Theory of pH-Stat Titration
Elena Ficara, Alberto Rozzi, Paola Cortelezzi
Dipartimento di Ing. Idraulica Amb. e del Rilev-Sez. Amb., Politecnico di
Milano, Milan, Italy; e-mail: alberto.rozzi@polimi.it
Received 5 February 2002; accepted 29 August 2002
DOI: 10.1002/bit.10541
Abstract: Innovative techniques are being studied to as-
sess the activity of bioreactors and to improve the per-
formance and operational stability of biological pro-
cesses. Among these techniques, the pH-stat titration is
applicable to any bioreaction involving pH variations. Up
to now, the main application of the pH-stat titration has
been for nitrification monitoring. In this article, we pre-
sent a theoretical model of pH-stat titration, which pre-
dicts the response to any reaction involving the produc-
tion or consumption of protons, hydroxyl ions, or inor-
ganic carbon chemical species (CO2, HCO3−, CO3=). This
model is a useful tool to understand pH-stat titrations, to
define their applicability and limits, and to select the best
experimental conditions for specific applications. Tests
have been performed to compare experimental pH-stat
titration rates in the presence of carbon dioxide and
HCO3− producing reactions to the values predicted by the
model and a very satisfying correspondence was found.
© 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 28–37,
2003.
Keywords: pH-stat; titration; theory; model; titrimetric
technics
INTRODUCTION
The pH-stat titration technique consists of the controlled
addition of dilute acidic or alkaline solutions to maintain a
constant pH in systems where a pH affecting reaction is
taking place. Under these conditions, the titration rate is
proportional to the reaction rate. In principle, this technique
is applicable to any biological or physico-chemical reaction
affecting the proton concentration, i.e., any reaction con-
verting neutral substrates into acidic or basic products, or
acidic or basic substrates into neutral products.
There are several bioreactions of environmental interest
which affect the pH of biological suspensions. Up to now,
research on pH-stat titrations has been mainly focused on
the assessment of protons producing or consuming reactions
(e.g., nitrification: Gernaey et al., 1998; Massone et al.,
1998; and denitrification: Bogaert et al., 1997; Foxon et al.,
in press; Massone et al., 1996; Rozzi et al., 1997). However,
H+ (used here instead of H3O+ for simplicity) or OH− pro-
duction or consumption is not the only way to affect the pH
Correspondence to: Alberto Rozzi
Contract grant sponsor: The Italian Ministry of University and Scientific
Research
Contract grant number: COFIN2000 MM08165741
© 2003 Wiley Periodicals, Inc.
in a reactor. For instance, any reaction involving the CO2/
HCO3−/CO3⳱ equilibria, such as reactions producing or
consuming CO2 or HCO3−, also result in a pH variation and
can be monitored by pH-stat titration. Heterotrophic degra-
dation of organic substrates produces CO2 (Ficara, 2001),
acetoclastic methanogenesis produces bicarbonate (Rozzi et
al., 2001; 2002), growth of chemolitotrophic organisms
(e.g. algae) consumes CO2, while the ANAMMOX both
consumes CO 2 and produces bicarbonate (Rozzi and
Remigi, 2001; Strous, 2000; van de Graaf et al., 1996).
In this article we present a theoretical model of the pH-
stat titration procedure, developed to predict the response of
this technique to any reaction involving the production or
consumption of protons and inorganic carbon species (CO2,
HCO3−, CO3⳱). Model predictions are discussed and theo-
retical pH-stat titration rates, in the presence of CO2 and
HCO3− producing reactions, are compared to experimental
data.
pH-STAT TITRATION MODEL
The pH-stat unit (Fig. 1, frame) consists of a bioreactor,
where the biomass is maintained at constant temperature
and mixed at a constant rate, a pH-meter, and a titration
control unit. According to the pH signal, a control unit
activates the addition of an appropriate titrant (a diluted acid
or alkaline solution) to maintain the pH constant at a pre-set
value (pHstat). The system is thus a fed-batch reactor with
liquid inflow (the titrant added) and no liquid outflow. Gas/
liquis mass-transfer processes, such as gas sparging or gas
production by the biomass, may also play a significant role.
When gas sparging is present, the system is defined as open;
in contrast, in a closed system, no liquid/gas mass transfer
takes place.
In the bioreactor, a generic reaction takes place which
converts substrate S into product P (assumed not to affect
the pH), with the concomitant production of protons, carbon
dioxide, bicarbonate, and carbonate. These products affect
the CO2/HCO3−/CO3⳱ equilibria and thus, the suspension
pH. This reaction is described by the following stoichiom-
etry:
sS → P + bHCO3− + cCO2 + dCO3⳱ + hH+ (1)
where the stoichiometric coefficients b, c, d, and h are posi-

