JoVE Logo
Faculty Resource Center

Sign In





Representative Results






The Use of Chemostats in Microbial Systems Biology

Published: October 14th, 2013



1Center for Genomics and Systems Biology, Department of Biology, New York University

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

The growth of cells is regulated by complex networks of interacting genetic and environmental factors1,2. The multifactorial regulation of cell growth necessitates a system-level approach to its study. However, the rigorous study of regulated cell growth is challenged by the difficulty of experimentally controlling the rate at which cells grow. Moreover, in even the simplest experiments extracellular conditions are frequently dynamic and complex as cells continuously alter their environment as they proliferate. A solution to these problems is provided by the chemostat: a method of culturing cells that enables experimental control of cell growth rates in def....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The principle of continuous culturing using a chemostat can be realized in a variety of implementations. In all chemostats it is essential to have 1) methods for maintaining sterility of all components, 2) a well-mixed culture, 3) appropriate aeration of the culture vessel and 4) a reliable means of media addition and culture removal. Here, we describe the use of a Sixfors bioreactor (Infors Inc) as a chemostat using methods that can be readily adapted to alternative setups.

1. Assembling the Ch.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

A major advantage of chemostats is the ability to control the growth rate of cells experimentally by varying the dilution rate. In the budding yeast, Saccharomyces cerevisiae, the morphology of a cell is informative of its phase in the cell division cycle. Populations with higher growth rates contain a higher proportion of actively dividing cells as determined by measuring the fraction of unbudded cells (Figure 5A). Analyses of global mRNA expression in chemostat cultures has shown that the expression of.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Chemostats enable the cultivation of microbes in growth-controlled steady-state conditions. The cells grow continuously at a constant rate resulting in an invariant external environment. This is in contrast to batch culture methods in which the external environment is continuously changing and the rate of cell growth is determined by the complex interaction of environment and genotype. Thus, a major advantage of culturing microbes in chemostats over batch cultures is the ability to experimentally control the growth rate .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This work was supported by start up funds form New York University. We thank Maitreya Dunham and Matt Brauer who initially developed the use of Sixfors bioreactors as chemostats.


Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Name of the reagent Company Catalogue number Comments (optional)
Infors-HT Sixfors Chemostat Appropriate Technical Resources, Inc.    
Glass Bottle 9.5 L Fisher Scientific 02-887-1 For Media Vessel and Hosing
Pinchcock Fisher Scientific 05-867 For Media Vessel and Hosing
Stopper, Size 12, Green Neoprene Cole-Palmer EW-62991-42 For Media Vessel and Hosing
Straight Connector Cole-Palmer EW-30703-02 For Media Vessel and Hosing
General purpose ties 4 in Fisher Scientific NC9557052 For Media Vessel and Hosing
Tubing, Silicone Rubber Small Parts B000FMWTDE For Media Vessel and Hosing
Tubing, Silicone, 3/8 in OD Fisher Scientific 02-587-1Q For Media Vessel and Hosing
Tubing, Silicone, 7/32 in OD Fisher Scientific 02-587-1E For Media Vessel and Hosing
Tubing, Stainless Steel, 3/16 in OD McMaster-Carr 6100K164 For Media Vessel and Hosing
Tubing, Stainless Steel, 3/8 in OD McMaster-Carr 6100K161 For Media Vessel and Hosing
Hook Connectors Fisher Scientific 14-66-18Q For Media Vessel and Hosing
Ratchet Clamp Cole-Palmer EW-06403-11 For Media Vessel and Hosing
Luer, Female Cole-Palmer EW-45512-34 For Media Vessel and Hosing
Luer, Male Cole-Palmer EW-45513-04 For Media Vessel and Hosing
Millipore Aervent MTGR05010 62 mm Filter, 0.2 μm Fisher Scientific MTGR05010 For Media Vessel and Hosing
PTFE Acrodisc CR 13 mm filters, 0.2 μm Fisher Scientific NC9131037 For Media Vessel and Hosing
Direct-Reading Flowtube for Air Cole-Palmer EW-32047-77 For Nitrogen Gas Setup
Direct-Reading Flowtube for Nitrogen Cole-Palmer EW-32048-63 For Nitrogen Gas Setup
Gas Proportioner Multitube Frames Cole-Palmer EW-03218-50 For Nitrogen Gas Setup
Regulator, Two-Stage Analytical Airgas Y12-N145D580 For Nitrogen Gas Setup
Hose Adaptor, Stainless Steel Airgas Y99-26450 For Nitrogen Gas Setup
Hose Male Adaptor Airgas WES544 For Nitrogen Gas Setup
Norprene Tubing US Plastics 57280 For Nitrogen Gas Setup
Tripod Base Cole-Palmer EW-03218-58 For Nitrogen Gas Setup
Valve Cartridges Cole-Palmer EW-03217-92 For Nitrogen Gas Setup
Carboy 10 L Fisher Scientific 02-963-2A For Media Preperation
Steritop Sterile Vacuum Bottle-Top Filters, 1,000 ml, PES membrane; for 45 mm neck size Fisher Scientific SCGP-T10-RE For Media Preperation
Media Bottle 100 ml, 45 mm neck size Fisher Scientific FB-800-100 For Media Preperation
calcium chloride·2H2O Fisher Scientific C79-500 Media Reagents
sodium chloride Fisher Scientific BP358-1 Media Reagents
magnesium sulfate·7H2O Sigma Aldrich 230391 Media Reagents
potassium phosphate monobasic Fisher Scientific AC424205000 Media Reagents
ammonium sulfate Fisher Scientific AC423400010 Media Reagents
potassium chloride Sigma Aldrich P9541 Media Reagents
boric acid Sigma Aldrich B6768 Media Reagents
copper sulfate·5H2O Sigma Aldrich 209198 Media Reagents
potassium iodide Sigma Aldrich 60400 Media Reagents
ferric chloride·6H2O Fisher Scientific I88-100 Media Reagents
manganese sulfate·H2O Sigma Aldrich 230391 Media Reagents
sodium molybdate·2H2O Sigma Aldrich M7634 Media Reagents
zinc sulfate·7H2O Fisher Scientific Z68-500 Media Reagents
biotin Fisher Scientific BP232-1 Media Reagents
calcium pantothenate Fisher Scientific AC24330-1000 Media Reagents
folic acid Sigma Aldrich F7876 Media Reagents
inositol (aka myo-inositol) Fisher Scientific AC12226-1000 Media Reagents
niacin (aka nicotinic acid) Sigma Aldrich N4126 Media Reagents
p-aminobenzoic acid Fisher Scientific AC14621-2500 Media Reagents
pyridoxine HCl Sigma Aldrich P9755 Media Reagents
riboflavin Sigma Aldrich R4500-25G Media Reagents
thiamine HCl Fisher Scientific BP892-100 Media Reagents
Leucine Sigma Aldrich L8000-100G Media Reagents
Uracil Sigma Aldrich U0750 Media Reagents
Dextrose Fisher Scientific DF0155-08-5 Media Reagents

