- Email: [email protected]
Determination of concentration of living immobilized yeast cells by fluorescence spectroscopy
Sensors and Actuators B 107 (2005) 126–134
Determination of concentration of living immobilized yeast cells by fluorescence spectroscopy O. Podrazky, G. Kuncova∗ Institute of Chemical Process Fundamentals, Academy of Sciences of the Czech Republic, Rozvojova 135, 16502 Prague, Czech Republic Received 19 April 2004; received in revised form 22 June 2004; accepted 16 August 2004 Available online 14 November 2004
Abstract Fluorescence spectroscopy is one of suitable methods for the direct monitoring of immobilized cells because it does not require (in certain arrangement) transparent environment like absorbance measurements. The aim of this work was to develop a method allowing measurements of concentrations of living immobilized cells using only intrinsic fluorescence of biogenic fluorophores in cells. Saccharomyces cerevisiae yeast cells were immobilized into alginate and into a mix of alginate with prepolymerized tetramethoxysilane (TMOS) and measured in flow through cuvettes. Transitions from aerobic to anaerobic conditions were made by stopping circulation of perfusing medium and 2-dimensional fluorescence (2-DF) spectra and time responses of nicotinamide adenine dinucleotide coenzymes (NAD(P)H) fluorescence intensity were measured. Fluorescence intensity of intracellular NAD(P)H, its changes during aerobic-anaerobic transitions and the time delay between halt of medium circulation and rise of NAD(P)H fluorescence intensity correlated with concentration of living immobilized yeast cells. In case of alginate–TMOS layers, only relative change of NAD(P)H fluorescence intensity corresponded to the content of active biomass in the whole range of measured concentrations. © 2004 Elsevier B.V. All rights reserved. Keywords: Immobilization; Cells; Sol–gel; Alginate; 2-D fluorescence spectroscopy
1. Introduction Whole cells are often used in immobilized form in today’s biotechnologies and biosensors. However, while the amount of cells to be immobilized can be controlled, nondestructible on-line methods have not been developed yet for the determination of surviving cells after the immobilization process. Nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H) take part in many metabolic processes in cells. Duysens and Amesz [1] described the monitoring of NAD(P)H in free cells by fluorescence as early as in 1957 and this method was described in many papers thereafter. The major conclusions summarized from these papers are the following [2,3]: ∗
Corresponding author. Tel.: +42 2 20920661; fax: +42 2 20390243. E-mail addresses: [email protected] (O. Podrazky), [email protected] (G. Kuncova). 0925-4005/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2004.08.031
(1) Concentration of NAD(P)H in cells can vary from less than 0.9 mol g−1 of dry weight for obligate aerobes, to more than 4.5 mol g−1 of dry weight for obligate anaerobes and members of the Lactobacillaceae and Saccharomyces cerevisiae. The concentration is not constant even in the same strain, depending on cultivation media and substrate composition. (2) Fluorescence of NAD(P)H in cells increases after the concentration of dissolved oxygen drops to zero, because the fluorescent reduced form of NAD(P)H cannot be oxidized in the respiration chain. (3) Biomass concentration can be evaluated by fluorescence of NAD(P)H when cells are in the exponential growth phase and all cultivation conditions such as pH, dissolved oxygen, initial substrate concentration, temperature and stirring speed remain constant. (4) In yeast cells, fluorescence of NAD(P)H quickly decreases after depletion of substrate (glucose, pyruvate, etc.).
O. Podrazky, G. Kuncova / Sensors and Actuators B 107 (2005) 126–134
Unlike NAD(P)H, other biogenic fluorophores such as aminoacids and pyridoxine, flavine and their derivatives are not used so often. These measurements require special nonfluorescent media because generally used nutrient-rich media show strong fluorescence background interfering with measurements. According to Horvath et al. [4,5] and Li et al. [6], it is possible to evaluate the concentration of biomass in suspension by fluorescence of tryptophan and pyridoxine even better than by fluorescence of NAD(P)H. Although a lot of papers deal with intrinsic fluorescence of cells, only a couple of them describe measurements of immobilized cells [7,8]. The aim of this work was to develop a method allowing the measurement of concentration of living immobilized cells using only intrinsic fluorescence of biogenic fluorophores in cells. We did not use any indicator because they might affect cell’s metabolism or opticaly interfere with other fluorophores present in cell. We measured fluorescence intensities by 2-D fluorescence spectroscopy [9] in the range of excitation wavelengths 220–650 nm and emission wavelengths 250–700 nm and monitored fluorescence of NAD(P)H at λEX /λEM = 330/440 nm during aerobic–anaerobic transition obtained by halt of circulation of aerated medium. This approach does not require genetically modified cells so it can be used almost for any type of cells and it allows us to characterize also the changes of fluorescence related to oxygen uptake rate.
