Optimisation of the biosensor for in situ fermentation monitoring of glucose concentration

Biosensors & Bioelectronics 6 (1991) 669-674

Optimisation of a biosensor for in situ fermentation monitoring of glucose concentration* Joanne

Bradley & Rolf D. Schmid

Department of Enzyme Technology, GBF (Gesellschaft fur Biotechnologische D-3300 Braunschweig, Germany

Forschung mbH).

(Received 23 July 1990, revised version received 11 March 1991; accepted 4 April 1991)

The optimisation of a mediated amperometric glucose biosensor designed for in siru bioprocess monitoring leading to improved stability (4 days of continuous use) and extended working range (up to 20 g 1-t) is described. An example of its application to fermentation monitoring is given in the model system of a pulse-fed baker’s yeast cultivation on defined medium. Abstract:

Keywords: in situ enzyme determination.

electrode,

INTRODUCTION For an effective control of bioprocesses it is necessary to determine every significant parameter as often as possible. Hence the determination of the concentration of glucose, which is often not only the main carbon source but also the growth-limiting substrate, is of particular importance, A limited range of on-line and in situ sensors are available for monitoring glucose concentration, the limits on their development being placed by the stringent requirements imposed: they must be (steam) sterilisable, they have to allow measurement over the range of interest, they should operate on-line without affecting or being affected by the bioprocess, they must respond rapidly to changes in analyte concentration, and their signals must *Paper presented at Biosensors May 1990. 09655663/91/$03.50

PO, Singapore. 2-4

fermentation

monitoring,

glucose

be easily interpretable and either continuously or frequently emitted. In situ designs are perhaps more acceptable to the fermentation industry, where dip-in probes have long been employed for the measurement of pH and dissolved gases, but external flow stream or on-line systems are in many ways the simpler option (Enfors & Molin, 1978). The problem of sensor sterilisation is avoided and any sample preparation required can easily be accomplished. However, there are disadvantages to on-line monitoring, in addition to the delay time involved; the flow stream must be dialysed to prevent microbial action on the sample during transport to the sensor (Cleland & Enfors, 1983; Mandenius et al., 1984) or an addition of metabolic inhibitor is required (Holst et al., 1988), and the technique is not applicable to small-scale batch fermentations or small-volume bioprocesses where the increase in cell density as a result of withdrawal of permeate is of importance (for

@ 1991 Elsevier Science Publishers Ltd.

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J Bradley & R D. Schmid

instance, in the case of physiological studies and bioprocess modelling). Numerous on-line detection systems have been documented, mostly based on the combination of immobilised enzyme columns with flow injection systems and various transducers (Garn et al., 1989; Dremel ec al., 1991; Filippini et al., 1991; Huangetal., 1991). The use ofamperometric enzyme electrodes for continuous glucose analysis in a by-pass line has been demonstrated by Geppert & Asperger (1987). Methods for the production of autoclavable enzyme electrodes by autoclaving of the electrode exclusive of the enzyme have been outlined (Enfors & Nilsson, 1979; Kok & Hogan, 1987). Enfors (1981) developed an immersible probe based on an oxygen electrode covered with a layer of immobilised glucose oxidase. A number of sterilisable glucose oxidase based electrodes for monitoring of Escherichia coli, baker’s yeast and Saccharomyces cerevisiae fermentations have been documented (Cleland & Enfors, 1983, 1984; Brooks et al., 1987; Bradley et al., 1989a, b and d; Filippini et al., 1991). The in situ sensors documented are all plagued with a number of limitations, the first of which is the inherent limited linear range because of the use of the immobilised enzyme (upper limit 3-O g 1-l (Cleland & Enfors, 1983) or 5.4g 1-l (Brooks etal., 1987). Cleland & Enfors (1984) have put forward a solution to this problem by the introduction of a constant buffer flow across the face of the electrode, which allows a linear range of up to 80 g 1-l to be achieved. A second problem is the often inadequate electrode stability in continuous use (13 h (Cleland & Enfors, 1983) 24 h (Cleland & Enfors, 1984) or 9 h (Brooks et al., 1987)) and a third major problem is the relatively long response times (3-9 min (Cleland & Enfors, 1983) or l-2.5 min (Brooks et al., 1987)). This presentation outlines the optimisation of an electrode based on the combination of the mediated amperometric electrode proposed by Brooks et al. (1987) together with the extension of linear range by continuous internal buffer flow (following Cleland & Enfors, 1984) in conjunction with the use of variation in membrane pore size. The electrode response specifications observed before the optimisation were: linear range up to 5.4 g 1-l; stability in continuous use 12 h; and response time 20 s4 min, depending on the mixing efficiency of the vessel (Bradley et al., 1989d). 670

