Deep-tank culture of blue-green algae in H2O and D2O


Taecker, R.G.; Crespi, H.L.; DaBoll, H.F.; Katz, J.J.

Biotechnology and Bioengineering 13(6): 779-793

1971


BIOTECHNOLOGY
AND
BIOENGINEERING,
VOL.
XIII,
PAGES
779-793
(1971)
Deep-Tank
Culture
of
Blue-Green
Algae
in
H
2
O
and
D
2
0
R.
G.
TAECKER,
H.
L.
CRESPI,
H.
F.
DABOLL,
and
J. J.
KATZ,
Chemistry
Division,
Argonne
National
Laboratory,
Argonne,
Illinois
60439
Summary
A
160-liter
stainless
steel
algal
growth
tank
has
been
constructed
and
has
been
used
essentially
continuously
for
over
three
years.
Filamentous
and
unicellular
blue-green
algae
as
well
as
a
photosynthetic
bacterium
have
been
cultured
using
both
ordinary
aand
heavy
water
(99.8
atom
%
'H).
By
using
a
recycling
technique,
yields
as
high
as
25
g/liter
of
2
H
2
0
have
been
obtained.
INTRODUCTION
One
of
the
more
interesting
consequences
that
arises
from
the
ability
to
culture
algae
and
other
microorganisms
in
99.8%
D20
is
the
availability
of
fully
deuterated
and
isotope
hybrid
biopolymers
containing
both
2
H
and
1H
for
studies
of
protein
and
enzyme
struc-
ture
and
function.
1-4
In
order
to
isolate
useful
amounts
of
some
of
the
protein
constituents
of
blue-green
algae,
it
becomes
necessary
to
culture
the
algae
on
a
very
large
scale.
Likewise,
large
amounts
of
fully
deuterated
algal
extracts
are
required
for
growing
hetero-
trophic
organisms
of
unusual
isotopic
composition.
We
have,
there-
fore,
put
into
operation
a
160-liter
deep-tank
algal
culture
apparatus
whose
design
and
operation
is
reported
here.
Our
experiments
are
aimed
at
achieving
a)
practical
growth
rates;
b)
efficient
use
of
D
2
0;
and
c)
continued
good
growth
in
the
presence
of
high
concentrations
of
exogenous
amino
acids.
Although
it
is
possible
to
cultivate
algae
on
a
large
scale
with
simpler
apparatus
(see,
for
example,
Lyman
and
Siegelman
5
),
the
more
elaborate
techniques
described
here
are
necessary
to
secure
the
degree
of
control
and
reproducibility
desired
779
©
1971
by
John
Wiley
&
Sons,
Inc.
780
TAECKER
ET
AL.
in
the
growth
of
organisms
extensively
substituted
with
isotopes
such
as
2
H,
"C,
and
"N.
A
culture
system
involving
very
high
in-
tensity
light,
for
example,
such
as
that
described
by
Matthern
et
a1.,
6
is
not
suited
to
our
purposes.
Most
species
of
algae
are
slow growing
in
H
2
O,
and
in
2
H20
a
depression
in
light
saturated
growth
rates
of
a
factor
of
3.5
6
is
encountered,
so
that
in
2
H
2
0
solarization
could
easily
become
a
severe
problem.
The
apparatus
we
describe
here
is
versatile
and
has
been
found
highly
suitable
for
the
culturing
of
blue-green
algae,
both
unicellular
and
filamentous
forms,
and
for
photosynthetic
bacteria.
The
ability
to
control
light,
intensity,
temperature,
and
other
variables,
and
the
instrumentation
for
automatically
recording
growth
rate
and
photosynthetic
activity
makes
this
apparatus
particularly
useful
for
investigations
on
algal
or
bacterial
physiology.
EQUIPMENT
AND
METHODS
Growth
Tank
Provisions
and
General
Characteristics
A
Type
316
stainless
steel
tank
of
dimensions
somewhat
similar
to
a
55-gal
drum
is
surrounded
by
an
integral
cooling
jacket.
(The
tank
with
fittings
and
the
drive
unit
was
fabricated
by
the
Kohler
Manufacturing
Co.,
Madison,
Wisc.)
Removable
bottom
and
top
end
plates
are
bored
on
approximately
3-in.
centers
to
receive
42
vertically
arranged
51-mm
(nominal
o.d.)
clear
glass
(Kimax)
tubes
into
which
fluorescent
tubes
may
be
inserted.
