Note: Descriptions are shown in the official language in which they were submitted.
CA 02576317 2007-01-25
r A;
METHOD OF OBTAINING INORGANIC SALT AND ACETATE SALT FROM CELLULOSIC
BIOMASS
[0001] The present invention relates to a method of obtaining inorganic salt
and
acetate salt from cellulosic biomass, more particularly to a method of
obtaining inorganic
salt and acetate salt produced from the enzymatic conversion of cellulosic
biomass to
sugar.
BACKGROUND OF THE INVENTION
[0002] Fuel ethanol is currently produced from feedstocks such as cornstarch,
sugar cane, and sugar beets. However, the production of ethanol from these
sources
cannot expand much further due to limited farmland suitable for the production
of such
crops and competing interests with the human and animal food chain. Finally,
the use of
fossil fuels, with the associated release of carbon dioxide and other products
in the
conversion process, is a negative environmental impact of the use of these
feedstocks
[0003] The possibility of producing fuel ethanol from cellulose-containing
feedstocks, such as agricultural wastes, grasses, forestry wastes, and sugar
processing
residues has received much attention due to the availability of large amounts
of these
inexpensive feedstocks, the desirability to avoid burning or landfilling
cellulosic waste
materials, and the cleanliness of ethanol as a fuel compared to gasoline. In
addition, a
byproduct of the cellulose conversion process, lignin, can be used as a fuel
to power the
cellulose conversion process, thereby avoiding the use of fossil fuels.
Studies have
shown that, taking the entire cycle into account, the use of ethanol produced
from
cellulose generates close to nil greenhouse gases.
[0004] The cellulosic feedstocks that may be used for ethanol production
include
(1) agricultural wastes such as corn stover, wheat straw, barley straw, canola
straw, and
soybean stover; (2) grasses such as switch grass, miscanthus, cord grass, and
reed canary
grass; (3) forestry wastes such as aspen wood and sawdust; and (4) sugar
processing
residues such as bagasse and beet pulp.
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[0005] Cellulose consists of a crystalline structure that is very resistant to
breakdown, as is hemicellulose, the second most prevalent component. The
conversion
of cellulosic fibers to ethanol requires: (1) liberating cellulose and
hemicellulose from
lignin or increasing the accessibility of cellulose and hemicellulose within
the cellulosic
feedstock to cellulase enzymes; (2) depolymerizing hemicellulose and cellulose
carbohydrate polymers to free sugars; and (3) fermenting the mixed hexose and
pentose
sugars to ethanol.
[0006] The feedstock is conveyed into the plant and the feedstock particles
are
typically reduced to the desired size to be suitable for handling in the
subsequent
processing steps. The next process step is a chemical treatment, which
generally involves
the use of steam or heated water along with acid or alkali to break down the
fibrous
material. The chemical treatment is carried out either as a direct conversion
process- acid
hydrolysis or alkali hydrolysis- or as a pretreatment prior to enzymatic
hydrolysis.
[0007] In the acid or alkali hydrolysis process, the feedstock is subjected to
steam
and acid or alkali under conditions sufficient to hydrolyze the cellulose to
glucose
(Grethlein, J. Appl. Chem. Biotechnol., 1978, 28:296-308). The glucose is then
fermented to ethanol using yeast, and the ethanol is recovered and purified by
distillation.
[0008] The enzymatic hydrolysis process involves pretreating the cellulosic
material in a process that is analogous to the acid or alkali hydrolysis
process described
above, but using milder conditions. This pretreatment process increases the
accessibility
of cellulose within the cellulosic fibers for subsequent conversion steps, but
results in
little conversion itself. In the next step, the pretreated feedstock is
adjusted to an
appropriate temperature and pH for enzymatic conversion of cellulose by
cellulase
enzymes. The reaction conditions for the pretreatment process are chosen to be
significantly milder than that in the acid or alkali hydrolysis process, such
that the
exposed cellulose surface area is greatly increased as the fibrous feedstock
is converted to
a muddy texture.
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[0009] In the case of acid pretreatment, much of the hemicellulose is
hydrolyzed,
but there is little conversion of the cellulose to glucose. The cellulose is
hydrolyzed to
glucose in a subsequent step that uses cellulase enzymes, and the hydrolysis
in this case is
known as pretreatment. Alkali pretreatment methods do not hydrolyze
hemicellulose, but
rather the base reacts with acidic groups present on the hemicellulose to open
up the
surface of the substrate. In addition, it has been reported that alkali alters
the crystal
structure of the cellulose so that it is more amenable to hydrolysis. The
cellulose is then
typically hydrolyzed to glucose in a subsequent step that uses cellulase
enzymes,
although it is possible to hydrolyze the cellulose, in addition to the
hemicellulose, using
acid hydrolysis after alkaline pretreatment.
[0010] The hydrolysis of the cellulose, whether by acid, alkali or by
pretreatment
followed by enzyme hydrolysis, may be followed by the fermentation of the
sugar to
ethanol, which is then recovered by distillation. Other fermentation products
that may be
produced include butanol and lactic acid.
[0011] The efficient conversion of cellulose from cellulosic material into
sugars
and the subsequent fermentation of sugars to ethanol or other valuable
products represent
a major challenge to the industry. In particular, a large amount of
impurities, including
salt, sugar degradation products, organic acids, soluble phenolic compounds,
and other
compounds are present in the sugar stream after the pretreatment. These
compounds
result from degradation of the feedstock or, in the case of the salts, from
the acids and
alkali added in the process. The presence of these impurities is highly
inhibitory to the
fermentation of the sugar by the yeast. In the absence of an efficient
fermentation of the
sugar in high yield, the production of ethanol from biomass is not
commercially viable.
Furthermore, the inability to recover acetic acid and salt from the sugar
streams, due to
the large amount of impurities present, represents a loss of potential revenue
in the
process.
[0012] The removal of toxic inhibitors, sulfuric acid and sulfate salts, and
acetic
acid and acetate salts from the sugar streams prior to fermentation has been
the subject of
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a significant amount of research. The processes studied include lime addition,
ion
exchange, and ion exclusion.
[0013] In lime addition, lime (calcium hydroxide), which is insoluble, is
added to
the sugar stream to precipitate impurities. The limed sugar solution has an
alkaline pH
and is neutralized with acid, typically phosphoric acid, sulfurous acid,
carbonic acid, or a
mixture thereof. Optionally, the lime cake is separated from the sugar by
filtration. A
second option is to filter the lime cake at alkaline pH and carry out a second
filtration to
remove material that precipitates during the acidification steps. Lime
treatment decreases
the toxicity of the sugar stream to yeast and other microbes. However, any
handling of
the lime cake is difficult and costly. In addition, the introduction of
calcium into the
stream increases the likelihood that calcium scale will deposit on
evaporators, distillation
columns, and other process equipment. The clean-up and avoidance of scale
increases
the cost of sugar processing. Furthermore, the introduction of lime makes the
recovery of
salt and acetic acid more difficult.
[0014] In ion exchange, the sugar stream is flowed through columns packed with
ion exchange resins. The resins are in a cation exchange or anion exchange
form, or a
combination of the two. In principle, cation-exchange resins remove cations
such as
sodium or potassium, while anion-exchange resins remove anions such as sulfate
and
acetate. For example, ion exchange has been investigated by Nilvebrant et al.
(App.
Biochem. Biotech., 2001, 91-93:35-49) in which a spruce hydrolyzate was
treated to
remove fermentation inhibitors, such as phenolic compounds, furan aldehydes
and
aliphatic acids. The separation was carried out using an anion exchanger, a
cation
exchanger and a resin without charged groups. The investigators found that
treatment at
pH 10.0 using an anionic exchanger removed phenolic inhibitors since at this
pH most of
the phenolic groups were ionized.
[0015] In practice, several factors limit the effectiveness of ion exchange
treatment to remove inhibitors. First, the multi-component nature of the
streams results
in an inefficient removal of some species at any single set of conditions.
Second, the
high ionic load demands very frequent and expensive regeneration of the resin.
Finally,
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not all of the inhibitors are ionic, and ion exchange is ineffective in
removing nonionic
compounds from sugar.
[0016] Ion exclusion uses ion exchange resins, but rather than bind target
ions in
solution, the charge on the resin matches that of the target ions in the
solution, thereby
excluding them from the resin. The excluded compounds then elute from the
column
readily, while uncharged compounds absorb into the resin and elute from the
column
more slowly. For example, a concentrated solution of sulfuric acid and glucose
has
hydrogen as the primary cation. A cation-exchange resin in the hydrogen form
will
exclude the acid, causing it to elute quickly. The glucose, which is
uncharged, is not
excluded from the resin and absorbs into the resin void, thereby eluting from
the column
more slowly than the acid.
[0017] Ion exclusion for detoxification of sugars from biomass streams has
been
described by various groups. For example, Wooley et al., (Ind. Eng. Chem.
Res., 1998,
37:3699-3709) teaches the removal of acetic acid and sulfuric acid from
biomass sugars
by pumping a product stream over a bed of cation exchange resin in the
hydrogen form.
The positive charge on the resin repels the hydrogen ion in the sulfuric acid,
thereby
causing the sulfuric acid to elute from the column very quickly. The uncharged
sugar
molecules are absorbed into the void space of the resin and elute from the
column more
slowly than the sulfuric acid. Fully associated acetic acid (non-ionic) is a
smaller
molecule than sugar or sulfuric acid and so elutes from the column more slowly
than
sulfuric acid or sugar. Also described is a Simulated Moving Bed (SMB) system
for
producing a glucose stream free of sulfuric acid and acetic acid. A
shortcoming of
Wooley's process is that the glucose recovery is only 92%. The 8% loss of
glucose
represents a significant cost in the system. The ion exclusion was carried out
at a pH of
between about 1-2 and, at such low pH values, significant degradation of
xylose is likely.
[0018] U.S. 5,560,827 and 5,628,907 (Hester et al.) disclose a process for
separating an ionic component (acid) from a non-ionic component (sugar) using
an SMB
arrangement, including a plurality of ion exclusion columns arranged in 4
zones. The
separations are run at a low pH using a cationic (or cation-exchange) resin in
the
CA 02576317 2007-01-25
hydrogen form. The methods of Hester incorporate various arrangements to
minimize
the dispersion and channeling effects. The sugar/acid solution is loaded onto
the column
and the acid elutes first while sugar is eluted later using water.
