on-line monitoring of glucose and penicillin by sequential
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ANALmcA
CHIMICA
ACM
Analytica Chimica Acta 320 (1996) 199-205LS VI R
On-line monitoring of glucose and penicillin by sequential
injection analysis
Rong Wei Min, Jens Nielsen * John Villadsen
Center for Process Biotechnology and Deparhn ent of Biotechnology, Techni cal Uni versity of Denmark, DK-2800 Lyngby, Denmark
Received 13 July 1995; revised 6 October 1995; accepted 8 October 1995
Abstract
A sequential injection analysis (SIA) system has been developed for on-line monitoring of glucose and penicillin during
cultivations of the filamentous fungus Penicil lium chrysogenum. The SL4 system consists of a peristaltic pump, an injection
valve, two piston pumps, two multi-position valves and a detector. The glucose analyzer is based on an enzymatic reaction
using glucose oxidase, which converts glucose to glucono-lactone with formation of hydrogen peroxide and subsequent
detection of H,O, by a chemiluminescence reaction involving luminol. The penicillin analysis is based on formation of
penicilloic acid by penicillinase. Penicilloic acid is detected either by a chemiluminescence method or by a decolorization
method. In the chemiluminescence method the penicilloic acid is quantified by its quenching effect on the chemilumines-
cence signal obtained when luminol reacts with iodine. In the decolorization method the penicilloic acid is detected
spectrophotometrically by the decrease in the absorbance of an iodine-starch complex.
Keywords: Enzymatic methods; Chemiluminescence; Glucose; Penicillin; On-line monitoring; Cultivation; Sequential injection analysis
1 Introduction
Flow injection analysis (FIA) is a powerful analy-
sis tool for on-line monitoring of cultivation pro-
cesses, especially in connection with quantification
of substrate uptake rates and product formation rates
[l]. Thus, in studies of penicillin production by the
filamentous fungus Penicillium chrysogenum it is
important to monitor glucose, which is the major
carbon and energy source, and penicillin which is the
majqr product.
A large number of different FIA systems have
been reported for the determination of glucose [2].
* Corresponding author.
Most of these are based on oxidation of a-o-glucose
to a-glucono-6lactone and H,O, by glucose oxi-
dase followed by detection of the formed hydrogen
peroxide either by reaction with phenol and 4-
aminophenazone [3-51, by a chemiluminescence re-
action [6,7], or by an electrode [8,9]. Several glucose
analysis methods have been implemented for on-line
monitoring of different cultivation processes [lo-121,
and the best method is probably the chemilumines-
cence method, which is based on oxidation followed
by detection of H,O, through a chemiluminescence
reaction involving luminol and K,Fe(CN), [13].
Several different FIA systems for measurement of
penicillin have been described. Most of them use
P-lactamase to hydrolyze the penicillin followed by
detection of the formed penicilloic acid by various
0003-2670/96/ 15.00 0 1996 Elsevier Science
B.V. All rights
reserved
SSDI 0003-2670(95)00523-4
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200 R. W. Min et al. /Analytica Chimica Acta 320 I 996) 199-205
methods: (i) reduction of molybdoarsenic acid to
molybdenum blue, which can be measured spec-
trophotometrically [14,15]; (ii) reduction of iodide to
iodine ions and measuring the iodine consumption
by the decrease in the absorbance of an iodine-starch
complex [15,16]; (“‘)11 measurement of the pH change
caused by hydrolysis of penicillin, either by use of
pH-dependent colour indicators [17,18] or by a pH-
electrode [15,19,20]; and (iv) measurement of the
reaction enthalpy of the enzymatic reaction by a
thermistor [21,22]. There are also several reports on
the integration of a penicillin biosensor into FIA
systems. These biosensors are either based on immo-
bilization of p-lactamase on a rapidly responding
pH-electrode [23-261 or on immobilization of peni-
cillin amidase on a pH-sensitive field effect transis-
tor [27]. In a comparison of three of the FIA systems
(molybdenum blue, iodometric and potentiometric)
Carlsen et al. [15] concluded that the iodometric
method is best suited for measurement of penicillin
in cultivation media, and it is an attractive alternative
to liquid chromatography (LC) due to the higher
analysis frequency. Of the above mentioned methods
three have been implemented for on-line monitoring
of penicillin V during penicillin cultivations: the
iodometric method [28]; the enzyme thermistor [29];
and the biosensor based on p-lactamase [28]. All
three methods give good results, but for industrial
monitoring the biosensor is to be preferred due to the
simple design of the FIA system [28]. However, a
drawback of the biosensor is that it measures the
combined sum of penicillin and penicilloic acid,
whereas the iodometric method permits both peni-
cillin and the combined sum of penicillin and peni-
cilloic acid to be measured [El.
