Download Stressrelated challenges in pentose fermentation to ethanol

Biotechnology
Journal
DOI 10.1002/biot.201000301
Biotechnol. J. 2011, 6, 286–299
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
2
Applied Microbiology, Lund University, Lund, Sweden
Chemical Engineering, Lund University, Lund, Sweden
Conversion of agricultural residues, energy crops and forest residues into bioethanol requires hydrolysis of the biomass and fermentation of the released sugars. During the hydrolysis of the hemicellulose fraction, substantial amounts of pentose sugars, in particular xylose, are released. Fermentation of these pentose sugars to ethanol by engineered Saccharomyces cerevisiae under industrial 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 arabinose fermenting capacity per se. Finally, the fermentation of a real lignocellulosic medium is discussed in terms of inhibitory effects of furaldehydes, phenolics and weak acids and the presence
of contaminating microbiota.
Received 6 October 2010
Revised 17 December 2010
Accepted 20 December 2010
Keywords: Contamination · Ethanol · Inhibitors · Pentose fermentation · Saccharomyces cerevisiae
1 Introduction
The hemicellulose fractions of many non-food biomass resources that can be used for sustainable
biofuel production – including major agricultural
residues such as corn stover, wheat straw and sugar cane bagasse – are pentose-rich (Table 1). Conversion of pentoses after hydrolysis can be
achieved using a variety of either natural fermenting or engineered microorganisms such as engineered Escherichia coli [1], Zymomonas mobilis [2],
thermophilic bacteria such as Thermoanaerobacterium 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: [email protected]
Fax: +46-46-149156
Abbreviations: HMF, 5-hydroxymethyl-2-furaldehyde; LAB, lactic acid bacteria; PPP, pentose phosphate pathway; SSF, simultaneous saccharification
and fermentation; SSL, spent sulphite liquor; XDH, xylitol dehydrogenase;
XI, xylose isomerase; XK, xylulokinase; XR, xylose reductase
286
cluding both natural pentose fermenting yeasts
such as Pichia stipitis, Candida shehatae or Pachysolen tannophilus [6] or metabolically engineered
Saccharomyces cerevisiae (recently reviewed by [7,
8]). This review discusses stress challenges in pentose fermentation of hydrolysates using recombinant S. cerevisiae.
2
Challenges in lignocellulosic fermentation
The biological conversion route of lignocellulose to
ethanol consists of a pretreatment step, an enzymatic hydrolysis step, a fermentation step and a final recovery of the formed ethanol [9]. A summary
of the challenges faced by the yeast S. cerevisiae
during the production of ethanol from lignocellulosic feedstocks is shown in Fig 1. The objective of
the pretreatment – normally a high temperature
** The authors contributed equally to this work.
** Current address: EMBRAPA Agroenergy, PqEB, Brasilia, 70770-901 DF,
Brazil
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Biotechnol. J. 2011, 6, 286–299
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Table 1. Reported composition (g/100 g dry matter) of some pentose-rich lignocellulosic feedstocks
Material
Corn stover (American)
Corn stover (Italian)
Sugar cane bagasse
Wheat straw
Barley straw
Rice straw
Switch grass
Salix
Glucan
Mannan
Galactan
Xylan
Arabinan
Lignin
Acetyl
Reference
36.1
36.8
43.3
41.2
32.6
36.8
34.2
36.6
34.2
41.5
1.8
0.3
NR
NR
2.5
2.9
NR
NR
0.8
2.2
NR
NR
1.5
2.1
21.4
22.2
24.3
26.1a)
20.1
17.2
24.5
16.1
23.3
15.0
3.5
5.5
2.0
–
3.3
5.3
NR
NR
2.0
1.8
17.2
21.2
22.8
19.1
26.5
14.3
11.9
14.9
19.9
25.2
3.2
1.7
2.0
4.2
2.0
NR
NR
NR
2.4
NR
[108]
[108]
[109]
[110]
[111]
[112]
[113]
[114]
[115]
[116]
NR
NR
NR
0.5
3.0
a) Including arabinan.
