Bioresource Technology 100 (2009) 356–361

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

The effect of glycerol as a sole and secondary substrate on the growth

and fatty acid composition of Rhodotorula glutinis
Emily R. Easterling, W. Todd French *, Rafael Hernandez, Margarita Licha
Dave C. Swalm School of Chemical Engineering, Box 9595, Mississippi State University, MS 39762, USA

a r t i c l e i n f o a b s t r a c t

Article history: Rhodotorula glutinis is a yeast that produces copious quantities of lipids in the form of triacylglycerols
Received 26 November 2006 (TAG) and can be used to make biodiesel via a transesterification process. The ester bonds in the TAG
Received in revised form 8 May 2008 are broken leaving behind two products: fatty acid methyl esters and glycerol that could provide an inex-
Accepted 8 May 2008
pensive carbon source to grow oleaginous yeast R. glutinis. Described here are the effects of different
Available online 9 July 2008
growth substrates on TAG accumulation and fatty acids produced by R. glutinis. Yeast cultured 24 h on
medium containing dextrose, xylose, glycerol, dextrose and xylose, xylose and glycerol, or dextrose
Keywords:
and glycerol accumulated 16, 12, 25, 10, 21, and 34% TAG on a dry weight basis, respectively. Lipids were
Glycerol
Lipid
extracted from R. glutinis culture and transesterified to form fatty acid methyl esters. The results show a
Oleaginous difference in the degree of saturation for the carbon sources tested. Cells cultivated on glycerol alone had
Yeast the highest degree of unsaturated fatty acids at 53% while xylose had the lowest at 25%. R. glutinis can be
Biodiesel cultivated on all sugars tested as single carbon substrates or in mixtures. Glycerol may be used as second-
ary or primary carbon substrate.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction
Biodiesel is one of the alternative fuels currently being pro-
duced in the United States and elsewhere around the World
(Van Gerpen, 2004; Sheehan et al., 1998). A chemical process
called transesterification is used to make biodiesel, this is a pro-
cess in which the glycerol is separated from the triacylglycerides
in fats or vegetable oils. (http://www.biodiesel.org/resources/bio-
diesel_basics/default.shtm). Glycerol is 10% of the product output
(Fortenbery, 2005), or 1 lb of glycerol for each gallon of biodiesel
fuel. The future supplies and usage of glycerol are expected to
increase as biodiesel plants increase production, and the output
will greatly outpace demand. Biodiesel production has already
had a significant impact on the price of refined glycerol (http://
www.virent.come/whitepapers/Biodiesel%20Whitepaper.pd). A ma-
jor concern of glycerol producers is the reduced price of glycerol
resulting from the increased production of biodiesel. Some alter-
native uses for this glycerol that have been investigated are sub-
strates for fermentation process or the production of
biosurfactants (Ashby et al., 2006; Solaiman et al., 2006). An-
other alternative use for glycerol is as a growth substrate for
the cultivation of oleaginous yeasts. Oleaginous yeasts are sin-
gle-celled fungi defined as having at least 20% of their dry
weight made up of lipids (Ratledge, 1977). Not only do these
yeasts contain membrane lipids, but they accumulate lipid in
* Corresponding author. Tel.: +1 662 325 4308; fax: +1 662 325 2482.
E-mail address: French@che.msstate.edu (W.T. French).
the form of triacylglycerol (TAG) (Gill et al., 1977; Davoli et al.,
2004). Rhodotorula glutinis is an oleaginous yeast which is able
to activate non-esterified fatty acids for the synthesis of triacyl-
glycerol (Gangar et al., 2001). In R. glutinis, fatty acids are acti-
vated in an ATP dependent manner prior to being used. Gangar
et al. (2002) have demonstrated that an enzyme, acyl–acyl car-
rier protein (ACP) plays a role in activating fatty acids for triac-
ylglycerol biosynthesis. There is plenty evidence to suggest that
this organism has the potential to be a source of fatty acids
for the production of biodiesel. Oleaginous yeasts have the abil-
ity to grow and accumulate lipids when grown on glycerol
(Meesters et al., 1996), have short generation times, and very
minimal nutrient requirements. While purified glycerol has
many possible uses, the crude glycerol produced during biodiesel
manufacturing contains macro elements such as calcium, potas-
sium, magnesium, sulfur and sodium (Thompson and He, 2005).
In order to minimize unknown variables introduced through the
use of crude glycerol, initial studies to determine whether or not
glycerol could be used as substrate or co-substrate for growth
were conducted using purified glycerol. Using the glycerol to
produce fatty acids to be used as biodiesel feedstock would pro-
vide an added bonus of offsetting costs of production.
The objectives of this work were: (1) determine the effect of
pure glycerol on the growth of the yeast R. glutinis, (2) assess the
effects of pure glycerol on the lipid accumulation of the yeast
and (3) determine the effect of using pure glycerol as a sole or sec-
ondary carbon source on the fatty acid methyl ester (FAMEs) con-
tent for R. glutinis.