Figure 1. Experimental set-up. The pH-stat unit is represented in the
frame.
tive when the corresponding chemical species is produced,
or negative when it is consumed. Coefficient s for the sub-
strate is always positive. For a correct pH-stat operation and
data interpretation, it is very important to determine the
relationship between the reaction rate (rp ⳱ d[P]/dt, where
[P] is the molar concentration of product P) and the pH-stat
titration rate, rt (moles of H+, if rt > 0, or moles of OH−, if
rt < 0, added by the pH-stat unit per unit of time and per unit
of suspension volume). Hence, this system is defined by
molar concentrations of the following components: S, P, H+,
OH−, CO2, HCO3−, and CO3⳱. The following assumptions/
simplifications were made:
1. The increase of biomass liquid volume due to titrant
addition is neglected and the system is modeled as a
constant volume batch reactor.
2. The effect of the ionic strength on concentrations is ne-
glected, i.e., the activity coefficient ␥ ⳱ 1.
3. The inorganic carbon (IC) species are assumed to be in
equilibrium at all times, i.e., the dynamics of the CO2/
HCO3−/CO3⳱ equilibria is assumed to be much faster
than the dynamics of the reaction in Eq. (1).
4. Carbonic acid concentration is negligible.
5. The buffer capacity is assumed to be due only to IC
species.
Assumption 1 is generally justified by the fact that the vol-
ume of titrant added can be minimized by selecting the most
Table I. System processes and related rates.
Process Reaction
1. Biological reaction sS → P + b HCO−3 + cCO2 + hH+ + dCO3⳱
2. Bicarbonate equilibrium CO2 + H2O ↔ HCO3− + H+
3. Carbonate equilibrium HCO3− ↔ CO3⳱ + H+
4. Liquid/gas exchange CO2l ↔ CO2g
5. Titrant addition +H+
appropriate titrant molarity. Assumption 2 was introduced
for the sake of simplicity; however, both dilution and ionic
strength could be accounted for in a more rigorous ap-
proach. Assumption 3 is justified by the fact that these
chemical reactions are generally faster than biologically
catalyzed ones. Assumption 4 is justified by the fact that
carbonic acid comprises ≅ 0.2% of [CO2] (Stumm and Mor-
gan, 1996).
Processes taking place in the pH-stat reactor and their
rates are reported in Table I. Equations describing the sys-
tem are indicated in Table II: the first six equations are mass
balances for the system components, Eq. (T2.7) is the ionic
product for water, while the last three equations are neces-
sary conditions for the system to be pH-stat. In particular,
Eqs. (T2.9–T2.10) are derived from IC species equilibria:
关HCO−3 兴 ⭈ 关H+兴
= KA1 or: 关CO2兴 = 10pKA1−pH ⭈ 关HCO−3 兴
关CO2兴
(2)
关CO=3 兴 ⭈ 关H+兴
= KA2 or: 关HCO−3 兴 = 10pKA2−pH ⭈ 关CO=3 兴
关HCO−3 兴
(3)
At constant pH, an increase/decrease in CO2 concentration
should be compensated by a directly proportional increase/
decrease in bicarbonate concentration [Eq. (2)]. Similarly,
an increase/decrease in [HCO3−] is, in turn, compensated by
a directly proportional increase/decrease in carbonate con-
centration [Eq. (3)]. Geometrically, in the tri-dimensional
space defined as: x ⳱ [HCO3−], y ⳱ [CO3⳱], z ⳱ [CO2],
at constant pH the system is forced to move along the
straight line defined by the intersection of the two planes
described by Eqs. (2–3), as shown in Figure 2. Altogether,
the system is described by nine first-order differential equa-
tions [Table II, Eqs. (T2.1–T2.6; T2.8–T2.10)] plus one
algebraic equation [Table II, Eq. (T2.7)], with the unknown
variables being the concentrations of the seven system com-
ponents, the bicarbonate and carbonate reaction rates (rb,rc),
and the pH-stat titration rate (rt). Therefore, the system can
be easily integrated. The assumed initial conditions for all
system components are summarized in Table III (3rd col-
umn).
Integration of Eqs. (T2.1–T2.6; T2.8–T2.10) allows
each component of the system to be evaluated as a function
of time, as summarized in the 4th column of Table III. The
titration rate function deserves special attention:
Reaction rate
rp
rb
rc
−KLa ⭈ ([CO2] − [CO2]eq)
rt
FICARA ET AL.: THEORY OF pH-STAT TITRATION 29
Table II. Equations which describe the system.

Equation Expression Equation Expression

T2.1 T2.6 Product mass balance

Bicarbonate d关HCOⳮ3 兴
d关P兴
mass balance = b ⭈ rp + rb − rc = rp
dt dt
T2.2 Proton d关H+兴 T2.7 Ionic product for water 关OH−兴关H+兴 = Kw
mass balance = h ⭈ rp + rb + rc + rt
dt
T2.3 Carbonate d关CO=3 兴 T2.8 pH-stat condition d 关H+兴
mass balance = d ⭈ rp + rc =0
dt dt
T2.4 Substrate d关S兴 T2.9 pH-stat condition for d关HCO−3 兴
mass balance =− s ⭈ rp CO2/HCO−3 equilibrium = 10pH−pKA1
dt d关CO2兴
T2.5 Carbon dioxide mass balance d关CO2兴
=+ c ⭈ rp − rb + T2.10 pH-stat condition for d关CO=3 兴
dt HCO−3/CO⳱3 equilibrium
= 10pH−pKA2
− KLa ⭈ 共关CO2兴 − 关CO2兴eq兲 d关HCO−3 兴
where [CO2]eq ⳱ CO2 equilibrium-
concentration with the initial gas-
phase composition

冋
rt = rp ⭈ 共b − h + 2 ⭈ d兲 − 共b + c + d兲
where KLa is the CO2 liquid/gas mass transfer coefficient
[see Eq. (20)]. It can be observed that the r(t) in Eq. (4) is

册
made of two terms:
10 pH−pKA1 ⭈ 共1 + 2 ⭈ 10 pH−pKA2兲
⭈
共1 + 10 pH−pKA1 ⭈ 共1 + 10 pH−pKA2兲兲
⭈ e−␣⭈t +

