1
SUPPLEMENTAL INFORMATION
METHODS
S1
.
Analysis of bacterial metabolic rate data from Hoehler et al. 2023, PNAS
,
and
∆
G ̊
’
of
PCN Facilitated Fermentation
, related to Figure 2.
Analysis of bacterial metabolic rate data from Hoehler et al. 2023, PNAS
Hoehler
e
t al.
1
recently presented a compendium of metabolic rate measurements across kingdoms of life to
estimate the metabolic rate of the total biosphere. Here, we aime
d to use a subset of this dataset to
contextualize our measurement of the extremely slow metabolic rate of PCN facilitated fermentation in
P
seudomonas
aeruginosa
, particularly the bacterial metabolic rate measurements that were classified as
Endogenous met
abolism
(“
Endogenous
”),
maintenance from continuous culture studies
(“
Maintenance
”),
and
fast growth
(“
Fast Growth
”).
Below we discuss and justify our analysis of this dataset. Sources for the values
used are cited at the end of the paragraph preceding the
values appearance.
Justification of electrons sec
-
1
units
The power units reported in the Hoehler et al. data table are Watts. However, the original primary
measurements are almost all in units of
[O
2
] [time]
-
1
[biomass]
-
1
. To convert to Watts, the
assumption used is
a conversion factor of 20 J (mL O
2
)
-
1
, which was also used previously by the prior metabolic rate meta
-
studies
they cite
2,3
. T
his value is well
-
established from studies of human and animals as the energy transformation for
oxygen. The value is derived from the joules released per volume oxygen consumed by combusting
carbohydrates in a bomb calorimeter, and while that is a reasona
ble source for quantifying the biochemical
energy conserved plus heat produced in an animal due to respiration, we are interested instead solely in the
ATP generated in a bacterium per amount O
2
consumed. C
ells cannot conserve all the energy released by a
combustion
(what
a calorimeter measures
)
into useful
biochemical
energy (i.e. ATP); if they did, overall
∆
G=0
and there would be no metabolic driving force.
This is exemplified by the following, where a typical value of 32 ATP generated glucose
-
1
conserves less than
half of the total energy of glucose combustion
4
:
C
6
H
12
O
6
+ 6 O
2
6 CO
2
+ 6 H
2
O
∆
G ̊
’=
-
2866
kJ/mol
32 x (ADP + P
i
ATP
∆G ̊’= 33.5 kJ/mol)
Overall
∆
G ̊
’ =
-
1794
kJ/mol (lost as heat)
We therefore sought an alternative unit of metabolic rate that would hold higher fidelity and intuition to the
underlying bioenergetics of ATP production in bacteria.
We present below that for the
glucose
metabolisms
and
aerobic respiration rates that compose the majority of this dataset,
units of electrons have a
n
approximate
equivalency to units of ATP.
We begin with glucose catabolized via aerobic respiration, the metabolism that predominates the
measurements within t
he Hoehler et al. dataset. In
Pseudomonas aeruginosa
, this reaction at the substrate
level is:
C
6
H
12
O
6
+ 3 ADP + 6 O
2
+ 3 P
i
6 CO
2
+ 3 ATP + 6 H
2
O
(1)
Which includes 1 net ATP from glycolysis (Entner
-
Doudoroff
pathway) and 2 from the TCA cycle. In this
process the metabolism also reduces and oxidizes 10 NAD(P)
+
molecules and 2 ubiquinone (UQ) molecules.
To convert the NAD(P)H and UQH
2
intermediates into ATP units, we assume a maximum energy conservation
efficie
ncy of the aerobic electron transport chain
5,6
(ETC):
10 H
+
out
/NADH (4H
+
/2e
-
from Nuo/NDH
-
1 NADH dehydrogenase, 2H
+
/2e
-
from
bc
1
complex, 4H
+
/2e
-
from
cbb
3
cytochrome oxidase)
2
6 H
+
out
/UQH
2
(2H
+
/2e
-
from
bc
1
complex, 4H
+
/2e
-
from
cbb
3
cytochrome oxidase)
4 H
+
in
/ATP (from ATP synthase)
This results in ATP ratios of:
2.5 ATP / NAD(P)H
1.5 ATP / UQH
2
We can now convert our overall reaction to
units of ATP.
