Biochemical Engineering Journal 65 (2012) 30–36

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Biochemical Engineering Journal

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Regular Article

Microbial conversion of biodiesel byproduct glycerol to triacylglycerols by

oleaginous yeast Rhodosporidium toruloides and the individual effect
of some impurities on lipid production
Jingyang Xu, Xuebing Zhao, Wencong Wang, Wei Du, Dehua Liu ∗
Institute of Applied Chemistry, Department of Chemical Engineering, Tsinghua University, Beijing 10084, PR China

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

Article history: With the development of biodiesel industry, the byproduct glycerol will become a considerable resource
Received 4 October 2011 as feedstock for production of many other chemicals. In present work, microbial conversion of crude
Received in revised form 26 March 2012 glycerol to triacylglycerols (microbial lipid) was proposed and investigated using the oleaginous yeast
Accepted 4 April 2012
Rhodosporidium toruloides (R. toruloides) by one-stage batch fermentation. Compared with glucose and
Available online 11 April 2012
refined glycerol, the crude glycerol could obtain significantly higher biomass concentration and lipid
yield. The highest biomass concentration of R. toruloides obtained from crude glycerol in a 5 L fermenter
Keywords:
reached 26.7 g/L with an intracellular lipid content of 70%. Further study was performed to investigate the
Batch processing
Bioconversion individual effect of five representative compounds which were present in crude glycerol as impurities. It
Glycerol was found that within the general concentration ranges, only methanol displayed somewhat inhibitive
Yeast effect, while others showed positive influence on lipid production. These results indicated that crude
Microbial lipid glycerol could be directly converted to triacylglycerols by R. toruloides without purification. Contrarily,
Biodiesel certain amount of salt and soap could promote the accumulation of biomass and lipid.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction Crude glycerol is a main byproduct of biodiesel production. As

Biodiesel industry has become important due to the shortage
of fossil oil and increasing awareness of environmental issue. For
its sustainable development, however, it is primarily necessary
to insure that triacylglycerol (TAG) can be produced continu-
ally without competing with food industry. Lipids derived from
microorganisms, known as microbial lipid, can be fast synthesized
and accumulated intracellularly, with fatty acids compositions
resembling these of vegetable oils. Since the production of micro-
bial lipid has many advantages over that of vegetable oils such as
short life cycle and no need of agricultural land, it has attracted
much interest in recent decades, and considered as a potential
non-food feedstock for biodiesel production. Importantly, how-
ever, during microbial conversion, abundance of carbon source
that can be effectively utilized by microorganisms is required.
Traditionally this conversion process is based on starch glucose.
Nevertheless high expense of glucose feedstock would significantly
limit the industrialization of microbial lipid production. For this
reason, renewable and affordable carbon source in abundance is
favored.
∗ Corresponding author. Tel.: +86 10 62772130; fax: +86 10 62772130.
E-mail address: dhliu@tsinghua.edu.cn (D. Liu).
the production of biodiesel grows, it will be generated in a con-
siderable quantity, and its utilization will become an urgent topic.
Although refined glycerol could be a valuable product, the purifi-
cation process is always costly and generally out of the range
of economic feasibility for small to medium-sized plants [1]. In
some European countries, crude glycerol is even simply treated
as industrial wastewater [2]. Nowadays, trials are underway and
researchers have already successfully converted crude glycerol not
only to useful chemicals such as 1,3-propanediol [3], phytase [4],
citric acid [3], but also single cell oil, which can further be trans-
formed to biodiesel [3].
In present work, Rhodosporidium toruloides (R. toruloides) was
employed as a promising candidate for prospective industrial appli-
cation. It can accumulate intracellular lipid up to 60% of its cellular
dry weight [5], which is mainly triacylglycerol, resembling that of
oils from food crops in terms of fatty acid composition, and has
been successfully converted to biodiesel [6]. Besides, R. toruloides
has already been proved to be capable to grow on a broad range
of substrates including but not limited to: sugars like glucose and
xylose, lignocellulosic hydrolysate, and excess sludge hydrolysate
[5,7]. Furthermore, it can simultaneously produce carotenoids as
by-products, which may have some potential values (data not
published yet). However, no related data on the conversion of
glycerol to microbial lipid by this robust yeast are found in the

