Biotechnology DOI 10.1002/biot.201000301 Biotechnol. J.

2011, 6, 286–299
Journal

Review

Stress-related challenges in pentose fermentation to ethanol

by the yeast Saccharomyces cerevisiae

João R. M. Almeida1*,**, David Runquist1*, Violeta Sànchez i Nogué1*, Gunnar Lidén2*,

Marie F. Gorwa-Grauslund1*
1 Applied Microbiology, Lund University, Lund, Sweden
2 Chemical Engineering, Lund University, Lund, Sweden

Conversion of agricultural residues, energy crops and forest residues into bioethanol requires hy-
Received 6 October 2010
drolysis of the biomass and fermentation of the released sugars. During the hydrolysis of the hemi- Revised 17 December 2010
cellulose fraction, substantial amounts of pentose sugars, in particular xylose, are released. Fer- Accepted 20 December 2010
mentation of these pentose sugars to ethanol by engineered Saccharomyces cerevisiae under in-
dustrial process conditions is the subject of this review. First, fermentation challenges originating
from the main steps of ethanol production from lignocellulosic feedstocks are discussed, followed
by genetic modifications that have been implemented in S. cerevisiae to obtain xylose and arabi-
nose fermenting capacity per se. Finally, the fermentation of a real lignocellulosic medium is dis-
cussed in terms of inhibitory effects of furaldehydes, phenolics and weak acids and the presence
of contaminating microbiota.

Keywords: Contamination · Ethanol · Inhibitors · Pentose fermentation · Saccharomyces cerevisiae

1 Introduction cluding both natural pentose fermenting yeasts

The hemicellulose fractions of many non-food bio-
mass resources that can be used for sustainable
biofuel production – including major agricultural
residues such as corn stover, wheat straw and sug-
ar cane bagasse – are pentose-rich (Table 1). Con-
version of pentoses after hydrolysis can be
achieved using a variety of either natural ferment-
ing or engineered microorganisms such as engi-
neered Escherichia coli [1], Zymomonas mobilis [2],
thermophilic bacteria such as Thermoanaerobac-
terium ethanolicus [3] or Th. saccharolyticum [4],
fungi such as Rhizopus oryzae [5] and yeasts in-
Correspondence: Professor Gunnar Lidén, Chemical Engineering, Lund
University, PO Box 124, 221 00 Lund, Sweden
E-mail: gunnar.liden@chemeng.lth.se
Fax: +46-46-149156
such as Pichia stipitis, Candida shehatae or Pachy-
solen tannophilus [6] or metabolically engineered
Saccharomyces cerevisiae (recently reviewed by [7,
8]). This review discusses stress challenges in pen-
tose fermentation of hydrolysates using recombi-
nant S. cerevisiae.
2 Challenges in lignocellulosic fermentation
The biological conversion route of lignocellulose to
ethanol consists of a pretreatment step, an enzy-
matic hydrolysis step, a fermentation step and a fi-
nal recovery of the formed ethanol [9]. A summary
of the challenges faced by the yeast S. cerevisiae
during the production of ethanol from lignocellu-
losic feedstocks is shown in Fig 1. The objective of
the pretreatment – normally a high temperature

Abbreviations: HMF, 5-hydroxymethyl-2-furaldehyde; LAB, lactic acid bacte-

ria; PPP, pentose phosphate pathway; SSF, simultaneous saccharification ** The authors contributed equally to this work.
and fermentation; SSL, spent sulphite liquor; XDH, xylitol dehydrogenase; ** Current address: EMBRAPA Agroenergy, PqEB, Brasilia, 70770-901 DF,
XI, xylose isomerase; XK, xylulokinase; XR, xylose reductase Brazil

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Table 1. Reported composition (g/100 g dry matter) of some pentose-rich lignocellulosic feedstocks

Material Glucan Mannan Galactan Xylan Arabinan Lignin Acetyl Reference

Corn stover (American) 36.1 1.8 2.5 21.4 3.5 17.2 3.2 [108]
Corn stover (Italian) 36.8 0.3 2.9 22.2 5.5 21.2 1.7 [108]
Sugar cane bagasse 43.3 NR NR 24.3 2.0 22.8 2.0 [109]
Wheat straw 41.2 NR NR 26.1a) – 19.1 4.2 [110]
32.6 0.8 20.1 3.3 26.5 2.0 [111]
Barley straw 36.8 NR 2.2 17.2 5.3 14.3 NR [112]
Rice straw 34.2 NR NR 24.5 NR 11.9 NR [113]
36.6 NR NR 16.1 NR 14.9 NR [114]
Switch grass 34.2 0.5 1.5 23.3 2.0 19.9 2.4 [115]
Salix 41.5 3.0 2.1 15.0 1.8 25.2 NR [116]
a) Including arabinan.
NR, not reported.

