Vol.
48,
No.
1
APPLIED
AND
ENVIRONMENTAL
MICROBIOLOGY,
JUlY
1984,
p.
114-118
0099-2240/84/070114-05$02.00/0
Copy igh
©D
1984,
Ame ican
Socie y
o
Mic obiology
Sus ained
Pho op oduc ion
o
Ammonia
om
Dini ogen
and
Wa e
by
he
Ni ogen-Fixing
Cyanobac e ium
Anabaena
sp.
S ain
ATCC
33047
JUAN
L.
RAMOS,
MIGUEL
G.
GUERRERO,*
AND
MANUEL
LOSADA
Depa amen o
de
Bioquimica,
Facul ad
de
Biologia
y
Conisejo
Suipe io
de
In es iga(Wiones
Cien i icas,
Uni e si y
o
Se ille,
Se ille,
Spaiin
Recei ed
30
Decembe
1983/Accep ed
10
Ap il
1984
Condi ions
ha e
been
de eloped
ha
leng hen
he
ime
du ing
which
pho osyn he ic
dini ogen
ixa ion
by
ilamen s
o
he
cyanobac e ium
Anabaena
sp.
s ain
ATCC
33047
p oceeds
eely,
whe eas
he
subsequen
con e sion
o
ammonia
in o
o ganic
ni ogen
emains
blocked,
wi h
he
esul ing
ammonia
eleased
o
he
ou e
medium.
When
L-me hionine-DL-sul oximine
was
added
e e y
20
h,
maximal
a es
o
ammonia
p oduc ion
(25
o
30
,umol/mg
o
chlo ophyll
pe
h)
we e
main ained
o
abou
50
h.
A e
his
ime,
ammonia
p oduc ion
ceased
due
o
a
de iciency
o
glu amine
and
o he
ni ogenous
compounds
in
he
ilamen s,
condi ions
which
inally
led
o
cell
lysis.
The
e ec i e
ammonia
p oduc ion
pe iod
could
be
u he
ex ended
o
abou
7
days
by
adding
a
small
amoun
o
glu amine
a
he
end
o
a
40-h
p oduc ion
pe iod
o
by
allowing
he
cells
o
eco e
o
8
h
in
he
absence
o
L-me hionine-DL-sul oximine
a e
e e y
40-h
pe iod
in
he
p esence
o
he
inhibi o .
A
mo e
p olonged
s eady
p oduc ion
o
ammonia,
las ing
o
longe
han
2
weeks,
was
achie ed
by
al e na ing
ea men s
wi h
he
glu amine
syn he ase
inhibi o s
L-me hionine-DL-sul oximine
and
phosphino h icin,
p o ided
ha
8-h
eco e y
pe iods
in
he
absence
o
ei he
compound
we e
also
al e na ed
h oughou .
The
biochemically
manipula ed
cyanobac e ial
ilamen s
hus
ep esen
a
sys em
ha
is
ela i ely
s able
wi h
ime
o
he
con e sion
o
ligh
ene gy
in o
chemical
ene gy,
wi h
he
ne
gene a ion
o
a
aluable
uel
and
e ilize
h ough
he
pho o educ ion
o
dini ogen
o
ammonia.
The
con e sion
o
sola
ene gy
in o
sui able
edox
ene gy
h ough
pho osyn hesis
o
he
wa e -spli ing
ype
ha
is
ca ied
ou
by
whole
o ganisms
is
a
p ocess
o
g ea
in e es
and
signi icance.
Ligh -d i en
syn hesis
o
ammonia
om
dini ogen
and
wa e
by
pho osyn he ic
o ganisms
ep e-
sen s
an
in e es ing
sys em
o
he
con e sion
o
ligh
ene gy
in o
s o ed
chemical
ene gy,
wi h
he
addi ional
in e es
ha
he
esul ing
compound
is
a
aluable
uel
and
e ilize
(4,
7).
Filamen ous
cyanobac e ia
(blue-g een
algae)
ha
a e
able
o
di e en ia e
he e ocys s
a e
unique
o ganisms
in
ha
hey
can
ix
N2
unde
ae obic
condi ions,
wi h
ligh
as
he
sole
sou ce
o
ene gy
and
wa e
as
he
ul ima e
educ an ,
acco ding
o
he
ollowing
global
equa ion:
6ce->P
ATP
N2
+
3H20
-'---P
2NH3
+
3/202
(AEo'
[pH
7]
=
-1.10
V;
lAG'
[pH
7]
=
+318
kJ/mol
o
ammonia)
whe e
AE(4
is
he
s anda d
edox
po en ial
change
and
AG'
is
he
s anda d
ee
ene gy
change.
