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| United States Patent Application |
20100124770
|
| Kind Code
|
A1
|
|
SABESAN; SUBRAMANIAM
; et al.
|
May 20, 2010
|
PROCESS FOR PRODUCING A CONCENTRATED SUGAR SOLUTION BY ENZYMATIC
SACCHARIFICATION OF POLYSACCHARIDE ENRICHED BIOMASS
Abstract
Methods for obtaining concentrated sugar solution from polysaccharide
enriched biomass by contacting biomass with water and at least one
nucleophilic base to produce a polysaccharide enriched biomass comprising
a solid fraction and a liquid fraction and then contacting the solid
fraction with saccharification enzyme consortium to produce a
saccharification product comprising at least about 7 percent by weight
sugars in 24 hours. The methods include optionally adding at least one
additive selected from the group consisting of polyethylene glycols,
fatty acid esters, fatty acid ethoxylates, nonionic surfactants derived
from polyethoxylated sorbitan and a fatty acid, sodium
lauriminodipropionate, sodium cocoamphoacetate, sodium tridecyl ether
sulfate and a combination of these, such that enzyme loading of the
saccharification enzyme consortium can be reduced.
| Inventors: |
SABESAN; SUBRAMANIAM; (Wilmington, DE)
; Spado; Christina Jacy; (Philadelphia, PA)
|
| Correspondence Address:
|
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1122B, 4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
| Assignee: |
E. I. DU PONT DE NEMOURS AND COMPANY
Wilmington
DE
|
| Family ID:
|
42172339
|
| Appl. No.:
|
12/621579
|
| Filed:
|
November 19, 2009 |
Related U.S. Patent Documents
| | | | |
|---|
|
| Application Number | Filing Date | Patent Number |
|---|
| | 61116382 | Nov 20, 2008 | |
| | 61116386 | Nov 20, 2008 | |
|
|
| Current U.S. Class: |
435/101 |
| Current CPC Class: |
C12P 19/02 20130101; C12P 2201/00 20130101; Y02E 50/16 20130101; C13K 1/02 20130101; C12P 19/14 20130101 |
| Class at Publication: |
435/101 |
| International Class: |
C12P 19/04 20060101 C12P019/04 |
Claims
1. A method of producing a concentrated sugar solution from biomass, the
method comprising: a) delignifying biomass comprising the substeps of i)
contacting with water and at least one nucleophilic base, a biomass
comprising lignin and having a glucan/xylan weight ratio G.sub.1/X.sub.1
to form a biomass slurry having a pH of about 12.5 to about 13.0; and ii)
maintaining the biomass slurry under reaction conditions such that the
slurry attains a pH of about 9.5 to about 10.0 and has a a glucan/xylan
weight ratio G.sub.2/X.sub.2 within about 15% of the value of
G.sub.1/X.sub.1, and wherein the slurry comprises a lignin-containing
liquid fraction and a solid fraction comprising a polysaccharide enriched
biomass; wherein G.sub.1 and G.sub.2 are grams of glucan per 100 grams of
biomass and biomass slurry respectively, and X.sub.1 and X.sub.2 are
grams of xylan per 100 grams of biomass and biomass slurry respectively;
and b) contacting with a saccharification enzyme consortium an aqueous
suspension of at least a portion of the solid fraction of the
polysaccharide enriched biomass, the solid fraction of the polysaccharide
enriched biomass being 13 weight percent to about 30 weight percent of
the aqueous suspension, at reaction conditions sufficient to produce a
saccharification product comprising at least about 7 percent by weight
sugars, based on the total weight of the saccharification product, in 24
hours of contact with the saccharification enzyme consortium.
2. The method of claim 1, further comprising: after delignifying, adding
an additive selected from the group consisting of polyethylene glycols,
fatty acid esters, fatty acid ethoxylates, nonionic surfactants derived
from polyethoxylated sorbitan and a fatty acid, sodium
lauriminodipropionate, sodium cocoamphoacetate, sodium tridecyl ether
sulfate, and a combination of these, such that enzyme loading of the
saccharification enzyme consortium is reduced relative to enzyme loading
of the saccharification enzyme consortium when none of the additives is
added.
3. The method of claim 1 or 2, wherein the at least one nucleophilic base
comprises a water soluble metal hydroxide, optionally in combination with
a metal carbonate or an organic hydroxide.
4. The method of claim 1 or 2, wherein the at least one nucleophilic base
comprises a water soluble metal hydroxide, optionally in combination with
a metal carbonate or an organic hydroxide, wherein the water soluble
metal hydroxide is selected from the group consisting of sodium hydroxide
or potassium hydroxide and the metal carbonate or the organic hydroxide
is selected from the group consisting of sodium carbonate, potassium
carbonate, ammonium hydroxides, and alkyl substituted ammonium hydroxide.
5. The method of claim 1 or 2, wherein the reaction conditions to produce
a polysaccharide enriched biomass include a temperature from about
20.degree. C. to about 110.degree. C. and a reaction time of about 4
hours to about 30 days.
6. The method of claim 1 or 2, wherein the value of G.sub.2/X.sub.2 is
within 10% of the value of G.sub.1/X.sub.1.
