Bioresource Technology 100 (2009) 356–361

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/ locate/bior tech

The effect of glycerol as a sole and secondary substrate on the growthand fatty acid composition of Rhodotorula glutinis

Emily R. Easterling, W. Todd French *, Rafael Hernandez, Margarita LichaDave 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:Received 26 November 2006Received in revised form 8 May 2008Accepted 8 May 2008Available online 9 July 2008

Keywords:GlycerolLipidOleaginousYeastBiodiesel

0960-8524/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.biortech.2008.05.030

* Corresponding author. Tel.: +1 662 325 4308; faxE-mail address: French@che.msstate.edu (W.T. Fre

Rhodotorula glutinis is a yeast that produces copious quantities of lipids in the form of triacylglycerols(TAG) and can be used to make biodiesel via a transesterification process. The ester bonds in the TAGare broken leaving behind two products: fatty acid methyl esters and glycerol that could provide an inex-pensive carbon source to grow oleaginous yeast R. glutinis. Described here are the effects of differentgrowth substrates on TAG accumulation and fatty acids produced by R. glutinis. Yeast cultured 24 h onmedium containing dextrose, xylose, glycerol, dextrose and xylose, xylose and glycerol, or dextroseand glycerol accumulated 16, 12, 25, 10, 21, and 34% TAG on a dry weight basis, respectively. Lipids wereextracted from R. glutinis culture and transesterified to form fatty acid methyl esters. The results show adifference in the degree of saturation for the carbon sources tested. Cells cultivated on glycerol alone hadthe highest degree of unsaturated fatty acids at 53% while xylose had the lowest at 25%. R. glutinis can becultivated on all sugars tested as single carbon substrates or in mixtures. Glycerol may be used as second-ary or primary carbon substrate.

� 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 processcalled transesterification is used to make biodiesel, this is a pro-cess in which the glycerol is separated from the triacylglyceridesin 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 biodieselfuel. The future supplies and usage of glycerol are expected toincrease as biodiesel plants increase production, and the outputwill greatly outpace demand. Biodiesel production has alreadyhad 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 glycerolresulting 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 ofbiosurfactants (Ashby et al., 2006; Solaiman et al., 2006). An-other alternative use for glycerol is as a growth substrate forthe cultivation of oleaginous yeasts. Oleaginous yeasts are sin-gle-celled fungi defined as having at least 20% of their dryweight made up of lipids (Ratledge, 1977). Not only do theseyeasts contain membrane lipids, but they accumulate lipid in

ll rights reserved.

: +1 662 325 2482.nch).

the form of triacylglycerol (TAG) (Gill et al., 1977; Davoli et al.,2004). Rhodotorula glutinis is an oleaginous yeast which is ableto 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. Gangaret 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 thatthis organism has the potential to be a source of fatty acidsfor 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 veryminimal nutrient requirements. While purified glycerol hasmany possible uses, the crude glycerol produced during biodieselmanufacturing contains macro elements such as calcium, potas-sium, magnesium, sulfur and sodium (Thompson and He, 2005).In order to minimize unknown variables introduced through theuse of crude glycerol, initial studies to determine whether or notglycerol could be used as substrate or co-substrate for growthwere conducted using purified glycerol. Using the glycerol toproduce 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 ofpure glycerol on the growth of the yeast R. glutinis, (2) assess theeffects of pure glycerol on the lipid accumulation of the yeastand (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.

E.R. Easterling et al. / Bioresource Technology 100 (2009) 356–361 357

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 Steris� (Mentor, Ohio) autoclavefor 15 min at 121 �C and 15 psi.

2.3. Stock culture medium

R. glutinis was cultured overnight on yeast peptone dextrosemedia (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 gKH2PO4, 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 BMbut was amended (per 1 l distilled water: 0.4 g KH2PO4, 0.3 g yeastextract, and 2.0 g NH4Cl) and contained the experimental carbonsources so that the C/N molar ratios were 10/1. For every 1 g ofNH4Cl, 20 g of glycerol, 32 g of xylose, or 39 g of glucose wouldbe added to the medium to achieve a 10/1 C/N molar ratio. ThepH 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 batchesof each culture medium. After six days of growth, the cultures werecentrifuged (20 min at 6000 rpm and 22 �C). The pellet was washedthree times with physiological saline (0.85% NaCl). The concentratedcells were used to inoculate a test media containing the appropriatecarbon source(s). The media was inoculated to a cell density of0.5 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 replicatecultures of R. glutinis with each of the six carbon sources were incu-

