Industrial Crops and Products 66 (2015) 7380

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Industrial Crops and Products

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Solid state fermentation by cellulolytic oleaginous fungi for direct

conversion of lignocellulosic biomass into lipids: Fed-batch and
repeated-batch fermentations
Benjamas Cheirsilp a, , Suleeporn Kitcha b
a
b

Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai Songkhla, 90110, Thailand
Program of Biology, Faculty of Science and Technology, Bansomdejchaopraya Rajabhat University, Bangkok 10600, Thailand

a r t i c l e

i n f o

Article history:
Received 18 September 2014
Received in revised form
28 November 2014
Accepted 17 December 2014
Available online 27 December 2014
Keywords:
Cellulase
Lignocellulosic material
Oleaginous fungi
Solid state fermentation
Xylanase

a b s t r a c t
Lignocellulosic wastes from palm oil mill are one of attractive feedstocks for microbial lipid production
by oleaginous microorganisms because of their low cost, renewable nature and abundance. In this study,
four lamentous fungi with cellulolytic activity were screened as potential oleaginous microorganisms
for direct conversion of these lignocellulosic wastes into lipid. Among them, Aspergillus tubingensis TSIP9
accumulated lipid at the highest amount of 39.5 2.2 mg per gram dry substrate (gds), and simultaneously
produced high activities of cellulase (2.35 0.22 U/gds) and xylanase (11.83 0.18 U/gds) through solid
state fermentation (SSF) of palm empty fruit bunches (EFB). The use of EFB mixed with palm kernel cake
(PK) promoted lipid production by the fungi up to 79.9 3.5 mg/gds. When the enzymes were extracted
from the rst batch and reused in the next batch, A. tubingensis TSIP9 produced much higher amount of
enzymes and accumulated lipid faster. Fed-batch SSF with intermittent adding of EFB could be applied
for lipid production but with a decrease in the enzyme activity. When repeated-batch SSF with 90%
replacement with new substrate was applied, both lipid and enzymes were efciently produced for long
period of fermentation. This new strategy for solid state fermentation may contribute greatly to the
commercialized enzyme and lipid productions from abundant lignocellulosic biomass.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Biomass-based biofuel production is now concerned as an
approach to face the challenges from high energy prices and potential depletion of crude oils reservoirs, to reduce greenhouse gas
emissions, and to enhance a sustainable economy. Biodiesel produced from plant oil is an attractive alternative biomass-based
biofuel because it is biodegradable, nontoxic, clean and having similar properties to the conventional diesel fuels. However, the use
of pure plant oil as a raw material for biodiesel production would
compete with its use as edible oil; thus, leading to an increase in
its food price. Therefore, new sources of biodiesel feedstocks have
been intensively searched. Microbial lipids produced by oleaginous microorganisms, are considered to be promising potential
biodiesel feedstock due to their plant-like oil composition (Zhu
et al., 2008). Among oleaginous microorganisms, oleaginous fungi

Corresponding author. Tel.: +66 898904205; fax: +66 74 55 6688.

E-mail address: benjamas.che@psu.ac.th (B. Cheirsilp).
http://dx.doi.org/10.1016/j.indcrop.2014.12.035
0926-6690/ 2014 Elsevier B.V. All rights reserved.

including both molds and yeasts, are increasingly been reported as

good lipid producers.
Lignocellulosic biomass (agricultural waste and forest biomass),
which contains 5565% carbohydrate, is widely recognized as a
promising feedstock for bio-fuel production (Chundawat et al.,
2011). It is also an attractive feedstock for microbial lipid production. However, the lignocellulosic biomass needs to be hydrolyzed
into fermentable sugars by a pool of cellulolytic enzymes including cellulase, xylanase, and other accessory enzymes. Filamentous
fungi are considered to be the most suitable source for cellulolytic
enzymes due to their high production yields and the ability to
utilize a wide range of inexpensive agro-residues (Dhillon et al.,
2011; Ncube et al., 2012; Liang et al., 2012). It was thought that if
oleaginous fungi with cellulolytic activity were screened, a direct
pathway for conversion of lignocellulosic biomass to sugars and
consequently lipid could be developed.
Solid-state fermentation (SSF) has been widely used for cultivation of lamentous fungi (Kunamneni et al., 2005; Dharani and
Kumaran, 2012; Pensupa et al., 2013; Saleem and Ebrahim, 2014).
This is because a solid medium simulates better natural habitat of
the fungi (Robinson et al., 2001). SSF has also experienced particular

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B. Cheirsilp, S. Kitcha / Industrial Crops and Products 66 (2015) 7380

