Fermentation monitoring

Fermentation monitoring Bengt Danielsson University of Lund, Lund, Sweden The dominating methodologies which are currently being applied to fermentation monitoring are high-performance liquid chromatography and flow injection analysis. The latter technique is generally combined with immobilized enzymes or biosensors. In an unexpectedly large number of studies, direct measurements on fermentation broth for establishing cell activities are made by flow calorimetry. Current Opinion in Biotechnology 1991, 2:17-22

Introduction

Sampling techniques

Fermentation monitoring covers a vast area, encompassing numerous analytical fields. Consequently, this presentation has had to be focused on bioanalydcal techniques, including biosensors and biosensors in combination with flow injection analysis (FIA). Although both biosensors and FIA are considered in separate reviews in this issue, they can not be omitted completely here because of their increasing importance in bioprocess monitoring and control. However, measurements of physical quantities, such as pH and redoxpotential have been largely excluded.

Apart from direct in situ monitoring, in which the analytical test is conducted directly in the bulk material, fermentat.ion monitoring generally requires removal of samples with or without some sort of filtration or dialysis (Fig. 1). Sampling may seem a trivial problem but, as described by Eitel in [1], it is associated with many requirements that are diflacult to fulfil satisfactorily. Finding a sensor that is suitable for fermentation monitoring is less of a problem than designing a complete analytical system that is compatible with the environment required for the process to take place in. As far as the fermentor operator is concerned, asepticity is the most important demand. This makes in situ probes, particularly those which can be sterilized whilst mounted in the tank, the easiest to use.

Sampling is a major problem in fermentation monitoring and, therefore, it will be considered in detail. A sample needs to be representative of the whole (with respect to site, stability in composition, and time), aseptic, and of the smallest possible volume. Thus, in situ probes are preferable even though they are associated with problems such as fouling and difficulties with calibration. A number of recent reports deal with direct monitoring, particularly of cell mass, and many of them are concemed with calorimetric studies. There have been far fewer applications of biosensors in direct measurements than expected, but this situation will probably change in the future. This review will first discuss sampling techniques, followed by monitoring of cell mass and activity, and the use of nucleic acid probes. The determination of lowmolecular-weight constituents, substrates and products is then considered. The various methods have been assigned to the following categories: direct methods, such as in situ fluorescence; probes and biosensors, apPlied both directly and in sample lines; flow injection analysis and other flow techniques in which biosensors may be used; and high-performance liquid chromatography (HPLC). Finally, techniques for determining larger molecules, such as proteins and hormones are described.

A commonly used and approved method of aseptic sampling involves opening and closing steam-sterilizable valves in order to draw a sample into a collection vessel. An automated system for this type of sampling, the MX-3 BioSampler, was presented recently by Webb et al. [2]. The prerequisites for on-line control by FIA have been outlined by Ogbomo et at [3 °] in a study that summarizes sampling techniques and analysis of both volatile compounds and dissolved substances. The same group describes the use of an electrodialysis unit to separate L-leucine from ct-ketoisocaproic acid in a sample stream from a Corynebacterium glutamicum fermentation, thereby enabling both compounds to be assayed with the same dehydrogenase [4]. This method may be used when charged substances are involved. Volatile compounds have been sampled through various gas diffusion membranes, such as silicone, potypropylene and porous Teflon membrane. Ktinnecke and Schmid [5°] have developed a gas diffusion FIA system with excellent performance for on-line monitoring of ethanol in

Abbreviations FET--field-effect transistor FIA--flow injection analysis; GC/MS~gas chromatography/mass spectrometry; HPLC--high-pefformance liquid chromatography. ~) Current Biology Ltd ISSN 0958-1669

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18 Analyticalbiotechnology Monitoring of cell mass and biological activity ,L,

Buffer

Fi& 1. A schematic representation of the alternative methods for removal of samples from fermentors. (a) in situ probe; (b) off-gas analysis; {c) headspace analysis; (d) non-invasive analysis (e.g. a fluorometric probe); (e) dialysis with dialyzer or dialysis probe inside fermentor; (f) whole broth pumped to sensor; (g) sampling through a crossflow filtration (or dialysis) unit; (h) manual or automated sampling through a steam-sterilized valve system; (i) sampling of gaseous or volatile compounds through a gas permeable membrane. S, sensor/sensing unit.

cultivation media. They have studied different membrane materials, as well as comparing the performances of two different alcohol oxidases, from Candt2ia boidinii and from Pic.bia pastoris Crossflow or tangential flow microfiltration through a sterile filter (that also constitutes a sterile barrier between the fermentor and the sample) provides a versatile means of bioprocess sampling. A high tangential flow rate over the membrane surface reduces fouling to an acceptable degree except in the most problematic broths, such as in penicillin fermentation. M/llipore/Waters has designed a special, automatic unit for on-line HPLC. A crossflow filtration unit equipped with standard 47 mm membranes for use with FIA systems for bioprocess monitoring has been constructed by Cram and co-workers [6.]. Their online analyzer was tested in various fermentations for the measurement of glucose, ethanol, phosphate and ammonia. The accuracy and reproducibility were comparable with that obtained by off-line analysis. The maximum frequency of analysis was 30 samples per hour, including up to 200-fold dilution of the sample.

