Appl Microbiol Biotechnol (2006) 69: 627–642
DOI 10.1007/s00253-005-0229-x
MINI-REVIEW
Yan Lin . Shuzo Tanaka
Ethanol fermentation from biomass resources: current state
and prospects
Received: 18 July 2005 / Revised: 21 October 2005 / Accepted: 22 October 2005 / Published online: 6 December 2005
# Springer-Verlag 2005
Abstract In recent years, growing attention has been devoted to the conversion of biomass into fuel ethanol, considered the cleanest liquid fuel alternative to fossil fuels.
Significant advances have been made towards the technology of ethanol fermentation. This review provides practical
examples and gives a broad overview of the current status of
ethanol fermentation including biomass resources, microorganisms, and technology. Also, the promising prospects of
ethanol fermentation are especially introduced. The prospects included are fermentation technology converting xylose to ethanol, cellulase enzyme utilized in the hydrolysis
of lignocellulosic materials, immobilization of the microorganism in large systems, simultaneous saccharification and
fermentation, and sugar conversion into ethanol.
Introduction
With the inevitable depletion of the world’s energy supply,
there has been an increasing worldwide interest in alternative sources of energy (Aristidou and Penttila 2000;
Jeffries and Jin 2000; John 2004; Kerr 1998; Wheals et al.
1999; Zaldivar et al. 2001). It is now understood that it is
important to use biomass energy as a means of providing
modern energy to the billions who lack it. It would complement solar, wind, and other intermittent energy sources
in the renewable energy mix of the future. One of the most
immediate and important applications of biomass energy
systems could be in the fermentation of ethanol from
biomass.
Biomass is seen as an interesting energy source for
several reasons. The main reason is that bioenergy can con-
Y. Lin . S. Tanaka (*)
Asian Center for Environmental Research,
Meisei University,
Tokyo, Japan
e-mail: tanakash@es.meisei-u.ac.jp
tribute to sustainable development (Van den Broek 2000;
Monique et al. 2003). Resources are often locally available,
and conversion into secondary energy carriers is feasible
without high capital investments. Moreover, biomass energy
can play an important role in reducing greenhouse gas
emissions; since CO2 that arises from biomass wastes would
originally have been absorbed from the air, the use of
biomass for energy offsets fossil fuel greenhouse gas emissions (Lynd 1996). Furthermore, since energy plantations
may also create new employment opportunities in rural
areas, it also contributes to the social aspect of sustainability.
In addition, application of agro-industrial residues in bioprocesses not only provides alternative substrates but also
helps solve their disposal problem. With the advent of
biotechnological innovations, mainly in the area of enzyme
and fermentation technology, many new avenues have
opened for their utilization.
Nearly all fuel ethanol is produced by fermentation of
corn glucose in the US or sucrose in Brazil (MacDonald
et al. 2001; Rosillo-Calle and Cortez 1998), but any country
with a significant agronomic-based economy can use
current technology for fuel ethanol fermentation. This is
possible because, during the last two decades, technology
for ethanol production from nonfood-plant sources has
been developed to the point at which large-scale production
will be a reality in the next few years. Therefore, agronomic
residues such as corn stover (corn cobs and stalks), sugarcane waste, wheat or rice straw, forestry, and paper mill
discards, the paper portion of municipal waste and dedicated energy crops—collectively termed “biomass”—can
be converted into fuel ethanol. In this field, although
bioethanol production has been greatly improved by new
technologies, there are still challenges that need further
investigations. A further understanding of the ethanol
fermentation needs to be reached.
This review will focus on the current status of ethanol
fermentation including biomass resources, microorganisms, technology, the practical examples, and especially the
promising prospects of ethanol fermentation.
628
Biomass resources
There are various forms of biomass resources in the world,
which can be grouped into four categories. Wood residues
are by far the largest current source of biomass for energy
production. It comes from the wood product industry which
includes paper mills, sawmills, and furniture manufacturing.
Municipal solid waste is the next largest, followed by
agriculture residues and dedicated energy crops. Among
these biomass resources including short-rotation woody
crops and herbaceous crops, primarily tall grasses, dedicated
energy crops seem to be the largest, most promising, future
resource of biomass. This is because of the ability to obtain
numerous harvests from a single planting, which significantly reduces average annual costs for establishing and
managing energy crops, particularly in comparison to conventional crops (Monique et al. 2003).
Fermentation processes from any material that contains
sugar could derive ethanol. The varied raw materials used in
the manufacture of ethanol via fermentation are conveniently
classified into three main types of raw materials: sugars,
starches, and cellulose materials. Sugars (from sugarcane,
sugar beets, molasses, and fruits) can be converted into
ethanol directly. Starches (from corn, cassava, potatoes, and
root crops) must first be hydrolyzed to fermentable sugars by
the action of enzymes from malt or molds. Cellulose (from
wood, agricultural residues, waste sulfite liquor from pulp,
and paper mills) must likewise be converted into sugars,
generally by the action of mineral acids. Once simple sugars
are formed, enzymes from microorganisms can readily
ferment them to ethanol.
The most widely used sugar for ethanol fermentation is
molasses which contains about 50 wt% of sugar and about
50 wt% of organic and inorganic compounds, including
water. It is a thick, dark-colored syrup produced during
refinement of sugar. Since molasses contains microorganisms which can disturb the fermentation, the molasses is
taken first to the sterilizer and then to the fermentor. Then it
is diluted with water to the mass fraction of 10±18% to
reduce its viscosity in the pipeline. In addition, a very high
concentration of sugar can give too much ethanol and results in a prolonged fermentation time and an incomplete
sugar conversion. After the pH of the mash is adjusted to
about 4–5 with mineral acid, it is inoculated with yeast or
bacteria, and the fermentation is carried out nonaseptically
at 20–32°C for about 1–3 days.
Most agricultural biomass containing starch can be used
as a potential substrate for the ethanol fermentation by
microbial processes. These substrates include corn (maize),
wheat, oats, rice, potato, and cassava. On a dry basis, corn,
wheat, sorghums (milo), and other grains contain around
60–75% (wt/wt) of starch, hydrolyzable to hexose with a
significant weight increase (stoichiometrically the starch to
hexose ratio is 9:10), and these offer a good resource in
many fermentation processes (Jackman 1987).
Fermentation of starch is somewhat more complex than
fermentation of sugars because starch must first be converted into sugar and then into ethanol. Starch is first
hydrolyzed by adding α-amylase to avoid gelatinization,
then cooked at high temperature (140–180°C). Next, the
liquefied starch is hydrolyzed to glucose with glucoamylase. The resulting dextrose is fermented to ethanol with the
aid of microorganisms producing CO2 as a coproduct.
During the process currently employed for industrial-scale
ethanol fermentation from starchy materials, high-temperature cooking (140–180°C) is very effective for fermentation
of starchy materials because it raises starch saccharification
efficiency and achieves high levels of ethanol production
under complete sterilization of harmful microorganisms.
However, production costs are high due to the high energy
consumption in the cooking process and the addition of
large amounts of amylolytic enzymes. So processes to
reduce the high production costs are required. To resolve
these difficulties, noncooking and low-temperature cooking
fermentation systems have been developed (Matsumoto et
al. 1985).
Industrial ethanol production has been reported using
various starchy materials such as corn, wheat, starch and
potatoes, cassava root (Lindeman and Rocchiccioli 1979),
corn stover (Kadam and McMillan 2003; Wilke et al.
1981), and starch (Maisch et al. 1979). Among many
starchy materials, cassava starch is an inexpensive fermentable source. It is a tropical root crop produced in more
than 80 countries (Sasson 1990). About 20% of the cassava
starch was incorporated into animal feed. A similar amount
was converted into starch for industrial use and another
portion used for human food in some developing countries.
The rest was lost since cassava is perishable after harvest.
Harnessing the lost portion in addition to gains from new
high-yielding varieties with outputs of 100 tons per hectare
could provide the fermentation industry with an abundance
of raw material (Anthony et al. 1996). Fresh cassava has a
very high starch content, up to 30%. The content of sucrose
is about 4%. Dried cassava has 80% fermentable substrate.
However, cassava waste processing is difficult because it
is high in toxic materials. The potential toxicity of cassava
is due to the presence of cyanogenic glycosides, linamarin,
and lotaustralin, which on hydrolysis yield hydrogen cyanide on its peel. Traditional methods of cooking like boiling and decanting remove cyanoglycosides to a certain
extent, but even then a certain amount of residual toxicity
remains in it (Westley 1980). Moreover, since starch
particles in cassava are bigger and there are some branched
structures, more glucoamylase has to be added into the
reactor. Furthermore, the nitrogen content of the cassava is
very low, so during the fermentation, nutrient has to be
added into the reactor to maintain the normal growth of the
microorganisms.
Among the three main types of raw materials, cellulose
materials represent the most abundant global source of
biomass and have been largely unutilized. The global production of plant biomass, of which over 90% is lignocellulose, amounts to about 200×109 tons per year, where about
8–20×109 tons of the primary biomass remains potentially
accessible. However, the effective utilization of the lignocellulosic feedstock is not always practical because of its
seasonal availability, scattered stations, and the high costs of
transportation and storage of such large amounts of organic
629
material (Polman 1994). Recently, the enzymatic hydrolysis
of biomass cellulose is considered to be the most promising
technology available (Ogier et al. 1999; Yu and Zhang
2004). However, despite the work done, the industrial scaleup of this process appears to be still hindered by technological issues or by the lack of a biomass refinery approach
in which ethanol is one of several products. In fact, because
raw material cost comprises more than 20% of the production cost (Brown et al. 2001; Kaylen et al. 2000; Zhuang
et al. 2001), the optimization of the cellulose conversion
should be accomplished by correct management and utilization of all process streams. A consequence of this situation is that even limited government intervention is still
crucial to maintaining ongoing research.
Furthermore, lignocellulose is a more complex substrate
than starch. It is composed of a mixture of carbohydrate
polymers (cellulose and hemicellulose) and lignin. The
carbohydrate polymers are tightly bound to lignin mainly
by hydrogen bonds but also by some covalent bonds. The
biological process for converting the lignocellulose to fuel
ethanol requires the following: delignification to liberate
cellulose and hemicellulose from their complex with lignin,
depolymerization of the carbohydrate polymers to produce
free sugars, and fermentation of mixed hexose and pentose
sugars to produce ethanol. Among the key processes described above, the delignification of lignocellulosic raw
materials is the rate-limiting and most difficult task to be
solved. Another problem is that the aqueous acid used to
hydrolyze the cellulose in wood to glucose and other
simple sugars destroys much of the sugars in the process.
Extensive research has been carried out in this field for
decades (Yu and Zhang 2004), and the first demonstration
plant using lignocellulosic feedstocks has been in operation
in Canada since April 2004 (Tampier et al. 2004). It is
expected that the cost of lignocellulosic ethanol can
undercut that of starch-based ethanol because low-value
agricultural residues can be used.
