Báo cáo Y học: Intracellular pH homeostasis in the filamentous fungus Aspergillus niger pdf

Intracellular pH homeostasis in the filamentous fungus
Aspergillus niger
Stephan J. A. Hesse
1
, George J. G. Ruijter
2
, Cor Dijkema
1
and Jaap Visser
2,
†
1
Department of Biophysics, Wageningen University, the Netherlands;
2
Department of Microbiology, section Fungal Genomics,
Wageningen University, the Netherlands
Intracellular pH homeostasis in the filamentous fungus
Aspergillus niger was measured in real time by
31
PNMR
during perfusion in the NMR tube of fungal biomass
immobilized in Ca
2+
-alginate beads. The fungus maintained
constant cytoplasmic pH (pH
cyt
) and vacuolar pH (pH
vac
)
values of 7.6 and 6.2, respectively, when the extracellular pH
(pH

ex
) was varied between 1.5 and 7.0 in the presence of
citrate. Intracellular metabolism did not collapse until a DpH
over the cytoplasmic membrane of 6.6–6.7 was reached
(pH
ex
0.7–0.8). Maintenance of these large pH differences
was possible without increased respiration compared to
pH
ex
5.8. Perfusion in the presence of various hexoses and
pentoses (pH
ex
5.8) revealed that the magnitude of DpH
values over the cytoplasmic and vacuolar membrane could
be linked to the carbon catabolite repressing properties of the
carbon source. Also, larger DpH values coincided with a
higher degree of respiration and increased accumulation of
polyphosphate. Addition of protonophore (carbonyl
cyanide m-chlorophenylhydrazone, CCCP) to the perfusion
buffer led to decreased ATP levels, increased respiration and
a partial (1 l
M
CCCP), transient (2 l
M
CCCP) or perma-
nent (10 l
M
CCCP) collapse of the vacuolar membrane
DpH. Nonlethal levels of the metabolic inhibitor azide (N

3
–
,
0.1 m
M
) caused a transient decrease in pH
cyt
that was closely
paralleled by a transient vacuolar acidification. Vacuolar
H
+
influx in response to cytoplasmic acidification, also
observed during extreme medium acidification, indicates a
role in pH homeostasis for this organelle. Finally,
31
PNMR
spectra of citric acid producing A. niger mycelium showed
that despite a combination of low pH
ex
(1.8) and a high acid-
secreting capacity, pH
cyt
and pH
vac
values were still well
maintained (pH 7.5 and 6.4, respectively).
Keywords: Aspergillus niger; intracellular pH; pH homeo-
stasis;
31
P NMR; perfusion.

During operation of cellular metabolism under aerobic
conditions net intracellular production of protons takes
place mainly by formation of tricarboxylic acid cycle acids,
CO
2
/H
2
CO
3
and protein synthesis [1]. Consequently, tight
control of proton fluxes, in combination with the ability to
maintain pH gradients across cellular membranes, is a
crucial aspect of cellular energetics. Large deviations of
cytoplasmic pH (pH
cyt
) need to be avoided to keep control
of fundamental intracellular processes which are sensitive to
pH, such as DNA transcription, protein synthesis and
enzyme activities [2]. In order to ensure optimal activity of
major metabolic pathways, constant removal of free
protons from the cytoplasm is required [3]. In lower
eukaryotes and plants this process is mediated through the
action of the plasma membrane P-ATPase at the expense of
ATP hydrolysis, which results in pH and electrical potential
differences across the plasma membrane. The P-ATPase is
involved in intracellular pH (pH
in
) regulation, maintenance
of a proper ion balance and generation of the electrochemi-
cal proton gradient (proton motive force, Dp) across the

cytoplasmic membrane which drives an array of secondary
transport systems [4]. In Saccharomyces cerevisiae, intracel-
lular pH is thought to be additionally regulated through the
action of alkali-cation/H
+
antiporters, such as the Nha1
antiporter with a H
+
/K
+
(Na
+
) exchange mechanism [5].
Intracellular pH homeostasis in the filamentous fungus
Neurospora crassa has been suggested to be achieved by
parallel operation of the H
+
-extruding P-ATPase and a
high-affinity proton symport uptake system for K
+
,
yielding a net 1 : 1 exchange of K
+
for cytoplasmic H
+
[6].
Other major ATPases in fungal cells are the vacuolar
membrane V-ATPase and the mitochondrial membrane
F
1

F
0
-ATPase. The action of the former, ATP-dependent
transport of protons into the vacuole, is thought to
contribute to cytoplasmic pH homeostasis as well [7]. The
resulting electrochemical proton potential is able to drive
amino acid and ion transport across the vacuolar mem-
brane, probably through proton antiport systems. The
F
1
F
0
-ATPase uses the proton motive force generated by the
electron transport chain across the inner mitochondrial
membrane to drive phosphorylation of ADP to ATP.
Consequently, intracellular pH and pH gradients are
directly linked to cellular energy levels and metabolism [8].
The response of intracellular pH values to different
conditions, especially variation in extracellular pH (pH
ex
),
reveals clues to mechanisms of pH regulation [9]. In a
previous study we reported on a system based on long-term
acquisition of
31
P NMR spectra of constantly perfused and
well-oxygenated immobilized mycelium for the determin-
ation of compartmental pH values in the filamentous fungus
Aspergillus niger [10]. A. niger is industrially important for
Correspondence to S. J. A. Hesse, Department of Biophysics,

