ALLANSCellulosic ethanol is ethanol (ethyl alcohol) produced from cellulose (the stringy fiber of a plant)

rather than from the plant's seeds or fruit . It is a biofuel produced from grasses, wood , algae, or other
plants. The fibrous parts of the plants are mostly inedible to animals, including humans, except for
ruminants (grazing, cud-chewing animals such as cows or sheep).

Considerable interest in cellulosic ethanol exists because it has the potential for strong economic
importance. Growth of cellulose by plants is a mechanism that captures and stores solar energy
chemically in nontoxic ways with resultant supplies that are easy to transport and store. Additionally,
transport may be unneeded anyway, because grasses or trees can grow almost anywhere temperate.
This is why commercially practical cellulosic ethanol is widely viewed as a next level of development for
the biofuel industry that could reduce demand for oil and gas drilling and even nuclear power in ways
that grain -based ethanol fuel alone cannot. Potential exists for the many benefits of carbonaceous
liquid fuels and petrochemicals (which today's standard of living depends on) but in a carbon cycle–
balanced and renewable way (recycling surface and atmosphere carbon instead of pumping
underground carbon up into it and thus adding to it). Commercially practical cellulosic alcohol could also
avoid one of the problems with today's conventional (grain-based) biofuels, which is that they set up
competition for grain with food purposes, potentially driving up the price of food. To date, what stands
in the way of these goals is that production of cellulosic alcohol is not yet sufficiently practical on a
commercial scale.

Overview

Cellulosic ethanol is a type of biofuel produced from

lignocellulose, a structural material that comprises much of the mass of plants. Lignocellulose is
composed mainly of cellulose, hemicellulose and lignin . Corn stover ,

Panicum virgatum (switchgrass), Miscanthus grass species, wood chips and the byproducts of lawn and
tree maintenance are some of the more popular cellulosic materials for ethanol production. Production
of ethanol from lignocellulose has the advantage of abundant and diverse raw material compared to
sources such as corn and cane sugars, but requires a greater amount of processing to make the sugar
monomers available to the microorganisms typically used to produce ethanol by fermentation.

Switchgrass and Miscanthus are the major biomass materials being studied today, due to their high
productivity per acre. Cellulose, however, is contained in nearly every natural, free-growing plant, tree,
and bush, in meadows, forests, and fields all over the world without agricultural effort or cost needed to
make it grow.

One of the benefits of cellulosic ethanol is it reduces

greenhouse gas emissions (GHG) by 85% over reformulated gasoline. [1] By contrast, starch ethanol
(e.g., from corn), which most frequently uses natural gas to provide energy for the process, may not
reduce GHG emissions at all depending on how the starch-based feedstock is produced. [2] According to
the National Academy of Sciences in 2011, there is no commercially viable bio-refinery in existence to
convert lignocellulosic biomass to fuel. [3] Absence of production of cellulosic ethanol in the quantities
required by the regulation was the basis of a United States Court of Appeals for the District of Columbia
decision announced January 25, 2013, voiding a requirement imposed on car and truck fuel producers in
the United States by the Environmental Protection Agency requiring addition of cellulosic biofuels to
their products. [4] These issues, along with many other difficult production challenges, led George
Washington University policy researchers to state that "in the short term, [cellulosic] ethanol cannot
meet the energy security and environmental goals of a gasoline alternative." [5]

History

The French chemist, Henri Braconnot, was the first to discover that cellulose could be hydrolyzed into
sugars by treatment with sulfuric acid in 1819. [6] The hydrolyzed sugar could then be processed to form
ethanol through fermentation. The first commercialized ethanol production began in Germany in 1898,
where acid was used to hydrolyze cellulose. In the United States, the Standard Alcohol Company opened
the first cellulosic ethanol production plant in South Carolina in 1910. Later, a second plant was opened
in Louisiana. However, both plants were closed after WWI due to economic reasons. [7]

The first attempt at commercializing a process for ethanol from wood was done 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 US gal (68 L) per ton). The Germans soon developed an industrial
process optimized for yields of around 50 US gallons (190 L) per ton of biomass. This process soon found
its way to the US, culminating in two commercial plants operating in the southeast during WWI. These
plants used what was called "the American Process" — a one-stage dilute sulfuric acid hydrolysis.
Though the yields were half that of the original German process (25 US gallons (95 L) of ethanol per ton
versus 50), the throughput of the American process was much higher. A drop in lumber production
forced the plants to close shortly after the end of WWI. In the meantime, a small but steady amount of
research on dilute acid hydrolysis continued at the USFS 's Forest Products Laboratory . [8][9][10] During
World War II, the US again turned to cellulosic ethanol, this time for conversion to butadiene to produce
synthetic rubber. The Vulcan Copper and Supply Company was contracted to construct and operate a
plant to convert sawdust into ethanol. The plant was based on modifications to the original German
Scholler process as developed by the Forest Products Laboratory. This plant achieved an ethanol yield of
50 US gal (190 L) per dry ton, but was still not profitable and was closed after the war. [11]

With the rapid development of enzyme technologies in the last two decades, the acid hydrolysis process
has gradually been replaced by enzymatic hydrolysis. Chemical pretreatment of the feedstock is
required to prehydrolyze (separate) hemicellulose, so it can be more effectively converted into sugars.
The dilute acid pretreatment is developed based on the early work on acid hydrolysis of wood at the
USFS 's Forest Products Laboratory . Recently, the Forest Products Laboratory together with the
University of Wisconsin–Madison developed a sulfite pretreatment to overcome the recalcitrance of
lignocellulose[12] for robust enzymatic hydrolysis of wood cellulose.

US President George W. Bush, in his State of the Union address delivered January 31, 2006, proposed to
expand the use of cellulosic ethanol. In his State of the Union Address on January 23, 2007, President
Bush announced a proposed mandate for 35 billion US gallons (130,000,000 m 3 ) of ethanol by 2017. It
is widely recognized that the maximum production of ethanol from corn starch is 15 billion US gallons
(57,000,000 m 3) per year, implying a proposed mandate for production of some 20 billion US gallons
(76,000,000 m 3) more per year of cellulosic ethanol by 2017. Bush's proposed plan includes $2 billion
funding (from 2007 to 2017?) for cellulosic ethanol plants, with an additional $1.6 billion (from 2007 to
2017?) announced by the USDA on January 27, 2007.
In March 2007, the US government awarded $385 million in grants aimed at jump-starting ethanol
production from nontraditional sources like wood chips, switchgrass, and citrus peels. Half of the six
projects chosen will use thermochemical methods and half will use cellulosic ethanol methods. [13]

The American company Range Fuels announced in July 2007 that it was awarded a construction permit
from the state of Georgia to build the first commercial-scale 100-million-US-gallon (380,000 m 3 )-per-
year cellulosic ethanol plant in the US. [14] Construction began in November, 2007. [15] The Range Fuels
plant was built in Soperton, GA, but was shut down in January 2011, without ever having produced any
ethanol. It had received a $76 million grant from the US Department of Energy, plus $6 million from the
State of Georgia, plus an $80 million loan guaranteed by the U.S. Biorefinery Assistance Program. [16]

Production methods

Bioreactor for cellulosic ethanol research.

The two ways of producing ethanol from cellulose are:

Cellulolysis processes which consist of hydrolysis on pretreated lignocellulosic materials, using enzymes
to break complex cellulose into simple sugars such as

glucose, followed by fermentation and distillation .

Gasification that transforms the lignocellulosic raw material into gaseous carbon monoxide and
hydrogen. These gases can be converted to ethanol by fermentation or chemical catalysis .

As is normal for pure ethanol production, these methods include distillation .

Cellulolysis (biological approach)

The stages to produce ethanol using a biological approach are: [12]

1. A "pretreatment" phase, to make the lignocellulosic material such as wood or straw amenable to
hydrolysis

2. Cellulose hydrolysis (that is, cellulolysis ) with

cellulases, to break down the molecules into sugars

3. Separation of the sugar solution from the residual materials, notably lignin

4. Microbial fermentation of the sugar solution

5. Distillation to produce roughly 95% pure alcohol

6. Dehydration by molecular sieves to bring the ethanol concentration to over 99.5%

In 2010, a genetically engineered yeast strain was developed to produce its own cellulose-digesting
enzymes. [17] Assuming this technology can be scaled to industrial levels, it would eliminate one or
more steps of cellulolysis, reducing both the time required and costs of production.

Pretreatment
Although lignocellulose is the most abundant plant material resource, its usability is curtailed by its rigid
structure. As a result, an effective pretreatment is needed to liberate the cellulose from the lignin seal
and its crystalline structure so as to render it accessible for a subsequent hydrolysis step. [18] By far,
most pretreatments are done through physical or chemical means. To achieve higher efficiency, both
physical and chemical pretreatments are required. Physical pretreatment is often called size reduction
to reduce biomass physical size. Chemical pretreatment is to remove chemical barriers so the enzymes
can have access to cellulose for microbial reactions.

To date, the available pretreatment techniques include

acid hydrolysis, steam explosion, ammonia fiber expansion, organosolv, sulfite pretreatment , [12]
AVAP® (SO2-ethanol-water) fractionation, [19] alkaline wet oxidation and ozone pretreatment. [20]
Besides effective cellulose liberation, an ideal pretreatment has to minimize the formation of
degradation products because of their inhibitory effects on subsequent hydrolysis and fermentation
processes. [21] The presence of inhibitors will not only further complicate the ethanol production but
also increase the cost of production due to entailed detoxification steps. Even though pretreatment by
acid hydrolysis is probably the oldest and most studied pretreatment technique, it produces several
potent inhibitors including furfural and hydroxymethyl furfural (HMF) which are by far regarded as the
most toxic inhibitors present in lignocellulosic hydrolysate. [22] Ammonia Fiber Expansion (AFEX) is a
promising pretreatment with no inhibitory effect in resulting hydrolysate. [23]

Most pretreatment processes are not effective when applied to feedstocks with high lignin content, such
as forest biomass. Organosolv , SPORL ('sulfite pretreatment to overcome recalcitrance of
lignocellulose') and SO2-ethanol-water (AVAP®) processes are the three processes that can achieve over
90% cellulose conversion for forest biomass, especially those of softwood species. SPORL is the most
energy efficient (sugar production per unit energy consumption in pretreatment) and robust process for
pretreatment of forest biomass with very low production of fermentation inhibitors. Organosolv pulping
is particularly effective for hardwoods and offers easy recovery of a hydrophobic lignin product by
dilution and precipitation. [24] AVAP® process effectively fractionates all types of lignocellulosics into
clean highly digestible cellulose, undegraded hemicellulose sugars, reactive lignin and lignosulfonates,
and is characterized by efficient recovery of chemicals. [25][26]

Cellulolytic processes

The cellulose molecules are composed of long chains of sugar molecules. In the hydrolysis of cellulose
(that is,

cellulolysis ), these chains are broken down to free the sugar before it is fermented for alcohol
production.

There are two major cellulose hydrolysis (cellulolysis) processes: a chemical reaction using acids, or an

enzymatic reaction use cellulases.

Chemical hydrolysis

In the traditional methods developed in the 19th century and at the beginning of the 20th century,
hydrolysis is performed by attacking the cellulose with an acid. [27] Dilute acid may be used under high
heat and high pressure, or more concentrated acid can be used at lower temperatures and atmospheric
pressure. A decrystalized cellulosic mixture of acid and sugars reacts in the presence of water to
complete individual sugar molecules (hydrolysis). The product from this hydrolysis is then neutralized
and yeast fermentation is used to produce ethanol. As mentioned, a significant obstacle to the dilute
acid process is that the hydrolysis is so harsh that toxic degradation products are produced that can
interfere with fermentation. BlueFire Renewables uses concentrated acid because it does not produce
nearly as many fermentation inhibitors, but must be separated from the sugar stream for recycle
[simulated moving bed (SMB) chromatographic separation, for example] to be commercially attractive.

Agricultural Research Service scientists found they can access and ferment almost all of the remaining
sugars in

wheat straw. The sugars are located in the plant’s cell walls, which are notoriously difficult to break
down. To access these sugars, scientists pretreated the wheat straw with alkaline peroxide, and then
used specialized enzymes to break down the cell walls. This method produced 93 US gallons (350 L) of
ethanol per ton of wheat straw. [1]

Enzymatic hydrolysis

Cellulose chains can be broken into glucose molecules by cellulase enzymes .

This reaction occurs at body temperature in the stomachs of ruminants such as cattle and sheep, where
the enzymes are produced by microbes. This process uses several enzymes at various stages of this
conversion. Using a similar enzymatic system, lignocellulosic materials can be enzymatically hydrolyzed
at a relatively mild condition (50 °C and pH 5), thus enabling effective cellulose breakdown without the
formation of byproducts that would otherwise inhibit enzyme activity. All major pretreatment methods,
including dilute acid, require an enzymatic hydrolysis step to achieve high sugar yield for ethanol
fermentation. [23] Currently, most pretreatment studies have been laboratory-based, but companies
are exploring means to transition from the laboratory to pilot, or production scale.

Various enzyme companies have also contributed significant technological breakthroughs in cellulosic
ethanol through the mass production of enzymes for hydrolysis at competitive prices.

The fungus Trichoderma reesei is used by Iogen Corporation to secrete "specially engineered enzymes"
for an enzymatic hydrolysis process. [28] Their raw material (wood or straw) has to be pre-treated to
make it amenable to hydrolysis.

Another Canadian company, SunOpta, uses steam explosion pretreatment, providing its technology to
Verenium (formerly Celunol Corporation)'s facility in

Jennings, Louisiana, Abengoa's facility in Salamanca, Spain , and a China Resources Alcohol Corporation
in

Zhaodong. The CRAC production facility uses corn stover as raw material. [29]

Genencor and Novozymes have received United States Department of Energy funding for research into
reducing the cost of cellulases, key enzymes in the production of cellulosic ethanol by enzymatic
hydrolysis. A recent breakthrough in this regard was the discovery and inclusion of lytic polysaccharide
monooxygenases . These enzymes are capable of boosting significantly the action of other cellulases by
oxidatively attacking a polysaccharide substrate. [30]
Other enzyme companies, such as Dyadic International ,

[31] are developing genetically engineered fungi which would produce large volumes of cellulase ,
xylanase and

hemicellulase enzymes, which can be used to convert agricultural residues such as corn stover, distiller
grains, wheat straw and sugarcane bagasse and energy crops such as switchgrass into fermentable
sugars which may be used to produce cellulosic ethanol.

In 2010, BP Biofuels bought out the cellulosic ethanol venture share of Verenium , which had itself been
formed by the merger of Diversa and Celunol, and with which it jointly owned and operated a 1.4-
million-US-gallon (5,300 m 3) per year demonstration plant in Jennings, LA, and the laboratory facilities
and staff in San Diego, CA. BP Biofuels continues to operate these facilities, and has begun first phases to
construct commercial facilities. Ethanol produced in the Jennings facility was shipped to London and
blended with gasoline to provide fuel for the Olympics.

KL Energy Corporation , [32] formerly KL Process Design Group, began commercial operation of a 1.5-
million-US-gallon (5,700 m 3) per year cellulosic ethanol facility in Upton, WY in the last quarter of 2007.
The Western Biomass Energy facility is currently achieving yields of 40–45 US gallons (150–170 L) per dry
ton. It is the first operating commercial cellulosic ethanol facility in the nation. The KL Energy process
uses a thermomechanical breakdown and enzymatic conversion process. The primary feedstock is soft
wood, but lab tests have already proven the KL Energy process on wine pomace, sugarcane bagasse,
municipal solid waste, and switchgrass.

Microbial fermentation

Main article: Ethanol fermentation

Traditionally, baker’s yeast ( Saccharomyces cerevisiae), has long been used in the brewery industry to
produce ethanol from hexoses (six-carbon sugars). Due to the complex nature of the carbohydrates
present in

lignocellulosic biomass , a significant amount of xylose and arabinose (five-carbon sugars derived from
the hemicellulose portion of the lignocellulose) is also present in the hydrolysate. For example, in the
hydrolysate of corn stover , approximately 30% of the total fermentable sugars is xylose. As a result, the
ability of the fermenting microorganisms to use the whole range of sugars available from the
hydrolysate is vital to increase the economic competitiveness of cellulosic ethanol and potentially
biobased proteins.

In recent years, metabolic engineering for microorganisms used in fuel ethanol production has shown
significant progress. [33] Besides Saccharomyces cerevisiae , microorganisms such as Zymomonas
mobilis and Escherichia coli have been targeted through metabolic engineering for cellulosic ethanol
production.

