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In Situ Fluorescence Cell Mass Measurements of Saccharomyces cerevisiae Using Cellular Tryptophan John J. Horvath,*Scott A. Glazier, and Christopher J. Spangler Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899
This work describes a new spectroscopic optical fiberhod technique for in situ real time measurement of cell mass and product concentrations in bioreactors using intrinsic fluorescence. The variable excitation/emission wavelength capability of this sensor allows for species-selective measurement during fermentations. Cell mass (tryptophan) and product concentrations (pyridoxine) have been measured during fermentations of Saccharomyces cerevisiae. The effects of varying substrate concentration and oxygen concentration on the observed cell mass signals are eliminated by direct measurement of cell mass, as opposed to indirect measurement schemes such as those using NADH fluorescence. The sensor is robust and able to undergo many cycles of in situ steam sterilization without degradation, and its fluorescence signal is linear with concentration for all species studied in this work. Tryptophan fluorescence from yeast is shown to be a better measure of cell mass than NADH fluorescence.
Introduction Recent biotechnological developments in the production of new drugs and chemicals require new measurement strategiesfor bioreactors. Bioprocessesrequire very closely controlled environments;these are normally realizable in batch processes. Such control is necessary due to the complex kinetics of most bioreactions, which involves separate periods of growth and product formation and biocatalyst degeneration, along with the mechanical difficulties of handling a rheologically complex material. Most monitoring techniques rely on indirect product measurements and off-line sampling with the inherent problems of contamination, long analysis time, and poor reproducibility. Therefore, the development of new on-line measurement techniques is critical for the future implementation of new bioprocesses. Many of the problems are best avoided using nonintrusive in situ measurement methods. Optical techniques provide the best potential for making rapid and selective measurements. Optical techniques, along with fiber optics, allow the measurement to be made in the sample rather than taking the sample to the measurement instrument with its resultant problems. One of the earliest reports using optical techniques for measurementson biomolecules was by Chance (1954),who used optical density (OD) changes in respiratory enzymes in living cell suspensions. The optical density changed when the aerobic cell suspension became anaerobic. The optical density measured was small (approximately 0.2 OD or less), and special absorption cells were required. The difficulties in measuring small absorbances in cell suspensions led other researchers (Duysens and Amesz, 1957; Chance et al., 1964;Harrison and Chance, 1970; Zabriskie and Humphrey, 1978)to investigate the use of fluorescencefor measurementsin cell culture. These early studies concentratad on the fluorescence from intracellular reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamidedinucleotide phosphate (NADPH), which fluoresces around 460 nm when excited with light between 350 and 370 nm. Duysens and Amesz (1957) observed fluorescence spectra from Photobacteriumphosphoreum, Photobacterium splendidum, and the alga Not subject to
Chlorella and determined that all three species showed fluorescence spectra primarily due to NADH. Chance et al. (1964)investigated sinusoidal oscillations of NADPH fluorescencein Saccharomyces carlsbergensis. The rapid cycling between the oxidized and reduced states of the intracellular pyridine nucleotide was observed upon the addition of glucose or in the aerobic-anaerobic transition of yeast. Harrison and Chance (1970)used a continuous culture of Klebsiella aerogenes to evaluate NADH fluorescence for process monitoring and control. The NADH fluorescence signal was found to be sensitive to the dissolved oxygen (DO) concentration. As the aeration rate was slowly lowered, there was a slow rise in the fluorescence signal, and as the DO content was reduced to zero, there was a rapid increase in the NADH fluorescence signal. The speed of the fluorescence response was greater than the DO electrode, with the fluorescence signal reaching 90% response in 1 s while the DO electrode required 1 min. The studies reported on the great sensitivity of NADH fluorescence to oxygen concentrations, especially those below the range of oxygen electrodes. Zabriskie and Humphrey (1978)used an excitation wavelength of 366 nm and an emission wavelength of 460 nm to study the application of culture fluorescence for measuring biomass concentration. Three aerobic batch fermentations were studied: S. cerevisiae,Streptomyces, and Thermoactinomyces. They approximated the amount of NADH responsible for yeast culture fluorescence by adding iodoacetate to an actively growing yeast culture in order to block the pathway for further production of intracellular energy. This caused a depletion of intracellular NADH reserves followed by cell death. The observed decrease in fluorescence, in comparison to prior calibrations,indicated that 50 % of the culture fluorophore content was intacellular NADH. A log-log-plot of fluorescence intensity vs biomass concentration for S. cerevisiae and Streptomyces fermentations was linear, and estimates of biomass were computed from the fluorescence data. The respective dispersions, calculated as coefficients of variation, were 15% for S. cerevisiae and 36% for Streptomyces.