0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2008.05.030
 E.R. Easterling et al. / Bioresource Technology 100 (2009) 356–361
2. Experimental procedures
2.1. Organism
The yeast R. glutinis (ATCC 204091) was used in all experiments
(American Type Culture Collection, Manassas, VA).
2.2. Media
All media was sterilized in a SterisÒ (Mentor, Ohio) autoclave
for 15 min at 121 °C and 15 psi.
2.3. Stock culture medium
R. glutinis was cultured overnight on yeast peptone dextrose
media (Fisher Scientific, hereinafter referred to as YPD) pH 6.5,
35 °C to maintain a stock culture.
2.4. Cell mass accumulation medium
Basal medium (BM) (containing per 1 l distilled water: 0.2 g
KH2PO4, 0.15 g yeast extract, and 8.0 g NH4Cl) based on ATCC Mini-
mal Medium protocols (http://www.atcc.org/common/documents/
mediapdfs/1199.pdf, http://www.atcc.org/common/documents/
mediapdfs/846.pdf) and contained experimental carbon source(s),
dextrose, xylose, glycerol, or mixtures of two carbon sources, at C/
N molar ratios of 10/1. This BM was used to culture R. glutinis to accu-
mulate cell mass in preparation for inoculating the test media.
2.5. Test medium
The test media used in the experiments was based on the BM
but was amended (per 1 l distilled water: 0.4 g KH2PO4, 0.3 g yeast
extract, and 2.0 g NH4Cl) and contained the experimental carbon
sources so that the C/N molar ratios were 10/1. For every 1 g of
NH4Cl, 20 g of glycerol, 32 g of xylose, or 39 g of glucose would
be added to the medium to achieve a 10/1 C/N molar ratio. The
pH of the media was adjusted to 6.0 with 10% HCl (vol/vol).
2.6. Inoculum preparation
R. glutinis subcultures were used to inoculate 3–500 mL batches
of each culture medium. After six days of growth, the cultures were
centrifuged (20 min at 6000 rpm and 22 °C). The pellet was washed
three times with physiological saline (0.85% NaCl). The concentrated
cells were used to inoculate a test media containing the appropriate
carbon source(s). The media was inoculated to a cell density of
0.5 mg/mL which correlated to an absorbance of 0.5 at 600 nm.
2.7. Experiment design
The carbon sources tested were: dextrose, xylose, and glycerol,
the carbon sources in mixtures tested were: dextrose plus xylose,
xylose plus glycerol, and glycerol plus dextrose. Three replicate
cultures of R. glutinis with each of the six carbon sources were incu-
Table 1
The effect of carbon source and mixtures of carbon sources on the cell mass accumulation of Rhodotorula glutinis when grown at 35 °C with agitation (112 rpm)
Carbon source 0 h (mg)
Dextrose 17.47 ± 1.42
Xylose 13.25 ± 2.03
Glycerol 20.21 ± 0.26
Dextrose/xylose 18.28 ± 0.25
Xylose/glycerol 18.87 ± 0.05
Glycerol/ dextrose 14.01 ± 0.34
357
bated at 35 °C on a New Brunswick Rotary Shaker/Incubator (Edi-
son, New Jersey) set at 112 rpm for 48 h.
2.8. Measurement of growth
Every 24 h, for 48 h a 1 mL sample was removed from each
experimental replicate and the absorbance of light at 600 nm
wavelengths was measured for each sample via Spectronic* Gene-
sys* 20 Spectrophotometer (Thermo*Electron, Waltman, MA). A
correlation of cell mass to optical density was generated by taking
optical density readings at A600 of known cell masses and plotting
that data on a line graph prior to the start of any experiment.
2.9. Lipid extraction and gravimetric analysis
To monitor lipid content of the cells, yeast grown on the test
media (Table 1) were extracted every 24 h as described by Bligh
and Dyer, 1959. Weights of the extracted lipids were measured
using a calibrated balance.
2.10. Gas chromatography/flame ionization detection of FAMEs
The lipid transesterification and fatty acid extractions were car-
ried out following the procedures described by Christie, 2003. The
