Appl. Environ. Microbiol. - Applied and Environmental Microbiology

AEM Accepts, published online ahead of print on 18 July 2014
Appl. Environ. Microbiol. doi:10.1128/AEM.01391-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Sorbic acid and acetic acid have distinct effects on the electrophysiology and metabolism of
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Bacillus subtilis
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J.W.A. van Beilena, M.J. Teixeira de Mattosb, K.J. Hellingwerfb, S. Brula#
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Molecular Biology & Microbial Food Safety, Swammerdam Institute for Life Sciences,
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University of Amsterdam, The Netherlandsa.
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Department of Molecular Microbial Physiology, Swammerdam Institute for Life Sciences,
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University of Amsterdam, The Netherlandsb.
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Running Head: Physiology of weak acid stress in Bacillus subtilis
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corresponding author: S. Brul, [email protected]
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Abstract
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Sorbic and acetic acid are amongst the most commonly used weak organic acid preservatives
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to improve the microbiological stability of foods. Both have a similar pKa value but sorbic
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acid is a far more potent preservative. Weak organic acids are most effective at low pH.
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Under these circumstances, they are assumed to diffuse across the membrane as neutral
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undissociated acids. We show here that the level of initial intracellular acidification depends
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on the concentration of undissociated acid and less on the nature of the acid. Recovery of the
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internal pH depends on the presence of an energy source, but acidification of the cytosol
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causes a decrease in glucose flux. Furthermore, sorbic acid is a more potent uncoupler of the
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membrane potential than acetic acid. Together these effects may also slow down the rate of
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ATP synthesis significantly and may thus (partially) explain sorbic acid’s effectiveness.
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Introduction
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Various small weak organic acids (WOA) have been used as food preservatives for a very
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long period of time. These weak acids slow down growth of various spoilage bacteria, yeasts
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and moulds without overt undesired effects on taste or being toxic to the consumer. The
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undissociated states of the WOA preservatives are more effective in slowing growth than the
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dissociated form, although the latter may have some level of toxicity. As such, WOAs are
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most effective when applied at low pH values, below their pKa value (1, 2). Under these
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conditions, the neutral acid is assumed to diffuse across the plasma membrane and dissociate
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in the cytosol which generally has a higher pH. In this way, the proton gradient over the
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membrane is depleted and the anion may accumulate to potentially toxic levels inside the
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cell. This is known as the classical “weak-acid preservative” theory (3). Commonly used
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WOA preservatives include sorbic and acetic acid, which have a similar pKa value of 4.76 but
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a dissimilar octanol: water partition coefficient (log Kow) of 1.33 and -0.17 respectively (4).
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This means that at a particular pH and the same total concentration, concentrations of both
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undissociated acids are the same, but sorbic acid has a higher affinity for a hydrophobic
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(membrane) environment. Sorbic acid is clearly the more potent preservative of the two, but
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the exact reason why is still not fully clear (5, 6).
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It is important to distinguish the different modes of action that WOAs may have on cell
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physiology. The classical “weak-acid preservative” theory (3) only assumes entry of the
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undissociated acid, dissociation in the cytosol and cytosolic acidification. While this is
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sometimes described as uncoupling, we will use this latter phrase for compounds that shuttle
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protons across the membrane and are thus protonophoric uncouplers. Others have also
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pointed to potential toxicity of accumulated anions (1, 7, 8). If we assume that only the
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undissociated acid diffuses across the membrane, it follows from the Henderson–Hasselbalch
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equation (9) that ∆
, and thus the internal concentration of the anion may
3
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become very high when a high ΔpH remains present. Also, specific binding of sorbate to
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cysteine (10) has been shown and is proposed as an explanation for the higher toxicity of
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sorbic acid.
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Several resistance mechanisms against WOAs have been reported for yeasts like
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Saccharomyces cerevisiae, Zygosaccharomyces baillii and Z. rouxii. These organisms induce
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the expression of H+-ATPases to regulate their cytosolic pH. S. cerevisiae uses a dedicated
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ATP binding cassette (ABC) transporter (Pdr12) to prevent accumulation of the anion (6).
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Also S. cerevisiae plasma membrane components are likely to play an important role in the
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modulation of the influx of lipophilic weak organic acids (11, 12). Furthermore, Z. rouxii and
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Aspergillus niger have been shown to degrade sorbic acid to 1,3-pentadiene (13, 14).
