E
ECOLOGY OF BACTERIA AND FUNGI IN FOODS
Contents
Effects of pH
Influence of Available Water
Influence of Redox Potential
Influence of Temperature
Effects of pH
E Coton, Université de Brest, Plouzané, France
I Leguerinel, Université de Brest, Quimper, France
Ó 2014 Elsevier Ltd. All rights reserved.
The Concept of pH and Its Relevance in Food Products
Also known as potential or power of hydrogen, pH, like other
factors (i.e., water activity and redox potential), is an intrinsic
and inherent characteristic of food and beverages. The concept
of pH was first introduced by the Danish chemist Søren
Sørensen in 1909. The pH value of a system is a direct function
of the free hydrogen ions (or protons) present in that system.
More precisely, the pH value corresponds to the negative log of
the hydrogen ion concentration (pH ¼ log [Hþ]). Note-
worthy, to represent the nature of the proton in an aqueous
solution, pH also is described as the negative log of the oxo-
nium ion (H3Oþ). Water protonation, however, can lead to
several other forms such as H5O2þ, H7O3þ, or H9O4þ. The
pH ¼ log [Hþ] definition corresponds to the fact that the pH
value of a given system decreases as the concentration of
hydrogen ions increases. For example, a system with a pH value
of 6 has a 106 (0.000001) mol l1 hydrogen ion concentra-
tion, while for a pH value of 4, the concentration of hydrogen
ions equals 104 (0.0001) mol l1. The pH scale ranges from
0 to 14 with pH 7 being neutral, based on the fact that pure
water at 25  C has a pH value of exactly 7. pH values under
7 are considered as acidic, and those above pH 7 are considered
basic or alkaline. The concentration of hydrogen ions in
a system is correlated to the nature and concentration of the
Encyclopedia of Food Microbiology, Volume 1 http://dx.doi.org/10.1016/B978-0-12-384730-0.00439-0
acids present. These acids can correspond to strong acids (i.e.,
hydrochloric acid), dissociating completely in water (the ioni-
zation of the compound leads to an equal molar amount of Hþ
and the negatively charged conjugate base), and weak acids
(i.e., lactic acid), which only partially dissociates, leading to an
equilibrium of both the dissociated and undissociated forms.
Hence, pH might be defined as the measure of acidity of
a product.
In food products and beverages, acids are either intrinsic,
originally present, or produced during food processing, such
as in the case of fermented products, or they can be added
during processing for product conservation (see the section
Use of pH as a Microbial Control Tool). The nature and the
concentration of the various compounds (especially acids)
found in a food will determine its pH. With a few exceptions
(i.e., egg white), food products are acidic (Table 1). Within
acidic foods, a pH value of 4.6 has been determined to
separate the high-acid and low-acid products. This pH, which
is based on the necessary value preventing Clostridium botu-
linum (responsible for botulism) from growing and
producing a deadly toxin, is of great importance in the food
industry. Indeed, a pH under this value is considered to
prevent growth of pathogenic bacteria. Yeasts and molds are
more acid tolerant than bacteria and hence can grow at lower
pH values.
577
578 ECOLOGY OF BACTERIA AND FUNGI IN FOODS j Effects of pH

Table 1 pH ranges of some common foods called a reference electrode; and the second one, called a glass
electrode, presents a variable potential correlated to the pH to
Dairy products be measured. Once immersed in the solution to be measured,
Butter 6.1–6.4
an electrochemical potential develops across the thin glass
Buttermilk 4.5
Cheese (American mild and cheddar) 4.9; 5.9 membrane proportional to the hydrogen ion concentrations
Cream 6.5 on the two surfaces. The difference of potential evolves pro-
Milk 6.3–6.5 portionally to the pH, according to the following formula:
Yogurt 3.8–4.2 DE ¼ a(pHa  pHb) þ b, where DE is the difference of poten-
Meat and poultry tial between the two electrodes, pHa is the pH of the system to
Beef (ground) 5.1–6.2 be measured, pHb is the pH of the reference solution, and
Chicken 6.2–6.4 a and b are intrinsic values of the apparatus determined
Ham 5.9–6.1 through the calibration. Indeed, to perform an accurate pH
Egg white 7.6–9.5
measurement, apparatus calibration using precalibrated
Veal 6.0
solutions (e.g., 4, 7, and 10) is required. Noteworthy, air
Egg yolks (white) 6.0–6.3
Fish and shellfish pressure and temperature both influence pH measurements;
Clams 6.5 therefore, food products should be tested at room tempera-
Crabs 7.0 ture, although most pH meters are equipped with a tempera-
Fish (most species) 6.6–6.8 ture sensor at the tip of a pH electrode to automatically correct
Oysters 4.8–6.3 the pH reading.
Salmon 6.1–6.3 As seen, the glass electrode is useful to measure pH in
Shrimp 6.8–7.0 solutions; however, food products exist in large variety of
Tuna fish 5.2–6.1 structures. For example, food products can vary from a very
White fish 5.5
homogenous (i.e., wine) to a more heterogeneous product (i.e.,
Fruits and vegetables
an egg is constituted of two parts exhibiting different specific-
Apple cider 3.6–3.8
Apples 2.9–3.3
ities and pHs), and they can be frozen or desiccated. To
Asparagus (buds and stalks) 5.7–6.1 measure the pH of foods, the food should be in liquid form or
Bananas 4.5–4.7 prepared as a puree in a blender. For homogeneous foods
Beans (string and lima) 4.6–6.5 (beverages, salad dressing), no special preparation is required
Beets (sugar) 4.2–4.4 as any portion is representative of the whole. For semisolid
Broccoli 6.5 foods, the addition of pure water (20%), which has no influ-
Brussels sprouts 6.3 ence on the measured pH, is generally performed for blending
Cabbage (green) 5.4–6.0 the samples. In the case of oily foods, the oil layer is removed
Carrots 4.9–5.2; 6.0
by decanting, skimming, or pouring to measure the non-oil
Cauliflower 5.6
phase. Cooling, freezing, and thawing also may be used to
Celery 5.7–6.0
Corn (sweet) 7.3
allow oil separation.