One of the most important factors to be determined during a fermentation process is the viable and total cell count. Kell et aL [7**] give an excellent overview of the problem associated with real-time monitoring of cellular biomass and discuss the physical approaches to its solution. The authors point out that most current on-line methods for the estimation of biomass in large-scale fermentors are indirect, being based on measurements of the rate of 0 2 uptake, o r C O 2 evolution, or the relative amount of any chemical that is produced or consumed by the cells at a constant rate. In spite of its limitations, this method has potential advantages, for instance in media where it is dit~cult to analyze biomass. This is illustrated by several of the studies found in this literature survey. Siegmund and Diekmann [8] have described a method for biomass determination in aerobic cultivation in media containing solid substrates. This method is based on off-line determination of O 2 uptake with an O2 electrode. When applied to a penicillin fermentation, using a suitable calibration factor, it was found to provide a better reference parameter for fermentation control than the net dry weight of the cells. A method for biomass estimation using on-line glucose monitoring by FIA has been developed by Valero et al [9]. A Minim (Millipore/Waters) crossflow microfiltratton unit provided a cell-free flow for the FIA system that was operated with soluble glucose oxidase and peroxidase. An extended Kalman filter was used in the calculations. A somewhat different example is seen in the on-line es~nation of algal growth in outdoor ponds, which is derived from the net O2 production rate [10]. This method was based on an analysis of the system's response to a perturbation of dissolved 0 2 concentration that was initiated every 3 h. Flow cytometry involves expensive instrumentation, but is a powerful technique that provides important information about cell size distribution and quality, such as RNA and protein content (after staining). A good example is given by Dalili and Ollis [ 11. ] who describe a flow cytometric kinetic study of hybridoma cell growth and antibody production. Culture fluorescence also provides information on biomass and can be measured by simpler instrumentation. Two such studies were carried out by Lid6n et al [12] and Greer et al [13o]. Both groups made parallel measurements by microcalorimetry. The authors used the FluoroMeasure system from BioChem Technology Inc., operated at 340 or 366nm (excitation) and 460nm (emission wavelength), reflecting the values of NADH fluorescence. The biphasic growth of SaccA~romyces cerevisiae could be followed by fluorescence detection, although it was found that recordings of small metabolic changes were far more reliable when combined with microcalorimetry [12]. Similarly, both of these techniques were found to be reliable indicators of biomass in a study on Xantbobacter autropbicu4 in which various on-line and off-line measurements were compared [13"].

Fermentation monitoring Danielsson 19 Plant cells impose special problems on biomass determination because they are much bigger than micro-organisms and grow in aggregates or as a callus. In a study on four different plant cell cultures [14o], linear relationships were found between the dry cell mass and the conductivity change in the cell-free medium in all cases. An instrument for non-invasive detection of low-level microbial activity by osclllometric conducfinaetry has been described in two articles by Blake-Coleman and Cossar [15.%16.']. Because it is a universal method for the assessment of microbial activity, calorimetry has been employed in many of the works published during the annual period of review. In the studies mentioned above [12,13.], ordinary LKB/ThermoMetric microcalorimeters were used, whereas in others, specially designed calorimeters have been employed. A bench-scale two litre calorimeter, marketed by Mettler Instruments AG, has been modUied, with special attention to improved aeration, for use as a chemostat [17]. In this study, heat measurements on S. cerev/s~e were used to control fed-batch fermentations. Stable control was achieved when the controller gain was set below the maximum specific cellular growth rate. Boe and Lovrien [18.] compared calorimetry with six other methods for measuring cell number during aerobic cultivations of Escber/~/a coil Spectrophotometry (turbidity measurement) was found to be the most rapid method, but it was seriously impaired by interference from pigments and foreign particles. Calorimetry, on the other hand, was capable of measuring cell numbers in opaque samples, although 10-30 min were required for the analysis of each sample. Detection of microbial contamination is an important topic in the fermentation industry. Gas chromatography/mass spectrometry (GC/MS) can detect extremely small amounts of bacteria, even in complex media, by the determination of specific chemical markers. By using GC/MS with selected ion monitoring, Elmroth and co-workers [19" ] were able to determine Enterobacter cloacae over the range 1-10000ppm in cultures of Leuconostoc mesenteroides

DNA probes Analyses based on nucleic acid hybridization reactions - - gene probes---offer a new alternative for characterizing cell populations and detecting infections. The application of gene probes to fermentation monitoring is still hampered by the long processing time required (several hours). However, two examples illustrating their potential usefulness have been selected for this review. Moore et a t [20] have developed a 'biosensing' systern based on immobilized single-stranded DNA for the determination of critical subpopulations in mixed culture biological systems. Johansson and B61ske [21o] have analyzed cell cultures for mycoplasma infections using a commercial DNA probe based on mycoplasm rRNA genes. DNA probes were found to be adequately sensitive in most cases.