NR, not reported.
treatment at 180–210°C for some minutes – is to facilitate the enzymatic hydrolysis by making the cellulose 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), organic solvents or oxygen facilitate partial solubilization of lignin [10]. The pH value must be adjusted after pretreatment, and this will lead to the
formation of salt, which can cause osmotic stress
for the yeast. The osmotic stress will trigger glycerol 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 materials 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 softwoods.The stress caused by acetic acid is thus more
important in xylose-rich hydrolysates. Furaldehydes, 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
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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 reviewed in [12]). The lignin fraction of the biomass
gives rise to different phenolics after pretreatment, 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]. Furthermore, the liquid fraction after pretreatment
may either be separated before the hydrolysis, or
treated together with the solid fraction. If the enzymatic hydrolysis takes place as a separate step, a
temperature of 40–50°C is used, in agreement with
the optimum temperature for the currently available cellulases. However, a lower temperature must
be used in the SSF to stay within the allowable temperature for the yeast. An improved yeast thermotolerance 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 development, ethanol concentrations in lignocellulose-based processes using pentoses seldom ex-
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 fermentation. In addition, the presence of highly contaminated 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 engineering of the yeast needed to convey pentose-utilising capacity adds genetic factors. The combination of the environmental and genetic factors result
in the physiological changes shown in Figure 1.
3
3.1
Metabolic engineering for efficient
fermentation and anaerobic growth
on pentose sugars
Xylose utilization
Utilization of the pentose sugar xylose has been
enabled in S. cerevisiae by expression of the heterologous enzymes xylose reductase (XR) and xylitol 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.
288
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Biotechnol. J. 2011, 6, 286–299
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followed to further improve xylose utilization, as
reviewed below.
3.1.1 Improvement of xylose utilization by non-target
approaches
Random mutagenesis and evolutionary engineering has been successfully applied to increase xylose utilization in a number of recombinant S. cerevisiae 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 using 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 heterologous 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 proteome analysis, which identified higher expression of
XR, XDH and the non-oxidative PPP in TMB3400
compared to the parental strain [23]. Transcriptome analyses of the strains TMB3400 and C1 were,
however, unsuccessful in identifying alterations at
a single control point, but rather suggested an additive effect of several small mutations in the
genome [24]. The strain RWB218 was not characterized at the transcriptome level, however increased 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 ultimately moving metabolic control points. A major
controlling step in xylose utilization is the expression level of the heterologous enzymes XR/XDH
and XI. Specifically, the low enzyme activity and
substrate affinity of XR and XI requires high intracellular xylose concentration to push the rate of the
reaction, which leads to decreased ethanol productivity as substrate is consumed [25]. Overexpression of XR somewhat alleviated this problem and
increased the rate of xylose consumption substantially [26]. Similarly, very high expression level of
XI encoding gene from episomal plasmids was required for anaerobic growth on xylose by the strain
RWB202-AFX (Fig. 3A) [27].
A
Notation
in Fig. 3
1
2
3
4
5
6
7
Strain
Relevant genotype
Additional step(s)
C1
TMB 3415
RWB202-AFX
TMB3421
TMB 3420
RWB 217
RWB 218
XYL1, XYL2, XKS1
XYL1 (K270R), XYL2, XKS1, PPP
XI multicopy
XYL1 (N272D P275Q), XYL2, XKS1, PPP
XYL1 (N272D P275Q), XYL2, XKS1, PPP
XI multicopy, XKS1, PPP
XI multicopy, XKS1, PPP
Chemical mutagenesis
–
–
–
Sequential batch selection
–
Batch and chemostat selection
Reference
[20]
[46]
[27]
[121]
[121]
[36]
[21]
B
0,6
re (g/gDW×h)
0,5
6
7
0,4
5
0,3
0,2
1
23
4
0,1
0
0
0,05
0,1
Specific growth rate (h-1)
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
0,15
Figure 3. (A) Engineered S. cerevisiae
strains capable of anaerobic growth on
xylose. (B) Relationship between the
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 xylulokinase (XK), and the PPP, respectively (Fig. 2).
Since XK uses ATP as a cofactor, the expression level of this enzyme needs to be carefully optimized
[28–30]. Overexpression of XK improved the conversion of xylulose [31]. However, without concurrent overexpression of XR, xylose utilization was decreased [28, 30]. The non-oxidative PPP follows after XK in the conversion of xylose to glycolytic intermediates (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 xylulose 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 overexpressed, the xylulose utilization increased, but xylose 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. cerevisiae strain [25]. However, when XR was overexpressed the control coefficient of transport increased [38], and in a strain where the PPP was
overexpressed, increased transport improved xylose utilization at least when the substrate concentration was below 4 g/L [39].
