOLISM 


lOI 


Sterols  appear  to  be  totally  lacking  in  the  Myxophyceae 


53 


NITROGENOUS   SUBSTANCES 

Little  is  known  of  the  proteins  of  algae  and  as  yet  only 
amino-acid  analyses  of  the  bulk  proteins  of  certain  species 
are  available.^^O'  i^^'  ^'^^  ^«'  ^^'  ^^^  The  analytical  results  pre- 
sented in  Table  8  are  not  all  of  equal  value.  In  Ulva, 
Laminaria,  Chondrus,  Microcystis  and  Phormidium,  not  all 
the  protein  nitrogen  is  accounted  for  and  the  protein  frac- 
tions examined  may  not  have  been  representative  of  the 
whole.  It  is  thus  possible  that  the  amino-acids  reported 
as  absent  in  these  algae  were,  in  fact,  present  and  in  this 

TABLE   8 

THE   AMINO-ACID    COMPOSITIONS    OF    THE    BULK    PROTEINS    OF 
VARIOUS   ALGAE   COMPARED    WITH    SIMILAR    DATA   FOR    HIGHER    PLANTS 

Amounts  are  given  as  g.  amino-acid  N/ioo  g.  protein  N 


CO 

e 

« 

00 

0 

0 
1  m 

0    Q 

0 

•r 

•« 

^ 

0 

S.8 

ea 

<3 

e 
ea 

d 

2 

s 

u 

0 

d 

CO 

<3 

0 

d 

en 
to 

1^ 

cystis  (Dt 
s)  aerugin 

*ds  wmpt 

"ds 

5 

K 
O 

.VJ 

5 

c 
0 

Micro 
cysu 

Anaba 
cylin 

Aspartic  acid 

6-4 

4-1 

51 

6-4 

1-9 

2-5 

4-6 

6-9 

0-9 

4-9-5 -4 

Glutamic  acid 

7-8 

7-6 

4-6 

4-9 

7-3 

8-2 

6-5 

5-6 

4-4 

6-6-7-8 

Serine 

3'3 

2-4 

4-2 

3'3 

2-4 

— 

— 

Threonine 

2-9 

4-0 

4-2 

— 

3-2 

5-7 

— 

30 

Glycine 

6-2 

0-8 

6-2 

6-1 

2-7 

2-1 

4*9 

5-5 

1-6 

0-4 

Alanine 

7-7 

6-5 

8-4 

6-5 

6-4 

3-7 

54 

60 

5-2 

4-4-5 -I 

Valine 

5*5 

S-2 

75 

7-5 

5-1 

2-8 

4-1 

7-0 

6-7 

3-3-4-2 

Leucine 

6i 

5-2 

6-4 

7-2 

2-5 

5-3 

4-2 

6-2 

2-1) 

7-1-8-8 

Isoleucine 

3'S 

4-1 

35 

2-2 

3-9 

—  ) 

Phenylalanine 

2-8 

2-3 

2-8 

3-4 

i-o 

1-5 

4-4 

2-9 

I-I 

2-5-26 

Tyrosine 

2-8 

o-o 

30 

I-Q 

19 

2-3 

1-6 

1-8 

2-3-2-S 

Proline 

7-2 

7-0 

6-1 

6-2 

7-6 

7-1 

3-2 

50 

7-0 

3-1 

Tryptophane 

2-1 

0-3 

1-8 

i-i 

i-i 

1-6 

X          10 

0-2 

I -8-2- 1 

Methionine 

1-4 

o-o 

1-4 

1-2 

O-O 

0-0 

1-7 

1-2 

2-0 

I -4-1 -6 

Cystine 

0-2 

1-8 

3-4 

1-6 



00 

1-3-1-5 

Arginine 

iS-8 

7-5 

159 

92 

i6-i 

IO-2 

II-7 

9-2 

13-7-14-3 

Histidine 

3'3 

1-2 

3-7 

2-8 

1-6 

1-8 

— 

25 

3-8 

3-6-3-7 

Lysine 

IO-2 

o-o 

QO 

8-3 

o-o 

4-0 

— 

6-6 

0-0 

6-3-6-6 

.\irude  N 

6-1 

6-5 

7-1 



80 

" 

4-7-5-3 

Total  N 

IOI-3 

49-S 

q8-q 

91-7 

58-6 

54-7 

47-7 

897 

46-0 

74-4 

(mean) 

102  THE    METABOLISM    OF    ALGAE 

connexion  it  should  be  noted  that  lysine,  methionine  and 
tyrosine,  reported  as  absent  in  Ulva,  are  present  in  Chlorella 
vulgaris,  the  other  species  of  Chlorophyceae  examined,  and 
similarly  cystine  and  lysine,  reported  as  absent  in  Phormi- 
diuni,  are  present  in  another  member  of  the  Myxophyceae.^^ 
It  thus  seems  most  probable  that  there  is  a  general  similarity 
in  amino-acid  composition  between  the  proteins  of  the 
various  classes  of  algae  and  of  higher  plants.  This  similarity 
cannot,  of  course,  be  taken  as  implying  that  the  individual 
proteins  of  these  various  forms  are  similar. 

Certain  amino-acids  other  than  those  mentioned  in  Table  8S 
may  be  characteristic  of  particular  classes.  All  representa- 
tives of  the  Myxophyceae  which  have  been  so  far  examined, 
i.e.  Anabaena  cylindrica,  Oscillatoria  sp.  and  Mastigocladus 
laminosus,  have  been  found  to  contain  a-£-diaminopimelic 
acid,  COOH.CH(NH2).(CH2)3.CH(NH2).COOH.  This 
amino-acid  appears  to  occur  otherwise  only  in  certain  bac- 
teria and  has  not  been  found  in  representatives  of  the 
Chlorophyceae,  Xanthophyceae,  Bacillariophyceae,  Eugle- 
nineae,  Phaeophyceae  and  Rhodophyceae  in  which  it  has 
been  sought. ^^^  The  presence  of  diiodotyrosine  has  been 
demonstrated  in  Laminaria  spp.  Diiodotyrosine  accounts 
for  only  a  small  proportion  of  the  iodine  to  be  found  in 
these  brown  seaweeds,  the  bulk  evidently  being  in  inor- 
ganic form.^^^" 

Water-soluble  peptides  occur  in  certain  marine  species 
of  Chlorophyceae,  Phaeophyceae  and  Rhodophyceae,  in 
amounts  comprising  up  to  0-73  per  cent  of  the  dry  weight 
of  the  alga.124, 126,  129, 120,  e?  j^.  ^g  not  clear  whether  these 

substances  are  comparable  in  origin  with  the  extracellular 
polypeptides  produced  by  blue-green  algae  (see  p.  90). 
They  are  not  present  in  all  marine  algae  and  there  is 
evidence  that  they  may  arise  as  a  result  of  lack  of  balance 
between  carbon  and  nitrogen  metabolism. ^^^  The  composi- 
tion of  these  peptides  varies  from  species  to  species,  e.g. 
in  Corallina  officinalis  a  pentapeptide  of  aspartic  acid  is 
present^^^'  ^^^  whereas  C.  squamata  contains  a  peptide  con- 
taining alanine  and  arginine.^^^  Only  for  Pelvetia  fastigiata 
has  the  constitution  of  the  peptide  been  established,  the 


THE    PRODUCTS    OF    METABOLISM  IO3 

material   isolated   being   L-pyrrolidonoyl-a-glutaminyl-L- 
glutamine:^^ 

CO  CO.NH2  CO.NH2 


CH2  CH2  CH2 

I  I.I 

CH2  CH2  CH2 

I  I  I 

NH— CH— CO— NH— CH— CO— NH— CH— COOH 

It  appears  possible  that  the  pyrrolidonoyl  ring  may  have 
been  formed  after  extraction  of  the  substance  and  that  in  the 
living  alga  there  is  present  tri-L-glutamine.^^ 

Other  nitrogenous  products  of  algae  have  received  little 
attention.  From  their  staining  reactions  it  is  evident  that 
algal  cells  contain  nucleic  acids  but  only  for  Polytomella 
caeca,  in  which  the  proportion  of  ribonucleic  acid  to  protein 
is  as  high  as  6  to  10  per  cent  in  actively  growing  cells,^^^ 
do  quantitative  investigations  appear  to  have  been  made. 
Cyanophycin,  a  conspicuous  reserve  product  of  the  Myxo- 
phyceae,  is  generally  stated  to  be  proteinaceous^'^^  but  has 
not  yet  been  examined  in  anything  more  than  a  superficial 
manner. 

THE  BIOCHEMICAL   CLASSIFICATION   OF   THE  ALGAE 

Many,  but  not  all,  of  the  chemical  components  of  algae 
are  characteristic  of  classes  rather  than  of  individual  species 
and  the  existence  of  biochemical  peculiarities  confirms  to 
a  remarkable  extent  the  classification  derived  on  morpho- 
logical grounds.^^^'  ^°^'  ^^^  The  photosynthetic  pigments,  in 
particular,  are  of  great  taxonomic  value,  often  affording  the 
simplest  and  most  certain  means  of  recognizing  represent- 
atives of  different  classes  (see  Table  2).  The  regular  occur- 
rence of  its  characteristic  pigments  in  different  species 
belonging  to  a  given  class  has  been  confirmed  in  several 
investigations.^^'  ^'^^  Ahhough  the  Chlorophyceae  has  been 
found  to  show  greater  variation  in  pigmentation  than  has 
generally  been  supposed,  the  variation  occurs  between 
orders  and  closely  related  species  contain  the  same  pig- 
ments.^"^ 
8 


104 


THE    METABOLISM    OF    ALGAE 


Although  often  chemically  ill-defined,  polysaccharides 
sometimes  have  a  characteristic  appearance  or  staining  reac- 
tion which  assists  in  the  identification  of  a  species.  In  some 
cases  the  characteristics  of  the  carbohydrate  metabolism  of 
algal  classes  can  be  defined  more  precisely.  The  Phaeo- 
phyceae  and  Rhodophyceae,  for  example,  are  characterized 

TABLE  9 

BIOCHEMICAL    CHARACTERISTICS    OF   THE   ALGAL    CLASSES 

(see  refs.  io6,  107  and  references  in  the  text) 


Class 

Resene 
products 

Cell  wall 
constituents 

Sterols 

Chlorophyceae 

Starch 

cellulose 

sitosterol 

fat 

pectin 

fucosterol 

chondrillasterol 

ergosterol 

Xanthophyceae 

fat 

pectin 
silica 
cellulose 
chitin  (?) 

sitosterol 

Chrysophyceae 

leucosin 

pectin 

fucosterol 

fat 

silica 

unidentified 
sterols 

Bacillario- 

fat 

silica 

fucosterol 

phvceae 

leucosin(?) 

pectin 

unidentified 

x^       • 

sterols 

Cr^-ptophyceae 

starch 

cellulose(?) 

Dinophyceae 

starch 

cellulose 

fat 

pectin 

Euglenineae 

paramylum 

none 

Phaeophyceae 

mannitol 

algin 

fucosterol 

laminarin 

fucoidin 
cellulose 

Rhodophyceae 

floridoside 

polygalactose- 

fucosterol 

mannoglycerate 

sulphate  esters 

sitosterol 

floridean 

cellulose 

unidentified 

starch 

sterols 

M\-xophyceae 

myxophycean 

pectin 

none 

starch 

cellulose 

cyanophycin 

Higher  plants 

starch 

cellulose 

sitosterol 

^               * 

fat 

pectin 

chondrillasterol 

lignin 

stigmasterol 

THE    PRODUCTS    OF    METABOLISM  I05 

by,  (i)  a  tendency  to  form  i  :  3  linkages  between  mono- 
saccharide residues  rather  than  i  :  4  Unkages  such  as  are 
found  in  other  algal  groups  and  higher  plants  and  animals; 
this  is  apparent  in  laminarin,  floridean  starch,  agar  and  car- 
ragheenin;  and  (2)  a  tendency  to  form  polysaccharide  sul- 
phate esters  as  in  fucoidin,  agar  and  carragheenin.  In  view 
of  differences  in  morphology  and  in  other  chemical  products, 
this  correspondence  between  the  two  classes  cannot  be 
taken  as  evidence  of  phylogenetic  affinity  and  perhaps  only 
reflects  the  similarity  of  the  environments  in  which  the  two 
types  have  evolved.  Other  noteworthy  peculiarities  of  the 
carbohydrate  metabolism  of  these  two  groups  are  the  for- 
mation of  a  reserve  polyglucose  with  /^-linkages  in  the 
Phaeophyceae  and  the  presence  of  an  a-glycoside,  florido- 
side,  in  the  Rhodophyceae.  Lack  of  ability  to  synthesize 
certain  carbohydrates  is  characteristic  of  some  classes.  For 
example,  starch  evidently  does  not  occur  in  the  Xantho- 
phyceae,  Chrysophyceae  or  Bacillariophyceae,  and  the  same 
three  classes  have  a  general  tendency  to  store  fats  as  reserve 
materials  rather  than  polysaccharides. 