U.S.Copyrlght. Published 1993 by American Chemical Society and Amrlcan Institute of Chemical Englneers
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A commercial instrument designed to measure NADH fluorescence was introduced by BioChem Technology (Fluor0 Measure) in the 19705, followed later by Ingold's Fluorosensor. These instruments use a mercury lamp with suitable filters for the excitation source and detect the NADH fluorescence near 460 nm. In some studies, good correlation between cell mass and the fluorescence signal was reported (Ristroph et al., 19771, or a casual relation was established between the fluorescencesignaland oxygen deprivation (Harrison and Chance, 1970). NADH fluorescence is only a good indicator of cell density when temperature, pH, and dissolvedoxygen conditions are held constant and the energy substrate is in excess. On the other hand, NADH culture fluorescence is a very rapid and good indicator of metabolic switches,energy substrate limiting conditions, and oxygen limitation (Einsele and Purhar, 1980). Over the years, roughly one-half dozen groups of investigators have actively pursued the use of on-line fluorometric measurements for monitoring bioreactors (Armiger et al., 1986; Meyer et al., 1984; Zabriskie and Humphrey, 1978; Scheper et al., 1987; Horvath and Semerjian, 1986). Their work has essentially involved the monitoring of NADH culture fluorescence, with the exception of the last work which used the intrinsic fluorescence of yeast (tryptophan). In this report, we describe our recent studies on the application of intrinsic fluorescence from yeast for in situ process monitoring in bioreactors (Horvath and Spangler, 1992). In these studies, we observe fluorescence from tryptophan-containing intracellular protein structures in S. cerevisiae (Undenfriend, 1962; Konev, 19671,as well as from pyridoxine secreted into the media. Materials and Methods Fluorometry. An SLM 8000C scanning spectrofluorometer, manufactured by SLM Aminco Instruments, 1nc.l (Urbana, IL),was used in this study. A detailed description of the experimental system has been reported elsewhere (Horvath and Spangler, 1992). For these measurements, one leg of a bifurcated optical fiber bundle (Fiberguide Industries, Stirling, NJ) was attached to the excitation monochromator, and the other leg was connected to the emission monochromator. The common leg of the fiber bundle was removably attached to an unclad optical rod (Heraeus Amersil, Inc., Duluth, GA) above the fermentor head plage, allowing the optical rod, which penetrated to within 4 cm of the bottom of the fermentor, to be autoclaved in situ with the vessel and nutrients (medium). The excitation and the fluorescencelight were transmitted through the distal end of the optical rod. Both the fiber bundle and the optical rod were made of fused silica. The optical rod had a diameter of 6 mm. There was no reference channel. A blank background noise spectrum was measured at the beginning of each fermentation experiment. For the yeast experiments, excitation wavelengths were as follows: 290 nm for yeast tryptophan; 320 nm for pyridoxine; and 345 nm for NADH. The fluorescence spectra were integrated over the ranges 320-360,380-420, and 440-480 nm for yeast, pyridoxine, and NADH, respectively. The fermentations were performed in a 3.3-L Certain commercial equipment, instruments, and materials are identified in this article in order to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the NIST, nor does it imply that the material or equipment is necessarily the best available for the purpose.
