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Appl Environ Microbiol, February 1998, p. 530-534, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Measurement of the Effects of Acetic Acid and
Extracellular pH on Intracellular pH of Nonfermenting, Individual
Saccharomyces cerevisiae Cells by Fluorescence
Microscopy
Lars Uhre
Guldfeldt* and
Nils
Arneborg
Department of Dairy and Food Science, Food
Microbiology, The Royal Veterinary and Agricultural University,
1958 Frederiksberg C, Denmark
Received 14 July 1997/Accepted 14 November 1997
 |
ABSTRACT |
The effects of acetic acid and extracellular pH (pHex)
on the intracellular pH (pHi) of nonfermenting, individual
Saccharomyces cerevisiae cells were studied by using a new
experimental setup comprising a fluorescence microscope and a perfusion
system. S. cerevisiae cells grown in brewer's wort to the
stationary phase were stained with fluorescein diacetate and
transferred to a perfusion chamber. The extracellular concentration of
undissociated acetic acid at various pHex values was
controlled by perfusion with 2 g of total acetic acid per liter at
pHex 3.5, 4.5, 5.6, and 6.5 through the chamber by using a
high-precision pump. The pHi of individual S. cerevisiae cells during perfusion was measured by fluorescence
microscopy and ratio imaging. Potential artifacts, such as fading and
efflux of fluorescein, could be neglected within the experimental time
used. At pHex 6.5, the pHi of individual S. cerevisiae cells decreased as the extracellular
concentration of undissociated acetic acid increased from 0 to 0.035 g/liter, whereas at pHex 3.5, 4.5, and 5.6, the
pHi of individual S. cerevisiae cells decreased
as the extracellular concentration of undissociated acetic acid
increased from 0 to 0.10 g/liter. At concentrations of undissociated
acetic acid of more than 0.10 g/liter, the pHi remained
constant. The decreases in pHi were dependent on the pHex; i.e., the decreases in pHi at
pHex 5.6 and 6.5 were significantly smaller than the
decreases in pHi at pHex 3.5 and 4.5.
| |
INTRODUCTION |
Acetic acid is a by-product formed
during yeast alcoholic fermentations; e.g., in wine and beer
fermentations the levels of acetic acid produced may be 1 to 2 g/liter
(13) and 150 to 280 mg/liter (8), respectively.
Furthermore, acetic acid is a potential inhibitor of yeast growth
(1, 14, 16). Acetic acid is believed to uncouple energy
generation from growth in Saccharomyces cerevisiae by
dissipating the proton motive force across the plasma membrane (15, 20). The uncoupling mechanism of acetic acid involves passive diffusion of acetic acid in its undissociated form across the
plasma membrane of S. cerevisiae. Once inside the cell, the undissociated acetic acid dissociates due to its pKa of
4.75 and the higher intracellular pH (pHi), causing
intracellular acidification. To counteract this acidification, protons
have to be pumped out of the cells by the plasma membrane ATPase, at
the expense of ATP (15, 20). Thus, the pHi seems
to be a crucial factor involved in the inhibitory effect of acetic acid
on S. cerevisiae. However, very little is known about the
effect of acetic acid on the pHi of S. cerevisiae. Pampulha and Loureiro-Dias (15) have
described the effects of acetic acid and extracellular pH
(pHex) on the pHi of fermenting S. cerevisiae cells as determined by using the distribution of
radioactively labelled propionic acid to measure pHi. These
authors concluded that the pHi of S. cerevisiae
decreases with increasing extracellular concentrations of undissociated acetic acid and that this decrease in pHi depends
exclusively on the extracellular concentration of undissociated acetic
acid and is independent of the pHex.
In this work we used a new experimental setup comprising a fluorescence
microscope and a perfusion system to study the effects of acetic acid
and pHex on the pHi of nonfermenting,
individual S. cerevisiae cells.
| |
MATERIALS AND METHODS |
Microorganism and growth conditions.
A commercial strain of
lager yeast, S. cerevisiae (catalog no. 2155), from the
Collection of Pure Cultures of Brewing Yeasts (Alfred Jørgensen
Laboratory Ltd., Copenhagen, Denmark) was grown in 2.5-liter European
Brewing Convention tubes at 14°C in brewer's wort with a specific
gravity of 10.8°P (1°P is equal to 1 g of sugar, as sucrose,
per 100 ml of wort at 20°C). The wort was oxygenated so that it
contained 10 ppm of dissolved oxygen before inoculation, and the rate
of inoculation was 106 cells × °P per ml of wort.