FAMEs were analyzed using an Agilent 6890 series Gas Chromato-
graph (Santa Clara, CA) equipped with a Supelco (St. Louis, MO) SP
2380 (100 m  0.25 mm  0.2 lm, model number Supelco 24317)
capillary column and a flame ionization detector (FID). The inlet
temperature was set at 260 °C with a pressure of 40 psi in split mode
(100:1). Helium served as the carrier gas with a flow of 126 mL/min
(total flow, 130.3 mL/min). The oven temperature program ramped
from 110 °C (held the initial temperature for 4 min) to 140 °C at
10 °C per min, from 140 °C to 220 °C at 2 °C per min, and finally from
220 °C to 240 °C at 2 °C per min. The temperature was held at 240 °C
for 40 min. The detector temperature was maintained at 260 °C with
a hydrogen flow of 40 mL/min and air flow of 450 mL/min with con-
stant makeup flow at 45 mL/min. A 10 lL Hamilton glass syringe was
used to inject a 1 lL sample of hexane containing the unknown
FAMEs and 1 mg/mL 1;3-dichlorobenzene as the internal standard.
3. Statistical method
The standard deviation (r) is a commonly used measure of the
confidence interval or variation. The standard deviation of a popu-
lationvofffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
observations is computed as shown (Eq. 2.3):
u n
uX ðX i  XÞ2
rX ¼t
i¼1
n1
4. Results and discussion
4.1. The effect of glycerol as a secondary substrate on the growth of R.
glutinis
When glycerol was provided as a carbon source along with dex-
trose, R. glutinis accumulated more cell mass after 48 h. (38% cell
24 h (mg) 48 h (mg) Overall cell mass increase (%)
17.86 ± 2.03 22.81 ± 0.26 24
13.05 ± 0.95 16.05 ± 0.17 18
22.15 ± 0.72 24.41 ± 1.08 18
25.63 ± 1.57 35.52 ± 1.46 49
21.40 ± 0.48 21.84 ± 0.49 14
17.96 ± 0.91 22.56 ± 0.95 38
358
mass increase) than when either carbon source was provided sin-
gly (Table 1). Compared to dextrose, glycerol is less favorable as
an energy source, which is verified by our findings that dextrose
grown R. glutinis increased cell density 24% when given dextrose
and 18% when given glycerol as the sole carbon source. Using xy-
lose and glycerol simultaneously as carbon sources resulted in
14% cell density increase which was a lower percent increase than
when the carbon sources were used alone (18% increase for both
xylose grown and glycerol grown).
The greatest cell mass increase of 49% was observed with dex-
trose plus xylose grown culture and was followed by a 38% increase
in cultures given glycerol plus dextrose. The results indicate that
growth on dextrose can be enhanced through the simultaneous
addition of glycerol. With the output of glycerol increasing as
new biodiesel plants come online and additional studies using
crude glycerol, processes of this type could make beneficial use of
the surplus glycerol. Biodiesel production has already had a signif-
icant impact on the price of refined glycerol (http://www.virent.
com/whitepapers/Biodiesel%20Whitepaper.pd).
4.2. The effect of glycerol on the lipid accumulation of R. glutinis
Although iron, zinc, phosphorus, nitrogen, and concomitant
limitations of nitrogen and phosphorus or magnesium have re-
sulted in lipid accumulation (Fortenbery, 2005; Granger et al.,
1993; Ykema et al., 1988) nitrogen limitation has been used
more extensively to create an environment conducive to lipid
hoarding. Carbon has been determined to be limiting at C/N
molar ratios of 4/1 and surfeit for oleaginous yeast Cryptococcus
curvatus at C/N molar ratios of 25/1 (Meesters et al., 1996). A C/
N ratio of 10/1 was provided in the test media used in these
R. glutinis batch culture experiments, therefore, rather than opti-
mizing lipid production, the goal was to observe the basic lipid
producing capability of the yeast when given different carbon
sources.
The lipid contents (as% dry cell weight) of the R. glutinis cultures