Recently, engineered yeasts have been described efficiently fermenting xylose, [34][35] and arabinose,
[36] and even both together. [37] Yeast cells are especially attractive for cellulosic ethanol processes
because they have been used in biotechnology for hundreds of years, are tolerant to high ethanol and
inhibitor concentrations and can grow at low pH values to reduce bacterial contamination.
Combined hydrolysis and fermentation

Some species of bacteria have been found capable of direct conversion of a cellulose substrate into
ethanol. One example is Clostridium thermocellum , which uses a complex cellulosome to break down
cellulose and synthesize ethanol. However, C. thermocellum also produces other products during
cellulose metabolism, including acetate and lactate , in addition to ethanol, lowering the efficiency of
the process. Some research efforts are directed to optimizing ethanol production by

genetically engineering bacteria that focus on the ethanol-producing pathway. [38]

Gasification process (thermochemical approach)

Fluidized Bed Gasifier in Güssing

Burgenland Austria

The gasification process does not rely on chemical decomposition of the cellulose chain (cellulolysis).
Instead of breaking the cellulose into sugar molecules, the carbon in the raw material is converted into

synthesis gas , using what amounts to partial combustion. The carbon monoxide, carbon dioxide and
hydrogen may then be fed into a special kind of

fermenter . Instead of sugar fermentation with yeast, this process uses Clostridium ljungdahlii bacteria.
[39] This microorganism will ingest carbon monoxide, carbon dioxide and hydrogen and produce ethanol
and water. The process can thus be broken into three steps:

1. Gasification — Complex carbon-based molecules are broken apart to access the carbon as carbon
monoxide, carbon dioxide and hydrogen

2. Fermentation — Convert the carbon monoxide, carbon dioxide and hydrogen into ethanol using the

Clostridium ljungdahlii organism

3. Distillation — Ethanol is separated from water

A recent study has found another Clostridium bacterium that seems to be twice as efficient in making
ethanol from carbon monoxide as the one mentioned above. [40]

Alternatively, the synthesis gas from gasification may be fed to a catalytic reactor where it is used to
produce ethanol and other higher alcohols through a thermochemical process. [41] This process can also
generate other types of liquid fuels, an alternative concept successfully demonstrated by the Montreal-
based company Enerkem at their facility in Westbury, Quebec. [42]

Hemicellulose to ethanol

Studies are intensively conducted to develop economic methods to convert both cellulose and
hemicellulose to ethanol. Fermentation of glucose, the main product of cellulose hydrolyzate, to ethanol
is an already established and efficient technique. However, conversion of xylose, the pentose sugar of
hemicellulose hydrolyzate, is a limiting factor, especially in the presence of glucose. Moreover, it cannot
be disregarded as hemicellulose will increase the efficiency and cost-effectiveness of cellulosic ethanol
production. [43]
Sakamoto (2012) et al. show the potential of genetic engineering microbes to express hemicellulase
enzymes. The researchers created a recombinant Saccharomyces cerevisiae strain that was able to:

1. hydrolyze hemicellulase through codisplaying endoxylanase on its cell surface,

2. assimilate xylose by expression of xylose reductase and xylitol dehydrogenase.

The strain was able to convert rice straw hydrolyzate to ethanol, which contains hemicellulosic
components. Moreover, it was able to produce 2.5x more ethanol than the control strain, showing the
highly effective process of cell surface-engineering to produce ethanol.

[43]

Economics

The shift to a renewable fuel resource has been a target for many years now. However, most of its
production is with the use of corn ethanol. In the year 2000, there was only 6.2 billion liters produced in
the United States and it has expanded over 800% to 50 billion litres in just a decade (2010). [44]
Government pressures to shift to renewable fuel resources has been apparent since the U.S
Environmental Protection Agency has implemented the 2007 Renewable Fuel Standard (RFS) to use a
percentage of renewable fuel in products or face penalties. The shift to cellulosic ethanol production
instead of corn has been strongly promoted by the US government. [45] Even with these policies in
place and the government attempting to create a market for cellulose ethanol, there was no commercial
production of this fuel in 2010 and 2011. [46] The Energy Independence and Security Act originally set
goals of 100 million, 250 million and 500 million gallons for the years 2010, 2011 and 2012 respectively.
However, as of 2012 it was projected that the production of cellulosic ethanol would be approximately
10.5 million far from its target. [46] In 2007 alone, the US government provided 1 billion US dollars for
cellulosic ethanol projects, while China invested 500 million US dollars into cellulosic ethanol research.
[47]

Due to the lack of existing commercialized plant data, it is difficult to determine the exact method of
production that will be most commonly employed. Model systems try to compare different technologies
costs, however these models cannot be applied to commercial-plant costs. Currently, there are many
pilot and demonstration facilities open that show cellulosic production on a smaller scale. These main
facilities are summarized in the table below .

Start-up costs for pilot scale lignocellulosic ethanol plants are high. On 28 February 2007, the U.S. Dept.
of Energy announced $385 million in grant funding to six cellulosic ethanol plants. [48] This grant
funding accounts for 40% of the investment costs. The remaining 60% comes from the promoters of
those facilities. Hence, a total of $1 billion will be invested for approximately 140-million-US-gallon
(530,000 m 3 ) capacity. This translates into $7/annual gallon production capacity in capital investment
costs for pilot plants; future capital costs are expected to be lower. Corn-to-ethanol plants cost roughly
$1–3/annual gallon capacity, though the cost of the corn itself is considerably greater than for

switchgrass or waste biomass. [49][50]

As of 2007, ethanol is produced mostly from sugars or starches, obtained from fruits and grains. In
contrast, cellulosic ethanol is obtained from cellulose, the main component of wood, straw, and much of
the structure of plants. Since cellulose cannot be digested by humans, the production of cellulose does
not compete with the production of food, other than conversion of land from food production to
cellulose production (which has recently started to become an issue, due to rising wheat prices.) The
price per ton of the raw material is thus much cheaper than that of grains or fruits. Moreover, since
cellulose is the main component of plants, the whole plant can be harvested. This results in much better
yields—up to 10 short tons per acre (22 t/ha), instead of 4-5 short tons/acre (9–11 t/ha) for the best
crops of grain. [ citation needed ]

The raw material is plentiful. An estimated 323 million tons of cellulose-containing raw materials which
could be used to create ethanol are thrown away each year in US alone. This includes 36.8 million dry
tons of urban wood wastes, 90.5 million dry tons of primary mill residues, 45 million dry tons of forest
residues, and 150.7 million dry tons of corn stover and wheat straw.

[51] Transforming them into ethanol using efficient and cost-effective hemi(cellulase) enzymes or other
processes might provide as much as 30% of the current fuel consumption in the United States. [ citation
needed ] Moreover, even land marginal for agriculture could be planted with cellulose-producing crops,
such as switchgrass, resulting in enough production to substitute for all the current oil imports into the
United States. [52]

Paper, cardboard, and packaging comprise a substantial part of the solid waste sent to landfills in the
United States each day, 41.26% of all organic municipal solid waste (MSW) according to California
Integrated Waste Management Board 's city profiles. [ citation needed ] These city profiles account for
accumulation of 612.3 short tons (555.5 t) daily per landfill where an average population density of
2,413 per square mile persists. All these, except gypsum board, contain cellulose, which is transformable
into cellulosic ethanol. [51] This may have additional environmental benefits because decomposition of
these products produces methane , a potent greenhouse gas. [53]

Reduction of the disposal of solid waste through cellulosic ethanol conversion would reduce solid waste
disposal costs by local and state governments. It is estimated that each person in the US throws away
4.4 lb (2.0 kg) of trash each day, of which 37% contains waste paper, which is largely cellulose. That
computes to 244 thousand tons per day of discarded waste paper that contains cellulose. [54] The raw
material to produce cellulosic ethanol is not only free, it has a negative cost—i.e., ethanol producers can
get paid to take it away. [55]

In June 2006, a U.S. Senate hearing was told the current cost of producing cellulosic ethanol is US $2.25
per US gallon (US $0.59/litre), primarily due to the current poor conversion efficiency. [ citation needed ]
At that price, it would cost about $120 to substitute a barrel of oil (42 US gallons (160 L)), taking into
account the lower energy content of ethanol . However, the Department of Energy is optimistic and has
requested a doubling of research funding. The same Senate hearing was told the research target was to
reduce the cost of production to US $1.07 per US gallon (US $0.28/litre) by 2012. "The production of
cellulosic ethanol represents not only a step toward true energy diversity for the country, but a very
cost-effective alternative to fossil fuels. It is advanced weaponry in the war on oil," said Vinod Khosla ,
managing partner of Khosla Ventures , who recently told a Reuters Global Biofuels Summit that he could
see cellulosic fuel prices sinking to $1 per gallon within ten years.

In September 2010, a report by Bloomberg analyzed the

European biomass infrastructure and future refinery development. Estimated prices for a litre of ethanol
in August 2010 are EUR 0.51 for 1g and 0.71 for 2g. [ clarification needed ] The report suggested Europe
should copy the current US subsidies of up to $50 per dry tonne. [56]

Recently on October 25, 2012, BP, one of the leaders in fuel products, announced the cancellation of
their proposed $350 million commercial-scale plant. It was estimated that the plant would be producing
36 million gallons a year at its location in Highlands County of Florida. BP has still provided 500 million
US dollars for biofuel research at the Energy Biosciences Institute. [57] General Motors (GM) has also
invested into cellulosic companies more specifically Mascoma and Coskata. [47] There are many other
companies in construction or heading towards it. Abengoa [2] is building a 25 million-gallon per year
plant in \ technology platform based on the fungus Myceliophthora thermophila to convert
lignocellulose into fermentable sugars. Poet is also in midst of producing a 200 million dollar, 25-million-
gallon per year in Emmetsburg, Iowa. Mascoma now partnered with Valero has declared their intention
to build a 20 million gallon per year in Kinross, Michigan.

[57] China Alcohol Resource Corporation has developed a 6.4 million liter cellulosic ethanol plant under
continuous operation. [57]

Also, since 2013, the Brazilian company GranBio is working to become a producer of biofuels and
biochemicals. The family-held company is commissioning a 82 million liters per year (22 MMgy)
cellulosic ethanol plant (2G ethanol) in the state of Alagoas, Brazil, which will be the first industrial
facility of the group. [58] GranBio's second generation ethanol facility is integrated to a first generation
ethanol plant operated by Grupo Carlos Lyra, uses process technology from Beta Renewables, enzymes
from Novozymes and yeast from DSM. Breaking ground in January 2013, the plant is in final
commissioning. According to GranBio Annual Financial Records, the total investment was 208 million US
Dollars. [59]

Enzyme-cost barrier

Cellulases and hemicellulases used in the production of cellulosic ethanol are more expensive compared
to their first generation counterparts. Enzymes required for maize grain ethanol production cost 2.64-
5.28 US dollars per cubic meter of ethanol produced. Enzymes for cellulosic ethanol production are
projected to cost 79.25 US dollars, meaning they are 20-40 times more expensive. [47] The cost
differences are attributed to quantity required. The cellulase family of enzymes have a one to two order
smaller magnitude of efficiency. Therefore, it requires 40 to 100 times more of the enzyme to be present
in its production. For each ton of biomass it requires 15-25 kilograms of enzyme. [60] More recent
estimates [61] are lower, suggesting 1 kg of enzyme per dry tonne of biomass feedstock. There is also
relatively high capital costs associated with the long incubation times for the vessel that perform
enzymatic hydrolysis. Altogether, enzymes comprise a significant portion of 20-40% for cellulosic
ethanol production. A recent paper [61] estimates the range at 13-36% of cash costs, with a key factor
being how the cellulase enzyme is produced. For cellulase produced offsite, enzyme production amounts
to 36% of cash cost. For enzyme produced onsite in a separate plant, the fraction is 29%; for integrated
enzyme production, the faction is 13%. One of the key benefits of integrated production is that biomass
instead of glucose is the enzyme growth medium. Biomass costs less, and it makes the resulting
cellulosic ethanol a 100% second-generation biofuel, i.e., it uses no ‘food for fuel’.

Feedstocks
In general there are two types of feedstocks: forest (woody) Biomass and agricultural biomass. In the
US, about 1.4 billion dry tons of biomass can be sustainably produced annually. About 370 million tons
or 30% are forest biomass. [62] Forest biomass has higher cellulose and lignin content and lower
hemicellulose and ash content than agricultural biomass. Because of the difficulties and low ethanol
yield in fermenting pretreatment hydrolysate, especially those with very high 5 carbon hemicellulose
sugars such as xylose, forest biomass has significant advantages over agricultural biomass. Forest
biomass also has high density which significantly reduces transportation cost. It can be harvested year
around which eliminates long term storage. The close to zero ash content of forest biomass significantly
reduces dead load in transportation and processing. To meet the needs for biodiversity, forest biomass
will be an important biomass feedstock supply mix in the future biobased economy. However, forest
biomass is much more recalcitrant than agricultural biomass. Recently, the USDA Forest Products
Laboratory together with the University of Wisconsin–Madison developed efficient technologies [12][63]
that can overcome the strong recalcitrance of forest (woody) biomass including those of softwood
species that have low xylan content. Short-rotation intensive culture or tree farming can offer an almost
unlimited opportunity for forest biomass production. [64]

Woodchips from slashes and tree tops and saw dust from saw mills, and waste paper pulp are common
forest biomass feedstocks for cellulosic ethanol production. [65]

The following are a few examples of agricultural biomass:

Switchgrass ( Panicum virgatum ) is a native tallgrass prairie grass. Known for its hardiness and rapid
growth, this perennial grows during the warm months to heights of 2–6 feet. Switchgrass can be grown
in most parts of the United States, including swamplands, plains, streams, and along the shores &
interstate highways . It is self-seeding (no tractor for sowing, only for mowing), resistant to many
diseases and pests, & can produce high yields with low applications of fertilizer and other chemicals. It is
also tolerant to poor soils, flooding, & drought; improves soil quality and prevents erosion due its type
of root system. [66]

Switchgrass is an approved cover crop for land protected under the federal Conservation Reserve
Program (CRP). CRP is a government program that pays producers a fee for not growing crops on land on
which crops recently grew. This program reduces soil erosion, enhances water quality, and increases
wildlife habitat. CRP land serves as a habitat for upland game, such as pheasants and ducks, and a
number of insects. Switchgrass for biofuel production has been considered for use on Conservation
Reserve Program (CRP) land, which could increase ecological sustainability and lower the cost of the CRP
program. However, CRP rules would have to be modified to allow this economic use of the CRP land.
[66]

Miscanthus × giganteus is another viable feedstock for cellulosic ethanol production. This species of
grass is native to Asia and is the sterile triploid hybrid of

Miscanthus sinensis and Miscanthus sacchariflorus . It can grow up to 12 feet (3.7 m) tall with little
water or fertilizer input. Miscanthus is similar to switchgrass with respect to cold and drought tolerance
and water use efficiency. Miscanthus is commercially grown in the European Union as a combustible
energy source.

Corn cobs and corn stover are the most popular agricultural biomass.
It has been suggested that Kudzu may become a valuable source of biomass. [67]

Environmental effects

The environmental impact from the production of fuels is an important factor in determining its
feasibility as an alternative to fossil fuels. Over the long run, small differences in production cost,
environmental ramifications, and energy output may have large effects. It has been found that cellulosic
ethanol can produce a positive net energy output. [68] The reduction in green house gas (GHG)
emissions from corn ethanol and cellulosic ethanol compared with fossil fuels is drastic. Corn ethanol
may reduce overall GHG emissions by about 13%, while that figure is around 88% or greater for
cellulosic ethanol. [69][70] As well, cellulosic ethanol can reduce carbon dioxide emissions to nearly
zero. [71]

Croplands

A major concern for the viability of current alternative fuels is the cropland needed to produce the
required materials. For example, the production of corn for corn ethanol fuel competes with cropland
that may be used for food growth and other feedstocks. [72] The difference between this and cellulosic
ethanol production is that cellulosic material is widely available and is derived from a large resource of
things. Some crops used for cellulosic ethanol production include switchgrass, corn stover, and hybrid
poplar. [69] These crops are fast-growing and can be grown on many types of land which makes them
more versatile. Cellulosic ethanol can also be made from wood residues (chips and sawdust), municipal
solid waste such as trash or garbage, paper and sewage sludge, cereal straws and grasses. [71] It is
particularly the non-edible portions of plant material which are used to make cellulosic ethanol, which
also minimizes the potential cost of using food products in production. [73]

The effectiveness of growing crops for the purpose of biomass can vary tremendously depending on the
geographical location of the plot. For example, factors such as precipitation and sunlight exposure may
greatly effect the energy input required to maintain the crops, and therefore effect the overall energy
output. [74] A study done over five years showed that growing and managing switchgrass exclusively as
a biomass energy crop can produce 500% or more renewable energy than is consumed during
production. [74] The levels of GHG emissions and carbon dioxide were also drastically decreased from
using cellulosic ethanol compared with traditional gasoline.

Corn-based vs. grass-based

See also: Environmental and social impacts of ethanol fuel in the U.S. , Indirect land use change impacts
of biofuels, and Low-carbon fuel standard

Summary of Searchinger et al.

comparison of corn ethanol and gasoline GHG emissions

with and without land use change

(Grams of CO 2 released per megajoule of energy in fuel) [75][76]

Fuel type

(U.S.)
Carbon

intensity

Reduction

GHG

Carbon

intensity

+ ILUC

Reduction

GHG

Gasoline 92 - 92 -

Corn ethanol 74 -20% 177 +93%

Cellulosic ethanol 28 -70% 138 +50%

Notes: Calculated using default assumptions for 2015 scenario for ethanol in E85 .