The
bottom
plate
is
fitted
with
a
total
of
6
small
gas
distributors
and
an
integrally
fabricated
valve
for
emptying
the
tank
contents.
The
top
plate
is
fitted
with
a
centrally
located
hub
with
Teflon
seal
and
guide
for
an
agitator
shaft,
and
a
plate
to
receive
up
to
4
threaded
gas
and
detector
fittings.
The
wall
of
the
tank
is
completely
surrounded
by
an
outer
blanket
of
Fiberglass
insulation
to
which
is
bonded
alumi-
num
foil
vapor
barrier.
The
plunger-type
agitator
is
composed
of
5
stainless
steel
plates
with
holes
bored
in
positions
corresponding
to
the
positions
of
the
glass
tubes,
stacked
at
6-in.
intervals
along
a
1-in.
(15.9
mm)
diameter
stainless
steel
shaft.
However,
the
holes
are
larger
than
the
glass
tubing
(21
in.
(63.5
mm)
diameter
compared
to
51
mm).
Thus,
as
the
plunger
is
moved
up
and
down,
the
growth
medium
with
sus-
pended
algae
passes
alternately
through
the
annular
orifices
formed
BIOTECHNOLOGY
AND
BIOENGINEERING,
VOL.
XIII,
ISSUE
6
DEEP-TANK
CULTURE
OF
ALGAE
781
by
the
glass
tubing
and
holes
of
the
plunger
plates.
Under
condi-
tions
of
drive
setting
to
provide
the
maximum
stroke
of
6
in.,
essen-
tially
all
of
the
supension
must
pass
alternately
from
upper
to
lower
zones
through
the
annuli,
and
hence,
within
a
mean
distance
of
approximately
g
in.
from
the
outer
walls
of
the
glass
tubes.
The
growth
tank,
agitator
drive,
and
other
directly
associated
accessories,
mounted
on
Unistrut
supports,
are
shown
in
Figure
1.
Exact
dimensions
and
fabrication
details,
including
those
of
the
flanges
and
"0"
rings
assemblies
for
sealing
the
glass
tubes
which
pierce
the
end
plates,
may
be
obtained
by
writing
the
authors.
A
simplified
schematic
drawing
is
given
in
Figure
2.
As
a
precaution
when
large
amounts
of
costly
2
1
-
1
2
0
are
used,
the
entire
tank
assem-
bly
is
set
into
a
circular
stainless
steel
trough,
which
is
5
in.
deep
and
5
ft.
in
diameter,
providing
a
reservoir
of
more
than
adequate
volume
to
catch
the
contents
of
the
tank
should
a
leak
occur.
Energy
for
photosynthesis
is
provided
by
up
to
38
Type
40TW
rapid
start
fluorescent
tubes,
arranged
in
banks
of
two,
which
may
be
freely
inserted
into
the
glass
tubes.
Of
the
remaining
4
holes,
2
holes
(top
end
plate)
are
used
for
access
to
the
vessel
and
inspection
during
operation;
and
2
holes,
complete
with
glass
tubes
but
without
fluorescent
lights,
are
used
for
optical
inspection
and
measurement
throughout
the
culture.
The
2
holes
in
the
bottom
end
plate
directly
opposite
the
2
holes
required
for
access
are
sealed
by
machined
Lucite
plugs
held
in
position
by
the
normal
gland
assembly
used
for
the
glass
tubes.
Gas,
as
carbon
dioxide,
nitrogen,
and
oxygen,
singly
or
in
any
of
several
combinations,
is
introduced
under
pressure
through
a
single
simple
distributor
in
the
center
of
the
bottom
plate,
and
through
1
or
more
of
5
radial
distributors.
Excess
and
generated
off-gas
is
dis-
charged
through
a
threaded
connection
in
the
top
plate,
or
through
a
reflux
condenser
attached
to
a
Lucite
closure
plug
of
an
inspection
hole
in
the
top
end
plate.
All
permanent
tubing
and
connectors
which
might
in
any
way
or
for
any
reason
become
partly
filled
with
culture
medium,
such
as
during
failure
of
gas
pressure
or
flow,
are
of
Type
316
stainless
steel.
Illumination
To
assure
easy
insertion
through
growth
tank
end
plates
and
relatively
stress-free
sealing
by
flange
and
"0"
ring
assemblies,
782
TAECKER
ET
AL.
_
#
*
.....
.
.P
.
o
.
..............
a.
t
Fig.
1.
The
160-liter
algae
tank
with
inserted
fluorescent
tubes,
oxygen
elec-
trode
with
stirrer
(top
front
of
tank),
recorder,
spill
retainer,
and
other
associated
equipment.