General process
Besides the initial removal of large and unsuitable items,
key components of an integrated residual waste treatment
system based on ethanol fermentation include recyclable
materials recovery and removal of contaminants via mechanical preprocessing, initial hydrolysis process (conversion to simpler compounds), fermentation of organics,
postfermentation purification of ethanol (by distillation or
filtration), gasification of solid residuals to provide process
heat, and treatment and disposal of wastewater.
Nearly all of the ethanol fermentation technologies use an
initial tipping floor removal of large or unsuitable materials,
followed by mechanical preprocessing to remove recyclables and contaminants, and shredding of the material. Then
the material is processed through vessels using various
systems for the purpose of hydrolysis (breaking down to
simpler compounds) of the material. Depending on the
technology, this may include high temperature, acid treatment, and/or high pressure. Following the initial hydrolysis
phase, the slurried material is then fermented to produce
alcohol, which is then purified through distillation and/or
filtration to produce the desired fuel-grade quality ethanol.
When cellulose was used as the raw material, the
cellulase responsible for enzymatic hydrolysis of pretreated
cellulosic biomass is strongly inhibited by hydrolysis
products: glucose and short cellulose chains. One way to
overcome cellulase inhibition is to ferment the glucose to
ethanol as soon as it appears in solution. Simultaneous
saccharification and fermentation (SSF) combines enzymatic hydrolysis with ethanol fermentation to keep the
concentration of glucose low (as shown in Fig. 1). The
accumulation of ethanol in the fermentor does not inhibit
cellulase as much as high concentrations of glucose, so SSF
is a good strategy for increasing the overall rate of cellulose
to ethanol conversion. In comparison to the process where
these two stages are sequential, the SSF method enables
attainment of higher (up to 40%) yields of ethanol by
removing end-product inhibition, as well as by eliminating
the need for separate reactors for saccharification and
fermentation (Bollók et al. 2000; Hari et al. 2001; Stenberg
et al. 2000). Other advantages of this approach are a shorter
fermentation time and a reduced risk of contamination with
external microflora, due to the high temperature of the
process, the presence of ethanol in the reaction medium,
and the anaerobic conditions (Emert and Katzen 1980;
Wyman 1994).
In spite of the obvious advantages presented by the SSF,
it has some drawbacks. These lie mainly in different
temperature optima for hydrolysis (45–50°) and fermentation (28–35°) (Ballesteros et al. 2004; Jeffries and Jin 2000;
Jeffries and Shi 1999). Besides, ethanol itself and some
toxic substances arising from pretreatment of the lignocellulose inhibit the action of fermenting microorganisms, as
well as the cellulose activity (Targonski and Achremowicz
1986; Yu and Zhang 2004). Achieving microorganism–
enzyme compatibility becomes a major issue in the SSF,
since some compounds (e.g., proteolytic enzymes) that are
released on cell lysis or are secreted by a particular strain
can degrade the cellulases, alternately, components in the
enzyme preparation, and reduce microbial viability leading
to cell lysis. On the whole, several process parameters must
be optimized: substrate concentration, enzyme to substrate
ratio, dosage of the active components (α-glucosidase to
glucanase ratio) in the enzymatic mixture, and yeast
concentration.
Size reduction
Residual solids
processing
Dilute acid
pretreatment
Enzyme
production
Simultaneous
saccharification and
fermentation
Ethanol
recovery
Fig. 1 Schematic diagram of the conversion of biomass feedstock
to ethanol fuel
630
Microorganisms related to ethanol fermentation
Ethanol fermentation is a biological process in which
organic material is converted by microorganisms to simpler
compounds, such as sugars. These fermentable compounds
are then fermented by microorganisms to produce ethanol
and CO2. During the whole process of ethanol fermentation, there are mainly two parts for microorganisms. One is
for the microorganisms which convert fermentable substrates into ethanol, and the other is to produce the enzyme
to catalyze chemical reactions that hydrolyze the complicate substrates into simpler compounds.
Microorganisms producing ethanol
Several reports and reviews have been published on
production of ethanol fermentation by microorganisms,
and several bacteria, yeasts, and fungi have been reportedly
used for the production of ethanol. Those microbes that are
capable of yielding ethanol as the major product are shown
in Tables 1 and 2.
As shown in Tables 1 and 2, there are some microorganisms which can accumulate high concentrations of
ethanol. Historically, the most commonly used microbe has
been yeast, among the yeasts, Saccharomyces cerevisiae,
which can produce ethanol to give concentration as high as
18% of the fermentation broth, is the preferred one for most
ethanol fermentation. This yeast can grow both on simple
sugars, such as glucose, and on the disaccharide sucrose.
Saccharomyces is also generally recognized as safe (GRAS)
as a food additive for human consumption and is therefore
ideal for producing alcoholic beverages and for leavening
bread.
As with many microorganisms, S. cerevisiae metabolizes glucose by the Embden–Meyerhof (EM) pathway.
Beside this, the Entner–Doudoroff (ED) pathway is an
additional means of glucose consumption in many bacteria,
such as Zymomonas. The high ethanol yield and productivity observed for Zymomonas are a consequence of its unique
physiology. Zymomonas is the only microorganism that
metabolizes glucose anaerobically using the ED pathway as
opposed to the EM or glycolytic pathway (Matthew et al.
2005). The ED pathway yields only half as much ATP per
mole of glucose as the EM pathway. As a consequence,
Zymomonas produces less biomass than yeast, and more
carbon is funneled to fermentation products. Also, as a
consequence of the low ATP yield, Zymomonas maintains a
high glucose flux through the ED pathway. All the enzymes
involved in fermentation are expressed constitutively, and
fermentation enzymes comprise as much as 50% of the
cells’ total protein (Sprenger 1996).
Zymomonas mobilis is an unusual Gram-negative microorganism that has several appealing properties as a biocatalyst for ethanol production. The microorganism has a
homoethanol fermentation pathway and tolerates up to
120 g/l ethanol. It has a higher ethanol yield (5–10% more
ethanol per fermented glucose) and has a much higher
specific ethanol productivity (2.5×) than Saccharomyces
sp. (Sprenger 1996). Furthermore, Z. mobilis is GRAS and
has simple nutritional needs. It is so well suited for ethanol
production that in the 1970s and 1980s, some researchers
advocated it as superior to S. cerevisiae. Despite its
advantages as an ethanologen, Z. mobilis is not well suited
for all of the biomass resources conversion because it
ferments only glucose, fructose, and sucrose. Moreover, for
Z. mobilis on synthetic media containing either glucose,
fructose or sucrose, the specific rates of sugar uptake and
ethanol production are at a maximum when utilizing the
glucose medium. In addition, S. cerevisiae is still preferred
by the industry because of the yeast hardiness.
Engineering Escherichia coli is another valuable bacterial resource for ethanol production. The construction of E.
coli strains to selectively produce ethanol (Millichip and
Doelle 1989) was one of the first successful applications of
metabolic engineering. E. coli has several advantages as a
biocatalyst for ethanol production, including the ability to
ferment a wide spectrum of sugars, no requirements for
complex growth factors, and prior industrial use (e.g., for
production of recombinant protein). The major disadvantages associated with using E. coli cultures are a narrow
and neutral pH growth range (6.0–8.0), less hardy cultures
compared to yeast, and public perceptions regarding the
danger of E. coli strains. The lack of data on the use of
residual E. coli cell mass as an ingredient in animal feed is
also an obstacle to its application.
Cellulose-to-ethanol biotransformation can be conducted
by various anaerobic thermophilic bacteria, such as Clostridium thermocellum (Ingram et al. 1987), as well as by
some filamentous fungi, including Monilia sp. (Saddler and
Chan 1982), Neurospora crassa (Gong et al. 1981), Neurospora sp. (Yamauchi et al. 1989), Zygosaccharomyces rouxii
(Pastore et al. 1994), Aspergillus sp. (Sugawara et al. 1994),
Trichoderma viride (Ito et al. 1990), and Paecilomyces sp.
(Gervais and Sarrette 1990). However, studies on the fermentation process utilizing these microorganisms have
shown this process to be very slow (3–12 days) with a
poor yield (0.8–60 g/l of ethanol), which most probably is
due to the low resistance of microorganisms to higher
concentrations of ethyl alcohol. Another disadvantage of this
process (particularly in the case of bacterial fermentation) is
the production of various by-products, primarily acetic and
lactic acids (Herrero and Gomez 1980; Wu et al. 1986).
Hydrolysis enzymes and the related microorganisms
In addition to polymeric carbohydrates, raw material for
ethanol fermentation contains varying amounts of polyphenolic lignin and other “extractables.” These compounds
are not directly fermentable by most yeasts, and they must
be pretreated to hydrolyze the complicate compounds to
simple sugars (Zertuche and Zall 1982). Development of an
ideal pretreatment process is difficult, given that “biomass”
includes such sources as hardwood and softwood trees,
agricultural residues such as corn stover and nonrecyclable
paper waste.
631
Table 1 Yeast species which produce ethanol as the main fermentation product
Strain-species
Temperature pH Carbon source and
(°C)
value concentration (g/l)
Nitrogen source and
concentration (g/l)
Incubation Concentration of
time (h) ethanol produced
(g/l)
References
27817Saccharomyces
cerevisiae
L-041-S.
cerevisiae
181-S.
cerevisiae
(aerobic)
UO-1-S.
cerevisiae
(aerobic)
V5-S. cerevisiae
30
5.5
Glucose (50–200)
Peptone (2) and ammonium
sulfate (4)
18–94
5.1–91.8
Vallet et al.
1996
30 or 35
–
Sucrose (100)
25–50
27
6.0
Glucose (10)
Urea (1) or ammonium sulfate 24
(1–2)
Peptone (5.0)
40–160
–
Leticia et al.