Gasoline is a combination of conventional and

reformulated gasoline . [76]

In 2008, there was only a small amount of switchgrass dedicated for ethanol production. In order for it
to be grown on a large-scale production it must compete with existing uses of agricultural land, mainly
for the production of crop commodities. Of the United States' 2.26 billion acres (9.1 million km2 ) of
unsubmerged land, [77] 33% are forestland, 26% pastureland and grassland, and 20% crop land. A study
done by the U.S. Departments of Energy and Agriculture in 2005 determined whether there were
enough available land resources to sustain production of over 1 billion dry tons of biomass annually to
replace 30% or more of the nation’s current use of liquid transportation fuels. The study found that
there could be 1.3 billion dry tons of biomass available for ethanol use, by making little changes in
agricultural and forestry practices and meeting the demands for forestry products, food, and fiber. [78]
A recent study done by the University of Tennessee reported that as many as 100 million acres (400,000
km 2 , or 154,000 sq mi) of cropland and pasture will need to be allocated to switchgrass production in
order to offset petroleum use by 25 percent. [ citation needed ]

Currently, corn is easier and less expensive to process into ethanol in comparison to cellulosic ethanol.
The Department of Energy estimates that it costs about $2.20 per gallon to produce cellulosic ethanol,
which is twice as much as ethanol from corn. Enzymes that destroy plant cell wall tissue cost 30 to 50
cents per gallon of ethanol compared to 3 cents per gallon for corn. [ citation needed ] The Department
of Energy hopes to reduce production cost to $1.07 per gallon by 2012 to be effective. However,
cellulosic biomass is cheaper to produce than corn, because it requires fewer inputs, such as energy,
fertilizer, herbicide, and is accompanied by less soil erosion and improved soil fertility. Additionally,
nonfermentable and unconverted solids left after making ethanol can be burned to provide the fuel
needed to operate the conversion plant and produce electricity. Energy used to run corn-based ethanol
plants is derived from coal and natural gas. The Institute for Local Self-Reliance estimates the cost of
cellulosic ethanol from the first generation of commercial plants will be in the $1.90–$2.25 per gallon
range, excluding incentives. This compares to the current cost of $1.20–$1.50 per gallon for ethanol
from corn and the current retail price of over $4.00 per gallon for regular gasoline (which is subsidized
and taxed). [79]

One of the major reasons for increasing the use of biofuels is to reduce greenhouse gas emissions. [80]
In comparison to gasoline, ethanol burns cleaner, thus putting less carbon dioxide and overall pollution
in the air [ citation needed ] . Additionally, only low levels of smog are produced from combustion. [81]
According to the U.S. Department of Energy, ethanol from cellulose reduces greenhouse gas emission by
86 percent when compared to gasoline and to corn-based ethanol, which decreases emissions by 52
percent. [82] Carbon dioxide gas emissions are shown to be 85% lower than those from gasoline.
Cellulosic ethanol contributes little to the greenhouse effect and has a five times better net energy
balance than corn-based ethanol. [81] When used as a fuel, cellulosic ethanol releases less sulfur,
carbon monoxide, particulates, and greenhouse gases. Cellulosic ethanol should earn producers carbon
reduction credits, higher than those given to producers who grow corn for ethanol, which is about 3 to
20 cents per gallon. [83]

It takes 0.76 J of energy from fossil fuels to produce 1 J worth of ethanol from corn. [84] This total
includes the use of fossil fuels used for fertilizer, tractor fuel, ethanol plant operation, etc. Research has
shown that fossil fuel can produce over five times the volume of ethanol from prairie grasses, according
to Terry Riley, President of Policy at the Theodore Roosevelt Conservation Partnership. The United
States Department of Energy concludes that corn-based ethanol provides 26 percent more energy than
it requires for production, while cellulosic ethanol provides 80 percent more energy. [85] Cellulosic
ethanol yields 80 percent more energy than is required to grow and convert it. [86] The process of
turning corn into ethanol requires about 1700 times (by volume) as much water as ethanol produced.
[ dubious ] Additionally, it leaves 12 times its volume in waste. [87] Grain ethanol uses only the edible
portion of the plant.

U.S. Environmental Protection Agency

Draft life cycle GHG emissions reduction results

for different time horizon and discount rate approaches

[88]

(includes indirect land use change effects )

Fuel Pathway

100 years +

2% discount

rate

30 years +
0% discount

rate

Corn ethanol (natural gas dry mill) (1) -16% +5%

Corn ethanol (Best case NG DM) (2) -39% -18%

Corn ethanol (coal dry mill) +13% +34%

Corn ethanol (biomass dry mill) -39% -18%

Corn ethanol (biomass dry mill with

combined heat and power)

-47% -26%

Brazilian sugarcane ethanol -44% -26%

Cellulosic ethanol from

switchgrass -128% -124%

Cellulosic ethanol from corn stover -115% -116%

Notes: (1) Dry mill (DM) plants grind the entire kernel and generally produce

only one primary co-product: distillers grains with solubles (DGS).

(2) Best case plants produce wet distillers grains co-product.

Cellulose is not used for food and can be grown in all parts of the world. The entire plant can be used
when producing cellulosic ethanol. Switchgrass yields twice as much ethanol per acre than corn. [85]
Therefore, less land is needed for production and thus less habitat fragmentation. Biomass materials
require fewer inputs, such as fertilizer, herbicides, and other chemicals that can pose risks to wildlife.
Their extensive roots improve soil quality, reduce erosion, and increase nutrient capture. Herbaceous
energy crops reduce soil erosion by greater than 90%, when compared to conventional commodity crop
production. This can translate into improved water quality for rural communities. Additionally,
herbaceous energy crops add organic material to depleted soils and can increase soil carbon, which can
have a direct effect on climate change, as soil carbon can absorb carbon dioxide in the air. [89][90] As
compared to commodity crop production, biomass reduces surface runoff and nitrogen transport.
Switchgrass provides an environment for diverse wildlife habitation, mainly insects and ground birds.
Conservation Reserve Program (CRP) land is composed of perennial grasses, which are used for cellulosic
ethanol, and may be available for use.

For years American farmers have practiced row cropping, with crops such as sorghum and corn. Because
of this, much is known about the effect of these practices on wildlife. The most significant effect of
increased corn ethanol would be the additional land that would have to be converted to agricultural use
and the increased erosion and fertilizer use that goes along with agricultural production. Increasing our
ethanol production through the use of corn could produce negative effects on wildlife, the magnitude of
which will depend on the scale of production and whether the land used for this increased production
was formerly idle, in a natural state, or planted with other row crops. Another consideration is whether
to plant a switchgrass monoculture or use a variety of grasses and other vegetation. While a mixture of
vegetation types likely would provide better wildlife habitat, the technology has not yet developed to
allow the processing of a mixture of different grass species or vegetation types into bioethanol. Of
course, cellulosic ethanol production is still in its infancy, and the possibility of using diverse vegetation
stands instead of monocultures deserves further exploration as research continues. [91]

A study by Nobel Prize winner Paul Crutzen found ethanol produced from corn had a "net climate
warming" effect when compared to oil when the full life cycle assessment properly considers the nitrous
oxide (N20) emissions that occur during corn ethanol production. Crutzen found that crops with less
nitrogen demand, such as grasses and woody coppice species, have more favourable climate impacts.
[92]

Cellulosic ethanol commercialization

Main article: Cellulosic ethanol commercialization

Cellulosic ethanol commercialization is the process of building an industry out of methods of turning
cellulose-containing organic matter into fuel. Companies such as

Iogen , POET, and Abengoa are building refineries that can process biomass and turn it into ethanol,
while companies such as DuPont , Diversa , Novozymes , and

Dyadic are producing enzymes which could enable a cellulosic ethanol future. The shift from food crop
feedstocks to waste residues and native grasses offers significant opportunities for a range of players,
from farmers to biotechnology firms, and from project developers to investors. [93]

The cellulosic ethanol industry developed some new commercial-scale plants in 2008. In the United
States, plants totaling 12 million liters (3.17 million gal) per year were operational, and an additional 80
million liters (21.1 million gal.) per year of capacity - in 26 new plants - was under construction. In
Canada, capacity of 6 million liters per year was operational. In Europe, several plants were operational
in Germany, Spain, and Sweden, and capacity of 10 million liters per year was under construction. [94]

Italy-based Mossi & Ghisolfi Group broke ground for its 13 MMgy cellulosic ethanol facility in
northwestern Italy on April 12, 2011. The project will be the largest cellulosic ethanol project in the
world, 10 times larger than any of the currently operating demonstration-scale facilities. [95]

Commercial Cellulosic Ethanol Plants in the U.S. [96][97]

(Operational or under construction)

Company Location Feedstock

Abengoa Bioenergy Hugoton, KS Wheat straw

BlueFire Ethanol Irvine, CA Multiple sources

Colusa Biomass Energy Corporation

Sacramento, CA Waste rice straw

Coskata Warrenville, IL

Biomass, Agricultural and Municipal wastes

DuPont Vonore, TN Corn cobs, switchgrass

DuPont Nevada, IA Corn stover

Fulcrum BioEnergy Reno, NV Municipal solid waste

Gulf Coast Energy Mossy Head, FL Wood waste

KL Energy Corp. Upton, WY Wood

Mascoma Lansing, MI Wood

POET-DSM Advanced Biofuels

Emmetsburg, IA

Corn cobs, husks, and stover [98]

Range Fuels [99] Treutlen County, GA Wood waste

SunOpta Little Falls, MN Wood chips

SweetWater Energy Rochester, NY Multiple Sources

US Envirofuels Highlands County, FL Sweet sorghum

Xethanol Auburndale, FL Citrus peels

See also

Renewable energy portal

Energy portal

Algae fuel

Biofuel

Biorefinery

Butanol

Butanol fuel

Candida tropicalis

Carbon Negative

Cellulosic ethanol commercialization

Cellulosome

China Resources Alcohol Corporation

Coskata

Clonostachys rosea f. rosea

Ethanol

Life cycle assessment

Iogen Corp.

Methanol

Methanol fuel

Non food crops

Range Fuels

SunOpta

Thermophilic

Thomas Friedman

Treethanol

Verenium Corporation

References

1. ^ Updated Energy and Greenhouse Gas Emissions Results of Fuel Ethanol

2. ^ "Clean cars, cool fuels" . 5 (2). Environment California. 2007. Retrieved 2007-11-28.

3. ^ National Research Council of the National Academies (2011), Renewable Fuel Standard: Potential
Economic and Environmental Effects of U.S. Biofuel Policy , Washington, D.C.: The National Academies
Press, p. 3 of "Summary", ISBN 978-0-309-18751-0

4. ^ Matthew L. Wald (January 25, 2013). "Court Overturns E.P.A.'s Biofuels Mandate" . The New York
Times . Retrieved January 26, 2013. "wishful thinking rather than realistic estimates"

5. ^ Somma D, Lobkowicz H, Deason JP (2010).

"Growing America's fuel: an analysis of corn and cellulosic ethanol feasibility in the United States" (PDF).
Clean Techn Environ Policy. 12: 373–380. doi :10.1007/s10098-009-0234-3 .

6. ^ Braconnot, H. Annalen der Physik. 1819, 63, 348.

7. ^ Solomon, B. D.; Barnes, J. R.; Halvorsen, K. E. Grain and cellulosic ethanol: History, economics, and
energy policy. Biomass and Bioenergy. 2007, 31, 416-425.

8. ^ Saeman JF. "Kinetics of wood saccharification: Hydrolysis of cellulose and decomposition of sugars in
dilute acid at high temperature", Industrial and Engineering Chemistry, 37(1): 43–52 (1945).
9. ^ Harris EE, Beglinger E, Hajny GJ, and Sherrard EC. "Hydrolysis of Wood: Treatment with Sulfuric Acid
in a stationary digester", Industrial and Engineering Chemistry , 37(1): 12–23 (1945)

10. ^ Conner AH, Lorenz LF. "Kinetic modeling of hardwood prehydrolysis. Part III. Water and dilute
acetic acid Prehydrolysis of southern red oak, Wood and Fiber Science, 18(2): 248–263 (1986).

11. ^ Katzen, R. and Schell, d.J., "Lignocellulosic feedstock Biorefinery: History and Plant Development
for Biomass Hydrolysis", pp 129-138 in Biorefineries - Industrial processes and Products, Volume 1,
Kamm, B., Gruber, P.R., and Kamm, M., eds. Wiley-VCH, Weinheim, 2006.

12. ^ a b c d Zhu JY, Pan XJ, Wang GS, Gleisner R (2009). "Sulfite pretreatment (SPORL) for Robust
enzymatic saccharification of spruce and red pine".

Bioresource Technology . 100 (8): 2411–2418.

doi :10.1016/j.biortech.2008.10.057 .

PMID 19119005 .

13. ^ Dirk Lammers (2007-03-04). "Gasification may be key to U.S. Ethanol" . CBS News. Archived from
the original on 2007-11-22. Retrieved 2007-11-28.

14. ^ "Range Fuels awarded permit to construct the nation's first commercial cellulosic ethanol plant" .
Range Fuels. 2007-07-02. Archived from the original on 2007-10-12. Retrieved 2007-11-28.

15. ^ Kathleen Schalch (2007-11-05). "Georgia plant is first for making ethanol from waste" . NPR.
Retrieved

2007-11-28.

16. ^ Green-energy plant sucks up subsidies, then goes bust

17. ^ Galazka, J.; Tian, C.; Beeson, W.; Martinez, B.; Glass, N.; Cate, J. (2010). "Cellodextrin Transport in
Yeast for Improved Biofuel Production". Science. 330 (6000): 84–86. Bibcode : 2010Sci...330...84G .

doi :10.1126/science.1192838 . PMID 20829451 .

18. ^ Mosier N, Wyman C, Dale BE, Elander R, Lee YY, Holtzapple M, Ladisch M (2005) Features of
promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 96:673-686

19. ^ Iakovlev, M.; van Heiningen, A. Efficient fractionation of spruce by SO2-Ethanol-Water (SEW)
treatment: closed mass balances for carbohydrates and sulfur. ChemSUSChem 2012, 5, 1625-1637. DOI
10.1002/cssc.201100600

20. ^ Klinke HB, Thomsen AB, Ahring BK (2004). "Inhibition of ethanol-producing yeast and bacteria by
degradation products produced during pre-treatment of biomass". Appl Microbiol Biotechnol . 66 (1):
10–26.

doi :10.1007/s00253-004-1642-2 . PMID 15300416 .

21. ^ Olsson L, Hahn-Hägerdal B (1996) Fermentation of lignocellulosic hydrolysates for ethanol

fermentation. Enzyme Microb Technol 18:312–331.
22. ^ Palmqvist E, Hahn-Hägerdal B (2000) Fermentation of lignocellulosic hydrolysates. I. Inhibition and
deoxification. Bioresour Technol 74:17–24

23. ^ a b Lynd LR (1996). Overview and evaluation of fuel ethanol from cellulosic biomass: technology,
economics, the environment, and policy. Annu Rev Energy Environ 21:403–465.

24. ^ Organosolv delignification of willow (2010), AL Macfarlane, MM Farid, JJJ Chen, Lambert Academic
Press, ISBN 978-3-8383-9155-7

25. ^ Iakovlev M. SO2-Ethanol-Water Fractionation of Lignocellulosics. Doctoral Thesis, Aalto University,

Finland, 2011. http://lib.tkk.fi/Diss/2011/isbn9789526043142/isbn9789526043142.pdf

26. ^ Retsina T., Pylkkanen V. US 8030039 B1, Method for the production of fermentable sugars and
cellulose from lignocellulosic material

27. ^ Wood Alcohol . Translation from E. Boullanger: Distillerie Agricole et Industrielle (Paris: Ballière,
1924).

28. ^ "Iogen technology makes it possible (process overview)" . Iogen Corp . 2005. Retrieved 2007-11-28.

29. ^ "Sunopta updates current cellulosic ethanol projects" . Sunopta press release, 2007.

30. ^ Frandsen, Kristian E H; Simmons, Thomas J; Dupree, Paul; Poulsen, Jens-Christian N; Hemsworth,
Glyn R; Ciano, Luisa; Johnston, Esther M; Tovborg, Morten; Johansen, Katja S. "The molecular basis of
polysaccharide cleavage by lytic polysaccharide monooxygenases" . Nature Chemical Biology .

doi :10.1038/nchembio.2029 .

31. ^ Dyadic International (DYAI)

32. ^ Blue Sugars

33. ^ Jeffries TW, Jin YS (2004). "Metabolic engineering for improved fermentation of pentoses by
yeasts". Appl Microbiol Biotechnol . 63 (5): 495–509.

doi :10.1007/s00253-003-1450-0 . PMID 14595523 .

34. ^ Brat D, Boles E and Wiedemann B (2009) Functional expression of a bacterila xylose isomerase in
Saccharomyces cerevisie. Appl. Environ. Microbiol.

doi :10.1128/AEM.02522-08

35. ^ Ohgren K, Bengtsson O, Gorwa-Grauslund MF, Galbe M, Hahn-Hagerdal B, Zacchi G (2006)

Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn
stover at high fiber content with Saccharomyces cerevisiae TMB3400. J Biotechnol. 126(4):488–98.

36. ^ Becker J, Boles E (2003). "A modified Saccharomyces cerevisiae strain that consumes L-Arabinose
and produces ethanol" . Appl Environ Microbiol. 69 (7): 4144–50. doi :10.1128/AEM.69.7.4144-
4150.2003 . PMC 165137 .

PMID 12839792 .
37. ^ Karhumaa K, Wiedemann B, Hahn-Hagerdal B, Boles E, Gorwa-Grauslund MF (2006) Co-utilization
of L-arabinose and D-xylose by laboratory and industrial

Saccharomyces cerevisiae strains. Microb Cell Fact. 10;5:18.

38. ^ University of Rochester Press Release: Genome Sequencing Reveals Key to Viable Ethanol
Production

39. ^ "Providing for a Sustainable Energy Future by producing clean RENEWABLE liquid energy and green
power" . Bioengineering Resources Inc. Retrieved

2007-11-28.

40. ^ "Formation of Ethanol from Carbon Monoxide via New Microbial Catalyst", Biomass & Energy v. 23
(2002), p. 487–493.

41. ^ "Power Energy Fuels Homepage" . Power Energy Fuels, Inc. Retrieved 2007-11-28.

42. ^ "Westbury, Quebec" . Retrieved 2011-07-27.

43. ^ a b Sakamoto, T.; Hasunuma, T.; Hori, Y.; Yamada, R.; Kondo, A. Direct ethanol production from
hemicellulosic materials of rice straw by use of an engineered yeast strain codisplaying three types of
hemicellulolytic enzymes on the surface of xylose-utilizing Saccharomyces cerevisiae cells. J. Biotechnol.
2012, 158, 203-210.

44. ^ Deepak, K., & Ganti, M. Impact of pretreatment and downstream processing technologies on
economics and energy in cellulosic ethanol production. Biotechnology for Biofuels, 4

45. ^ Wald, Matthew L. (2012-01-09). "Companies Face Fines for Not Using Unavailable Biofuel" . The
New York Times .

46. ^ a b U.S. Will Be Hard-Pressed to Meet Its Biofuel Mandates | MIT Technology Review

47. ^ a b c Sainz, M. B. (2011). Commercial cellulosic ethanol: the role of plant-expressed enzymes.
Biofuels, 237-264.

48. ^ "DOE Selects Six Cellulosic Ethanol Plants for Up to $385 Million in Federal Funding" . United States
Department of Energy . 2007-02-28.

49. ^ "Feasibility Study for Co-Locating and Integrating Ethanol Production Plants from Corn Starch and
Lignocellulosic Feedstocks" (PDF). United States Department of Energy. January 2005.

50. ^ "Determining the Cost of Producing Ethanol from Corn Starch and Lignocellulosic Feedstocks"
(PDF). U.S. Department of Agriculture and U.S. Department of Energy. October 2000.

51. ^ a b "Biomass Resource Estimates" .

52. ^ "Switchgrass Fuel Yields Bountiful Energy: Study" . Reuters. January 10, 2008. Retrieved

2008-02-12.

53. ^ National Geographic Magazine , 'Carbon's New Math', October 2007

54. ^ [300 million people × 4.4 lb (2.0 kg) per person × 37% is paper / 2000 lb per ton]

55. ^ "Solid Waste Generation" (PDF).

56. ^ Boyle, Harry et al. Next-generation ethanol and biochemicals page 16, Bloomberg New Energy
Finance, 14 September 2010. Retrieved: 14 September 2010.

57. ^ a b c A Canceled BP Plant Calls into Question Cellulosic Ethanol Economics | MIT Technology
Review

58. ^ http://www.slideshare.net/RonDavisPress/reportagem-granbio-folha-de-sp-092013

59. ^ http://www.slideshare.net/RonDavisPress/balano-contbil-granbio-2013

60. ^ Yang, B., & Wyman, C. E. (2007). Pretreatment: the key to unlocking low‐cost cellulosic ethanol.
Biofuels, Bioproducts and Biorefining, 2(1), 26-40.

61. ^ a b Johnson, Eric (18 Feb 2016). "Integrated enzyme production lowers the cost of cellulosic
ethanol" . Biofuels, Bioproducts & Biorefining .

doi :10.1002/bbb.1634 . Retrieved 3 June 2016.

62. ^ Perlack, et al. 2005. Biomass as feedstock for a bioenergy and bioproducts Industry: the technical
feasibility of a billion-ton annual supply. Oar Ridge National Laboratory Report ORNL/TM-2005/66, US
Dept. of Energy, Oak Ridge, TN

63. ^ Wang GS, Pan XJ, Zhu JY, Gleisner R (2009). "Sulfite pretreatment to overcopme recalcitrabce of
lignocellulose (SPORL) for robust Enzymatic Saccharification of hardwoods". Biotechnology Progress .

25 (4): 1086–1093. doi : 10.1002/btpr.206 .

PMID 19551888 .

64. ^ Foody, B.E., Foody, K.J., 1991. Development of an integrated system for producing ethanol from
biomass. In: Klass, D.L. (Ed.), Energy from Biomass and Waste. Institute of Gas Technology, Chicago, pp.
1225-1243

65. ^ Better Bug to Make Cellulosic Ethanol | MIT Technology Review

66. ^ a b Rinehart, L. (2006). "Switchgrass as a Bioenergy Crop. National Sustainable Agriculture

Information Service" (PDF). Retrieved 2007-12-10.

67. ^ Richard G. Lugar, R. James Woolsey. The New Petroleum. Foreign Affairs. 1999. Vol. 78, No 1. p. 88.

68. ^ Farrell, A. E.; Plevin, R. J.; Turner, B. T.; Jones, A. D.; O’Hare, M.; Kammen, D. M. Ethanol can
contribute to energy and environmental goals. Science. 2006, 311, 506–508

69. ^ a b Huanga, H.; Ramaswamya, S.; Al-Dajania, W.; Tschirnera, U.; Cairncrossb, R. A. Effect of biomass
species and plant size on cellulosic ethanol: A comparative process and economic analysis. Biomass
Bioenergy. 2009, 33, 234-246
70. ^ Wanga, M. Q.; Han, J.; Haq, Z.; Tyner, W. E.; Wua, M.; Elgowainy, A. Energy and greenhouse gas
emission effects of corn and cellulosic ethanol with technology improvements and land use changes.
Biomass Bioenergy. 2011, 35, 1885-1896

71. ^ a b Solomon, B. D.; Barnes, J. R.; Halvorson, K. E. Grain and cellulosic ethanol: History, economics,
and energy policy. Biomass Bioenergy. 2007, 31, 416–425

72. ^ Brown, L. R. Plan B 2.0: rescuing a plant under stress and a civilization in trouble. New York, USA:
W.W. Norton & Company; 2006

73. ^ Vasudevan, P. T.; Gagnon, M. D.; Briggs, M. S.; Environmentally Sustainable Biofuels – The Case for
Biodiesel, Biobutanol and Cellulosic Ethanol. Sustainable Biotechnology. 2010, 43-62

74. ^ a b Schmer, M. R.; Vogel, K. P.; Mitchell, R. B.; Perrin, R. K. Net energy of cellulosic ethanol from
switchgrass. Proc. Natl. Acad. Sci. 2008, 105(2), 464-469

75. ^ Timothy Searchinger; et al. (2008-02-29). "Use of U.S. Croplands for Biofuels Increases Greenhouse
Gases Through Emissions from Land-Use Change" .

Science. 319 (5867): 1238–1240.

Bibcode : 2008Sci...319.1238S . doi :10.1126/science.1151861 . PMID 18258860 . Retrieved

2008-05-09. Originally published online in Science Express on 7 February 2008. See Letters to Science by
Wang and Haq. There are critics to these findings for assuming a worst-case scenario.

76. ^ a b Searchinger; et al. (2008). "Supporting Materials for Use of U.S. Croplands for Biofuels Increases
Greenhouse Gases Through Emissions from Land Use Change" (PDF). Princeton University . Retrieved
2009-06-11. Data taken from Table 1B, pp. 21.

77. ^ The World Fact Book, www.cia.org, 01 May 2008

78. ^ "Cellulosic Ethanol: Benefits and Challenges. Genomics: GTL" . U.S. Department of Energy Office of
Science. 2007. Retrieved 2007-12-09.

79. ^ "Cellulosic ethanol: fuel of the future?" (PDF). ILSR Daily. 2007.

80. ^ "Cellulosic ethanol: Fuel of the future?" . Science Daily. 2007. Retrieved 2007-12-10.

81. ^ a b Demain A, Newcomb M, Wu D (March 2005).

"Cellulase, Clostridia, and Ethanol. Microbiology" .

Molecular Biology Reviews . 69 (1): 124–154.

doi :10.1128/MMBR.69.1.124-154.2005 .

PMC 1082790 . PMID 15755956 .

82. ^ Alternative Fuels Data Center: Ethanol Benefits and Considerations

83. ^ Weeks, J (2006). "Are We There Yet? Not quite, but cellulosic ethanol may be coming sooner than
you think" . Grist Magazine. Retrieved 2007-12-08.
84. ^ Farrell AE; Plevin RJ; Turner BT; Jones AD; O’Hare M; Kammen DM (2006-01-27). "Ethanol can
contribute to energy and environmental goals". Science. 311 (5760): 506–508. Bibcode :
2006Sci...311..506F . doi :10.1126/science.1121416 . PMID 16439656 .

85. ^ a b The numbers behind ethanol, cellulosic ethanol, and biodiesel in the U.S. | Grist

86. ^ Ratliff E (2007). "One molecule could cure our addiction to oil". Wired Magazine . 15 (10).

87. ^ Marshall B (October 2007). "Gas from the grass".

Field and Stream: 40–42.

88. ^ "EPA Lifecycle Analysis of Greenhouse Gas Emissions from Renewable Fuels - Technical Highlights"
(PDF). Retrieved 2009-06-07. See Table 1 .

89. ^ Nelson R (2007). "Cellulosic ethanol: bioethanol in Kansas" (PDF). Retrieved 2007-12-09.

90. ^ Charles W. Rice (2002). "Storing Carbon in Soil: Why and How?" . Retrieved 2008-11-10.

91. ^ BIES L (2006) The Biofuels Explosion: Is Green Energy Good for Wildlife? Wildlife Society Bulletin:
Vol. 34, No. 4 pp. 1203–1205

92. ^ Crutzen, PJ, Mosier AR, Smith KA, Winiwarter W. "Nitrous oxide release from agro-biofuel
production negates global warming reduction by replacing fossil fuels", Atmospheric Chemistry and
Physics , 8(2): 389–395 (2008).

93. ^ Pernick, Ron and Wilder, Clint (2007). The Clean Tech Revolution p. 96.

94. ^ REN21 (2009). Renewables Global Status Report: 2009 Update p. 16.

95. ^ Kris Bevill (April 12, 2011). "World's largest cellulosic ethanol plant breaks ground in Italy" . Ethanol
Producer Magazine .

96. ^ Decker, Jeff. Going Against the Grain: Ethanol from Lignocellulosics , Renewable Energy World ,
January 22, 2009. Retrieved February 1, 2009.

97. ^ Building Cellulose

98. ^ Matthew L. Wald (July 6, 2011). "U.S. Backs Project to Produce Fuel From Corn Waste" . The New
York Times . Retrieved July 7, 2011. "The Energy Department plans to provide a $105 million loan
guarantee for the expansion of an ethanol factory in Emmetsburg, Iowa, that intends to make motor
fuelMohammad J. Taherzadeh * and Keikhosro Karimi

School of Engineering, University of Borås, 501 90 Borås, Sweden

Department of Chemical Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran

* Author to whom correspondence should be addressed Tel. +46-33-4355908, Fax: +46-33-4354008

Received: 5 March 2008; in revised form: 27 August 2008 / Accepted: 1 September 2008 / Published: 1
September 2008
Abstract: Lignocelluloses are often a major or sometimes the sole components of different waste
streams from various industries, forestry, agriculture and municipalities. Hydrolysis of these materials is
the first step for either digestion to biogas (methane) or fermentation to ethanol. However, enzymatic
hydrolysis of lignocelluloses with no pretreatment is usually not so effective because of high stability of
the materials to enzymatic or bacterial attacks. The present work is dedicated to reviewing the methods
that have been studied for pretreatment of lignocellulosic wastes for conversion to ethanol or biogas.
Effective parameters in pretreatment of lignocelluloses, such as crystallinity, accessible surface area, and
protection by lignin and hemicellulose are described first. Then, several pretreatment methods are
discussed and their effects on improvement in ethanol and/or biogas production are described. They
include milling, irradiation, microwave, steam explosion, ammonia fiber explosion (AFEX), supercritical
CO and its explosion, alkaline hydrolysis, liquid hot-water pretreatment, organosolv processes, wet
oxidation, ozonolysis, dilute-and concentrated-acid hydrolyses, and biological pretreatments.

Keywords: Pretreatment; biogas; methane; ethanol; lignocellulose; waste; biodegradation

1. Introduction

Production of waste materials is an undeniable part of human society. The wastes are produced by
several sectors including industries, forestry, agriculture and municipalities. The accumulation of waste
and the “throw-away philosophy” result in several environmental problems, health issues and safety
hazards, and prevent sustainable development in terms of resource recovery and recycling of waste
materials. A perspective aimed at promoting greater sustainable development and resource recovery
has influenced solid waste management practices, and is gradually becoming implemented through
policy guidelines at national levels in a number of industrialized and even developing countries.
Guidelines and directives to reduce waste generation and promote waste recovery are laid down
according to the “waste management hierarchy”, in which waste prevention, reuse, recycling and
energy recovery are designed to minimize the amount of waste left for final, safe disposal [ 1].

Ethanol is now the most important renewable fuel in terms of volume and market value [ 2 ]. Nowadays
it is produced from sugar-and starch-based materials such as sugarcane and corn. However, the second
generation production of ethanol derived from lignocellulosic materials is now being tested in pilot
plants [ 3 , 4 ]. The present work paves the way for integrating waste streams into the raw materials for
ethanol plants, by reviewing different methods for their pretreatment for improved production of
ethanol.

Biogas is another energy source that is used as car fuel, or for production of heat or electricity in
different countries [ 5 , 6 ]. Biogas production from activated sludge is an old and almost established
process. It has also recently been produced on industrial scales from municipal solid waste (MSW) and
some homogeneous wastes such as manures. Forestry and agriculture residues and MSW are by nature
heterogeneous in size, composition, structure, and properties. Sugars, starches, lipids and proteins
present in MSW are among the materials easily degradable by microorganisms, while some other
fractions such as lignocelluloses and keratin are more difficult to degrade [ 7 ]. Biological degradations of
these polymers are carried out by several enzymes such as amylase, cellulase, protease, keratinase and
lipase, before further fermentation or digestion to e.g. ethanol or biogas. However, these polymers
should be accessible to the enzymes for biodegradation.
Pretreatment by physical, chemical or biological means is a well-investigated process for ethanol
production from lignocellulosic materials. Furthermore, there have been some efforts to pretreat waste
materials for biogas production. The pretreatment can enhance the bio-digestibility of the wastes for
ethanol and biogas production and increase accessibility of the enzymes to the materials. It results in
enrichment of the difficult biodegradable materials, and improves the yield of ethanol or biogas from
the wastes ( Figure 1).

Figure 1. Pretreatment of lignocellulosic materials prior to bioethanol and biogas production

The present work deals with reviewing the pretreatment processes used in ethanol and biogas
processes from waste materials. However, since wastes comprise a wide range of materials, we dedicate
this review only to lignocellulosic part of waste materials in order to shorten the discussion.

2. Lignocelluloses among waste materials

In addition to inorganic wastes, different types of polymers are available in various waste materials.
Natural materials such as starch, lipids, glycogen, elastin, collagen, keratin, chitin and lignocelluloses, as
well as synthetic polymers such as polyesters, polyethylene and polypropylene, are among these
polymers. In this work, we focus on treatment of lignocellulosic polymers that are resistant to biological
degradation.

Lignocelluloses ( Figure 2 ) comprise a large fraction of municipal solid waste (MSW), crop residues,
animal manures, woodlot arisings, forest residues, or dedicated energy crops [ 5 ]. Lignocelluloses are
composed of cellulose, hemicellulose, lignin, extractives, and several inorganic materials [ 8 ]. Cellulose
or β-1-4-glucan is a linear polysaccharide polymer of glucose made of cellobiose units [ 9 , 10 ]. The
cellulose chains are packed by hydrogen bonds in so-called ‘elementary and microfibrils’ [ 11]. These
fibrils are attached to each other by hemicelluloses, amorphous polymers of different sugars as well as
other polymers such as pectin, and covered by lignin. The microfibrils are often associated in the form of
bundles or macrofibrils [ 9 ] ( Figure 2 ). This special and complicated structure makes cellulose resistant
to both biological and chemical treatments. Cellulose is available in waste streams in the form of
lignocelluloses, or partly purified in the form of e.g. papers or pure cellulose such as cotton, or mixed
with other materials, in e.g. citrus wastes [ 12 ].

Figure 2. Effect of pretreatment on accessibility of degrading enzymes

The dominant sugars in hemicelluloses are mannose in softwoods and xylose in hardwoods and
agriculture residues [ 13 – 15 ]. Furthermore, these heteropolymers contain galactose, glucose,
arabinose, and small amounts of rhamnose, glucuronic acid, methyl glucuronic acid, and galacturonic
acid. In contrast to cellulose, which is crystalline and strong, hemicelluloses have a random, amorphous,
and branched structure with little resistance to hydrolysis, and they are more easily hydrolyzed by acids
to their monomer components [ 8 , 10 , 16 – 18 ].

Lignin is a very complex molecule constructed of phenylpropane units linked in a three-dimensional

structure which is particularly difficult to biodegrade. Lignin is the most recalcitrant component of the
plant cell wall, and the higher the proportion of lignin, the higher the resistance to chemical and
enzymatic degradation. Generally, softwoods contain more lignin than hardwoods and most of the
agriculture residues. There are chemical bonds between lignin and hemicellulose and even cellulose [ 19
, 20 ]. Lignin is one of the drawbacks of using lignocellulosic materials in fermentation, as it makes
lignocellulose resistant to chemical and biological degradation.

3. Effective parameters in pretreatment of lignocelluloses

The inherent properties of native lignocellulosic materials make them resistant to enzymatic attack. The
aim of pretreatment is to change these properties in order to prepare the materials for enzymatic
degradation. Since lignocellulosic materials are very complicated, their pretreatment is not simple
either. The best method and conditions of pretreatment depend greatly on the type of lignocelluloses.
For instance, pretreatment of bark from poplar trees or corn leaf with a dilute-acid process seems to be
promising, but this method is not effective for treating the bark from sweetgum or corn stalks [ 21 , 22 ].

The crystallinity of cellulose, its accessible surface area and protection by lignin and hemicellulose,
degree of cellulose polymerization, and degree of acetylation of hemicelluloses are the main factors
considered as affecting the rate of biological degradation of lignocelluloses by the enzymes [ 23 ]. These
factors will be discussed briefly below.

3.1. Crystallinity

The cellulose microfibrils have both crystalline and amorphous regions, and the crystallinity is given by
the relative amounts of these two regions. The major part of cellulose (around 2/3 of the total cellulose)
is in the crystalline form [ 24 ]. It was shown that cellulase readily hydrolyzes the more accessible
amorphous portion of cellulose, while the enzyme is not so effective in degrading the less accessible
crystalline portion. It is therefore expected that high-crystallinity cellulose will be more resistant to
enzymatic hydrolysis, and it is widely accepted that decreasing the crystallinity increases the digestibility
of lignocelluloses [ 25 ].

On contrary, there are some studies [ 23 , 26 ] that show more digestibility of more crystalline
lignocelluloses. This conflict in the reports might appear, while the effects of other factors are ignored.
Grethlein [ 26 ] pretreated hard-and softwoods by mild acid hydrolysis and determined their pore size
distribution. Regardless of the substrate, the initial rate of hydrolysis was shown to be linearly correlated
with the pore volume of the substrate accessible to the size of the cellulase. However, it was also shown
that the crystallinity index has no relationship to the rate of hydrolysis. Kim and Holtzapple [ 27 ] found
that the degree of crystallinity of corn stover slightly increased from 43% to 60% through delignification
with calcium hydroxide, which was related to removal of amorphous components (lignin, hemicellulose).
However, an increase in crystallinity of pretreated material did not negatively affect the yield of
enzymatic hydrolysis. Fan et al. [ 25 ] studied the effect of ball milling on surface area and crystallinity of
cellulose. They observed an increase in crystallinity of cellulose by reducing the size of cellulose by
milling. It is believed that recrystallization during water swelling may increase the crystallinity of highly
ball-milled cellulose.