These  and  other  biochemical  characteristics  are  sum- 
marized in  Table  9.  It  will  be  seen  from  this  table  and  that 
giving  the  characteristic  pigments  of  the  algal  classes 
(Table  2)  that  there  are  resemblances  between  certain  of  the 
classes.  These  biochemical  similarities,  together  with  mor- 
phological evidence,  indicate  an  affinity  between  the  Xantho- 
phyceae,  Chrysophyceae  and  Bacillariophyceae,  which  are 
consequently  grouped  together  as  the  Chrysophyta,  and 
between  the  Dinophyceae  and  Cryptophyceae,  which  are 
grouped  together  as  the  Pyrrophyta.^^^ 


CHAPTER    VII 
GROWTH   AND   METABOLISM 

Metabolism  and  growth  are  interdependent.  It  has  already 
been  noted  that  abiUty  to  assimilate  a  particular  substrate 
may  depend  on  the  previous  history  of  an  alga,  but  the 
clearest  examples  of  this  interdependence  are  to  be  found 
in  the  relation  which  exists  between  the  physiological  con- 
dition of  the  organism  and  the  products  of  its  metabolism 
and  it  is  with  this  relationship  that  this  chapter  must  be 
chiefly  concerned. 

An  alga  varies  in  chemical  composition,  most  notably  in 
its  content  of  major  fractions  such  as  protein,  fat  and  carbo- 
hydrate, according  to  the  stage  of  growth  which  it  has 
reached  and  to  the  conditions  under  which  growth  has 
taken  place.  Growth  and  multiplication  in  any  organism  are 
contingent  upon  the  development  of  particular  structures 
which  in  turn  depends  on  particular  chemical  conditions 
which  are  likely  to  be  more  circumscribed  the  more  complex 
the  structures  involved.  The  greatest  variation  in  chemical 
composition  is  accordingly  found  in  the  simplest  organisms. 
In  the  fungi  and  bacteria  this  variation  is  less  evident  than 
it  otherwise  might  be  because  products  of  metabolism  which 
are  not  required  for  protoplasmic  synthesis  may  be  liberated 
into  the  surrounding  medium.  In  algae,  in  which  excretion 
appears  generally  to  be  reduced  to  a  minimum,  changes  in 
intracellular  and  extracellular  conditions  are  reflected  to  a 
greater  extent  in  the  composition  of  the  cell  material.  In 
Chlorella,  for  example,  growth  of  which  is  possible  under 
a  wide  range  of  conditions,  the  chemical  composition  of  the 
cells  may  vary  to  an  extreme  extent  (see  Table  ii).  In  the 
structurally  more  complex  algae  the  range  within  which 
variation  of  this  sort  is  possible  is  reduced  as  requirements 
for  growth  and  reproduction  become  more  exacting.  The 
cases  of  simple   and   structurally   complex   algae   are  for 

1 06 


GROWTH    AND    METABOLISM  I07 

this  and  other  reasons  most  conveniently  considered 
separately. 

GROWTH   AND   METABOLISM    IN   SIMPLE   ALGAE 

Many  studies  have  been  made  of  the  growth  of  simple 
algae  in  laboratory  culture  (e.g.  ^^o,  225,  88,  254,  302,  i48)  and  it 
is  possible  to  give  a  generalized  picture  of  the  growth  cycle 
under  these  conditions.  Following  inoculation  of  an  appro- 
priate medium  with  the  alga  and  its  exposure  to  suitable 
conditions  of  light  and  temperature,  growth  in  cell  nuni- 
bers  may  begin  immediately,  as  is  generally  the  case  in 
Chlorella  spp.  and  other  Chlorophyceae,^^*^.  225  qj.  after  a  lag 
period,  as  in  Anabaena  cylindrica.^^  Growth  is  usually  expo- 
nential in  the  first  few  days,  the  cell  numbers  per  unit 
volume  of  medium  (««)  at  any  time  {t)  being  given  by  the 
expression: 

tit^riQe'^.  .  .  .      (24) 

where  n^  is  the  number  of  cells  per  unit  volume  of  medium 
at  zero  time,  e  the  base  of  natural  logarithms,  and  r  the 
relative  growth  constant.  This  expression  may  be  converted 
to  the  form: 

log««=logwo+^^    •  •  •      (25) 

and  the  accuracy  with  which  it  represents  experimental 
results  may  be  demonstrated  by  the  closeness  of  fit  of  the 
points  to  a  straight  line  when  log  rit  is  plotted  against  t. 
Following  the  exponential  phase  is  a  period  in  which  the 
relative  growth  constant  declines  continuously  and  finally  a 
stationary  phase  is  reached  in  which  there  is  no  further 
increase  in  cell  numbers  (Fig.  15).  When  other  measures 
of  growth,  such  as  bulk  or  dry  weight  of  alga  per  unit 
volume  of  medium,  are  used,  the  pictur^es  obtained  are 
generally  similar  to  that  just  described  but  differ  in  detail 
since  cell  volume  and  relative  dry  weight  are  not  constant 
throughout  the  growth  cycle  (Figs.  12  and  15). 

Algae  growing  under  natural  conditions  show  phases  of 
growth  similar  to  that  observed  in  culture,  for  example  the 
growth  of  phytoplankton  populations  during  the  spring 
'outburst'  in  lakes  generally  follows  a  nearly  exponential 


io8 


THE    METABOLISM    OF    ALGAE 


20 


)  5  10  IS 

DAYS     OF    GROWTH 

FIG.  15.  Growth  of  Chlorella  vulgaris  in  culture.  A,  log.  cell  number 
per  mm.^  of  medium;  B,  log.  volume  of  cells  (mm.^)  in  lo  ml. 
of  medium;  C,  log.  dry  weight  of  cells  as  mg.  per  10  ml.  of 
medium  ;  D,  relative  dry  weight  as  mg.  per  10^  cells;  E, 
mean  cell  diameter  in  ft  (after  ref.  225). 

course,^^'^  but  growth  rates  may  alter  in  response  to  changes 
in  conditions  other  than  those  occurring  as  a  result  of 
growth  of  the  algae  themselves. ^^^ 

A  general  feature  of  the  growth  of  microscopic  algae 
which  requires  comment  is  that  its  rate  is  generally  less 
than  that  of  the  growth  of  non-photosynthetic  micro- 
organisms. For  example,  even  after  allowing  for  the  effects 
of  differences  in  optimum  temperatures,  the  maximum 
relative  growth  rate  recorded  for  Chlorella  pyrenoidosa,  one 
of  the  most  rapidly  growing  algae  used  in  experimental 
studies,  is  less  than  one  half  as  great  as  that  of  a  yeast  or  a 
bacterium  of  the  same  order  of  size. 200  It  is  evidently  not 
carbon  assimilation  which  thus  limits  the  growth  rate  of  an 
alga.2^^  The  explanation  may  perhaps  lie  in  the  fact  that  the 
photosynthetic  apparatus  of  an  alga,  which,  as  simple 
microscopical  examination  shows,  represents  a  considerable 


_  GROWTH    AND    METABOLISM  IO9 

proportion  of  the  protoplast,  contains  a  high  proportion  of 
materials  such  as  pigments  and  other  lipoids,  which  are  inert 
in  so  far  as  they  are  directly  concerned  in  growth.  The  rela- 
tive growth  rate  of  C.  pyrenoidosa^  for  example,  depends  on 
the  protein  content  of  the  cells,  not  upon  lipoid.^"  Thus, 
whereas  under  optimum  conditions  the  substances  assimi- 
lated by  a  non-photosynthetic  cell  are  largely  used  for  the 
synthesis  of  more  material  capable  of  growth  (i.e.  'autosyn- 
thetic',  see  ref.  145),  a  large  part  of  the  synthetic  capacity 
of  an  alga  is  diverted  to  the  production  of  the  non-growing 
material  of  the  chromatophore  and  it  may  be  because  of  this 
that  the  alga  has  the  lower  rate  of  growth  under  otherwise 
comparable  circumstances.  However,  were  the  situation  as 
simple  as  this,  it  would  be  expected  that  colourless  algae 
would  show  higher  growth  rates  than  the  pigmented  species 
from  which  they  are  derived  but  this  does  not  appear  to 
be  so. 

The  chemical  kinetics  underlying  the  growth  sequence  in 
simple  algae  are  presumably  similar  to  those  which  have 
been  postulated  to  occur  in  bacteria.^^^  In  material  which 
is  not  actively  growing,  enzymes  may  have  denatured  and 
the  concentrations  of  essential  metabolites  may  have  fallen, 
so  that  a  period  of  reconstitution  is  necessary  before  expo- 
nential growth  can  begin.  In  agreement  with  this  view  it 
has  been  found  that  in  Anabaena  cylindrica  a  marked  in- 
crease in  the  amount  of  nitrogenous  substances  per  unit 
h  volume  of  algal  material  occurs  during  the  lag  and  that  the 
length  of  this  phase  increases  with  the  age  of  the  inoculum. ^^ 
The  duration  of  the  lag  in  A.  cylindrica  is  dependent  upon 
light  intensity,  being  longer  the  higher  the  intensity.  ^*^  It  is 
possible  that  the  establishment  of  the  level  of  concentra- 
tion of  nitrogenous  constituents  necessary  for  exponential 
growth  is  directly  retarded  as  carbon  assimilation  becomes 
more  intense  but  other  explanations  of  this  phenomenon 
are  possible,  e.g.  photo-oxidation  of  some  essential  meta- 
bolite may  occur  at  higher  light  intensities.  Cases  in  which 
exponential  growth  of  algae  evidently  awaits  the  develop- 
ment of  enzymes  adapted  to  particular  substrates  have 
already  been  mentioned  (p.  56). 


no  THE    METABOLISM    OF    ALGAE 

It  would  appear  that  during  exponential  growth  the  dif- 
ferent enzyme  systems  concerned  must  increase  at  equal 
rates.i^^  This  does  not  necessarily  mean  that  the  chemical 
composition  of  an  alga  should  remain  the  same  as  long  as  the 
relative  growth  factor  remains  constant.  In  fact,  the  chemi- 
cal composition  of  Chlorella  vulgaris  may  vary  during  expo- 
nential growth"-^  and  during  exponential  growth  of  Anabaena 
cylindrica  the  nitrogen  content  per  unit  volume  of  cell  ma- 
terial falls  considerably.^^  Such  variation  may  be  partly  due 
to  changes  in  amount  of  substances  not  directly  concerned 
in  growth,  particularly  reserve  products  and  cell  wall  con- 
stituents. The  variations  both  in  the  total  amount  and  in  the 
relative  proportions  of  various  fractions  of  ribonucleic  acid 
which  have  been  found  to  occur  during  exponential  growth 
of  Polytomella  caeca  are  less  easily  explained,  particularly 
since  it  has  been  reported  that  in  yeast  there  is  a  linear  rela- 
tionship between  relative  growth  rate  and  ribonucleic  acid 
content. ^^^  However,  when  exponential  growth  is  continued 
for  long  periods  then  a  constant  chemical  composition  of  cell 
material  is  approached.  In  an  apparatus  in  which  a  constant 
cell  population  per  unit  volume  of  medium  is  maintained 
indefinitely  in  exponential  growth  by  continuous  automatic 
dilution  with  fresh  medium,  cells  are  produced  having 
nearly  constant  chemical  and  physiological  characteris- 
tics.2^^»  2^2  The  type  of  algal  cell  characteristic  of  exponen- 
tial growth  that  has  continued  for  some  time  is  small,  with 
thin  cell  walls  and  little  reserve  material,  and  having 
vacuoles  in  the  protoplast  either  absent  or  inconspicuous 
and  the  chromatophores  relatively  undeveloped.  Such  cells 
have  a  high  nitrogen  content  and  it  is  evident  that  their 
metabolism  is  chiefly  directed  to  protein  and  protoplasmic 
synthesis. ^^^  Chlorella  pyrenoidosa  in  this  state  growing 
under  a  set  of  conditions  in  which  light  intensity  was  limit- 
ing has  been  found  to  have  the  following  elementary  com- 
position: C  53-0  per  cent,  H  7-5  per  cent,  O  28-5  per  cent, 
N  10-8  per  cent;^^^  this  corresponds  to  a  protein  content 
of  about  67-5  per  cent  of  the  dry  organic  matter  of  the  cells. 
From  this  analysis,  a  'formula'  for  Chlorella  may  be  ob- 
tained and,  since  extracellular  organic  products  are  negli- 


