Biochemical Engineering Journal 17 (2004) 147–151

Software sensors based on titrimetric techniques for the monitoring and

control of aerobic and anaerobic bioreactors
Heiko Feitkenhauer∗ , Ulrich Meyer
Institut für Chemie- und Bioingenieurwissenschaften, ETH Zürich, Hönggerberg, CH-8093 Zürich, Switzerland
Received 9 January 2003; accepted after revision 28 May 2003

Abstract
Microbial activity influences the vicinity of a microbial cell in a way that changes the pH value of the surrounding medium. Key
processes are the production or degradation of charged substances (e.g. volatile fatty acids) and the production of carbon dioxide (or
hydrogen carbonate). In a technical system, the shift of the pH value may be prevented by a pH control system. It is proposed in this paper
that the resulting base (or acid) consumption rates can be used as input for “software sensors” for substrate and biomass concentrations.
This titrimetric technique was successfully applied to extreme thermophilic, aerobic bioprocesses (growth substrates phenol, aliphatic
hydrocarbons) for the estimation of biomass and substrate concentrations (formation/degradation rates). In anaerobic experiments, the
formation of volatile fatty acids (VFA) was monitored and the reduction of the VFA formation rate in the presence of an inhibitory
compound investigated. The results show the versatility of software sensors based on titrimetric techniques and demonstrate the potential
for process control in applications in which more sophisticated sensors are not available or affordable.
© 2003 Elsevier B.V. All rights reserved.
Keywords: Aerobic processes; Anaerobic processes; Control; Software sensor; Thermophilic microorganisms

1. Introduction metabolites, e.g. fatty acids. Near the neutral pH most of

Both software sensor and titration techniques have gained
increasing popularity in recent years [1,2]. Reasons are their
robustness and lower price compared to other, more sophis-
ticated equipment. The latter is based on lower investment
costs as well as on the relatively simple maintenance (and
hence no need to employ additional, well trained techni-
cians). Furthermore, the equipment necessary is installed
in many plants (or laboratory bioreactors), but not used for
automatic process control purposes.
1.1. Bioprocess fundamentals of the process control
techniques
Nearly all activities of microorganisms affect their sur-
roundings in a way that changes the pH value in the vicinity
of the individual cell (or a microbial floc/pellet). Such a pH
alteration can be, for example, expected during the aerobic
degradation of uncharged substrates such as aliphatic hydro-
carbons. Cells metabolize hydrocarbons and oxygen while
they produce carbon dioxide and minor quantities of other
∗ Corresponding author. Tel.: +41-1-632-3064; fax: +41-1-632-1074.
E-mail address: feitkenhauer@tech.chem.ethz.ch (H. Feitkenhauer).
the carbon dioxide will be converted to hydrogen carbon-
ate and severely influence the pH value, while the rest will
leave the reactor as gaseous carbon dioxide. The amount of
carbon dioxide that leaves the reactor depends not only on
the pH value, but also on the mass transfer characteristics
within the treatment system. Therefore, not only microbial
activities influence the pH value, but also process variables
such as the aeration rate or stirrer speed. A further pH-shift
is encountered when cells degrade compounds which in-
fluence the pH value of the solution, e.g. the pH increases
when an acidic compound is degraded or incorporated. One
important example of that kind is also the incorporation of
ammonium into growing cells.
A similar pH change in the treatment system occurs in
denitrification processes [3]. A very intense change is also
found in two-phase anaerobic digestion systems. In this case
no oxygen is involved in the reaction. In the first bioreactor
(also called acidification reactor) the wastewater constituents
are transformed into volatile fatty acids (and carbon diox-
ide), while the same fatty acids are converted into biogas
in the second reactor (methane reactor). Therefore, a strong
change in pH accompanies the biological activities in both
reactors. Concluding, in a characterized biological system
it is not necessary to monitor the cells themselves (or their

1369-703X/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S1369-703X(03)00150-5
148 H. Feitkenhauer, U. Meyer / Biochemical Engineering Journal 17 (2004) 147–151
substrates) to detect the microbial activity, but it is sufficient
to monitor the surroundings of the cells, e.g. by monitoring
the pH.