1997
Todor and
Tsonka 2002
30
5.0
Sucrose (20)
Ammonium sulfate (1)
60–96
–
24
–
Glucose (250)
–
36
–
ATCC 24860-S.
cerevisiae
Bakers’ yeast-S.
cerevisiae
Bakers’ yeast-S.
cerevisiae
Fiso-S. cerevisiae
30
4.5
Molasses (1.6–5.0)
Ammonium sulfate (0.72–2.0) 24
30
4.5
Sugar (150–300)
–
28
5.0
Sucrose (220)
30
5.0
A3-S. cerevisiae
30
5.0
L52-S. cerevisiae 30
5.0
GCB-K5-S.
cerevisiae
GCA-II-S.
cerevisiae
KR18-S.
cerevisiae
CMI237-S.
cerevisiae
2.399-S.
cerevisiae
24860-S.
cerevisiae
27774Kluyveromyces
fragilis
30017-K.fragilis
30
5–18.4
192
53 (max)
96
96.71
60
4.8–40
60
4.8–36.8
60
2.4–32.0
6.0
Peptone(5) and ammonium
dihydrogen phosphate (1.5)
Galactose (20–150) Peptone, ammonium sulfate
and casamino acid (10)
Galactose (20–150) Peptone, ammonium sulfate
and casamino acid (10)
Galactose (20–150) Peptone, ammonium sulfate
and casamino acid (10)
Sucrose (30)
Peptone (5)
72
27
30
6.0
Sucrose (30)
Peptone (5)
72
42
30
6.0
Sucrose (30)
Peptone (5)
72
22.5
30
4.5
Sugar (160)
Ammonium sulfate (0.5)
30
70 (max)
30
5.5
Glucose (31.6)
Urea (6.4)
30
13.7 (max)
–
–
Glucose (150)
27
48 (max)
30
5.5
Glucose (20–120)
Ammonium dihydrogen
phosphate (2.25)
Peptone (2) and ammonium
sulfate (4)
18–94
48.96 (max)
30
5.5
Glucose (20–120)
18–94
48.96 (max)
30016-Kluyvero- 30
myces marxianus
30091-Candida 30
utilis
ATCC-32691
30
Pachysolen
tannophilus
5.5
18–94
44.4 (max)
18–94
44.4 (max)
100
7.8 (max)
5.5
4.5
Peptone (2) and ammonium
sulfate (4)
Glucose (100)
Peptone (2) and ammonium
sulfate (4)
Glucose (100)
Peptone (2) and ammonium
sulfate (4)
Glucose (0–25) and Peptone (3.6) and ammonium
xylose (0–25)
sulfate (3)
CamachoRuiz et al.
2003
Virginie et al.
2001
Ergun and
Mutlu 2000
Roukas 1996
Caylak and
Vardar 1996
da Cruz et al.
2003
da Cruz et al.
2003
da Cruz et al.
2003
Kiran et al.
2003
Kiran et al.
2003
Kiran et al.
2003
Navarro et al.
2000
Yu and Zhang
2004
Ghasem et al.
2004
Vallet et al.
1996
Vallet et al.
1996
Vallet et al.
1996
Vallet et al.
1996
Sanchez et al.
1999
632
Table 2 Bacterial species which produce ethanol as the main
fermentation product
Mesophilic
organisms
Mmol ethanol produced per References
mmol glucose metabolized
Clostridium
sporogenes
Clostridium
indoli
(pathogenic)
Clostridium
sphenoides
Clostridium
sordelli
(pathogenic)
Zymomonas
mobilis
(syn. Anaerobica)
Zymomonas
mobilis subsp.
pomaceas
Spirochaeta
aurantia
Spirochaeta
stenostrepta
Spirochaeta
litoralis
Erwinia
amylovora
Escherichia
coli KO11
up to 4.15a
Miyamoto 1997
1.96a
Miyamoto 1997
1.8a (1.8)b
Miyamoto 1997
1.7
Miyamoto 1997
1.9
Miyamoto 1997
1.7
Miyamoto 1997
1.5 (0.8)
Miyamoto 1997
0.84 (1.46)
Miyamoto 1997
1.1 (1.4)
Miyamoto 1997
1.2
Miyamoto 1997
Escherichia
coli LY01
Leuconostoc
mesenteroides
Streptococcus
lactis
Klebsiella
oxytoca
Klebsiella
aerogenes
Mucor sp. M105
Fusarium sp. F5
0.7–0.1
Dien et al. 2003;
Matthew et al.
2005
40–50 g ethanol produced/l Dien et al. 2003
1.1
Miyamoto 1997
1.0
Miyamoto 1997
0.94–0.98
24 g ethanol produced/l
Matthew et al.
2005
Ingram et al. 1998
–
–
Ingram et al. 1998
Ingram et al. 1998
tirely if a fully enzymatic process (yet to be developed) is
implemented instead.
Traditionally, starch was, and still is, hydrolyzed to low
molecular weight dextrins and glucose using acid, but
enzymes have several advantages. First, the specificity of
enzymes allows the production of sugar syrups with welldefined physical and chemical properties. Second, the
milder enzymatic hydrolysis results in few side reactions
and less “browning.” Indeed, for the production of glucose
syrups from starch, enzymic hydrolysis is essential. A
summary of starch degrading enzymes is shown in Fig. 2
(Hsu 1996).
There have been several reports about yeasts that could
produce extracellular α-amylase and glucoamylase. These
include Candida tsukubaensis CBS 6389 (Aktinson and
Mavituna 1991), Filobasisium capsuligenum (Aktinson
and Mavituna 1991), Lipomyces kononenkoae (de Mot
and Verachtert 1985), Lipomyces starkeyi (SpencerMartins and Van Uden 1979), Saccharomycopsis bispora
(formerly Endomycopsis bispora) (Kelly et al. 1985),
Saccharomycopsis capsularis, Saccharomycopsis fibuligera (Ebertova 1966; Stepanov et al. 1975), Schwanniomyces alluvius (Gasperik et al. 1985), Schwanniomyces
castelli (Simoes-Mendes 1984), and Trichosporon pullulans (Silla et al. 1984).
In addition, for the production of cellulolytic enzymes to
be used in the hydrolysis, the lignocellulose-degrading
fungus Trichoderma reesei can be used (Sharma 2000).
This fungus is able to metabolize pentose and hexose
sugars and also oligomers, and it is insensitive to inhibitors
generated from the lignocellulosic material, because these
are normally present in its natural environment.
In this field, it was investigated whether the cellulolytic
fungus T. reesei could degrade inhibitory compounds
present in a hemicellulose hydrolysate obtained after steam
pretreatment of willow and thereby decrease its inhibitory
effect on the ethanolic fermentation by S. cerevisiae. It was
also investigated whether the inhibitor containing fraction
could be used as a carbon source for the production of highquality cellulolytic enzymes to be used in the hydrolysis.
Kinetic models
These diverse feedstocks have caused researchers to test
numerous pretreatment processes ranging from hot water
and steam explosion treatments, to alkaline and solvent
pretreatments, to many useful versions of acid pretreatment
(Kaar and Holtzapple 2000; Maiorella 1985; Sun and
Cheng 2002). However, they acknowledge that detoxification of acid-hydrolyzed lignin and other “extractables” in
the sugar hydrolysate will present additional costs for the
total hydrolysis process, costs that could be avoided en-
Generally, economic restrictions force industrial processes
to work in a very small range of operating conditions. For
some batch processes which have long operating times in
each cycle and depend strongly on the operating variables,
it is very important to define the optimum conditions to
achieve sufficient profitability. Kinetic models describing
the behavior of microbiological systems can be a highly
appreciated tool and can reduce tests to eliminate extreme
possibilities.
Various kinetic models have been proposed in the literature for freely suspended cells in either batch or continuous operation (Ramon-Portugal et al. 1997; Reynders
et al. 1996; Tan et al. 1996). Unstructured models give the
most fundamental observations concerning microbial metabolic processes and can be considered a good approxi-
633
Fig. 2 A summary of starch
degrading enzymes
Endo-α-1,4-glucanase
Exomaltohexahydrolase
Exomaltotetrahydrolase
α-1,4-Glucanases
Exo-α-1,4-glucanases
Starch-degrading
enzymes
β-Amylase
Glucoamylase
Isopullulanase
Endo-α-1,6-glucanases
Pullulanase
Isoamylase
Exo-α-1,6-glucanase
Exopullulanase
α-1,6-Glucanases
mation when the cell composition is time dependent or
when the substrate concentration is high compared to the
saturation constant (Sonnleitner et al. 1997). Control
models for routine operation of industrial fermentations
are often based on simple, unstructured models since the
process computer will adjust the model parameters based
on the response of the system to disturbances.
When cultured in glucose media, unstructured models
have been found effective for describing the exponential
phase of the batch fermentation kinetics of cell growth and
ethanol production for strains of Z. mobilis ZM4 and ATCC
10988 (Moser 1985). These models, incorporated with the
bottleneck model approach, provide a base for establishing
a structured model that can describe the transient behavior
of a batch fermentation. An additional parameter, reflecting
the quality of the inoculum, is adjusted to match the model
prediction with the corresponding experimental result. In
continuous culture, the experimental findings suggest that
the specific substrate uptake rate is not linearly dependent
upon the specific growth rate, μ. A structured two-compartment model was introduced by Jobses et al. (1985) to
describe the fermentation of Z. mobilis. According to this
model, the specific substrate (glucose) uptake rate in
steady-state continuous culture is a nonlinear second-order
function of μ.
Gulnur et al. (1998) investigated the mathematical
description concerned with the basic metabolic processes
of S. cerevisiae in immobilized form. Glucose utilization,
ethanol production, and growth pattern of yeast cells
immobilized in calcium alginate gel beads were determined
in a stirred batch system using four different initial substrate concentrations. Eleven different mathematical models taking into account the possibility of glucose or ethanol
inhibition on both yeast cell growth and ethanol production
were studied. The batch performance curves predicted by
the models were compared with the experimental data, and
the results were analyzed in terms of the possible effects of
initial condition (Doruker et al. 1995).
During the simulation of batch alcoholic fermentation
with the different initial conditions employed 11 different
models: the models of Monod, Moser, and Teissier were
used to represent inhibition-free substrate limitation kinetics; the models of Andrews and Noack, Aiba and Luong
include substrate inhibition effects, whereas the models of
Levenspiel, Aiba, Jerusalimsky, Ghose and Tyagi, and
α-Amylase
Hinshelwood include product inhibition effects. The models proposed by Monod and Hinshelwood were found to be
more appropriate for describing the batch growth and
ethanol production of immobilized S. cerevisiae at low and
high initial glucose concentrations, respectively (Gulnur et
al. 1998).
Structured models describing culture kinetics are
important in the control of bioreactors, as they provide a
mathematical description of the mechanism of the process
which are required for optimization and control. The objective of structured modeling is to obtain expressions that
quantitatively describe the behavior of the process under
consideration. A wide variety of models have been proposed for the kinetics of the process; these range from very
simple models (Mori et al. 1970; Namba et al. 1984) to
more complex global models (Park and Toda 1990; Park et
al. 1990, 1991), which take into account the activating and
inhibiting effects of the substrate (glucose and oxygen) and
the product (ethanol and acetic acid; Oh et al. 2000).
However, none of these studies have put forward a general
model sufficiently well developed to permit the design of a
good simulator which is capable of performing simulations
with batch processes.
Moreover, structured models have been used to predict
the influence of operating parameters on cell concentration,
substrate utilization rate, and ethanol production rate.
These models may lead to the development of better strategies for the optimization of the fermentation process to
ensure its economic viability. Although four factors (substrate limitation, substrate inhibition, product inhibition,
and cell death) are known to affect ethanol fermentation,
none of these models accounts for these kinetic factors
simultaneously. Monod’s (1950) equation accounts only
for substrate limitation. The models of Hinshelwood (1946),
Holzberg et al. (1967), Egamberdiev and Jerusalimsky
(1968), Nagatani et al. (1968), Ghose and Tyagi (1979),
Hoppe and Hansford (1982), and Lee (1988) account only
for ethanol inhibition. The models of Aiba et al. (1968),
Aiba and Shoda (1969), and Luong (1985) include only
substrate limitation and substrate inhibition terms. An
appropriate ethanol fermentation model should therefore
account for the four kinetic factors.
