Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF BIOMASS PRETREATMENT
Background of the Invention
1. Field of the Invention
The invention relates to methods for the pretreatment of a lignin
containing biomass to render the biomass amenable to digestion. Pretreatment
comprises the addition of calcium hydroxide and water to the biomass to form
a mixture, and maintaining the mixture at a relatively high temperature.
Alternatively, an oxidizing agent, selected from the group consisting of
oxygen
and oxygen-containing gasses, may be added under pressure to the mixture.
The invention also relates to the digested products of the pretreated biomass
which includes useful feedstocks, fuels, and chemicals such as sugars,
ketones,
fatty acids and alcohols, and to a method for the recovery of calcium from the
pretreated biomass.
2. Description of the Background
Biomass can be classified in three main categories: sugar-, starch-
and cellulose-containing plants. Sugar-containing plants (e.g. sweet sorghum,
sugarcane) and starch-containing plants (e.g. corn, rice, wheat, sweet
potatoes)
are primarily used as food sources. Cellulose-containing plants and waste
products (e.g. grasses, wood, bagasse, straws) are the most abundant forms of
biomass. Although they are not easily converted to useful products, a well
engineered process to convert them to feedstock may potentially be economical
since the costs of feedstock are much less than those of sugar- and starch-
containing biomass.
Cellulose-containing materials are generally referred to as
lignocellulosics because they contain cellulose (40% - 60%), hemicellulose
(20% - 40%) and lignin (10% -25%). Non-woody biomass generally contains
less than about 15-20% lignin. Cellulose, a glucose polymer, can be hydrolyzed
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to glucose using acid, enzymes or microbes. Glucose can serve as a feedstock
for fuel alcohol and single-cell protein production. Microbial hydrolysis
produces cellular biomass (single-cell protein) and metabolic waste products
such as organic acids. Acid hydrolysis, although simple, produces many
undesirable degradation products. Enzymatic hydrolysis is the cleanest and
most preferred approach. However, production of enzymes, mainly cellulase
and cellobiase, can be an expensive step. Apart from alcohol production,
lignocellulose can be used as inexpensive cattle feed. Since raw
lignocellulose
cannot be easily digested by cattle, it must be processed to improve its
digestibility before it can be fed to ruminants. Also, anaerobic fermentation
using rumen microorganisms can produce low molecular weight volatile fatty
acids.
Cellulose is the world's most. abundant biological material.
Approximately 40% to 45% of the dry weight of wood species is cellulose. The
degree of polymerization ranges from 500 to 20,000. Cellulose molecules are
completely linear, unbranched and have a strong tendency to form inter- and
intra-molecular hydrogen bonds. Bundles of cellulose molecules are thus
aggregated together to form microfibrils in which highly ordered (crystalline)
regions alternate with less ordered (amorphous) regions. Microfibrils make
fibrils and finally cellulose fibers. As a consequence of its fibrous
structure and
strong hydrogen bonds, cellulose has a very high tensile strength and is
insoluble in most solvents.
Hemicellulose is the world's second most abundant carbohydrate
and comprises about 20% to 30% of wood dry weight. Hemicelluloses,
although originally believed to be intermediates in cellulose biosynthesis,
are
formed through biosynthetic routes different from cellulose. Hemicellulose are
heteropolysaccharides and are formed by a variety of monomers. The most
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common monomers are glucose, galactose and mannose (the hexoses) and
xylose and arabinose (the pentoses). Most hemicelluloses have a degree of
polymerization of only 200. Hemicelluloses can be classified in three
families,
xylans, mannans and galactans, named for the backbone polymer.
Lignin is the world's most abundant non-carbohydrate
biomaterial. It is a three dimensional macromolecule of enormously high
molecular weight. Since its units are extensively cross-linked, it is
difficult to
define an individual molecule. Lignin provides strength by binding cellulose
fibrils together. Being hydrophobic in nature, it prevents water loss from the
vascular system and, being highly resistant to enzymatic degradation, it
protects
plants from insects and microbial attack.
Phenylpropane, an aromatic compound, is the basic structural
unit of lignin. The monomers not only cross-link with each other, but also
covalently bond to hemicellulose. A great constraint to cellulose and
hemicellulose accessibility is the presence of lignin. It has been shown that
decreased lignin content causes increased digestibility. Lignin can be removed
by physical, chemical, or enzymatic treatments. It must be decomposed to
smaller units that can be dissolved out of the cellulose matrix. There are
several well developed pulping methods that disintegrate and remove lignin,
leaving the cellulose fairly intact. Conventional pulping processes, such as
Kraft and sulfite pulping, are too costly as bioconversion pretreatments.
Also,
economical use of the removed lignin is difficult because its chemical
structure
and size distribution are highly heterogeneous.
Another major deterrent to enzymatic cellulosic hydrolysis is the
highly ordered molecular packing of its crystalline regions. Cellulolytic
enzymes readily degrade the more accessible amorphous portions of cellulose,
but are unable to attack the less accessible crystalline material. Thus,
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enzymatic hydrolysis rates increase with decreasing crystallinity index
measured
by X-ray diffraction methods.
The moisture content of cellulose fibers influences enzymatic
degradation. Cellulosic materials are effectively protected from deterioration
by enzymes or microbes provided the moisture content is maintained below a
critical level characteristic of the material and the organism involved. In
general, this critical level is slightly above the fiber saturation point,
approximately 40% of dry weight. Moisture plays three major roles: (1) it
swells the fibers by hydrating cellulose molecules, thus opening up the fine
structure and increasing enzyme access, (2) it. provides a diffusion medium
for
enzymes and for partial degradation products, and (3) it is added to cellulose
during hydrolytic cleavage of the glycosidic links of each molecule.
The surface area of lignocellulose is another important factor
that determines susceptibility to enzymatic degradation. It is important
because contact between enzyme molecules and the cellulose surface is a
prerequisite for hydrolysis to proceed. A few other factors that also
influence
susceptibility include size and diffusibility of enzyme molecules in relation
to
size and surface properties of capillaries, unit cell dimensions of cellulose
molecules, and conformation and steric rigidity of hydro-glucose units.
To enhance susceptibility to enzymatic hydrolysis, lignocellulose
pretreatment is an essential requirement. The heterogeneous enzymatic
degradation of lignocellulosics is primarily governed by its structural
features
because (1) cellulose possesses a highly resistant crystalline structure, (2)
the
lignin surrounding the cellulose forms a physical barrier and (3) the sites
available for enzymatic attack are limited. An ideal pretreatment, therefore,
would reduce lignin content, with a concomitant reduction in crystallinity and
increase in surface area. Pretreatment methods can be classified into
physical,
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chemical, physicochemical, and biological, depending on the mode of action.
The literature available on this subject is voluminous. The various
pretreatment methods that have been used to increase cellulose digestibility
are summarized in Table 1.
5 Biological pretreatments employ fungi for microbial de-
lignification to make cellulose more accessible. Major biological lignin
degraders are the higher fungi, Ascomycetes and Basidiomycetes. Fungal
degradation is a slow process and most fungi attack not only lignin, but
cellulose also, thus resulting in a mixture of lignin fragments and sugars.
Improvements may require developing more specific and efficient microbes.
Physical Pretreatments
Physical pretreatments can be classified in two general categories:
mechanical (involving all types of milling) and nonmechanical (involving high-
pressure steaming, high energy radiation and pyrolysis). During mechanical
pretreatments, physical forces, (e.g. shearing, compressive forces) subdivide
lignocellulose into finer particles. These physical forces reduce
crystallinity,
particle size and degree of polymerization and increase bulk density. These
structural changes result in a material more susceptible to acid and enzymatic
hydrolysis. However, due to enormously high operating costs associated with
the high energy requirements, low yields and large time requirements, these
mechanical pretreatments are not practical. Nonmechanical physical
pretreatment methods also increase digestibility, but have similar
disadvantages
and thus are not economical for real processes.
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Physicochemical Pretreatments
Steam explosion and Ammonia Fiber Explosion (AFEX) are the
main physicochemical pretreatments. Steam explosion heats wetted
lignocellulose to high temperatures (about 250 C) and releases the pressure
instantly. Due to rapid decompression, which flashes the water trapped in
fibers, physical size reduction occurs. The high temperatures remove acetic
acid from hemicellulose, so this process results in some autohydrolysis of the
biomass. These changes result in better digestibilities, but the severe
conditions also produce degradation products that inhibit hydrolysis and
fermentation. These products are removed by washing with water which results
in a loss of water soluble hemicellulose. Thus, although digestibilities are
improved, biomass degradation and protein denaturization limits the use of
steam explosion.
