Appl Biochem Biotechnol (2014) 172:21972204

DOI 10.1007/s12010-013-0651-y

Bioconversion of Corncob Acid Hydrolysate into Microbial

Oil by the Oleaginous Yeast Lipomyces starkeyi
Chao Huang & Xue-Fang Chen & Xiao-Yan Yang &
Lian Xiong & Xiao-Qing Lin & Juan Yang & Bo Wang &
Xin-De Chen

Received: 30 July 2013 / Accepted: 15 November 2013 /

Published online: 17 December 2013
# Springer Science+Business Media New York 2013

Abstract For the first time, corncob acid hydrolysate was used for microbial oil production by
the oleaginous yeast Lipomyces starkeyi. After hydrolysis by dilute sulfuric acid, corncob
could turn into an acid hydrolysate with a sugar concentration of about 42.3 g/L. Detoxified by
overliming and absorption with activated carbon, the corncob hydrolysate could be used by L.
starkeyi efficiently that a total biomass of 17.2 g/L with a lipid content of 47.0 % (corresponding to a lipid yield of 8.1 g/L) and a lipid coefficient of 20.9 could be obtained after
cultivation on the corncob hydrolysate for 8 days. Therefore, L. starkeyi is a promising strain
for microbial oil production from lignocellulosic biomass. Glucose and xylose were used by L.
starkeyi simultaneously during lipid fermentation while arabinose could not be utilized by it.
Besides, the lipid composition of L. starkeyi was similar to that of vegetable oils; thus, it is a
promising feedstock for biodiesel production.
Keywords Lipomyces starkeyi . Microbial oil . Corncob acid hydrolysate . Biodiesel

Introduction
Recently, microbial oil is considered as one attractive lipid feedstock for biodiesel production
due to its advantages such as a short life cycle; less required labor; little limited by location,

Chao Huang and Xue-Fang Chen have the same contribution and are co-first authors.

C. Huang : L. Xiong : X.<Q. Lin : J. Yang : X.<D. Chen

Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou 510640, Peoples
Republic of China
C. Huang : X.<F. Chen : X.<Y. Yang : L. Xiong : X.<Q. Lin : J. Yang : B. Wang : X.<D. Chen (*)
Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, No.2 Nengyuan Road, Tianhe
District, Guangzhou 510640, Peoples Republic of China
e-mail: cxd_cxd@hotmail.com
X.<F. Chen : X.<Y. Yang : B. Wang
University of Chinese Academy of Sciences, Beijing 100049, Peoples Republic of China

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Appl Biochem Biotechnol (2014) 172:21972204

season, and climate; and easier to scale up [1]. To make this bioconversion economically
feasible, various low-cost substrates were used for microbial oil production [24] and lignocellulosic biomass is regarded as a potential feedstock for its many advantages such as great
availability in nature and renewable feature [5].
Various oleaginous microorganisms have been used for microbial oil production [68].
Among them, the oleaginous yeast Lipomyces starkeyi is considered as attractive for microbial
oil production from various low-cost substrates such as wastewaters [9], sewage sludge [4], etc.
The potential of L. starkeyi to accumulate lipid on lignocellulosic hydrolysates was raised since
this yeast could utilize both glucose and xylose for microbial oil production [6]. However, to
our knowledge, no work has used real lignocellulosic hydrolysate for microbial oil production
by L. starkeyi. Compared with a synthetic medium, the composition of lignocellulosic hydrolysates was much more complex, and many by-products such as organic acid or aldehyde would
influence the lipid fermentation of oleaginous yeast [10, 11]. Thus, it is necessary to carry out
lipid fermentation by L. starkeyi on some actual lignocellulosic hydrolysates.
Among various lignocellulosic hydrolysates, corncob acid hydrolysate was shown as a
promising substrate due to its high sugar concentration especially xylose concentration for
fermentation [12]. Therefore, in this work, for the first time, corncob acid hydrolysate was used
for microbial oil production by L. starkeyi in order to evaluate the possibility of this bioconversion process.