A developed mathematical model capable of predicting
the cell, substrate, and ethanol concentrations during the
continuous anaerobic fermentation is necessary. However,
634
it cannot be expected that any kinetic model will be directly
applicable to a real process situation. Therefore, mathematical modeling should start with the simplest type, but it
must be reiterated, modified, and extended until it eventually leads to an adequate process kinetic model.
Pilot plants producing ethanol
Approximately 80% of the ethanol produced in the world is
still obtained from fermentations; the remainder comes
largely by synthesis from the petroleum product, ethylene.
The alcohol produced in the US is primarily used in
alcoholic beverages, but this is not always the case elsewhere in the world. Brazil has embarked on a major
program to produce ethanol for fuel and thereby diminish
petroleum imports. As of 1984, approximately 7.9 million
tons of ethanol was produced by fermentation in Brazil,
with sucrose from sugarcane as the carbon source. The US
is also substantially increasing its fuel alcohol production,
originally because of the rapid increase in petroleum costs
during the 1970s, and the subsequent need for developing
alternative energy sources.
In spite of extensive research on fuel ethanol production
from biomass (shown in Table 3), until 1995, not a single
plant capable of converting cellulosic feedstock to ethanol,
via biological processing on the industrial scale, has been
put into operation anywhere in the world, although some
pilot scale plants have been commissioned (Szczodrak and
Fiedurek 1996).
During World War II, when wartime conditions changed
economic conditions and priorities, several ethanol-fromcellulose (EFC) plants were built and operated in various
countries to provide an alternative fuel source. These
countries include Germany, Russia, China, Korea, Switzerland, and the US among others. Since the end of the war,
competition from synthetically produced ethanol has forced
many of these plants to close (Badger 2002). Since April
2004, the first demonstration plant using lignocellulosic
feedstocks in Canada has been in operation (Tampier et al.
2004). The target volume of 100 million liters of ethanol,
anticipated by 2006, will likely be met or exceeded by 2007.
There is also progress on pretreatment of softwood residues
and pentose fermentation (Natural Resources Canada’s
management team 2005).
Currently, some countries in locations with higher ethanol and fuel prices are producing ethanol from cellulosic
feedstocks. It is only recently that cost-effective technologies for producing EFC in the US have started to emerge
(Badger 2002). In Canada, Iogen Corporation built a small
commercial-scale cellulose–ethanol plant using proprietary
enzymatic hydrolysis technology. In 1997, they partnered
with Petro-Canada to produce cellulose–ethanol beginning
with a 1-million-gallon-per-year ethanol demonstration facility, located at Iogen’s headquarters in Ottawa, using corn
stover and switchgrass (Energy & Environmental Research
Center, 2001). In summer 2005, a Swedish plant in
Örnsköldsvik started to produce ethanol from sawdust.
The production is still in a start-up phase, but the optimism is
high. In a not so distant future, Sweden could become selfsufficient of ethanol from wood and wood residues, which
would be a much more sustainable way of supplying
ethanol to the Swedish market (Advanced course in LCA
2005).
Nowadays, in the field of sugar and starch utilization, the
large-scale application of modern bioenergy conversion
technologies has already occurred in a number of countries,
both in the industrialized and developing worlds. In the US,
the Minnesota Pollution Control Agency (MPCA) has
scheduled a public information meeting in early 2005 to
discuss the proposed Heron Lake BioEnergy ethanol project. The proposed plant would cover 37 acres at a site about
1 mile northeast of the city of Heron Lake in Jackson
County. It would process 21.7 million bushels of corn
annually to produce 55 million gallons of ethanol and
193,300 tons of distiller dried grains (Sullivan 2005).
Another example is that of Brazil, a country that has committed itself to the development of its modern bioenergy
Table 3 The lists of pilot plants for ethanol production from biomass
Year
Place
Substrates
1976
1981
1983
1983
US
Canada
–
Japan
–
Grain
Cellulose
–
1984
1988
1993
2001
2002
2003
2005
Canada
France
US
US
US
Canada
US
–
Cellulose
Concentrated sweet whey
Corn
MSW
Lignocellulosic
–
Capacity
(ton/day)
Production
(l ethanol/day)
References
1
960
2,000
720
–
27,400–220,000
57,750
150–200
1
96
7.5
–
–
–
–
160–190 kg/1,000 kg wood
5,178
155,000
10,360,000
41,500
570,000
Emert and Katzen 1980; Emert et al. 1980
Robert 2004
Emert et al. 1983
Morikawa et al. 1985a,b;
Morikawa and Tadokoro 1987
Bente 1984
Ballerini et al. 1994; Nativel et al. 1992
National Renewable Energy Laboratory 1996
Gary 2002
Badger 2002
Tembec 2003
MN Pollution Control Agency 2005
–
635
potential. Its sugarcane-based ethanol industry annually
produces around 15 billion liters from about 350 distilleries
and satisfies over 33% of the country’s gasoline needs
(Agama Energy 2003).
For the Global ethanol market, Brazil has more than 300
plants, producing 15 billion liters per year and supplying 3
million cars with pure ethanol. In the US, there are more
than 80 plants producing 10 billion liters per year, which it
intends to increase to 19 billion liters by 2010. China could
create 3 billion liters of ethanol per year. India’s annual
production of ethanol is 2.7 billion liters, and Eastern
Europe’s 2.5 billion liters. Western Europe’s production
ability is 2 billion liters and in Canada, 0.24 billion liters
could be achieved and possibly expanded to 1.4 billion
liters (Klein 2005).
Moreover, a fuel tax exemption is necessary for ethanol
to compete with gasoline. Biodiesel from waste vegetable
oil is already nearly competitive with conventional diesel,
which cannot be said of biodiesel made from far more
expensive virgin oils. It is foreseen that within the next 5–
10 years, renewable, alternative transportation fuels from
biomass and wastes will be competitive with fuels derived
from petroleum at about US $ 0.2 per liter.
Generally, economic restrictions force industrial processes to work in a very small range of operating conditions.
For some batch processes which have long operating times
in each cycle and depend strongly on the operating
variables, it is very important to define the optimum conditions to achieve sufficient profitability. Kinetic models
describing the behavior of microbiological systems can be a
highly appreciated tool and can reduce tests to eliminate
extreme possibilities.
Most promising prospects
Ethanol fermentation involves significantly greater challenges, owing to the necessity of converting xylose as well
as glucose to ethanol in the process, the microorganism–
enzyme compatibility in SSF, and the low rates of cellulose
hydrolysis. Recently, research has concentrated on the
development of improved processes; however, there are
still challenges that need further investigations.
Fermentation technology converting xylose to ethanol
Major fermentable sugars in hydrolyzate from cellulose
and hemicellulose are glucose and xylose. Glucose fermentation to ethanol can be carried out efficiently by S.
cerevisiae. In contrast, xylose fermentation is challenging
because only a few traditional ethanol-producing microorganisms can readily ferment xylose, though many microorganisms utilize xylose as a carbon source. Efforts were
made to improve ethanol fermentation from xylose (Jeffries
and Shi 1999; Ho et al. 1999; Ingram et al. 1987; Zhang et
al. 1995).
However, low ethanol yields, by-product formation,
neutral pH requirement for growth, and intolerance to high
ethanol concentration are disadvantages in using bacteria in
large-scale fermentation (Bothast et al. 1999). Currently, the
bacterial conversion of xylose to ethanol has been studied
mostly with utilizing the recombinant microorganisms.
The recombinant E. coli was used for ethanol production
from xylose, and this ethanologenic strain (KO11) was able
to convert glucose and xylose to ethanol at yields of 103–
106% of theoretical value (Gonzalez et al. 2003; Tao et al.
2001). The extra ethanol was thought to arise from fermentation of carbohydrates present in the rich medium that
was not accounted for in the sugar balance. Moreover,
KO11 grows faster on xylose-containing medium than its
parent strain ATCC11303. Comparison of global gene
expression by microarray technology demonstrated that
KO11 overexpresses xylose metabolism genes (Tao et al.
2001). During the combination, two genes are needed, one
for pyruvate decarboxylase and another for alcohol dehydrogenase. These enzymes working together in the cell will
divert pyruvate away from other fermentation products to
ethanol. This would convert E. coli into an ethanologenic
microorganism. The steps by initial E. coli and ethanologenic E. coli in alcoholic fermentation are shown in Fig. 3
(Gottschalk 1986; Matthew et al. 2005).
Similarly, the ethanol-producing bacterium Z. mobilis
was metabolically engineered to broaden its range of
fermentable substrates to include the pentose sugar xylose.
Two operons encoding xylose assimilation and pentose
phosphate pathway enzymes were constructed and transformed into Z. mobilis. The recombinant efficiently
fermented both glucose and xylose, which is essential for
economical conversion of lignocellulosic biomass to ethanol (Ingram and Doran 1995; Lynd et al. 2002; McMillan
et al. 1999; Sun and Cheng 2002; Zhang et al. 1995).
Currently, bacteria modified by this approach must operate
at neutral pH where control of invasion by other organisms
is more difficult than at the more acidic pH levels typical of
most yeasts.
Moreover, Tolan and Finn (1987) transformed Klebsiella
planticola ATCC 33531 with multicopy plasmids containing the pdc gene inserted from Z. mobilis, and expression of
the gene markedly increased the yield of ethanol to 1.3 mol
per mole of xylose, or 25.1 g/l. Concurrently, there was
significant decrease in the yield of other organic byproducts (i.e., formate, acetate, lactate and butanediol).
There have also been several yeast strains which were
capable of fermenting xylose to produce ethanol in batch
culture. Fein et al. (1984) isolated seven strains which were
capable of fermenting xylose to produce ethanol from
crude wood hydrolyzate in batch culture. Xylitol was
found to be one of the major by-products, and the amount
of xylitol varied depending on the strain used. Candida
tropicalis showed the greatest potential for ethanol production from xylose. The crude acid hydrolyzate was
inhibitory to all strains of yeast, even at dilute hydrolyzate
concentrations. Strain acclimatization and chemical pretreatment resulted in a marked increase in utilization of
substrates in acidic crude hydrolyzate. In an attempt to
develop a xylose-fermenting yeast for industrial ethanol
production, UV light-induced mutants of Pachysolen
636
1/2 Glucose
(100 mol glucose)
A
2004; Jeewon 1997; Palmqvist and Hahn-Hagerdal 2000).
Xylose-fermenting yeasts do not grow under anoxic
conditions and do not ferment when fully aerobic. Therefore, development of fermentation glucose and xylose
efficiently is required for large-scale industrial application.