Cucumbers 3.8 To facilitate pH measurements in the agrifood context,
Grapefruit (juice) 3.0 different probes have been designed. Examples of these appa-
Grapes 3.4–4.5 ratus include puncture probes for semisolid food (cheese and
Honeydew melons 6.3–6.7 meat) testing, a flat membrane combination pH electrodes for
Lettuce 6.0 surface measurements, and knife probes for pH analysis in
Limes 1.8–2.0 frozen meat.
Olives (green) 3.6–3.8 Beyond the actual food structure, people are consuming
Onions (red) 5.3–5.8 more and more ready-to-eat (RTE) preparations (i.e., sand-
Oranges (juice) 3.6–4.3
wiches, RTE meals) that consist of various food products
Parsley 5.7–6.0
Parsnip 5.3
(ingredients) each exhibiting a specific pH. In this context,
Plums 2.8–4.6 microorganisms present will not have the same behavior in the
Potatoes (tubers and sweet) 5.3–5.6 various parts of the food preparation. Therefore, it is crucial to
Pumpkin 4.8–5.2 establish the pH of each ingredient. This is especially important
Rhubarb 3.1–3.4 when one wants to evaluate the growth potential of a food-
Spinach 5.5–6.0 borne pathogen. Microbiological food challenge-testing corre-
Squash 5.0–5.4 sponds to the inoculation of a pathogen in a food preparation
Tomatoes (whole) 4.2–4.3 to evaluate its ability to grow and therefore determines the risk
Turnips 5.2–5.5
for the consumer. These tests are always performed in the
Watermelons 5.2–5.6
fraction of a food preparation, presenting the least strict
intrinsic conditions, especially pH.
Finally, although the pH of the final food product usually
The pH of food usually is determined using a pH meter is stable, pH may vary considerably during production. In this
consisting of a probe presenting a thin-walled glass bulb at its context, microorganisms have to cope with the encountered
tip and a numerical converter. The probe is composed of two pH dynamics. For example, in beef meat, the animal flesh
electrodes: The first one, which displays a constant potential, is prior to slaughter exhibits a pH value close to 7.0. After
 ECOLOGY OF BACTERIA AND FUNGI IN FOODS j Effects of pH 579

slaughtering, the glycogen in the meat turns into lactic acid. As situated between 6.7 and 7.0; neutralophilic bacteria have pHi
a result, the postmortem pH declines, reaching a pH value values of 7.5–8.0, and alkaliphiles exhibit pH values of 8.4–
around 5.5, 24 h after slaughter. The pH then rises again to 9.0. In eukaryotes, due to cell compartmentalization, several
reach 6.5. Another example is fermented foods (wine, cheese, pHs can be observed according to the considered organelle.
olives, and sauerkraut) in which the technological microbiota While the vacuole is acidic (4.8–5.4), mitochondria are
lowers the pH, hence protecting the final product. Actually, alkaline. Yeast and mold cytosols display a circumneutral pHi.
this is one of the first conservation methods discovered by In both prokaryotes and eukaryotes, the cytoplasm is the siege
humans. of the majority of metabolic reactions and hence the need for
fine-tuning the pH conditions for optimal cell function
(enzyme activity, reaction rates, protein stability, nucleic acid
Effect of pH on Food Microorganisms structure). The process allowing for pHi stability is called pH
homeostasis.
The pH in a given environment has a profound effect on the
physiological state of all microorganisms (growth and survival,
and, in some cases, sporulation and germination). Thus, in pH Homeostasis
foods, this directly affects food product conservation and
safety. According to their taxonomical position, microorgan- To be able to react to pH variations, microorganisms must
isms exhibit different pH ranges and optima for growth sense external as well as internal changes to initiate mecha-
(Table 2). In regard to prokaryotes in a food context, Gram- nisms that will correct the pHi. In both prokaryotic and
negative bacteria grow between pH 4.0 and 8.5 and Gram- eukaryotic organisms, passive and active homeostasis mecha-
positive bacteria grow between 4.5 and 9.0. According to these nisms exist. If some of these mechanisms are specific to
parameters, bacteria can be classified into three categories particular species or groups, common mechanisms also are
according to their adaptation to more acidic, neutral, or alka- exerted. This section will focus only on these aspects.
line environments. They are named acidophiles, neu- Concerning bacteria, these microorganisms possess trans-
tralophiles, and alkaliphiles, respectively. In food, the main membrane proton pumps expelling in an unregulated manner
bacteria encountered, especially spoilage and pathogenic Hþ ions from the cytoplasm. Although natural diffusion caused
bacteria are neutralophiles; however, in fermented products, by a concentration gradient would lead to the reentry of the
acidophiles are also found. For eukaryotes, the pH ranges for protons and the electrostatic force would cause the Hþ to
growth are much larger, between 2.5 and 8.5 for yeast and diffuse down the electrical potential, the low permeability to
between 1.5 and 9.5 for filamentous fungi (molds). ions of the bacterial bilipidic membrane counteracts these
To survive in a specific environment, microorganisms have effects. Hence, a transmembrane proton gradient is established
to be able to maintain their internal pH (pHi) in a relatively and associated with an electrochemical gradient. This gradient
narrow range. The pHi of acidophiles has been shown to be is constituted with a chemical gradient of protons (DpH;
interior alkaline) and a transmembrane electrical component
(DJ; interior negative). This electrochemical gradient provides
Table 2 Examples of cardinal pH values for different the driving force for the production of adenosine triphosphate
microorganisms (ATP) through the entry of Hþ via membrane bound ATPases
described as a proton-motive force (PMF).
