JOURNAL OF BACTERIOLOGY, Sept. 1973, p.

937-942
Copyright 0 1973 American Society for Microbiology

Vol. 115, No. 3

Printed in U.S.A.

Efficiency of Energy Conversion by Aerobic

Glucose Metabolism in Aphanocapsa 6714
RICHARD A. PELROY AND JAMES A. BASSHAM

Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley,

California 94720
Received for publication 5 March 1973

Efficiency of adenosine triphosphate (ATP) formation from glucose oxidation

in Aphanocapsa 6714 was estimated by quantitative measurement of phosphorylated intermediary metabolites and glycogen (polyglucose) formed from 14C_
glucose. P/2e ratios based on oxygen uptake ranged from 2.62 to 3.08, whereas
those based on 14CO2 evolution ranged from 1.66 to 1.72. The synthesis of
glycogen, which is the dominant energy-consuming process in resting cells
exposed to exogenous glucose, was almnost totally inhibited under anaerobic
conditions, and the cellular concentration of ATP decreased steadily. Thus, both
net synthesis of ATP and the steady-state concentration of ATP are obligatorily
linked to respiration in this heterotrophic unicellular blue-green alga.

Aphanocapsa 6714 is a facultative unicellular

blue-green alga capable of chemoheterotrophic
growth (in the dark) on glucose. However, dark
growth is slow in comparison to photosynthetic
growth (8), decreasing by almost an order of
magnitude. This may be the result of inefficient
coupling of catabolism to adenosine triphosphate (ATP) synthesis. Because glucose is metabolized by the reactions of the oxidative
pentose pathway (6, 7), a high yield of potential
energy is available in the form of reduced
nicotinamide adenine dinucleotide phosphate,
i.e., six equivalents for each turn of a cyclic
pathway. Thus, a low yield of ATP from respiratory oxidation of reduced pyridine nucleotides
would necessarily imply a low efficiency of
energy coupling.
In the present study, our purpose was to
estimate the efficiency of energy conversion, or
P/O ratio, by an indirect method. Resting cell
suspensions, incubated in the dark and fed
glucose, convert most of the assimilated carbon
into a polyglucose or glycogen-like polymer (6,
7). Since the energy requirements for this process are known, i.e., two ATP per glucosyl unit, a
lower limit on the amount of ATP synthesized
per equivalent of glucose oxidized can be determined. This was done by measurements of
oxygen uptake and CO2 production (parameters
of catabolism) versus incorporation of glucose
into phosphorylated intermediates and polyglucose (parameters of anabolism).
In Aphanocapsa 6714, the glucose-6-phosphate and 6-phosphogluconate dehydrogenases
9,37

are both nicotinamide adenine dinucleotide

phosphate-linked enzymes. Thus, the primary
dehydrogenation reaction of catabolism in this
organism are also directly coupled to reduced
nicotinamide adenine dinucleotide phosphate
metabolism (7).
The use of the oxidative pentose pathway for
catabolism would almost certainly require a
respiratory mechanism. However, it is possible
that Aphanocapsa 6714 also is capable of synthesizing ATP by substrate phosphorylation
reactions and fermentation. Two lines of evidence suggest that this is not the case. First,
cell-free extracts contain little or no phosphofructokinase, 6-phosphogluconate dehydratase,
and 2-keto-3-deoxy-6-phosphogluconate aldolase activity (7). Thus, key enzymes of glycolysis and the Entner-Doudoroff pathway are
probably absent in the intact organism. If this is
so, then the two main bacterial pathways of
hexose fermentation are also absent. The second line of evidence is the observation that cells
maintained in the dark, in the absence of air,
are incapable of long-term survival (8). Thus, a
second objective of this work was to determine,
if possible, the quantitative effect of anaerobiosis on the capacity of Aphanocapsa 6714 to
synthesize ATP.
MATERIALS AND METHODS
Organism. Aphanocapsa 6714 is a unicellular bluegreen alga of typological group II A (10).
Media and culture conditions. Cells were grown
photoautotrophically in an inorganic medium, BG-11,