BIOTECHNOLOGY
AND
BIOENGINEERING,
VOL.
XIII,
ISSUE
6
DEEP-TANK
CULTURE
OF
ALGAE
783
B
E
F
G
H
A
D
C
_
B
Fig.
2.
Left:
A
schematic
of
the
algae
tank
showing
(A)
drive
shaft
linkage
and
central
hub
(B)
with
Teflon
sleeve
(C)
and
cap
(D).
In
practice,
the
Teflon
sleeve
is
very
loose
fitting,
but
an
easily
replaceable,
beveled
Teflon
bushing
is
fitted
beneath
cap
D
and
kept
to
a
snug
fit
with
this
cap.
At
E
we
show
glass
tubes
in
radial
alignment,
although
the
bulk
of
the
tube
holes
are
radially
stag-
gered.
At
F
is
the
cooling
water
jacket,
G
shows
agitator
plates,
H
is
the
drain
valve,
and
I
indicates
2
of
the
4
Unistrut
"feet"
with
leveling
bolts.
Right:
A
drawing
of
a
glass
tube
projecting
from
the
base
plate
of
the
algae
tank,
with
flange
and
"0"
ring
(A)
and
a
"cage"
for
holding
a
fluorescent
tube
(B).
the
glass
tube
outside
diameters
are
held
to
a
maximum
of
51.0
mm
by
selection
with
a
ring
gauge.
Tube
length
is
48
in.
(nominal).
The
"cage"
illustrated
in
Figure
2
offers
positive
support,
align-
ment,
and
ventilation
for
the
fluorescent
tubes.
They
are
conven-
iently
installed
and
removed,
and
are
rather
readily
fabricated
from
784
TAECKER
ET
AL.
3
/
3
2
in.
(2.3
mm)
diameter
stainless
steel
welding
rod
into
which
are
mounted,
with
a
dab
of
epoxy
resin,
General
Electric
ALF
275-01)
lampholders.
Lower
cages
are
bent
slightly
near
their
ends
to
support
the
fluorescence
tubes.
To
mate
with
the
slightly
bent
prongs
of
the
lower
cages,
one
end
of
each
glass
tube
is
heated,
drawn
in
slightly,
and
fire-polished.
Care
is
exercised
so
the
final
minimum
diameter
is
not
less
than
the
sum
of
a
fluorescent
tube
diameter
and
2
prong
diameters,
and
the
bead
formed
around
the
edge
of
the
glass
tube
during
fire-
polishing
is
rolled
outwards
rather
than
inwards.
However,
if
inner
diameters
cannot
be
kept
to
uniform
minima,
inner
sides
of
the
cage
prongs
may
be
ground
or
filed
slightly
to
form
easy
fits
with
the
fluorescent
tubes
in
place.
Proper
fits
are
essential
to
eliminate
the
possibility
of
glass
or
fluorescent
tube
breakage
during
installa-
tion
and
use,
as
determined
by
ease
of
complete
interchangeability
of
fluorescent
tubes
with
their
slightly
variable
diameters.
Upper
cage
prongs
are
not
bent,
as
they
serve
only
to
keep
fluorescent
tubes
aligned
and
contain
the
electrical
sockets.
These
ends
of
the
glass
tubes
are
fire-polished
only,
with
beads
inclined
slightly
outward,
as
they
are
not
required
for
support.
To
provide
for
maximum
illumination,
and
particularly
for
maxi-
mum
flexibility
in
lighting
configurations,
wiring
and
accessory
parts
are
installed
for
all
possible
glass
tube
positions
in
the
growth
tank.
Thus,
wiring,
fluorescent
tube
sockets,
and
cages
are
provided
for
42
positions,
and
21
sets
of
fuse-switch-pilot
lamp
combinations
are
installed
at
the
control
panel.
Wires
from
the
fluorescent
tube
sockets
are
joined
through
distribution
centers
equipped
with
barrier
terminals.
Light
attenuation
as
a
function
of
algal
concentration
(S.
lividus)
was
determined
with
a
"unit
cell"
that
consisted
of
a
cylindrical
chamber
through
which
projected
a
single
fluorescent
tube
assembly
Light
intensity
measurements
were
taken
with
a
cadmium
sulfide
photo
resistance
cell
at
i-in.
intervals.
Agitation
Agitation
at
fixed
frequency
(10
cycles/min)
but
variable
ampli-
tude
(6
to
3
in.)
is
accomplished
by
the
plunger.