Microorganism Minimum Optimum Maximum The low membrane permeability toward protons is actually
Prokaryotes a passive mechanism avoiding a passive influx of protons in the
Bacillus cereus 4.9 6.0–7.0 8.8 case of a pH decrease. A second passive mechanism corre-
Campylobacter spp. 4.9 6.5–7.5 9 sponds to the nature of the cytoplasm. Indeed, the cytoplasm is
Clostridium botulinum 4.6 8.5 composed of various molecules, including both organic
Clostridium perfringens 5.5–5.8 7.2 8.0–9.0 (amino acids, protein with ionizable groups, polyamines) and
Enterohemorrhagic Escherichia coli 4.4 6.0–7.0 9 inorganic molecules (polyphosphate, inorganic phosphate),
Lactobacillus spp. 3.8 5.5–6.5 8.0 which provide buffering capacities. The term buffer corre-
Listeria monocytogenes 4.4 7.0 9.4 sponds to the ability of a solution to maintain its pH in the case
Salmonella spp. 4.2 7.0–7.5 9.5
of small additions of acid or base and also in the case of
Shigella spp. 4.9 9.3
Staphylococcus aureus 4.0 6.0–7.0 10.0
dilution. According to the species considered, buffering
Vibrio parahaemolyticus 4.8 7.8–8.6 11.0 capacity (b) ranges typically from 50 to 200 mM protons per
Vibrio vulnificus 5.0 7.8 10.2 pH unit shift.
Yersinia enterocolitica 4.2 7.2 9.6 Beyond low membrane permeability toward protons and
Eukaryotes the cytoplasm buffering capacity, active homeostasis mecha-
Aspergillus flavus 2.1 7.5 11.2 nisms are established by the bacterial cell to cope with external
Byssochlamys fulva 2.0 3.0 9.0 pH changes during acid shock. The bacterium Escherichia coli
Debaryomyces hansenii 2.0 6.0 10.0 was studied extensively due to its ease of grow and manipu-
Geotrichum candidum 3.0 5.0–5.5 11.0 lation. In the food context, this bacterium is used as a hygiene
Penicillium crustosum 2.2 4.5 9.0–10.0
marker (fecal contamination indicator) in daily routine food
Penicillium roqueforti 3.0 6.0 10.0
Saccharomyces cerevisiae 1.6–2.0 4.0 8.6
analyses and therefore will be used as the main example
throughout this section (Figure 1).
580 ECOLOGY OF BACTERIA AND FUNGI IN FOODS j Effects of pH
Figure 1 Mechanisms involved in pH homeostasis in E. coli.
The membrane is the first cell component in contact with
the external environment to detect various changes. During
an acid shock, the production of cyclopropane fatty acids
(CFAs), which usually is associated with late exponential and
early stationary growth phases of E. coli, is observed. The CFA
synthase gene (cfa) is upregulated by low pH (change of
external pH) and by acetate, a weak acid encountered in
foods (change of internal pH). The same type of regulation
was observed in the food pathogen Salmonella enterica and
various lactic acid bacteria. Moreover, E. coli cells with
a mutated cfa gene lose the ability to survive an acid shift
from neutral pH to pH 3. During acid shock, CFA synthesis is
regulated by the s38 sigma factor; an rpoS gene product also
implicated in the regulation of other acid-related cell
responses (i.e., gad regulon). CFAs have been shown to
reduce membrane permeability to Hþ and to enhance the
ability to extrude protons. In the food pathogen Listeria
monocytogenes, when confronted with a low pH, a decrease in
the ratio of branched chain and saturated straight fatty acids
of total lipids and the total lipid phosphorus has been
observed. Membrane-bound proteins are also regulated
during acid shock and contribute to acid tolerance response
(ATR). The most studied correspond to the ompF and ompC
porins. Porin proteins are channels controlling the perme-
ability of low-molecular-weight hydrophilic polar solutes
across the outer membrane. The porin encoding genes (ompF
and ompC) are under the control of a two-component regu-
latory system EnvZ/OmpR. The ompC gene expression
increases in E. coli (high levels of OmpR-P) cells grown in
acidic pH, whereas the expression of ompF (low levels of
OmpR-P) is higher in alkaline conditions.
An essential level of response in pH homeostasis corre-
sponds to the direct active efflux or influx of protons through
various dedicated systems. Primary proton pumps are the first
of these systems. Under acid stress, E. coli (respiratory neu-
tralophile) modulates the efflux and influx of protons by
upregulating the expression of the respiratory chain complexes
extruding protons out of the cell and by downregulating the
expression of the ATP synthase associated with proton entry for
ATP production. In non-respiratory neutralophiles (i.e., Strep-
tococcus mutans, Enterococcus hirae), in acidic conditions, F1F0-
ATPase expression and activity are increased to promote
ATP-dependent Hþ efflux. The second type of system corre-
sponds to inorganic ion transporters corresponding mainly to
cation/proton (Naþ/Hþ and Kþ/Hþ) and anion/proton (Cl/
Hþ) antiporters. The cation/proton antiporters show a central
role in non-respiratory and respiratory neutralophiles at alka-
line pH, whereas anion/proton antiporters are associated with
acidic conditions. The Naþ/Hþ antiporter NhaA has been
studied extensively and is essential for homeostasis in E. coli in
an alkaline environment. NhaA has been shown to be inactive
at pH 6.5 and active at pH 8.5. This is an efficient system with
high transport capacity exchanging two Hþ ions for every Naþ
exiting the cell. In alkaline conditions, F1F0-ATPase contributes
to pHi acidification through its influx of Hþ (ATP synthesis).