2. Materials and methods 2.1. Cells and media S. cerevisiae wild-type strain SP-4 (␣, leu1 , arg4 ) [10] obtained from Prof. T. Bilinski (Rzesz´ow Pedagogical University, Poland) and DBM60 (S. chevalieri) obtained from Department of Biochemistry and Microbiology of Institute of Chemical Technology in Prague (Czech Republic) were cultivated in liquid YPD medium (20 g l−1 d-glucose, 10 g l−1 peptone, 10 g l−1 yeast extract) at 30 ◦ C for 18 h. After cultivation, cells were centrifuged at 2000 × g for 5 min, washed with physiological solution (9 g l−1 NaCl), centrifuged again and resuspended in non-fluorescent Horvath–Spangler (HS) [4] medium without glucose. Resuspended cells in HSmedium were cultivated for another 2 h without glucose and then stored at 4 ◦ C overnight. Before the measurements, about one half of cells stored in HS-medium were placed into 80 ◦ C water bath for 30 min in order to obtain dead cells for mixtures.
measuring optical density of suspension at 650 nm (OD650 ) and calculated using empirical formula: X = 1.6632 × OD650 . Cells were centrifuged and resuspended in physiological solution (PS) to give a concentration of dry cell mass 72 g l−1 . Living and dead cells were mixed together to form suspensions with various living cells content but the same total dry mass concentration of 12 g l−1 . The concentration was chosen according to results of preliminary experiments [11], 1 ml of each suspension was mixed with 5 ml of 3.2% (w/v) sterile solution of sodium alginate (Sigma-Aldrich, type IV, practical grade), put into syringe and dripped through flat-end needle (inner diameter 1 mm) into sterile solution of CaCl2 (7 g l−1 ). Regular beads were formed having diameter about 3 mm. They were left in stirred CaCl2 solution for another 30 min, washed with HS-medium without glucose and stored at 4 ◦ C. 2.3. Alginate–TMOS planar films Tetramethoxysilane (TMOS), Fluka product no. 87682, was mixed with distilled water and HCl in TMOS:H2 O:HCl mol ratio = 1:5:10−2 to form a clear solution and left to prepolymerize for 24 h at 4 ◦ C. Concentrations of DBM60 cells in films ranged from 20 to 80 g l−1 of dry mass. Lower concentrations did not give reproducible results [12]. Two types of films were prepared: (1) films containing mixture of living and dead cells, maintaining concentration of total biomass constant, (2) films containing only living cells in different concentrations. ad 1: Suspensions of living and dead cells were mixed together into a centrifugation cuvette in different ratios to form a total volume of 10 ml of suspension with various concentrations of living cells but with the same total dry mass concentration. The suspension was centrifuged, and cells were resuspended in 0.5 ml of PS. The cells (0.5 ml) were mixed together with 3.2% (w/v) sodium–alginate (0.125 ml), 0.05MNaOH (0.25 ml) and prepolymerized TMOS (0.125 ml). This biocomposite solution was homogenized and 0.314 ml was pipetted onto quartz glass slide (surface area 3.14 cm2 ) which then served as a front face of the flow through cuvette (Fig. 1). Immediately after gelation, the film was bathed in CaCl2 so-
2.2. Immobilization procedures 2.2.1. Alginate beads Total dry mass concentration (X [g l−1 ]) of S. cerevisiae strain SP-4 cells stored in HS-medium was determined by
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Fig. 1. Flow-through cuvette for composite alginate–TMOS layers.
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lution (7 g l−1 ) for 30 min. After that, flow-through cuvette was assembled and measurements took place. ad 2: 10 ml of cells stored in HS-medium were centrifuged, cells were resuspended with volumes of PS, ranging from 0.5 to 2.0 ml in order to obtain various concentrations of cells. Concentrations of dry biomass in resuspended cells were determined by measurement of optical density at 650 nm and calculated using empirical formula: X = 0.1523 × OD650 . Half milliliters of the suspension was taken for immobilization procedure and composite alginate–TMOS film was created and measured in the same way as described above. In order to prepare films with thickness in the range 0.5–2 mm, volumes of biocomposite suspension (dry biomass concentration 75 g l−1 of biocomposite) in range 0.157–0.628 ml were pipetted onto the quartz glass.
2.5. Reproducibility of measurements We verified the reproducibility of NAD(P)H fluorescence and its change during a halt of medium flow: - by filling the cuvette 5 times with alginate beads with the same concentration of living cells, - by preparing four alginate–TMOS films with the same concentration of living cells, - by measurement of NAD(P)H fluorescence during repetitive halts and runs of medium flow in one alginate–TMOS film (Fig. 5).
3. Results and discussion 3.1. Reproducibility of measurements
2.4. Fluorescence measurements Twenty-five alginate beads were placed into flow-through cuvette with quartz-glass window (Fig. 2), the cuvette was installed into Hitachi’s F-4500 fluorescence spectrophotometer in front-face arrangement and perfused with aerated HSmedium without glucose at 12 ml min−1 . After equilibration of NAD(P)H fluorescence at λEX = 340 nm and λEM = 440 nm medium circulation was stopped and changes in NAD(P)H fluorescence intensity were measured. 2-DF spectra (λEX = 220–650 nm, λEM = 250–700 nm) were scanned at “aerobic state”, before the circulation of aerated medium was stopped, and “anaerobic state”, after dissolved oxygen in cuvette was depleted and NAD(P)H fluorescence intensity stabilized. These spectra were subtracted (Fig. 3) and all wavelength pairs in “aerobic”, “anaerobic” and subtracted 2-DF spectra were correlated with concentrations of dry mass of living cells in beads, using Microsoft Excel “CORREL” function. Wavelength pairs with highest correlation coefficients were located using “correlation 2-D spectra”, where fluorescence intensities were replaced by correlation coefficients (Fig. 4). Composite alginate–TMOS films were measured in the same way as the alginate beads.