interest due to its many advantages in comparison to submerged

fermentation, e.g., smaller bioreactor volume, reduced downstream
processing costs, higher productivity, simpler technique, reduced
energy requirement, and low wastewater output (Mitchell et al.,
2006). The hyphal development of the fungi also allows them to
effectively colonize and penetrate the solid substrate. Furthermore,
they can utilize the bound water of their substrates; and thus, grow
in the absence of free water (Krull et al., 2010).
To date research work that has been associated with the direct
conversion of lignocellulosic biomass into microbial lipid has
been limited. One research group has focused on the isolation of
oleaginous fungi with cellulase activity (Peng and Chen, 2007).
However, the cellulase activities of their strains were very low at
only 0.310.69 lter paper units (FPU) and the lipid yield were
1942 mg/g of the substrate, dry wheat straw. Therefore, there was
a need to add exogenous cellulases to boost the lipid yield to as
much as 74 mg/g dry wheat straw (Peng and Chen, 2008). Recently,
the direct conversion of wheat and rice straws into lipid by cellulase producing fungi was attempted (Lin et al., 2010; Dey et al.,
2011). However, there was no available information on xylanase
production by their fungi and its relationship to lipid production.
Currently, the palm oil industry has considerably expanded its
production in Thailand and this has produced much lignocellulosic
wastes including 13.5% palm pressed ber (PPF), 22% palm empty
fruit bunches (EFB) and 5.5% palm kernel cake (PK) (Pua et al., 2013).
These wastes, therefore could be suitable lignocellulosic raw materials for microbial lipid production. The aim of this study was to
directly convert such lignocellulosic wastes from palm oil mill into
lipid by cellulolytic oleaginous fungi. Firstly, oleaginous fungi with
high cellulolytic activity were isolated from soils and wastes associated with palm oil mill. The production of lipid and enzymes by
these isolated oleaginous fungi was then tested through solid state
fermentation (SSF) of palm empty fruit bunches (EFB) added with
palm kernel cake (PK) as an alternative nitrogen source. The process development for solid state fermentation including reuse of
crude enzymes, fed-batch strategy with intermittent adding of a
carbon source and repeated-batch strategy with various percent
replacements, was attempted for sustainable production of lipid
and enzymes from lignocellolusic biomass.

2. Materials and methods

2.1. Lignocellulosic wastes from palm oil mill
EFB and PK were obtained from Thai Taro and Oils Co., Ltd., Surat
Thani, Thailand and sun-dried for 2 days. The waste was cut into
small pieces of about 1 cm in length. To remove the lignin, EFB were
soaked with 10% NaOH at a ratio of solid to liquid of 10% and boiled
at 100 C for 15 min (Boonsawang et al., 2012). The pretreated solids
were then washed with tap water to nearly a neutral pH. After pretreatment, the materials were dried to constant weight at 60 C,
stored in plastic bags and kept at room temperature before use.
The hemicellulose, cellulose and lignin contents of EFB and PK were
determined by standard method (A.O.A.C., 1999). The lipid content
was determined by the method of Folch et al. (1957). The hemicellulose, cellulose, lignin and lipid contents of EFB used in this study
were 18.2 0.9% (w/w), 60.1 0.3% (w/w), 14.3 1.0% (w/w) and
2.13 0.21% (w/w) or 21.3 2.1 mg/g dry substrate, respectively.
After pretreatment, EFB became softer and the cellulose content
of EFB was increased up to 76.9 0.8% (w/w), while the hemicellulose and lignin contents decreased to 3.3 0.1% (w/w) and
11.6 0.3% (w/w), respectively. The lipid content of pretreated EFB
was 1.11 0.49% (w/w) or 11.1 4.9 mg/g dry substrate. The hemicellulose, cellulose and lignin contents of PK used in this study
were 18.9 1.0%, 40.5 0.6% and 15.7 1.2% (w/w), respectively.

The lipid content of PK was 4.89 0.2% (w/w) or 48.9 2.0 mg/g
dry substrate.
2.2. Media preparation
The enrichment medium for isolation consisted of a mineral salts solution (MS solution), 20 g/L palm ber and 1.0 g/L
yeast extract. The MS solution contained (NH4 )2 SO4 1.7 g, KH2 PO4
2.0 g, MgSO4 7H2 O 0.5 g, CaCl2 2H2 O 0.2 g, FeSO4 7H2 O 0.01 g,
ZnSO4 7H2 O 0.01 g, MnSO4 4H2 O 0.001 g, CuSO4 5H2 O 0.0005 g,
0.1% Tween-80 (w/v) and 1000 mL of deionized water, with the pH
adjusted to 5.5. The carboxymethyl cellulose (CMC) agar medium
consisted of 20 g/L CMC and the MS solution with the pH adjusted to
5.5. The Potato dextrose agar (PDA) used as a pre-culture medium
contained 200 mL of potato soup (boiled potato 1 kg and water 1 L
for 15 min), dextrose 20 g/L and agar 20 g/L with the pH adjusted to
5.5.
2.3. Isolation and screening of oleaginous fungi
The oleaginous fungi were isolated from soils and wastes of palm
oil mill in southern region of Thailand. One gram of soils and wastes
sample was enriched in 5 mL of enrichment medium. Then, 0.1 mL
of a diluted culture was inoculated onto the carboxymethyl cellulose (CMC) agar medium containing 0.0001% chloramphenicol,
using the spread-plate technique and incubated for 5 days at room
temperature. Fungal strains were stained with the Sudan black B
technique (Patnayak and Sree, 2005) to screen for fungi with high
lipid content. Sudan Black B is a lysochrome (fat-soluble dye) diazo
dye used for staining lipid. The stained fungi were observed with a
phase contrast microscope using oil immersion to detect the presence of blue or grayish-colored lipid globules within the cells. The
strains with large lipid globules were selected and puried on PDA
medium.
The selected oleaginous fungi were identied based on their
18S rDNA sequences of their internal transcribed spacer regions
(ITS) as described by White et al. (1990). Briey, mycelia were harvested from liquid culture after 3 days of growth by ltration and
were transferred to sterile mortar and liquid nitrogen was added.
Mycelia were ground into a ne powder. After grinding with liquid nitrogen the genomic DNA was extracted by CTAB method. The
ITS region (including ITS1, 5.8SrDNA and ITS2) was amplied using
the universal primers ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) and
ITS5 (5 -GAAGTAAAAGTCGTAACAAGG-3 ). PCR amplication was
carried out under the following conditions: initial denaturation at
94 C (1 min); followed by 30 cycles of 94 C denaturation (1 min),
55 C annealing (70 s), and 72 C extension (1 min) and a nal
extension at 72 C for 10 min. The obtained sequences were BLAST
searched against the National Center for Biotechnology Information
database.
2.4. Solid state fermentation (SSF)
One gram of dried lignocellulosic materials was added to 50 mL
cotton plugged Erlenmeyer asks and supplemented with 1 mL of
MS solution. After sterilization, the media was cooled and the water
content was then adjusted to 65% by adding 0.85 mL of spore suspension containing 107 spores/gds (Ismaili-Alaoui et al., 2003). The
culture was incubated at 28 C for 5 days. For time course studies,
the whole ask replicates were collected as sample time points.
In the fed-batch solid state fermentation, the SSF was rst operated for 3 days, and 1.43 g of new substrate after water content
adjusted to 65% (this contained 0.5 g dry substrate) was added every
3 days. Repeated-batch solid state fermentation was carried out by
continuously repeating different cycles of batch fermentation. The
fermented biomass was replaced with new substrate every 3 days.

B. Cheirsilp, S. Kitcha / Industrial Crops and Products 66 (2015) 7380

The percent replacement was varied at 50%, 70% and 90% by harvesting 1.43 g, 1.99 g and 2.57 g of fermented biomass and adding
same amount of new substrate after water content adjusted to 65%,
which contained dry substrate of 0.5 g, 0.7 g and 0.9 g, respectively.
2.5. Determination of cellulase and xylanase activities
The qualitative cellulolytic activity of fungal strains was determined by their ability to grow and form clear zones around colonies
on the CMC agar medium. The plates were incubated at 28 C for
5 days. Then, 0.1% Congo Red was added to the plate surface and
incubated for 15 min. The dye was then removed and 1 M NaCl solution was added, followed by incubation for a further 10 min. The
clear zone around the colony was observed (Doolotkeldieva and
Bobusheva, 2011).
The quantitative analysis for xylanase and cellulase activities of
fungi strains was determined by their ability to hydrolyze birchwood xylan and CMC, respectively. The xylan and CMC were
prepared in 50 mM acetate buffer pH 5 with 0.1 mL of appropriately
diluted enzyme. The enzyme-substrate mixture was incubated at
50 C for 5 min. The released reducing sugars were determined
by the 3,5-dinitrosalicylic acid (DNS) method using xylose or glucose as standards for xylanase and cellulase activities, respectively
(Ncube et al., 2012). One unit of xylanase or cellulase is dened as
the amount of enzyme that liberates 1 mol of xylose or glucose
equivalents per minute. Filter paper unit (FPU) of fungal cellulases
was determined according to the method described by Santos et al.
(2012). One milliliter of a sodium citrate buffer solution with pH of
4.8 at 50 mM, 0.5 mL of enzyme solution and a lter paper (Whatman No. 1) strip were added to the tube containing the reaction
assay. The mixture was incubated at 50 C for 1 h. The released
reducing sugars were determined by the 3,5-dinitrosalicylic acid
(DNS) method. One FPU is dened as the amount of enzyme that
liberates 1 mol of glucose from lter paper per minute.
2.6. Determination of biomass and lipid yield and productivity
Fungal biomass in submerged fermentation was harvested by
ltration, washed twice with distilled water, and then was dried at
60 C to constant weight for 2 days. The dried fungal biomass in submerged fermentation was expressed as gram dry weight per liter.
The whole fermented lignocellulosic material and fungal biomass
in SSF were harvested and washed twice with distilled water using
vacuum ltration, and then was dried at 60 C to constant weight
for 2 days (Yao et al., 2012). The whole fermented lignocellulosic
material and fungal biomass was used for lipid extraction. The
lipid was extracted by the method of Folch et al. (1957). Briey,
the dry biomass was ground and added with 10 mL of chloroform:
methanol (2:1). The mixture was sonicated for 30 min and then
ltered to obtain the liquid phase containing lipid. This step was
repeated twice to ensure complete extraction, and all liquid phase
from the same sample were combined. The solvent was removed by
evaporation and the total lipid was measured gravimetrically. The
lipid content of the cells in submerged fermentation was expressed
percentage of gram lipid per gram dry cell weight (%). The lipid
yield in SSF was expressed as milligram lipid per gram dry substrate (mg/gds). The lipid in raw material was subtracted from the
total lipid extracted before calculation. The productivity of lipid and
enzyme per day were calculated by multiplying the production per
gram dry substrate with the productivity of the fermented biomass.
2.7. Determination of fatty acid composition
The method for fatty acid methyl ester (FAME) production
from the extracted lipid involved hydrolysis of the lipid followed
by esterication (Jham et al., 1982). FAME was analyzed using a

75

HP6850 Gas Chromatograph equipped with a cross-linked capillary

FFAP column (length 30 m, 0.32 mm I.D, 0.25 m lm thickness)
and a ame ionization detector. The operating conditions were as
follows: inlet temperature at 290 C, detector temperature at 300 C
and initial oven temperature at 210 C held for 12 min, then ramped
to 250 C at a rate of 20 C/min and held for 8 min. Fatty acids were
identied by comparison of their retention times with those of standard ones and calculated as a percentage based on their respective
peak areas using a standard mixture of FAME.

