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



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.



R. W. Min et al. /Analytica Chimica Acta 320 1996) 199-205

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



202


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



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.



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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Documents

On-line Monitoring of Glucose and Penicillin by Sequential