Biotechnol. Prog. 1991, 7, 21-27
21
Monitoring Cell Concentration and Activity by Multiple Excitation Fluorometry J.-K. Li, E. C. Asali, and A. E. Humphrey* Center for Molecular Bioscience and Biotechnology, Lehigh University, Bethlehem, Pennsylvania 18015
J. J. Horvath Chemical Process Metrology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899
Four key cellular metabolic fluorophores-tryptophan, pyridoxine, NAD(P)H, and riboflavin-were monitored on-line by a multiple excitation fluorometric system (MEFS) and a modified SLM 8000C scanning spectrofluorometer in three model yeast fermentation systems-bakers’ yeast growing on glucose, Candida utilis growing on ethanol, and Saccharomyces cereuisiae RTY 110/pRB58 growing on glucose. T h e measured fluorescence signals were compared with cell concentration, protein concentration, and cellular activity. T h e results indicate t h a t the behavior and fluorescence intensity of various fluorophores differ in the various fermentation systems. Tryptophan fluorescence is the best signal for t h e monitoring of cell concentration in bakers’ yeast and C. utilis fermentations. Pyridoxine fluorescence is the best signal for the monitoring of cell concentration in the S. cereuisiae R T Y 110/pRB58 fermentation. In bakers’ yeast fermentations t h e pyridoxine fluorescence signal can be used t o monitor cellular activity. T h e NAD(P)H fluorescence signal is a good indicator of cellular activity in the C. utilis fermentation. For this fermentation NAD(P)H fluorescence can be used t o control ethanol feeding in a fed-batch process.
Introduction Our ability to control and perform on-line optimization of fermentation processes is limited by our ability to monitor these processes. We would like to be able to measure on-line substrate, product, and cell concentrations, as well as cellular activities within the fermentor, in a noninvasive manner. Techniques exist to achieve this, based upon enzyme or immunological probe sensors. However, none of these probes can withstand in situ sterilization. None are particularly robust devices. Presently, two approaches to monitoring cell concentration on-line are used. One involves the use of indirect measurements by material balancing around the bioreactor; specifically measuring oxygen uptake rates and utilizing simple metabolic models to estimate cell growth, substrate uptake, and product formation rates. Unfortunately, the models are based on conditions of defined media and single substrate limitations. As such, these models may not apply in many real situations. For example, some antibiotic fermentations use starch as the energy substrate for the growth phase but switch to an oil-based substrate for the product formation phase. During the changeover period it is most difficult to predict the various concentrations and rates from the single substrate limitation model. I t is in this varying period where control is the most important. The second approach involves optical density and /or light reflectance. This measurement is reliable only for dilute nonmycelium cultures growing on clear, non-particulate-containing
* To whom correspondence should be addressed. 8756-7938/91/3007-0021$02.50/0
0 1991 American
media, conditions that are aenerallv uncommon in most industrial fermentations. But the future for fermentation monitoring is not necessarily bleak. In recent years a number of significant advances have been made in spectroscopic analyses, specifically fluorescence and infrared spectroscopy. This, coupled with advances in fiber optics and computer-aided analysis, has encouraged a number of biotechnologists to look at various in situ spectroscopic analyses for monitoring bioreactors. The biotechnology laboratory at Lehigh University has chosen to investigate various aspects of fluorometry as a potential bioreactor monitoring tool. I t was 20 years ago, when our group, then located a t the University of Pennsylvania and interacting with Dr. B. Chance, began using fluorometry to monitor intracellular NAD(P)H levels (Harrison and Chance, 1970). This technique involved exciting cultures with light in the 330-370-nm range and measuring the backscattered fluorescence in the 440-480nm range (Harrison et al., 1972). We first applied in situ fluorometry to monitoring bioreactors in 1969. Since that time two commercial products (BioChem Technology and Ingold) have appeared deriving from our original work. Unfortunately, NAD(P)H fluorometry has encountered a number of problems. The interpretation of NAD(P)H fluorescence signal for whole culture broths is very complex and involved (Ristroph et al., 1977; Zabriskie and Humphrey, 1978). However, the NADH fluorescence signal is a measure of intracellular redox and has value in determining the activity of a fermentation if it can be quantified. Depending upon the fermentation medium and metabolic parameters such as the extent of oxygen and