Cells were harvested in the stationary phase (i.e., after 146 h of
fermentation).
Staining of cells with FD.
The total cell number was
determined by using a Neubauer counting chamber. A cell suspension was
centrifuged at 3,000 × g for 4 min at 4°C. The
supernatant was discarded, and the cells were resuspended in cold
phosphate-buffered saline (PBS) (containing [per liter] 8 g of
NaCl, 0.2 g of KCl, 1.44 g of
Na2HPO4, and 0.24 g of
KH2PO4; pH 5.6) to a final concentration of
2.5 × 106 cells/ml. Fluorescein diacetate (FD)
(catalog no. F-7378; Sigma) was added to the cell suspension from a
stock solution (2.4 mM FD in acetone) to a final concentration of 12 µM, and the preparation was mixed thoroughly for 10 s (final
acetone concentration, 0.5% [vol/vol]). To minimize photobleaching,
the cell suspension was incubated for 10 min in the dark at 40°C and
immediately transferred to ice, where it remained for at least 10 min.
FD is a nonfluorescent prefluorochrome that is taken up by passive
diffusion by S. cerevisiae cells (3). Once inside
a cell, FD is hydrolyzed by unspecified esterases, which results in the
fluorescent compound fluorescein (10). Fluorescein is
accumulated within the cell due to its polarity (17). Before
microscopic analysis the cells were resuspended in PBS having the same
pH as the perfusion solution used in the experiment (see below).
Perfusion system for dynamic studies of individual cells.
The perfusion system used is shown in Fig.
1A. The perfusion chamber (model RC-21A
cell culture-perfusion chamber; Warner Instrument Corporation, Hamden,
Conn.) was assembled with a poly-D-lysine (catalog no.
P-6407; Sigma)-coated bottom coverslip (24 by 32 mm; Knittel
Gläser, Bie & Berntsen, Rødovre, Denmark) and a top coverslip
(diameter, 15 mm; Warner Instrument Corporation). Previous experiments
in our laboratory had shown that poly-D-lysine was a useful
agent for immobilizing yeast cells on the bottom coverslip during
perfusion (data not shown). The chamber was sealed by using silicon
grease. The volume of the perfusion chamber was 0.25 ml. The stained
cell suspension was placed in the assembled perfusion chamber and
allowed to settle and immobilize on the polylysine-coated bottom
coverslip. Subsequently, the chamber was mounted on an appropriate
platform (type PH1; Warner Instrument Corporation) and placed on the
stage of a microscope (Zeiss model Axiovert 135 TV; Brock & Michelsen
A/S, Birherød, Denmark). Solutions (see below) were perfused through
the inlet of the chamber at a rate of 0.6 µl/s by using a modified
Alitea XV pump (Microlab Aarhus A/S, Aarhus, Denmark) and were removed
from the outlet of the chamber by using a model 101U pump (Watson
Marlow, Wilmington, Mass.). The perfusion solutions consisted of PBS
(pH 3.5, 4.5, 5.6, and 6.5) and 2 g of total acetic acid per liter
in PBS (pH 3.5, 4.5, 5.6, and 6.5) at 25°C. In each experiment the
perfusion chamber was filled with newly stained cells, and perfusion
was initiated at time zero with a perfusion solution having the same pH
as the cell suspension.
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FIG. 1.
Schematic diagrams showing the perfusion system (A) and
the fluorescence microscope (B). CCD, charge-coupled device.
|
|
Measurement of pHi of individual cells.
The
pHi of an individual cell was measured by ratio imaging by
using a fluorescence microscope as shown in Fig. 1B. During perfusion,
stained cells were excited at 490 and 435 nm as described by Slavik
(19) with exposure times of 1,000 and 500 ms, respectively. The excitation source was an optical fiber-connected monochromator with
a 75-W short-arc xenon lamp (Monochromator B; T.I.L.L. Photonics GmbH,
Planegg, Germany). A 50% transmission neutral filter was installed
between the optical fiber and the microscope. Emission was collected at
wavelengths between 515 and 565 nm by using a band-pass emission filter
(Zeiss type BP 515-565; Brock & Michelsen A/S), a beam splitter
(Zeiss model BSP 510; Brock & Michelsen A/S), and a cooled slow-scan
frame transfer charge-coupled device camera (EEV 512 × 512 12-bit
frame-transfer CCD; Princeton Instruments Inc., Trenton, N.J.). Ratio
imaging of emission signals collected from excitation at 490 and 435 nm
was performed by using the software package MetaFluor, version 2.0 (Universal Imaging Corporation, West Chester, Pa.). Before ratio
imaging, cells were focused and selected as regions of interest under
bright-field illumination, to avoid subjective selection based on
fluorescence, by using a Zeiss Fluar oil objective (magnification,
×100; numerical aperture, 1.30). One region of interest comprised an
individual cell; i.e., the emission obtained from a region was the
average value for an individual cell. Ratio imaging was initiated at
time zero of perfusion. Images were recorded at 10-s intervals, and
each experiment was terminated within 10 min. In each experiment
between 20 and 30 randomly selected individual S. cerevisiae
cells were analyzed. Each experiment was repeated at least twice, and
the results obtained were conclusive.