A general drawback of FIA is that it is a single
component analysis, and often one requires simulta-
neous measurement of several components. For this
purpose a further development of FIA called sequen-
tial injection analysis (SIA) has been introduced. The
concept of SIA is based on sequential aspiration of
the sample and reagent by switching a multi-position
valve, followed by dispersing the sample and reagent
into a detector [30,31]. This allows for measurement
of more than one compound in the same manifold, as
described in [32] for off-line measurements of glu-
cose, lactate and penicillin during penicillin cultiva-
tions. SIA is therefore well suited for simultaneous
on-line measurements during cultivation processes.
In the past the use of SIA for monitoring of single
components has been demonstrated [33-361, but to
our knowledge there have been no reports on simul-
taneous on-line measurement of more than one com-
ponent.
Power
multiplexing
Amplitier
. . . . . . . *.*.;
0;
I Muithpositlon
i vahm II
: Reauorcoil
i
(Dummyremctor)
.i Detector
h@-
Sample cdection
Injection valve
Fig. 1. Experimental set-up for the SIA system.
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201
In this study we describe a SIA system for on-line
monitoring of glucose and penicillin during peni-
cillin cultivations. The glucose analyzer is based on
glucose oxidase and the chemiluminescence reaction.
Two different methods for penicillin analysis have
been tested. Both are based on degradation of peni-
cillin to penicilloic acid by penicillinase. In the first
method penicilloic acid is measured by a chemilumi-
nescence method, which is based on measuring the
quenching effect of penicilloic acid on a chemilumi-
nescence signal obtained by reaction between iodine
and luminol. In the other the amount of penicilloic
acid is measured by the classical iodimetric method
in which iodine is reduced to iodide ions.
2. Experimental
2 1
Equipment
An overview of the SIA system used for on-line
monitoring of glucose and penicillin is shown in Fig.
1. An in situ membrane module (ABC, Munich) is
placed directly in the bioreactor and is used for
withdrawing cell free samples. The SIA system con-
sists of a peristaltic pump (Watson-Marlow), two
piston pumps (Microlab 940, Hamilton), one multi-
position valve with 6 positions (Rheodyne), one
multiposition valve with 10 positions (Valco) and an
injection valve (Rheodyne). A straight piece of PTFE
tubing with a volume of 0.25 ml (0.5 m) connects
the bioreactor with the 6-position valve which is
used to choose between sample and buffer. The
volume of the sample loop of the injection valve is
0.30 ml. The piston pump is driven by a step motor
with 1000 steps. The whole system is operated by a
PC with in-house developed software. Piston pump I
is used for both glucose and penicillin analysis,
whereas piston pump II is used only for penicillin
analysis with the chemiluminescence method. Two
detectors are used: (1) a chemiluminescence detector
(in house design) for glucose analysis and analysis of
penicillin by the chemiluminescence method; and (2)
a spectrophotometer detector (Hitachi, U-l 100) for
penicillin analysis by the decolorization method. The
inner volume of the chemiluminescence detector is
0.008 ml, and of the spectrophotometer 1 ml. The
holding coil (with a volume of 0.8 ml> is connected
to the multiposition valve II by a straight piece of
tubing (with a volume of 0.075 ml). The reaction
coil is placed in front of the detector to gain a final
intimate mixing of the sample and the reagents. The
holding coil and the reaction coil are tightly knit in a
meander winding on a metal plate to give an almost
complete radial mixing of the aspirated zones. All
tubing is made of PTFE (0.8 mm i.d.1.
The enzyme reactors are prepared as described in
[32,37,38] by immobilization of glucose oxidase
(GOD) and penicillinase, respectively, on a piece of
nylon tube using the o-alkylation method. Each en-
zyme reactor is 1 m long with an inner diameter of 1
mm. To ensure a high degree of conversion of
glucose in the GOD reactor the sample is dispersed
and aspirated twice in the reactor. This gives a high
residence time in the reactor, and the radial disper-
sion ensures an intimate contact of the sample with
the enzyme which is immobilized on the tube wall.
The software is written in Turbo-Pascal and it
contains subroutines for control of the injection valve,
the two multiposition valves, the piston pumps, data
acquisition, calibration and data analysis.
2.2. Reagents
Reagents for the glucose and the penicillin ana-
lyzer based on the chemiluminescence methods were
prepared as described in [32]. For the penicillin
analyzer based on the decolorization method an io-
dine stock solution containing 10 mM iodine in 10
mM KI was stored at 4°C. A fresh 2.8 mM solution
was prepared daily by dilution with phosphate buffer
at pH 6.5. A stock starch solution was prepared as
follows: A strong starch solution with 10 g starch
dissolved in 50 ml water was prepared. This solution
was diluted up to 500 ml with a boiling solution of
distilled water containing 4.3 PM KI. The stock
starch solution could be kept at least for one week. A
starch solution was prepared by 5 times dilution with
a 4.3 PM RI solution.