This discussion may indicate that the crystallinity is an important factor in digestibility of lignocelluloses.
However, it is not the only factor in effective enzymatic hydrolysis of these materials, due to the
heterogeneous nature of celluloses and the contribution of other components such as lignin and
hemicellulose.

3.2. Effect of accessible surface area

Several studies have shown a good correlation between the pore volume or population (accessible
surface area for cellulase) and the enzymatic digestibility of lignocellulosic materials. The main reason
for improvement in enzymatic hydrolysis by removing lignin and hemicellulose is related to the cellulose
accessible surface area. The effect of this area may correlate with crystallinity or lignin protection or
hemicellulose presentation or all of them. Therefore, many researchers have not considered the
accessible surface area as an individual factor that affects the enzymatic hydrolysis [ 23 ]. The first part
of enzymatic hydrolysis consists of [ 23 , 28 ]: (I) adsorption of cellulase enzymes from liquid phase onto
the surface of cellulose (solid), (II) biodegradation of cellulose to simple sugars, mainly cellobiose and
oligomers, and (III) desorption of cellulase to the liquid phase. Thus, the reaction is a heterogeneous
catalytic reaction and direct physical contact between the cellulytic enzymes’ molecules and cellulose is
a prerequisite for enzymatic hydrolysis. As a result, the accessible surface area in lignocellulosic material
and its interaction with the enzymes can be limiting in enzymatic hydrolysis [ 25 , 28 , 29 ].

Lignocellulosic materials have two different types of surface area: external and internal. The external
surface area is related to the size and shape of the particles, while the internal surface area depends on
the capillary structure of cellulosic fibers. Typically, dry cellulosic fibers have small size, about 15 to 40
μm, and therefore they possess a considerable external specific surface area, e.g. 0.6–1.6 m /g.
However, the internal surface area of dried cellulosic fibers is smaller than the external surface area.
Swelling of lignocelluloses with water and polar solvents creates a very large internal surface area [ 25 ].
Drying of fibers can result in irreversible collapse and shrinking of the capillary and thus reduce the
accessible surface area. Presence of water has a significant effect on the specific surface area of natural
cellulose. The specific surface area is known to increase with wetting. Water is known to increase the
crystallinity of cellulose, due to a re-crystallization of highly amorphous cellulose.

The accessible surface area changes during enzymatic hydrolysis. The rate of hydrolysis is usually very
high at first, and then decreases in the later stages. The specific surface area, or accessible surface area
per gram of substrate (m /g), sharply increases during the initial stage. However, it was shown that the
cellulose surface area is not a major limiting factor for hydrolysis of pure cellulose [ 25 ]. In other words,
the slowdown of hydrolysis in the later stages is not due to a lack of associable surface area, but to the
difficulty in hydrolysis of crystalline part of cellulose. Therefore, one may expect a lower rate of
hydrolysis after hydrolysis of the amorphous cellulose [ 25 ].

3.3. Effect of lignin

The cellulose and hemicellulose are cemented together by lignin. Lignin is responsible for integrity,
structural rigidity, and prevention of swelling of lignocelluloses. Thus, lignin content and distribution
constitute the most recognized factor which is responsible for recalcitrance of lignocellulosic materials
to enzymatic degradation by limiting the enzyme accessibility; therefore the delignification processes
can improve the rate and extent of enzymatic hydrolysis. However, in most delignification methods, part
of the hemicellulose is also hydrolyzed, and hence the delignification does not show the sole effect of
lignin [ 23 ]. Dissolved lignin due to e.g. pretreatment of lignocelluloses is also an inhibitor for cellulase,
xylanase, and glucosidase. Various cellulases differ in their inhibition by lignin, while the xylanases and
glucosidase are less affected by lignin [ 30 ].

The composition and distribution of lignin might also be as important as the concentration of lignin.
Some softwoods are more recalcitrant than hardwoods. This might be related to the lignin type, since
softwoods have mainly guaiacyl lignin while hardwoods have a mix of guaiacyl and syringyl lignin. It has
been suggested that guaiacyl lignin restricts fiber swelling and enzyme accessibility more than syringyl
lignin [ 31 ].

In some investigations (e.g. [ 32 ]), the inhibitory role of lignin has been related to its effect on cellulose
swelling. On the other hand, the swelling can be achieved without removal of lignin, and it does not
increase the pore size or the extent of hydrolysis. However, it was shown that lignin still has a significant
effect on enzymatic digestibility, even in cases where it no longer prevents fiber swelling. The reason for
improved rate of hydrolysis by removal of lignin might be related to a better surface accessibility for
enzymes by increasing the population of pores after removing of lignin.

3.4. Effect of hemicellulose

Hemicellulose is a physical barrier which surrounds the cellulose fibers and can protect the cellulose
from enzymatic attack. Many pretreatment methods were shown to be able to remove hemicelluloses
and consequently improve the enzymatic hydrolysis. But most of these processes partly remove the
lignin as well, so the improvement is not the result of removal of hemicellulose alone [ 23 ]. The
accessible surface for enzymatic attack may be related to cellulose crystallinity, lignin, and hemicellulose
content. Hemicellulose can be hydrolyzed by enzymatic hydrolysis by hemicellulase. However, a suitable
pretreatment, e.g. dilute-acid treatment which removes the hemicellulose, eliminates or reduces the
need for use of hemicellulase enzyme mixtures for degrading of biomass [ 33 ].

4. Pretreatment methods for lignocellulose wastes

To achieve enzymatic degradation in production of ethanol by enzymatic hydrolysis or in order to

improve formation of biogas, a pretreatment process is necessary. An effective and economical
pretreatment should meet the following requirements: (a) production of reactive cellulosic fiber for
enzymatic attack, (b) avoiding destruction of hemicelluloses and cellulose, (c) avoiding formation of
possible inhibitors for hydrolytic enzymes and fermenting microorganisms, (d) minimizing the energy
demand, (e) reducing the cost of size reduction for feedstocks, (f) reducing the cost of material for
construction of pretreatment reactors, (g) producing less residues, (h) consumption of little or no
chemical and using a cheap chemical.

Several methods have been introduced for pretreatment of lignocellulosic materials prior to enzymatic
hydrolysis or digestion. These methods are classified into “Physical pretreatment”, “Physico-chemical
pretreatment”, “Chemical pretreatment”, and “Biological pretreatment” [ 23 , 30 , 34 – 56 ]. The
methods of pretreatment of lignocellulosic materials are summarized in Table 1. In this section, we
review these methods, although not all of them have yet developed enough to be feasible for
applications in large-scale processes.

Table 1. Pretreatment processes of lignocellulosic materials

4.1. Physical pretreatment

Physical pretreatment can increase the accessible surface area and size of pores, and decrease the
crystallinity and degrees of polymerization of cellulose. Different types of physical processes such as
milling (e.g. ball milling, two-roll milling, hammer milling, colloid milling, and vibro energy milling) and
irradiation (e.g. by gamma rays, electron beam or microwaves) can be used to improve the enzymatic
hydrolysis or biodegradability of lignocellulosic waste materials.
4.1.1. Milling

Milling can be employed to alter the inherent ultrastructure of lignocelluloses and degree of crystallinity,
and consequently make it more amenable to cellulase [ 57 ]. Milling and size reduction have been
applied prior to enzymatic hydrolysis, or even other pretreatment processes with dilute acid, steam or
ammonia, on several lignocellulosic waste materials, MSW and activated sludge [ 57 – 60 ]. Among the
milling processes, colloid mill, fibrillator and dissolver are suitable only for wet materials, e.g. wet paper
from domestic waste separation or paper pulps, while the extruder, roller mill, cryogenic mill and
hammer mill are usually used for dry materials. The ball mill can be used for either dry or wet materials.
Grinding with hammer milling of waste paper is a favorable method [ 61 ].

Milling can improve susceptibility to enzymatic hydrolysis by reducing the size of the materials [ 62 ],
and degree of crystallinity of lignocelluloses [ 25 ], which improves enzymatic degradation of these
materials toward ethanol or biogas. Without any pretreatment, corn stover with sizes of 53–75 μm was
1.5 times more productive than larger corn stover particles of 425–710 μm [ 62 ]. Sidiras and Koukios
[ 63 ] showed that due to crystallinity reduction by ball milling, saccharification of more than 50% of
straw cellulose with minimal glucose degradation becomes possible at mild hydrolytic conditions. The
crystallinity index of Solka Floc by ball milling changed from 74.2 to 4.9% [ 25 ]. The milling process has
been studied prior to and in combination with enzymatic hydrolysis, where mechanical actions, mass
transport and enzymatic hydrolysis are performed simultaneously in order to improve the hydrolysis
process. The attrition mill bioreactor [ 64 ] and the intensive mass transfer reactor including
ferromagnetic particles and two ferromagnetic inductors [ 65 ] are two examples of these processes.

Mais et al. [ 57 ] used a ball mill reactor for the pretreatment and hydrolysis of α-cellulose and SO
-impregnated steam-exploded Douglas fir wood chips. They reported the number of ball beads as an
effective parameter to improve enzymatic hydrolysis of α-cellulose. They obtained up to 100% hydrolysis
of lignocellulosic substrate with a relatively low enzyme loading (10 filter paper units/g of cellulose)
when the materials were pretreated with the mills. Jim and Chen [ 66 ] studied superfine grinding, i.e. in
the order of 60 μm, of steam-exploded rice straw. Rice straw was cut to 5–8 cm and steam exploded at
180, 195, 210 and 220 °C for 4–5 min separately by saturated steam. The steam-treated material was
then pulverized using a vegetation disintegrator, and was put into superfine grinding using a fluidized-
bed opposed jet mill. The enzymatic hydrolysis of the superfine ground straw gained the highest
hydrolytic rate and yielded a very high reducing sugar.

It was shown that smaller particles were better digested in biogas production, but size reduction would
have been more efficient if combined with other pretreatments. A study for improvement of biogas
production from rice straw showed that a combination of grinding, heating, and ammonia treatment
(2%) resulted in the highest biogas yield [ 59 ]. In another study, grinding of municipal solid waste from
2.2 to 1.1 mm had no effect on mesophilic digestion, but improved thermophilic digestion by 14% [ 6 ].
Similar work on waste-activated sludge showed a substantial improvement (16–110%) in volatile solid
destruction as the effect of mechanical shear [ 60 ]. However, this effect was sludge-dependent.

Ball milling involves significant energy costs. It was suggested to use a continuous stirred tank reactor
(CSTR) located between a ball mill and a hollow-fiber cartridge, in order to decrease the energy costs
[ 67 ]. Another disadvantage of milling is its inability to remove the lignin which restricts the access of
the enzymes to cellulose and inhibit cellulases [ 30 , 68 ].
4.1.2. Irradiation

Irradiation by e.g. gamma rays, electron beam and microwaves can improve enzymatic hydrolysis of
lignocelluloses. The combination of the radiation and other methods such as acid treatment can further
accelerate enzymatic hydrolysis [ 69 , 70 ]. Irradiation has enhanced enzymatic degradation of cellulose
into glucose. However, pre-irradiation is more effective in air than in acid solution [ 70 ]. Kumakura and
Kaetsu [ 71 ] studied the effect of irradiation for pretreatment of bagasse prior to its enzymatic
hydrolysis. The pretreated bagasse resulted in double yield of glucose by the hydrolysis compared to the
untreated one. The cellulose component of the lignocellulose materials can be degraded by irradiation
to fragile fibers and low molecular weight oligosaccharides and even cellobiose [ 71 ]. It could be due to
preferential dissociation of the glucoside bonds of the cellulose molecular chains by irradiation in the
presence of lignin. A very high irradiation, above 100 MR, can lead to the decomposition of
oligosaccharides and the glucose ring structure [ 71 ]. The enzymatic hydrolysis of filter paper with no
lignin was not improved by irradiation pretreatment. Furthermore, enzymatic hydrolysis of newspapers
with small amounts of lignin was slightly improved by irradiation. Therefore, the effect of radiation
should be correlated with the presence of lignin as well as the structure such as crystallinity and density
[ 71 – 74 ]. However, the irradiation methods are expensive and have difficulties in industrial application.

Ultrasound is a means used for pretreatment in biogas production. It can be used for disintegration of
waste-activated sludge and aquaculture effluents [ 75 – 78 ]. In this method, the sludge flocculi are
disintegrated and the bacterial cells’ walls are disrupted [ 78 ]. Several factors such as ultrasonic density
and intensity, sludge pH and sludge concentration have impact on the disintegration [ 79 ]. In addition to
sonication, other methods such as cavitation, repeated freezing and defreezing, and heating at low
temperatures of e.g. 60–170°C for 5–30 min or high temperatures of 180–200 °C for 10 s, can improve
cell disruption and lysing [ 80 , 81 ]. The other physical methods such as γ-irradiation [ 82 ], microwaves [
83 – 85 ] and electrical pulses [ 86 ] have also been used to improve formation of biogas from waste
materials.

4.2. Physico-chemical pretreatment

Pretreatments that combine both chemical and physical processes are referred to as physico-chemical
processes [ 87 ]. We review the most important processes of this group in this section.

4.2.1. Steam explosion (autohydrolysis)

Among the physico-chemical processes, steaming with or without explosion (autohydrolysis) has
received substantial attention in pretreatment for both ethanol and biogas production. The
pretreatment removes most of the hemicellulose, thus improving the enzymatic digestion. In steam
explosion, the pressure is suddenly reduced and makes the materials undergo an explosive
decompression. High pressure and consequently high temperature, typically between 160 and 260 °C,
for a few seconds (e.g. 30 s) to several minutes (e.g. 20 min), were used in steam explosion [ 49 , 88 – 99
]. The steam explosion process is well documented and was tested in lab-and pilot processes by several
research groups and companies. Its energy cost is relatively moderate, and it satisfies all the
requirements of the pretreatment process.

The process of steam explosion was demonstrated on a commercial scale at the Masonite plants [ 24 ].
Increase in temperature up to a certain level can effectively release hemicellulosic sugars. However, the
sugars loss steadily increases by further increasing the temperature, resulting in a decrease in total
sugar recovery [ 100 ]. Ruiz et al. [ 100 ] studied steam explosion for pretreatment of sunflower stalks
before enzymatic hydrolysis at a temperature in the range of 180–230 °C. The highest glucose yield was
obtained in steam-pretreated sunflower stalks at 220 °C, while the highest hemicellulose recovery was
obtained at 210 °C pre-treatment temperature. Using a steam explosion process for pretreatment of
poplar ( Populus nigra ) biomass, at 210 °C and 4 min, resulted in cellulose recovery above 95%,
enzymatic hydrolysis yield of about 60%, and 41% xylose recovery in the liquid fraction. Furthermore,
the large particles can be used for poplar biomass, since no significant effect of particle size on
enzymatic hydrolysis was observed [ 101 ].

Ballesteros et al. [ 92 ] applied steam explosion for production of ethanol from several lignocellulosic
materials with

Kluyveromyces marxianus . The poplar and eucalyptus chips were treated at 210°C for 4 min; wheat
straw at 190 °C for 8 min;

Brassica carinata residue at 210 °C at 8 min; and sweet sorghum bagasse at 210 °C for 2 min. Steam
explosion extensively solubilized the hemicellulosic sugars and decreased 75–90% of xylose content,
depending on the substrate. It is possible to combine steaming and mechanical treatment to effectively
disrupt the cellulosic structure. Several combination technologies have been developed [ 24 , 102 ,

103 ].

Steam explosion and thermal pretreatments are widely investigated for improving biogas production
from different dedicated materials such as forest residuals [ 104 ] and wastes of e.g. activated sludge
[ 105 – 108 ], cattle manure [ 109 ] or municipal solid wastes [ 110 ]. However, there are several
investigations on combining “thermal” pretreatment with addition of bases such as NaOH, which usually
give a better result than individual thermal or chemical pretreatment (e.g. [ 111– 113 ]).

Special care should be taken in selecting the steam explosion conditions in order to avoid excessive
degradation of the physical and chemical properties of the cellulose. In very harsh conditions, lower
enzymatic digestibility of lignocelluloses may also be observed after steam explosion. For instance,
generation of condensation substances between the polymers in steam explosion of wheat straw may
lead to a more recalcitrant residue [ 114 ].

4.2.2. Steam explosion with addition of SO

Steam pretreatment can be performed with addition of sulfur dioxide (SO ), while the aim of adding this
chemical is to improve recovering both cellulose and hemicellulose fractions. The treatment can be
carried out by 1–4% SO (w/w substrate) at elevated temperatures, e.g. 160–230 °C, for a period of e.g.
10 min [ 53 ]. Eklund et al. [ 53 ] studied steam pretreatment of willow with the addition of SO or H SO in
order to recover both cellulose and hemicellulose. The maximum glucose yield, 95%, was obtained when
the willow was treated with 1% SO at 200 °C. However, the yield of xylose recovery by SO was not as
high as pretreatment with dilute sulfuric acid.

4.2.3. Ammonia fiber explosion (AFEX)

AFEX is one of the alkaline physico-chemical pretreatment processes. Here the biomass is exposed to
liquid ammonia at relatively high temperature (e.g. 90–100 °C) for a period of e.g. 30 min, followed by
immediate reduction of pressure. The effective parameters in the AFEX process are ammonia loading,
temperature, water loading, blowdown pressure, time, and number of treatments [ 46 ]. The AFEX
process produces only a pretreated solid material, while some other pretreatments such as steam
explosion produce a slurry that can be separated in a solid and a liquid fractions [ 115 ].