Although homeostasis also concerns growth at alkaline pH, in
food, this is less relevant due to the rather acidic nature of food
products as shown in Table 1.
 ECOLOGY OF BACTERIA AND FUNGI IN FOODS j Effects of pH
Another major pH homeostasis mechanism relies on meta-
bolic enzymes producing or consuming protons. These enzymes
correspond to different families: decarboxylases, deaminases,
and hydrogenases. Amino-acid decarboxylases and deaminases
as well as their associated transporters play a central role in pH
homeostasis in an amino acid rich environment like food
products. In acidic conditions, decarboxylases (lysine, glutamate,
arginine, serine decarboxylases) offer a pHi regulation mecha-
nism through the consumption of a proton (COOH function),
associated with the diffusion of CO2, while producing a biogenic
amine (alkaline product) that is either maintained in the cyto-
plasm or exported to allow for the entry of the decarboxylase
precursor. For example, in E. coli, genes associated with the
decarboxylation of lysine (cadA) and the export of the resulting
metabolite cadaverine (cadB) are upregulated at low pH. In this
context, after sensing the pH via an extracellular domain, the
associated two-component regulator (CadC) induces the
expression of the cadA and cadB. As part of an adaptive response
to acidic conditions, CadC also reduces the production of the
OmpC and OmpF porins, hence modulating the outer
membrane permeability. The same observations were made in
the pathogen S. enterica Typhimurium. Biogenic amines–
producing pathways have been extensively studied in food-
related lactic acid bacteria, in which they mainly are carried by
mobile elements (plasmids, genomic islands) transferred by
horizontal gene transfer events and may contribute to acid pH
resistance as well as to the production of energy through a PMF.
Under low pH (pH 2–2.5) conditions, the glutamate-dependent
Gad system, under the control of RpoS (s38), enables E. coli to
survive for several hours. In this context, glutamate decarbox-
ylase (GadB; activated by chloride ions) consumes protons
through the production of g-aminoglutarate (GABA); the
conversion of cytoplasmic glutamate (net charge 1) to GABA
(net charge 0) might contribute to the reverse DJ observed in
these conditions, thus preventing proton leakage into the cells.
Then, the exchange of GABA for more glutamate via the GadC
antiporter allows for decarboxylation to continue. In the same
pH conditions and anaerobiosis, the E. coli enzyme hydroge-
nase-3, responsible for H2 (hydrogen gas) production from
cytoplasmic Hþ, is also upregulated. While decarboxylases
generate an alkalinizing adaptive response to an acid challenge,
deaminases (i.e., TnaA-tryptophan deaminase and TnaB-trypto-
phan transporter) associated with organic acid production are
upregulated in alkaline conditions. In other species, other
enzymes (i.e., urease for Helicobacter pylori) are used for pHi
maintenance. Signaling and acid tolerance gene regulation is
performed by a complex cascade involving various signaling
proteins and activators (i.e., EvgS/EvgA, SafA, PhoQ/PhoP, IraM,
RpoS, gadE) and enforcing the acid-resistant phenotype.
Interestingly, in acidic conditions, bacteria establish mech-
anisms not only to tolerate this stress but also to flee from it
through modulation of their motility. In food, this might be of
importance according to the homogeneous or heterogeneous
nature of the product.
Concerning fungi, like other eukaryotic cells, their pHi is
only slightly sensitive to large changes of external pH, which is
in agreement with the fact that they can grow in a much wider
pH range than bacteria. As stated, the cellular organization in
these microorganisms is different than bacteria; cell compart-
mentalization is associated with different organelles
581
(cytoplasm, mitochondria, vacuole, nucleus), each exhibiting
a specific pH critical for the organelle roles in the cell. In pH
homeostasis, fungi have developed several cell processes
allowing for fine pHi control. The best studied microorganisms,
Aspergillus nidulans and Saccharomyces cerevisiae (microbial food
culture or spoilage microorganism in food; Figure 2) will be
used as examples in this chapter.
In unicellular (yeast) and pluricellular (molds) fungi,
common mechanisms have been observed. When grown on
glucose, fungal metabolism leads to the large production of
organic acids and CO2, which are the main source of intracel-
lular protons. In this context or when confronted with acidic
conditions, fungal cells rely on the activity of various proton
transporters to maintain the various pH gradients in the cells. A
key component of pH homeostasis machinery in fungi is
a plasma membrane bound and ATP-dependent proton pump
(named Pam1p in S. cerevisiae), expelling protons out of the
cells at the expense of energy (ATP). This proton pump, as
observed in bacteria, is responsible for the formation of a PMF.
Another important primary proton pump in pH homeostasis
corresponds to the vacuolar ATPase (V-ATPase), which trans-
locates cytosolic Hþ to the vacuole, hence leading to its acidi-
fication via the consumption of ATP. Pma1p functions in
tandem with V-ATPases to control the cytosolic pH. Alkaline
cation (Naþ and Kþ)/proton exchangers also play a role,
although considered secondary, in pHi maintenance. The
plasma membrane Naþ/Hþ antiporter (nha1 gene) in yeast is
responsible for cell growth on high concentrations of KCl and
NaCl in acidic external pH values, while in alkaline conditions,
the Ena1 ATPase is active. If Nha1 is associated with pH
homeostasis, however, it is not regulated by pH change
(constitutive expression).