Vertical
oscillatory
motion
of
the
plunger
is
imparted
by
a
walking
beam
connected
at
BIOTECHNOLOGY
AND
BIOENGINEERING,
VOL.
XIII,
ISSUE
6
DEEP-TANK
CULTURE
OF
ALGAE
785
the
driven
end
by
a
connecting
rod
to
the
face
wheel
of
an
electric
motor
powered
speed
reducer.
Apparent
(submerged)
weight
of
the
plunger
is
counterbalanced
by
a
lead-loaded
cylinder
which
is
made
part
of
the
connecting
rod.
Each
of
the
lower
2
plunger
plates
has
slots
milled
into
the
edge,
approximately
on
a
diameter
which
is
orthogonal
to
the
walking
beam,
that
loosely
engage
guide
rails
welded
to
the
lower
inner
wall
of
the
growth
tank.
The
guide
arrangement
serves
primarily
to
avoid
rotation
of
the
plunger,
as
a
self-aligning
(as
opposed
to
sleeve)
bearing
is
used
to
connect
the
plunger
shaft
to
the
walking
beam
linkage.
Secondarily,
it
serves
to
prevent
possible
tube
breakage
should
the
tank
be
seriously
out-of-level
or
should
the
central
hub
with
Teflon
sleeve,
through
which
the
plunger
rod
passes,
be
mis-
aligned.
If
the
tank
is
level
and
the
central
hub
with
Teflon
sleeve
is
properly
aligned,
the
edges
of
the
slots
in
the
plunger
plates
touch
the
guide
rails
only
rarely.
In
motion,
the
plunger
imparts
a
gentle
streaming
action
to
the
medium,
as
it
is
alternatively
forced
up
and
down
through
the
plunger-type
annuli.
Cooling
and
Heating
Cooling
is
accomplished
by
circulating
cold
water
through
the
growth
tank
jacket.
A
blower
induced
down
draft
flow
of
air
in
the
annular
space
between
the
fluorescent
tubes
and
the
inner
glass
tube
walls
assists
in
removing
heat
generated
by
the
fluorescent
tubes,
and
helps
to
prolong
fluorescent
tube
life.
For
ambient
temperatures
of
30-32°C,
heating
is
rarely
required
except
during
early
stages
of
growth
when
light
intensities
are
minimal.
A
technique
that
is
generally
satisfactory
is
to
install
temporarily
4
to
8
fluorescent
tubes
which
are
wrapped
in
a
spirally
spaced
manner
with
electrical
tape.
With
the
blower
turned
off,
the
several
fluorescent
tubes,
wrapped
to
reduce
light
emission
by
about
80%,
transfer
suff
icient
heat
energy
to
the
medium
to
achieve
and
maintain
elevated
temperatures
without
exceeding
light
intensity
limits
of
dilute
cultures.
Gas
Flow
System
Gases
used
in
the
photosynthetic
apparatus
are
carbon
dioxide,
oxygen,
and
nitrogen.
Connections
and
interconnections
are
pro-
vided
for
readily
routing
and
rerouting
each
gas
and
for
mixing
them
786
TAECKER
ET
AL.
in
appropriate
proportions.
In
addition,
as
it
is
assumed
that
the
photosynthetic
apparatus
might
at
a
future
date
be
operated
at
slightly
above
or
below
atmospheric
pressure,
with
or
without
rare
stable
or
unstable
isotopic
gaseous
constituents,
stainless
steel
tube
fittings
and
accessories
are
used
and
a
2-channel
pressure
indicator—
recorder
is
provided.
Required
carbon
dioxide
rates
during
algae
growth
rarely
exceed
150
ml/min
(5
psig
and
30°C)
when
introduced
as
pure
gas
through
the
center
distributor
(with
a
nitrogen
flow
of
2000
ml/min
through
the
radial
distributor)
or
3000
ml/min
(5
psig
and
30°C)
introduced
at
5%
CO2-95%
N2
through
the
radial
distri-
butors.
Oxygen
is
required
during
part
or
all
of
any
dark
period
at
a
rate
of
at
least
120
ml/min
(5
psig
and
30°C).
Growth
Rate
Growth
rate
is
measured
indirectly
as
the
change
in
intensity
with
time
of
light
from
a
given
constant
light
source
transmitted
through
a
fixed
thickness
of
algal
suspension.