The reproducibility of NAD(P)H fluorescence intensity and the t parameter is summarized in Table 1. Relative standard errors of “steps” during repetitive halts and runs of medium flow in one alginate–TMOS film (Fig. 5) were 0.5% for NAD(P)H fluorescence intensity and 5.5% for its relative change. Slow decrease of NAD(P)H fluorescence intensity can be ascribed to stresses and limited living conditions of immobilized cells (absence of carbon source, vitamins, etc. in medium). 3.2. Alginate beads The highest correlation coefficients were found at λEX /λEM = 350/430 nm for anaerobic conditions, at λEX /λEM = 350/440 nm for aerobic conditions and at λEX /λEM = 330/440 nm for subtraction spectra (Fig. 4). These wavelengths correspond to the position of maximum of NAD(P)H fluorescence in 2-DF spectra [13]. Relations of fluorescence intensity at these wavelengths to dry mass of living cells were plotted (Fig. 6) showing a linear relation between these parameters. The experiments showed that fluorescence intensity of NAD(P)H can be used for the evaluation of active cells after immobilization in conditions where environmental factors such as dissolved O2 , temperature, substrate concentration and pH remain constant. This is in accordance with papers describing a good correlation between NAD(P)H fluorescence and concentration of free cells in liquid media [14–16]. Table 1 Relative standard errors of measurements of NAD(P)H fluorescence intensity Immobilization matrix
Fig. 2. Flow-through cuvette for alginate beads.
Alginate beads Alginate–TMOS films
Relative standard error I0 (%)
(I1 − I0 )/I0 (%)
t (%)
10 2.5
15 20
5 5
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Fig. 3. 2-DF spectra of yeast cells in aerobic and anaerobic state and their subtraction.
Changes in NAD(P)H fluorescence intensity during halts of medium flow were characterized by the following parameters (Fig. 7): - I0 : average fluorescence intensity during the 60 s before the halt, - I1 : average fluorescence intensity during the last 60 s of equilibrated signal, - (I1 − I0 )/I0 : relative change of fluorescence intensity,
- t: time delay between the halt of medium flow and a rise of the fluorescence signal. Similarly to fluorescence intensities of NAD(P)H in aerobic and anaerobic states, (I1 − I0 )/I0 ratio can represent active biomass too (Fig. 8). An interesting finding was the relation between the concentration of living cells and t (Fig. 9). Unlike the fluorescence intensities, which were affected only by the cells
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Fig. 4. “Correlation 2-D spectrum” of yeast cells for aerobic state.
lying in the optical path of the instrument, we can suppose that t parameter depend on the oxygen uptake rate which is affected by all cells present in the cuvette. Consequently, this parameter should provide information about total amount of immobilized active biomass present in the cuvette.
3.3. Alginate–TMOS films Compared with the alginate beads, which could be stored overnight at 4 ◦ C, alginate–TMOS films had to be measured right after preparation. After overnight storage we did not
Fig. 5. NAD(P)H fluorescence intensities during repetitive halts and runs of medium flow in one alginate–TMOS film with living immobilized DBM60 cells (80 g l−1 ).
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Fig. 6. Relation between fluorescence intensity and dry mass of living cells in alginate beads for the best correlating wavelengths.
Fig. 7. A time dependence of NAD(P)H fluorescence intensity at λEX /λEM = 340/440 nm during a halt of medium flow.
observed any response of NAD(P)H fluorescence to halt of medium flow. In the cuvette flowed through with oxygen saturated medium, yeast cells survived overnight, but the content of the cells on the side of the film fixed to the quartz slide decreased. When the circulating medium contained a carbon source (e.g. glucose), the cells inside the film in the cuvette were growing. The amount of surviving cells depended on matrix properties [12]. Since experiments with alginate beads revealed that NAD(P)H is a suitable biogenic fluorophore for immobilized yeast cells, only time scans of NAD(P)H fluorescence intensity were measured for composite alginate–TMOS films. In films prepared only with living cells NAD(P)H fluorescence intensities (I0 , I1 ) increased proportionally to dry biomass concentration (Fig. 10). However, (I1 − I0 )/I0 ratio and t (Fig. 11) dropped down with increasing dry biomass concentration. In films containing mixtures of dead and living cells, NAD(P)H fluorescence intensities (I0 , I1 ) were proportional to concentrations of dry active biomass up to cca 55 g l−1 (Fig. 12). The decrease of fluorescence intensity of the last
sample containing 80 g l−1 of living cells is probably caused by absence of dead cells which could serve as a carbon source in the three previous samples containing a mixture of living and dead cells.
Fig. 8. Relation between relative change in NAD(P)H fluorescence intensity (I1 − I0 )/I0 and the dry mass of living cells immobilized into alginate beads.
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Fig. 9. Relation between time delay (t) and dry mass of living cells immobilized into alginate beads.
Fig. 12. Relation between fluorescence intensities in aerobic state (I0 ), anerobic state (I1 ) and their relative change (I1 − I0 )/I0 and amount of living cells in composite alginate–TMOS layers containing mixture of living and dead cells. L-dry mass of living cells (g l−1 ); T-total immobilized biomass (g l−1 ).
Fig. 10. Relation between fluorescence intensities in aerobic state (I0 ), anerobic (I1 ) and their relative change (I1 − I0 )/I0 and dry mass of living cells in composite alginate–TMOS layers containing only living cells. Fig. 13. Relation between time delay (t) and dry mass of living cells in composite alginate–TMOS layers containing mixture of living and dead cells.
Fig. 11. Relation between time delay (t) and dry mass of living cells in composite alginate–TMOS layers containing only living cells.