The 490 nm/435 nm ratios for individual cells were calibrated to pH
values by using an in vitro calibration curve (Fig.
2).
The calibration curve was prepared by
using 10 µM fluorescein
(catalog no. F-7505; Sigma) in PBS adjusted
to various pH values
with 2 N HCl and 2 N NaOH. The perfusion chamber
was filled with
the fluorescein solutions, and ratio imaging was
performed as
described above.
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FIG. 2.
Relationship between the logarithm of the 490 nm/435 nm
ratio and pH. The in vitro calibration curve was prepared by using 10 µM fluorescein in PBS adjusted to various pH values as described in
Materials and Methods.
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|
Calculation of the undissociated acetic acid concentrations
during perfusion.
The concentrations of undissociated acetic acid
in the perfusion solutions containing 2 g of total acetic acid per
liter at pHex 3.5, 4.5, 5.6, and 6.5 were calculated by
using the Henderson-Hasselbalch equation: pHex = pKa + log([Ac
]PS/[HAc]PS)
and [HAc]PS + [Ac]PS = [HAc]total,PS = 2 g/liter, where the pKa of
acetic acid is 4.75 and [HAc]PS is the concentration of
undissociated acetic acid in the perfusion solution.
In order to calculate the concentration of undissociated acetic acid in
the perfusion chamber at a given time during perfusion,
the flow of
perfusion solution through the chamber was characterized.
This
characterization was performed by using 5 µM fluorescein
in PBS (pH
5.6). At a fluorescein concentration of 5 µM, artifacts,
such as
concentration quenching, were avoided; i.e., the fluorescence
intensity
was linearly correlated with the fluorescein concentration
(data not
shown). The chamber was filled with PBS (pH 5.6), and
at time zero
perfusion with 5 µM fluorescein in PBS (pH 5.6) was
initiated at a
rate of 0.6 µl/s. Emission signals from excitation
at 435 nm were
measured by using MetaFluor, version 2.0, software
(Universal Imaging
Corporation). The exposure time was 1,000 ms,
and the 50% transmission
neutral filter was omitted. Images were
recorded from time zero of
perfusion at 10-s intervals until the
maximum fluorescence intensity
was reached. The changes in fluorescence
intensity in the chamber with
time during perfusion are depicted
in Fig.
3.
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FIG. 3.
Characterization of the flow of perfusion solution
through the perfusion chamber. The chamber was filled with PBS (pH
5.6). At time zero, perfusion with 5 µM fluorescein in PBS (pH 5.6)
at a rate of 0.6 µl/s was initiated. Fluorescence intensity after
excitation at 435 nm for 1,000 ms was measured as described in
Materials and Methods.
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|
The concentration of undissociated acetic acid in the chamber at a
given time during perfusion was calculated by using the
following
equation: [HAc]
T = [HAc]
PS × [(
I435,T 
I435,min)/(
I435,max I435,min)], where [HAc]
T is the
concentration of undissociated
acetic acid at time T,
[HAc]
PS is the concentration of undissociated
acetic acid
in the perfusion solution,
I435,T is the
fluorescence
intensity after excitation at 435 nm at time T (Fig.
3),
and
I435,min and
I435,max
are the minimum and maximum fluorescence intensities,
respectively,
after excitation at 435 nm (Fig.
3).
| |
RESULTS |
Perfusion with PBS.
Efflux (2) and photobleaching
(3) of fluorescein have previously been reported to occur in
S. cerevisiae cells. In order to determine the influence of
these potential artifacts on our results, stained S. cerevisiae cells were suspended in PBS at pHex 3.5, 4.5, 5.6, and 6.5 and perfused with PBS having the same pHex as the cell suspension. The fluorescence intensities
after excitation at 490 and 435 nm of individual S. cerevisiae cells were not affected by perfusion with PBS at
pHex 4.5 (Fig. 4A and B). The
same results were observed at pHex 3.5, 5.6, and 6.5 (data not shown), indicating that the influence of efflux and photobleaching of fluorescein on our results could be ignored under the conditions used.