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Bioflo fermentation system (New Brunswick Scientific, New Brunswick, NJ) with the optical fiber/rod immersed to 4 cm from the fermentor bottom. Previous work (Horvath and Spangler, 1992) found this position to minimize the effects of varying agitation speed and aeration rate. Varying the agitation speed from 300 to 800 rpm and the aeration rate from 1.5 to 5.0 L/m only reduced the fluorescence signal by 5%. The fluorometer was programmed to acquire spectra automatically during the fermentation. S.cerevisiaeGrowth Medium. In order to determine the validity of using cell tryptophan fluorescence as a fermentation cell mass monitor, without the complications of a fluorescent background from the media, a synthetic, very low fluorescing medium previously developed in this laboratory was used (Horvath and Spangler, 1992). This medium was found to display essentially no fluorescence a t any of the excitation wavelengths and zero absorbance from 200 to 600 nm. S. cerevisiae Growth Conditions. All yeast fermentations were carried out using the Bioflo I1 fermentation system with a 3.3-L vessel containing 2.8 L of medium. The temperature and pH were microprocessor-controlled at 32 "C and 6.0,respectively. The Bioflo I1 controlled the pH of the fermentation by adding NaOH or HC1 solutionsas required. The dissolved oxygen (DO) electrode was calibrated a t 0% with Nz and at 100% with 2.0 L/m air at an agitation rate of 300 rpm. The agitation and aeration rates were kept constant at 300 rpm and 2.0 L/m, respectively. This allowed the DO level to respond to cell metabolism changes. A nutrient-starved condition was created to allow the observation of one of the yeast cell's metabolic constituents, NADH. This condition was characterized by a total absence of glucose in the fermentation broth. This was ascertained by the use of a quantitative enzymatic test for glucose [Sigma Diagnostics (St. Louis, MO), Glucose Trinderl. The effects of oxygen deprivation were studied by the replacement of oxygen by nitrogen a t the same flow rate. These techniques are similar to those of other researchers (Duysens and Amesz, 1957; Siano and Mutharasan, 1989).
Results To determine the fluorescent species contained and produced in the fermentation, fluorescence spectra of test chemicals in buffer (0.2 M potassium phosphate, pH 6) were compared with the observed fluorescence in the fermentation broth. The fluorescent species determined to occur in this fermentation are tryptophan [the internal fluorescingcomponent in yeast (Konev, 196711,pyridoxine, and NADH. Figure 1 shows the individual spectra of 1 X 10-5M solutions overlaid, indicating regions of spectral overlap and areas of measurement for each. During a fermentation, the actual concentrations of each one will differ widely. For example, immediately after the inoculum was added, there was no measurable fluorescence from the yeast. After 50 h, near the end of a batch fermentation, we observed a large fluorescencesignal from the yeast culture. Fluorescence spectra were obtained automatically at fixed time intervals under computer control. Figure 2 shows the time evolution of our measured parameters during the course of the fermentation. The three fluorescence measurements were taken sequentially on (a) yeast cell mass (tryptophan signal from intracellular proteins in yeast), (b)production of product (pyridoxinevitamin &), and (c) cell mass measurement (intracellular NADH). The other two measurements were (d) the dry mass of yeast produced and (e) the oxygen uptake
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Incubation Time (Hours) Figure 2. Growth profile for a S. cereuisiae fermentation. Integrated fluorescence intensities taken with listed excitation (Lz) and emission (Lm) wavelength ranges for each species (determined from pure solution measurements): (a, top) tryptophan fluorescence; (b, 2nd from top) pyridoxine fluorescence; (c, middle) NADH fluorescence; (d, 2nd from bottom) dry cell mass; (e, bottom) dissolved oxygen content. (utilization) by the yeast cell culture during the course of the fermentation. The yeast dry weight curve demonstrates the classic fermentation stages: (1)0-22-h incubation stage, no cell mass growth; (2) 22-48-h exponential growth, steady increase of cell mass; and (3) 49-68-h stationary phase when cell mass stays relatively constant. The dissolved oxygen content plot indicates the activity of the culture. In the incubation stage there are few yeast cells. Their low activity is indicated by the low oxygen uptake. However, when the yeast is actively growing,
oxygen utilization increases. The yeast cells continue to grow and increase their oxygen uptake until the nutrient (glucose) is depleted, and oxygen demand decreases rapidly. In Figure 2 the signals of plots a and c, the fluorescence of tryptophan and NADH, follow the yeast dry mass curve (Figure 2d), indicating that both signals are a reliable measurement for monitoring yeast cell growth. However, the tryptophan signal (Figure 2a) is an order of magnitude greater than the NADH signal (Figure 2c), resulting in a much higher signal-to-noise ratio than the NADH signal. The signal from the product pyridoxine (Figure2b) appears to follow the dry cell mass curve (Figure 2d) with twice the signal of the tryptophan (Figure 2a). The oxygen utilization curve, Figure 2e, is inversely representative of cell and product formation but is a direct measure of cell activity. In Figure 3, the effect of glucose addition to a starved yeast culture on NADH and tryptophan fluorescence is shown. In the first 100 min, there is a relatively noisy constant NADH signal and 100% DO, indicating a resting state of low metabolic activity. Upon the addition of 3 g of glucose at t = 120 min, the DO drops dramatically and the NADH fluorescence increases by approximately 167%. When the tryptophan fluorescence is monitored, in the first 100 min there is a stable tryptophan fluorescence and 100%DO, indicating a starved resting state. Upon the addition of 3 g of glucose a t 120 min, the DO once again drops dramatically, while the tryptophan signal remains constant.