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The responses and potential resistance mechanisms of bacteria against weak acids have not
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been so well-described as for yeasts (15, 16). The level of growth reduction has been
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modelled some 30 years ago (1) and the effect of sorbic acid on the membrane potential in
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Escherichia coli membrane vesicles has been described (17). More recently Ter Beek et al.
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(2) performed a microarray study on B. subtilis exposed to sorbic acid. Their results showed a
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broad transcriptomic response resembling a pattern typical for cells responding to nutrient
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limitation. The authors observed an upregulation of genes encoding potential efflux pumps as
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well as genes involved in remodelling of the plasma membrane.
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Bacillus subtilis has been the Gram positive model organism for decades, because it is
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Generally Recognized As Safe (GRAS), it is genetic accessibility and has a fully sequenced
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genome. It also forms heat resistant spores and is as such a recognized spoilage organism
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(18). Spores of several related bacterial species are of great concern to the food industry
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because they are highly resistant to most preservation techniques and, once germinated, can
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cause food spoilage through growth of vegetative cells that may produce toxins (16).
4
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To comprehensively elucidate the physiological effects of WOAs on B. subtilis, we measured
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the effects of sorbic and acetic acid on the chemical (ΔpH) and electrical (ΔΨ) component of
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the proton motive force (PMF). With a depleted PMF, we speculated that the cell might alter
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its energy needs in terms of glucose consumption and availability of its terminal electron
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acceptor (i.e. O2), which were hence determined. We assessed the rate and extent of change
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in pHi caused by these two weak acids using B. subtilis cells that were either directly exposed
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to both WOAs or had been pre-exposed to sorbic acid and re-exposed to WOAs. The latter
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experiment was done because we inferred from the previously collected micro-array data that
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cells elicit an adaptive response to these compounds (2).
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Methods
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General growth conditions and strains
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For general purposes B. subtilis PB2 strains were grown in Lysogeny Broth (LB). For weak
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acid stress experiments and fluorescence measurements, B. subtilis strains were grown in
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defined liquid medium (M3G; (19) set at pH 5.5, 6.4 (buffered with 80 mM MES), as well as
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7.0 and 7.4 (buffered with 80 mM MOPS). The medium contained 5 mM glucose, 10 mM
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glutamate, and 10 mM NH4Cl as carbon and nitrogen sources. All cultures were grown at
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37°C, under continuous agitation at 200 rpm. The wild-type strain used (B. subtilis PB2) was
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obtained from C.W. Price and A. Ter Beek (2). The strain expressing IpHluorin (B. subtilis
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PB2 PptsG-IpHluorin) was constructed as described (20). When required, 50 µg/ml
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spectinomycin was added to the medium.
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Calibration of pHluorin and Internal pH measurements
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The internal pH was measured as described (20). All strains were grown in M3G at pH = 6.4.
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For rapid-exposure WOA stress experiments, potassium sorbate (K-S) and potassium acetate
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(K-Ac) were used at 250 mM, dissolved in M3G medium without glucose. Of these solutions,
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2-10 µl were injected into the cell suspensions at 310 µl/s using the injector of the FluoStar
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Optima (BMG Labtech, Germany). As a control experiment, the same concentrations of
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either KCl or NaCl were injected into the microtiter wells. For 50% and 80% growth
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inhibition experiments, 3 and 11 mM K-sorbate or 25 and 80 mM K-acetate were used,
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respectively.
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Cell counts and protein measurements
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In order to compare results per cell from the different experiments, cell counts were
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performed with a CASY counter (Roche, Germany) equipped with a 60 µm tube. The number
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of cells counted were 4-10x104 cells per ml. Protein concentrations were determined using a
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BCA kit (Thermo Scientific) according to the manufacturer’s instructions. Finally, the OD600
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of the cultures was measured with a FluoStar Optima.