In eukaryotic cells, the cell wall is the first part of the cell in
contact with the external pH; it has been shown that various
signaling systems actually are based on monitoring cell wall
integrity. For example, in S. cerevisiae, the Mid2p cell wall
sensor is mainly responsible for the activation of the protein
kinase C (PKC) pathway, which mediates tolerance to acidifi-
cation of the extracellular environment via the Bck1 and Slt2
proteins (members of the mitogen-activated protein kinase
(MAPK) cascade). Using the same cascade, the Wsc1 cell wall
surface sensor signals alkaline pH stress in S. cerevisiae. At low
pH, S. cerevisiae shows modification of its cell wall, inducing
resistance to b 1,3-glucanase through alkali-sensitive linkage of
cell wall proteins to b 1,3-glucan (hog1 gene-dependent). In
acidic conditions, the human opportunistic fungal pathogen,
Candida glabrata, modulates the structure of its cell wall by
reducing total b-glucan levels under the effect of the CgYps1
regulator.
Concerning the regulation of fungal pH homeostasis, the
most extensively studied regulatory systems correspond to
the pal (A. nidulans) and rim (S. cerevisiae) signaling pathways.
The effector (PacC/Rim101) corresponds to a three Cys2His2
zinc finger transcription factor (DNA binding protein), which
upon pH-dependent cleavage, regulates gene expression in
neutral and alkaline conditions. Under acidic conditions, PacC
is held in a closed tridimensional form, making it inaccessible
to protease action. In these conditions, a number of genes (i.e.,
pacA and gabA encoding an acid phosphatase and a GABA
permease, respectively) are expressed. On the contrary, in
582 ECOLOGY OF BACTERIA AND FUNGI IN FOODS j Effects of pH
Figure 2 Mechanisms involved in pH homeostasis in S. cerevisiae.
neutral and alkaline conditions, a complex pathway is acti-
vated. First, a plasma membrane complex constituted of two
plasma membrane sensors (palH/Rim21 and PalI/Rim9) will
transmit the environmental alkaline pH signal by triggering the
ubiquitination of the palF/Rim8 arrestin. Endocytosis is then
observed and allows for the formation of an endosome
membrane complex consisting of the PalC/YGR122W, PalA/
Rim20, and the PalB/Rim13 proteins. In this context, the PacC/
Rim101 effector bound to PalA/Rim20 will be cleaved by the
PalB/Rim13 cysteine protease to release an open conformation
truncated Pac protein. Unlike Rim101, the truncated PAC will
undergo a second pH-independent proteolysis for full activation
by a processing protease (proteasome). The active form of the
PacC/Rim101 effector is then transported to the nucleus where it
represses the expression of acid-expressed genes (i.e., pacA and
gabA) and activates the expression of alkaline-expressed genes
(i.e., palD and prtA encoding an alkaline phosphatase and
alkaline protease, respectively). In S. cerevisiae, in addition to the
rim pathway, it has been shown that exposure to alkaline pH
initiates a strong calcium influx, resulting in the activation of
the calcium-activated phosphatase calcineurin and of the calci-
neurin-regulated transcription factor Crz1/Tcn1. As stated, due
to the acidic nature of food products, these alkaline adaptive
pathways are not as relevant. During pH adaptation, genes
regulated by ambient pH also include those encoding intra-
cellular enzymes participating in the syntheses of exported
products (i.e., antibiotic, mycotoxins), as well as secreted
enzymes and permeases.
Effect of pH According to the Physiological State
Bacteria and fungi exhibit different physiological states during
their life cycle. Indeed, they can be in lag phase, exponential
growth, or a stationary state, and some can exhibit resistance or
dissemination forms (bacterial spores and fungal spores, respec-
tively) and when adequate conditions are encountered can
germinate. In combination with other food environmental or
intrinsic parameters (temperature, O2 content, water activity -aw-,
composition), pH will influence this physiological state. In foods,
these parameters are also used as a technological lever to ensure
food safety and quality (i.e., pasteurization and sterilization,
drying, modified atmosphere packaging, and salting). For pH,
this will be done through acidification using permeant weak acid
that will not only decrease pH but also interfere with the micro-
bial metabolism.
Concerning bacterial growth, while bacteria can survive at
low pH, the pH range in which bacteria are able to grow is
much narrower. For example, while E. coli can survive for
several hours at low pH (2–2.5) using some of the mecha-
nisms presented above (Gad system and hydrogenase 3), the
lowest pH allowing growth is situated around 4.0. Indeed,
although pH homeostasis occurs, neutralophilic bacterial
growth rate is affected when pH and pHi decrease. For
example, E. coli exhibits 50% growth reduction at pHi 7.2
and almost complete inhibition at pHi 6.6. Some studies on
E. coli showed a linear relation between growth rate and
hydrogen ion concentration (E. coli being unable to grow at
 ECOLOGY OF BACTERIA AND FUNGI IN FOODS j Effects of pH
pH 3.7) and growth rate and undissociated lactic acid
concentration (E. coli being unable to grow above 8.32 mM
undissociated lactic acid). The same effect has been observed
on pathogenic food bacteria. For example, the maximal
growth rate h1 (mmax) of Salmonella Enteritidis reduces to
0.99 in a medium at pH 4.35 compared with 1.20 at pH 7.1.
Listeria monocytogenes growth rates were also decreased with
a linear correlation to undissociated acetic acid and sodium
benzoate. In this bacterium, survival at pH 2.5 was increased
by six orders of magnitude upon entry into a stationary
phase compared with the growth phase, underlining the fact
that the physiological state of cells also influences the
adaptive response to pH.