Wageningen University, Dreijenlaan 3, 6703 HA Wageningen,
the Netherlands. Fax: +31 317 484 011, Tel.: +31 317 484 692,
E-mail:
Abbreviations: CCCP, chlorophenylhydrazone.
Present address: Postbus 396, 6700 AJ Wageningen, the Netherlands.
(Received 12 April 2002, accepted 30 May 2002)
Eur. J. Biochem. 269, 3485–3494 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03042.x
large-scale production of organic acids (e.g. citric acid) due
to a high intrinsic excretion capacity for this compound [11].
Despite this capacity, quantitative analysis of metabolism
using kinetic models and metabolic engineering, comple-
mentary to traditional strain improvement, is still a
promising approach to increase citric acid production or
to shorten fermentation times [12]. However, a more
predictive accuracy for kinetic (mechanistic) models requires
a more detailed description of the conditions under which
the enzymes involved operate in vivo. With the perfusion
system developed, problems related to fungal morphology
during long-term in vivo NMR measurements have been
overcome. With this method it is now possible to obtain
more information on intracellular pH, one of the key
parameters affecting enzyme activity, and its homeostasis.
Also, the ability of A. niger to acidify its medium to pH
values below 2.0 during production of large quantities of
organic acids implies that a very efficient pH-homeostatic
system exists in these cells. As protein synthesis and
intracellular enzyme activities are sensitive to pH, mainten-
ance of intracellular pH under extreme conditions (especi-
ally low pH
ex

) is crucial to ensure optimal cellular activity
during (industrial) fermentations. So far, however, data on
intracellular pH in filamentous fungi have been scarce. A
few reports have dealt with cytoplasmic pH of citric acid-
producing A. niger mycelium [13,14]. More detailed know-
ledge about intracellular pH homeostasis in filamentous
fungi is to date only available for N. crassa [1,15,16]. To
investigate the ability of A. niger to maintain cellular energy
levels, cells were subjected to several stresses like low pH
ex
,
citric acid producing conditions, increased proton permeab-
ility by an uncoupler and inhibition of ATP synthesis by
sodium azide. Also tested was the effect of different carbon
sources on steady-state DpH values. Together our results
provide reliable data on intracellular pH under various
extracellular conditions, and demonstrate the ability of the
fungus to maintain cellular energetics under extreme
conditions with a formidable tolerance towards extracellular
acidity.
EXPERIMENTAL PROCEDURES
Strain, immobilization of conidia and culture conditions
Condiospores of A. niger NW131 (cspA1 goxC17) lacking
glucose oxidase activity [17] were propagated at 30 °C on
complete medium [18] solidified by 1.5% agar and contain-
ing 1%
D
-glucose. Conidiospores were harvested with a
solution containing 0.9% NaCl and 0.05% (v/v) Tween-80.
Immobilization of conidia in Ca

2+
-alginate beads (diameter
1 mm) was performed as described previously [10]. A 2.5%
solution of Manugel DJX (ISP Alginates, Tadworth,
Surrey, UK) was used in all immobilization experiments.
After harvesting and washing with demineralized water,
immobilized conidia were cultured in 500-mL Erlenmeyer
flasks. Immobilized mycelium was obtained by incubating
the beads (10 g) for 40–44 h in a rotary shaker at 250 r.p.m
and 30 °C in 100 mL minimal medium (2 gÆL
)1
NH
4
NO
3
,
1.5 gÆL
)1
KH
2
PO
4
,0.5gÆL
)1
KCl, 0.5 gÆL
)1
MgSO
4
Æ7H
2

O
pH 6.0), supplemented with 1.5%
D
-glucose, 0.02% (v/v) of
a trace element solution [19] and 0.05% yeast extract.
To obtain immobilized mycelium under citric acid-
producing conditions a 500-mL bubble column reactor
was filled with 125 mL immobilized conidia and 375 mL
medium optimized for citric acid production (140 gÆL
)1
decationized glucose, 0.2 gÆL
)1
KH
2
PO
4
,1.25gÆL
)1
(NH
4
)
2
SO
4
,0.25gÆL
)1
MgSO
4
Æ7H
2

O, 1.3 mgÆL
)1
ZnSO
4
Æ
7H
2
O, 6.5 mgÆL
)1
FeSO
4
Æ7H
2
O). Under these conditions no
growth of biomass outside of the beads occurred. The
culture pH was not regulated and was initially set at 3.5.
Cultures were sparged with 1.5 LÆmin
)1
air, and after 24 h
15 lL of a solution containing 30% (v/v) polypropylene-
glycol in alcohol was added to the reactor to prevent
excessive foaming. The reactors were run for 2 or 7 days at
30 °C. The fermentation volume was periodically adjusted
to 0.5 L with double-distilled water.
Perfusion conditions
Immobilized biomass from shake-flask cultures was har-
vested, washed with a buffer (30 °C) containing 25 m
M
sodium citrate pH 5.8, 0.25 gÆL
)1

NH
4
NO
3
,0.2gÆL
)1
KCl,
0.2 gÆL
)1
MgSO
4
Æ7H
2
O, 0.2 m
M
KH
2
PO
4
,0.3m
M
CaCl
2
,
and perfused within the NMR tube with 1 L of the same
buffer saturated with oxygen. The extracellular pH was
varied by using perfusion buffer with pH values ranging
from 1.0 to 7.0 or by direct titration of the buffer reservoir
with 2
M

HCl. The same buffer supplemented with 50 m
M
Tris was used under alkaline extracellular conditions (pH
ex
,
7.0–9.0). The effect of the presence of various sugars on
intracellular pH values was tested after a 2-h transfer of
immobilized biomass to 150 mL of perfusion buffer pH 5.8
supplemented with 10 m
M
of sugar (
D
-glucose,
D
-fructose,
D
-xylose or
L
-arabinose). Subsequently, the beads were
perfused with the same buffer saturated with oxygen for 3 h.
Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and
sodium azide (NaN
3
) were directly added to the buffer
reservoir (pH 5.8) from a 10-m
M
stock solution in ethanol
and a 10% stock solution in water, respectively. Oxygen
consumption during perfusion (DO
2

