Appl Biochem Biotechnol (2009) 159:591–604

DOI 10.1007/s12010-008-8491-x

Screening of Oleaginous Yeast Strains Tolerant

to Lignocellulose Degradation Compounds

Xi Chen & Zihui Li & Xiaoxi Zhang & Fengxian Hu &

Dewey D. Y. Ryu & Jie Bao

Received: 5 September 2008 / Accepted: 15 December 2008 /

Published online: 21 January 2009
# Humana Press 2009

Abstract High cost of triacylglycerol lipid feedstock is the major barrier for commercial
production of biodiesel. The fermentation of oleaginous yeasts for lipid production using
lignocellulose biomass provides a practical option with high economic competitiveness. In
this paper, the typical oleaginous yeast strains were screened under the pressure of
lignocellulose degradation compounds for selection of the optimal strains tolerant to
lignocellulose. The inhibitory effect of lignocellulose degradation products on the
oleaginous yeast fermentation was carefully investigated. Preliminary screening was
carried out in the minimum nutritious medium without adding any expensive complex
ingredients then was carried out in the lignocellulosic hydrolysate pretreated by dilute
sulfuric acid. Seven typical lignocellulose degradation products formed in various
pretreatment and hydrolysis processing were selected as the model inhibitors, including
three organic acids, two furan compounds, and two phenol derivatives. The inhibition of
the degradation compounds on the cell growth and lipid productivity of the selected
oleaginous yeasts were examined. Acetic acid, formic acid, furfural, and vanillin were
found to be the strong inhibitors for the fermentation of oleaginous yeasts, while levulinic
acid, 5-hydroxymethylfurfural, and hydroxybenzaldehyde were relatively weak inhibitors.
Trichosporon cutaneum 2.1374 was found to be the most adopted strain to the lignocellulose
degradation compounds.

Keywords Triacylglycerol lipid . Oleaginous yeasts . Trichosporon cutaneum . Screening .

Lignocellulose degradation compounds

X. Chen : Z. Li : F. Hu : J. Bao (*)

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology,
130 Meilong Road, Shanghai 200237, China
e-mail: jbao@ecust.edu.cn

X. Zhang
Jilin Fuel Alcohol Co. Ltd, PetroChina Corporation,
Jilin Economic Development Zone, Jilin 132101, China

D. D. Y. Ryu
Biochemical Engineering Program, University of California, One Shields Ave, Davis, CA 95616, USA
592 Appl Biochem Biotechnol (2009) 159:591–604

Introduction

Biodiesel is a diesel-equivalent fatty acid alkyl esters produced through transesterification

reaction of triacylglycerol lipids with methanol or ethanol. It is an ideal substitute of the
conventional vehicles diesel for its biodegradable, nontoxic, and typically produces about
60% less net carbon dioxide emissions than petroleum-based diesel. Generally, the cost of
the triacylglycerol such as vegetable oils, animal fats, or recycled greases and other oil
resources is about 70–75% of the total biodiesel production [1, 2]. The meaningful
replacement of biodiesel to petroleum based diesel on the transportation fuel market
requires the sustainable and stable supply of cheap triacylglycerol lipid to release the
pressure on the ever-increasing prices of soybean oil and rapeseed oils [3–5]. Two
resolutions have been exploited, the first one is to grow oil plants in the dry, mountain, or
saline lands; the second one is through the fermentation of oleaginous microorganisms in
the bioreactor [6]. Oleaginous microorganisms capable of metabolizing lignocellulose-
derived sugars could provide a practical option with high economic competitiveness [7].
The key technical barrier to this goal is the high of fermentation process and one of the
major resolution is using cheap fermentation medium, such as fermentation waste fluents,
lignocellulose hydrolysate without the addition of expensive fermentation ingredients such
as yeast extract, peptone, etc.
Lignocellulose such as agriculture stover, forest residues, and energy plants is the most
abundant and sustainable biomass on earth with low commercial values currently [8]. The
cost for production of triacylglycerol lipid could be considerably reduced if lignocellulosic
biomass is used as carbon source. To obtain the fermentable sugars from the lignocellulose,
a harsh physical or chemical process called pretreatment such as steam explosion or dilute
acid pretreatment is applied to break the lignin or hemicellulose shell and transform the
crystalline cellulose to amorphous cellulose. Various degradation compounds are released
from lignocellulose degradation during the pretreatment and will enter the downstream
hydrolysis and fermentation processes. These degradation products in the hydrolysate
include sugar degradation products such as furfural from xylose and 5-hydroxymethylfur-
fural (5-HMF) from glucose [9]; lignin degradation products such as vanillin, syringalde-
hyde, and 4-hydroxybenzaldehyde [10]; organic acids such as acetic acid from acetyl
group, formic acid from xylose oxidation, and levulinic acid from glucose oxidation [11].
The composition of the degradation products depends on the type of lignocellulosic
materials used, as well as the chemistry and the nature of the pretreatment process. It has
been well realized and carefully investigated that the lignocellulose degradation products
are strong inhibitors to ethanol fermentation [12]. Up to now, very little knowledge was
known about the fermentation performance and bio-lipid accumulation of oleaginous
microorganisms under the stress and tolerance of lignocellulose degradation compounds.
Oleaginous microorganisms such as yeast, mould, bacterium, and algae can be used to
produce a great deal of lipid, up to 70% lipid of the total dry cell weight under the certain
conditions [6, 7, 13]. Among these microorganisms, the eucaryotic yeasts, fungi, and algae
synthesize polyunsaturated fatty acid triacylglycerol similar to vegetable oil, while the
prokaryotic bacterium synthesizes special lipids. In this work, oleaginous yeasts were
chosen as the fermentation strains for production of lipid using corn stover hydrolysate as
the carbon source. The common oleaginous yeasts are including: Rhodotorula glutinis,
Trichosporon cutaneum, Rhodotorula rubra, Rhodosporidium toruloides, Lipomyces
starkeyi, Cryptococcus albidum [14]. Most of the oleaginous yeasts were covered in this
study. The basis principles for screening include: (1) the fatty acid triacylglycerol produced
by the oleaginous yeasts meets the requirement as biodiesel feedstock, that is, the carbon
Appl Biochem Biotechnol (2009) 159:591–604 593