[0019] U.S. 5,407,580 (Hester et al.) discloses a process for separating an
ionic
component (acid) from a non-ionic component (sugar) using a preparative-scale
ion
exclusion system. The system includes a floating head distribution plate to
prevent
evolution of a dilution layer caused by the shrinkage of the resin bed. The
columns can
be operated over a range of process conditions to produce separate and
distinct elution
profiles for the acid and sugar. Acceptable conditions for carrying out the
process are at
a sulfuric acid concentration of 1.0 to 20.0%, a feed volume of 1.0 to 5.0, a
flux rate of
0.1 to 2.0 and using a divinylbenzene resin with a percent crosslinking of
between 1.0
and 15.
[0020] U.S. 5,580,389 and 5,820,687 (Farone et al.) teach a method of
producing
and separating sugars. The two-step method involves decrystallizing and
hydrolyzing
biomass using acid, then pressing the hydrolyzate and collecting the liquid,
which
contains acid and sugars. The liquid is loaded onto a cross-linked strong
cation exchange
resin run at low pH, where the sugars adsorb to the resin. The resin is purged
with gas,
pushing the acid out of the resin; the resin is then washed with water,
producing a sugar
stream.
[0021] U.S. 5,968,362 (Russo et al.) discloses a method of separating sugars
and
acid by ion exclusion chromatography using an anion exchange resin. The sugars
elute
through the column, and may contain residual acid and heavy metals. The heavy
metals
can be removed and the acid neutralized using a lime treatment. The acid
adsorbs to the
resin and is retained; it is eluted from the resin with water.
[0022] Nanguneri et al. (Sep. Sci. Tech., 1990, 25(13-15):1829-1842) simulated
the separation of sugars from acids using a modified mathematical model and
compared
the results obtained with experimental data. Separation performances at
different process
parameters were then analyzed to determine optimal processing conditions. The
simulated process would result in an acid-rich stream eluting first, followed
by a dilute
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acid/sugar interface stream and then a sugar-rich stream. Nanguneri et al.
performed an
economic analysis at the optimal processing conditions and concluded that ion
exclusion
is highly feasible for the processing of lignocellulosic feedstocks to produce
ethanol.
However, a drawback of the method of Nanguneri et al. is that the dilute
acid/sugar
interface stream is costly to separate and recover.
[0023] U.S. 6,663,780 (Heikkila et al.) discloses a method in which product
fractions, such as sucrose, betaine and xylose, are separated from molasses
that are
obtained from a variety of sources, including beet and cane molasses, as well
as
hydrolyzates produced from biomass. The process involves treating the molasses
with
sodium carbonate (pH 9) to precipitate calcium followed by removing the
resulting
precipitate. The filtrate is then subjected to a simulated moving bed (SMB)
process
which is carried out using at least two SMB systems packed with a strongly
acid cation
exchange resin. Sucrose is recovered in a first system and betaine is
recovered in a
second system. The sucrose obtained from the first system may be crystallized
and the
crystallization run-off applied to the second system. Also described is a
process for
recovering xylose from sulphite cooking liquor using two systems. Prior to
fractionation
in the first system, the sulphite cooking liquor, having a pH of 3.5, is
filtered and diluted
to a concentration of 47% (w/w). The xylose fractions obtained from the first
system are
crystallized and, after adjustment to pH 3.6 with MgO, the run-off is fed to
the second
system. In the second system, a sequential SMB is used to separate xylose from
the
crystallization run-off.
[0024] A disadvantage of the separation technique disclosed in U.S. 6,663,780
(Heikkila et al.) is that the inclusion of two SMB systems is costly and adds
to the
complexity of the process. In addition, sugars present in a hydrolyzate
produced by the
processing of lignocellulosic biomass are much more difficult to crystallize
than sucrose
in a beet process. The initial sucrose purification by crystallization in U.S.
6,663, 780 is
not successful with glucose in biomass systems.
[0025] Various groups have reported the separation of sucrose from molasses
obtained from sugar cane using ion exclusion chromatography or ion exchange.
For
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example, U.S. 4,359,430 (Heikkila et al.) discloses a method of recovering
betaine from
inverted molasses. The molasses are first diluted with water to a
concentration of 35-
40% and then applied to a column containing a cation exchange resin. On
elution with
water, a first non-sugar waste fraction is obtained, followed by a second
sugar-containing
fraction, and a third fraction containing betaine. The betaine is recovered by
evaporation
and crystallization. Although high levels of betaine are recovered, the patent
does not
address the recovery of sucrose from the sugar-containing fraction.
[0026] U.S. 6,482,268 (Hyoky et al.) also discloses a method of separating
sucrose and betaine from beet molasses by a simulated moving bed (SMB)
process.
Similar to U.S. 6,663,780, the method of Hy6ky et al. involves first
precipitating calcium
from the beet molasses by adding sodium carbonate and filtering the resulting
calcium
carbonate by filtration. The beet molasses are next applied to a column packed
with a
strong cation exchanger resin with a divinylbenzene backbone. A sucrose
fraction is
eluted first, followed by a betaine fraction, which is then concentrated and
further
fractionated to yield a second sucrose fraction and a second betaine fraction
containing
some sucrose. The second sucrose and betaine fractions are combined with the
sucrose
and betaine fractions obtained from the initial fractionation. Although Hyoky
et al.
describe the separation of sucrose and betaine from beet molasses, in a
biomass
conversion process, these components would not be present.
[0027] A method of separating sugar from molasses using ion exclusion
chromatography is taught in GB 1,483,327 (Munir et al.). The ion exclusion
column
comprises two types of cation exchange resins used in the salt form to help
prevent
shrinkage of the column bed. Sugar adsorbs to the column and is eluted using
decarbonized water adjusted to a pH of greater than 9.
[0028] WO 95/17517 (Chieffalo et al.) discloses a method of processing
municipal solid waste to recover reusable materials and to make ethanol.
Cellulosic
material is shredded and pre-treated with acid and lime to remove heavy
metals, then
treated with concentrated acid (sulfuric) to produce sugars. The sugars and
the acid are
separated on a strong acidic cation ion exchange resin.
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[0029] U.S. 4,101,338 (Rapaport et al.) discloses a method of separating salts
and
sucrose present in blackstrap molasses obtained from sugar cane by ion
exclusion
chromatography. Prior to ion exclusion chromatography, the molasses are
treated by
removing organic non-sugar impurities and colour. Various methods are
suggested for
removing these impurities, including a preferred method utilizing
precipitation with iron
salts, such as ferric chloride or ferric sulfate, to form flocs. The insoluble
flocs are then
removed from the molasses stream and the soluble iron salts are removed by the
addition
of lime and phosphoric acid or inorganic phosphate salts, which raises the pH
to above
7Ø The molasses stream is then applied to the ion exchange column to produce
fractions
containing sucrose and separated salts. A disadvantage of this process is
that, upon
addition of ferric ions, the molasses has a pH that is in the range of 2.0 to
3Ø At such a
low pH, degradation of xylose could occur. Furthermore, Rapaport et al. do not
address
the separation of acetic acid from sugars.
[0030] Organic non-carbohydrate impurities within a lignocellulosic system
cannot be removed by the methods of Rapaport et al. According to the method of
Rapaport, the amount of solids precipitated by iron salts or ethanol is modest
and no
solids are removed by centrifugation. By contrast, the sugar streams produced
during the
processing of lignocellulosic feedstock have a much higher level of organic
non-
carbohydrate impurities and inorganic salts. Rapaport et al. do not address
the processing
of such concentrated streams.
[0031] U.S. Patent No. 6,709,527 (Fechter et al.) discloses a process of
treating
an impure cane-derived sugar juice to produce white sugar and white strap
molasses. The
process involves subjecting the sugar juice to microfiltration/ultrafiltration
to decrease the
levels of impurities. The sugar juice is next subjected to ion exchange with a
strong acid
cation exchange resin in the hydrogen form and then to ion exchange with an
anion
exchange resin in the hydroxide form. After ion exchange, the resulting sugar
solution is
concentrated to produce syrup which is then crystallized to produce impure
crystallized
sugar and white strap molasses. Although the process results in the removal of
impurities
from the sucrose solution, it would be subject to the limitations associated
with ion
exchange chromatography described above.
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[0032] U.S. 4,631,129 (Heikkila et al.) teaches a method of purifying sugar
from
a sulfite pulping spent liquor stream. The process involves two steps, in
which, during
the first step, the pH of the spent sulfite liquor is adjusted to below 3.5
and the stream is
passed through a strongly acidic ion exclusion resin to recover two
lignosulfonate-rich
raffinate fractions and a product stream containing the sugar and consisting
of 7.8%-55%
lignosulfonate. In the second step, the product stream is adjusted to pH 5.5-
6.5. The
product stream is then filtered, and applied to a second ion exclusion column
to further
purify the sugar by separating it from the large amount of lignosulfonates in
this stream.
A problem with this process is that the use of two ion exclusion systems is
costly and
adds to the complexity of the process. Moreover, Heikkila et al. do not
quantify or
address the separation of compounds present during the processing of biomass
such as
inorganic salts, including sulfate salts, and acetic acid and other organic
acids.
[0033] Bipp et al. (Fresenius J. Anal. Chem., 1997, 357:321-325) describes the
analytical determination and quantification of sugar acids and organic acids
from whey
powder hydrolyzates by ion exclusion chromatography. The elution was carried
out with
0.005 M sulfuric acid (pH of 2.3) at a temperature of 45 C and 0.05 M (pH of
1.30) and a
temperature of 10 C. Although the analysis demonstrated that the method was
suitable
for the determination and quantification of organic acids, including sugar
acids and acetic
acid, the temperatures required for the separation would not be practical in
an industrial
application. Furthermore, such low pH values would likely result in the
production of
degradation products.
[0034] There is a need for an economical system for obtaining inorganic salt
and
acetate salt from processing of a cellulosic biomass. The development of such
a system
remains a critical requirement for the overall process to convert
lignocellulosic
feedstocks to glucose and subsequently to ethanol or other products.