For S. cerevisiae strains employing the oxido-reductive 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+ dependent, the cell creates an excess of NAPD+ and a deficiency 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 engineering for balanced cofactor usage of the two enzymes
[41–44]. In this way the ethanol productivity, and
consequently the anaerobic growth rate, was drastically 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-epimerase (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 considerably less efficient than xylose, it shares several control points with xylose utilization since both heterologous pathways end with the formation of xylulose (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 arabinose [49, 50]. Thus, in S. cerevisiae strains combining the oxidation/reduction pathway for xylose and
the isomerization pathway for arabinose-utilization, almost all consumed arabinose was converted
to arabitol (arabinitol) by XR (Fig. 2) [51–53]. Arabitol formation can be avoided by using the XIbased 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-utilization (Fig. 2), so that arabitol can be further metabolized to xylitol and ethanol. An oxidation/reduction pathway for arabinose metabolism has thus
been described [56, 57] and its performance was recently 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 undetoxified pentose-rich lignocellulosic hydrolysates (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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Biotechnol. J. 2011, 6, 286–299
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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
fermentation conditions
Ethanol concentration and
xylose conversion
424A(LNH-ST)
(XR/XDH/XK)
Sweet sorghum
bagasse
AFEX pretreatment (2:1 ammonia to
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
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)
(XR/XDH/XK)
Poplar
Comparison of many different
pretreatments including AFEX,
oxidative alkali, SO2 catalysed steam
pretreatment, followed by enzymatic
hydrolysis. Initial liquid concentrations (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
Highest ethanol concentration reached [84]
after 50 h: 40 g/L (lime pretreatment)
with about 90% conversion xylose;
SO2 steam explosion yielded 26 g/L
ethanol, about 90% xylose conversion.
TMB3400
(XR/XDH/XK +
mutagenesis and
selection)
Corn stover
SSF
Steam pretreatment at 200°C, 5 min,
SO2 catalysed. Liquid composition:
glucose 7.2, xylose 36, acetic acid,
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.
Ethanol concentration of 36.8 g/L
[78]
reached at 11% WIS using fed-batch
addition. Residual xylose concentration
in liquid about 10 g/L after 96 h
TMB3400
(XR/XDH/XK)
Wheat straw
SSF
Steam pretreatment, dilute H2SO4
catalyst, 210°C, 2.5 min. Liquid
composition after pretreatment:
glucose 10.9 g/L, xylose 51.2 g/L,
acetic acid 5 g/L, HMF 0.6 g/L,
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
A final ethanol concentration of 38 g/L
and a xylose conversion about 40%
in fed-batch up to 9% WIS.
Corresponding for fed-batch at 7%:
ethanol concentration of 34 g/L and
a xylose conversion of 74%.
[118]
TMB3400
(XR/XDH/XK)
Sugar cane
bagasse
SSF
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.
Conversion of xylose about 40% at
7.5%WIS (Ethanol concentration
20 g/L, and xylose conversion 88%
at 5%WIS).
[119]
TMB3400
(XR/XDH/XK)
Sugar cane
bagasse
Steam pretreatment at 190°C, 5 min,
SO2 catalysed. Liquid concentrations:
15 g/L xylose, 4 g/L glucose.
Initial yeast concentration 5 g/L
Only hemicellulose part fermented.
Highest xylose conversion for liquid
from pretreatment 65% after 20 h.
[109]
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Reference
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Table 2. Continued
Strain
Raw material
Pretreatment and
fermentation conditions
Ethanol concentration and
xylose conversion
Reference
MA-R4
Eucalyptus
(XR/XDH/XK
introduced into
flocculating industrial
strain IR-2)
Primarily mechanical pretreatment
Ethanol concentration 37.2 g/L.
(ball milling) followed by enzymatic
Conversion of 90% of the xylose
hydrolysis at high cellulase loadings.
after 48 h
Liquid composition: 61.1 g/L glucose,
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
[120]
RWB2182
(XI)
Strain improved
by evolution
Wheat straw
Steam explosion pretreated followed
by enzymatic hydrolysis, batch
fermentation. Liquid composition:
47.8 g/L glucose, 20.7 g/L xylose,
3 g/L acetic acid.