1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bej.2012.04.003
 J. Xu et al. / Biochemical Engineering Journal 65 (2012) 30–36
literatures. Besides, for effective utilization of biodiesel byprod-
uct, detailed knowledge is required to comprehensively evaluate
its fermentability and also the effects of impurities. Therefore, the
objective of present work is to find out whether the crude glycerol
can be directly used as carbon source for microbial lipid production,
and how the impurities present in crude glycerol affect the growth,
lipid accumulation and as well as the lipid fatty acids composition
of R. toruloides.
2. Materials and methods
2.1. Microorganism and chemicals
The oleaginous microorganism used in the experiments was
R. toruloides AS2.1389, which was obtained from China General
Microbiological Culture Collection Center (CGMCC). The yeast was
maintained at 4 ◦ C on yeast extract peptone dextrose (YEPD) slopes
containing: 20 g/L glucose, 10 g/L peptone, 10 g/L yeast extract, and
20 g/L agar. For preparing inocula, the organism was pre-cultured
overnight in the medium containing 20 g/L glucose (or 15 g/L glyc-
erol), 10 g/L peptone and 10 g/L yeast extract at 30 ◦ C and 200 rpm
in an air-bath shaker.
The chemicals and reagents in all the experiments were analyt-
ically pure and purchased locally. The crude glycerol samples were
provided by Hunan Rivers Bioengineering Co., Ltd., Hunan, China.
2.2. Fermentation process
Batch fermentation experiments with crude glycerol as car-
bon substrate were conducted in either flasks or 5-L fermenters.
In flask fermentation, the experiments were conducted in 500 mL
flasks with 100 mL liquid medium containing 50 g/L glycerol (for
crude glycerol, the addition amount was decided by its exact glyc-
erol concentration which was tested by HPLC), 0.1 g/L (NH4 )2 SO4 ,
0.75 g/L yeast extract, 0.4 g/L KH2 PO4 , and 1.5 g/L MgSO4 ·7H2 O. The
pH value of the liquid medium was adjusted to 6.0. Followed by
sterilization at 115 ◦ C for 15 min, 10 mL inocula were added to the
medium, and the culture was conducted at 30 ◦ C in an air-bath
shaker at 200 rpm for 5–6 days. Before inoculation, the biomass
concentration of the inocula was carefully controlled at 0.5–0.6 g/L.
Each culture was performed by duplicate test.
For culture in 5-L fermenters, the experiments were conducted
with 4 L culture volume. The initial glycerol concentration was
60 g/L and other nutrients concentrations were the same as those in
flask culture. pH was controlled at 6.0 by feeding 40 wt% NaOH. Dis-
solved oxygen was controlled at 20–30% saturation by automatic
adjustment of stirring speed with 2 vvm aeration. For comparison,
glucose was also used as carbon source at the same initial concen-
tration.
2.3. Analytical methods
The quantitative analysis of methanol, glucose, and glycerol
was performed with a Shimadzu10AVP HPLC system (Shimadzu
Corp., Japan) equipped with a RID-10A refractive index detec-
tor, Aminex HPX-87H column (300 mm × 7.8 mm, Bio-Rad, USA) at
65 ◦ C with 5 mM H2 SO4 as the eluent at a flow rate of 0.8 mL/min.
The estimation of glyceride was performed with a Shimadzu
HPLC system (Shimadzu, Kyoto, Japan) with an ELSD-LT II low
temperature-evaporative light scattering detector. Before analysis,
2 ␮L sample and 1 mL acetone were mixed thoroughly, and 20 ␮L
of the aforementioned mixture was injected. A C18 column (5 ␮m,
250 mm × 4.6 mm) (Dikma Technology, PLATISIL ODS, China) and
a gradient elution program by acetonitrile and dichloromethane at
1.5 mL/min was employed, respectively. The column temperature
31
was 40 ◦ C. The drift pipe temperature was 70 ◦ C, and the nitro-
gen pressure was 320 kPa. The ash content of crude glycerol was
determined by burning the glycerol sample in muffle furnace until
a constant weight was reached.
The yeast biomass concentration was expressed as the dry
weight concentration of cell biomass (CB , g/L). After the fermenta-
tion broth was centrifuged, the wet cells were washed with deioned
water and dried at 105 ◦ C to a constant weight. The dry weight
concentration of cell biomass was then calculated according to fol-
lowing equation:
dry weight of cell biomass (g)
CB (g/L) =
volume of fermentation broth (L)
Total crude intracellular lipid was extracted by acid-heating
procedure with mixture of chloroform and methanol as extrac-