The AFEX pretreatment process soaks lignocellulose in liquid
ammonia at high pressure and then explosively releases the pressure.
Pretreatment conditions (30 C - 100 C) are less severe than steam explosion.
An increase in accessible surface area coupled with reduced cellulose
crystallinity (caused by ammonia contacting) result in increased enzymatic
digestibility. However, use of ammonia (a hazardous chemical) and the high
.20 pressure release makes the process quite complex and energy intensive.
Chemical Pretreatments
Many chemical treatments have been used for lignin removal and
destruction of the lignin crystalline structure. Of these chemicals, acids;-
gases,
oxidizing agents, cellulose solvents, and solvent extraction agents, are all
able
to increase digestibility, but are not as popular as alkalis. Economics,
simpler
processes and less degradation favor alkalis as chemical pretreatment agents.
However, most of these are process for paper pulping and involves the
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complete or nearly complete destruction of lignin, and a corresponding
destruction of cellulose. Although unimportant in pulping, these pulping
process are quite severe and not useful as pretreatments for biomass.
Furthermore, the traditional pulping processes used by the paper industry are
too expensive as lignocellulose pretreatment methods.
U.S. Patent No. 4,644,060 to Chou is directed to the use of super-
critical ammonia to increase lignocellulose digestibility.
U.S. Patents Nos. 4,353,713 and 4,448,588 to Cheng are directed
to the gasification of biomass or coal which is an endothermic process. These
patents also relate to a method for adding the required thermal energy by
reacting lime with carbon dioxide which is an exothermic reaction.
U.S. Patent No. 4,391,671 to Azarniouch is directed to a method
for calcining calcium carbonate in a rotary kiln. The reference relates to the
paper/pulp industry where the calcium carbonate would be contaminated with
waste biomass. The waste biomass is burned to provide the needed heat of
reaction.
U.S. Patent No. 4,356,196 to Hulquist is directed to, treating
biomass with ammonia.
U.S. Patent. No. 4,227,964 to Kerr is directed to the use. of
ammonia to promote the kinking of pulp fiber to increase paper strength, not
to break down the fibers.
U.S. Patent No. 4,087,317 to Roberts is directed to the use of
lime and mechanical beating to convert pulp into a hydrated gel. There is no
mention of lime recovery or enzymatically hydrolyzing the hydrated gel.
U.S. Patent No. 4,064,276 to Conradsen directed to a process
where biomass is covered with a tarp and then ammoniated with ammonia,
which is allowed to dissipate into the atmosphere.
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U.S. Patent No. 3,939,286 to Jelks is directed to oxidizing
biomass with high-pressure oxygen under elevated temperature and pressure
in the presence of an acid catalyst, and a metal catalyst, ferric chloride, to
break lignin bonds and to increase digestibility. The catalysts are described
as
essential to the process and calcium hydroxide is utilized as a neutralizing
agent to adjust the resulting pH of the hydrolyzed biomass.
U.S. Patent No. 3,878,304 to Moore is directed to production of
slow-release nonprotein nitrogen in ruminant feeds. An amide, urea, is reacted
with waste carbohydrates in the presence of an acid catalyst. The resulting
material is pelleted and used as animal feed. Since the nitrogen is released
slowly in the rumen, it is nontoxic to the animal.
U.S. Patent No. 3,944,463 to Samuelson et al. is directed to a
process for producing cellulose pulp of high brightness. The cellulose is
pretreated with an alkaline compound at a temperature of between about 60 C
to about 200 C so as to dissolve between 1 and 30% of the dry weight of the
material in the pretreatment liquor. The pretreatment liquor preferably
contains sodium carbonate, sodium bicarbonate or mixtures thereof, or possible
sodium hydroxide.
U.S. Patent No. 3,639,206 to Spruill is directed to the treatment
of waste water effluent derived from a pulping process with calcium oxide or
hydroxide to reduce the fiber and color content of the effluent.
U.S. Patent No. 4,048,341 to Lagerstrom et al. is directed to a
process for increasing the feed value of lignocellulosic material by
contacting
the material with an alkaline liquid, specifically, sodium hydroxide. The
alkaline liquid, supplied in excess, is allowed to run off the material before
any
essential alkalization effect has been reached. After the liquid absorbed in
the
material has provided its effect, an acid solution is added to the material to
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neutralize the excess alkali. The reference does not disclose the
interrelationship of temperature and time of alkali treatment, nor does it
disclose the optimal amounts of the sodium hydroxide and water.
U.S. Patent No. 4,182,780 to Lagerstrom et al. is directed to a
process for increasing the feed value of lignocellulosic materials by alkali
treatment and subsequent neutralization of the materials with an acid in a
closed system under circulation of the treating agents.
U.S. Patent No. 4,515,816 to Anthony is directed to a process in
which lignocellulose is treated with dilute acid in an amount of about 1.5 to
2.5% of the dry weight of lignocellulose. The mixture is then stored at
ambient conditions for 5 to 21 days in an air-free environment.
U.S. Patent No. 4,842,877 to Tyson is directed to a process for
the delignification of non-woody biomass (<20% lignin). In this process, non-
woody biomass is treated with a chelating agent, to prevent unnecessary
oxidation, and maintained at high pH and high temperatures (150 F to 315 F)
in the presence of hydrogen peroxide and pressurized oxygen. Hydrogen
peroxide is stated to cause a reaction on the cell walls to allow the
hemicellulose and lignin to solubilize and be removed through a subsequent
hydrolysis process. Oxygen is added to initiate and accelerate the activation
of hydrogen peroxide.
The conditions and results of studies reported in the literature
using ammonia (gaseous, anhydrous liquid, or NH4OH) and sodium hydroxide
as pretreatment agents are listed in Table 2 and Table 3, respectively. The
literature available on the use of these two chemicals to enhance
lignocellulose
digestibility of ruminant feeds, as well as for hydrolysis to glucose, is
extensive.
The literature on calcium hydroxide pretreatment processes is considerably
less
compared to that for sodium hydroxide and ammonia. The conditions and
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results of studies reported in the literature using calcium hydroxide are
shown
in Table 4.
The references cited in the Tables and below are:
Anderson, D.C.; Ralston, A.T. J. Anim. Sci. 1973; 37, 148.
5 Baker, A.J.; Millett, M.A.; Satter, L.D. ACS Symposium Series 1975; 10,
75.
Brown, W.F.; Phillips, J.D.; Jones, D.B. J. Anim. Sci. 1987; 64, 1205.
Dawish, A.; Galal, A.G. In Proc. Conf. Anim. Feeds Trop. Subtrop.
Origin 1975.
10 Felix, A.; Hill, R.A.; Diarra, B. Anim. Prod. 1990; 51, 47.
Feist, W.C.; Baker, AJ.; Tarkow, H. J. Anim. Sci. 1970; 30, 832.
Gharib, F.H.; Meiske, J.C.; Goodrich, R.D.; El Serafy, A.M. J. Anim.
Sci. 1975; 40(4), 734.
Hulquist, J.H. U.S. Patent No. 4,356,296; 1982.
Kellens, R.D.; Herrera-Saldana, R.; Church, D.C. I. Anim. Sci. 1983;
56(4), 938.
Mandels, M.; Hontz, J.R.; Kystrom, J. Biotech. Bioeng. 1974; 16, 1471.
Millet, M.A. et al., J. Anim. Sci. 1970; 31(4), 781.
Millet, M.A.; Baker, A.J.; Satter, L.D. In Biotech. Bioeng. Symp. 1975;
5, 193.
Moore, W.E.; Effland, M.J.; Medeiros, J.E. J. Agr. Food Chem. 1972;
20(6), 1173.
Morris, PJ.; Movat, D.N. Can. J. Anim. Sci. 1980; 60, 327.
Playne, M. J. Biotech. Bioeng., 1984; 26, 426.
Rounds, W.; Klopfenstein, T. J. Anim. Sci. 1974; 39, 251 (abst.).
Turner, N.D.; Schelling, G.T.; Greene, L.W.; Byers, P.M. J. Prod. Agric.
1990; 3(1), 83.
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Villareal, E.R. Ph.D. Thesis Texas A&M Univ. College Station, TX
1988.