) was expressed as the
percentage of oxygen removed from an oxygen saturated
buffer after passage through the immobilized cell plug.
Immobilized citric acid producing mycelium from 2- and
7-day-old fermentations was directly transferred from the
bubble column reactor to the NMR tube and perfused with
filtered culture medium (500 mL) from 2-day- and 7-day-
old fermentations, respectively. In all cases, a 4-cm high plug
of immobilized biomass (± 12.5 mL beads) was perfused at
arateof15mLÆmin
)1
.
31
P NMR spectroscopy
31
P NMR spectra were recorded at 121.5 MHz at 30 °C on
a AMX300 wide-bore spectrometer (Bruker, Germany),
equipped with a 20-mm switchable
31
P/
13
C probe tuned at
the
31
P nucleus, and collected in 15, 20 or 60-min blocks
(4500, 5700 or 18000 FIDs, respectively) using acquisition
parameters described previously [10]. Methylene diphos-
phonic acid (0.2
M
, pH 8.9), contained in an in situ capillary,

was used as an internal reference, resonating at 16.92 p.p.m.
relative to 85% H
3
PO
4
(0 p.p.m).
Analyses
Cytoplasmic and vacuolar pH values were determined by
comparing the pH-sensitive chemical shifts of cytoplasmic
3486 S. J. A. Hesse et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(P
cyt
) and vacuolar inorganic phosphate (P
vac
)witha
calibration curve for inorganic phosphate (P
i
). This curve
has been referred to in many other NMR studies on both
yeasts and fungi [16,20,21], and shows the pH dependence
of the chemical shift of P
i
measured in a medium made up to
approximate concentrations of the major cationic compo-
nents in yeast [22]. The perfusion buffer pH (pH
ex
)was
monitored by pH electrode measurements. Intracellular pH
values in the presence of various carbon sources and in
mycelium producing high levels of citric acid were deter-

mined from 12–16 and 5–7 different spectra, respectively;
final values represent mean values that were statistically
analysed by a two-tailed Student’s t-test (a ¼ 5%). Relative
increases in polyphosphate levels were determined from
31
P
NMR spectra by relating the integral of the polyphosphate
peak to the integral of the internal reference peak. Citrate,
ammonium and phosphate levels in culture filtrate samples
of citric acid fermentations were determined as described
before [17]. Perfusion buffer and transfer medium samples
were analysed for sugars and organic acids by HPLC using
an HPX-87H column (Bio-Rad) eluted with 25 m
M
HCl at
50 °C with UV (210 nm) and refractive index detection, and
for polyols using a Carbopac MA-1 column (Dionex) eluted
with 480 m
M
NaOH at 20 °C with pulsed amperometric
detection. Dry weight determinations on immobilized
biomass were carried out by dissolving the Ca
2+
-alginate
beads in a 100-m
M
solution of the Ca
2+
-scavenger sodium
hexametaphosphate (Fluka AG, Buchs, Switzerland).

Mycelium was then collected by filtration, washed with
demineralized water, frozen in liquid nitrogen, lyophilized
and weighed.
RESULTS
The dependence of intracellular pH values on ambient pH
A. niger has the ability to acidify its environment to values
as low as pH 1.5 [17]. To investigate to what extent
extracellular pH (pH
ex
) affects intracellular pH (pH
in
)
values, we determined pH
cyt
and pH
vac
as a function of
ambient pH using
31
P NMR as described previously [10].
Surprisingly, the cells were able to maintain pH
cyt
and pH
vac
at 7.6 and 6.2, respectively, when pH
ex
was varied between
1.5 and 7.0, implying that a very steep DpH over the
cytoplasmic membrane of 6.1 can be sustained by the cells
(Fig. 1A). The DpH over the vacuolar membrane was

maintained at a constant value of 1.4. At pH
ex
1.5, this
gradient was about 0.1 pH unit larger due to a slightly more
acidic vacuole. Further acidification to pH
ex
1.0 caused
pH
cyt
to drop to pH 7.4, in parallel to an even larger
vacuolar acidification than at pH
ex
1.5. A comparable
observation was made at pH
ex
8.0: pH
cyt
was still reason-
ably well regulated, whereas the vacuoles became more
alkaline (pH
vac
6.5). Interestingly, in the pH
ex
range of 1.0–
7.0 in the presence of 25 m
M
citrate, cells consumed the
same amount of oxygen once a steady-state was reached
(DO
2

¼ 8–9%). Cellular metabolism collapsed and O
2
consumption rapidly dropped to 0% when pH
ex
reached a
value of about 0.7–0.8. Cells only moderately increased their
oxygen consumption (from 9 to 11%) just before the
collapse. A representative
31
P NMR spectrum of immobi-
lized A. niger mycelium perfused in the presence of 25 m
M
citrate pH 1.5 and 0.2 m
M
P
i
isshowninFig.1B.
Fig. 1. The dependence of A. niger NW131 cytoplasmic pH (pH
cyt
) and vacuolar pH (pH
vac
) on extracellular pH (pH
ex
) in the presence of oxygen-
saturated buffer (A) and typical
31
P NMR spectrum of immobilized A. niger NW131 mycelium (B). (A) Buffer contained 25 m
M
citrate (pH
ex

1.0–7.0)
or 25 m
M
citrate and 50 m
M
Tris (pH
ex
7.0–8.5). Values are the mean of two experiments. (B) Mycelium cultured for 42 h and perfused with
oxygen-saturated buffer containing 25 m
M
citrate at pH
ex
1.5. Abbreviations and chemical shifts of the assignments: SME: sugar phospho-
monoesters, 4.9 p.p.m.; SDE: sugar phosphodiesters, 4.5 p.p.m.; P
cyt
: cytoplasmic inorganic phosphate, 2.9 p.p.m.; P
vac
: vacuolar inorganic
phosphate, 1.2 p.p.m.; P
ex
: extracellular inorganic phosphate, 0.6 p.p.m.; c-ATP: ) 4.9 p.p.m.; P
2
: pyrophosphate and terminal phosphate of
polyphosphate, )5.8 p.p.m.; a-ATP: )9.9 p.p.m.; NAD(H): )10.6 p.p.m.; UDPG: uridine diphosphoglucose, )10.6 and )12.3 p.p.m.; P
3
and P
4
:
penultimate phosphates of polyphosphate, )17.7 and )19.7 p.p.m., respectively; a-ATP: )18.6 p.p.m.; P
n