SUMMARY OF THE INVENTION
[0035] The present invention relates to a method of obtaining inorganic salt
and
acetate salt from cellulosic biomass, more particularly to a method of
obtaining inorganic
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salt and acetate salt produced from the enzymatic conversion of cellulosic
biomass to
sugar.
[0036] It is an object of the present invention to provide a method of biomass
conversion having improved performance.
[0037] This process overcomes the disadvantages of the prior art by operating
an
ion exclusion at a much higher pH range than that previously reported for
process streams
arising from biomass conversion processes. The present invention is based on
the
discovery that at pH values of about 5-10, inorganic salts produced during
processing of
the cellulosic biomass and acetate salts arising from the pretreatment are
excluded by the
cation exclusion resin. This results in a similar elution of the inorganic and
acetate salts
at pH 5 to 10, thereby eliminating the need for using two streams to recover
inorganic
acid and acetic acid that is required at more acidic conditions. This, in
turn, decreases the
complexity of the system.
[0038] Therefore, the invention offers significant advances in obtaining
inorganic
and acetate salts during the conversion of lignocellulosic feedstocks.
[0039] The present invention provides a process (A) for obtaining an inorganic
salt and acetate salt by processing a cellulosic biomass, the process
comprising:
a) pretreating the cellulosic biomass by adding a base to the cellulosic
biomass to
produce a pretreated cellulosic biomass comprising the acetate salt;
b) adding an acid to the pretreated cellulosic biomass to adjust the
pretreated
cellulosic biomass to a pH of about 4.0 to about 6.0, thereby producing a
neutralized
cellulosic biomass;
c) hydrolyzing the neutralized cellulosic biomass with cellulase enzymes to
produce a crude sugar stream;
d) separating insoluble residue from the crude sugar stream to produce a
clarified
sugar stream; and
e) feeding an aqueous stream obtained from the pretreated cellulosic biomass,
an
aqueous stream obtained from the neutralized cellulosic biomass, the clarified
sugar
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stream, or a combination thereof, to an ion exclusion chromatographic
separation to
produce a salt stream comprising inorganic salt and acetate salt, which ion
exclusion
separation is performed at a pH range between about 5.0 and about 10.0 using a
cation
exchange resin, and obtaining the inorganic salt and acetate salt.
[0040] The present invention also provides a process as described above
(process
A), wherein the clarified sugar stream is fed to the ion exclusion
chromatographic
separation, and, wherein, the ion exclusion separation further produces a
purified sugar
stream. Furthermore, after the chromatographic separation, the purified sugar
stream
may be fermented, for example to produce to produce ethanol, lactic acid or
butanol.
[0041] The present invention is also directed to a process as described above
(process A), wherein the aqueous stream obtained from the pretreated
cellulosic biomass
maybe produced after a step of washing the pretreated biomass, in this case,
the aqueous
stream is then subjected to an acid addition step, or the aqueous stream
obtained from the
pretreated cellulosic biomass may be produced after a step of removing
insoluble solids
from the pretreated biomass, and the aqueous stream then subjected to an acid
addition
step.
[0042] The present invention also pertains to the process as described above
(process A), wherein the aqueous stream obtained from the neutralized
cellulosic biomass
is produced after a step of washing the neutralized biomass, or the aqueous
stream
obtained from the neutralized cellulosic biomass is produced after a step of
removing
insoluble solids from the neutralized biomass.
[0043] The present invention provides a process as described above (process
A),
further comprising a step of recovering the salt stream comprising inorganic
salt and
acetate salt. The inorganic salt from the salt stream may also be recovered
for use as a
fertilizer.
[0044] The present invention provides a process as described above (process
A),
wherein the cellulosic biomass is obtained from a feedstock selected from the
group
consisting of an agricultural waste, corn stover, wheat straw, barley straw,
canola straw,
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oat straw, rice straw, soybean stover, a grass, switch grass, miscanthus, cord
grass, reed
canary grass, a forestry residue, aspen wood or sawdust, a sugar residue,
bagasse and beet
pulp. Additionally, the acid may be sulfuric acid and the inorganic salt
comprises a
sulfate salt. The dosage of the cellulase enzymes may be from about 5 to about
50 IU per
gram of cellulose. The process may further comprise the addition of at least
one xylanase
enzyme during the step of hydrolyzing.
[0045] The present invention pertains to a process as described above (process
A), wherein in the step of pretreating (step a)), the base is a soluble base.
For example,
the soluble base may be selected from the group consisting of sodium
hydroxide,
potassium hydroxide, ammonia and ammonium hydroxide.
[0046] The present invention includes a process as described above (process
A),
wherein the ion exclusion chromatography is performed at a pH of between about
6.5 and
about 10.
[0047] The present invention provides a process (process B) for obtaining an
inorganic salt and acetate salt by processing a cellulosic biomass, the
process comprising:
a) pretreating the cellulosic biomass by adding a base to the cellulosic
biomass to
produce a pretreated cellulosic biomass comprising the acetate salt;
b) adding an acid to the pretreated cellulosic biomass to adjust the
pretreated
cellulosic biomass to a pH of about 4.0 to about 6.0, thereby producing a
neutralized
cellulosic biomass;
c) hydrolyzing the neutralized cellulosic biomass with cellulase enzymes to
produce a crude sugar stream;
d) fermenting the sugar stream to produce a fermentation broth comprising
ethanol;
e) distilling the fermentation broth to produce concentrated ethanol and still
bottoms; and
f) feeding the still bottoms to an ion exclusion chromatographic separation to
produce a salt stream comprising inorganic salt and acetate salt, which ion
exclusion
separation is performed at a pH range between about 5.0 and about 10.0 using a
cation
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exchange resin, and obtaining the inorganic salt and acetate salt.
[0048] The present invention provides a process as described above (process
B),
wherein wherein the ion exclusion separation step (f) is performed on an
aqueous stream
obtained from the still bottoms.
[0049] The present invention provides a process (process C) for obtaining
ethanol
by processing a cellulosic biomass, the process comprising:
a) pretreating the cellulosic biomass by adding a base to the cellulosic
biomass to
produce a pretreated cellulosic biomass comprising acetate salt;
b) adding an acid to the pretreated cellulosic biomass to adjust the
pretreated
cellulosic biomass to a pH of about 4.0 to about 6.0, thereby producing a
neutralized
cellulosic biomass;
c) hydrolyzing the neutralized cellulosic biomass with cellulase enzymes to
produce a crude sugar stream;
d) separating insoluble residue from the crude sugar stream to produce a
clarified
sugar stream; and
e) feeding the clarified sugar stream to an ion exclusion chromatographic
separation to produce a salt stream comprising inorganic salt and acetate salt
and a
purified sugar stream, which ion exclusion separation is performed at a pH
range between
about 5.0 and about 10.0 using a cation exchange resin;
f) fermenting the purified sugar stream to produce ethanol in a fermentation
broth; and
g) isolating the ethanol produced in step (f).
[0050] This summary of the invention does not necessarily describe all
necessary
features of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
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[00511 These and other features of the invention will become more apparent
from
the following description in which reference is made to the appended drawings
wherein:
[0052] Figure 1 shows a representation of the zones and liquid flows in a
Simulated Moving Bed (SMB) system.
[0053] Figure 2 shows separation of glucose and sodium sulphate and sodium
acetate using ion exclusion chromatography at different pH values. Figure 2A
shows the
elution of sodium sulfate, sodium acetate, and glucose at pH 8. Figure 2B
shows the
elution of sodium bisulfate, acetic acid, and glucose at pH 3.
[0054] Figure 3 shows separation of sugar (glucose, xylose and arabinose),
sodium sulphate and sodium acetate in a biomass conversion clarified sugar
stream using
ion exclusion chromatography performed at pH 8.
[0055] Figure 4 shows the elution of xylose and salts in a biomass conversion
process stream using ion exclusion chromatography performed at pH 7.
[0056] Figure 5 shows the separation of xylose from salts in a biomass
conversion
process using ion exclusion chromatography performed at pH 5. Figure 5A shows
the
elution of ammonium sulfate and xylose. Figure 5B shows the elution of sulfate
ions,
ammonium ions and xylose. Figure 5C shows the elution of ammonium sulfate,
xylose
and acetic acid.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0057] The present invention relates to a method of obtaining inorganic salt
and
acetate salt from cellulosic biomass, more particularly to a method of
obtaining inorganic
salt and acetate salt produced from the enzymatic conversion of cellulosic
biomass to
sugar.
[0058] The following description is of a preferred embodiment.
CA 02576317 2007-01-25
[0059] The process of the present invention allows for the removal of acetate
salt
and inorganic salts from streams that originate during the conversion of a
lignocellulosic
feedstock to sugar.
[0060] The sugar stream is the product of the conversion of a cellulosic
feedstock
to sugar by hydrolysis. By the term "lignocellulosic feedstock",
"lignocellulosic
biomass", "cellulosic biomass", or "biomass feedstock", it is meant any type
of biomass
comprising cellulose. Cellulosic biomass can consist of an entire plant or a
portion
thereof, or a mixture of plants or portions thereof, whichever may be the
source of crude
sugar for the process. The process of the invention is effective on a wide
variety of
biomass feedstocks, including: (1) agricultural wastes such as corn stover,
corn fiber,
wheat straw, barley straw, canola straw, oat straw, rice straw, and soybean
stover; (2)
grasses such as switch grass, miscanthus, cord grass, and reed canary grass;
(3) forestry
residues such as aspen wood and sawdust; (4) sugar residues, such as bagasse
or beet
pulp; and any combination thereof.
[0061] Cellulosic biomass comprises cellulose in an amount greater than about
20%, more preferably greater than about 30%, more preferably greater than
about 40%
(w/w), still more preferably greater than 50% (w/w). For example, the
lignocellulosic
material may comprise from about 20% to about 50% (w/w) cellulose, or more, or
any
amount therebetween. The cellulosic biomass may also contain lignin at an
amount
between greater than about 10%, or, more typically, in an amount greater than
about 15%
(w/w).
