Handbook of plant based biofuels - Chapter 11 potx

159
11
Bioethanol from
Lignocellulosic Biomass
Part III Hydrolysis
and Fermentation
Ramakrishnan Anish and Mala Rao
ABSTRACT
Lignocellulose is the most abundant natural renewable resource and is one of the
preferred choices for the production of bioethanol. As a substrate for bioethanol
production it has a barrier in its complex structure, which resists hydrolysis. For
lignocellulose to be amenable to fermentation, treatments are necessary that release
CONTENTS
Abstract 159
11.1 Introduction 160
11.2 Hydrolysis of Lignocellulosic Biomass 160
11.2.1 Acid Hydrolysis 160
11.2.1.1 Dilute Acid Hydrolysis 160
11.2.1.2 Concentrated Acid Hydrolysis 162
11.3 Enzymatic Hydrolysis of Lignocellulosic Biomass 163
11.3.1 Factors Governing Enzymatic Hydrolysis 164
11.3.2 Detoxication 166
11.3.2.1
Biological Detoxication Methods 166
11.3.2.2 Physical Detoxication Methods 167
11.3.2.3
Chemical Detoxication Methods 167
11.4 Fermentation of Lignocellulosic Biomass to Ethanol 167
11.4.1 Separate Hydrolysis and Fermentation (SHF) 167
11.4.2 Direct Microbial Conversion (DMC) 167
11.4.3 Simultaneous Saccharication and Fermentation (SSF) 168

11.5 Recombinant DNA Approaches 168
11.6 Conclusions and Future Prospects 169
References 170
© 2009 by Taylor & Francis Group, LLC
160 Handbook of Plant-Based Biofuels
monomeric sugars, which can be converted to ethanol by microbial fermentation. The
current state of the art on acid and enzymatic hydrolysis of lignocellulose and subse-
quent microbial fermentation to ethanol are described in this chapter. Approaches for
detoxication of the lignocellulose hydrolysate for effective fermentation to ethanol
are also discussed.
11.1 INTRODUCTION
The rapid depletion of fossil fuels coupled with the increasing demands for transpor-
tation fuels has necessitated research focus on alternative renewable energy sources.
Lignocellulose is the most abundant renewable resource, abundantly available for
conversion to fuels. On a worldwide basis, terrestrial plants produce 1.3 × 10
10
metric
tons of wood per year (equivalent to 7 × 10
9
metric tons of coal) or about two-thirds
of the world’s energy requirement (Demain, Newcomb, and Wu 2005). Agriculture
and other sources provide about 180 million tons of cellulosic feedstock per year.
Furthermore, tremendous amounts of cellulose are available as municipal and indus-
trial wastes causing pollution problems. Lignocellulosic biomass includes materi-
als such as agricultural and forestry residues, municipal solid waste, and industrial
wastes. Herbaceous and woody crops can also be used as a source of biomass. Ligno-
cellulosic biomass can be used as an inexpensive feedstock for production of renew-
able fuels and chemicals.
Lignocellulosic biomass is made up of cellulose, hemicellulose, and a cementing
material, lignin. Cellulose is a linear polymer of glucose, whereas hemicellulose is a