treatment at 180–210°C for some minutes – is to fa-
cilitate the enzymatic hydrolysis by making the cel-
lulose more accessible for enzymatic degradation.
Acid catalysts (e.g. sulphuric acid, SO2 or carboxylic
acids) improve the hydrolysis of hemicellulose,
whereas base catalysts (NaOH, ammonia, lime), or-
ganic solvents or oxygen facilitate partial solubi-
lization of lignin [10]. The pH value must be ad-
justed after pretreatment, and this will lead to the
formation of salt, which can cause osmotic stress
for the yeast. The osmotic stress will trigger glyc-
erol formation, affect membrane transport pro-
cesses and slow down growth [11]. Acetyl groups
cleaved off from the hemicelluloses will be found in
the hydrolysate as acetate/acetic acid. The acetyl
content is normally higher in pentose-rich materi-
als than in materials with a low pentose content
(such as softwoods). In particular glucuronoxylans
in hardwoods are acetylated to a higher degree
than the galactoglucomannans typical of soft-
woods.The stress caused by acetic acid is thus more
important in xylose-rich hydrolysates. Furaldehy-
des, primarily 2-furaldehyde from pentoses and
5-hydroxymethyl-2-furaldehyde (HMF) from hex-

Figure 1. Challenges faced by the yeast S. cerevisiae during the production of ethanol from lignocellulosic feedstocks.

© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 287

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oses, are formed by dehydration of the liberated
sugar monomers at high temperatures and acidic
conditions, and a fraction of the furaldehydes is
also likely to be further degraded to other organic
acids – e.g. levulinic acid and formic acid (as re-
viewed in [12]). The lignin fraction of the biomass
gives rise to different phenolics after pretreat-
ment, e.g. phenylpropanoid derivatives, such as
p-coumaric and ferulic acid, or phenolics without
the propanoid side chain [13].The hydrolysis of the
pretreated material may take place together with
the fermentation, i.e. the so-called simultaneous
saccharification and fermentation (SSF) [14]. Fur-
thermore, the liquid fraction after pretreatment
may either be separated before the hydrolysis, or
treated together with the solid fraction. If the enzy-
matic hydrolysis takes place as a separate step, a
temperature of 40–50°C is used, in agreement with
the optimum temperature for the currently avail-
able cellulases. However, a lower temperature must
be used in the SSF to stay within the allowable tem-
perature for the yeast. An improved yeast thermo-
tolerance would thus be beneficial from a process
point of view.
Also the end-product of the fermentation,
ethanol, is in itself inhibitory at high levels (see [15]
for a recent review), but in its present stage of de-
velopment, ethanol concentrations in lignocellu-
lose-based processes using pentoses seldom ex-
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Biotechnol. J. 2011, 6, 286–299
ceed 5% by weight, and inhibition by ethanol is
therefore minor compared to that of many other
compounds.
As described above, almost each process step
gives rise to environmental stress factors that may
impact the ethanol yield or productivity in the fer-
mentation. In addition, the presence of highly con-
taminated feedstock and non-sterile conditions
(see e.g. [16]) introduce an additional challenge of
competition with contaminating microorganisms
for the available sugars. Finally, the metabolic engi-
neering of the yeast needed to convey pentose-util-
ising capacity adds genetic factors. The combina-
tion of the environmental and genetic factors result
in the physiological changes shown in Figure 1.
3 Metabolic engineering for efficient
fermentation and anaerobic growth
on pentose sugars
3.1 Xylose utilization
Utilization of the pentose sugar xylose has been
enabled in S. cerevisiae by expression of the het-
erologous enzymes xylose reductase (XR) and xyl-
itol dehydrogenase (XDH) or xylose isomerase (XI)
[7, 17] (Fig. 2). Both non-targeted and targeted
metabolic engineering approaches have then been
Figure 2. Xylose and arabinose pathways expressed in
recombinant S. cerevisiae. The utilization of cofactors and ATP
in the central carbon metabolism is depicted schematically.
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Biotechnol. J. 2011, 6, 286–299
followed to further improve xylose utilization, as
reviewed below.
3.1.1 Improvement of xylose utilization by non-target
approaches
Random mutagenesis and evolutionary engineer-
ing has been successfully applied to increase xy-
lose utilization in a number of recombinant S. cere-
visiae strains. The strains H2490-4 [18], TMB3400
[19] and C1 [20] were constructed based on the
oxido-reductive XR/XDH pathway (Fig. 2) and were
selected during prolonged chemostat cultivation
following chemical mutagenesis (TMB3400 and C1)
(Fig. 3A). The strain RWB218 was constructed us-
ing the XI pathway (Fig. 2) and was selected in
chemostat followed by sequential batch cultivation
(Fig. 3A) [21].All strains had significantly improved
ethanol productivity and growth rate on xylose,
compared to their parental strains. Enzymatic
analysis revealed increased activity of the heterol-
ogous enzymes XR and XDH in the strains C1 and
TMB3400, whereas the activity of several enzymes
in the pentose phosphate pathway (PPP) (Fig. 2)
was increased in the strain H2490-4 [18, 22]. These
observations were further corroborated by prote-
ome analysis, which identified higher expression of
XR, XDH and the non-oxidative PPP in TMB3400
compared to the parental strain [23]. Transcrip-
tome analyses of the strains TMB3400 and C1 were,
A
Notation Strain
in Fig. 3
1 C1
2 TMB 3415
3 RWB202-AFX
4 TMB3421
5 TMB 3420
6 RWB 217
7 RWB 218
B
0,6
0,5
6
re (g/gDW×h)
0,4
5
0,3
0,2 4
1 23
0,1
0 xylose. (B) Relationship between the
0 0,05
Specific growth rate (h-1)
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
however, unsuccessful in identifying alterations at
a single control point, but rather suggested an ad-
ditive effect of several small mutations in the
genome [24]. The strain RWB218 was not charac-
terized at the transcriptome level, however in-
creased transport affinity was detected in kinetic
studies, which possibly resulted from increased
expression of high-affinity native transporters
[21].
3.1.2 Improvement of xylose utilization by rational
pathway design
Rational design of the xylose utilization pathway in
S. cerevisiae is based on identifying, altering and ul-
timately moving metabolic control points. A major
controlling step in xylose utilization is the expres-
sion level of the heterologous enzymes XR/XDH
and XI. Specifically, the low enzyme activity and
substrate affinity of XR and XI requires high intra-
cellular xylose concentration to push the rate of the
reaction, which leads to decreased ethanol produc-
tivity as substrate is consumed [25]. Overexpres-
sion of XR somewhat alleviated this problem and
increased the rate of xylose consumption substan-
tially [26]. Similarly, very high expression level of
XI encoding gene from episomal plasmids was re-
quired for anaerobic growth on xylose by the strain
RWB202-AFX (Fig. 3A) [27].