are shown Fig. 1. The data demonstrates differences in oil accumu-
50%
45%
40%
Lipid C oncentration (% dry cell weight )
35%
30%
25%
20%
15%
10%
5%
0%
Fig. 1. The effect of glycerol and culture duration on the total lipid production of Rhodotorula glutinis when grown at 35 °C with agitation (112 rpm).
E.R. Easterling et al. / Bioresource Technology 100 (2009) 356–361
lation by R. glutinis between those cultures provided single carbon
sources and those provided mixtures of carbons sources. When
comparing the 24 and 48 h data for all experiments, lipid content
of the glycerol grown R. glutinis increased on average 12.97% while
dextrose grown and xylose grown R. glutinis decreased 8.56% and
9.08%, respectively. The dextrose plus xylose grown culture in-
creased 1.11% from 24 to 48 h. While the data suggests that using
glycerol as a sole carbon source may result in greater lipid produc-
tion by the oleaginous yeast R. glutinis the standard error shows
there is not sufficient evidence to determine whether using glyc-
erol in conjunction with a six or five carbon sugar will cause the
oleaginous yeast R. glutinis to produce more lipid than when the
carbon sources are used individually. It can be submitted, however,
that glycerol grown R. glutinis accumulates more lipid under these
experimental conditions than dextrose grown and xylose grown
cultures.
The lipid accumulating properties of R. glutinis has been ex-
plored extensively (Gangar et al., 2002; Granger et al., 1992;
Solaiman et al., 2006; Yoon and Rhee, 1983) especially when
considered a source of fatty acids in the nutritional supplement
arena. The use of glycerol as a carbon substrate, due to its pro-
spective surplus and decreased value, has been appraised (Gill
et al., 1977; Noureddini et al., 1998; Papanikolaou et al., 2002).
The implications of recycling glycerol for use as a substrate on
which to grow oleaginous yeasts for the production of lipids
should be investigated more thoroughly, for its potential is out-
standing. While over 70% of biodiesel production costs are due
to the expense of feedstocks such as soybean and rapeseed oil
(Haas et al., 2006), using a renewable lipid source such as that
from oleaginous yeast could also help to cut the costs of
production.
The research presented here compares the lipid accumulating
capabilities of R. glutinis when grown on substrates such as dex-
trose, xylose, glycerol and their combinations has not been previ-
ously considered. Our findings validate that glycerol is a viable
carbon source for the production of lipid by the oleaginous yeast
R. glutinis.
Dextrose
Xylose
Glycerol
Dextrose + Xylose
Xylose + Glycerol
Dextrose + Glycerol
24 48
time
 E.R. Easterling et al. / Bioresource Technology 100 (2009) 356–361
4.3. The effect of different growth substrates used on the FAME
saturation of R. glutinis
Fig. 2a shows the results of the fatty acid saturation content of
R. glutinis grown on each of the six carbon source(s) tested. After
24 h of incubation, at least 50% of the total FAMES present in each
culture were saturated in nature except in the case of the glycerol
and dextrose grown R. glutinis which had a content of 48%
(SD = 0.82) saturated FAMEs. The xylose grown and the glycerol
grown R. glutinis contained the greatest fraction of saturated
FAMEs at 69% (SD = 1.88) and 68% (SD = 9.00), respectively.
At 24 h when glycerol was present as a secondary carbon
source, there was an increase in polyunsaturated fats for the xylose
plus glycerol grown R. glutinis compared to the fatty acid composi-
tion of only xylose grown R. glutinis. An increase in monounsatu-
rated fatty acids in the fatty acid composition for R. glutinis was
observed when the cells grown on dextrose plus glycerol when
