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The 4th International Federation of Automatic Control Conference on Management and Control of Production and Logistics
September 27-30, Sibiu - Romania



M. Mironescu, *C. Posten, **K. Kriger, **R. Tzoneva

„Lucian Blaga“ University,Sibiu, Romania / *Karlsruhe University, Germany /

**Peninsula Technikon, South Africa

Abstract: H. mediterranei is an extreme haloplylic archaeon producing extracellular

polysaccharides with special properties. For the rigorous command and control of
the bioprocess, a new control and command system was developed using the
programming environment LabVIEW. The system was called „Biolab” and allows
the regulation of temperature, pH and dissolved oxygen with very good results, the
application reacting fast at the biosystem changes. The feeding function allows the
maintaining of various substrate concentrations for long time and can be used for
helping to find the optimal cultivation conditions for biomass or polysaccharide
production. Copyright © 2007 IFAC

Keywords: command and control systems, biotechnology, temperature control, pH

control, PID control.

1. INTRODUCTION cultivation meaning the presence inside the

bioreactor of the entire quantity of substrate and
Biotechnology is an emerging field on the edge microbial cells from the beginning, without other
between biology and engineering. In the last years, inputs and/or outputs. The batch system is cheaper,
valuable metabolites like enzymes (Aehle, 2004) or but the growth and the metabolites production are
vitamins (Shimizu, 2001) are produced on inhibited by the high substrate concentration
biotechnological way by using microorganisms. needed. This problem can be avoided using the fed-
Development of a bioprocess involves its control batch cultivation system who allows the
and command. The control strategy is strongly maintaining of a constant substrate level, by
dependent on the bioprocess. For example, introducing new substrate in bioreactor, frequently
(Mailleret et al., 2004) use an adaptive control after an exponential feeding function (Posten and
strategy at the wastewater treatment processes, the Rinas, 2000).
controlled variable being the dissolved oxygen and For the control and command of batch and fed-
the manipulated variable being the aeration rate. batch bioprocesses, a new system called “BioLab”
Feed-back control strategy is used for the based on the LabVIEW-software package was
production of yeast biomass (Heinzle and Saner, designed. This system is the result of a
1991) or for recombinant proteins production collaborative project between the Department of
(Akesson 1999), the controlled variable being the Biotechnology from University of Karlsruhe,
respiratory coefficient (in the first case) or acetate Germany and the Department of Electrical
as secondary product (in the second case); the Engineering, Peninsula Technikon, South Africa
manipulated variable is the glucose feeding rate for (Kriger et al., 2005).
both cases. This paper shows the practical results obtained
The cultivation systems used at the cultivation of by using “BioLab” for the command and control
microorganisms are batch, fed-batch and of fed-batch cultivation of a microorganism. The
continuous. The batch system is closed, the
strain chosen to test the command and control Taking into account these requirements, the
system is a new discovered archaeon called technical solution is presented schematically in
Haloferax mediterranei. The microorganism is figure 1.
presented in the literature as producer of
extracellular polysaccharides (EPS) (Oren, 2002).
These polysaccharides show interesting properties Serial NI Lab PC- 1200
connexion Card
as ions binding capacity (Mironescu and Signals
Mironescu, 2004a) or stability of viscosity in a conditioning

large domain of pH, temperature and salinity Mo

(Mironescu, 2006; Anton et al., 1988). Due to these Air
properties, EPS could find applications in the food Temp
industry as viscosity agent in extreme conditions or DO2

as ion binder. Analysis of

Little literature on the optimisation of biomass Warm

and/or polysaccharide production by H. water

mediterranei is available (Mironescu, 2006; Cold

Mironescu et al., 2004b, Rodriguez-Valera et al., Fed- Acid Base Inoculum Bioreactor
1991). For this reason, researches on the factors
influencing the bioprocess as temperature, pH, Feeding bioreactor with liquid media
Water circulation system for temperature control
feeding strategy are necessary. The goal of this
Gases circulation
research is to establish if “BioLab” could be used
Signals from the sensors
for the control of physical factors influencing the Signals to the execution elements
cultivation of H. mediterranei (temperature, pH,
dissolved oxygen) and for the command of feeding Fig. 1. Design of cultivation plant
in the fed-batch phase of the bioprocess. The
results will help to optimise the cultivation and to
identify the conditions for maximal EPS synthesis. 1.3. The application “BioLab”