3 ATP from substrate
-
level phosphorylation (SLP)
+
10 NAD(P)H == 25 ATP from OxPhos
+
2 UQH
2
== 3 ATP from OxPhos
=
3
1
ATP glucose
-
1
.
If glucose is completely oxidized, 24 electrons are ultimately transferred from the glucose molecule to ox
ygen.
31
ATP
glucose
−
1
24
electrons
glucose
−
1
=
ퟏ
.
ퟑ
퐀퐓퐏
퐞퐥퐞퐜퐭퐫퐨퐧
Note that in deriving this value, the maximum ETC energy conservation efficiency and complete oxidation of
glucose was assumed. The real
in vivo
value is therefore likely less than this estimate.
As another example, in
E. coli
that takes a different metabolic route, the overall reaction (1) is mostly the same
except there is no net ATP produced via glycolysis (
Embden
-
Meyerhof
-
Parnas pathway
)
and
the
ETC energy
conservation efficiency
is different. In
E. coli
5
:
8 H
+
out
/NADH (4H
+
/2e
-
from Nuo/NDH
-
1 NADH dehydrogenase, 4H
+
/2e
-
from
b
o
3
oxidase)
4 H
+
out
/UQH
2
(4H
+
/2e
-
from
bo
3
oxidase
)
3.33 H
+
in
/ATP (from ATP synthase)
This results in ATP ratios of:
2.4 ATP / NAD(P)H
1.2 ATP / UQH
2
We can now convert our overall reaction to units of ATP.
2 ATP from SLP
+
10 NAD(P)H == 24 ATP from OxPhos
+
2 UQH
2
== 2.4 ATP from OxPhos
=
28.4
ATP glucose
-
1
.
And therefore 1.2 ATP electron
-
1
in
E. coli
.
Now we turn to compare this to the PCN facilitated fermentation metabolism (Figure 1B). This metabolism has
an upstream glycolytic arm with the equation:
C
6
H
12
O
6
+ 2 NAD(P)
+
+ ADP + P
i
2 C
3
H
3
O
3
-
+ 2 NAD(P)H + ATP + 4 H
+
(2)
3
This reaction is presumed to be redox
-
balanced by a succinate
-
producing pathway. Succinate has been
previously shown to be produced during
P
.
aeruginosa
anaerobic survival in glucose or pyruvate minimal
media with and without phenazine redox
-
cycling
7,8
, and accordingly we find that the total PCN r
educed during
facilitated fermentation is stoichiometric to the total acetate produced (Figure 1E and main text). This
succinate
-
producing pathway has the equation:
C
3
H
3
O
3
-
+ HCO
3
-
+ ATP + NADH + FADH
2
+ H
+
C
4
H
4
O
4
-
2
+ NAD
+
+ FAD + ADP + P
i
+ 2 H
2
O
(3)
In this equation, FADH
2
(the covalently
-
bound cofactor of the succinate ubiquinone oxidoreductase that
produces succinate in this pathway
9
) is listed as a placeholder for
an unknown electron donor. Typically,
fumarate reductase activity that produces succinate and promotes growth in other organisms is driven by a
specific fumarate reductase that oxidizes a menaquinol
10
, a redox intermediate that
P
.
aeruginosa
does not
synthesize
11
. Instead, the reverse reaction of succinate dehydrogenase is posited to explain succinate
production, as has been shown to occur in
E. coli
12
. Therefore, while the mechani
stic details of succinate
production are unknown, we assume that reducing equivalents produced during glycolysis can be balanced by
this pathway (i.e., NADH = FADH
2
placeholder in the equation) and explain the equimolar production of acetate
and reduced PC
N during PCN facilitated fermentation.
Together so far, glycolysis and the succinate
-
producing pathway produce no net ATP and no net reducing
equivalents while producing one pyruvate to be oxidized by the acetate
-
producing pathway, which has the
equation:
C
3
H
3
O
3
-
+ NAD
+
+ ADP + P
i
+ 2 H
2
O
C
2
H
3
O
2
-
+ NADH + ATP + HCO
3
-
+ 2 H
+
(4)
Lastly, the reducing equivalent produced by this acetate
-
producing pathway is oxidized by PCN:
NADH + PCN + H
+
NAD
+
+ PCNH
2
(5)
Adding up all the reactions above (reactions
2, 3, 4, and 5), we have a net reaction of:
C
6
H
12
O
6
+ ADP + P
i
+ PCN
C
4
H
4
O
4
-
2
+ C
2
H
3
O
2
-
+ ATP + PCNH
2
+ 3 H
+
(6)
During this reaction, 2 electrons are ultimately transferred from the glucose molecule to PCN. Therefore:
1
ATP
glucose
−
1
2
electrons
glucose
−
1
=
ퟎ
.
