Oil Production from Yarrowia lipolytica Po1g Using Rice Bran Hydrolysate

Hindawi Publishing Corporation
Journal of Biomedicine and Biotechnology
Volume 2012, Article ID 378384, 10 pages
doi:10.1155/2012/378384

Research Article
Oil Production from Yarrowia lipolytica Po1g Using
Rice Bran Hydrolysate

Yeshitila Asteraye Tsigie,1 Chun-Yuan Wang,1 Novy S. Kasim,1 Quy-Do Diem,1
Lien-Huong Huynh,1, 2 Quoc-Phong Ho,2 Chi-Thanh Truong,2 and Yi-Hsu Ju1
1 Department of Chemical Engineering, National Taiwan University of Science and Technology, 43 Keelung Road, Section 4,
Taipei 106, Taiwan
2 Department of Chemical Engineering, Cantho University, 3-2 Street, Cantho City, Vietnam

Correspondence should be addressed to Yi-Hsu Ju, yhju@mail.ntust.edu.tw

Received 22 September 2011; Revised 31 October 2011; Accepted 3 November 2011

Academic Editor: Isabel S´ -Correia
a

Copyright © 2012 Yeshitila Asteraye Tsigie et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

The purpose of this study was to produce microbial oil from Yarrowia lipolytica Po1g grown in defatted rice bran hydrolysate. After
removing oil from rice bran by Soxhlet extraction, the bran is subjected to acid hydrolysis with various sulfuric acid concentrations
(1–4% v/v), reaction times (1–8 h), and reaction temperatures (60–120◦ C). The optimal conditions for maximum total sugar
production from the hydrolysate were found to be 3% sulfuric acid at 90◦ C for 6 h. Glucose was the predominant sugar (43.20 ±
0.28 g/L) followed by xylose (4.93 ± 0.03 g/L) and arabinose (2.09 ± 0.01 g/L). The hydrolysate was subsequently detoxified by
neutralization to reduce the amount of inhibitors such as 5-hydroxymethylfurfural (HMF) and furfural to increase its potential as
a medium for culturing Y. lipolytica Po1g. Dry cell mass and lipid content of Y. lipolytica Po1g grown in detoxified defatted rice
bran hydrolysate (DRBH) under optimum conditions were 10.75 g/L and 48.02%, respectively.

1. Introduction result in a shortage in edible oils and leads to the soar of
food price. Adoption of animal fat, used frying oil, and waste
The increasing industrialization and motorization of the cooking oil as feedstock is a good strategy to reduce the cost.
world has led to a steep rise for the demand of petroleum- However, these limited resources cannot meet the increasing
based fuels which are obtained from limited reserves. These needs for clean renewable fuels [3, 4].
finite reserves are highly concentrated in certain regions
Recently, there has been an increasing interest in looking
of the world. Therefore, those countries not having these
for new oil sources for biodiesel production. Among them,
resources are facing energy/foreign exchange crisis, mainly
microbial oils, namely single cell oils (SCOs), have attracted
due to the import of crude petroleum. Hence, it is necessary
to look for alternative fuels which can be produced from great attention worldwide. Oils from oleaginous microor-
resources available locally within the country such as alcohol, ganisms including bacteria, yeasts, moulds, and microalgae
biodiesel, and vegetable oils [1]. [5] are now considered as promising candidates due to their
Biodiesel is a clean, biodegradable, renewable, and specific characteristics such as being unaffected either by
nontoxic fuel which contributes no net carbon dioxide or seasons or by climates, having high lipid contents, ability to
sulfur to the atmosphere and emits less pollutants than be produced from a wide variety of sources with short period
conventional diesel. However, the high cost of raw material of time, and their similar fatty acid compositions to that
(70–75%) for biodiesel production has become one of the of vegetable oils [6, 7]. However, the high production cost
major obstacles for its development and wide applications of SCO makes microbial oils less economically competitive.
[2]. On the other hand, consumption of a large amount of As a result, the production of microbial oils from wastes or
vegetable oils as raw material for biodiesel production would renewable materials is significantly important [8].