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Membrane potential measurements
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B. subtilis PB2 and B. subtilis PptsG-IpHluorin were grown as described in M3G, pH = 6.4, to
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OD600 = 0.4. Cells were harvested by centrifugation and resuspended in 1/10 volume M3G
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without glucose. To inhibit growth and protein synthesis between experiments, 10 µg/ml
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chloramphenicol was added. Cells were stored at 37°C. The membrane potential (ΔΨ) was
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measured using a Tetra Phenyl Phosphonium ion (TPP+) electrode (World Precision
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Instruments, Inc., USA) filled with 1 mM TPP+. All measurements were performed in a
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warmed (37°C), 2-ml measuring cell, containing a TPP+ electrode, reference electrode and an
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oxygen sensor (see below). The cell suspension was stirred magnetically and aerated by a
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continuous flow of compressed air, so that at least 120 µM oxygen was present when Δψ was
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measured. One ml of cell suspension was added to 1 ml medium with glucose; subsequently,
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weak organic acids and a TPP+ calibration series (1, 1, 2, 4 and 8 µl of 1 mM TPP+) were
129
added. The addition of weak organic acid did affect the offset, but not the slope of the
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calibration response. Stress conditions tested were: 3 and 11 mM K-S and 25 and 80 mM K-
131
Ac. These conditions were compared to non-exposed cells. The membrane potential was
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calculated as described (21–24) using de-energized cells (incubated for 10 min at 70°C) as
133
reference for non-specific binding of the probe.
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The electric potential was calculated using the following equation (21, 24):
∆
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1
·
1
1
Where
2.303 ·
136
137
Δψ = transmembrane electrical potential (mV, ψin – ψout)
138
Z = Conversion factor
139
F = Faraday’s constant (96.6 J mV-1 mol-1)
140
R = universal gas constant (8.31 J K-1 mol-1)
141
n = charge of the translocated ion
142
T = absolute temperature (K)
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C0 = Probe concentration in the medium without addition of cells
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Ce = Extracellular probe concentration
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fcm = Ratio of fractional cytoplasmic membrane and intracellular volume
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Kcm = Cytoplasmic membrane partition coefficient
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x = Fractional internal volume
148
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The factor fcmKcm was determined by a probe binding assay (21) to be 14. In this (simplified)
150
approach one assumes that the amount of TPP+ bound to extra-cytoplasmic components of
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the cell (like parts of the cell wall) is equal to the amount of probe that binds to intracellular
152
components like nucleic acids. At the end of the experiment, 1.5-ml samples were collected
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for protein quantification as described above. Three biological replicates were measured for
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each condition.
155
156
Oxygen consumption measurements
157
Oxygen consumption rates were measured simultaneously with the Δψ, in the same
158
measuring cell using a Neofox fiber optic oxygen sensor (Ocean Optics Inc., USA). Oxygen
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concentrations were measured every second from the maximally aerated to the fully oxygen
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depleted state. The slope of the straight part of the plot (at least 15 seconds) was used to
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derive the oxygen consumption rate. At the end of the experiment, 1.5-ml samples were
162
collected for protein quantification as described above. Rates were normalized to protein
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content.
164
165
Glucose consumption and metabolite measurements
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B. subtilis PB2 and B. subtilis PptsG-IpHluorin were grown as described in M3G, pH = 6.4, to
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OD600 = 0.8. The cultures were split and exposed to different stress conditions (3 and 11 mM
168
K-sorbate or 25 and 80 mM K-Ac), and 10 mM glucose was added to each culture. A high
169
OD600 was required to observe a significant decrease in glucose concentrations within the
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timeframe of the experiment.
171
Samples, taken every 30 minutes, were snap frozen in liquid nitrogen. The protein
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concentration of each sample was measured as described above. Samples were further
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processed for HPLC analysis; a 1 ml sample was mixed with 100 µl 35% perchloric acid and
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subsequently 55 µl 7 M KOH was added. Filtered supernatants were analysed for glucose
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consumption levels by the cells as well as the presence of fermentation end-products.
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Glucose, succinate, lactate, acetate, 2,3-butanediol and ethanol contents were determined by
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HPLC (LKB) with a REZEX organic acid analysis column (Phenomenex) at 45°C with 7.2
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mM H2SO4 as the eluent, using an RI 1530 refractive index detector (Jasco) and AZUR
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chromatography software for data integration. All measurements were performed with two
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biological replicates. From the obtained metabolite fluxes, a carbon balance was calculated
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with the assumption that similar molar amounts of CO2 were produced per amount of O2
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consumed plus similar molar amounts of CO2 were produced per mole of acetate produced
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and per mole of 2,3-butanediol, 2 moles of CO2 were produced. Oxygen consumption was
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corrected for directly related acetate and 2,3-butanediol production.
%
100% · 2
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, ,
/6
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Results
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Internal pH during growth under WOA stress remains low
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Weak organic acids have been shown to lower the internal pH of microorganisms (25, 26).