ퟓ
퐀퐓퐏
퐞퐥퐞퐜퐭퐫퐨퐧
While we do not observe measurable cell growth during this facilitated fermentation in our survival assay
(Figure 3D, F), anabolic pathways may still be active to replace damaged molecules during maintenance. This
val
ue therefore is likely an overestimate.
We conclude that across bioenergetically disparate metabolisms relevant to this study, metabolic rates in units
of electrons have an approximate equivalency to units of ATP. In this dataset, this conclusion carries
an ATP
error discrepancy between PCN facilitated fermentation and oxygen respiration measurements of about 2
-
3x.
While reporting metabolic rates in units of ATP sec
-
1
cell
-
1
would lower the metabolic rate measured for the
PCN facilitated fermentation and raise the metabolic rates for most of the measurements in the Hoehler et al.
dataset, enhancing their difference, we choose to instead report the metabolic rate in units of e
lectrons
because it carries the least assumptions across all the measurements.
4
Justification of cell
-
1
units
The goal of our analysis was to use the already
-
curated list of bacterial metabolic rate publications from the
Hoehler
et al. dataset and, using the primary publication
’
s reported values and units, convert to units of
e
lectrons
sec
-
1
cell
-
1
and
e
lectrons
sec
-
1
(
g cell dry weight
)
-
1
. Normalizing by gCDW is sensical: metabolic
rate is known to scale with the total biomass
(
i.e.
enzymes) carrying it out. This value has the further benefit of
usually being directly measured in the studies. However, implicit in this normalization is the assumption that the
total abundance of enzymes is rate limiting, which may not be the case f
or a starved or slow
-
growing cell; one
can reasonably imagine a significant subset of total enzyme present regulated to be inactive, aggregated, or
operating below their V
max
because of substrate limitations that may vary study to study. Since we cannot
co
nfidently and sufficiently know the physiological state of the cells in all measurements reported, the more
intuitively interpretable
e
lectrons
sec
-
1
cell
-
1
is preferred in this analysis and reported as such in the main text.
However, use of cell
-
1
units becomes weaker when considering bacterial cells of an unusually large size (e.g.
Thiomargarita namibiensis
) where a sizable fraction of their overall metabolic capability would need to be
inactive for values normalized by gCDW to approach those norm
alized per cell. We therefore chose to omit
these few datapoints of very large species in the Hoehler et al. dataset (gCDW cell
-
1
> 10
-
11
g,
n=4
) as outliers
from our analysis.
We also note that variation in a bacterial species
’
cell size across condition
s is typically less than an order of
magnitude
13
. We used the same dry weight cell
-
1
values collated by Hoehler et al. Cell dry weight values, if not
reported in the primary
publication, were estimated from other sources that measured those cells in similar
conditions. Otherwise, cell sizes from a reference source such as Bergey
’
s Manual were used. Given most
measurements of bacterial size are usually taken while the cell is
in a growth state (i.e. the state associated
with its largest sizes) these values likely lean toward being overestimates relative to non
-
growth conditions
such as endogenous metabolic rates, the state most relevant to our analysis. This would therefore lea
d to
underestimations of the true
e
lectrons
sec
-
1
cell
-
1
.
Conversion factors
The temperature and mass conversion factors remained the same between the Hoehler et al. analysis and our
analysis, and stem from justifications originally made by Makarieva
et al.
3
.
Temperature
Conversion
The data was normalized to a standard 25 ̊C using a Q
10
conversion:
푊
(25
°C
) =
푊
(
푇
)
∗
푄
10
(25 ̊C
-
T)/10 ̊C
W
here W is the metabolic
rate
,
Q
10
is the fold
-
change in metabolic
rate
for a 10°C temperature
increase, and T
is the temperature of measurement.
We use a Q
10
value of 2.0 for the bacterial measurements here
3
.
Mass
Conversion
To convert from protein weight to cell dry weight a
protein
-
to
-
dry weight ratio of 0.5
was used
(
https://bionumbers.hms.harvard.edu
, ID: 115319)
14
.