2. MATERIALS AND METHODS “BioLab” is a flexible and user-friendly LabVIEW

application, having the main characteristics:
1.1 Microorganism, cultivation media and - bioprocess is commanded by using an adjustable
determinations feeding function during the fed-batch phase of a
The microorganism Haloferax mediterranei ATCC - temperature is controlled by on/off real-time
33500 from DSMZ (Deutsche Sammlung für control of valves for cold or warm water;
Mikroorganismen und Zellkulturen, Germany) was - pH is controlled by on/off real-time control of
used. pumps for acid or base;
The cultivation media contained: 200g/l NaCl; - dissolved oxygen is controlled using an auto-
50g/l MgCl2x6H2O; 5g/l K2SO4; 0.133g/l CaCl2x tuning PID controller from LabVIEW, which
2H2O; 0.025g/l KH2PO4; 5g/l yeast extract; 5g/l command the agitation (motor speed);
peptone. - display graphically the physical variables in
Glucose was used as carbohydrate source, in real-time and logs data to allow easier
quantity of 3.5-5 g/l during the batch stage and interpretation and extraction of results for analysis;
(0.1-0.2)-2 g/l in the fed-batch stage of the - allows changing the minimum, maximum and
bioprocess. set point values of variables as desired and
Biomass formed was determined gravimetrically displays errors and alarms (Kriger et al., 2005).
after drying (Mironescu et al., 2003a). Glucose and The software algorithm is structured on three
EPS were measured spectrofotometrically levels: process interface, control level and user
(Mironescu et al., 2003b). interface as presented detailed in (Kriger et al.,
2005; Mironescu, 2006).
The feeding function is exponential, adapted after
1.2. Cultivation (Posten and Rinas, 2000) having the form:

µ set ⋅ c X ⋅ V R
On designing the cultivation plant, some f S _ LabView = K LabView ⋅ ⋅ e µ set ⋅t . (1)
requirements were important: y XS ⋅ ( c S _ in − c S )
- The recipients and the pipes must be resistant
to the corrosion action of cultivation substrate; where:
- A command and control system for pH, KLabView – coefficient;
temperature and substrate feeding during the µ set – specific growth rate, set for the fed-batch
fed-batch phase is necessary; phase, h-1;
- A system for monitoring dissolved oxygen and cX – biomass concentration, g/l;
gases evacuated from the bioreactor (O2 and VR – liquid volume in bioreactor at the beginning
CO2) was demanded; of the fed-batch phase, l;
- The control of oxygen dissolved in the yXS – theoretical biomass yield, g/g;
cultivation broth (DO2) using agitation as cS_in – substrate (glucose) concentration in the
manipulated variable is intended. feeding medium, g/l;

cS – substrate (glucose) concentration in bioreactor, 3.2. Control of pH
The coefficient KLabView has variable values, Practically was observed that the microorganism
depending on cS_in and on the initial feeding pump produces acids during normal growth and in the
rotation (Mironescu et al., 2004b); cX is established absence of stress factors. When the substrate
practically for each cultivation; VR is always 5 l; yXS become limited (as in the case of fed-batch
= 0.5 g biomass/g substrate (glucose) (Korz et al., cultivation with limited glucose concentration) or
1995); µset was fixed 0.05 h-1 based on other stress factors appear, the cells are producing
experimental results (Mironescu et al., 2004b); cS_in bases. Based on these practical observations, the
was 50 and 100 g/l; cS was varied between 0.1-0.2 control of pH during cultivation is imposed.
(at the limit of the quantification method) and 2 g/l. Figure 3 presents a fed-batch cultivation where two
As presented in (Kriger et al., 2005), the pH value were tested: 7.1 and 7.2, both in the
parameters are introduced easily by user (in this optimal pH range of H. mediterranei.
case, the biotechnologist). As figure 3 shows, “BioLab” is efficient in
controlling pH during the batch phase of
3. RESULTS AND DISCUSSIONS cultivation, the variations around the setpoints
being in the range ±0.02. When setpoint is changed,
3.1. Control of temperature the system reacts very fast, attiring in short time the
new pH value.
The evolution of temperature during a fed-batch For the cultivation presented in figure 3 the lag
cultivation is presented in figure 2. Two setpoint phase was around 24 hours and is not shown.
values in the optimal temperature range of this
microorganism were tested: 38°C and 45°C.
46 7,22

45 7,2

44 7,18
Start fed-batch
43 7,16
Start fed-batch
Temperature, °C



37 7,06 batch
36 7,04

batch 24 30 36 42 48 54 60 66

12 18 24 30 36 42 48 54 60 Cultivation time, h

Cultivation time, h Fig. 3. Use of “BioLab” for the control and

Fig. 2. Use of “BioLab” for the control and command of pH during the cultivation of H.
command of temperature during the cultivation mediterranei. Two setpoints for the pH values
of H. mediterranei. Two setpoints for (7.1 and 7.2) were tested.
temperature (38°C and 45°C) were tested.