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cence signal became nonlinear from the effects of reabsorption and light scattering at a yeast dry mass concentration of 0.5 g/L. The optical fiberhod, which measures in the back-scatter configuration with a variable path length, can monitor the yeast fluorescence at higher optical densities than measurements in a 90° cuvette. At low conentrations and optical densities, the path length will be long (4 cm). As optical density increases, the pathlength shortens, maintaining light intensity and linear response at higher concentrations. Upon comparing Figure 2a,d, tryptophan fluorescence and dry cell mass, we see very good agreement between the two signals. However, the NADH plot, Figure 2c, with an overall signal that is an order of magnitude lower than that of the tryptophan (Figure 2a) fluorescence, exhibits more noise and larger fluctuations when compared to the yeast dry mass (Figure 2d). This may be partially due to changes in local fermentor conditions, such as glucose and oxygen conditions, yielding a change in the internal NADH fluorescence without any change in the cell mass. Also, because of the lower fluorescence of the NADH signal, it may be more affected by background and scattering noise, whereas the tryptophan signal, which is an order of magnitude stronger, is less affected. In Figure 2, the NADH fluorescence (part c) parallels the tryptophan signal (part a), until at 28 h it decreases a t a point where the DO is approximately 85% At 45 h and a DO concentration of 20 % , the NADH fluorescence jumps up and oscillates until the DO returns to 100% at 56 h, indicating glucose depletion, whereupon it attains a reduced but stable level. The tryptophan signal smoothly reflects the yeast dry mass plot (Figure 2d) and is not affected by DO or glucose changes. The tryptophan fluorescence signal is directly related to cell mass concentration, whereas NADH fluorescence is perturbed by environmental changes. The effect of these changes on the NADH fluorescence is shown in Figures 3 and 4 and has also been studied by various researchers (Harrison and Chance, 1970; Zabriskie and Humphrey, 1978; Ristroph et al., 1977; Armiger et al., 1986). The effects of adding glucose to a starved yeast culture are shown in Figure 3. The tryptophan signal is unaffected while the NADH signal increases by almost 20%. This increase in NADH fluorescence upon the addition of glucose to a starved yeast cell culture has been studied by many researchers (Zabriskie and Humphrey, 1978;Armiger et al., 1986; Srinivas and Mutharasan, 1987;Humphrey et al., 1989), who found that during glucose starvation the low respiration rate causes a reduction in the NADH levels in the culture. The resultant oxidized form, NAD, is not fluorescent; however, when glucose is added the observed fluorescence rises due to the increased respiration rate. The poorer signal quality of the NADH is also demonstrated by its large fluctuations under both starved and excess glucose conditions, whereas the stronger (approximately 6 times) tryptophan signal stays constant. Tryptophan fluorescence, reflecting cell mass, will not be affected by metabolic state shifts: it will only be changed by cell growth, which is very small over this time scale. Indirect measures of cell mass, such as NADH fluorescence, can also be influenced by the gaseous environment as well. Figure 4 illustrates the effect on NADH and tryptophan fluorescence as oxygen is replaced by nitrogen. With the oxygen removed, the yeast cells become anaerobic, increasing the observed NADH fluorescence with no increase in cell mass. The tryptophan fluorescence signal is not affected by the lack of oxygen due to ita inclusion in the yeast cell walls; therefore, it is a more reliable