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Thus, we measured pHi during growth with a number of WOA stresses. To reduce growth
191
rate by approximately 50%, 3 mM K-sorbate or 25 mM K-acetate was used. To reduce
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growth rate by 85%, 10 and 80 mM were used, respectively.
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Growth and pHi were monitored for 6 hours (Figures 1A and 1B respectively). The internal
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pH of non-stressed cultures dropped from pHi = 7.5 to 7.2 during this time. The internal pH
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of sorbic acid-stressed cultures continued to drop from t = 0 until the end of the experiment.
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With 80 mM K-Ac, this is not seen, and pHi remained stable around pH = 7.3. These results
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show that there is considerable acidification of the cell during growth and that there is no
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recovery of the pHi during extended exposure to WOAs, not even when growth resumes.
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Weak organic acids cause rapid drop in internal pH
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Long term (6 h) exposure to WOAs as described above, showed no long-term recovery of the
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pHi of the cells. The largest drop in pHi upon weak acid exposure occurs within the first
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minutes after addition. To investigate the influx rate of weak organic acid preservatives and
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their dependence on the metabolic activity of the cell, we investigated the short term effects
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of WOA injections into the medium.
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Before acid injection, the pHi of starved wild-type B. subtilis was 7.30 ± 0.05. Injection of
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either KCl or NaCl at identical concentrations as the weak acids had no effect on pHi within
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one minute after injection (not shown). Upon glucose injection, the pHi rapidly increased to
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7.5 within 1 minute (not shown). When WOAs were injected, the pH dropped to its lowest
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point within 1-4 s. In starved cells, this pH remained stable, but in the presence of glucose,
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pHi recovered quickly to a new equilibrium (Figures 2A-D). The acidification of starved cells
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was fitted with a first order kinetic equation
0
1
·
213
where (Bi) is the amplitude factor (ΔpH/mM), indicating the intracellular buffering capacity,
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[HA] is the concentration of undissociated weak acid (in mM), (ki) is the rate constant (s-1)
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and pHi(0) is the offset (pHi at t=0, before injection). Using this equation, values for (Bi) and
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(ki) were determined for sorbic and acetic acid. The value for ΔpHi or the amplitude of
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acidification has a linear relation with the concentration of undissociated acid in the medium
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(Figure 4). These data (with starved cells) show that sorbic acid causes a similar change in
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pHi as acetic acid does. The rate of acidification is high and similar for both acids, 1.29 ±
10
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0.09 s-1 for sorbic acid and 1.27 ± 0.33 s-1 for acetic acid. However, with a measuring
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frequency of one per second, this rate is most likely set by our detection system and the actual
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rate is likely higher. Cells pre-exposed to sorbic acid had similar values as non-stressed cells
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for Bi and ki for either WOA, showing no sign of adaptation at this level (Supplementary
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table 1 and our unpublished data).
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With glucose added to the cultures, recovery of the pHi started immediately after the injection
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of weak acid. This curve too was fitted, but with an additional factor describing recovery.
0
1
1
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Where Br is the amplitude of recovery and kr is the rate constant of the pH recovery.
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Recovery kinetics do not seem to change between unexposed and pre-exposed cultures. There
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is however a clear difference between sorbic and acetic acid. The pHi of acetic acid stressed
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cultures recovers to a higher pH than sorbic acid stressed cultures (Figure 3 and
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supplementary table 1). The amplitude of recovery also appears to have a linear relation with
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the acid concentration. The acquired constants allow predictions for both acidification and
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subsequent recovery (Supplementary figure 1).