7. The method of claim 1 or 2, wherein the saccharification product
comprises at least about 18 percent by weight sugars in 168 hours.
8. The method of claim 2, wherein the additive is from about 0.1 weight
percent to about 5 weight percent, based on the weight of the solid
fraction of the polysaccharide enriched biomass solid fraction in the
aqueous suspension.
9. The method of claim 8, wherein the additive is a polyethylene glycol
of molecular weight 500 to 50,000 Daltons, a fatty acid ester selected
from the group consisting of methyl esters of C.sub.12 to C.sub.30 fatty
acid esters, fatty acid ethoxylates of C.sub.12 to C.sub.30 fatty acids,
nonionic surfactants derived from polyethoxylated sorbitan and a C.sub.12
to C.sub.30 fatty acid, and a combination of these.
10. The method of claim 1 or 2 wherein at least about 70 percent of the
lignin in the biomass is delignified in the solid fraction of the
polysaccharide enriched biomass.
11. The method of claim 1 or 2, wherein the concentration of the solid
fraction in the aqueous suspension is from about 21 weight percent to
about 30 weight percent, and the saccharification product comprises
sugars corresponding to at least a 65% saccharification yield based on
the sum of glucan and xylan in the polysaccharide enriched biomass.
12. The method of claim 1 or 2, wherein the sugars comprise at least one
sugar monomer selected from the group consisting of glucose, arabinose,
xylose, mannose, and galactose, and a combination of these.
13. A method to produce a concentrated sugar solution from biomass, the
method comprising: a) providing biomass having undergone a
delignification process to become a delignified biomass comprising
greater than about 85 percent polysaccharides on a dry weight basis; b)
contacting with a saccharification enzyme consortium an aqueous
suspension of the delignified biomass, the concentration of the
delignified biomass in the aqueous suspension being from about 13 weight
percent to about 30 weight percent, at reaction conditions sufficient to
produce a saccharification product comprising at least about 7 percent by
weight sugars, based on the total weight of the saccharification product,
in 24 hours of contact with the saccharification enzyme consortium.
14. The method of claim 13, further comprising: adding an additive
selected from the group consisting of polyethylene glycols, fatty acid
esters, fatty acid ethoxylates, nonionic surfactants derived from
polyethoxylated sorbitan and a fatty acid, sodium lauriminodipropionate,
sodium cocoamphoacetate, sodium tridecyl ether sulfate, and a combination
of these, such that enzyme loading of the saccharification enzyme
consortium is reduced relative to enzyme loading of the saccharification
enzyme consortium when none of the additives is added.
15. A method for delignifying a biomass to produce a polysaccharide
enriched biomass, the process comprising: a) contacting with water and at
least one nucleophilic base, a biomass comprising lignin and having a
glucan/xylan weight ratio G.sub.1/X.sub.1 to form a biomass slurry having
a pH of about 12.5 to about 13.0; and b) maintaining the biomass slurry
under reaction conditions such that the slurry attains a pH of about 9.5
to about 10.0 and has a a glucan/xylan weight ratio G.sub.2/X.sub.2
within about 15% of the value of G.sub.1/X.sub.1, and wherein the slurry
comprises a lignin-containing liquid fraction and a solid fraction
comprising a polysaccharide enriched biomass; wherein G.sub.1 and G.sub.2
are grams of glucan per 100 grams of biomass and biomass slurry
respectively, and X.sub.1 and X.sub.2 are grams of xylan per 100 grams of
biomass and biomass slurry respectively.
16. A polysaccharide enriched biomass produced by the process of claim
16.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority from Provisional
Application No. 61/116382 filed Nov. 20, 2008, and from Provisional
Application No. 61/116386 filed Nov. 20, 2008. This application hereby
incorporates by reference Provisional Application Nos. 61/116382 and
61/116386 in their entirety.
FIELD OF THE INVENTION
[0002] Methods for treating biomass to obtain concentrated sugar solutions
are provided. Specifically, polysaccharide enriched biomass is obtained
by the pretreatment of biomass with at least one nucleophilic base in a
manner which retains the glucan/xylan weight ratio of the untreated
biomass. Concentrated sugar solutions are obtained by enzymatic
saccharification of the polysaccharide enriched biomass, optionally in
the presence of at least one additive.
BACKGROUND
[0003] Cellulosic and lignocellulosic feedstocks and wastes, such as
agricultural residues, wood, forestry wastes, sludge from paper
manufacture, and municipal and industrial solid wastes, provide a
potentially large renewable feedstock for the production of valuable
products such as fuels and other chemicals. Cellulosic and
lignocellulosic feedstocks and wastes, composed of carbohydrate polymers
comprising cellulose, hemicellulose, and lignin are generally treated by
a variety of chemical, mechanical and enzymatic means to release
primarily hexose and pentose sugars, which can then be fermented to
useful products.
[0004] Pretreatment methods are used to make the carbohydrate polymers of
cellulosic and lignocellulosic materials more readily available to
saccharification enzymes. Standard pretreatment methods have historically
utilized primarily strong acids at high temperatures; however due to high
energy costs, high equipment costs, high pretreatment catalyst recovery
costs and incompatibility with saccharification enzymes, alternative
methods are being developed, such as enzymatic pretreatment, or the use
of acid or base at milder temperatures where decreased hydrolysis of
biomass carbohydrate polymers occurs during pretreatment, requiring
improved enzyme systems to saccharify both cellulose and hemicellulose.