For fungi, if temperature and aw are recognized as the
most important parameters for determining fungal growth,
pH also influences fungal development. For S. cerevisiae,
while the maximum specific growth rate is observed at pH 4,
modification of the environment below or above this value
will reduce both the growth rate and the maximal pop-
ulation reached. As observed with bacteria, under low
external pH conditions, a relationship between pHi and cell
proliferation activity was shown in both the brewer’s and
baker’s yeasts. For Zygosaccharomyces rouxii, an osmophilic
bakery product spoilage yeast, optimal growth is observed in
the range of pH 3.5 to 5. A reduction in pH to pH 2.5
induced a 30% decrease in growth rate. Filamentous fungi
(molds) are less affected by environmental pH values than
bacteria. For example, Penicillium roqueforti, a dairy product
contaminant, shows a large tolerance to several pH values
tested from 4.5 to 7.5. For Penicillium glabrum, radial growth
rate is almost constant in the pH range 2.0–7.0 (optimal pH
5.0), but growth rate is affected at pH 1 (50% reduction) and
even more at alkaline pH values (90% reduction at pH 11).
The minimal pH conditions for growth seemed to be
between 0.5 and 1.0. The higher sensitivity of P. glabrum
toward alkaline conditions compared with acidic ones has
also been observed for various Penicillium species: Penicillium
citreonigrum and Penicillium jensenii. The latter was not shown
to be sensitive to pH ranges from 3.5 to 7.1; however, an
important decrease in fungal growth is observed at pH values
below pH 3.3.
The external pH value influences not only fungal growth
rate but also metabolism. For example, in the yeast S. cerevisiae,
alcoholic fermentation is affected by pH, and in molds,
mycotoxinogenesis is also affected. Aspergillus flavus isolates
produces more aflatoxins when the external pH becomes
increasingly acidic. In the case of the cereal pathogen Fusarium
graminearum, trichothecene production is induced only under
acidic pH conditions.
Food-related microorganisms are not always in a growth
phase as some microorganisms may produce resistance forms
(sporulated bacteria, such as Bacillus spp. and Clostridium spp.)
or dissemination forms (fungal spores). In these conditions,
pH can also affect the various physiological states (i.e., sporu-
lation and germination).
Concerning bacteria, spore sporulation, and germination
are highly affected by the decrease of external pH. For instance,
some authors have observed that bacterial sporulation was
limited to environmental conditions allowing bacterial growth.
For example, the impact of the pH factor was quantified in
583
Geobacillus stearothermophilus, Bacillus licheniformis, and Bacillus
weihenstephanensis. Sporulation rates and spore populations are
maximal in optimal growth conditions. Moreover, bacterial
spores formed at optimal growth conditions show the highest
heat resistance. Environmental pH influences bacterial spores’
germination and presents an optimal pH value directly affected
by incubation temperature changes. The magnitude of the pH
effect varies among species and strains. For example, among 12
different Bacillus cereus strains, minimum germination pH varied
from pH <3.8 to 5, according to strain, and was affected by the
type of molecule triggering germination. The nature of the acidic
compound present, however, does not have a significant effect
on germination rate. For C. botulinum, germination rate also is
affected by external pH but at different levels according to the
bacterial strain. For instance, for C. botulinum reference strains
62A and 12885A, more than 90% of spores germinated at pH
6.5–7 while only 5–10% of spores were able to germinate at pH
5.4–5.7. For C. botulinum strain 53B, however, no clear inhibi-
tion appears at lower pH values.
In filamentous fungi, the optimal pH for sporulation
usually is close to the optimal growth pH. For Didymella bryo-
niae (watermelon pathogen), pH 4–6 stimulated growth, with
the highest sporulation observed at pH 6, while pH in the range
5–6 stimulated conidial germination. In Rhizopus stolonifer and
Gilbertella persicaria (stone and pome fruit pathogens), spor-
angiospore germination as well as mycelial growth was optimal
at pH 3–10, while complete inhibition was observed at pH 2.5.
For Penicillium expasum, a fungi causing decay in various plants
(i.e., apples), spore cultures in media with pH values at 2, 5,
and 8 showed that spore germination was inhibited at pH
2 and 8.
A last effect of pH on microorganisms concerns the selective
aspect that is of particular interest in fermented foods. As
noted, microorganisms are adapted to specific pH. Variation of
pH in combination with other parameters (nutrients, ethanol,
decrease of inhibiting molecules) tends to favor adapted
microorganism. For example, in black olive fermentations in
brine, the pH decreases quickly between day 0 and day 15
(from pH 6.7 to 4.9) and then decreases slowly to reach 4.5 at
day 180. In wine, after the alcoholic fermentation, the pH will
increase during the malolactic fermentation carried out by
lactic acid bacteria. These changes in composition and the
characteristics of the food matrix will exert a selective effect on
the microbial communities, thereby explaining the sequential
emergence of specific microbial groups or species in these
products.
Use of pH as a Microbial Control Tool
Permeant acids (including fermentation acids and weak acids
used as preservatives) can drastically affect the pH homeostasis
of a system, leading to growth retardation and even cell death.
This ability is directly used in the food industry (use of weak
acids) to optimize food conservation and maintain not only
food product safety (action against pathogens) but also
organoleptic qualities (action against spoilage microorgan-
isms). Some of the oldest conservation methods (i.e., fermen-
tation and pickling), although based at the time on empirical
observations, rely on this ability.
584 ECOLOGY OF BACTERIA AND FUNGI IN FOODS j Effects of pH
Organic acids are present in a wide variety of foods derived
from plants or animals, fresh, frozen, or canned. The inhibi-
tory effect of organic acids is due to the decrease in pH in
relation to their concentrations and to specific molecular
inhibitory effects. Traditionally, it is recognized that undisso-
ciated forms of organic acids can easily cross the cell
membrane and enter into the cell cytoplasm, where they
dissociate into anions and protons, hence reducing the
bacterial pHi. The decrease in medium pH by organic acid
addition increases the concentrations and proportion of the
non-dissociated form, which has a bactericidal effect. Anti-
bacterial mechanisms are not yet fully understood. To main-
tain the pH in the cytoplasm near neutrality, bacterial export
of excess protons outside the cell requires energy (cellular
ATP) and may result in the depletion of cellular energy.