GROWTH    AND    METABOLISM  III 

gible,  an  over-all  equation  for  the  metabolism  at  this  stage 
can  be  written  as  follows: 

i-oNH4++57C02+3'4H20  -> 

C5.7H9.802.3Ni.o+6-2502+PoH+     (26) 

The  value  of  the  photosynthetic  quotient  required  by  this 
equation,  i.e.  i-io,  is  in  good  agreement  with  that  observed, 
i.e.  I -06.^^^  The  relative  growth  rate  and  composition  of 
cells  in  exponential  growth  alter,  of  course,  according  to  the 
conditions  of  culture,  e.g.  increase  in  light  intensity  pro- 
duces an  increase  in  the  proportion  of  protein. ^^'^'  ^* 

Cells  of  the  type  just  described  show  characteristic  changes 
in  metabolism  when  subjected  to  conditions  other  than  those 
under  which  growth  was  taking  place. ^^^  Chlorella  pyre- 
noidosa  cells  starved  of  carbohydrate  by  aerobic  incubation 
in  a  complete  inorganic  nutrient  medium  in  the  dark  lose 
all  detectable  starch  in  the  course  of  about  ten  hours  and 
corresponding  with  this  the  rate  of  respiration  falls  and 
approaches  a  constant  value.  The  respiratory  quotient 
characteristic  of  such  starved  cells  indicates  that  carbo- 
hydrate rather  than  protein  or  fat  forms  the  substrate  for 
respiration.  The  rate  of  photosynthesis  in  starved  cells  is 
only  about  20  per  cent  less  under  conditions  of  light  and 
carbon  dioxide  saturation  than  it  is  under  similar  conditions 
in  normal  cells.®*  It  is  algae  in  this  starved  condition  that 
have  usually  been  used  in  studies  on  oxidative  assimilation 
and  further  information  regarding  their  reactions  may  be 
found  under  this  heading  in  Chapter  III.  Cells  starved 
of  carbohydrate  are  incapable  of  assimilating  nitrate  or 
ammonium  nitrogen  (see  pp.  72  and  76)  and  on  return  to 
conditions  permitting  growth  only  show  nitrogen  assimi- 
lation after  a  period  of  photosynthesis  which  presumably 
restores  the  original  carbon /nitrogen  balance. ^^^ 

Nitrogen-deficient  cells  of  Chlorella  may  be  produced  by 
exposure  to  intensities  of  light  saturating  photosynthesis  or, 
more  effectively,  by  transference  to  a  medium  containing 
no  source  of  nitrogen.  Such  cells,  although  showing  a  re- 
duced capacity  for  photosynthesis^**  continue  to  increase 
in  dry  weight  if  exposed  to  light  and  come  to  have  a  low 


112  THE    METABOLISM    OF    ALGAE 

nitrogen  content. ^^^  On  return  to  a  medium  suitable  for 
growth  nitrogen-starved  cells  show  an  unusually  high  rate 
of  nitrogen  assimilation^^^'  -^-'  ^^-'  ^'^^  a  phenomenon  that 
has  already  been  discussed  in  Chapter  IV. 

Cells  of  various  species  of  Chlorella  and  Scefiedesmiis  have 
been  found  to  show  abnormally  high  respiratory  quotients, 
i.e.  AC02/-A02  =  i*2  to  2-0,  when  transferred  from  a 
medium  containing  glucose  and  an  ample  nitrogen  supply, 
in  which  they  were  growing,  to  one  deficient  in  available 
nitrogen. ^^®  There  is  no  evidence  that  these  high  values 
result  from  the  oxidation  of  organic  acids  but  they  may  be 
the  result  of  the  formation  of  more  reduced  products,  such 
as  fats,  from  stored  carbohydrate.  This  behaviour  is  evi- 
dently similar  to  that  of  diatoms  such  as  Pimmlaria  spp., 
which  accumulate  conspicuous  amounts  of  fat  in  the  course 
of  48  hours  following  transference  from  a  natural  environ- 
ment to  glucose  or  sucrose  solutions. ^^^ 

Determinations  of  photosynthetic  quotient  have  generally 
been  made  with  material  transferred  from  growing  cultures 
to  conditions  of  higher  light  intensity  and  the  values  avail- 
able in  the  literature  give  some  indication  of  the  direction 
of  metabolism  in  nitrogen-starved  cells  as  well  as  provid- 
ing evidence  that  algae  of  different  classes  show  similar 
metabolic  behaviour  under  these  conditions.  The  values 
given  in  Table  10  are  all  close  to  unity,  indicating  that  a 

TABLE   10 

PHOTOSYNTHETIC      QUOTIENTS      (Qi'=A02/-AC02),      WITH      THEIR 

STANDARD    DEVIATIONS,     OF    VARIOUS    ALGAE    EXPOSED    TO 

HIGH   LIGHT    INTENSITIES 

Class  Species  Qp 

Chlorophyceae     .      .      .    Chlorella  sp.^^'  i-o9±o-oo8 

C.  pyrefioidosa^^^  i-09±o-05 

HorfTiidium  flacddum-^^  i  -oi  ±0-034 

Bacillariophyceae       .      .    Nitzschia  closteriutn^'^  i-04±o-03 

N.palea-^  i-03±o-o3 
Euglenineae    ....    Englena  gracilis  var. 

bacillaris^^-'  I'oSs 

Dinophyceae.      .      .      .   Per idi fiitmi  sp.-^  i-03±o-oi 

Rhodophyceae      .      .      .    Gelidium  cartilagineum-^*  0-95 ±0-049 

Myxophyceae       .      .      .   Synechococcus  sp.^"^"  i-o8±o-03 


GROWTH    AND    METABOLISM  II3 

carbohydrate  or  similar  compound  is  the  principal  product 
of  metabolism.  A  photosynthetic  quotient  corresponding  to 
the  production  of  fat  only,  e.g.  Qp  for  the  formation  of 
glycerol  tripalmitate  =  i-42,  has  apparently  never  been  ob- 
served in  this  type  of  experiment,^^^  but  the  tendency  for 
the  observed  values  to  exceed  unity  suggests  that  a  small 
proportion  of  fat  or  protein  is  normally  produced.  Only  one 
of  the  values  given  in  Table  10,  that  for  Chlorella  sp.,  differs 
by  a  statistically  significant  amount  from  the  mean  value, 
1-045,  of  all  those  given  and  it  is  clear  that  this  deviation 
is  not  one  characteristic  of  the  Chlorophyceae  generally. 
Table  10  shows  that  representatives  of  classes  having  quite 
different  storage  products  (see  Table  9)  nevertheless  form 
photosynthetic  products  of  similar  composition  when  ex- 
posed to  similar  conditions.  This  provides  further  evidence 
for  the  view  that  the  mechanism  of  photosynthesis  is  funda- 
mentally the  same  in  all  types  of  algae. 

Nitzschia  and  Chlorella  spp.  when  deprived  of  phosphate 
show  a  pattern  of  behaviour  similar  to  that  characteristic 
of  nitrogen  defiijency.  Carbon  assimilation  continues  in 
phosphorus-deficient  cells  and  on  return  to  a  complete 
medium  there  is  an  enhanced  rate  of  phosphorus  uptake. ^^^ 
Phosphorus-deficient  cells  of  Polytomella  caeca  have  been 
found  to  show  accelerated  protein  synthesis  immediately 
following  addition  of  excess  of  phosphate  to  the  culture. 
This  is  accompanied  by  a  decrease  in  ribonucleic  acid  con- 
tent but  subsequently  there  is  an  increase  in  ribonucleic 
acid  content  the  rate  of  which  far  exceeds  that  of  protein 
synthesis. ^^*  These  effects  a^re_perhaps  to  be  interpreted^  a^s 
the  result  of  inorganir  phosphatelBeing  immediately  avail- 
ablefor  the  energy  transfer  mechanisms  of  protein  synthesis 
whereas  its  incorporation  into  nucleotides  is  a  slower 
process. 

In  cultures  made  in  a  limited  volume  of  medium  expo- 
nential growth  of  algae  sooner  or  later  ceases.  The  factors 
bringing  this  about  are  various,  the  following  being  the  more 
important:  (i)  Exhaustion  of  a  nutrient  substance  from  the 
medium;  culture  media  generally  contain  all  essential  nutri- 
ent substances  in  quantities  sufficient  for  luxuriant  growth 


114  THE    METABOLISM    OF    ALGAE 

but  trace  elements  such  as  iron  may  be  precipitated  in  an 
unavailable  form  in  alkaline  media  and  frequently  it  is  short- 
age of  this  element  which  brings  the  growth  of  algae  in 
culture  to  a  standstill.  If  precautions  are  taken  to  maintain 
trace  elements  in  an  available  form  by  means  of  complex- 
forming  agents,  much  higher  population  densities  may  be 
achieved  in  culture.^^*  In  species  with  unrecognized  re- 
quirements for  trace  elements  or  organic  growth  factors 
growth  may  cease  after  a  short  time  as  the  traces  of  the  par- 
ticular substance  concerned  introduced  with  the  inoculum 
or  by  contamination  become  exhausted.  (2)  Preferential  ab- 
sorption of  a  particular  ion  may  result  in  the  reaction  of  the 
medium  becoming  inimical  to  growth.  Effects  of  this  sort 
produced  by  ammonium  nitrogen  have  already  been  men- 
tioned, another  example  is  that  of  Chilomonas,  which  by 
absorption  of  weak  organic  acids  without  equivalent  intake 
of  cations  causes  the  medium  in  which  it  is  placed  to  become 
too  alkaline  to  support  growth. ^^'  (3)  Accumulation  of 
growth  inhibiting  products  of  metabolism;  autoinhibition 
of  growth  by  metabolic  products  accumulating  in  the  me- 
dium has  been  demonstrated  for  Nostoc  punctiforme}^'^  a 
strain  of  Chlorella  'vidgaris^'^^  and  Nitzschia  palea.^^  The 
autoinhibitor  of  Chlorella,  'chlorellin',  may  be  a  product  of 
the  photo-oxidation  of  fatty  acids  but  rigorous  proof  of 
this  is  lacking.^®^  Those  of  Nostoc  and  Nitzschia  do  not 
appear  to  have  been  studied  in  detail.  (4)  The  increasing 
density  of  the  culture  may  reduce  the  penetration  of  light 
into  the  bulk  of  the  medium  so  that  photosynthesis  be- 
comes insufficient  to  maintain  exponential  growth. 