In order to analyse the results, the control program 3.3. Control of dissolved oxygen
must be correlated with the cells growth phases. In
the first 12 hours the H. mediterranei cells are in The DO2-values during fed-batch cultivation
the lag phase, they adapts to the conditions in the without DO2-control and constant agitation is
bioreactor; cells don’t grow and don’t produce presented in figure 4.
metabolites, the control being not representative for 100 600
the bioprocess. Approximately 12 hours after the 90
start of cultivation, the cells begin to multiply and 80
the population develops, being in the exponential 70
growth phase. During growth, H. mediterranei 400
Agitation. rpm

produces heat. Preliminary calculations showed
DO 2, %

50 300
that 3 g/l biomass produce 18.75 cal/h
(corresponding to warming the cultivation media 200
with 0.0187°C/h). So, “BioLab” acts more to Start fed-batch
decrease the temperature in bioreactor. As the 100
results show, “BioLab” works quite well at the batch
control of temperature, the fluctuations around 0 0
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90
38°C being in the range ±0.4°C. Cultivation time, h
After the change of setpoint to 45°C the biosystem Fig. 4. Evolution of DO2 (points) and agitation
(consisting on substrate and cells) attires in very (line) during fed-batch cultivation of H.
short time the new temperature (figure 2). mediterranei with 1l/min air flow rate and
After 45 hours of cultivation begin, the fed-batch without control of dissolved oxygen in the fed-
stage of the cultivation is started. As figure 2 batch stage
shows, in the fed-batch stage the variations of
temperature are not so high. In this stage the cell The quantity of oxygen dissolved in the cultivation
metabolism is no more oriented on biomass broth (DO2) influences strongly the cultivation of
production and less heat is produced.
calculated parameters are presented in (Kriger et
al., 2005).
The calibration of PID parameters is necessary
H. mediterranei, which is aerobic (Anton et al, each cultivation. As figure 5 shows, sometime new
1988).The solubility of oxygen is depending on the calibrations are necessary during the same
composition of the substrate an on the metabolites. cultivation. The process takes hours, some future
The medium used at the cultivation of this improvements being necessary to reduce the
particular microorganism has very high ionic calibration time.
strength (I ≅ 421) because of its salts content and Another disadvantage is that during calibration,
reduces the solubility of oxygen. Peptone and yeast agitation varies very strongly. These fluctuations
extract decrease DO2 with arround 30% (Zarnea et
don’t stress the microorganism, the cells and
al., 1980). This could be the main reason why the
bioprocess being not visible influenced by changes
value 100% (air saturated with oxygen) of DO2 is
of agitation in the limit 300-600 rpm imposed by
maintained for short time at the beginning of
cultivation and decreases at 90% in 6 h. biotechnologists.
As figure 4 shows, DO2 decrease during the batch
stage from 90% to around 30% in the exponential
growth phase. This phase begins after around 18 h 3.4. Command of feeding
of cultivation and ends after 40 h.
After start of the fed-batch stage, many fluctuations The analysis of the influence of glucose
of DO2 are observed. These variations are caused concentration in the fed-batch phase on the biomass
by the cells metabolism, but the presence of and EPS formation allows finding the optimal fed-
polysaccharides can influence the values of batch cultivation conditions for EPS production.
dissolved oxygen. EPS modify the viscosity of For this purpose, the bioreactor was feed with
cultivation substrate and so the oxygen transfer substrate containing salts and glucose using the
coefficient. The viscosity measurements in “BioLab” system.
bioreactor showed that the cells-EPS mixture has The results of two cultivations with different
high viscosity, especially after increase of glucose concentrations are presented in figures 6
polysaccharides content to values higher as 5 g/l. and 7. In both cases, small quantity of biomass is
This concentration is attired in the fed-batch phase, formed in the batch stage of cultivation, necessary
the broth behaving like a pseudoplastic fluid (data for producing EPS. Depending on the glucose
not shown). content intended to be maintained, the fed-batch
Taking into account these observations, the control stage begins by starting the function “FEED” in
of dissolved oxygen at a constant value in the fed- “BioLab”.
batch stage of cultivation is required.
The DO2-values during fed-batch cultivation with 15
-control using agitation is presented in figure
DO2100 600
5. 14 Glucose, g/l
Steps I, II, III Step IV 13 Biomass, g/l
500 EPS, g/l
80 11
Start fed-batch
Agitation, rpm