2.3. Standards
Glucose and penicillin standards were prepared in
10 mM phosphate buffer solution adjusted to pH 6.5.
A 2 g/l penicilloic acid stock solution was kept at
4°C and standards were prepared by dilution with 10
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202
R. W. Min et al. /Analytica Chimica Acta 320 1996) 199-205
Table 1
Arrangements of ports of multiposition valve II
Port Glucose Penicillin
Penicillin
analysis analysis A
analysis B
1 0.010 g/l 0.100 g/l 0.100 g/l
2
0.100 g/l
0.500 g/l 0.500 g/l
3 1.000 g/l
1.000 g/l 1.000 g/l
4
7.000 g/l 1.800 g/l
1.800 g/l
5
Sample Sample Sample
6 Detector Detector
Detector
7 Enzyme reactor Iodine
Iodine
(GOD)
8 Luminol
Buffer Starch
9
K,Fe(CN),
Buffer
Buffer
10
EDTA Buffer Buffer
mM phosphate buffer at pH 6.5. All standards were
prepared daily.
2.4.
LC analysis
For off-line analysis of penicillin a reversed-phase
LC method was used as described in [39].
2.5. Cult iuat ions
Batch and continuous cultivations were carried
out using an industrial strain of
Penicillium chryso-
genum. The
procedure was as described in [401. All
cultivations were carried out using a defined medium.
In the batch cultivations sucrose was used as carbon
and energy source, whereas the continuous cultures
were carried out using glucose as carbon and energy
source.
2.6. Procedur e
The ports of the multiposition valve II were ar-
ranged as specified in Table 1. For multiposition
valve I only two ports were used: port 1 connected to
buffer and port 2 connected to the sample stream.
The other ports were also connected to buffer to
prevent formation of air bubbles.
For on-line measurement the sample was pumped
by the peristaltic pump through multi-position valve
I to the sample loop of the injection valve. When the
injection valve was switched from the load position
to the injection position, the sample was aspirated
into the holding coil by piston pump I. After aspira-
tion of the sample the injection valve was switched
back to the load position and a new sample filled
into the sample loop. Analysis of one sample took 4
min corresponding to a sample frequency of 15
samples per hour. The analyzer was calibrated with
two standards (each measured three times) after mea-
surement of 15 samples. During calibration multipo-
sition valve I was switched to port 1 and the injec-
tion valve was switched to the load position resulting
in washing of the sample loop with buffer. This also
ensured washing of the penicillinase enzyme reactor,
which was placed between the peristaltic pump and
multiposition valve II. If the sample signal was
outside the calibration range the analyzer was in-
stantly calibrated with a new set of standards (again
two standards, each measured three times). After this
new calibration the above mentioned procedure with
measurement of 15 samples followed by calibration
was restarted.
For off-line measurements an automatic sample
collection system was used. When the peristaltic
pump feeding the injection valve was closed another
peristaltic pump was used to pump sample to a
sample collector kept in a refrigerator at 4°C.
2.7.
Gl ucose analy zer
The procedure of the glucose analyzer was as
described in [32]. However, to increase the degree of
conversion in the enzyme reactor the sample was
held in the reactor for 5 s whereafter it was aspirated
and again dispensed into the enzyme reactor where it
was held for another 5 s before it was aspirated back
into the holding coil. The linear range of the glucose
analyzer can be varied by changing the sample vol-
ume. In this work three different sample volumes
have been used: 200 ~1 (0.010-0.100 g/l); 30 ~1
(0.10-1.00 g/l); and 2 ~1 (1.0-7.0 g/l). This en-
abled measurements of glucose between 0.01 and 7.0
g/l.
2.8. Penicillin analyzer
The penicillin analyzer is based on an enzymatic
conversion of penicillin to penicilloic acid, and two
different methods have been applied for measure-
ment of the formed penicilloic acid. In both methods
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R. W . M in et al. A pica Chim ica Act a 320 (1996) 199-205
203
S-sunpk
I - lodlne
L-Luminol
B-Surfer
ST-StNdl
(A)
(B)
Fig. 2. Measurement of penicillin by the decolorization method. (A) Measuring the sample signal. (B) Measuring the reference signal.
the sample is pumped through an enzyme reactor
containing penicillinase (see Fig. 1) before it is
aspirated into the SIA system. With a flow rate
below 0.2 ml/min there is 100% conversion in the
reactor. For calibration of the SIA system standards
with known concentration of penicilloic acid are
used, and as described for the glucose analyzer the
linear range can be changed by varying the sample
volume. For both methods three sample volumes
have been used: 50 ~1 (0.10-0.50 g/l); 25 ~1 (0.