In  natural  environments  interactions  between  different 
species  almost  invariably  occur  and  it  is  rarely  possible  to 
ascribe  the  cessation  of  growth  of  a  particular  alga  to  the 
operation  of  any  single  physico-chemical  factor.  However, 
in  an  example  which  has  been  examined  with  particular 
thoroughness,  that  of  Asterioiiellu  forniosa  in  the  .Engli.sh. 
lakes,  it  appears  that  it  is  depletion  of  silica  in  the  water 
which  usually  limits  growth  in  the  spring.^^^ 

When  exponential  growth  ceases  the  course  of  metabolism 
of  an  alga  alters  considerably.  An  example  which  may  be 


GROWTH    AND    METABOLISM 


115 


conveniently  taken  first  is  that  of  Chlorella  vulgaris  growing 
under  conditions  in  which  neither  carbon  nor  nitrogen 
assimilation  is  limited. ^^^  The  changes  occurring  in  the 
amounts  of  various  components  in  cultures  of  this  alga  are 
shown  in  Fig.  16.  At  the  end  of  the  exponential  phase  (8 


DAYS      OF     GROWTH 

FIG.  16.  Amounts  per  culture  of  different  components  of  Chlorella 
vulgaris,  plotted  logarithmically  and  adjusted  as  to  level  for 
comparative  purposes.  A,  disaccharide;  B,  starch;  C,  insoluble 
carbohydrate;  D,  protein  (after  ref.  225). 

days)  the  relative  rates  of  accumulation  of  both  protein  and 
carbohydrate  decline  but  the  former  decreases  more  than 
the  latter.  Later,  when  the  stationary  phase  is  reached  (16 
days),  protein  synthesis  ceases  altogether  but  starch  con- 
tinues to  be  accumulated.  As  a  result,  cells  harvested  in  the 
stationary  phase  are  of  different  chemical  composition  to 
those  taken  at  the  end  of  the  exponential  phase,  e.g.  at 
20  days  15-3  per  cent  of  the  dry  matter  is  protein  and  21-5 
per  cent  is  starch  as  compared  with  24-4  per  cent  of  protein 
and  15-5  per  cent  of  starch  at  7  days.  Corresponding  with 


Il6  THE    METABOLISM    OF    ALGAE 

this,  the  microscopical  appearance  of  the  cells  changes,  the 
characteristic  form  at  the  beginning  of  the  stationary  phase 
being  large,  with  vacuolated  protoplasm,  extensive  dark 
green  chromatophore,  and  thick  walls.  The  composition  as 
well  as  the  total  amounts  of  the  major  chemical  fractions 
may  show  changes.  Thus  the  end  of  the  exponential  phase 
is  marked  by  a  replacement  of  hexose  by  disaccharide  in  the 
soluble  carbohydrate  fraction  and  in  senescent  cultures  there 
is  a  tendency  towards  hydrolysis  of  starch  with  a  corre- 
sponding increase  in  disaccharide  (see  Fig.  i6).  The  com- 
position of  the  protein  of  C.  'vulgaris  growing  under  the 
same  conditions  does  not  change  to  any  great  extent  after 
the  cessation  of  exponential  growth;  the  most  marked  effect 
being  an  increase  in  histidine  content  as  cultures  age.^'^ 
Similar  changes  have  been  observed  in  other  algae.  In 
Chlorella  pyrenoidosa,  growing  in  a  purely  mineral  medium 
which   in   the    later   stages   of   growth   became    nitrogen 
deficient,  the  protein  content  has  been  observed  to  fall 
from  over  60  per  cent  of  the  dr}'  weight  in  the  exponential 
phase  to  less  than  10  per  cent  in  the  post-exponential  phase. ^'^ 
In  another  organism,  Polytomella  caeca,   exponential  in- 
crease in  ribonucleic  acid  continues  for  a  while  after  the 
cessation  of  exponential  increase  in  protein  with  the  result 
that  cells  at  the  beginning  of  the  stationary  phase  have  a 
high  ribonucleic  acid  content.^^^ 

The  accumulation  of  lipide  in  algae  which  have  ceased 
active  growth  has  been  noted  frequently.  Beijerinck  first 
showed  by  qualitative  methods  that  deficiency  of  nitrogen 
is  the  principal  factor  promoting  fat  accumulation  in 
diatoms-®  but  detailed  quantitative  information  is  so  far 
available  only  for  Chlorella  pyretioidosa.^^'^^  ^^^'  ^"  The  lipide 
content  of  this  alga  can  be  varied  experimentally  by  changes 
in  any  one  of  several  environmental  conditions,  but  the  extent 
of  the  variation  is  limited  primarily  by  the  concentration  of 
available  nitrogen  in  the  medium.  The  effect  of  the  initial 
concentration  in  the  medium  of  ammonium  nitrogen  on 
the  lipide  content  of  C.  pyrenoidosa  is  shown  in  Fig.  17; 
only  if  this  concentration  becomes  reduced  below  o-ooi  M 
in  the  course  of  growth  is  lipide  accumulation  considerable. 


GROWTH    AND    METABOLISM 


117 


Somewhat  more  lipide  is  accumulated  if  ammonium  rather 
than  nitrate  nitrogen  is  suppUed  and  high  light  intensities 
and  low  concentrations  of  free  oxygen  also  favour  fat  for- 
mation. Other  factors,  e.g.  phosphate  concentration,  tem- 
perature and  carbon   dioxide  supply,   are  without  great 


/•5  3-0  4-5  6-0  7-5 

M/LL/ MOLES  OF  AMMONIUM  CHLORIDE  PER  LITRE 


90 


FIG.  17.  Effect  of  the  initial  nitrogen  concentration  in  the  culture 
medium  on  the  lipide  content  of  Chlorella  pyrenoidosa  (after 
ref.  264). 

effect. 264  Extremely  high  proportions  of  lipide  are  stored 
in  cells  subjected  to  prolonged  incubation  under  conditions 
of  nitrogen  deficiency,  as  is  shown  in  Table  11,  but  such 
cells  are  not  degenerate  and  will  resume  growth  if  trans- 
ferred to  a  suitable  medium. ^^^ 


ii8 


THE    METABOLISM    OF    ALGAE 


TABLE    11 
COMPOSITION  OF  Chlorella  pyrenoidosa  growtst  under  different 

CONDITIONS 

(data  from  ref.  264) 


Initial  cone. 

of  N  available 
in  medium 

Age  in 
days 

%  of  dry  weight 

Protein 

Carbohydrate 

Lipide 

0-0250  M 
0-00225  M 
0-00308  M 

17 
15 
15 
63 

5S 

50 
26 

9 

37 
32 

25 
6 

5 
18 

49 
85 

The  proportion  which  fatty  acids  form  of  the  total  lipide 
increases  as  the  lipide  content  of  C.  pyrenoidosa  increases 
and  there  is  a  corresponding  decrease  in  the  proportion  of 
unsaponifiable  material  (Table  12).  Chlorophyll  degrada- 
tion products  account  for  about  half  the  water-soluble 
saponification  products  from  the  samples  with  the  lower 


TABLE    12 

ANALYSIS    OF    LIPIDE    FRACTIONS    OF    SAMPLES    OF    Chlorella 

pyrenoidosa  of  different  total  lipide  contents ^^^ 


Lipide  as  ^0  of  dry  \vt. 

23-4 

33-2 

63-0 

75-5 

Fatty  acids  as  %  of  lipide 

28-0 

49-5 

830 

86-8 

Unsaponifiable  material  as  % 

I2-0 

7-7 

3-3 

3-3 

of  lipide 

Water-soluble  saponification 

600 

42-8 

13-7 

99 

products  as  %  of  lipide 

total  lipide  contents  but  the  chlorophyll  content  of  Chlorella 
decreases  rapidly  as  the  lipide  content  increases.  As  the 
lipide  content  of  Chlorella  increases  there  is  a  decrease  in 
the  degree  of  unsaturation  of  the  fatty  acids. ^^* 

The  conclusion  based  upon  microscopical  examination 
that  fat  accumulation  is  characteristic  of  certain  classes  of 
algae  is  borne  out  by  the  little  quantitative  information  that 


GROWTH    AND    METABOLISM  II9 

is  available.  High  lipide  contents  have  been  recorded  for 
representatives  of  the  Xanthophyceae^*  and  the  Bacilla- 
riophyceae^^^  whereas  the  Phaeophyceae,^^^'  2'^»  ^^"^  Rhodo- 
phyceae^^^  and  Myxophyceae^*  rarely  have  a  total  lipide 
content  of  more  than  5  per  cent.  In  view  of  the  results 
obtained  with  Chlorella  pyrenoidosa  and  since  various 
species  of  Chlorophyceae  and  a  diatom  have  been  found  to 
contain  approximately  equal  amounts  of  lipide  after  growth 
under  similar  conditions,^^''  it  has  been  suggested  that  the 
accumulation  of  lipide  which  is  regarded  as  characteristic 
of  classes  such  as  the  Bacillariophyceae  is  dependent  more 
on  the  environmental  conditions  under  which  such  species 
habitually  grow  than  on  genetically  determined  peculiari- 
ties of  metabolism.^^^  That  inherent  differences  in  capacity 
to  accumulate  lipide  do,  nevertheless,  exist  among  the  algae 
is  shown  by  the  fact  that  certain  Myxophyceae  do  not 
accumulate  lipide  even  under  conditions  of  nitrogen 
deficiency.^* 

The  general  explanation  of  the  phenomena  just  described 
lies  in  the  fact  that  carbon  assimilation  may  continue  in  an 
alga  after  cell  division  has  been  brought  to  a  standstill. 
This  much  was  pointed  out  by  Beijerinck  in  1904^^  but  it 
is  now  possible  to  put  forward  more  detailed  evidence  in 
support  of  this  idea  and  to  suggest  mechanisms  for  some  of 
the  observed  effects. 

Many  of  the  factors  acting  to  bring  about  the  cessation  of 
exponential  grov^h  are  without  direct  effects  on  photo- 
synthesis or  on  the  chemotrophic  assimilation  of  carbon. 
Thus,  synthesis  of  fresh  protoplasm  cannot  occur  in  the 
absence  of  a  sufficient  supply  of  nitrogen  whereas  chemo- 
trophic assimilation  of  carbon  can  continue  under  these 
conditions.  Photosynthesis  is  reduced  but  not  totally  in- 
hibited by  nitrogen  deficiency.^**'  ^^^'  ^^^  Similarly  defi- 
ciency of  most  other  nutrient  substances  except  carbon 
dioxide  or  the  organic  carbon  source  affects  protoplasmic 
synthesis  more  directly  than  carbon  assimilation.  Photo- 
synthesis has  been  found  not  to  be  directly  affected  by 
changes  in  reaction  within  widely  separated  limits, '^^'  ^^^'  ^^ 
whereas  the  growth  of  many  algae  can  only  take  place  within 

9 


120  THE    METABOLISM    OF    ALGAE 

a  more  restricted  range  of  hydrogen  ion  concentra- 
tion (e.g.  ^^'  2^).  The  autoinhibitor  produced  by  Nitzschia 
palea  appears  specifically  to  block  mitosis  without  affecting 
assimilation.^^  This  does  not  mean  that  the  capacity  to 
assimilate  carbon  does  not  decrease  as  cultures  age.  A  con- 
tinuous increase  in  the  minimum  quantum  requirement 
has,  in  fact,  been  shown  to  occur  as  cultures  of  Chlorella 
3ggi63  2^^(^  ^]^g  maximum  rate  of  photosynthesis  of  which 
Chlorella  cells,  taken  in  the  stationary  phase,  are  capable 
has  been  found  to  be  from  a  quarter  to  less  than  one-tenth 
of  that  which  cells  in  exponential  growth  can  achieve. ^^^'  ^^^ 
A  water-soluble  substance  inhibiting  photosynthesis,  which 
may  perhaps  be  the  agent  bringing  about  this  decrease  in 
efficiency,  has  been  extracted  from  cells  from  old  cultures  of 
Chlorella?^^  Nevertheless,  the  results  of  many  different 
workers  (e.g.  ^^^'  ®^'  ^°^'  ^®'*)  show  that  photosynthesis  and 
chemotrophic  assimilation  of  carbon  continues  in  cultures 
in  which  all  cell-division  has  ceased. 

It  has  already  been  emphasized  that  assimilated  carbon 
enters  directly  into  a  system  of  intermediates  common  to 
the  major  metabolic  cycles  and  that  therefore  it  may  be  used 
directly  for  the  synthesis  of  carbohydrate,  lipide  or  protein. 
When  cell-division  is  no  longer  possible,  carbon  which 
would  otherwise  be  used  for  the  synthesis  of  protoplasmic 
constituents  will  flow  along  other  channels  and  accumulate 
as  reserve  products.  In  cells  in  which  grouth  has  ceased  as 
a  result  of  deficiency  of  nitrogen,  the  reserve  materials 
formed  will  necessarily  be  carbohydrate  or  lipide  rather 
than  protein  and,  in  general,  protein  synthesis  appears  to 
be  more  sensitive  to  adverse  conditions  than  synthesis  of 
lipide  or  carbohydrate. 