-
CIhemical Society and American Institute of Chemical Engineers
22
energy substrate limitation, the fraction of the fluorometric signal due solely to NAD(P)H can vary from less than 10% to up to 50% of the total backscattered fluorometric signal (Humphrey e t al., 1989). Another problem in real fermentations is that cells as well as growth media , contain a number of fluorophores. Most significant are the aromatic amino acids, pyridoxine, and riboflavin in addition to NAD (P)H. I t was this latter fact, i.e., that several fluorophores in cells are key metabolites, coupled with the fact that some of the specific spectral lines of a Hg arc lamp are a t or near the optimal wavelength for exciting these fluorophores, that encouraged us to investigate multiple excitation fluorometry as an on-line bioreactor monitoring tool. This activity has been further stimulated by the fact that bioreactors are available with sterilizable optic wells that can be fitted with quartz optical fiber bundles with high efficiency for light transmittance in the UV region, i.e., 200-400 nm. Additionally, there is a long-term attractiveness to fluorometry, particularly if phase fluorometry can be developed into a relatively simple analytical tool for fluorophore concentration quantification (Burdock et al., 1990). It seems evident to us, therefore, that fluorometry will eventually become a powerful on-line monitoring device for bioreactor and downstream bioprocesses control. However, except for pure component fluorescence, the knowledge base for creating such a fluorescence monitoring system is very limited. The exception is NAD(P)H fluorometry. Approximately 50 such devices have been sold to the biotechnology industry and over 100 publications on NAD(P)H fluorometry have appeared in the past decade (Humphrey et al., 1989). The evidence is accumulating to show that this relatively simple fluorometry device, which excites whole culture broth with light either from the 334- or 365-nm Hg arc spectral lines and monitors the backscattered culture fluorescence over the 440-520nm range, can be useful in on-line optimization of fedbatch bioreactors and multistage waste treatment systems (W. B. Armiger, BioChem Technology Inc., personal communication, 1989). In cooperation with the Chemical Process Metrology Division of the National Institute of Standards and Technology (NIST), we decided to begin creating a knowledge base for monitoring the four major cellular fluorophores-tryptophan, pyridoxine, NAD(P)H, and riboflavin-in whole broth cultures of various fermentation systems. The reasons for selecting these particular four fluorophores were severalfold. First, and most important, these fluorophoresare key metabolic components. Second, these four fluorophores are optimally or near optimally excited by Hg arc lamp spectral lines of 289,313,334,365, and 404 nm (Figure 1). Third, these four fluorophores, when excited at selected wavelengths, fluoresce in separate and distinct regions with little or no overlap (Figure 2). For example, the fluorescence emission spectra of NAD(P)H and riboflavin overlap if both of them are excited at the wavelength of 365 nm. However, NAD(P)H does not fluoresce when excited a t 404 nm while riboflavin strongly fluoresces. Therefore, an excitation wavlength (404 nm) can be selected where NAD(P)H does not interfere with the emission spectrum of riboflavin. When riboflavin fluorescence is relatively strong and NAD(P)H fluorescence is relatively weak, riboflavin will grossly interfere with the NAD(P)H signal when excited a t 365 nm. In this case, NAD(P)H can be excited a t 334 nm, which also excites pyridoxine but excites riboflavin only minimally. Monitoring the fluorescence a t 450-480 nm, where pyridoxine
Biotechnol. Prog., 1991, Vol. 7, No. 1
' k
WAVELENGTH ("mi
WAVELENGTH (MI
Pyridoxine
a
a
>,,
111
1%
1 In
YAVELENCT" (m)
1
I,,
I!.