938

PELROY AND BASSHAM

described elsewhere (10). Cultures were aerated

with air containing about 0.03% CO2 (wt/vol).
'IC tracer experiments. Late log-phase cells (optical density of 230 klett units) were harvested at room
temperature by centrifugation at 29,000 x g for 20
min and resuspended in BG-11 plus 50 mM sodium
bicarbonate. The cell concentration was kept at
approximately 2% hard packed cells (wet weight)
(wt/vol). The cell slurry was then kept at low light
intensity until used in experiments.
Uniformly labeled "4C-glucose (New England Nuclear Corp.) was added to unlabeled carrier glucose to
give a final sp act of 20 nCi per nmol. The cell
suspension of Aphanocapsa 6714 was then added to
the main chamber of the steady-state apparatus,
previously described (5), and aerated vigorously.
Temperature was maintained at 25 C by means of a
circulating water bath. The pH of the algal suspension
was 9.2. Just before beginning.the experiment, the
system was closed to the atmosphere by a two-way
stopcock, and the endogenous rate of 02 uptake was
monitored automatically with a Beckman model F-3
oxygen analyzer, connected to a chart recorder. When
the system had stabilized, the endogenous rate of 02
consumption was less than 0.02 nmol per min per mg
of algae. The room lights were then turned off and
"4C-glucose was added to the algae through a serum
stopper by syringe. The initial concentration of glucose in the system was 1 mM. At 2-min intervals,
0.5-ml samples of algal suspension were removed by
syringe and killed by the addition of 2 ml of methanol.
From each of these samples, 250 ml was removed and
analyzed by two-dimensional paper chromatography,
as described in detail elsewhere (1, 5, 6).
Soluble intermediates, after being separated by
paper chromatography and identified by autoradiography, were counted by means of the "automatic
spot counter" developed by Moses et al. (4). Glycogen, which remained at the origin of the chromatograms, was eluted by acid hydrolysis with 2 N HCI at
100 C for 2 h, taken to dryness in a vacuum desiccator, and resuspended in 25 mM sodium bicarbonate.
The hydrolysate was then chromatographed by twodimensional paper chromatography. Material migrating as free glucose was determined by autoradiography and counted as described above.
During the course of the incubation of the cells with
"4C-glucose in the steady-state apparatus, alternate
0.5-ml samples were removed by syringe at 2-min
intervals, placed in Warburg flasks, and quickly
sealed with rubber stoppers. The cells were immediately killed by dumping 1.0 ml of 2 N HCI from the
side arm of the vessel. The center wells contained 0.4
ml of Protosol (New England Nuclear Corp.), which
was used to absorb CO2 released by acidification of
the algal suspension. Because of the high pH of the
system, the vapor pressure of CO2 was negligible, and
virtually all CO2 formed from glucose was retained in
solution until the acid was added.
After 10 h with occasional agitation, the contents of
the center wells were removed by syringe and placed
in Aquasol (New England Nuclear Corp.). Each
center well was rinsed several times with Aquasol to
insure the complete removal of radioactive material.
"4CO2 trapped in the center wells was then deteras

J. BACTERIOL.

mined by counting in a Packard Tri Carb scintillation

spectrometer.
32P tracer experiments. In certain experiments,
inorganic 32P-labeled phosphate was used to label
radioactive material. The procedure for subsequent
radioautography and 32P determination in metabolites was modified as described previously (5, 6). Cells
were harvested and resuspended in BG-11, as described above. 32P-labeled inorganic phosphate was
then added at a concentration of approximately 2 ,Ci
(wet weight) of algae per mg, and the cells were
incubated in the steady-state apparatus for 8 h at low
light intensity with continuous aeration. Cells were
then removed into 10-ml round-bottom flasks and
sealed with serum stoppers.
Anaerobic experiments. A nitrogen stream was
first passed through an Oxsorbent (Burrel Corp.) trap
and then through a second trap containing a methylene-blue indicator. When the dye was in the leuco
form, indicating removal of the last traces of oxygen,
the nitrogen stream was connected to a syringe needle
extending through a serum stopper, down into an
algal suspension in a round-bottom flask. A second
needle was used as an outlet for the nitrogen stream.
After 1 h of gassing with N2, glucose was added to the
system to start the experiment. Samples were withdrawn through the serum stopper with a syringe and
for chromatography, as described above. The system
was kept under a positive pressure of nitrogen during
the experiment to minimize introduction of oxygen
during sampling.