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Sorbic acid affects both ΔpH and ΔΨ
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Proton translocation by the electron transport chain through the cell membrane results in both
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a gradient in the chemical potential of protons (i.e. ΔpH), as well as an electrical potential
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(ΔΨ). This electrochemical proton gradient exerts an inward-directed proton motive force or
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PMF (27, 28). The PMF can drive protons back into the cell via the F1F0-ATPase for ATP
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synthesis, and is also required for various membrane transport processes and for flagellar
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rotation. The PMF is defined as (e.g. (29):
∆
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·
∆ψ
·∆
Where
11
2.303 ·
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Δp= Proton motive force, PMF, (mV)
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ΔµH+ = transmembrane electrochemical proton potential (J/mol)
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F = Faraday’s constant (96.6 J mV-1 mol-1)
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Δψ = transmembrane electrical potential (mV, ψin – ψout)
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Z = Conversion factor
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ΔpH = transmembrane pH gradient (pHin – pHout)
249
R = universal gas constant (8.31 J K-1 mol-1)
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T = absolute temperature (K)
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n = charge of the translocated ion
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253
In our experiments, which were performed at 37°C, Z = 61.4 mV. Our measurements with
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TPP+ show that under non-stressed conditions with extracellular pH = 6.4, Δψ = 103 ± 9 mV
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and the pHi = 7.78 ± 0.05. This thus gives a Z*ΔpH value of 84 ± 3 mV and a total PMF of
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188 ± 9 mV (Figure 5). This value for Δψ is similar to earlier reported values (23, 30) and the
257
pHi value is slightly higher (30). Sorbic acid has a severe effect on Δψ, reducing it by 64%
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even at 3 mM K-S. Acetic acid, however, does not deplete Δψ as strongly. With 25 mM K-
259
Ac, the Δψ is 17% lower and with 80 mM only a 45% reduction is seen. It appears that sorbic
260
acid acts more as an uncoupler than acetic acid does.
261
Together, Δψ - Z*ΔpH comprise the proton motive force. In total, WOA stress causes a
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dissipation from 188 ± 9 mV in non-stressed cells to 83 ± 5 mV with 11 mM K-S and 108 ± 7
263
mV with 80 mM K-Ac. This too shows that sorbic acid has a stronger effect on the
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electrochemical gradient for protons than acetic acid does at concentrations that lead to
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similar growth inhibition (Supplementary table 2).
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Oxygen consumption is reduced upon addition of high WOA concentrations
268
Because of the depleted PMF, the cell may experience energy depletion stress, since the F1F0-
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ATPase depends on the proton gradient for its activity. It is possible that the cell tries to
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compensate for the decrease in PMF by increasing the flux through the electron transfer chain
271
and increasing proton pumping activity. This also requires a terminal electron acceptor (e.g.
272
O2). We therefore measured the oxygen levels during our PMF experiments and made sure
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that at least 120 µM O2 was measured when Δψ was measured. Non-stressed cells consumed
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oxygen at a rate of 7.4 mmol s-1 (mg protein)-1. Sorbic acid stress reduced the rate of oxygen
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consumption to 3.9 and 1.5 mmol s-1 (mg protein)-1 with 3 and 11 mM of K-sorbate
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respectively. 25 mM of K-acetate had a non-significant effect on the oxygen consumption
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rate, but 80 mM reduced it to 1.1 mmol s-1 (mg protein)-1 (Figure 6). These effects took place
278
immediately after addition of the acid. These observations show that the cell consumes less
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O2 when faced with weak organic acid stress.
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Glucose metabolism is affected by weak organic acid stress
282
Because weak organic acids partially dissipate the PMF, we speculated that B. subtilis might
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alter its metabolism
284
Glucose consumption was highest for non-stressed cells, reaching 7.8 mmol s-1 (mg protein)-
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1
. Addition of weak acids lowered glucose consumption rates, with 80 mM K-Ac resulting in
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1.45 mmol s-1 (mg protein)-1 (Figure 7 and supplementary table 3).
287
Acetate was produced under all conditions tested and already present in low amounts at the
288
start of the experiment. Non-stressed cultures produced acetate at the highest rate, together
289
with the K-sorbate exposed cells. Addition of K-sorbate lowered the synthesis rate of acetate.
290
Acetic acid-stressed cells had the lowest rate of acetate production. These cultures re-directed
13
291
fermentation routes by switching to 2,3-butanediol fermentation. Some 2,3-butanediol was
292
also found in the final sample of non-stressed cells, but not with sorbic acid-stressed cells.
293
294
Carbon flux
295
When confronted with a lack of electron acceptor, B. subtilis employs a mixed acid
296
fermentation, and has been shown to produce lactate, acetate, acetoin, ethanol, 2,3-butanediol
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and succinate as fermentation products when grown on glucose (31), but also produces a lot
298
of acetate as a result of overflow metabolism (32). In our experiments, only acetate and 2,3-
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butanediol were found.