: polyphosphate, )22.5 p.p.m. Data were
collected in a 60-min block. The internal reference is not shown.
Ó FEBS 2002 Intracellular pH homeostasis in A. niger (Eur. J. Biochem. 269) 3487
Different bioenergetic states with different sugars
For the yeast Candida tropicalis it was demonstrated by
31
P
NMR that cells aerobically metabolizing glucose were
more energized than xylose-fed cells [23]. Besides higher
UDPG and polyphosphate levels and higher rates of P
i
assimilation, cells metabolizing glucose had a slightly
higher pH
cyt
and a slightly lower pH
vac
. To investigate to
what extent different nutritional conditions result in
different steady-state intracellular pH values in A. niger,
mycelium was perfused for 3 h with perfusion buffer
pH 5.8 containing one of the following sugars:
L
-arabi-
nose,
D
-xylose,
D
-fructose or
D
-glucose. In this sequence

these sugars represent poor to good carbon sources for
A. niger. The initial oxygen consumption (DO
2
)ofperfused
biomass was 10–15% (using buffers saturated with O
2
).
No steady oxygen consumption was reached for any of the
sugars tested during 3 h of data acquisition. Instead, a
gradual increase in oxygen consumption was observed. In
the absence of sugar, citrate was the only available carbon
source, and under these circumstances the initial oxygen
consumption was lower and remained constant throughout
the experiment (8–9%). Catabolism of glucose, fructose
and xylose resulted in a more pronounced cytoplasmic
alkalinization compared to arabinose or citrate only,
whereas a clearly stronger vacuolar acidification was
observed only in the presence of glucose and fructose
(Table 1). Although the differences between the deter-
mined pH
in
values were only small, an increased oxygen
consumption coincided with higher pH
cyt
and lower pH
vac
values. As inferred from the
31
P NMR spectra, however,
no significant differences in ATP levels could be observed

for the conditions tested (spectra not shown). Replacement
ofcitratebya25m
M
Mes buffer (pH 5.8) resulted in
similar pH values in the case of glucose (results not
shown), indicating that the effect of citrate on pH
in
is only
minor. HPLC analysis of perfusion buffer samples showed
that no polyols or organic acids were excreted during 3 h
of perfusion, ensuring constant extracellular conditions
during data acquisition. Besides generating ATP and
establishing pH gradients, an alternative way for the cells
to store energy generated by catabolism may be polyphos-
phate synthesis. The relative increase in polyphosphate
levels during 3 h of perfusion appeared to coincide with
increased pH gradients over the cytoplasmic and vacuolar
membrane (Table 1). Younger mycelium (18–24 h old)
contained hardly any polyphosphate (spectra not shown).
The effect of CCCP on intracellular pH
A strong argument in favour of Mitchell’s chemiosmotic
theory [24] was the fact that it could explain the mode of
action of lipid-soluble weak acids that are able to carry out
electrogenic proton transport across biological and artificial
membranes, thereby dissipating both the electrical mem-
brane potential (DY) and the proton gradient (DpH). These
uncouplers abolish the tight coupling of electron transport
to oxidative phosphorylation and allow respiration to
proceed without control by phosphorylation. The decreased
ability of the cytoplasmic and vacuolar membrane to

maintain transmembrane proton gradients in vivo in the
presence of increasing extracellular concentrations of the
uncoupler CCCP is shown in Fig. 2. It should be noted that
after transfer of the cells to the perfusion system, pH
in
values
(especially pH
vac
) reached their steady-state values only
after 1.5–2 h of perfusion. To shorten experimental times,
CCCP (and azide, see below) were added before this time
point, resulting in slightly different initial pH
cyt
and pH
vac
values in Figs 2 and 3. Addition of 1 l
M
CCCP to the
perfusion buffer caused pH
cyt
to drop from 7.6 to 7.1 and
pH
vac
to increase from 6.3 to 6.6 during the first 45 min after
addition (Fig. 2A). At the same time, ATP and vacuolar
phosphate levels decreased and cytoplasmic phosphate
levels increased (spectra not shown). During this period
oxygen consumption increased from approximately 9 to
21%. A complete collapse of the vacuolar membrane pH
gradient did not occur. The cells started to recover 60–

75 min after the addition. At this point initial ATP levels
were nearly restored. The absolute pH
in
values determined
after recovery were slightly higher than initial values, in
particular pH
cyt
. Consequently, the re-established pH
gradient over the vacuolar membrane (1.5) was somewhat
higher than before addition (1.3). In the presence of 2 l
M
CCCP essentially the same changes were observed as
described for 1 l
M
CCCP. In this case, however, the
vacuolar membrane pH gradient was completely dissipated
(Fig. 2B). After 30 min no distinct P
cyt
or P
vac
could be
recorded. Instead, both peaks had merged into one large
intracellular phosphate resonance, corresponding to an
intracellular pH value of 6.9. The cells reacted to the
Table 1. Steady-state pH
in
values, increase in oxygen consumption and relative increase in polyphosphate levels during 3 h of perfusion in the presence
of oxygen-saturated buffer containing 25 m
M
citrate pH 5.8, supplemented with 10 m