Initial yeast concentration: 1.5 g/L.
Ethanol concentration: 38.1 g/L.
About 90% of xylose converted
after 55 h. Almost stoichiometric
yield based on consumed sugars
[17]
RWB218
(XI)
Corn stover
Steam explosion pretreated
(190°C, 5 min), followed by enzymatic
hydrolysis. Liquid composition:
40 g/L glucose, 9 g/L xylose,
4 g/L acetic acid. Fed-batch + batch
fermentation.
Ethanol concentration about 18 g/L.
About 90% of xylose converted
after 48 h.
[17]
SSF, simultaneous saccharification and fermentation; WIS, water insoluble solids; AFEX, ammonia fibre expansion.
a long period of time. The specific xylose consumption rates obtained in hydrolysates were also clearly 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 concentration of various aldehydes, such as furfural [59–61].
Among 60 identified phenolic compounds in lignocellulosic 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 enzymes in the central carbon metabolism [64–67].
This results in reduction of fermentation rate, cessation of growth, extension of lag-phase of yeast,
and reduction of ethanol productivity and/or yield
[12, 63]. Understanding and improving the mechanisms by which microorganisms respond to aldehydes is clearly important for the development of
strains with increased tolerance towards lignocellulosic hydrolysates [12, 68].
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4.1.1 Metabolically engineered microorganisms
for aldehyde reduction
A fundamental observation is that as long as certain aldehydes – e.g. furfural, HMF, vanillin, veratraldehyde – are present in the medium, yeasts are
not able to grow and show reduced metabolic activity [61, 69–71]. However, at permissive levels, the
yeast can usually slowly convert the aldehydes to
less toxic compounds and growth and metabolic activity are restored [61, 72–74]. S. cerevisiae, for instance, can reduce furfural and HMF to their corresponding alcohols; 2-furan methanol (FM or furfuryl 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 furaldehyde reduction, and their positive effect upon overexpression of the corresponding gene has been
confirmed [68, 75]. Overexpression of the transcription factor encoding gene YAP1, known to be
involved in oxidative stress response, was also
shown to give an increased tolerance to hydrolysate and specific model inhibitors – and an increased reduction capacity of furaldehydes [76].
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More recently an isolate of the gram positive
bacteria Cupriavidus basilensis (HMF14) could aerobically completely metabolize HMF and furfural
[77], which may open new possibilities for the microbial detoxification of hydrolysates.
4.1.2 Interactions between xylose and furaldehyde
pathways
S. cerevisiae keeps its redox balance tightly regulated, essentially by regulating the activity of glycolytic 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 respect to generation/consumption of the co-factor
pairs NADH/NAD+ (and NADPH/NADP+). However, during the fermentation of lignocellulosic hydrolysates 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 reduction reactions using NADH and NADPH. Consequently, both xylose metabolism and detoxification reactions will affect the redox (or more correctly 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 substantial 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 hydrolysates 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 regenerates NAD+ necessary for xylitol oxidation by XDH.
Thereby glycerol and/or xylitol yields may decrease [81].
The simultaneous conversion of aldehyde compounds and xylose was investigated in an XR/XDH
strain overexpressing either the NADH-dependent
ADH1-S110P-Y295C encoding gene or the NADPHdependent Adh6 encoding gene [82]. NADPH usage during HMF reduction slightly reduced xylitol
formation, whereas the usage of NADH in HMF reduction considerably decreased both xylitol and
glycerol production and increased biomass yield
[82].A metabolic flux analysis indicated that the reduction 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 feedstocks 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 hemicellulose fraction, whereas formic and levulinic acids
are formed during the breakdown of HMF and furfural [83]. The innate levels of acetic acid are highly 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 concentrations 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 pretreatment of poplar [84].
Weak acids are also produced during the fermentation stage, since acetic acid is a minor byproduct of yeast fermentation. However, acetic and
lactic acids are mainly generated from the contamination by lactic acid bacteria (LAB) and other bacteria (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 concentration for growth of 100 mM (6 g/L), as compared to 278 mM (25 g/L) for lactic acid in nonbuffered defined medium [86].