Bactericidal concentrations of organic acids may be due to the
combination of dissipation and the inability to maintain pHi
followed by the denaturation of acid-sensitive proteins and
DNA. Moreover, organic acids can interfere with cytoplasmic
membrane and protein structures in the electron transport
chain, which disrupts ATP production and PMF. Acid anion
accumulation in the cell increases osmotic pressure inside the
cell and induces feedback inhibition effects toward important
metabolic pathways. These effects also concern organic acid
salt concentrations present in the medium.
Organic acids traditionally are used in a wide variety of
foods and commonly are used as preservatives to efficiently
limit microbial development. Their nature and use varies
depending on the type of food, targeted pathogenic, or spoilage
microorganisms as well as legislation. Weak acids are applied
widely to decontaminate meat carcasses. Aqueous solutions of
1.5–2.5% lactic acid or acetic acid are sprayed on beef carcasses
at all stages of the cutting process. The same decontamination
methods are used in poultry slaughterhouses. For packaged
meats, stored under refrigeration or in cold-storage conditions,
soaking solutions containing 2.5–5% of either lactic acid, acetic
acid, acetate salts, or sodium sorbate can be used against
L. monocytogenes. Lactic acid is used for meat product conser-
vation, such as sausages, ham, or dry meats. Lactic acid may
also be added as a salt or is produced by lactic acid bacteria
during fermentation and mainly presents a bacteriostatic effect.
On seafood products, organic acids can be applied by
dipping or spraying foods to limit microbial growth and
increase shelf life during cold storage. The effects are highly
variable, however, and concentrations are limited to avoid any
negative effects in regard to product quality (i.e., organoleptic
characteristics).
Lactic, citric, acetic, malic, and tartaric acids were shown to
be strong antimicrobial agents on fresh fruits and vegetables.
For example, citric and ascorbic acids were used to reduce the
microbial load of salads. Organic acids are used in combina-
tion with other methods (i.e., modified atmosphere packaging)
to limit bacterial growth.
Fruit juices present low pH values and naturally contain
high concentrations of organic acid, such as malic acid in apple
juice or citric acid and ascorbic acid in citrus fruits. In these fruit
juices, benzoic acid often is added to increase their conserva-
tion and limit yeast and mold growth that are able to grow at
low pH values.
Fermented milks, dairy products, and most cheeses present
low pH values due to lactic acid or propionic acid production
by bacteria, hence limiting pathogenic flora development in
cheese or yogurt.
In canned foods, pH is a criterion that guides the intensity
of the heat treatment and sterilization value (F0) to be
applied. The heat treatments are different between foods
having a pH greater than or less than 4.6. This critical pH value
corresponds to the growth limit for C. botulinum. For acidic
foods with a pH lower than 4.6, low heat treatments can be
applied. Concerning canning of low acid foods with a pH
above 4.6, F0 values higher than 3 are recommended or
mandatory, according to state legislation. A decrease in pH
allows microbial loads to greatly reduce, especially bacterial
spores that are otherwise heat resistant during heat treat-
ments. For example, for Bacillus cereus ADQP407 spores iso-
lated from shrimp, a decrease in pH from 7 to 5.4 led to
a 10-fold reduction in their heat resistance or decimal
reduction time. The pH decrease in canned foods usually is
carried out by the addition of organic acids. This method not
only allows reducing heat treatments (economic impact) but
also maintains organoleptic and nutritional food qualities
while ensuring complete inactivation of pathogenic and
spoilage microflora. In canned vegetables, citric acid or acetic
acids usually are added.
Modeling of pH and Weak Acids Incidence
on Microbial Growth
Predictive microbiology is a discipline that was developed in
the 1980s. It aims to predict the evolution of populations of
microorganisms using mathematical models. During the past
decades, many approaches and mathematical models have
been developed to describe and quantify the effect of the main
environmental factors on the growth capacity or the survival of
pathogenic or food spoiler microorganisms. These mathemat-
ical tools are currently used to determine food shelf life or to
optimize food formulations. This section focuses on models
involving the pH and acid concentration effects on microbial
growth.
In the literature, many polynomial models describing the
effect of pH on bacterial growth have been proposed. The
models, however, lack robustness and provide poor predictive
quality. In 1992, a modular approach was proposed in which
the effect of each environmental factor was described inde-
pendently by simple functions. These functions then are
combined into a global model taking into account each of the
considered factors. This approach to predictive microbiology,
named gamma concept (eqns [1] and [2]), predicts the
maximum growth rate of microorganism as a function of
environmental conditions (temperature, pH, acid concentra-
tion, water activity) and the optimal growth rate (mopt)
obtained in optimal conditions in a given matrix for the
studied microbial species or strain.
mmax ¼ g$mopt [1]
Gamma function describes and quantifies the relative
inhibitory effect of the studied factors on mopt.
 ECOLOGY OF BACTERIA AND FUNGI IN FOODS j Effects of pH 585

1.2 0.9
0.8
1.0
0.7
0.8 0.6

µmax (h–1)
µmax (h–1)

0.5
0.6
0.4
0.4 0.3
0.2
0.2
0.1
0.0 0.0
4 5 6 7 8 9 10 11 0 10 20 30 40 50
pH Acetic acid (mM)
Figure 3 Effect of pH on the growth rate of L. monocytogenes. Figure 4 Effect of acid concentration at pH 5.1 (-) and pH 5.4 (:)
Comparison between fitted model (eqn [3]) and experimental data. on the growth rate of L. monocytogenes. Comparison between fitted model
Adapted from Le Marc, Y., Huchet, V., Bourgeois, C.M., Guyonnet, J.P., (eqn 4) and experimental data. Adapted from Coroller, L., Guerrot, V.,
Mafart, P., Thuault, D., 2002. Modelling the growth kinetics of Listeria as Huchet, V., Le Marc, Y., Mafart, P., Sohier, D., Thuault, D., 2005.
a function of temperature, pH and organic acid concentration. Interna- Modelling the influence of single acid and mixture on bacterial growth.
tional Journal of Food Microbiology 73 (2–3), 219–237. International Journal of Food Microbiology 100 (1–3), 167–178.