M
sugar.
Sugar pH
cyt
pH
vac
Increase in
O
2
consumption
a
[polyphosphate]
b
Glucose 7.76 ± 0.02 6.05 ± 0.04 24% 1.82
Fructose 7.73 ± 0.02 6.08 ± 0.02 21% 1.48
Xylose 7.72 ± 0.03 6.15 ± 0.04 17% 1.39
Arabinose 7.64 ± 0.02 6.19 ± 0.03 10% 1.31
— 7.58 ± 0.01 6.21 ± 0.02 0% 1.02
a
Expressed as the percentage of oxygen removed from an oxygen-saturated buffer after passage through the immobilized cell plug. Initial
oxygen consumption in the presence of sugar: 10–15%; in the presence of citrate only: 8–9%.
b
Relative increases in polyphosphate levels
were determined from
31
P NMR spectra by relating the integral of the polyphosphate peak to the integral of the internal reference peak at
t ¼ 0 and t ¼ 3h.
3488 S. J. A. Hesse et al. (Eur. J. Biochem. 269) Ó FEBS 2002
imposed stress condition once more by increasing their
oxygen consumption from 9 to 30% within 60 min of

addition. Surprisingly, 90 min after CCCP was added the
large intracellular phosphate resonance split up again into
two separate resonances: P
cyt
, shifting to the left (indicating
alkalinization within that compartment) and P
vac
,shifting
to the right (indicating acidification within that compart-
ment). Again, the onset of pH
in
recovery coincided with the
(partial) restoration of original ATP levels (spectra not
shown). With 10 l
M
CCCP, an irreversible collapse of the
vacuolar membrane pH gradient was observed within
15 min (Fig. 2C). The intracellular pH, deduced from the
chemical shift of a large intracellular phosphate resonance,
became 6.7 and remained around that value during another
105 min of perfusion. The oxygen consumption increased
during the first 5 min following the CCCP addition, but
then rapidly dropped to 0%, indicating cell death. Con-
comitantly, a rapid loss of ATP was observed, whereas
UDPG, cofactor and polyphosphate levels gradually
decreased during the 120 min that spectra were acquired.
A complete collapse of the residual DpH across the
cytoplasmic membrane (approximately 0.9 pH unit) did
not occur, even when CCCP levels were doubled to 20 l
M

.
The effect of azide on intracellular pH
The inhibitory mode of action of azide on cellular ATP
synthesis is twofold by inhibiting both the mitochondrial
F
1
F
0
-ATPase and cytochrome-c-oxidase in the terminal part
of the electron transport chain. The effect of transient and
permanent depletion of ATP due to azide addition on pH
in
is
shown in Fig. 3. In the former case (0.1 m
M
N
3
–
,Fig.3A)a
transient decrease in pH
cyt
was observed with a minimal
value of 7.0 after 30 min. In contrast with CCCP, the drop in
pH
cyt
was paralleled by a decrease in pH
vac
from 6.3 to 5.9.
Spectra showed that ATP levels dropped sharply, whereas
both cytoplasmic and vacuolar phosphate levels were slightly

higher compared to the original situation (Fig. 4A and B). At
the same time, cells increased their oxygen consumption from
9 to 23%. Interestingly, the observed rise in both pH
cyt
and
pH
vac
after 45 min was accompanied by a small increase in
ATP level (Fig. 4C). In the new steady-state, pH
in
values
were identical to those observed before the addition of the
inhibitor, although ATP and UDPG levels were somewhat
lower (Fig. 4D). Polyphosphate levels remained unchanged
during 2.5 h of perfusion. Cells lost all of their ATP
permanently when 0.5 m
M
azide was used. Moreover, an
effective and permanent dissipation of the vacuolar mem-
brane pH gradient was observed (Fig. 3B), visualized in the
spectra as one large intracellular phosphate resonance (data
not shown). Lethal azide concentrations were much more
effective in dissipating the pH gradient across the cytoplasmic
membrane than lethal doses of uncoupler. The residual pH
gradient observed 2 h after azide addition (0.5 m
M
) was 0.3–
0.4 pH unit, which is considerably lower than when CCCP
was used (0.9 pH unit). Eventually, pH
in

became equal to
Fig. 2. The effect of CCCP addition on cytoplasmic pH (pH
cyt
) and
vacuolar pH (pH
vac
) in immobilized A. niger NW131 mycelium, grown
for 42 h and perfused with oxygen-saturated buffer containing 25 m
M
citrate pH 5.8. CCCP was added at t ¼ 0, and the applied concen-
trations were 1 l
M
(A), 2 l
M
(B) and 10 l
M
(C). Addition occurred
before pH
cyt
and pH
vac
reached steady-state values, resulting in slightly
different initial pH values (see text). Data points represent the mean of
two experiments.
Ó FEBS 2002 Intracellular pH homeostasis in A. niger (Eur. J. Biochem. 269) 3489
pH
ex
after 8 h of perfusion. During this whole period only a
very small breakdown of polyphosphate could be observed.
The bioenergetic state of citric acid producing mycelium

A. niger has the capacity to produce high levels of citric acid
from hexoses and disaccharides in traditional citric acid
producing processes when two important criteria are met
[25]: a low pH (< 2) and absence of manganese ions
(Mn
2+
). Low pH is necessary to avoid production of
gluconic acid and oxalic acid. In our experiments interfer-
ence by gluconic acid production was prevented by using an
A. niger N400 derivative, strain NW131, lacking glucose
oxidase activity. The amount of citric acid accumulated
from glucose in 7 days by immobilized biomass in a bubble
column reactor was approximately 50 gÆL
)1
.Inatypical
fermentation the pH dropped from 3.5 to 1.8 in  3days,
and remained around that value. The cells consumed all
NH
4
+
and PO
4
3–
during the first 24 h (data not shown),
and no citrate was produced in this period. Dry weight
determinations of 2- and 7-day-old cultures indicated a
small decrease in biomass content during the fermentation
(17.4 and 16.1 gÆL
)1
, respectively). It was decided to