number of the major fatty acid is C16–18. (2) High growth rates on wide varieties of
substrates and utilize cheap raw materials. The purpose of this work is to screen the most
adopted oleaginous strains tolerant to the lignocellulose degradation compounds based on
their inhibitory performance. The tolerance and stress of the degradation inhibitors on the
sugar utilization of lignocellulose-derived glucose and xylose, the cell growth, and lipid
accumulation properties were quantitatively studied. The results show that the fermentation
performance under the tolerance of degradation compounds varies significantly depending
on the oleaginous yeast species, inhibitor types, and fermentation conditions. A best
inhibitor tolerant strain, T. cutaneum 2.1374, was screened and fermented in the
lignocellulose hydrolysate with the minimum nutrients for production of triglycerol lipid.

Materials and Methods

Strains and Media

All oleaginous yeast strains used in the experiments were listed in Table 1. The strains were
purchased from China General Microbiological Culture Collection Center, Beijing, China.
The strains were transferred and maintained on YPD-agar plates containing 10 g/L yeast
extract, 20 g/L peptone, 20 g/L glucose, and 20 g/L agar–agar. The inoculum medium
contains 20 g/L glucose; 0.5 g/L yeast extract; 5 g/L (NH4)2SO4; 1 g/L KH2PO4; 0.5 g/L
MgSO4·7H2O. The fermentation medium is nitrogen-limited which contains only minimum
nutrients without the addition of any complex ingredients such as yeast extract, peptone,
etc. The composition of the nitrogen-limited or minimum nutrition was fixed in all the
culture experiments to 30 g/L glucose; 20 g/L xylose; 2 g/L (NH4)2SO4; 1 g/L KH2PO4;
0.5 g/L MgSO4·7H2O, unless mentioned otherwise.

Reagents

Furfural and 5-HMF were from Acros Organics (New Jersey, USA). Levulinic acid was
from Alfa Aesar (Ward Hill, MA, USA). Vanillin, 4-hydroxybenzaldehyde, sodium
hydroxide, sodium acetate were all from Sinopharm Chemical Reagent (Shanghai, China).
Glucose, formic acid, acetic acid, and ammonia sulfate were purchased from other local

Table 1 Oleaginous microorganisms used.

Microorganisms CGMCC ID Short names

Rhodotorula glutinis 2.107 R. glutinis 2.107

Rhodotorula glutinis 2.703 R. glutinis 2.703
Rhodotorula glutinis 2.704 R. glutinis 2.704
Trichosporon cutaneum 2.571 T. cutaneum 2.571
Trichosporon cutaneum 2.1374 T. cutaneum 2.1374
Rhodotorula rubra 2.1515 R. rubra 2.1515
Rhodosporidium toruloides 2.1389 R. toruloides 2.1389
Rhodosporidium toruloides 2.1609 R. toruloides 2.1609
Lipomyces starkeyi 2.1390 L. starkeyi 2.1390
Lipomyces starkeyi 2.1608 L. starkeyi 2.1608

All strains were from CGMCC

CGMCC China General Microbiological Culture Collection Center
594 Appl Biochem Biotechnol (2009) 159:591–604

chemical reagent companies in Shanghai, China. Two industrial cellulase enzymes used
were Spezyme CP from Genencor International (Rochester, NY, USA), and Novozyme 188
from Novo Industrial A/S (purchased from Sigma–Aldrich, St Louis, MO, USA).