Unfortunately, a shifting pH influences the cells them-
selves as well as their growth medium. An example for the
latter is acetic acid, which can pass into microbial cells only
in the neutral (protonated) form. In general, the bioavailabil-
ity of many compounds changes with the pH. An example
for the influence of the pH on the cells themselves is the
distribution of the individual volatile fatty acids produced
in an acidification reactor, which is dependent on the pH
in the growth medium [4]. If a base (or acid) is supplied
to the process to keep the pH value constant (e.g. by a pH
controller), the amount of titrant (base/acid) added per unit
time can be used as the relevant information instead of the
pH value. This direct titrimetric technique will be used and
discussed in this paper for the estimation of concentrations
of biomass or degraded substrate.
A pH-measurement and an online correction system for
the pH value (pH-stat) is available in most laboratory scale
wastewater treatment plants and is also used in many full
scale (industrial) installations. However, online measure-
ments of biomass concentrations are not as frequently used,
as they are costly and require some maintenance. The same
holds for online measurements of substrate concentrations or
wastewater sum parameters. Offline measurements are time
consuming and hence not very well suited for control pur-
poses. Furthermore, it is not very easy to determine the best
sampling times, especially in batch-wise operated reactors.
Also sampling may be required during the night (un-staffed
operating hours). Therefore, it would be beneficial to use
data that are easily available online such as the base con-
sumption to maintain the pH constant.
In this paper, the “construction” of simple software sen-
sors based on titration techniques is described. Three ex-
amples will be used to demonstrate the versatility of this
technique in more detail:
(a) Control of aerobic fermentation: estimation of biomass
and substrate (phenol) concentrations.
(b) Control of (anaerobic) acidification reactors: estimation
of metabolite concentrations.
(c) Control of (anaerobic) acidification reactors: estimation
of inhibitory effects.
2. Materials and methods
2.1. Aerobic bioreactor and control system
The artificial wastewater either contained phenol or
eicosane as the carbon source (concentrations indicated in
the results). Furthermore, it contained the following mineral
salts (in g/l): (NH4 )2 SO4 , 1.0; MgCl2 ·6H2 O, 0.1; CaCl2 ·
2H2 O, 0.1; KCl, 0.04; FeSO4 ·7H2 O, 0.001; and 10 ml/l
trace element solution according to DSMZ, Medium 141 [5].
It was sterilized in the treatment system (20 min at 121 ◦ C
bioreactor). The phosphate buffer system (final concentra-
tion: Na2 HPO4 , 0.42 g/l and NaH2 PO4 , 0.2 g/l) was adjusted
to the desired pH and autoclaved separately and added be-
fore the inoculation with the indicated Bacillus strain (phe-
nol degradation: Bacillus thermoleovorans A2 [6]; eicosane
degradation: B. thermoleovorans Hamburg I [7]). The 2 l
bioreactor (liquid volume of 1.6 l, Typ VSF, Bioengineering,
Wald, Switzerland) was equipped with three “disc turbine”
stirrers on the axis and operated at 1500 rpm. The aeration
rate (0.5 vvm) and temperature (65 ◦ C) remained unchanged
during the experiment. The system was equipped with an
oxygen probe and indicator (Bioengineering). The oxygen
saturation was recorded and was above 5% air saturation at
all times of the experiments. The system was also equipped
with a pH-probe (Endress und Hauser, Weil am Rhein, Ger-
many) and a pH controller. The pH was kept at the desired
value by this controller connected to a peristaltic pump (us-
ing 1 N NaOH solution). The base supply flask was placed
on scales, which were read at the indicated times.