In contrast to films prepared only with living cells (I1 − I0 )/I0 ratios increased with content of living cells in the whole range of measured concentrations. We can explain the difference in results for films containing only living cells and mixture of dead and living cells by the following mechanism—in films with mixed living and dead cells, concentration of total biomass was constant, so optical conditions were constant as well despite of content of living cells. On
the other hand, in films containing only living cells, total dry biomass concentration varied—the biocomposites containing less cells had lower optical density so excitation light could penetrate deeper into the film and absorption and scattering of emitted light by cells were lower than in films with higher cells concentrations. t decreased with concentrations of dry active biomass similarly in both types of films (Figs. 11 and 13) as it is not dependent on optical density of biocomposite. In contrast to films, where cells in direct contact with circulating medium (i.e. source of oxygen) were separated from the most illuminated cells, in cuvette filled with alginate beads these cells were also washed by the medium. Thus, in films, diffusion and oxygen consumption inside the biocomposite had stronger influence on oxygen concentration in the vicinity of illuminated cells, than in the alginate beads. While the NAD(P)H fluorescence intensity is increasing with the film thickness up to 1.5 mm, trend of the relative change of fluorescence intensity after the medium halt, (I1 − I0 )/I0 , was opposite due to limitation of oxygen diffu-
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On the other hand, on-line monitoring of immobilized cells can be used in whole-cell optical biosensors for monitoring of e.g. environmental pollution.
Acknowledgements Funding for this research was provided by the Grant Agency of the Czech Republic through the grant no.104/01/461 and by the Ministry of Education, Youth and Sports of CR grant OC 840.10.
References Fig. 14. Relation between fluorescence intensity in aerobic state (I0 ) and its relative change (I1 − I0 )/I0 and layer thickness.
sion in thicker films (Fig. 14). Less oxygen was available for the cells near the quartz glass window of the cuvette because it was consumed inside the film. This effect became stronger in thicker films (alginate–TMOS) so cells located deeper than 1–1.5 mm were lacking oxygen.
4. Conclusions Measurements of NAD(P)H fluorescence intensity and its changes during oxygen depletion well express concentration of active immobilized biomass in non-transparent matrices. Fluorescence intensities well represented active immobilized biomass in systems where the cells situated near the surface were predominantly measured. Monitoring of changes in fluorescence during oxygen depletion allowed evaluation of active immobilized biomass also in films, in which measurement of intensities did not provide satisfactory results due to effects of diffusion and oxygen consumption inside the biocomposite. Time delay between halt of medium circulation and rise of NAD(P)H fluorescence (i.e. time needed for depletion of oxygen in cuvette) also provided information about amount of living cells in the cuvette and it was reciprocally proportional to the concentration of active biomass in alginate beads. The measurements of films with different thickness allow us to examine the conditions in inner layers of immobilization matrices because the cells, which are the most illuminated by excitation light are separated from medium by the film itself. However, the method described in this paper requires calibration for each type of cells, medium, cuvette and immobilization matrix, it can be useful for optimization of immobilization techniques for bioprocesses. Unlike the other methods it allows in situ measurements in non-transparent matrices and it does not require any dyes or agents, which can interfere with cells. It is possible to measure cells inside the matrix and its disintegration, which can affect the viability of the cells, is not necessary.
[1] L.N.M. Duysens, J. Amesz, Fluorescence spectrophotometry of reduced phosphopyridine nucleotide in intact cells in the nearultraviolet and visible region, Biochem. Biophys. Acta 24 (1957) 19–26. [2] G. Rao, S.B. Bambot, S.C.W. Kwong, H. Szmacinski, J. Sipior, R. Holavanahali, G. Carter., Application of fluorescence sensing to bioreactors, in: J.R. Lakowicz (Ed.), Fluorescence spectroscopy, probe design and chemical sensing, 4, Plenum Press, New York, 1994, pp. 417–448. [3] J. London, M. Knight, Concentrations of nicotinamide nucleotide coenzymes in micro-organisms, J. Gen. Microbiol. 44 (1966) 241–254. [4] J.J. Horvath, C.J. Spangler, In Situ on-line optical fiber sensor for fluorescence monitoring in bioreactors, in: P. Todd, S. Sikdar, M. Bier (Eds.), Frontiers in Bioprocessing II, American Chemical Society, Washington DC, 1992, pp. 99–115. [5] J.J. Horvath, S.A. Glazier, C.J. Spangler, In situ fluorescence cell mass measurements of Saccharomyces cerevisiae using cellular tryptophan, Biotechnol. Progr. 9 (1993) 666–670. [6] J.-K. Li, E.C. Asali, A.E. Humphrey, Monitoring cell concentration and activity by multiple excitation fluorometry, Biotechnol. Progr. 7 (1991) 21–27. [7] K.F. Reardon, T. Scheper, J.E. Bailey, In situ fluorescence monitoring of immobilized clostridium acetobutylicum, Biotechnol. Lett. 8 (11) (1986) 817–822. [8] W. M¨uller, G. Wehnert, T. Scheper, Fluorescence monitoring of immobilized microorganisms in cultures, Anal. Chim. Acta 213 (1988) 47–53. [9] S. Marose, C. Lindemann, T. Scheper, Two-dimmensional fluorescence spectroscopy: a new tool for on-line bioprocess monitoring, Biotechnol. Prog. 14 (1998) 63–74. [10] T. Bilinski, J. Lukaszkiewicz, A. Sledziewski, Demonstration of anaerobic catalase synthesis in the cz1 mutant of Saccharomyces cerevisiae, Biochem. Biophys. Res. Commun. 83 (1978) 1225–1233. [11] O. Podrazky, G. Kuncova, A. Krasowska, K. Sigler, Monitoring the growth and stress responses of yeast cells by two-dimensional fluorescence spectroscopy: first results, Fol. Microb. 48 (2003) 189. [12] G. Kuncova, O. Podrazky, S. Ripp, J. Tr¨ogl, G.S. Sayler, K. Demnerova, R. Vankova, Monitoring of the viability of cells immobilized by sol–gel process, J. Sol–gel Sci. Technol. (2004), accepted for publication. [13] O.S. Wolfbeis, The fluorescence of oragnic natural products, in: S.G. Schulman (Ed.), Molecular luminescence spectroscopy methods and applications: part1, chemical analysis, 77, Wiley, New York, 1985, pp. 167–370. [14] D.W. Zabriskie, A.E. Humphrey, Biomass concentration by measuring culture fluorescence, Biotechnol. Bioeng. 35 (1978) 337–343. [15] W.B. Armiger, J.F. Forro, L.M. Montalvo, J.F. Lee, D.W. Zabriskie, The interpretation of on-line process measurements of intracellular
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nadh in fermentation processes, Chem. Eng. Commun. 45 (1986) 197–206. [16] T. Scheper, K. Sch¨ugerl, Culture fluorescence studies on aerobic continuous cultures of Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 23 (1986) 440–444.