  1. Ingraham, J. L., Maaloe, O., Neidhardt, F. C. . Growth of the Bacterial Cell. , (1983).
  2. Hall, M. N., Raff, M. C., Thomas, G. . Cell Growth: Control of Cell Size. , (2004).
  3. Monod, J. La technique de culture continue, theorie et applications. Ann. Inst. Pasteur. 79, 390-410 (1950).
  4. Novick, A., Szilard, L. Description of the chemostat. Science. 112, 715-716 (1950).
  5. Kjeldgaard, N. O., Maaloe, O., Schaechter, M. The transition between different physiological states during balanced growth of Salmonella typhimurium. J. Gen. Microbiol. 19, 607-616 (1958).
  6. Maaloe, O., Kjeldgaard, N. O. Control of macromolecular synthesis. , (1966).
  7. Saldanha, A. J., Brauer, M. J., Botstein, D. Nutritional Homeostasis in Batch and Steady-State. Culture of Yeast. Mol. Biol. Cell. 15, 4089-4104 (2004).
  8. Boer, V. M., Crutchfield, C. A., Bradley, P. H., Botstein, D., Rabinowitz, J. D. Growth-limiting intracellular metabolites in yeast growing under diverse nutrient limitations. Mol. Biol. Cell. 21, 198-211 (2010).
  9. Boer, V. M., de Winde, J. H., Pronk, J. T., Piper, M. D. The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J. Biol. Chem. 278, 3265-3274 (2003).
  10. Brauer, M. J., et al. Coordination of growth rate, cell cycle, stress response, and metabolic activity in yeast. Mol. Biol. Cell. 19, 352-367 (2008).
  11. Gresham, D., et al. Adaptation to diverse nitrogen-limited environments by deletion or extrachromosomal element formation of the GAP1 locus. Proc. Natl. Acad. Sci. U.S.A. 107, 18551-18556 (2010).
  12. Regenberg, B., et al. Growth-rate regulated genes have profound impact on interpretation of transcriptome profiling in Saccharomyces cerevisiae. Genome Biol. 7, R107 (2006).
  13. Castrillo, J. I., et al. Growth control of the eukaryote cell: a systems biology study in yeast. J. Biol. 6, 4 (2007).
  14. Cipollina, C., et al. Revisiting the role of yeast Sfp1 in ribosome biogenesis and cell size control: a chemostat study. Microbiology. 154, 337-346 (2008).
  15. Gresham, D., et al. System-level analysis of genes and functions affecting survival during nutrient starvation in Saccharomyces cerevisiae. Genetics. 187, 299-317 (2011).
  16. Levy, S. F., Ziv, N., Siegal, M. L. Bet hedging in yeast by heterogeneous, age-correlated expression of a stress protectant. PLoS Biol. 10, e1001325 (2012).
  17. Kao, K. C., Sherlock, G. Molecular characterization of clonal interference during adaptive evolution in asexual populations of Saccharomyces cerevisiae. Nat. Genet. 40, 1499-1504 (2008).
  18. Gresham, D., et al. The repertoire and dynamics of evolutionary adaptations to controlled nutrient-limited environments in yeast. PLoS Genet. 4, e1000303 (2008).
  19. Wenger, J. W., et al. Hunger Artists: Yeast Adapted to Carbon Limitation Show Trade-Offs under Carbon Sufficiency. PLoS Genet. 7, e1002202 (2011).
  20. Dunham, M. J., et al. Characteristic genome rearrangements in experimental evolution of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 99, 16144-16149 (2002).
  21. Ronen, M., Botstein, D. Transcriptional response of steady-state yeast cultures to transient perturbations in carbon source. Proc. Natl. Acad. Sci. U.S.A. 103, 389-394 (2006).
  22. Kresnowati, M. T. A. P., et al. When transcriptome meets metabolome: fast cellular responses of yeast to sudden relief of glucose limitation. Mol. Sys. Biol. 2, 49 (2006).
  23. Tu, B. P., Kudlicki, A., Rowicka, M., McKnight, S. L. Logic of the yeast metabolic cycle: temporal compartmentalization of cellular processes. Science. 310, 1152-1158 (2005).
  24. Tzur, A., Kafri, R., LeBleu, V. S., Lahav, G., Kirschner, M. W. Cell Growth and Size Homeostasis in Proliferating Animal Cells. Science. 325, 167-171 (2009).
  25. Conlon, I., Raff, M. Size control in animal development. Cell. 96, 235-244 (1999).
  26. Conlon, I. J., Dunn, G. A., Mudge, A. W., Raff, M. C. Extracellular control of cell size. Nat. Cell Biol. 3, 918-921 (2001).
  27. Fussmann, G. F., Ellner, S. P., Shertzer, K. W., Hairston, N. G. Crossing the hopf bifurcation in a live predator-prey system. Science. 290, 1358-1360 (2000).
  28. Cohen, E. P., Eagle, H. A simplified chemostat for the growth of mammalian cells: characteristics of cell growth in continuous culture. J. Exp. Med. 113, 467-474 (1961).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved