Journal of Scientific & Industrial Research
918
Vol. 67, November 2008, pp.918-926
J SCI IND RES VOL 67 NOVEMBER 2008
Brazilian potential for biomass ethanol: Challenge of using hexose and pentose cofermenting yeast strains
Boris U Stambuk1*, Elis C A. Eleutherio2, Luz Marina Florez-Pardo3 Ana Maria Souto-Maior4 and Elba P S Bon5
1
2
Laboratório de Biologia Molecular e Biotecnologia de Leveduras, Centro de Ciências Biológicas,
Universidade Federal de Santa Catarina, Florianópolis, SC, Brasil
Laboratório de Investigação de Fatores de Estresse, Instituto de Química, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, RJ, Brasil
3
Departamento Sistemas de Produccion, Universidad Autonoma de Occidente, Cali, Colombia
4
5
Departamento de Antibióticos, Universidade Federal de Pernambuco, Recife, PE, Brasil
Laboratório de Tecnologia Enzimática, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil
Received 15 July 2008; revised 16 September 2008; accepted 01 October 2008
This paper reviews Brazilian scenario and efforts for deployment of technology to produce bioethanol vis-à-vis recent
international advances in the area, including possible use of hexose and pentose co-fermenting yeast strains.
Keywords: Biomass ethanol, Brazilian ethanol production, Ethanologenic yeasts, Hexose-pentose co-fermentation
Introduction
Brazil produces annually 5 x 109 gallons of ethanol
from sugarcane grown on 5 million ha. Sugarcane is
processed in 365 sugar/ethanol producing units, and it
is forecasted that 86 new distilleries will be built before
2015. Sugarcane juice can go either directly to
fermentation vats for ethanol production, or be used to
produce sugar (crystal sucrose, and its residue
molasses), depending on which product is priced more
favorably. As a consequence, 75 million tonnes of
bagasse are produced annually. In each sugar and/or
ethanol mill, approx. 85% of bagasse is burned for the
production of steam (heat) and power/electricity
generation.
Bioethanol from Sugarcane
Brazil has been a front-runner to substitute liquid fuel
gasoline by renewable ethanol1-5. State of São Paulo is
biggest sugarcane producer (68% of total sugarcane
plantations in Brazil). Out of 365 sugar/ethanol
producing units in Brazil, 240 produce sugar and
*Author for correspondence
E-mail: [email protected]
ethanol, 109 produce only ethanol and 16 produce only
sugar. Brazil will produce around 27 billion litre of ethanol
in 2008, and plans to build 41 new distilleries before 2010,
and 45 more until 20156. Land use for energy crops as
compared to food crops has been opted for renewable
fuels with use of biomass polysaccharide part for the
production of fuel ethanol7-9. A simple mass balance
(Fig. 1) for harvested sugarcane indicates that
lignocellulosic-derived fuel ethanol could add up to > 50%
bonus over total amount of ethanol currently produced
from sugarcane, considered that the amount of sugars
found in sugarcane biomass could be recovered and
fermented at ~90% efficiency4,10-12.
Structure of lignocellulosic biomass (LB) is represented
by physicochemical interaction of cellulose, with
hemicellulose and lignin13-15. Cellulose is a highly ordered
polymer of cellobiose whose length may present 10,000
glycosyl units in cellulose chain that form fibrils.
Hemicelluloses are shorter (degree of polymerization
within 100-200), highly branched heteropolymers of
xylose, arabinose, mannose, galactose and glucose as well
as uronic acids. Depending on predominant sugar type,
hemicelluloses are referred to as mannans, xylans or
galactans. Pentose and hexose sugars, linked through 1,3,
STAMBUK et al: BRAZILIAN BIOETHANOL
919
Fig. 1—Bioethanol production from sugarcane [current technology of sugarcane processing and fuel ethanol production from sucrose
(black arrows) is illustrated at the right side of the figure, while the required technological achievements for bioethanol production
from sugarcane bagasse and straw (biomass pre-treatment, hydrolysis and pentose fermentation, gray arrows) are shown on the right
side of the figure]
1,6 and 1,4 glycosidic bonds and often acetylated, form
a loose and very hydrophilic structure that acts as a
glue between cellulose and lignin.
Parallel to the implementation of Brazilian Proálcool
Program, in 1970s, several research institutions and
companies have carried out research for the production
of bioethanol, using primarily and municipal cellulosic
solid wastes. The first pilot-scale facility was built in
1981 at Fundação de Tecnologia Industrial (FTI,
Lorena, SP) to run using the Scholler-Madison process
based on concentrated sulphuric acid to hydrolyze
Eucalyptus panicutata to produce 500 l ethanol per day.