2 Journal of Biomedicine and Biotechnology

The oleaginous yeast Y. lipolytica is one of the most hydrolysate as a nutrient source for Y. lipolytica Po1g for
extensively studied “nonconventional” yeasts found in envi- microbial oil production. The effects of acid concentration,
ronments rich in hydrophobic substrates, such as alkanes reaction time, and temperature on the hydrolysis of DRB
or lipids, and has developed sophisticated mechanisms for were investigated. The effects of different types of acid
the efficient use of hydrophobic substrates as its sole carbon hydrolysates of rice bran on the growth and lipid content
source [9]. One of the most striking features of this yeast were also studied. To the best of our knowledge, this is the
is the presence in its genome of several multigene families first report to use defatted rice bran hydrolysate to culture Y.
involved in these metabolic pathways. The complexity and lipolytica Po1g for microbial oil production.
multiplicity of these genes enable Y. lipolytica to use and
valorize a wide range of HS. Using these mechanisms, 2. Materials and Methods
this yeast is able to accumulate large amount of lipids
[10]. Studies about the uses of Y. lipolytica showed its 2.1. Materials. All solvents and reagents were either high-
potential application for the production of reserve lipids performance liquid chromatography (HPLC) or analytical
with composition resembling that of cocoa butter [11], lipids reagent grade, obtained from commercial sources. For
with particular structures (e.g., oils enriched in essential HPLC analysis, all the standards were purchased from
polyunsaturated fatty acids), and nonspecific oils for use as Acros Organics (USA) and Sigma Aldrich (USA). Thin
renewable starting materials for the synthesis of biofuels [12]. layer chromatography (TLC) aluminium plates (20 × 20 cm)
Lipid biosynthesis from sugars and related substrates were obtained from Merck KGaA (Darmstadt, Germany).
is a secondary anabolic activity, conducted after essential Qualitative filter papers (grade no. 2, 0.26 mm thickness,
nutrient (usually nitrogen) depletion in the medium. Due 80% collection efficiency) were acquired from Advantec,
to this exhaustion, the carbon flow is directed towards the MFS Inc. (Dublin, CA).
accumulation of intracellular citric acid that is used as acetyl-
CoA donor in the cytoplasm. Acetyl-CoA generates cellular
fatty acids and subsequently triacylglycerols. SCO produced 2.2. Defatted Rice Bran Hydrolysate (DRBH) Preparation
by Y. lipolytica cultivated under specific growth conditions 2.2.1. Raw Material and Pretreatment. Fresh rice bran was
could be directly converted into biodiesel, as its fatty acid purchased from a local rice mill in Taoyuan County, Taiwan.
composition is similar to the one from common vegetable Bran collected from the mill was stored in a freezer at 4◦ C
oils [13]. Studies related to the production of specialty before use. Prior to defatting, it was dried at 50◦ C for 24 h
lipids by the yeast Yarrowia lipolytica during growth on until weight was constant. Defatting of rice bran was carried
various fatty agroindustrial residues utilized as substrates out in a Soxhlet extractor with hexane as the solvent at 60◦ C
showed that the potential for growth on stearin (a low-cost for 6 h. The defatted rice bran was kept at 4◦ C for future use.
industrial derivative of tallow composed of saturated free
fatty acids) resulted in significant biomass production. This
was accompanied by notable intracellular accumulation of 2.2.2. Acid Hydrolysis of Defatted Rice Bran. The defatted rice
lipid which occurred as a primary anabolic activity regardless bran was hydrolyzed, following the method of Zhu et al. [4]
of the extracellular nitrogen availability in the medium [14]. with minor modification, by using H2 SO4 (1%, 2%, 3%, or
In order to reduce the cost of microbial oil production 4%, v/v) with a bran to acid ratio of 1 : 8 (g/mL). The effect
from Y. lipolytica, low-cost raw materials, such as rice hull of temperature on hydrolysis was investigated at 60, 70, 80,
[15], industrial fats [16], industrial sugars [17], lignocellu- 90, 100, and 120◦ C. The effect of hydrolysis time was also
losic residues [18], and raw glycerol [19, 20], have been used investigated. After hydrolysis, the mixture was subjected to
as substrates. Rice hull, one of the agricultural residues, is vacuum filtration to obtain the defatted rice bran hydrolysate
used for microbial oil production. According to Economou (DRBH).
et al. [15], acid hydrolysis of rice hull using sulfuric acid
resulted in hydrolysate that was used as feedstock for micro- 2.3. Detoxification of DRBH. To reduce the concentration
bial lipids production with the oleaginous fungus Mortierella of inhibitors in DRBH, neutralization with Ca(OH)2 was
isabellina. Results from kinetic experiments showed the employed. Ca(OH)2 was slowly added to the DRBH by
maximum oil accumulation into fungal biomass to be 64.3% stirring the mixture at room temperature until the pH was
and suggested rice hull as cheap source of carbon. adjusted to 6.5. Then, the hydrolysate was repeatedly vacuum
Rice bran is also one of the most abundant agricultural filtrated until the precipitated particles were removed. After
by-products in the world. Typical rice bran is composed its composition was analyzed by HPLC, the detoxified
of about 15–19.7% lipids, 34.1–52.3% carbohydrates, 7– hydrolysate was stored in a refrigerator at 4◦ C for further use
11.4% fiber, 6.6–9.9% ash, and 10–15% proteins. After oil as nutrient for microbial fermentation.
is being removed, the residual defatted rice bran (DRB)
powder contains significant amount of starchy and cellulosic 2.4. Fermentation
polysaccharides. Enzymatic and chemical hydrolysis is used
to break the polysaccharides into smaller molecules which 2.4.1. Microorganism. Y. lipolytica Po1g cells were obtained
will be used as carbon sources for microorganisms [21]. from YEASTERN Biotech Co. Ltd. (Taipei, Taiwan). The
The purpose of this study was to investigate the possi- strain is a derivative of the wild-type strain W29 (ATCC
bility of using cheap and easily available defatted rice bran 20460) by a series of genetic modifications. The cells were