(4) 冋
rt共1兲 = rp ⭈ 共b − h + 2 ⭈ d兲 − 共b + c + d兲

+ ␣ ⭈ 10 pH−pKA1 ⭈ 共1 + 2 ⭈ 10 pH−pKA2兲 ⭈ 共 关CO2兴0

− 关CO2兴eq兲 ⭈ 共e−␣⭈t 兲 ⭈
10pH−pKA1 ⭈ 共1 + 2 ⭈ 10pH−pKA2兲
共1 + 10pH−pKA1 ⭈ 共1 + 10pH−pKA2兲兲
⭈ e−␣⭈t 册 (6)

where: [CO2]eq is the [CO2] at equilibrium with the initial and

gas phase composition (defined by Henry’s law) and ␣ is a
constant parameter equal to: rt共2兲 = ␣ ⭈ 10pH−pKA1 ⭈ 共1 + 2 ⭈ 10pH−pKA2兲 ⭈ 共关CO2兴0
− 关CO2兴eq兲 ⭈ 共e−␣⭈t兲 (7)
K La
␣= (5)
共1 + 10 pH−pKA1
⭈ 共1 + 10 pH−pKA2兲兲 The first term, rt(1), describes the pH-stat titration response
to the production of protons, carbon dioxide, bicarbonate,
and carbonate due to the reaction in Eq. (1). The second
term, rt(2), represents the response of the pH-stat unit to a
CO2 dissolving/stripping process originated by an initial
[CO2] unbalance. As a matter of fact, a variation of [CO2]
should be compensated by a variation of [HCO3−] and
[CO3⳱], as required by the pH-stat conditions [Table II,
Eqs. (T2.9) and (T2.10)]. Therefore, if [CO2]0 ⫽ [CO2]eq, a
titrant demand will be observed even when rp ⳱ 0. In
particular, when [CO2]0 > [CO2]eq, CO2 will be stripped and
its dissolved concentration will decrease; therefore [HCO3−]
and [CO3⳱] will diminish. This is illustrated in Figure 2: If
the system is initially at A, where CO2 is oversaturated with
respect to its equilibrium concentration (represented by
[CO2]B), a decrease in [CO2] due to gas stripping forces the
system to move along the line at constant pH (i.e., pH-stat
titration occurs), toward point B. The latter is characterized
by: [HCO3−]B < [HCO3−]A and [CO3⳱]B < [CO3⳱]A. Thus,
the pH-stat unit adds acid titrant to decrease [HCO3−] and
[CO 3 ⳱ ] by ([HCO 3 − ] A − [HCO 3 − ] B ) and ([CO 3 ⳱ ] A
Figure 2. System evolution in the space x ⳱ [HCO3−]; y ⳱ [CO3⳱]; z − [CO3⳱]B), respectively.
⳱ [CO2]. Normally, pH-stat titration is used to assess rp from its

30 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 82, NO. 1, APRIL 5, 2003

Table III. System variables, initial conditions and evolution over time.

Eq.
number Variable Initial condition Variable time evolutiona

T3.1 Carbon dioxide 关CO2兴t=0 = 关CO2兴0 关CO2兴 = 关CO2兴eq + 共关CO2兴0 − 关CO2兴eq兲 ⭈ e−␣⭈t +
concentration 共b + c + d兲
[CO2] + rp ⭈ 共1 − e−␣t兲
KLa
T3.2 Bicarbonate con- 关HCO−3 兴t=0 = 关HCO−3 兴0 关HCO−3 兴 = 关CO2兴 ⭈ 10pH−pKA1
centration
=关CO2兴⭈0 ⭈ 10pHstat−pKA1
[HCO−3]
T3.3 Carbonate concen- 关CO=3 兴t=0 =关CO=3 兴0 关CO=3 兴 = 关CO2兴 ⭈ 102pH−pKA1−pKA2
tration [CO⳱
3 ] ⭈
= 关CO2兴0 ⭈ 10 2pHstat−pKA1−pKA2

T3.4 Proton concentra- 关H+兴t=0 = 10−pHstat 关H+兴 = 10−pHstat

tion [H+]
T3.5 Hydroxyl ion con- 关OH−兴t=0 = 10pHstat−pKw 关OH−兴 = 10pHstat−pKw
centration [OH−]
T3.6 Substrate concen- 关S兴t=0 = 关S兴0 关S兴 = 关S兴0 − s ⭈ rp ⭈ t
tration [S]
T3.7 Product concentra- 关P兴t=0 = 关P兴0 关P兴 = 关P兴0 + rp ⭈ t
tion [P]

冋 册
T3.8 Titrant addition rt,t=0 = 0 rt = rp ⭈
rate rt 10pH−pKA1 ⭈ 共1 + 2 ⭈ 10pH−pKA2兲
共b − h + 2 ⭈ d兲 − 共b + c + d兲 ⭈ ⭈e−␣⭈t
共1 + 10pH−pKA1 ⭈ 共1 + 10pH−pKA2兲兲
−␣⭈t
+ ␣ ⭈ 10 pH−pKA1
⭈ 共1 + 2 ⭈ 10 pH−pKA2
兲 ⭈ 共关CO2兴0 − 关CO2兴eq兲 ⭈ 共e 兲
T3.9 Carbon dioxide
→ Bicarbonate
rb,t=0 = 0
rb = 102pH−pKA1−pKA2 ⭈ 冉 − ␣ ⭈共关CO2兴0 − 关CO2兴eq⭈兲

冊
reaction rate rb
共b + c + d兲
+ rp ⭈ ⭈ e−␣⭈t − d ⭈ rp
1 + 10pH−pKA1 ⭈ 共1 + 10pH−pKA2兲
T3.10 Bicarbonate →
Carbonate re-
rc,t=0 = 0
rc = 10pH−pKA1 ⭈ 共1 + 10pH−pKA2兲 ⭈ 冉 −␣ ⭈ 共关CO2兴0 − 关CO2兴eq兲

冊
action rate rc
共b + c + d兲
+ rp ⭈ ⭈ e−␣⭈t −共b + d兲 ⭈ rp
1 + 10pH−pKA1 ⭈ 共1 + 10pH−pKA2兲