[0062] It is preferred that the feedstocks do not comprise molasses or spent
sulfite
liquor. Greater than about 80%, preferably greater than about 85% or 90% of
the sugar in
the sugar stream is the result of hydrolysis of cellulose and hemicellulose in
the cellulosic
feedstock. For example, 80, 82, 85, 87, 90, 92, 95, 97, or 100% of the sugar
in the sugar
stream may be derived from cellulose and hemicellulose. Furthermore, it is
preferred
that at least 50, 55, 60, 65, 70, 75, 80, 85 or 90 wt% of the cellulose in the
biomass is
converted to glucose.
16
CA 02576317 2007-01-25
[0063] The sugars may be produced by any method known in the art, for
example, but not limited to, subjecting the feedstock to acid or alkali
hydrolysis (e.g. as
disclosed in Brennan et al, Biotech,. Bioeng. Symp. No. 17, 1986, which is
incorporated
herein by reference). The acid or alkali hydrolysis may be carried out to
convert the
cellulose to glucose, convert a portion of the cellulose to glucose, convert
the
hemicellulose to its monomeric sugars of xylose, arabinose, galactose,
mannose, convert
a portion of the hemicellulose to its monomeric sugars, or a combination
thereof.
[0064] The acid used for acid hydrolysis may be any type of suitable acid
known
in the art, including, but not limited to, sulfuric acid. Sulfuric acid may be
used in a
dilute form from about 0.1% to about 5% on weight of feedstock or any amount
therebetween, or the sulfuric acid may be used in a concentrated form, for
example,
submersing the feedstock in from about 30% to about 80%, or any amount
therebetween,
solution of acid, by weight. It is preferred that the crude sugar stream is
characterized as
having a lignosulfonate content of less than 10% of the total dry solids of
the crude sugar
stream. For example, crude sugar streams characterized as having an amount of
lignosulfonate from about 0 to about 10% of the total dry solids of the crude
sugar
stream, or about 10, 8, 6, 4, 2, 1 or 0% of the total dry solids of the crude
sugar stream,
may be used in the process described herein.
[0065] The cellulosic biomass may be subjected to a pretreatment to increase
the
susceptibility of the cellulosic biomass to hydrolysis by cellulase enzymes.
This may
involve subjecting the biomass feedstock to an acidic pretreatment process to
convert
hemicellulose, a portion of the cellulose, a portion of the hemicellulose, or
any
combination thereof, to sugar, and the remaining cellulose may then be
converted to
glucose by enzymatic hydrolysis with cellulase enzymes. The acidic
pretreatment is
carried out to convert at least a portion of hemicellulose to xylose,
arabinose, galactose,
mannose and a small portion of the cellulose to glucose, and the remaining
cellulose is
then converted to glucose by enzymatic hydrolysis with cellulase enzymes. A
non-
limiting example of such a treatment includes steam explosion, as described in
U.S.
4,461,648 (Foody; which is incorporated herein by reference). Generally,
acidic
pretreatment conditions for lignocellulosic feedstocks comprise a temperature
in the
17
CA 02576317 2007-01-25
range of about 170 C to about 260 C, or any amount therebetween, for a period
of about
0.1 to about 30 minutes or any amount therebetween, and at a pH of about 0.4
to about
2.0 or any amount therebetween.
[0066] Examples of other acid pretreatment processes that are suitable for the
practice of this invention, which are not to be considered limiting, include
those
described in U.S. 5,536,325; U.S. 4,237,226; and Grethlein, J. Appl. Chem.
Biotechnol.,
1978, 28:296-308; which are incorporated herein by reference. After
pretreatment, the
pH of the material is adjusted to the appropriate range prior to enzymatic
hydrolysis.
[0067] The low pH for acid pretreatment may involve the addition of dilute
acid,
added to achieve a final concentration in the feedstock from about 0.02%(w/v)
to about
3%(w/v), or any amount therebetween. The acid used for pretreatment may be any
type
of suitable acid known in the art, including, but not limited to, sulfuric
acid, or
phosphoric acid. Sulfuric acid is preferred due to its low cost and, following
recovery, its
use in fertilizer in the form of sulfate salts. The term "sulfate salts"
includes bisulfate
salts that are also present, at a concentration that depends on the pH.
[0068] Alternatively, the pretreatment involves the addition of base to
produce an
alkali pretreated feedstock. Without wishing to be bound by theory, alkali
pretreatment
typically does not hydrolyze hemicellulose, but rather the base reacts with
acidic groups
present on the hemicellulose to open up the surface of the substrate and
produce acetate
salt. In addition, the base may alter the crystal structure of the cellulose
so that it is more
amenable to hydrolysis.
[0069] Alkali that may be used in the pretreatment includes ammonia, ammonium
hydroxide, potassium hydroxide, and sodium hydroxide. The base is preferably
soluble
in water, which excludes lime or magnesium hydroxide.
[0070] An example of a suitable alkali pretreatment is Ammonia Freeze
Explosion, or Ammonia Fiber Explosion ("AFEX" process). According to this
process,
the cellulosic biomass is contacted with ammonia or ammonium hydroxide in a
pressure
vessel. The contact is maintained for a sufficient time to enable the ammonia
or
18
CA 02576317 2007-01-25
ammonium hydroxide to swell (i.e., decrystallize) the cellulose fibers. The
pressure is
then rapidly reduced which allows the ammonia to flash or boil and explode the
cellulose
fiber structure. (See U.S. Patent Nos. 5,171,592, 5,037,663, 4,600,590,
6,106,888,
4,356,196, 5,939,544, 6,176,176, 5,037,663 and 5,171,592 which are
incorporated herein
by reference.) The flashed ammonia may then be recovered according to known
processes.
[0071] The step of pretreating the feedstock with ammonia or ammonium
hydroxide may be performed at a temperature between about 20 C to about 200 C,
or
any temperature therebetween. For example, the temperature may be 20, 40, 60,
80, 100,
120, 140, 160, 180, or 200 C. The pH is typically from about pH 9.5 to about
pH 12, or
any pH therebetween. For example, the pH of the feedstock may be 9.5, 9.8,
10.0, 10.2,
10.4, 10.6, 10.8, 11.0, 11.2, 11.4, 11.6, 11.8 or 12Ø The treatment time may
be from 2
minutes to about 20 minutes, or any amount of time therebetween. For example,
the
duration of the pretreatment may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18,
19 or 20 minutes. The moisture content of the feedstock may be between 50% and
70%,
or any range therebetween; for example, the moisture content may be 50, 52,
54, 56, 58,
60, 62, 64, 66, 68 or 70%. The ammonia or ammonium hydroxide is added to
achieve a
concentration which is generally about 0.5 to about 2.5 times the mass of the
feedstock
on a dry basis, or any amount therebetween. For example, the ammonia
concentration
may be about 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4 or 2.5
times the mass of
the feedstock on a dry basis.
[0072] If the feedstock is pretreated with sodium hydroxide or potassium
hydroxide, the temperature may be between about 120 C to about 220 C, or any
temperature range therebetween. For example, the temperature may be 120, 130,
140,
150, 160, 170, 180, 190, 200, 210 or 220 C. The pH is typically between about
10 to
about 13, or any pH range therebetween. For example, the pH may be 10.0, 10.5,
11.0,
11.5, 12.0, 12.5 or 13Ø The treatment time may be from about 15 minutes to
about 120
minutes, or any range therebetween. In a non-limiting example, the treatment
time is 15,
20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 minutes.
19
CA 02576317 2007-01-25
[0073] The pretreated cellulosic biomass is enzymatically hydrolyzed with
cellulase enzymes. Cellulase enzymes can typically tolerate a range of pH of
about 4 to
6; therefore, the pretreated feedstock is generally neutralized prior to
enzymatic
hydrolysis. A pH more favorable to the cellulase enzymes is, for example,
within the
range of about 4.5 to about 5.0, or any value therebetween. If the feedstock
is pretreated
with base, the enzymatic hydrolysis may further comprise the addition of
xylanase
enzymes.
[0074] The term "base" is meant to encompass any soluble species that, when
added to water, gives a solution with a pH that is more than 7, and which is
suitable for
neutralizing the feedstock after acid pretreatment to a pH value that is
compatible with
enzymes used during enzymatic hydrolysis.
[0075] In the case of acid pretreatment, the pH adjustment is preferably
carried
out using a soluble base. By the term "soluble base", it is meant a base that
has a
solubility in water that is at least 0.1 M at 20 C. This term is meant to
exclude salts that
are slightly soluble or insoluble. Examples of insoluble bases that are
excluded from the
definition of soluble base are CaCO3 and Ca(OH)2. Non-limiting examples of
soluble
bases include sodium hydroxide, potassium hydroxide, ammonia, and ammonium
hydroxide.
[0076] When an alkali pretreatment is performed, the pH adjustment involves
addition of an acid. Non-limiting examples of acids that may be used are
sulfuric acid,
sulfurous acid, sulfur dioxide, phosphoric acid, carbonic acid, carbon
dioxide,
hydrochloric acid, or a combination thereof In the case of a pretreatment
carried out
with ammonia or ammonium hydroxide, the pH may be adjusted with sulfuric acid,
phosphoric acid, hydrochloric acid, carbon dioxide/carbonic acid or sulfurous
acid which
produces the inorganic salts ammonium sulfate, ammonium phosphate, ammonium
chloride, ammonium carbonate or ammonium sulfite, respectively. If potassium
hydroxide is used in the pretreatment, the feedstock may be neutralized with
phosphoric
acid to produce potassium phosphate. These inorganic salts may be used
directly as a
fertilizer or, in the case of ammonium sulfate or ammonium carbonate,
subjected to
CA 02576317 2007-01-25
degradation reactions to produce ammonia, which, in turn, may be recovered
and/or
recycled in the process.
[0077] For example, ammonium carbonate may be decomposed to produce
ammonia and carbon dioxide. The decomposition may involve thermal treatment to
liberate the ammonia and carbon dioxide. The ammonia and/or the carbon dioxide
may
then be recovered, for example, by distillation, stripping or evaporation. The
recovered
ammonia may, in turn, be recycled to the alkaline pretreatment step.