I
1
WAVELENGTE
Y$
M
(-1
Figure 1. Percent of maximum fluorescence output of the four major cellular fluorophores at various excitation wavelengths. Riioilavi" LXLD
%
400 E
350 366 258
I
s
ZBB
ise 188
3ee 325 356 37s 4uu 42s 4se 475
sue 52s ssu
57s 666 NM
WAVELENCTH Inmi
Figure 2. Fluorescence emission spectra of the four fluorophores excited by Hg arc lamp spectral lines.
has little fluorescence intensity, allows correction of riboflavin interference with the NAD(P)H fluorescence signal. Similar situations exist with the other cellular fluorophores. To date, no one has clearly identified whether the culture fluorescence signal comes from intracellular or extracellular fluorophores, or both. Many factors, especially inner filter and cascade effects, can also affect fluorescence signal output (Li and Humphrey, 1990). The quantum yield of a fluorophore may change when it combines with other compounds. For example, the quantum yield of tryptophan in protein can be either higher, lower, or about the same as that of the free amino acid. Its quantum yield can vary from 0.05 for y-globulin to nearly 0.48 for bovine serum albumin (Teale, 1960). For these reasons it is almost impossible to obtain accurate fluorophore concentrations from the backscattered culture fluorescence signal alone. However, by using fluorescence signal time profiles for the various cellular fluorophores, we believe significant information can be obtained for fermentation control purposes. It was with this idea in mind that we began looking a t the fluorometric behavior of various fermentations in cooperation with researchers a t the NIST.
23
Blotechnol. Rog., 1991, Vol. 7, No. 1
Table I. Medium Composition for Bakers' Yeast Culture.
NADH Probe
L Spectrum
component glucose (NH412S04 K2HP04 CaC12 MgSO4 FeS04 ZnS04
concn 10 g/L 5 g/L 0.5 g/L 1 g/L 0.5 g/L 0.005 g/L 0.005 g/L a Nonfluorescence medium.
comDonent concn 0.005 g/L COS04 inositol 2.0 mg/L 0.4 mg/L niacin 0.4 mg/L pantothenic acid thiamine 0.4 mg/L 0.2 mg/L PABAh biotin 0.004 mg/L p-Aminobenzoic acid.
Table 11. Medium Composition for C. utilis Culture.
Figure 3. Multiple excitation fluorometric system (MEFS).
component ethanol KH2P04 (NH4)2S04 K2HPO4 MgSO4.7H20 a
Materials and Methods Fluorometric Probes. For the Lehigh University work, a multiple excitation fluorometric system (MEFS) was built (Figure 3). This system used a bifurcated quartz optical fiber bundle placed in an optical well in an 1.25-L Bioflo 111 fermenter. An Oriel Co. 200-W Hg arc lamp provided the light source. Five UV band-pass filters (289, 313,334,365, and 404 nm) with half-height widths of 10 nm in a computer-activated filter wheel were used to produce the various narrow-band excitation wavelengths. The transmission efficiencies of the filters were 13, 13.5, 28, 21.5, and 37?, respectively. A Guided-Wave, Inc., spectrum analyzer coupled to an IBM-AT computer was used to analyze the various fluorescence spectra. The commercial NADH probe (BioChem Technology Inc., FluroMeasure system) was also used in Lehigh work for comparison purposes. For the NIST work a modified SLM 8000C scanning spectrofluorometer, manufactured by SLM Instrument, Inc. was utilized. This instrument uses a 450-W Xe lamp with a diffraction grating as the monochromatic excitation source. T o measure the fluorescence, fermentation broth was pumped to a flow cuvette where the fluorescence signal of the broth was measured a t a 90' angle. Culture Systems. Three different fermentation systems-bakers' yeast, Candida utilis (ATCC 26387), and Saccharomyces cereuisiae auxotroph, RTY 110/ pRB58 (studied a t N1ST)-were used in this work. S. cereuisiae RTY 110/pRB58 is a modified bakers' yeast containing the plasmid pRR58. This particular strain of bakers' yeast without the plasmid is sucrose, leucine, and uracil negative. Since the plasmid contains the necessary genes for sucrose and uracil synthesis, but not for leucine, the plasmid containing yeast requires leucine for growth (Williams e t al., 1985). I t excretes invertase, which contains tryptophan. Thus it offers a specific challenge for monitoring tryptophan fluorescence. Media. The media compositions for the three cultures are listed in Tables 1-111. Note that no pyridoxine and riboflavin were added in media 1and 2, and no pyridoxine was added in medium 3, in order to minimize background fluorescence. Growth Conditions. The bakers' yeast and C. utilis fermentations were performed in a 1.25-L Bioflo I11 fermenter (New Brunswick Scientific, Inc.) a t 28 "C, pH = 5.5 and 5.2, respectively. The S. cereuisiae RTYllO/ pRB58 runs were performed under controlled conditions of 30 "C, pH = 6.0, and DO > 90% saturation in a 2.5-L Marubishi fermenter.