The
achie emen
o
e ec i e
p oduc ion
o
ammonia
om
dini ogen
by
N2- ixing
cyanobac e ia
equi es
(i)
p e en ion
o
he
inco po a ion
o
ammonia
o
ca bon
skele ons,
which
akes
place
mainly
h ough
he
glu amine
syn he ase-glu a-
ma e
syn hase
pa hway
(18),
and
(ii)
o e coming
he
an ago-
nis ic
e ec s
o
ammonia
on
dini ogen
ixa ion
(4).
Bo h
equisi es
ha e
been
simul aneously
ul illed
by
in e e ing
*
Co esponding
au ho .
P esen
add ess:
Uni
o
Ni ogen
Fixa ion,
Uni e si y
o
Sussex,
B igh on
BN1
9RQ,
Uni ed
Kingdom.
wi h
he
ope a ion
o
he
main
ammonia
assimila ion
pa h-
way
by
using
glu ama e
analogs,
such
as
MSX
(L-me hio-
nine-DL-sul oximine)
o
PT
(phosphino h icin
[2-amino-4-
(me hylphosphinyl)-bu anoic
acid])
(6,
9,
12,
17).
To
be
conside ed
p ac ical,
a
sys em
o
he
con e sion
o
sunligh
ene gy
mus
ope a e
a
a
conside able
a e
and
e iciency
and
mus
be
ela i ely
s able
wi h
ime.
The
sys em
cons i u ed
by
MSX-
o
PT- ea ed
Anabaena
sp.
s ain
ATCC
33047
ilamen s
has
been
shown
o
p oduce
ammonia
a
high
a es,
in
he
ange
o
25
o
30
pLmol/mg
o
chlo ophyll
pe
h
(6,
9,
12),
wi h
ela i ely
high
e iciency,
ca.
10%
o
i s
heo e ical
maximum,
bu
un il
now
he
p ocess
has
been
only
ca ied
ou
o
sho
pe iods
o
ime.
This
communica ion
epo s
he
achie emen
o
s eady
pho op oduc ion
o
ammonia
om
dini ogen
by
he
cyano-
bac e ium
Anabaena
sp.
s ain
ATCC
33047,
las ing
o
longe
han
2
weeks.
MATERIALS
AND
METHODS
G ow h
o
cells.
Anabaena
sp.
s ain
ATCC
33047
was
g own
pho oau o ophically,
unde
con inuous
illumina ion
(Syl ania
day
ligh
luo escen
ubes,
wi h
an
i adiance
alue
o
25
W/m2
a
he
su ace
o
he
cul u e
essels)
a
40°C
on
a
syn he ic
medium
modi ied
om
BG11
medium
as
desc ibed
p e iously
(16),
excep
ha
25
mM
NaHCO3
was
added.
A
s eam
o
2%
( ol/ ol)
CO,
in
ai
was
bubbled
h ough
he
cul u e
a
a
low
a e
o
1.2
li e s/li e
o
cell
suspension
pe
h.
Ammonia
p oduc ion
expe imen s.
Anabaena
sp.
s ain
ATCC
33047
ilamen s
om
2-day-old
cul u es,
con aining
20
o
25
1Lg
o
chlo ophyll
a
(Chl)
pe
ml,
we e
used.
The
ilamen s
we e
ha es ed,
washed
wi h
cul u e
medium,
and
inally
esuspended
in
he
same
medium
o
each
a
cell
densi y
o
8
o
10
pLg
o
Chl
pe
ml.
The
ilamen
suspensions
we e
incuba ed
o
60
o
90
min
in
he
ligh
unde
s anda d
114
SUSTAINED
AMMONIA
PRODUCTION
FROM
DINITROGEN
cul u e
condi ions.
Expe imen s
we e
s a ed
by
adding
enough
MSX
o
he
cell
suspension
o
achie e
a
a io
o
3.5
o
4
nmol
o
MSX
pe
,ug
o
Chl.
Assays
o
enzyme
ac i i ies.
Ni ogenase
ac i i y
was
mea-
su ed
in
whole
cells
by
he
ace ylene
educ ion
echnique
(9).
Glu amine
syn he ase
( ans e ase)
ac i i y
was
de e -
mined
in
oluenized
ilamen s
(12).
Alanine
dehyd ogenase
was
es ima ed
in
cell- ee
ex ac s
as
p e iously
desc ibed
(13).
Ex ac ion
o
cellula
me aboli es.
Pe chlo ic
acid
was
added
o
a
sample
o
he
cell
suspension
o
gi e
a
concen a-
ion
o
0.6
N.
A e
10
min
a
0WC,
he
samples
we e
cen i uged
a
40,000
x
g
o
15
min,
and
he
supe na an
was
neu alized
a
0°C
wi h
KOH.