[0078] Preferably, the alkali pretreatment comprises addition of ammonia or
ammonium hydroxide, followed by neutralization with sulfuric acid to produce
ammonium sulfate. It will be understood by those of skill in the art that the
ammonia
may be provided in anhydrous form. The ammonium sulfate produced during the
neutralization may be used directly as a fertilizer, or, alternatively, may be
subjected to
thermal decomposition according to the method of co-pending U.S. application
entitled
"Process for Producing Ammonia and Sulfuric Acid from a Stream Comprising
Ammonium Sulfate" (Curren et al.) to produce sulfuric acid and sulfate salts,
such as
ammonium sulfate.
[0079] The acid or alkali requirement for pretreatment may be decreased by
removing salts from the feedstock prior to pretreatment. Salts may be removed
by
washing, leaching, or a combination of these processes. One form of the
leaching
process is taught in WO 02/070753 (Griffin et al., which is incorporated
herein by
reference). This process involves contacting the feedstock with water for two
minutes or
longer, then separating the solids from the aqueous phase ("leachate") by
pressing or
filtration. After leaching, the aqueous phase contains potassium and other
salts and trace
elements. This process may not only decrease costs, but may also decrease the
degradation of xylose in the pretreatment process.
[0080] By the term "cellulase enzymes", "cellulase", or "enzymes", it is meant
enzymes that catalyse the hydrolysis of cellulose to products such as glucose,
cellobiose,
and other cellooligosaccharides. Cellulase is a generic term denoting a
multienzyme
mixture, produced by a number of microorganisms, comprising exo-
cellobiohydrolases
21
CA 02576317 2007-01-25
(CBH), endoglucanases (EG) and 0-glucosidases ((.3G). Among the most widely
studied,
characterized, and commercially produced cellulases are those obtained from
fungi of the
genera Aspergillus, Humicola, and Trichoderma, and from the bacteria of the
genera
Bacillus and Thermobifida. In a non-limiting example, the pretreated feedstock
may be
submitted to hydrolysis by cellulase enzymes produced by Trichoderma.
[0081] The cellulase enzyme dosage is chosen to convert the cellulose of the
feedstock to glucose. For example, an appropriate cellulase dosage can be
about 5.0 to
about 50.0 Filter Paper Units (FPU or IU) per gram of cellulose, or any amount
therebetween. For example, the cellulase dosage may be about 5, 8, 10, 12, 15,
18, 20,
22, 25, 28, 30, 32, 35, 38, 40, 42, 45, 48, or 50 FPU, or any amount
therebetween. The
FPU is a standard measurement which is familiar to those skilled in the art
and is defined
and measured according to Ghose (Pure and Appl. Chem., 1987, 59:257-268).
[0082] The sugar stream produced by the enzymatic hydrolysis is preferably
clarified. Any suitable method for removing insoluble residue from the crude
sugar
stream to produce a clarified sugar stream can be employed as would be known
by one of
skill in the art. This includes, but is not limited to, microfiltration, plate
and frame
filtration, crossflow filtration, pressure filtration, vacuum filtration,
centrifugation and the
like.
[0083] The soluble compounds in the clarified sugar stream may include
monomeric sugars such as glucose, xylose, arabinose, galactose, mannose, and
oligomers
of these sugars; acetic acid, sulfuric acid, lactic acid, oxalic acid, among
other organic
acids, and the salts of these acids; cations including sodium, calcium,
potassium,
ammonium, magnesium, and others; anions, in addition to the organic acids
named
above, including silicate, phosphate, and carbonate. Preferably, the solids in
the clarified
sugar stream are comprised of at least 30 wt% sugar; for example, the solids
in the
clarified sugar stream may comprise more than 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80,
85, 90, or 95 wt% sugar. It is also preferred that the minimum concentration
of acetic
acid and acetate salts in the clarified sugar stream is about 5 g/L. A variety
of other
compounds are present in the clarified sugar stream, including sugar
degradation products
22
CA 02576317 2007-01-25
such as furfural and hydroxymethyl furfural, and soluble phenolic compounds
derived
from lignin. Organic extractive compounds, such as soaps and fatty acids, are
also
present.
[0084] As described in more detail below, a stream obtained during processing
of
the cellulosic biomass (referred to herein as a "feed stream") is treated by
ion exclusion
chromatography to separate sugars or other nonionic compounds from inorganic
salts and
other ionic compounds. The ion exclusion chromatography is carried out in the
range of
about 5.0 to about 10.0, or any pH value therebetween, for example at a pH
from about
6.0 to about 10.0, a pH from about 6.5 to about 10, a pH from about 6 to about
8, or at a
pH of about 5.0, 5.2, 5.5, 5.7, 6.0, 6.2, 6.5, 6.7, 7.0, 7.2, 7.5, 7.7, 8.0,
8.2, 8.5, 8.7, 9.0,
9.2, 9.5, 9.7, 10Ø To maintain the desired pH range of 5 to 10, the feed
stream may be
adjusted to this pH range. Those of skill in the art will be aware of
chemicals suitable for
adjusting the pH of the feed stream, for example but not limited to, ammonium
hydroxide, sodium hydroxide, potassium hydroxide, ammonia or sulfuric acid.
[0085] The feed stream for the ion exclusion separation may arise from a
variety
of different streams produced during the processing of the cellulosic biomass
to produce
sugar. Examples of such streams are set forth in more detail below.
[0086] For example, the feed stream may be an aqueous stream obtained from the
pretreated cellulosic biomass. This aqueous stream may be produced after a
step of
washing the pretreated biomass. Alternatively, the aqueous stream is obtained
after
removing solids from the pretreated biomass, for example, by centrifugation,
microfiltration, plate and frame filtration, crossflow filtration, pressure
filtration, vacuum
filtration and the like.
[0087] For acid pretreatment, this aqueous stream may be treated with base to
produce inorganic salt. Optionally, this stream may be treated with a strong
acid cation
exchange resin to remove metal cations from the aqueous stream prior to the
treating the
stream with the base.
23
CA 02576317 2007-01-25
[0088] In the case of a basic pretreatment, this aqueous stream may then be
treated with one or more acid to produce inorganic salt. Alternatively, this
stream may be
treated with a strong acid cation exchange resin. This may involve passage of
the stream
through a column packed with a sulfonated polystyrene resin cross-linked with
divinyl
benzenes in an alkali/alkaline earth metal form. An alkali regenerant, such as
sodium
hydroxide, is then added to the column to produce the inorganic salt. The
inorganic salt
may also arise from salts that are native to the cellulosic biomass. In
addition, this stream
will also contain acetate or acetic acid arising from the pretreatment.
[0089] In another embodiment of the invention, the feed stream to the
chromatographic separation is an aqueous stream obtained from the neutralized
cellulosic
biomass. In this case, the aqueous stream may be produced after a step of
washing the
neutralized biomass. Alternatively, this aqueous stream is obtained after
removing solids
from the neutralized biomass using any of the separation techniques described
previously.
This stream will comprise inorganic salt produced by addition of acid or base
to the
pretreated cellulosic biomass.
[0090] In the case of a base pretreatment, the streams obtained from the
neutralized feedstock or from the pretreated feedstock will comprise a small
amount of
monomeric sugars, and nonionic compounds may be present. If an acid
pretreatment is
employed, these streams will typically comprise sugars produced as a result of
the
hydrolysis of hemicellulose.
[0091] In yet another embodiment of the invention, the clarified sugar stream
is
subjected to the chromatographic separation of the present invention. By
operating at the
pH range of between 5 and 10, sugars, and most other nonionic compounds
present, are
collected in a high-binding sugar stream, and the inorganic salt and acetate
salt are
collected in a separate low-binding raffinate or "salt stream". The purified
sugar stream
can then be fermented by microbes to produce ethanol, lactic acid, butanol or
other
fermentation products.
[0092] Prior to the chromatographic separation, the sugar stream may be
fermented to produce a fermentation broth comprising ethanol. The fermentation
broth
24
CA 02576317 2007-01-25
may then be distilled to produce concentrated ethanol and still bottoms. The
still bottoms
stream, which comprises unfermented sugar, acetate and inorganic salts, may
then be
used as the feed to the chromatographic separation. Typically, the still
bottoms stream is
clarified prior to the chromatographic separation to remove yeast cells. This
clarification
step may be conducted either before or after distillation. Preferred methods
for carrying
out the fermentation and subsequent distillation are set forth in more detail
below.
[0093] The separation of sugar from sodium sulfate and sodium acetate at an
alkaline pH following methods of the present invention is shown in Figure 2A
(Example
1). In addition, the separation of sugar from sodium bisulfate and acetic acid
that would
be present in a sugar stream arising from biomass conversion was carried out
at pH 3
following the methods of Example 2. With reference to Figure 2B, at pH 3, the
separation of sugar from sodium bisulfate and acetic acid leads to undesirable
separation
performance under these conditions as acetic acid co-elutes with the sugar
product.
[0094] The ion exclusion system of the present invention may be operated in a
temperature range of about 20 C to about 90 C, preferably at a temperature
between
about 45 C to about 80 C, or any value therebetween, for example, at a
temperature of
about 60 C to about 70 C, or at about 45, 47, 50, 52, 55, 57, 60, 62, 65, 67,
70, 72, 75,
77, 80 C.
[0095] The stream fed to the chromatographic separation of the present
invention
may contain water as a primary component. For example, the amount of water
present in
the crude sugar stream may be an amount in the range of about 40% to about 95%
(w/w),
or any amount therebetween. Preferably, the crude sugar stream may comprise
from
about 50% to about 85% (w/w) water, or any amount therebetween, arising from a
step of
concentration.
[0096] The feed stream may be concentrated using any technique known to those
of skill in the art. For example, concentration may be carried out by
subjecting the feed
stream to, membrane filtration, evaporation, or a combination thereof. Without
being
limiting, microfiltration (with a pore size of 0.05 to 5 microns) may be
carried out to
remove particles, followed by ultrafiltration (500-2000 mw cut off) to remove
soluble
CA 02576317 2007-01-25
lignin and other large molecules and reverse osmosis to increase the solids to
a
concentration of about 12 to about 20%, or any amount therebetween, followed
by
evaporation.
[0097] The feed stream should not contain any significant amount of insoluble
compounds, as the insoluble compounds foul the ion exclusion chromatography
system.