300
Although batch-cultures are not ideally suited for accurate carbon flux determinations, our
301
data do seem to be in line with reported values for wild-type B. subtilis (33). With the
302
assumptions made previously regarding O2 utilization, our data (Supplementary table 3) for
303
WOA stressed cells show reasonably close C-balances for all stresses, apart from 25 mM K-
304
Ac, which appears to use about 3.5x as much O2 as estimated based on carbon fluxes based
305
solely on glycolysis and fermentation.
306
The calculated qATP based on qAcetate and q2,3-Butanediol (qATP = 2*qAcetate + 2*q2,3-
307
Butanediol) through glycolysis and fermentation displayed a similar WOA concentration
308
dependent behaviour as the qGlucose. That is, increasing concentrations of weak acid cause a
309
reduction in fluxes. This behaviour seems analogous to the effect on pHi, which depends
310
more on the concentration than the nature of the acid (as shown above). Indeed, there appears
311
to be a linear relation between pHi and qGlucose (Figure 8), as well as between pHi, and the
312
calculated amounts of ATP generated via production of qAcetate and q2,3-Butanediol (our
313
unpublished calculations). When looking at correlations between Δψ and qGlucose, such a
314
correlation does not exist for both WOA. Only for sorbic acid might a linear correlation be
315
observed, but more experiments will be needed to confirm this relationship. The relation
14
316
between qO2 and decrease in pHi seems rather constant, while a decreasing qO2 seems to
317
correlate with a decreasing ΔΨ. However, more experiments will be needed for an accurate
318
description.
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321
Discussion and conclusions
322
To reduce the growth rate to a similar level, more acetic acid is required than sorbic acid at an
323
identical external pH value. Both sorbic and acetic acid lower pHi and reduce the growth rate
324
of B. subtilis. When growing with WOA stress, the internal pH is lower than that of cells not
325
exposed to WOAs during the early exponential phase. Even though in the first minute after
326
WOA exposure some recovery takes place, the internal pH does not seem to recover fully of
327
this acidification, even after 6 h of growth. Given that the pKa for both weak acids is almost
328
equal, this makes sorbic acid a more effective preservative.
329
Homeostasis of the pHi is crucial for cell physiology. Optimal proton extrusion depends
330
clearly on the presence of an energy source (i.e. glucose) in the medium, be it that recovery of
331
the pHi does not reach the original values. It appears that a new ΔpH equilibrium is
332
established within a few minutes, i.e. before most transcriptomic changes take effect. The
333
level of cytosolic acidification after weak acid injection into the medium is very similar for
334
both acids without glucose present. This suggests that in starved cells, an equilibrium for acid
335
dissociation is established. Since the only factor measured in this regard is pH or [H+], the
336
anion accumulation level is unknown. It can, however, be estimated if it is assumed that
337
∆
338
reduce anion concentrations, but high enough to allow metabolism. As is clear from the data,
339
the ability to restore pHi differs between sorbic acid and acetic acid-treated cells.
. The cell has to establish a new equilibrium with a ΔpH low enough to
15
340
For sorbic acid, the inability to recover pHi during acid stress reflects the ability of sorbate to
341
act as a classic uncoupler, shuttling protons over the membrane whereas the less lipophilic
342
acetate is believed to do this to a lesser extent. The latter is corroborated by the greater effect
343
that sorbic acid has on the membrane potential, while acetic acid only carries bulk volume
344
protons across the membrane until a steady state is reached, allowing Δψ to remain relatively
345
unaffected. Even though some authors have pointed to the similar effects of the uncoupler
346
2,4-dinitrophenol and sorbic acid on growth and metabolism (10, 34, 35), to our knowledge
347
no quantitative measurements of the effect that sorbic acid has on Δψ have been reported thus
348
far. While relatively little research has been published on the effects that WOA preservatives
349
have on the membrane potential of microorganisms (7, 17, 30, 36, 37), the depletion of Δψ
350
may have a plethora of effects. The consequences may range from reduced transporter
351
activity (54) to destabilization of the bacterial cytoskeleton (55), although we have not seen
352
evidence for the latter effect (our unpublished results). Anion efflux from the cytosol would
353
be driven by the remaining Δψ. This effect was shown clearly for the permeant ion picrate,
354
which mainly acts as an uncoupler in everted cell membranes when a high Δψ was
355
established (38). It has also been observed that lipophilic compounds such as ethanol can
356
stimulate leakage of protons over the membrane of S. cerevisiae (39, 40). However, recent
357
results show this not to be the case for sorbic or acetic acid (6).
