The Impacts of NaF on Respiration in Yeast

Chloe Black

17 November 2022

Introduction

Glycolysis, the first step in cellular respiration and fermentation, involves splitting one

glucose molecule into two molecules of pyruvate (Alfarouk et al., 2014). If there is no oxygen

present to move onto the next step of cellular respiration, the two pyruvate molecules enter

fermentation (Alfarouk et al., 2014). There are specific enzymes involved in every step of

glycolysis, all of which are used to catalyze the reaction (Li et al., 2015). These enzymes play a

key role in substrate-level phosphorylation, which attaches a phosphate group from a substrate

directly to ADP, forming high-energy ATP (Li et al., 2015). The purpose of glycolysis is to

generate two molecules of pyruvate that can be later used in respiration to produce a surplus of

ATP, or in fermentation to produce ethanol or lactic acid (Alfarouk et al., 2014). Enolase is an

essential glycolytic enzyme responsible for the conversion of D-2-phosphoglycerate (PGA) and

phosphoenolpyruvate (PEP), two vital metabolic pathways in both eukaryotic and prokaryotic

organisms, which needs a magnesium cofactor to function correctly (Lopez-Lopez et al., 2018).

Fluorine is the most electronegative element, making it highly reactive (Banerjee and

Roychoudhury, 2019). The negative fluorine ion strongly associates with other positive ions,

which decreases the electronegativity of pure fluorine; this decrease in fluorine reactivity lessens

the toxicity of fluorine in glycolysis (Banerjee and Roychoudhury, 2019). Enolase is inhibited by

pure fluorine, which stops the process of glycolysis (Banerjee and Roychoudhury, 2019). The

fluorine-induced inhibition of glycolytic enzymes suppresses respiration rates up to 50% in yeast

cells (Banerjee and Roychoudhury, 2019).

 The most common form of yeast used for fermentation is Baker’s Yeast Saccharomyces

cerevisiae, which causes bread dough to rise by converting sugar to carbon dioxide gas (Lahue et

al., 2020). S. cerevisiae is crucial in the fermentation of foods, and has been found in

environments such as soil and trees (Lahue et al., 2020). The presence of S. cerevisiae in a range

of environments forces it to be highly adaptable, which indicates a wide variety of strains and

genetic changes throughout time (Lahue et al., 2020). S. cerevisiae is scientifically valuable

because of its numerous genetic changes that have adapted to current bread dough, and how the

variation of wild strains can be used to improve baking strains (Lahue et al., 2020).

The purpose of this project was to determine if magnesium could ameliorate fluorine

toxicity. The project tested two hypotheses: sodium fluoride will affect carbon dioxide

production in yeast, and magnesium chloride will ameliorate the impacts of sodium fluoride

treatments. We predict that adding magnesium to fluorine treated yeast will have higher growth

than yeast treated exclusively with fluorine.

Methods

Baker’s Yeast, Saccharomyces Cerevisiae was obtained from Fleiscmans rapid rise

instant yeast. This yeast was activated by adding the yeast packet to 300 ml of warm tap water.

All treatment groups received 3 mL of the yeast solution. The experiment tested five different

treatments containing either distilled water, glucose, sodium fluoride and/or magnesium chloride.

The first treatment, the negative control, contained yeast along with 6 ml of distilled

water. The next three treatments all contained 3 ml of 5% glucose, with the second also

containing 3 ml of distilled water. The third treatment contained 3 ml of 0.05 M NaF along with

the 3 ml of glucose. The fourth treatment contained 3 ml of 0.5 M NaF along with the 3 ml of
glucose. The fifth and final treatment contained 3 ml of 0.05 M NaF, along with 3 ml of 0.07 M

MgCl. There were 40 replicates for each treatment group.

After filling each test tube with the correct solutions, a respiration chamber was created

by lowering a larger test tube over the smaller test tube as far as it could go. These test tubes

were inverted very quickly so that no fluid was lost from the small test tube. After the test tubes

were inverted, the airspace in the small test tube was measured with a ruler in millimeters. After

recording the initial airspace, the test tubes were then placed in the 37 ℃ water bath. After 45

minutes, the test tubes were taken out of the water bath and the final airspace was measured in

millimeters. The initial airspace was subtracted by the final airspace to find the difference in

growth. All data were tested for normality and variance. Treatments were compared using an

ANOVA (a=0.05).

Results

Yeast growth decreased when treated with high and low concentrations of NaF. The

results of the ANOVA showed there is a significant difference in gas production between

glucose and 0.05 M NaF treatments, with glucose having greater growth (p-value < 0.001, Figure

1). The results of the ANOVA showed there is a slight significant difference in gas production

between water and 0.5 M NaF treatments, with 0.5 NaF having greater growth (p-value = 0.039,

Figure 1). A high concentration of 0.5 M NaF had less gas production than a lower concentration

of 0.05 M NaF.
 Figure

1: Gas Production Under Different Ion Concentrations

MgCl at a concentration of 0.07 M ameliorated the impacts of NaF on yeast growth, but

not entirely. The results of the ANOVA showed there is a significant difference in gas

production between glucose and 0.07 MgCl + 0.05 NaF treatments, with glucose having greater

growth (p-value < 0.001, Figure 1). The results of the ANOVA showed there is a significant

difference in gas production between 0.05 M NaF and 0.05 M NaF + 0.07 MgCl, with 0.05 M

NaF + 0.07 MgCl having greater growth (p-value < 0.01, Figure 1).