0.50-1.00 g/l); 10 ~1 (1.00-1.80 g/l). For the
chemiluminescence method the procedure was as
described in [32]. Just like the chemiluminescence
method, the decolorization method is based on reac-
tion of penicilloic acid with iodine, but here the
reaction is quantified by the classical iodimetric
method where the iodine consumption is detected
spectrophotometrically by the decrease in the ab-
sorbance of an iodine-starch complex. First sample
(or buffer) is aspirated together with iodine (80 ~1)
and starch (100 ~1) into the holding coil, and there-
after the combined sample and reagent plug is dis-
pensed into the detector (see Fig. 2). The reference
signal is obtained by aspirating buffer instead of the
sample.
3. Results
3 1 On-line monitoring of glucose
The SIA system has been used for on-line moni-
toring of glucose during several batch cultivations of
Penicitlium chrysogenum, and Fig. 3 shows the re-
sults from one of these experiments. In the batch
cultivation the initial medium contained 25 g/l su-
crose. During sterilization of the medium the sucrose
was partly hydrolyzed to glucose and fructose result-
ing in an initial glucose concentration of 700 mg/l.
The batch cultivation was inoculated with spores,
and when they germinated after approximately 15 h
the fungus expressed invertase activity resulting in
hydrolysis of sucrose to glucose and fructose 1411.
The rate of hydrolysis was faster than the uptake and
metabolism of glucose, and consequently the glucose
concentration increased. After 27 h the glucose con-
centration reached a level of 3.5 g/l, whereafter it
decreased to almost zero over the next 10 h.
Throughout the batch cultivation the on-line mea-
surements of glucose corresponded very well with
the off-line measurements using an YSI analyzer.
3.2.
On-line monitoring of penicillin
The two penicillin analyzers have been applied
for on-line monitoring of penicillin during continu-
ous cultivations (Figs. 4 and 5). Each continuous
cultivations was started as a batch culture as de-
scribed in [40], and when the glucose and fructose
Fig. 3. On-line measurement of glucose during a batch cultivation
of P. chrysogenum compared with off-line measurements using
an YSI analyzer. (-1 SIA, (A 1 YSI.
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204
R. W. Min et al /Analytica Chimica Acta 320 1996) 199-205
204 * I
” ” r ’
0
50
100
150 200 250 300 350 400 450
Cultivation time hour)
Fig. 4. On-line measurement of penicillin with the chemilumines-
cence method during a continuous cultivation with a dilution rate
of 0.025 h-l. Off-line measurements by LC are shown for
comparison. (-1 SIA, (A ) LC.
were exhausted feed addition was initiated. The .?+eed
contained the precursor phenoxyacetic acid for pro-
duction of penicillin V, and when the feed was added
the cells started to produce penicillin. After approxi-
mately two residence times a steady state was ob-
tained in the penicillin concentration. After approxi-
mately five residence times at steady state the peni-
cillin concentration decreased due to the formation
of lower producing mutants [40]. With both methods
there is a very good correspondence between the
on-line measurements by the SIA system and the
off-line LC measurements ( Figs. 4 and 5), but the
decolorization method gives the best results since the
noise level is very low. This method also has the
advantage of using only one piston pump. However,
h
5,
I
i 4. 5
s 1
0 SO 100
150 200 250 300 350 400
Cultivation time hour)
Fig. 5. On-line measurement of penicillin with the decolorization
method during a continuous cultivation with a dilution rate of
0.058 h-l. Off-line measurements by LC are shown for compari-
son. (-_) SIA, (A ) LC.
with both analyzers it is possible to obtain a good
estimate of the productivity in the bioreactor from
the large number of measurements. With both sys-
tems it was necessary to correct for a background
signal, which was determined by aspirating a buffer
solution which was subsequently pumped through
the enzyme reactor. During the cultivations there was
some degradation of penicillin to penicilloic acid,
and the on-line measurements with the SIA systems
were therefore compared with the sum of these two
compounds, which are both measured by the applied
LC method.
4. Conclusion
In the present work, a glucose analyzer and two
penicillin analyzers have been tested for on-line
monitoring of glucose and penicillin during peni-
cillin cultivations. A good agreement between the
on-line measurements and off-line measurements is
obtained. Compared with traditional FIA on-line sys-
tems, the SIA analysis system is simpler, e.g., a
dilution system is not required since the sample
volume can be adjusted. Furthermore, the running
cost of SIA is much smaller than for FIA, since only
a small amount of reagent is spent for each analysis,
e.g., in the glucose analyzer only 50 ~1 luminol and
50 ~1 K,Fe(CN), is used per sample. Finally, the
required sample is much smaller than a FIA system,
which is of importance for on-line monitoring of
laboratory bioreactors.
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