Biographies is a research worker in the Immobilized Biomaterials and Optical Sensors group at the Institute of Chemical Process Fundamentals, Academy of Sciences of the Czech Republic. He graduated in 1998 from the Institute of Chemical Technology, Prague with MS in water technology and
environmental engineering. He received his PhD in environmental chemistry and technology in the same institute in 2003. Now, he is concerned with optical biosensors and immobilization of cells.
Gabriela Kuncova is the head of the Immobilized Biomaterials and Optical Sensors group at the Institute of Chemical Process Fundamentals, Academy of Sciences of the Czech Republic, Prague. She graduated in 1974 from Institute of Chemical Technology, Prague with MS in technology of silicates. In 1979 she obtained PhD in organic chemistry at the Institute of Chemical Process Fundamentals, Czechoslovak Academy of Sciences, Prague. Following 10 years (1979–1989) she worked on development of drawing and coating of optical fibers for telecommunication and quartz capillaries for chromatography in Joint Laboratory for Silicates CAS and ICT at Prague.
Determination of concentration of living immobilized yeast cells by fluorescence spectroscopy O. Podrazky, G. Kuncova∗ Institute of Chemical Process Fundamentals, Academy of Sciences of the Czech Republic, Rozvojova 135, 16502 Prague, Czech Republic Received 19 April 2004; received in revised form 22 June 2004; accepted 16 August 2004 Available online 14 November 2004
Abstract Fluorescence spectroscopy is one of suitable methods for the direct monitoring of immobilized cells because it does not require (in certain arrangement) transparent environment like absorbance measurements. The aim of this work was to develop a method allowing measurements of concentrations of living immobilized cells using only intrinsic fluorescence of biogenic fluorophores in cells. Saccharomyces cerevisiae yeast cells were immobilized into alginate and into a mix of alginate with prepolymerized tetramethoxysilane (TMOS) and measured in flow through cuvettes. Transitions from aerobic to anaerobic conditions were made by stopping circulation of perfusing medium and 2-dimensional fluorescence (2-DF) spectra and time responses of nicotinamide adenine dinucleotide coenzymes (NAD(P)H) fluorescence intensity were measured. Fluorescence intensity of intracellular NAD(P)H, its changes during aerobic-anaerobic transitions and the time delay between halt of medium circulation and rise of NAD(P)H fluorescence intensity correlated with concentration of living immobilized yeast cells. In case of alginate–TMOS layers, only relative change of NAD(P)H fluorescence intensity corresponded to the content of active biomass in the whole range of measured concentrations. © 2004 Elsevier B.V. All rights reserved. Keywords: Immobilization; Cells; Sol–gel; Alginate; 2-D fluorescence spectroscopy
1. Introduction Whole cells are often used in immobilized form in today’s biotechnologies and biosensors. However, while the amount of cells to be immobilized can be controlled, nondestructible on-line methods have not been developed yet for the determination of surviving cells after the immobilization process. Nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H) take part in many metabolic processes in cells. Duysens and Amesz [1] described the monitoring of NAD(P)H in free cells by fluorescence as early as in 1957 and this method was described in many papers thereafter. The major conclusions summarized from these papers are the following [2,3]: ∗
Corresponding author. Tel.: +42 2 20920661; fax: +42 2 20390243. E-mail addresses: [email protected] (O. Podrazky), [email protected] (G. Kuncova). 0925-4005/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2004.08.031
(1) Concentration of NAD(P)H in cells can vary from less than 0.9 mol g−1 of dry weight for obligate aerobes, to more than 4.5 mol g−1 of dry weight for obligate anaerobes and members of the Lactobacillaceae and Saccharomyces cerevisiae. The concentration is not constant even in the same strain, depending on cultivation media and substrate composition. (2) Fluorescence of NAD(P)H in cells increases after the concentration of dissolved oxygen drops to zero, because the fluorescent reduced form of NAD(P)H cannot be oxidized in the respiration chain. (3) Biomass concentration can be evaluated by fluorescence of NAD(P)H when cells are in the exponential growth phase and all cultivation conditions such as pH, dissolved oxygen, initial substrate concentration, temperature and stirring speed remain constant. (4) In yeast cells, fluorescence of NAD(P)H quickly decreases after depletion of substrate (glucose, pyruvate, etc.).