Production of Corn Stover Hydrolysate

Corn stover was harvested in fall 2006 from Northeast Province Jilin, China. The corn
stover was milled and fractionated though a sieve with the pore diameter of 5 mm. The
chipped corn stover was presoaked with 2.5% sulfuric acid for 1.5 h at ambient
temperature. The hot steam (3 MPa) was jetted into the 2.5 L pretreatment reactor and
kept jetting for 5 min at 180 °C. The pretreated corn stover was separated into a solid
fraction and a liquid prehydrolysate. The prehydrolysate was over-limed using calcium
hydroxide to pH 5 at 50 °C for 30 min. Then, the over-limed prehydrolysate was mixed
with the pretreated solid then to the enzymatic hydrolysis step for production of hydrolysate
used in the fermentation. Spezyme CP and Novozyme 188 were added at the amount of
7 FPU and 15 IU/g of dried solid fraction, respectively, in the enzymatic hydrolysis process.
The hydrolysis was carried out at 50 °C for 48 h. The water insoluble solid in the
hydrolysate was separated by centrifugation and the liquid hydrolysate was used to culture
the oleaginous yeasts. The major compositions in lignocellulosic hydrolysate include
glucose 84.65 g/L, xylose 36.39 g/L, acetic acid 11.58 g/L, levulinic acid 1.49 g/L, formic
acid 2.56 g/L, furfural 0.32 g/L, 5-HMF 1.01 g/L, vanillin 0.061 g/L, and hydroxyben-
zaldehyde 0.103 g/L.

Culture Conditions and Lipid Recovery

A large single colony of the selected oleaginous yeast strains was picked up from YPD-agar
plates and inoculated into 20 mL of inoculum medium for 24 h for the seeding culture; 5-
mL seed suspension was inoculated into the 250-mL conical flasks containing 50 mL of
fermentation medium in which the nitrogen was regulated by ammonia sulfate addition.
The pH of all media was adjusted to 5 with 2 M NaOH and autoclaved at 115 °C for
20 min. All cultures were incubated at an orbit agitation rate of 180 rpm and the
temperature of 30 °C. The concentration of sugars and degradation compounds, the dry cell
mass, and the lipid composition were sampled periodically. The yeast cells were harvested
by centrifugation at 10,000 rpm for 5 min and then washed with deionized water twice to
remove the extracellular fat from the cell surface and superfluous nutrient substances. The
dry cell mass (DCM) was determined after the cell was dried at 105 °C for 24 h. One gram
of the wet cells was disrupted and homogenized in 6 mL of 4 M hydrochloric acid for
30 min. The cell debris was socked for 30 min, heated at 100 °C for 10 min, and then
quenched at −20 °C. Frozen cells were stirred with 10 mL methanol–chloroform mixture
(methanol–chloroform=1:2 by volume ratio) for 30 min. The extracted lipid was
centrifuged to give a clear organic solvent and anhydrous sodium sulfate was added to
remove any residual moisture [15]. The solvent was removed by evaporation and the total
lipid was measured by gravimetric method.

Analysis of Sugars, Inhibitors, and Lipids

Glucose, xylose, and lignocellulose degradation compounds were analyzed by high

performance liquid chromatography (LC-20AD, refractive index detector RID-10A,
Shimadzu, Japan) with a Bio-rad Aminex HPX-87H column at the column temperature
Appl Biochem Biotechnol (2009) 159:591–604 595

65 °C. The mobile phase was 0.005 M H2SO4 at the rate of 0.6 mL/min. All samples were
centrifuged to remove the cell mass and other water insoluble substances, and then filtered
through a 0.22-μm filter before the analysis.
The fatty acid composition of the lipid was analyzed using gas chromatography-mass
spectrometry (GC-MS). The Clarus500 gas chromatograph (PerkinElmer) used a PE-5
column (30 m×0.25 mm×0.25 μm). The carrier gas was He at the rate of 1 mL/min; the
initial oven temperature was 80 °C, increased at the rate of 16 °C/min, and the final
temperature reached 280 °C. First, the lipid was transesterified with methanol into its fatty
acid methyl ester form using 10% boron trifluoride as catalytic agent; then the ester was
heated to 60 °C for 10 min; finally, it was injected into the GC-MS. The fatty acid
composition was found using NIST MS Search 2.0.

Results

Preliminary Screening of Oleaginous Yeasts in the Minimum Nutritious Medium

The cell survival and metabolism in the lignocellulose hydrolysate environment are the first
priority during the fermentation. The screening was carried out in the minimum nutritious
medium under the nitrogen-limited condition. The medium was prepared with glucose and
xylose as the sole carbon sources, ammonia sulfate as the sole nitrogen source, without
adding any expensive ingredients such as yeast extract, peptone, and other complex
substances. Six oleaginous yeast strains including T. cutaneum 2.1374, R. toruloides
2.1389, L. starkeyi 2.1390, L. starkeyi 2.1608, R. glutinis 2.704, and R. glutinis 2.107 were
found to grow well in the designed medium, and the growth profile was shown in Fig. 1.
The growth of the rest four strains including R. glutinis 2.703, T. cutaneum 2.571, R. rubra
2.1515, and R. toruloides 2.1609 was not observed, indicating that the growth activity of
the four strains were weak in the nitrogen-limiting or minimum nutritious medium.
The DCM, the lipid in medium (g/L), and the lipid in cells (wt.%) of these six
oleaginous yeast strains after cultured 96 h was shown in Table 2. The lipid in medium was
ranged from 0.52 to 2.29 g/L, and the lipid in cells was ranged from 13.0% to 39.8%. The
fatty acid chains on the yeast lipid produced was characterized using GC-MS, and the fatty

Fig. 1 The growth of six yeasts 10

in the nitrogen-limited medium T. cutaneum 2.1374
versus fermentation time. The L. starkeyi 2.1608
nitrogen-limited medium 8 R. glutinis 2.107
including 50 g/L glucose; 2 g/L R. toruloides 2.1389
(NH4)2SO4; 1 g/L KH2PO4; L. starkeyi 2.1390
0.5 g/L MgSO4·7H2O. All 6 R. glutinis 2.704
DCM (g/L)

cultures were incubated in an

orbital shaker at an agitation rate
of 180 rpm and incubation 4
temperature 30 °C

0
0 24 48 72 96
Time(hr)
596 Appl Biochem Biotechnol (2009) 159:591–604

Table 2 The DCM, lipid in medium, lipid in cells and lipid yield of the six oleaginous yeast strains after
cultured 96 h in the nitrogen-limited medium.