[0005] Teixeira, L., et al. (Appl. Biochem. and Biotech. (1999)
77-79:19-34) disclosed a series of biomass pretreatments using
stoichiometric amounts of sodium hydroxide and ammonium hydroxide, with
very low biomass concentration. The ratio of solution to biomass is 14:1.
[0006] Elshafei, A. et al. (Bioresource Tech. (1991) 35:73-80) examined
the pretreatment of corn stover utilizing NaOH. Kim, T. and Y. Lee
(Bioresource Technology (2005) 96:2007-2013) report the use of high
amounts of aqueous ammonia for the pretreatment of corn stover.
[0007] Int'l. Pat. App. Pub. No. WO2004/081185 discusses methods for
hydrolyzing lignocellulose, comprising contacting the lignocellulose with
a chemical; the chemical may be a base, such as sodium carbonate or
potassium hydroxide, at a pH of about 9 to about 14, under moderate
conditions of temperature, pressure and pH.
[0008] U.S. Pat. Nos. 5,916,780 and 6,090,595, describe a pretreatment
process wherein a specified ratio of arabinoxylan to total nonstarch
polysaccharides (AX/NSP) is assessed and used to select the feedstock.
[0009] U.S. Pat. No. 7,354,743 discloses methods for degrading a
lignocellulosic material, comprising treating the lignocellulosic
material with an effective amount of one or more cellulolytic enzymes in
the presence of at least one surfactant; the presence of the surfactant
increases the degradation of lignocellulosic material compared to the
absence of the surfactant.
[0010] Borjesson, J. et al. (Enzyme and Microbial. Technology (2007)
40:754-762) focused on the enzymatic hydrolysis of the softwood substrate
spruce lignocellulose and aimed to give further understanding of the
mechanism behind the enhancing effect on the conversion by addition of
ethylene oxide based surfactants and polymers. No effect of PEG was seen
on a delignified substrate.
[0011] Int'l. Pat. App. Pub. No. W02008134037 added surfactants in the
pretreatment step to enhance the removal of lignin in corn stover biomass
in an effort to increase the digestibility of the delignified biomass.
However, not considered was a reduction in enzyme loading nor reported
was a saccharification product comprising at least about 7 percent by
weight sugars in a 24 hour period after contact with an enzyme
consortium.
[0012] Most pretreatments such as the ones described above either result
in a pretreated biomass depleted of lignin and hemicellulose or the
partial depletion of hemicellulose with retention of most of the lignin.
Therefore a method is needed to selectively remove only lignin without
significant loss of either hemicellulose or cellulose from the biomass,
as these constitute the source of sugars for fermentation. Thus, none of
these references relates to the unpredicted mechanism recited herein:
retaining hemicellulose or cellulose in the biomass such that the
saccharification product comprises at least about 7 percent by weight
sugars in a 24 hour period after contact with an enzyme consortium.
[0013] In order to be economically competitive, a commercial process for
the production of sugars from a renewable resource biomass requires the
hydrolysis of carbohydrates in lignocellulosic biomass to provide high
yields of sugars at high concentrations using low amounts of chemicals.
SUMMARY
[0014] Described herein are methods to produce a concentrated sugar
solution from polysaccharide enriched biomass containing both
hemicellulose and cellulose. The described methods involve a pretreatment
step wherein biomass is contacted with water and at least one
nucleophilic base, with subsequent change in pH that may range about
12.5-13.0 to about 9.5-10. During pretreatment, the lignin is solubilized
and the glucan/xylan weight ratio in the insoluble biomass is largely
retained, compared to that for untreated biomass. The solid fraction of
the resulting polysaccharide enriched biomass is contacted as an aqueous
suspension with a saccharification enzyme consortium, and optionally with
at least one additive, to produce a saccharification product comprising
at least about 7 percent by weight sugars in 24 hours.
[0015] One method described herein is a method of producing a concentrated
sugar solution from biomass, the method comprising: [0016] a)
delignifying biomass comprising the substeps of [0017] i) contacting
with water and at least one nucleophilic base, a biomass comprising
lignin and having a glucan/xylan weight ratio G.sub.1/X.sub.1 to form a
biomass slurry having a pH of about 12.5 to about 13.0; and [0018] ii)
maintaining the biomass slurry under reaction conditions such that the
slurry attains a pH of about 9.5 to about 10.0 and has a a glucan/xylan
weight ratio G.sub.2/X.sub.2 within about 15% of the value of
G.sub.1/X.sub.1, and wherein the slurry comprises a lignin-containing
liquid fraction and a solid fraction comprising a polysaccharide enriched
biomass; [0019] wherein G.sub.1 and G.sub.2 are grams of glucan per 100
grams of biomass and biomass slurry respectively, and X.sub.1 and X.sub.2
are grams of xylan per 100 grams of biomass and biomass slurry
respectively; and [0020] b) contacting with a saccharification enzyme
consortium an aqueous suspension of at least a portion of the solid
fraction of the polysaccharide enriched biomass, [0021] the solid
fraction of the polysaccharide enriched biomass being 13 weight percent
to about 30 weight percent of the aqueous suspension, [0022] at
reaction conditions sufficient to produce a saccharification product
comprising at least about 7 percent by weight sugars, based on the total
weight of the saccharification product, in 24 hours of contact with the
saccharification enzyme consortium.