2.2. Anaerobic bioreactor and control system
The synthetic wastewater and system for the control of
the anaerobic acidification reactor has been described before
[8]. Briefly, it consists of a 7 l bioreactor with equipment
for online sampling and a purpose build titration cell for
the determination of volatile fatty acids and carbonate. The
actual titration was performed using a commercial tirator
(719, Metrohm, Herisau, Switzerland). All equipment was
connected to a PC (Dell Optiplex, Austin, USA) using the
serial interfaces of the computer (RS 232) or a multi-purpose
I/O card (MIO 16E4, National Instruments, Austin, USA).
The data acquisition and control software was written in
Labview 5.1 (National Instruments). A pH controller kept
the pH value in this reactor constant via a peristaltic pump
(using 2 N NaOH solution). The time of operation of the
base pump was recorded and converted into base amounts
by the computer using a calibration curve.
The same computer was also used in control of the first
(acidification) reactor in a biogas system. This 2 l bioreac-
tor was similar to the bioreactor described above, but in
this case the titrator (Metrohm 719) was directly connected
to the reactor for pH control. The pH value and amount
of base (0.5 N NaOH) added were read every 90 s by the
computer. The reactor was emptied and cleaned after every
experiment to avoid cell growth on the reactor wall. The
simplified wastewater contained the substrate glucose and
the following mineral salts (g/l): NH4 Cl, 0.15; MgCl2 , 0.05;
CaCl2 , 0.05; FeSO4 ·7H2 O, 0.02; HNa2 PO4 ·12H2 O, 0.35;
KH2 PO4 , 0.35; yeast extract 0.02 NaCl as indicated in the
experiment (adjusted to pH 6 with 0.5 N NaOH after inoc-
ulation). It was inoculated with 160 mg/l (cell dry weight)
of a mixed culture obtained from a continuously operated
stirred bioreactor (substrate: starch). All cultures for inocu-
lation were taken from a continuously operated bioreactor
 H. Feitkenhauer, U. Meyer / Biochemical Engineering Journal 17 (2004) 147–151
at the same time and stored at 4 ◦ C before the start of each
experiment.
3. Results and discussion
3.1. Control of an aerobic fermentation: estimation of
biomass and substrate concentrations
There are two main effects that can be exploited for
measurements in aerobic systems: (a) Cells degrade (or
incorporate) compounds which influence the pH value of
the solution. One important example is the incorporation of
ammonium into cell mass during the growth on a carbon
source. (b) Cells release intermediates and products of the
aerobic degradation in the wastewater, e.g. carbon diox-
ide/hydrogen carbonate. These two processes are related to
the metabolic activity of the cells and hence in most cases
to the growth on a certain pollutant. Hence these data can
be used to estimate the biomass or the degraded substrate
concentrations. In both cases the initial concentration can-
not be derived with a titrimetric technique. If required, they
must be determined offline. In laboratory measurements
they are often known from the set-up of the experiment
(e.g. the start concentration of a substrate is known from
the amount of substrate added to the reactor).
3.1.1. Estimation of substrate (phenol) concentrations
Phenol was degraded as sole carbon and energy source
by B. thermoleovorans A2 at 65 ◦ C in a batch-wise operated
reactor (Fig. 1). In general, higher concentrations of phe-
nol are very inhibitory to microorganisms (e.g. >200 mg/l),
while lower concentrations are degraded fast and efficiently.
Hence an online control of the degradation of phenolic com-
5 0
4.5
Phenol [mmol l ], OD x 5 [-]
4 -2.5
3.5
3 -5
-1
2.5
2 Phenol
1.5 OD
1 Base
0.5
0 -12.5
0 10 20 30
Time [h]
Fig. 1. Phenol degradation and microbial growth of the thermophilic B.
thermoleovorans A2 growing at 65 ◦ C and a pH of 6 in a 2 l bioreactor.