branched heteropolymer of -xylose, L-arabinose, D-mannose, D-glucose, D-galac-
tose and D-glucuronic acid. Lignin is a complex, hydrophobic, cross-linked aromatic
polymer that interferes with the hydrolysis process. Current processes for the conver-
sion of biomass to ethanol involve chemical and/or enzymatic hydrolysis of cellulose
and hemicellulose to the respective sugars and subsequent fermentation to ethanol.
Enzymatic processes are highly specic and are carried out under mild conditions of
temperature and pH and do not create a corrosion problem. The process requires the
use of expensive biocatalysts. Dilute acid hydrolysis is fast and easy to perform but
is hampered by nonselectivity and by-product formation.
11.2 HYDROLYSIS OF LIGNOCELLULOSIC BIOMASS
The most commonly considered hydrolysis processes are the concentrated hydro-
chloric acid process, the two-step dilute acid hydrolysis, and enzymatic hydroly-
sis. During the hydrolysis of lignocellulosic materials a wide range of compounds
are released which are inhibitory to microbial fermentation. The composition of the
inhibitors differs depending on the type of lignocelluIosic hydrolysates.
11.2.1 ac i d Hy d r o l y S i S
11.2.1.1 Dilute Acid Hydrolysis
Dilute acid hydrolysis of biomass is, by far, the oldest technology for converting
biomass to ethanol. The rst attempt at commercializing a process for producing
© 2009 by Taylor & Francis Group, LLC
Hydrolysis and Fermentation of Lignocellulose 161
ethanol from the wood was carried out in Germany in 1898. It involved the use of
dilute acid to hydrolyze the cellulose to glucose, and was able to produce 7.6 liters of
ethanol per 100 kg of wood waste (18 gal per ton).
The hydrolysis occurs in two stages to accommodate the differences between
the hemicellulose and the cellulose (Harris et al. 1985) and to maximize the sugar
yields from the hemicellulose and cellulose fractions of the biomass. The rst stage
is operated under milder conditions to hydrolyze the hemicellulose, while the sec-
ond stage is optimized to hydrolyze the more resistant cellulose fraction. The liquid
hydrolysates are recovered from each stage, neutralized, and fermented to ethanol.

The National Renewable Energy Laboratory (NREL), a facility of the U.S.
Department of Energy (DOE) operated by Midwest Reseach Institute, Bettelle, out-
lined a process whereby the hydrolysis is carried out in two stages to accommodate
the differences between hemicellulose and cellulose. The rst stage can be operated
under milder conditions, which maximize yield from the more readily hydrolyzed
hemicellulose. The second stage is optimized for hydrolysis of the more resistant
cellulose fraction. NREL has reported the results for a dilute acid hydrolysis of soft-
woods in which the conditions of the reactors were as follows: Stage 1, 0.7% sulfuric
acid, 190°C, and a 3-minute residence time; Stage 2, 0.4% sulfuric acid, 215°C, and
a 3-minute residence time. The liquid hydrolysates are recovered from each stage
and fermented to alcohol. Residual cellulose and lignin left over in the solids from
the hydrolysis reactors serve as boiler fuel for electricity or steam production. These
bench-scale tests conrmed the potential to achieve yields of 89% for mannose, 82%
for galactose, and 50% for glucose. Fermentation with Saccharomyces cerevisiae
achieved ethanol conversion of 90% of the theoretical yield (Nguyen 1998).
The degradation of the lignocellulosic structure often requires two steps, rst, the
prehydrolysis in which the hemicellulose structure is broken down, and second, the
hydrolysis of the cellulose fraction in which lignin will remain as a solid by-product.
The two hydrolyzed streams are fermented to ethanol either together or separately,
after which they are mixed together and distilled (Figure
11.1). During the degrada-
tion of the lignocellulosic structure, not only fermentable sugars are released, but a
Lignocellulosic Material
Prehydrolysis
Fermentation
Pentose
Hydrolysis
Fermentation
Hexose
Lignin

Distillation
Ethanol
Hemicellulose fraction
Cellulose fraction
FIGURE 11.1 Flow chart for ethanol production from lignocellulosic biomass.
© 2009 by Taylor & Francis Group, LLC
162 Handbook of Plant-Based Biofuels
broad range of compounds, some of which might inhibit the fermenting microorgan-
ism. The prehydrolysis process can be performed by physical, chemical, or biologi-
cal methods such as steam pretreatment, milling, freeze explosion, acid treatment
(hydrochloric acid, phosphoric acid, sulfuric acid, sulfur dioxide), alkaline treatment
(sodium hydroxide, ammonia), or treatment with organic solvents (ethanol, ethylene
glycol) or white rot fungi (Vallander and Eriksson 1990; Saddler, Ramos, and Breuil
1993). In the prehydrolysis step, the hemicellulose is liqueed, resulting in a mixture
of mono- and oligosaccharides. The hydrolysis of the cellulose is usually performed
by weak acids or by enzymes (Olsson and Hahn-Hägerdal 1996).
11.2.1.2 Concentrated Acid Hydrolysis
This process is based on concentrated acid decrystallization of the cellulose followed
by dilute acid hydrolysis to sugars at near theoretical yields. The separation of acid
from the sugars, acid recovery, and acid reconcentration are critical operations. The
fermentation converts sugars to ethanol. A process was developed in Japan in which
the concentrated sulfuric acid was used for the hydrolysis. The process was commer-
cialized in 1948. The remarkable feature of their process was the use of membranes
to separate the sugar and acid in the product stream. The membrane separation, a
technology that was way ahead of its time, achieved 80% recovery of acid (Wenzl
1970).
The concentrated sulfuric acid process was also commercialized in the former
Soviet Union. However, these processes were only successful during times of national
crisis, when economic competitiveness of ethanol production could be ignored. Con-
centrated hydrochloric acid has also been utilized and in this case, the prehydrolysis