Any suitable method for removing insoluble residue from the feed streams to
produce a
clarified feed stream can be employed as would be known by one of skill in the
art. This
includes, but is not limited to, microfiltration, plate and frame filtration,
crossflow
filtration, pressure filtration, vacuum filtration, centrifugation, and the
like.
[0098] It is preferred that the feed stream is characterized as having a
lignosulfonate content of less than 4% of the total dry solids of the
clarified sugar stream.
For example, the feed stream may be characterized as having an amount of
lignosulfonate
from about 0 to about 4% of the total dry solids of the clarified sugar
stream.
[0099] The process of ion exclusion chromatography may involve the use of one,
or more than one, column filled with ion exchange resin, as is evident to one
of skill in
the art. For the sake of simplicity, the operation of a single column will be
illustrative,
but the use of more than one column is also considered to be within the scope
of the
present invention. The ion exchange resin is a cation exchange resin.
Preferably, the
resin is a strong cation exchange resin, for example, which is not to be
considered
limiting, with a polystyrene backbone and 4-8 % divinylbenzene crosslinking.
These
resins have sulfonate functional groups and are available commercially in the
sodium
form, or, less preferably, in the hydrogen, potassium or ammonium form. The
resins are
preferably of diameter of from about 0.1 to about 1.0 mm. Cationic exchange
resins are
available from several vendors, including Dow or Mitsubishi.
[00100] The column may be prepared prior to carrying out the separation by
converting it into the desired cation-exchange form. This may involve washing
a volume
of the clarified sugar stream through the column. The volume may be equal to
from
about 2 to about 5 times the volume of the resin in the column, or the washing
may be
carried out until the effluent pH matches the pH of the clarified sugar
stream.
26
CA 02576317 2007-01-25
Alternatively, the column may be prepared by washing it with a volume of
solution
containing cations corresponding to those that would be present in the
clarified sugar
stream.
[00101] Once the column is in the appropriate cation-exchange form, the feed
stream is applied onto the column.
[00102] A quantity of the feed stream equal to about 0.05 to about 0.3 times
the
volume of the column, or any amount therebetween, is applied. However, the
amount of
the feed stream to be applied may differ from that just disclosed, and it may
be readily
determined based on experimentation to determine column capacity and
separation. A
desired liquid flow rate is also selected as may be readily determined by one
of skill in
the art, for example, but not limited to, a liquid flow rate corresponding to
about 5% to
about 70%, or any amount therebetween, of the column volume per hour.
[00103] As the feed stream is applied, the charged ions in the salts and other
charged compounds are excluded from the resin and flow through the column. The
sugar
or nonionic compounds present in the feed stream are not repelled by the
charged resin,
and penetrate the pores of the resin. The sugar or nonionic compounds are
thereby
retained by the resin and elute the colunm more slowly than the ionic
compounds.
[00104] After the desired volume of the feed stream is injected, the feed is
switched to water, which may have been previously softened to decrease the
concentration of multivalent cations. The ionic compounds contain inorganic
salts such
as the inorganic salts of the base or acid used for pH adjustment and the
inorganic salts of
the acid or base used in pretreatment, as well as acetic acid and other
organic acids
originating from the cellulosic biomass. The ionic compounds flow through the
colunm
and are collected in one or more than one stream. This one or more than one
stream is
designated as a "raffinate stream" or "salt stream" (or one or more than one
raffinate) and
contains the majority of the inorganic and acetate salts, and trace amounts of
sugar or
other nonionic compounds. The one or more than one raffinate stream is
followed by the
elution of sugars arising from the processing of the cellulosic biomass or
nonionic
compounds, which are collected separately from the one or more than one
raffinate. The
27
CA 02576317 2007-01-25
purified sugar stream contains most of the sugar and little of the salt and
other ionic
components.
[00105] In a preferred embodiment, the ion exclusion chromatography is carried
out by a Simulated Moving Bed (SMB) device. An SMB contains ion exchange resin
similar to that in an ion exclusion system described above, and performs the
same type of
separation of sugars and nonionic compounds in the product stream and salts
and other
ionic compounds in the raffinate stream. For a given feed stream, an SMB is
run at the
same pH and temperature as an ion exclusion system.
[00106] However, an SMB system has distinct locations for feeding of the
clarified
sugar stream, feeding of dilution water, and withdrawal of sugar product and
of the one
or more than one raffinate streams. For example, which is not to be considered
limiting,
four flow locations equally spaced apart may be used on one or more than one
column.
The order of the locations is, arbitrarily starting from the feed inlet, 1)
the clarified sugar
stream feed, 2) the raffinate withdrawal, 3) the dilution water feed, and 4)
the product
withdrawal. If a single column is used, the outlet from the top of the column
may be used
to feed the bottom, thus completing a circle. If more than one column is used,
which is
typical, the outlet of each column feeds the next column, again producing a
circle of flow.
The SMB is therefore much more of a fully continuous operation than a single-
column
ion exclusion system. Additional flow locations may be included if more than
one
raffinate stream is to be collected.
[00107] Another difference between an SMB and a single-column ion exclusion
system is that the SMB has a recirculation flow that supplements and is co-
current with
all of the other flow streams. This recirculation flow is carefully chosen,
along with the
other flows, to provide the optimum separation between the sugar and salt
streams.
[00108] Additionally, an SMB system simulates movement of the resin bed in a
direction opposite to that of the liquid flow. With reference to Figure 1, the
simulated
movement is carried out by periodically shifting the four flow locations by
some fraction
of the total bed. For example, if an SMB system is visualized as a circular
system 10, for
example a clock, then liquid 20 flows counterclockwise in this system. The 12
hourly
28
CA 02576317 2007-01-25
positions on the clock can symbolize an SMB with 12 zones, with the feed 30
set
arbitrarily at 12 O'clock. As the liquid flows around, the raffinate 40, which
has a low
affinity for the resin 50, is withdrawn at 9 O'clock. Dilution water 60 is
added at 6
O'clock at a flow rate that is from about 1.0 to about 4.0 fold the feed
application rate. In
a preferred embodiment, the dilution water flow is added at from about 1.0 to
about 1.5
times the feed flow rate. The bound compounds do not have a high enough
affinity to
remain bound at the high flow rates present after the dilution water 60 is
added. These
compounds are washed off the resin 50 at the product stream withdrawa170,
positioned at
3 O'clock. After the product withdrawa170, a relatively clean stream flows
back up to 12
O'clock to continue the cycle.
[00109] At a chosen interval of perhaps 10 minutes to 4 hours, preferably 15
minutes to 2 hours, the stream positions (flow locations) are shifted
clockwise to simulate
movement of the bed. If the positions are shifted by 1 hour in location, the
feed 30 is
then at 1 O'clock, product 70 at 4 O'clock, dilution water at 7 O'clock, and
raffinate 40 at
O'clock, and this system has shifted 1/12 in position.
[00110] The bed positions are shifted at a frequency and to a degree that are
chosen to optimize the separation of interest which depends on the affinity
that the sugar
and salt have for the resin, the liquid flow rates, and the cost of such a
switching system.
A typical SMB rotates the positions by about 1/16 to about 1/4 of the extent
of the cycle,
thereby defining from about 16 to about 4 zones, respectively. The 4- to -16
zones can be
carried out on a single column. In a preferred embodiment, one column is used.
This
simplifies the demarcation of zones and allows for a given column to be
brought off line,
for cleaning or maintenance without overly disturbing the operation. For
example, which
is not to be considered limiting, from about 4 to about 16 columns may be
used. In a
more preferred embodiment, about 4 to about 8 columns are used. However, the
number
of columns may be adjusted as required.
[00111] Improved SMB ("ISMB") systems (available for example from Eurodia
Industrie S.A., Wissous, France; Applexion S.A., Epone, France; or Amalgamated
Research Inc., Twin Falls, Idaho) may also be used as described herein. ISMB
systems
29
CA 02576317 2007-01-25
include variable flow rates of feed, dilution water, product, raffinate, or a
combination
thereof, or sequential periods with one or more streams closed off, with or
without re-
circulation of the liquid in the columns, or a combination of two or more of
these
features. The present invention can be practiced with ISMB or SMB operations.
[00112] The sugar and feed streams, the purified sugar stream obtained after
ion
exclusion chromatography, or both streams, may be concentrated. Any suitable
method
may be utilized for concentrating these streams. This includes the methods
described
above.
[00113] The purified sugar stream obtained following ion exclusion
chromatography is readily fermented. Prior to fermentation, the product sugar
stream
may be adjusted to a pH from about 4 to about 6, as desired for the particular
fermentation. The product sugar stream may be concentrated by evaporation,
filtration,
or other methods familiar to those skilled in the art, prior to fermentation.
[00114] In a preferred embodiment, the sugar in the purified sugar stream is
fermented to ethanol. Fermentation may be carried out by yeast, bacteria or
other
microbes capable of fermenting the product stream to a desired efficiency and
yield. In a
preferred embodiment, the fermentation is carried out using a genetically
engineered
yeast, for example, but not limited to, Saccharomyces or Pichia, or bacteria,
for example,
but not limited to, Zymomonas or E. coli capable of fermenting the pentose
sugars xylose,
arabinose, or a combination thereof, in addition to the hexose sugars glucose,
mannose,
galactose, or a combination thererof. Alternatively, the sugar in the product
sugar stream
is fermented to lactic acid. Those skilled in the art are familiar with the
requirements in
fermentation of sugar to produce ethanol, lactic acid or other products.
[00115] The inorganic salt in the raffinate stream or salt stream may be
crystallized, dried or subjected to electrodialysis or agglomeration and
granulation, and
used as desired, for example, as a solid fertilizer. Alternatively, the
inorganic salt may be
concentrated as a wet slurry and used in a liquid form, for example, as a
liquid fertilizer.
Processing of inorganic salt stream may be carried out as described in co-
pending U.S.
CA 02576317 2007-01-25
patent application entitled "Recovery of Inorganic Salt During Processing of
Lignocellulosic Feedstock", which is incorporated herein by reference.