The growth was monitored using the optical density OD at 570 nm as
a parameter. On the second y-axis, the consumption of NaOH is plotted
(negative values refer to the level of the NaOH reservoir and were
not converted to make the good correlation with the phenol data more
obvious).
149
pounds would be very beneficial, but traditional systems are
often expensive and complicated. Offline measurements are
time consuming and hence not very well suited for control
purposes.
In the experimental system, the pH was maintained con-
stant by a pH controller, which pumped 1 N NaOH solution
into the reactor. A variation of about 0.04 pH units was
observed due to the “on/off” type controller used in the
experiment. The amount of base used was measured on a
balance (Mettler-Toledo, Switzerland, 0.1 g steps). A good
correlation between the phenol degradation and the base
consumption was found in this experiment (Eq. (1)). The
following linear correlation was derived from these data to
estimate the phenol consumption from the base consump-
tion data (in ml1 N NaOH /lbioreactor ):
Phenol degradation (mmol/l)
= 0.3884 × base consumption (ml/l)
(correlation coefficient R2 = 0.99) (1)
This correlation can be used to monitor online a degrada-
tion experiment or to control continuous degradations (pH
difference between feed and reactor have to be considered
in this case). Deviations of measurement and correlation
are caused by the relatively large steps of the balance
(0.1 g) and the on/off type of control. The simple linear
relationship also suggests that microbial ammonium uptake
(proportional to phenol uptake) was equally important to
carbon dioxide dissolution (where, e.g. saturation effects
could yield a non-linear relation). Due to the use of NaOH
insoluble sodium salts were probably produced from the
dissolved carbon dioxide.
Several types of biosensors for phenolic compounds are
in use in different areas, including process technology and
medical applications [9,10]. Most sensors for process con-
trol are based either on immobilized enzymes [11] or more
sophisticated extraction and detections methods, e.g. em-
ploying solid phase extractions and spectroscopic measure-
1 N NaOH [ml l ]
-1
ments [10]. All these methods have the advantage of being
more specific for phenolic compounds. On the other hand,
their drawback is the considerably high input in resources
-7.5
(investment, maintenance) to get reliable results.
-10 3.1.2. Estimation of biomass concentrations
Similar correlations can be derived for wastewater treat-
ment plants or for software sensors that estimate the cell
40 density (optical density, OD) from the base consumption
data. The following equation was derived for eicosane degra-
dation by B. thermoleovorans at 65 ◦ C and pH 7 [12] and
was confirmed also in experiments with hexadecane as the
growth substrate:
OD = 0.24× base consumption (in ml1 N NaOH /lbioreactor )
(correlation coefficient R2 = 0.94) (2)
150 H. Feitkenhauer, U. Meyer / Biochemical Engineering Journal 17 (2004) 147–151
There are several types of sensors for the monitoring of
cell/sludge densities in research bioreactors and industrial
applications. Many of them are based on optical measure-
ments. Problems are often encountered with air-bubbles and
biofilms on the sensor, or, like in the above present appli-
cation, with a second organic phase (aliphatic hydrocarbons
like eicosane dissolve only in minor quantities in the aque-
ous medium and hence form a second liquid phase even at
low concentrations). An example for the development of an
advanced sensor in this field is the multi-wavelength fluo-
rescence sensor [13], which measures also substrate concen-
trations. However, these methods are more complicated and
expensive than the proposed use of titrimetric data.
3.2. Control of (anaerobic) acidification reactors:
estimation of metabolite concentrations
The offline control of anaerobic digestion processes by
buffer capacity measurements is widely used. In samples,
the volatile fatty acid (VFA) content and carbonates can be
distinguished and this method is available for online use [8].
A simpler way to monitor the acidification rate is presented
here. It was shown that the base consumption correlates
very well with the VFA concentration (as measured online
with the above mentioned method and verified by a GC
method with minor deviation). The base consumption was
determined by measuring the operation time of the pump
connected to the pH controller (and using a calibration curve
for this pump).