3. Results and discussion

3.1. Screening of oleaginous fungi with cellulolytic activity
Fifty one fungal strains were isolated from soils and wastes of
palm oil mill in southern region of Thailand. They were stained with
Sudan black B to detect the presence of blue or greyish lipid globules within the cells. There were 18 isolates showing lipid globules
within the cells but only 10 fungal isolates showed large lipid globules indicating high accumulation of lipid. These 10 fungal isolates
were then selected as potential lipid producers. The cellulolytic
activity of the fungal strains was determined according to their
ability to grow and form clear zones around fungal colonies in a
CMC agar medium. All selected fungal isolates formed a clear zone
around the colonies. To quantitatively determine the cellulolytic
activity, they were then inoculated into CMC broth medium.
The cell growth, lipid production, and cellulolytic activity of fungal isolates that were submerge-cultivated in CMC broth medium
are shown in Fig. 1. Among the strains tested, SRT5, GG6, TT2 gave
the highest biomass followed by PSU-WRS1, TSIP7 and TSIP12.
However, SRT5 produced the highest lipid by far at 265 mg/L
(Fig. 1a). The lipid productions by other strains were in the range
of 100164 mg/L. The strains that accumulated lipid higher than
20% of their dry weight were SRT5 (21.8%), TSP11 (22.9%), TSIP9
(20.5%) and GG4 (20.0%). For the cellulolytic activity (Fig. 1b),
TT2 produced the highest cellulase activity of 1.34 U/mL, followed by TSIP12 (0.96 U/mL), GG6 (0.96 U/mL), TSP11 (0.90 U/mL)
and TSIP7 (0.90 U/mL). The isolate TT2 also produced the highest xylanase activity of 1.91 U/mL, followed by TSIP9 (1.86 U/mL),
TSIP12 (1.77 U/mL) and TSIP11 (1.67 U/mL). TCH1 possessed the
lowest cellulolytic activity with corresponding to its lowest growth
ability on CMC medium. As a consequence, four fungal strains:
SRT5, TSIP9, TSIP11, and TT2, were selected based on their high
lipid accumulation with high cellulolytic activity for further studies. Fig. 2 shows lipid globules detected within the cells of those
selected strains.
The fatty acid compositions of the lipids produced by these fungi
are shown in Table 1. The fungal lipids mainly consisted of longchain fatty acids of 16 and 18 carbon atoms, and the four major
fatty acids were oleic acid (C18:1), palmitic acid (C16:0), linoleic
acid (C18:2) and stearic acid (C18:0). Oleic acid was the most predominant fatty acid found in all strains: SRT5 (41.5%), TSIP9 (35.8%),
TSIP11 (32.8%) and TT2 (32.8%) followed by palmitic acid in the
case of SRT5 (30.3%), TSIP9 (21.5%) and TSIP11 (24.9%) but followed
by linoleic acid in the case of TT2 (27.6%). Lauric acid (C12:0) and
myristic acid (C14:0) were found in TSIP9 and TSIP11 at 4.36.2%.
It was of interest that, only the TT2 lipid was rich in linolenic acid
(1.9%). In addition, arachidic acid (C20:0), which is a saturated fatty
acid found in sh oil and peanut oil (1.11.7%), was also found in
TSIP9 and TSIP11.
The fatty acids found in this study were similar to those found in
Mortierella isabellina that were 4954% oleic acid, 2435% palmitic
acid, 211% linoleic acid, 0.42% linolenic acid, 12% palmitoleic
acid and 3.58.0% stearic acid (Economou et al., 2010) and those
found in Microsphaeropsis sp. that were 46.1% oleic acid, 27.3%

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B. Cheirsilp, S. Kitcha / Industrial Crops and Products 66 (2015) 7380

1.4

(a)
a

Biomass
a

1.2

Lipid production

250

Biomass (g/L)

bcd

200

bc
c bcd

c bc

150

de

cde

de

0.6

100

0.4
50

0.2

0.0

Cellulase (U/mL) and xylanase activity

(U/mL)

SRT5

2.0

Lipid productionm (mg/L)

b
1.0
0.8

300

GG6

TT2

PSU-WRS1

TSIP7

TSIP12

GG4

(b)

TSP11

Cellulase

TCH1

Xylanase

ab

ab

1.6

TSIP9

ab

a
c

c
b

1.2

bcd

bcd

bc
d

de

0.8

cde

0.4

e
g

0.0

SRT5

GG6

TT2

PSU-WRS1

TSIP7

TSIP12

GG4

TSIP9

TSP11

TCH1

Strains
Fig. 1. Biomass and lipid production (a), cellulase and xylanase activity (b) of ten isolated fungi submerge-cultivated in CMC based medium for 5 days. Different lowercase
letters on the bars indicate signicant differences between strains (P < 0.05).