concn 10 g/L 4 g/L 3 g/L 0.5 g/L 0.5 g/L
comDonent inositol niacin pantothenic acid thiamin biotin
concn 2.0 mg/L 0.4 mg/L 0.4 mg/L 0.4 mg/L 0.004 mg/L
Nonfluorescence medium.
Table 111. Medium Composition for S. cerevisiae RTY 1 IO/DRRS~
component glucose bacto-yeast nitrogen base w/o amino acids leucine inosito1 calcium pantothenate
10.0 g/L 6.7 g/L
concn
component niacin thiamin
0.3 g/L 2.0 mg/L 0.4 mg/L
PABA riboflavin biotin
concn 0.4 mg/L 0.4 mg/L
0.2 mg/L 0.2 mg/L 0.002 mg/L
Analysis. The excitation and fluorescence emission monitoring wavelengths are summarized in Table IV for the Lehigh work and Table V for the NIST work. Fluorescence intensity was defined by the area under the fluorescence peak in certain wavelength ranges (Table IV) and measured in relative and arbitrary fluorescence units (AFU) for the Lehigh work. For the NIST work, the fluorescence signal was normalized relative to the excitation light intensity. The fluorescence intensity of the commercial NADH probe was expressed in terms of normalized fluorescence units (NFU) as defined by Armiger (1987). Ethanol and acetic acid concentrations were analyzed by gas chromatography (HP-5830A). The column was a 50% carbowax 20 M, 80/ 120 glass column. The operating conditionswere such that oven temperature was increased from 90 to 210 "C a t a rate of 5 "C/min. The mobile phase flow rate was 20 mL/min. Cell concentrations were estimated by measuring the optical density a t 660 nm and converted to dry weight for the Lehigh work. For the NIST work, cell concentrations were measured directly by dry weight. Glucose concentrations were measured by YSI glucose analyzer (Model 23A). For protein analysis cells were disrupted by a sonifier (Branson Sonic Power Co., Model W-350). Protein concentrations of the resulting suspension were assayed by using a Bio-Rad protein assay dye reagent. A leastsquares method was used to fit the data.
Results and Discussion Bakers' Yeast Cultured on Glucose-Mineral SaltsVitamin Medium. Figure 4 illustrates typical fluorescence patterns obtained for the four major cellular fluorophores for bakers' yeast fermentations. Pyridoxine fluorescence gave the strongest signal. Riboflavin fluorescence was very weak. The NAD(P)H fluorescence profiles from two different excitation wavelengths (334 and 365 nm) were very different. The fluroescence from the 334-nm excitation was much stronger than that from
Biotechnol. Prog., 1991, Vol. 7, No. 1
24
Table IV. Excitation and Fluorescence Monitoring Wavelengths ( n m ) for the Lehigh Work
3'0E+04
f'luorophore
excitation
integrated signal range
tryptophan pyridoxine
289 313 365 404
335-365 385-415 450-480 500-530
NADH riboflavin
Table V. Excitation and Fluorescence Monitoring Wavelengths ( n m ) for the NIST Work fluorophore
excitation
integrated signal range
tryptophan pyridoxine
290 325
NADH
345 395
320-360 380-420 440-480 510-540
riboflavin
2.5€+034
,
I
,
I
1
0.1
Cell concentration (g/L)
"FLOC.
