APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1978, p. 615-617
0099-2240/78/0036-0615$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 36, No. 4 Printed in U.S.A.
Flow Microfluorometry Study of Diauxic Batch Growth of Saccharomyces cerevisiae MAUREEN F. GILBERT,' DONALD N. McQUITTY,2 AND JAMES E. BAILEY'* Departments of Chemical Engineering' and Biology,2 University of Houston, Houston, Texas
77004
Received for publication 4 April 1978
Flow microfluorometry reveals complex changes in types and relative numbers of different Saccharomyces cerevisiae cell forms during glucose-limited diauxic batch growth.
Growth of the budding yeast Saccharomyces cerevisiae produces a heterogeneous population that changes throughout the growth cycle (6) and can be altered by environmental conditions (10). Population distributions from samples grown on solid media or from broth cultures have been analyzed previously from photomicrographs (e.g., 7, 16) or with Coulter Counters (5, 8, 13). Developments in flow microfluorometer (FMF) instrumentation now permit rapid measurements (3,000 cells per s) of protein and nucleic acid contents of single microbial cells (1, 2, 11, 12, 14), allowing statistically significant measurement of composition distributions in the microbial population. In this work, FMF measurements have been employed to observe the changing state of an S. cerevisiae population during batch growth. A monoclonal strain of S. cerevisiae derived from commercial Fleischmann's yeast which was grown to stationary phase was used as the inoculum for 2.5 liters of von Meyenburg medium (15) modified by the substitution of 0.4 mg of nicotinic acid per liter and 0.2 mg of p-aminobenzoic acid per liter for the yeast extract. Growth and substrate utilization data are shown in Fig. 1. Figure 2 illustrates the normalized yeast population density as a function of cellular protein content, as deterrnined by FMF analysis of samples withdrawn from the batch culture at the indicated times after inoculation. The abscissa, channel number, is proportional to fluorescein isothiocyanate and thus to the cellular protein content. The corresponding ordinate gives the corresponding population density of the S. cerevisiae culture. The interesting features in Fig. 2 are best discussed in the context of the overall growth and substrate utilization pattern (Fig. 1). As observed previously for batch cultivation of S. cerevisiae under glucose limitation (3, 9), growth is diauxic, consisting of an initial "aerobic fermentation" stage, where glucose is consumed
and ethanol is formed, and a subsequent second growth phase in which ethanol is employed as the carbon source. At h 5, about midway in the first exponential growth phase, the population protein density is unimodal. Microscopic examination of the samples taken from the culture at this time reveals that the predominant yeast forms are cells at various stages of budding; few nonbudding cells were observed. As the second growth phase begins (h 13), a bimodal distribution clearly emerges. A subpopulation of cells consisting of relatively small protein content (peak near channel no. 30) increases through the second exponential (h 15) and stationary (h 21) stages of growth. Microscopic counts of the yeast subpopulations at h 13, 15, and 21 indicate an increase in the fraction of the population consisting of cells without buds, with this cell form predominating at h 21. These results are consistent with the FMF results in Fig. 2 and provide a means for identifying the yeast subpopulations which contribute to the overall population protein density. The shoulder and longer tail appearing in the right-hand side of the protein density data for h 15 and 21 (Fig. 2) reflect the presence of pleomorphic yeast types, which are mostly double cells with one or two buds; the identity and relative numbers of this subpopulation were verified by microscopic examinations. Clearly FMF measurements have revealed significant changes in the S. cerevisiae population which are coupled to the metabolic shift from glucose consumption in the first growth phase to ethanol utilization in the second. It would be extremely difficult to obtain such information on microbial populations by other methods with anything approaching the statistical significance which can be achieved using the FMF. FMF results typically reflect measurements on more than 105 individual cells, and FMF sample preparation and analysis require only approximately 3 h for 10 samples. Also,
615
616
APPL. ENVIRON. MICROBIOL.
NOTES
--'~v.v
v.
2
4
8
6
10 12 14 TIME (HRS)
16
18
20 22
24
FIG. 1. S. cerevisiae growth (0), glucose concentration (5), and ethanol concentration (A) during batch cultivation in glucose-limited medium. T = 30°C, pH 5.5, agitation 500 rpm, 2.5-liter working volume in a New Brunswick Microferm 5-liter vessel, aeration rate = 2.5 vol/vol/min. Glucose and ethanol were assayed using the Worthington Glucostat and Sigma enzymatic methods, respectively. 0.02
HR 5
HR 13
0.01 z
0
W
0.02
"_
'
.
_
_
HR
HR 9
z
6
.
15
o
We are especially indebted to James A. Oro and M. J. Correia for assistance, and we have benefitted from discussions with J. Fazel-Madjlessi, J. E. Evans, A. Bartel, L. Y. Lee, and J. C. AMred. This work was supported by the National Science Foundation, the Camille and Henry Dreyfus Foundation, and the University of Houston.