Two
"optical
density"
measurement
systems
are
in
service:
a
selenium
photo
potential
device
which,
through
an
external
sampling
cell,
"sees"
a
tungsten
lamp;
and
a
cadmium
sulfide
photo
resistance
device
which,
through
the
internal
algae
suspension,
"sees"
a
fluorescent
"source
tube."
The
selenium
device
of
the
external
system
has
a
long
lag
period
as
it
approaches
equilibrium
after
an
abrupt
change
in
light
intensity.
On
the
other
hand,
this
system
is
extremely
sensitive
to
growth
changes
in
very
dilute
cultures,
is
totally
isolated
from
the
internal
illumination
pattern,
and
is
readily
coupled
to
a
millivoltmeter
type
indicator—controller
in
parallel
with
a
potentiometric
recorder.
The
cadmium
sulfide
device
of
the
internal
system
is
prompt
in
response
to
light
changes,
however
abrupt,
and
is
extremely
sensitive
to
growth
changes
in
dilute
cultures.
However,
as
the
fluorescent
source
tube
is
surrounded
by
other
fluorescent
tubes
which
may
be
brought
into
use,
scattered
light
from
the
neighboring
tubes
may
upset
the
calibration.
Both
the
external
and
internal
systems
be-
come
less
sensitive
to
changes
as
algal
concentration
increases.
Both
optical
sensors
were
calibrated
by
means
of
dry
weight
measurements
and
all
cell
densities
reported
here
are
in
terms
of
dry
weight.
Oxygen
Monitoring
The
dissolved
oxygen
concentration
was
monitored
by
a
Delta
Scientific
Series
322
oxygen
monitor
with
continuous
recording.
BIOTECHNOLOGY
AND
BIOENGINEERING,
VOL.
XIII,
ISSUE
6
DEEP-TANK
CULTURE
OF
ALGAE
787
Stirrer
Fall-Safe
Because
of
the
powerful
drive
unit
(1
hp,
1725
rpm
motor
through
a
1
to
200
gearbox),
instantaneous
decoupling
of
the
face
wheel
from
the
gearbox
is
required
and
is
accomplished
by
replacing
the
steel
key
of
the
face
wheel
and
gearbox
output
shaft
with
a
short
key
(approx.
a
in-long)
machined
from
Teflon
key
stock.
In
the
event
of
shearing
of
the
Teflon
key,
the
face
wheel
is
dislodged
inward,
activating
a
normally
closed
microswitch
which,
through
relays,
interrupts
power
to
the
fluorescent
tubes.
Simultaneously,
power
to
a
two-way
solenoid
valve
is
interrupted,
and
gas
flow
is
diverted
from
the
peripheral
spargers
to
the
top
freeboard
of
the
tank.
Re-
routing
the
nitrogen
(or
N2—0O2)
gas
to
sweep
the
surface
tends
to
conserve
the
dissolved
oxygen.
If
a
fail-safe
of
more
than
a
few
hours
is
desired,
a
low
rate
of
oxygen
may
be
supplied
through
the
center
bottom
hole
along
with
the
CO
2
feed.
At
high
light
intensities
(high
heat
load)
fail-safe
temperature
control
may
be
obtained
by
continuously
bleeding
cooling
water
through
the
tank
jacket.
Flow
rates
are
adjusted
so
as
to
be
just
insufficient
to
cool
the
culture
below
the
set
temperature.
In
case
of
failure
of
"house"
nitrogen,
a
pressure
switch
activates
a
normally
closed
solenoid
valve
to
allow
flow
from
standing
nitrogen
cylinders.
A
check
valve
prevents
backflow
into
the
"house"
nitrogen
system.
Nutrients
The
Ac
nutrient
medium
described
earlier
8
has
been
routinely
used
for
all
three
blue-green
algae
listed
above.
In
runs
involving
many
harvest
cycles,
nitrate
concentrations
were
maintained
above
250
ppm.
It
was
determined
by
flask
culture
experiments
that
the
alga
S.
lividus
would
grow
to
nitrate
exhaustion
(3
ppm),
so
the
nitrate
concentration
has
been
used
as
the
key
to
nutrient
makeup.
After
each
harvest,
FeSo4
7H20
in
the
amount
of
0.004
g/liter
and
microelements
at
4
the
standard
level'
were
added
to
the
culture
medium.
Algal
Species
The
blue-green
algae
Synechococcus
lividus,
Phormidium
luridum,
and
Fremyella
diplosiphon
8
have
been
tank
cultured.
S.
lividus
was
cultured
at
45°C,
P.
luridum
at
35°C,
and
F.
diplosiphon
at
30°C.