-Ex. a t 289 nm for tryptophan -Ex. ot 365 n m far NADH . . . . I Ex. a t 404 nm for riboflavin
Figure 5. Logarithm of fluorescence signals vs logarithm of cell concentration for the bakers' yeast culture.
.. a 25E+C3
"..
, , , , , , , , , , , , , , , , ,, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 0 10 20 30 40 50
*cm
Fermentation time ( hr )
Ex Ex Ex
a! 289 nm f o r Tryptophan at 313 nm f o r Pyridoxine at 3 3 4 nm f o r NADH
Ex at 365 nm f o r NADH
mH Ex 90494
Ex
ot 365 n m f o r Riboflavin at 404 nm for Riboflavin
a 6 w 8 B
2
10
Figure 4. Time course of fluorescence patterns for the bakers' yeast culture. the 365-nm excitation. This is because the strong pyridoxine fluorescence interferes with the NAD(P)H fluorescence when excited at 334 nm. Hence, in this particular system, exciting the broth a t 365 nm gives a better indication of NAD(P)H levels, particularly since the riboflavin fluorescence is so weak. There was no difference for riboflavin fluorescence profiles excited at 365 and 404 nm since the NAD(P)H fluorescence signal was relatively weak. This resulted in little interference between NAD( P ) H and riboflavin fluorescence. The tryptophan fluorescence profile closely followed cell growth. Upon plotting the logarithm of fluorescence signal vs cell concentration, linear correlations were obtained for tryptophan, NADH, and riboflavin fluorescence (Figure 5 ) . In this particular fermentation, the tryptophan fluorescence signal is much better than other fluorescence signals for indicating cell concentration since it was the most sensitive to cell concentration changes. In Figure 6 the pyridoxine fluorescence signal pattern is compared with that for glucose utilization and those for cell, ethanol, and acetic acid concentrations. The pyridoxine fluorescence profile appears to be indicative of the culture respiratory activity. On the basis of the pyridoxine fluorescence and substrate utilization patterns, the fermentation can be divided into five stages. Stage 1 is characterized by the rapid increase of the fluorescence intensity until it reaches a maximum at 15h. In this stage the pyridoxine profile follows the cell growth curve. Stage 2 is characterized by the sharp decrease of the fluorescence signal. In this stage significant conversion of glucose to ethanol occurs. Ethanol concentration reaches a maximum a t the end of this stage and the cell concentration continues
Fermentation t i m e (hr) Oeeeo Cell conc.
-Glucose conc. HU. Ethanol conc.
HH. Ex. at 313 nm for Pyridoxine **km Acetic
acid conc.
Figure 6. Time course of the bakers' yeast fermentation in comparison to the pyridoxine fluorescence signal. to increase until it reaches a stationary phase. The decrease of pyridoxine fluorescence is indicative of a shift of metabolic pathway to ethanol fermentation. I t has been previously shown that there is a correlation between respiratory activity and pyridoxine content of yeast cells (Nakamura et al., 1976). This is consistent with the suggestion that reduction of the fluorescence signal is concomitant with the respiratory activity reduction. Ethanol consumption and conversion to acetic acid is observed in stage 3. The fluorescence intensity is constant in this stage. Stage 4 is characterized by the utilization of acetic acid. During this stage the fluorescence signal increases. This phenomenon could be due to the respiratory activity of acetate metabolism. In the last stage, acetic acid is consumed. The fluorescence intensity remains constant since the cells are metabolically inactive. On the basis of these studies we believe that, with this particular culture and medium, tryptophan fluorescence can be used as an indicator of cell concentration and pyridoxine fluorescence as an indicator of cellular metabolic activity. The challenge now will be to undertake careful measurements with this system in order to confirm this belief. A further study is being conducted to look into the relationship between the pyridoxine fluorescence profile and cell metabolic stages, especially in connection with heme synthesis and cytochrome content of the cells, which
Biotechnol. Prog., 1991. Vol. 7, No. 1 10
'
25
LtO
2.0EC04
I
-1
200
OE+04
5'
v
I EiP 5
E
t
Y
2.OE+O3
-Ex. -NADH
cm;Protein concentration ot 289 nm for Tryptophon Ex. at 31 3 nm for -Ex. at 365 nm far 00000 E x . ot 404 nm, for Riboflavin WBeO Cell concentration -EX.