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Unlike NAD(P)H, other biogenic fluorophores such as aminoacids and pyridoxine, flavine and their derivatives are not used so often. These measurements require special nonfluorescent media because generally used nutrient-rich media show strong fluorescence background interfering with measurements. According to Horvath et al. [4,5] and Li et al. [6], it is possible to evaluate the concentration of biomass in suspension by fluorescence of tryptophan and pyridoxine even better than by fluorescence of NAD(P)H. Although a lot of papers deal with intrinsic fluorescence of cells, only a couple of them describe measurements of immobilized cells [7,8]. The aim of this work was to develop a method allowing the measurement of concentration of living immobilized cells using only intrinsic fluorescence of biogenic fluorophores in cells. We did not use any indicator because they might affect cell’s metabolism or opticaly interfere with other fluorophores present in cell. We measured fluorescence intensities by 2-D fluorescence spectroscopy [9] in the range of excitation wavelengths 220–650 nm and emission wavelengths 250–700 nm and monitored fluorescence of NAD(P)H at λEX /λEM = 330/440 nm during aerobic–anaerobic transition obtained by halt of circulation of aerated medium. This approach does not require genetically modified cells so it can be used almost for any type of cells and it allows us to characterize also the changes of fluorescence related to oxygen uptake rate.
2. Materials and methods 2.1. Cells and media S. cerevisiae wild-type strain SP-4 (␣, leu1 , arg4 ) [10] obtained from Prof. T. Bilinski (Rzesz´ow Pedagogical University, Poland) and DBM60 (S. chevalieri) obtained from Department of Biochemistry and Microbiology of Institute of Chemical Technology in Prague (Czech Republic) were cultivated in liquid YPD medium (20 g l−1 d-glucose, 10 g l−1 peptone, 10 g l−1 yeast extract) at 30 ◦ C for 18 h. After cultivation, cells were centrifuged at 2000 × g for 5 min, washed with physiological solution (9 g l−1 NaCl), centrifuged again and resuspended in non-fluorescent Horvath–Spangler (HS) [4] medium without glucose. Resuspended cells in HSmedium were cultivated for another 2 h without glucose and then stored at 4 ◦ C overnight. Before the measurements, about one half of cells stored in HS-medium were placed into 80 ◦ C water bath for 30 min in order to obtain dead cells for mixtures.
measuring optical density of suspension at 650 nm (OD650 ) and calculated using empirical formula: X = 1.6632 × OD650 . Cells were centrifuged and resuspended in physiological solution (PS) to give a concentration of dry cell mass 72 g l−1 . Living and dead cells were mixed together to form suspensions with various living cells content but the same total dry mass concentration of 12 g l−1 . The concentration was chosen according to results of preliminary experiments [11], 1 ml of each suspension was mixed with 5 ml of 3.2% (w/v) sterile solution of sodium alginate (Sigma-Aldrich, type IV, practical grade), put into syringe and dripped through flat-end needle (inner diameter 1 mm) into sterile solution of CaCl2 (7 g l−1 ). Regular beads were formed having diameter about 3 mm. They were left in stirred CaCl2 solution for another 30 min, washed with HS-medium without glucose and stored at 4 ◦ C. 2.3. Alginate–TMOS planar films Tetramethoxysilane (TMOS), Fluka product no. 87682, was mixed with distilled water and HCl in TMOS:H2 O:HCl mol ratio = 1:5:10−2 to form a clear solution and left to prepolymerize for 24 h at 4 ◦ C. Concentrations of DBM60 cells in films ranged from 20 to 80 g l−1 of dry mass. Lower concentrations did not give reproducible results [12]. Two types of films were prepared: (1) films containing mixture of living and dead cells, maintaining concentration of total biomass constant, (2) films containing only living cells in different concentrations. ad 1: Suspensions of living and dead cells were mixed together into a centrifugation cuvette in different ratios to form a total volume of 10 ml of suspension with various concentrations of living cells but with the same total dry mass concentration. The suspension was centrifuged, and cells were resuspended in 0.5 ml of PS. The cells (0.5 ml) were mixed together with 3.2% (w/v) sodium–alginate (0.125 ml), 0.05MNaOH (0.25 ml) and prepolymerized TMOS (0.125 ml). This biocomposite solution was homogenized and 0.314 ml was pipetted onto quartz glass slide (surface area 3.14 cm2 ) which then served as a front face of the flow through cuvette (Fig. 1). Immediately after gelation, the film was bathed in CaCl2 so-
2.2. Immobilization procedures 2.2.1. Alginate beads Total dry mass concentration (X [g l−1 ]) of S. cerevisiae strain SP-4 cells stored in HS-medium was determined by
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Fig. 1. Flow-through cuvette for composite alginate–TMOS layers.
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lution (7 g l−1 ) for 30 min. After that, flow-through cuvette was assembled and measurements took place. ad 2: 10 ml of cells stored in HS-medium were centrifuged, cells were resuspended with volumes of PS, ranging from 0.5 to 2.0 ml in order to obtain various concentrations of cells. Concentrations of dry biomass in resuspended cells were determined by measurement of optical density at 650 nm and calculated using empirical formula: X = 0.1523 × OD650 . Half milliliters of the suspension was taken for immobilization procedure and composite alginate–TMOS film was created and measured in the same way as described above. In order to prepare films with thickness in the range 0.5–2 mm, volumes of biocomposite suspension (dry biomass concentration 75 g l−1 of biocomposite) in range 0.157–0.628 ml were pipetted onto the quartz glass.
2.5. Reproducibility of measurements We verified the reproducibility of NAD(P)H fluorescence and its change during a halt of medium flow: - by filling the cuvette 5 times with alginate beads with the same concentration of living cells, - by preparing four alginate–TMOS films with the same concentration of living cells, - by measurement of NAD(P)H fluorescence during repetitive halts and runs of medium flow in one alginate–TMOS film (Fig. 5).