It  may  be  supposed  in  the  first  instance  that  in  those 
forms  which  characteristically  store  lipide,  lipide-synthe- 
sizing  enzymes  are  more  effective  in  competing  for  the  pri- 
mary products  of  photosynthesis  than  are  those  which 
synthesize  carbohydrate.  Nevertheless,  there  is  the  con- 
tradictor\'  fact,  already  commented  upon  (p.  113),  that 
the  photosynthetic  quotient  under  conditions  of  relative 
nitrogen   deficiency  of  algae  of  this  type,   e.g.   diatoms, 


GROWTH    AND    METABOLISM  121 

corresponds  to  the  formation  of  carbohydrates  as  the  prin- 
cipal photosynthetic  products.  Because  of  this  it  has  been 
supposed  that  Hpide  formation  does  not  take  place  directly 
from  the  primary  products  of  photosynthesis^^  but,  as  we 
have  already  seen  (p.  44),  this  does  not  appear  to  be  so. 
Measurements  of  photosynthetic  quotients  appear  never  to 
have  been  made  using  material  known  with  certainty  to  be 
capable  of  accumulating  lipide  in  quantity  and  it  seems 
likely  that  the  explanation  of  the  apparently  discrepant 
values  is  that  diatoms  transferred  from  growing  cultures  to 
a  medium  deficient  in  nitrogen  are  not  in  a  condition  for  the 
immediate  rapid  synthesis  of  lipide.  It  is  to  be  expected 
that  in  a  growing  organism  the  enzymic  equipment  will  be 
adapted  to  produce  protoplasmic  constituents  and  that  this 
system,  if  transferred  to  conditions  under  which  growth 
cannot  take  place,  will  not  immediately  be  reorganized  and 
will  meanwhile  produce  such  temporary  reserve  products 
as  can  be  synthesized  with  the  enzymes  available.  This  sup- 
position is  not  necessarily  inconsistent  with  the  idea  that  the 
high  respiratory  quotient  of  growing  cells  transferred  to 
nitrogen  deficient  conditions  indicates  fat  formation  (p.  112). 
Fat  formation  might  proceed  at  the  same  rate  in  photo- 
synthesizing  cells  but  at  high  light  intensities  its  effect  on 
the  photosynthetic  quotient  would  be  slight.  On  prolonged 
incubation  under  nitrogen  deficient  conditions  or  when  the 
transition  to  conditions  under  which  growth  cannot  occur  is 
less  abrupt,  as  during  the  ageing  of  a  culture,  there  is  oppor- 
tunity for  the  enzymic  equipment  to  be  reorganized  for  the 
production  of  high  proportions  of  lipide  from  excess  primary 
products  of  photosynthesis. 

This  hypothesis  receives  support  from  the  finding  that 
different  nitrogen  fractions,  including  enzymes,  in  micro- 
organisms are  affected  to  different  extents  by  nitrogen- 
starvation^^^  and  from  determinations  of  the  relationship 
between  the  lipide  and  total  nitrogen  contents  of  various 
algae.  ^*  In  Fig.  18  the  fatty  acid  contents  of  six  species  have 
been  expressed  as  a  percentage  of  the  total  content  of  re- 
serve material  Ctaken  as  dry  material  other  than  protein) 
and  plotted  as  a  function  of  nitrogen  content.  From  this  it 


122 


THE    METABOLISM    OF    ALGAE 


0  l23'f-56789 

N/TROGEN     AS    PER     CENT     OF    DRY    WEIGHT 

FIG.  1 8.  Production  of  fatty  acids  by  various  algae  as  a  function  of 
their  nitrogen  contents  (after  ref.  94). 


will  be  seen  that  not  only  is  the  fatty  acid  production  of  the 
various  species  different  when  compared  at  corresponding 
nitrogen  contents  but  that  the  capacity  of  a  species  to  syn- 
thesize fat  is  not  the  same  at  all  levels  of  cell  nitrogen.  The 
manner  in  which  the  capacity  for  fat  synthesis  varies  in 
relation  to  nitrogen  content  is  different  in  different  species. 


GROWTH    AND    METABOLISM  I23 

In  Chlorella  vulgaris^  at  least  within  certain  limits  and 
under  the  particular  experimental  conditions  used,  the  pro- 
portion which  fatty  acids  form  of  the  total  reserve  material 
changes  little  as  the  nitrogen  content  of  the  cell  varies.  In 
Monodus,  Trihonema  and  C.  pyrenoidosa,  however,  a  decrease 
in  nitrogen  content  produces  a  considerable  alteration  in 
favour  of  lipide  synthesis,  whereas  in  Anahaena  cylindrica 
and  Oscillatoria  sp.  there  is  a  less  marked  trend  in  the 
opposite  direction.  From  this  it  appears  that  algae  which 
characteristically  store  lipide  are  able  to  develop  the 
mechanism  for  converting  the  primary  products  of  photo- 
synthesis into  lipide  on  nitrogen  starvation  whereas  in  other 
algae  the  balance  of  the  lipide  and  carbohydrate  synthe- 
sizing systems  is  not  altered  to  any  great  extent  by  nitrogen 
deficiency. 

GROWTH  AND   METABOLISM   IN   THE   HIGHER  ALGAE 

The  changes  in  metabolism  which  occur  during  the 
growth  of  a  structurally  complex  plant  are  in  a  general  way 
similar  to  those  which  have  just  been  described  as  occurring 
in  cultures  of  simple  algae. ^^^  However,  in  most  multi- 
cellular organisms  the  situation  is  complicated  by  the 
specialization  of  tissues  for  particular  functions.  Thus,  while 
a  growing  point  of  one  of  the  higher  algae  may  show  the 
same  type  of  metabolism  as  a  unicellular  alga  in  the  expo- 
nential phase  of  growth,  the  metabolism  of  other  tissues 
will  be  more  equivalent  to  that  of  a  population  of  single 
cells  in  the  stationary  phase.  The  activities  of  the  two  types 
of  tissue  cannot  be  considered  entirely  separately  since 
in  the  intact  plant  the  one  will  be  interrelated  with  the 
other. 

Studies  of  the  growth  and  metabolism  ofthe  larger  algae, 
most  of  which  are  seaweeds  belonging  to  the  classes 
Chlorophyceae,  Phaeophyceae  or  Rhodophyceae,  have  been 
restricted  because  of  the  difficulty  of  growing  these  plants 
under  controlled  conditions.  Detailed  accounts  of  growth 
appear  to  exist  only  for  certain  Laminariaceae  and 
Fucaceae.  (e.g.  ^^2, 220^^  Since  a  fair  amount  of  information 
on  both  growth  and  chemical  constitution  is  available  for 


124  THE    METABOLISM    OF    ALGAE 

Laminaria  saccharina,  this  alga  has  been  selected  for  de- 
tailed consideration  here. 

Lamiimria  saccJiarina,  which  grows  at  and  just  below  low 
tide  level,  consists  of  a  broad  ribbon-like  frond  arising  from 
a  stipe  terminating  in  the  holdfast  by  means  of  which  it  is 
attached  to  the  substratum.  This  plant  is  the  sporophyte; 
the  microscopic  gametophyte  generation  with  which  it 
alternates  need  not  concern  us  here.  Sporophytes  are  pro- 
duced at  all  times  of  the  year  but  generally  it  is  only  spring 
sporophytes  which  persist  until  maturity.  On  the  British 
coast  their  life  span  does  not  exceed  three  years.  Growth  of 
the  plant  is  continuous  throughout  life  but  shows  seasonal 
variation,  being  most  rapid  from  January  to  June.  Gro\\th 
also  changes  with  the  age  of  the  sporophyte,  the  maximum 
both  in  rate  and  total  amount  being  reached  during  the 
second  period  of  rapid  growth.  Tissue  is  worn  away  con- 
tinuously from  the  distal  end  of  the  frond  so  that  normally 
the  age  of  the  oldest  frond  tissue  is  not  more  than  5  to 
7  months.  Reproductive  tissue  develops  when  the  plants 
are  from  8  to  12  months  old.^'^^ 

Analyses  of  L.  saccharina  have  been  made  using  samples 
taken  from  naturally  occurring  populations  and  including 
plants  of  all  ages.  The  changes  which  can  be  followed  are 
thus  seasonal  ones  in  average  chemical  composition  rather 
than  those  which  occur  in  the  life  cycle  of  the  individual 
plant.  During  the  period  of  rapid  growth  both  the  mean 
fresh  weights  and  mean  total  protein  content  of  the  plants 
increase  in  spite  of  the  continuous  wearing  away  of  the 
fronds.  Protein  content  expressed  as  a  percentage  of  dry 
weight  is  highest  at  the  beginning  of  this  phase  (Fig.  19) 
but  falls  progressively  as  the  nitrate  in  the  seawater  becomes 
exhausted.  The  contents  of  mannitol  and  laminarin  show 
reciprocal  changes  to  those  of  protein  and,  in  general, 
laminarin  is  almost  completely  absent  when  growth  is  most 
rapid.  There  is  a  decrease  in  growth  rate  in  June  to  July, 
coinciding  with  the  minimum  of  phosphate  and  nitrate 
concentrations  in  the  seawater,  and  loss  of  material  from 
the  fronds  then  equals  or  exceeds  the  gains.  Photosynthesis 
continues  at  a  high  rate  for  about  two  months  after  this 


GROWTH    AND    METABOLISM 


125 


^^ 


^O 


i 

Q 
O 


36-. 


32 


28- 


2^- 


8 


O 


20-       y""\ 


16 


I2r- 


:  ASH 


/O      12       2 
!94  7 


10      12        2 
1943 
MONTH   OF  YEAR 


8      /O 


FIG.  19.  Seasonal  variation  in  the  amounts  of  various  chemical  con- 
stituents in  whole  plants  of  Laminaria  saccharina  from  a 
Scottish  loch  (after  ref.  37). 


126  THE    METABOLISM    OF    ALGAE 

point,  as  is  shown  by  the  continued  increase  in  mannitol 
and  laminarin  contents.  A  temporary  fall  in  mannitol  con- 
tent which  is  sometimes  observed  in  August  may  be  due 
to  the  effect  of  high  temperatures  in  increasing  the  rate  of 
respiration  relative  to  that  of  photosynthesis.  In  the  autumn, 
decreasing  light  intensity  brings  about  a  reduction  in  the 
rate  of  photosynthesis  but  the  concentration  of  nitrate 
available  is  then  rising  so  that  the  mannitol  and  laminarin 
contents  fall  whereas  that  of  protein  increases. ^^'  ^^'  ^^ 

The  extent  of  the  variations  in  the  chemical  composition 
of  L.  saccharina  depends  on  environmental  conditions,  being 
least  in  the  open  sea  and  greatest  in  lochs  and  other  situa- 
tions where  temperature  and  the  composition  of  the  sea- 
water  are  less  constant.^'  In  general  the  mannitol  content 
increases  with  depth  of  immersion  of  the  weed  down  to 
6  to  10  metres  and  decreases  below  this.  The  maximum  in 
mannitol  content  appears  to  correspond  w^ith  a  maximum 
in  intensity  of  photosynthesis.  Laminarin  content  tends  to 
decrease  progressively  with  depth  but  other  constituents, 
such  as  alginic  acid,  protein  and  ash,  remain  at  a  level  which 
is  more  or  less  independent  of  depth  of  immersion. ^^ 

These  variations  in  chemical  composition  shown  by  L. 
saccharina  are  less  extreme  than  those  shown  by  unicellular 
algae.  For  example,  the  extreme  recorded  limits  of  variation 
of  the  protein  content  of  L.  saccharina  and  other  brown  sea- 
weeds are  3  and  15  per  cent  of  the  dry  weight^^'  ^^'  ^'^  which 
is  a  much  narrower  range  than  that  shown  by  Chlorella 
pyrenoidosa  (Table  11).  However,  the  variations  just  des- 
cribed for  L.  saccharina  are  those  taking  place  in  whole 
plants.  If  blades  and  stipes  are  analysed  separately  it  is 
found  that  the  variations  occurring  in  the  blades  cover  a 
wider  range  than  those  of  the  plant  as  a  whole.  Fluctuations 
in  the  chemical  composition  of  the  stipes  follow  those  in 
the  blades  but  their  range  is  smaller.  Laminarin  is  absent 
from  the  stipes  throughout  the  year.^^'  ^'  This  state  of 
affairs  is  to  be  expected  since  growth  and  photosynthesis 
are  more  intense  in  the  blade.  Appreciable  translocation  of 
material  can  evidently  occur  in  species  of  Laminariales 
and  in  the  largest,  e.g.  Macrocystis  spp.,  the  holdfast  and 