KLa
a
␣=
共1 + 10pH−pKA1 ⭈ 共1 + 10pH−pKA2兲兲

stoichiometric relationship with rt(1). Therefore, rt(1)
should be derived from rt by subtracting rt(2), which is the
expression for CO2 equilibration. It is important to note that
rt(2) is an interfering process and is only present when
liquid/gas exchange takes place, i.e., in open systems.
In the following sections, the response of pH-stat titration
to production (or consumption) of each of the following
species: protons, bicarbonates, carbonates or carbon diox-
ide, is analyzed separately. Since the titration rate of CO2
stripping/dissolution, rt(2), is additive to any pH-stat titra-
tion, its effect is addressed separately. However, this type of
interference occurs during any pH-stat test performed under
non-equilibrated [CO2] conditions and should be taken into
account in such cases.
Production/Consumption of Protons
When only protons are produced and the stoichiometric co-
efficients b, c, and d equal to zero in Eq. (6), rt(1) becomes:
rt(1) ⳱ [−h ⭈ rp]
From Eq. (8) one can observe that the titration rate is lin-
early proportional to the reaction rate, the proportionality
depending on the stoichiometric coefficient (h). This very
simple relationship explains why pH-stat titrations have
been successfully applied to measure the activity of ammo-
nia-oxidizing microorganisms (Massone et al., 1998; Rama-
dori et al., 1980), since this reaction produces two moles of
protons per mole of oxidized ammonium (NH4+ + 3/2 O2 →
NO2− + 2H+ + H2O). Although accurate results have gen-
erally been obtained for this application, residual titration
rates have been frequently observed, even after complete
substrate (ammonium) consumption. This residual titration
is likely due to unequilibrated initial [CO2] and its rate is
equal to term rt(2). During nitrification monitoring by pH-
stat titrations, this residual titration rate has generally been
considered to be a constant “background rate” (Gernaey et
al., 1998; Massone et al., 1998). However, the assumption
of a constant rt(2) leads to incorrect estimations for rt(1)
because, as one may note from Eq. (7), rt(2) is an exponen-
tial function of time. Therefore, its value, measured after
(8) complete substrate consumption, is different from the value

FICARA ET AL.: THEORY OF pH-STAT TITRATION 31

of rt(2) during the nitrification phase. The best way to avoid The molar ratio between the CO2 titration rate and the CO2
the interference due to the above described nonlinear titra- production rate is defined as fCO2 and is given in eq. (12):
tion term is to perform pH-stat titrations only after the dis-
solved CO2 concentration has reached its equilibrium value rt 10pH−pKA1 ⭈ 共1 + 2 ⭈ 10pH−pKA2兲
(i.e., [CO2]0 ⳱ [CO2]eq). fCO2 = =− , (12)
c ⭈ rp 共1 + 10pH−pKA1 ⭈ 共1 + 10pH−pKA2兲兲

Production/Consumption of CO2
The pH-stat titration of a CO2-producing reaction is now
considered. First, the case of an open system (i.e., KLa ⫽ 0)
is discussed. If [CO2]0 ⳱ [CO2]eq, a CO2-producing reac-
tion induces a decrease in the system pH, leading to addition
of OH− by the pH-stat unit to maintain a constant pH. Bi-
carbonate and carbonate are thus produced according to the
following reactions:
This ratio represents the titration efficiency in closed sys-
tems and depends only on the working pH. In Figure 3, the
relationship between fCO2 and pH is plotted. Within the
usual pH working range for most biological reactions (7.5 to
8.5), |fCO2| ranges from 0.93 to 1.01 and indicates that the
CO2 produced is almost totally titrated.
Production/Consumption of Bicarbonate

CO2 + OH− → HCO3− → CO3⳱ + H+ (9) The pH-stat titration rate of bicarbonate production in an
open system is obtained by setting h ⳱ c ⳱ d ⳱ 0 in Eq.
Therefore, [HCO3−] and [CO3⳱] increase, as indicated by (6), which simplifies to:
the path A → C in Figure 2. As already noted, any increase
in [HCO3−] and [CO3⳱] under pH-stat conditions corre- rt共1兲 = b ⭈ rp ⭈
sponds with a proportional increase in [CO2]. Therefore, a
pHstat-dependent fraction of the CO2 produced remains in
solution and is not titrated. The higher [CO2] makes CO2
冋 1−
10pH−pKA1 ⭈ 共1 + 2 ⭈ 10pH−pKA2兲
共1 + 10 pH−pKA1
⭈ 共1 + 10
pH−pKA2
兲兲
⭈ 共e␣⭈t兲 册
transfer to the gas phase increasingly efficient, inducing a (13)
loss of CO2. Therefore, a fraction of the CO2 produced by
From Eq. (13) one may note that rt can be either positive or
the reaction in Eq. (1) is titrated and converted into bicar-
negative depending upon pH. The pH value at which the
bonate and carbonate, a fraction remains in solution and a
titration unit transitions from acid to base is found by im-
fraction is transferred to the gas phase. Titrant addition stops
posing rt(1) ⳱ 0.
completely when [CO2] reaches the value at which the CO2
For t ⳱ 0, this value is: pH ⳱ (pKA1 + pKA2)/2 ≅ 8.3 (at
stripping rate equals the CO2 production rate (i.e., KLa ⭈
25°C, pKA1 ⳱ 6.3 and pKA2 ⳱ 10.3). In fact, at pH ⳱ 8.3,
([CO2] – [CO2]eq) ⳱ c ⭈ rp and the steady state CO2 con-
a small fraction (≈1%) of the bicarbonate produced is con-
centration is: [CO2] ⳱ [CO2]eq + c ⭈ rp/KLa).
verted into carbonate, with concomitant acidity production
The CO2 titration rate vs. time is obtained by setting b ⳱
(HCO3− → H+ + CO3⳱), while an equal fraction is con-
d ⳱ h ⳱ 0 in Eq. (6):
verted into CO2, with concomitant acidity consumption
10pH−pKA1 ⭈ 共1 + 2 ⭈ 10pH−pKA2兲 (HCO3− + H+ → CO2 + H2O). Therefore, at pH ⳱ 8.3,
rt 共1兲 =− c ⭈ rp ⭈ ⭈ 共e−␣⭈t 兲 bicarbonate production has no effect on the suspension pH.
共1 + 10pH−pKA1 ⭈ 共1 + 10pH−pKA2兲兲 For pH > (pKA1 + pKA2)/2, the term in square brackets in
(10) Eq. (13) is negative, indicating that bicarbonate conversion
From Eq. (10) it is clear that the titration rate, rt(1), is to CO2 is more relevant at the beginning of the reaction
directly proportional to the CO2 production rate, rp, only
when t ⳱ 0. For t → ⬁, rt → 0 because the CO2 produced
by the reaction in Eq. (1) is completely transferred to the gas
phase. Moreover, the rate at which rt(1) decreases with time
is proportional to ␣ [Eq. (5)]. This parameter increases in
proportion with KLa, because a more efficient liquid/gas
mass transfer occurs. Additionally, it decreases with in-
creasing pH because at higher pH a higher fraction of the
CO2 produced is converted into bicarbonate and carbonate.
If the system is closed (i.e., KLa ⳱ 0; ␣ ⳱ 0), then rt ⳱
rt(1) and remains constant during pH-stat titration. For this
case, the titration rate approaches the CO2 production rate in
function of pH, as shown in Eq. (11):
Figure 3. Dependence, from the working pH, of the ratio between titra-
10pH−pKA1 ⭈ 共1 + 2 ⭈ 10pH−pKA2兲 tion rate and reaction rate when no gas exchange is allowed. Case of CO2
rt =− c ⭈ rp ⭈ (11) producing reactions: fCO2; HCO3− producing reactions: fHCO3−; CO3⳱ pro-
共1 + 10pH−pKA1 ⭈ 共1 + 10pH−pKA2兲兲 ducing reactions: fCO3⳱.