The effects of weak acids are strongly pH dependent. At pH value below the pKa-value of the
acid, the undissociated form of weak acids predominates. 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 maintain 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 decreases. In addition, high levels of weak acids may
result in the intracellular accumulation of anions
(Fig. 2) [90], which has been shown to be inhibitory to several glycolytic enzymes [91]. ATP is also
needed for the active efflux of the accumulating
dissociated acids, which has been suggested to occur 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 XRXDH or the XI pathway [84, 93–96]. A high level of
acetic acid with a low pH caused dramatic reduction 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 engineered 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 recombinant XI-strain at pH 3.5 and in the presence of
3 g/L acetic acid by applying a continuous low feeding of glucose to the medium [93].
Acetic acid decreases ethanol productivity but
its effect on ethanol yield is less clear. In some reports, the ethanol yield was shown to increase as
the metabolism was redirected from anabolic (biomass) to dissimilatory (ethanol) pathway in the
presence of acetic acid [93, 94]. In contrast, a lower
ethanol yield from glucose and xylose was also reported 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 undissociated 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 (Section 4.3).
Recently, S. cerevisiae was metabolically engineered allowing acetate to replace dihydroxyacetone phosphate (DHAP) as a redox sink during
anaerobic growth in a glycerol mutant strain. This
was achieved by introducing a bacterial NAD+-dependent acetylating acetaldehyde dehydrogenase,
by which acetyl-CoA may be reduced to acetaldehyde (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 competition 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 recurrently 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 contamination are in SSL, which is the waste stream of cellulose 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, including the xylose-utilising yeast Pichia membranaefaciens, the acetic acid bacteria Acetobacter tropicalis
and A. syzygii, the homofermentative bacteria Lactobacillus 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]. During 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
www.biotechnology-journal.com
4.3.2 Effects of microbial contamination on yeast
fermentation
5
LAB compete with yeast for sugar utilization. Homofermentative bacteria are able to consume hexose 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 fermentation by reducing biomass formation and
ethanol yield.
Both targeted and non-targeted approaches have
been applied to generate efficient pentose-fermenting S. cerevisiae strains. The strength of the
non-targeted approach is that it requires less a priori knowledge of the pathway and is generally
straight forward and efficient. The targeted approach on the other hand may require tedious
identification of specific traits that enhance metabolism, however once identified these traits can be
directly transferred between strains. Both approaches have been successful in the generation of
S. cerevisiae strain that can grow anaerobically on
xylose, a trait which is shown to be linearly correlated to ethanol productivity (Fig. 3B). Still the xylose consumption rate remains three to sixfolds
lower than the glucose consumption rate. Generating 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-contaminant in the process.
A central issue in converting pentoses from hydrolysates is also the presence of aldehydes – not
least the furaldehydes. Recent identification of enzymes with aldehyde reduction activity has enabled
targeted approaches for improving the in situ reduction capacity of S. cerevisiae strains. Similarly genetic 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.
4.3.3
Strategies to control a contamination
In the sugar cane ethanol industry, acid wash treatment 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 addition 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 temporarily 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, personal communication).
The use of hops extract to control bacterial contamination in lignocellulosic fermentation plants is
also discussed. Hop that gives a bitter taste and acts
as antiseptic agent, is well-known in beer production. 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. cerevisiae strains with xylose consumption rates that
are sufficiently high to compete with LAB for the
utilization of all sugars present in the lignocellulosic hydrolysates, may provide another solution to
the bacterial contamination issue.
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Concluding remarks
The authors wish to thank the Swedish Energy
Agency (Energimyndigheten) for financial support.
The authors have declared no conflict of interest.
6
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Gunnar Lidén received his Ph.D. in
Chemical Reaction Engineering from
Chalmers University, Göteborg, Sweden in 1993, where he remained until
1999. At that time he moved to Lund
university to take on his current position as professor in chemical engineering. His main research focus concerns
biotechnology for the production of
biofuels.In particular, he worked on improving fermentation technology for efficient lignocellulose conversion. 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 Microbiology in 1999. Her current research projects involve the identification, development and engineering of biocatalysts for the production
of fuels and bulk chemicals and for stereo-selective whole cell biotransformations.
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