GROWTH    AND    METABOLISM  I27 

lower  part  of  the  stipe  are  entirely  dependent  on  products 
of  photosynthesis  translocated  from  above. ^^'^ 

Both  seasonal  and  diurnal  fluctuations  of  the  mannitol 
content  of  Phaeophyceae  are  less  extreme  than  those  in 
laminarin  content. ^^^'  ^^'  ^'^'  ^^  The  enzymic  interconver- 
sion  of  mannitol  and  laminarin  does  not  appear  to  have  been 
demonstrated,  but  it  seems  from  this  behaviour  that  they 
constitute  a  reserve  substance  system  in  which  their  roles 
are  analogous  to  those  of  sugar  and  starch  respectively  in 
higher  plants.  Fluctuations  in  the  amount  of  another  carbo- 
hydrate, alginic  acid,  however,  parallel  those  in  protein 
content  rather  than  those  in  mannitol  and  laminarin,  apart 
from  a  tendency  to  a  second  maximum  in  autumn  (Fig.  19). 
Two  maxima  in  the  amounts  of  cellulose  expressed  upon  a 
dry  weight  basis  occur  during  the  year.  The  cellulose  con- 
tent of  L.  saccharina  is  at  a  maximum  in  spring,  falling  to  a 
minimum  during  the  period  of  rapid  growth,  reaching  a 
second  maximum  in  autumn  and  falling  again  in  the 
winter.^®  These  fluctuations  in  cellulose  and  alginic  acid 
are  evidently  due  to  the  rate  of  deposition  of  these  cell  wall 
constituents  being  considerably  slower  than  that  of  forma- 
tion of  protoplasmic  material  during  the  period  of  rapid 
growth.  The  ash  constituents  of  L.  saccharina  are  present 
in  the  living  plant  as  salts  accumulated  by  the  protoplasts 
and  bases  associated  with  the  intercellular  sulphuric  acid 
esters.  It  is  to  be  expected  therefore  that  the  seasonal  varia- 
tion in  their  amount  should  follow  those  of  protein  and 
alginic  acid  (Fig.  19).  The  substances  that  have  so  far  been 
mentioned  in  this  account  of  variations  in  chemical  com- 
position of  L.  saccharina  together  make  up  over  90  per  cent 
of  its  dry  weight.  Fluctuations  in  minor  constituents  such 
as  lipides  do  not  appear  to  have  been  studied. 

The  pattern  of  metabolism  in  other  Phaeophyceae 
appears  to  be  substantially  the  same  as  that  which  has  been 
described  for  L.  saccharina^  both  in  sub-littoral,  e.g.  L. 
digitata,  L.  cloustoni  2Lnd  Saccorhiza  bulbosa,^^'  ^^  and  littoral 
species,  e.g.  Ascophyllum  nodosum  and  Fucus  spp.^^'  ^^'  ^^® 
In  the  littoral  species  the  tendency  seen  in  the  algae  of 
the  sub-littoral  zone  towards  more  than  one  maximum  in 


128 


THE    METABOLISM    OF    ALGAE 


mannitol  and  laminarin  contents  during  the  year  is  more 
pronounced  (Fig.  20).  These  fluctuations  are  due  to  the  inter- 
actions of  a  number  of  factors;  degree  of  exposure,  temper- 


o 

i 


X 

^ 


3        4       S        6        7       8       9 
MONTH     OF     YEAR 

FIG.  20  Seasonal  variation  in  the  amounts  of  laminarin  in  various 
littoral  algae.  The  algae  occur  in  the  following  order  upwards 
from  low  tide  level:  Fucus  serratus,  F.  vesiculosus,  F.  spiralis 
and  Pelvetia  canaliculata  (data  from  ref.  35). 


GROWTH    AND    METABOLISM  129 

ature  and  formation  of  fructifications  being  perhaps  of  most 
importance.  The  minimum  in  laminarin  content  occurring 
in  July  and  August  is  almost  certainly  the  result  of  the 
reduction  in  photosynthetic  activity  which  is  known  to  be 
brought  about  by  increased  desiccation.^^^  The  increase 
in  this  effect  with  degree  of  exposure  is  shown  in  Fig.  20, 
from  which  it  will  be  seen  that  the  early  summer  maximum 
occurs  sooner  and  the  summer  minimum  is  more  pro- 
nounced the  higher  the  position  which  the  species  occupies 
on  the  shore.  Judging  by  the  behaviour  of  simple  algae  as 
described  earlier  in  this  chapter,  it  seems  unlikely  that  ex- 
haustion of  nutrients  such  as  nitrate  and  phosphate  from 
the  seawater  would  affect  photosynthesis  directly  and  lead 
to  the  summer  minimum  in  laminarin  and  mannitol,  as  has 
been  suggested. ^^  Differences  in  lipide  metabolism  of 
various  Phaeophyceae  are  also  correlated  with  depth  of 
immersion,  e.g.  Pelvetia  canaliculata,  which  grows  at  high- 
water  mark,  has  been  found  to  contain  6-2  per  cent  of  fat, 
whereas  Laminaria  digitata,  which  grows  below  low  water 
mark,  has  been  found  to  contain  only  o-i6  per  cent.^^^ 
These  differences  may  depend  on  the  decreasing  amounts 
of  nitrogen  available  to  plants  growing  higher  up  the  shore 
but  other  factors  besides  this  are  almost  certainly  involved. 
Few  studies  of  seasonal  variation  in  chemical  constitution 
have  been  made  with  representatives  of  the  Rhodophyceae. 
In  conformity  with  the  results  obtained  with  Phaeophyceae 
the  carbohydrate  content  of  Chondrus  crispus  has  been  found 
to  be  lowest  in  early  spring  and  highest  from  June  to 
September.  ^1  The  agar  extracted  from  Gigartina  in  autumn 
gives  a  gel  of  greater  strength  than  that  extracted  in 
spring,!^^  a  variation  that  is  perhaps  related  to  seasonal 
changes  in  the  type  of  sulphuric  esters 'present,  such  as 
have  been  observed  in  Chondrus. ^^ 


CHAPTER    VIII 

SUMMARY   AND    CONCLUSIONS 

Of  the  great  variety  of  organisms  classified  together  as  algae 
only  a  few  species  have  been  examined  from  the  physio- 
logical and  biochemical  points  of  view.  Thus  the  account 
of  the  mechanisms  of  carbon  and  nitrogen  assimilation  in 
algae  given  in  this  book  is  largely  based  on  the  results  of 
studies  with  unicellular  green  algae,  whereas  for  the  con- 
sideration of  the  chemistry  of  the  final  products  of  meta- 
bolism most  information  is  available  for  the  brown  and  red 
seaweeds.  In  spite  of  this  incompleteness  in  our  knowledge 
it  seems  possible  to  draw  certain  general  conclusions 
regarding  algal  metabolism. 

There  is  no  reason  to  suppose  that  the  patterns  of  such 
major  metabolic  processes  in  these  organisms  as  photo- 
synthesis, respiration  and  nitrogen  assimilation,  differ  in 
any  fundamental  way  from  those  occurring  in  other  forms 
of  life.  The  algae  do,  however,  show  a  considerable  amount 
of  variety  in  metabolism  of  a  kind  which  is  also  found 
among  the  bacteria  and  which  appears  to  be  one  character- 
istic of  the  most  primitive  organisms.  This  is  best  exempli- 
fied in  photosynthesis,  the  characteristic  method  of  carbon 
assimilation  in  algae.  In  higher  plants  this  process  is  stereo- 
typed in  that  the  photosynthetic  pigments  are  always  of  the 
same  kind  and  that  the  hydrogen  needed  for  the  reduction 
of  the  carbon  dioxide  is  always  derived  from  water.  In  algae 
the  principal  photosynthetic  pigment  appears  to  be  the 
same,  i.e.  chlorophyll  a,  as  it  is  in  higher  plants.  This, 
together  with  other  evidence,  indicates  that  the  fundamental 
reaction  of  photosynthesis,  in  which  light  energy  is  trans- 
formed into  chemical  energy  available  for  the  reduction  of 
carbon  dioxide,  is  the  same  in  all  algae  and  in  higher  plants. 
However,  the  accessory  pigments  associated  with  chloro- 
phyll a  differ  both  in  kind  and  proportions  in  the  various 

130 


SUMMARY    AND    CONCLUSIONS 


131 


algal  classes.  Light  absorbed  by  certain  of  these  pigments 
can  be  transferred  to  chlorophyll  a  and  used  in  photo- 
synthesis so  that  the  efficiency  and  manner  of  utilization 
of  light  of  different  wavelengths  varies  considerably  from 
class  to  class.  Similarly,  while  it  appears  that  the  primary 
photochemical  reaction  in  photosynthesis  always  results  in 
the  splitting  of  water,  the  ultimate  hydrogen  donor,  although 
usually  water  as  in  higher  plants,  may,  in  many  algae  under 
certain  conditions,  be  elementary  hydrogen  or  hydrogen 
sulphide.  There  is  also  a  related  tendency  among  algae  to 
utilize  reduced  inorganic  substrates  such  as  these  as  a 
source  of  energy  for  growth,  a  tendency  which  is  shown  by 
no  higher  green  plants. 

Another  characteristic  of  the  metabolism  of  algae  is  its 
flexibility,  which  is  apparent  both  in  the  variety  of  sub- 
strates which  can  be  assimilated  and  the  considerable 
variation  which  can  occur  in  the  proportions  of  the  various 
products  of  metabolism  accumulating  within  the  organism.. 
Many  algae,  although  capable  of  photosynthesis,  can  also 
grow  in  the  dark  if  provided  with  a  suitable  substrate  and 
for  many  algae  these  substrates  may  be  of  very  varied 
chemical  nature.  A  similar  flexibility  is  evident  in  the 
assimilation  of  nitrogen.  Among  the  final  products  of  meta- 
bolism the  proportions  of  such  materials  as  fats,  carbo- 
hydrates and  proteins,  which  accumulate  within  the  cells, 
may  vary  within  wide  limits  according  to  the  stage  of 
growth  attained  and  the  conditions  under  which  it  has  taken 
place.  During  growth  the  products  of  photosynthesis  or  of 
the  assimilation  of  organic  substances  are  used  for  the 
synthesis  of  protoplasm  but  on  cessation  of  growth  the 
continuation  of  assimilation  results  in  the  accumulation  of 
storage  products  the  nature  of  which  varies  according  to 
the  species  concerned  and  to  other  circumstances.  The 
limits  within  which  the  chemical  composition  of  an  alga 
may  vary  as  a  result  of  this  are  particularly  wide  in  the 
simpler  forms;  as  a  general  rule  structurally  more  complex 
algae  are  more  exacting,  both  in  their  internal  and  external 
environmental  requirements,  and  this  limits  the  amount 
of  variation  which  can  occur. 


132  THE    METABOLISM    OF    ALGAE 

This  flexibility  is  understandable  when  it  is  considered 
that  the  reactions  in  which  the  intermediates  of  metabolism 
are  involved  are  mostly  reversible  and  that  the  major  meta- 
bolic processes  share  common  intermediates.  That  this  is 
true  for  carbohydrate,  acid  and  nitrogen  metabolism,  has 
become  apparent  from  studies  of  other  organisms,  but  the 
idea  that  photosynthesis  is  not  apart  from  the  rest  of  meta- 
bolism but  intermeshes  with  other  processes  at  an  early 
stage  has  arisen  as  the  result  of  studies  with  algae.  Material 
may  be  introduced  into  the  metabolic  system  at  many  points 
and  does  not  follow  any  unique  pathway,  but  appears  as 
ultimate  products  the  nature  of  which  varies  according  to 
the  conditions  to  which  the  system  has  been  exposed  in 
the  past  as  well  as  to  those  to  which  it  is  exposed  at  the 
moment.  The  variation  in  chemical  composition  of  the  cell 
material  which  results  is  perhaps  more  marked  in  algae 
than  in  comparable  bacteria  or  fungi  because  algae  have  a 
marked  tendency  towards  conservation  of  material  within 
their  cells  rather  than  of  excretion  of  surplus  substances 
into  the  surrounding  medium. 