A e
emo al
o
KCl04
by
cen i uga ion
(10,000
x
g
o
10
min),
he
supe na an s
we e
used
o
he
es ima ion
o
me aboli es.
Analy ical
me hods.
Ammonia
was
de e mined
by
he
phenol-hypochlo i e
me hod
as
desc ibed
p e iously
(15).
Ca bohyd a es
we e
es ima ed
by
he
phenol-sul u ic
me h-
od
(5).
Chl
was
de e mined
spec opho ome ically
in
me h-
anolic
ex ac s
by
using
he
ex inc ion
coe icien
gi en
by
MacKinney
(8).
E hylene
was
measu ed
by
using
a
PYE
UNICAM
204
gas
ch oma og aph
equipped
wi h
a
column
illed
wi h
Po apak
Q
and
a
lame
ioniza ion
de ec o .
Glu ama e
and
glu amine
we e
de e mined
acco ding
o
Be gmeye
(3).
y-Glu amil
hyd oxama e
was
es ima ed
a e
i s
eac ion
wi h
FeCl3
in
acid
medium
(14).
Cellula
p o ein
was
de e mined
by
he
Low y
p ocedu e
as
modi ied
by
Bailey
(2),
by
p e ea men
o
he
ilamen s
wi h
10%
(w /
ol)
ichlo oace ic
acid.
Fo
d y-weigh
de e mina ion,
30-ml
samples
o
he
cell
cul u e
we e
il e ed
h ough
p eweighed
d y
il e s
(Wha -
man
GF/C).
The
il e s
we e
washed
wi h
2
olumes
o
dis illed
wa e
and
d ied
a
90°C
un il
cons an
weigh
was
eached.
Rela i e
phycocyanin
le els
we e
es ima ed
spec-
o luo ime ically
by
using
an
Aminco-Bowmnan
spec o-
pho o luo ime e
equipped
wi h
a
HTV
pho omul iplie
ube,
ype
R44GS.
Fo
luo escence
measu emen s,
ilamen
suspensions
(9
pLg
o
Chl
pe
ml)
we e
illumina ed
wi h
g een
ligh
(590
nm,
5
W/m2),
which
is
mainly
abso bed
by
phycocyanin,
wi h
he
ene gy
hen
ans e ed
o
chlo o-
phyll.
Chlo ophyll
luo escence
is
p opo ional
o
he
le el
o
phycocyanin
in
he
ilamen
suspensions.
Ligh
in ensi y
measu emen s
we e
done
wi h
a
YSI-Ke e ing
model
65A
adiome e .
Chemicals.
MSX
was
pu chased
om
Sigma
Chemical
Co.,
S .
Louis,
Mo.
PT
(6)
was
kindly
supplied
by
P.
J.
Lea
(Ro hams ed,
Uni ed
Kingdom).
O he
chemicals
we e
p od-
uc s
o
E.
Me ck
AG,
Da ms ad ,
Fede al
Republic
o
Ge many.
RESULTS
AND
DISCUSSION
The
addi ion
o
MSX
o
suspensions
o
Anabaena
sp.
s ain
ATCC
33047
ilamen s
ha
we e
g owing
on
ai
(78%
N2)
as
he
ni ogen
sou ce
caused
a
apid
inac i a ion
o
cellula
glu amine
syn he ase,
wi h
cell
g ow h
consequen ly
p e en ed.
Ni ogenase
ac i i y
inc eased
g adually
in
e-
sponse
o
MSX,
eaching
a
s able
le el,
which
was
a ound
wo old
highe
han
he
ini ial
le el,
abou
4
h
a e
i s
addi ion.
Mos
o
he
ammonia
(ca.
90%)
esul ing
om
N2
ixa ion
by
he
cells
was
eleased
and
accumula ed
in
he
ou e
medium
(9;
see
also
Fig.
1).
The
a e
o
ammonia
p oduc ion
inc eased
concomi an ly
wi h
he
inc ease
in
ni ogenase
ac i i y
men ioned
abo e,
un il
i
eached
a
maximum
in
co espondence
wi h
he
es ablishmen
o
a
s able
high
ni ogenase
le el.
The
maximal
a es
o
ammonia
p oduc ion
unde
hese
condi ions
we e
25
o
30
p.mol/mg
o
Chl
pe
h,
and
he
p ocess
con inued
o
be
ope a i e
o
abou
20
h
(Fig.
1).
Figu e
1
also
shows
ha
abou
20
h
a e
MSX
addi ion
glu amine
syn he ase
ac i i y
s a ed
o
eco -
e
and
accumula ion
o
ammonia
in
he
medium
ceased.