The AFEX process can either modify or effectively reduce the lignin fraction of the lignocellulosic
materials, while the hemicellulose and cellulose fractions may remain intact. At optimum conditions,
AFEX can significantly improve the enzymatic hydrolysis. The optimum conditions for AFEX depend on
the lignocellulosic materials. For example, the optimum conditions in pretreatment of switchgrass were
reported to be about 100°C, ammonia loading of 1:1 kg of ammonia per kg of dry matter, and 5 min
retention time [ 43 ]. One of the major advantages of AFEX pretreatment is no formation of some types
of inhibitory by-products, which are produced during the other pretreatment methods, such as furans in
dilute-acid and steam explosion pretreatment. However, part of phenolic fragments of lignin and other
cell wall extractives may remain on the cellulosic surface. Therefore, washing with water might be
necessary to remove part of these inhibitory components, although increasing the amount of
wastewater from the process [ 116 ]. However, there are some disadvantages in using the AFEX process
compared to some other processes. AFEX is more effective on the biomass that contains less lignin, and
the AFEX pretreatment does not significantly solubilize hemicellulose compared to other pretreatment
processes such as dilute-acid pretreatment. Furthermore, ammonia must be recycled after the
pretreatment to reduce the cost and protect the environment [ 23 , 28 ,

117 ].

4.2.4. CO explosion

Supercritical carbon dioxide has been considered as an extraction solvent for non-extractive purposes,
due to several advantages such as availability at relatively low cost, non-toxicity, non-flammability, easy
recovery after extraction, and environmental acceptability [ 118 ]. Supercritical carbon dioxide displays
gas-like mass transfer properties, besides a liquid-like solvating power [ 119 ]. It was shown that in the
presence of water, supercritical CO can efficiently improve the enzymatic digestibility of aspen
(hardwood) and southern yellow pine (softwood) [ 120 ]. The delignification with carbon dioxide at high
pressures can be improved by co-solvents such as ethanol–water or acetic acid–water, and can
efficiently increase the lignin removal [ 121 ]. Carbon dioxide molecules should be comparable in size to
those of water and ammonia, and should be able to penetrate small pores accessible to water and
ammonia molecules.

Simultaneous pretreatment by CO explosion and enzymatic hydrolysis in one step has also been of
interest [ 118 , 122 ]. Park et al. [ 122 ] obtained 100% glucose yield, while applying supercritical CO and
enzymatic hydrolysis of cellulose simultaneously. The cellulase was sustained at pressures of up to 160
bar for 90 min at 50 °C under supercritical carbon dioxide. They found that kinetic constants of
hydrolysis under supercritical conditions were increased, compared to those under atmospheric
conditions. Zheng and Tsao [ 118 ] showed cellulase enzyme to be stable in supercritical CO at a
temperature of 35 °C. Only a slight decay indicates a loss of activity of about 10% after 5 days.

Explosion pretreatments of the cellulosic materials by supercritical carbon dioxide were studied by
Zheng et al. [ 119 ]. Upon an explosive release of the carbon dioxide pressure, the disruption of the
cellulosic structure should increase the accessible surface area of the substrate for enzymatic hydrolysis.
Temperature is an important factor in the cellulosic hydrolysis. The experiments can be carried out at
either supercritical or subcritical temperature (respectively above and below 31.1 °C). Experimental
results indicated that subcritical carbon dioxide is less effective than supercritical [ 119 ]. A reason for
such retardation in subcritical carbon dioxide is likely to be low diffusion in liquid carbon dioxide. In
comparison with supercritical temperatures, carbon dioxide molecules at subcritical conditions find it
relatively hard to penetrate the pores in the cellulosic structures, and then disrupt them when the
carbon dioxide pressure is released suddenly. The higher pressure of carbon dioxide resulted in the
higher glucose yield, which indicates that higher pressure is desirable for faster penetration of the
carbon dioxide molecules into the cellulosic pores [ 119 ]. Apart from these advantages, the supercritical
CO process might be too expensive for industrial application.

4.2.5. Liquid hot-water pretreatment

Cooking of lignocellulosic materials in liquid hot water (LHW) is one of the hydrothermal pretreatment
methods applied for pretreatment of lignocellulosic materials since several decades ago in e.g. pulp
industries. Water under high pressure can penetrate into the biomass, hydrate cellulose, and remove
hemicellulose and part of lignin. The major advantages are no addition of chemicals and no requirement
of corrosion-resistant materials for hydrolysis reactors in this process. The feedstock size reduction is a
highly energy-demanding operation for the huge bulk of materials on a commercial scale; there could be
no need for size reduction in LHW pretreatment. In addition, the process has a much lower need of
chemicals for neutralization of the produced hydrolyzate, and produces lower amounts of neutralization
residues compared to many processes such as dilute-acid pretreatment. Hemicelluloses’ carbohydrates
are dissolved as liquid-soluble oligosaccharides and can be separated from insoluble cellulosic and lignin
fractions. LHW can enlarge the accessible and susceptible surface area of the cellulose and make it more
accessible to hydrolytic enzymes [ 62 ].

Pretreatments with steam and LHW are both hydrothermal pretreatments. Higher pentosan recovery
and lower formation of inhibitory components are the main advantages of LHW pretreatment compared
to steam explosion. For instance, treating of de-starched corn fiber with hot water at 160 °C for 20 min
dissolved 75% of the xylan [ 123 ]. At higher temperatures, e.g. 220 °C, LHW can dissolve hemicelluloses
completely and remove lignin partially within 2 min with no chemicals used [ 124 ].

Xylan removal via percolation reactor, or by base addition (adjusting the pH) during the process, has
been suggested to reduce the formation of inhibitors such as furfural and degradation of xylose [ 98 ].
The pH, processing temperature, and time should be controlled in order to optimize the enzymatic
digestibility by LHW pretreatment [ 23 , 115 , 125 , 126 ]. An optimized condition for LHW pretreatment
of corn stover was reported to be 190 °C for 15 min, in which 90% of the cellulose conversion was
observed by subsequent enzymatic hydrolysis [ 125 ]. LHW pretreatment at 160 °C and a pH above 4.0
can dissolve 50% of the fibers from corn fibers in 20 min [ 126 ]. The results showed that the
pretreatment enabled the subsequent complete enzymatic hydrolysis of the remaining polysaccharides,
mainly cellulose, to the corresponding monomers. The LHW pretreatment resulted in 80% soluble
oligosaccharides and 20% monosaccharides with less than 1% of the carbohydrates lost to degradation
products.

Laser et al. [ 98 ] compared the performance of LHW and steam pretreatments of sugarcane bagasse,
which was subsequently used in ethanol production by SSF. They performed the treatments in a 25-l
reactor at 170–230 °C for 1–46 min with 1% to 8% solids concentration. The results showed that both
methods can significantly improve the hydrolysis; however, the LHW resulted in much better xylan
recovery compared to steam pretreatment. Under the optimum conditions, the results of LHW
pretreatment were comparable with dilute-acid pretreatment processes, besides having no requirement
for acid or production of neutralization wastes. They also showed that the process favored high
temperatures (above 220 °C), short residence times (less than 2 min) and low solid concentration (less
than 5%).

The hot water processing removes mainly hemicellulose. A two-stage process which combines the hot
water for hemicellulose removal and a treatment for delignification (e.g. ammonia treatment) was also
suggested for further improvement of enzymatic hydrolysis [ 27 , 127 ].

4.2.6. Microwave-chemical pretreatment

The microwave/chemical pretreatment resulted in a more effective pretreatment than the conventional
heating chemical pretreatment by accelerating reactions during the pretreatment process [ 128 , 129 ].
Zhu et al. [ 129 ] examined three microwave/chemical processes for pretreatment of rice straw –
microwave/alkali, microwave/acid/alkali and microwave/acid/alkali/H O – for its enzymatic hydrolysis
and for xylose recovery from the pretreatment liquid. They found that xylose could not be recovered
during the microwave/alkali pretreatment process, but could be recovered as crystalline xylose during
the microwave/acid/alkali and microwave/acid/alkali/H O pretreatment. The enzymatic hydrolysis of
pretreated rice straw showed that the pretreatment by microwave/acid/alkali/H O had the highest
hydrolysis rate and glucose content in the hydrolyzate.

4.3. Chemical pretreatment

4.3.1. Alkaline hydrolysis

Alkali pretreatment refers to the application of alkaline solutions such as NaOH, Ca(OH) (lime) or
ammonia to remove lignin and a part of the hemicellulose, and efficiently increase the accessibility of
enzyme to the cellulose. The alkali pretreatment can result in a sharp increase in saccharification, with
manifold yields (e.g. [ 130 ]). Pretreatment can be performed at low temperatures but with a relatively
long time and high concentration of the base. For instance, when soybean straw was soaked in ammonia
liquor (10%) for 24 h at room temperature, the hemicellulose and lignin decreased by 41.45% and
30.16% respectively [ 131 ]. However, alkaline pretreatment was shown to be more effective on
agricultural residues than on wood materials.

Vaccarino et al. [ 132 ] studied the effects of SO , Na CO , and NaOH pretreatments on the enzymatic
digestibility of grape marc, and the greatest degrading effects were obtained by pretreatment with 1%
NaOH solution at 120°C. Silverstein et al. [ 133 ] studied the effectiveness of sulfuric acid, sodium
hydroxide, hydrogen peroxide, and ozone pretreatments for enzymatic conversion of cotton stalks. They
found that sodium hydroxide pretreatment resulted in the highest level of delignification (65% with 2%
NaOH in 90 min at 121°C) and cellulose conversion (60.8%). Zhao et al. [ 134 ] reported that
pretreatment with NaOH could obtain a higher enzymatic conversion ratio of cellulose compared with H
SO pretreatment. Compared with acid or oxidative reagents, alkali treatment appears to be the most
effective method in breaking the ester bonds between lignin, hemicellulose and cellulose, and avoiding
fragmentation of the hemicellulose polymers [ 135 ].
The alkaline pretreatment was also used as a pretreatment method in biogas production. A
pretreatment with bases such as Ca(OH) could be a solution, when high loads of e.g. lipids and phenolic
compounds are subjected to the digestion. Olive mill effluent is an example of seasonal waste with low
pH (about 4.3), and high lipid (ca 13 g/l) and phenolic compounds concentration (ca 8 g/l). Addition of
lime and bentonite greatly improves the digestion of olive mill effluents with more than 91% removal of
COD [ 136 ]. In another work, a pretreatment with ultrasound and NaHCO was reported to improve the
digestibility of newsprint wastes [ 79 ]. A treatment of waste-activated sludge with 0.3 g NaOH/g volatile
solids (VS) at 130°C for 5 min resulted in 40–50% solubilization of VS and more than 200% improvement
in methane production compared to the control experiment [ 137 , 138 ]. Furthermore, treatment of the
sludge with dilute NaOH (e.g. 1.6 g/l) at room or low temperature (25–55 °C) is able to improve the VS
removal by 40–90% [ 139 , 140 ]. A similar treatment by 5 g/kg NaOH on municipal solid waste has also
improved the formation of biogas by 35% [ 6 ].

4.3.2. Alkaline peroxide

Alkaline peroxide is an effective method for pretreatment of biomass. In this method, the lignocelluloses
are soaked in pH-adjusted water (e.g. to pH 11–12 using NaOH) containing H O at room temperatures
for a period of time (e.g. 6–24 h). The process can improve the enzymatic hydrolysis by delignification.
Saha and Cotta [ 141 ] showed that by using such alkaline peroxide pretreatment, wheat straw can be
converted to fermentable sugars with an excellent yield (97%) by enzymatic saccharification. In another
report [ 142 ], they showed that diluted alkaline peroxide treatment (7.5% H O , v/v; pH 11.5; 35 °C; 24
h) is an efficient method for pretreatment of rice hulls, resulting in almost complete conversion (96%) of
rice hulls to sugars after enzymatic hydrolysis. No measurable furfural and hydroxymethylfurfural (HMF)
were detected in the process, which makes it more fermentable/digestable compared to e.g. in dilute-
acid pretreatment. Mishima et al. [ 143 ] examined twenty chemical pretreatments in order to improve
the efficiency of enzymatic hydrolysis of water hyacinth and water lettuce. It was shown that the
alkaline/oxidative pretreatment, in which NaOH and H O were used, was the most effective method for
improving the enzymatic hydrolysis.

Sun et al. [ 114 ] studied a two-stage process based on steam explosion pretreatment followed by
alkaline peroxide post-treatment. Wheat straw was first steamed at 200–220 °C and 15–22 bar. The
washed fiber was then delignified by 2% H O at 50 °C for 5 h under pH 11.5. The steam explosion
pretreatment resulted in a significant loss in hemicelluloses, and about 11–12% lignin removal, while the
alkaline peroxide post-treatment resulted in 81–88% removal of the original lignin, which altogether
removed 92–99% of the original lignin from wheat straw. They also showed that the basic structure
remaining is of lignin nature only, which is important for those who plan biomass strategies especially in
terms of chemical uses of lignin. Curreli et al. [ 144 ] suggested two steps, mild alkaline/oxidative
pretreatment at low temperature (25–40 °C) and low concentration of chemicals. Alkaline pretreatment
(1% NaOH for 24 h) in the first step solubilizes hemicellulose, and a second alkaline/oxidative step (1%
NaOH and 0.3% H O for 24 h) in order to solubilize and oxidize lignin. The pretreatment is also useful in
removing waxes, silica, and the waterproof cutins that coat plant tissue.

4.3.3. Organosolv process

Organosolv can be used to provide treated cellulose suitable for enzymatic hydrolysis, using an organic
or aqueous organic solvent to remove or decompose the network of lignin and possibly a part of the
hemicellulose [ 144 –
147 ]. In this process, lignocellulose is mixed with organic liquid and water and heated to dissolve the
lignin and part of the hemicellulose, leaving reactive cellulose in the solid phase. In addition, a catalyst
may be added either to reduce the operating temperature or to enhance the delignification process
[ 24 ]. Lignin in the biomass can be extracted from the solvent for e.g. generation of electricity, process
heat, lignin-based adhesives and other products, due to its high purity and low molecular weight [ 148 ].

In organosolv pretreatment of lignocellulosic materials, a large number of organic or aqueous-organic

solvents at temperatures of 150–200 °C can be used with or without addition of catalysts such as oxalic,
salicylic, and acetylsalicylic acid. Furthermore, the solvent may accompany acetic acid released from
acetyl groups developed by hydrolysis of hemicelluloses. A variety of organic solvents such as alcohols,
esters, ketones, glycols, organic acids, phenols, and ethers have been used. However, the price of
solvent and simplicity in recovery of solvent should also be considered. The applied solvents should be
separated by e.g. evaporation and condensation, and recycled to reduce the operational costs of the
process. Removal of solvents from the pretreated cellulose is usually necessary because the solvents
might be inhibitors to the enzymatic hydrolysis and fermentation or digestion of hydrolyzate [ 28 ].
Araque et al. [ 149 ] studied the organosolv acetone–water for pretreatment. They found the highest
ethanol yield to be 99.5% after pretreatment at 195 °C, 5 min, pH 2.0, and 1:1 ratio of acetone-water.
For economic reasons, the use of low-molecular-weight alcohols such as ethanol and methanol has been
favored over alcohols with higher boiling points, e.g. ethylene glycol, tetrahydrofurfuryl alcohol [ 24 ,
28 , 41 ]. Ethanol is a common solvent, although it inhibits hydrolytic enzymes [ 23 ]. It should therefore
be removed from the solid fraction before enzymatic hydrolysis. The main advantage of the use of
solvents over other chemical pretreatments is that relatively pure, low-molecular-weight lignin is
recovered as a by-product [ 28 , 103 ].

Organosolv can be used together with acid hydrolysis to separate hemicellulose and lignin in a two-stage
fractionation. Papatheofanous et al. [ 150 ] suggested such a system for pretreatment of biomass.
Lignocellulosic raw material can first be treated with dilute aqueous acid (0.5–2.5 N sulfuric acid) at
about 100°C for 10–60 min in order to selectively hydrolyze the hemicellulosic fraction. The aim of the
second stage of the process is delignification of the pretreated lignocellulose by acidic conditions (2 N
sulfuric acid) at about 81°C for 90 min. In this stage, ethanol is added (62.5–87.5%) to the system to
provide the medium for dissolving and recovery of lignin generated under the acidic conditions.
Negligible cellulose loss (less than 2% w/w of original cellulose) and high lignin removal (more than 70%
w/w of original lignin) makes the two-stage low-temperature acid-catalyzed process interesting for
laboratory pretreatment of lignocellulose before enzymatic hydrolysis.

4.3.4. Wet oxidation

Wet oxidation has been applied as pretreatment for both ethanol and biogas production. In this process,
the materials are treated with water and air or oxygen at temperatures above 120°C (e.g. 148–200°C)
for a period of e.g. 30 min [ 151 – 153 ]. The temperature, followed by reaction time and oxygen
pressure, are the most important parameters in wet oxidation [ 154 ]. The process is exothermic, and
therefore it becomes self-supporting with respect to heat while the reaction is initiated [ 154 ]. Wet
oxidation of the hemicellulose fraction is a balance between solubilization and degradation. This process
is an effective method in separating the cellulosic fraction from lignin and hemicellulose [ 24 ,

155 ]. Oxygen participates in the degradation reactions and allows operation at comparatively reduced
temperatures by enhancing generation of organic acids. However, the control of reactor temperature is
critical because of the fast rates of reaction and heat generation [ 153 ]. The main reactions in wet
oxidation pretreatment are the formation of acids from hydrolytic processes, as well as oxidative
reactions. All three fractions of lignocellulosic materials are affected in this process. The hemicelluloses
are extensively cleaved to monomeric sugars; the lignins undergo both cleavage and oxidation; and
cellulose is partly degraded. The cellulose becomes highly susceptible to enzymatic hydrolysis [ 156 ].

Bjerre et al. [ 157 ] combined wet oxidation and alkaline hydrolysis for wheat straw pretreatment. The
process resulted in a relatively highly convertible cellulose (85% conversion yield) and hemicellulose.
However, addition of some alkaline agent such as sodium carbonate had only a small effect on the
concentration of solubilized hemicellulose. Meanwhile, alkaline pretreatment conditions significantly
decreased the degradation of hemicellulose to inhibitors, e.g. furfural [ 158 ]. Martin et al. [ 159 ]
studied wet oxidation as a pretreatment method for enhancing the enzymatic convertibility of
sugarcane bagasse. The pretreatment at 195 °C for 15 min solubilized 93–94% of hemicelluloses and 40–
50% of lignin. The alkaline wet oxidation pretreatment at 185 °C for 5 min solubilized only 30% of
hemicelluloses and 20% of lignin; meanwhile the alkaline condition reduced the formation of furans. The
highest sugar yield in the liquid fraction was obtained at 185 °C, 5 min and acidic pH. The acidic wet
oxidation pretreatment at 195 °C for 15 min resulted in the highest formation of carboxylic acids,
phenols and furans, resulting in loss of a significant part of the polysaccharides due to degradation and
formation of the by-products.