Relevant genotype Additional step(s) Reference
XYL1, XYL2, XKS1 Chemical mutagenesis [20]
XYL1 (K270R), XYL2, XKS1, PPP – [46]
XI multicopy – [27]
XYL1 (N272D P275Q), XYL2, XKS1, PPP – [121]
XYL1 (N272D P275Q), XYL2, XKS1, PPP Sequential batch selection [121]
XI multicopy, XKS1, PPP – [36]
XI multicopy, XKS1, PPP Batch and chemostat selection [21]
7
Figure 3. (A) Engineered S. cerevisiae
strains capable of anaerobic growth on
0,1 0,15
ethanol productivity (re) and the anaerobic
growth rate on xylose.
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Additional control points in xylose metabolism
are the endogenous phosphorylating enzyme xylu-
lokinase (XK), and the PPP, respectively (Fig. 2).
Since XK uses ATP as a cofactor, the expression lev-
el of this enzyme needs to be carefully optimized
[28–30]. Overexpression of XK improved the con-
version of xylulose [31]. However, without concur-
rent overexpression of XR, xylose utilization was de-
creased [28, 30]. The non-oxidative PPP follows af-
ter XK in the conversion of xylose to glycolytic in-
termediates (Fig. 2). Compared to glycolysis, the PPP
is kinetically constrained since the PPP reactions
are less thermodynamically favourable and operate
close to the equilibrium [32]. Fermentation of xylu-
lose to ethanol in native S. cerevisiae is thus orders
of magnitude slower than that of glucose [31, 33].
When all genes of the non-oxidative PPP were over-
expressed, the xylulose utilization increased, but xy-
lose conversion was not improved unless XR and
XDH activities were raised [34, 35]. A considerably
higher ethanol productivity was also observed when
high XI level was combined with high expression of
XK and non-oxidative PPP encoding genes in
strains RWB 217 and RWB 218 (Fig. 3A) [27, 36].
In S. cerevisiae, xylose is transported via the
hexose transporters, however with a much lower
affinity, which limits its utilization in the presence
of glucose [37]. On xylose medium however, it was
initially shown that xylose transport did not control
the flux of the pathway in a recombinant S. cere-
visiae strain [25]. However, when XR was over-
expressed the control coefficient of transport in-
creased [38], and in a strain where the PPP was
overexpressed, increased transport improved xy-
lose utilization at least when the substrate concen-
tration was below 4 g/L [39].
For S. cerevisiae strains employing the oxido-re-
ductive xylose pathway, an additional control point
is the imbalanced co-factor preference of the two
recombinant enzymes XR and XDH. Since XR
prefers NADPH and XDH is strictly NAD+ depend-
ent, the cell creates an excess of NAPD+ and a de-
ficiency in NADH which leads to the production of
xylitol as a byproduct (Fig. 2) and lower ethanol
yield [25, 40]. The cofactor specificity of XR and/or
XDH has thus been modified by protein engineer-
ing for balanced cofactor usage of the two enzymes
[41–44]. In this way the ethanol productivity, and
consequently the anaerobic growth rate, was dras-
tically improved for strains TMB 3415, TMB 3420
and TMB 3421 utilizing mutant XRs (Fig. 3) [45, 46].
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3.2 Arabinose utilization
Arabinose utilization has been investigated to less
extent than xylose utilization due to the lower
abundance of arabinose in lignocellulosic biomass
(Table 1). Arabinose can be utilized by S. cerevisiae
via the introduction of either an isomerization
pathway consisting of L-arabinose isomerase (AI),
L-ribulokinase (RK) and L-ribulose-5-P 4-epimer-
ase (RE) or a reduction/oxidation-based pathway
consisting of XR, L-arabinitol 4-dehydrogenase
(LAD), L-xylulose reductase (LXR) and XDH (Fig.
2) [7]. Although arabinose utilization is consider-
ably less efficient than xylose, it shares several con-
trol points with xylose utilization since both het-
erologous pathways end with the formation of xy-
lulose (Fig. 2). Arabinose utilization thus benefits
from high expression of the genes from the initial
heterogonous arabinose-catabolizing enzymes
[47], the endogenous XK, the non-oxidative PPP as
well as high affinity native transporters [48].
In practice, the fermentation of the arabinose
fraction in lignocellulose hydrolysates cannot be
disconnected from the fermentation of xylose,
which raises a set of new challenges. For instance,
XR displays a similar affinity for xylose and arabi-
nose [49, 50]. Thus, in S. cerevisiae strains combin-
ing the oxidation/reduction pathway for xylose and
the isomerization pathway for arabinose-utiliza-
tion, almost all consumed arabinose was converted
to arabitol (arabinitol) by XR (Fig. 2) [51–53]. Ara-
bitol formation can be avoided by using the XI-
based pathway for xylose utilization, which does
not include XR. Nevertheless this approach proved
difficult since an elaborate selection procedure was
required to enable arabinose utilization without
lowering the capacity of the same strain for xylose
utilization [54, 55]. Arabitol formation can also be
reduced by employing the oxidation/reduction
pathways for both xylose- and arabinose-utiliza-
tion (Fig. 2), so that arabitol can be further metab-
olized to xylitol and ethanol. An oxidation/reduc-
tion pathway for arabinose metabolism has thus
been described [56, 57] and its performance was re-
cently improved using a suitable combination of
enzymes [58].
4 Pentose fermentation in hydrolysates
In reported studies in which pentose-fermenting
strains of S. cerevisiae have been evaluated in un-
detoxified pentose-rich lignocellulosic hydrolysa-
tes (Table 2), the maximum ethanol concentrations
obtained did not exceed 45 g/L and in many cases,
the xylose conversion was not complete even after
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Table 2. Some studies in which pentose fermenting S. cerevisiae has been used for the conversion of lignocellulosic hydrolysates