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Figure 4. Effects of oxygen deprivation of a carbon-starved fermentationon oxygen concentration,NADH fluorescence,and tryptophan (yeast) fluorescence. Figure 4 illustrates the effect of changing DO concentration on the NADH and tryptophan fluorescence signals of a starved yeast culture. A starved yeast culture is shown in Figure 4 at 100% DO with a small NADH signal level with slight fluctuations (noise). At approximately137 min, the air is replaced by nitrogen, and the NADH fluorescence rises by approximately 17%. When the air purge is resumed, the DO value returns to loo%,and the NADH fluorescence signal returns to its initial level. Figure 4 also shows that the DO and tryptophan fluorescence were both a t constant levels with the DO at 100%. When air is replaced with nitrogen, the DO value drops to zero while the tryptophan signal remains unchanged. Return of the air brings DO back to 100% while the tryptophan fluorescence remains unchanged.
Discussion S. cerevisiae. The bifurcated optical fiberhod fluorescence measurement system was shown to be a simple, rugged, and sensitive method for real time, in situ measurements in bioreactors. The optical rod has undergone multiple steam sterilization cycles with no degradation or loss of sensitivity, allowingmeasurements with no chance of contamination. Figure 2 presents the fluorescence measurements, cell mass, and DO concentration during a yeast fermentation obtained with the optical fiber/rod sensor. In Figure 2a, we observe the fluorescence signalfrom the yeast increase using the optical fibedrod throughout the fermentation up to a dry cell mass of 2.68 g/L. In our earlier work with a flow-through cuvette (Horvath and Spangler, 1992), the yeast fluores-
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measure of cell mass. The excreted product, pyridoxine, also was not affected by changes in the oxygen or carbon source (Horvath and Spangler, 1992). Pyridoxine fluorescence, under non-pH-controlled conditions, was found to show excitation/emission pH-dependent behavior possibly useful for development of an optical pH meter (Horvath and Spangler, 1992). The effects of changing glucose and DO levels are also seen in Figure 2 in the fermentation growth profiles. Figure 2d (yeast dry mass) shows a constant increase until the glucose is depleted at approximately 50 h, which is also reflected in the rapid DO increase to 100%. The NADH fluorescence increases linearly between 30 and 44 h, while the DO concentration changes from approximately 80% to 30% along with the tryptophan and pyridoxine fluorescence signals. After 44 h, the NADH signal increases abruptly and fluctuates until 50 h when the DO returns to 100% and the NADH signals decrease and become stable. During this time (44-50 h), the tryptophan and pyridoxinesignalsslowly increased until 48 h, when glucose depletion occurred,and then leveled off toa constant value.
Summary A new spectroscopic technique using an optical fiber/ rod sensor for in situ fluorescence measurements was used for cell mass and product measurements. In a fermentation of S.cerevisiae,yeast cell mass and product (vitamin Bg)were measured using the sensor. Cell mass measured by tryptophan fluorescence was correlated with dry yeast mass. The production of pyridoxine was also found to increase with cell mass. Spectra were obtained in real time (2-3 min), and the active sensor (the optical rod) can be sterilized within the bioreactor without loss of sensitivity. Measurement of intrinsic (tryptophan) cell fluorescence was shown to be more accurate than NADH fluorescence for cell mass measurements. The data demonstrate the ability of fluorescence spectra to detect fermentation products by selective utilization of excitation and emission wavelengths and to monitor their production for process control and measurement applications. Future studies will use the techniques presented here in a bacterial fermentation for the measurement of cell mass and antibiotic production in media containing commonly used commercial feedstocks, e.g., soybean flour, corn meal, etc. Literature Cited Armiger, W. G.; Forro, J. R.; Montalvo, J. F.; Lee, J. F.; Zabriskie, D. W. The Interpretation of On-Line Process Measurements of Intracellular NADH in Fermentation Processes. Chem.Eng. Commun. 1986,45,197-201. ATCC Catalog of Bacteria and Bacteriophages, 17th ed., 1989, p 290. Chance, B. Spectrophotometry of Intracellular Respiratory Pigments. Science 1954,120,767-775.