HUI
&dHOXine
at 385 nm for NPSH -Acetic acid conc CWI Ethanol conc. proba data
Figure 8. Time course of the C. utilis fermentation in comparison to the NAD(P)H fluorescence signal.
Figure 7. Time course of fluorescence patterns for C. utilis growing on ethanol. could be closely related to pyridoxine content of the cell. Cavinato et al. (1990)has used cytochrome and cytochrome oxidase absorbency profiles to determine the metabolic state of cells during ethanol fermentation. This suggests that the pyridoxine fluorescence profile may have similar value since pyridoxine is a precursor of the cytochromes. C. utilis Growing on an Ethanol-Mineral SaltsVitamin Medium. The time course of the fluorescence signals from a C.utilis fermentation utilizing an ethanolmineral salts-vitamin medium is shown in Figure 7. The tryptophan fluorescence gave the strongest signal, followed by pyridoxine fluorescence. Tryptophan fluorescence had a significant increase after a 5-h lag phase. This corresponds to the exponential growth phase of the yeast. The signal began to decrease at about 21 h, although the cell concentration did not yet decrease. The total culture protein concentration also began to decrease at this time. This could be an explanation for tryptophan fluorescence intensity decrease at 21 h. At this time, the ethanol had been used up and cells began to use acetate, resulting in a change in the metabolic state of cells (Figure 8). Also, since the time history profiles of tryptophan fluorescence and protein concentrations are similar, tryptophan fluorescence may be a good indicator of protein concentration during the fermentation. The fluorescence signal time profiles for other cellular fluorophores were similar to that for tryptophan but not so intense, particularly during the exponential phase of growth. All four fluorophore signals can be linearly related to cell concentration during the exponential growth phase for this particular organism and medium (Figure 9). Tryptophan, however, is the best fluorophore to be used for estimating cell concentration since its fluorescence signal had the biggest change. Again, riboflavin gave a very weak fluorescence signal. In the Candida yeast fermentation, when ethanol is oxidized NADH is formed from NAD+. When ethanol is used up and acetate is utilized, the NADH concentration will decrease (Watteeuw et al., 1979). The NAD(P)H fluorescence signal reflects this behavior (Figure 8). The fluorescence intensity decreased sharply when ethanol was used up, and cells started to use acetic acid as carbon source. When acetic acid was used up, the NAD(P)H fluorescence again increased. Since in a fed-batch fermentation process ethanol should be added when acetate is used up in order to optimize the yield, the NADH
10 a 0.1
Cell concentration (g/L) -Ex. at U H U Ex. a t -Ex. at Ppppp Ex. a t
289 nm for Tryptophan 3 1 3 n m f o r Pyridoxine 3 6 5 nm f o r NADH 404 nm f o r Riboflavin
Figure 9. Comparison of cell concentration with various fluorescence signals for the C. utilis fermentation. fluorescence offers a signal that can be used to control ethanol feeding. The fluorescence signal profile from the commercial NADH probe was found to be similar to that of NAD(P)H fluorescence from the MEFS probe during the exponential growth phase of the cell. However, it did not track the NAD(P)H fluorescence measured by the MEFS probe after the exponential growth phase. Clearly, the fluorescence signal from the commercial NADH probe is a complex mixture of several fluorophore signals. During the exponential growth phase, all cellular fluorophore signals had a similar profile to cell concentration. After the exponential growth phase, the various fluorophore signals exhibited different behavior so that the fluorescence signal profile from the commercial NADH probe did not track any of the individual fluorophore signals. The sharp decrease of the signal from the commercial NADH probe a t about 21 h was due to the sharp decrease of NAD(P)H fluorescence. Unlike the NAD(P)H fluorescence from the MEFS, the fluorescence signal from the commercial NADH probe did not continuously decrease. This is because the pyridoxine fluorescence continuously increased during that period (refer to Figure 7). This illustrates one of the possible problems that can be encountered in using existing commercial NADH probes. The fluorescence spectrum profile of the whole culture excited at 289 nm during the fermentation process is similar to that for pure tryptophan in buffer. This was somewhat
Biotechnol. Prog., 1991, Vol. 7, No. 1
26
A
I
I
'I-
-Ex.