Biosensors& Bioelectnmics 6 (1991) 669-674

MATERIALS

AND METHODS

In situ fermenter probe design and operation The fermenter probe was a modification of that described by Bradley et al. (1989a); the two-part construction was comprised of a stainless steel outer housing and a mediated amperometric enzyme electrode (Fig. 1). The outer housing was closed by a polycarbonate membrane of O-015 pm pore size (Nucleopore Corporation, Pleasanton, USA) and a metal membrane of 2 pm pore size (the kind gift of Pall Filtrationstechnik GmbH, Dreieich, FRG). The polycarbonate membrane provided a sterile barrier between the fermentation broth and the electrode, and the metal membrane provided a rigid support for the polycarbonate membrane which results in a decrease in signal noise. The electrode held four graphite working electrodes (3.05 mm X 2 mm pellets, Ringsdorff-Werke GmbH, Bonn-Bad Godesberg, FRG), which press fitted into recesses in the electrode face and were held against the polycarbonate membrane, poised at +220 mV against a centrally located silver/silver chloride reference element. One of the working electrodes was used -to provide a baseline signal and held only the mediator (l,l’-dimethylferrocene, Aldrich Chem. Co., Steinheim, FRG); the other three were immobilised with glucose oxidase (Grade II from Aspetgi1Zu.s niger, 5000 U mg-‘, Boehringer, Mannheim, FRG) according to the published procedure (Bradley ef al., 1989~). The cavities between the outer housing and the enzyme electrode and that surrounding the reference element were filled with a continuous stream of 0.01 M sodium phosphate buffer (pH 7-O) supplemented with 0.1 M potassium chloride (flow rate 0.38 ml min-‘). The buffer flow, and glucose injections into this flow to provide in situ calibration, were controlled using a simple flow injection analysis (FIA) system (developed in-house) consisting of pumps (Jungkeit, Bovenden, FRG) and calibrant injection valve (Tecator, Hogan&, Sweden) with an injection volume of 100~1. The relationship between the response to internal calibrant and external glucose concentration was constant and was dependent on the membranes and buffer flow rates selected. The potential was poised potentiostat (Bank using a four-channel Elektronik, Gbttingen, FRG) and data were collected using a four-channel chart recorder (Linseis, Selb, FRG) for manual analysis.

Biosensors & Bioelectronics 6 (1991) 669-674

Fermentation monitoring of glucose concentration

Bioprocess Following successful 3 day sterility/stability trials of the electrode in a 10 1 vessel (b10 bioreactor, Giovanola, Monthey, Switzerland), the 1

-2

cultivation was inoculated with a commercially available baker’s yeast (inoculum concentration 4.2 g 1-l wet weight). After a 4 h period in which the culture was allowed to stabilise, glucose was introduced in pulses to approximately 4, 10 and 2og1-‘. Bioprocess conditions applied were: temperature control at 30-31°C; stirrer speed at 200 rev min-’ , with aeration at 0.2 N m3 h-‘; and pH control at 5.0 using 1 M sodium hydroxide. The medium used consisted of: (NH&SO4 15 g l-‘, (NH&HP04 0.63 g l-‘, MgS04.7Hz0 O-11 g l-‘, yeast extract 3 g l-‘, FeS04*7H20 0.05 g l-‘, ZnS04.7H20 0.03 g l-‘, MnS04*H20 0.0035 g l-‘, CuS04.5H20 0.0008 g 1-l. Off-line analysis of glucose concentration was performed using a YSI Model 27 glucose analyser, in accordance with manufacturer’s instructions.

RESULTS AND DISCUSSION

-3

y4

The use of a polycarbonate membrane of 0.015pm pore size in combination with a constant internal buffer flow rate of 0.38 ml min- ’ allowed a linear range of up to 10 g 1-l (correlation coefticient 0995, n = 5) and a working range of up to 20 g 1-l to be achieved (see Figs. 2(a) and (b)). Unfortunately, a sacrifice required by the extension in linear range is the relatively slow response time (90 s to 95% steadystate response). A further advantage of the use of constant internal buffer flow in addition to the improvements in linear range it afforded was the resulting improvement in stability (to approximately 4 days of continuous use with only a 15% loss in response), perhaps as a result of the practically constant ionic strength and pH environment it provided. Parts of the glucose profile of the baker’s yeast cultivation are shown in Figs. 3(a) and (b). The