compared to cells grown on dextrose or glycerol solely. The addi-
tion of glycerol as a carbon source along with dextrose or xylose re-
sulted in a decrease in the amount of saturated fatty acids.
At 48 h (Fig. 2b) the xylose grown and the dextrose plus xylose
grown cultures contained the greatest percent saturated FAMEs at
76% (SD = 7.97) and 71% (SD = 5.96), respectively, with the dex-
trose grown R. glutinis following at 70% (SD = 5.58) (Fig 2b). The
glycerol grown R. glutinis and the glycerol and dextrose grown R.
glutinis contained 46% (SD = 4.26) and 46% (SD = 3.15) saturated
fatty acids, respectively. The degree of fatty acid saturation still de-
creased when comparing single carbon sources to mixed sugar
sources, except in the case of the dextrose plus xylose grown R.
glutinis.
4.4. The effect of time on fatty acid saturation
When comparing the 24 h profiles to the 48 h (Tables 2 and
3) compositions, the saturated fatty acids derived from glycerol
grown R. glutinis decreased from 68% (SD = 9.0) to 46%
(SD = 4.26). The saturation of the FAMEs from the xylose plus
dextrose grown yeast increased 62% (SD = 7.9) to 71%
90
80
70
% of Total Fatty Acids
60
50
40
30
20
10
0
Dextrose Xylose
Fig. 2a. The effect of carbon source on the saturation of fatty acids produced by Rhodotorula glutinis after 24 h of agitated (112 rpm) incubation at 35 °C.
359
(SD = 5.96) and xylose grown cultures increased in saturation
69% (SD = 1.88) to 76% (SD = 7.97). The FAME saturation of the
dextrose grown, glycerol plus dextrose grown, and xylose and
glycerol grown R. glutinis cultures did not change significantly
over time.
When glycerol was used along with dextrose as carbon sub-
strates, the saturation of the FAMEs derived from R. glutinis was
less than when dextrose was used alone, but was comparable to
the saturation levels of glycerol grown R. glutinis. The xylose grown
yeast produced the most saturated FAMEs and the effect of adding
glycerol as a secondary carbon substrate in the media was that the
unsaturated FAME’s increased.
Our results indicate that mixing glycerol with either of the other
two carbon sources serves to decrease the saturation of FAMEs pro-
duced by R. glutinis and that with time the saturation of the glyc-
erol grown cultures decrease. One possible explanation is that
glycerol metabolism activates the expression of different enzymes
used in fatty acid synthesis. Microarray analysis would be able to
confirm or disprove this theory.
4.5. The effect of glycerol on the FAME profile of R. glutinis grown
Five fatty acids commonly reported in the literature for differ-
ent oils are the saturated fatty acids, palmitic acid (C16:0) and
stearic acid (C18:0); the monounsaturated fatty acid, oleic acid
(C18:1); and the polyunsaturated fatty acids linoleic acid (C18:2)
and linolenic acid (C18:3) (26,28,29,30) (Hassan et al., 1993; Davoli
et al., 2004). The fatty acids produced by R. glutinis in the above
experiments were predominantly palmitic acid and stearic acid.
Compared to soybean oil and rapeseed oil, which are used in the
US and the EU, respectively as feedstocks for biodiesel production,
the FAMEs derived from R. glutinis were more saturated. Soybean
oil contains mostly C18:2 (53.7%) and C18:1 (23.3%) while rape-
seed oil consists of mainly the same fatty acids at 23.3% and
64.4%, respectively (O’Brien, 1988).
When considering whether biodiesel derived from yeast lipids
is suitable for use as a fuel, several factors should be analyzed.
The fatty acid structures of the alky esters that make up biodiesel
Saturated
Monounsaturated
Polyunsaturated
Glycerol Glycerol- Xylose- Dextrose-
Dextrose Glycerol Xylose
Carbon Source(s)
360 E.R. Easterling et al. / Bioresource Technology 100 (2009) 356–361