Mathematical gamma functions have been developed to inhibitory effect of the undissociated forms of the organic
model the effect of each environmental factor temperature, pH, acids used.
water activities, or acid concentrations on microbial growth
!a
rate. ½AH
g½AH ¼ 1  [4]
g ¼ gðTÞ$gðpHÞ$gðaw Þ$gð½AHÞ [2] MICU

For each factor, g function equals 0 when no growth is

observed and g function equals 1 for optimal growth rate.
Cardinal models, as gamma function, have been developed to
quantify the influences of temperature, pH, and aw using as
parameters the minimum, optimum, and maximum growth
values (cardinal values) corresponding to each factor. Cardinal
model parameter values are associated and characteristic of
bacterial strains, thus pHmin values depend only on the bacte-
rial strain, while mopt values depend on bacterial strains and
food matrix.
Concerning the pH effect (Figure 3), the gamma function is
given by the following equation:
½acid
½AH ¼ [5]
1 þ 10pHpKa
In this model, [AH] is the concentration of the undissoci-
ated form of the acid used at a given pH, the MICU value is the
minimum inhibitory concentration of the undissociated form
of the acid, and the a parameter corresponds to a shape
parameter.
The effects of acid mixtures can be calculated by considering
that the inhibitory effects of each acid are multiplicative.

( 0
pH < pHmin ; ðpH  pHmin ÞðpH  pHmax Þ
gðpHÞ ¼ pHmin < pH < pHmax ; gðpHÞ ¼ [3]
ðpH  pHmin ÞðpH  pHmax Þ  ðpH  pHopt Þ2
pH > pHmax ;
0

This gamma function has been developed to model the

effect of pH on bacterial growth rate but also can be applied to
fungal development.
The influence of organic acid concentrations on bacterial
growth (Figure 4) can be described and quantified by the
following gamma function, taking into account the
gAH ½AH ¼ gacid 1 ½AHacid 1   gacid 2 ½AHacid 2  [6]
Other models have been developed taking into account the
preponderant weight of the acid with the highest inhibitory
potential. For different species and strains, pH cardinal values
and the MICU for different acids are available in the literature
586 ECOLOGY OF BACTERIA AND FUNGI IN FOODS j Effects of pH

Table 3 Examples of MIC values (mM) and their associated Further Reading
a values for different acids and different bacterial species
Álvarez-Ordóñez, A., Prieto, M., Bernardo, A., Hill, C., López, M., 2012. The acid
Microorganism Acid MICU a
tolerance response of Salmonella spp.: an adaptive strategy to survive in stressful
environments prevailing in foods and the host. Food Research International 45,
L. monocytogenes acetic 6.2–18.9 0.67
482–492.
S. typhimurium acetic 7.5 0.53 Bignell, E., 2012. The molecular basis of pH sensing, signaling, and homeostasis in
L. innocua acetic 21.5 0.42 fungi. Advances in Applied Microbiology 79, 1–18.
S. aureus acetic 7.1 0.83 Coroller, L., Guerrot, V., Huchet, V., Le Marc, Y., Mafart, P., Sohier, D., Thuault, D.,
L. monocytogenes capric 0.04–0.07 0.39 2005. Modelling the influence of single acid and mixture on bacterial growth.
L. monocytogenes citric 0.2–3.6 1.02 International Journal of Food Microbiology 100, 167–178.
S. typhimurium citric 0.6 2.3 Gahan, C.G.M., Hill, C., 1999. The relationship between acid stress responses and
L. monocytogenes lactic 3.6–5.7 1.07 virulence in Salmonella typhimurium and Listeria monocytogenes. International
Journal of Food Microbiology 50, 93–100.
L. innocua lactic 6.4 1.68
Krulwich, T.A., Sachs, G., Padan, E., 2011. Molecular aspects of bacterial pH sensing
S. enteritidis lactic 5.0–6.0 1.98
and homeostasis. Nature Reviews Microbiology 9, 330–343.
L. monocytogenes lauric 0.008–0.012 0.43 Myers, R.J., 2010. One-hundred years of pH. Journal of Chemical Education 87,
L. monocytogenes propionic 4.0–8.0 0.56 30–32.
L. innocua propionic 8.9 0.43 Peñalva, M.A., Arst Jr., H.N., 2002. Regulation of gene expression by ambient pH in
E. coli propionic 8.3 0.33 filamentous fungi and yeasts. Microbiology and Molecular Biology Reviews 66,
S. typhimurium propionic 3.6 0.34 426–446.
S. aureus propionic 6.9 0.32 Slonczewski, J.L., Fujisawa, M., Dopson, M., Krulwich, T.A., 2009. Cytoplasmic pH
measurement and homeostasis in bacteria and archaea. Advances in Microbial
Physiology 55, 1–79. 317.
Theron, M.M., Lues, J.F.R., 2010. Organic Acids and Food Preservation. CRC Press.
Piper, P.W., 2011. Resistance of yeasts to weak organic acid food preservatives.
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See also: Ecology of Bacteria and Fungi: Influence of Available
Water; Ecology of Bacteria and Fungi in Foods: Influence of
Temperature; Ecology of Bacteria and Fungi in Foods:
Influence of Redox Potential.