Fig. 3. The effect of sodium azide addition on cytoplasmic pH (pH
cyt
)
and vacuolar pH (pH
vac
) in immobilized A. niger NW131 mycelium,
grown for 42 h and perfused with oxygen-saturated buffer containing
25 m
M
citrate pH 5.8. Azide was added at t ¼ 0, and the applied
concentrations were 0.1 m
M
(A) and 0.5 m
M
(B). Addition occurred
before pH
cyt
and pH
vac
reached steady-state values, resulting in slightly
different initial values. Data points represent the mean of two
experiments.
Fig. 4.
31
P NMR spectra of immobilized A. niger NW131 mycelium in
the presence of 25 m
M
citrate pH 5.8, showing the effect of addition of
0.1 m
M

sodium azide. (A) The situation before addition. P
cyt
,cyto-
plasmic inorganic phosphate resonance; P
vac
, vacuolar inorganic
phosphate resonance. The internal reference is not shown. (B) After
30 min both P
cyt
and P
vac
had moved to a lower chemical shift (i.e.
compartmental acidification), accompanied by a sharp decrease in
intensities of the c-ATP and the a-ATP peaks as indicated by the
arrows. (C) After 45 min ATP levels started to restore again, and P
cyt
and P
vac
moved to a higher chemical shift again (i.e. compartmental
alkalinization). (D) In the new steady-state (t ¼ 120 min) compart-
mental pH values were identical to those observed before addition. See
Fig. 3A for corresponding pH values.
3490 S. J. A. Hesse et al. (Eur. J. Biochem. 269) Ó FEBS 2002
investigate a younger (2-day old) and an older (7-day old)
culture. To mimic citric acid production conditions as
closely as possible, immobilized cell mass was perfused with
filtered medium of 2-day- and 7-day-old cultures, respect-
ively, saturated with oxygen. The specific citric acid
production rates of mycelium after transfer to the perfusion
set up were 0.20 gÆLh

)1
(2-day-old mycelium) and
0.32 gÆLh
)1
(7-day-old mycelium), whereas in the bioreactor
values of 0.37 gÆLh
)1
(2-day-old mycelium) and 0.19 gÆLh
)1
(7-day-old mycelium) were obtained. The values obtained
for perfused mycelium confirmed that spectra were acquired
under true citric acid producing conditions, although
transfer to the perfusion system appeared to have an effect
upon specific production rates.
31
P NMR spectra of 2- and
7-day-old-citric acid-producing mycelium showed a remark-
able constancy with respect to metabolite levels (including
polyphosphate) and pH
in
values during the first 4–6 h of
perfusion (spectra not shown). For correct assignment of
P
cyt
and P
vac
,CCCP(2l
M
) was used to partially collapse
the DpH between these two compartments. No difference

could be detected between steady-state pH
cyt
values of
2- and 7-day-old mycelium (7.53 ± 0.05 and 7.54 ± 0.04,
respectively) although cytoplasmic phosphate, sugar phos-
phate and ATP levels were clearly higher in 2-day-old
mycelium. Compared to pH
cyt
values obtained in the
presence of various carbon sources (Table 1) or under
extreme extracellular acidity (Fig. 1A, pH
ex
1.5–2.0), citric
acid-producing mycelium appeared to have only a slightly
more acidic cytoplasm. The vacuoles of 2-day-old mycelium
were relatively alkaline (pH 6.41 ± 0.03), whereas an even
higher pH
vac
was found in vacuoles of 7-day-old mycelium
(pH 6.50 ± 0.04).
DISCUSSION
The proper functioning of cells relies on maintenance of
their intracellular pH within relatively narrow limits, as
large deviations of pH from normal values would be
severely inhibitory to metabolism based on pH optima of
cytoplasmic enzymes [1]. Our results show that A. niger is
indeed capable of tightly maintaining its intracellular pH
values within a narrow range. In the presence of various
carbon sources at pH
ex

5.8, pH
cyt
varied only from 7.58
(citrate) to 7.76 (glucose), whereas pH
vac
ranged from 6.05
(glucose) to 6.19 (citrate) (Table 1). As no significant
differences in ATP levels could be observed in the
31
P
spectra, an energetically more favourable carbon source
may lead to a larger availability of ATP to the P-ATPase
and V-ATPase due to increased ATP turnover, reflected by
a higher oxygen consumption and larger pH gradients. Of
the carbon sources tested, glucose has the largest carbon
catabolite repressing effect in A. niger,andcitratethe
lowest (glucose > fructose > xylose > arabinose > cit-
rate; G. J. G. Ruijter, Wageningen, the Netherlands,
personal communication). Thus, the carbon catabolite
repressing capacity of the carbon source could be linked
to steady-state DpH values.
Interestingly, larger pH gradients and a higher oxygen
consumption coincided with increased polyphosphate syn-
thesis in A. niger (Table 1). Polyphosphate has been
suggested to function as a cellular phosphate or high-energy
reserve. Although polyphosphate may have additional
functions (e.g. chelation of cations, or a pH-homeostatic
function as sequestrator or donor of protons), its exact
physiological role is not yet fully understood. Our results
showed that with increased cellular energy levels polyphos-

phate accumulated to higher levels, suggesting a role in
cellular energy storage for the polymer. Recent studies on
E. coli cells revealed a more regulatory role for polyphos-
phate [26]. Cells deficient in polyphosphate were unable to
express many genes that are needed for adaptation to
deficiencies and environmental stresses during the stationary
phase, and lost their viability relatively quickly. Increased
polyphosphate synthesis may therefore greatly enhance the
chances of survival in stationary phase cells. If so, it is
crucial for cells to accumulate as much polyphosphate as
possible in times of energy excess. Our results are in
agreement with this hypothesis as polyphosphate accumu-
lation was maximal in the presence of glucose at pH
ex
5.8. In
the presence of a poor carbon source at the same pH
ex
(citrate), no accumulation of polyphosphate was observed.
In the presence of glucose at pH
ex
1.0, no increase in
polyphosphate levels occurred either (results not shown). A
key role for polyphosphate in stationary phase cells is
further corroborated by the fact that polyphosphate was
practically absent in younger (18–24-h-old) mycelium.
In N. crassa, the ratio of polyphosphate to orthophosphate
in vacuoles increased from 2.4 in early log phase cells to
13.5 in stationary phase cells [15]. When early log phase cells
were exposed to a hypo-osmotic shock, both pH
cyt