Journal of Biomedicine and Biotechnology 3

maintained on a sterilized yeast-, peptone-, and dextrose- 80◦ C, and nitrogen flow rate was 2 mL/min. The column
(YPD-) agar plate containing 10 g/L yeast extract (Bacto, temperature was 25◦ C. Acetonitrile: water (80 : 20, v/v) was
France), 10 g/L peptone (Bacto, France), 20 g/L glucose the eluent mobile phase with a flow rate of 1 mL/min, and
(Acros Organics, USA), and 20 g/L agar (Acros Organics, the injection volume was 25 µL.
USA) at 4◦ C for further microbial fermentation. The concentrations of 5-hydroxymethylfurfural (5-
HMF) and furfural in the hydrolysate were determined
2.4.2. Media, Inoculum Preparation, and Large-Scale Fermen- by HPLC (Jasco, Japan) equipped with a PU-2089 pump
tation. The preculture of Y. lipolytica Po1g was incubated on combined with degasser, an UV 2077 UV detector, and a
a sterilized YPD-agar plate and rejuvenated by incubation Luna C-18 column (5 µm particle size, 250 mm × 4.6 mm,
in 25 mL YPD medium containing yeast extract (10 g/L), Phenomenex, USA). The sample was diluted appropriately
peptone (10 g/L), and D-glucose (20 g/L) for 24 h at 26◦ C with deionized water, filtered through a 0.22 µm PVDF
in an orbital shaker incubator model LM-570 (Chemist syringe filter (Test high), and then injected into the column
Scientific Corp, Taiwan) and then inoculated to cultures in under the following conditions: 25◦ C column temperature,
500 mL Erlenmeyer flasks at an inoculum to medium ratio acetonitrile : water : acetic acid (11 : 88 : 1, v/v/v) with a flow
of 1 : 10 (v/v). rate of 0.8 mL/min, injection volume of 25 µL, and the
Fermentation was carried out in several 500 mL Erlen- absorption wavelength was 276 nm. The concentrations of
meyer flasks each containing 250 mL detoxified DRBH with these compounds were calculated by using calibration curves
initial pH 6.5. To investigate the effect of different nitrogen obtained from standard D-glucose, D-xylose, L-arabinose,
sources on growth of cells and lipid content, either urea furfural, and 5-HMF solutions.
(5 g/L, Acros Organics, USA) or peptone (5 g/L) was added The amount of total reducing sugars in the hydrolysate
to the DRBH. The effect of the concentration of fermentable was measured by the dinitrosalicylic acid (DNS) method
sugars in DRBH on microbial growth and lipid content based on a colorimetric reaction between the sugars and
was studied by diluting the sugar concentration obtained dinitrosalicylic acid. DNS reagent (0.5 mL) was added to the
at optimum conditions (48.41 g/L) and adjusted to 20 g/L, appropriately diluted sample (0.5 mL) in a capped brown
30 g/L, or 40 g/L. bottle to prevent DNS from being affected by light. Then, the
The flasks were then incubated in an orbital shaker mixture was heated to 100◦ C for 5 min, and after cooling to
incubator at 150 rpm and 26◦ C. Cell biomass was harvested room temperature in a cold water bath, the absorbance was
by centrifugation at 3500 g for 30 min. The cell mass monitored with a spectrophotometer at 540 nm. The results
was washed twice with deionized water and dried in an were calculated based on calibration curve of standard D-
oven at 50◦ C until constant weight. All media and flasks glucose.
were autoclaved at 121◦ C for 30 min before the microbial
fermentation. 2.5.3. Lipid Analysis. Extraction of total lipid was performed
by using Soxhlet extractor with hexane and methanol (1 : 1,
2.5. Analytical Methods v/v) for 4 h. The extracted lipid was subjected to silica gel
thin layer chromatography (TLC) analysis to identify its
2.5.1. Cell Concentration Determination. For monitoring neutral lipid content. After that, the crude microbial lipid
yeast growth, optical density (OD) of the diluted fermen- was dewaxed and degummed according to the methods
tation broth was measured at 600 nm using a UV/Vis spec- described by Rajam et al. [23], and Vandana et al. [24].
trometer V-550 model (Jasco, Japan), and cell concentration Crude microbial oil was dissolved in hot water at 60◦ C, and
was determined from a calibration curve of absorbance the water soluble fraction was separated from the insoluble
versus dry cell weight. fraction by vacuum filtration. The insoluble fraction was
then dissolved in acetone and kept at 60◦ C for 1 h to
2.5.2. Analysis of Protein, Sugar, and Inhibitor Concentration obtain clear solution. After allowing the content to cool
in DRBH. The solubilized protein content of the hydrol- to room temperature, the solution was then kept at 5◦ C
ysates was determined by the Bradford method [22]. for 24 h to crystallize the remaining waxes. The insoluble
Absorbance at 595 nm was measured by using a UV-VIS fraction was separated by vacuum filtration. The dewaxed
spectrophotometer V-550 model (Jasco, Japan), and then and degummed lipid was analyzed by gas chromatography
comparison to a standard curve provided a relative measure- (GC-17A, Shimadzu, Japan) for its neutral lipid composition
ment of protein concentration. as well as its fatty acid profile. The GC was equipped with a
Concentrations of D-glucose, D-xylose, and L-arabinose flame ionization detector and a DB-17HT capillary column
in the hydrolysates were determined by HPLC (Jasco, Japan) (0.25 cm × 30 m, Agilent Technologies Inc., USA). The
equipped with a PU-1580 pump, a DG-4400 degasser, an column temperature was programmed to increase from 80◦ C
Alltech 2000 ELSD detector, and a Zorbax NH2 column to 365◦ C at 10◦ C/min and kept at 365◦ C for 29 min. Nitrogen
(5 µm particle size, 250 mm × 4.6 mm, Agilent Technologies, was used as the carrier gas at a flow rate of 0.80 mL/min.
USA). The sample was diluted appropriately with deionized The split ratio was 1 : 50 (v/v). The temperatures of injector
water, filtered through a 0.22 µm PVDF syringe filter (Test and the detector were both maintained at 370◦ C. Twenty
high) and then injected into the HPLC under the following milligrams sample was dissolved in 1 mL ethyl acetate, and
conditions. The temperature of the ELSD detector was 0.5 µL sample was taken and injected into the GC. Standards


Table 1: Composition of DRBH with different reaction time and H2 SO4 concentration at 90◦ C.