Strain Raw material Pretreatment and Ethanol concentration and Reference

fermentation conditions
424A(LNH-ST) Sweet sorghum AFEX pretreatment (2:1 ammonia to
(XR/XDH/XK) bagasse biomass loading, 140°C, 30 min)
followed by enzymatic hydrolysis.
Approximate liquid concentrations:
glucose 65 g/L, xylose 33 g/L.
Initial yeast concentration 0.3 g/L
xylose conversion
Highest ethanol concentration [117]
reached 42.3 g/L, with a xylose
conversion of 55.6% (after about 75 h).
Very little xylitol formation.

424A(LNH-ST) Poplar Comparison of many different Highest ethanol concentration reached [84]
(XR/XDH/XK) pretreatments including AFEX, after 50 h: 40 g/L (lime pretreatment)
oxidative alkali, SO2 catalysed steam with about 90% conversion xylose;
pretreatment, followed by enzymatic SO2 steam explosion yielded 26 g/L
hydrolysis. Initial liquid concentra- ethanol, about 90% xylose conversion.
tions (AFEX): glucose 62.3 g/L,
xylose 16.2 g/L, acetic acid 3.5 g/L.
Initial liquid concentrations (steam
pretreatment SO2) glucose 33.2 g/L,
xylose 25.8 g/L, acetic acid 6.2 g/L.
Initial yeast concentration about 5 g/L

TMB3400 Corn stover SSF Ethanol concentration of 36.8 g/L [78]

(XR/XDH/XK + Steam pretreatment at 200°C, 5 min, reached at 11% WIS using fed-batch
mutagenesis and SO2 catalysed. Liquid composition: addition. Residual xylose concentration
selection) glucose 7.2, xylose 36, acetic acid, in liquid about 10 g/L after 96 h
2.2 g/L, HMF 0.2 g/L g, furfural 1.5 g/L.
Substantial amounts of xylan remaining
in the solid material (6.6–9.2%), which
was degraded during SSF.
Initial yeast concentration: 5 g/L.