Fig. 2. Phase contrast microscope photographs of lipid globules in the isolated strain SRT5 (a), TSIP9 (b), TSI11 (c) and TT2 (d). The isolated fungi were cultivated using CMC
as a carbon source, stained, and observed under a phase contrast microscope and 1000 oil immersion lens.
Table 1
Percentage of fatty acid composition of fungal lipids.
Lipid source

C12:0

C14:0

C16:0

C16:1

C18:0

C18:1

C18:2

C18:3

C20:0

SFA

USFA

SRT5
TSIP9
TSIP11
TT2

0.0
5.4
4.3
0.0

0.8
6.1
6.2
1.0

30.3
21.5
24.9
26.3

1.0
0.2
0.3
2.3

9.9
9.6
13.0
8.1

41.5
35.8
32.8
32.8

16.5
18.8
14.0
27.6

0.0
0.5
0.2
1.9

0.0
0.5
0.5
0.0

41.0
42.7
48.4
35.4

59.0
55.3
47.4
64.6

Fatty acids including lauric (C12:0), myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3) and arachidic acid
(C20:0).
SFA: Saturated fatty acid; USFA: Unsaturated fatty acid.

palmitic acid, 20.0% linoleic acid, 3.5% palmitoleic acid and 3.0%
stearic acid (Peng and Chen, 2008). Whereas, the lipid of Aspergillus

oryzae A-4 was mainly composed of palmitic acid at 32.95% followed by linoleic acid at 27.74% and oleic acid at 22.64% (Lin et al.,

B. Cheirsilp, S. Kitcha / Industrial Crops and Products 66 (2015) 7380

3.2. Lipid yield and enzyme production in solid state

fermentation of palm empty fruit bunch

3.3. Lipid yield and enzyme production in solid state

fermentation of EFB added with palm kernel cake

3.4. Effect of crude enzymes addition

As the direct conversion of lignocellulosic, biomass rely on the
cellulolytic activity of the fungi to break down cellulose and hemicelluloses into available sugar monomers for uptake, the addition
of the enzymes at initial would facilitate the fungi growth and lipid
storage at the early stage. Therefore, the crude enzymes eluted
from the rst SSF were concentrated and added into the next SSF.
Fig. 4 shows the SSF of EFB mixed with PK at 50% ratio without and
with addition of crude enzymes. With addition of crude enzymes,
the lipid and enzymes were produced rapidly. The xylanase was
produced with the productivity of 58.6 1.5 U/gds/day (Fig. 4b)
which was about 3 times higher than that without addition of crude
enzymes (14.9 3.6 U/gds/day, Fig. 4a). Consequently, the lipid productivity was also as high as 15.5 0.7 mg/gds/day which twice
that without addition of crude enzymes (9.37 0.86 mg/gds/day).
Whereas, the cellulase productivity was only slightly higher in
the SSF with addition of crude enzymes. The time course results

100

25

a a

80

20

a
b

60

15

c b
40

10

d
c

20

Cellulase and xylanase (U/gds)

SSFs of EFB added with MS solution by four selected fungi

were performed at 28 C for 5 days. Among them, TSIP9 produced the highest lipid yield of 39.5 2.2 mg/gds, followed
by SRT5 (29.4 1.3 mg/gds), TSIP11 (28.9 0.1 mg/gds) and TT2
(21.1 1.7 mg/gds). One possible explanation for high lipid yield
would be because TSIP9 produced the highest cellulolytic activities
(11.83 0.18 U/gds for xylanase and 2.35 0.22 U/gds for cellulase). These results show that the lipid producing ability of the fungi
did correlate to their cellulolytic activity. Therefore, the amount of
the enzymes produced by the fungi would be an important indicator for possible use in bioconversion of lignocellulosic wastes into
lipid. The lipid yield of the selected fungi in this study was comparable to that of A. oryzae A-4 cultivated on wheat straw and bran
mixture (36.6 mg lipid/gds) (Lin et al., 2010). Since TSIP9 could efciently converted EFB into lipid and also produced high activity of
cellulase and xylanase, it was then chosen and identied using 18S
rRNA gene sequence and NCBI blast search. The results showed
a 99% sequence identity of the TSIP9 with the genus Aspergillus
tubingensis (accession no. AB981202).

and enzyme production by A. tubingensis TSIP9 (Fig. 3). The ratio of

PK was varied from 0 to 100%. The enzyme production increased
with increasing the PK ratio up to 100% (the use of PK as a sole
substrate). The results also showed that the PK was the crucial factors for the enzyme production. Only small amount of the enzymes
was produced when using only EFB (PK 0%). This could be because
PK contained sufcient nutrients such as 0.53 0.05% nitrogen,
0.99 0.01% phosphorus and 0.52 0.01% potassium that are usually required for cell growth and enzyme production. In addition, as
PK also serves as a carbon source it was possible that the increased
enzyme production was also due to the more easily digestible structure of the PK compared to the EFB.
The lipid yield increased with increasing the PK ratio up to 50%.
The maximum lipid yield of 79.9 3.5 mg/gds was obtained at this
PK ratio. Although the use of only PK (100%) produced the highest enzyme activity, it did not give the highest amount of lipid. It
was also possible that at the beginning of the SSF the lipid yield
greatly depended on the excretion of the cellulolytic enzymes, but
when the enzymes reached its minimum requirement the lipid
yield might be limited by other factors such as the amount of available carbon source and the ratio of carbon to nitrogen. Usually,
the condition for lipid production requires an excess carbon source
with a limiting nitrogen source, while that for enzyme production
is rich in nitrogen source. It should be also noted that some oleaginous microorganisms can use oil as substrate and accumulate it in
an unchanged or modied form (Fickers et al., 2005). Therefore, the
increased lipid yield with the addition of PK would also come from
the possibility that the fungi might use the oil in PK as substrate for
their growth and lipid accumulation.