Determination of low-molecular-weight constituents Direct methods

More direct methods used for fermentation monitoring, which do not require reagents, have often been based on fluorometric probes, many of them registering NADHfluorescence. This topic has been reviewed by Luong and Mulchandani in [1]. Other non-invasive photometric methods now emerging include near-infrared spectroscopy and laser Raman spectrometry. Cavinato et a t [22*] used short-wavelength near-irffrared spectroscopy (in the range 7(Kl-ll00nm) for the determination of ethanol during fermentation, by connecting a diode army spectrometer with a fiber-optic probe placed outside the fermentor glass wall. Ethanol concentrations could be predicted with errors of less than 0.4%. A quantitative laser Raman spectrometric method, which employs fiber-optics for monitoring alcoholic fermentations, has also been described [23]. Ethanol, glucose and fructose could be determined remotely with a precision of about 10g per litre. This type of sensor is gaining more and more recognition as the number of metabolites that it can measure and its precision and sensitivity improve. One important advantage is the potential for non-invasive, remote multicomponent analyses. In [1], Wolfbeis gives a good overview of fiber-optic sensors in bioprocess control. Gases and volatiles are more easily accessible for measurements than substances in solution. Monitoring of volatiles in alcoholic fermentations by a gas_chromatography unit connected via a gas membrane has been investigated by Groboillot and co-workers [24.]. Acetaldehyde, ethyl acetate and alcohols were measured. Fatibello-Filho etal, [25] found that a piezoelectric quartz crystal coated with tetrakis(hydroxyethyl)ethylenediaminewas a good monitor for COz in fermentations. The sensor could be used over a 2-week period with linear response within the range 1.8-16% (volume-to-volume). Another use for piezoelectric crystals in fermentation monitoring is as a viscosity meter in dextran fermentations [26]. A miniature fuel cell equipped with a porous Teflon membrane interface, designed to measure dissolved H2 continuously, has been tested under anaerobic digestion conditions [27*]. In water, the detector responded quickly to variations in H2 concentrations in the range 80-770 nM, The response was also fast and reliable in a methanogenic reactor. In our laboratory [28.], we have established another method for determining H2, using a highly sensitive palladium metal-oxide-semiconductor (Pd-MOSFET). This study also employs an enzyme thermistor calorimeter to monitor the influence of ampicillin on E. coli fermentations. On-line gas analysis by mass spectrometry is very powerful and allows several compounds to be monitored simultaneously. Heinzle et al. [29"'] have made a very good presentation of the limitations and applications of the technique.

20

Analytical biotechnolokn/ One of the few examples of biosensors designed for direct use in a fermentor is the mediator electrode for the detection of glucose (at less than 16raM), which can be operated in fermentation media for 12 weeks, or longer [30°]. The stability of this type of electrode has been achieved by incorporating the electron mediator (ferrocene or tetrathiafulvalene) in a graphite-epoxy resin matrix, and coating it with immobilized glucose oxidase. Further information on sensors for measurements in broth samples can be found in [1] in the chapters on electrochemical sensors and electrochemical biosensors.

Applications of flow injection analysis and other flow techniques HPLC is a very powerful and commonly used technique in off-line analysis for following fermentations. The analysis time is decreasing and special analyses can presently be done in less than two to three minutes [ 1]. An obvious advantage of HPLC is its capability for multicomponent analysis. For instance, in one run, a long row of common carbohydrates in complex media may be determined following some sample clean-up [31]. The technique that clearly dominates bioprocess monitoring and the one that currently appears to be the most practical is FIA. It can exist in a variety of forms, usually in combination with immobilized enzymes and with detecting systems such as biosensors. Several studies [3.,4,5,,6 o] have already been mentioned in this review and, because FIA and biosensors have each been allotted separate reviews in this issue (Hall, pp 9-16 and Guilbault, pp 3-8, respectively), others will only be mentioned briefly. One important publication is by Nielsen et a/. [32 oo] who have designed a complete FIA-system for on-line monitoring of lactic acid fermentations. It comprises glucose, lactose, galactose, and lactate determinations using oxidase enzyme reactors with chemiluminescent hydrogen peroxide detection. Protein analysis based on the Biuret reaction and photometric biomass detection complete the system. A four-channel enzyme thermistor has been designed by Hundeck et al. [33"] and applied to the on-line deterruination of different sugars, using immobilized enzymes and entrapped micro-organisms. Thermistor probes have been reviewed further by Jespersen in [ 1] and by myself [34°]. A most promising type of enzyme-modified bio-fieldeffect transistor (FET) has been presented recently by Brand and co-workers [35"]. Determinations of urea, glucose and 13-1actams, using this technique were reported. Of particular interest was the on-line monitoring of glucose during cultivation of E. cola In cases where antibodies are a~ilable, automated enzyme immunoassay procedures can also be developed for sensitive on-line determination of smaller molecules present in process solutions. An example of this is an automated homogeneous enzyme-linked binding assay for

folate and biotin in broth samples [36"]. In this case, specific binding proteins were used, but the same principle would apply to antibody binding.