4[H]
2[H]
Phosphoenol
Pyruvate
Succinate
10.7
CO2
Lactate
79.5
Pyruvate
Cellulase enzyme
2[H]
Acetyl-CoA
Formate
2.4
2[H]
Acetaldehyde
CO + H
88.0 75.0
Acetyl-P
2[H]
Acetate
36.5
Ethanol
49.8
1/2 Glucose
(100 mol glucose)
B
4[H]
2[H]
Phosphoenol
Pyruvate
Succinate
0.4
Pet operon
2[H]
Pyruvate
Lactate
5.7
Acetaldehyde
PDC
+CO
2[H]
Acetyl-CoA
Formate
ADH
Ethanol
206
CO + H
2[H]
Acetaldehyde
Ethanol
Acetyl-P
Acetate
0.7
Fig. 3 a Typical fermentation products made by a K12 Escherichia
coli fermenting glucose. Products are in moles produced per 100 mol
fermented glucose (Dien et al. 2003; Gottschalk 1986) with 91% of
the carbon accounted for as fermentation products. b Transforming
E. coli with pet operon diverts almost all glucose to ethanol. This
strain (KO11) also carries a mutation that blocks succinate production. Amount of each fermentation product is shown per 100 mol
glucose (Dien et al. 2003; Ohta et al. 1991). Moles of CO2 produced
was not measured, but should be 206 mol based on ethanol
production
tannophilus have been isolated, which can grow faster on
xylose. Several other yeast strains for xylose utilization
have been reported (Jeewon 1997).
On the other hand, S. cerevisiae traditionally has been
used for ethanol production, such as beer and wine fermentation. This yeast does not exhibit many of the limitations encountered with bacteria. However, S. cerevisiae is
not able to ferment xylose. Therefore, metabolic engineering of xylose fermentation in S. cerevisiae is an attractive
approach (Sonderegger and Sauer 2003).
Although some significant progress can be noted in this
field, there are still some problems which exist. One of
them is ethanol inhibition. Ethanol inhibition of yeasts and
other microorganisms has received much attention in
microbial conversion of xylose to ethanol (Ghasem et al.
Using lignocellulosic materials such as agricultural
residues, grasses, forestry wastes, and other low-cost biomass can significantly reduce the cost of raw materials
(compared to corn) for ethanol production. A reduction of
the cost of ethanol production can be achieved by reducing
the cost of either the raw materials or the cellulase enzymes. It was predicted that the use of genetically engineered raw materials with higher carbohydrate content
combined with the improvement of conversion technology
could reduce the cost of ethanol by US $0.11 per liter over
the next 10 years (Wooley et al. 1999).
Xylose metabolism employs pathways distinctly different from those involved in the utilization of glucose. With
most yeast, xylose metabolism requires aerobic conditions
at which cellular respiration is promoted; however, xylose
is fermented to ethanol in poor yields and at low rates. To
get around this problem, it has been proposed that the
xylose fraction first be converted to readily fermentable
xylulose, i.e., enzyme-mediated fermentation of xylose to
ethanol using the bacterial enzyme, xylose isomerase.
Reducing the cost of cellulase enzyme production is a
key issue in the enzymatic hydrolysis of lignocellulosic
materials. Genetic techniques have been used to clone the
cellulase coding sequences into bacteria, yeasts, fungi, and
plants to create new cellulase production systems with
possible improvement of enzyme production and activity.
Riley et al. (1996) and Wood et al. (1997) reported the
expression of recombinant endoglucanase genes from
Erwinia chrysanthemi P86021 in E. coli KO11, and the
recombinant system produced 3,200 IU endoglucanase/l
fermentation broth (IU, international unit, defined as a
micromole of reducing sugar as glucose released per
minute using carboxymethyl cellulose as substrate). The
thermostable endoglucanase E1 from Acidothermus cellulolyticus was expressed in Arabidopsis thaliana leaves
(Ziegler et al. 2000), potato (Dai et al. 2000), and tobacco
(Hooker et al. 2001).
Immobilization
As in the case of microalgae culture in open ponds,
microecological engineering techniques will need to be
developed to maintain such strains in large systems which
could be subject to invasion and contamination by potentially much faster growing wild microbes. Such microecological techniques would relieve the constraints of
having to maximize the amounts and activities of the
enzymes used in this process and/or maintain strictly
aseptic conditions which are not economical. If intact mi-
637
crobial cells are directly immobilized, the removal of
microorganisms from downstream product can be omitted,
and the loss of intracellular enzyme activity can be kept to a
minimum level (Najafpour 1990).
Use of biofilm reactors for ethanol production has been
investigated to improve economics and the performance of
fermentation processes (Vega et al. 1988). Immobilization
of microbial cells for fermentation has been developed to
eliminate inhibition caused by high concentration of
substrate and product and also to enhance productivity
and yield of ethanol. The work on ethanol production in an
immobilized cell reactor (ICR) showed that production of
ethanol using Z. mobilis was doubled (Ghasem et al. 2004;
Takamitsu et al. 1993). The immobilized recombinant Z.
mobilis was also successfully used with high concentrations of 12–15% sugar (Yamada et al. 2002).
Recently, immobilized biomass activity has been given
more attention since it has been acknowledged to play a
significant role in bioreactor performance (Gikas and
Livingston 1997; Yamada et al. 2002). Frequently, immobilized cells are subjected to limitations in the supply of
nutrients to the cells. Thus, because of the presence of
heterogeneous materials such as immobilized cells, there is
no convective flow inside the beads and the cells can
receive nutrients only by diffusion (Riley et al. 1996).
Immobilization of cells to a solid matrix is an alternative
means of high biomass retention. The cells divide within
and on the core of the matrix (Senthuran et al. 1997).
too low to be useful. There is a group of microorganisms
(Clostridium, Cellulomonas, Trichoderma, Penicillium,
Neurospora, Fusarium, Aspergillus, etc.) showing a high
cellulolytic and hemicellulolytic activity, which are also
highly capable of fermenting monosaccharides to ethanol.
It may be possible, within this group of microorganisms, to
produce “superstrains” via genetic engineering capable of
hydrolyzing cellulose and xylan along with fermentation of
glucose and xylose to ethanol.
Moreover, to make the SSF process more effective, it has
also been found necessary to search for thermostable
strains capable of producing substantial amounts of ethyl
alcohol at temperatures optimal for saccharification and
suitably resistant to ethanol (Szczodrak and Targonski
1988).
Roychoudhury et al. (1992) have developed a notable
way of eliminating the negative effects which excessive
concentrations of ethanol have on yeast activity and
cellulased within the SSF system. They used a vacuum
cycling reactor where the concentration of ethanol was kept
at a relatively low level by its removal from the flash
chamber.
However, more efforts have to be made in the development of microorganisms for industrial ethanol production.
In addition, it is important to keep the rate-limiting step in
mind. In SSF, the ethanol production rate is controlled by
the cellulase hydrolysis rate and not the glucose fermentation, and hence, steps to increase the rate of hydrolysis
will lower the cost of ethanol production via SSF.
Simultaneous saccharification and fermentation
Sugar conversion
Simultaneous saccharification and fermentation (SSF)
gives higher reported ethanol yields and requires lower
amounts of enzyme because end-product inhibition from
cellobiose and glucose formed during enzymatic hydrolysis is relieved by the yeast fermentation (Banat et al. 1998;
McMillan et al. 1999). However, it is not easy to meet all
the requirements of industry due to their low rates of
cellulose hydrolysis, which is the stage limiting the rate of
alcohol production. Another problem arises from the fact
that most microorganisms used for converting cellulosic
feedstock cannot utilize xylose, a hemicellulose hydrolysis
product. Moreover, SSF requires that enzyme and culture
conditions be compatible with respect to pH and temperature. T. reesei cellulases, which constitute the most active
preparations, have optimal activity at pH 4.5 and 55°C. For
Saccharomyces cultures, SSF are typically controlled at
pH 4.5 and 37°C.
To overcome the problems related to SSF, many species
of yeasts, as well as the bacterium Z. mobilis, have been
tested with cellulases produced by T. reesei mutants
(Chaudhuri and Sahai 1993; Haltrich et al. 1994; Spindler
et al. 1992). The currently most promising ethanologenic
bacteria for industrial exploitation are E. coli, Klebsiella
oxytoca, and Z.mobilis (Matthew et al. 2005). Genetic
engineering made it possible to transfer cellulose genes
from Trichoderma to S. cerevisiae (Shoemaker 1984).
However, the cellulases were produced at a concentration
Since sugars are already available in a degradable form and
yeast cells can metabolize sugars directly, these substrates
require the least costly preparation. The other carbohydrates must be hydrolyzed to sugars before they can be
metabolized. Several studies have dealt with the economic
assessment of using cellulose hydrolysate, either from
waste (Cysewski and Wilke 1976; Green et al. 1989;
Maiorella et al. 1984; Wilke et al. 1976) or from wood
(Hinman et al. 1992; Marco et al. 2002). One disadvantage
with the application of these materials is their low sugar
content resulting in low cell and ethanol concentrations.
Hence, although starchy or cellulosic materials are cheaper
than sugar-containing raw materials, the requirement for
converting the starchy or cellulosic materials to fermentable sugars is a disadvantage of these substrates (Lynd et al.
2001).
Moreover, microorganisms used in industry are selected
to provide the best possible combination of characteristics
for the process and equipment being used. The selected
strains should have tolerance to high concentrations of
sugar and ethanol (Keim 1983; Oh et al. 2000).
Ethanol inhibition of yeasts and other microorganisms
has received much attention (Casey and Ingledew 1986) in
microbial production of ethanol. Lucas and van Uden
(1985) investigated the effects of temperature on ethanol
tolerance and thermal death of Candida shehatae and
638
determined that it was more tolerant of ethanol at lower
temperatures. Du Preez et al. (1987) quantitatively evaluated the effects of ethanol on the growth of the xylosefermenting yeasts C. shehatae and Pichia stipitis using
Luong kinetics. The effect of ethanol on metabolic rate has
been examined with ethanol added exogenously. Both
Lucas and van Uden (1985) and du Preez et al. (1987)
placed cells into media containing different concentrations
of ethanol and measured the specific growth rate which
ensued. Unfortunately, less inhibition is observed with
exogenous ethanol than with the same concentration of
ethanol produced endogenously (Hoppe and Hansford
1982; Novak et al. 1981; du Preez et al. 1987). Some have
claimed that the apparently greater inhibition by endogenously produced ethanol reflects the tendency of actively
fermenting cells to accumulate ethanol intracellularly
(Casey and Ingledew 1986; Ghasem et al. 2004); however,
the yeast plasma membrane is known to be very permeable
to ethanol, which casts doubt on this hypothesis. Whatever
the reason for the different effects of externally added and
internally generated ethanol, realistic assessments of ethanol inhibition ought to involve ethanol generated in situ.