tant [8]. Crude lipid content was expressed as gram lipid per gram
of dry biomass. For determination of fatty acid compositions, the
crude lipid was first converted to fatty acid methyl ester (FAME)
with excess methanol in tert-butanol system under the catalysis of
excess loading of immobilized lipase (NS 435 and NS 40044, pro-
duced by Novozymes) [9]. The obtained FAME was then analyzed
with a GC-14B gas chromatography (Shimadzu, Japan) equipped
with a CP-FFAP CB capillary column (25 m × 0.32 m × 0.30 ␮m) pro-
duced by Agilent. Heptadecanoic acid methyl ester was used as
internal standard. The column temperature was kept at 180 ◦ C for
0.5 min and heated to 250 ◦ C at 10 ◦ C/min, and then maintained for
6 min. The temperatures of the injector and detector were set at
245 ◦ C and 260 ◦ C, respectively.
For describing the efficiency of the microbial lipid production,
several response variables, namely, lipid concentration (CL ), lipid
yield (YL ), and lipid content (CnL ) are defined as follows, respec-
tively:
weight of lipid (g)
CL (g/L) =
volume of fermentation broth (L)
YL (g/100 g carbon substrate)
weight of lipid (g)
= × 100
weight of carbon substrate consumed (g)
weight of lipid (g)
CnL (%) = × 100%
weight of cell biomass (g)
3. Results and discussion
3.1. Lipid production by R. toruloides in flask fermentation
Generally, in the biodiesel industry, about 0.1 t of crude glycerol
is produced for every 1 t of biodiesel. Some soap and alcohol (usu-
ally methanol) together with small amount of fat soluble substance
are also present in the glycerol phase depending on the feedstock
and production processes. Two types of crude glycerol samples
used in present work were analyzed, and their main components
are shown in Table 1.
Glycerol A contained 85% glycerol, a small amount of glyceride
and fatty acid methyl ester (FAME). Notably, after burning the sam-
ple to a constant weight, the content of ash residue reached 6.5%,
most of which was soluble in water. The ash generally consisted
of sodium salts from the catalyst (e.g. sodium hydroxide) of trans-
esterification reaction. Therefore, ash content can be considered
as the total percentage of salt and soap components in the crude
glycerol sample.
Glycerol B was originated from lipase-catalyzed transesterifica-
tion of palm oil. It contained 32.97% glycerol, a small amount of
glyceride and fatty acid methyl ester, and a little ash. However, no
32 J. Xu et al. / Biochemical Engineering Journal 65 (2012) 30–36
Table 1
Main components of crude glycerol samples used in present work.
Samplea Glycerol (%) Methanol (%)
A 85.19 ± 2.16 – 0.09 ± 0.00
B 32.97 ± 0.96 14.89 ± 1.10
a
Crude glycerol A was from an alkaline-catalyzed biodiesel production process; crude glycerol B was from an enzyme-catalyzed biodiesel production process. Both two
samples had a brown color with a pH around 6–7 and the rest component was water.
distillation pretreatment was performed, and the methanol con-
centration was as high as 14.89%.
To evaluate whether R. toruloides can grow on crude glycerol, we
first examined the growth of this yeast on 50 g/L crude glycerol. For
comparison, glucose and refined glycerol were also used as carbon
source for culture. The experimental data after 160 h incubation are
summarized in Table 2.
It can be known that R. toruloides could well utilize refined glyc-
erol for lipid accumulation. Furthermore, R. toruloides grew better
in crude glycerol than in refined glycerol. For instance, the biomass
concentration obtained by crude glycerol A was 50% higher than
that obtained by refined glycerol. Corresponding lipid concentra-
tion and yield were also higher. For the scenario of crude glycerol B,
similar results were obtained. It was also interesting to find that the
yeast even grew better on crude glycerol than on glucose, which
shows that crude glycerol from biodiesel production process is a
promising feedstock for microbial lipid production. However, the
experimental results should be further verified by larger scale cul-
tivations.
3.2. Lipid production by R. toruloides in 5-L bioreactors
To further increase the cell growth and production of lipids,
batch fermentation was carried out in a 5-L fermentor equipped
with pH control and aeration systems. The time courses of cell
biomass concentration, substrate consumption and lipid concen-