Brazilian company Dedini, in Piracicaba (SP), began
in 1987 the development of a biomass-to-ethanol
production technology (called Dedini Hidrolise Rapida,
DHR), in partnership with Copersucar (presently Centro
de Tecnologia Canavieira) and State of São Paulo
Research Supporting Foundation (FAPESP). DHR
technology for ethanol production is an organosolv
hydrolysis single-stage process16 that employs very
diluted H 2 SO 4 for hydrolysis of cellulose and
hemicellulose to sugars, and an organic solvent for
lignin extraction. DHR’s unique feature is reduced
hydrolysis reaction time (few min) in a continuous highthroughput process, with quick cooling of hydrolysate.
However, all projects that studied acid hydrolysis
observed low sugar yields, formation of inhibitors for
subsequent ethanol fermentation step, and corrosion of
equipments. Besides, acidic pH of sugar hydrolysate
requires a neutralization step prior to fermentation.
Considering detrimental aspects of acid hydrolysis,
studies focused on enzyme-based route that involves
biomass pretreatment because LB are structured for
strength and resistance to biological, physical and
chemical attack. Pretreatments17,18 (steam explosion,
hydrothermolysis or using catalytic amounts of acid),
render raw cellulose digestible by cellulases. Sugarcane
bagasse was pretreated via steam explosion using a 1.6 l
reactor19. These studies paved way for development of
sugarcane bagasse steam explosion process used to date
in Brazil for the production of cattle feed.
Presently, “Bioethanol Project: Bioethanol Production
through Enzymatic Hydrolysis of Sugarcane Biomass”,
initiated in 2006, is supported by Brazilian Ministry of
Science and Technology through its Research and Projects
Financing agency (FINEP), and is carried out by a
network of more than 20 institutions, including universities
and research institutes. This project aims to develop a
viable technology for conversion of sugarcane bagasse
and straw into fuel ethanol, minimizing expansion of
sugarcane fields. It is expected that Brazilian sugar mills
will be gradually converted into biorefineries, able to
eventually process all fractions of sugarcane. This
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J SCI IND RES VOL 67 NOVEMBER 2008
technology will use all already existing facilities in sugar
and alcohol plants including juice/sugars syrup treatment
system, fermentation & distillation, cogeneration,
wastewater & residues treatment and recycling,
instrumentation and automatic control systems,
management, and commercialisation. Biomass sugar
syrups will be blended with molasses or with sugarcane
juice prior to fermentation carried out by S. cerevisiae
yeast strains. As a result, lower biomass ethanol
production cost is expected compared with plants
dedicated to produce biomass ethanol solely. For
enzymatic hydrolysis of pretreated biomass, the project
is developing enzyme blends in situ for sugarcane
bagasse.
To produce ethanol from LB economically, it is essential
to have a biocatalyst able to ferment hexose and pentose
sugars under adverse conditions of an industrial
environment. Yeast S. cerevisiae, applied successfully
in industrial production of ethanol from sugarcane juice
and molasses in Brazil, is able to ferment hexoses rapidly
and efficiently20, and exhibits high ethanol tolerance,
GRAS status, and tolerance to low pH. However, S.
cerevisiae for use in a second generation process
(lignocellulose-to-fuel ethanol process) cannot metabolize
pentoses, although development of S. cerevisiae pentoseutilizing strains could be integrated into existing ethanol
plants. Engineering industrial strains that would be able
to co-ferment efficiently all sugars in lignocellulose
hydrolysates to ethanol is, however, still a challenge for
molecular biologist and engineers worldwide21,22.
Pentose Fermentation
In 1922, Willaman and co-workers showed that
Fusarium lini could ferment xylose into ethanol. Since
then, many bacteria have been found to metabolize and
ferment hexose and pentose sugars, but all produced an
unwanted mixture of fermentation products. Although,
homo-ethanol pathway is found in Z. mobilis, substrate
range this organism can metabolize (glucose, fructose
and sucrose) is very limited23,24. Many yeasts able to
ferment xylose were also found, including several
Candida, Pichia, Hansenula, Debaromyces,
Schwanniomyces, etc. If xylose was converted to
xylulose, several more yeast could ferment it directly to
ethanol, including S. cerevisiae24,25. Thus, although many
yeasts are able to ferment xylose, large-scale utilization
is hampered by their low tolerance to ethanol and
hydrolysate inhibitors, requirement for microaerophilic
conditions, and an inability to ferment xylose at low pH26.
Best yeast strains24,27,28 known for fermenting xylose
are P. stipitis, P. tannophilus and C. shehatae. Yeast
P. stipitis is capable of converting xylose and all major
lignocellulosic sugars to ethanol (yield, 0.3-0.44 g/g of
substrate). Although this is suitable for some waste
streams, commercial fermentation for fuel ethanol
requires higher performance24,28.
