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Abstract

Introduction

Protocol

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Materials

References

Biology

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Published: September 30th, 2018

DOI:

10.3791/58192

1Department of Chemistry, Universidad Autónoma de Querétaro, 2Department of Biochemical Engineering, Instituto Tecnológico de Morelia, 3Department of Biochemical Engineering, Instituto Tecnológico Superior de Ciudad Hidalgo

Here we present a protocol to estimate the respiratory and fermentative metabolism by fitting the exponential growth of Saccharomyces cerevisiae to the exponential growth equation. Calculation of the kinetic parameters allows for the screening of influences of substances/compounds on fermentation or mitochondrial respiration.

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The fermentable carbon concentration mainly governs how the yeast cells generate ATP; thus, the variation in fermentable carbohydrate levels drives the energetic metabolism of S. cerevisiae. This paper describes a high-throughput method based on exponential yeast growth to estimate the effects of concentration changes and nature of the carbon source on respiratory and fermentative metabolism. The growth of S. cerevisiae is measured in a microplate or shaken conical flask by determining the optical density (OD) at 600 nm. Then, a growth curve is built by plotting OD versus time, which allows identification and selection of the exponential phase, and is fitted with the exponential growth equation to obtain kinetic parameters. Low specific growth rates with higher doubling times generally represent a respiratory growth. Conversely, higher specific growth rates with lower doubling times indicate fermentative growth. Threshold values of doubling time and specific growth rate are estimated using well-known respiratory or fermentative conditions, such as non-fermentable carbon sources or higher concentrations of fermentable sugars. This is obtained for each specific strain. Finally, the calculated kinetic parameters are compared with the threshold values to establish whether the yeast shows fermentative and/or respiratory growth. The advantage of this method is its relative simplicity for understanding the effects of a substance/compound on fermentative or respiratory metabolism. It is important to highlight that growth is an intricate and complex biological process; therefore, preliminary data from this method must be corroborated by the quantification of oxygen consumption and accumulation of fermentation byproducts. Thereby, this technique can be used as a preliminary screening of compounds/substances that may disturb or enhance fermentative or respiratory metabolism.

Saccharomyces cerevisiae growth has served as a valuable tool to identify dozens of physiological and molecular mechanisms. Growth is measured primarily by three methods: serial dilutions for spot testing, colony-forming unit counting, and growth curves. These techniques can be used alone or in combination with a variety of substrates, environmental conditions, mutants, and chemicals to investigate specific responses or phenotypes.

Mitochondrial respiration is a biological process in which growth kinetics has been successfully applied for discovering unknown mechanisms. In this case, the supplementation of growth media with non-fer....

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1. Culture Media and Inoculum Preparation

  1. Prepare 100 mL of 2% yeast extract-peptone-dextrose (YPD) liquid medium (add 1 g of yeast extract, 2 g of casein peptone, and 2 g of glucose to 100 mL of distilled water). Dispense 3 mL of the media into 15 mL sterilizable conical tubes. Autoclave the media for 15 min at 121 °C and 1.5 psi.
    NOTE: The media can be stored for up to one month at 4–8 °C.
  2. Inoculate a conical tube filled with 3 mL of cool sterile 2% YPD broth with 250 μ.......

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Growth curves can be used to preliminarily discriminate between respiratory and fermentative phenotypes in the S. cerevisiae yeast. Therefore, we performed batch cultures of S. cerevisiae (BY4742) with different glucose concentrations that have been reported to induce fermentative growth: 1%, 2%, and 10% (w/v)9. Cultures showing a fermentative phenotype have a small lag phase and an exponential phase with a high growth rate (F.......

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A long time has passed since J. Monod10 expressed that the study of the growth of bacterial cultures is the basic method of microbiology. The advent of the molecular tools delays the usage and study of the growth as a technique. Despite the complexity of growth which involves numerous interrelated processes, its underlying mechanisms can be described by using mathematical models11. This is a robust approach that can be used as a complementary tool to elucidate the most intr.......

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This project was supported by grants of the Consejo Nacional de Ciencia y Tecnología (grant number 293940) and Fundación TELMEX-TELCEL (grant number 162005585), both to IKOM.

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Name Company Catalog Number Comments
Orbital Shaker Thermo Scientific 4353 For inoculum incubation or conical fask cultures
Bioscreen  Growth curves C MBR For batch cultures in microplates
Glucose Sigma  G7021 For YPD broth preparation
Peptone from casein, enzymatic digest Sigma  82303 For YPD broth preparation
Yeast extract Sigma  09182-1KG-F For YPD broth preparation
Bacteriological Agar Sigma  A5306 For YPD agar preparation
NaH2PO4 Sigma  S8282 For SC broth preparation
(NH4)2SO4 Sigma  A4418 For SC broth preparation
Yeast nitrogen base without amino acids and ammonium sulfate Sigma  Y1251 For SC broth preparation
Yeast synthetic drop-Out medium supplements Sigma  Y1501 For SC broth preparation
Ammonium sulfate granular J.T. Baker 0792-R For medium supplementation example
Resveratrol Sigma  R5010 For medium supplementation example
Galactose Sigma  G8270 For medium supplementation example
Sucrose Sigma  S7903 For medium supplementation example
Absolut ethanol Merck 107017 For medium supplementation example
Glycerol J.T. Baker 2136-01 For medium supplementation example
GraphPad Prism GraphPad Software For data analysis
Honeycomb microplates Thermo Scientific 9502550 For microplate cultures

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