H2 SO4 Reaction Concentration (g/L)
(%v/v) time (h) Glucose Xylose Arabinose HMF Furfural
2 13.36 ± 0.89 1.88 ± 0.42 1.13 ± 0.28 0.05 ± 0.02 0.004 ± 0.003
4 16.25 ± 0.94 2.34 ± 0.28 1.46 ± 0.19 0.16 ± 0.01 0.012 ± 0.002
2%
6 21.26 ± 0.90 3.20 ± 0.28 1.97 ± 0.35 0.21 ± 0.02 0.020 ± 0.003
8 25.57 ± 0.76 3.84 ± 0.11 2.41 ± 0.16 0.28 ± 0.02 0.029 ± 0.020
2 26.87 ± 0.64 4.61 ± 0.11 2.82 ± 0.07 0.13 ± 0.01 0.006 ± 0.002
4 36.67 ± 0.32 5.45 ± 0.043 2.23 ± 0.01 0.25 ± 0.01 0.015 ± 0.001
3%
6 43.20 ± 0.28 4.93 ± 0.03 2.09 ± 0.01 0.32 ± 0.05 0.025 ± 0.003
8 40.12 ± 0.49 3.89 ± 0.05 1.46 ± 0.07 0.41 ± 0.02 0.041 ± 0.003
2 28.80 ± 0.15 4.26 ± 0.02 2.83 ± 0.01 0.23 ± 0.01 0.011 ± 0.002
4 37.98 ± 0.19 4.60 ± 0.02 2.07 ± 0.81 0.40 ± 0.01 0.030 ± 0.003
4%
6 43.64 ± 0.49 4.37 ± 0.16 1.87 ± 0.12 0.60 ± 0.08 0.053 ± 0.009
8 36.75 ± 1.79 3.78 ± 0.28 1.32 ± 0.21 0.88 ± 0.07 0.078 ± 0.005

of saturated and unsaturated fatty acids (Sigma-Aldrich, optimum conditions resulted in glucose (43.20 ± 0.28 g/L),
USA) were used for the identification of fatty acids in the xylose (4.93 ± 0.03 g/L), arabinose (2.09 ± 0.01 g/L), HMF
lipid. All data were averages of triplicate determinations. (0.32 ± 0.05 g/L), and furfural (0.025 ± 0.003 g/L). Compared
to the total fermentable sugars from SARSH (35.2 g/L), our
results showed that rice bran has higher concentration of
3. Result and Discussion fermentable sugars than sulfuric-acid-pretreated rice straw
hydrolysate. On the other hand, the highest monosaccharide
3.1. Acid Hydrolysis of DRB. Huang et al. [25] studied the
sugar concentration obtained in this study (43.20 ± 0.28 g/L)
production of microbial oil from T. fermentans grew in
is glucose, while that of SARSH (25.5 g/L) is xylose. This
sulfuric-acid-treated rice straw hydrolysate (SARSH). The
indicates rice bran is a better source of glucose than rice straw
hydrolysate (SARSH) contained a maximum of 35.2 g/L
for microbial fermentation.
fermentable monosaccharides including glucose, xylose, and
The total sugar obtained in this work was also much
arabinose, and the concentration of pentoses was about six
higher than that of enzymatic hydrolysis of DRB which
times higher than hexose.
resulted in 37 g/L total sugars when defatted rice bran was
In this study, the composition of the defatted rice
subjected to saccharification with a mixture of amylase
bran hydrolysate (DRBH) obtained by using 1% H2 SO4
and cellulase [26]. Therefore, dilute acid hydrolysis breaks
at different temperature (60◦ C–120◦ C) for 1 h showed that
down the lignocelluloses better than enzymes and can be
glucose was the predominant sugar in the hydrolysate. This
considered as an efficient method to hydrolyze the DRB to
result suggests the presence of a higher proportion of starch
obtain sugars for utilization in microbial fermentation.
and glucans in DRB along with other minor pentoses
The concentration of monosaccharides increases as time
such as xylose and arabinose, which are mainly derived
increases from 2 h to 8 h using 2% acid concentration.
from xylans and arabinoxylans, respectively. The maximum
However, at an acid concentration of 3% or 4%, glucose
fermentable sugars concentration from DRBH under these
concentration increased with time until 6 h and then started
conditions was 5.96 ± 0.43 g/L out of which glucose, xylose,
to decrease. The same trend was observed for xylose and ara-
and arabinose are 3.16 ± 0.22 g/L, 1.36 ± 0.12 g/L, and
binose. The rapid increase in the concentration of inhibitors
0.74 ± 0.01 g/L, respectively. However, the results suggest that
with longer hydrolysis time indicates the decomposition of
hydrolysis of DRB for 1 h with 1% H2 SO4 was not efficient
monosaccharides into less desirable compounds under the
to hydrolyze the DRB to produce fermentable sugars. The
severe acid hydrolysis condition (4% H2 SO4 ). A similar work
optimum temperature was 90◦ C, and this was used as a
also reported that sugar concentration increases with longer
basis for the next hydrolysis reactions with different acid
hydrolysis time and higher acid concentration until the
concentrations.
optimum conditions were reached and decreases thereafter
Table 1 shows the effects of H2 SO4 concentrations (2%,
[27].
3%, and 4%) and hydrolysis time (2–8 h) on the composition
of DRBH at 90◦ C. The concentration of total fermentable
sugars using 2% H2 SO4 was much lower than that of 3% and 3.2. Effect of Detoxification on the Composition of DRBH.
4%, which shows that higher acid concentration could break The presence of inhibitory substances in the fermentation
down the polymerized structure of hemicellulose, amylase, medium can restrict both sugar utilization and growth
and amylopectin more efficiently. The highest total released of microorganisms. It has been reported that oleaginous
sugar concentration (50.22 ± 0.34 g/L) was obtained when microorganisms could not grow and reproduce well in the
DRB was hydrolyzed with 3% H2 SO4 at 90◦ C for 6 h. These rice straw hydrolysate obtained by acid hydrolysis without