90

80

70 Saturated

Monounsaturated
% of Total Fatty Acids

60
Polyunsaturated
50

40

30

20

10

0
Dextrose Xylose Glycerol Glycerol- Xylose-Glycerol Dextrose-Xylose
Dextrose
Carbon Source(s)

Fig. 2b. The effect of carbon source on the saturation of fatty acids produced by Rhodotorula glutinis after 48 h of agitated (112 rpm) incubation at 35 °C.

Table 2
A comparison of the total percent of selected fatty acid methyl esters produced by Rhodotorula glutinis when grown on different carbon sources at 24 h

Carbon source Dextrose Xylose Glycerol Gly + Dex Xyl + Gly Dex + Xyl
C16:0 palmitic 26.30 ± 4.65 30.06 ± 1.80 12.35 ± 6.87 18.81 ± 0.74 11.09 ± 1.66 12.17 ± 7.84
C18:0 stearic 35.37 ± 7.64 24.88 ± 1.02 38.63 ± 7.02 19.80 ± 1.18 39.65 ± 7.76 45.42 ± 8.62
C18:1 oleic 18.76 ± 4.25 14.5 ± 4.25 6.58 ± 2.91 28.46 ± 4.58 3.07 ± 2.62 7.42 ± 1.34
C18:2 linoleic 3.04 ± 1.93 8.01 ± 0.87 4.04 ± 1.64 3.80 ± 0.71 4.35 ± 0.8 4.32 ± 1.52
C18:3 linoleic 0 0 1.74 ± 0.66 0.72 ± 0.08 3.80 ± 0.71 3.05 ± 1.62

Table 3
A comparison of the total Percent of selected fatty acid methyl esters produced by Rhodotorula glutinis when grown on different carbon sources at 48 h

Carbon source Dextrose Xylose Glycerol Gly + Dex Xyl + Gly Dex + Xyl
C16:0 palmitic 24.88 ± 3.27 38.24 ± 1.05 16.01 ± 0.92 17.48 ± 1.28 12.22 ± 6.56 18.02 ± 2.22
C18:0 stearic 32.30 ± 1.19 28.04 ± 3.29 21.86 ± 3.56 20.96 ± 3.51 39.05 ± 8.40 49.54 ± 6.31
C18:1 oleic 24.76 ± 4.88 7.37 ± 3.77 18.05 ± 3.02 27.62 ± 4.36 8.97 ± 12.92 8.17 ± 5.53
C18:2 linoleic 0.26 ± 0.02 5.02 ± 1.97 15.91 ± 4.25 2.71 ± 1.99 5.91 ± 4.41 2.64 ± 1.05
C18:3 linoleic 0.42 ± 0.21 0 1.76 ± 0.50 0.86 ± 0.26 2.71 ± 1.99 1.61 ± 0.33

lend specific properties to the fuel that must conform to standards
set by American Society of Testing and Materials (ASTM). The ce-
tane number (CN), cloud point, pour point, heat of combustion,
and lubricity are some of the properties influenced by fatty acid
structure and the standards for 100% biodiesel are outlined in the
method ASTM D 6751 (http://wetestit.com/ASTM%20D6751.htm).
Saturated fatty acids tend to give favorable properties to the bio-
diesel such as increased cetane value, decreased NOX emissions,
shorter ignition delay time, and oxidative stability; while unsatu-
rated fatty acids lend better cold flow and pour point values
(http://wetestit.com/ASTM%20D6751.htm; Knothe, 2005).
This study has demonstrated that pure glycerol can be com-
bined with other carbon sources such as dextrose and xylose to
serve as a carbon and energy source for the oleaginous yeast R. glu-
tinis. The results of this study also indicate the fatty acid composi-
tion can be affected from the type of carbon source provided. The
ability of R. glutinis to convert pure glycerol into fatty acids and
TAG is the first step needed to using the crude form of glycerol pro-
duced during biodiesel production.
Acknowledgements
This material is based upon work supported by the US
Department of Energy under award number DE-FG36-
04G014251. The financial and facilities support of Mississippi
State University is gratefully acknowledged. The authors thank
Dr. Earl Alley and Jimmie Cain for their valuable technical assis-
tance and advice, and Mallory Bricka and Parisa Toghiani for
their help in the laboratory.
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