Waiss, AC. et al., J. Anim. Sci. 1972; 35(1). 109.
Waller, J.C.; Klopfenstein, T. J. Anim. Sci. 1975, 41 424 (abstract).
Playne (1984) investigated the effects of alkali treatment and
steam explosion on baggase digestibility. The digestibility of untreated
bagasse
was 190 g organic matter (OM)/kg bagasse dry matter. It was raised to: 733
g organic matter by using NaOH (and also by using Ca(OH)2 with Na2CO3);
to 430 g OM using NH3; and to 724 g OM using Ca(OH)2. When Ca(OH)2
alone was used, a high loading (about 180 - 300 g Ca(OH)2 kg bagasse) was
used. Gharib et al. (1975) used calcium oxide for in vitro evaluation of
chemically treated poplar bark. They reported that calcium oxide increased in
vitro true digestibility from 38% to,, 52% for a 150-day treatment, although
little
improvement was found for a 1-day treatment. Rounds and Klopfenstein
(1974) studied the effects of NaOH, KOH, NH4OH and Ca(OH)2 on in vivo
digestibility of corn cobs by feeding to lambs and on in vitro digestibility
using
an artificial rumen. Ca(OH)2 alone was unable to increase the in vitro
digestibility, although rations treated with Ca(OH)2 + NaOH resulted in higher
daily gain and feed efficiency for lambs. Waller and Riopfenstein (1975) used
various combinations of NaOH, Ca(OH)2 and NH4OH for treating feed for
lambs and heifers and reported that the highest daily gain and lowest
feed/gain
was obtained for the 3% NaOH + 1% Ca(OH)2 rations. Darwish and Galal
(1975) used maize cobs treated with 1.5% Ca(OH)2 in a milk production ration
and found no significant change in milk output. Felix et al. (1990) evaluated
the effects of ensiling and treating soya-bean straw with NAOH, Ca(OH)2 and
NH4OH on ruminant digestibility. Results indicate that there was no
significant
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improvement due to alkali treatment of dry and unensiled straw, although
alkali treatment improved digestibility of ensiled straw.
Although the use of calcium hydroxide as a pretreatment agent
has been demonstrated, considerably less work has been done employing this
chemical compared to other alkalis. Most of the previous work has been
performed by animal scientists trying to develop a very simple process to
increase the lignocellulose digestibility of animal feed. All these studies
were
done at room temperature or below, at lower water loadings, for very long
periods and without any mixing. These processes required very long treatment
times which is very expensive since the reactors must be very large. There is
thus a need to improve the currently existing methods of pretreating
lignocellulose containing material to render it amenable to enzymatic
digestion.
Summary of the Invention
One embodiment of the invention is directed to an economical
process for pretreating a lignocellulose-containing biomass to render the
biomass amenable to digestion. Pretreatment comprises adding calcium
hydroxide and water to the biomass, and subjecting the biomass to relatively
high temperatures for a period of time. The pretreated biomass is digested to
produce a useful product.
Another embodiment of the invention is directed to an
economical pretreatment method comprising adding calcium hydroxide and
water to the biomass to form a mixture, adding an oxidizing agent, selected
from the group consisting of oxygen and oxygen-containing gasses, to the
mixture under pressure, and subjecting the biomass to relatively high
temperatures for a period of time. The pretreated biomass is digested to
produce a useful product.
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Another embodiment of the invention is directed to a process for
recovering calcium from a biomass. After pretreatment, the biomass is
carbonated with a carbonating agent to form calcium carbonate. Calcium
carbonate is recovered from the pretreated mixture or recovered after
digestion and can be reconverted into calcium hydroxide.
Brief Description of the Drawings
Figure 1 is a schematic diagram of Reactor System 3.
Figure 2 is a glucose calibration curve for the filter paper assay.
Figure 3 is an enzyme calibration curve for the filter paper assay.
Figure 4 is a calibration curve for the DNS assay.
Figure 5 is a flow diagram for continuous calcium hydroxide recovery.
Figure 6 is a techematic diagram of one embodiment of using a
hydroclone to separate lime solids from lime solution.
Figure 7 is a schematic diagram of another embodiment of using a
hydroclone to separate lime solids from lime solution.
Figure 8 is a schematic diagram showing different possible permutations
of pretreating biomass with lime.
Figure 9 shows the lime treatment process for ruminant animal feed
production.
Figure 10 is a graph of oxygen pressure verses yield to show the effect
of oxygen on the hydrolysis of newspaper.
Figure 11 is a graph of pretreatment time verses yield to show the effect
of treatment time on the hydrolysis of newspaper.
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Description of the Invention
The present invention comprises economical methods for the
pretreatment of a biomass. Pretreatment comprises the addition of calcium
hydroxide and water to a biomass to render the biomass susceptible to
degradation. Calcium hydroxide is inexpensive and much cheaper than other
alkalis. It is safe to handle and, unlike the sodium residue, the calcium
residue
is little or no problem for animal feed. In an artificial rumen, calcium
hydroxide produces calcium acetate which is also safe and nontoxic. Calcium
hydroxide (Ca(OH)2) as the lignocellulose pretreatment agent is therefore very
.10 economical. Further, there was also no significant difference in
digestibility
between Ca(OH)2 pretreated material and NH3 or NaOH pretreated material
in animals.
The operating conditions of the pretreatment methods of the
invention are a significant improvement over the existing literature. Previous
researchers restricted their operating temperature to ambient and below in
order to create a very simple process without heaters. These simple processes
required extremely long treatment times typically ranging from 8 to 150 days.
Raising the treatment temperature could decrease the treatment time, but runs
the risk of degrading the lignocellulose. High-temperature treatment
conditions have been identified that did not degrade the lignocellulose and
resulted in treatment times that were orders of magnitude shorter. The
economic impact is significant since the reactor can be orders of magnitude
smaller.
Further, previous calcium hydroxide (lime) pretreatment methods
used very low water loadings. Since their processes operated at room
temperature, good heat transfer was not an issue. They could operate with
very little water since the thermal insulating properties of air were not
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detrimental. However, when operating at a higher temperature, the process
benefits by including about ten times more water since its high heat capacity
and heat transfer coefficient ensure a uniform temperature. Higher water
loadings also provides a medium into which the lime can more uniformly
5 disperse, but are unattainable using conventional procedures. Consequently,
most of the previous research in lime treatment used relatively low lime
loadings whereas we were able to realistically consider higher loadings can be
realistically considered because a lime recovery process is also incorporated
into the invention.
10 In one embodiment, the invention is directed to a method for
pretreating a lignocellulose-containing biomass to render the biomass amenable
to digestion and comprises providing a lignocellulose-containing biomass,
adding calcium hydroxide and water to the biomass to form a mixture, and
maintaining the mixture at an elevated temperature and for a period of time
15 sufficient to render the biomass of the mixture amenable to digestion.
Types
of useful biomass include grass, wood, bagasse, straw, paper, plant material,
and combinations thereof. Lignocellulose-containing biomass to which the
process of the invention is directed is preferably biomass containing greater
than 15% lignin and more preferable biomass containing greater than 20%
lignin.
Preferably, biomass to be pretreated is fed into a chipper,
grinder, chopper, shredder or the like, to be reduced in size. Resulting
biomass chips or particles are preferable about one-half inch or smaller. The
biomass particles are then combined with calcium hydroxide and water to form
an alkaline biomass mixture. The mixture contains between about 6 to about
19 grams of water per gram of dry biomass and preferably about 16 grams of
water per gram of dry biomass. The mixture also contains between about 2 to
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about 50 grams of calcium hydroxide per 100 grams of dry biomass and
preferably contains about 30 grams of calcium hydroxide per 100 grams of dry
biomass. Depending on the type of biomass, the preferable amount may be
more or less. Calcium hydroxide may be added before or after the water or
as an aqueous solution or dispersion.
The aqueous calcium hydroxide/biomass mixture is maintained
in reaction chambers, which are preferably stainless steel, at between about
40 C to about 150 C, preferably between about 100 C to about 140 C, and
more preferably at about 120 C. Depending on the type of biomass, the
temperature range may be between about 70 C to about 110 C, between about
110 C to about 150 C, or between about 50 C to about 65 C. The
temperature is maintained for between about 1 to about 36, preferably between
about 1 to about 20 hours, more preferably about 3 hours. Again, depending
on the type of biomass, the time period may be longer or shorter such as
between about 15 to about 25 hours.