Table 1The effect of carbon source and mixtures of carbon sources on the cell mass accumulation

Carbon source 0 h (mg) 24 h (mg)

Dextrose 17.47 ± 1.42 17.86 ± 2.03Xylose 13.25 ± 2.03 13.05 ± 0.95Glycerol 20.21 ± 0.26 22.15 ± 0.72Dextrose/xylose 18.28 ± 0.25 25.63 ± 1.57Xylose/glycerol 18.87 ± 0.05 21.40 ± 0.48Glycerol/ dextrose 14.01 ± 0.34 17.96 ± 0.91

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 eachexperimental replicate and the absorbance of light at 600 nmwavelengths was measured for each sample via Spectronic* Gene-sys* 20 Spectrophotometer (Thermo*Electron, Waltman, MA). Acorrelation of cell mass to optical density was generated by takingoptical density readings at A600 of known cell masses and plottingthat 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 testmedia (Table 1) were extracted every 24 h as described by Blighand Dyer, 1959. Weights of the extracted lipids were measuredusing 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. TheFAMEs were analyzed using an Agilent 6890 series Gas Chromato-graph (Santa Clara, CA) equipped with a Supelco (St. Louis, MO) SP2380 (100 m � 0.25 mm � 0.2 lm, model number Supelco 24317)capillary column and a flame ionization detector (FID). The inlettemperature 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 rampedfrom 110 �C (held the initial temperature for 4 min) to 140 �C at10 �C per min, from 140 �C to 220 �C at 2 �C per min, and finally from220 �C to 240 �C at 2 �C per min. The temperature was held at 240 �Cfor 40 min. The detector temperature was maintained at 260 �C witha 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 wasused to inject a 1 lL sample of hexane containing the unknownFAMEs and 1 mg/mL 1;3-dichlorobenzene as the internal standard.

3. Statistical method

The standard deviation (r) is a commonly used measure of theconfidence interval or variation. The standard deviation of a popu-lation of observations is computed as shown (Eq. 2.3):

rX ¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiXn

i¼1

ðXi � XÞ2

n� 1

vuut

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

of Rhodotorula glutinis when grown at 35 �C with agitation (112 rpm)

48 h (mg) Overall cell mass increase (%)

22.81 ± 0.26 2416.05 ± 0.17 1824.41 ± 1.08 1835.52 ± 1.46 4921.84 ± 0.49 1422.56 ± 0.95 38

358 E.R. Easterling et al. / Bioresource Technology 100 (2009) 356–361

mass increase) than when either carbon source was provided sin-gly (Table 1). Compared to dextrose, glycerol is less favorable asan energy source, which is verified by our findings that dextrosegrown R. glutinis increased cell density 24% when given dextroseand 18% when given glycerol as the sole carbon source. Using xy-lose and glycerol simultaneously as carbon sources resulted in14% cell density increase which was a lower percent increase thanwhen the carbon sources were used alone (18% increase for bothxylose 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% increasein cultures given glycerol plus dextrose. The results indicate thatgrowth on dextrose can be enhanced through the simultaneousaddition of glycerol. With the output of glycerol increasing asnew biodiesel plants come online and additional studies usingcrude glycerol, processes of this type could make beneficial use ofthe 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 concomitantlimitations 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 usedmore extensively to create an environment conducive to lipidhoarding. Carbon has been determined to be limiting at C/Nmolar ratios of 4/1 and surfeit for oleaginous yeast Cryptococcuscurvatus 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 theseR. glutinis batch culture experiments, therefore, rather than opti-mizing lipid production, the goal was to observe the basic lipidproducing capability of the yeast when given different carbonsources.

The lipid contents (as% dry cell weight) of the R. glutinis culturesare shown Fig. 1. The data demonstrates differences in oil accumu-

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

24

Lip

idC

once

ntra

tion

(%dr

yce

llw

eigh

t)

Dextrose

Xylose

Glycerol

Dextrose + Xylose

Xylose + Glycerol

Dextrose + Glycerol

Fig. 1. The effect of glycerol and culture duration on the total lipid productio

lation by R. glutinis between those cultures provided single carbonsources and those provided mixtures of carbons sources. Whencomparing the 24 and 48 h data for all experiments, lipid contentof the glycerol grown R. glutinis increased on average 12.97% whiledextrose grown and xylose grown R. glutinis decreased 8.56% and9.08%, respectively. The dextrose plus xylose grown culture in-creased 1.11% from 24 to 48 h. While the data suggests that usingglycerol as a sole carbon source may result in greater lipid produc-tion by the oleaginous yeast R. glutinis the standard error showsthere is not sufficient evidence to determine whether using glyc-erol in conjunction with a six or five carbon sugar will cause theoleaginous yeast R. glutinis to produce more lipid than when thecarbon sources are used individually. It can be submitted, however,that glycerol grown R. glutinis accumulates more lipid under theseexperimental conditions than dextrose grown and xylose growncultures.