Lissens et al. [ 160 ] used wet oxidation to improve anaerobic biodegradability and methane yields of
several raw biowastes (food waste, yard waste, and digested biowaste treated in a full-scale biogas
plant). Wet oxidation temperature (185–220°C) and oxygen pressure (0–12 bar) for 15 min were used to
evaluate their effect on the methane yield. The wet oxidation process reportedly increased methane
yields by approximately 35–70% from raw and digested lignocellulosic biowastes.

Similar to many other delignification methods, the lignin produced by wet oxidation cannot be used as a
fuel, since a major part of the lignin undergoes both cleavage and oxidation. This phenomenon
considerably reduces the income from this by-product at industrial scale for ethanol production from
lignocellulosic materials [ 161 ].

Wet oxidation can also be performed by oxidation agents such as hydrogen peroxide (H O ). Azzam [ 162
] showed that the pretreatment with hydrogen peroxide greatly enhanced the susceptibility of cane
bagasse to enzymatic hydrolysis. About 50% of the lignin and most hemicellulose were solubilized by
treating the biomass with 2% H O at 30°C within 8 h, giving 95% efficiency of glucose production from
cellulose enzymatic hydrolysis.

4.3.5. Ozonolysis pretreatment

Pretreatment of lignocellulosic materials can be performed by treatment with ozone, referred to as

“ozonolysis” pretreatment. This method can effectively degrade lignin and part of hemicellulose. The
pretreatment is usually carried out at room temperature, and does not lead to inhibitory compounds
[ 163 ]. However, ozonolysis might be expensive since a large amount of ozone is required [ 28 ]. The
main parameters in ozonolysis pretreatment are moisture content of the sample, particle size, and
ozone concentration in the gas flow. Among these parameters, an essential factor is the percentage of
water in the feed, and it has the most significant effect on the solubilization. The optimum water
content was found to be around 30%, corresponding to the saturation point of the fibers. This is an
attractive pretreatment method since it does not leave acidic, basic, or toxic residues in the treated
material [ 164 ].

Ozonolysis pretreatment for biogas production was investigated to improve digestion of several wastes
such as sewage-activated sludge [ 165 – 167 ] and olive mill waste [ 168 ]. The ozone also reduced the
phenolic compounds present in olive mill waste, which are toxic to methanogenic bacteria, and resulted
in improvement of the digestion.

4.3.6. Acid hydrolysis pretreatment

Treatment of lignocellulosic materials with acid at a high temperature can efficiently improve the
enzymatic hydrolysis. Sulfuric acid is the most applied acid, while other acids such as HCl and nitric acid
were also reported [ 3 ]. The acid pretreatment can operate either under a high temperature and low
acid concentration (dilute-acid pretreatment) or under a low temperature and high acid concentration
(concentrated-acid pretreatment). The lower operating temperature in concentrated-acid pretreatment
(e.g. 40 °C) is a clear advantage compared to dilute-acid processes. However, high acid concentration
(e.g. 30–70%) in the concentrated-acid process makes it extremely corrosive and dangerous. Therefore,
this process requires either specialized non-metallic constructions or expensive alloys. The acid
recovery, which is necessary in the concentrated-acid process for economical reasons, is an energy-
demanding process. On the other hand, the neutralization process produces large amounts of gypsum.
The high investment and maintenance costs also reduce the commercial interest in this process as a
commercial option [ 23 , 99 , 169 ].

Dilute-acid hydrolysis is probably the most commonly applied method among the chemical
pretreatment methods. It can be used either as a pretreatment of lignocellulose for enzymatic
hydrolysis, or as the actual method of hydrolyzing to fermentable sugars. Different types of reactors
such as batch, percolation, plug flow, countercurrent, and shrinking-bed reactors, for either
pretreatment or hydrolysis of lignocellulosic materials by the dilute-acid processes, have been applied.
These processes and different aspects of dilute-acid hydrolysis and pretreatment have recently been
reviewed [ 3 , 4 ]. At an elevated temperature (e.g. 140–190 °C) and low concentration of acid (e.g. 0.1–
1% sulfuric acid), the dilute-acid treatment can achieve high reaction rates and significantly improve
cellulose hydrolysis. Almost 100% hemicellulose removal is possible by dilute-acid pretreatment. The
pretreatment is not effective in dissolving lignin, but it can disrupt lignin and increases the cellulose’s
susceptibility to enzymatic hydrolysis [ 23 , 170 ].

Dilute-acid pretreatment can be performed either in short retention time (e.g. 5 min) at high
temperature (e.g. 180 °C) or in a relatively long retention time (e.g. 30–90 min) at lower temperatures
(e.g. 120 °C). Sun and Cheng [ 171 ] pretreated rye straw and Bermuda grass for ethanol production by
enzymatic hydrolysis at 121°C with different sulfuric acid concentrations (0.6, 0.9, 1.2 and 1.5%, w/w)
and residence times (30, 60, and 90 min). Emmel et al. [ 15 ] pretreated

Eucalyptus grandis impregnated with 0.087 and 0.175% (w/w) H SO at 200–210°C for 2–5 min. The best
conditions for hemicellulose recovery were obtained at 210°C for 2 min, while a lower pretreatment
temperature of 200°C was enough to obtain the highest yield of cellulose conversion (90%) by enzymatic
hydrolysis.
The optimum conditions for the highest hemicellulosic sugars recovery do not necessarily mean the
most effective conditions for enzymatic hydrolysis. Cara et al. [ 172 ] reported the maximum hemi-
cellulose recovery (83%) of olive tree biomass to be obtained at 170 °C and 1% sulfuric acid
concentration, but the enzyme accessibility of the corresponding pretreated solid was not very high. The
maximum enzymatic hydrolysis yield (76.5%) was obtained when pretreated at 210 °C with 1.4% acid
concentration. The maximum total sugars, 75% of all sugars present in olive tree biomass, were
obtained when the feedstock was pretreated by dilute acid at 180 °C with 1% sulfuric acid
concentration. This indicates that the highest overall sugars, higher hemicellulose recovery and higher
enzymatic hydrolysis yield can be achieved under respectively different conditions.

Pretreatment with acids such as acetic and nitric were also used to remove lignin from waste newsprints
[ 173 ] and activated sludge [ 174 ] for biogas production. Cellulose-lignin association is considered to be
the major limiting factor on long-term anaerobic digestion of newsprint. Pretreatment of bagasse and
coconut fibers with HCl improved the formation of biogas from these materials by 31% and 74%,
respectively [ 175 ]. Acetic acid cannot dissolve lignin even at a very high concentrations, e.g. as high as
80%, at elevated temperature (in a boiling water bath) for 30 min. In order to effectively dissolve
significant amounts of lignin, nitric acid should be added. A treatment of newsprints with 30% acetic acid
and 2% nitric acid resulted in removing 80% of the lignin and increasing the cellulose/lignin ratio from
1.6 to 9.9. This treatment gave improved digestion of the newsprints. The production of biogas
increased by three times within 60 days incubation, from 97 ml CH /g VS for the untreated newsprints to
364 ml CH /gVS for the treated ones. A portion of the nitric acid might be replaced by another strong
acid like hydrochloric acid; however, a longer reaction time may be required for pretreatments with
lower concentrations of nitric acid [ 173 ].

Dilute-acid hydrolysis can be combined with other chemical treatments. Azzam [ 176 ] studied
pretreated bagasse in a solution of ZnCl and 0.5% hydrochloric acid, heated at 145°C for 10 min, cooled
and precipitated with acetone. The pretreated biomass was highly hydrolysable (yield of 93%) by
cellulase.

The major drawback of some pretreatment methods, particularly at low pH is the formation of different
types of inhibitors such as carboxylic acids, furans and phenolic compounds [ 4 , 19 ]. These chemicals
may not affect the enzymatic hydrolyses, but they usually inhibit the microbial growth and
fermentation, which results in less yield and productivity of ethanol or biogas [ 3 ]. Therefore, the
pretreatments at low pH should be selected properly in order to avoid or at least reduce the formation
of these inhibitors.

4.4. Biological pretreatment

Microorganisms can also be used to treat the lignocelluloses and enhance enzymatic hydrolysis. The
applied microorganisms usually degrade lignin and hemicellulose but very little part of cellulose, since
cellulose is more resistance than the other parts of lignocelluloses to the biological attack. Several fungi,
e.g. brown-, white- and soft-rot fungi, have been used for this purpose. White-rot fungi are among the
most effective microorganisms for biological pretreatment of lignocelluloses [ 28 ].

Taniguchi et al. [ 177 ] evaluated biological pretreatment of rice straw using four white-rot fungi
( Phanerochaete chrysosporium, Trametes versicolor, Ceriporiopsis subvermispora, and
Pleurotus ostreatus ) on the basis of quantitative and structural changes in the components of the
pretreated rice straw as well as susceptibility to enzymatic hydrolysis. Pretreatment with P. ostreatus
resulted in selective degradation of the lignin rather than the holocellulose component, and increased
the susceptibility of rice straw to enzymatic hydrolysis. Some bacteria can also be used for biological
pretreatment of lignocellulosic materials. Kurakake et al. [ 178 ] studied the biological pretreatment of
office paper with two bacterial strains, Sphingomonas paucimobilis and Bacillus circulans, for enzymatic
hydrolysis. Biological pretreatment with the combined strains improved the enzymatic hydrolysis of
office paper from municipal wastes. Under optimum conditions, the sugar recovery was enhanced up to
94% for office paper.

Biological treatments with microorganisms or enzymes are also investigated to improve digestion in
biogas production. The biological pretreatment might be used not only for lignin removal, but also for
biological removal of specific components such as antimicrobial substances. Solid-state fermentation of
orange peels by fungal strains of Sporotrichum, Aspergillus, Fusarium and Penicillum enhanced the
availability of feed constituents and reduced the level of the antimicrobial substances [ 179 ]. In a similar
work, cultivation of white-rot fungi was used to detoxify olive mill wastewater and improve its digestion
[ 180 ].

Low energy requirement, no chemical requirement, and mild environmental conditions are the main
advantages of biological pretreatment. However, the treatment rate is very low in most biological
pretreatment processes [ 28 ].

5. Concluding remarks

Several pretreatment methods have been presented for lignocelluloses and waste materials in order to
improve ethanol or biogas production. All these methods should make the lignocelluloses available to
the enzymatic attack, where crystallinity of cellulose, its accessible surface area and protection by lignin
and hemicellulose are the main factors in order to obtain an efficient hydrolysis. In addition, the efficient
utilization of the hemicelluloses is an opportunity to reduce the cost of ethanol or biogas production.
Diverse advantages have been reported for most of the pretreatment methods, which make them
interesting for industrial applications. While methods such as dilute acid, hot water, AFEX, ammonia
recycle percolation, and lime are capital-intensive [ 117 ], some other methods such as biological
pretreatment are extremely slow [ 28 ]. Furthermore, some technological factors such as energy
balance, solvent recycling and corrosion, as well as environmental factors such as wastewater
treatment, should be carefully considered for the selected method.

Acknowledgments

This work was financially supported by the Swedish Excellent Center “Waste Refinery” and Sjuhärad
Association of Local Authorities in Sweden.

References

solvent with a boiling point higher than the cooking temperature. Then the impregnated material is
immersed in a liquid which is immiscible with the solvent of the solution.

U.S. Pat. No. 4,520,105 (Sinner) describes a process involving a chemical pretreatment with a mixture of
water and lower alcohols or acetone, after which the residue is separated and then treated with a
similar solvent mixture at elevated temperature. However, alcohol or acetone mixtures with water
cannot be separated into two phases. Also, separation of the lignin from dissolved sugars would require
further processing through extensive washing.

U.S. Pat. No. 4,594,130 (Chang) describes a cooking process, in the absence of oxygen, at elevated
temperatures with a neutral or acidic mixture of alcohol and water containing a magnesium, calcium or
barium salt as a catalyst. The catalyst is for the purpose of aiding retention of the hemicellulose in the
cellulosic cake.

European Patent Application 86305606.5 (Biodyne) describes a process for digesting lignocellulosic
material with an ester, an organic lignin solvent and water. The lignin solvent is either an organic acid or
alcohol or mixtures thereof, and it is miscible in both the ester and the water. Cooling of the liquor
apparently results in some phase separation, but a centrifuge is also required.

There has not heretofore been described an efficient process involving the use of a single phase cooking
liquor which can be readily separated into two liquid phases when desired.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a process for separating wood and herbaceous biomass into its
three major components for further processing.

It is an object of the invention to provide a convenient and efficient process for separating lignocellulosic
material into lignin, cellulose, hemicellulose and sugars.

It is another object of the invention to provide a process for separating lignocellulosic material into its
three major components using either a batch process or a continuous process.

It is yet another object of the invention to provide a very pure lignin stream in a process for fractionating
lignocellulosic material.

It is still another object of this invention to provide a cellulose stream free of re-precipitated lignin in a
process for fractionating lignocellulosic material.

Additional objects, advantages, and novel features of the invention shall be set forth in part in the
description that follows and in part will become apparent to those skilled in the art upon examination of
the following or may be learned by the practice of the invention. The objects and the advantages of the
invention may be realized and attained by means of the instrumentalities and in combinations
particularly pointed out in the appended claims.

To achieve the foregoing and other objects and in accordance with the purpose of the present invention,
as embodied and broadly described herein, the improved methods may comprise separating
lignocellulosic material into lignin, (b) cellulose, and (c) hemicellulose and sugars. In one embodiment
the method comprises:

(a) digesting the lignocellulosic material in a single-phase mixture of alcohol, water and a water-
immiscible organic solvent selected from the group consisting of ketones;

(b) adjusting the amount of water in said mixture to cause phase separation;
(c) separating said mixture into first, second and third phases; wherein said first phase comprises lignin
and said organic solvent, said second phase comprises solid cellulosic cake, and said third phase is an
aqueous alcoholic mixture and comprises hemicellulose and dissolved sugars.

The methods and techniques provided by this invention enable wood or herbaceous biomass to be
fractionated very efficiently in a single phase, after which the pulping mixture can be separated into
separate phases wherein the lignin is present in a homogeneous organic phase, the hemicellulose and
sugars are present in the aqueous phase, and cellulose is present as a solid cellulosic cake. The organic
phase can be separated, and the lignin can be isolated by evaporation of the organic solvent. The
isolated lignin is substantially free of sugars.

The separate components of the wood and herbaceous biomass can be used for further desired
processing. For example, the cellulose can be used for making paper and paperboard products or
ethanol. It can also be used for making cellulose derivatives such as cellulose esters. The lignin can be
used as a feedstock for phenolics, enhanced oil recovery surfactants, or fuel additives. The hemicellulose
can be used making ethanol or other chemicals.

Other advantages of the methods of this invention will be apparent from the following detailed
description.

DETAILED DESCRIPTION OF THE INVENTION

The improved methods of this invention involve placing the lignocellulosic material (e.g., wood or
herbaceous biomass) in a suitable reactor, after which a mixture of solvents and water are added. It is
necessary to obtain a single phase mixture.

A water-insoluble or water-immiscible organic solvent is used which is a ketone. A water-soluble or

miscible alcohol and water are also used. Preferably the ketone is an aliphatic ketone having at least 4
carbon atoms (and may have as many as 10 carbon atoms). The alcohol preferably has less than about 4
carbon atoms to assure that it will be water-miscible.

Useful aliphatic ketones include, for example, methyl ethyl ketone, methyl isopropyl ketone, methyl
propyl ketone, methyl butyl ketone, methyl isobutyl ketone, methyl isoamylketone, diethyl ketone, ethyl
isopropyl ketone, ethyl propyl ketone, and ethyl isobutyl ketone. Useful alcohols include methanol,
ethanol, propanol, isopropanol and butanol.

Typically the ketone is present in the solvent system in an amount of about 7 to 65% by weight, and
water is present in an amount of about 10 to 65% by weight. The alcohol is typically present in an
amount of about 25 to 35% by weight. The weight ratio of ketone to water is preferably in the range of
about 1:9 to 6.5:1, so long as a single phase of digesting liquid is obtained.

Typically, the weight ratio of liquor to wood or biomass is at least about 4:1 and could be much greater if
desired, e.g., 8:1 or 10:1.

The digestion is preferably carried out at an elevated temperature. Typically the digestion mixture is
heated in the reactor to a temperature in the range of about 100° to 220° C. Some types of biomass can
be digested more quickly than other types. If desired, a concept called severity may be used to
determine the length of time required to obtain complete digestion of a particular biomass. The term
severity involves use of an empirically derived equation relating time at temperature and the
temperature above a base temperature at which reaction does not occur. The concept and the equation
are explained in Organosolv Pretreatment for Enzymatic Hydrolysis of Poplars. 2. Catalyst Effects and the
Combined Severity Parameter, H. L. Chum, D. K. Johnson, and S. K. Black, I & EC Research, 1990, 29, 156-
162, incorporated herein by reference.

After the digestion has been completed, the single phase can be easily converted into two liquid phases
upon the addition of either water or water-immiscible solvent. The two phases have very little cross-
contamination of components from one phase into the other. The lignin stream is very pure and is easily
isolated by evaporation of the organic solvent which is water-immiscible. The cellulosic stream obtained
is free of re-precipitated lignin because the lignin and other dissolved materials remain in solution at all
temperatures of the reaction. The lignin and hemicellulose are dissolved away from the wood chips
leaving an insoluble cellulosic cake.