TMB3400 Wheat straw SSF A final ethanol concentration of 38 g/L [118]

(XR/XDH/XK) Steam pretreatment, dilute H2SO4 and a xylose conversion about 40%
catalyst, 210°C, 2.5 min. Liquid in fed-batch up to 9% WIS.
composition after pretreatment: Corresponding for fed-batch at 7%:
glucose 10.9 g/L, xylose 51.2 g/L, ethanol concentration of 34 g/L and
acetic acid 5 g/L, HMF 0.6 g/L, a xylose conversion of 74%.
furfural, 1.7 g/L. Xylan content in
fibre about 3.5%.
Fed-batch fermentation at a total solids
loading of 9%WIS. Initial yeast
concentration 4 g/L

TMB3400 Sugar cane SSF

(XR/XDH/XK) bagasse Steam pretreatment at 190°C, 5 min,
SO2 catalysed. Liquid composition
after pretreatment: glucose 4.6 g/L,
xylose 39.5 g/L, arabinose 2.7 g/L,
furfural 0.9 g/L, acetic acid 4.2 g/L.
Yeast concentration 4 g/L.
Ethanol concentration 26 g/L. [119]
Conversion of xylose about 40% at
7.5%WIS (Ethanol concentration
20 g/L, and xylose conversion 88%
at 5%WIS).

TMB3400 Sugar cane Steam pretreatment at 190°C, 5 min, Only hemicellulose part fermented. [109]
(XR/XDH/XK) bagasse SO2 catalysed. Liquid concentrations: Highest xylose conversion for liquid
15 g/L xylose, 4 g/L glucose. from pretreatment 65% after 20 h.
Initial yeast concentration 5 g/L

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Table 2. Continued

Strain Raw material Pretreatment and Ethanol concentration and Reference

fermentation conditions xylose conversion
MA-R4 Eucalyptus Primarily mechanical pretreatment Ethanol concentration 37.2 g/L. [120]
(XR/XDH/XK (ball milling) followed by enzymatic Conversion of 90% of the xylose
introduced into hydrolysis at high cellulase loadings. after 48 h
flocculating industrial Liquid composition: 61.1 g/L glucose,
strain IR-2) 13.0 g/L xylose, 1.2 g/L galactose,
1.0 g/L mannose, 0.6 g/L arabinose,
10 g/L yeast extract added (rich medium).
Low inhibitor concentration
Relatively high yeast density –
final value 14.3 g dw/L

RWB2182 Wheat straw Steam explosion pretreated followed Ethanol concentration: 38.1 g/L. [17]
(XI) by enzymatic hydrolysis, batch About 90% of xylose converted
Strain improved fermentation. Liquid composition: after 55 h. Almost stoichiometric
by evolution 47.8 g/L glucose, 20.7 g/L xylose, yield based on consumed sugars
3 g/L acetic acid.
Initial yeast concentration: 1.5 g/L.

RWB218 Corn stover Steam explosion pretreated Ethanol concentration about 18 g/L. [17]
(XI) (190°C, 5 min), followed by enzymatic About 90% of xylose converted
hydrolysis. Liquid composition: after 48 h.
40 g/L glucose, 9 g/L xylose,
4 g/L acetic acid. Fed-batch + batch
fermentation.
SSF, simultaneous saccharification and fermentation; WIS, water insoluble solids; AFEX, ammonia fibre expansion.

a long period of time. The specific xylose consump-
tion rates obtained in hydrolysates were also clear-
ly lower than those on synthetic media.
4.1 Effect of aldehyde inhibitors on xylose
fermentation
The toxicity of lignocellulosic hydrolysates has
been correlated with the presence and concentra-
tion of various aldehydes, such as furfural [59–61].
Among 60 identified phenolic compounds in ligno-
cellulosic hydrolysates, the phenylaldehydes were
found to be more toxic than the phenolic acids and
alcohols [62, 63] underlining the importance of the
aldehydes as inhibitors.
Aldehydes can interact with cellular structures,
generate reactive oxygen species and inhibit en-
zymes in the central carbon metabolism [64–67].
This results in reduction of fermentation rate, ces-
sation of growth, extension of lag-phase of yeast,
and reduction of ethanol productivity and/or yield
[12, 63]. Understanding and improving the mecha-
nisms by which microorganisms respond to alde-
hydes is clearly important for the development of
strains with increased tolerance towards lignocel-
lulosic hydrolysates [12, 68].
4.1.1 Metabolically engineered microorganisms
for aldehyde reduction
A fundamental observation is that as long as cer-
tain aldehydes – e.g. furfural, HMF, vanillin, vera-
traldehyde – are present in the medium, yeasts are
not able to grow and show reduced metabolic ac-
tivity [61, 69–71]. However, at permissive levels, the
yeast can usually slowly convert the aldehydes to
less toxic compounds and growth and metabolic ac-
tivity are restored [61, 72–74]. S. cerevisiae, for in-
stance, can reduce furfural and HMF to their corre-
sponding alcohols; 2-furan methanol (FM or fur-
furyl alcohol) and furan 2,5-dimethanol (FDM or
HMF alcohol). A few alcohol dehydrogenases, i.e.
Adh6, Adh7 and a mutated Adh1 enzyme, as well as
the P. stipitis XR (Ps-XR) and the YKL071w gene
product have been identified as catalyzing furalde-
hyde reduction, and their positive effect upon over-
expression of the corresponding gene has been
confirmed [68, 75]. Overexpression of the tran-
scription factor encoding gene YAP1, known to be
involved in oxidative stress response, was also
shown to give an increased tolerance to hy-
drolysate and specific model inhibitors – and an in-
creased reduction capacity of furaldehydes [76].