[0023] In the above method described herein, the at least one nucleophilic
base may comprise a water soluble metal hydroxide, optionally in
combination with a metal carbonate or an organic hydroxide. The water
soluble metal hydroxide is selected from the group consisting of sodium
hydroxide or potassium hydroxide and the metal carbonate or the organic
hydroxide is selected from the group consisting of sodium carbonate,
potassium carbonate, ammonium hydroxides, and alkyl substituted ammonium
hydroxide. The reaction conditions to produce a polysaccharide enriched
biomass include a temperature from about 20.degree. C. to about
110.degree. C. and a reaction time from about 4 hours to about 30 days.
The value of G.sub.2/X.sub.2 may be within 10% of the value of
G.sub.1/X.sub.1.
[0024] The composition of the solid fraction of the polysaccharide
enriched biomass solid fraction, on a dry weight basis, may be greater
than about 85% polysaccharide.
[0025] Another method described herein comprises: [0026] a) providing
biomass having undergone a delignification process to become a
delignified biomass and comprising greater than about 85 percent
polysaccharides on a dry weight basis; [0027] b) contacting with a
saccharification enzyme consortium an aqueous suspension of the
delignified biomass, the concentration of the delignified biomass in the
aqueous suspension being from about 13 weight percent to about 30 weight
percent, at reaction conditions sufficient to produce a saccharification
product comprising at least about 7 percent by weight sugars, based on
the total weight of the saccharification product, in 24 hours of contact
with the saccharification enzyme consortium.
[0028] In any method described herein, at least one additive may be added,
such that enzyme loading of the saccharification enzyme consortium is
reduced relative to enzyme loading of the saccharification enzyme
consortium when none of the additives is added. The additive is from
about 0.1 weight percent to about 5 weight percent, based on the weight
of the isolated polysaccharide enriched biomass solid fraction in the
aqueous suspension. The additive is selected from the group consisting of
polyethylene glycols, fatty acid esters, fatty acid ethoxylates, nonionic
surfactants derived from polyethoxylated sorbitan and a fatty acid,
sodium lauriminodipropionate, sodium cocoamphoacetate, sodium tridecyl
ether sulfate, and a combination of these. The polyethylene glycol may
have a molecular weight between 500 to 50,000 Daltons. The fatty acid
ester may be selected from the group consisting of methyl esters of
C.sub.12 to C.sub.30 fatty acid esters.
[0029] Another method described herein is a method for delignifying a
biomass to produce a polysaccharide enriched biomass, the process
comprising: [0030] a) contacting with water and at least one
nucleophilic base, a biomass comprising lignin and having a glucan/xylan
weight ratio G.sub.1/X.sub.1 to form a biomass slurry having a pH of
about 12.5 to about 13.0; and [0031] b) maintaining the biomass slurry
under reaction conditions such that the slurry attains a pH of about 9.5
to about 10.0 and has a a glucan/xylan weight ratio G.sub.2/X.sub.2
within about 15% of the value of G.sub.1/X.sub.1, and wherein the slurry
comprises a lignin-containing liquid fraction and a solid fraction
comprising a polysaccharide enriched biomass; [0032] wherein G.sub.1 and
G.sub.2 are grams of glucan per 100 grams of biomass and biomass slurry
respectively, and X.sub.1 and X.sub.2 are grams of xylan per 100 grams of
biomass and biomass slurry respectively.
[0033] Also described is a polysaccharide enriched biomass produced by the
process described above.
BRIEF DESCRIPTION OF THE FIGURES
[0034] The methods described herein are described with reference to the
following figures.
[0035] FIG. 1 is a graphical representation of the results for Example 2
showing the amount of xylose and glucose produced upon saccharification
of corn cob delignified with 5.1 weight percent, 8.0 weight percent, and
20.0 weight percent sodium hydroxide (relative to the weight of the cob).
[0036] FIG. 2 is a graphical representation of the results for Example 5
showing the effect of additives PEG 2000 and NINEX.RTM. MT-610 with
varying enzyme loading on the saccharification yield of delignified corn
cob.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0037] The methods described herein are described with reference to the
following terms.
[0038] As used herein, where the indefinite article "a" or "an" is used
with respect to a statement or description of the presence of a step in a
process of this invention, it is to be understood, unless the statement
or description explicitly provides to the contrary, that the use of such
indefinite article does not limit the presence of the step in the process
to one in number.
[0039] As used herein, when an amount, concentration, or other value or
parameter is given as either a range, preferred range, or a list of upper
preferable values and lower preferable values, this is to be understood
as specifically disclosing all ranges formed from any pair of any upper
range limit or preferred value and any lower range limit or preferred
value, regardless of whether ranges are separately disclosed. Where a
range of numerical values is recited herein, unless otherwise stated, the
range is intended to include the endpoints thereof, and all integers and
fractions within the range. It is not intended that the scope of the
invention be limited to the specific values recited when defining a
range.