Not taking into account the initial concentration of VFA
(which cannot be determined by direct titration), the VFA
concentration in the system can be calculated from the base
consumption by
VFA (mmol/l) = base consumption (mmol/l)/0.91
(correlation coefficient R2 = 0.99) (3)
The factor in this equation is not one because a part of the
organic acids is not dissociated at pH 6. On the other hand,
some carbon dioxide produced by the cells was dissolved in
the wastewater and changed the pH of the medium (at pH
6 the predominant species is carbon dioxide, but hydrogen
carbonate has still a considerably share of the total carbonate
alkalinity). Hence, when establishing such a simple correla-
tion always similar conditions to the subsequent process con-
trol must be used (especially pH, temperature and to some
extent salinity). If these conditions change considerably, the
whole process has to be modeled and the factors influencing
the base consumption have to be incorporated in this model.
There is an array of alternative methods to measure the
VFA content in anaerobic bioreactors, most of them using
more sophisticated techniques. One of the techniques with
a lower investment necessary is the online titration of sam-
ples [8]. At the other end of the spectrum, there are several
techniques using simplified and sophisticated mass spec-
trometers [14,15]. The advantage of these techniques is the
80
0 g/l NaCl"
70
40 g/l NaCl"
Base [ml 0.5 N NaOH]
60
50 80 g/l NaCl"
40
30 0 g/l NaCl
10 g/l NaCl
20
40 g/l NaCl
10 80 g /l NaCl
0
0 1500 3000 4500
Time [min]
Fig. 2. Determining the acidification rate of glucose (5 g l−1 ) in the
presence of increasing NaCl concentrations in a 2 l bioreactor.
possibility to discriminate between different VFA species
or at least between VFA and hydrogen carbonate (titration
of samples). The main disadvantages are the much higher
installation and maintenance costs, including the need for
trained staff.
3.3. Control of (anaerobic) acidification reactors:
estimation of inhibitory effects
The examples described above relate to cases in which
concentrations were determined directly from measured
data. These data can also be used to gain information on in-
hibitory substances [16]. One simple example is the follow-
ing: the increase of the VFA concentration in a time interval
is calculated from titration data. From this data, acidification
rates in the presence of increasing concentrations of an in-
hibitor (increasing NaCl concentrations) can be calculated.
These decreasing rates (decreasing rates of base addition)
can be used to calculate an inhibition constant (Fig. 2).
A related technique has been used by Rozzi et al. [17]
to determine inhibitory effects on methanogenic bacteria.
In this case not the degradation, but the formation of an
intermediate in the presence of an inhibitor substance was
successfully studied.
4. Conclusions
Software sensors based on titrimetric techniques were
successfully used to determine key concentrations and
rates in wastewater treatment processes. In all cases useful
correlations were established which are valid for specific
systems and processes. The equations were obtained from
experiments that can be performed relatively fast in other
biosystems to establish the correlations in these biopro-
cesses (specific correlations have to be established for each
process to be controlled or monitored).
 H. Feitkenhauer, U. Meyer / Biochemical Engineering Journal 17 (2004) 147–151
In an aerobic process, the metabolism is mainly related to
the degradation of a substrate and the formation of biomass.
Both the substrate (shown for phenol) and biomass con-
centration were described with a satisfactory accuracy. In
anaerobic digestion, the metabolism of the involved groups
of microorganisms is mainly a metabolism of volatile
fatty acids, either production (acidification) or degradation
(methanogenesis). In this case, the activity of the biomass
can be determined using software sensors, e.g. for the acid-
ification rate. It was also possible to relate the inhibitory
effects to concentrations of inhibitory substances. For the
further expansion of the utilization of this technique, we
are suggesting a combination of titrimetric techniques with
oxygen saturation and/or conductivity measurements and
the use of more detailed models (to make this titration-based
techniques also applicable in cases were, e.g. no linear
relationships can be established).
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