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FIG. 4.
Fluorescence intensity after excitation at 490 nm (A)
and 435 nm (B), and pHi (C) of five representative S. cerevisiae cells during perfusion with PBS (pHex 4.5)
at a rate of 0.6 µl/s. Fluorescent staining of cells and ratio
imaging were performed as described in Materials and Methods.
|
|
Moreover, the pH
i of individual
S. cerevisiae
cells was not affected by perfusion with PBS at pH
ex 4.5 (Fig.
4C). The same
results were observed at pH
ex 3.5, 5.6, and 6.5 (data not shown).
Hence, any potential buffer interference
could be ignored in the
experiments performed with acetic acid in the
perfusion solution.
The pH
i values of individual
S. cerevisiae cells
ranging between 5.6 and 6.2 (Fig.
4C) are consistent with
pH
i values previously
found for brewer's yeast (
6,
10-12,
18). Furthermore, the
pH
i values presented in
Fig.
4C demonstrate that the asynchronous
yeast population used in this
study was highly heterogeneous with
respect to pH
i. These
results agree with previously reported results
obtained with
asynchronous
S. cerevisiae cultures (
3,
6,
9).
Perfusion with undissociated acetic acid.
In order to
determine the effects of acetic acid and pHex on the
pHi of S. cerevisiae cells, stained cells were
suspended in PBS at pHex 3.5, 4.5, 5.6, and 6.5 and
perfused with 2 g of total acetic acid per liter in PBS having the
same pHex as the cell suspension. During perfusion with
2 g of total acetic acid per liter at pHex 6.5, the
average pHi of S. cerevisiae cells decreased
from 6.2 to 5.9 as the extracellular concentration of undissociated
acetic acid increased from 0 to 0.035 g/liter (Fig. 5A). During perfusion
with 2 g of total acetic acid per liter at pHex 5.6, the average pHi of S. cerevisiae cells decreased from 5.8 to 5.4 as the extracellular concentration of undissociated acetic acid increased from 0 to 0.10 g/liter, and at concentrations of
undissociated acetic acid higher than 0.10 g/liter, the pHi remained constant at 5.4 (Fig. 5B). During perfusion with 2 g of
total acetic acid per liter at pHex 4.5 and 3.5, the
average pHi of S. cerevisiae cells decreased
from 5.6 to 4.4 as the extracellular concentration of undissociated
acetic acid increased from 0 to 0.10 g/liter, and at concentrations of
undissociated acetic acid higher than 0.10 g/liter, the pHi
remained constant at 4.4 (Fig. 5C and D). The decreases in the
pHi values of individual S. cerevisiae cells at
pHex 5.6 and 6.5 were smaller than the decreases in the pHi values at pHex 3.5 and 4.5 (0.3 to 0.4 and
1.2 pH units, respectively) (Fig. 5).
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FIG. 5.
Relationship between pHi of individual
S. cerevisiae cells and extracellular concentration of
undissociated acetic acid. The cells were perfused with 2 g of
total acetic acid per liter in PBS at pHex 6.5 (A),
pHex 5.6 (B), pHex 4.5 (C) and pHex
3.5 (D) at a rate of 0.6 µl/s. The pHi values presented
are averages from 20 to 30 randomly selected cells. The calculated
standard deviations are shown by error bars. The insets show the
relationship between pHi and the full range of extracellular concentrations of undissociated acetic acid
used in the experiments. The time intervals between data points are
20 s (A) (for clarity) and 10 s (B through D). Fluorescent
staining of cells, ratio imaging, and calculation of the undissociated
acetic acid concentrations during perfusion were performed as described
in Materials and Methods.
|
|
| |
DISCUSSION |
In this study we investigated the effect of acetic acid on the
pHi of individual S. cerevisiae cells at various
pHex values by using a new experimental setup comprising a
fluorescence microscope and a perfusion system (Fig. 1). The
extracellular concentration of undissociated acetic acid at various
pHex values was controlled by perfusing 2 g of total
acetic acid per liter at pHex 3.5, 4.5, 5.6, and 6.5 through a perfusion chamber by using a high-precision pump, and the
pHi values of individual S. cerevisiae cells
during perfusion were measured by fluorescence microscopy and ratio
imaging.