358
Other commonly used preservatives such as lactic, benzoic and propionic acid are also
359
expected to diffuse and dissociate in agreement with the Henderson – Hasselbalch equation
360
(9). Future experiments should establish to what extent these and other preservatives have
361
uncoupling properties, and what the quantitative effect of Δψ depletion is on growth rate. It is
362
likely that their ability to cross the membrane barrier (log KOW) into the aqueous phase will
363
play a role, as seems to be the case with sorbic and acetic acid (41, 42).
16
364
With a low pHo, the balance of the PMF is shifted towards ΔpH, so the effectiveness of weak
365
organic acids at low pH is two-fold: The concentration of undissociated acid is higher and
366
they act on the dominant component of the PMF. In the search for food preservatives that are
367
also active near neutral pH, the food industry should consider preferably uncoupler-like
368
molecules similar to or better than sorbate. At an even higher pH, permeable cations might be
369
useful to deplete the membrane potential. In food with low glucose concentrations, the
370
recovery of pHi might be less, hence less growth is observed with lower preservative
371
concentrations.
372
We observed a decrease in O2 consumption rate with WOA-stressed cells. This suggests that
373
there are fewer electrons to feed the electron transport chain and that metabolism may be
374
affected by acidification of the cytosol. Because weak organic acids partially dissipate the
375
PMF, the amount of ATP that the cells can generate through the F1F0-ATPase is considerably
376
smaller. So, to produce a sufficient amount of ATP to restore pH homeostasis and proliferate,
377
the cell might alter its glucose metabolism to generate ATP through substrate level
378
phosphorylation. Because increased energy needs have been observed in WOA stressed yeast
379
(43, 44) and a starvation-like response was reported for sorbic acid-stressed B. subtilis (2), we
380
measured the glucose consumption rate as well as the production of fermentation products.
381
Glucose metabolism slows down in the presence of WOA, depending on the concentration of
382
undissociated acid, as has also been described for E. coli and Saccharomyces cerevisiae (25,
383
45, 46). This results in a linear relation between pHi and qGlucose. The observation that
384
acetic acid continues to be produced when cells are exposed to sorbic acid (2 mmol h-1 (mg
385
protein)-1 with 11 mM K-sorbate), may be an extra stress factor for cells growing under these
386
conditions, and may be part of the explanation for the observed continued decrease in pHi
387
during sorbic acid stress. Addition of K-sorbate lowered the synthesis rate of acetate, possibly
388
due to growth reduction or a decrease in glycolytic activity
17
389
Glycolytic flux can be controlled, amongst other factors by pH (47), affecting
390
phosphofructokinase (Pfk) activity (48), or specifically in the case of Bacillus species,
391
phosphoglycerate mutase activity (49), which is also very sensitive to pH changes. Glucose is
392
generally assumed to be the preferred carbon and energy source for B. subtilis which is taken
393
up via the phosphoenolpyruvate-sugar phosphotransferase system (PTS). It is subsequently
394
converted into fructose 1,6-bisphosphate (FBP) by Pfk. FBP is required for phosphorylation
395
of CcpA, one of the main repressors in carbon catabolite repression (CCR) in B. subtilis. This
396
way, FBP availability causes CCR (50). Also, CCR inhibits expression of citric acid cycle-
397
enzymes when glucose concentrations are high. When a decrease of the internal pH reduces
398
the activity of Pfk, the concentration of FBP is reduced, CcpA (a global regulator (inhibitor)
399
of many carbon metabolism routes(51)) activity is reduced and CCR is released. This may
400
explain how in earlier studies (2) a starvation-like response could be observed in B. subtilis
401
upon sorbic acid stress.
402
With high concentrations of acetic acid, it is likely that fermentation routes towards acetate
403
are diverted to 2,3-butanediol. Synthesis of 2,3-butanediol depends on bdhA, encoding
404
acetoin reductase (31), a gene that has SigB-controlled expression. Acetic acid stress can
405
trigger stressosome activity and indeed results in up regulation of acetoin reductase
406
(Unpublished results by A. Ter Beek, (52)). Therefore, when acetate is present as a
407
preservative, putative organoleptic properties of 2,3-butanediol should be considered when B.