0.01
z
!
specific acriflavine stain and the stain for doublestranded nucleic acids, propidium iodide (4).
0
0.02
HR21
HRII
-J
0.
0 0.01 0. 01 01%
I
5C
C^
.1
100 150 0 0.^
I=^
.
50
C^
.^ IVW
150
IC^
CHANNEL NUMBER (RELATIVE PROTEIN CONTENT)
FIG. 2. FMF data showing S. cerevisiae population protein densities at different times during batch growth. The data have been normalized so that the area under each density function is unity. Samples withdrawn from the culture were fixed in 70% ethanol and stained with the protein-specific fluorochrome fluorescein isothiocyanate prior to FMF analysis.
sensitivity to fine details, as revealed by peak sharpness and occasional unusually shaped distributions in these and other FMF studies of microorganisms (1, 2, 11, 12, 14), appears to be much greater than has been achieved with Coulter cell volume measurements (5, 8, 13). Moreover, FMF methods can be applied to and based upon other cell constituents for which suitable fluorescent dyes are available, including DNA-
LITERATURE CITED 1. Bailey, J. E. 1976. Structural cellular dynamics as an aid to improved fermentation processes, p. 95-98. In J. L. Gainer (ed.), Proceedings of the Conference on Enzyme Technology and Renewable Resources. University of
Virginia, Charlottesville, 2. Bailey, J. E., J. Fazel-Madjelessi, D. N. McQuitty, L. Y. Lee, J. C. Allred, and J. A. Oro. 1977. Characterization of bacterial growth by means of flow microfluorometry. Science 198:1175-1176. 3. Beck, C., and H. D. von Meyenburg. 1968. Enzyme pattern and aerobic growth of Saccharomyces cerevisiae under various degrees of glucose limitation. J. Bacteriol. 96:479-486. 4. Crissman, H. A., P. F. Mullaney, andJ. A. Steinkamp. 1975. Methods and applications of flow systems for analysis and sorting of mammalian cells. Methods Cell Biol. 9:179-246. 5. Gordon, C. N., and S. G. Elliott. 1977. Fractionation of Saccharomyces cerevisiae cell populations by centrifugal elutriation. J. Bacteriol. 129:97-100. 6. Hartwell, L. H. 1974. Saccharomyces cerevisiae cell cycle. Bacteriol. Rev. 38:164-198. 7. Hayashibe, M., N. Sando, and H. Abe. 1973. Increase in cell size as a measure of growth of Saccharomyces cerevisiae. J. Gen. Appl. Microbiol. 19:287-303. 8. Lloyd, D., L. John, M. Hamill, C. Philips, J. Kader, and S. W. Edwards. 1977. Continuous flow cell cycle fractionation of eukaryotic microorganisms. J. Gen. Microbiol. 99:223-227. 9. Maxon, W. D., and M. J. Johnson. 1953. Aeration studies on propagation of Bakers yeast. Ind. Eng. Chem. 45:2554-2560. 10. Miiler, I., and B. Brunn. 1969. Zellvolumen and Trock-
VOL. 36, 1978 engewicht von homound heterozygoten Stammen von Saccharomyces cerevisiae im Verlauf des Wachstums und unter verschiedenen Bedingungen. Arch. Microbiol. 64:327-337. 11. Paau, A. S., J. R. Cowles, and J. Ore. 1977. Flow microfluorometric analysis of Escherichia coli, Rhizobium meliloti, and Rhizobium japonicum at different stages of the growth cycle. Can. J. Microbiol. 23:1165-1169. 12. Paau, A. S., D. Lee, and J. R. Cowles. 1977. Comparison of nucleic acid content in populations of free-living and symbiotic Rhizobium meliloti by flow microfluorometry. J. Bacteriol. 129:1156-1158. 13. Sebastian, J., B. L. A. Carter, and H. 0. Halvorson.
NOTES
617
1971. Use of yeast populations fractionated by zonal centrifugation to study the cell cycle. J. Bacteriol. 108:1045-1050. 14. Slater, M. L., S. 0. Sharrow, and J. J. Gart. 1977. Cell cycle of Saccharomyces cerevisiae in populations growing at different rates. Proc. Natl. Acad. Sci. U.S.A. 74:3850-3854. 15. von Meyenburg, H. K. 1969. Energetics of the budding cycle of Saccharomyces cerevisiae during glucose limited aerobic growth. Arch. Microbiol. 66:289-303. 16. Vrana, D., and K. Beran. 1977. Cytomorphological characterization of Saccharomyces cerevisiae and Candida utilis and an index of the physiological state of the culture. Mikrobiologiya 46:134-137.