Another embodiment of the invention is directed to a method for
converting a lignocellulose-containing biomass into a useful product and
comprises providing a lignocellulose-containing biomass, adding calcium
hydroxide and water to the biomass to form a mixture, oxygenating the mixture
with pressurized oxygen, maintaining the mixture at an elevated temperature
and for a period of time sufficient to render the biomass of the mixture
amenable to digestion, and digesting the biomass of the mixture to convert the
biomass into the useful product. Oxygen is relatively inexpensive and readily
available as pressurized oxygen gas, pressurized air, and other pressurized
oxygen-containing gasses. Oxygen is also non-toxin and non-polluting to the
environment. Calcium hydroxide is added to a biomass as described above to
form a mixture. To the mixture is added an oxidizing agent under pressure
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selected from the group consisting of oxygen and oxygen-rich gasses.
Preferably, the added oxygen-containing gas has a pressure of between about
20 to about 500 psig (pounds per square inch gauge), preferably greater than
about 50 psig, and more preferably greater than about 100 psig.
After either of the above-described embodiments, the pretreated
biomass is digested by hydrolysis such as acid hydrolysis, enzymatic action,
fermentation, or a combination of digestion methods. The digested biomass
comprises material which are useful products such as alcohols, acids such as
organic acids, sugars, ketones, starches, fatty acids, or combinations
thereof.
These products can be made into feedstocks such as chemical feedstocks, fuels,
and other useful products. Due to the relatively gentle pretreatment
conditions, the useful products are obtained in higher quantities and are of a
higher quality than products obtained after other pretreatment methods. The
maximum amount of material is converted into useful product with as little
waste as possible. Further, no toxins or harmful chemicals are introduced into
the biomass therefore none need to be removed or even tested for in the final
product.
Another embodiment of the. invention is directed to a method for
recovering calcium from a biomass pretreatment process comprising pretreating
the biomass with calcium hydroxide and water to form a mixture, optionally
adding an oxidizing agent selected from the group consisting of oxygen and
oxygen-containing gasses to the mixture under pressure, and maintaining the
mixture at an elevated temperature and for a period of time sufficient to
render the biomass of the mixture amenable to digestion, carbonating the
mixture or the liquid portion thereof to precipitate calcium carbonate, and
recovering the precipitated calcium carbonate. The pH of the carbonated
mixture is between about 8.5 and about 10.5, and preferably between about 9.0
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and about 10. The calcium carbonate is precipitated in the mixture and can
be recovered by filtration, hydroclone separation, sedimentation,
centrifugation,
or by combinations of these methods. The calcium carbonate may also be
heated and converted into carbon dioxide and calcium oxide, and the calcium
recovered as calcium oxide.
Alternatively, the pretreated mixture is treated with a carbonating
agent, which is preferably carbon dioxide gas which is bubbled into the
mixture,
forming calcium carbonate. The pretreated and carbonated biomass is digested
and the useful product separated from the remaining mixture or residual
mixture. The residual mixture, comprising lignin and calcium carbonate, is
heated, for example in a kiln, preferably a lime kiln, to convert the calcium
carbonate into calcium hydroxide. The heat supplied to the kiln may be
derived from the burning of the lignin, making for a highly economical overall
process.
The following examples are offered to illustrate embodiments of
the invention, but should not be viewed as limiting the scope of the
invention.
Examples
Example 1 Sample Preparation
Raw bagasse was collected from the Animal Science Department
of Texas A&M University. For three years, it was lying in the open, covered
only by a plastic sheet. Thus, it was first thoroughly washed with water and
then dried in an oven (at 80 C) for about 8 hours. Also, because bagasse
deteriorates with storage time, the bagasse used in the present study was more
recalcitrant than that used by Holtzapple et al., Appl. Biochem. Biotech.
28/29,
59 (1991). The wheat straw was already clean and did not require washing.
Softwood newspaper was the Bryan/College Station, Texas, Eagle newspaper.
It was first shredded in a paper shredder. All materials were ground by a
CA 02615904 2008-01-23
19
Wiley mill to 1 x 1 mm particle size and then passed through a 40 mesh sieve.
Dry weight analysis was done by placing a small sample in an oven, at 80 C for
24 hr and measuring the weight loss due to water evaporation.
Example 2 Calcium Hydroxide Pretreatment
Calcium hydroxide pretreatment involves reacting biomass with
calcium hydroxide in the presence of water at a relatively high temperature.
The effectiveness of the pretreatment process was studied for several
different
reaction conditions. Process variables studied were lime loading (2 to 30 g
Ca(OH)2/100g dry biomass), water loading (6 to 19 g water/g dry biomass),
treatment temperature (50 C to 145 C) and treatment time (1 hour to 36
hours). The following three types of reactor systems were employed.
Reactor System 1: For initial pretreatment experiments, 500-mL glass
Erlenmeyer flasks were used as reactors. The flasks were sealed with rubber
stoppers and placed in a 100-rpm shaking water-bath. This method was limited
to 65 C, the highest temperature attainable by the water bath.
Reactor System 2: Erlenmeyer flasks were placed in an oven, with
periodic manual shaking. Only one experiment, at 65 C, was performed by this
method. This method resulted in lower yields, thus demonstrating that
continuous shaking was required for effective mass transfer.
Reactor System 3: Steel reactors were used for experiments in the 65 C
and 145 C temperature range. To resist the corrosive nature of calcium
hydroxide solutions, the reactors were constructed of 304 stainless steel. The
reactors were, 1.5 "I.D. x 5" long, cylindrical nipples, with end caps on both
ends. To provide mixing inside the reactors, a rotating device was fabricated
in the Texas A & M Chemical Engineering Department's machine shop. A
schematic diagram of this reactor system is shown in Figure 1. This device
holds the reactors (R) inside the oven (0) and, by rotating them continuously,
CA 02615904 2009-05-04
provides tumble mixing of the contents. It has a steel rod (R1) supported from
both ends on two ball bearings (B). The bearings are bolted on a steel frame
(SF) that can be placed inside the oven. Six holes are drilled through this
rod
to hold the clamps (C) that hold the reactors. Set screws hold the clamps in
place during rotation. Through the back side of the oven, a 1" hole is
drilled.
A small rod (R2) supported by a ball bearing (bolted on the oven wall) passes
through this hole. A pulley (P) is mounted on the end that is outside the
oven.
A variable speed AC/DC motor (M) mounted on the top of the oven rotates
R2. The steel rod that holds the reactors (RI) has a splined end (a small nut
(N)) that couples with the pulley (via a socket (S)). This coupling
arrangement
allow the reactors to be placed in, and removed from, the oven with ease.
To perform the pretreatment experiments, the reactors were
prepared by winding at least four layers of Teflon tape on both ends. One end
was closed by placing the nipple in a vice and tightening the end cap by a
pipe
wrench. The reaction mixture was prepared by placing the measured quantities
of biomass (7.5 g dry weight) and Ca(OH)2 (according the lime loading) inside
the reactors. The material was thoroughly mixed inside the reactors using a
spatula. Measured amounts of water were then added to this dry mixed
sample. The end cap was placed on the other end of the nipple and tightened.
The reactors were then placed in boiling water for 5 to 15 minutes (depending
on the pretreatment temperature) to pre-warm them. Prewarming the reactors
is necessary to rapidly bring them to higher temperatures. They were then
clamped and fixed on the rotating device and placed in the oven maintained
at the desired pretreatment temperature. The motor was turned on and the
system was left for the desired pretreatment time. After the treatment time
elapsed, the reactors were removed from the oven and transferred to a water
bath to rapidly lower the temperature to ambient temperature. Samples were
CA 02615904 2008-01-23
21
them removed from the reactors for hydrolysis. A complete step-by-step
procedure is given below.
Optionally, oxygen-containing gas is introduced into the reactor
from a high-pressure gas container or tank attached to the system. The gas
may be pure pressurized oxygen, compressed air (which is very economical) or
any oxygen-containing gas under pressure. The pressure of the oxygen-
containing gas can be determined from a pressure gauge mounted on the gas
supply container or on the reactor.
Example -3 Calcium Hydroxide Pretreatment -- Reactor System 3
TM
1. Remove the old Teflon tape and clean the threads at both ends.
TM
Wrap (clockwise) at least four layers of fresh Teflon tape.
2. Label and number all the reactors. 2 or 4 or 6 reactors can be
run each time.