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More recently an isolate of the gram positive
bacteria Cupriavidus basilensis (HMF14) could aer-
obically completely metabolize HMF and furfural
[77], which may open new possibilities for the mi-
crobial detoxification of hydrolysates.
4.1.2 Interactions between xylose and furaldehyde
pathways
S. cerevisiae keeps its redox balance tightly regu-
lated, essentially by regulating the activity of gly-
colytic and fermentative enzymes. The anaerobic
glucose catabolism (when not considering draining
the pathway of anabolic precursors), is balanced in
the glycolysis and fermentation pathways with re-
spect to generation/consumption of the co-factor
pairs NADH/NAD+ (and NADPH/NADP+). Howev-
er, during the fermentation of lignocellulosic hy-
drolysates there are additional compounds which
will be reduced and/or oxidized: (i) the conversion
of xylose by the XR/XDH pathway from P. stipitis is
based on the use of NADPH and NAD+ (Fig. 2);
(ii) the detoxification of aldehydes is based on re-
duction reactions using NADH and NADPH. Con-
sequently, both xylose metabolism and detoxifica-
tion reactions will affect the redox (or more cor-
rectly the co-factor) balance of the cell.
Ethanolic fermentation with S. cerevisiae strains
overexpressing the XR/XDH pathway in defined
mineral medium containing xylose often gives sub-
stantial yields of xylitol as a result of the co-factor
imbalance between the NADPH-preferring XR and
the NAD+-dependent XDH (cf. Fig. 2). However,
fermentation of xylose in lignocellulosic hydroly-
sates by engineered S. cerevisiae strains showed
considerably lower xylitol yields than during the
fermentation of xylose in defined mineral medium
[78, 79]. Most likely this is due to aldehydes and
other compounds in the lignocellulosic hydrolysate
which act as redox sinks [80, 81]. For instance, an
NADH-dependent reduction of furfural regener-
ates NAD+ necessary for xylitol oxidation by XDH.
Thereby glycerol and/or xylitol yields may de-
crease [81].
The simultaneous conversion of aldehyde com-
pounds and xylose was investigated in an XR/XDH
strain overexpressing either the NADH-dependent
ADH1-S110P-Y295C encoding gene or the NADPH-
dependent Adh6 encoding gene [82]. NADPH us-
age during HMF reduction slightly reduced xylitol
formation, whereas the usage of NADH in HMF re-
duction considerably decreased both xylitol and
glycerol production and increased biomass yield
[82].A metabolic flux analysis indicated that the re-
duction of HMF replaced the regeneration of NAD+
in the glycerol pathway and provided NAD+ for the
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oxidation of xylitol so that more carbon became
available for biomass production.
4.2 Pentose fermentation in the presence
of weak acids
Ethanol fermentation from lignocellulosic feed-
stocks is also challenged by the presence of weak
acids in the medium, at concentrations that can be
inhibitory to S. cerevisiae metabolism. Acetic acid
originates from the deacetylation of the hemicellu-
lose fraction, whereas formic and levulinic acids
are formed during the breakdown of HMF and fur-
fural [83]. The innate levels of acetic acid are high-
ly dependent on the raw material. Reported acetyl
contents in some pentose rich materials are in the
range 1.7–4.2 g/100 g DM (Table 1). The concentra-
tions of weak acids obtained in the fermentation
broth will vary even more widely depending on
both the method of pretreatment as well as the
solids loading used in the pretreatment. It can be
estimated that the concentration of acetic acid may
easily be above 10 g/L after a typical steam pre-
treatment of poplar [84].
Weak acids are also produced during the fer-
mentation stage, since acetic acid is a minor by-
product of yeast fermentation. However, acetic and
lactic acids are mainly generated from the contam-
ination by lactic acid bacteria (LAB) and other bac-
teria (see also Section 4.3). For example, acetic and
lactic acid levels above 12 and 16 g/L, respectively,
have been reported in a pilot-scale study using
corn fibre as substrate (LAB) [16, 85].
4.2.1 Effect of weak acids on yeast metabolism
Both acetic and lactic acid are known to increase
the lag phase and reduce the specific growth rate of
S. cerevisiae, thereby affecting ethanol productivity
(see e.g. [86–89]. Acetic acid is more inhibitory than
lactic acid, with a reported minimal inhibitory con-
centration for growth of 100 mM (6 g/L), as com-
pared to 278 mM (25 g/L) for lactic acid in non-
buffered defined medium [86].
The effects of weak acids are strongly pH de-
pendent. At pH value below the pKa-value of the
acid, the undissociated form of weak acids predom-
inates. It is this form which enters the cell and once
inside it dissociates (due to the higher intracellular
pH), thereby releasing protons. In order to main-
tain a proper intracellular pH, protons must be
transported out of the cells with the help of the
plasma membrane ATPase and at the expense of
ATP (Fig. 2). Whereas low levels of acids activate
the glycolytic rate by stimulating ATP production
[89], higher levels become inhibitory due to the
acidification of the cytosol after depletion of the
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Journal
available ATP. Also, as less ATP becomes available
for biomass formation, ethanol productivity de-
creases. In addition, high levels of weak acids may
result in the intracellular accumulation of anions
(Fig. 2) [90], which has been shown to be inhibito-
ry to several glycolytic enzymes [91]. ATP is also
needed for the active efflux of the accumulating
dissociated acids, which has been suggested to oc-
cur via Pdr12p [92].
4.2.2 Xylose fermentation in the presence
of weak acids
The effect of acetic acid on xylose fermentation has
been investigated in strains carrying either the XR-
XDH or the XI pathway [84, 93–96]. A high level of
acetic acid with a low pH caused dramatic reduc-
tion of xylose consumption rate for an XR-XDH
strain, whereas a change in pH alone showed little
effects on the xylose consumption [84]. Growth and
ethanol production from xylose were also impaired
in a recombinant XI-expressing strain at low pH
(3.5) and in the presence of 3 g/L acetic acid [93],
whereas the glucose consumption rate was only
slightly affected under similar conditions.
The lower rate of xylose consumption, typically
three to sixfold lower than for glucose in engi-
neered S. cerevisiae [93, 94] affects the maximum
specific ATP production rate, which explains why
weak acids have a more dramatic effect on yeast
growth on xylose than on glucose [93–95]. Indeed
xylose fermentation could be restored in a recom-
binant XI-strain at pH 3.5 and in the presence of
3 g/L acetic acid by applying a continuous low feed-
ing of glucose to the medium [93].
Acetic acid decreases ethanol productivity but
its effect on ethanol yield is less clear. In some re-
ports, the ethanol yield was shown to increase as
the metabolism was redirected from anabolic (bio-
mass) to dissimilatory (ethanol) pathway in the
presence of acetic acid [93, 94]. In contrast, a lower
ethanol yield from glucose and xylose was also re-
ported in the presence of a more complex medium
such as spent sulphite liquor (SSL) [96]. However,
it is unclear whether the reported yield relates to
the consumed or the initial sugar levels, since the
tested XR and XDH-engineered strains displayed
low xylitol and unchanged glycerol yields in acetic
acid rich-hydrolysate media [84, 96].
4.2.3 Engineering approaches to overcome the weak
acid inhibition
One straightforward strategy to overcome or limit
the inhibition by acids is to run fermentations at a
pH value sufficiently high to keep the undissociat-
ed acids at low levels. In practice however, it may be
difficult to implement this strategy as fermentation
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Biotechnol. J. 2011, 6, 286–299