[0040] As used herein, the terms "comprises," "comprising," "includes,"
"including," "has," "having," "contains" or "containing," or any other
variation thereof, are intended to cover a non-exclusive inclusion. For
example, a composition, a mixture, process, method, article, or apparatus
that comprises a list of elements is not necessarily limited to only
those elements but may include other elements not expressly listed or
inherent to such composition, mixture, process, method, article, or
apparatus. Further, unless expressly stated to the contrary, "or" refers
to an inclusive or and not to an exclusive or. For example, a condition A
or B is satisfied by any one of the following: A is true (or present) and
B is false (or not present), A is false (or not present) and B is true
(or present), and both A and B are true (or present).
[0041] The term "invention" or "present invention" as used herein is a
non-limiting term and is not intended to refer to any single variation of
the particular invention but encompasses all possible variations
described in the specification and recited in the claims.
[0042] As used herein, the term "about" modifying the quantity of an
ingredient or reactant of the invention employed refers to variation in
the numerical quantity that can occur, for example, through typical
measuring and liquid handling procedures used for making concentrates or
use solutions in the real world; through inadvertent error in these
procedures; through differences in the manufacture, source, or purity of
the ingredients employed to make the compositions or carry out the
methods; and the like. The term "about" also encompasses amounts that
differ due to different equilibrium conditions for a composition
resulting from a particular initial mixture. Whether or not modified by
the term "about", the claims include equivalents to the quantities. The
term "about" may mean within 10% of the reported numerical value,
preferably within 5% of the reported numerical value.
[0043] As used herein, the term "biomass" refers to any cellulosic or
lignocellulosic material and includes materials comprising cellulose, and
optionally further comprising hemicellulose, lignin, starch,
oligosaccharides and/or monosaccharides. Biomass may also comprise
additional components, such as protein and/or lipid. Biomass may be
derived from a single source, or biomass can comprise a mixture derived
from more than one source; for example, biomass could comprise a mixture
of corn cobs and corn stover, or a mixture of grass and leaves. Biomass
includes, but is not limited to, bioenergy crops, agricultural residues,
municipal solid waste, industrial solid waste, sludge from paper
manufacture, yard waste, wood and forestry waste or a combination
thereof. Examples of biomass include, but are not limited to, corn grain,
corn cobs, crop residues such as corn husks, corn stover, grasses, wheat,
wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste
paper, sugar cane bagasse, sorghum, soy, components obtained from milling
of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs
and bushes, vegetables, fruits, flowers, and animal manure or a
combination thereof. Biomass that is useful for the invention may include
biomass that has a relatively high carbohydrate value, is relatively
dense, and/or is relatively easy to collect, transport, store and/or
handle. In one embodiment of the invention, biomass that is useful
includes corn cobs, corn stover, sawdust, and sugar cane bagasse.
[0044] As used herein, the term "lignocellulosic" refers to a composition
comprising both lignin and cellulose. Lignocellulosic material may also
comprise hemicellulose.
[0045] As used herein, the term "cellulosic" refers to a composition
comprising cellulose.
[0046] As used herein, by "dry weight" of biomass is meant the weight of
the biomass having all or essentially all water removed. Dry weight is
typically measured according to American Society for Testing and
Materials (ASTM) Standard E1756-01 (Standard Test Method for
Determination of Total Solids in Biomass) or Technical Association of the
Pulp and Paper Industry, Inc. (TAPPI) Standard T-412 om-02 (Moisture in
Pulp, Paper and Paperboard).
[0047] As used herein, the terms "target chemical" and "target product"
are interchangeable and refer to a chemical, fuel, or chemical building
block produced by fermentation. Chemical or product is used in a broad
sense and includes molecules such as proteins, including, for example,
peptides, enzymes, and antibodies. Also contemplated within the
definition of target product are ethanol and butanol.
[0048] As used herein, the term "saccharification" refers to the
hydrolysis of polysaccharides to their constituent monomers and/or
oligomers.
[0049] As used herein, the term "polysaccharide enriched biomass" means
biomass that has been subjected to pretreatment prior to saccharification
such that the noncarbohydrate component of the biomass is significantly
reduced.
[0050] As used herein, "readily saccharifiable biomass" means biomass that
is carbohydrate-enriched and made more amenable to hydrolysis by
cellulolytic or hemi-cellulolytic enzymes for producing monomeric and
oligomeric sugars. The term "readily saccharifiable biomass" as used
herein is interchangeable with the term "solid fraction of the
polysaccharide enriched biomass".
[0051] As used herein, the term "carbohydrate-enriched" as used herein
refers to the biomass produced by the process treatments described
herein. The terms polysaccharide enriched and carbohydrate-enriched are
interchangeable. in one embodiment the readily saccharifiable
carbohydrate-enriched biomass produced by the processes described herein
have a carbohydrate concentration of greater than or equal to about 85%
of the biomass carbohydrate as compared to biomass prior to pretreating
as described herein while removing 75% or greater of the biomass lignin.
[0052] As used herein, the term "loading of the enzyme consortium" and
"enzyme loading" are interchangeable and refer to a ratio of the amount
total weight of protein in the enzyme consortium relative to the weight
of polysaccharide enriched biomass.