The results reported in this study demonstrate that at pHex
3.5, 4.5, and 5.6 the pHi values of individual S. cerevisiae cells decreased as the concentration of undissociated
acetic acid increased from 0 to 0.10 g/liter (Fig. 5). At
concentrations of undissociated acetic acid higher than 0.10 g/liter
the pHi remained constant (Fig. 5). These results agree
with the results reported by Warth (21) obtained with
benzoic acid, which showed that the pHi of S. cerevisiae decreased as the benzoic acid concentration increased from 0 to 0.13 g/liter and that at benzoic acid concentrations higher
than 0.13 g/liter the pHi remained constant. Our finding that the pHi of individual S. cerevisiae cells
remains constant at concentrations of undissociated acetic acid higher
than 0.10 g/liter may be explained by the presence of a constant level
of accumulated acetic acid in S. cerevisiae, as suggested
previously for benzoic acid (21). Alternatively, the plasma
membrane ATPase activity of S. cerevisiae may be activated
at concentrations of undissociated acetic acid higher than 0.10 g/liter. This should result in an increased efflux of protons, thereby
compensating for the acidification of the cytosol. In the present
study, however, the experiments were carried out without glucose or any
other energy source; i.e., the ATP pools within the cells may have been depleted, and the plasma membrane ATPase may not have functioned.
Pampulha and Loureiro-Dias (15) found that acetic
acid-induced decreases in the pHi in S. cerevisiae depend exclusively on the extracellular concentration
of undissociated acetic acid and are independent of the
pHex. These results are not consistent with our results
which show that the decreases in pHi induced by
undissociated acetic acid are dependent on the pHex. The
difference between the results may be explained by the different
experimental approaches used. Pampulha and Loureiro-Dias
(15) investigated the effects of acetic acid and
pHex on the pHi of S. cerevisiae by
using the distribution of radioactively labelled propionic acid to
measure the pHi. In this technique (i) impermeability of
the anion of acetic acid is assumed (22), (ii) cells are incubated for 85 min in the presence of undissociated acetic acid, and
(iii) cell suspensions are used. With our technique (i) no assumptions
concerning impermeability of the anion of acetic acid have to be made,
(ii) measurements are carried out at 10-s intervals, and (iii) the
pHi of individual cells is measured. Furthermore, Pampulha
and Loureiro-Dias (15) studied fermenting S. cerevisiae cells, whereas we studied nonfermenting cells.
The fact that the pHex affects acetic acid-induced
decreases in pHi in S. cerevisiae (Fig. 5)
indicates that the mechanisms underlying these decreases in
pHi may be sensitive to pHex. The pHex-sensitive mechanisms may include plasma membrane
permeability to undissociated acetic acid, which has been reported to
decrease in S. cerevisiae with decreasing pHex
(4, 5), and the buffering capacity of the cytosol, which for
Zygosaccharomyces bailii has been reported to increase with
decreasing pHex (7). The results obtained in
this work, however, are opposite the results which could be anticipated
from the mechanisms mentioned above; i.e., the pHi is lower
at low pHex values compared with high pHex
values (Fig. 5), whereas it should be higher due to a lower passive
diffusion rate of undissociated acetic acid (4, 5) and a
higher buffering capacity of the cytosol (7). Our results
cannot be explained by plasma membrane permeability to undissociated
acetic acid and the buffering capacity of the cytosol. Thus, this
subject needs further investigation.
In conclusion, our results show that at pHex 3.5, 4.5, and
5.6 the pHi values of individual S. cerevisiae
cells decrease as the concentration of undissociated acetic acid
increases from 0 to 0.10 g/liter. At concentrations of undissociated
acetic acid higher than 0.10 g/liter the pHi remains
constant. Furthermore, our results demonstrate that the decreases in
pHi strongly depend on the pHex. In future
experiments we will attempt to elucidate the mechanisms underlying the
changes in the pHi values of S. cerevisiae cells
induced by undissociated acetic acid.
| |
ACKNOWLEDGMENTS |
This work was financially supported by Alfred Jørgensen
Laboratory Ltd., Copenhagen, Denmark, and by the FØTEK program
sponsored by the Danish Ministry of Research through the LMC-Centre for Advanced Food Studies.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Dairy and Food Science, Food Microbiology, The Royal Veterinary and
Agricultural University, Rolighedsvej 30, 1958 Frederiksberg C,
Denmark. Phone: 45 35283286. Fax: 45 35283214. E-mail:
lgm{at}kvl.dk.
| |
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Appl Environ Microbiol, February 1998, p. 530-534, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