Methods and Materials

Corncob Acid Hydrolysate
Detoxified corncob acid hydrolysate was obtained from Zhongke New Energy Co., Ltd.
(Ying-Kou, China). According to Zhongke New Energy Co., Ltd., dilute sulfuric acid was
used for hydrolysis and the resulted hydrolysate was detoxified by overliming and absorption
with activated carbon according to the previous studies [13, 14]. The sugar composition
(grams per liter) of this hydrolysate was as follows: glucose 2.0, xylose 36.3, and arabinose
4.0. Without adding any nutrient (nitrogen source and trace element), this substrate was used
for microbial oil production by L. starkeyi.
Microorganism and Microbial Oil Production
The oleaginous yeast L. starkeyi CH010 (Laboratory of Energy and Biochemical Engineering,
Guangzhou Institute of Energy Conversion) was used for microbial oil production.
The yeast was cultured first on a pre-cultivation medium (initial pH 6.0) containing (grams per
liter) xylose 20, peptone 10, and yeast extract 10, at 28 C and 150 rpm for 36 h. Then, 8 % (v/v) seed
culture was inoculated into the corncob hydrolysate (initial pH 6.5). Cultivation was performed in
250-mL conical flasks containing 50 mL of hydrolysate in a rotary shaker at 28 C and 150 rpm. To
evaluate the effect of nitrogen source concentration (C/N ratio) on the lipid production by L. starkeyi,
different concentrations (1, 2, 3, and 4 g/L) of yeast extract were added into the corncob acid
hydrolysate, and the lipid fermentation was carried out with the same condition.
Analytical Methods
Biomass was harvested by centrifugation and determined by its dry cell weight after drying at
105 C for 24 h. Extraction of lipid from dry biomass was performed according to the modified

Appl Biochem Biotechnol (2014) 172:21972204

2199

procedure of Folch et al. [15], with a mixture of chloroform/methanol (2:1, v/v). The extracted
lipid was centrifuged to obtain a clear supernatant, and the solvent was removed by vacuum
evaporation. Lipid yield is expressed as the amount of lipid extracted from the cells in per liter
fermentation broth (grams per liter), and lipid content is defined as the percentage of lipid to
dry biomass (percent, w/w). Besides, lipid coefficient (percent, w/w) is referred as the amount
of lipid produced in each 100 g total sugar in the substrate consumed.
The fatty acid profile was determined by measuring the corresponding fatty acid methyl
ester composition using GC according to the method used in our previous work [12]. Sugar
concentrations (D-glucose, D-xylose, and L-arabinose) in the corncob hydrolysate were analyzed by HPLC according to the procedure described in our previous work [16]. Furfural and
5-hydroxymethylfurfural (HMF) were measured by HPLC, and acetic acid was detected by
GC according to the method described in our previous study [12]. Total nitrogen was evaluated
by Hach DR2700 Water Quality Analyzer (Hach Company, USA).