[0053] As used herein, the terms "delignification" refers to any process
by which lignin is either partly, mostly or wholly removed from
cellulosic materials. Generally, this process is by means of chemical
treatment. The residue that remains consists of cellulose,
hemicelluloses, and other carbohydrate materials. Any residue having
undergone a delignification is described herein as "delignified". As used
herein, "lignin" refers generally to a polymer found extensively in the
cell walls of all woody plants.
[0054] As used herein, the term "cellulase" refers to
polysaccharide-hydrolyzing enzymes that can exhibit an activity, such as
cellulose degradation, that may be several enzymes or a group of enzymes
having different substrate specificities. Thus, a cellulase from a
microorganism may comprise a group of enzymes, all of which may
contribute to the cellulose-degrading activity.
[0055] As used herein, the terms "nucleophile" and "nucleophilic base"
refer to a Lewis base (as that term is used in the art) that is a reagent
that forms a chemical bond to its reaction partner, the electrophile, by
donating both bonding electrons. Most bases are also nucleophiles. (See
for example Organic Chemistry, 7.sup.th Edition, Morrison, Robert
Thornton; Boyd, Robert N., (1998) Publisher: (Prentice Hall, Englewood
Cliffs, N.J.). For example, in the methods described herein, the
nucleophile NaOH reacts and forms chemical bonds with lignin and its
components.
Pretreatment (Delignification)
[0056] In the methods described herein, biomass is contacted with water
and at least one nucleophilic base to form a biomass slurry having an
initial pH of about 12.5 to about 13.0. The biomass has a glucan/xylan
weight ratio G.sub.1/X.sub.1, where G.sub.1 is the grams of glucan per
100 grams of biomass and X.sub.1 is the grams of xylan per 100 grams of
biomass. Glucan and xylan content of biomass can be determined by methods
known in the art. The source of the biomass is not determinative of the
invention and the biomass may be from any source.
[0057] Once processed the biomass slurry is maintained at a temperature
and for a reaction time sufficient to produce a polysaccharide enriched
biomass having a glucan/xylan weight ratio G.sub.2/X.sub.2, where G.sub.2
is the grams of glucan per 100 grams of polysaccharide enriched biomass
and X.sub.2 is the grams of xylan per 100 grams of polysaccharide
enriched biomass. In contrast to other pretreatment methods, the
polysaccharide enriched biomass is produced without selective loss of
xylan, as evidenced by a comparison of the values of the ratios
G.sub.2/X.sub.2 and G.sub.1/X.sub.1. Similarity of the numerical values
for the glucan/xylan weight ratios of the treated and the untreated
biomass indicate that both glucan and xylan are retained in about the
same relative amounts in the polysaccharide enriched biomass as were
present in the biomass before pretreatment. In one of the described
methods, the value of G.sub.2/X.sub.2 is within about 15% of the value of
G.sub.1/X.sub.1. In another, the value of G.sub.2/X.sub.2 is within about
10% of the value of G.sub.1/X.sub.1. Avoiding preferential loss of xylan
during the pretreatment step provides higher xylose yield after
saccharification and contributes to improved sugar yields overall and
higher sugar concentrations.
[0058] The pretreated biomass is referred to as "polysaccharide enriched
biomass" or "carbohydrate-enriched biomass" because the pretreatment
described above, and in more detail below, solubilizes the lignin
contained in the biomass. The glucan and xylan remain insoluble. Physical
separation of the lignin-containing liquid fraction from the solid
fraction removes lignin and provides solid polysaccharide enriched
biomass.
[0059] Delignifying biomass prior to enzymatic hydrolysis
(saccharification) is advantageous as lignin can bind non-specifically to
saccharification enzymes. Removal of lignin before saccharification
enables the use of lower enzyme loadings, which provides cost savings
with regard to enzyme usage. Removing lignin before saccharification can
also improve saccharification rate, titer, and yield. Furthermore, as
lignin can contribute to increased viscosity of biomass and biomass
slurry, removal of lignin can provide reduced viscosity of biomass and
slurries containing biomass, thereby enabling very high loading, for
example, greater than about 20 percent, of the biomass in order to
produce concentrated sugar syrup.
[0060] The biomass may be used directly as obtained from the source, or
energy may be applied to the biomass to reduce the size, increase the
exposed surface area, and/or increase the availability of cellulose,
hemicellulose, and/or oligosaccharides present in the biomass to the
nucleophilic base and to saccharification enzymes and/or additive used in
the saccharification step. Energy means useful for reducing the size,
increasing the exposed surface area, and/or increasing the availability
of cellulose, hemicellulose, and/or oligosaccharides present in the
biomass include, but are not limited to, milling, crushing, grinding,
shredding, chopping, disc refining, ultrasound, and microwave. This
application of energy may occur before or during pretreatment, before and
during saccharification, or any combination thereof.
[0061] In general, it is often required to mill the biomass before and/or
after pretreatments in order to reduce the particle size and to produce
high surface area and porous particles for effective enzymatic
saccharification. In the current invention, we unexpectedly find that
this energy intensive milling process can be avoided, as the nucleophilic
base treatment under selected conditions results in chemical milling to
provide delignified biomass of substantially reduced particle size.