32 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 82, NO. 1, APRIL 5, 2003

and acidity is produced. On the other hand, when pH <
(pKA1 + pKA2)/2, bicarbonate production makes the suspen-
sion pH increase and the pH-stat unit doses the acid titrant
from the beginning of the test. However, as the reaction
proceeds, bicarbonate titration leads to CO2 production in-
dependently of the working pH. Consequently, [CO2] in-
creases, as well as [HCO3−] and [CO3⳱] (e.g., the system
moves from point A toward point C in Fig. 2). Thus, only a
fraction of the bicarbonate produced by the reaction in Eq.
(1) is titrated, while the rest remains in solution. Moreover,
the increase in [CO2] makes CO2 stripping more efficient,
and over time, increasing fractions of the CO2 produced by
bicarbonate titration are transferred to the gas phase. Thus,
the alkalizing effect of CO2 stripping compensates and, fi-
nally, overcomes the acidifying effect due to carbonate for-
mation, resulting in a net acid demand. Moreover, the frac-
tion of in-solution bicarbonate decreases because of CO2
stripping, while the titrated fraction increases. When the
CO2 production rate, due to bicarbonate protonation, equals
the CO2 stripping rate (i.e., when: [CO2] ⳱
[CO2]eq + b ⭈ rp/KLa), the titration rate, rt(1), equals the
bicarbonate production rate, b rp. Finally, it may be ob-
served that rt(1) approaches b ⭈ rp more rapidly the higher
the value of ␣, which means the higher the KLa and the
lower the working pH.
If no gas exchange is allowed (KLa ⳱ 0), the molar ratio
between the titration rate and the bicarbonate production
rate, defined as fHCO3−, is given in Eq. (14):
fHCO−3 =
rt
b ⭈ rp
= 冉 1 − 10pH−pKA1 ⭈ 10pH−pKA2
共1 + 10 pH−pKA1
⭈ 共1 + 10 pH−pKA2
Figure 3 shows the relationship between fHCO3− and pH.
Within the usual pH working range for biological
reactions (7.5 to 8.5), rt is only a small fraction of the
bicarbonate production rate (fHCO3− < 0.07). Therefore, if
no gas exchange is allowed while working within the
above indicated pH range, the pH-stat titration technique
is not suitable to monitor bicarbonate producing reactions
in closed systems.
Production/Consumption of Carbonate
The pH-stat titration rate of carbonate production in an open
system is obtained by setting h ⳱ b ⳱ c ⳱ 0 in Eq. (6),
which becomes:
rt共1兲 = d ⭈ rp
冉 冊
10pH−pKA1 ⭈ 共1 + 2 ⭈ 10pH−pKA2兲
⭈ 2−
共1 + 10pH−pKA1 ⭈ 共1 +10pH−pKA2兲兲
In this case, carbonate production always has an alkalizing
effect, as rt (1) is always positive. Therefore, the pH-stat unit
always doses the acid titrant, leading to:
CO=3 + H+ → HCO−3 (16)
HCO−3 +
+ H → CO2 + H2O (17)
Initially, part of the bicarbonate produced by the first reac-
tion remains in solution, while the rest is converted into CO2
according to the reaction in Eq. (17) to satisfy the pH-stat
condition [Eq. (T2.9) in Table II)]. Therefore, the molar ratio
between the carbonate produced and the protons added by
the pH-stat unit (rt/(d ⭈ rp)), is between 1 and 2. Since
carbonate titration leads to CO2 and bicarbonate production,
the system moves toward increasing [HCO3−] and [CO2].
Therefore, CO2 stripping becomes increasingly efficient,
until [CO2] reaches a value corresponding with the com-
plete stripping of the CO2 produced by the reaction in Eq.
(17). From this moment onwards, [CO2], and therefore the
concentration of all IC species, remains constant and the
CO3⳱ produced is completely converted into CO2, accord-
ing to:
CO3⳱ + 2H+ → CO2 + H2O (18)
The CO2 produced by the reaction in Eq. (18) is completely
transferred to the gas phase. The reaction in Eq. (18) indi-
cates that, under these conditions, the molar ratio between
CO3⳱ production rate and titration rate is equal to 2, as
indicated by Eq. (15) when t → ⬁.
In a closed system, the molar ratio between rt and the
carbonate production rate is defined as fCO⳱3⳱:
fCO3⳱ = rt Ⲑ 共d ⭈ rp兲
兲兲
冊 (14)
= 冉 2−
10pH−pKA1 ⭈ 共1 + 2 ⭈ 10pH−pKA2兲
共1 + 10pH−pKA1 ⭈ 共1 + 10pH−pKA2兲兲
冊 (19)
The IC species concentration increases throughout the
whole test, while fCO3⳱ remains constant at a value defined
by the working pH value. Figure 3 shows that for pH ⳱ 7.5
to 8.5, fCO3⳱ ⳱ 0.99 to 1.07, indicating that rt is very close
to the carbonate production rate in closed systems.
EXPERIMENTAL VALIDATION
To experimentally validate the mathematical model pre-
sented, pH-stat titration tests were performed and the theo-
retical and experimental titration rates compared. The first