3. Close the reactors by placing the cap on one end. Hold the
nipple in the vice and tighten the cap using a pipe wrench.
4. Weigh ground and sieved material that has 7.5 g dry weight.
Using a funnel, pour it in the labeled reactors.
5. Weigh calcium hydroxide, according to the desired lime loading,
and pour into the reactors with the biomass.
6. Using a spatula, mix Ca(OH)2 and biomass thoroughly. This dry
mixing is essential to ensure a uniform reaction.
7. Pour water according to desired water loading.
7A (Optionally) Open a the valve connecting the reactor to a container
of oxygen gas under pressure. Close the valve when a specified gauge pressure
within the reactor has been achieved.
8. Close the ends of the reactors.
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9. Place the reactors in boiling water for about 5 min, for a 50 C
run, to about 15 min, for a 135 C run. The water boiler takes about 30 min to
heat up, so it must be turned on before hand.
10. Heat the oven to the desired pretreatment temperature. The
oven takes about 1 hour to reach a stable temperature. Keep the rotating
device inside the oven during heating so that it gets prewarmed.
11. Clamp the reactors, making sure that the clamps are in the
center of the reactors so that there is no blocking during rotation.
12. Place the clamps in the slots of the rotating rod and tighten the
set screws.
13. Place the device in the oven and couple it with the motor using
the coupling arrangement.
14. Turn on the motor and keep the rotation speed at the minimum
possible. Make sure that the motor does not stall.
15. Observe the temperature of the oven.
16. After the pretreatment time has elapsed, take out the reactors
and place them in a cold tap water bath. Let them cool for about 10 min.
17. Perform enzymatic hydrolysis.
Example 4 Filter Paper Assay
The filter paper assay is commonly used to quantitatively study
cellulose hydrolysis and measure cellulose activity. Filter paper is used
since
it is a readily available and reproducible substrate and is neither too
susceptible nor too resistant to cellulase enzymes. The filter paper is
incubated
with various amounts of cellulose enzyme for 1 hour at 50 C and pH of 4.8.
The amount of reducing sugars released in 1 hour is measured by the
Dinitrosalicylic Acid (DNS) assay. (Also see below). The amount of enzyme
CA 02615904 2008-01-23
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that produces .2 mg of reducing sugar (expressed as glucose) in 1 hour is
equal
to 0.185 International Units (1 IU = 1 mmole glucose/min).
Calibrate DNS reagent using glucose (along with filter paper):
1. Using a 500mg/dl (5mg/mL) glucose standard, prepare 0.5 mL
samples in pairs of test tubes according to Table 5.
2. Add 1.0 mL of pH 4.8, 0.05 M citrate buffer.
3. Add 1 x 6 cm filter paper strip (Whitman #1, rolled in a curl).
Vortex.
4. Incubate at 50 C for 1 hour (use capped test tubes). Prevent any
shaking
5. Add 3.0 mL of DNS to each test tube.
6. Boil samples for 15 minutes in water bath.
7. Add 10 mL water and vortex.
8. Filter through 0.45 um nylon membrane filter.
9. Measure the absorbance at 550 nm and prepare a calibration
curve of absorbance vs. glucose concentration as shown in Figure 2.
Measure enzyme activity:
1. Add 0, 5, 10, 15, 20 mg enzyme to 10 mL, 0.05 M citrate buffer, pH 4.8,
and vortex.
2. Pipet 0.5 mL of prepared enzyme samples into pairs of test tubes.
3. Repeat steps 2 to 8 performed during calibration curve preparation.
4. Measure the absorbance at 550 nm.
Measure sugars in the enzyme:
1. Pipet 0.5 mL of 20 mg/mL enzyme sample into pairs of test tubes A.
2. Pipet 0.5 mL of distilled water into pairs of test tubes B.
3. Add 1.0 mL of pH 4.8, 0.05 M citrate buffer to test tubes A and B.
4. Repeat steps 5 to 7 performed during calibration curve preparation.
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5. Measure absorbance at 550 nm.
6. Calculate absorbance correction factor (ACF) as follows.
ACF = (Abs A - Abs B)
(20 mg enzyme/10 mL) x 0.5 mL
Calculate specific enzyme activity:
1. Using the glucose calibration curve, calculate absorbance, the
absorbance for 2 mg glucose weight (see Figure 2).
2. Apply absorbance correction factor (AC) to absorbance data from the
enzyme results.
Absca = Abs - ACF x E (where E = mg enzyme in 0.5 mL)
3. Plot Abscor vs. E to get Figure 3.
4. Find E' corresponding to Abs' using Figure 3.
5. Calculate specific activity:
Activity (IU/mg) = 2 mg glucose x 1 hour x mmole
E' mg h 60 mm 0.18 mg glucose
Example 5 Enzymatic Hydrolysis Procedure
The pretreated material (7.5 g dry weight) was transferred from
the reactors to 500-mL Erlenmeyer glass flasks. The operating pH and
temperature for the enzyme system were kept at 4.8 and 50 C respectively.
The pH was reduced from about 11.5 to 4.8 by adding acetic acid. The total
liquid volume was increased to 150 mL by adding distilled water to obtain a
50 g/L slurry of biomass. The required amounts of cellulase and cellobiose we
added to the mixture, and the flasks were stoppered and kept in a 100-rpm
shaking air bath at 50 C for 3 days. The enzymatic hydrolysis of control
materials was performed using 150 mL of 0.05 M, pH 4.8 citrate buffer.
After 3 days, 1 mL liquid samples were withdrawn from each
flask using a 1000-mL Eppendorf pipet. The samples were boiled in capped
CA 02615904 2008-01-23
test tubes for 30 minutes to denature the enzyme, thus avoiding further
hydrolysis. The boiled samples were filtered through 0.45-um nylon membrane
filters. The reducing sugar concentration was measured using the DNS assay
(Miller, G.L., Anal. Chem. 1959, 31, 462) with glucose as the calibration
standard. Thus, the sugar yields are reported as equivalent glucose/g dry
biomass. Both cellulase and cellobiose contain sugars. To measure these
sugars, enzymes were added to 150 mL of water in. the same concentration as
used previously, but without any biomass. One mL samples were taken to
measure the sugar concentration. This measured sugar in the enzyme amounts
to a correction of 45 mg eq. glucose/g dry biomass that was subtracted from
the 3-day reducing sugar yields from the pretreated biomass. The hydrolysis
samples were diluted from 13 to 33 times to bring the concentration within the
assay range (0.1 - 1.0 mg/mL).
A detailed hydrolysis procedure is given below:
1. Open one end of the reactors and empty the contents (as much
as possible) into the labeled 500-mL Erlenmeyer flasks.
2. To completely transfer the biomass, use water to wash the
reactors. Pour this water and biomass mixture into the flasks. Add enough
water such that the total liquid volume (water added during the wash + water
added during pretreatment) is 140 mL.
3. Add glacial acetic acid to the mixture until the pH reaches 4.8.
During acetic acid addition, continuously monitor the pH and stir using a
magnetic bar. Note the volume of acetic acid added. If the pH goes below
4.8, use Ca(OH)2 to raise it to 4.8.
4. Add more water to bring the total liquid volume to 150 mL.
TM
5. Add 0.259 g cellulose powder "Cytolase 300 P" (filter paper
activity, 215 IU/g powder) and 0.652 mL cellobiose "Novozyme' (activity 250
CA 02615904 2008-01-23
26
CBU/mL). CytolaseM300 P was supplied by Genecor, Inc. (South San
Francisco, CA) and cellobiose was supplied by Novo Laboratories (Wilton,
CT). The cellulase loading was 7.4 IU/g dry pretreated lignocellulose and the
cellobiose loading was 22 CBU/g dry lignocellulose.
6. Place the flasks inside the 100-rpm shaking air bath at 50 C.
7. Close flasks with rubber stoppers after flasks have been warmed
for 10 min.
8. Keep flasks in the bath for 3 days.
9. Withdraw 1 mL of sample and boil them for 30 min. in capped
test tubes.
10. Filter samples through 0.45 um nylon membrane filter. Perform
DNS assay to measure reducing sugars as explained below.
Example 6 Dinitrosalicylic Acid (DNS) Assay
The DNS assay is the most commonly used technique for
measuring reducing sugars released by cellulose hydrolysis. A glucose standard
is used for the calibration, thus the reducing sugars are measured as
"equivalent glucose."