[0062] The biomass is contacted with water sufficient to wet the entire
biomass and at least one nucleophilic base comprising a water soluble
metal hydroxide, such as sodium hydroxide or potassium hydroxide. The
water soluble metal hydroxide may be used alone or in combination with a
metal carbonate, such as sodium carbonate or potassium carbonate, or an
organic hydroxide, such as ammonium or alkyl substituted ammonium
hydroxides. The nucleophilic base is combined as an aqueous solution or
as a solid with the biomass and water to form biomass slurry having an
initial pH of about 12.5 to about 13.0. As the delignification proceeds,
some of the base is consumed and the pH of the biomass slurry is reduced
to a range of about 9.5 to about 10.0. A sufficient concentration of base
should be used such that the pH does not drop lower, which would result
in insufficient delignification. The extent of delignification can depend
at least in part on the choice of reaction conditions and the type of
biomass used. For example, in the case of corn cob, about 8 weight
percent of NaOH relative the weight of the corn cob has been found to
provide optimum delignification. At least about 70 percent of the lignin
in the provided biomass may be delignified in the isolated polysaccharide
enriched biomass. At least about 80 percent of the lignin in the provided
biomass may be delignified in the isolated polysaccharide enriched
biomass. At least about 90 percent of the lignin may be delignified in
the isolated polysaccharide enriched biomass.
[0063] The amount of water in the biomass slurry may be from about 25
weight percent to about 90 weight percent, for example from about 50
weight percent to about 90 weight percent, or from about 75 weight
percent to about 90 weight percent based on the combined weight of the
biomass, the water, and the nucleophilic base. The water in the biomass
slurry refers to the total water from all sources and includes any water
contained in or on the biomass, water contained in an aqueous solution of
the nucleophilic base, and water added separately.
[0064] In the methods described herein, the dry weight of biomass in the
biomass slurry may be at an initial concentration from about 10 weight
percent to about 75 weight percent, or for example from about 10 weight
percent to about 50 weight percent, or for example from about 10 weight
percent to about 25 weight percent, based on the combined weight of the
biomass, the water, and the nucleophilic base. The biomass concentration
may be maximized to the extent possible to minimize the volume of the
reaction vessel. The high biomass concentration also reduces the total
volume of pretreatment material, making the process more economical. From
a practical viewpoint, high ratios of the weight of biomass to the weight
of the basic solution may be limited by the ability to provide sufficient
mixing, or intimate contact, for pretreatment to occur at a practical
rate.
[0065] The biomass slurry is maintained at a temperature of from about
20.degree. C. to about 110.degree. C., for example from about 80.degree.
C. to about 110.degree. C.
[0066] The contacting of the biomass with water and at least one
nucleophilic base is carried out for a period time from about 4 hours to
about 30 days, for example from about 4 hours to about 1 day. Longer
periods of pretreatment are possible, however a shorter period of time
may be preferable for practical, economic reasons. Typically a period of
contact may be about 24 hours or less and may be determined by the time
required for the pH of the biomass slurry to drop from a range of about
12.5 to 13.0 to a range of about 9.5 to 10.0.
[0067] The delignification of biomass with water and at least one
nucleophilic base may be performed at a relatively high temperature for a
relatively short period of time, for example at from about 90.degree. C.
to about 100.degree. C. for about 24 hours to about 16 hours.
Alternatively, the biomass-nucleophilic base contacting process may be
performed at a lower temperature for a longer period of time, for example
from about 50.degree. C. to about 80.degree. C. for about 140 hours to
about 100 hours. Moreover, the biomass-acid contacting process may be
performed at room temperature (approximately 22-25.degree. C.) for a
period of time up to about 300 hours. Other temperature and time
combinations intermediate to these may also be used.
[0068] For the contacting of the biomass with water and at least one
nucleophilic base, the temperature, reaction time, base concentration,
weight percent of total water, the biomass concentration, the biomass
type, and the biomass particle size are related; thus these variables may
be adjusted as necessary to obtain sufficient delignification rate in a
controllable manner and to obtain an optimal product for saccharification
to sugars.
[0069] The pretreatment may be performed in any suitable vessel, such as a
batch reactor a continuous reactor. The suitable vessel may be equipped
with a means, such as impellers, for agitating the biomass/acid mixture.
Reactor design is discussed in Lin, K.-H., and Van Ness, H. C. (in Perry,
R. H. and Chilton, C. H. (eds), Chemical Engineer's Handbook, 5.sup.th
Edition (1973) Chapter 4, McGraw-Hill, NY). The pretreatment may be
carried out as a batch process, or as a continuous process.
Alternatively, the biomass, water and nucleophilic base may be combined
in one vessel, then transferred to another reactor. Also biomass may be
pretreated in one vessel, then further processed in another reactor.
[0070] In order to obtain sufficient quantities of sugars from biomass,
the biomass may be pretreated with water and at least one nucleophilic
base either once or several times. Likewise, a saccharification reaction
may be performed one or more times. Both pretreatment and
saccharification processes may be repeated if desired to obtain higher
yields of sugars. To assess performance of the pretreatment and
saccharification processes, separately or together, the theoretical yield
of sugars derivable from the starting biomass may be determined and
compared to the measured yields.
