Rates of Respiration for Different Carbon Sources

Saccharomyces cerevisiae, commonly utilized for fermenting in baking and brewing, is a eukaryotic fungus that can metabolize sugars for energy through cellular respiration. Fungi and animals share a common ancestor in the tree of life, which suggests studying single-celled yeasts is an effective way to identify and study complex systems in animal cells (Freeman, et al.

, 2017). S. cerevisiae was the first eukaryotic organism that had its genome mapped completely, is simple to maintain because of its small size and low nutrient requirements, and it reproduces rapidly with great numbers (Freeman, et al., 2017). These characteristics make S. cerevisiae an excellent model organism.

In the presence of oxygen and a carbon source, S. cerevisiae metabolizes complex energy-rich molecules into usable energy via cellular respiration, producing water and carbon dioxide (Freeman, et al., 2017). S. cerevisiae can respire using many carbon sources such as the ones used in this experiment: glucose, sucrose, lactose, glycerol, and water as a control (Bartlett, 2018). Measuring the amount of CO₂ produced by the S.

cerevisiae cultures fed with different fuel sources, will give an indication as to which carbon source S. cerevisiae metabolizes optimally. Determining the optimal carbon source for S. cerevisiae metabolism leads to a better understanding of the biochemistry involved in cellular respiration, as well as finding the maximum rate of CO₂ production can have many applications.

Samples of S. cerevisiae cultures are mixed with 5% solutions of each carbon source, then analyzed using fresh respiration chambers and a CO₂ sensor that was left to return to ambient conditions.

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The enzymes that catalyze the processes of cellular respiration work optimally at 30° C, a water bath is used to control for this variable and keep the temperature constant (Bartlett, 2018). To eliminate confounding variables or interferents when detecting the rate of carbon dioxide production, temperature, amount of carbon source used, and status of equipment is unchanged.

Hypothesis

Since glucose enters cellular respiration immediately and requires no extra processing, the measured rate of CO₂ production for glucose by S. cerevisiae will be higher than that for other carbon sources.

Methods

To determine the rate of CO₂ produced per minute via cellular respiration by S. cerevisiae, five carbon source solutions were mixed with S. cerevisiae cultures, incubated in a water bath for 10 minutes, and then placed in a respiration chamber to collect the amount of CO₂ produced over a 4-minute period. First, five different culture tubes with 2.0 mL each of S. cerevisiae solution were prepared, as well as a 30° C water bath. Before any carbon-source solution was added to the S. cerevisiae cultures, LoggerPro was loaded onto the computer for data collection and connected to a carbon dioxide gas sensor probe. The LoggerPro software was formatted to collect data at a rate of 6 samples per minute for a total of 4 minutes. Additionally, a clean, empty respiration chamber and a test tube rack were put in the water bath. As soon as the equipment was set up, 2.0 mL of deionized water was added to one of the S. cerevisiae culture tubes and immediately placed in the test tube rack in the water bath for a 10-minute incubation period at 30°. Once the incubation period was over, 1.0 mL of the sample was placed in the respiration chamber previously added to the water bath with a pipette, the CO₂ gas sensor was secured to the respiration chamber, and data collection began. Data was then collected on the other previously prepared 2.0 mL S. cerevisiae culture tubes, by adding 2.0 mL of 5% solutions of glucose, sucrose, lactose, and glycerol using the same methods to set up each round of data collection. Once data collection was completed, the rate of CO₂ production was determined by calculating a line of best fit with an interval of where the graph was increasing. These rates were determined for all 5 samples, as well as repeated in five other experiments to determine the average rate of CO₂ gas in parts per million per minute.

Results

When Saccharomyces cerevisiae metabolized energy sources provided in this experiment, glucose had the highest rate of respiration, measured in ppm of CO₂(g)/min. Glucose was respired at a rate of 631.2 ppm CO₂/min (Fig 1). Figure 2 also shows that the disaccharide molecules sucrose and lactose when consumed by baker’s yeast, demonstrated an average rate of carbon dioxide production of fewer than 100ppm of CO2/min. Glycerol, an alcohol, also followed this trend. Overall, glucose yielded respiration rates around 10 times greater than the other 5% solutions. Standard deviations for the average rates of carbon dioxide (Fig 2) for 5 lab groups document that rates of CO₂ production were variable between groups.

Discussion

Utilizing glucose as a carbon source for metabolism by S. cerevisiae yielded the highest rate of Carbon dioxide gas production. This suggests that glucose is the optimal source of fuel for S. cerevisiae cells, thus supporting the hypothesized result. After glucose, the average respiration rate in decreasing order was sucrose, then glycerol and finally lactose. One example study suggests an explanation for why disaccharides can be metabolized similarly to hexoses excluding the first few steps: maltose cannot enter the plasma membrane through diffusion like glucose can, it must bind to an energy-dependent permease membrane protein to enter glycolysis. (Daran-Lapujade, et al., 2003). However, maltose is easily utilized because hydrolysis breaks it into 2 glucose molecules, meanwhile, lactose and sucrose are comprised of one glucose each, plus an additional galactose and fructose respectively (Freeman, et al., 2017). Galactose and fructose must be processed further by enzymes to convert them to glucose, which cannot occur until those enzymes are made available (Bartlett, 2018) (Freeman, et al., 2017). In any case, cellular respiration as a metabolic pathway is optimized for glucose and requires little energy investment; glucose can enter the catabolic pathway directly, but non-glucose molecules may require energy input, enzymatic processing, and more time to be utilized (Bartlett, 2018).

Overall, glucose had the highest rate of respiration, which suggests that glucose is the optimum carbon source for S. cerevisiae. This was expected because glucose is the molecule that cellular respiration is based upon. The other carbon sources were inefficient because of their structure. Lactose and Sucrose are both disaccharides that must be separated into their monomer subunits before they can be utilized for metabolism. Another inefficiency when using lactose and sucrose is that part of their monomer units are non-glucose molecules which require further processing before they can be utilized in cellular respiration. Glycerol is a poor carbon source because of the few carbon and hydrogen bonds that store energy.

All of the carbon sources tested were of different molecular structures, i.e. an alcohol, disaccharides, and a monosaccharide. Of the monosaccharides other than glucose, other hexoses could be the second best in terms of metabolic efficiency. If enzymatic processing of non-glucose molecules are required, a lack of nutrients, such as metal ions or other compounds necessary for protein synthesis may be a limiting factor in those pathways. Perhaps measuring the efficiency of metabolism during specific times in S. cerevisiae’s life cycle is another way that glucose uptake could be optimized.

References

1. Bartlett, M. (2018). Principles of Biology: Bi 214 Laboratory Manual. Principles of Biology I. Portland State University.
2. Daran-Lapujade, P., Jansen, M. L., Daran, J.-M., van Gulik, W., de Winde, J. H., & Pronk, J. T. (2003). Role of Transcriptional Regulation in Controlling Fluxes in Central Carbon Metabolism of Saccharomyces cerevisiae, a Chemostat Culture Study. Delft: Journal of Biological Chemistry.
3. Freeman, S., Quillin, K., Allison, L., Black, M., Podgorski, G., Taylor, E., & Carmichael, J. (2017). Biological Science (6 ed.). Hoboken: Pearson Higher Education.