Prepare DNS reagent:
1. Dissolve 10.6 g of 3,5-dinitrosilicylic acid crystals and 19.8 g
NaOH in 1416 mL of distilled water.
2. Add 306 g Na-K tartrate (Rochelle salts).
3. Melt phenol crystals under a fume hood at 50 C using a water
bath. Add 7.6 mL of phenol to above mixture.
4. Add 83 g sodium meta-bisulfite.
5. Add NaOH, if required, to the solution obtained to adjust pH to
12.6.
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Calibrate DNS reagent:
1. Using a 200 mg/dL (2mg/mL) glucose standard, prepare 1 mL
samples in pairs of test tubes according to Table 5.
2. Take 0.5 mL of each sample.
3. Dispense 1.5 mL of DNS regent into each test tube using a 5-mL
Brinckmann dispensette.
4. Place the caps on the tubes and vortex.
5. Boil samples in a water bath for 15 minutes.
6. Cool the test tubes for a few minutes. Add 8 mL of distilled
water and vortex.
7. Zero the spectrophotometer at 550 run with distilled water (Note:
to stabilize the spectrophotometer it should be turned on for at least 1 hour
before using).
8. Measure the absorbance.
9. Prepare a calibration curve as shown in Figure 4.
Measure reducing sugars of samples:
1. Dilute the filtered sample into a pair of test tubes' such that the sugar
concentration lies between 0.1 to 1.0 mg/mL.
2. Vortex the diluted sample.
3. Pipette 0.5 mL of each diluted sample.
4. Dispense 1.5 mL DNS reagent into each test tube.
5. Repeat steps 4 to 8 used in preparation of calibration curve.
6. Calculate sugar concentration from the absorbance of the samples using
the calibration curve.
7. Calculate the reducing sugar yield by the following expression:
Y = S x D x 20 Y = reducing sugar yield (mg eq. glucose/g dry biomass)
S = sugar concentration in sample (mg eq. glucose/mL)
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D = dilution factor
20 = 150 mL liquid volume/7.5 g dry biomass
Example 7 Calcium Hydroxide Recovery
Two factors motivate recovery of calcium hydroxide from the
pretreated biomass. First, an inexpensive recovery and recycle process will
reduce the pretreatment costs. Second, high calcium residues have a
detrimental effect on its use as cattle feed. Thus, reducing the calcium
content
results in a more utilizable material. The method for recovering Ca(OH)2 is
to wash the pretreated material with water, and to contact or react this wash
water containing lime with carbon dioxide. This converts soluble Ca(OH)2 to
insoluble CaCO3 that can be removed by precipitation. The CaCO3 can then
be heated to produce CaO and CO2. The CaO is hydrated to Ca(OH)2 which
can be reused as the lignocellulose treatment agent. Carbon dioxide can, in
turn, be reused for lime recovery. Thus, ideally, it is a system capable of
total
recycling.
The carbonate concentration is quite low when the pH is below
9.5. Thus, to form and precipitate more CaCO3, the pH was maintained above
9.5.
All the recovery experiments were done using bagasse. The
experiments to study the recovery process were conducted by two different
approaches: Continuous Recovery and Batch Recovery.
1. Continuous Recovery
A systematic flow diagram of the continuous recovery
experimental apparatus is shown in Figure 5. The pretreated bagasse was
packed in a 1" I.D. x 8.5" high glass column. Rubber stoppers at both ends had
connections for the inlet (bottom) and the outlet (top). Filters (nylon cloth)
were glued on both stoppers. A peristaltic pump (Watson-Marlow, 502S)
CA 02615904 2008-01-23
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pumped water through the column. The average volumetric flow rate was 20
ml/min. The outlet from the column went to a 300-mL flask. Carbon dioxide
was bubbled thought the lime-saturated liquid in this flask to produce CaCO3.
A pH probe was placed in this flask to continuously monitor the pH. The pH
was maintained near 95 by bubbling only as much CO2 as was required to
lower the pH from about 12.0 to 9.5. However, because good pH control was
lacking, when the pH dropped below 9.5, the required amount of NH4OH
(about 1 or 2 mL) was added to bring the pH back to 9.5. The overflow from
the flask went to a filter assembly. Although most of the CaCO3 remained in
the flask, filtration was required to remove the CaCO3 in the overflow.
A glass fiber filter (G6, Fisher Scientific, Inc.) was placed between the
fritted
glass filter and glass jar. A clamp was used to hold it and provide a good
seal.
The pump suction was the driving force for filtration. The filters were
replaced periodically when they clogged with the CaCO3 paste. The filtered
water was then pumped back to the column for washing, thus completing the
cycle.
The washing was stopped after about one hour. The wash water
left in the glass jar clamped to the filter, was transferred to the flask and
left
for 24 hour to let CaCO3 settle. Clear liquid (1 mL) was taken from the top
of the flask. The clear liquid was decanted slowly and collected in another
beaker. The bottom portion containing much higher amounts of CaCO3
discarded after measuring its volume. The same volume of fresh water was
added to the system so that the liquid volume before and after precipitation
and decantation remained the same. Further recovery of the lime remaining
in the biomass was performed with this batch of decanted water for about 45
minutes. After the wash, the CaCO3-saturated water was again left for
CA 02615904 2009-12-03
precipitation and a sample of clear liquid was taken after this second
precipitation.
To measure the calcium concentration in the water during
washing, 1-mL samples were periodically taken from the column inlet and
outlet. The calcium concentration was measured by the atomic absorption
apparatus available in the Kinetics Group of the Texas A&M Chemical
Engineering Department. Depending on the calcium concentration, the
samples were diluted over the temperature range 11 C to 135 C since the best
yields were obtained
between 65 C and 100 C, this wide range ensured that the optimal
temperature was found. The pretreatment times of 1 to 36 hours were logical
choices since longer times were difficult to justify economically. Since
mixing
would ensure uniformity of the reaction mixture and probably result in a
better
pretreatment, continuous shaking was always employed except in one study that
used periodic shaking.
In all the experiments, the 3-day reducing sugar yields were used
as the measure of enzymatic susceptibility of lime-treated bagasse. The
reducing sugar yields were calculated as mg equivalent glucose/g dry bagasse.
Typically, 50% and 85% of the 3-day sugars were released in 6 hours and 24
hours, respectively.
Table 7 summarizes the conditions and the reactor systems used
in the various bagasse experiments.
The experiment at 50 C (Bagasse Experiment, Example 2), lime
loading = 5 g Ca(OH)Z/100 g dry bagasse, water loading = 10 g water/g dry
baggase, and treatment times of 1 and 24 hours was repeated thrice to find the
error involved in measuring the 3-day reducing sugar yields. For the 1 hour
run, the yields were 112, 125, 111 mg eq. glucose/g dry bagasse, showing a
standard deviation of 7.8 mg eq. glucose/g dry bagasse. For the 24 hour run,
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31
the yields were 273, 256, 268 mg eq. glucose/g dry bagasse, showing a standard
deviation of 8.7 mg eq. glucose/g dry bagasse. These standard deviations can
be generalized to apply to the rest of the experiments, and thus other
experiments were not repeated.
Example 8 Calcium Acetate Inhibition Experiment
High calcium acetate concentrations are present in the hydrolysis
mixture since acetic acid (about 5 mL glacial acetic acid for a sample treated
with a lime loading of 30 g Ca(OH)2/100 g dry bagasse) is used to neutralize
the lime for pH adjustment. To measure the calcium acetate inhibition of
enzymes, an experiment was performed in which group bagasse was
ammoniated at the reported optimum conditions. These ammoniation
conditions were: temperature = 93 C; treatment time = 30 min., water loading
0.25 g water/g dry bagasse, ammonia loading = 1.5 g NH3/g dry bagasse,
particle size = 40 mesh.