RESULTS
Carbon dioxide production during aerobic
glucose metabolism was approximately twice
the rate of 02 uptake (Fig. 1). However, no
correction was made for possible differences in
the specific activity of the individual carbon
atoms of glucose. Uniformly labeled glucose,
commercially produced by photosynthesis with
"CO2, is often uniform with respect to 14C only
in the sense that all carbon atoms contain the
tracer. Carbon atoms 1 to 3 may contain about
60% of the isotope (New England Nuclear Corp.
technical bulletin). Because of the asymmetry
of glucose oxidation due to use of the oxidative
pentose pathway (6, 7), most of the carbon
released as CO2 comes from this portion of the
glucose molecule. Thus, the estimate of CO2
based on the average specific activity per carbon atom may be as much as 20% too high.
Accordingly, the actual rate of CO2 evolution
must be between 1.6 to 2.0 times the rate for 02
The rate of glucose disappearanxie is approximately linear for the first 30 min and then
decreases much more slowly until an undetectable concentration of the free sugar is reached.
Glycogen formation is approximately linear for
the same time period and constitutes from 70 to
85% of the glucose taken up by the cells (Fig. 2).
The soluble phosphorylated intermediates (Fig.

20

10

50
30 40
TIME (min.)

60

'

120

FIG. 1. CO2 production and 02 consumption by

dark cells of Aphanocapsa 6714 exposed to 14Cglucose.

ag

939

EFFICIENCY OF ENERGY CONVERSION

VOL. 115, 1973

assumption that each CO2 molecule formed was

the result of two dehydrogenation reactions.
The corresponding values of P/2e were nearly
constant at about 1.7 (Table 3). Thus, for each
electron pair generated by dehydrogenation
reactions, from 1.7 to 3.0 ATP molecules were
synthesized. The smaller limit is probably a low
estimate because of the non-equivalency expected in the individual carbon atoms of 14Cglucose, as described above.
The excess CO2 production over 02 consumption means that an alternate electron acceptor is in the system. The most likely candidate for this is the NO,- ion, which is present in the suspending medium at a fairly high
concentration and which is also the nitrogen
donor for the growing culture. It is possible,
therefore, that we are overestimating the efficiency of ATP synthesis if 0, consumption is
used as the basis of measurement. Some ATP
synthesis might be coupled to NO, reduction
or to the reduction of other acceptor molecules,

16

IE

a
0

12

It

0
a

Glucose
10

20

50
40
30
TIME (min.)

* G6P
F6P
o S7P

E
v

60

120

FIG. 2. Concentration of free 14C-glucose and 14Cglycogen from dark cells of Aphanocapsa 6714.

Eo
C

3, 4, and 5), glutamate and CO2, account for the

remaining carbon (Table 1).
The mole ratio of 2 consumed to glycogen
plus phosphorylated metabolites produced is
nearly constant at between 0.04 to 0.05. Thus, 4
to 5% of the glucose metabolized is coupled to
aerobic respiration. This yields an estimate of
P/2e ranging from 2.6 to 3.0 (Table 2, 3). This
estimate is based on the assumption that each
phosphorous group incorporated into phosphorylated metabolites requires the expenditure of
one ATP, and each glucose residue in glycogen
requires two ATP. The synthesis of 3-phosphoglycerate yields an ATP, so the net utilization
was assumed to be zero for this compound. Both
the enzymes leading to glycogen synthesis from
glucose, i.e., hexokinase and adenosine diphosphate (ADP)-glucose pyrophosphorylase, have
been demonstrated in cell-free extracts of
Aphanocapsa 6714.
It is also possible to base a P/2e ratio on the
rate of CO, production. To do this we made the

10

20

30
40
TIME (min.)

FIG. 3. Concentration of 14C in glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and sedoheptulose-7-phosphate (S7P), in dark cells of Aphanocapsa 6714.

*0 16
0'

E
E

-*

12

FDP

* SDP

E0

f
0

10

20

50
30 40
TIME (min.)