and hydrolysis are carried out in one step. Generally, acid hydrolysis procedures give
rise to a broad range of compounds in the resulting hydrolysate, some of which might
negatively inuence the subsequent steps in the process. A weak acid hydrolysis pro-
cess is often combined with a weak acid prehydrolysis.
In 1937, the Germans built and operated commercial concentrated acid hydroly-
sis plants based on the use and recovery of hydrochloric acid. Several such facilities
were successfully operated. During World War II, researchers at the U.S. Department
of Agriculture’s Northern Regional Research Laboratory in Peoria, Illinois, further
rened the concentrated sulfuric acid process for corn cobs. They conducted pro-
cess development studies on a continuous process that produced a 15 to 20% xylose
sugar stream and a 10 to 12% glucose sugar stream, with the lignin residue remain-
ing as a by-product. The glucose was readily fermented to ethanol at 85 to 90% of
theoretical yield. Research and development based on the concentrated sulfuric acid
process studied by the USDA (and which came to be known as the “Peoria Process”)
picked up again in the United States in the 1980s, particularly at Purdue University
and at the Tennessee Valley Authority (TVA) (Broder, Barrier, and Lightsey 1992).
Among the improvements added by these researchers were recycling of dilute acid
from the hydrolysis step for pretreatment, and improved recycling of sulfuric acid.
Minimizing the use of sulfuric acid and recycling the acid cost effectively are criti-
cal factors in the economic feasibility of the process. (see rgy.
gov/biomass/printable_versions/concentrated_acid.html). The conventional wisdom
© 2009 by Taylor & Francis Group, LLC
Hydrolysis and Fermentation of Lignocellulose 163
in the literature suggests that the Peoria and TVA processes cannot be economical
because of the high volumes of acid required (Wright and d’Agincourt 1984). The
improvements in the acid sugar separation and recovery have opened the door for
commercial application. Two companies, Arkenol and Masada, in the United States
are currently working with DOE and NREL to commercialize this technology by
taking advantage of niche opportunities involving the use of biomass as a means of
mitigating waste disposal or other environmental problems (rgy.