Microorganisms DCM Lipid in Lipid in Lipid productivity Lipid yield

(g/L) medium (g/L) cells (wt.%) (g/L h−1) (g/100 g glucose)

T.cutaneum 2.1374 2.75 1.09 39.8 0.011 10.1

L. starkeyi 2.1608 9.35 2.04 21.8 0.097 10.5
L. starkeyi 2.1390 6.16 2.29 37.2 0.021 10.6
R. glutinis 2.107 4.01 0.52 13.0 0.042 4.92
R. glutinis 2.704 5.49 0.92 16.7 0.057 2.78
R. toruloides 2.1389 4.26 1.67 39.3 0.044 12.6

acid composition showed that the major fatty acids were palmitic acid, stearic acid, oleinic
acid, linoleic acid, and palmitoleic acid which are all C16 and C18 fatty acids, either
saturated or unsaturated, which were the suitable feedstocks for production of biodiesel.

Screening of the Oleaginous Yeasts Under the Inhibition of Organic Acids

Acetic acid, formic acid, and levulinic acid are the major organic acid components in the
lignocellulose hydrolysate. Acetic acid is formed during the hydrolysis of hemicellulose in
which the acetyl group of hemicellulose linked to the lignin or cellulose is released and
hydrolyzed to form acetic acid. Levulinic acid is the terminal product of oxidation of D-
glucose and D-mannose. There are two formation pathways for formic acid formation, one
is the terminal product of xylose oxidation and another one is the byproduct of D-glucose
and D-mannose oxidation to levulinic acid, both occurred mainly during the chemical
pretreatment step [16, 17]. These three compounds were selected to be the representative
inhibitors to investigate the inhibitory performance of organic acids released from
lignocellulose on the six prescreened oleaginous yeast fermentation.
The effect of acetic acid on the cell culture of T. cutaneum 2.1374 was shown in Fig. 2.
The growth of all six strains except T. cutaneum 2.1374 ceased completely when 5 g/L
acetic acid was added to the nitrogen-limited or minimum nutritious medium. For T.

Fig. 2 Time course of the T. 10 0.16

cutaneum 2.1374 fermentation Acetic acid
DCM (with acetic acid) 0.14
under the acetic acid inhibition. DCM
The medium includes 30 g/L 8
Sugar consumption (g h L )

Glucose (with acetic acid)

-1

0.12
DCM and acetic acid (g/L)

glucose, 20 g/L xylose, 5 g/L Glucose

-1

Xylose (with acetic acid)

acetic acid, 2 g/L (NH4)2SO4, Xylose
6 0.10
1 g/L KH2PO4, 0.5 g/L
MgSO4·7H2O. All cultures were 0.08
incubated in an orbital shaker at
an agitation rate of 180 rpm and 4
0.06
incubation temperature 30 °C
0.04
2
0.02

0 0.00
0 24 48 72 96
Time (hr)
Appl Biochem Biotechnol (2009) 159:591–604 597

cutaneum 2.1374, the cell growth (indicated by DCM) and the glucose or xylose
consumption rate decreased considerably when 5 g/L of acetic acid was added to the
medium. Figure 2 also indicated that T. cutaneum 2.1374 was able to metabolize acetic acid
slowly simultaneously with the glucose or xylose consumption. Probably this was at least
the partial reason of the high acetic acid tolerance of T. cutaneum 2.1374. At the end of the
96-h cultivation, the maximum cell mass (3.68 g/L) was greater than that without acetic
acid addition (2.75 g/L). The lipid productivity with the acetic acid addition (1.12 g/L) was
also bit greater than that without acetic acid addition (1.09 g/L). The phenomenon indicated
that acetic acid participated the lipid fermentation for production of lipid under the range of
T. cutaneum 2.1374.
Similar to acetic acid, the growth of all six selected strains were strongly inhibited under
the existence of formic acid with the only exception of T. cutaneum 2.1374. The result was
shown in Table 3. When the concentration of formic acid reached to 3 g/L, the growth of L.
starkeyi 2.1390, L. starkeyi 2.1608 and R. glutinis 2.107 completely ceased, and of course
there were almost no lipid produced. For R. glutinis 2.704 and R. toruloides 2.1389, there
were only a little growth with almost no lipid production. All these five strains completely
ceased to grow when the concentration of formic acid reached to 5 g/L. T. cutaneum 2.1374
showed the best tolerance to formic acid, which was almost not affected by formic acid
addition. The growth of T. cutaneum 2.1374 was even enhanced when the formic acid
concentration was 1 g/L at which the maximum DCM was 4.85 g/L.
The inhibition effect of levulinic acid on the selected oleaginous yeast culture was
different with that of acetic acid and formic acid. As shown in Table 3, all six strains were
not significantly affected by the existence of levulinic acid under the experimental range up
to 10 g/L, which was twice as high as the acetic and formic acids tested. On the contrary,
the growth of some strains such as T. cutaneum 2.1374 was enhanced by the existence of
levulinic acid. The maximum dry cell mass and lipid for T. cutaneum 2.1374 were 4.34 and
1.19 g/L, relatively, which appeared in the levulinic acid concentration of 10 g/L. For the
other five strains, the growth and sugar consumption decreased but not ceased to grow with
the increasing concentration of levulinic acid. For R. toruloides 2.1389, the maximum
DCM and lipid were 4.60 and 1.40 g/L, relatively, which appeared in the levulinic acid
concentration of 5 g/L (the data was not showed).