Table 2: Chemical composition of DRBH before and after detoxification. The hydrolysate used for detoxification was obtained by using 3%
H2 SO4 at 90◦ C for 6 h.

Type of Concentration (g/L)
DRBH Glucose Xylose Arabinose HMF Furfural
Before detoxification 25.57 ± 0.76 3.84 ± 0.11 2.41 ± 0.16 0.28 ± 0.02 0.029 ± 0.020
2%
After detoxification 25.40 ± 0.87 3.53 ± 0.43 2.03 ± 0.11 0.22 ± 0.01 0.023 ± 0.001
3% Before detoxification 43.20 ± 0.28 4.93 ± 0.03 2.09 ± 0.01 0.32 ± 0.05 0.025 ± 0.003
Before detoxification 43.64 ± 0.49 4.37 ± 0.16 1.87 ± 0.12 0.60 ± 0.08 0.053 ± 0.009
4%

detoxification. Low lipid content was obtained after culturing 22 10
T. fermentans for 8 days owing to both sugar utilization 20
and lipid accumulation being inhibited by various inhibitors 18
8
[25]. Therefore, detoxification of DRBH is necessary to

Sugar concentration (g/L)
16
reduce the amount of inhibitors and achieve higher fer-
14

Biomass (g/L)
mentability of sugars. Various methods such as overliming, 6
neutralization, ion exchange, steam stripping, and treatment 12
with activated carbon have been applied to detoxify the 10
lignocellulosic hydrolysate. However, the most economical 8 4
way to detoxify lignocellulosic hydrolysate was neutralization 6
by calcium hydroxide [28]. 4 2
Detoxification of DRBH by using Ca(OH)2 was applied
2
since it can easily remove the residual SO4 2− by forming
0
CaSO4 precipitate and efficiently reducing the level of 0
inhibitors simultaneously. The decrease in the concentra- 0 1 2 3 4 5 6
tions of HMF and furfural is due to the reaction between Fermentation time (day)
calcium ions and furans, producing complex ions [29].
During the hydrolysis of DRB, the concentrations of Figure 1: Effect of different types of DRBH on the growth of Y.
both HMF and furfural increased with increasing acid lipolytica Po1g. Culture conditions: growth in Erlenmeyer flasks;
concentrations. Using 2% sulfuric acid, the concentrations of initial total sugar concentration: 20 g/L; pH: 6.5; incubation tem-
HMF and furfural were 0.28 g/L and 0.029 g/L, respectively, perature: 26◦ C; agitation speed = 150 rpm; biomass concentration:
( ) 2%, (◦) 3%, ( ) 4%; sugar consumption: ( ) 2%, (•) 3%, ( )
and when the acid concentration increased from 3% to
4%.
4%, the concentration of HMF and furfural also increased
from 0.32 g/L to 0.60 g/L and from 0.025 g/L to 0.053 g/L,
respectively.
The maximum concentration of total released sugars Three different types of detoxified DRBHs were obtained
from nondetoxified DRBH at optimum conditions was from rice bran hydrolysis with three different acid concen-
50.22 ± 0.34 g/L, out of which the concentration of glucose trations (2%, 3%, and 4%) and named 2%, 3%, and 4%,
was 8.76 times higher than that of xylose. HMF and respectively. The DRBHs were then used as the basic media
furfural were the main inhibitors in the hydrolysate before for the growth of Y. lipolytica Po1g. The concentrations
neutralization. As shown in Table 2, the concentration of of total fermentable sugars in all three DRBH media were
monosaccharides before and after detoxification by Ca(OH)2 made constant (20 g/L) by appropriately diluting the corre-
did not show a significant change. However, the concen- sponding initial detoxified DRBHs with deionized water. The
trations of HMF and furfural decreased by 25.0% and protein contents of DRBHs obtained by using 2%, 3%, and
24.0% (in the 3% DRBH), respectively, which indicates that 4% sulfuric acid were analyzed to be 1.09, 2.85, and 3.05 g/L,
detoxification by lime can effectively reduce the amount of respectively. To study the possibility of using the hydrolysates
inhibitors and can provide a potential alternative medium for as direct nutrient sources, comparison of the growth profile
better microbial fermentation. of Y. lipolytica Po1g in these media was made, and the
lipid contents of the maximum biomass concentrations were
3.3. Growth of Y. lipolytica Po1g in Different Types of DRBH determined.
Media. DRB is rich in carbohydrates, fiber, and proteins and As can be seen in Figure 1, yeast growth was poor
can be regarded as a potential nutrient source for microbial (maximum biomass concentration = 4.43 g/L, 3rd day) in the
oil production [21]. Therefore, detoxified DRBH can be a 2% DRBH medium. The corresponding cellular lipid content
potential nutrient source for growing Y. lipolytica Po1g. obtained was 21.66%.