Over the past 20 years, metabolic engineering has
been used to construct bacterial and yeast strains with
advantageous traits for lignocellulosic ethanol
production26,29-31. Efforts are concentrated on three most
promising microbial platforms23,29-33 (S. cerevisiae, Z.
mobilis, and E. coli). Since S. cerevisiae has been used
for mature industrial technologies of ethanol production
from cane and grain sugars, several strategies that have
been used to engineer this yeast towards pentose
fermentation (Fig. 2) include insertion of bacterial and
yeast/fungal pentose-utilization genes34-39, selection or
overexpression of the own S. cerevisiae xylose utilization
pathway40,41, improvement of xylulose consumption
through overexpression of xylulokinase and pentosephosphate pathway enzymes42-47, engineering redox
cofactor regeneration to reduce by-product formation,
in particular xylitol48-51, and application of random
mutagenesis and evolutionary engineering to select for
improved traits52-55.
Unfavorable kinetic properties of enzymes with an
imperfect match of cofactor, and an inadequate pentose
phosphate pathway, apparently limit ability of recombinant
yeast to ferment xylose28,55. Despite development of
recombinant strains with improved xylose fermentation
performance, high products yields and increased product
concentrations are key targets that have yet to be
achieved. Recent transcriptome and proteome analysis
have indicated that xylose is not perceived as a
fermentable sugar by S. cerevisiae56,57 probably because
signaling pathways that ensure efficient utilization of
hexoses 58-60 do not recognize pentoses xylose or
arabinose. Thus, genetic modifications that could have
greatest impact on economic feasibility of these
fermentations should include amplification and
deregulation of rate-limiting reactions61.
Sugar uptake from medium is major rate limiting step
for fermentation of even naturally fermentable sugars
(glucose, fructose, maltose, maltotriose) by
S. cerevisiae62-65. In general, overexpression of cognate
sugar transporters, which can mediate facilitated diffusion
or active transport of sugar across plasma membrane
STAMBUK et al: BRAZILIAN BIOETHANOL
921
Fig. 2—Pentose fermentation by yeasts [Simplified scheme of xylose and arabinose metabolic pathways present in yeasts
and fungi (black lettering), as well as the bacterial arabinose pathway that has been successfully expressed in S. cerevisiae
(gray lettering), and their integration into the main glycolytic pathway towards ethanol production. Abbreviations: AR, Larabinose(aldose) reductase; LAD, L-arabinitol 4-dehydrogenase; LXR, L-xylulose reductase; XR, D-xylose(aldose)
reductase; XDH, xilitol dehydrogenase; XI, D-xylose isomerase; XK, D-xylulokinase; AI, L-arabinose isomerase; RK, Lribulokinase; RPE, L-ribulose-5-P 4-epimerase]
(Fig. 3), improves significantly fermentation performance
of cells66-69. Xylose and arabinose uptake across plasma
membrane is also rate-limiting for pentose fermentation
by S. cerevisiae, specially because this yeast transports
xylose and arabinose with very low affinity, compared
with uptake of other fermentable sugars34,36,47,70,71. All
yeast species that use xylose and/or arabinose have both
low- and high-affinity pentose transport activities,
covering a wide range of sugar concentrations extending
to at least 2-3 orders of magnitude. Affinities for xylose
are always lower than those for glucose. While most
strains present both high-affinity arabinose/xylose-H+
symport and low-affinity facilitated transport of
sugars72,73 (Fig. 3) in S. cerevisiae, these pentoses are
transported solely by facilitated transport mediated by
hexose permeases. Expression of any of the major HXT
transporters (HXT1-HXT7 and GAL2) allows growth of
an hxt-null strain on xylose74,75. Of these transporters,
mid-to-high-affinity glucose permeases (HXT4, HXT5,
HXT7 and GAL2) were more effective arabinose and
xylose transporters36,71,74,75, which is in good agreement
with analysis of mutated and evolved S. cerevisiae cells
showing improved pentose fermentation due to an
increased expression of HXT5, HXT7 and GAL2
permeases 36,51-54.
In P. stipitis, xylose uptake is also considered ratelimiting step for xylose utilization76. Although genome of
this yeast indicates presence of a large family of sugar
transporter genes 77 , only three sugar permeases
characterized in P. stipitis are high affinity glucose
permeases that transport xylose with significantly lower
affinity78. This study also revealed first example of sugar
permeases whose expression is regulated by oxygen
availability, which may be also present in other yeasts
and fungi72,79. Although pentose transport activities are
described in several other yeasts, only other known
xylose/glucose permease genes were isolated from
C. intermedia through functional expression in an
hxt-null S. cerevisiae strain80. While there are still many
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J SCI IND RES VOL 67 NOVEMBER 2008
Fig. 3—Sugar transport by yeasts [Illustration of sugar transport mechanisms present in yeasts, including the high
capacity sugar facilitated diffusion transporters (which in general have affinity for hexoses in the mM range, and in the
M range for pentoses), and the active sugar-H+ permeases (which in general have affinity in the ¼M range for
monosaccharides, and for disaccharides in the mM range) driven by the proton motive force (created by the plasma
membrane H+-ATPase) that allows the intracellular accumulation of the sugar]
limitations towards functional expression of heterologous
active pentose permeases in S. cerevisiae 71,81 ,
overexpression of a xylose/glucose facilitated diffusion
transporter from P. stipitis is reported to significantly
enhance xylose fermentation by a S. cerevisiae strain
engineered to metabolize this sugar82.