[00116] Ammonium, potassium, sulfate, and phosphate salts in the raffinate
stream
are typically of value. Other compounds present, including salts of sodium and
sulfite
salts, may be of less value in fertilizer. However, these salts can be
converted to forms of
higher value. For example, which is not to be considered limiting, sodium
salts can be
converted to ammonium salts or potassium salts by the use of ion exchange,
which is
familiar to those skilled in the art. In this example, sodium hydroxide may be
used for
some or all of the neutralization of sulfuric acid during the processing of a
lignocellulosic
feedstock, and the sodium ion exchanged with ammonium or potassium using a
cation
exchange resin. The resulting ammonium or potassium salt may then be of more
value as
a fertilizer. Additionally, sulfite salts can be converted to sulfate salts by
oxidation with
air or other oxidizing agent, for example, sulfurous acid or sulfur dioxide.
[00117] The present invention will be further illustrated in the following
examples.
EXAMPLES
Example 1: Ion exclusion separation of sodium sulfate, sodium acetate, and
glucose
[00118] The effectiveness of ion exclusion at pH 8 as a process for the
removal of
sodium acetate and sodium sulfate salts from glucose is illustrated by this
example.
[00119] A fixed bed ion exclusion column was filled with Mitsubishi Chemical
resin #UBK530. Prior to filling the column, 135 ml of the resin was suspended
in 1 liter
of deionized water and allowed to settle. The supernatant was decanted and the
procedure carried out three times, which was sufficient to remove all visible
fine
particles. After decanting of the third supernatant, two resin volumes of
deionized water
were added to the resin, and the slurry was poured onto the 127 ml column. The
column
contains a hot water jacket, but the jacket was not used during the resin-
loading
procedure. The top of the column was sealed with a rubber stopper attached to
a water
pump. Care was taken to ensure that the seal was airtight.
31
CA 02576317 2007-01-25
[00120] The packed column was washed with 300 ml of degassed, deionized
water. This removed dissolved gases and minimized resin channeling. If the
column
became overloaded with air bubbles, the resin was back-washed and the column
repacked.
[00121] Once the column was degassed satisfactorily, water at 70 C was
circulated
through the water jacket. The column was washed with water until the
temperature of the
water bath reached 70 C.
[00122] At this point, the resin was prepared with the feed solution
(clarified sugar
stream). For this experiment, the feed solution was a synthetic sugar stream
of 1% acetic
acid, 10% sodium sulfate, and 2.5% glucose (w/w) dissolved in deionized water
and
adjusted to pH 8.0 with lON sodium hydroxide. The resulting concentration of
sodium
ions was 32.4 g/L. A volume of 200 ml of feed solution was fed onto the column
at a
flow rate of 1 ml/min. The effluent from the column was collected and
discarded. If
suspended solids formed in the feed, the feed was filtered and the flow
restarted.
[00123] Once the feed volume of 200 ml was achieved, the column was washed
with deionized water. The conductivity of the eluent was measured and the
water wash
deemed complete when the eluent conductivity matched that of the water feed.
At this
point, any excess water present on top of the column bed was removed by using
a pipette.
A weight of 6.4 grams of feed was then added to the top of the bed, and the
column
sealed with a stopper as before. The pump was started to pump water at a rate
of 1
ml/minute. The stopcock was opened at the base of the column and 4 ml
fractions
collected over 4 minutes. The water feed and fraction collection were
continued until 30
fractions had been collected. After the collection of the 30th fraction, the
column was
washed with 300 ml deionized water prior to the next run. Care was taken to
avoid
drying of the resin during overnight storage.
[00124] The product fractions were analyzed for sodium sulfate, sodium acetate
and glucose. For the acetate determination, the samples were adjusted to pH 3-
3.5 with
dilute sulfuric acid prior to injection into a gas chromatograph and detection
as acetic
acid.
32
CA 02576317 2007-01-25
[00125] The results of the elution are shown in Figure 2A. The sodium sulfate
and
sodium acetate elute with a large degree of overlap, followed by the elution
of glucose.
The separation was good, in that there was little glucose with the salts and
little salt with
the glucose.
Example 2: Comparative example - Separation of glucose from acetic acid at pH
3
[00126] This example illustrates the use of ion exclusion for the separation
of
sugar from acetic acid and sodium bisulfate at pH 3. The separation of acetic
acid from
glucose at pH 3.0 is poor as these two components co-elute (see Figure 2B).
The use of
the methods as described herein, provide superior separation of acetic acid
and sugar.
[00127] A fixed bed ion exclusion column was filled with Mitsubishi Chemical
resin #UBK530. Prior to filling the column, 135 ml of the resin was suspended
in 1 liter
of deionized water and allowed to settle. The supernatant was decanted and the
procedure carried out three times, which was sufficient to remove all visible
fine
particles. After decanting of the third supernatant, two resin volumes of
deionized water
were added to the resin, and the slurry was poured onto the 122-ml column. The
column
contains a hot water jacket, but the jacket was not used during the resin-
loading
procedure. The top of the column was sealed with a rubber stopper attached to
a water
pump. Care was taken to ensure that the seal was airtight.
[00128] The packed column was washed with 300 ml of degassed, deionized
water. This removed dissolved gases and minimized resin channeling. If the
column
became overloaded with air bubbles, the resin was back-washed and the column
repacked.
[00129] Once the column was degassed satisfactorily, water at 70 C was
circulated
through the water jacket. The column was washed with water until the
temperature of the
water bath reached 70 C.
[00130] At this point, the resin was equilibrated with the feed solution. For
this
experiment, the feed solution was a synthetic sugar stream of 25 g/L acetic
acid, 150 g/L
sulfuric acid, and 75 g/L glucose dissolved in deionized water and adjusted to
pH 3.0
33
CA 02576317 2007-01-25
with lON sodium hydroxide. The resulting concentration of sodium bisulfate was
about
190 g/L. A volume of 200 ml of feed solution was fed onto the column at a flow
rate of
1.6 ml/min. The effluent from the column was collected and discarded. If
suspended
solids formed in the feed, the feed was filtered and the flow restarted.
[00131] Once the feed volume of 200 ml was achieved, the column was washed
with deionized water. The conductivity of the eluent was measured and the
water wash
deemed complete when the eluent conductivity matched that of the water feed.
At this
point, any excess water present on top of the column bed was removed by using
a pipette.
A weight of 6.4 grams of feed was then added to the top of the bed, and the
column
sealed with a stopper as before. The pump was started to pump water at a rate
of 1.6
ml/minute. The stopcock was opened at the base of the column and 4.8 ml
fractions
collected over 3 minutes. The water feed and fraction collection were
continued until 30
fractions had been collected. After the collection of the 30th fraction, the
column was
washed with 300 ml deionized water prior to the next run. Care was taken to
avoid
drying of the resin during overnight storage.
[00132] The product fractions were analyzed for sodium bisulfate
concentration,
acetic acid, glucose, and dry weight. For the acetic acid determination, the
samples were
adjusted to pH 3-3.5 with dilute sulfuric acid prior to injection into a gas
chromatograph.
[00133] The results of the elution are shown in the Figure 2B. Most of the
sodium
bisulfate elutes before glucose is recovered. However, a portion of the sodium
bisulfate
co-elutes, and is present in samples comprising glucose. Furthermore, the
separation of
glucose and acetic acid is poor, and a significant portion of acetic acid co-
elutes with
glucose. This is attributed to the non-ionic nature of acetic acid at pH 3,
which makes it
difficult to separate from glucose at this pH. This is in contrast with
Examples 1 and 3
that show a significantly improved separation of sodium sulfate and sodium
acetate from
glucose at an alkaline pH, for example, but not limited to, pH 8Ø
Example 3: Elution of biomass sugars
[00134] A feed sample of biomass sugars was made by subjecting wheat straw to
pretreatment with sulfuric acid at conditions described in U.S. 4,461,648. The
pH of
34
CA 02576317 2007-01-25
pretreatment was 1.4 and the resulting pretreated feedstock was adjusted to pH
5 with
sodium hydroxide. The neutralized cellulosic biomass was subjected to
enzymatic
hydrolysis by cellulase enzymes made by the fungus Trichoderma to produce a
crude
sugar stream. The crude sugar stream was separated from the insoluble residue,
which is
primarily lignin, by using plate and frame filtration. The clarified sugar
stream was
evaporated to 44% total solids, with a concentration of 109 g/L sulfate salts
of sodium
and potassium, 300 g/L glucose, 44 g/L xylose, 5.3 g/L arabinose, 10.9 g/L
sodium
acetate (measured as acetic acid), and various trace metals. The clarified
sugar stream
was evaporated, adjusted to a pH of 8 and then fed to the column and eluted as
described
in Example 1. The results are shown in Figure 3.
[00135] The ion exclusion system provides a good separation of the sugar from
the
sodium acetate and sodium sulfate. Almost all of the sugar is in the sugar
stream, and
almost all of the salt is in the salt stream. This is the case even when the
sugar stream is
from a biomass conversion process.
Example 4: Large scale purification of sugars from cellulose and fermentation
to produce
ethanol
[00136] A feed stream of sugars from cellulose was prepared using the
procedures
from Example 2 with a concentration of 145 g/L sulfate salts of sodium and
potassium,
153 g/L glucose, 49 g/L xylose, 7.3 g/L arabinose, 9.1 g/L sodium acetate
(measured as
acetic acid), various trace metals, and a significant amount of unidentified
impurities.
This feed stream was divided into two parts. The first part was diluted 1:3 in
water and
set aside for fermentation, with a concentration of 48.4 g/L sulfate salts of
sodium and
potassium, 51 g/L glucose, 16 g/L xylose, 2.6 g/L arabinose, 3.1 g/L sodium
acetate. The
second part was subjected to large scale ion exclusion chromatography.
[00137] The chromatography was carried out on an Improved Simulated Moving
Bed (ISMB) system (Eurodia Industrie S.A. of Wissous, France, available
through
Ameridia, Somerset, New Jersey) of volume 6700 liters, packed with cation
exchange
resin from Mitsubishi Chemical, resin #UBK530. The ISMB system consists of 4
CA 02576317 2007-01-25
columns with 4 bed shifts per cycle and was operated with the feed stream
maintained at
pH 7.5 to 8Ø The system was maintained at 70 C as was the sugar feed and the
dilution
water. The sugar stream was fed at an average rate of 4 liters per minute and
dilution
water was added at a ratio of 4:1 with the sugar feed. Product and raffinate
streams were
collected, with the product stream containing 1.6 g/L sulfate salts, 66 g/L
glucose, 22 g/L
xylose, 3.3 g/L arabinose, and 0.09 g/L acetate (measured as acetic acid).