Increases in biomass concentration were observed when Table 3: Effects of sugar concentration on microbial oil production
Y. lipolytica Po1g grew in the DRBH obtained by hydrol- by Y. lipolytica Po1g.
ysis using 3% H2 SO4 . The highest biomass concentration
Sugar
(8.76 g/L) was observed on the 4th day. The cellular lipid Maximum Lipid content Lipid yield
concentration
content obtainable is 30.13% and is relatively higher than the biomass (g/L) (%) (g/L)
(g/L)
one from the medium that contained the DRBH from 2%
20 8.76 ± 0.13 30.13 ± 0.56 2.64 ± 0.08
H2 SO4 . The sugar concentration in the medium prepared
by hydrolysis of rice bran with 3% H2 SO4 also decreased 30 10.75 ± 0.19 48.02 ± 0.61 5.16 ± 0.15
faster due to an increase in biomass. As the biomass increases, 40 9.33 ± 0.16 40.97 ± 0.74 3.82 ± 0.13
sugar consumption also increases. On the other hand, both
biomass and lipid contents decreased slightly to 7.51 g/L and
27.73%, respectively, when Y. lipolytica Po1g was cultivated Zhu et al. [4] investigated cell growth and lipid accumu-
in the DRBH obtained by hydrolysis with 4% H2 SO4 . lation of T. fermentans in molasses with different total sugar
According to a study conducted by Economou et al. [15], concentrations ranging from 5% to 30%. Both biomass and
rice hulls hydrolysate was used as the sole carbon and energy lipid content increased with the total sugar concentration,
source for oleaginous fungus M. isabellina. In their study, and the maximum biomass (29.9 g/L) and lipid content
an increase in acid concentration from 0.03 M to 0.09 M (31.8%) were attained at 15% total sugar concentration.
increased the extractable oil amount from 36% to 64.3%, and Further rise in the total sugar concentration beyond 15%
it was observed that in the rice hulls hydrolysate obtained led to the decrease in biomass and lipid content. The same
from sulfuric acid concentration higher than 1 M, the fungus trend was observed when Y. lipolytica Po1g was cultivated in
failed to grow, probably due to the high concentrations of DRBH with different sugar concentrations which increased
toxic compounds in the culture medium. The same trend was both biomass and lipid content when sugar concentration
observed in our study. The highest acid concentration (4%) was raised from 20 g/L to 30 g/L. But further increase to
might have led to higher concentration of inhibitors during 40 g/L resulted in lowering of biomass concentration and
hydrolysis. This shows that neutralization of this hydrolysate lower lipid content. This may be due to substrate inhibition
with Ca(OH)2 alone did not reduce the inhibitors concen- in which excess sugar concentration in the DRBH media
tration, and these inhibitors affected the growth of the yeast inhibits the growth of the microorganism during the early
cells. stages of growth.
A review about lipid accumulation properties of Y. Another study on the biochemical behavior of wild-type
lipolytica shows that the study of de novo lipid accumulation or genetically modified Y. lipolytica strains cultivated on
as a result of the conversion of carbohydrate substrates, commercial glucose and nitrogen-limited cultures showed
such as glucose, provides insight into the regulation of the that carbon-excess conditions favored the secretion of
entire lipid synthesis pathway. The accumulation of lipids organic acids into the growth medium [17]. Cultivation of
produced from these substrates is triggered by nutrient Y. lipolytica Po1g using 40 g/L in this study also resulted
limitation. Nitrogen limitation is the principal type of in reduction of biomass concentration, which may most
limitation governing lipid accumulation [30]. Our results probably be due to the secretion of organic acids.
also suggested that limited nitrogen content in the media
(3% DRBH) led to better growth and lipid accumulation 3.5. Effect of Addition of Different Nitrogen Sources on Growth
while the protein content (1.09 g/L) in the medium from 2% and Microbial Oil Production. Factors such as carbon and
H2 SO4 was not enough for the growth of the cells, which nitrogen sources have significant influences on cell growth
limited biomass concentration to 4.43 g/L. and lipid accumulation of oleaginous microorganisms [31].
To study the effect of addition of different nitrogen sources
3.4. Effect of DRBH Sugar Concentration on Biomass and on biomass concentration and microbial oil production,
Microbial Oil Content. To investigate the effect of sugar three different DRBH media with the same total fermentable
concentrations on microbial growth and lipid accumulation, sugar concentration (30 g/L) and the same initial protein
three different concentrations of total fermentable sugars content (2.82 g/L) were prepared. No external nitrogen
(20 g/L, 30 g/L, and 40 g/L) were prepared by appropriate source was added to the first one, but either peptone (5 g/L)
dilution of the detoxified DRBH (type 3%, Table 2) with or urea (5 g/L) was added to the other two media.
an initial total sugar concentration of 48.41 g/L. The total As it can be seen from Figure 2, the medium without
protein content in each of these media was adjusted to 5 g/L additional nitrogen source was the best for yeast cells to
by addition of appropriate amount of yeast extract. accumulate the highest lipid content (48.02%) with a lipid
As shown in Table 3, the lowest cellular lipid content yield of 5.16 g/L. The addition of urea did not significantly
(30.13%) was obtained when the Y. lipolytica Po1g was affect (P-value = 0.27) biomass production (10.85 g/L) but
incubated in the medium with the lowest sugar concentra- caused reduction in cellular lipid accumulation to 14.19%.
tion (20 g/L). However, the highest lipid content (48.02%) Compared to urea, Y. lipolytica Po1g grew better, and the
and lipid yield (5.16 g/L) were observed when the sugar maximum biomass concentration was 13.53 g/L when the
concentration in the medium was 30 g/L. Both biomass nitrogen source was peptone. The cellular lipid content
concentration and cellular lipid contents decreased when the (26.43%) obtained when cells grew in peptone was much
total sugar concentration was 40 g/L. higher than the one with urea, indicating that urea is not a