tration are shown in Fig. 1. As shown in the figure, the growth of
R. toruloides on glucose substrate was fast at first, but gradually
speeded down as the fermentation proceeded. The final biomass
concentration was 16.7 g/L with lipid concentration of 11 g/L. On
refined glycerol substrate, glycerol consumption was obviously
slower than that of glucose; the lipid production, however, was
comparable. On crude glycerol substrates, the kinetics of biomass
growth, glycerol consumption and lipid production showed some
difference depending on the characteristics of the crude glycerol.
On crude glycerol B, the growth and glycerol consumption slowed
down after the 150 h, and the final biomass reached 18 g/L with
Fig. 1. Cultivation of R. toruloides in a 5 L fermenter with crude glycerol as carbon
substrate. (A) Biomass concentration and (B) lipid concentration.
Biodiesel and glyceride (%) Ash content (%)
6.52 ± 0.16
1.81 ± 0.00 0.11 ± 0.00
lipid concentration of 13.4 g/L. On crude glycerol A, the biomass
concentration, glycerol consumption, and lipid production were
impressively fast at an almost stable rate. The final biomass and
lipid concentrations reached 26.7 g/L and 18.5 g/L, respectively,
with lipid yield of as high as 17.5 g/100 g consumed glycerol. Com-
pared with refined glycerol substrate, crude glycerol A could obtain
42.0% higher biomass concentration and 68.2% higher lipid concen-
tration. This performance indicated a remarkable advantage when
using crude glycerol as carbon source.
Compared with the some reported data from the literatures
shown in Table 3, crude glycerol seems to be effectively utilized
by this yeast, and led to similar or higher biomass and lipid pro-
ductivity, which demonstrates that crude glycerol from biodiesel
production could be a promising feedstock for microbial lipid pro-
duction.
According to the literatures, palmitic acid (C16:0), stearic acid
(C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid
(C18:3) constitute the majority of fatty acids in microbial triacyl-
glycerols (TAGs) [10,11]. We thus further determined the fatty acid
compositions of R. toruloides lipids derived from different carbon
sources as shown in Table 4. It was found that the predominant
fatty acids were palmitic acid, stearic acid, oleic acid, and linoleic
acid; and palmitic acid and oleic acid accounted for about 70% of
the total fatty acid composition. There was no significant difference
observed for the lipids obtained from refined glycerol and crude
glycerol substrates. However, the oleic acid and linolenic acid con-
tents of the lipid obtained from glucose were a little higher than that
obtained from glycerol, correspondingly palmitic acid and stearic
acid contents were somewhat lower.
Above experimental results showed that crude glycerol could be
well utilized by R. toruloides with obvious advantages over refined
glycerol and even glucose. Scott et al. [12] also demonstrated such
advantages when they used crude glycerol from fish oil production
as carbon source to produce oil rich in DHA by a thraustochytrid.
Since the crude glycerol from biodiesel plant unavoidably contains
some impurities such as salts, methanol and soap, which might
exert influences on the yeast growth and lipid accumulation, we
thus further performed experiments to investigate the individual
effects of these impurities on the fermentation process.
3.3. Individual effects of some impurities on lipid production by R.
toruloides
Some compounds contained in crude glycerol were added into
the refined glycerol medium to study the individual effects of
the impurities on microbial lipid production. Taking into account
that the concentrations of those substances in real crude glycerol
composition, we selected the compounds and their concentration
ranges as follows: sodium oleate (0–2 g/L), methyl oleate (0–2 g/L),
glyceryl monooleate (0–2 g/L), sodium chloride (0–16 g/L), and
methanol (0–16 g/L). In most cases, the real concentrations of the
impurities do not exceed the upper limit as set.
3.3.1. Effects of soap
Oleic acid is one of the main fatty acid compositions of biodiesel
feedstock oil [6,13]. In an alkali (for example, sodium hydroxide)
catalyzed transesterification reaction, sodium oleate is produced
 J. Xu et al. / Biochemical Engineering Journal 65 (2012) 30–36 33