While  many  algae  are  thus  able  to  utilize  a  wide  range  of 
substrates  and  are  capable  of  synthesizing  for  themselves 
all  the  metabolites  which  they  need,  others  have  more 
limited  metabolic  capacities  and  are  dependent  upon  the 
presence  of  particular  substances  in  their  environment 
either  as  energy  sources  or  as  growth  factors.  An  inability 
to  utilize  a  given  substrate  as  energy  source  may  be  due  to 
impermeability  of  the  plasma  membrane  towards  the  sub- 
stance, to  the  absence  of  the  specific  enzyme  necessary  for 
its  entry  into  metabolism,  or  to  less  well-defined  causes. 
Obligate  phototrophy,  an  inability  to  maintain  growth  ex- 
cept when  carrying  out  photosynthesis  which  is  perhaps 
common  among  algae,  is  possibly  due  to  the  absence  of  a 
mechanism  for  the  synthesis  of  a  specific  substance  but 
has  not  yet  been  satisfactorily  explained.  In  the  other 
direction,  the  capacity  for  photosynthesis  may  be  lost  and 
many  colourless  organisms  absolutely  dependent  on  organic 
substances  as  energy  sources  have  clearly  been  derived  from 
photosynthetic  algae  by  loss  of  chromatophores.  Loss  of 


SUMMARY    AND    CONCLUSIONS  I33 

enzymes  responsible  for  the  synthesis  of  specific  meta- 
boHtes  leads  to  the  appearance  of  nutritionally  exacting 
forms  similar  to  those  well  known  among  bacteria,  fungi  and 
other  organisms.  A  few  algae,  both  naturally  occurring 
strains  and  artificially  induced  mutants,  have  been  shown 
to  have  a  requirement  for  thiamine  and  it  seems  likely  that 
when  a  more  thorough  investigation  is  made,  the  growth  of 
many  algae  will  be  found  to  be  dependent  on  the  exogenous 
supply  of  specific  organic  factors.  Such  growth  factor  re- 
quirements occur  quite  independently  of  the  presence  or 
absence  of  a  capacity  for  photosynthesis. 

Many  of  the  algal  classes  are  characterized  by  the  general 
occurrence  in  their  members  of  particular  metabolic  pro- 
ducts. This  is  especially  evident  with  the  photosynthetic 
pigments,  each  class  having  its  distinctive  accessory  pig- 
ments. Peculiarities  in  carbohydrate  metabolism  occur  in 
several  classes.  The  Phaeophyceae  and  Rhodophyceae,  for 
example,  tend  to  produce  carbohydrates  containing  i  :  3 
linkages,  rather  than  i  :  4  linkages  such  as  are  formed  by 
other  algae  and  higher  plants,  and  polysaccharide  sulphate 
esters.  Those  algae  grouped  together  in  the  Chrysophyta 
seem  unable  to  synthesize  starch  and  generally  accumu- 
late fat  rather  than  carbohydrate  reserves.  The  occurrence 
of  these  and  other  biochemical  characteristics  confirms  to 
a  remarkable  degree  the  classification  of  algae  derived  on 
morphological  grounds. 

Its  possession  of  such  features  justifies  the  consideration 
of  the  algal  type  of  metabolism  as  a  distinct  field  of  study 
which  has  much  to  contribute  to  comparative  biochemistry 
and  our  understanding  of  the  economy  of  nature. 


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INDEX 


absorption  spectra,  19,  20,  22-7 
acetate,  8,  9,  43,  44,  51,  54,  55,  58, 
60,  63-6 

—  organisms,  51,  55 
acetylcoenzyme  A,  65 
adaptation,  29-30,  56,  57,  109,  121 
adenosine  diphosphate,  7, 1 1,  80,  81 

—  triphosphate,  7,  11,  79-81 
adenylic  acid,  81 

agar,  96,  97,  105 

alanine,  36,  37,  43,  79,  82,  loi,  102 

Alaria,  14,  98,  99 

algin,  95,  104 

alginic  acid,  2,  95,  96,  125,  127 

amide,  76,  77,  79-83.  9°,  loi 

amino-acids,  43,  44,  50,  70,  71,  77- 

82,  84,  85,  loi,  102 
/>-amino-benzoic  acid,  86,  87 
ammonia,  16,  43,  47,  68,  70-83,  85, 

90,  III,  114,  116,  117 
amylase,  93 
amylopectin,  93,  94 
amylose,  93,  94 
Anabaena,  15,  46,  59,  69,  70,  74, 

75,    90,    loi,    102,    107,    109, 

no,  122,  123 
Anabaeniopsis,  15,  69 
anaerobic  conditions,  6,  30-2,  36, 

63,  76 
Ankistrodesmiis,  13,  32,  74,  82 
Aphanizomenon,  15,  69 
apochlorosis,  61,  62,  132 
arginine,  78,  79,  loi,  102 
Ascophyllutn,  14,  32,  94,  127 
ash  constituents,  96,  125,  127 
asparagine,  43,  82 
aspartic  acid,  36,  37,  43,  70,  79,  82, 

84,  85,  loi,  102 
Astasia,  14,  62,  86 
Asterionella,  13,  114 
Athiorhodaceae,  15 
Aulosira,  15,  69 
autotrophy,  12,  16,  68-83,  84 
Azotobacter,  68,  70 

Bacillariophyceae,  5,  9,  13,  20-4, 
32,  59,  92,  95.  99.  102,  104, 
105,  n2,  116,  119,  121 


Bacillus,  48 

bacteria,  compared  with  algae,  i, 
18,  30-2,  48,  49,  55,  59,  68, 
70,  79,  82,  86,  90,  96,  102, 
106,  108,  109,  130,  132,  133 

Beggiatoa,  15,  49 

bicarbonate,  33-5 

Bostrychia,  15,  92 

butyric  acid,  51 

calcareous  algae,  35 

Calothrix,  15,  69 

carbohydrate    deficient    cells,    67, 

72,  73,  82,  III 
—  synthesis,  36,  42,  43,  63-7 
carbohydrates,  91—8,  106,  112,  115, 

118,  120,  121,  131,  133 
carbon  dioxide  and  photosynthesis, 

17,  18,  30,  33-41,  58,  60,  117 

,  'extra',  71,  72 

carbon   monoxide,    inhibition   by, 

10,  70 
carbonate,  33,  35 
carbonic  anhydrase,  35,  60 
carboxylation,  see  decarboxylation 
carotene,  20,  21 
carotenoids,  19,  20,  24-6,  28 
carragheenin,  97,  105 
cell  walls,  95-8,  104,  no,  116,  127 
cellobiose,  56,  57 
cellulose,  95,  104,  127 
Ceramiales,  15,  93 
chemical  composition  of  algae,  91— 

103,  106,  109-29,  131,  132 
chemolithotrophy,   16,  48-50,  60 
chemo-organotrophy,  16,  50-67 
chemosynthesis,  16,  48 
chemotrophy,    15,   48-67,   71,   74, 

119,  120,  131,  132 
Chilomonas,  14,  86,  94,  114 
chitin,  98,  104 
Chlamydomonas,  13,  31,  32,  49,  58- 

60,  62,  85-7 
Chlorella  3-7,  9-1 1,  13,  16,  18, 
26,  28,  31-6,  39,  40,  42,  46, 
54,  56-64,  66,  67,  69,  71-7, 
79-82,  89,  93,  98-102,  106-20, 
122,  123,  126 


H5 


146 


THE    METABOLISM    OF    ALGAE 


Chlorococcales,  12,  13 

Chlorogonium,  13.,  51 

Chlorophyceae,  6,  9—13,  20-2,  32, 
50,  51,  69,  79,  83,  89-93,  95, 
99,  100,  102-4,  107,  112,  113, 

119,  123,  130 
chlorophyll,   17-21,  23-9,   32,  44, 

61,  118,  130 
Chlorophyta,  13 
chondrillasterol,  100,  104 
Chondrus,  15,  97,  loi,  129 
Chorda,  14,  94 
chromatic  adaptation,  29 
chromatophore,  22,  23,  26,  61,  109, 

1 10,  1 16,  132 
Chroococcales,  15,  69 
Chroococcus,  15,  26,  27 
Chrysophyceae,    14,   21,    95,    104, 

105 
Chrysophyta,  13,  105,  133 
citrate,  8,  9,  36,  42,  43,  54,  60,  65 
Cladophora,  13,  99,  100 
classification,  12-16,  103-5,  ^33 
Clostridium,  68 
co-carboxylase,  9 
Coccomyxa,  13,  89 
coenzyme     II,     see     triphospho- 

pyridinenucleotide 
Coilodesme,  14,  25,  26 
colourless  algae,  13,  61-3 
Corallina,  14,  35,  102 
Cryptomonas,  14,  87 
Cryptonemiales,  14,  93 
Cryptophyceae,    14,    20,    51,    94, 

104,  105 
culture  of  algae,  3,  5,  50,  68,  75, 

84,  87-90,   107,  108,   1 10-18, 

120,  121 

cyanide,  inhibition  by,  10,  66,  74, 

76 
cyanophycin,  103,  104 
Cyanophyta,  15 

Cylindrospermum,  6,  9,  15,  32,  69 
cystine,  loi,  102 
Cystococcus,  see  Trehoiixia 
cytochrome,  10,  66 
—  oxidase,  10 

deamidation,  83 
deamination,  47,  82,  83 
decarboxylation,  9,  36,  38-40,  44, 

47,  64,  65,  86 
dehydrogenases,  7,  9,  10,  78 
diaminopimelic  acid,  102 
diatoms,  see  Bacillariophyceae 


diiodotyrosine,  102 

dinitrophenol,  81 

Dinophyceae,  14,  21,  95,  104,  105, 

112 
disaccharides,  52,  77,  93,  94,  115, 

116 
Dityliiim,  13,  87 
dulcitol,  92 

Ectocarpus,  14,  94 

energy  sources,  15,  17,  48,  49,  59, 

60,  131,  132 
Enter omorpha,  13,  87 
enzymes,   5,   52,   55,   56,   85,   109, 

no,  121,  132,  133 
ergosterol,  100,  104 
ethyl  alcohol,  63 
Eiiglena,  10,  11,  14,  15,  51,  56,  61, 

62,  71,  78,  84-6,  112 
Euglenineae,  11,  14,  20,  2i,  33,  51, 

86,  93,  102,  104,  112 
Euglenophyta,  14 
excretion,  54,  89-91,  102,  106,  no, 

132 
exponential  growth,  52,  53,  56,  58, 

107-16,  120,  123 

fat  synthesis,   36,  43,  44,  65,  67, 

116-23 
fats,  see  lipides 
fatty  acids,   15,  43-5,   50,  51,  55, 

98,  99,  118,  121-3 
fermentation,  6,  63,  76 
flavoproteins,  10 
floridean  starch,  93,  104,  105 
floridoside,  92,  104,  105 
fluorescence,  19,  20,  28,  29 
freshwater  algae,   36,   71,   87,   90, 

93,  99,  107,  114 
fructose,  51,  52,  56,  92 
Fucaceae,  123 
fucoidin,  97,  104,  105 
fucose,  98 
fucosterol,  100,  104 
fucoxanthin,  19,  21,  24,  25 
Fucus,  10,  14,  94,  98,  99,  127,  128 
fumarate,  8,  36,  42,  60 
fungi,  compared  with  algae,  i,  16, 

90,  98,  100,  106,  132,  133 

galactose,  52,  53,  92,  96,  97 
Gelidium,  6,  7,  9,  14,  36,  96,  112 
Gigartina,  15,  28,  97,  129 
Gigartinales,  15,  93 
Gloeocapsa,  15,  69 


INDEX 


147 


Gloeotrichia,  15,  88 

glucose,  II,  30,  33,  51-8,  60,  63-6, 

70,  76-8,  80,  82,  91-5,  112 
glucose- 1 -phosphate,  11,  56,  94 
glutamate,  36,  43,  44,  46,  47,  70, 

78-80,  82,  86,  loi 
glutamic  dehydrogenase,  78 
glutamine,  43,  47,  79,  80,  82,  102, 