Screening of Oleaginous Yeasts Under the Inhibition of Furan Derivatives

from Sugar Degradation

Furfural and 5-HMF are the two major degradation products from xylose and glucose,
respectively, and have been well-recognized as the major fermentation inhibitors on the
ethanol fermentation. The inhibition effect of these two furan compounds on the lipid
fermentation was investigated using the minimum nutritious medium under nitrogen-limited
conditions. The results also showed in Table 3. Table 3 showed that furfural strongly
inhibited the cell growth and lipid production of all the selected six oleaginous yeast strains
under the experimental range. T. cutaneum 2.1374 showed the relatively better tolerance to
furfural, but the cell almost ceased to grow at the furfural concentration greater than 2 g/L.
The maximum DCM was only 2.94 at 0.5 g/L of furfural and the lipid seemed increase
little. R. glutinis 2.107 and L. starkeyi 2.1390 grew only at the minimum furfural (less than
1 g/L); L. starkeyi 2.1608, R. glutinis 2.704, and R. toruloides 2.1389 ceased the growth
even at the minimum concentration in the experimental range (0.5 g/L).
5-HMF showed a significant different inhibition performance comparing to furfural.
Table 3 showed that 5-HMF inhibited the cell growth and lipid production weakly in the
598 Appl Biochem Biotechnol (2009) 159:591–604

Table 3 Performance of six strains under the inhibition of six inhibitors in the nitrogen-limited medium.

Microorganisms Inhibitor concentration (g/L) DCM Lipid in Lipid Lipid yield

(g/L) medium in cells (g/100 g
(g/L) (wt.%) glucose)

T. cutaneum 2.1374 Formic acid 1.0 4.85 1.04 21.5 6.00

3.0 3.58 0.63 17.5 5.30
5.0 3.02 0.60 20.0 6.30
Levulinic acid 1.0 2.88 0.79 27.5 6.00
5.0 4.20 1.19 28.3 6.30
10 4.34 1.08 25.0 5.40
Furfural 0.5 2.94 1.25 42.5 12.3
1.0 1.76 0.54 30.6 6.76
5-HMF 0.5 1.78 0.83 46.8 10.1
1.0 2.58 1.14 44.2 12.0
2.0 2.37 1.04 43.8 10.1
Vanillin 0.5 3.39 1.24 36.6 10.4
2.0 2.50 0.98 39.2 10.2
Hydroxybenzaldehyde 0.5 2.30 0.78 34.0 7.98
1.5 1.32 0.48 36.5 5.64
L. starkeyi 2.1608 Formic acid 1.0 6.31 0.98 15.5 10.2
Levulinic acid 1 7.93 0.74 9.33 5.22
10 2.98 0.24 8.05 5.05
Furfural 0 9.35 2.04 21.8 10.5
5-HMF 0.5 9.50 2.00 23.8 11.2
1.0 8.44 2.08 24.6 11.9
Vanillin 0.5 7.60 1.58 20.8 9.62
Hydroxybenzaldehyde 0.5 10.6 2.93 27.7 11.1
L. starkeyi 2.1390 Formic acid 1.0 0.27 0.078 28.9 5.61
Levulinic acid 1 2.99 0.48 16.1 4.41
5 2.90 0.30 10.3 2.89
Furfural 0.5 5.41 1.64 30.3 9.26
5-HMF 0.5 7.09 2.22 31.3 8.95
1.0 5.51 2.00 36.3 6.69
Vanillin 0 6.16 2.29 37.2 10.6
Hydroxybenzaldehyde 0.5 6.00 2.30 38.3 10.3
R. glutinis 2.704 Formic acid 1 2.13 0.06 2.82 0.69
3 1.23 0.04 3.25 0.69
Levulinic acid 1 4.13 0.52 12.6 3.44
5 4.36 0.44 10.1 2.87
10 3.91 0.34 8.70 2.86
Furfural 0 7.42 0.68 9.16 2.78
5-HMF 0.5 5.29 0.34 6.43 2.03
1.0 3.86 0.24 6.22 1.96
2.0 3.12 0.14 4.49 1.40
Vanillin 0.5 2.96 0.46 15.5 4.40
1.0 1.65 0.27 16.4 6.03
Hydroxybenzaldehyde 0.5 5.01 0.30 5.99 2.41
R. glutinis 2.107 Formic acid 1 2.38 0.12 5.04 2.28
Levulinic acid 1 4.91 0.40 8.15 3.02
5 5.12 0.36 7.03 2.27
10 3.56 0.26 7.30 2.30
Furfural 0.5 3.63 0.20 5.51 2.05
Appl Biochem Biotechnol (2009) 159:591–604 599