of lignocellulosic hydrolysates are preferably run
at a low pH to limit bacterial contaminations (Sec-
tion 4.3).
Recently, S. cerevisiae was metabolically engi-
neered allowing acetate to replace dihydroxyace-
tone phosphate (DHAP) as a redox sink during
anaerobic growth in a glycerol mutant strain. This
was achieved by introducing a bacterial NAD+-de-
pendent acetylating acetaldehyde dehydrogenase,
by which acetyl-CoA may be reduced to acetalde-
hyde (and CoA). The engineered strain was able to
grow anaerobically and acetate was reduced to
ethanol in the process [97]. Such approach may pave
the way to other strategies enabling the conversion
of the inhibitory acetate during fermentation.
4.3 The contamination issue
Large scale plants for the production of ethanol
from lignocellulosic biomass are not yet available.
However, one can anticipate that microbial compe-
tition will become an important problem, due to the
presence of a large amount of pentose sugars that
are still not, or only poorly, utilized by S. cerevisiae.
4.3.1 Microbial contaminants in lignocellulosic
hydrolysates
Systematic and acute bacterial infections are re-
currently observed in large scale facilities during
the ethanol production from hexose sugars, with
sugar cane in Brazil [98] and corn in United States
of America [99]. However, little is known on the
contaminant microbiota in lignocellulosic ethanol
pilot plants. The most studied cases of contamina-
tion are in SSL, which is the waste stream of cellu-
lose pulp that has been obtained by treating the
wood mechanically and chemically. The SSL has a
high sugar content which can be used as feedstock
for various microorganisms.
Both yeast and bacterial contaminants have
been isolated and identified in SSL plants, includ-
ing the xylose-utilising yeast Pichia membranaefa-
ciens, the acetic acid bacteria Acetobacter tropicalis
and A. syzygii, the homofermentative bacteria Lac-
tobacillus plantarum and L. pentosus as well as the
heterofermentative L. buchneri [100] (C. Larsson
and E. Albers, personal communication; V. Sànchez
et al., unpublished). Lactobacillus contaminants
have also been identified in a pilot-plant where
corn fibres were used to produce ethanol [16]. Dur-
ing episodes of contamination in sugar cane
ethanol plants, non-S. cerevisiae strains were also
found as main contaminants [101].
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnol. J. 2011, 6, 286–299
4.3.2 Effects of microbial contamination on yeast
fermentation
LAB compete with yeast for sugar utilization. Ho-
mofermentative bacteria are able to consume hex-
ose and pentose sugars to produce lactic acid,
whereas heterofermentative bacteria produce
acetic acid in addition to lactic acid [102]. Finally
acetic acid bacteria produce acetic acid from
ethanol [103]. An immediate consequence of the
contamination is that less sugar and essential
growth factors become available for S. cerevisiae
and the ethanol yield decreases (see e.g. [104]). In
addition, the production of considerable amount of
acids decreases the pH which inhibits yeast fer-
mentation by reducing biomass formation and
ethanol yield.
4.3.3 Strategies to control a contamination
In the sugar cane ethanol industry, acid wash treat-
ment is used as a common strategy for reducing
acute episodes of contamination [105]. Pulses of
sulphuric acid are made, leading to a temporary pH
drop on the system. The bacterial population is
then reduced due to its high sensitivity for low pH
[106].
Various pasteurization procedures and the ad-
dition of biocides, such as penicillin, virginiamycin
and nisin, have also been tested in a series of batch
and continuous fermentation runs in a corn fibre
based pilot plant. All these methods reduce tem-
porarily the level of contamination but none has a
permanent effect [16]. Also, the usage of biocides to
control contamination is not economically feasible
and not advisable from an environmental point of
view.
Recent studies demonstrated that the addition
of NaCl and ethanol on softwood hydrolysate could
inhibit the growth of LAB whereas baker’s yeast
was still able to grow (C. Larsson and E.Albers, per-
sonal communication).
The use of hops extract to control bacterial con-
tamination in lignocellulosic fermentation plants is
also discussed. Hop that gives a bitter taste and acts
as antiseptic agent, is well-known in beer produc-
tion. Hop cones have a high content of α-acids, for
which Gram-positive bacteria are sensitive to [106,
107].
In the near future, the development of S. cere-
visiae strains with xylose consumption rates that
are sufficiently high to compete with LAB for the
utilization of all sugars present in the lignocellu-
losic hydrolysates, may provide another solution to
the bacterial contamination issue.
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
5 Concluding remarks
Both targeted and non-targeted approaches have
been applied to generate efficient pentose-fer-
menting S. cerevisiae strains. The strength of the
non-targeted approach is that it requires less a pri-
ori knowledge of the pathway and is generally
straight forward and efficient. The targeted ap-
proach on the other hand may require tedious
identification of specific traits that enhance metab-
olism, however once identified these traits can be
directly transferred between strains. Both ap-
proaches have been successful in the generation of
S. cerevisiae strain that can grow anaerobically on
xylose, a trait which is shown to be linearly corre-
lated to ethanol productivity (Fig. 3B). Still the xy-
lose consumption rate remains three to sixfolds
lower than the glucose consumption rate. Generat-
ing tolerant S. cerevisiae with even higher xylose
consumption rate would of course benefit ethanol
productivity, but it would also enable the cells to
better cope with the ATP-demanding acid stress
and outcompete any pentose-utilizing LAB-con-
taminant in the process.
A central issue in converting pentoses from hy-
drolysates is also the presence of aldehydes – not
least the furaldehydes. Recent identification of en-
zymes with aldehyde reduction activity has enabled
targeted approaches for improving the in situ re-
duction capacity of S. cerevisiae strains. Similarly ge-
netic engineering approaches are emerging to cope
with the problem of weak acid inhibition. In both
cases, however, it is important to understand how
these modifications impact pentose metabolism.
The authors wish to thank the Swedish Energy
Agency (Energimyndigheten) for financial support.
The authors have declared no conflict of interest.
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Gunnar Lidén received his Ph.D. in
Chemical Reaction Engineering from
Chalmers University, Göteborg, Swe-
den in 1993, where he remained until
1999. At that time he moved to Lund
university to take on his current posi-
tion as professor in chemical engineer-
ing. His main research focus concerns
biotechnology for the production of
biofuels.In particular, he worked on im-
proving fermentation technology for efficient lignocellulose conver-
sion. His research group is currently involved in two large European
research projects within FP7 (NEMO and BIOLYFE) concerning these
issues.
Marie Gorwa-Grauslund is professor of
Applied Microbiology, Lund University
since 2009. After a Ph.D. in Molecular
and Cellular Genetics in 1996 at INSA
Toulouse, France and a post-doctoral
fellowship at Lesaffre (France) on the
development of stress-tolerant yeast,
she joined the Division of Applied Mi-
crobiology in 1999. Her current re-
search projects involve the identifica-
tion, development and engineering of biocatalysts for the production
of fuels and bulk chemicals and for stereo-selective whole cell bio-
transformations.
strain using evolutionary engineering. Biotechnol. Biofuels
2010, 3, 13.
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