Also, there was no explosion (as used in Ammonia Fiber
Explosion process) since the pressure was slowly released. This pretreated
material was hydrolyzed in enzyme solutions containing various calcium acetate
concentrations. The calcium acetate solutions were prepared by adding various
amounts of Ca(OH)2 (according to the lime loadings used for the
pretreatment) to 150 mL water, and then adding acetic acid to reduce the pH
to 4.8. Thus, the calcium acetate concentration in these solutions was the
same
as for lime-pretreated materials. The enzymes were added to solutions only
after the pH was brought to 4.8, thus there was no loss of enzyme acidity due
to high pH. The 3-day sugar yield obtained from this ammonia-treated material
clearly shows that increased calcium hydroxide loadings decrease sugar yields
due to calcium acetate inhibition of the enzyme. For the sample hydrolyzed
without addition of Ca(OH)2 to the saccharifictions flask, the sugar yield was
CA 02615904 2010-11-25
32
390 mg eq. glucose/g dry bagasse. This yield is 1.16, 1.14, 1.16, 1.15, 1.25
and
1.22 of that obtained from samples hydrolyzed in the solutions with 2, 5, 10,
15,
20 and 30 g Ca(OH)2/100 g dry bagasse, respectively. In the subsequent
experiments using lime pretreatments, these factors were used to correct the
sugar yields. Since this approach is simplified and only approximately
corrects
for calcium acetate inhibition, the original data (without the correction
factor)
are reported in addition to the corrected data.
Example 9 Overview of Data (Bagasse)
The 3-day reducing sugar yield for untreated bagasse sample
(used as a control) was 40 mg eq. glucose/g dry bagasse which is only about
6% of the theoretical yield. Many different conditions as listed in Table 7
produced high sugar yields. Six high yielding conditions are tabulated in
Table
8. The choice of conditions to be used industrially will depend not only on
the
sugar yields, but also on the expense associated with conditions.
Example 10 Softwood Newspaper Study
Softwood newspaper is the largest fraction of most residential
municipal solid waste. Profitable utilization of softwood newspaper could help
solve the ever-growing trash disposal problem. The composition of softwood
newspaper is about 70% polysaccharides and 30% lignin, thus, the theoretical
yield is bout 750 mg eq. glucose/g dry newspaper. The purpose of this study
was to check the feasibility of using Ca(OH)2 to pretreat newspaper. The
conditions used for pretreating softwood newspaper are summarized in Table
9.
Example 11 Overview of Data (Newspaper)
The 3-day reducing sugar yield from an untreated softwood
newspaper sample, used as control, was 240 mg eq. glucose/g dry newspaper.
Pretreatment processes that work well for many lignocellulosics do not work
CA 02615904 2008-01-23
33
well for softwood newspaper. This probably results from its high lignin
content. The yield improvements with Ca(OH)2 pretreatment found in this
present study are comparable to other pretreatments.
Of all the conditions tested, the best yield was obtained for
120 C, 24 hours, 30 g Ca(OH)2/100 g dry newspaper and 16 g water/g dry
newspaper. This yield was 344 mg eq. glucose/g dry newspaper (corrected: 430
mg/g). An interesting observation is that the pretreatment works better for
either very severe conditions (120 C, 24 hours) or very mild conditions (65 C,
1 hour).
Example 12 Wheat Straw Study
Wheat straw is one of the most abundant agricultural crop
residues. In the United States, about 20% of cropland produces wheat, thus
large quantities of wheat straw are generated. Typically, based on dry weight,
wheat straw is 39% cellulose, 36% hemicellulose, and 10% lignin. According
to this composition, the maximum theoretical yield is about 800 mg eq.
glucose/g dry wheat straw.
The results for bagasse were used to guide the selection of
treatment conditions. The lime loadings were 5, 10, 15 and 20 g Ca(OH)2/100
g dry wheat straw. Only two water loadings were used. Treatment
temperatures of 50, 65, 85 and 125 C, and treatment times of 1, 3 and 24
hours, were studied. A summary of the treatment conditions is tabulated in
Table 10.
Example 13 Overview of Data
As in the case of bagasse, several different conditions produced
good yields. Since treatment time and temperature play the most important
role in process economics, the selection of conditions will not depend
strictly
CA 02615904 2008-01-23
34
on sugar yields. Eight conditions that produced good yields are tabulated in
Table 11.
Example 14 Calcium Recovery Study
The best yields for both bagasse and wheat straw were obtained
for a lime loading of 10 to 20 g Ca(OH)2/100 g dry material. Thus, an
industrial process using such loadings would need significant amounts of lime.
A process to recover and recycle lime can reduce the total of lime
requirement. Since calcium hydroxide is quite cheap, one main requirement
for a recovery process is that it should be simple and inexpensive.
A new recovery process was developed in this invention.
Ca(OH)2 was leached or washed out of the pretreated biomass. The lime-
saturated wash water was carbonated to convert Ca(OH)2 to insoluble CaCO3
that was subsequently settled. The recovered CaCO3 can be calcinated to form
CaO, which can be hydrated to Ca(OH)2, and thus reused. In this study, the
washing, carbonation and precipitating steps were performed. The experiments
were conducted by two different approaches, namely, continuous and batch
recovery processes.
Example 15 Continuous Recovery Process
Three runs were performed to recover Ca(OH)2 from bagasse by
the continuous recovery method. The lime treatment conditions used in the
continuous lime recovery study are tabulated in Table 12. The recovery results
for the corresponding samples are tabulated in Table 13.
The calcium content in the raw bagasse sample was 0.4 g Ca/100
g dry bagasse. The residual calcium content in the bagasse sample was brought
down to, about 2 g Ca/100 g dry bagasse from 5.4 g Ca/100 g dry bagasse
(Samples A and C) and to 2.3 g Ca/100 g dry bagasse from 8.1 g Ca/100 g dry
bagasse (Sample B). This shows that 68% of the added calcium was removed
CA 02615904 2008-01-23
from Samples A and C and 75% of added calcium was removed from Sample
B. The reduction in a content showed that the recovery process is working
fairly well. However, if the pretreated lignocellulosics are to be used as
cattle
feed, this residual calcium concentration might be slightly high (1 to 2 g
5 Ca/ 100 g dry biomass is desired).
One of the main drawbacks observed with the continuous
recovery experiment was that there was some channeling inside the bagasse-
filled column. Thus, the wash water was not contacting all of pretreated
material. This definitely would lower the process effectiveness. With the use
10 of some packing material and an efficient column design, it might be
possible
to increase the continuous recovery process efficiency. However, this
invention
used a second approach (i.e. batch recovery) that provides good contact by
mixing the wash water and biomass.
Example 16 Batch Recovery Process
15 In this experiment, instead of packing the pretreated material in
a column, a glass beaker was used to mix the biomass with wash water. This
wash water was saturated with CaCO3 and had a pH of 8.7. The lime-
saturated wash water obtained after filtration was contacted with CO2 and the
resulting CaCO3 containing solution was allowed to settle. The pretreatment
20 conditions and the corresponding calcium contents are given in Table 14 and
Table 15, respectively.
The first three samples (D, E, and F) used six washings whereas
the other samples used ten washings. For a lime loading of 10 g Ca(OH)2/100
g dry bagasse (Samples D and E), with six washings, the calcium concentration
25 was reduced to about 1.7 g Ca/100 g dry bagasse, and for a lime loading of
15
g Ca(OH)2/100 g dry bagasse (sample P) with six washings, the calcium
CA 02615904 2008-01-23
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concentration was reduced to about 2.2 g Ca/100 g dry bagasse. Thus about
75% of added calcium was removed.
Sample F and G received the same lime treatment. Whereas
sample F was washed six times Sample G received four additional washings.
These additional washings reduced the residual calcium content from 2.2 to 1.5
g Ca/ 100 g dry bagasse. Thus ten washings were able to remove 86% of the
added calcium.
Since it is possible that the calcium atoms are chemically bound
to the cellulose and other macromolecules of bagasse, simple washing with
water may not work beyond a certain limit. To explore the possibility that
bound calcium ions (+2), could be replaced by NH4+ ions, the lime-treated
bagasse was washed with an ammonium hydroxide solution. The ammonium
hydroxide concentration varied from 0.2 to 8.7 g NH4OH/100 g water. The
KH4OH was a 30% (w/w) solution of ammonia in water.
These experiments shoved that the ammoniated wash water was
not able to further recover calcium.
In all the previous samples, ammonia was added to the
carbonated water to adjust the pH to 9.5. This ensured that the carbonate ions
dominated, thus enhancing the precipitation of CaCO3. For Samples R and I,
samples for calcium analysis were drawn from settled wash water that was
carbonated to pH 6.7, and also from settled carbonated wash water that has
been raised to pH 9.5 by adding ammonia. It was found that it was essential
to have the pH about 9.5 in order to effectively recover the CaCO3. Although
this experiment used ammonia to adjust the pH to 9.5, industrially this would
be achieved by having a good control on the CO2 addition. After lime
treatment, the pH is about 11.5. Only the amount of CO2 needed to reduce the
pH to 9.5 would be added.