10 mechanism, are able to accumulate huge amounts of lipid
50 when growth is carried out on various hydrophobic carbon
sources as substrates, since accumulation of lipid from fat
8 materials is a completely different process from that from
Biomass (g/L), lipid content (%)

40 sugars [13].

Lipid yield (g/L)
6 3.6. Cellular Lipid Analysis. Crude microbial oil from Y.
30
lipolytica Po1g cells grown in a medium with detoxified
DRBH (3%) was investigated by TLC, and the contents were
4 determined gravimetrically. After dewaxing and degum-
20
ming, the neutral lipid content and the fatty acid profile were
determined by GC 17A.
10 2 The crude microbial oil consisted of gum (52.77%),
neutral lipids (41.92%), and wax (5.31%). The results of
analysis of lipids isolated from the biomass of the yeast C.
0 0 lipolytica grown in medium with methanol or glucose as
No external nitrogen Urea Peptone the only carbon source also showed that polar lipids make
added
more than half of the total cell lipids for both carbon sources
Biomass (52.3 and 64.2%, resp.) [32]. This is in agreement with the
Lipid content results of our work. On the other hand, Makri et al. [19]
Lipid yield found that neutral lipids are the major components in Y.
lipolytica when grown in a glycerol medium. According to
Figure 2: Effects of nitrogen addition on microbial oil production their study, lipid composition in Y. lipolytica showed specific
by Y. lipolytica Po1g grown in DRBH medium. Culture conditions: trends with time that largely reflect the physiological role
growth in Erlenmeyer flasks; initial total sugar concentration: of individual lipids. The increase of neutral lipids during
30 g/L; pH: 6.5; incubation temperature: 26◦ C; agitation speed = lipogenic phase and their subsequent decrease in citric acid
150 rpm. production phase may explain the physiological role of
neutral lipids, which is energy storage in times of plenty and
energy provision in times of shortage. In fact, during lipid
good nitrogen source for microbial oil production from Y. turnover phase, neutral lipids are preferentially degraded,
lipolytica Po1g grown in detoxified DRBH medium. while some quantities of polar lipids are synthesized.
The GC chromatograms (Figures 3(a) and 3(b)) show
Microbial oil production with both nitrogen supple-
that the major components of neutral lipid are free fatty
ments showed similar tendencies in which the microorgan-
acids (FFA, 79.56%), monoacyl glycerides (MAG, 8.91%),
isms did not accumulate more lipids but produced more
diacyl glycerides (DAG, 2.15%), and triacyl glycerides (TAG,
biomass than the one without external nitrogen source. 8.59%). Media, type of strain, and cultivation time have
Nitrogen is essential for the syntheses of proteins and nucleic significant effect on the amount of FFA in lipids from Y.
acids which are required for cellular proliferation. However, lipolytica. As reported by Papanikolaou et al. [16], growth of
this synthesis will be repressed when nitrogen source is lim- Y. lipolytica on medium containing hydrophobic compounds
ited, and carbon source will be channeled to lipid synthesis as (co)substrates resulted in the accumulation of storage lipid
causing accumulation of lipid in the cells [32]. Therefore, containing considerable quantities of FFA (30 to 40 wt% of
nitrogen limitation is effective in enhancing the accumula- total lipids). They also found that large quantities of FFA did
tion of lipid in Y. lipolytica Po1g grown in DRBH media. not result in lethality or growth deficit, which is similar to
Similar results by Zhu et al. [4] also showed that the our result. According to Juliano [21], rice bran is composed
cellular lipid content of T. fermentans grown in a medium of about 15–19.7% lipids, which depends on factors such as
with urea as the nitrogen source is 2.4 times lower than that type and origin of rice. The solvent used or extraction time
when peptone was the nitrogen source. The result of this used in this study might have an effect in which some amount
study shows that the yeast tended to channel sugar into cellu- of oil has not been removed from rice bran. The excess oil
lar lipid production when it was cultivated on the nitrogen- may remain in the medium and contribute to the increase
limited detoxified DRBH resulting in the highest cellular in the FFA amount that can be extracted from Y. lipolytica
lipid content. On the other hand, according to Papanikolaou Po1g. The reason for the high FFA content needs further
et al. [17], growth of either wild-type or genetically modified investigation.
strains of Y. lipolytica under nitrogen-limited culture con- The fatty acid profile of the neutral lipid consisted of
ditions, with glucose or similarly metabolized compounds oleic acid (C18 : 1, 59.91%), palmitic acid (C16 : 0, 20.46%),
used as substrates, was not accompanied by significant palmitoleic acid (C16 : 1, 11.41%), and stearic acid (C18 : 0,
lipid accumulation. Carbon-excess conditions favored the 5.39%), with 2.83% as unidentified fatty acids. The main
secretion of organic acids into the growth medium. cellular fatty acid of Y. lipolytica lipid produced during
In general, various Y. lipolytica strains, despite being inca- growth on glucose or glycerol media also showed similar
pable of producing lipid via the de novo lipid accumulation results, and oleic acid, palmitic acid, palmitoleic acid, and