3. Results and discussion 3.1. Reproducibility of measurements
2.4. Fluorescence measurements Twenty-five alginate beads were placed into flow-through cuvette with quartz-glass window (Fig. 2), the cuvette was installed into Hitachi’s F-4500 fluorescence spectrophotometer in front-face arrangement and perfused with aerated HSmedium without glucose at 12 ml min−1 . After equilibration of NAD(P)H fluorescence at λEX = 340 nm and λEM = 440 nm medium circulation was stopped and changes in NAD(P)H fluorescence intensity were measured. 2-DF spectra (λEX = 220–650 nm, λEM = 250–700 nm) were scanned at “aerobic state”, before the circulation of aerated medium was stopped, and “anaerobic state”, after dissolved oxygen in cuvette was depleted and NAD(P)H fluorescence intensity stabilized. These spectra were subtracted (Fig. 3) and all wavelength pairs in “aerobic”, “anaerobic” and subtracted 2-DF spectra were correlated with concentrations of dry mass of living cells in beads, using Microsoft Excel “CORREL” function. Wavelength pairs with highest correlation coefficients were located using “correlation 2-D spectra”, where fluorescence intensities were replaced by correlation coefficients (Fig. 4). Composite alginate–TMOS films were measured in the same way as the alginate beads.
The reproducibility of NAD(P)H fluorescence intensity and the t parameter is summarized in Table 1. Relative standard errors of “steps” during repetitive halts and runs of medium flow in one alginate–TMOS film (Fig. 5) were 0.5% for NAD(P)H fluorescence intensity and 5.5% for its relative change. Slow decrease of NAD(P)H fluorescence intensity can be ascribed to stresses and limited living conditions of immobilized cells (absence of carbon source, vitamins, etc. in medium). 3.2. Alginate beads The highest correlation coefficients were found at λEX /λEM = 350/430 nm for anaerobic conditions, at λEX /λEM = 350/440 nm for aerobic conditions and at λEX /λEM = 330/440 nm for subtraction spectra (Fig. 4). These wavelengths correspond to the position of maximum of NAD(P)H fluorescence in 2-DF spectra [13]. Relations of fluorescence intensity at these wavelengths to dry mass of living cells were plotted (Fig. 6) showing a linear relation between these parameters. The experiments showed that fluorescence intensity of NAD(P)H can be used for the evaluation of active cells after immobilization in conditions where environmental factors such as dissolved O2 , temperature, substrate concentration and pH remain constant. This is in accordance with papers describing a good correlation between NAD(P)H fluorescence and concentration of free cells in liquid media [14–16]. Table 1 Relative standard errors of measurements of NAD(P)H fluorescence intensity Immobilization matrix
Fig. 2. Flow-through cuvette for alginate beads.
Alginate beads Alginate–TMOS films
Relative standard error I0 (%)
(I1 − I0 )/I0 (%)
t (%)
10 2.5
15 20
5 5
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Fig. 3. 2-DF spectra of yeast cells in aerobic and anaerobic state and their subtraction.
Changes in NAD(P)H fluorescence intensity during halts of medium flow were characterized by the following parameters (Fig. 7): - I0 : average fluorescence intensity during the 60 s before the halt, - I1 : average fluorescence intensity during the last 60 s of equilibrated signal, - (I1 − I0 )/I0 : relative change of fluorescence intensity,
- t: time delay between the halt of medium flow and a rise of the fluorescence signal. Similarly to fluorescence intensities of NAD(P)H in aerobic and anaerobic states, (I1 − I0 )/I0 ratio can represent active biomass too (Fig. 8). An interesting finding was the relation between the concentration of living cells and t (Fig. 9). Unlike the fluorescence intensities, which were affected only by the cells
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Fig. 4. “Correlation 2-D spectrum” of yeast cells for aerobic state.
lying in the optical path of the instrument, we can suppose that t parameter depend on the oxygen uptake rate which is affected by all cells present in the cuvette. Consequently, this parameter should provide information about total amount of immobilized active biomass present in the cuvette.
3.3. Alginate–TMOS films Compared with the alginate beads, which could be stored overnight at 4 ◦ C, alginate–TMOS films had to be measured right after preparation. After overnight storage we did not
Fig. 5. NAD(P)H fluorescence intensities during repetitive halts and runs of medium flow in one alginate–TMOS film with living immobilized DBM60 cells (80 g l−1 ).
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Fig. 6. Relation between fluorescence intensity and dry mass of living cells in alginate beads for the best correlating wavelengths.
Fig. 7. A time dependence of NAD(P)H fluorescence intensity at λEX /λEM = 340/440 nm during a halt of medium flow.
observed any response of NAD(P)H fluorescence to halt of medium flow. In the cuvette flowed through with oxygen saturated medium, yeast cells survived overnight, but the content of the cells on the side of the film fixed to the quartz slide decreased. When the circulating medium contained a carbon source (e.g. glucose), the cells inside the film in the cuvette were growing. The amount of surviving cells depended on matrix properties [12]. Since experiments with alginate beads revealed that NAD(P)H is a suitable biogenic fluorophore for immobilized yeast cells, only time scans of NAD(P)H fluorescence intensity were measured for composite alginate–TMOS films. In films prepared only with living cells NAD(P)H fluorescence intensities (I0 , I1 ) increased proportionally to dry biomass concentration (Fig. 10). However, (I1 − I0 )/I0 ratio and t (Fig. 11) dropped down with increasing dry biomass concentration. In films containing mixtures of dead and living cells, NAD(P)H fluorescence intensities (I0 , I1 ) were proportional to concentrations of dry active biomass up to cca 55 g l−1 (Fig. 12). The decrease of fluorescence intensity of the last
sample containing 80 g l−1 of living cells is probably caused by absence of dead cells which could serve as a carbon source in the three previous samples containing a mixture of living and dead cells.