Lipid yield (mg/gds)

2010). Fakas et al. (2009) have reported that the production of

polyunsaturated fatty acid was related to the age of the mycelia.
Their fungal strains produced linoleic acid and linolenic acid as the
key fatty acids during the initial stages of growth, but when the
lipid accumulation exceeded 20%, oleic and palmitic acids were
predominant. The high content of unsaturated acids found in all
fungal lipids in this study would provide excellent fuel properties
at low temperatures that would be an advantage for operations in
winter.
Three important chemical properties of biodiesel attributed to
the fatty acid proles are iodine value (IV), saponication value (SV)
and high heating value (HHV) of the fungal lipids were predicted
following the theoretical calculation taking into consideration of
ve fatty acids, namely, C16:0, C18:0, C18:1, C18:2 and C18:3
(Gopinath et al., 2009). The predicted IVs (g I2 /100 g oil) of fungal
lipids from SRT5 (74.9), TSIP9 (77.7), TSIP11 (71.5) and TT2 (87.9)
are below the European biodiesel standards (<130) (). The predicted
SVs (200.9, 206.8, 200.8 and 206.9 for SRT5, TSIP9, TSIP11 and TT2,
respectively) are within the range of SVs of plant oils commonly
used for biodiesel production (180210) (Gopinath et al., 2009). The
predicted HHVs (40.1, 39.8, 39.9 and 39.9 for SRT5, TSIP9, TSIP11
and TT2, respectively) are also close to those of plant oils (3941)
(Gopinath et al., 2009). In addition, it is also known that the fatty
acid proles impact on the cetane number (CN) of the biodiesel.
Considering the percentage distribution of fatty acids shown in
Table 1 and the empirical equation (Krisnangkura, 1986), the lipids
from SRT5, TSIP9, TSIP11 and TT2 could produce biodiesel with the
CN values in the range of 5761, 5660, 5761 and 5459, respectively. These values are higher than the minimal requirement for
CN values in the biodiesel standards ASTMD 6751 (USA), DIN 51606
(Germany) and EN 14214 (European Organization), those have been
set at 47, 49 and 51, respectively. The similarity of the fatty acids of
fungal lipids to those of plant oils and the important chemical properties that meet the biodiesel standards indicate their potential use
as biodiesel feedstocks.

77

0
PK 0% PK 20% PK 50% PK100%

In this experiment, the palm kernel cake (PK) that contained

a considerable amount of nitrogen (0.53 0.05%) was used as a
sole substrate and a co-substrate to EFB without the addition of
ammonium sulfate. The PK was mixed with EFB and used for lipid

Fig. 3. Effect of palm kernel cake ratio on lipid yield (white bar), cellulase (gray bar)
and xylanase (black bar) production by selected fungus A. tubingensis TSIP9. The
fungi were incubated at room temperature (28 2 C) for 5 days. Different lowercase
letters on the bars indicate signicant differences between treatments (P < 0.05).

78

B. Cheirsilp, S. Kitcha / Industrial Crops and Products 66 (2015) 7380

(b) With addition of crude enzymes

120

60

90

40

60

20

30

0
2

3 4 5
Time (days)

Lipid yield

Cellulase

150

80

120

60

90

40

60

20

30

Lipid yield (mg/gds)

80

100

0
0

Xylanase

Cellulase and xylanase (U/gds)

Cellulase and xylanase (U/gds)

Lipid yield (mg/gds)

(a) Without addition of crude enzymes

150
100

3 4 5
Time (days)

Lipid yield

Cellulase

7
Xylanase

Fig. 4. Effect of addition of crude enzymes on the solid state fermentation of EFB mixed with PK at 50% ratio.

Table 2
Fed-batch solid state fermentation by oleaginous fungus A. tubingensis TSIP9.
Day

PK ratio

Production per gram dry substrate

Lipid yield (mg/gds)

03
46
79
1012

100%
50%
33%
25%

59.0
86.6
80.1
71.9

2.6
3.1
2.7
3.7

Productivity (per day)

a

Cellulase (U/gds)
11.5
22.1
24.5
19.0

1.1
0.8
0.5
1.0

Lipid yield
(mg/day)

1.3
1.7
1.6
0.6

20.3
43.7
11.9
6.79

Xylanase (U/gds)
114.0
78.9
71.7
65.6

Cellulase (U/day)

3.1
2.6
2.1
2.0

0.2
16.3
15.7
9.53

0.1
1.1
0.1
0.7

Xylanase (U/day)
38.0
21.9
43.9
28.3

4.6
1.3
1.2
1.2

showed that the xylanase activity decreased after reached its maximum level at day 2 and the lipid yield also decreased after reached
its maximum level at day 3. It was possible that the decrease in the
xylanase activity might cause the lower available sugar monomers
for the fungi uptake and lead to the lipid degradation. Similarly,
Lin et al. (2010) found that the lipid degradation did occur in the
fermentation of A. oryzae A-4 in accordance with the decrease in its
own cellulase activity. They also proved that the addition of exogenous cellulase could prevent the lipid degradation by the fungi.
3.5. Fed-batch solid state fermentation

150

200

+EFB

+EFB

120

160

90

120

60

80

30

40

0
0

To increase the lipid productivity, the fed-batch solid state fermentation in which the new substrate is intermittently added, was
attempted. Since the results in Fig. 3 showed that the use of only
PK produced the highest enzyme activity, the SSF using PK 100%
was rst operated for 3 days to produce sufcient amount of the
enzymes and 0.5 g of EFB was added every 3 days to increase the
availability of the carbon source for lipid production. The proles
of lipid yield and enzyme production are shown in Fig. 5 . The production per gram substrate and productivity are summarized in
Table 2. The xylanase was rapidly produced up to 114.0 1.3 U/gds
at day 3 with a relatively low lipid yield of 59.0 2.6 mg/gds. The

+EFB

+PK

Lipid yield

6
8
10
Time (days)
Total lipid
Cellulase

Cellulase and xylanase (U/gds)

Lipid yield (mg/gds), Total lipid (mg)

The fermentation started from 0.5 g PK and 0.5 g EFB was added every 3 days.
a
The maximum enzyme production at day 1 after EFB addition.