Table 2
Lipid production by R. toruloides in flask fermentation for 160 h.

Carbon source CB (g/L) CL (g/L) YL (g/100 g carbon source) CnL (wt %)

Glucose 14.4 ± 0.1 7.2 ± 0.1 13.4 ± 0.2 50.0 ± 0.4

Refined glycerol 12.8 ± 0.6 5.6 ± 0.0 13.9 ± 0.6 43.4 ± 1.0
Crude glycerol A 19.2 ± 0.4 9.2 ± 0.8 14.9 ± 1.5 47.7 ± 2.0
Crude glycerol B 20.1 ± 1.0 8.6 ± 0.9 14.4 ± 1.8 42.9 ± 2.0

Table 3
Comparison of lipid production by R. toruloides with those of some other microorganisms.

Strain Carbon source Biomass Lipid content (%) Lipid concentration Cultivation Reference
concentration (g/L) (g/L)

Cunninghamella echinulata Glucose 15 46 6.9 Batch in flask [27]

Thraustochytrium sp. Glucose 23.9 30.1 7.2 Batch in flask [12]
Rhodosporidium toruloides Glucose 18.2 76.1 13.9 Batch in flask [28]
Mortierella isabellina Crude glycerol 8.5 51 4.4 Batch in fermenter [3]
Cryptococcus curvatus Crude glycerol 32.9 52.9 18 Fed-batch in fermenter [2]
Yarrowia lipolytica Crude glycerol 8.1 43 3.5 Continuous in fermenter [26]
Rhodosporidium toruloides Glucose 16.7 66.9 11.2 Batch in fermenter This work
Pure glycerol 18.8 58.7 11.0 Batch in fermenter This work
Crude glycerol A 26.7 69.5 18.5 Batch in fermenter This work
Crude glycerol B 18.0 74.1 13.4 Batch in fermenter This work

Table 4
Fatty acid composition of R. toruloides lipid obtained from different carbon sources.