Table 3 (continued)

Microorganisms Inhibitor concentration (g/L) DCM Lipid in Lipid Lipid yield

(g/L) medium in cells (g/100 g
(g/L) (wt.%) glucose)

5-HMF 0.5 5.11 0.56 11.0 4.27

1.0 3.05 0.20 6.56 2.12
2.0 2.93 0.24 8.19 2.31
Vanillin 0.5 3.58 0.30 8.38 3.21
Hydroxybenzaldehyde 0.5 3.44 0.38 11.0 3.65
R. toruloides 2.1389 Formic acid 1 2.71 0.20 7.38 1.92
3 1.73 0.16 9.25 2.17
Levulinic acid 1 4.2 1.38 32.9 7.75
5 4.60 1.40 30.4 5.92
10 3.83 0.73 19.1 4.11
Furfural 0 4.26 1.67 39.2 12.6
5-HMF 0.5 3.39 0.76 22.4 6.2
1.0 2.42 0.40 16.5 4.02
2.0 1.90 0.28 14.7 3.25
Vanillin 0.5 1.92 0.36 18.8 4.46
Hydroxybenzaldehyde 0.5 2.21 0.34 15.4 3.82
1.0 5.93 1.68 28.3 8.15

Under the inhibitor concentration above the list value, the strains do not grow

tested concentration range. No obvious inhibition of 5-HMF on T. cutaneum 2.1374 was

observed in the experimental range. The cell growth and lipid production on the other five
strains decreased with increasing 5-HMF concentration, but the deduction was not as
significant as that by furfural. Only L. starkeyi 2.1608 and 2.1390 ceased to growth in the
experimental range of 5-HMF.

Screening of the Oleaginous Yeasts Under the Inhibition by Phenol Derivatives from Lignin

Lignin is a complicated polymer of phenol compounds, composed of three monomers of

p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol through the alkyl–aryl, alkyl–
alkyl, and aryl–aryl ether bonds. Lignin could be converted into various phenol derivatives
in the pretreatment processing, especially in the chemical-based pretreatment such as dilute
acid pretreatment. Phenol compounds from lignin degradation had been long recognized as
the inhibitors for ethanol fermentation, but its inhibitory performance on oleaginous yeast
was not yet well characterized. In this work, the typical lignin degradation compounds,
vanillin and hydroxybenzaldehyde, were selected as the model inhibitors to investigate the
inhibition effect of lignin degradation products on oleaginous yeasts.
Table 3 shows the results of the effect of vanillin to minimum nutritious medium. For
vanillin, the minimum amount of it can significantly inhibit the cell growth and lipid
production with the only exception of T. cutaneum 2.1374. L. starkeyi 2.1608, R. toruloides
2.1389, and R. glutinis 2.107 ceased to grow under the very low concentration of vanillin
(less than 1 g/L) or even died when the smallest dosage of vanillin was added in the
experimental range (0.5 g/L). R. glutinis 2.704 performed a better vanillin tolerance and
ceased to grow when vanillin was greater than 2 g/L. No obvious inhibition of vanillin on T.
cutaneum 2.1374 in the experimental range. Oppositely the cell growth and lipid production
600 Appl Biochem Biotechnol (2009) 159:591–604

of T. cutaneum 2.1374 reached the maximum when vanillin was added to 0.5 g/L, in which
the DCM and lipid were 3.41 and 1.17 g/L, respectively.
For hydroxybenzaldehyde, Table 3 showed that the cell growth and lipid production of
the selected six strains decreased with the increasing concentration of hydroxybenzaldehyde
in the experimental range. However the inhibition of hydroxybenzaldehyde was not as
strong as that of vanillin. Again, T. cutaneum 2.1374 showed the best tolerance and
resistance to hydroxybenzaldehyde, and the only strain among the selected six to grow in
the maximum concentration of hydroxybenzaldehyde, although its growth decreased with
the increasing hydroxybenzaldehyde concentration. The other strains ceased to grow at the
1.5 g/L of hydroxybenzaldehyde.
The cumulative inhibition on T. cutaneum 2.1374 by the major inhibitors, acetic acid,
furfural, and 5-HMF, was added and the result was shown in Fig. 3. The major compo-
sitions in medium include glucose 28.18 g/L, xylose 19.08 g/L, acetic acid 1.24 g/L,
levulinic acid 2.66 g/L, furfural 0.25 g/L, 5-HMF 0.71 g/L. Figure 3 showed that under the
cumulative inhibition of acetic acid, furfural, and 5-HMF, the cell growth of T. cutaneum
2.137, glucose or xylose consumption, and inhibitor consumption was similar to that under
the inhibition of individual inhibitors.