12
Xylanase

Fig. 5. The performance of fed-batch solid state fermentation by oleaginous fungus

A. tubingensis TSIP9. The fermentation started from 100% PK and EFB was added at
day 3, 6 and 9.

1st addition of EFB increased the lipid yield up to 86.6 3.1 mg/gds.
The lipid productivity also increased from 20.3 3.1 mg/day to
43.7 2.6 mg/day (Table 2). This could be due to the more available
carbon source for lipid accumulation.

Table 3
Repeated-batch solid state fermentation by oleaginous fungus A. tubingensis TSIP9.
Replacement (%)

Productivity of
fermented biomass
(gds/d)

Production per gram dry substrate

Lipid yield
(mg/gds)
50
70
90

0.17
0.23
0.30

91.9 2.2
91.6 2.8
86.7 2.6

Cellulase
(U/gds)
18.4 0.2
18.0 1.8
17.5 0.2

Productivity (per day)

Xylanase
(U/gds)

Lipid yield
(mg/day)

Cellulase
(U/day)

Xylanase
(U/day)

119.0 1.5
115.1 2.4
107.6 1.3

15.3 1.1
21.4 1.5
26.0 1.1

3.06 0.11
4.21 0.12
5.24 0.25

19.8 0.4
26.9 1.0
32.3 1.8

The fermentation started from EFB mixed with PK at 50% ratio and 50%, 70% and 90% of the fermented biomass were replaced with new substrate every 3 days.

B. Cheirsilp, S. Kitcha / Industrial Crops and Products 66 (2015) 7380

79

Table 4
Lipid and cellulase production through solid-state fermentation of lignocellulosic biomass by oleaginous fungi.
Lignocellulosic biomass

Strain

Mode

Lipid yield

Cellulase

References

Wheat straw and bran

Wheat straw and bran
Wheat straw and bran
Rice straw and wheat bran
Rice straw and wheat bran
Rice straw and wheat bran
Rice straw and wheat bran
Rice straw
Palm empty fruit bunch and
palm kernel cake
Palm empty fruit bunch and
palm kernel cake
Palm empty fruit bunch and
palm kernel cake

Microsphaeropsis sp.
Microsphaeropsis sp.
A. oryzae A-4
Colletotrichum sp.
Colletotrichum sp.
Alternaria sp.
Alternaria sp.
M. elongate PFY
A. tubingensis TSIP9

Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch

1942 (mg/gds)
74 (mg/gds)
62.9 (mg/gds)
68.2 (mg/gds)
84.3 (mg/gds)
60.3 (mg/gds)
81.7 (mg/gds)
70.7 (mg/gds)
79.9 (mg/gds)

Peng and Chen (2007)

Peng and Chen (2008)
Lin et al. (2010)
Dey et al. (2011)
Dey et al. (2011)
Dey et al. (2011)
Dey et al. (2011)
Yao et al. (2012)
This study

A. tubingensis TSIP9

Fed-batch
after 1st addition
Repeated-batch with 50%
replacement

86.6 (mg/gds)

0.310.69 (FPU/gds)
+Exogenous cellulasea
1.69 (FPU/gds)
1.84 (FPU/gds)
+Crude cellulaseb
1.21(FPU/gds)
+Crude cellulaseb
-c
11.1 (U/gds)
or 0.97 (FPU/gds)
22.1 (U/gds)
or 2.25 (FPU/gds)
18.4 (U/gds)
or 1.81 (FPU/gds)

This study

Exogenous cellulase was added at 10 FPU/gds.

The substrate was treated with crude cellulase extract before use.
Not available.

2.5

100

Lipid yield (mg/gds)

Recovery weight (g)

120

2.0
1.5
1.0

Total
weight
(a) 50%
Replacement
PK33%
PK50%

200

PK25%

160
PK100%

80

120
60
80
40
40

20

0.5

0
20

0.0
0

120

2.5

100

Lipid yield (mg/gds)

3.0

2.0
1.5
1.0
0.5

0
42

64
86
108
Time (days)
Time (days)

TotalReplacement
weight
(b) 70%
PK33%
PK50%

1210

12
200

PK25%

160
PK100%

80

120
60
80
40
40

20

0.0

120

2.5

100

Lipid yield (mg/gds)

3.0

2.0
1.5
1.0
0.5

0
20

42

64
86
108
Time (days)
Time (days)

Total
weight
PK33%
(c) 90%
Replacement
PK50%

1210

12
200

PK25%

160
PK100%

80

120
60
80
40
40

20

0.0

0
0

0
20

Lipid yield

42

64
86
108
Time (days)
Time (days)
Recovery weight

1210
Cellulase

Cellulase and xylanase (U/gds)