and
pH
vac
increased and cells lost 95% of their total polyphos-
phate content. In contrast, hypo-osmotic shock of station-
ary phase cells did not cause any changes in intracellular pH
or polyphosphate levels, showing that these cells were much
more effective in handling osmotic stress.
A striking observation was the ability of A. niger to
maintain constant intracellular pH values during extracel-
lular acidification to pH values as low as 1.0 without having
to change its steady-state oxygen consumption. In yeast,
filamentous fungi and higher plant cells, the proton
pumping activity of the plasma membrane P-ATPase has
been recognized as the major factor responsible for pH
cyt
homeostasis [27–29], possibly in combination with high
affinity potassium uptake in symport with protons. Oper-
ating in parallel in N. crassa, these two systems yield a net
1 : 1 exchange of K
+
for cytoplasmic H
+
[6]. In N. crassa,
changes of pH
ex
between 3.9 and 9.3 affect pH
cyt
linearly
with a slope of approximately 0.1 unit pH

cyt
per unit pH
ex
[1]. In S. cerevisiae, both pH
cyt
and pH
vac
became more
acidic at pH
ex
3.5 compared with pH
ex
6.5 whether glucose
was present or not [30]. Intracellular pH homeostasis in
respiring Escherichia coli cells was good (pH
cyt
7.6 ± 0.2)
over a pH
ex
range of about 5.5–9.0 [31]. Finally, in sycamore
(Acer pseudoplatanus L) cells pH
cyt
and pH
vac
values were
maintained when pH
ex
was varied from 4.5 to 7.5 [29].
Oxygen consumption measurements of these cells in a
perfusion setup revealed a progressive acceleration of the

rate of O
2
consumption towards the uncoupled O
2
uptake
rate (+1l
M
FCCP) as pH
ex
decreased from 6.5 to 4.5. Our
results clearly show that after addition of CCCP (2 l
M
,
pH
ex
5.8) a much higher oxygen consumption could be
achieved (DO
2
¼ 25–30%) than the steady-state oxygen
consumption (DO
2
¼ 8–9%) observed in the presence of
citrate only (25 m
M
,pH
ex
1.0–7.0). Apparently A. niger can
maintain its intracellular pH while keeping its oxygen
uptake far from the uncoupled value. An obvious
Ó FEBS 2002 Intracellular pH homeostasis in A. niger (Eur. J. Biochem. 269) 3491

explanation for this high tolerance towards extreme
extracellular acidity would be to contribute this behaviour
to plasma membranes with an unusual lipid composition,
rendering them highly impermeable to protons. For acido-
philic prokaryotes (both bacteria and archaea) it has been
shown that a link exists between the lipid composition of
their plasma membranes and an acidophilic mode of
existence [32]. The proton permeability (P, cmÆs
)1
)in
biological membranes has been found to be extremely pH
dependent, with values ranging from 10
)3
to 10
)6
cmÆs
)1
[33]. Based on results obtained by Sanders and Slayman [1],
Burgstaller argued that the proton permeability of N. crassa
plasma membranes is probably much lower than 10
)3
to
explain their results [33]. Using lipid bilayer membranes
composed of bacterial phosphatidylethanolamine, Gutkn-
echt found a 10
6
times lower P at pH 2 compared to pH 7,
indicating much lower values for P at low pH [34]. Using a
value for P of 10
)6

cmÆs
)1
for A. niger at pH
ex
1.5, and
assuming hydrolysis of 1 ATP/H
+
expelled by the P-
ATPase, the ATP requirement to maintain a pH
cyt
value of
7.6 can be calculated from the passive proton flux J
H
+
(molÆgdw
)1
Æs
)1
) ¼ PÆAÆD[H
+
]. For A (m
2
Ægdw
)1
), a value
of 2.9 has been found for A. niger NW131 (B. R. Poulsen,
Department of Microbiology, Fungal Genomics Section,
Wageningen University, the Netherlands, personal commu-
nication). Using these values the ATP turnover for pH
homeostasis is 10

)6
molÆgdw
)1
Æs
)1
. This value is within the
same range of ATP turnover necessary for cellular main-
tenance (2 · 10
)6
molÆgdw
)1
Æs
)1
), assuming a maintenance
coefficient m of 0.034 g glucoseÆgdw
)1
Æh
)1
and 38 mol ATP
formed per mole glucose (B. R. Poulsen, personal commu-
nication). Although the exact value for P in fungal
membranes at low pH is not known, these values suggest
that with a low intrinsic proton permeability the energy
costs to maintain such large DpHs are relatively low. This
means that physical protection by the cytoplasmic mem-
brane alone may be sufficient to keep pH
cyt
close to
neutrality in an extremely acidic environment. If we assume
DY to be 0 mV, then the proton motive force (Dp) generated

at pH
ex
1.0 would be around )400 mV (Dp ¼
DY ) ZDpH). This is near the theoretical limit if we assume
the maximal amount of energy available to the P-ATPase to
result from ATP hydrolysis and if a stoichiometry of 1 H
+
expelled per ATP hydrolysed is assumed [35].
Compared to the pH-homeostatic properties of the
organisms mentioned above, A. niger behaves like a typical
acidophile. So far, bacteria and archaea have been the main
focus of studies on intracellular pH homeostasis in acido-
philes. Comparable to A. niger, the acidophile Thiobacillus
ferrooxidans is capable of maintaining its intracellular pH
constant at a value of 6.5 over a range of pH
ex
from 1.0 to
8.0 [36]. Acidophiles are able to sustain such large
cytoplasmic DpH values by counteracting the large inwardly
directed H
+
gradient with a positive-inside DY that results
from Donnan or H
+
diffusion potentials [37]. Thus, the
proton motive force across the cytoplasmic membrane is
reduced in order to decrease the proton leak into the cells
and to decrease the back-pressure for H
+
extrusion by the

