Tasmin Roupp, Rebekah Church, Sara Church, and Oshene Kelly

Background Information
• Glycerol and citric acid are used in everyday life
• Microbes produce Citric Acid
• Alternative energy source
Pichia spp. Gram Stain, 1000x Magnification with oil
Common Citric Acid Producing Yeast
• Saccharomyces cerevisiae
• Yarrowia lipolytica
•
Rhodotorula spp.
• Pichia spp.
Yarrowia lipolytica cells, Methylene blue stain, magnification 400 x, notice the filamentous structures (pseudohyphae) https://commons.wikimedia.org/wiki/File:Yarrowia_lipolytica_400x_img60.jp
g
Saccharomyces cerevisiae. Gram stain, 1000x Magnification with oil

•
Rhodotorula spp . and Pichia spp . obtained from organic pear
• Saccharomyces cerevisiae common name Baker’s Yeast www.cookingforengineers.com
www.organicfacts.net

Experimental Design
•
Independent Variable : Strains of yeast
•
Dependent Variable : Production of Citric Acid
• Standardized Variable www.nudenicotine.com
: Media, temperature, incubation time, titration method www.al-tar.com
http://fphoto.photoshelter.com/

Experimental Design
•
Collection of samples
•
Culturing sample
• Initial plating on YGC and Dextrose plates
•
Isolation
• Gram stain
• Pure Culture on Dextrose plate
•
Inoculation of glycerol medium with colony
•
Titration and pH
Pichia spp. Gram Stain
1000X, oil Immersion

Rhodotorula spp.
Pichia spp.
Saccharomyces cerevisiae

●
●
●
●
Titration was performed using 40 g/L NaOH
Phenolphthalein used as the color indicator
The titrants used were the samples of yeast grown in the glycerol-containing media
The change in color indicated the moles of citric acid produced by the sample
Rhodotorula spp.
Pichia spp.
Saccharomyces cerevisiae
Sterile Medium
Initial Moles of Acid Moles of Acid on Day 3
------
------
------
.0336 mol/L
.00373 mol/L
.000933 mol/L
.00392 mol/L
.00168 mol/L
Moles of Acid on Day 7
.00280 mol/L
.00560 mol/L
.00280 mol/L
.00187 mol/L

●pH electrode was used to measure the level of acidity
●The pH meter was used to supplement the titration measurements
Rhodotorula spp.
Pichia spp.
Saccharomyces cerevisiae
Sterile Medium
Initial pH
------
------
------
5.4
pH Day 3
5.5
5.4
5.4
5.3
pH Day 7
5.73
4.76
5.33
5.34

Identification
● Biochemical tests were performed in order to determine the species of the unknown yeast samples
● Fungal staining was also performed to complement the results found from the biochemical tests
Glucose
Sucrose
Lactose
Urea
Starch
Colony Color
Colony Morphology
Cell Size
Rhodotorula spp.
Negative
Indeterminate
Negative
Positive
Negative
Pink/Red
Round, Convex
5.3µm
Pichia spp.
Positive
Positive
Negative
Negative
Negative
Creamy White
Round, Convex
4.24µm

•
Pichia spp. produced the greatest yield of citric acid as compared to the other two strains of yeast
•
Saccharomyces cerevisiae produced citric acid at the fastest rate
• When the
Rhodotorula spp . was subjected to a colder temperature (4°C), the red color of the colonies gradually became darker.
37 ℃ 4 ℃

•Hypothesis Rejected
•Null/Alternative Hypothesis: Grown under optimum conditions,
Pichia spp.
will yield the most citric acid in comparison to the other strains used in this experiment

Suggestions and Complications
•
Only use YGC plates that contain an antibacterial agent
• Dextrose plates do not select for fungi; thus, bacteria predominantly grew.
•
Obtain the yeast strains from a certified laboratory
• Difficulty obtaining yeast from samples
•
Titration did not serve as an accurate method for testing citric acid production
• Alternative method: Microassay
• Measure a growth curve for fungal development
• Rule out/establish growth pattern as contributing factor
• Identify the strains of yeast prior to titration

References
Acknowledgements
Kamzolova, S. V., Fatykhova A. R., Dedyukhina, E. G.,
Anastassiadis, S. G., Golovchenko, N. P. and Morgunov I. G.
Citric Acid Production by Yeast Grown on Glycerol-Containing
Waste from Biodiesel Industry . Food Technol. Biotechnol.
(2011) 49 (1) 65–74.
Smazenko, Martin. What’s Your Temperature?
2012.
Web. 9 Dec 2015. Retrieved from: http://www.whatsyourtemperature.com/common-alternativeuses-citric-acid/
"Viticulture & Enology." Rhodotorula Glutinis . The Regents of the
University of California, 13 Aug. 2014. Web. 11 Dec. 2015.
Ghosh, Swapan. "Study of Yeast Flora from Fruit of Syzygium Cumini
(linn) Skeel." ABJNA Agriculture and Biology Journal of North
America 2.8 (2011): 1166-170. Web. 11 Dec. 15.
<http://scihub.org/ABJNA/PDF/2011/8/ABJNA-2-8-1166-1170.pdf>.
•
Dr. Stephen F. Baron
• Research Assistance
•
Bridgewater College Chemistry
Department
• Advice and titration supplies
•
Dr. Ed Lickey
• Yeast Confirmation
•
Bridgewater College Biology
Department
• Instruments and Supplies