0099-2240/78/0036-0615$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 36, No. 4 Printed in U.S.A.
Flow Microfluorometry Study of Diauxic Batch Growth of Saccharomyces cerevisiae MAUREEN F. GILBERT,' DONALD N. McQUITTY,2 AND JAMES E. BAILEY'* Departments of Chemical Engineering' and Biology,2 University of Houston, Houston, Texas
77004
Received for publication 4 April 1978
Flow microfluorometry reveals complex changes in types and relative numbers of different Saccharomyces cerevisiae cell forms during glucose-limited diauxic batch growth.
Growth of the budding yeast Saccharomyces cerevisiae produces a heterogeneous population that changes throughout the growth cycle (6) and can be altered by environmental conditions (10). Population distributions from samples grown on solid media or from broth cultures have been analyzed previously from photomicrographs (e.g., 7, 16) or with Coulter Counters (5, 8, 13). Developments in flow microfluorometer (FMF) instrumentation now permit rapid measurements (3,000 cells per s) of protein and nucleic acid contents of single microbial cells (1, 2, 11, 12, 14), allowing statistically significant measurement of composition distributions in the microbial population. In this work, FMF measurements have been employed to observe the changing state of an S. cerevisiae population during batch growth. A monoclonal strain of S. cerevisiae derived from commercial Fleischmann's yeast which was grown to stationary phase was used as the inoculum for 2.5 liters of von Meyenburg medium (15) modified by the substitution of 0.4 mg of nicotinic acid per liter and 0.2 mg of p-aminobenzoic acid per liter for the yeast extract. Growth and substrate utilization data are shown in Fig. 1. Figure 2 illustrates the normalized yeast population density as a function of cellular protein content, as deterrnined by FMF analysis of samples withdrawn from the batch culture at the indicated times after inoculation. The abscissa, channel number, is proportional to fluorescein isothiocyanate and thus to the cellular protein content. The corresponding ordinate gives the corresponding population density of the S. cerevisiae culture. The interesting features in Fig. 2 are best discussed in the context of the overall growth and substrate utilization pattern (Fig. 1). As observed previously for batch cultivation of S. cerevisiae under glucose limitation (3, 9), growth is diauxic, consisting of an initial "aerobic fermentation" stage, where glucose is consumed
and ethanol is formed, and a subsequent second growth phase in which ethanol is employed as the carbon source. At h 5, about midway in the first exponential growth phase, the population protein density is unimodal. Microscopic examination of the samples taken from the culture at this time reveals that the predominant yeast forms are cells at various stages of budding; few nonbudding cells were observed. As the second growth phase begins (h 13), a bimodal distribution clearly emerges. A subpopulation of cells consisting of relatively small protein content (peak near channel no. 30) increases through the second exponential (h 15) and stationary (h 21) stages of growth. Microscopic counts of the yeast subpopulations at h 13, 15, and 21 indicate an increase in the fraction of the population consisting of cells without buds, with this cell form predominating at h 21. These results are consistent with the FMF results in Fig. 2 and provide a means for identifying the yeast subpopulations which contribute to the overall population protein density. The shoulder and longer tail appearing in the right-hand side of the protein density data for h 15 and 21 (Fig. 2) reflect the presence of pleomorphic yeast types, which are mostly double cells with one or two buds; the identity and relative numbers of this subpopulation were verified by microscopic examinations. Clearly FMF measurements have revealed significant changes in the S. cerevisiae population which are coupled to the metabolic shift from glucose consumption in the first growth phase to ethanol utilization in the second. It would be extremely difficult to obtain such information on microbial populations by other methods with anything approaching the statistical significance which can be achieved using the FMF. FMF results typically reflect measurements on more than 105 individual cells, and FMF sample preparation and analysis require only approximately 3 h for 10 samples. Also,
615
616
APPL. ENVIRON. MICROBIOL.
NOTES
--'~v.v
v.
2
4
8
6
10 12 14 TIME (HRS)
16
18
20 22
24
FIG. 1. S. cerevisiae growth (0), glucose concentration (5), and ethanol concentration (A) during batch cultivation in glucose-limited medium. T = 30°C, pH 5.5, agitation 500 rpm, 2.5-liter working volume in a New Brunswick Microferm 5-liter vessel, aeration rate = 2.5 vol/vol/min. Glucose and ethanol were assayed using the Worthington Glucostat and Sigma enzymatic methods, respectively. 0.02
HR 5
HR 13
0.01 z
0
W
0.02
"_
'
.
_
_
HR
HR 9
z
6
.
15
o
We are especially indebted to James A. Oro and M. J. Correia for assistance, and we have benefitted from discussions with J. Fazel-Madjlessi, J. E. Evans, A. Bartel, L. Y. Lee, and J. C. AMred. This work was supported by the National Science Foundation, the Camille and Henry Dreyfus Foundation, and the University of Houston.