788
TAECKER
ET
AL.
Start-
Up
Initial
inocula,
usually
5
liters
of
culture
at
a
cell
density
of
2
g/liter,
were
taken
from
rocking-box
cultures.'
Inoculation
was
into
nutrient
medium
at
0.5
the
salt
strength
listed
by
DaBoll
et
al.
8
The
dissolved
oxygen
in
the
tank
was
adjusted
to
from
6-10
ppm
before
inoculation,
and
the
inoculum
allowed
to
stand
in
the
dark
for
10-20
minutes.
At
this
pint,
the
oxygen
feed
is
stopped
and
the
CO
2
feed
brought
to
150
ml/min
(the
N2
feed
is
at
a
rate
of
2000
ml/min).
As
the
oxygen
concentration
decreases,
5
light
banks
are
turned
on.
Oxygen
evolution
begins
at
once
and
the
oxygen
level
stabilizes
rather
quickly.
After
a
lag
of
2-3
hr,
growth
is
registered
by
the
optical
growth
sensors.
Harvest
Cells
are
harvested
at
a
cell
density
of
about
1
g/liter.
At
harvest,
15
liters
of
culture
are
removed
and
aerated
vigorously,
the
tank
is
darkened,
oxygen
is
introduced
at
a
rate
of
about
1000
ml/min
and
the
CO2
feed
is
stopped.
The
tank
is
pressurized
to
3.5
psig
with
nitrogen
gas
and
the
algal
suspension
fed
through
a
Sharples
Super-
speed
Centrifuge
(the
centrifuge
is
cooled
with
tap
water)
at
a
flow
rate
of
1
liter/40
to
50
sec.
Centrifugation
is
complete
in
2
hr.
The
harvested
algae
are
removed
from
the
centrifuge
bowl,
packed
into
muslin
bags
and
immediately
frozen
in
liquid
nitrogen.
The
supernatant
solution
is
pressure
pumped
back
to
the
culture
tank.
The
previously
removed
15
liters
of
culture
are
returned
to
the
tank,
along
with
additional
inorganic
salts
and
any
necessary
2
1120
to
return
to
volume.
At
this
point,
the
dissolved
oxygen
level
is
usually
at
6-10
ppm.
The
tank
may
then
be
brought
to
the
desired
temperature
by
feeding
hot
water
through
the
tank
jacket,
the
N2
-
spargers
are
brought
to
2000
ml/min,
CO
2
is
fed,
02
feed
is
stopped,
and
as
the
oxygen
concentration
falls,
5
light
banks
are
turned
on.
After
the
initial
lag
period
and
some
hours
of
growth,
the
light
intensity
is
increased
to
11
light
banks
and
as
the
cell
density
in-
creases
beyond
0.1
g/liter
the
full
18-20
light
banks
are
used.
Nitrate
Exhaustion
In
these
experiments,
all
salts
were
added
at
normal
levels,
but
only
0.5
g/liter
of
KNO
3
(approx.
240
ppm
of
nitrate
ion)
was
added
at
each
cycle,
sufficient
to
yield
almost
1
g/liter
of
algae.
At
15-20
BIOTECHNOLOGY
AND
BIOENGINEERING,
VOL.
XIII,
ISSUE
6
20
I0
8
6
5
4
3
2
Co
nce
n
tra
t
io
n
(p
e
rce
n
t)
DEEP-TANK
CULTURE
OF
ALGAE
789
hr
before
harvest
(nitrate
level
at
about
50
ppm)
15
liters
of
culture
were
removed
and
stored
at
32°C
in
dim
light
with
vigorous
bubbling
of
air.
This
material
served
as
the
inoculation
for
the
next
growth
cycle.
Addition
of
Amino
Acids
Amino
acids
were
dissolved
directly
into
the
nutrient
solutions.
During
the
first
growth
cycle,
the
amino
acid
level
was
brought
to
a
reasonably
high
level,
as
judged
by
the
effect
on
growth
rate,
dis-
solved
oxygen,
and
previous
experience
in
rocking
trays.'
In
succeeding
cycles,
levels
were
maintained
or
increased,
depending
on
the
response
of
the
algae.
RESULTS
AND
DISCUSSION
Figure
3
illustrates
the
characteristic
growth
rate
of
S.
lividus
in
H
2
O
and
D
2
0.
In
general,
the
illumination
of
D20
cultures
is
conservative,
leading
to
growth
rates
in
D
2
0
that
gave
an
average
production
of
20-30
g
of
algae
per
day.