60

120

FIG. 4. Concentration of 14C in fructose-1,6diphosphate (FDP) and sedoheptulose-1, 7-diphosphate (SDP), in dark cells of Aphanocapsa 6714.

aL)
0

J . BACTrERIOL.

PELROY AND BASSHAM

940

16 F

Cr

TABLE 2. Concentration of carbon compounds

derived from "IC-glucose and estimated A TP required
for their synthesis by dark aerobic cells of
Aphanocapsa 6714a

PGA

* Glutomate

E 12

ATP (nmol) required

for synthesis

Concn (nmol)
Concnponmold

Compound
-8
E

5 min 10 min 20 min 5 min 10 min 20 min

_~~~~~~

I
0

10

20

F-

/J

40
50
30
TIME (min.)

60

120

FIG. 5. Concentration of 14C in 3-phosphoglycerate

(PGA) and glutamate, in dark cells of Aphanocapsa
6714.
TABLE 1. Recovery of 'IC from "C-glucose
metabolized by aerobic dark cells of Aphanocapsa
6714a
Time
(min)

Glucose
utilized

Soluble
compounds

Glycogen

5
10
20

50
88
154

27.4
31.9
33.3

26
60
114

plus CO,

Recovery

107
105
96

a The values in the columns are expressed in

ng-atoms of "4C. Taken from Fig. 1 to 5.

2.80 7.20 13.0

2.06 1.70 0.88
0.20 0.20 0.09
0.60 0.50 0.38
0.53 0.97 1.04
0.25 0.34 0.50
0.40 0.57 0.80
4.33 10.00 19.40

C02
G6P
S7P
F6P
FDP
SDP
PGA
Glycogen'

2.06 1.70 0.88

0.20 0.20 0.09
0.60 0.50 0.38
1.06 1.94 2.08
0.50 0.68 1.00
0.00 0.00 0.00
8.66 20.00 38.80

a
Data taken from Fig. 1 to 5; see figure legends for
compound abbreviations. Nanomole equivalents estimated by dividing the ng-atoms of 14C in a given
compound by the number of carbon atoms in that
compound.
bGlycogen expressed in terms of glucose equivalents.

TABLE 3. Efficiency of energy conversion (P/2e ratios)

for aerobic glucose metabolism by aerobic dark cells
ofAphanocapsa 6714a
ATPd

Time

(min)

Oxygen

CO,

taken upb evolvedc

synthesized

P/2e'

P/2ee

(nmol)

e.g., via fermentation. These possibilities are

2.62 1.72
5.0
3.8
13.08
5
made unlikely by the results of the following ex2.84 1.74
25.02
10
8.8
7.2
periments, where the effect of anaerobiosis on
3.08 1.66
43.23
20
13.0
14.0
energy production is measured.
a Data taken from Table 2.
As can be seen (Fig. 6), glycogen synthesis
was almost totally blocked in the absence of air,
Nanogram-atoms of oxygen from Fig. 1.
c Nanogram-atoms of CO2 from Fig. 1.
and CO2 production was reduced to 14% of the
d Nanogram-atoms of ATP required for biosyntheaerobic rate. However, phosphorylation reacsis
from columns 4, 5, and 6, Table 2.
in
the
tions consuming ATP were occurring
absence of air (Fig. 7).
At 14 min, 02 was admitted to the system to
10- Anaerobic
10 0IV
Aerobic
insure that the cells were still capable of energy
metabolism after the long incubation in the w, 8
8Ea)
a
absence of oxygen (see Materials and Methods).
C02
O
6
of
02
to
the
the
addition
Immediately after
Glycogen
system, there was a rapid increase in the rate of Ea 6 CO2 formation and a surge of labeled material
c0
into soluble pools followed by a high rate of 0oN 4
260E
glycogen synthesis (Fig. 6, 7).
4o
In a separate experiment, the concentrations lo'
c)
of ATP and ADP were also measured directly
_
(Fig. 8, 9). In the absence of oxygen, the level of
ATP decreased rapidly after the addition of
4
24
20
0
8
12
16
glucose, presumably as the result of phosphorylTIME (min.)
ation of glucose to glucose-6-phosphate, which
FIG. 6. CO2 production and glycogen synthesis by
was then converted to other metabolites. In the anaerobic dark cells of Aphanocapsa 6714 exposed to
presence of oxygen, the concentration of ATP 14C-glucose.
a