CA 02615904 2008-01-23
37
All the recovery experiments show that except for time loading,
the other pretreatment conditions (i.e. treatment time, temperature and water
loading) do not affect the recovery process. To determine if there are
pretreatment reactions that hinder the recovery process, lime was recovered
from untreated material, i.e. a physical mixture of bagasse and Ca(OH)2. The
calcium concentration in the wash water had a pattern similar to the other
samples. The residual calcium content was 1.3 g Ca/100 g dry bagasse which
is the lowest obtained from all the recovery experiments. Thus, there seems
to be greater calcium binding to the biomass resulting from pretreatment
process. However, the effect is small since ten washings of pretreated
material
had residuals of about 1.5 to 1.7 g Ca/100 g dry bagasse. Thus, there is not
much difference in the recovery process for the pretreated material and for
the
untreated material.
An important question is how far can one atmosphere CO2 lower
the pH. Trichoderma reesei cellulase operates at pH 4.8. The pH of pretreated
biomass is about 11.5, so an acid must be added to lower the pH. During
alcohol fermentation processes, much C02 is generated, so it will be the
cheapest acid for pH adjustment. To answer this question, a simple
experiment was performed in which a few lime-saturated wash water samples
were bubbled with 1 atmosphere CO2 for about 15 min. The minimum pH that
was reached was about 6.5. Thus a stronger acid than CO2 will be required to
lower the pH to 4.8. It would be.more desirable to use a cellulase system that
operates at pH 7 such as those in bacteria (e.g. Clostridium thermocellum).
Thus it is seen that calcium hydroxide is an excellent
pretreatment agent. Many different conditions produced high sugar yields.
There was no significant effect of water loading on sugar yields, although 10
g water/g dry material produced slightly higher yields. Lime loadings of 10
CA 02615904 2008-01-23
38
and 15 g Ca(OH)2/100 g dry material worked well. It was generally found that
the lower temperatures (50 C, 65 C) required longer times (24 hours), whereas
higher temperatures (135 C) needed shorter times (1 hour) to produce high
yields.
The recovery process was able to reduce the calcium content in
biomass from 8.1 to about 1S g Ca/100 g dry material, thus recovering 86%
of the added calcium.
Example 17 Circulation Methods
To prevent the addition of excess lime, the biomass may be
contacted with a solution of hot lime water. This may be accomplished in a
number of ways:
Method 1: A solution of hot (temperature range of from about 40 C
to about 150 C) saturated lime water is circulated through a packed bed of
biomass for a time period of from about 1 hour to about 36 hours. As the
solution exits the bed, it is heated to replace heat loss in the packed bed.
The
solution of saturated lime water can be prepared by mixing excess lime with
water and separating the excess solid lime from the water phase by filtration,
hydroclone separation (Figure 6), settlement, or centrifugation.
Method 2: A solution of hot (temperature range of from about 40 C
to about 150 C), saturated lime water is circulated through a stirred slurry
of
biomass for a time period of from about 1 hour to about 36 hours. The
biomass is separated from the solution by a filter, hydroclone (Figure 7),
settler, or centrifuge. As the solution exits the hydroclone, it is heated to
replace heat loss in the stirred slurry of biomass. The solution of saturated
lime water can be prepared by mixing excess lime with water and separating
the excess solid lime from the water phase by filtration, hydroclone
separation
(Figure 7), settlement, or centrifugation.
CA 02615904 2008-01-23
39
In either Method 1 or Method 2, the lime solids must always be
kept in contact with hot water at the highest temperature in the loop, such as
between 40 C and 150 C. This is required because lime is relatively more
soluble in cold water than in hot water. if the water contacting the lime
solids
were cold, it would dissolve too much lime. Then, if the lime solution were
heated later, lime would either precipitate out and foul the heat exchanger or
deposit on the biomass. This explains why the water is heated before it
contacts lime solids rather than after.
Example 18 Different Permutations
The materials needed for this invention include: Biomass, lime,
or calcium hydroxide, and water. The manipulative procedures for this
invention include: Mixing, heating, and simultaneous mixing and heating.
Thus, these materials and procedures are capable of a number of permutations,
as diagrammatically shown in Figure 8. The notations used in Figure 8 are: L -
lime; W - water; B - biomass; M - mixing; H - heating; and M & H -
simultaneous mixing and heating. An arrow in Figure 8 signifies adding,
introducing, or a step to be performed.
Example 19 Lime Treatment Process for Ruminant Animal Feed Production
Figure 9 shows the lime treatment process for ruminant animal
feed production. The lime may be added directly to the biomass, as shown.
Alternatively, the biomass may be contacted with a circulating solution of
saturated lime water to avoid excess lime addition. Regardless of the addition
method, the raw biomass is soaked in hot (ca. 65 C) lime water for about 24
hours. Then, after the reaction is complete, the lime water is circulated
through an adsorption column where it is contacted with carbon dioxide. The
carbon dioxide reacts with the lime to form insoluble calcium carbonate which
is filtered out and sent to a lime kiln. The calcium carbonate is heated to
CA 02615904 2008-01-23
about 1200 C in the lime kiln which drives off the carbon dioxide. The hot
exit
gases (primarily carbon dioxide with some nitrogen from the combustion air)
are cooled in a countercurrent beat exchanger recovering high-pressure steam
that may be used for electricity production and/or process heat. The cooled
5 carbon dioxide is recycled to the absorption column where the carbon dioxide
again reacts with lime water. Inert, such as nitrogen, will exit the
absorption
column. Make-up carbon dioxide must be added to replace any losses.
The circulating lime water will contain free sugars and protein
extracted from the biomass. In addition, there will be some calcium acetate
10 produced from the acetyl groups on the hemicellulose. A bleed stream will
be
taken off from the circulating loop that will be acidified with sulfuric acid
so
the calcium ions are precipitated as gypsum. The free sugars, protein, and
acetic acid (HAc) will be concentrated by an appropriate technology (e.g
reverse osmosis or multi-effect evaporation). It may be sold as monogastric
15 (e.g. chickens, pigs) animal feed.
The treated biomass will emerge from the process in a wet state.
If the ruminant animals are located close to the processing plant, they may
eat
it directly in the wet state. If it must be stored for awhile before it is
consumed, it may be dried in the steam driers we have previously described in
20 past disclosures.
Example 20 Pretreatments with Calcium Hydroxide and Pressurized Oxygen
Oxygen is believed to partially oxidizes the lignin which opens the
biomass structure making it more enzymatically digestible. Oxygen from high-
pressure oxygen tanks was added to samples of newspaper under increasing
25 pressure (Figure 10). Treatment conditions were consistent with those
previously described. Temperature of the biomass was 120 C for 24 hours with
30 g calcium hydroxide/ 100 g dry newspaper, and water at 16 g/g dry
CA 02615904 2008-01-23
41
newspaper. Increasing amounts of oxygen-containing gas was introduced to
samples of newspaper from high-pressure tanks. After pretreatment, samples
were digested enzymatically and the yield of glucose determined after three
days. Newspaper treated with only lime resulted in a yield of 418 mg eq.
glucose/g dry newspaper. For comparison purposes, untreated raw newspaper
yields 240 mg eq. glucose/g dry newspaper. With 20 pounds/square inch gauge
(psig), glucose yield increased over 10%. With increasing oxygen pressure up
to 100 psig, glucose yield increased to over 500 mg eq.
Useful oxygen pressures are between about 20 psig to about 500
psig and preferable are between about 100 to about 400 psig. Oxygen is
supplied to the biomass from high-pressure sources such as pure oxygen gas,
oxygen-containing gas, or compressed air.
In a second series of experiments (Figure 11), samples of
newspaper were pretreated with lime at 30 g/100 g dry newspaper, water at 6
g/g dry newspaper, and oxygen at 100 psig. These sample were pretreated for
up to 24 hours. The pretreated samples were enzymatically digested and the
sugar content determined. Maximal yield with oxygen pressures of 100 psig
was observed at three hours of pretreatment - 580 mg eq. of glucose/g of dry
newspaper.
Other embodiments and uses of the invention will be apparent
to those skilled in the art from consideration of the specification and
practice
of the invention disclosed therein. It is intended that the specification and
examples be considered exemplary only, with the true scope and spirit of the
invention being indicated by the following claims.
CA 02615904 2008-01-23
42
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