In
H
2
O,
algae
could
be
produced
easily
at
a
rate
of
30-40
g
per
day.
Harvest
of
all
the
blue-
90
1
1 1
1
1 1 1 1 I 1
_
1.5g/1
30
70
50
40
0.5
g/2
100%
=
2.45
grams
(dry
wt.)/liter
of
medium
I I
1
I
1
1 1 1
I
1
0
2
3
4
5
6
Days
7
8
9
10
Fig.
3.
A
semi-log
plot
showing
the
characteristics
course
of
growth
of
S.
lividus
in
99.8%
D
2
0.
In
H
2
O
one
would
observe
a
similar
curve
on
a
somewhat
shorter
time
scale.
A
concentration
of
100%
represents
2.4
g(dry)/liter.
790
TAECKER
ET
AL.
green
algae
was
at
a
cell
density
of
about
1
g/liter
for
several
reasons.
As
cultures
increased
in
cell
density
above
1
g/liter,
there
was
an
increasing
tendency
for
the
algae
to
stick
to
the
glass
and
steel
surfaces,
especially
with
P.
luridum
and
F.
diplosiphon
cultures.
(The
photosynthetic
bacterium
Rhodospirillum
rubrum
has
also
been
successfully
cultured
(H
2
0)
in
this
apparatus.)
As
the
culture
density
increases
beyond
1.5
g/liter,
the
growth
rate
begins
to
fall
and
production
begins
inefficient.
We
formed
the
impression
that
best
results
on
recycling
cultures
were
obtained
when
the
region
of
decreasing
growth
rates,
and
thus
perhaps
dead
cells,
was
avoided.
Figure
4
illustrates
the
distribution
of
light
within
the
culture
tank
at
a
concentration
of
algae
equal
to
1.7
g/liter.
At
this
cell
density,
the
cells
are
exposed
to
a
very
low
light
intensity
for
a
large
fraction
of
the
time
and
the
previously
linear
growth
rate
falls
exponentially.
Figure
5
shows
the
effective
volume
fractions
of
culture
receiving
certain
minimum
light
intensities
(300
and
500
ft-candles)
as
a
function
of
light
intensity.
This
plot
indicates
that
when
all
cells
'a
'1
1
Fig.
4.
A
CALCOMP
plot
of
light
intensity
distribution
in
a
culture
at
a
cell
density
of
1.7
g/liter.
The
No.
1
indicates
a
light
intensity
greater
than
1000
ft-candles;
No.
2,
between
500
and
600
ft-candles;
No.
3,
between
300
and
400
ft-candles.
BIOTECHNOLOGY
AND
BIOENGINEERING,
VOL.
XIII,
ISSUE
6
DEEP-TANK
CULTURE
OF
ALGAE
791
I
.0
0.9
0.8
0.7
0
t
0.6
0.5
E
Ti
0.4
0.3
0.2
0.I
0.4
0.8
1.2
1.6
2.0
2.4
Grams
per
liter
Fig.
5.
Curves
showing
the
fraction
of
the
culture
volume
receiving
300
or
more
and
500
or
more
ft-candles
of
light
intensity
(effective
volume
fraction)
as
a
function
of
cell
density.
The
fraction
of
the
culture
receiving
at
least
500
ft-candles
of
light
falls
off
very
rapidly
in
the
range
of
0.6
to
1.2
g/liter.
received
at
least
300
ft-candles
of
light
intensity,
growth
is
exponen-
tial.
At
a
cell
density
of
0.5
g/liter
growth
becomes
linear,
and
in
the
region
of
cell
concentration
of
1.4-1.5
g/liter
where
the
linear
growth
rate
begins
to
fall,
the
volume
fraction
receiving
at
least
300
ft-candles
is
falling
most
rapidly.
Above
cell
densities
of
1.5
g/liter,
then,
the
production
of
algae
in
this
apparatus
becomes
increasingly
inefficient.
Of
particular
interest
has
been
the
culture
of
S.
lividus
for
the
extraction
and
purification
of
three
low
molecular
weight
proteins:
cytochrome
c,
ferredoxin,
and
a
flavoprotein.
These
three
proteins
can
be
isolated
easily
and
in
high
purity.
Table
I
lists
yields
of
these
proteins
obtained
under
various
conditions.
It
has
been
found
that
in
H
2
O
yields
can
be
increased
considerably
by
allowing
the
culture
to
grow
6-10
hr
beyond
nitrate
exhaustion
before
harvest.