Determination of larger molecules During femaentation and downstream processing it is often necessary to follow the concentration of larger molecules, such as hormones, enzymes or antibodies, which could be either the desired product or contaminants. Assay procedures found in the literature are generally based on automated enzyme immunoassays or continuous-flow (FIA) methods for enzyme activity deterruination. An example of the latter is the automated FIA determination of cellulase activity in bioreactor preparations [37]. This technique is based on the determination of reducing sugars produced by the action of ceUulase on carboxymethylcellulose. The limit of detection was O.1 units per ml and samples were taken at a rate of five per hour. In principle, this method could be used with any carbohydrase which acts on soluble substrates to produce reducing sugars. The usefulness of combinations of FIA and enzymelinked immunosorbam assays for bioprocess monitoring and control has been discussed by Mattiasson et al. [38°]. In particular, they describe automated versions of flow injection binding assays with cycle times as short as 70 s. The streaming potential that can be registered over a small column filled with a specific adsorbent, when the sample is passed through it, has been exploited in the construction of a new affinitysensor [39]. The device was tested with immunoglobulins G and M, and with monoclonal antibodies during culture. The lowest concentrat/on of sample recorded was 0.1 taM. Finally, in comparison with process control using automated HPLC, other chromatographic procedures can be automated for monitoring larger molecules, such as hormones. A fully automatic system for on-line monitoring of insulin-like growth factor-1 secreted during fermentation of E coli has been presented by Lundstr6m et al. [40]. It is based on a BIOPEM (from B Braun, Melsungen AG) sampling device and affinity chromatography on immunoglobulin G Sepharose (fast flow) followed by integrated/ultraviolet recording. This system is reported to be highly reproducible and can be used over a broad sample concentration range.

Conclusions In recent years, the bioprocess industry has recognized the advantages of making specific measurements as close to the process as possible. In order to make use of increasing knowledge about the control of processes, new analytical methods have to be invented and applied. One current trend is automation of techniques such as HPLC

Fermentation monitoring Danielsson

and FIA, bringing them closer to the process itself. Another is the acceptance of automated on-line sampling, even though it sometimes still only involves automation of manual procedures and equipment. Another ongoing development in bioprocesses is the design and application of comparatively cheap, but specific, biosensors of various kinds, including enzyme electrodes, thermal probes, optrodes and semiconductor sensors, such as bio-FETs. This trend began with the rapidly expanding use of immobilized enzymes in FIA procedures, and it will certainly become increasingly important in the future. Non-invasive techniques like near-infrared spectroscopy are also expected to attract greater attention.

References and r e c o m m e n d e d reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest .• of outstanding interest 1.

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BOE I, LOVRmNR: Cell Counting and Carbon Utilization Velocities via Microbial Calorimetry. Biotechnol Bioeng 1990, 35:1-7. Cell number estimation through calorimetry is compared with that achieved by spectrophotometry, Kjeldahl nitrogen, biuret protein, dry weight, microscopy direct counting and viable colony counting. Both calorimetry and spectrophotometry are found to have advantages over the other methods.

KELLDB, MARKXGH, DAVEYCL, TODD RW: Real-time Monitoring of Cellular Biomass: Methods and Applications. Trends Anal Chem 1990, 9:190-194. Various physical approaches to the problem of devising a real-time biomass probe are reviewed. The best solution involves direct measurement of the dielectric permitivity of cell suspensions at radio frequencies.

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8.

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9.

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7. ..

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33. •

HUNDECKH-G, SAAUERBREIA, HOBNERU, SCHEPERT, SCHI[IGERL If, KOCH R, AN'IXANIKIANG: Four-channel Enzyme Thermistor System for Process Monitoring and Control in Bioteclmology. Anal Cbim Acta 1990, 238:211-221. A description of the design of a new enzyme thermistor system and its application to bioprocess monitoring. 34. DANIELSSONB: Calorimetric Biosensors. J Biotechnol 1990, • 15:187-200. A general up-to-date review. 35.

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•

36. •

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B Danielsson, Department of Pure and Applied Biochemistry, University of Lund, PO Box 124, Lund S-221 00, Sweden.