103 
glycerol,  43,  52,  53,  60,  63,  65,  92, 

98,  113 
glycine,  38,  43,  44,  82,  85,  loi 
glycogen,  11,  54,  94 
glycollate,  38,  43,  65 
glycolysis,  6,  11,  41,  42,  47,  64,  79 
growth,  3,  52,  90,  106-29,  131 

—  factors,    15,    16,   48,    59,    84-8, 

114,  132,  133 

—  inhibitors,  86,  114,  120 
Gymnodinium,  14,  87 

Haematococcus,  13,  82 

Harvey ella,  14,  63 

hentriacontane,  100 

heterotrophy,  12,  16,  82,  84-8 

hexokinase,  55 

hexose,  6,  40,  42,  51,  52,  92,  116 

—  phosphates,  6,  7,  11,  37,  42,  43, 

56,  64 
higher  plants,  compared  with  algae, 
I,  2,  18,  20,  22,  30,  70,  78,  79, 
93,  99,  loi,  102,  104,  105,  130, 

131,  133 
Hill  reaction,  39,  41,  60,  73 
histidine,  85,  loi,  116 
holozoic  nutrition,  50 
Hormidium,  13,  58,  82,  112 
hydrocarbons,  99,  100 
hydrogen  ion  concentration,  effects 

on    metabolism,   33,    34,    55, 

64,  70-2,  74,  75,  82,  114,  119, 

120 
— ,  molecular,  30,  33,  70,  131 

—  sulphide,  32,  33,  49,  131 
hydrogenase,  31,  70 
hydroxylamine,  31,  70,  74 

immersion,  effect  on  composition 

of  seaweeds,  126,  128 
inulin,  51 

Iridaea,  15,  28,  92,  97 
iron,  114 
iso-citrate,  8,  46 
isoleucine,  loi 
isotopic  tracers,  36-47,  65,  69,  78 


ketoglutarate,  8,  43,  47,  78,  79 
Krebs  cycle,  see  tricarboxylic  acid 
cycle 

lactate,  6,  51,  54,  63 

lactose,  57 

lag  phase,  56,  57,  107,  109 

Laminaria,  14,  94,  98,  99,  10  r,  102, 

124-7 
Laminariaceae,  123 
Laminariales,  14,  126 
laminaribiose,  94 
laminarin,  94,  104,  105,  124-9 
leucine,  79,  loi 
leucosin,  95,  104 
light,   effects  on  metabolism,   35, 

46,  57-61,  109,  III,  117 
lipides,    98-101,     104-6,     111-13, 

116-23,  127,  129,  131,  133 
lipoids,  99-101,  109,  118 
lysine,  78,  79,  loi,  102 

Macrocystis,  14,  126 

malate,  8,  36-40,  42,  43,  60 

'malic'  enzyme,  40 

malonate,  inhibition  by,  9,  40 

maltase,  56 

maltose,  52,  56 

manganese,  74 

mannitan,  92 

mannitol,  52,  92,  104,  124-9 

mannoglycerate,  93 

mannose,  92 

mannuronic  acid,  95 

marine  algae,  i,  2,  4,  14,  29,  31,  35, 

50,  87,  90,  92-103,  123-9 
Mastigocladus,  15,  69,  102 
mesotrophy,  16,  75 
metabolic  pool,  41,  43,  64,  8r,  120, 

132 
methionine,  loi,  102 
methyl-^-D-glucoside,  57 
Microcystis,  15,  69,  10 1 
molybdenum,  70,  74 
Monodus,  13,  122,  123 
Monostroma,  13,  26 
mutants,  60-2,  86,  87,  133 
Myelophycus,  6,  7,  9,  14 
Myxophyceae,  9,    15,  21,  22,  32, 

35.  49,  68,  69-71,  90,  92-5, 

101-4,  112,  119 

Navicula,  13,  19,  24,  25,  28,  51,  54, 

lOI 

Nitella,  13,  99,  100 


148 


THE    METABOLISM    OF    ALGAE 


nitrate,  16,  68,  70-4,  90,  iii,  117, 

124,  126,  129 
nitrite,  74 
nitrogen  assimilation,  68-85,  ^2, 

130,  131 
• —  fixation,  68-71,  90 
nitrogen-deficient  cells,  71,  75-8, 

81,  111-13,  116,  117,  119-23 
Nitrosomonas,  16 
Nttzschia,  13,  28,  32,  36,  55,  86,  98, 

99,  1 12-14,  120 
Nostoc,  15,  32,  51,  69,  70,  78,  114 
nucleic  acid,  86,  103,  no,  113,  116 

Ochromonas,  14,  50 

Oedogonium,  13,  99,  100 

organic  substances,  assimilation  by 

algae,  50-67,  81-8,  131 
ornithine,  78,  79 
Oscillatoria,  12,  15,  28,  32,  49,  94, 

102,  122,  123 
Oscillatoriaceae,  15,  69 
oxaloacetate,  8,  9,  39,  43,  47,  65, 

66,  79 
oxidative  assimilation,  4,  63-7,  in 
oxyhydrogen  reaction,  30,  48,  49 

palmitic  acid,  98,  113 
paramylum,  93,  104 
pectin,  98,  104 

Pelvetia,  14,  92,  94,  102,  128,  129 
pentose,  52,  64,  90,  92,  98 
peptides,  102,  103 
Peridinium,  14,  59,  75,  112 
permeability,  33,  34,  54,  55,  71-3, 

89,  132 
peroxidase,  1 1 

Phaeophyceae,  9,   11,   21,   23,  25, 
32,  50,  92,  94-6,  98-100,  102, 
104,  105,  119,  123-9,  130.  133 
Phaeophyta,  14 
o-phenanthroline,  31 
phenylalanine,  44,  85,  loi 
Phormidium,  15,  69,  loi,  102 
phosphate  deficient  cells,  113 
phosphoglyceraldehyde,  7,11 
phosphoglyceric  acid,  6,  7,  11,  37- 

43,  51.  59,  64 
phosphorylation,  6,  11,  44,  56,  64, 

66,  79,  96 
photolithotrophy,  15,  33 
photo-organotrophy,  15,  33 
photoreduction,  30,  32,  49 
photosynthesis,  i,  2,  4,  5,  10,  12, 

17-47,    57-61,    71,    73,    108, 

119-21,  130,  132,  133 


photosynthesis,  acceptor  in  carbon 
dioxide  fixation,  38-40,  43,  44 
action  spectra,  22,  23,  25—7 
dark  reactions,  18,  36-41 
hydrogen  donors,  15,  17,  18,  30- 

3,  130,  131 
inhibitors,  32,  41,  120 
light  absorption,  17,  22-30,  131 
oxygen  evolution,  17,  22,  31,  32, 

39,  58,  72,  73 
photochemical  reaction,   15,  18, 
28,  31,  39-41,  44,  60,  61,  73, 

74,  131 
products,  17,  18,  36-47,  73,  79, 

113,  119-23,  127,  132 
quantum  efficiency,   17,  22,  24, 

25,  27,  31,  120 
rate,  35,  108,  in,  114,  119,  120, 

124,  126,  129 
photosynthetic    bacteria,     15,     18, 

30,  68 

—  pigments,  2,  10,  17-30,  48,  50, 

61-3,  99,  103,  105,  109,  130, 

133 

—  quotient,    17,    72,    73,    111-13, 

120,  121 
phototrophy,     15,     17-47,    57-^1, 

70,  71,  74,  86 
— ,  obligate,  54,  59-61,  85,  132 
phycobilins,  19,  20,  22,  26—9 
phycocyanin,  20—2,  27 
phycoerythrin,  20-2,  27 
Pinmdaria,  13,  32,  112 
polypeptides,  90,  102 
polyphenol  oxidase,  1 1 
polysaccharides,    67,    77,    78,    80, 

93-8,  104,  133 
Polytoma,  13,  62,  85 
Polytomella,  10,  12,  13,  56,  62,  85, 

93,  103,  no,  113,  116 
Porphyra,  10,  14,  27,  28,  32 
Porphyridium,  14,  28,  32 
proline,  loi 
propionic  acid,  63 
Prorocentrum,  14,  59,  75 
protein,   101-3,   106,   109-n,  113, 

115,  116,  118,  120,  121,  124- 

7,  131 

—  synthesis,    36,   43,   44,   47,   67, 

73,  81,  no,  113,  115,  120 
proteinase,  55 
Prototheca,  4,  6,  9,  13,  16,  51,  54, 

55,  60,  62,  63,  65,  66,  86 
pyranose  structure,  91 
pyrimidine,  85,  86 


INDEX 


149 


L-pyrrolidonoyl-a-glutaminyl-L- 

glutamine,  103 
Pyrrophyta,  14,  105 
pyruvate,  6,  37-43,  47,  5 1,  54,  55, 

60,  64-6,  79,  86 
pyruvic  dehydrogenase,  9 

relative  growth  rate,  52,  53,  107- 11 
reserve     products,     91-5,     103-5, 

110,  113,  119-23,  127 
respiration,  5-12,  36,  44-7,  52,  54, 

58,   60,   63-7,  71,  76,  79-81, 

111,  126,  130 

respiratory  quotient,  iii,  112,  121 
Rhizohium,  68,  70 
Rhodophyceae,  9,   11,   14,  21,  22, 

27,  32,  35,  50,  92,  93,  95,  96, 
99,   100,   102,   104,   105,   112, 

119,  123,  129,  130,  133 
Rhodophyta,  14 
Rhodymenia,  15,  96,  99 
riboflavin,  10 

Saccorhiza,  14,  127 

Scenedesmus,  6,  7,  11,  13,  18,  31- 

3,  35-7,  41,  42,  44,  45,  48,  49, 

51-3,  55-7,  63,  65,  67,  78,  82, 

89,  93,  98,  100,  112 
seasonal    variations    in    seaw^eeds, 

124-9 
serine,  43,  loi 

sewage  organisms,  49-51,  82 
silica,  104,  114 
Siphonales,  13,  20,  21,  35 
sitosterol,  100,  104 
soil  algae,  3,  50,  62,  71,  87 
—  extract,  87,  88 
sorbitol,  92 
starch,  11,  51,  54,  93,  94,  96,  104, 

105,  III,  115,  116,  127,  133 
stationary   phase,    107,    115,    116, 

120,  123 
sterols,  99-101,  104 
Stichococcus,  13,  82 
streptomycin,  62 
succinamide,  82 

succinate,  8,  9,  36,  42,  43,  51,  54, 

60,  66 
succinic  dehydrogenase,  9 
sucrose,  37,  42,  52,  56,  93,  112 


sulphanilamide,  86,  87 

sulphuric  esters,   96-8,    105,    127, 

129,  133 
symbiotic  algae,  50,  71 
Synechococcus,  15,  32,  112 
Synechocystis,  15,  32 
Synura,  14,  87 

tartaric  acid,  51 

Tetrachloris,  13,  62 

thiamine,  9,  65,  66,  85,  86,  133 

thiazole,  85,  86 

Thiohacillus,  59 

threonine,  44,  loi 

Tolypothrix,  15,  69 

transamination,  79,  83,  85 

translocation,  126 

Trebouxia,  13,  51 

trehalose,  93 

Tribonema,  13,  95,  loi,  122,  123 

tricarboxylic  acid  cycle,  7-10,  42-4, 

46,  47,  65,  66,  79,  86 
triose  phosphates,  6,  37,  42,  43 
triphosphopyridine  nucleotide,  39, 

40 
tryptophane,  loi 
tyrosine,  44,  loi,  102 

Ulva,  6,  7,  9,   13,  26,  32,  50,  87, 

loi,  102 
utilization  of  algae,  4,  5,. 96 

valine,  loi 

Valonia,  12,  34,  95 
Vaucheria,  10,  13 
vitamin  Bi,  see  thiamine 

—  B12,  86 

—  requirements  of  algae,  85-8 
Volvocales,  13,  51 

Xanthophyceae,  10,  13,  21,  95,  98, 

100,  102,  104,  105,  119 
xanthophylls,  20-2 
xylose,  52,  96 

yeast,  5,  6,  11,  12,  65,  87,  108,  110 

zeaxanthin,  21,  22 
zonation  of  algae,  29,  129 
Zygnema,  13,  82 


Printed  in  Great  Britain  by 

Butler  &  Tanner  Ltd., 

Frome  and  London