408
subtilis or related species may be present. Weak organic acid stress also causes an up
409
regulation of citric acid cycle enzymes (2, 52). This route may also consume added acetic
410
acid, thereby providing an extra carbon source and eliminating this weak organic acid. The
411
high oxygen consumption rate that we observed in the presence of 25 mM and 80 mM K-Ac,
412
may be explained by the utilization of acetate. While growth rates are reduced by 80% with
413
both 11 mM K-sorbate and 80 mM K-acetate, the glucose consumption rate with 80 mM K-
18
414
acetate is almost half of that with 11 mM K-sorbate. Future experiments should be conducted
415
in a turbidostat setup and include CO2 measurements to allow a direct measurement of the
416
carbon balance under these conditions. Therefore, knowledge of the pHi can be used to
417
predict glycolysis and this knowledge might be useful for modelling purposes in food
418
fermentation (47, 53).
419
In summary, oxidative phosphorylation is an important source of ATP for aerobic, non-
420
stressed B. subtilis cells. The O2 consumption rates follow a similar trend as qGlucose,
421
qAcetate and q2,3-Butanediol, apart from the 25 mM K-Ac stress. The level of WOA stress
422
may leave too little PMF to allow F1F0-ATPase to produce ATP. Sorbic acid has a similar
423
effect as acetic acid on pHi, but is more effective in depleting Δψ. This may partially explain
424
the observation that sorbic acid is a more potent preservative than acetic acid. In the quest for
425
preservatives active at near neutral pH, industry should best focus on ‘uncoupler-like’
426
molecules that act similar to or better than sorbic acid.
427
428
Acknowledgements
429
Jos Arents is thanked for his assistance with the membrane potential measurements. Gertien
430
Smits is acknowledged for critically reading initial versions of the manuscript.
431
432
433
434
435
436
437
438
19
439
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24
589
Lengends to the figures:
590
591
Figure 1. Growth curve (A) and internal pH (B) of B. subtilis PB2 pDG-IpHluorin monitored
592
for 6 h under various stress conditions. Medium pH = 6.4. In M3G, the growth rate is highest,
593
but a continuous decrease in pHi is seen. Sorbic acid stress causes the growth rate to be
594
reduced as well as a continuous decrease in internal pH. Acetic acid stress has a similar effect
595
on growth rate, but pHi remains constant.
596
597
Figure 2. Acidification and recovery upon weak organic acid addition to B. subtilis PB2
598
PptsG-IpHluorin. Sorbic acid (A and B) or acetic acid (C and D) is injected at t = 0 s. Medium
599
pH = 6.4. The internal pH drops to its lowest point within seconds (A and C) and recovers
600
fast when glucose is available (B and D). The pH recovers to higher values with acetic acid
601
stress. Data from typical examples is shown.
602
603
Figure 3. Acidification and recovery upon weak organic acid addition to B. subtilis PB2
604
PptsG-IpHluorin. Acetic or sorbic acid is injected at t = 10s. Medium pH = 6.4. The internal
605
pH drops to its lowest point within seconds and recovers fast when glucose is available. With
606
glucose available, the pHi at t = 0 is also higher. The pH recovers to higher values with acetic
607
acid stress. Data from a typical example is shown
608
609
Figure 4. Amplitude of acidification by addition of sorbic or acetic acid. A linear trend can
610
be observed between ΔpH and –log[HA] ([HA] in M). Data from experiments at an
611
extracellular pH=5.5 and 6.4 are combined. Error bars indicate standard deviation.
612
613
25
614
Figure 5. Differential effects of sorbic and acetic acid on the proton motive force. Sorbic acid
615
stress affects both ΔpH as well as Δψ. Acetic acid affects ΔpH, but has little effect on Δψ.
616
Data are from cultures exposed to weak acids for approximately 5 min. Results are based on
617
3 biological replicates. Error bars indicate standard deviation.
618
619
Figure 6. Weak organic acid stress reduces respiration. Respiration was monitored for 1 min
620
or the time it took to reduce O2 levels to 0 µmol. Results are based on 3 biological replicates.
621
Error bars indicate standard deviation.
622
623
Figure 7. Increased weak organic acid stress lowers glucose consumption rate. The rate of
624
glucose consumption compared to the rate of acetate and 2,3-butanediol production. Results
625
are based on two biological replicates. Error bars indicate standard deviation.
626
627
Figure 8. Glucose flux versus internal pH due to weak organic acid stress (no stress, 3 and 11
628
mM K-S, 25 and 80 mM K-Ac). A decrease in pHi due to WOA stress is accompanied by a
629
decrease in glucose flux.
630
26

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