Results and Discussion

Growth and Lipid Accumulation of L. starkeyi
The sugar composition of corncob acid hydrolysate was analyzed by HPLC, and the dominant
sugar present in this hydrolysate was xylose, suggesting that hemicellulose was the main
component hydrolyzed by dilute sulfuric acid. Overall, the corncob acid hydrolysate was
detoxified by overliming and absorption before lipid fermentation [13, 14]. For overliming, the
pH of the hydrolysate was increased to 10.0 by adding Ca(OH)2. After 1 h at pH 10.0, the
hydrolysate was filtrated to remove the sediment. Then, the resulted hydrolysate was acidified
to pH 5.5 and filtrated again to remove the sediment after 1 h of sedimentation. As for
adsorption, activated charcoal was washed with water and equilibrated with 0.4 M HCl and
then mixed with the corncob hydrolysate (1:5, w/v). The mixture was incubated at 50 C and
200 rpm for 1 h. Finally, the activated charcoal was taken out by filtration, and the hydrolysate
pH was then adjusted to a fermentation pH value. After detoxification, there were still some
inhibitors such as furfural, HMF, and acetic acid presenting in the hydrolysate; their concentration was 0.06, 0.30, and 0.04 g/L, respectively [12]. Without adding any nutrient (nitrogen
source and trace element), this hydrolysate was used as a substrate for microbial oil production
by L. starkeyi.
The time courses of growth and lipid accumulation of L. starkeyi were shown in Fig. 1. After
the first day of fermentation, the biomass of L. starkeyi was merely about 0.9 g/L. It seems that
an obvious lag phase existed during the beginning of fermentation, which was different to our
previous work where no clear lag phase was observed [17]. It is possible that the inhibitor
presenting in the corncob hydrolysate showed a negative effect to the growth of L. starkeyi, and
thus, the lag phase existed during its fermentation. Also, the fermentation pH value decreased a
little after fermentation for 1 day, possibly due to the secretion of acidic materials. Since the
biomass of L. starkeyi was extremely low, its lipid content was hard to detect after 1 day of
fermentation. After that, the biomass of L. starkeyi began to increase and the lipid content
became detectable from the second day of fermentation. Initially, the lipid content (10.8 %) of L.
starkeyi was low after 2 days of fermentation. However, its lipid content increased from then on,
indicating the carbon source has fluxed into the lipid accumulation process.
After 5 days of fermentation, the growth of L. starkeyi became much slower possibly due to
the exhaustion of assimilate sugars. However, it continued to accumulate lipid until the eighth
day of fermentation, suggesting that its lipid accumulation might be later than its growth.

20

50

16

40

12

30

20

10

0
1

9 10 11

Lipid content (%)

Appl Biochem Biotechnol (2014) 172:21972204

Biomass (g/L) / lipid yield (g/L)

2200

Fermentation time (d)

Fig. 1 Cell growth and lipid accumulation of L. starkeyi on the corncob acid hydrolysate medium. Biomass
(white triangle), lipid content (white circle), and lipid yield (white square)

Overall, the highest biomass (17.1 g/L) and lipid content (47.0 %) of L. starkeyi were achieved
at the eighth day. After that, its lipid content decreased quicker than its biomass, indicating that
it might use the cellular lipid to maintain its growth. This phenomenon, the so-called lipid
turnover was also observed by other researchers [2, 5, 16]. It is worth noting that the pH value
increased to about 8.0 from the first day to the second day of fermentation. After that, the pH
value maintained at near 8.0. Again, lignocellulosic hydrolysate (corncob acid hydrolysate)
shows a buffer capacity similar to that from previous works [17].
It is normal that the lipid composition would change during lipid fermentation [15]. In this
work, the lipid composition of L. starkeyi was detected throughout the fermentation (Table 1).
As shown in Table 1, as for unsaturated fatty acids, oleic acid (C18:1), palmitoleic acid
(C16:1), and linoleic acid (18:2) were three main ones. During the early phase of fermentation
(days 1 and 2), the ratio of oleic acid was low (about 40 %). However, this ratio increased as
the fermentation went on. This phenomenon was in contrast to our previous work with
oleaginous Trichosporon dermatis whose ratio of oleic acid was higher during the beginning

Table 1 Lipid composition of L.

starkeyi during lipid fermentation
process on the corncob acid
hydrolysate

Others were C8:0, C10:0, C12:0,

C14:0, C14:1; C18:3, C20:0,
C22:0, C22:1, C24:0, and C24:1

Fermentation time Lipid composition of L. starkeyi (%)