0.01
z
!
specific acriflavine stain and the stain for doublestranded nucleic acids, propidium iodide (4).
0
0.02
HR21
HRII
-J
0.
0 0.01 0. 01 01%
I
5C
C^
.1
100 150 0 0.^
I=^
.
50
C^
.^ IVW
150
IC^
CHANNEL NUMBER (RELATIVE PROTEIN CONTENT)
FIG. 2. FMF data showing S. cerevisiae population protein densities at different times during batch growth. The data have been normalized so that the area under each density function is unity. Samples withdrawn from the culture were fixed in 70% ethanol and stained with the protein-specific fluorochrome fluorescein isothiocyanate prior to FMF analysis.
sensitivity to fine details, as revealed by peak sharpness and occasional unusually shaped distributions in these and other FMF studies of microorganisms (1, 2, 11, 12, 14), appears to be much greater than has been achieved with Coulter cell volume measurements (5, 8, 13). Moreover, FMF methods can be applied to and based upon other cell constituents for which suitable fluorescent dyes are available, including DNA-
LITERATURE CITED 1. Bailey, J. E. 1976. Structural cellular dynamics as an aid to improved fermentation processes, p. 95-98. In J. L. Gainer (ed.), Proceedings of the Conference on Enzyme Technology and Renewable Resources. University of
Virginia, Charlottesville, 2. Bailey, J. E., J. Fazel-Madjelessi, D. N. McQuitty, L. Y. Lee, J. C. Allred, and J. A. Oro. 1977. Characterization of bacterial growth by means of flow microfluorometry. Science 198:1175-1176. 3. Beck, C., and H. D. von Meyenburg. 1968. Enzyme pattern and aerobic growth of Saccharomyces cerevisiae under various degrees of glucose limitation. J. Bacteriol. 96:479-486. 4. Crissman, H. A., P. F. Mullaney, andJ. A. Steinkamp. 1975. Methods and applications of flow systems for analysis and sorting of mammalian cells. Methods Cell Biol. 9:179-246. 5. Gordon, C. N., and S. G. Elliott. 1977. Fractionation of Saccharomyces cerevisiae cell populations by centrifugal elutriation. J. Bacteriol. 129:97-100. 6. Hartwell, L. H. 1974. Saccharomyces cerevisiae cell cycle. Bacteriol. Rev. 38:164-198. 7. Hayashibe, M., N. Sando, and H. Abe. 1973. Increase in cell size as a measure of growth of Saccharomyces cerevisiae. J. Gen. Appl. Microbiol. 19:287-303. 8. Lloyd, D., L. John, M. Hamill, C. Philips, J. Kader, and S. W. Edwards. 1977. Continuous flow cell cycle fractionation of eukaryotic microorganisms. J. Gen. Microbiol. 99:223-227. 9. Maxon, W. D., and M. J. Johnson. 1953. Aeration studies on propagation of Bakers yeast. Ind. Eng. Chem. 45:2554-2560. 10. Miiler, I., and B. Brunn. 1969. Zellvolumen and Trock-
VOL. 36, 1978 engewicht von homound heterozygoten Stammen von Saccharomyces cerevisiae im Verlauf des Wachstums und unter verschiedenen Bedingungen. Arch. Microbiol. 64:327-337. 11. Paau, A. S., J. R. Cowles, and J. Ore. 1977. Flow microfluorometric analysis of Escherichia coli, Rhizobium meliloti, and Rhizobium japonicum at different stages of the growth cycle. Can. J. Microbiol. 23:1165-1169. 12. Paau, A. S., D. Lee, and J. R. Cowles. 1977. Comparison of nucleic acid content in populations of free-living and symbiotic Rhizobium meliloti by flow microfluorometry. J. Bacteriol. 129:1156-1158. 13. Sebastian, J., B. L. A. Carter, and H. 0. Halvorson.
NOTES
617
1971. Use of yeast populations fractionated by zonal centrifugation to study the cell cycle. J. Bacteriol. 108:1045-1050. 14. Slater, M. L., S. 0. Sharrow, and J. J. Gart. 1977. Cell cycle of Saccharomyces cerevisiae in populations growing at different rates. Proc. Natl. Acad. Sci. U.S.A. 74:3850-3854. 15. von Meyenburg, H. K. 1969. Energetics of the budding cycle of Saccharomyces cerevisiae during glucose limited aerobic growth. Arch. Microbiol. 66:289-303. 16. Vrana, D., and K. Beran. 1977. Cytomorphological characterization of Saccharomyces cerevisiae and Candida utilis and an index of the physiological state of the culture. Mikrobiologiya 46:134-137.