C18:1
C16:0
FFA

FID response
FID response

C16:1
TAG C18:0
MAG DAG O
O

5 7.5 10 12.5 15 17.5 20 22.5 25 27.5
5 7.5 10 12.5 15 17.5 20 22.5 25 27.5
Retention time (min)
Retention time (min)
O = others

(a) (b)

Figure 3: GC chromatograms of neutral lipid before saponification (a) and fatty acid profile after saponification with KOH and ethanol (b).

Table 4: Lipid contents of different strains of Y. lipolytica grown in different agroindustrial sources.

Lipid
Biomass Lipid yield
Strain Carbon source content Reference
(g/L) (g/L)
(%)
Y. lipolytica Industrial derivative of animal fat 12.5 54 3.8 [16]
C. lipolytica 8% molasses NA 59.9 NA [33]
Y. lipolytica LGAM (7)1 Teucrium polium L. aqueous extract 9.3 33 3.12 [34]
Y. lipolytica LGAM (7)1 Glucose 9.3 25 2.3 [34]
Y. lipolytica Industrial fats 8.7 44.0 3.8 [11]
Y. lipolytica ACA-YC 5033 Commercial glucose 5.5 14 NA [17]
C. lipolytica Mixtures of stearin, glucose, and glycerol 11.4 30 3.4 [35]
C. lipolytica 1% methanol NA 4.9 NA [32]
Y. lipolytica Po1g Sugarcane bagasse hydrolysate 11.42 58.5 6.68 [18]
Y. lipolytica Po1g Defatted rice bran hydrolysate 10.75 48.02 5.16 This study

stearic acid were the major fatty acids [17, 19]. The combined when compared to the results of different strains of Y.
content of saturated and monounsaturated fatty acid is lipolytica.
more than 97.17%, making neutral lipids obtained from the In our previous study, Y. lipolytica Po1g cells were
culturing of Y. lipolytica Po1g in rice bran hydrolysate ideal cultivated in sugarcane bagasse hydrolysate, and the maxi-
feedstock for biodiesel production. mum biomass, lipid content, and lipid yield attainable were
Table 4 gives comparison of maximum biomass concen- 11.42 g/L, 58.5%, and 6.68 g/L, respectively [18]. Therefore,
tration, lipid content, and lipid yield of Y. lipolytica Po1g under different media, different strains of Y. lipolytica have
grown in DRBH media with those from different strains of different lipid contents and lipid yields.
Y. lipolytica grown in different nutrient sources. In terms of
biomass concentration, DRBH is a better nutrient source 4. Conclusion
for the Y. lipolytica Po1g compared to commercial glucose
used to cultivate Y. lipolytica ACA-YC 5033. This indicates Defatted rice bran is an undervalued agricultural by-product
the potential of defatted rice bran as a nutrient source for which can be hydrolyzed by dilute acids not only to
oleaginous microorganisms. release carbon sources from starch and lignocellulose but
According to Karatay and D¨ nmez [33], C. lipolytica can
o also enough nitrogen sources for growing microorganisms.
accumulate lipids (59%, w/w) when grown in 8% molasses Detoxification of the defatted rice bran hydrolysate is
medium. However, the maximum lipid concentration of Y. required to reduce the level of inhibitors in the fermentation
lipolytica Po1g in detoxified DRBH is 48.02%. On the other medium. Detoxified DRBH was found to be an effective
hand, our work shows better results compared to that of Y. medium for cultivating Y. lipolytica Po1g, and high cellular
lipolytica cultivated in methanol and industrial fats which lipid content was obtained. The composition of the neutral
resulted in a lipid content of 4.9% and 44%, respectively. lipid obtained is similar to those of most staple vegetable oils
Lipid yield of Y. lipolytica Po1g (5.6 g/L) is more promising indicating its potentials for biodiesel production.


Acknowledgment Applied Microbiology and Biotechnology, vol. 58, no. 3, pp. 308–
312, 2002.
This work was supported by a Project (NSC 98-2221-E-011- [17] S. Papanikolaou, A. Chatzifragkou, S. Fakas et al., “Biosynthe-
046-MY3) from the National Science Council of Taiwan. sis of lipids and organic acids by Yarrowia lipolytica strains
cultivated on glucose,” European Journal of Lipid Science and
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