P-ATPase. In this way H
+
influx eventually becomes self-
limiting with an increasing positive-inside DY.AK
+
/H
+
symport uptake system operating in parallel with the
P-ATPase, combined with a low intrinsic cation permeab-
ility of the plasma membrane, offers the acidophile a
possibility to generate and sustain a positive-inside DY.
Indications for such a mechanism have been reported
for two acidophilic eukaryotes: the alga Dunaliella
acidophila and the yeast Metschnikowia reukaufii [38].
Whether A. niger relies on generation of a positive-inside
DY, a low plasma membrane proton permeability at
low pH, or a combination of both, still remains to be
investigated.
The capacity of the various energy-transducing mem-
branes to maintain proton gradients appeared to be quite
different. CCCP was much more efficient in dissipating
DpH across the vacuolar membrane than across the
cytoplasmic membrane, a finding that had already been
reported for S. cerevisiae [39]. This could mean that the
ability of the V-ATPase to be stimulated by increased
proton leak is rather poor. ATP levels (and pH gradients)
could be maintained or restored as long as the respiratory
rate could still be stimulated after uncoupler addition, even
when a temporary collapse of the vacuolar membrane DpH
occurred. A complete collapse of DpH across the cytoplas-

mic membrane in the presence of lethal CCCP levels
(10–20 l
M
) could not be observed. A similar result has been
reported for S. cerevisiae [39], and the authors suggested
that besides ATP-dependent ion pumps an additional role in
pH homeostasis may be reserved for the cytoplasmic
buffering capacity. However, Sanders and Slayman [1] have
clearly shown that a large involvement of the cytoplasmic
buffering capacity in intracellular pH homeostasis is not
very likely. A more obvious explanation would be that,
although true uncouplers are able to reduce the H
+
electrochemical potential difference across a membrane to
zero when the concentration applied is high enough, the pH
difference can only be dissipated if the charge imbalance as a
result of the H
+
transport is compensated by the movement
of other ions [40]. Hence, if the permeability of the A. niger
plasma membrane for other ions is low, a lack of
compensating charge fluxes could account for the observed
residual DpH. Lethal levels of azide resulted in a smaller
residual cytoplasmic membrane DpH than lethal levels of
CCCP (0.3 and 0.9 pH units, respectively). Besides
cytoplasmic acidification due to specific inhibition of ATP
synthesis, azide has been suggested to have additional
uncoupling abilities [41]. This combined effect may account
for the observed difference. However, as lethal doses of
CCCP also lead to a complete loss of ATP, it is more

tempting to speculate that azide, upon uptake into the cell, is
able to alter ion conductivity in the cytoplasmic membrane.
As a consequence, larger compensating ion fluxes may
occur that allow a larger dissipation of cytoplasmic
membrane DpH. Indeed, azide was found to have a specific
effect on ion transport (probably K
+
/H
+
exchange) in
plasma membranes of S. cerevisiae [42].
In the presence of nonlethal azide concentrations, chan-
ges in pH
vac
followed a course that was similar to pH
cyt
.
Vacuolar H
+
influx in response to increased cytoplasmic
H
+
levels indicates a role in pH
cyt
homeostasis for this
organelle. These results are in accordance with observations
made in S. cerevisiae [30] and in higher plant cells (Acer
pseudoplatanus L.) [29].
The observed recovery of A. niger from nonlethal CCCP
levels, even under conditions of complete vacuolar mem-

brane DpH dissipation, may be attributed to induced
expression of membrane-bound ATP-dependent transpor-
3492 S. J. A. Hesse et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ters of the ATP-binding cassette superfamily involved in
multidrug resistance. Transcription of two ATP-binding
cassette transporter encoding genes (atrAandatrB) in
A. nidulans was shown to be enhanced within a few
minutes of treatment with several drugs, including antibio-
tics, azole fungicides and plant defence toxins [43]. A
subsequent energy-dependent drug efflux activity may also
confer such resistance to A. niger after CCCP addition,
provided that a certain minimal ATP level can be
maintained within the cell.
Industrial-scale production of citric acid is performed
mainly by fermentation processes using A. niger [12].
Previously reported values for pH
cyt
in citric acid producing
A. niger mycelium vary from 6.5 [13] to 6.8 [14]. We report
here a considerable higher pH
cyt
of 7.5 (in both 2- and
7-day-old-mycelium) under fully oxygenated conditions.
The low pH
cyt
values reported earlier probably resulted
from difficulties to keep the mycelium well-energized during
spectra acquisition. With metabolic engineering and mod-
elling studies as a promising approach to improve produc-
tivity [44], a detailed description of pH

in
during citric acid
production provides valuable additional information about
the conditions under which the enzymes involved operate
in vivo. To reach a higher degree of accuracy for Aspergillus
kinetic models, more information on the internal metabolic
changes that take place during the transition to citric acid
producing mycelium is needed in the future. Our results
have shown that, at least with respect to intracellular pH,
these changes are minor: the combination of low pH
ex
and a
high acid-secreting capacity only led to a slightly lower
cytoplasmic pH.
ACKNOWLEDGEMENTS
The authors acknowledge financial support from the EC in the
framework of the Eurofung Cell Factory project (QTRK3-1999–
00729).
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