gov/biomass/concentrated_acid.html). Minimizing the use of the sulfuric acid and
recycling the acid cost effectively are the critical factors in the economic feasibility
of the process. U.S. Patent 5,366,558 (Brink 1994) describes the use of two “stages”
to hydrolyze the hemicellulose sugars and the cellulosic sugars in a countercurrent
process using a batch reactor, which results in poor yields of glucose and xylose
using a mineral acid. Further, the process scheme is complicated and the economic
potential on a large scale to produce inexpensive sugars for fermentation is low. U.S.
Patent 5,188,673 employs concentrated acid hydrolysis which has the benet of high
conversion of biomass, but suffers from low product yields due to degradation and
the requirement of acid recovery and recycling. Sulfuric acid concentrations used
are 30 to 70 weight percent at temperatures less than 100°C. Although 90% hydro-
lysis of the cellulose and hemicellulose is achieved by this process, the concentrated
acids are toxic, corrosive, and hazardous and require reactors that are resistant to
corrosion. In addition, the concentrated acid must be recovered after the hydrolysis
to make the process economically feasible (Von Sivers and Zacchi 1995). A multi-
function process for hydrolysis and fractionation of lignocellulosic biomass to sepa-
rate hemicellulosic sugars using mineral acids like sulfuric acid, phosphoric acid,
or nitric acid has been described (Torget et al., U.S. Patent 6,022,419). A process
for treatment of hemicellulose and cellulose in two different congurations has also
been described (Scott and Piskorz, U.S. Patent 4,880,473). Hemicellulose is treated
with dilute acid in a conventional process. The cellulose is separated out from the
prehydrolysate and then subjected to pyrolysis at high temperatures. Further, the
process step between the hemicellulose and cellulose reactions requires a drying step
with a subsequent high-temperature pyrolysis step at 400 to 600°C for conversion of
the cellulose to fermentable products. A 70% yield of glucose was obtained from the
hydrolysis of lignocellulose under extremely low acid and high temperature condi-
tions by autohydrolysis (Ojumu and Ogunkunle 2005).
11.3 ENZYMATIC HYDROLYSIS OF LIGNOCELLULOSIC BIOMASS
The enzymatic hydrolysis or saccharication of lignocellulosic biomass is preceded
by a pretreatment process in which the lignin component is separated from the cel-

lulose and hemicellulose to make it amenable to the enzymatic hydrolysis. The lignin
interferes with hydrolysis by blocking the access of the cellulases to the cellulose
and by irreversibly binding the hydrolytic enzymes. Therefore, the removal of the
lignin can dramatically increase the hydrolysis rate (McMillan 1994). For the ef-
cient enzymatic hydrolysis of lignocellulosic biomass a pretreatment step is neces-
sary. Various pretreatment processes and the enzymes involved in hydrolysis have
been described in different chapters.
© 2009 by Taylor & Francis Group, LLC
164 Handbook of Plant-Based Biofuels
11.3.1 fa c t o r S Go v e r n i n G en z y m a t i c Hy d r o l y S i S
There are different factors that affect the enzymatic hydrolysis of cellulose, namely,
substrates, cellulase activity, and reaction conditions (temperature, pH, as well as
other parameters). To improve the yield and rate of enzymatic hydrolysis, research
has been focused on optimizing the hydrolysis process and enhancing the cellulase
activity. The yield and initial rate of enzymatic hydrolysis of cellulose is affected
mainly by the substrate concentration. At low substrate levels, an increase of sub-
strate concentrations yields an increase in the reaction rate of the hydrolysis and the
products (Cheung and Anderson 1997). However, substrate inhibition is caused at
high substrate concentration, which considerably lowers the rate of hydrolysis. The
ratio of the enzyme to substrate in the hydrolysis reaction is crucial to establish the
level of substrate inhibition (Huang and Penner 1991). The hydrolysis of cellulosic
substrates by the enzymes depend to a large extent on the structural features of the
substrate, such as cellulose crystallinity, degree of cellulose polymerization, surface
area, and content of lignin (Table 11.1).
The yield and rate of hydrolysis of the cellulosic substrate can be increased to a
certain extent by increasing the dosage of the cellulases in the process, but that would
signicantly increase the cost of the process. Cellulase dosage of 10 FPU/g cellulose
is often used in laboratory studies because it provides a hydrolysis prole with high
levels of glucose yield in a reasonable time (48–72 h) at a reasonable enzyme cost
(Gregg and Saddler 1996). Depending on the type and concentration of the sub-