(days)
C16:0 C16:1 C18:0 C18:1 C18:2 Othersa
2

38.4

3.5

5.3

40.4

6.9

5.4

39.8

4.2

6.3

42.5

3.9

3.3

37.8

4.4

5.9

46.9

2.6

2.4

38.5

4.8

5.5

46.7

2.1

2.4

37.6

5.1

5.3

47.4

2.4

2.2

7
8

38.4
37.7

5.0
4.8

5.2
5.1

47.2
48.1

2.0
2.3

2.2
2.0

37.3

5.0

5.0

48.4

2.3

2.0

10

38.5

5.0

4.6

47.7

2.3

1.8

11

37.8

5.0

4.2

48.3

2.7

2.0

Appl Biochem Biotechnol (2014) 172:21972204

2201

of fermentation [17]. As for saturated fatty acids, palmitic acid (C16:0) and stearic acid (18:0)
were two main ones, and their ratios were relatively stable. Overall, the lipid composition of L.
starkeyi is similar to that of vegetable oils; thus, it is a promising feedstock for biodiesel
production.
Sugar and Nitrogen Metabolisms of L. starkeyi
To elucidate the sugar metabolism of L. starkeyi, the time courses of sugar utilization by L.
starkeyi on the corncob acid hydrolysate were measured (Fig. 2). As shown in Fig. 2, merely
small amount of sugars were consumed by L. starkeyi during the first day of fermentation. This
further proved the existence of the lag phase during the lipid fermentation of L. starkeyi on the
corncob acid hydrolysate. After the adaptation to the hydrolysate environment, L. starkeyi
began utilizing different sugars for its growth and lipid accumulation. Interestingly, glucose
and xylose were used by L. starkeyi simultaneously, which was different from other yeast such
as Trichosporon fermentans that will utilize hexose first and then pentose [5]. Since merely
about 2.0 g/L glucose was present in the corncob acid hydrolysate, it was used up at the second
day of fermentation. In contrast, xylose was not exhausted until the seventh day of fermentation for its high concentration in the hydrolysate. It is worth noting that arabinose could be
hardly utilized by L. starkeyi throughout the fermentation. Finally, after fermentation, there
was about 3.6 g/L arabinose still present in the corncob acid hydrolysate. Although arabinose
could not be used by L. starkeyi, the remaining arabinose could be recovered and make profits
by its high price. Overall, the lipid coefficient of L. starkeyi (lipid yield on sugar consumption)
was about 20.9, suggesting its excellent capacity of lipid production on lignocellulosic
hydrolysates.
Generally, there are two main kinds of lipid synthesis in the cell of oleaginous microorganisms, namely de novo lipid accumulation and ex novo lipid accumulation [18]. Different
from the lipid fermentation on hydrophobic substrates (ex novo lipid accumulation), de novo
lipid accumulation is on the hydrophilic substrates such as corncob acid hydrolysate like this
work. Limited nutrients (usually nitrogen source) are required for de novo lipid accumulation
and C/N ratio is also necessary for lipid fermentation in many cases [19]. However, adding
nitrogen sources in lignocellulosic hydrolysate showed a negative effect on the lipid

Sugar concentration (g/L)

45
40

Glucose
Xylose
Arabinose
Total sugars
Total nitrogen

35
30
25
20
15
10
5
0
0

9 10 11

Fermentation time (d)

Fig. 2 Time courses of sugar and nitrogen utilization by L. starkeyi on the corncob acid hydrolysate

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Appl Biochem Biotechnol (2014) 172:21972204