tests were designed to perform pH-stat titration of dissolved
CO2 stripping due to unequilibrated CO2, theoretically de-
scribed by term rt(2) in Eq. (7). Then, experiments were
designed to simulate carbon dioxide- and bicarbonate-
producing reactions. Tests were performed under abiotic
conditions; therefore, CO2 and HCO−3 were not produced by
a biological reaction, but were added as carbon dioxide- and
bicarbonate-concentrated solutions to a saline solution con-
⭈ 共e−␣⭈t兲 tained in the pH-stat unit reactor to allow for the precise
control of CO2 and HCO3− production rates. Theoretical
(15) titration rates were calculated and compared with the cor-
responding experimental values. The validity of the pH-stat
titration model for proton-producing reactions has been pre-
viously proven when applied to monitoring ammonium oxi-
dation (Massone et al., 1998) and is not considered here.
FICARA ET AL.: THEORY OF pH-STAT TITRATION 33
MATERIALS AND METHODS
Instrument Description
The pH-stat unit used for these experiments was similar to
the instrument described by Massone et al. (1998). The
system consisted of a reactor containing a saline solution
and a titration system. The reactor was a 2 L vessel that was
completely stirred and thermostated (temperature oscilla-
tions were kept within 0.2°C). When gas exchanges were
allowed, air was sparged by a bubbling system at a constant
air flow rate in the range 1 to 100 Lair(Lreactor ⭈ h)−1. The
titration system consisted of a pH probe (Mettler Toledo,
U402-S7/200), which activated the addition of incremental
volumes (50 to 100 ␮L each) of titrant (NaOH or HCl
0.1M). The pH of the saline solution was kept within a fixed
range pHstat ± ⌬pH, where pHstat is the selected working pH
and ⌬pH is the system tolerance, set equal to 0.02 pH units.
All solutions were prepared using reagent-grade chemicals.
Experiments Description
pH-Stat Titration of CO2 or HCO3− Production
The experimental set-up used to simulate CO2 or HCO3−
production is presented in Figure 1. A CO2 or HCO3− con-
centrated solution was added at constant rate by a peristaltic
pump to the pH-stat reactor which contained 1 L of saline
solution. Experiments were performed in the presence or
absence of air sparging, to allow or avoid liquid/gas mass
transfer and simulate open and closed systems, respectively.
Experiments were repeated at various pH and KLa values.
Experimental conditions are summarized in Table IV. The
composition of the solutions is described below:
● In-reactor saline solution: 17 mM NaCl; 0.36 to 14.4 mM
NaHCO3 or 1 to 5 mM HCl, depending on the selected
working pH.
● Carbon dioxide-saturated solution was obtained by con-
tinuously sparging pure CO2 into a saline solution (same
concentration of salts as the in-reactor solution). The
measurement of pH enabled the degree of CO2 saturation
to be determined from Eq. (2).
● Bicarbonated-concentrated solution was composed by 17
mM NaCl; 178 to 1000 mM NaHCO3.

pH-Stat Titration of [CO2] Equilibration
Tests were designed to perform a pH-stat titration of dis-
solved CO2 stripping due to unequilibrated CO2 concentra-
tions. A 50% CO2–50% N2 gas mixture was sparged in 1 L
of a saline solution (NaCl ⳱ 17 mM; NaHCO3 ⳱ 0.25 mM)
to obtain a dissolved CO2 concentration that was oversatu-
rated with respect to the atmosphere. As soon as the desired
pH and, thus, the desired [CO2] was reached [Eq. (2)], gas
sparging was replaced with air bubbling. A pH-stat titration
was then started and the resulting alkaline titration was
monitored until [CO2] equilibration was reached. Two tests
were performed at different pH values (test 1 at pH ⳱ 7.74,
alkalinity ⳱ 2.2 mM; test 2 at pH ⳱ 7.98, alkalinity ⳱ 5
mM) while KLa was kept constant at 0.6 min−1.
The bicarbonate concentration was measured for all solu-
tions by pH potentiometric titration.
KLa Estimation
Tests were performed to assess the actual KLa value re-
quired to calculate theoretical titration rates. Temperature,
gas sparging, and mixing conditions of the in-reactor saline
solution were held constant during each set of experiments.
After pH equilibration at atmospheric CO2 partial pressure,
a known amount of HCl (3 to 5 mM) was added to the saline
solution, leading to CO2 release [Eq. (17)] and, consequently,
to CO2 oversaturation. The pH of the saline solution was
then monitored during CO2 stripping, until pH equilibration
was again achieved. Then, [CO2] was calculated from Eq.