strates, cellulase enzyme could be used in the hydrolysis (7–33 FPU/g substrate). The
adsorption of the cellulase enzymes onto the surface of the cellulose, the biodegrada-
tion of cellulose to fermentable sugars, and desorption of the cellulase are three steps
involved in enzymatic hydrolysis of the cellulose. The cellulase activity decreases
during hydrolysis because of the irreversible adsorption of the cellulase on the cel-
lulose (Converse et al. 1988). The cellulose surface property can be modied and the
irreversible binding of the cellulase can be minimized by the addition of surfactants
during the hydrolysis. The ionic surfactants Q-86W (cationic) at high concentration
and Neopelex F-25 (anionic) have been shown to have an inhibitory effect (Ooshima,
TABLE 11.1
Structural Properties Potentially Limiting Enzymatic Hydrolysis of
Cellulosic Fibers at Different Structural Levels
Structural Level Substrate Factor
Microbril Degree of polymerization
Crystallinity
Cellulose lattice structure
Fibril Structural composition (lignin content and distribution)
Particle size (bril dimension)
Fiber Available surface area
Degree of ber swelling
Pore structure and distribution
From Manseld et al. 1999. Biotechnol. Progr. 15: 804–816. With permission.
© 2009 by Taylor & Francis Group, LLC
Hydrolysis and Fermentation of Lignocellulose 165
Sakata, and Harano 1986), hence, the nonionic surfactants such as Tween 20, 80 (Wu
and Ju 1998), polyoxyethylene glycol (Park et al., 1992), Tween 81, Emulgen 147,
amphoteric Anhitole 20BS, cationic Q-86W (Ooshima, Sakata, and Harano 1986),
sophorolipid, rhamnolipid, and bacitracin (Helle, Duff, and Cooper 1993) have been
used to enhance the cellulose hydrolysis. Cellulose conversion with 2% (w/v) F68
and 2 g/l cellulase reached 52%, compared to 48% conversion with 10 g/l cellulase

in a surfactant-free system (Wu and Ju 1998). However, Tween 20 was highly inhibi-
tory to D. clausenii even at a low concentration of 0.1%. Use of a cellulase mixture
from different microorganisms, or a mixture of cellulases and other enzymes, in the
hydrolysis of cellulosic materials was studied by Excofer, Toussaint, and Vignon
(1991). The addition of β-glucosidases into the Trichoderma reesei cellulases sys-
tem achieved better saccharication than the system without β-glucosidases. The
β-glucosidase hydrolyzes the cellobiose, which is an inhibitor of the cellulase activ-
ity. The saccharication of the cellulose is reported to be faster when supplemented
with additional β-glucosidase. There are few organisms that secrete complete cel-
lulase, for example, Penicillium funiculosum with high β-glucosidases activity (Rao,
Seeta, and Deshpande 1983). A mixture of hemicellulases or pectinases with cellu-
lases exhibited a signicant increase in cellulose conversion (Beldman et al. 1984).
A 90% enzymatic saccharication of 8% alkali-treated sugarcane bagasse has been
reported when a mixture of the cellulases (dose, 1.0 FPU/g substrate) from Aspergil-
lus ustus and Trichoderma viride was used (Mononmani and Sreekantiah 1987). The
use of the cellulase mixture of the commercial Cellucast and Novozyme prepara-
tions has achieved a nearly complete saccharication of steam-explosion pretreated
Eucalyptus viminalis chips (substrate concentration of 6% and enzyme loading of
10 FPU/g cellulose) (Ramos, Brueil, and Saddler 1993). Baker, Adney, and Nieves
(1994) reported a new thermostable endoglucanase from Acidothermus cellulolyti-
cus E1 and an endoglucanase from T. fusca E5 that exhibited striking synergism with
T. reesei CBH1 in the saccharication of the microcrystalline cellulose. The cellu-
lases can be recovered from the liquid supernatant or the solid residues and recycled.
Enzyme recycling can effectively increase the rate and yield of the hydrolysis and
lower the enzyme cost (Mes-Hartree, Hogan, and Saddler 1987). The efciency of
cellulose hydrolysis gradually decreases with each recycling step (Ramos, Brueil,
and Saddler 1993).
Recently, the enzymatic hydrolysis of lignocellulosic biomass has been opti-
mized using enzymes from different sources and mixing in an appropriate propor-
tion using a statistical approach of factorial design. A twofold reduction in the total