Cultivation of T. cutaneum 2.1374 in Corn Stover Hydrolysate

The corn stover hydrolysate was obtained using the method in the “Materials and Methods”
section. The inhibition effect of the combinational degradation products from lignocellulose
on T. cutaneum 2.1374 was carried out in the corn stover hydrolysate as the culture
medium. The hydrolysate was diluted by the synthetic medium to 80%, 60%, 40%, and
20% (hydrolysate content). The time courses of the cell growth and the consumption of
glucose, xylose, and acetic acid were shown in Fig. 4.
The obvious lag phase was observed in the fermentation profile of each dilution
medium. The lag time increased with the increasing content of hydrolysate: the
fermentation started with almost no lag time when the hydrolysate was diluted to 20%
and 40%; lag time was 24, for the media with 60%. The fermentation did not happen for the
media with 80% dilutions, respectively. Only 20% had xylose consumption. Figure 4b
showed an interesting phenomenon for acetic acid metabolism. Obviously acetic acid

Fig. 3 Time course of the 6 0.20

T. cutaneum 2.1374 fermentation DCM
under the cumulative effect of the Acetic acid
5
Dry cell mass and inhibitors (g/l)

major inhibitors. The major 5-Hydormethylfunan

Sugar consumption (g L h )
-1

compositions in medium include Furaldehyde 0.15

-1

glucose 28.18 g/L, xylose 4 Levulinic acid

19.08 g/L, acetic acid 1.24 g/L, Glucose
levulinic acid 2.66 g/L, furfural Xylose
0.25 g/L, 5-HMF 0.71 g/L. All 3 0.10
cultures were incubated in an
orbital shaker at an agitation rate 2
of 180 rpm and incubation
0.05
temperature 30 °C
1

0 0.00
0 24 48 72 96
Time (hr)
Appl Biochem Biotechnol (2009) 159:591–604 601

Fig. 4 Time course of the 14

T. cutaneum 2.1374 in lignocel- (a) DCM
12
lulosic hydrolysate fermentation.
Symbols indicate the different 10

DCM (g /L)..
dilutioin ratio lignocellulosic 8
hydrolysate with the nitrogen-
6
limited medium whose sugar and
nitrogen composition were same 4
with hydrolysate. a–d The 2
concentration change of DCM,
0
acetic acid, glucose, xylose verse
14
fermentation time, respectively. (b) Acetic acid utilization
e Lipid production at the end of 12
fermentation. Symbols indicate 10
Acetic acid (g/L)..

the different dilution ratio ligno-

8
cellulosic hydrolysate with the
nitrogen-limited medium whose 6
sugar and nitrogen composition
4
were the same with hydrolysate
2

0
120
(c) Glucose utilization
100
Glucose (g /L)

80

60

40

20

0
60
(d) Xylose utilization
50

40
Xylose (g /L)

30
Dilution percentage
20 20%
40%
10 60%
80%
0
0 24 48 72 96 120
Time(hr)
1.2 12
(e) Lipid productioin at the end of fermentation
1.0 Lipid in the medium 10
Lipid in the medium (g/L)

Lipid in the cells (wt.%).

Lipid in the cells

0.8 8

0.6 6

0.4 4

0.2 2

0.0 0
20 40 60 80
Diluted percent (%)
602 Appl Biochem Biotechnol (2009) 159:591–604

participated the metabolism of T. cutaneum 2.1374 for production of lipid and seemed that
there was correlation between the fermentation lag time and the acetic acid. In other words,
the fermentation and lipid production started when the residual acetic acid was consumed to
below a certain value (about 2–3 g/L). The result was in agreement with that acetic acid as
the sole inhibition (“Screening of the Oleaginous Yeasts under the Inhibition of Organic
Acids”). Figure 4e showed that lipid in medium and lipid in cell were much lower than the
fermentation in minimum nutritious medium. And with the increasing of hydrolysate
content, the inhibition was much stronger. When the hydrolysate content was to 80%, the T.
cutaneum 2.1374 creased to grow completely.