Table IV. Experimental titration tests: Main experimental parameters.

(c ⭈ rp) or (b ⭈ rp)
Test pHstat Temperature (°C) (mM/min) KLa (min−1)

CO2 titration tests 1 6.41 24.2 0.02 0.02

2 7.59 24.2 0.04 0.04
3 7.60 23.1 0.02 0.21
4 8.51 23.5 0.02 0.02
5 10.13 24.1 0.02 0.02
6 6.27 21.7 0.16 0.00
7 9.45 22.6 0.16 0.00
8 8.45 23.9 0.14−0.08−0.04 0.00
HCO3− titration tests 9 8.06 23.2 0.17 0.93
10 7.63 22.0 0.18 0.93
11 7.57 21.9 0.18 0.21
12 7.63 21.8 0.18 0.55
13 3.93 25.0 0.25 0.00
14 6.03 24.8 0.25 0.00
15 7.24 23.6 0.25 0.00
16 10.01 23.7 0.25 0.00

34 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 82, NO. 1, APRIL 5, 2003

(2) as a function of time using measured pH values. Bicar-
bonate concentration was analytically measured at the end
of the test and assumed to remain constant during the whole
CO2 stripping process. This assumption is justified by the
fact that [HCO3−] variations, due to IC species equilibration
to pH variations (10−6 to 10−8 M), were always at least 2
orders of magnitude lower than the initial [HCO3−] (10−4 to
10−3 M).
The well-known mass-transfer equation based on the
two-film theory (Tchobanoglous and Burton, 1991) was
used to model dissolved CO2 stripping:
d关CO2兴
=− KLa ⭈ 共关CO2兴 − 关CO2兴eq兲 (20)
dt
Integration of Eq. (30) leads to:
关CO2兴 − 关CO2兴eq
1n =− KLa ⭈ t (21)
关CO2兴0 − 关CO2兴eq
Therefore, KLa was estimated by linear regression using
calculated CO2 concentration values derived from experi-
mental pH data.
RESULTS AND DISCUSSION
pH-Stat Titration of [CO2] Equilibration
Results obtained from the pH-stat titration of [CO2] equili-
bration tests, performed at different initial pH values, are
shown in Figure 4. Experimental titration rates are plotted
together with the expected theoretical values, predicted by
rt(2) in Eq. (7). A very satisfying correspondence between
experimental data and the mathematical model can be ob-
served. As expected, the titration rate is maximum at the
beginning of the test and decreases over time, as [CO2]
approaches its equilibrium value. Again, it is worth noting
that rt(2) is not constant. A simplified approach may be to
assume for rt(2) a linear decrease or increase over time in
case of CO2 stripping or redissolution, respectively. The
lower the value of parameter ␣ (i.e., the lower the KLa or the
higher the working pH), the lower the linearization error.
Figure 4. Comparison between theoretical (thick lines) and experimental
(thin lines) titration rates of CO2 stripping.
Finally, it can also be observed that for high values of ␣, or
when the bioreaction to be monitored lasts long enough to
allow for CO2 re-equilibration (e.g., for tests lasting more
than 200 min, Fig. 4), rt(2) may not be appreciable at the
end of the experiment. In this case, it is difficult to estimate
rt(2) during the course of the test and thus, to accurately
assess rt(1) from rt.
pH-Stat Titration of CO2 Production
Titration tests were performed to compare theoretical and
experimental values for rt(1) in CO2-producing reactions
under different experimental conditions. First, tests were
performed at different pHstat values while KLa was main-
tained almost constant (test 1, 2, 4, and 5 in Table IV). The
results are plotted in Figure 5. It may be observed that, for
pH < (pKA1 + pKA2)/2 ≅ 8.3, the titration rate efficiency, i.e.
the ratio rt/(c ⭈ rp) was lower than 100% and it decreased
significantly with time. On the contrary, for pH > (pKA1 +
pKA2)/2 ≅ 8.3, the ratio rt/(c ⭈ rp) was higher than 100% and
it did not decrease significantly with time, due to the low
value of ␣ for such pH values. These results are all in
agreement with theoretical predictions.
A second series of tests were performed to analyze the
effect of KLa on the titration efficiency (tests 2 and 3 in
Table IV) and results are plotted in Figure 5. As predicted
by the theoretical model, the initial titration efficiency was
the same for both tests, since its value depends only on the
working pH. On the contrary, the higher the KLa value, the
greater the rate of the rt decrease. Again, experimental data
were in agreement with what was expected from the theo-
retical model.
Finally, a third series of tests was performed without air
sparging (closed system). First, the carbon dioxide-addition
rate was varied during the test to observe changes in rt (test
8, in Table IV). At the working pH adopted (pH ⳱ 8.45),
the titration model predicted a very good agreement be-
tween rt and CO2 production rate (fCO2 ≅ 1). Experimental
data are plotted in Figure 6, which also shows a good cor-
Figure 5. Comparison between theoretical (thick lines) and experimental
(thin lines) CO2 titration efficiency (rt/c ⭈ rp) at different working pH (KLa
⳱ 0.02 min−1) and at different KLa (pH ⳱ 7.6).
FICARA ET AL.: THEORY OF pH-STAT TITRATION 35
Figure 6. Comparison between theoretical (thick lines) and experimental
(thin lines) CO2 titration rates at different CO2 production rates, closed
system.
respondence between rt and the CO2 production rate. Then,
the effect of the working pH was analyzed (tests 6 and 7 in
Table IV). Again, a very good agreement between theoret-
ical and experimental values of fCO2 was observed, as
shown in Figure 7. However, a higher instability was ob-
served for fCO2 at pH ⳱ 9.45 than at pH ⳱ 6.27. Oscilla-
tions in titration rates originate from discontinuous addi-
tions of titrant into a buffered solution, which negatively
affects the sensitivity of the pH-stat technique. However,
when the time constant of the discontinuous titrant additions
is lower than the time constant of the reaction to be moni-
tored, a moving window regression of titration data can be
used to obtain accurate results.
Titration Tests for HCO3− Producing Reactions
Tests were performed to compare the theoretical and ex-
perimental rt(1) of HCO3− producing reactions at different
experimental conditions. First, tests were performed at dif-
ferent working pH values by varying [HCO3−] in the in-
reactor saline solution, while aeration rate and HCO3− pro-
duction rate (b ⭈ rp in Table IV) were held constant (tests 9
and 10 in Table IV). The results are plotted in Figure 8. It
Figure 7. Comparison between theoretical (thick lines) and experimental
(thin lines) CO2 titration efficiency (fCO2) at different pH values, closed
system.
36 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 82, NO. 1, APRIL 5, 2003
Figure 8. Comparison between theoretical (thick lines) and experimental
(thin lines) HCO3− titration efficiency (rt/b ⭈ rp) at different working pH.
may be observed that the titration rate efficiency, rt/b ⭈ rp,
increases with time and tends toward 100%. Additionally,
this increase is faster at lower pH values, as expected from
the theoretical model.
A second series of tests were performed to assess the
effect of KLa on the titration efficiency (tests 10, 11, and 12
in Table IV) and results are presented in Figure 9. As pre-
dicted by the theoretical model, the initial titration effi-
ciency was practically the same for the three tests, since its
value depends only on the working pH. In contrast, at higher
KLa value, the rt(1) approached the bicarbonate production
rate faster. Again, experimental data were found to be in
agreement with those predicted by the model. Finally, tests
were performed without air sparging at different pH values
(Fig. 10; tests 13–16 in Table IV). As expected, the titration
efficiency decreased at increasing pH, being only around
20% for pH ⳱ 7.2. Moreover, when pH < (pKA1 + pKA2)/2
(tests 13, 14, and 15), the pH-stat unit dosed the acid titrant,
while the alkaline titrant was required when pH > (pKA1 +
pKA2)/2 (test 16). This is in agreement with the model pre-
diction.
CONCLUSIONS
The mathematical expressions that describe the pH-stat ti-
tration response to reactions producing/consuming protons,
Figure 9. Comparison between theoretical (thick lines) and experimental
(thin lines) HCO3− titration efficiency (rt/b ⭈ rp) at different KLa values
(min−1).