protein required to reach glucan to glucose and xylan to xylose hydrolysis targets
(99% and 88% conversion, respectively), thereby validating this approach toward
enzyme improvement and process cost reduction for lignocellulose hydrolysis (Kim,
Kang, and Lee 1997, Berlin et al. 2005).
Many studies have been presented over the years aiming to understand the inhib-
iting factors in enzymatic hydrolysis of lignocellulose substrates. Reasons for low
yield of fermentable sugars in enzymatic conversion include reduced accessible sur-
face area of cellulose in the lignocellulose complex, leading to restricted access for
enzymes; restricted pore volume of the substrate (Eklund et al. 1990; Mooney et al.
1998); slow enzyme kinetics for crystalline cellulose (Fan et al. 1980); and obstacles
© 2009 by Taylor & Francis Group, LLC
166 Handbook of Plant-Based Biofuels
in the structure of cellulose leading to unproductive enzyme binding (Eriksson,
Karlsson, and Tjerneld 2002; Väljamäe et al., 1998). Lignin has also been identied
to have a high binding afnity for cellulase proteins (Lu et al. 2002; Berlin et al.
2005). Both addition of lignin (Sewalt et al. 1997) and the composition of lignin have
been shown to be responsible for inhibitory factors for the degradation of cellulose.
It was recently found that cellulases lacking cellulose binding module (CBM) also
have a high afnity for lignin, indicating the presence of lignin-binding sites on the
catalytic module (Berlin et al. 2005).
An enhancement in enzymatic hydrolysis of softwood lignocellulosic by non-
ionic surfactants and polymers was observed. It was suggested that ethylene oxide
containing surfactants and polymers such as polyethylene glycol bind to lignin by
hydrophobic interaction and hydrogen bonding and helps to reduce the unproductive
binding of enzymes, thus yielding more fermentable sugars (Börjesson, Peterson,
and Tjerneld 2007).
11.3.2 de t o x i f i c a t i o n
Biological, physical, and chemical methods have been employed for detoxication
(the specic removal of inhibitors prior to fermentation) of lignocellulosic hydro-
lysates (Olsson and Hahn-Hägerdal, 1996). The methods of detoxication change

depending on the source of the lignocellulosic hydrolysate and the microorganism
being used. The lignocellulosic hydrolysates vary in their degree of inhibition and
different microorganisms have different inhibitor tolerances. Several reports on
adaptation of yeasts to inhibiting compounds in lignocellulosic hydrolysates are
found in the literature (e.g., Amartey and Jeffries 1996; Buchert, Puls, and Poutanen
1988; Nishikawa, Sutcliffe, and Saddler 1988).
11.3.2.1
Biological Detoxification Methods
Biological methods of treatment make use of specic enzymes or microorgan-
isms that act on the toxic compounds present in hydrolysates and change their
composition. Treatment with the enzymes peroxidase and laccase, obtained from
the ligninolytic fungus Trametes versicolor, has been shown to increase maxi-
mum ethanol productivity in a hemicellulose hydrolysate of willow two to three
times due to their action on acid and phenolic compounds (Jönsson et al. 1998).
The lamentous soft-rot fungus Trichoderma reesei has been reported to degrade
inhibitors in a hemicellulose hydrolysate obtained after steam pretreatment of wil-
low, resulting in around three times increased maximum ethanol productivity and
four times increased ethanol yield (Palmqvist et al. 1997). Acetic acid, furfural,
and benzoic acid derivatives were removed from the hydrolysate by treatment with
T. reesei. The use of microorganism has also been proposed to selectively remove
inhibitors from lignocellulose hydrolysates. Adaptation of a microorganism to the
hydrolysate is another interesting biological method for improving the fermenta-
tion of hemicellulosic hydrolysate media.
© 2009 by Taylor & Francis Group, LLC
Hydrolysis and Fermentation of Lignocellulose 167
11.3.2.2 Physical Detoxification Methods
Hydrolysate concentration by vacuum evaporation is a physical detoxication method
for reducing the concentration of volatile compounds such as acetic acid, furfural,
and vanillin present in the hydrolysate. However, physical detoxication increases
moderately the concentration of nonvolatile toxic compounds and consequently the