On the other hand, since the distillation cost per unit
amount of ethanol produced is substantially higher at low
ethanol concentrations (Zacchi and Axelsson 1989), several investigators have dealt with the idea of concentrating
sugar solutions prior to fermentation (Cysewski et al. 1976;
Iraj et al. 2002; Oh et al. 2000; Zacchi and Axelsson 1989).
Clearly, it is necessary to solve the problem between the
concentration of ethanol produced and sugar added if an
economically sustainable system is to be created using this
method.
References
Advanced course in LCA (2005) How to decrease environmental
impact by choice of car fuel. http://www.infra.kth.se/fms/utbildning/
lca/project%20reports/Group%201%20-%20E85.pdf
Agama Energy (2003) Employment potential of renewable energy in
South Africa. Research commissioned by SECCP. http://www.
agama.co.za/pdf/EPRESAFinalNov2003.pdf
Aiba S, Shoda M (1969) Reassessment of the product inhibition in
alcohol fermentation. J Ferment Technol 47:790–803
Aiba S, Shoda M, Nagatani M (1968) Kinetics of product inhibition
in alcoholic fermentation. Biotechnol Bioeng 11:846–864
Aktinson B, Mavituna F (1991) Upstream processing. In:
Biochemical engineering and biotechnology. Stockton, New
York, pp 525
Anthony O, Ejiofor, Yusuf C, Murray MY (1996) Culture of
Saccharomyces cerevisiae on hydrolyzed waste cassava starch
for production of baking-quality yeast. Enzyme Microb
Technol 18:519–525
Aristidou A, Penttila M (2000) Metabolic engineering applications
to renewable resource utilization. Curr Opin Biotechnol
11:187–198
Badger PC (2002) Ethanol from cellulose: a general review. In:
Janick J, Whipkey A (eds) Trends in new crops and new uses.
American Society for Horticultural Science (ASHS) Press,
Alexandria, VA, USA
Ballerini D, Desmarquest JP, Pourquie J, Nativel F, Rebeller H
(1994) Ethanol production from lignocellulosics: large scale
experimentation and economics. Bioresour Technol 50:17–23
Ballesteros M, Oliva JM, Negro MJ, Manzanares P, Ballesteros I
(2004) Ethanol from lignocellulosic materials by a simultaneous saccharification and fermentation process (SSF) with
Kluyveromyces marxianus CECT 10875. Process Biochem
39:1843–1848
Banat IM, Nigam P, Singh D, Marchant P, McHale AP (1998)
Ethanol production at elevated temperatures and alcohol
concentrations. Part I: Yeasts in general. World J Microbiol
Biotechnol 14:809–821
Bente PF (1984) Ethanol from cellulose. In: Bente PF (ed)
International bioenergy directory and handbook. The BioEnergy Council, Washington, DC
Bollók M, Réczey K, Zacchi G (2000) Simultaneous saccharification and fermentation of steam-pretreated spruce to ethanol.
Appl Biochem Biotechnol 84–86:69–80
Bothast RJ, Nichols NN, Dien BS (1999) Fermentations with new
recombinant organisms. Biotechnol Prog 15:867–875
Van den Broek R (2000) Sustainability of biomass electricity
systems—an assessment of costs, macro-economic and environmental impacts in Nicaragua, Ireland and the Netherlands.
Utrecht University, p 215
Brown RC, Radlein D, Piskorz J (2001) Pretreatment processes to
increase pyrolytic yield of levoglucosan from herbaceous
feedstocks. In: Bosell JJ (ed) American chemical society
symposium series no. 784. American Chemical Society,
Washington DC, USA, pp 123–134
Camacho-Ruiz L, Perez-Guerra N, Roses RP (2003) Factors
affecting the growth of Saccharomyces cerevisiae in batch
culture and in solid sate fermentation. Electron J Environ Agric
Food Chem 2(5):531–542
Casey GP, Ingledew WM (1986) Ethanol tolerance in yeasts. Crit
Rev Microbiol 13:219–280
Caylak B, Vardar SF (1996) Comparison of different production
processes for bioethanol. Turk J Chem 22:351–359
Chaudhuri BK, Sahai V (1993) Production of cellulose enzyme from
lactose in batch and continuous cultures by a partially
constitutive strain of Tricholerma reesei. Enzyme Microb
Technol 15:513–518
da Cruz SH, Batistote M, Ernandes JR (2003) Effect of sugar
catabolite repression in correlation with the structural complexity of the nitrogen source on yeast growth and fermentation.
J Inst Brew 109(4):349–355
Cysewski GR, Wilke CR (1976) Utilization of cellulosic materials
through enzymatic hydrolysis. I. Fermentation of hydrolysate to
ethanol and single cell protein. Biotechnol Bioeng 18:1297–
1313
Dai ZY, Hooker BS, Anderson DB, Thomas SR (2000) Improved
plant-based production of E1 endoglucanase using potato:
expression optimization and tissue targeting. Mol Breed 6:277–
285
Dien BS, Cotta MA, Jeffries TW (2003) Bacteria engineered for fuel
ethanol production current status. Appl Microbiol Biotechnol
63:258–266
Doruker O, Onsan ZI, Kirdar B (1995) Ethanol fermentation by
growing S. cerevisiae cells immobilized in small Ca-alginate
beads. Turk J Chem 19:37–42
Ebertova H (1966) Amylolytic enzymes of Endomycopsis capsularis. II. A study of properties of isolated a-amylase,
amyloglucosidase and maltose trans glucosidase. Folia Microbiol
11:422–438
Egamberdiev NB, Jerusalimsky A (1968) Continuous cultivation of
microorganisms. Czechoslovak Academy of Sciences, Prague
Emert GH, Katzen R (1980) Gulf’s cellulose-to-ethanol process.
Chemtech 10:610–614
Emert GH, Katzen R, Fredrickson RE, Kaupisch KF (1980)
Economic update of the Gulf cellulose alcohol process. Chem
Eng Prog 76:47–52
Emert GH, Katzen R, Fredrickson RE, Kaupisch KF, Yeats CE
(1983) Update on the 50 T/D cellulose-to-ethanol plant (in Proc
Cellulose Conf. 1982 Part 2). J Appl Polym Sci Appl Polym
Symp 37:787–795
639
Ergun M, Mutlu SF (2000) Application of a statistical technique to
the production of ethanol from sugar beet molasses by
Saccharomyces cerevisiae. Bioresour Technol 73:251–255
Fein JE, Tallim SR, Lawford GR (1984) Evaluation of D-xylose
fermenting yeasts for utilization of a wood-derived hemicellulose hydrolysate. Can J Microbiol 30:682–690
Gary D (2002) Developing Manitoba’s ethanol industry. http://www.
gov.mb.ca/est/energy/ethanol/
Gasperik H, Hostinova E, Zeinka J (1985) Production of extracellular amylase by Endomycopsis fibuligera on complex starch
substrates. Biologia (Bratisl) 40:1176–1194
Gervais P, Sarrette M (1990) Influence of age of mycelia and water
activity on aroma production by Trichoderma viride. J Ferment
Bioeng 69:46–50
Ghasem N, Habibollah Y, Ku S, Ku I (2004) Ethanol fermentation in
an immobilized cell reactor using Saccharomyces cerevisiae.
Bioresour Technol 92:251–260
Ghose TK, Tyagi RD (1979) Rapid ethanol fermentation of cellulose
hydrolysate II. Product and substrate inhibition and optimization of fermentor design. Biotechnol Bioeng 21:1401–1420
Gikas P, Livingston AG (1997) Specific ATP and specific oxygen
uptake rate in immobilized cell aggregates: experimental results
and theoretical analysis using a structured model of immobilized cell growth. Biotechnol Bioeng 55:660–672760
Gong CS, Maun CM, Tsao GT (1981) Direct fermentation of
cellulose to ethanol by a cellulolytic filamentous fungus
Monilia sp. Biotechnol Lett 3:77–82
Gonzalez R, Tao H, Purvis JE, York SW, Shanmugam KT, Ingram
LO (2003) Gene Array-Based identification of changes that
contribute to ethanol tolerance in ethanologenic Escherichia
coli: comparison of KO11 (Parent) to LY01 (resistant mutant).
Biotechnol Prog 19:612–623
Gottschalk G (1986) Bacterial metabolism, 2nd edn. Springer, New
York Berlin Heidelberg, p 237
Green M, Kimchie S, Malester I, Shelef G (1989) Ethanol
production from municipal solid waste via acid hydrolysis.
In: Klas DL (ed) Energy from biomass and wastes XIII.
Institute of Gas Technology, Chicago, pp 1281–1293
Gulnur B, Pemra D, Betul K, Ilsen O, Kutlu U (1998) Mathematical
description of ethanol fermentation by immobilized Saccharomyces Cererisiae. Process Biochem 33(7):763–771
Haltrich D, Laussamayer B, Steiner W, Nidetzky B, Kulbe D (1994)
Cellulolytic and semicellulolytic enzymes of Sclerotium rolfsii:
optimization of the culture medium and enzymatic hydrolysis
of lignocellulosic material. Bioresour Technol 50:43–50
Hari KS, Janardhan RT, Chowdary GV (2001) Simultaneous
saccharification and fermentation of lignocellulosic wastes to
ethanol using thermotolerant yeast. Bioresour Technol 77:193–
196
Herrero AA, Gomez RF (1980) Development of ethanol tolerance in
Clostridium thermocellum: effect of growth temperature. Appl
Environ Microbiol 40:571–577
Hinman ND, Schell DJ, Riley CJ, Bergeron PW, Walter PJ (1992)
Preliminary estimate of the cost of ethanol production for SSF
technology. Appl Biochem Biotechnol 34/35:639–49
Hinshelwood CN (1946) Kinetics of the bacterial cell. Oxford
University Press, London, UK
Ho NWY, Chen Z, Brainard AP, Sedlak M (1999) Successful design
and development of genetically engineered Saccharomyces
yeasts for effective cofermentation of glucose and xylose from
cellulosic biomass to fuel ethanol. Adv Biochem Eng
Biotechnol 65:163–192
Holzberg I, Finn RF, Steinkraus KH (1967) A kinetic study of the
alcoholic fermentation of grape juice. Biotechnol Bioeng
9:413–423
Hooker BS, Dai Z, Anderson DB, Quesenberry RD, Ruth MF,
Thomas SR (2001) Production of microbial cellulases in
transgenic crop plants. In: Himmel ME, Baker JO, Saddler
JN (eds) Glycosyl hydrolases for biomass conversion. American Chemical Society, Washington, DC, pp 55–90
Hoppe GK, Hansford GS (1982) Ethanol inhibition of continuous
anaerobic yeast growth. Biotechnol Lett 4:39–44
Hsu T (1996) Pretreatment of biomass. In: Wyman C (ed) Handbook
on bioethanol: production and utilization. Taylor & Francis,
Washington DC, pp 179–212
Ingram LO, Conway T, Clark DP, Sewell GW, Preston JF (1987)
Genetic engineering of ethanol production in Escherichia coli.