DO 2 , %

50 300 7
20 Start fed-batch 3
batch 2
0 0 0
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90
12 18 24 30 36 42 48 54 60 66 72
Cultivation time, h Cultivation time, h

Fig. 5. Evolution of DO2 (points) and agitation Fig. 6. Consumption of glucose and formation of
(line) during fed-batch cultivation of H. biomass and EPS during fed-batch cultivation
mediterranei with control of dissolved oxygen of H. mediterranei with control of DO2 (25%
by agitation in the fed-batch stage using in the fed-batch stage), pH (7.2 during entire
“BioLab”. DO2 setpoint was 30%; air flow rate cultivation) and temperature (38°C during
was 1l/min during the entire cultivation. cultivation) and command of glucose feeding
using “BioLab”. Parameters of the
The change of agitation determines the variation of exponential feeding function were: VR=5 l;
air bubbles dimension and facilitates the access of mX =11.65 g; cS_in=100 g/l glucose; cS=0.1
oxygen to cells (Bailey and Ollis, 1986). g/l glucose; KLabView=18; µ=0.05 h-1. Aeration
In the results presented in figure 5, at the start of 1 l/min
fed-batch stage, “BioLab” was used to control DO2
at the value 30%. For the calibration, the PID In the fed-batch cultivation presented in figure 6 is
automatic control provided by LabVIEW was used. intended to start the feeding at the end of batch
The values for PID parameters were calculated in stage, when quite all glucose is consumed by the
many steps (four in the case presented in figure 5), microorganism and to maintain the glucose
each step having 10 to 15 cycles. Examples of concentration at very low values, around 0.1 g/l. As
figure 6 shows, the feeding function works well.
The substrate introduced in bioreactor is consumed cultivation, the parameters that influence the
rapidly by H. mediterranei, the glucose process are pH and temperature during the entire
concentration being maintained at 0.1-0.2 g/l cultivation and dissolved oxygen during the fed-
during the fed-batch stage. Due to feeding with batch stage.
glucose limitation, biomass and metabolite register For the maintaining of the control parameters in
different evolutions. The biomass concentration certain limits during cultivation and for the
increases at the beginning of the fed-batch stage command of feeding during the fed-batch stage, the
and then remains constant at values around 5 g/l, “BioLab” system was build using the LabVIEW-
whereas EPS concentration increases very much software package. The results show a good control
from 5.69 g/l at the beginning to 14.37 after 23 h of of temperature, pH and dissolved oxygen, the
feeding with substrate. application reacting fast at changes in the
It can be concluded that the microbial metabolism biosystem consisting on H. mediterranei cells,
is oriented on polysaccharide formation when polysaccharides and substrate rich in salts in
glucose is limiting in the nutritive substrate. different growth phases.
15 The analysis of the DO2 evolution in bioprocesses
14 Glucose, g/l without and with control offered valuable
13 informations on the biosystem, influenced by the
EPS, g/l
Biomass, g/l presence of EPS which modifies the oxygen
9 The feeding function build in this work is
8 exponential and allows identifying the limiting
7 Start fed-batch
effect of glucose during the cultivation and to find
the optimal cultivation conditions for biomass
4 and/or EPS production. So, the production of
3 polysaccharide is maximised when the glucose
concentration is very low in the fed-batch stage of
cultivation. Both bioprocesses (biomass production
18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 and EPS synthesis) are stimulated when the
Cultivation time, h concentration of glucose is high.
Fig. 7. Consumption of glucose and formation of
biomass and EPS during fed-batch cultivation
of H. mediterranei with control of DO2 (25% ACKNOWLEDGEMENTS
in the fed-batch stage), pH (7.2 during entire
cultivation) and temperature (38°C during This research work was done under the
cultivation) and command of glucose feeding collaborative research project “Optimisation of
using “BioLab”. Parameters of the batch fermentation process in food industry”
exponential feeding function were: VR= 5 l; funded by the International Bureau of BMBF-IB
mX = 7.65 g; cS_in = 100 g/l glucose; cS = 2 g/l Germany and The National Research Foundation
glucose; KLabView = 27; µ = 0.05 h-1. Aeration NRF South Africa.
1 l/min

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