Chance, B.; Estabrook, R. W.; Ghosh, A. Damped Sinusoidal Oscillations of Cytoplasmic Reduced Pyridine Nucleotide in Yeast Cells. Proc. Natl. Acad. Sei. U.S.A. 1964, 51, 12441451. Darken, M. A.; Berenson, H.; Shirk, R. J.; Sjolander, N. 0. Production of Tetracycline by Streptomyces aureofaciens in Synthetic Media. Appl. Microbiol. 1960,8, 46-51. Duysens, L. N. M.; Amesz, J. Fluorescence Spectrophotometry of Reduced Phosphopyridine Nucleotide in Intact Cells in the Near-Ultraviolet and VisibleRegion. Biochem. Biophys. Acta 1957,24,19-26. Einsele, A.; Purhar, E. On-line Erfassung von Fluoreszenz und Kohlendioxidpartialdruck in Bioreaktoren. Acta Biotechnol. 1980,10,33-37. Harrison, D. E. F.; Chance, B. Fluorometric Technique for Monitoring Changes in the Level of Reduced Nicotinamide Nucleotidesin Continuous Cultures of Microorganisms. Appl. Microbiol. 1970, 19, 446-450. Horvath, J. J.; Semerjian, H. G. LIF for Sensing in Bioreactors, Presented at the International Symposium on Biosensors, 192nd National Meeting of the American Chemical Society, Anaheim, CA, September, 1986, MBTD 21. Horvath, J. J.; Spangler, C. J. In Frontiers in Bioprocessing ZI; Todd, P., Sikdar, S., Bier, M., Eds.; American Chemical Society: Washington, D.C., 1992; pp 99-115. Humphrey, A. E.; Brown, K.; Horvath, J. J.; Semerjian, H. G. Bioproducts and Bioprocesses; Fiechter, A,, Okada,T., Tanner, R., Eds.; Springer-Verlag: Berlin, 1989; pp 309-320. Konev, S. V. Fluorescence and Phosphorescence ofProteins and Nucleic Acids; Plenum Press: New York, 1967. Meyer, H. P.; Beyeler, W.; Flechter, A. Experiences with the On-Line Measurement of Culture Fluorescence During Cultivation of Bacillus subtilis, Escherichia coli, and Sporotrichum thermophile. J . Biotechnol. 1984,1, 340-341. Ristroph, D. L.; Watteeuw, C. M.; Arminger, W. B.; Humphrey, A. E. Experience in the Use of Culture Fluorescence for Monitoring Fermentations. J. Ferment. Technol. 1977,55, 599-608. Scheper, T.; Gebauer, A.; Schugerl, K. Monitoring of NADHdependent Culture Fluorescence During the Cultivation of Escherichia coli. Chem. Eng. J. 1987,34, B7-Bl2. Siano, S. A.; Mutharasan, R. NADH and Flavin Fluorescence Responses of Starved Yeast Cultures to Substrate Additions. Biotechnol, Bioeng. 1989, 34, 660470. Simon, H. J.; Yin, E. J. Microbioassay of Antimicrobial Agents. Appl. Microbiol. 1970, 19, 573-579. Srinivas, S. P.; Mutharasan, R. Inner Filter Effects and Their Interferences in the Interpretation of Culture Fluorescence. Biotechnol. Bioeng. 1987,30, 769-774. Undenfriend, S. Fluorescence Assay in Biology and Medicine; Academic Press: New York, 1962. Zabriskie, D. W.; Humphrey, A. E. Biomass Concentration by Measuring Culture Fluorescence. Appl. Environ. Microbiol. 1978,35,337-343. Accepted July 8, 1993.' ei Abstract published in Advance ACS Abstracts, September 1, 1993.