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 whenconsidered a source of fatty acids in the nutritional supplementarena. The use of glycerol as a carbon substrate, due to its pro-spective surplus and decreased value, has been appraised (Gillet al., 1977; Noureddini et al., 1998; Papanikolaou et al., 2002).The implications of recycling glycerol for use as a substrate onwhich to grow oleaginous yeasts for the production of lipidsshould be investigated more thoroughly, for its potential is out-standing. While over 70% of biodiesel production costs are dueto the expense of feedstocks such as soybean and rapeseed oil(Haas et al., 2006), using a renewable lipid source such as thatfrom oleaginous yeast could also help to cut the costs ofproduction.

The research presented here compares the lipid accumulatingcapabilities 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 viablecarbon source for the production of lipid by the oleaginous yeastR. glutinis.

48time

n of Rhodotorula glutinis when grown at 35 �C with agitation (112 rpm).

E.R. Easterling et al. / Bioresource Technology 100 (2009) 356–361 359

4.3. The effect of different growth substrates used on the FAMEsaturation of R. glutinis

Fig. 2a shows the results of the fatty acid saturation content ofR. glutinis grown on each of the six carbon source(s) tested. After24 h of incubation, at least 50% of the total FAMES present in eachculture were saturated in nature except in the case of the glyceroland dextrose grown R. glutinis which had a content of 48%(SD = 0.82) saturated FAMEs. The xylose grown and the glycerolgrown R. glutinis contained the greatest fraction of saturatedFAMEs at 69% (SD = 1.88) and 68% (SD = 9.00), respectively.

At 24 h when glycerol was present as a secondary carbonsource, there was an increase in polyunsaturated fats for the xyloseplus 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 wasobserved when the cells grown on dextrose plus glycerol whencompared 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 xylosegrown cultures contained the greatest percent saturated FAMEs at76% (SD = 7.97) and 71% (SD = 5.96), respectively, with the dex-trose grown R. glutinis following at 70% (SD = 5.58) (Fig 2b). Theglycerol grown R. glutinis and the glycerol and dextrose grown R.glutinis contained 46% (SD = 4.26) and 46% (SD = 3.15) saturatedfatty acids, respectively. The degree of fatty acid saturation still de-creased when comparing single carbon sources to mixed sugarsources, 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 and3) compositions, the saturated fatty acids derived from glycerolgrown R. glutinis decreased from 68% (SD = 9.0) to 46%(SD = 4.26). The saturation of the FAMEs from the xylose plusdextrose grown yeast increased 62% (SD = 7.9) to 71%

0

10

20

30

40

50

60

70

80

90

Dextrose Xylose Glycerol

Carb

% o

f T

otal

Fat

ty A

cids

Saturated

Monouns

Polyunsa

Fig. 2a. The effect of carbon source on the saturation of fatty acids produced b

(SD = 5.96) and xylose grown cultures increased in saturation69% (SD = 1.88) to 76% (SD = 7.97). The FAME saturation of thedextrose grown, glycerol plus dextrose grown, and xylose andglycerol grown R. glutinis cultures did not change significantlyover time.

When glycerol was used along with dextrose as carbon sub-strates, the saturation of the FAMEs derived from R. glutinis wasless than when dextrose was used alone, but was comparable tothe saturation levels of glycerol grown R. glutinis. The xylose grownyeast produced the most saturated FAMEs and the effect of addingglycerol as a secondary carbon substrate in the media was that theunsaturated FAME’s increased.

Our results indicate that mixing glycerol with either of the othertwo 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 thatglycerol metabolism activates the expression of different enzymesused in fatty acid synthesis. Microarray analysis would be able toconfirm 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) andstearic 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; Davoliet al., 2004). The fatty acids produced by R. glutinis in the aboveexperiments were predominantly palmitic acid and stearic acid.Compared to soybean oil and rapeseed oil, which are used in theUS and the EU, respectively as feedstocks for biodiesel production,the FAMEs derived from R. glutinis were more saturated. Soybeanoil 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% and64.4%, respectively (O’Brien, 1988).