C14 C16 C16:1 C18:0 C18:1 C18:2 C18:3

Glucose 1.5 ± 0.2 26.1 ± 0.8 0.0 ± 0.0 13.0 ± 1.5 46.4 ± 1.8 9.2 ± 0.5 3.8 ± 0.4
Glycerol 1.6 ± 0.2 28.7 ± 1.1 0.2 ± 0.0 15.3 ± 1.0 41.5 ± 0.6 10.1 ± 0.2 2.6 ± 0.1
Crude glycerol A 1.6 ± 0.0 29.1 ± 0.6 1.0 ± 0.1 17.8 ± 1.0 38.1 ± 1.5 9.7 ± 0.0 2.6 ± 0.1
Crude glycerol B 1.3 ± 0.3 29.2 ± 0.0 1.0 ± 0.1 13.9 ± 0.7 41.4 ± 0.3 10.4 ± 0.4 2.9 ± 0.0

and remains in the reaction system. As shown in Fig. 2, positive
effect was observed when sodium oleate was added, and all of
the response variables were increased with sodium oleate concen-
tration. For control experiments, the biomass, lipid concentrations
and lipid yield were 12.8 g/L, 5.6 g/L and 13.9%, respectively. How-
ever, the addition of 0.5–2 g/L sodium oleate resulted in increases of
biomass concentration by 25–41%, lipid concentration by 34–59%
and lipid yield by 13–43%, respectively.
The effect of sodium oleate, as a representative of soap, deserves
further discussion. Soap plays a role as surfactant, which may
provide some influences in fermentation process. It has been
reported by Pyle et al. [14] that soap showed a negative effect on
Schizochytrium limacinum for DHA production, and the inhibition
was pronounced at higher levels. They suggested that this phe-
nomenon should relate to the function of soap as a surfactant, and
the inhibition is caused by the complex interaction between the cell
wall (membrane) and the surfactants. In our experiment, it was
found that with the addition of 2 g/L sodium oleate, the medium
could be emulsified to a great extent. Thus it can be assumed
that sodium oleate should function as a surfactant in the culture
medium. However, no significantly negative effect on cell growth
and lipid production was observed when sodium oleate concen-
tration was increased to 4 g/L. Contrarily, the presence of sodium
oleate in culture medium led to an increase of biomass concentra-
tion with associated increase of lipid concentration and yield, which
is in accordance with some researches that a proper concentration
of surfactants benefits the microbial fermentation process [15–17].
It is very likely that some surfactants can interfere with the per-
meability of the cell membrane, and enhance the nutritional input
from the surroundings to the cells [18].

