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

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.

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

Wageningen University, Dreijenlaan 3, 6703 HA Wageningen,
the Netherlands. Fax: +31 317 484 011, Tel.: +31 317 484 692,
E-mail: [email protected]
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

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, 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

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 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-

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

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

: 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

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

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

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

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

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

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

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

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

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

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

)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

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-

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-

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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