o t 280 nm
-Ex. a t 325 nm W E x . a t 345 nm o t 395 nm *Ex. 120
-
m
E
s 6
40
0.1
0.1
3D
20
4
Fermentation time ( -Ex. *Ex. -Cell
a t 325 nm a t 345 n m
hr )
To
conc.
Figure 10. Time cause of pyridoxine and NAD(P)Hfluorescence signals for the S.cereuisiae RTYllO/pRB58 fermentation. surprising because in many cases tryptophan fluorescence exhibits a blue shift when it is incorporated into a protein (Chen, 1973). No fluorescent spectrum shift was observed for pyridoxine during the fermentation. However, the NADH fluorescence spectrum had a 20-nm blue shift and the riboflavin fluorescence spectrum had a 12-nm blue shift in the whole broth culture. S. cerevisiaeRTY 110/pRB58 Growing on GlucoseYeast Nitrogen Base without Amino Acids-Leucine and Vitamins Medium. The results of the S. cereuisiae RTYllO/pRB58 fermentation growing on glucose-yeast nitrogen base medium are presented in Figure 10. This figure depicts the fluorescence signal behavior for whole culture broth. The pyridoxine fluorescence signal behavior over time is very similar to that for cell concentration. It appears to be very sensitive to cell concentration. For example, it increases nearly 40-fold during the 20-40-h growth period, while the dry weight only increases 10fold. The NAD(P)H fluroescence signal similarly tracks the cell concentration, but the pyridoxine signal is clearly stronger. This suggests that for this particular yeast system pyridoxine fluorescence may be a very sensitive means of monitoring the cell concentration. The sensitivity of the relationship between cell concentration and fluorescence signal for this fermentation is illustrated in Figure 11,where the relative fluorescence intensity for each cellular fluorophore is plotted against the cell dry weight. Approximate linear relationships are obtained for the pyridoxine and NAD(P)H fluorescence. The riboflavin fluorescence signal is very weak and therefore not particularly useful. The tryptophan fluorescence signal bears little relation to cell concentration. It would appear from these results that for this yeast and medium system pyridoxine is the best fluorophore to use for cell concentration estimations. The tryptophan fluorescence behavior suggests some protein excretion information may be contained in the signal. This point, however, needs to be further investigated.
Conclusions While these results are based only on three different cell-medium systems, they are sufficiently encouraging to suggest that the multiple excitation fluorometric systems (MEFS) have the potential for monitoring cell concentration and cellular activity, particularly for systems where control is based on trend analyses. Whether NAD(P)H fluorescence or another fluorophore fluorescence may be the best signal to use depends upon the particular
0 1
3
4
Cell Concentration (g/L)
Figure 11. Relationship between fluorescence signals and cell concentration of the S . cereuisiaeRTYllO/pRB58 fermentation.
fermentation. In most cases, the fluorescence intensity of various cellular fluorophores linearly corresponds to cell concentration during the exponential phase of cell growth. With respect to using fluorescence for specifically monitoring cell concentration, the best fluorophore to monitor will vary with different fermentation systems. In general, pyridoxine and NAD(P)H fluorescence signals appear to be good indicators of cellular activity. Riboflavin gave very weak fluorescence signals for all systems investigated. This implies that riboflavin has the potential to be used as a mixing time and gas hold-up tracer in fermentation processes, particularly since it strongly fluoresces at very low concentration. The fluorescence signal from the commercial NADH probe is a mixture of fluorescence signals from several cellular fluorophores. However, since the fluorescence intensity of various cellular fluorophores linearly relates to cell concentration during the exponential phase of cell growth, the NADH probe gives a relatively good correlation between its signal and cell concentration during the exponential phase of cell growth.