When considering whether biodiesel derived from yeast lipidsis suitable for use as a fuel, several factors should be analyzed.The fatty acid structures of the alky esters that make up biodiesel

Glycerol-Dextrose

Xylose-Glycerol

Dextrose-Xylose

on Source(s)

aturated

turated

y Rhodotorula glutinis after 24 h of agitated (112 rpm) incubation at 35 �C.

0

10

20

30

40

50

60

70

80

90

Dextrose Xylose Glycerol Glycerol-Dextrose

Xylose-Glycerol Dextrose-Xylose

Carbon Source(s)

% o

f T

otal

Fat

ty A

cids

Saturated

Monounsaturated

Polyunsaturated

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 2A 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 ± 7.84C18:0 stearic 35.37 ± 7.64 24.88 ± 1.02 38.63 ± 7.02 19.80 ± 1.18 39.65 ± 7.76 45.42 ± 8.62C18:1 oleic 18.76 ± 4.25 14.5 ± 4.25 6.58 ± 2.91 28.46 ± 4.58 3.07 ± 2.62 7.42 ± 1.34C18:2 linoleic 3.04 ± 1.93 8.01 ± 0.87 4.04 ± 1.64 3.80 ± 0.71 4.35 ± 0.8 4.32 ± 1.52C18:3 linoleic 0 0 1.74 ± 0.66 0.72 ± 0.08 3.80 ± 0.71 3.05 ± 1.62

Table 3A 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 ± 2.22C18:0 stearic 32.30 ± 1.19 28.04 ± 3.29 21.86 ± 3.56 20.96 ± 3.51 39.05 ± 8.40 49.54 ± 6.31C18:1 oleic 24.76 ± 4.88 7.37 ± 3.77 18.05 ± 3.02 27.62 ± 4.36 8.97 ± 12.92 8.17 ± 5.53C18:2 linoleic 0.26 ± 0.02 5.02 ± 1.97 15.91 ± 4.25 2.71 ± 1.99 5.91 ± 4.41 2.64 ± 1.05C18:3 linoleic 0.42 ± 0.21 0 1.76 ± 0.50 0.86 ± 0.26 2.71 ± 1.99 1.61 ± 0.33

360 E.R. Easterling et al. / Bioresource Technology 100 (2009) 356–361

lend specific properties to the fuel that must conform to standardsset 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 acidstructure and the standards for 100% biodiesel are outlined in themethod 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 toserve 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. Theability of R. glutinis to convert pure glycerol into fatty acids andTAG 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 USDepartment of Energy under award number DE-FG36-04G014251. The financial and facilities support of MississippiState University is gratefully acknowledged. The authors thankDr. Earl Alley and Jimmie Cain for their valuable technical assis-tance and advice, and Mallory Bricka and Parisa Toghiani fortheir help in the laboratory.

References

American Type Culture Collection. <http://www.atcc.org/common/documents/mediapdfs/1199.pdf> (accessed August 2006).

American Type Culture Collection, <http://www.atcc.org/common/documents/mediapdfs/846.pdf> (accessed September 2006).

Ashby, R.D., Solaiman, D., Foglia, T.A., 2006. New uses for glycerol: fermentationsubstrates for value-added product synthesis [abstract]. Annual Meeting andExpo of the American Oil Chemists’ Society. p. 72.

ASTM D6751. Specification for biodiesel blend stock (B 100) for distillate fuels.<http://wetestit.com/ASTM%20D6751.htm>. (accessed July 2006).

E.R. Easterling et al. / Bioresource Technology 100 (2009) 356–361 361

Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction andpurification. Canadian Journal of Biochemistry and Physiology 37 (8), 911–917.

Christie, W., 2003. Lipid Analysis, third ed. VCH, Berlin.Davoli, P., Mierau, V., Weber, R.S.W., 2004. Carotenoids and fatty acids in ard yeasts

Sporoblomyces rosus and Rhodotorula glutinis. Applied Biochemistry andMicrobiology 40, 392–397.

Fortenbery, T.R., 2005. Biodiesel feasibility study: An evaluation of biodieselfeasibility in Wisconsin, Department of Agribusiness, University of Wisconsin– Madison. <http://64.233.187.104/search?q=cache:dlPIj5Su9>.

Gangar, A., Karande, A.A., Rajasenkharan, R., 2001. Purification and characterizationof acyl-acyl carrier protein synthetase from oleaginous yeast and its role intryacylglycerol biosynthesis. Biochemical Journal 360, 471–479.