These
changes
we e
ollowed
by
he
u iliza ion
o
he
ammonia
ha
was
eleased
by
he
ilamen s,
wi h
g ow h
s a ing
again.
In
pa allel
wi h
he
occu ence
o
ammonia
up ake,
he
ni ogenase
ac i i y
le el
dec eased
sligh ly.
Since
unde
s anda d
cul u e
condi ions
and
in
he
ab-
sence
o
ilamen s
MSX
keeps
ac i e
o
a
leas
48
h,
he
cessa ion
o
he
e ec
o
he
inhibi o
on
ammonia
p oduc-
ion
could
be
ela ed
o
i s
ans o ma ion
by
he
cyanobac-
e ial
cells
a he
han
o
i s
spon aneous
deg ada ion.
The
empo a y
in e up ion
o
ammonia
p oduc ion
is
appa en ly
due
o
he
eco e y
o
glu amine
syn he ase
ac i i y
a he
han
o
he
induc ion
o
any
o he
ammonia
assimila ion
pa hway.
Acco dingly,
o
leng hen
he
pe iod
o
ammonia
p oduc ion,
esh
MSX
a
i s
op imal
a io
wi h
espec
o
he
amoun
o
cells
(3.5
o
4
nmol/Lg
o
Chl)
was
added
o
suspensions
o
Anabaena
sp.
s ain
ATCC
33047
in
which
he
MSX
e ec
had
been
los .
This
addi ion
esul ed
again
in
inac i a ion
o
p eexis ing
glu amine
syn he ase
and
allowed
anew
maximal
a es
o
ammonia
p oduc ion
(da a
no
E
I
n
w
I
z
I
D
0
I
I
4
C-)
.-
z
cm
0
I-J
-J
w
C-)
100
w
.s
ZA
0
4
w
E
50
8
x
_
x
b-
E
0
30
w
In
04-
w
I
O i
Z
_
)
=
15
w
c
z
E
4
D
PI-
%-'o
0-
0
0
0
12
24
TIME
(h)
FIG.
1.
Time
cou se
o
he
e en s
leading
o
ammonia
p oduc-
ion
and
i s
la e
cessa ion
in
MSX- ea ed
Anabaena
sp.
s ain
ATCC
33047
ilamen s.
A
ilamen
suspension
(150
ml)
wi h
a
densi y
o
9
,ug
o
Chl
pe
ml
ha
was
supplemen ed
wi h
35
,uM
MSX
was
incuba ed
unde
s anda d
cul u e
condi ions.
Samples
we e
wi hd awn
a
he
imes
indica ed,
and
ammonia
in
he
medium
(A),
densi y
o
he
ilamen
suspension
(0),
and
cellula
ac i i ies
o
ni ogenase
(0)
and
glu amine
syn he ase
(A)
we e
de e mined.
VOL.
48,
1984
115
116
RAMOS,
GUERRERO,
AND
LOSADA
shown).
Following
his
app oach,
successi e
addi ions
o
MSX
o
he
ilamen
suspension
e e y
20
h
allowed
maximal
a es
o
ammonia
p oduc ion
o
be
main ained
o
abou
48
h
(Fig.
2).
Du ing
his
pe iod,
glu amihe
syn he ase
emained
ully
inac i e,
and
he
le el
o
ni ogenase
ac i i y
was
a
leas
wo old
highe
han
ha
in
un ea ed
ilamen s.
Ne e -
hele,ss,
a e
48
h
in
he
p-esence
o
MSX,
he
ammonia
p oduc ion
a e
dec eased
p og essi ely,
in
spi e
o
he
ac
ha
glu amine
syn he ase
emained
inac i e.
This
cessa ion
in
ammonia
p oduc ion
seems
o
be
a
consequence
o
a
loss
o
ni ogenase
ac i i y
(da a
no
shown).
Cell
lysis
o
he
suspension
was
obse ed
sho ly
a e wa ds.
To
de e mine
he
cellula
si ua ion
leading
o
he
cessa ion
o
ammonia
p oduc ion
by
Anabaena
sp.
s ain
ATCC
33047,
he
le els
o
a ious
cell
componen s,
enzyma ic
ac i i ies
ela ed
o
ni ogen
me abolism,
and
amino
acids
in ol ed
in
he
glu amine
syn he ase-glu ama e
syn hase
pa hway
we e
measu ed
ih
ilamen s
which
had
been
ea ed
wi h
MSX
o
36
h
and
we e
compa ed
wi h
hose
in
un ea ed
ilamen s.
Table
1
shows
he
esul s
ob ained.
The
alues
o
he
chlo ophyll
and
p o ein
con en
we e
only
sligh ly
lowe
in
MSX- ea ed
ilamen s
han
i n
he
un ea ed
ones.
