1. OlukonyinsolaOluwole
Candidate of BachelorsinBiology
StudentUniversityof MassachusettsDartmouth
Fatty Acid Methyl Esters
Abstract
Biodiesel is clean-burning fuel that is environmentally friendly and has the ability to be economically
sustainable because of its renewable quality. Biodiesel has many renewable sources such as recycled
cooking oil, soy bean oil, and animal fats. Recent research has found that fungi, bacteria, and yeast are
other potential sources for biodiesel. To date,there has not been a feasible cost effective solution for a
mass-scale production of biodiesel. This project aims to determine if yeast strains, can be used as cost
effective resource for the commercial production of biodiesel.
Literature Review
The reckless consumption of crude oil has resulted in the clear environmental consequences that people
are conflicted with today. Last year,the U.S. consumed approximately 6.95 billion barrels of petroleum
products. That is an average of 19.05 million barrels a day (eia.gov 2015). These actions have put a strain
on the environment and the finite commodity that is fossil fuels. The solution to this problem is biodiesel.
Biodiesel is a clean-burning renewable fuel that reduces greenhouse gas emission by 57% to 87% when
compared to petroleum diesel (Biodisel.org 2015). Biodiesel is created by a variety of renewable sources
such as recycled cooking oil, animal fats and algae and fungi bi-products. Biodiesel is not only safer for
the environment, but it can also reduce the dependence on oil and boost the economy by creating new
jobs. Only 0.34 billion barrels of the 6.95 billion barrels consumed by the U.S. last year were biodiesel
(eia.gov 2015). Biodiesel has not made the transition to the majority fuel source because an economically
feasible solution has not yet been discovered to meet the needs of the high demand of commercial
production. Research has found that fungi, specifically yeast,is a potential feedstock for the use of
commercial biodiesel. To obtain biodiesel, triglycerides (TAG) produced by yeast are chemically
converted to fatty acid methyl esters (FAME). FAME can then be used as fuel. Yeast distinguishes itself
from other candidates because of its economic characteristics. Yeast is known for its short life cycle,
simple maintenance, and ability to grow in a variety of environments (Li et al. 2008). The goal of the
proposed research is to determine if yeast strains can be utilized as a feedstock for mass production of
biodiesel
Research Goals
This project will test the hypothesis that oleaginous yeast strains can be used as a feedstock for
commercial production of biodiesel because of its high output of lipids. The objectives of the project are
optimizing biomass growth relative to TAG accumulation, optimizing C/N ratios for TAGaccumulation,
reducing the oxygen sparing rate, and monitoring of the ability for cell wall lysis, the separation
processes, and the catalytic upgrading of the resulting TAG’s after fermentation. It is expected that these
objectives will be met, and an optimal condition for aerobic cultivation of yeast strains will be developed.
Materials & Method
The project will be conducted in the Chemical Engineering department of the Massachusetts Institute of
Technology during the summer of 2015. Lipomyces Starkeyi will be grown using fermentation protocols
provided by NREL. YPD media consisting of Bacto yeast, Bacto peptone, glucose and deionized water
will be used to feed yeast strains (NREL Fermentation Protocols 2015). Methods used for this project are
the flask screening method, batch fermentation method and the fed-batch fermentation method. The flask
screening method will be performed by transferring seed cultures into test tubes with YPD. When
exponential growth is reach and optical density requirements are met seed cultures will be centrifuged,
washed and placed in a baffle shake flask. The baffle flasks will contain YPD 5% or modified YNB. Next
the culture is incubated. Optical density and glucose measurements will be taken daily. Cells will be
harvested for FAME analysis 24 hours after glucose is consumed or glucose uptake ceases. The batch
fermentation method will be performed in the Sartorius BIOSTAT A-plus fermenter. The culture media

2. that will be used YPD. First the yeast strain is transferred to a shake flask with medium then incubated.
Once the incubation period is over the culture will be inoculated in a baffle flask containing YPD 5%.
Next the culture will be used to inoculate the fermenter. The fermenter will contain YPD media and
glucose for the L.starkeyi. The fermenter will run for 96 hours after inoculation. Once this time has passed
the fermentation broth will be harvested,centrifuged and washed with deionized water. The fed-batch
fermentation method will also be performed in the Sartorius BIOSTAT A-plus fermenter. The inoculation
procedure is the same as the batch fermentation process. YP with glucose will be used to grow cells in the
batch phase. There will be two types of fed-batch fermentation methods used in this project. The pH-stat
fed-batch fermentation method maintains a desired homeostatic environment by increasing glucose or pH
when needed. The DO-stat fed batch fermentation method uses oxygen consumption as a signal when
glucose is exhausted. DO rises and sugar is supplied with a deeding pump until DO drops below a
threshold. Both fermentation process run until glucose is consumed. All methods will analyze lipid
content, lipid titer, yield and productivity using the percent FAME analysis. The lipid profile will be
analyzed by the Gas chromatography-Mass Spectrometry (GC-MS). After lipids have been converted to
fatty acid methyl esters (FAME) by transesterification. Cell growth will be determined using optical
density. Samples will be transferred to a centrifuged, washed with deionized water and dried in a
lyophlizer for FAME analysis. Sugar concentrations and organic acids will be measure using the HPLC.
Justification
The successfulchange to biodiesel as an alternative fuel source could stop the dependence on crude oil,
create a sustainable source of energy, and significantly benefit the economy and environment. Research of
yeast as a potential feedstock for biodiesel is a necessary inquiry that is much overdue.

3. References
1. Li Q, Du W, Liu D. Perspectivesof microbial oilsforbiodiesel production.AppliedMicrobiologyand
Biotechnology.2008;80(5):749-756.
2. AnschauA, XavierM, HernalsteensS,FrancoT. Effectof feedingstrategiesonlipidproductionbyLipomyces
starkeyi.Bioresource Technology.2014;157:214-222.
3. Biodiesel.org.Biodiesel Basics - Biodiesel.org.2015 [accessed2015 Jun9]. http://biodiesel.org/what-is-
biodiesel/biodiesel-basics
4. Eia.gov.How muchoil is consumedinthe UnitedStates? - FAQ- U.S. EnergyInformationAdministration
(EIA).2015 [accessed2015 Jun 9]. http://www.eia.gov/tools/faqs/faq.cfm?id=33&t=6
5. Ravenbiotech.com.RavenBiotechInc.- Online methanolmonitoringandcontrol inPichiapastoris
fermentation- FermentationTips.2015 [accessed2015 Jun 13].
http://www.ravenbiotech.com/fermentation_tips.php