production in some situations [20]. In this work, adding other nitrogen sources show little
effect on the lipid yield of L. starkeyi in corncob acid hydrolysate (the lipid yields are close,
data not shown). Considering the cost of nitrogen sources, addition of no other nitrogen source
was better. The total nitrogen was merely about 0.1 g/L in the corncob acid hydrolysate; thus,
the C/N ratio was suitable for lipid accumulation. Throughout the lipid fermentation process,
the total nitrogen in the medium was around 0.1 g/L (Fig. 2), indicating that the assimilable
nitrogen sources have been utilized.
Comparison with Other Lipid Production on Various Substrates
The oleaginous yeast L. starkeyi has been applied for lipid production since long time ago [21]. It
shows great potential of utilizing low-cost substrates especially wastewaters for microbial oil
production, e.g., sewage sludge and olive oil mill wastewaters [4, 9] could be used efficiently to
accumulate lipid by L. starkeyi. The possibility of using lignocellulosic hydrolysates for microbial
oil production by L. starkeyi has been stated for its capacity of using both glucose and xylose as
carbon sources [6]. Although L. starkeyi showed great capacity for lipid production [22], no work
actually used this strain for microbial oil production on real lignocellulosic hydrolysate. In this work,
for the first time, one kind of lignocellulosic hydrolysate, namely corncob acid hydrolysate, was
shown as one potential raw material for lipid production by L. starkeyi.
Recently, microbial oil production on lignocellulosic hydrolysates is a hot topic focused by
many researchers. The theoretical yield of lipids on glucose and xylose (lipid coefficient) is 33
and 34 %, respectively [23]. However, the lipid coefficient of oleaginous microorganism was
not high (usually lower than 20 %) on different lignocellulosic hydrolysates [12, 13, 17]. It is
worth noting that the lipid coefficient of L. starkeyi in this work was even higher than 20 % in
spite that it could not metabolize arabinose, again showing its great potential for lipid
production on lignocellulosic hydrolysates.
Finally, the lipid production in this work was compared with others which utilized various
low-cost substrates such as wastewaters, tapioca starch, industrial fats, whey, glycerol, molasses, corncob waste liquor, etc. (Table 2). Obviously, the capabilities of utilizing different agroindustrial residues varied with microbial strains. As shown in Table 2, L. starkeyi showed

Table 2 Lipid production on different carbon sources by various microorganisms

Strain

Carbon source

Biomass Lipid content Lipid yield Reference

(g/L)
(%)
(g/L)

R. glutinis

Monosodium glutamate wastewater

25.0

20.0

5.0

[3]

Mucor sp. RRL001

Y. lipolytica

Tapioca starch
Industrial fats

28.0
8.7

17.8
44.0

5.0
3.8

[24]
[25]

M. isabellina

Cheese whey

23.1

17.3

4.0

[26]

T. capitatum

Cane molasses

17.3

37.6

6.5

[2]
[27]

C. echinulata

Glycerol

6.2

53.2

3.3

R. glutinis

Glycerol

20.2

25.0

5.1

[28]

Aspergillus sp.

Corncob waste liquor

2.0

22.1

0.4

[29]

L. starkeyi

Glucose and xylose

20.5

61.5

12.6

[6]

L. starkeyi
L. starkeyi

Olive oil mill wastewaters

Sewage sludge

11.0
9.4

22.4
68.0

2.5
6.4

[9]
[4]

L. starkeyi

Corncob acid hydrolysate

17.2

47.0

8.1

This work

Appl Biochem Biotechnol (2014) 172:21972204

2203

bigger capacity of lipid production than other oleaginous microorganisms. Although L.

starkeyi could have a higher biomass and lipid content on the synthetic medium containing
glucose and xylose [6], the sugar concentration of this medium was higher than that
of the corncob acid hydrolysate; thus, the lipid coefficient in this medium was lower
than that in this work. On the other hand, although the lipid content (68 %) of L.
starkeyi in the sewage sludge was higher than that in this work, its lipid yield was
lower compared with the lipid fermentation on the corncob acid hydrolysate. Overall, oleaginous yeast L. starkeyi has great potential for lipid production on various low-cost substrates
especially lignocellulosic hydrolysates.

Conclusions
Although no nitrogen source or trace element has been added, the corncob acid hydrolysate
could be used for microbial oil production by the oleaginous yeast L. starkeyi efficiently; thus,
it is a promising strain for the production of microbial oil from lignocellulosic materials.
Acknowledgments The authors acknowledge the financial support from a project of Guangzhou Science and
Technology (2013J4300031), the Support Plan Project of the National Science and Technology
(2012BAD32B07), the National Natural Science Foundation of China (51378486, U1261116), the Natural
Science Foundation of Guangdong Province (S2012040007546), and the Foundation of Director of Guangzhou
Institute of Energy Conversion, Chinese Academy of Sciences (y107rf1001).

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