degree of fermentation inhibition.
11.3.2.3
Chemical Detoxification Methods
Chemical detoxication includes precipitation of toxic compounds and ionization of
some inhibitors under certain pH values, the latter being able to change the degree of
toxicity of the compounds (Mussatto 2002). Toxic compounds may also be adsorbed
on activated charcoal (Dominguez, Gong, and Tsao 1996; Mussatto and Roberto
2001), on diatomaceous earth (Ribeiro et al. 2001) and on ion exchange resins (Lars-
son et al. 1999; Nilvebrant et al. 2001).
11.4 FERMENTATION OF LIGNOCELLULOSIC
BIOMASS TO ETHANOL
The hydrolysis of lignocellulosic biomass yields reducing sugars. Once the sugars
are available, its fermentation to ethanol is not a difcult task as many technologies
have been developed. Essentially, there are three different types of processes by
which this can be achieved, namely,
1. Separate hydrolysis and fermentation (SHF)
2. Direct microbial conversion (DMC)
3. Simultaneous saccharication and fermentation (SSF)
SSF has been shown to be the most promising approach to biochemically convert
cellulose to ethanol in an effective way (Wright, Wyman, and Grohmann 1988).
11.4.1 Se P a r a t e Hy d r o l y S i S a n d fe r m e n t a t i o n (SHf)
This is a conventional two-step process where the lignocellulose is hydrolyzed using
enzymes to form reducing sugars in the rst step and the sugars thus formed are fer-
mented to ethanol in the second step using Saccharomyces or Zymomonas (Bisaria
and Ghose 1981; Philippidis 1996). The advantage of this process is that each step
can be carried out at its optimum conditions.
11.4.2 di r e c t mi c r o B i a l co n v e r S i o n (dmc)
This process involves three major steps, namely, enzyme production, hydrolysis
of the lignocellulosic biomass, and the fermentation of the sugars, all occurring in
one step (Hogsett et al. 1992). The relatively lower tolerance of the ethanol is the

main disadvantage of this process. A lower tolerance limit of about 3.5% has been
reported as compared to 10% of ethanologenic yeasts. Acetic acid and lactic acid are
© 2009 by Taylor & Francis Group, LLC
168 Handbook of Plant-Based Biofuels
also formed as by-products in this process in which a signicant amount of carbon
is utilized (Klapatch et al. 1994). Neurospora crassa is known to produce ethanol
directly from cellulose/hemicellulose, because it produces both cellulase and xyla-
nase and also has the capacity to ferment the sugars to ethanol anaerobically (Desh-
pande et al. 1986).
11.4.3 Si m u l t a n e o u S Sa c c H a r i f i c a t i o n a n d fe r m e n t a t i o n (SSf)
The saccharication of lignocellulosic biomass by enzymes and the subsequent fer-
mentation of the sugars to ethanol by yeast such as Saccharomyces or Zymomonas
take place in the same vessel in this process (Glazer and Nikaido 1995). The com-
patibility of both saccharication and fermentation processes with respect to various
conditions, such as pH, temperature, substrate concentration, etc., is one of the most
important factors governing the success of the SSF process. The main advantages of
using SSF for ethanol bioconversion are:
Enhanced rate of lignocellulosic biomass (cellulose and hemicellulose) due •
to removal of the sugars that inhibit cellulase activity
Lower enzyme loading•
Higher product yield•
Reduced inhibition of the yeast fermentation in case of continuous recovery •
of the ethanol
Reduced requirement for aseptic conditions, resulting in increasing eco-•
nomics of the process (Deshpande, Siva Raman, and Rao 1984; Schell et al.
1988; Wright, Wyman, and Grohmann 1988; Philippidis and Smith 1995).
Because several inhibitory compounds are formed during hydrolysis of the raw
material, the hydrolytic process has to be optimized so that inhibitor formation can
be minimized. When low concentrations of inhibitory compounds are present in
the hydrolysate, detoxication is easier and fermentation is cheaper. The choice of