Gangar, A., Raychaudhuri, S., Rajasekhran, R., 2002. Alteration in the cytosolictriacylglycerol biosynthetic machinery leads to decreased cell growth andtriacylglycerol synthesis in oleaginous yeast. Biochemistry Journal 365, 577–589.

Gill, C.O., Hall, M.J., Ratledge, C., 1977. Lipid accumulation in oleaginous yeast(Candida 107) growing on glucose in a single stage continuous culture. Appliedand Environmental Microbiology 33 (2), 235–239.

Granger, L.-M., Perlot, P., Goma, G., Pareilleux, A., 1992. Kinetics of growth and fattyacid production of Rhodotorula glutinis. Applied Microbiology andBiotechnology 37, 13–17.

Granger, L.-M., Perlot, P., Goma, G., Pareilleux, A., 1993. Efficiency of fatty acidsynthesis by oleaginous yeast: prediction of yield and fatty acid cell contentfrom consumed C/N ratio by simple method. Biotechnology and Bioengineering42, 1151–1156.

Haas, M.J., McAloon, A.J., Yee, W.C., Foglia, T.A., 2006. A process model to estimatebiodiesel production costs. Bioresource Technology 97, 671–678.

Hassan, M., Blanc, P.J., Granger, M., Pareilleux, A., Goma, G., 1993. Lipid productionby an unsaturated fatty acid auxotroph of the oleaginous yeast Apiotrichumcurvatum grown in a single stage continuous culture. Applied Microbiology andBiotechnology 40, 483–488.

Knothe, G., 2005. Dependence of biodiesel fuel properties on the structure of fattyacid alkyl esters, fuel processing technology 86, 1059–1070.

Noureddini, H., Dailey, W.R., Hunt, B.A., 1998. Production of ethers of glycerol fromcrude glycerol- the byproduct of biodiesel production. Papers in BiomaterialsUniversity of Nebraska, Lincoln.

Meesters, P.A., Huijberts, G.N.M., Egglink, G., 1996. High-cell-density cultivation ofthe lipid accumulating yeast Cryptococcus curvatus using glycerol as a carbonsource. Applied Microbiology and Biotechnology 45, 575–579.

O’Brien, R.D., 1988. Fats and Oils, first ed. Lancaster Technomic Publishing Co.,United Kingdom. pp. 206–207.

Papanikolaou, S., Munilglia, L., Chevalot, I., Aggelils, G., Marc, I., 2002. Yarrowialipolytica as a potential producer of citric acid from raw glycerol. Journal ofApplied Microbiology 92, 737.

Ratledge, C., 1977. In: Rehm, H.J., Reed, G. (Eds.), Microbial Lipids in Biotechnology,vol. 7. VCH, Germany, pp. 133–197.

Sheehan, J., Dunahay, T., Beneman, J., Roessler, P., 1998. A Look Back at the USDepartment of Energy Aquatic Species Program: Biodiesel from Algae, NationalRenewable Energy Laboratory.

Solaiman, D., Ashby, R.D., Zerkowski, J.A., Hotchkiss, T.A., Foglia, T.A., Marmer, W.N.,2006. Bioconversion of soy-based feedstocks into biopolymers andbiosurfactants [abstract]. Annual Meeting and Expo of the American OilChemists’ Society, 14.

The official Site of the National Biodiesel Board. Biodiesel. <http://www.biodiesel.org/resources/biodiesel_basics/default.shtm> (accessed July2006).

Thompson, J.C., B.B. He, B.B., 2005. Characterization of Crude Glycerol from BiodieselProduction from Multiple Feedstocks. Food and Process Engineering InstituteDivision of ASABE.

Van Gerpen, J., 2004. Business Management for Biodiesel Producers, IowaState University Under Contract with the National Renewable EnergyLaboratory.

Virent. Conversion of Glycerol Stream in a Biodiesel Plant Background. <http://www.virent.com/whitepapers/Biodiesel%20Whitepaper.pd> (accessed July2006).

Ykema, A., Verbree, E.C., Kater, M.M., Smit, H., 1988. Optimization of lipidproduction in the oleaginous yeast Apiotrichum curvatum in whey permeate.Applied Microbiology and Biotechnology 29, 211–218.

Yoon, S.H., Rhee, J.S., 1983. Lipid from yeast fermentation: effects ofculture conditions on lipid production and its characteristics ofRhodotorula glutinis. Journal of the American Oil Chemists’ Society 60(7), 1281–1286.