Howe e ,
ema kable
di e ences
we e
ound
ega d-
ing
he
le els
o
ca bohyd a es
and
phycocyanin.
The
ca bo-
hyd a e
and
phycocyanin
le els
in
MSX- ea ed
ilamen s
we e
h ee old
highe
and
i e old
lowe ,
espec i ely,
han
hose
in
un ea ed
ilamen s.
The
educ ion
in
he
phyco-
cyanin
le el
in
MSX- ea ed
cells
was
also
e iden
om
he
change
in
he
abso bance
a
628
nm/abso bance
a
680
nm
a io
o
he
cell
suspensions,
which
dec eased
om
0.91
o
0.72
as
a
consequence
o
he
p olonged
ea men
wi h
MSX.
The
di e ences
in
ca bohyd a e
and
phycocyanin
con en
we e
in e p e ed
as
being
due
o
con inued
pho osyn he ic
CO2
ixa ion
in
he
p esence
o
MSX,
which
o he wise
o-'
E
%-.
0
Lii
0
Q
0-
0
6
4
2
0
20
40
60
TIME(h
)
FIG.
2.
Con inuous
ammonia
pho op oduc ion
by
MSX- ea ed
Anabaena
sp.
s ain
ATCC
33047
ilamen s.
Condi ions
we e
he
same
as
hose
desc ibed
in
he
legend
o
Fig.
1,
excep
ha
MSX
(35
,uM)
was
eadded
e e y
20
h.
The
ammonia
in
he
medium
was
de e mined
a
he
imes
indica ed.
TABLE
1.
E ec
o
MSX
on
he
le els
o
di e en
cell
componen s
in
Anabaena
sp.
s ain
ATCC
33047"
Cell
componen
Time
(h)
and
ea men
Chlb
Phyco-
P o einh
Ca bo-
cyaniin"
hyd a esb
0
31
100
700 79
36
(wi hou
MSX)
30
100
650
94
36
(wi h
MSX)
26
20
550
284
'
A
suspension
o
Anabaena
sp.
s ain
ATCC
33047
ilamen s
con aining
7.9
,ug
o
Chl
(250
pLg
[d y
weigh ])
pe
ml
was
di ided
in o
wo
hal es,
o
one
o
which
MSX
(35
FM)
was
added
a
ze o
ime
and
20
h
la e .
A e
36
h
o
incuba ion
unde
s anda d
g ow h
condi ions,
he
cell
densi y
alues
eached
we e
37.0
jig
o
Chl
(1,214
,ug
[d y
weigh ])
pe
ml
and
7.8
,g
o
Chl
(304
,ug
[d y
weigh ])
pe
ml
o
he
MSX- ee
and
MSX-con aining
suspensions,
espec i ely.
Values
a e
in
mic og ams
pe
millig am
(d y
weigh ).
Values
a e
pe cen ages
o
he
con ol
a
ze o
ime.
p e en ed
he
inco po a ion
o
ammonia
de i ed
om
N2
ixa ion
in o
ca bon
skele ons.
Ac ually,
he
C/N
a io
inc eased
in
MSX- ea ed
Anabaena
sp.
s ain
ATCC
33047
ilamen s
om
4.2
a
ze o
ime
o
6.8
a
36
h.
The
MSX-
ea ed
cells
hus
appea
o
be
ni ogen
s a ed
(1).
Such
an
induc ion
o
ni ogen
s a a ion
by
MSX
has
been
also
epo ed
o
N2- ixing
Anabaena
cylind ica
(19),
bu
i
is
in
con as
wi h
he
si ua ion
no iced
in
MSX- ea ed
Anacys is
cells
p oducing
ammonia
om
ni a e,
which
did
no
exhibi
enhanced
phycocyanin
deg ada ion
(10,
11).
MSX- ea ed
Anabaena
sp.
s ain
ATCC
33047
ilamen s
lacked
glu amine
syn he ase
ac i i y
and
exhibi ed
low,
al hough
ep oducible,
le els
o
NADH-alanine
dehyd oge-
nase
(ca.
14
mU/mg
o
p o ein),
abou
wo old
highe
han
hose
ound
in
un ea ed
ilamen s.
This
ac i i y
migh
be
esponsible
o
he
assimila ion
o
low
amoun s
o
ammonia,
since
he
ni ogen
de iciency
was
mo e
se e e
in
MSX-
ea ed
Anabaena
sp.
s ain
ATCC
33047
kep
unde
a gon
a mosphe e
han
in
ha
main ained
unde
ai .
In
ac ,
a e
36
h
o
ea men ,
he
o me
had
los
mos
o
i s
phycocya-
nin,
whe eas
he
la e
s ill
e ained
ca.