Appl Environ Microbiol 53:2420–2425
Ingram LO, Doran JB (1995) Conversion of cellulosic materials to
ethanol. FEMS Microbiol Rev 16:235–241
Ingram LO, Gomez PF, Lai X, Moniruzzaman M, Wood BE,
Yomano LP, York SW (1998) Metabolic engineering of bacteria
for ethanol production. Biotechnol Bioeng 58(2,3):204–214
Iraj N, Giti E, Lila A (2002) Isolation of flocculating Saccharomyces
cerevisiae and investigation of its performance in the fermentation of beet molasses to ethanol. Biomass Bioenergy 23:481–
486
Ito K, Yoshida K, Ishikawa T, Kobayashi S (1990) Volatile
compounds produced by fungus Aspergillus oryzae in rice
koji and their changes during cultivation. J Ferment Bioeng
70:169–172
Jackman EA (1987) Industrial alcohol. In: Bu’lock JD, Christiansen
B (eds) Basic biotechnology. Academic, London, pp 309–336
Jeewon L (1997) Biological conversion of lignocellulosic biomass
to ethanol. J Biotechnol 56:1–24
Jeffries TW, Jin YS (2000) Ethanol and thermotolerance in the
bioconversion of xylose by yeasts. Adv Appl Microbiol
47:221–268
Jeffries TW, Shi NQ (1999) Genetic engineering for improved
xylose fermentation of yeasts. Adv Biochem Eng Biotechnol
65:117–161
Jobses IML, Egberts GTC, van Baalen A, Roels JA (1985)
Substrate-limited continuous culture results at all growth rates
and showed a slight downward. Biotechnol Bioeng 27:984–995
John T (2004) Biofuels for transport. http://www.task39.org/
Kaar WE, Holtzapple MT (2000) Using lime pretreatment to
facilitate the enzymatic hydrolysis of corn stover. Biomass
Bioenergy 18:189–199
Kadam KL, McMillan JD (2003) Availability of corn stover as a
sustainable feedstock for bioethanol production. Bioresour
Technol 18:17–25
Kaylen ML, Van Dyne D, Choi YS, Blase M (2000) Economic
feasibility of producing ethanol from lignocellulosic feedstocks.
Bioresour Technol 72:19
Keim CR (1983) Technology and economics of fermentation alcohol
—an update. Enzyme Microb Technol 5:103–114
Kelly CT, Moriarty ME, Fogarty WM (1985) Thermostable
extracellular a-amylase and α-glucosidase of Lipomyces
starkeyi. Appl Microbiol Biotechnol 22:352–358
Kerr RA (1998) The next oil crisis looms large—and possibly close.
Science 281:1128–1131
Kiran S, Sikander A, Lkram-ul-Haq (2003) Time course study for
yeast invertase production by submerged fermentation. J Biol
Sci 3(11):984–988
Klein K (2005). Economic and social implications of bio-fuel use
and production in Canada. http://www.biocap.ca/images/pdfs/
conferenceSpeakers/Klein_K.pdf
Lee JM (1988) Computer simulation in ethanol fermentation. In:
Fofer SS, Zaborsky OR (eds) Biomass conversion processes for
energy and fuels. Plenum, New York
Leticia P, Miguel C, Humberto G, Jaime AJ (1997) Fermentation
parameters influencing higher alcohol production in the tequila
process. Biotechnol Lett 19(1):45–47
Lindeman LR, Rocchiccioli C (1979) Ethanol in Brazil; brief
summary of the sate of the industry in 1977. Biotechnol Bioeng
21:1107–1119
Lucas C, van Uden N (1985) The temperature profiles of growth,
thermal death and ethanol tolerance of the xylose-fermenting
yeast Candida shehatae. J Basic Microbiol 25:547–550
Luong JHT (1985) Kinetics of ethanol inhibition in alcohol
fermentation. Biotechnol Bioeng 27:280–285
Lynd LR (1996) Overview and evaluation of fuel ethanol production
from cellulosic biomass: technology, economics, the environment, and policy. Annu Rev Energy Environ 21:403–465
640
Lynd LR, Lyford K, South CR, van Walsum GP, Levenson K (2001)
Evaluation of paper sludges for amenability to enzymatic
hydrolysis and conversion to ethanol. Tappi J 84(2):50
Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial
cellulose utilization: fundamentals and biotechnology. Microbiol
Mol Biol Rev 66(3):506–577
MacDonald T, Yowell G, McCormack M (2001) Staff report. US
ethanol industry production capacity outlook. California energy
commission. Available at http://www.energy.ca.gov/reports/
2001-08-29_600-01-017.PDF
Maiorella BL (1985) Ethanol. In: Comprehensive biotechnology, vol
3. Pergamon, Oxford, pp 861–909
Maiorella BL, Blanch HW, Wilke CR (1984) Economic evaluation
of alternative ethanol fermentation processes. Biotechnol
Bioeng 26:1003–1025
Maisch WF, Sobolov M, Petricola AJ (1979) Distilled beverages. In:
Peppler HJ, Perlman D (eds) Microbial technology. Academic,
New York, pp 79
Marco DL, Cristiano PB, Tito LMA (2002) Economic analysis of
ethanol and fructose production by selective fermentation
coupled to pervaporation: effect of membrane costs on process
economics. Desalination 147:161–166
Matsumoto N, Yoshizumi H, Miyata S, Inoue S (1985) Development of the non-cooking and low temperature cooking systems
for alcoholic fermentation of grains. Nippon Nogeikagaku
Kaishi 59:291–299
Matthew H, Ashley O, Brian K, Alisa E, Benjamin JS (2005) Wine
making 101. Available at http://www.arches.uga.edu/∼matthaas/
strains.htm
McMillan JD, Newman MM, Templeton DW, Mohagheghi A (1999)
Simultaneous saccharification and cofermentation of dilute-acid
pretreated yellow poplar hardwood to ethanol using xylosefermenting Zymomonas mobilis. Appl Biochem Biotechnol 77/
79:649–655
Millichip RJ, Doelle HW (1989) Large-scale ethanol production
from Milo (Sorghum) using Zymomonas mobilis. Process
Biochem 24:141–145
Miyamoto K (1997) Renewable biological systems for alternative
sustainable energy production. http://www.fao.org/docrep/w7241e/
w7241e00.htm #Contents
MN Pollution Control Agency (2005). http://www.me3.org/issues/
ethanol/
Monique H, Faaij A, van den Broek R, Berndes G, Gielen D,
Turkenburg W (2003) Exploration of the ranges of the global
potential of biomass for energy. Biomass Bioenergy 25:119–
133
Monod J (1950) The growth of bacterial culture. Ann Rev Microbiol
3:371
Mori A, Konno N, Terui G (1970) Kinetic studies on submerged
acetic acid fermentation. I. Behaviors of Acetobacter rancens
cells towards dissolved oxygen. J Ferment Technol 48:203–212
Morikawa Y, Tadokoro T (1987) Alcohol as a fuel. New
manufacturing method by fermentation and its problems.
Nenryo Kyokaishi 66:982–991
Morikawa Y, Kawamori M, Ado Y, Shinsha Y, Oda F, Takasawa S
(1985) Production of ethanol from biomasses. Part 1. Improvement of cellulose production in Trichoderma reesei. Agric
Biol Chem 49:1869–1871
Morikawa Y, Takasawa S, Masunaga I, Takayama K (1985) Ethanol
production from D-xylose and cellobiose by Kluyveromyces
cellobiovorus. Biotechnol Bioeng 27:509–513
Moser A (1985) Kinetics of batch fermentations. In: Rehm HJ, Reed
G (eds) Biotechnology. VCH Verlagsgesellschaft mbH, Weinheim, pp 243–283
de Mot R, Verachtert H (1985) Purification and characterization of
the extracellular amylolytic enzymes for the yeast Filobasidium
capsuligenum. Appl Environ Microbiol 50:1474–1482
Nagatani M, Shoda M, Aiba S (1968) Kinetics of product inhibition
in alcoholic fermentation. J Ferment Technol 46:241–249
Najafpour GD (1990) Immobilization of microbial cells for the
production of organic acids. J Sci Islam Repub Iran 1:172–176
Namba A, Tamura A, Nagai S (1984) Synergistic effects of acetic
acid and ethanol on the growth of Acetobacter sp. J Ferment
Technol 62(6):501–505
National Renewable Energy Laboratory (1996). http://es.epa.gov/
techinfo/facts/nu-rctor.html
Nativel F, Pourquie J, Ballerini D, Vandecasteele JP, Renault PH
(1992) The biotechnology facilities at Soustons for biomass
conversion. Int J Sol Energy 11:219–229
Natural Resources Canada’s management team (2005). http://www2.
nrcan.gc.ca/dmo/aeb/English/ReportDetail.asp?x=265&type=rpt
Navarro AR, Sepulveda MC, Rubio MC (2000) Bio-concentration
of vinasse from the alcoholic fermentation of sugar cane
molasses. Waste Manag 20:581–585
Novak M, Strehaiano P, Moreno M, Gama G (1981) Alcoholic
fermentation: on the inhibitory effect of ethanol. Biotechnol
Bioeng 23:201–211
Ogier JC, Ballerini D, Leygue JP, Rigal L, Pourquie J (1999)
Ethanol production from lignocellulosic biomass. Oil Gas Sci
Technol 54:67–94
Oh KK, Kim SW, Jeong YS, Hong SI (2000) Bioconversion of
cellulose into ethanol by nonisothermal simultaneous saccharification and fermentation. Appl Biochem Biotechnol 89:15–30
Ohta K, Beall DS, Mejia JP, Shanmugam KT, Ingram LO (1991)
Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes
encoding pyruvate decarboxylase and alcohol dehydrogenase
II. Appl Environ Microbiol 57:893–900
Palmqvist E, Hahn-Hagerdal B (2000) Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification and II:
inhibitors and mechanisms of inhibition. Bioresour Technol 74
(1):17–33
Park YS, Toda K (1990) Simulation study on bleed effect in cellrecycle culture of Acetobacter aceti. J Gen Appl Microbiol
36:221–233
Park YS, Ohtake H, Toda K (1990) A kinetic study of acetic acid
production by liquid-surface culture of Acetobacter aceti. Appl
Microbiol Biotechnol 33:259–263
Park YS, Kiyoshi T, Fukaya M, Okumura H, Kawamura Y (1991)
Production of a high concentration acetic acid by Acetobacter
aceti using a repeated fed-batch culture with cell recycling.