Current Fuel Ethanol Technology in Brazil:
Challenges and Opportunities for Bioethanol
Industrial production of fuel ethanol in Brazil uses raw
sugarcane juice and/or molasses as substrate. Sugarcane
juice contains significant amounts of yeast and bacteria,
depending on different factors mainly related to cane
variety and age, time lag between cane harvesting and
processing, etc.83-86. While bacterial infections can be
controlled by antibiotics and acid treatment of the yeast
cells, yeast contaminations can have a significant impact
in fermentation performance of distillery. In wine
fermentations, D. bruxellensis, major contaminant in
industrial fuel ethanol plants in Brazil, can cause
decreased productivity as well as other operational
problems85,86. Thus, to ensure high productivities and
efficiency in industrial process, currently fuel ethanol
plants (> 80%) work with an adaptation of Melle-Boinot
process87-89. This process is a fed-batch fermentation
that uses high cell densities (~10 g yeast/l) allowing very
short residence times for fermentation to be completed
(<8-10 h), and thus up to 3 fermentation runs can be
performed per day, contributing to overall process
success. Normally, final sugar concentration employed
is also high (~180 g/l), with high conversion yields (up to
92%) due to high cell density process that limits cell
growth. Crucial to the performance of this process is
constant recycling of yeast slurry into fermentors, after
treatment with H2SO4, a procedure that ensures less
bacterial and wild yeast contaminations. Consequently,
yeast fitness for industrial process includes high tolerance
to fermentative stresses (high temperatures, ethanol and
osmotic pressure, low pH, production interruptions, etc.),
and yeast should also dominate fermentation tanks during
whole crop season. Indeed, analysis of yeast population
dynamics during fuel ethanol production in Brazil has
revealed existence of selected yeast strains that tend to
dominate a given industrial process90. Actually, some
yeast strains (PE-2, CAT-1) are commercially available
as
starters
for
fuel
ethanol
industry
(www.fermentec.com/en/index.html), showing high
alcohol productivity and increased resistance to stressful
STAMBUK et al: BRAZILIAN BIOETHANOL
conditions found in this industrial process5,87, again
contributing significantly to overall process economical
success.
Regarding introduction of sugarcane biomass
hydrolysates into actual fuel ethanol production setup,
one major concern is that even with the best performing
S. cerevisiae yeast strains22,82, rates of pentose uptake
by cells (<0.25 g sugar/g yeast/h) are far slower than
rate of fermentable sugar uptake by industrial yeast
strains (2-4 g sugar/g yeast/h). This would mean either
longer fermentation cycles (affecting the overall process
productivity), or presence and persistence for longer
periods of pentose sugars in fermentor would favor
contaminant bacteria and wild yeasts that use these
sugars more efficiently91, probably affecting whole
fermentation process. Thus, even if pentose fraction in
sugarcane represents only ~14% of total sugar available
for fermentation from this energy crop (Fig. 1), all these
sugars need to be used by S. cerevisiae cells present in
fermentor in order to not increase the risk of
contaminations in fuel ethanol process.
Since transport systems for hexoses present in this
yeast also transport pentoses with lower affinity, xylose
and arabinose fermentation can only take place after
hexoses have been significantly depleted from medium92.
An interesting alternative for Brazilian sugarcane fuel
industry, which uses sucrose-rich broths (Fig. 1), could
be to use S. cerevisiae yeast strains that would not
hydrolyze outside the cells the sucrose present in cane
juice, but sugar would be transported directly into
cytoplasm and hydrolyzed intracellularly. Efforts towards
this goal are already in development in Brazil93,94. Finally,
selected industrial yeast strains mentioned above should
be seriously considered for metabolic engineering of
pentose fermentation pathways (Fig. 2) and other desired
phenotypic characteristic required for bioethanol
production from sugarcane biomass22, and indeed recent
data indicates feasibility of such strains for genetic
transformation95, paving the way towards implementing
bioethanol production processes in Brazil.
Acknowledgments.
Authors thank Brazilian funding agencies CNPq,
CAPES, and FINEP for financial support. Ms. A M
Klinkowstrom is also acknowledged for editorial
assistance.
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