Fig. 8. Relation between relative change in NAD(P)H fluorescence intensity (I1 − I0 )/I0 and the dry mass of living cells immobilized into alginate beads.
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Fig. 9. Relation between time delay (t) and dry mass of living cells immobilized into alginate beads.
Fig. 12. Relation between fluorescence intensities in aerobic state (I0 ), anerobic state (I1 ) and their relative change (I1 − I0 )/I0 and amount of living cells in composite alginate–TMOS layers containing mixture of living and dead cells. L-dry mass of living cells (g l−1 ); T-total immobilized biomass (g l−1 ).
Fig. 10. Relation between fluorescence intensities in aerobic state (I0 ), anerobic (I1 ) and their relative change (I1 − I0 )/I0 and dry mass of living cells in composite alginate–TMOS layers containing only living cells. Fig. 13. Relation between time delay (t) and dry mass of living cells in composite alginate–TMOS layers containing mixture of living and dead cells.
Fig. 11. Relation between time delay (t) and dry mass of living cells in composite alginate–TMOS layers containing only living cells.
In contrast to films prepared only with living cells (I1 − I0 )/I0 ratios increased with content of living cells in the whole range of measured concentrations. We can explain the difference in results for films containing only living cells and mixture of dead and living cells by the following mechanism—in films with mixed living and dead cells, concentration of total biomass was constant, so optical conditions were constant as well despite of content of living cells. On
the other hand, in films containing only living cells, total dry biomass concentration varied—the biocomposites containing less cells had lower optical density so excitation light could penetrate deeper into the film and absorption and scattering of emitted light by cells were lower than in films with higher cells concentrations. t decreased with concentrations of dry active biomass similarly in both types of films (Figs. 11 and 13) as it is not dependent on optical density of biocomposite. In contrast to films, where cells in direct contact with circulating medium (i.e. source of oxygen) were separated from the most illuminated cells, in cuvette filled with alginate beads these cells were also washed by the medium. Thus, in films, diffusion and oxygen consumption inside the biocomposite had stronger influence on oxygen concentration in the vicinity of illuminated cells, than in the alginate beads. While the NAD(P)H fluorescence intensity is increasing with the film thickness up to 1.5 mm, trend of the relative change of fluorescence intensity after the medium halt, (I1 − I0 )/I0 , was opposite due to limitation of oxygen diffu-
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On the other hand, on-line monitoring of immobilized cells can be used in whole-cell optical biosensors for monitoring of e.g. environmental pollution.
Acknowledgements Funding for this research was provided by the Grant Agency of the Czech Republic through the grant no.104/01/461 and by the Ministry of Education, Youth and Sports of CR grant OC 840.10.
References Fig. 14. Relation between fluorescence intensity in aerobic state (I0 ) and its relative change (I1 − I0 )/I0 and layer thickness.
sion in thicker films (Fig. 14). Less oxygen was available for the cells near the quartz glass window of the cuvette because it was consumed inside the film. This effect became stronger in thicker films (alginate–TMOS) so cells located deeper than 1–1.5 mm were lacking oxygen.
4. Conclusions Measurements of NAD(P)H fluorescence intensity and its changes during oxygen depletion well express concentration of active immobilized biomass in non-transparent matrices. Fluorescence intensities well represented active immobilized biomass in systems where the cells situated near the surface were predominantly measured. Monitoring of changes in fluorescence during oxygen depletion allowed evaluation of active immobilized biomass also in films, in which measurement of intensities did not provide satisfactory results due to effects of diffusion and oxygen consumption inside the biocomposite. Time delay between halt of medium circulation and rise of NAD(P)H fluorescence (i.e. time needed for depletion of oxygen in cuvette) also provided information about amount of living cells in the cuvette and it was reciprocally proportional to the concentration of active biomass in alginate beads. The measurements of films with different thickness allow us to examine the conditions in inner layers of immobilization matrices because the cells, which are the most illuminated by excitation light are separated from medium by the film itself. However, the method described in this paper requires calibration for each type of cells, medium, cuvette and immobilization matrix, it can be useful for optimization of immobilization techniques for bioprocesses. Unlike the other methods it allows in situ measurements in non-transparent matrices and it does not require any dyes or agents, which can interfere with cells. It is possible to measure cells inside the matrix and its disintegration, which can affect the viability of the cells, is not necessary.
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Biographies is a research worker in the Immobilized Biomaterials and Optical Sensors group at the Institute of Chemical Process Fundamentals, Academy of Sciences of the Czech Republic. He graduated in 1998 from the Institute of Chemical Technology, Prague with MS in water technology and
environmental engineering. He received his PhD in environmental chemistry and technology in the same institute in 2003. Now, he is concerned with optical biosensors and immobilization of cells.
Gabriela Kuncova is the head of the Immobilized Biomaterials and Optical Sensors group at the Institute of Chemical Process Fundamentals, Academy of Sciences of the Czech Republic, Prague. She graduated in 1974 from Institute of Chemical Technology, Prague with MS in technology of silicates. In 1979 she obtained PhD in organic chemistry at the Institute of Chemical Process Fundamentals, Czechoslovak Academy of Sciences, Prague. Following 10 years (1979–1989) she worked on development of drawing and coating of optical fibers for telecommunication and quartz capillaries for chromatography in Joint Laboratory for Silicates CAS and ICT at Prague.