VOL. 115, 1973

EFFICIENCY OF ENERGY CONVERSION

Anaerobic

Aerobic

16
w

o 12

Monophosphates

941

On the other hand, respiration is evidently an

efficient means of maintaining cellular energy,
as the concentration of ATP actually increases

in the dark when glucose is available as a

substrate over the comparable levels in photosynthesizing cells. The P/2e ratios estimated by
8
quantitative determination of energy-consumIn
ing process of intermediary metabolism gave
E
values of 1.6 to 3.1, depending on the parameter
0
used to estimate glucose dehydrogenation.
Thus, there is more than one coupling site, and
4
possibly as many as three sites in the respira8
12
16
20 24
C
2
20tory
chain. These data suggest efficient linkage
between respiration and ATP synthesis. It has
TIME
(mOl.)
FIG. 7. Incorporation of "C-glucose into metabo- been suggested that the coupling between pyrilites by anaerobic dark cells of Aphanocapsa 6714.
dine nucleotide oxidation and electron transport in the blue-green algae and chemolithotrophic bacteria may be inherently defective as
a means of ATP synthesis (2, 3, 9). This is
32
0)
clearly not the case for Aphanocapsa 6714.
0
0'
At present we know nothing of the electron
24
carriers involved in respiration. It is possible
that the respiratory chain of Aphanocapsa 6714
E
_.,
shares elements in common with the photosynA >,
a- 16 i
E

&* fDP hh
o SDP
PGA

8IM (mi6.)

N
0

AT P
5

15
10
25
20
30
TIME (min.)
FIG. E8. Anaerobic concentrations of ATP and ADP
measureed by 32P, before and after the addition of 5
mM glutcose.

increasled about 20% after the addition of glucose.

If glzycogen synthesis and the formation of
phosph orylated metabolites are used to estimate ALTP synthesis in the presence and absence of oxygen, we conclude that the net energy
production was reduced more than 90% under

anaerobic conditions. Also, under anaerobic

conditions, the cell is unable to maintain ATP
levels due to the added energy demand for the
incorporation of glucose into metabolite pools
(Fig. 8). Thus, it seems reasonable to conclude
that fermentation and anaerobic respiration
(electron flow to nitrate) contribute little if
anything to the catabolic requirements of the
cell.

thetic electron transport system. Alternately,

the organism may synthesize independent systems for photosynthesis and respiratory electron
transport.
The

oxidative

pentose

pathway

in

Aphanocapsa 6714 would appear to be well

suited for preserving the cells' energy balance in
the dark. It is not as certain that that pathway
functions as well for the biosynthesis of required
carbon precursors for growth. As shown elsewhere, the rate of protein synthesis along with
the overall rate of metabolism is reduced in
dark cells (manuscript in preparation). It has
also been shown (8) that the mean generation
time in dark-growing cultures increases to about
110 h from a value of 10 h for photosynthetically
32k
ATP

C 24
E

16
C-i
ADP

r_

DISCUSSION
In the dark, metabolism of glucose by
Aphanocapsa 6714 is linked to aerobic respiration. Under anaerobic conditions, the rate of
ATP synthesis in severely reduced and the
concentration of the triphosphate falls steadily.

15
20
25
30
10
TIME (min.)
FIG. 9. Aerobic concentrations of ATP and ADP
measured by 32p, before and after the addition of 5
mM glucose.

942

PELROY AND BASSHAM

growing cells (exposed to glucose). It would

appear likely that factors other than the efficiency of energy production limit the growth of
Aphanocapsa 6714 in the dark.

1.
2.

3.
4.

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5:11-17.

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Light-dark transients in levels of intermediate com-

6.
7.

8.
9.
10.

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Rippka, R. 1972. Photoheterotrophy and chemoheterotrophy among unicellular blue-green algae. Arch. Mikrobiol. 87:93-98.
Smith, A. J., J. London, and R. Y. Stanier. 1967.
Biochemical basis for obligate autotrophy in blue-green
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