An acid catalyst is added to reduce the reaction temperature from about 200° C. for uncatalyzed cooks
to 140° C. for a catalyzed cook. It also reduces the amount of time required. The catalysts used are
mineral acids such as sulfuric or phosphoric acid. Nitric acid may also be used but it is not as effective.
The amount of catalyst used varies with the feedstock but is generally in the range of 0.025M to 0.2M
(0.2 to 2 wt % of the liquor used).

The processes of this invention are useful for fractionating all types of lignocellulosic material into
separate components. For example, the processes may be used in connection with wood and
herbaceous derived materials such as sugar cane bagasse, switch grass, and legumes.

EXAMPLE 1

Poplar wood chips (13.7 g oven-dried equivalent) were charged into a 200 ml batch reactor. A single
phase pulping liquor composed of 24% water, 44% methyl isobutyl ketone (MIBK) and 32% ethanol with
a 0.05 M H2 SO4 catalyst was added in a ratio of 10 parts liquor to 1 part wood. The reactor was placed
in a preheated heating block. The reactor was held at 140° C. for 56 minutes after a 34 minute heat-up
time. Severity of the reaction was 4.3. The resulting pulp was fiberized in a Waring blender and washed
with fresh neutral liquor. The oven-dried equivalent yield of pulp was 64%. Kappa number for this pulp
was measured at 72.

Water was added to the liquor in a ratio of 1.3 parts water to 1 part liquor to cause phase separation of
the insoluble MIBK component. Lignin was isolated from the MIBK phase by evaporation with a yield of
18%. Klason lignin analysis of this lignin gave a 88% purity.

Dissolved sugars composed mostly of hemicellulose were contained in the combined alcohol-aqueous
fraction in a 18% yield based on the wood charged.

EXAMPLE 2

Under the same conditions as above, 14.4 g of oven-dried equivalent weight poplar was charged into a
batch reactor. A H2 SO4 catalyst at a 0.1M concentration was added. The yield of pulp was 53% with a
Kappa number of 46. The lignin yield was 23% with a purity of 92%. The yield of the hemicellulose
fraction was 24%.

EXAMPLE 3
Aspen chips (193 g, oven-dried equivalent) were charged into a 1.7 liter percolation reactor. The reactor
was filled with a MIBK/ethanol/water mixture containing 16% MIBK, 34% ethanol and 50% water
containing 0.025M H2 SO4. The reactor was heated to 140° C. over 34 minutes without flow of solvent.
When the pulping temperature was reached, pulping solvent of the same composition was pumped
through the chip bed at a flow rate of 28 ml/min. for 56 minutes. The chips were then washed in the
reactor with neutral solvent at the same flow rate for 60 minutes without heating. Total severity for the
reaction was 4.27. The reactor was drained, the chips fiberized and the lignin separated as described
above. The pulp yield was 52% with a Kappa of 28. Lignin was isolated in a 17% yield. Hemicellulose yield
was 31%.

EXAMPLE 4

Poplar chips from undebarked logs (189 g, oven-dried equivalent) were pulped under the same
conditions as example 3 except for a 0.1 M H 2 SO4 catalyst concentration. Severity for this run was
4.27. The pulp yield was 45% with a Kappa of 42. Lignin was isolated in a 22% yield at a purity of 93%.
Hemicellulose yield was 33%.

EXAMPLE 5

Depithed sugar cane bagasse (72 g, oven-dried equivalent) was pulped under the same conditions as
example 4. The solvent mixture used was the same as that of example 1. Severity was 4.23. The yield of
pulp was 49% with a Kappa of 8. The yield of lignin was 32%. Yield of hemicellulose was 19%.

The foregoing is considered as illustrative only of the principles of the invention. Further, since
numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit
the invention to the exact construction and operation shown and described. Accordingly, all suitable
modifications and equivalents may be resorted to falling within the scope of the invention as defined by
the claims which follow. The embodiments of the invention in which an exclusive property or privilege is
claimed are defined as follows:

PATENT CITATIONS

Cited Patent Filing date Publication date Applicant Title

US1594389 * Dec 26, 1924 Aug 3, 1926 Applic Ind Des Brevets Peufail Process for retting or steeping flax
and other vegetable fibers

US1888025 * Feb 15, 1928 Nov 15, 1932 Hercules Powder Co Ltd Method of extracting resinous material
from plant tissue

US2024689 * Jan 22, 1935 Dec 17, 1935 Celanese Corp Production of cellulose from ligno-cellulosic
materials

US2037001 * Jul 18, 1934 Apr 14, 1936 Northwest Paper Company Alcoholic treatment of ligneous
cellulosic material

US2042705 * Jan 4, 1935 Jun 2, 1936 Dreyfus Henry Manufacture of cellulose from ligno-cellulosic
products

US3585104 * Jul 29, 1968 Jun 15, 1971 Kleinert Theodor N Organosolv pulping and recovery process
US3932207 * Mar 5, 1973 Jan 13, 1976 Societe De Promotion Et D'exploitation Industrielles De Procedes
De Brevets S.A.

Process for obtaining cellulose from ligno-cellulosic raw materials

US3951734 * Oct 11, 1974 Apr 20, 1976 Weyerhaeuser Company Ammonia-ketone pulping process

US4520105 * Aug 10, 1983 May 28, 1985 Bau- Und Forschungsgesellschaft Thermoform Ag

Process for production of sugars and optionally cellulose and lignin from lignocellulosic raw materials

US4594130 * Jul 7, 1983 Jun 10, 1986 Chang Pei Ching Pulping of lignocellulose with aqueous alcohol
and alkaline earth metal salt catalyst

EP0211558A2 * Jul 22, 1986 Feb 25, 1987 Biodyne Chemicals, Inc. Process for digesting lignocellulosic
material

* Cited by examiner

REFERENCED BY

Citing Patent Filing date Publication date Applicant Title

US7396434 * Jun 3, 2004 Jul 8, 2008 Jose Antonio Rodriguez Rivera

Catalytic reactor process for the production of commercial grade pulp, native lignin and unicellular
protein

US7419809 Sep 25, 2001 Sep 2, 2008 Iogen Energy Corporation Method for glucose production with a
modified cellulase mixture

US7465791 Jan 30, 2008 Dec 16, 2008 Lignol Innovations Ltd. Continuous counter-current organosolv
processing of lignocellulosic feedstocks

US7666637 Feb 1, 2007 Feb 23, 2010 Xuan Nghinh Nguyen

Integrated process for separation of lignocellulosic components to fermentable sugars for production of
ethanol and chemicals

US8012721 Mar 5, 2003 Sep 6, 2011 Iogen Energy Corporation Method for glucose production using
endoglucanase core protein for improved recovery and reuse of enzyme

US8013195 Jun 15, 2007 Sep 6, 2011 Uop Llc Enhancing conversion of lignocellulosic biomass

US8038840 * Mar 6, 2008 Oct 18, 2011 Rongxiu Li Process for refining and producing cellulose, lignin
and xylose from biomass material

US8193324 Nov 26, 2008 Jun 5, 2012 Lignol Innovations Ltd. Continuous counter-current organosolv
processing of lignocellulosic feedstocks

US8227004 * Nov 10, 2008 Jul 24, 2012 Lignol Innovations Ltd. Continuous counter-current organosolv
processing of lignocellulosic feedstocks
US8528463 Jul 23, 2012 Sep 10, 2013 Lignol Innovations Ltd. Modular system for organosolv
fractionation of lignocellulosic feedstock

US8529731 Sep 5, 2008 Sep 10, 2013 Council Of Scientific & Industrial Research

Process for fractionating sugarcane bagasse into high α-cellulose pulp, xylan and lignin

US8545633 Sep 19, 2011 Oct 1, 2013 Abengoa Bioenergy New Technologies, Inc.

Method for producing ethanol and co-products from cellulosic biomass

US8603295 Apr 23, 2010 Dec 10, 2013 Greenfield Specialty Alcohols Inc.

Separation of reactive cellulose from lignocellulosic biomass with high lignin content

US8772427 May 30, 2012 Jul 8, 2014 Lignol Innovations Ltd. Continuous counter-current organosolv
processing of lignocellulosic feedstocks

US8778084 Jul 29, 2008 Jul 15, 2014 Abengoa Bioenergy New Technologies, Llc. Method and apparatus
for treating a cellulosic feedstock

US8900370 Jul 29, 2008 Dec 2, 2014 Abengoa Bioenergy New Technologies, Llc. Method and apparatus
for conveying a cellulosic feedstock

US8911557 Jul 29, 2008 Dec 16, 2014 Abengoa Bioenergy New Technologies, Llc. Method and apparatus
for conveying a cellulosic feedstock

US8915644 Apr 12, 2012 Dec 23, 2014 Abengoa Bioenergy New Technologies, Llc. Method and
apparatus for conveying a cellulosic feedstock

US9004742 Jan 28, 2009 Apr 14, 2015 Abengoa Bioenergy New Technologies, Llc. Method and apparatus
for conveying a cellulosic feedstock

US9010522 Jul 29, 2008 Apr 21, 2015 Abengoa Bioenergy New Technologies, Llc Method and apparatus
for conveying a cellulosic feedstock

US9033133 Jan 28, 2009 May 19, 2015 Abengoa Bioenergy New Technologies, Llc. Method and
apparatus for conveying a cellulosic feedstock

US9127325 Jan 26, 2011 Sep 8, 2015 Abengoa Bioenergy New Technologies, Llc. Method and apparatus
for treating a cellulosic feedstock

US9187862 Nov 4, 2011 Nov 17, 2015 Greenfield Specialty Alcohols Inc.

Bagasse fractionation for cellulosic ethanol and chemical production

US9335043 Aug 24, 2010 May 10, 2016 Abengoa Bioenergy New Technologies, Inc.

Method for producing ethanol and co-products from cellulosic biomass

US9421477 Aug 8, 2014 Aug 23, 2016 Green Extraction Technologies Biomass fractionation and
extraction apparatus

US9481760 Mar 24, 2016 Nov 1, 2016 Resinate Materials Group, Inc.
High recycle content polyols from thermoplastic polyesters and lignin or tannin

US9487712 * Nov 8, 2011 Nov 8, 2016 Wisconsin Alumni Research Foundation

Method for producing liquid hydrocarbon fuels directly from lignocellulosic biomass

US20020069987 * Feb 9, 2002 Jun 13, 2002 Pye Edward Kendall Integrated processing of biomass and
liquid effluents

US20040051200 * Aug 13, 2001 Mar 18, 2004 Henryk Struszczyk Process for producing fibres, film,
casings and other products from modified soluble cellose

US20040053373 * Sep 25, 2001 Mar 18, 2004 Brian Foody Method for glucose production with
improved recovery and reuse of enzyme

US20050269048 * Jun 3, 2004 Dec 8, 2005 Fortune Forest & Lignin S.A. De C.V.

Novel catalytic reactor process for the production of commercial grade pulp, native lignin & unicellular
protein

US20060008885 * Mar 5, 2003 Jan 12, 2006 Daphne Wahnon Method for glucose production using
endoglucanase core protein for improved recovery and reuse of enzyme

US20080057555 * Feb 1, 2007 Mar 6, 2008 Xuan Nghinh Nguyen

Integrated process for separation of lignocellulosic components to fermentable sugars for production of
ethanol and chemicals

US20080295980 * Jan 18, 2008 Dec 4, 2008 Lignol Innovations Ltd. Continuous counter-current
organosolv processing of lignocellulosic feedstocks

US20080299628 * Aug 15, 2007 Dec 4, 2008 Lignol Energy Corporation Continuous counter-current
organosolv processing of lignocellulosic feedstocks

US20080299629 * Jan 30, 2008 Dec 4, 2008 Lignol Innovations Ltd. Continuous counter-current
organosolv processing of lignocellulosic feedstocks

US20080312479 * Jun 15, 2007 Dec 18, 2008 Mccall Michael J Enhancing Conversion of Lignocellulosic
Biomass

US20090117226 * Nov 10, 2008 May 7, 2009 Lignol Innovations Ltd. Continuous counter-current
organosolv processing of lignocellulosic feedstocks

US20090118477 * Nov 26, 2008 May 7, 2009 Lignol Innovations Ltd. Continuous counter-current
organosolv processing of lignocellulosic feedstocks

US20090176286 * Nov 15, 2006 Jul 9, 2009 O'connor Ryan P Process for Fractionating Lignocellulosic
Biomass into Liquid and Solid Products

US20100152509 * Mar 25, 2008 Jun 17, 2010 Reac Fuel Ab Fuel Produced From a Biomass

US20100200182 * Mar 6, 2008 Aug 12, 2010 Shanghai Jiaotong University Process for refining and
producing cellulose, lignin and xylose from biomass material
US20100269990 * Apr 23, 2010 Oct 28, 2010 Greenfield Ethanol Inc. Separation of reactive cellulose
from lignocellulosic biomass with high lignin content

US20100276093 * Sep 5, 2008 Nov 4, 2010 Anjanikumar Jyotiprasad Varma

Process for fractionating sugarcane bagasse into high a-cellulose pulp, xylan and lignin

US20100313882 * Apr 23, 2010 Dec 16, 2010 Greenfield Ethanol Inc. Fractionation of biomass for
cellulosic ethanol and chemical production

US20110094505 * Feb 9, 2006 Apr 28, 2011 Oxiteno S.A. Industria E Comercio

Acid hydrolysis process of cellulosic and lignocellulosic materials

US20110143411 * Nov 10, 2010 Jun 16, 2011 Fpinnovations Biomass fractionation process for
bioproducts

US20130217869 * Oct 28, 2011 Aug 22, 2013 Annikki Gmbh Method for production of lignin

US20130276778 * Mar 4, 2013 Oct 24, 2013 Virdia Ltd Methods and Systems for Processing
Lignocellulosic Materials and Related Compsitions

US20130305594 * Nov 8, 2011 Nov 21, 2013 Li Shuai Method for producing liquid hydrocarbon fuels
directly from lignocellulosic biomass

US20140186901 * Dec 27, 2013 Jul 3, 2014 Api Intellectual Property Holdings, Llc

Processes and apparatus for producing fermentable sugars, cellulose solids, and lignin from
lignocellulosic biomass

US20150141628 * Nov 10, 2014 May 21, 2015 Virdia, Inc. Methods for treating lignocellulosic materials

US20160002851 * Feb 26, 2014 Jan 7, 2016 Bio-Sep Limited Process Operations for Biomass
Fractionation

CN101524634B Dec 30, 2008 Dec 8, 2010 淮阴师范学院 Maize straw core absorbing material and
preparing method thereof

CN102733223A * Jun 18, 2012 Oct 17, 2012 中国科学院广州能源研究所

Pulping method by catalysis of low boiling point solvent with diluted acid generated from xylose residue
and furfural residue

CN102733223B * Jun 18, 2012 Mar 18, 2015 中国科学院广州能源研究所

Pulping method by catalysis of low boiling point solvent with diluted acid generated from xylose residue
and furfural residue

CN104164801A * Aug 6, 2014 Nov 26, 2014 复旦大学 Method for separating and recycling cellulose,
semicellulose and lignin in lignocellulosic biomass at one time

EP2150572A1 * Apr 25, 2008 Feb 10, 2010 Lignol Innovations Ltd. Continuous counter-current
organosolv processing of lignocellulosic feedstocks
EP2158296A1 * Jun 11, 2008 Mar 3, 2010 Uop Llc Enhancing conversion of lignocellulosic biomass

EP2489780A1 Feb 16, 2011 Aug 22, 2012

Rheinisch-Westfälisch-Technische Hochschule Aachen

Integrated process for the selective fractionation and separation of lignocellulose in its main
components

WO2008017145A1 * Aug 1, 2007 Feb 14, 2008 Emicellex Energy Corporation

Process for recovery of holocellulose and near-native lignin from biomass

WO2008121043A1 * Mar 25, 2008 Oct 9, 2008 Reac Fuel Ab A fuel produced from a biomass

WO2009031164A1 * Sep 5, 2008 Mar 12, 2009 Council Of Scientific & Industrial Research

A process for fractionating sugarcane bagasse into high a-cellulose pulp, xylan and lignin

WO2010045576A2 * Oct 16, 2009 Apr 22, 2010 Mascoma Corporation Production of pure lignin from
lignocellulosic biomass

WO2010045576A3 * Oct 16, 2009 Jul 22, 2010 Mascoma Corporation Production of pure lignin from
lignocellulosic biomass

WO2010058185A1 Nov 23, 2009 May 27, 2010 Bio-Sep Limited Processing of biomass

WO2011066487A1 * Nov 28, 2010 Jun 3, 2011 Applied Biorefinery Sciences, Llc

Biorefinery process for extraction, separation and recovery of fermentable saccharides, other useful
compounds, and yield of improved lignocellulosic material from plant biomass

WO2012110231A1 Feb 15, 2012 Aug 23, 2012 Rheinisch-Westfälische Technische Hochschule

Integrated process for the selective fractionation and separation of lignocellulose in its main
components

WO2013165962A2 * Apr 30, 2013 Nov 7, 2013 Bp Corporation North America Inc. Lignocellulosic
conversion process with tissue separation

WO2013165962A3 * Apr 30, 2013 Jul 24, 2014 Bp Corporation North America Inc. Lignocellulosic
conversion process with tissue separation

WO2014132055A1 Feb 26, 2014 Sep 4, 2014 Bio-Sep Limited Biomass process optimisation

WO2014132056A1 Feb 26, 2014 Sep 4, 2014 Bio-Sep Limited Process operations for biomass
fractionation

WO2014139515A3 * Mar 6, 2014 Nov 13, 2014 Studiengesellschaft Kohle Mbh Method for breaking
down lignocellulosic biomass

WO2016033456A1 * Aug 28, 2015 Mar 3, 2016 University Of Tennessee Research Foundation
Comprehensive process for selectively separating lignocellulosic biomass into purified components with
high yield

* Cited by examiner

CLASSIFICATIONS

U.S. Classification 162/16 , 127/37 , 162/77 , 162/72

International Classification D21C11/00 , D21C5/00 , D21C3/20

Cooperative Classification D21C3/20 , D21C11/0007 , D21C5/00

European Classification D21C11/00B, D21C5/00, D21C3/20

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