Appl Microbiol Biotechnol 35:149–153
Pastore GM, Park YK, Min DB (1994) Production of a fruity aroma
by Neurospora from beiju. Mycol Res 98:25–35
Polman K (1994) Review and analysis of renewable feedstocks for
the production of commodity chemicals. Appl Biochem
Biotechnol 45:709–722
du Preez JC, Bosch M, Prior BA (1987) Temperature profiles of
growth and ethanol tolerance of the xylose-fermenting yeasts
Candida shehatae and Pichia stipitis. Appl Microbiol Biotechnol
25:521–525
Ramon-Portugal F, Delia-Dupuy ML, Pingaud H, Riba JP (1997)
Kinetic study and mathematical modeling of the growth of S.
cerevisiae 522D in presence of K2 killer protein. J Chem
Technol Biotechnol 68:195–201
Reynders MB, Rawlings DE, Harrison STL (1996) Studies on the
growth, modeling and pigment production by the yeast Phaffia
rhodozyma during fed-batch cultivation. Biotechnol Lett 18
(6):649–654
Riley MR, Muzzio FJ, Buettner HM, Reyes SC (1996) A simple
correlation for predicting effective diffusivities in immobilized
cell systems. Biotechnol Bioeng 49:223–227
Robert G (2004) Profiles of successful proponents. http://www.
nrcan-rncan.gc.ca/media/newsreleases/2004/200402b_e.htm
Rosillo-Calle F, Cortez L (1998) Towards proalcohol II: a review of
the Brazilian bioethanol programme. Biomass Bioenergy
14:115–124
Roukas T (1996) Ethanol production from non-sterilized beet
molasses by free and immobilized Saccharomyces cerevisiae
cells using fed-batch culture. J Eng 27:87–96
Roychoudhury PK, Ghose TK, Ghosh P (1992) Operational
strategies in vacuum-coupled SSF for conversion of lignocellulose to ethanol. Enzyme Microb Technol 14:581–585
641
Saddler JN, Chan MKH (1982) Optimization of Clostridium
thermocellum growth on cellulose and pretreated wood
substrates. Eur J Appl Microbiol Biotechnol 16:99–104
Sanchez S, Bravo V, Castro E, Moya AJ, Camacho F (1999)
Comparative study of the fermentation of D-glucose/D-xylose
mixtures with Pachysolen tannophilus and Candida shehatae.
Bioprocess Eng 21:525–532
Sasson A (1990) Feeding tomorrow’s world. UNESCO, Paris,
pp 500–510
Senthuran A, Senthuran V, Mattiasson B, Kaul R (1997) Lactic acid
fermentation in a reactor using immobilized Lactobacillus
casei. Biotechnol Bioeng 55:841–853
Sharma SK (2000) Saccharification and bioethanol production from
sunflower stalks and hulls. PhD thesis, Department of Microbiology. Punjab Agricultural University, Ludhiana, India
Shoemaker SP (1984) Cellulase system of Trichoderma reesei:
trichoderma strain improvement and expression of Trichoderma
cellulases in yeast. World Biotech Rep 2:593–600
Silla AM, Zygora PSJ, Stewart GG (1984) Characterization of Sch.
Castellii mutants with increased productivity of amylase. Appl
Microbiol Biotechnol 20:124–128
Simoes-Mendes B (1984) Purification and characterization of the extracellular amylase of the yeast Sch. Alluvius. Can J Microbiol
30:1163–1170
Sonderegger M, Sauer U (2003) Evolutionary engineering of
Saccharomyces cerevisiae for anaerobic growth on xylose.
Appl Environ Microbiol 69(4):1990–1998
Sonnleitner B, Rothen AS, Kuriyama H (1997) Dynamics of glucose
consumption in yeast. Biotechnol Prog 13:8–13
Spencer-Martins I, Van Uden N (1979) Extracellular amylolytic
system of the yeast Lipomyces kononenkoae. Eur J Appl
Microbiol Biotechnol 6:241–250
Spindler DD, Wyman CE, Grohmann K, Philippidis GP (1992)
Evaluation of the cellobiose-fermenting yeast Brettanomyces
custersii in the simultaneous saccharification and fermentation
of cellulose. Biotechnol Lett 14:403–407
Sprenger GA (1996) Carbohydrate metabolism in Zymomonas
mobilis: a catabolic highway with some scenic routes. FEMS
Microbiol Lett 145:301–307
Stenberg K, Bollók M, Réczey K, Galbe M, Zacchi G (2000) Effect
of substrate and cellulase concentration on simultaneous
saccharification and fermentation of steam-pretreated softwood
for ethanol production. Biotechnol Bioeng 68(2):201–210
Stepanov AI, Afanaseva VP, Zaitseva GV, Mednokova AP,
Lupandina IB (1975) Regulation of the biosynthesis of the
enzyme of amylolytic acomplex of Endomycopsis fibuligera.
Prikl Biohim Mikrobiol 11:682–685
Sugawara E, Hashimoto S, Sakurai Y, Kobayashi A (1994)
Formation by yeast of the HEMF (4-hydrpxy-2 (or 5)-ethyl-5
(or 2)-methyl-3 (2H)-furanone) aroma components in Miso
with aging. Biosci Biotechnol Biochem 58:1134–1135
Sullivan J (2005) Information meeting February 17 on environmental review, air and water permits for Heron Lake Ethanol
Project.
http://www.pca.state.mn.us/news/data/newsRelease.
cfm?NR=266105&type=2
Sun Y, Cheng J (2002) Hydrolysis of lignocellulosic materials for
ethanol production: a review. Bioresour Technol 83(1):1–11
Szczodrak J, Fiedurek J (1996) Technology for conversion of
lignocellulosic biomass to ethanol. Biomass Bioenergy 10
(5):367–375
Szczodrak J, Targonski Z (1988) Selection of thermotolerant yeast
strains for simultaneous saccharification and fermentation of
cellulose. Biotechnol Bioeng 31:300–303
Takamitsu I, Izumida H, Akagi Y, Sakamoto M (1993) Continuous
ethanol fermentation in molasses medium using Zymomonas
mobilis immobilized in photo-cross linkable resin gels. J
Ferment Bioeng 75:32–35
Tampier M, Smith D, Bibeau E, Beauchemin PA (2004) Identifying
environmentally preferable uses for biomass resources. http://
www.cec.org/files/PDF/ECONOMY/Biomass-Stage-I-II_en.pdf
Tan Y, Wang Z, Marchall KC (1996) Modelling substrate inhibition
of microbial growth. Biotechnol Bioeng 52:602–608
Tao H, Gonzalez R, Martinez A, Rodriguez M, Ingram LO, Preston
JF, Shanmugam KT (2001) Engineering a homo-ethanol
pathway in Escherichia coli: increased glycolytic flux and
levels of expression of glycolytic genes during xylose fermentation. J Bacteriol 183:2979–2988
Targonski, Achremowicz B (1986) The effect of aromatic monomeric
derivatives of lignin on the biosynthesis and activity of
cellulolytic enzymes from Fusarium oxysporum. Acta Microbiol
Pol 35:69–76
Tembec (2003) http://tembec.com/DynamicPortal?key=web&lng=enUS&crit=about_rnd&page=tpl_about
Todor D, Tsonka UD (2002) Influence of the growth conditions on
the resistance of Saccharomyces cerevisiae, strain NBIMCC
181, by freeze–drying. J Cult Collect 3:72–77
Tolan JS, Finn RK (1987) Fermentation of D-xylose to ethanol by
genetically modified Klebsiella planticola. Appl Environ
Microbiol 53(9):2039–2044
Vallet C, Said R, Rabiller C, Martin ML (1996) Natural abundance
isotopic fractionation in the fermentation reaction: influence of
the nature of the yeast. Bioorg Chem 24:319–330
Vega JL, Clausen EC, Gaddy JL (1988) Biofilm reactors for ethanol
production. J Enzyme Microb Technol 10:390–402
Virginie AG, Bruno B, Sylvie D, Jean-Marie S (2001) Stress effect
of ethanol on fermentation kinetics by stationary-phase cells of
Saccharomyces cerevisiae. Biotechnol Lett 23:677–681
Westley J (1980) Rhodanase and sulfane pool. In: Jakoby WB
(ed) Enzymatic basis of detoxification, 2. Academic, New
York, pp 245–262
Wheals AE, Basso LC, Alves DMG, Amorim HV (1999) Fuel
ethanol after 25 years. Trends Biotechnol 17:482–486
Wilke CR, Cysewski GR, Yang RD, von Stockar U (1976)
Utilization of cellulosic materials through enzymatic hydrolysis. II. Preliminary assessment of an integrated processing
scheme. Biotechnol Bioeng 18:1315–1323
Wilke CR, Yang RD, Scamanna AF, Freitas RP (1981) Raw material
evaluation and process development studies for conversion of
biomass to sugars and ethanol. Biotechnol Bioeng 23:163–183
Wood BE, Beall DS, Ingram LO (1997) Production of recombinant
bacterial endoglucanase as a co-product with ethanol during
fermentation using derivatives of Escherichia coli KO11.
Biotechnol Bioeng 55(3):547–555
Wooley R, Ruth M, Glassner D, Sheehan J (1999) Process design
and costing of bioethanol technology: a tool for determining the
status and direction of research and development. Biotechnol
Prog 15:794–803
Wu JF, Lastick SM, Updegraff DM (1986) Ethanol production from
sugars derived from plant biomass by a novel fungus. Nature
321:887–888
Wyman CE (1994) Ethanol from lignocellulosic biomass: technology economics, and opportunities. Bioresour Technol 50:3–15
Yamada T, Fatigati MA, Zhang M (2002) Performance of
immobilized Zymomonas mobilis 31821 (pZB5) on actual
hydrolysates produced by Arkenol technology. Appl Biochem
Biotechnol 98:899–907
Yamauchi H, Akita O, Obata T, Amachi T, Hara S, Yoshizawa K
(1989) Production and application of a fruity odor in a solidstate culture of Neurospora sp. using pregelatinized polish rice.
Agric Biol Chem 53:2881–2888
Yu ZS, Zhang HX (2004) Ethanol fermentation of acid-hydrolyzed
cellulosic pyrolysate with Saccharomyces cerevisiae. Bioresour
Technol 93:199–204
Zacchi G, Axelsson A (1989) Economic evaluation of meconcentration in products of ethanol from dilute sugar solutions.
Biotechnol Bioeng 34:223–233
Zaldivar J, Nielsen J, Olsson L (2001) Fuel ethanol production from
lignocellulose: a challenge for metabolic engineering and
process integration. Appl Microbiol Biotechnol 56:17–34
642
Zertuche L, Zall RR (1982) A study of producing ethanol from
cellulose using Clostridium thermocellum. Biotechnol Bioeng
24:57–68
Zhang M, Eddy C, Deanda K, Finkestein M, Picataggio S (1995)
Metabolic engineering of a pentose metabolism pathway in
ethanologenic Zymomonas mobilis. Science 267:240–243
Zhuang XY, Zhang HX, Yang JZ, Qi HY (2001) Preparation of
levoglucosan by pyrolysis of cellulose and its citric acid
fermentation. Bioresour Technol 79:63–66
Ziegler MT, Thomas SR, Danna KJ (2000) Accumulation of a
thermostable endo-1,4-b-D-glucanase in the apoplast of Arabidopsis thaliana leaves. Mol Breed 6:37–46