Since the optimal PK ratio for lipid yield in the SSF was 50%
(Fig. 3), the repeated-batch SSF of EFB mixed with PK at 50%
ratio was attempted. The percent replacement of the fermented
biomass with new medium every 3 days was 50%, 70% and 90%.
The proles of lipid yield and enzyme production are shown in
Fig. 6. Table 3 shows the kinetic performance of the repeatedbatch solid state fermentation. It was of interest that the enzymes
and lipid were effectively produced during 4 times repeated-batch.
This was likely because the suitable carbon to nitrogen ratio could
be maintained, and this hence encouraged both enzymes and
lipid production. The maximum enzymes and lipid production per
gram dry substrate were slightly decreased with increasing percent
replacement up to 90% (Fig. 6c). However, it should be noted that
higher percent replacement gives higher recovery weight of the fermented biomass (Fig. 6) and hence higher productivity (Table 3).
The productivity of lipid and enzyme per day were calculated by
multiplying the production per gram dry substrate with the productivity of the fermented biomass. From these calculations, it was
found that the productivity of the enzymes and lipid increased
with increasing the percent replacement. With this repeated-batch
strategy, it was possible to effectively and continuously produce
enzymes and lipid by solid state fermentation without the timeconsuming inoculums preparation for each batch.
Table 4 compares lipid and cellulase production through solidstate fermentation of lignocellulosic biomass by oleaginous fungi.
Peng and Chen (2007) rst attempted to directly produce lipid from

3.0

Cellulase and xylanase (U/gds)

3.6. Repeated-batch solid state fermentation

wheat straw and bran mixture by Microsphaeropsis sp. But due to

the low cellulase activity (0.310.69 FPU/gds) of their strain, the

Cellulase and xylanase (U/gds)

When EFB was added, the enzyme activity was rst reduced
due to the increase in the total volume of the substrate before
increased up to the maximum level at day 4 but decreased thereafter. The maximum level of xylanase activity also decreased with
increasing numbers of EFB addition. This could be due to the limited nitrogen source since the intermittent adding of EFB at day
3, 6 and 9 reduced the PK ratio from 100% to 50%, 33% and 25%,
respectively. After the 2nd and 3rd addition of EFB, the lipid yield
decreased to 80.1 2.7 mg/gds and 71.9 3.7 mg/gds, respectively.
The lipid productivity was also as low at 11.9 2.1 mg/day and
6.79 2.0 mg/day (Table 2). Although high carbon to nitrogen ratio
is suitable for lipid accumulation, the enzyme activity is crucial factor for bioconversion of lignocellulosic biomass into lipid. It was
likely that the decrease in the enzyme activity lower than the minimum requirement could not supply sufcient amount of sugars
for uptake and this decreased the lipid yield in the 2nd and 3rd EFB
addition. However, with this fed-batch strategy the total lipid was
about 10 folds increased from the initial (Fig. 5).

Recovery weight (g)

91.9 (mg/gds)

Recovery weight (g)

a
b

A. tubingensis TSIP9

This study

12
Xylanase

Fig. 6. The performance of repeated solid state fermentation by oleaginous fungus A.

tubingensis TSIP9. The fermentation started from EFB mixed with PK at 50% ratio and
50%, 70% and 90% of the fermented biomass were replaced with the new medium
every 3 days.

80

B. Cheirsilp, S. Kitcha / Industrial Crops and Products 66 (2015) 7380

lipid yield was only 1942 mg/gds. When exogenous cellulase was
added at 10 FPU/gds, the lipid yield increased up to 74 mg/gds (Peng
and Chen, 2008). Lin et al. (2010) found that A. oryzae A-4 could produce high amount of lipid yield (62.9 mg/gds) by its own cellulase
(1.69 FPU/gds). Dey et al. (2011) compared lipid production by two
endophytes Colletotrichum sp. and Alternaria sp. They were able to
produce high cellulase activity of 1.84 and 1.21 FPU/gds and lipid
yield of 68.2 and 60.3 mg/gds, respectively. Their lipid yields were
increased up to 84.3 and 81.7 mg/gds, respectively, when the substrate was treated with their crude cellulase extract before use. M.
elongate PFY was another strain that has been reported for its lipid
yield of 70.7 mg/gds from rice straw but with no available information of its enzyme activity (Yao et al., 2012). In this study, A.
tubingensis TSIP9 could produce comparable lipid and cellulase to
those previously reported in the same batch mode. It should be
noted that this is the rst time reporting that the lipid yield and
enzyme activity could be signicantly improved by fed-batch and
repeated-batch modes.
4. Conclusions
Oleaginous fungus with high cellulolytic enzymes was screened
and identied as A. tubingensis TSIP9. The fungi exhibited satisfactory enzymes and lipid production in a solid state fermentation of
palm empty fruit bunch mixed with palm kernel cake. The reuse of
crude enzymes, fed-batch, and repeated-batch solid state fermentation did enhance the enzyme and lipid production by the selected
fungi. The repeated solid state fermentation with 90% replacement
with new medium was the most suitable process to continuously
produce enzyme and lipid with the highest productivity. This new
strategy for solid state fermentation would be a useful tool for
direct conversion of lignocellulosic materials into valuable products including enzymes and microbial lipid. This would also greatly
contribute toward the economics of biofuel production from lignocellulosic biomass.
Acknowledgments
This work was nancial supported by Prince of Songkla University and Thai Governmentunder Grant No. AGR570076S. The second
author was supported by the Postdoctoral Fellowship from Prince
of Songkla University and the Higher Education Research Promotion and National Research University Project of Thailand, Ofce of
the Higher Education Commission. Also thanks to Dr. Brian Hodgson Faculty of Pharmaceutical Science, PSU for assistance with the
English.
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