20%
o
he
ini ial
le el.
As
a
consequence
o
he
inac i a ion
o
MSX
o
glu amine
syn he ase,
he
glu amine
le el
in
ammonia-p oducing
ila-
men s
dec eased
apidly
wi h
ime
and
became
p ac ically
negligible
9
h
a e
he
addi ion
o
he
inhibi o .
This
beha -
io
con as ed
wi h
ha
o
un ea ed
ilamen s,
which,
unde
he
same
condi ions,
main ained
hei
ini ial
glu amine
le el.
The
le el
o
glu ama e
in
MSX- ea ed
Anabaena
sp.
s ain
ATCC
33047
ilamen s
also
dec eased
wi h
ime
bu
did
so
much
mo e
slowly
han
ha
o
glu amine,
i s
alue
a e
36
h
being
75%
o
ha
in
un ea ed
ilamen s
(Fig.
3).
This
amino
acid
analysis
indica es
ha
he
Anabaeha
sp.
s ain
ATCC
33047
ilamen s
ha
we e
subjec ed
o
ea men
wi h
MSX,
in
addi ion
o
su e ing
om
gene al
ni ogen
s a a ion,
became
speci ically
de icien
in
glu amine
and
p obably
also
in
glu amine
de i a i es.
Such
a
me abolic
si ua ion
migh
e en ually
lead
o
he
cessa ion
o
ammonia
p oduc ion.
To
o e come
he
MSX-p omo ed
de iciencies
and
hus
p olong
he
ammonia
p oduc ion
pe iod,
wo
di e en
a -
emp s
we e
made.
One
o
hem
was
he
addi ion
o
he
cell
suspension
o
glu amine
(0.2
mM)
simul aneously
wi h
he
no mal
eaddi ion
o
MSX
40
h
a e
he
expe imen
had
been
ini ia ed.
The
da a
in
Fig.
4
show
ha
his
glu amine
ea men
esul ed
in
sus ained
ammonia
p oduc ion
a
a
cons an
a e
o
an
ex a
pe iod
o
a
leas
32
h,
a
phenome-
non
ha
did
no
occu
in
a
con ol
ha
ecei ed
he
no mal
/
I
0
*/
0
a
/
APPL.
ENVIRON.
MICROBIOL.
SUSTAINED
AMMONIA
PRODUCTION
FROM
DINITROGEN
117
'a
E
C
w
z
4
0
12
24
36
0-%
2
c
u
1
S
O"
0
E
I.
CD
TIME
(h)
FIG.
3.
Cellula
le els
o
glu amine
and
glu ama e
in
MSX-
ea ed
and
un ea ed
Anabaena
sp.
s ain
ATCC
33047
ilamen s.
An
ai -g own
suspension
o
ilamen s
con aining
9
,ug
o
Chl
pe
ml
was
ans e ed
ei he
o
a
medium
ee
o
combined
ni ogen
sou ce
(open
symbols)
o
o
he
same
medium
supplemen ed
wi h
35
,uM
MSX
(closed
symbols).
Samples
we e
wi hd awn
a
he
imes
indica ed,
and
he
cellula
con en
o
glu amine
(ci cles)
and
glu a-
ma e
( iangles)
was
es ima ed.
O he
condi ions
we e
he
same
as
hose
desc ibed
in
he
legend
o
Fig.
2.
addi ion
o
MSX
(35
,uM)
bu
did
no
ecei e
glu amine.
The
glu amine
ea men
did
no
esul
in
cell
g ow h
(measu ed
as
an
inc ease
in
Chl)
o
in
any
inc ease
in
glu amine
syn he ase
ac i i y
bu
allowed
he
main enance
o
he
high
ni ogenase
ac i i y
le el.
Glu ama e
(0.2
mM)
could
no
eplace
glu amine
in
sus aining
ammonia
p oduc ion,
a
esul
which
sugges s
ha
cessa ion
o
he
p ocess
is
ela ed
o
glu amine
de iciency.
The
second
app oach
o
ex end
he
ammonia
p oduc ion
pe iod
was
o
allow
he
ilamen s
o
eco e
om
hei
E
0
w
-
cD
0
0.
c
0-
z
0
S
18
9
0
24
48
72
TIME
(h)
FIG.
4.
E ec
o
glu amine
addi ion
on
he
p olonga ion
o
he
ammonia
p oduc ion
pe iod
by
MSX- ea ed
Anabcue naJ
sp.
s ain
ATCC
33047
ilamen s.
Condi ions
we e
he
same
as
hose
de-
sc ibed
in
he
legend
o
Fig.