3.3.2. Effects of glycerides and fatty acid methyl esters

Fig. 2. The effect of sodium oleate on cell growth, lipid production and lipid yield
of R. toruloides cultivated with refined glycerol as carbon source for 7 days.
After transesterification reaction, biodiesel is separated out as
product through pH adjustment and settlement or centrifugation.
However, unavoidably there are still some glycerides and fatty acid
methyl esters (biodiesel) remaining in the aqueous glycerol phase.
Since oleic acid is always the main fatty acid component in most of
biodiesel feedstocks and monoglyceride is more or less present in
the solution, in this work we selected methyl oleate and glyceryl
monooleate as the representatives to investigate their effects on
cell growth and lipid accumulation. The results are shown in Figs.
3 and 4. It can be known that both two esters showed somewhat
positive influence on cell growth and lipid production. For methyl
oleate, the biomass was increased by 9–12%, and lipid production
was increased by 7–22%. For glyceryl monooleate, the biomass was
increased by 8–13%, and lipid production was increased by 10–16%,
respectively.
Some yeasts have specific metabolic pathways to utilize
hydrophobic substrate such as alkanes, fatty acids and oils. For
example, a widely studied yeast Yarrowia lipilytica can utilize oils
34 J. Xu et al. / Biochemical Engineering Journal 65 (2012) 30–36
Fig. 3. The effect of methyl oleate on cell growth, lipid production and lipid yield of
R. toruloides cultivated with refined glycerol as carbon source for 7 days.
like glycerides by first hydrolyzing the lipid into free fatty acids,
and then taking up by the cell as carbon source [19,20]. On the
other hand, hydrophobic compounds are known to partition into
cell membranes, interfering with the bilayer structure, and mod-
ifying its fluidity [16]. Considering that glycerides and fatty acid
methyl esters have similar structures to that of sodium oleate while
differ in the hydrophilic group, it is likely that such chemicals play
a role as a “week” surfactant (compared with soap), and may also
have some interactions with cell membranes as well as the culture
mediums. However, since the effect of hydrophobic compounds on
R. toruloides is not clear yet, corresponding mechanisms should be
further investigated.
3.3.3. Effect of inorganic salt
In alkali-catalyzed production of biodiesel, alkali like sodium
hydroxide is used as the catalyst. After transesterfication, pH
adjustment, and separation process, part of the sodium remains
in glycerol phase in the form of salts. It has been reported
that a suitable salt content provides a positive effect on oleagi-
nous microorganisms for cell growth and lipid production. Many
microalgae even can utilize salt streams, and produce lipids under
high salt concentrations [21,22]. However, excessive amount of salt
may cause osmotic pressure. For this reason, sodium chloride was
added to culture medium until its concentrations were 4 g/L, 8 g/L,
and 16 g/L, respectively, to investigate its effect on cell growth and
lipid production.
Fig. 4. The effect of glyceryl monooleate on cell growth, lipid production and lipid
yield of R. toruloides cultivated with refined glycerol as carbon source for 7 days.
Fig. 5. The effect of sodium chloride on cell growth, lipid production and lipid yield
of R. toruloides cultivated with refined glycerol as carbon source for 7 days.
As shown in Fig. 5, the addition of sodium chloride led to obvious
increase of biomass concentration, lipid concentration and yield.
It indicated that the concentration of salt in crude glycerol was
not high enough to cause an osmotic pressure. Contrarily, with
the addition of 4–16 g/L sodium chloride, the biomass concentra-
tion, lipid concentration and lipid yield were increased by 12–40%,
20–48%, and 9–20%, respectively. This result was in accordance
with the reported phenomena that salt can lead to a higher intracel-
lular lipid content and biomass concentration if added in optimized
amount [23–25]. It is probably that a low concentration of salt con-
tributes to the physiological state in favor of lipid synthesis, in
which circumstance cells can adapt to the salts with an increased
production of carbonhydrates and lipids as the osmoprotectants.
3.3.4. Effect of methanol
Methanol is largely used for transesterification reaction; there-
fore the unreacted methanol remains in the aqueous phase.
Depending on separation process, methanol concentration in the
aqueous phase is varied. To cover the methanol concentration
ranges of most crude glycerol, in this work, methanol was added
to the culture medium until its concentration was 4 g/L, 8 g/L, and
16 g/L, respectively.
In our experiments, methanol was found to be somewhat
inhibitive to R. toruloides. As shown in Fig. 6, the biomass, lipid con-
centrations and lipid yield was decreased by about 0–5%, 6–24%,
and 6–23%, respectively, when methanol was added. Whereas, it
also can be observed that the inhibitory effect did not become even
Fig. 6. The effect of methanol on cell growth, lipid production and lipid yield of R.
toruloides cultivated with refined glycerol as carbon source for 7 days.
 J. Xu et al. / Biochemical Engineering Journal 65 (2012) 30–36 35

Table 5
Fatty acid composition of lipid obtained from R. toruloides growing on refined glycerol in the present of selected crude glycerol impurities.