[00138] Both the diluted feed stream and the product stream were pumped into
fermentation vessels in liquid volumes of 100 liters and total volume 200
liters. The
fermenters were inoculated with 4 g/L yeast strain 1400-LNHST obtained from
Purdue
University. This strain has been developed to ferment glucose and xylose to
ethanol, as
described in U.S. 5,789,210. The yield of ethanol for both treated and
untreated product
streams are provide in Table 1.
Table 1: Ethanol yields from treated and untreated sugar streams
Sugar stream Ethanol (/gL) Ethanol Yield
after 48 hrs (g/ initial itial glucose and xylose)
Diluted, untreated sugars 12.2 0.182
Ion exclusion-treated sugars 37.9 0.431
[00139] The ion exclusion treated sugar stream was essentially completely
fermented by the yeast. Without wishing to be bound by theory, the reduced
yield of
ethanol produced using the untreated sugar stream may be a result of
inhibitors present in
this feed stream. The ion exclusion treated stream resulted in a much higher
yield of
ethanol as shown in Table 1, possibly due to reduced amounts of inhibitors in
the ion
exclusion-treated stream. This is a demonstration of the detoxification of the
sugar
stream and the removal of acetate salts, and possibly other inhibitors, by the
ion exclusion
treatment.
Example 5: Separation of salts from xylose at pH 7
36
CA 02576317 2007-01-25
[00140] Wheat straw was leached according to the methods described in WO
02/070753 (Griffin et al.) to remove inorganic salts. A feedstock sample of
biomass
sugars was then produced by subjecting the leached wheat straw to pretreatment
with
sulfuric acid at conditions described in US 4,461,648 (Foody). The pH of the
pretreatment was 1.4 and the pH was adjusted with ammonium hydroxide to a pH
value
of between 4.5 and 5Ø The pretreated feedstock was subjected to enzymatic
hydrolysis
by cellulase enzymes made by the fungus Trichoderma to produce a crude sugar
stream.
[00141] The resulting crude sugar stream was separated from the unhydrolyzed
residue, which is primarily lignin, by using plate and frame filtration. After
filtering, the
clarified sugar stream was evaporated under vacuum at a temperature of between
65 to
75 C to increase the solids content by 3-4 fold. The concentrated hydrolyzate
was then
filtered by plate and frame filtration. The glucose in the clarified sugar
stream was
fermented to ethanol with Saccharomyces cerevisiae yeast. While the glucose is
easily
fermentable, xylose sugars present in the hydrolyzate are more difficult to
ferment.
[00142] After fermentation, the fermentation broth was filtered and then
centrifuged to remove yeast cells. The pH was then adjusted to 7.0 with
ammonium
hydroxide. The fermentation broth was distilled to produce fuel grade ethanol
and still
bottoms, which were then evaporated to 13% total solids (w/w). The
concentrated still
bottoms contained 44 g/L sulfate salts of sodium, potassium and ammonium, 0.4
g/L
glucose, 12.1 g/L xylose, 0.3 g/L arabinose, 9.3 g/L acetic acid, and various
trace metals.
The still bottoms were then adjusted to pH 7 with a small volume of 1 M NaOH
and
filtered. Ion exclusion chromatography of the concentrated still bottoms was
performed
as in Example 1, except that ammonium sulfate was passed through the column
prior to
addition of the sugar stream to convert the resin into the ammonium form.
[00143] The total solids content of each fraction were measured by placing 1
ml
from each fraction in a pre-weighed aluminum tray and allowing them to
evaporate in an
oven at 100 C for at least an hour. The trays were allowed to cool briefly
before being
re-weighed, and the mass difference was divided by the volume to obtain the
concentration of total dissolved solids in g/L.
37
CA 02576317 2007-01-25
[00144] For xylose analysis, an aliquot of each fraction that contained
dissolved
solids was assayed for reducing sugars using the DNS (3,5-dinitrosalicylic
acid) method
described by Miller, G.L. (Anal. Chem., 1959, 31:426).
[00145] The results of the elution are shown in Figure 4. Salt eluted first,
followed
by the elution of xylose. The separation was good in that little xylose eluted
with the
salts and little salt eluted with the xylose.
[00146] The purified xylose is then fermented to ethanol by a yeast strain
that can
convert xylose to ethanol. An example of such a strain is that described in
U.S.
5,789,210 (Ho et al.).
Example 6: Separation of salts from xylose at pH 5
[00147] A feed stream of sugars from cellulosic biomass was prepared using the
procedures from Example 5 except that the pH of the sugar stream was
maintained at pH
prior to feeding it to the ion exclusion column.
[00148] Concentrations of sulfate were measured by ion exchange chromatography
and concentrations of ammonium were measured by colourimetric assay. Total
solids
were measured as described in Example 5. The results of the elution at pH 5
are shown
in Figures 5A, 5B and 5C.
[00149] As shown in Figure 5A, the ammonium sulfate elutes first, followed by
the elution of xylose. The separation was good in that there was very little
bleeding of
salts into the xylose peak with both the ammonium sulfate concentration and
the
concentration of xylose reaching close to zero between the two peaks.
[00150] Figure 5B shows the sulfate ion, ammonium ion and xylose content of
select pulse test fractions at pH 5. The sulfate and ammonia elution peaks
overlapped
and were followed by an elution peak containing xylose.
[00151] Figure 5C compares the elution of ammonium sulfate, xylose and acetic
acid at pH 5. At this pH, "acetic acid" includes 1/3 acid and 2/3 acetate
salts. As can be
seen from Figure 5C, acetic acid eluted at the end of the ammonium sulfate
peak, but
38
CA 02576317 2007-01-25
without bleeding into the xylose peak. The acetic acid removal from the xylose
is
acceptable. A larger bleeding of acetic acid into the xylose stream would be
expected at
a lower pH, as was observed at pH 3 in Example 2B.
Example 7: Separation of salts from a cellulosic biomass pretreated with base
[00152] An alkaline pretreatment followed by obtaining inorganic salt and
acetate
salt may be carried out as follows.
[00153] Wheat straw is received in bales and chopped into pieces having a size
of
about 20 mesh and smaller. The chopped straw is slurried in water to reach a
moisture
content of about 70%. The wet straw is added to a reactor with pressurized
ammonia
slurry heated to 120 C to reach a pressure of 300 psia. The mass of ammonia
equals the
mass of straw on a dry basis. The temperature is maintained for 20 minutes,
after which
the pressure is released quickly, which flashes off about 99% of the ammonia.
The flash
cools the reactor contents down to ambient temperature. The slurry is then
adjusted to
about pH 5.0 with concentrated sulfuric acid.
[00154] Upon acid addition, the soluble salt of ammonium sulfate is formed.
The
insoluble salt, calcium sulfate, is also formed.
[00155] The neutralized, cooled pretreated slurry is then added to a
hydrolysis
reactor and the reactor is mixed. The slurry will consist of 4.5% undissolved
solids, and
the undissolved solids will consist of 35% cellulose. Once the pretreated
slurry is added
to the hydrolysis reactor, cellulase and hemicellulase enzyme from Trichoderma
reesei
are added. The enzyme dosage is 35 mg protein per gram cellulose, which
corresponds
to a cellulase activity of 35.6 Filter Paper Units (FPU) per gram of cellulose
and a
xylanase activity of 275 xylanase units per gram of solids.
[00156] The hydrolysis will run for 2 days, at which point over 90% of the
cellulose is converted to glucose and over 90% of the xylan is converted to
xylose. The
expected final glucose concentration is 6.0 to 8.0 g/L, with an average of 7.5
g/L. The
hydrolysis slurry is then filtered by using a vacuum filter to separate the
unhydrolyzed
solid residue from the aqueous stream. The unhydrolyzed solid residue contains
39
CA 02576317 2007-01-25
primarily lignin, unhydrolyzed cellulose and silica, but also the insoluble
salts such as
calcium sulfate. The filtrate is essentially free of insoluble particles and
contains glucose,
xylose, and arabinose sugar; the soluble salts ammonium sulfate, potassium
sulfate,
magnesium sulfate and a small amount of dissolved calcium sulfate, and acetic
acid,
soluble lignin, and other dissolved organics.
[00157] The process stream is evaporated to increase the solids concentration
ten-
fold. The expected concentrations of materials in the evaporated stream are 62
g/L
glucose, 20 g/L xylose, and 2.0 g/L acetic acid. The feed stream will also
contain sulfate
salts of ammonium and potassium, various trace metals, and a significant
amount of
unidentified impurities. This feed stream will be subjected to large scale ion
exclusion
chromatography.
[00158] The chromatography will be carried out on an Improved Simulated
Moving Bed (ISMB) system (Eurodia Industrie S.A. of Wissous, France, available
through Ameridia, Somerset, New Jersey) of volume 6700 liters, packed with
cation
exchange resin from Mitsubishi Chemical, resin #UBK530. The ISMB system will
consist of 4 columns with 4 bed shifts per cycle and will be operated with the
feed stream
maintained at pH 7.5 to 8Ø The system will be maintained at 70 C, as will be
the sugar
feed and the dilution water. Product and raffinate streams will be collected,
with the
product stream containing significantly reduced concentrations of sulfate
salts and acetate
salts (measured as acetic acid).
[00159] The product stream will be pumped into fermentation vessels in liquid
volumes of 1001iters and total volume 2001iters. The fermenters will be
inoculated with
4 g/L yeast strain 1400-LNHST obtained from Purdue University. This strain has
been
developed to ferment glucose and xylose to ethanol, as described in U.S.
5,789,210.
[00160] At the conclusion of the fermentation, the yeast cells are removed by
centrifugation. The dilute beer is distilled to recover the ethanol from the
aqueous
solution, leaving still bottoms behind.
CA 02576317 2007-01-25
[00161] All citations are hereby incorporated by reference.
[00162] The present invention has been described with regard to one or more
embodiments. However, it will be apparent to persons skilled in the art that a
number of
variations and modifications can be made without departing from the scope of
the
invention as defined in the claims.
41