2,
excep
ha
glu amine
(0.2
mM)
was
added
a e
40
h
(a ow)
o
one-hal
o
he
suspension
(@).
wi h
he
o he
hal
used
as
a
con ol
wi hou
glu amine
addi ion
(0).
=
2
E
E
(
0
2
~~~~~~00
0
4
40
80
120
160
TIME
(h)
FIG.
5.
E ec
o
he
ecu en
addi ion
and
emo al
o
MSX
on
he
pho op oduc ion
o
ammonia
by
Anihbaeni
sp.
s ain
ATCC
33047
ilamen s.
A
ilamen
suspension
(150
ml)
wi h
a
densi y
o
9.8
p.g
o
Chl
pe
ml
was
main ained
o
40
h
in
he
s anda d
cul u e
medium
supplemen ed
wi h
35
,uM
MSX.
A e
his
ime
cells
we e
ha es ed
and
washed
wi h
esh
medium
lacking
MlSX.
A e
8
h
in
he
absence
o
he
inhibi o ,
and
once
he
cell
load
had
been
co ec ed
o
10
+
0.2 p.g
o
Chi
pe
ml,
MSX
(35
F.M
inal
concen a ion)
was
eadded.
Successi e
pe iods
o
ammonia
p o-
duc ion
(ca.
40
h)
and
cell
eco e y
(ca.
8
h)
(a ows)
we e
al e na ed
as
indica ed.
de iciencies
by
hemsel es.
This
was
achie ed
by
al e na ing
exposu e
o
MSX
wi h
pe iods
in
which
he
glu amine
syn he ase
inac i a o
was
emo ed.
Figu e
5
shows
ha
his
sequen ial
ea men
allowed
con inuous
ammonia
p o-
duc ion
o
a
leas
7
days.
The
long
pe iod
in
he
p esence
o
MSX
(a ound
40
h)
esul ed
in
ne
ammonia
p oduc ion,
whe eas
he
sho e
pe iod
in
i s
absence
(a ound
8
h)
did
no .
Du ing
he
eco e y
pe iods,
glu amine
syn he ase
8
0
E
E
0
w
u
i
0
4
6
4
2
0
3
6
9
12
15
18
TIME
(days)
FIG.
6.
E ec
o
he
al e nance
o
MSX
and
PT
on
he
pho op o-
duc ion
o
ammonia
by
Anabaena
sp.
s ain
ATCC
33047
ilamen s.
Condi ions
we e
he
same
as
hose
desc ibed
in
he
legend
o
Fig.
5.
excep
ha
he
successi e
pe iods
o
ammonia
p oduc ion
p oceed-
ed
al e na ely
in
he
p esence
o
MSX
(ca.
40
h)
and
o
PT
(ca.
20
h).
wi h
eco e y
pe iods
(ca.
8
h)
in
he
absence
o
any
inhibi o
be ween
hem
(a ows).
/
"I
si ~~~~/
0~~~~~~~
~ ~ ~
.
VOL.
48,
1984
118
RAMOS,
GUERRERO,
AND
LOSADA
ac i i y
inc eased
om
0
mU
o
140
o
280
mU/mg
o
p o ein
(7
o
14%
o
he
le el
in
no mal
cells),
allowing
glu amine
syn hesis,
inc ease
o
he
phycocyanin
le el
( om
20
o
25%
o
50
o
80%
o
he
ini ial
one),
and
sligh
g ow h
( om
9.8
o
abou
10.5
p.g
o
Chl
pe
ml
in
8
h).
Su p isingly,
a e
7
days
o
ammonia
p oduc ion,
esis ance
o
MSX
appea ed.
This
phenomenon
was
p obably
due
o
an
adap a ion
o
he
ilamen s
o
MSX
and
made
i
necessa y
o
inc ease
he
MSX
concen a ion
up
o
100
o
150
FM
o
again
achie e
comple e
inac i a ion
o
cellula
glu amine
syn he ase.
The
e ec
on
he
du a ion
o
he
ammonia
p oduc ion
p ocess
o
al e na ing
ea men s
wi h
wo
di e en
glu a-
mine
syn he ase
inhibi o s,
namely,
MSX
and
PT,
has
also
been
es ed.
PT
is
ano he
glu ama e
analog
which
e ec i e-
ly
inhibi s
glu amine
syn he ase
and
allows
ammonia
p oduc-
ion
a
high
a es
by
Anabaenca
sp.
s ain
ATCC
33047
(6).
The
ea men
including
he
use
o
bo h
MSX
and
PT
consis ed
o
a
i s
pe iod
in
he
p esence
o
MSX
(ca.
40
h),
ollowed
a e
a
sho
in e al
o
eco e y
(ca.
8
h)
by
a
second
pe iod
(a ound
20
h)
in
he
p esence
o
PT.