Impurities C14 C16 C16:1 C18:0 C18:1 C18:2 C18:3 Saturated FAs Unsaturated FAs

Control 1.3 ± 0.1 26.3 ± 1.4 2.7 ± 0.0 11.7 ± 0.6 45.1 ± 2.4 10.8 ± 0.5 2.1 ± 0.2 39.3 ± 2.5 60.7 ± 3.0
Sodium chloride 1.2 ± 0.0 25.9 ± 0.1 0.9 ± 0.0 12.1 ± 0.0 46.3 ± 0.0 11.7 ± 0.0 2.0 ± 0.0 39.2 ± 0.1 60.8 ± 0.1
Methanol 1.3 ± 0.0 25.4 ± 0.2 2.3 ± 0.2 12.6 ± 0.0 45.4 ± 0.6 11.3 ± 0.0 1.8 ± 0.0 39.4 ± 0.2 60.6 ± 0.2
Sodium oleate 1.6 ± 0.2 27.2 ± 0.7 2.1 ± 0.2 12.1 ± 0.1 44.1 ± 0.3 11.3 ± 0.5 1.6 ± 0.3 40.9 ± 1.0 59.1 ± 1.0
Methyl oleate 1.7 ± .0.2 25.2 ± 0.2 3.2 ± 0.1 10.8 ± 0.5 43.1 ± 0.3 13.4 ± 0.5 2.6 ± 0.2 37.7 ± 0.2 62.3 ± 0.2
Glyceryl momooleate 1.6 ± 0.1 23.5 ± 0.1 3.5 ± 0.1 11.0 ± 0.5 47.1 ± 0.8 11.0 ± 0.5 2.3 ± 0.1 36.1 ± 0.3 63.9 ± 0.2

severer when the methanol concentration was increased to as high Acknowledgments

as 16 g/L.
This observation accords with some literatures that methanol
can lead to inhibitory effects on cell growth and lipid synthesis
of some oleaginous microorganisms at high concentrations. For
instance, Pyle et al. [14] reported that when methanol concentra-
tion is above 10 g/L in the medium, several important objective
variables including biomass productivity, growth yield, DHA con-
tent, DHA yield and DHA productivity go down. Liang et al. also
found that methanol could harm the cell growth of S. limacinum,
but the inhibitory effect was not significant during the fed-batch
fermentation process with a methanol concentration no more than
8 g/L [26]. However, the exact mechanism of methanol to oleagi-
nous microorganisms is not clear yet. It has been hypothesized that
methanol might be uptake into the cell and even utilized [2]. In
this sense, it is probably that the methanol metabolism could exert
some influence on the cell growth and lipid synthesis.
Anyway, the experimental results indicate that although
methanol can pose somewhat inhibitory effect on R. toruloides, a
small amount of methanol remaining in the crude glycerol does
not show severe impact on the fermentation process. Additionally,
methanol is easy to be separated from crude glycerol through evap-
oration process, and should be recovered for recycling. Therefore,
the effect of methanol actually can be neglected.
3.3.5. Effects of impurities on fatty acid compositional profiles of
the yeast lipid
To further investigate whether the impurities could affect the
lipid synthesis pathways, the fatty acid composition of microbial
lipid was analyzed as shown in Table 5. It can be known that the
major fatty acid composition of lipids was C16:0 (palmitic acid),
C18:0 (stearic acid), C18:1 (oleic acid) and C18:2 (linoleic acid),
which accounted for about 26%, 12%, 45% and 11% of the total
fatty acids, respectively. The addition of the compounds did not
result in significant change of fatty acid compositions. This result
was in accordance with the aformentioned conclusion that refined
glycerol and crude glycerol led to similar fatty acid compositions.
Additionally, it also implied that the impurities of crude glycerol
did not affect the fatty acid biosynthetic pathways.
4. Conclusion
This study has shown that oleaginous yeast R. touloides had
great potential for converting biodiesel byproduct glycerol to valu-
able lipids, although some impurities such as soap, glycerides, fatty
acid methyl esters, inorganic salt and methanol were present in
crude glycerol. It was found that some of those impurities could
even increase biomass concentration and lipid accumulation, and
the impurities did not affect the fatty acid compositions of the
obtained lipid. The results demonstrated that biodiesel byprod-
uct glycerol could be directly used as carbon source for microbial
lipid production and even more that it could induce higher biomass
concentration and lipid yield than glucose.
This work is supported by International Cooperation
Project of the Ministry of Science and Technology of China
(No. 2010DFB40170), and Tsinghua Research Funding (No.
20121080046), which are greatly appreciated by the authors.
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