Discussion

The hypothesis that NaF would reduce carbon dioxide production in yeast cultures was

tested. The results showed a similar gas production in the water treatment compared to 0.5 M

NaF (Figure 1), which supported the hypothesis. Furthermore, the results showed a significantly

higher gas production in glucose treatments than 0.05 M treatment (p-value<0.001, Figure 1),

which also supported the hypothesis. All treatments with NaF had a lower gas production than
glucose. To determine if fluorine was negatively impacting the Mg cofactor of enolase, Mg was

added to yeast cultures to find out if Mg could ameliorate the effects of F toxicity. The results

showed that treatments receiving 0.05 M NaF along with 0.07 M MgCl had significantly higher

gas production than treatments only receiving 0.05 M NaF (p-value<0.01, Figure 1). The results

supported the hypothesis that Mg will ameliorate the toxicity of fluorine in gas production.

The observed differences in gas production between glucose, 0.05 M NaF, and 0.5 M

NaF were caused by the toxicity of fluorine in glycolysis. Fluorine inhibits the Mg cofactor of

enolase, which shuts down glycolysis and greatly reduces carbon dioxide production in yeast.

The higher concentration of 0.5 M NaF had a lower gas production than 0.05 M NaF because

fluorine is working at a higher concentration, therefore inhibiting enolase and shutting down

glycolysis more rapidly. Earlier studies have similarly proved the negative impacts of fluorine on

enolase. A study published in 2019 described the effects of fluorine on plants, describing that

even at low concentrations, fluorine causes physiological complications such as leaf damage and

inhibition in development (Banerjee and Roychoudhury, 2019). Similarly, Banerjee and

Roychoudhury (2019) found that fluorine stress on the magnesium cofactor of enolase

significantly reduced photosynthetic activity, resulting in decreased growth and development of

the plant and the degradation of plant pigments. This study’s results closely compare with the

outcome of our experiment, proving the toxicity of fluorine in glycolysis.

The differences between glucose, 0.05 M NaF and 0.07 M MgCl were very significant.

The glucose treatment had ideal growth, while the 0.05 M NaF and 0.07 M MgCl treatment

contained fluorine toxicity that significantly lowered gas production. Even with the presence of

Mg, F inhibited the Mg cofactor of enolase therefore shutting down glycolysis. However, Mg

significantly ameliorated the impacts of fluorine toxicity by competing with F to bind to enolase.
A study done in 2018 described the effects of Mg on rabbit muscle enolase (Lopez-Lopez et al.,

2018). This study indicated that Mg protects against a compound-induced deterioration of rabbit

muscle enolase (Lopez-Lopez et al., 2018). The positive impacts of Mg on the inhibition of

enolase compares to our findings that Mg ameliorated the impacts of F toxicity. This indicates

that Mg is a very efficient treatment for F toxicity in glycolysis.

There were a few outliers in the experiment possibly due to failure to pretreat with MgCl

before cells were exposed to NaF. These outliers are potential errors that have slightly influenced

our results. Our growth time of 45 minutes provided for accurate data that could be studied. An

improvement that could be made to the experiment is to provide more concentrations of MgCl.

Since our experiment only used one concentration of MgCl, our results did not show if higher

concentrations of Mg could better ameliorate F toxicity. This would add another aspect to the

experiment that could be accurately studied. Our results could be improved in the future by

creating a slightly larger sample size and testing additional concentrations of NaF and MgCl.

The importance of this study goes beyond the scope of carbon dioxide production in

yeast. The issue of fluorine polluted soils could potentially be solved by our findings. Our

experiment, along with similar studies, proves that Mg ameliorates F toxicity in yeast and

possibly plants. Plant pollution is an urgent issue that affects the air we breathe and our world’s

survival. Our understanding of what is polluting the plants is important, but our knowledge of

how to fix the pollution is vital. Our results are important in improving humans' understanding of

how Mg can better F toxicity, and help provide a better knowledge of how to depollute our

plants.
Literature Cited:

Alfarouk, KO, D Verduzco, C Raunch, AK Muddathir, HHB Adil, GO Elhassan, ME Ibrahim,

JDP Orozco, RA Cardone, SJ Reshkin and S Harguindey. 2014. Glycolysis, tumor metabolism,

cancer growth and dissemination. A new pH-based etiopathogenic perspective and therapeutic

approach to an old cancer question. Onoscience 1 (12): 777-802.

Banerjee, A and A Roychoudhury. 2019. Fluorine: a biohazardous agent for plants and

phytoremediation strategies for its removal from the environment. Biologia Plantarum 63: 104-

112.

Lahue, C, AA Madden, RR Dunn and CS Heil. 2020. History and domestication of

Saccharomyces cerevisiae in bread baking. Frontiers in Genetics 11: 584718.

Li, X, J Gu and Q Zhou. 2015. Review of aerobic glycolysis and its key enzymes- new targets

for lung cancer therapy. National Library of Medicine 6 (1): 17-24.

Lopez-Lopez, MJ, IC Rodriguez-Luna, EE Laura-Ramirez, M Lopez-Hidalgo, CG Benitez-

Cardoza and X Guo. 2018. Biochemical and biophysical characterization of the enolase from

Helicobacter pylori. Biomed Research International 2018: 9538193.