Acknowledgment We express our appreciation to the National Institutes of Standards and Technology, Division of Chemical Process Metrology, for support of this work.
Literature Cited Armiger, W. B. The FluroMeasure System User's Manual; BioChem Technology Inc.: Malvern, PA, 1987. Burdick, D. S.; Tu, X. M.; McGown, L. B.; Millican, D. W. Resolution of Multicomponent Fluorescent Mixtures by Analysis of the Excitation-Emission-Frequency Array. J . Chemom. 1990, 4 , 15-28. Cavinato, A. G.; Mayes, D. M.; Ge, 2.; Gallis, J. B. A Non-invasive Sensor for on-line Monitoring of Fermentation Processes. Presented at the 199th National Meeting of the American Chemical Society, Boston, MA, April 22-27, 1990. Chen, R. F. In Practical Fluorescence, Theory, Methods, and Techniques; Guilbault, Ed.; Marcel Dekker, Inc.: New York, 1973; pp 467-541. Gibson, K. D.; Laver, W. G.; Neuberger, A. Initial Stages in the Biosynthesis of porphyrins. Biochem. J . 1958, 70, 71-81. Harrison, D. E. F.; Chance, B. Fluorometric Technique for Monitoring Changes in the Level of Reduced Nicotinamide Nucleotides in Continuous Cultures of Microorganisms. A p p l . Microbiol. 1970, 19, 446-450. Harrison, D. E. F.; Harmes, C. S.; Humphrey, A. E. Control of Culture Systems for Ultimate Process Optimization. Process Biochem. 1972, 4 , 13-16.
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Humphrey, A. E.; Brown, K.; Horvath, J. J.; Semerjian, H. In Bioproducts a n d Bioprocesses; Fiechter, Okada, and Tanner, Eds.; Springer-Verlag: Berlin, 1989; pp 309-320. Li, J.-K.; Humphrey, A. E. Use of Fluorometry for Monitoring and Control of a Bioreactor. Submitted for publication in Riotechnol. Rioeng., 1990. Nakamura, I.; Nishikawa, Y.; Kamihara, T.; Fukui, S. ThiamineInduced Reversible Deficiency in Respiratory Activity of Saccharomyces carlsbergensis: Respiratory Adaptation caused by Pyridoxine. FEBS Lett. 1976, 62, 354-358. Ristroph, D.; Watteeuw, C. M.; Armiger, W. B.; Humphrey, A. E. Experience in the Use of Culture Fluorescence for Monitoring Fermentations. J. Fermedt. Technol. 1977, 55, 599608. Teale, F. W. J. Ultraviolet Fluorescence of Proteins in Natural Solution. Biochem. J. 1960, 76, 381-388.
27
Watteeuw, C. M.; Armiger, W. B.; Ristroph, D. L.; Humphrey, A. E. Production of Single Cell Protein from Ethanol by FedBatch Process. Biotechnol. Bioeng. 1979, 21, 1221-1237. Williams, R. S.; Trumby, R. J.; MacColl, R.; Trimble, R. B.; Maley, F. Comparative Properties of Amplified External and Internal Invertase from the Yeast SUC2 Gene. J.Biol. Chem. 1985,260, 13334-13341. Zabriskie, D. W.; Humphrey, A. E. Estimation of Fermentation Biomass Concentration by Measuring Culture Fluorescence. Appl. Enuiron. Microbiol. 1978, 35, 337-343. Accepted October 25, 1990.
Registry No. NADH, 58-68-4; L-tryptophan, 73-22-3; pyridoxine, 65-23-6; riboflavin, 83-88-5; ethanol, 64-17-5.