Discussion

In this paper, the most adopted oleaginous strains were screened based on their inhibitory
performance of the lignocellulose degradation compounds. The tolerance and stress of the
degradation inhibitors on the sugar utilization of lignocellulose-derived glucose and xylose,
the cell growth, and lipid accumulation properties were quantitatively studied. Based on the
results, an optimal strain T. cutaneum 2.1374 tolerant to the degradation products was
screened for the use of lignocellulose hydrolysate fermentation. Seven typical lignocellu-
lose degradation products formed in various pretreatment and hydrolysis processing were
selected as the model inhibitors, including three organic acids: acetic acid, formic acid, and
levulinic acid; two furan compounds: furfural and 5-HMF; two phenol derivatives: vanillin
and hydroxybenzaldehyde. These compounds represent the major degradation products from
lignocellulose. The effect of these compounds on the ethanol fermentation strains were already
well characterized, but the effect on oleaginous yeast strains remained almost untouched.
The minimum nutritious medium under the nitrogen-limiting condition were applied to
carry out all the experiments without adding any expensive complex ingredients such as
yeast extract, peptone, etc. The addition of these expensive ingredients certainly improved
the fermentation performance and lipid productivity, but the high cost made the lipid
produced too expensive to be used as biodiesel feedstock. We clearly and consistently keep
on using the minimum nutritious medium in which glucose and xylose were the sole carbon
source, ammonia sulfate was the sole nitrogen source, and two inorganic salts (KH2PO4,
MgSO4). The selected oleaginous yeast strains were prescreened using the minimum
nutritious medium under the nitrogen limiting conditions. Four of the ten oleaginous strains
were deleted because of their poor performance on the cell growth and lipid production
in the minimum nutritious medium. The rest of the six strains were selected for the
further inhibitory effect experiment, including T. cutaneum 2.1374, R. toruloides 2.1389,
L. starkeyi 2.1390, L. starkeyi 2.1608, R.glutinis 2.704, and R. glutinis 2.107.
For the inhibitory effect of organic acids, acetic acid was found to be the strongest
inhibitors among the three selected organic acids. T. cutaneum 2.1374 was the only survival
among the six candidates under 5 g/L of acetic acid. It should be noticed that acetic acid is
the inevitable compound from the hemicellulose hydrolysis, and 5 g/L acetic acid is just a
low index for the lignocellulose hydrolysate (depending on the different pretreatment, the
concentration of acetic acid may reach as high as 10–20 g/L [18]). Formic acid is also a
strong inhibitor, and again, T. cutaneum 2.1374 showed the best tolerance to it. The
inhibition on the selected oleaginous yeasts by levulinic acid was weak compared to acetic
acid and formic acid. For T. cutaneum 2.1374, no obvious inhibitory effect was observed on
its cell growth and lipid production.
Appl Biochem Biotechnol (2009) 159:591–604 603

For the inhibitory effect of furan compounds, the xylose degradation furfural was found
to be a strong inhibitor. Most strains ceased to grow under the minimum presence of
furfural with the only exception of T. cutaneum 2.1374. On the other hand, 5-HMF, the
glucose degradation product and a furan derivative with similar molecular structure to
furfural, was found not a strong inhibitor, although the cell growth and lipid production
decreased with the increasing 5-HMF concentration. No obvious inhibitory effect for
T. cutaneum 2.1374 by 5-HMF was observed on its cell growth and lipid production.
For the inhibitory effect of phenol derivatives, vanillin was found to be a strong inhibitor
similar to acetic acid and furfural. All strains ceased to grow except T. cutaneum 2.1374.
Hydroxybenzaldehyde was a weak inhibitor to the selected strains, and again, T. cutaneum
2.1374 showed the best tolerance to hydroxybenzaldehyde.
Based on the results of the inhibitory experiment above, T. cutaneum 2.1374 always was
the most adopted strain for each group of the inhibitors from lignocellulose degradation
compounds, either organic acid, furan compounds, or phenol derivatives, among the
selected oleaginous yeast strains. The strain was further tested in the real corn stover
hydrolysate for production of lipid, and the results showed that the combinational inhibitory
by the full spectrum of lignocellulose degradation products in the hydrolysate were
strengthened comparing to that of the single inhibition, indicated by the lag time before the
lipid fermentation started. It was worth notifying that T. cutaneum 2.1374 was able to
metabolize acetic acid, formic acid, levulinic acid slowly simultaneously with the glucose
or xylose consumption. In the hydrolysate fermentation, the fermentation lag time was
found to be closely related to the concentration of acetic acid. Only acetic acid was reduced
to a certain value, the fermentation could start and the lipid began to accumulate. However,
although acetic acid and other organic acids participated the metabolism, the lipid did not
increased correspondingly to the organic acid consumption rate, indicating the metabolic
pathway of organic acids for T. cutaneum 2.1374 is complicated than that of the sugars.
As an environmental organism usually used for the treatment of waste water, the
performance of T. cutaneum 2.1374 for the tolerance of lignocellulose degradation products
was understandable. The outstanding tolerance to inhibitors, the efficient uptake of the
minimum nutritious substances, and the sufficient utilization of glucose and xylose which
were the major fermentable sugars from the hydrolysis of lignocellulosic biomass made T.
cutaneum 2.1374 to be one of the candidates of industrial importance for lipid production
using lignocellulose material. The further research on T. cutaneum 2.1374 and its mutant
strains is going on in our laboratory for production of lipid using lignocellulose hydrolysate
as carbon source. A very preliminary estimation is made as following: the current market
price of diesel or biodiesel is about $1100/ton. Since the lipid oil cost generally takes 70–
80% of the total biodiesel cost, the price for the lipid for production of biodiesel should be
around $900/ton. On the other hand, the cost for fermentable sugars from the
lignocellulosic feedstock is approximately $180/ton based on our hydrolysis experiment
(data not shown). The current yield from sugars to lipid in this study is approximately 10%,
such the cost for the lipid is approximately $1,800/ton. That means that at the present
fermentation level, the cost of oleaginous yeast lipid is almost doubled than the current
economic price of the lipid in biodiesel production. To serve the meaningful biodiesel
feedstock, the oleaginous yeast lipid should cut at least 50% of its production cost.

Acknowledgement This research was supported by Ministry of Education of China (Grant No. 107123),
PetroChina Co. Ltd (Grant No. 060511-4-7), and China National Special Fund for State Key Laboratory of
Bioreactor Engineering (Grant No. 2060204).
604 Appl Biochem Biotechnol (2009) 159:591–604

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