detoxication method has to be based on the degree of microbial inhibition caused
by the compounds. As each detoxication method is specic to certain types of
compounds, better results can be obtained by combining two or more different
methods. Another factor of great importance in the fermentative processes is the
cultivation conditions, which, if inadequate, can stimulate the inhibitory action of
the toxic compounds.
SSF seems to offer a better option for commercial production of ethanol from
lignocellulosic biomass. Penicillium funiculosum cellulase and Saccharomyces
uvarum cells have been reported to be used for SSF (Deshpande et al. 1981).
11.5 RECOMBINANT DNA APPROACHES
Recombinant DNA methods are being used currently for lignocellulosic hydrolysis
and fermentation to ethanol. Genetic manipulations of Saccharomyces cerevisiae
and Z. mobilis have been explored for improving their ability to utilize lignocel-
lulosic biomass. S. cerevisiae has been engineered with arabinose metabolizing
© 2009 by Taylor & Francis Group, LLC
Hydrolysis and Fermentation of Lignocellulose 169
genes from yeasts such as Candida aurigiensis (Jeffries and Shi, 1999) and xylose
transporting gene from P. stipitis. Z. mobilis, an ethanologenic microorganism has
been engineered to utilize glucose through the Entner-Doudoroff pathway and pos-
sess elevated levels of glycolytic and ethanologenic enzymes (pyruvate decarbox-
ylase, PDC, and alcohol dehydrogenase, ADH), resulting in high ethanol yields,
around 97% of theoretical value (Zhang et al. 1995). A recombinant strain of Z.
mobilis has been constructed wherein the xylose and arabinose utilization genes
have been inserted.
11.6 CONCLUSIONS AND FUTURE PROSPECTS
The effective hydrolysis of cellulosic biomass requires the synergistic action of cel-
lulases such as exocellulase, endocellulase, and β-glucosidase. Even though soluble
substrates have been developed for measuring endoglucanase and β-glucosidase
activities there are very few substrates available for the estimation of exoglucanase
activity. The hydrolysis data from soluble substrates cannot yield useful informa-

tion on the hydrolysis of insoluble substrates. The heterogeneity of the cellulosic
biomass, the dynamic interactions between insoluble substrates, and the complexity
of cellulase components result in formidable problems in extrapolating the activity
measured on one solid substrate to other solid substrates, especially those with sig-
nicance for biorenery processes. This point is critical to the eventual improvement
of cellulases for the conversion of pretreated plant cell walls in energy crops and
agricultural residues. Realistic methods must be based on physically and chemically
relevant industrial substrates.
For an economically viable bioconversion process it is necessary to utilize both
the cellulosic and hemicellulosic fractions of biomass. Extensive work has been
conducted on xylose fermenting yeasts, such as Pachysolen tannophilus, Candida
shehatae, and Pichia stiptis. However, low ethanol tolerance and catabolite repres-
sion in xylose conversion due to glucose need to be addressed. Another potential
ethanologen is recombinant Zymomonas mobilis in view of its ability to ferment
both xylose and glucose. An Escherichia coli strain developed by Ingram’s group
deserves special recognition, as it not only ferments all ve sugars (glucose, xylose,
arabinose, mannose, and galactose) present in synthetic sugar mixtures to ethanol
but also performs competently in real hydrolysates like that of Pinus. Saccharomy-
ces LNH-ST is another promising recombinant capable of fermenting dilute acid-
treated corn ber hydrolysates.
Although bioethanol production has been greatly improved by new technologies
there are still challenges that need further investigation. These challenges include
maintaining a stable performance of the genetically engineered yeasts in commer-
cial-scale fermentation operations and integrating the optimal components into eco-
nomic ethanol production systems.
Metabolic engineering and other classical techniques such as random muta-
genesis address the further enhancement of microorganism capabilities by adding
or modifying traits such as tolerance to ethanol and inhibitors, efcient hydroly-
sis of cellulose/hemicellulose, thermotolerance, reduced need for nutrient supple-
mentation, and improvement of sugar transport. The improvement achieved in the

© 2009 by Taylor & Francis Group, LLC
170 Handbook of Plant-Based Biofuels
fermentation step with the help of metabolic engineering is just one of the aspects
of an integrated process. Keeping a realistic perspective one can conclude that
several pieces still remain to be properly assembled and optimized before an ef-
cient industrial conguration is acquired.
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