T
o convert cell nitrogen
weight
to
cell
dry weight a nitrogen
-
to
-
dry weight ratio of 0.1
was used
15
.
To convert from wet mass to dry mass, a dry
-
wet ratio of 0.3, or 70%
water content, was used
16
.
5
To convert carbon in biomass to cell dry weight, a carbon
-
to
-
dry weight ratio of 0.5 was used
17
.
Conversion to units of electrons
To convert mL O
2
to electrons, the ideal gas law was used to assume 1 mol O
2
= 22.4 L O
2
.
Therefore, 4.46 x 10
-
5
mol O
2
(mL O
2
)
-
1
.
Multiplying by Avogadro
’
s number, we get 2.69 x 10
19
molecules O
2
(mL O
2
)
-
1
.
Multiplying by 4 electrons per oxygen molecule reduced to water, we get 1.08 x 10
20
electrons (mL O
2
)
-
1
.
For measurements of C consumption, 4 electrons per C atom oxidized was assumed (complete oxidation to
CO
2
).
For
measurements of S consumption, 8 electrons per S atom oxidized was assumed (complete oxidation to
sulfate).
Notes on Collation
During the analysis, some duplicate values were found in the dataset and removed.
In the case of multiple measurements
reported in a single publication for a single species (e.g., different
strains), the lowest reported value was collated.
In the case where both carbon consumption rate and oxygen consumption rate are reported in maintenance
energies from Tijhuis (1993), o
xygen consumption was preferentially used when available. This matches the
majority of endogenous metabolism measurements and incorporates the possibility of endogenous electron
donor contributions to the metabolism.
In the case where metabolic rates were
found to be derived from a model instead of directly measured, they
were excluded.
One metabolic rate was added to the Hoehler et al. collection: the metabolic rate of
Pseudomonas aeruginosa
during fast aerobic growth in continuous culture, which was 1.5
x 10
6
electrons sec
-
1
cell
-
1
at 25 ̊C
18
.
∆
G ̊
’
of PCN Facilitated Fermentation
Here we estimate the
∆
G ̊
’
for PCN facilitated fermentation. As described above and shown
in Figure 1E, PCN
is only (or otherwise predominantly) involved in redox
-
balancing the acetate
-
producing portion of the
fermentation. Therefore, to consider how much energy can be conserved from PCN reduction, only the
oxidation of pyruvate to acetyl
-
CoA
is considered
19,20
:
pyruvate + CoA
acetyl
-
CoA + CO
2
+ H
+
+ 2 e
-
E ̊
’ =
-
480
mV
PCN
ox
+ 2 H
+
+ 2 e
-
PCN
red
E ̊
’ =
-
140
mV
pyruvate + CoA + PCN
ox
+ H
+
acetyl
-
CoA + PCN
red
+ CO
2
∆
E ̊
’ = +340
mV
We then use the equation:
∆
퐺
̊
′
=
−
∆
퐸
̊
′
푛
푒
퐹
where
n
e
is the number of electrons transferred and
F
is Faraday
’
s constant.
∆
퐺
̊
′
=
−
(
0
.
340
푉
)
∗
2
∗
96
,
500
퐶
푚표푙
−
1
=
−
ퟔퟓ
.
ퟔ
풌푱
풎풐풍
−
ퟏ
Using this
∆
G ̊
’,
Avogadro
’
s number, and
n
e
we derive the conversion value:
6
65
.
6
푘퐽
푚표푙
∗
푚표푙
푃퐶푁
6
.
02
푥
10
23
푚표푙푒푐푢푙푒푠
푃퐶푁
∗
푚표푙푒푐푢푙푒
푃퐶푁
2
푒
−
=
5
.
45
푥
10
−
20
퐽
(
푒
−
)
−
1
This conversion value and our gCDW cell
-
1
measurement (2.0 x 10
-
13
) can then be used to convert our cell
-
specific metabolic rate to units of W (g C)
-
1
:
1
.
6
푥
10
3
푒
−
sec
∗
푐푒푙푙
∗
5
.
45
푥
10
−
20
퐽
푒
−
∗
푐푒푙푙
2
.
0
푥
10
−
13
푔퐶퐷푊
∗
푔퐶퐷푊
0
.
5
푔
퐶
=
ퟖ
.
ퟕ
풙
ퟏퟎ
−
ퟒ
푾
(
품
푪
)
−
ퟏ