Figure 10. Comparison between theoretical (thick lines) and experimen-
tal (thin lines) HCO3− titration efficiency (fHCO3−) at different working pH,
closed system.
carbon dioxide, bicarbonate, and carbonate were rigorously
derived. The main factors influencing the titration effi-
ciency, defined as the ratio between the titration rate and the
reaction rate, were identified through analysis of the derived
equations.
For proton producing or consuming reactions in open
systems (i.e., when gas/liquid mass transfer is allowed), the
titration rate is proportional to the reaction rate only if the
pH-stat titration is carried out after dissolved carbon dioxide
has reached its equilibrium concentration with the gas
phase. Otherwise, the additional titration of CO2 equilibra-
tion (stripping or redissolution) should be taken into ac-
count. This titration term follows a negative exponential
trend and may be difficult to discriminate from the titration
term due to the protons production rate. This interference is
avoided by preventing gas/liquid mass transfer (closed
system).
For carbon dioxide producing or consuming reactions,
mass transfer of CO2 from the liquid to the gas phase should
be minimized to maximize the fraction of CO2 titrated.
When mass transfer is avoided (closed system), titration rate
is directly proportional to the CO2 produced/consumed and
their molar ratio is almost 1 for pH ⳱ 7.5 to 8.5.
For bicarbonate-producing or consuming reactions, the
titration rate is initially lower than the bicarbonate produc-
tion rate. Then, once the CO2 concentration reaches an equi-
librium value between its production rate (originated from
bicarbonate acid titration) and its stripping rate (due to gas
sparging), titration rate equals bicarbonate production rate.
Therefore, it is convenient to maximize the efficiency of
CO2 mass transfer to the gas phase. On the contrary, in
closed systems at pH ⳱ 7.5 to 8.5, more than 90% of the
bicarbonate produced remains in solution, while only the
residual fraction is titrated.
Finally, a pH-stat titration of carbonate-producing reac-
tions can be performed, at pH values between 7.5 and 8.5,
either in the presence or in the absence of gas exchange. In
open systems, the titration rate is initially equal to the car-
bonate production rate, then it increases until it becomes
twice the carbonate production rate; this occurs when the
CO2 produced by acid titration of carbonate is completely
stripped. In closed systems, the titration rate remains con-
stant in the range from 100 to 200% of the carbonate-
production rate, depending on the working pH.
Experimental pH-stat titration of CO2- and HCO3−-
producing reactions were performed in abiotic conditions.
Theoretical titration rates were found to compare well with
experimental results.
In conclusion, pH-stat titration can be applied to monitor
proton-, carbon dioxide-, bicarbonate- and carbonate-
producing reactions. However, it is necessary to select the
best working conditions in terms of initial CO2 concentra-
tion, pH, and gas/liquid mass-transfer rate to maximize the
titration efficiency.
Useful comments and revision of the manuscript by Dr. Nancy
Love (Virginia Tech, Blacksburg, USA) are gratefully acknowl-
edged.
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FICARA ET AL.: THEORY OF pH-STAT TITRATION 37