-5 Fig. 1. Fermenter probe configuration. (1) Reference electrode (~AgCl), (2) sensor insert, (3) heat shrink tubing (provides elecm’cal isolation), (4) platinum contact, (5) sensor head, (6) bayonet Jitting end-cap (retains membranes), (7) polycarbonate membrane, (0.015 pm pore size) and, between the fermenter broth and the polycarbonate membrane, a metal membrane (2 urn pore size), (8) working electrode (graphite pellet, 3.05 mm diameter X 2 mm depth), (9) outer housing, (IO) calibrant flow path. 671

Biosensors & Bioelectronics 6 (1991) 669-614

J Bradley & R D. Schmid

Glucose concentration (g/l) 'Internal pulsed' 'External injected'

Current (nA) 100

‘Internal 0.0

pulsed’

glucose

concentration

2.0

1.0

(g/l)

0

4.0

3.0

5.0

100.0,

2

5

1

, 60.0--

'i 5 0

40.0.20.0--

(b)

“’4

0.0

!‘.’

'External Injected’

10.0 glucose

I

20.0

15.0 concsntmtion

(g/l)

0

Fig. 2.(a) “Internal pulsed” and “external injected” calibration. “Internal pulsed” calibration was achieved by injection of 100 ul aliquots of calibration standards into the continuous internal bufferjow (0.01 M sodium phosphate buffer (PH 7.0) supplemented with 0. I Mpotassium chloride at aflow rate of 038 ml mitt-‘). ‘External injected”calibration was obtained by the injection ofglucose standard into 0.01 Msodium phosphate buffer (pH 7.0) supplemented with 0.1 Mpotassium chloride. (b) Calibration curves obtained on ‘internal pulsed” and “external injected” calibration. Data derived from (a).

electrode clearly detected 4, 10 and 20 g 1-l glucose pulses, and the correlation with off-line analysis was considered acceptable. Figures 3(b) and 4 depict the electrode response profiles obtained on injection of different glucose concentrations both before and during yeast cultivation. The response time on calibration before the cultivation (to 95% steadystate response) was less than 90 s; however, 672

during the bioprocess, the response time (to maximum registered signal) was considerably longer, and varied not only with respect to that observed on pre-cultivation calibration but also with respect to the glucose concentration injected. Additionally, the form of the response profile varied with glucose concentration, and the response to 10 and 20 g 1-l additions showed a considerably enhanced sigmoidal profile as

Fermentation monitoring of glucose concentration

Biosensors & Bioelectronics 6 (1991) 669-614

observed during the cultivation), and glucose injections were made via the same reactor port, this excludes the explanation that the variation in profiles was due to inefficient mixing. Also, on examination of the electrode membranes on completion of the cultivation, no signs of membrane fouling were found. A possible explanation is that the change in response profile is due to more complex factors, such as metabolic activity variations and an increase in cell density during the cultivation. A more detailed knowledge of metabolic status-for instance, that which would be revealed by dissolved oxygen, carbon dioxide and ethanol profiles-would have helped to validate this hypothesis.

CONCLUSION

(b)

Time (min.) elapsed since injection

Fig. 3.(a) Continuous in situ monitoring of a bakers yeast cultivation on dejined medium. The projile of two feed pulses (of approximately 10 and 20g 1-t). (b) The response proJie of a 4gl-’ glucose pulse carried out within the bioprocess and its comparison with fermenter probe response on “external injected” glucose calibration (to 4 g 1-1) co).-

0

1 Time

4

3

2 (min.)

elapsed

since

5

The optimisation of the previously published glucose biosensor in terms of increased stability and working range greatly extends its field of application. The improvements permit its application in the monitoring of glucose profiles during other bioprocesses, such as the fermentation of Aspetgillus niger for the production of glucose oxidase (Bradley ef al., 1990) where the batch process has an initial glucose concentration of 2og1-‘. The data obtained from fermentation monitoring indicate.the importance of rapid and continuous measurement for accurate analysis of the glucose concentration in bioreactors. The continuous signal obtained from the electrode and its ease of analysis means that it can easily be used for bioreactor control and bioprocess modelling; investigations in this direction are now under development.

REFERENCES

injection

Fig. 4. Responseprofiles of thefermenterprobe to injections ofglucose to various concentrations. (0) “External injected” calibration (to 4 g 1-t) before the bioprocess, (0) 4 g I-‘, (A) IOg l-t, (A) 20 g 1-l glucose injection during the bioprocess.

compared with that observed to a 4 g 1-l addition. As both bioprocess and calibration were carried out under the same environmental conditions (i.e. with the same stirring rate and air flow rate, and no increase in viscosity was

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