(Either
the
protein
content
increases
or
the
cells
are
more
completely
extracted.)
Although
the
growth
rate
drops
suddenly
at
nitrate
792
TAECKER
ET
AL.
exhaustion,
the
rate
of
oxygen
evolution
falls
off
only
slowly.
As
monitored
optically,
cell
density
continues
to
increase
after
nitrate
exhaustion,
but
at
0.25
the
rate
of
increase
observed
just
before
nitrate
exhaustion.
The
concentration
of
dissolved
oxygen,
however,
is
maintained
to
within
10-15%
of
that
before
nitrate
exhaustion.
We
have
not
yet
determined
whether
or
not
the
algae
are
fixing
nitrogen
during
this
period
or
extended
this
approach
to
D20
cultures.
TABLE
I
Yield
of
Purified
Proteins
from
S.
lividus
mg/100
g
dry
algae
H
2
0,
nitrate
Protein
H
2
O
depletion
D
2
0
Cytochrome
c
15-20
30-40
15-20
Ferredoxin
10-20
20-30
10-20
Flavoprotein
10-20
20-30
20-50
Three
runs
in
99.8%
D
2
0
have
been
successfully
carried
out
from
which
7,
12,
and
21
harvests
have
been
obtained.
In
these
runs,
the
blue-green
alga
S.
lividus
was
cultured
in
the
presence
of
the
exog-
enous
amino
acids
leucine,
phenylalanine,
threonine,
and
cystine.
Algae
grown
under
these
nutritional
conditions
are
then
a
source
of
isotope
hybrid
compounds,
as
described
by
Crespi
et
al.
2
Essentially
no
isotopic
dilution
is
observed
over
many
weeks
of
culturing,
a
re-
flection
of
the
large
amount
of
D
2
0
involved.
The
successful
appli-
cation
of
high
resolution
nuclear
magnetic
resonance
techniques
to
the
study
of
biopolymers
depends
to
a
large
degree
on
the
simplification
of
spectra
by
deuterium
substitution.
9
The
ability
to
grow
large
quantities
of
both
fully
deuterated
and
isotope
hybrid
algae
at
relatively
low
cost
makes
this
technique
a
practical
possibility.
It
is
our
judgment
that
in
the
absence
of
mechanical
failure
or
overdose
of
toxic
exogenous
amino
acids,
D20
can
be
cycled
through
15-20
harvests,
thus
greatly
lowering
the
cost
of
isotopic
substitution.
When
one
considers
that
the
charge
of
heavy
water
is
essentially
completely
recoverable,
the
cost
of
algae
grown
in
D
2
0
is
comparable
to
the
cost
of
algae
grown
in
I-1
2
0.
The
procedures
for
the
culture
of
blue-green
algae
described
here
are
much
more
economical
than
the
BIOTECHNOLOGY
AND
BIOENGINEERING,
VOL.
XIII,
ISSUE
6
DEEP-TANK
CULTURE
OF
ALGAE
793
single
batch
method
described
in
our
previous
work,'
as
a
liter
of
heavy
water
will
yield
in
the
order
of
20
g
of
algae.
This
work
was
performed
under
the
auspices
of
the
U.
S.
Atomic
Energy
Commission.
The
authors
would
like
to
thank
Mr.
James
Leipper
of
this
Laboratory
for
his
help
in
following
the
construction
of
the
growth
tank
and
Mssrs.
Ronald
Turski,
Paul
Howard,
and
Richard
Leedy
of
the
Argonne
Summer
Engineering
Practice
School
for
their
study
of
light
distribution
within
the
tank.
References
1.
H.
L.
Crespi
and
J. J.
Katz,
Nature,
224,
560
(1969).
2.
H.
L.
Crespi,
H.
F.
DaBoll,
and
J.
J.
Katz,
Biochim.
Biophys.
Acta,
200,
26
(1970).
3.
R.
M.
Rosenberg,
H.
L.
Crespi,
and
J. J.
Katz,
Biochim.
Biophys.
Acta,
175,
31
(1969).
4.
I.
Putter,
J.
L.
Markley,
and
0.
Jardetzky,
Proc.
Natl.
Acad.
Sci.,
65,
395
(1970).
5.
H.
Lyman
and
H.
W.
Siegelman,
J.
Protozool.,
14,
279
(1967).
6.
R.
0.
Mattern,
J.
A.
Kostick,
and
I.
Okada,
Biotechnol.
Bioeng.,
11,
863
(1969).
7.
H.
L.
Crespi,
S.
M.
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