Figu e
6
shows
ha
his
sequen ial
ea men
wi h
MSX
and
PT
pe mi ed
con inuous
ammonia
p oduc ion
o
longe
han
2
weeks,
p o ided
ha
eco e y
pe iods
in
he
absence
o
ei he
inhibi o
we e
also
al e na ed
h oughou .
The
p ocess
o
biological
ammonia
pho op oduc ion
om
N2
by
adequa ely
ea ed
li ing
cyanobac e ial
cells
ep e-
sen s
he
ne
gain
o
a
aluable
compound
om
abundan
and
inexpensi e
subs a es,
namely,
ai
and
wa e ,
a
he
expense
o
only
sunligh
as
he
sou ce
o
ene gy.
Since
he
sys em
ope a es,
once
i
has
been
s abilized
as
desc ibed
he ein,
a
a
conside able
a e
(see
e e ence
4
o
a
compa i-
son
o
ammonium
p oduc ion
a es
by
di e en
biological
sys ems)
and
wi h
a
easonable
ene gy
con e sion
e iciency
(a ound
2%),
i
could
p o e
e en ually
aluable
o
p ac ical
pu poses.
ACKNOWLEDGMENTS
This
wo k
was
suppo ed
by
g an s
om
Fundacidn
Ram6n
A eces
and
Comisi6n
Aseso a
In es igacion
(Spa in).
We
hank
An onia
F iend
and
Pepa
Pe ez
de
Leon
o
help ulI
assis ance.
We
a e
g a e ul
o
Pe e
J.
Lea
(Ro hams ed,
Uni ed
Kingdom)
o
a
gene ous
gi
o
phosphino h icin.
LITERATURE
CITED
1.
Allen,
M.
M.,
and
A.
I.
Smi h.
1969.
Ni ogen
chlo osis
in
blue-
g een
algae.
A ch.
Mic obiol.
69:114-120.
2.
Bailey,
J.
L.
1967.
Techniques
in
p o ein
chemis y.
2nd
ed.,
p.
340.
Else ie /No h-Holland
Biomedical
P ess.
Ams e dam.
3.
Be gmeye ,
H.
U.
1974.
Me hoden
de
Enzyma ischen
Analyse.
2nd
ed.
Ve lag
Chemie.
Weinheim.
Fede al
Republic
o
Ge ma-
ny.
4.
Gue e o,
M.
G.,
J.
L.
Ramos,
and
M.
Losada.
1982.
Pho osyn-
he ic
p oduc ion
o
ammonia.
Expe ien ia
38:53-58.
5.
Koche ,
G.
1978.
Ca bohyd a e
de e mina ion
by
he
phenol-
sul u ic
me hod,
p.
95-97.
In
J.
A.
Hellebus
and
J.
S.
C aigie
(ed.),
Handbook
o
physiological
me hods.
Physiological
and
biochemical
me hods.
Camb idge
Uni e si y
P ess,
Camb idge.
6.
Lea,
P.
J.,
K.
W.
Joy,
J.
L.
Ramos,
and
M.
G.
Gue e o.
1984.
The
ac ion
o
2-amino-4-(me hylphosphinyl)bu anoic
acid
(phosphino h icin)
and
i s
2-oxo-de i a i e
on
he
me abolism
o
plan s.
Phy ochemis y
23:1-6.
7.
Losada,
M.
1979.
Pho op oduc ion
o
ammonia
and
hyd ogen
pe oxide.
Bioelec ochem.
Bioene g.
6:205-225.
8.
Mackinney,
G.
1941.
Abso p ion
o
ligh
by
chlo ophyll
solu-
ions.
J.
Biol.
Chem.
140:315-322.
9.
Ramos,
J.
L.,
M.
G.
Gue e o,
and
M.
Losada.
1981.
Pho op o-
duc ion
o
ammonia
om
dini ogen
by
whole
cells
o
blue-
g een
algae,
p.
707-717.
In
G.
Akoyunoglou
(ed.),
Pho osyn he-
sis,
ol.
6.
P oceedings
o
he
Fi h
In e na ional
Cong ess
on
Pho osyn hesis.
Balaban
In e na ional
Science
Se ices,
Phila-
delphia.
10.
Ramos,
J.
L.,
M.
G.
Gue e o,
and
M.
Losada.
1982.
Pho op o-
duc ion
o
ammonia
om
ni a e
by
Anacys is
nidiul(ans
cells.
Biochim.
Biophys.
Ac a
679:323-330.
11.
Ramos,
J.
L.,
M.
G.
Gue e o,
and
M.
Losada.
1982.
Sus ained
pho op oduc ion
o
ammonia
om
ni a e
by
AnacYs is
nidi-
lIns.
Appl.
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