Saccharification
[0071] Following pretreatment of the provided biomass with water and at
least one nucleophilic base, the polysaccharide enriched biomass
comprises a mixture of nucleophilic base, water, partially degraded
biomass, lignin, polysaccharides, and monosaccharides. The mixture
comprises a solid (insoluble) fraction and a liquid (soluble) fraction.
The solid fraction comprises biomass in which the non-carbohydrate
component has been significantly reduced. The liquid fraction is composed
of lignin and its fragments as its metal salt, along with the excess base
and salts related to the nucleophilic base. Prior to saccharification, at
least a portion of the solid fraction of the polysaccharide enriched
biomass may be isolated in order to physically separate it from the
lignin-containing liquid fraction. Isolation of as much of the solid
fraction as possible is advantageous, as this allows higher yield of
sugars to be obtained after saccharification. In the methods described
herein, the composition of the isolated solid fraction of the
polysaccharide enriched biomass, on a dry weight basis, is greater than
about 75% polysaccharide. The composition of the isolated solid fraction
of the polysaccharide enriched biomass, on a dry weight basis, may be
greater than about 80% polysaccharide or greater than about 85%
polysaccharide or greater than about 90% polysaccharide.
[0072] Methods for separating the solid fraction from the liquid fraction
include, but are not limited to, decantation, filtration, and
centrifugation. Methods of filtration include, for example, belt
filtration, vacuum filtration, and pressure filtration. Optionally, at
least a portion of the solid fraction may be recycled to the pretreatment
reactor. The solid fraction may optionally be washed with an aqueous
solvent (e.g., water) to remove adsorbed lignin prior to being recycled
to the pretreatment reactor. The solid fraction may then be re-subjected
to additional treatment with at least one nucleophilic base as described
above for pretreatment, followed by saccharification with a
saccharification enzyme consortium.
[0073] The liquid fraction may optionally be used as an energy source, or
some of the desirable components contained in it may be isolated for
additional uses.
[0074] In the methods described herein, an aqueous suspension of the solid
fraction of the polysaccharide enriched biomass is contacted with a
saccharification enzyme consortium, and optionally with at least one
additive, at a pH and a temperature sufficient to produce a
saccharification product comprising at least about 7 percent by weight
sugars in 24 hours of contact with the saccharification enzyme
consortium. The concentration of the solid fraction of the polysaccharide
enriched biomass in the aqueous suspension may be from about 13 weight
percent to about 30 weight percent, for example, from about 21 weight
percent to about 30 weight percent, or for example, from about 15 weight
percent to about 25 weight percent. The at least one additive is selected
from the group consisting of polyethylene glycols, fatty acid esters,
fatty acid ethoxylates, nonionic surfactants derived from polyethoxylated
sorbitan and a fatty acid, and a combination of these.
[0075] Alternatively, delignified biomass comprising greater than about 85
percent polysaccharides on a dry weight basis can be used in place of the
isolated solid fraction of polysaccharide enriched biomass obtained as
described above. This delignified biomass can be obtained by an
alternative method.
[0076] Prior to saccharification, the aqueous suspension of the solid
fraction of the polysaccharide enriched biomass may be treated to alter
the pH, composition or temperature such that the enzymes of the
saccharification enzyme consortium will be active. The pH may be altered
through the addition of acids in solid or liquid form. Alternatively,
carbon dioxide (CO.sub.2), which may be recovered from fermentation, may
be utilized to lower the pH. For example, CO.sub.2 may be collected from
a fermenter and fed, such as by bubbling, into the aqueous suspension of
the isolated polysaccharide enriched biomass while monitoring the pH,
until the desired pH is achieved. The temperature may be brought to a
temperature that is compatible with saccharification enzyme activity, as
noted below. Any cofactors required for activity of enzymes used in
saccharification may be added.
[0077] At least a portion of the isolated solid fraction of the
polysaccharide enriched biomass is then further hydrolyzed in the
presence of a saccharification enzyme consortium to release
oligosaccharides and/or monosaccharides in a hydrolyzate.
Saccharification enzymes and methods for biomass treatment are reviewed
in Lynd, L. R., et al. (Microbiol. Mol. Biol. Rev. (2002) 66:506-577).
[0078] The saccharification enzyme consortium comprises one or more
enzymes selected primarily, but not exclusively, from the group
"glycosidases" which hydrolyze the ether linkages of di-, oligo-, and
polysaccharides and are found in the enzyme classification EC 3.2.1.x
(Enzyme Nomenclature 1992, Academic Press, San Diego, Calif. with
Supplement 1 (1993), Supplement 2 (1994), Supplement 3 (1995, Supplement
4 (1997) and Supplement 5 [in Eur. J. Biochem. (1994) 223:1-5, Eur. J.
Biochem. (1995) 232:1-6, Eur. J. Biochem. (1996) 237:1-5, Eur. J.
Biochem. (1997) 250:1-6, and Eur. J. Biochem. (1999) 264:610-650,
respectively]) of the general group "hydrolases" (EC 3.). Glycosidases
useful in the present method can be categorized by the biomass component
that they hydrolyze. Glycosidases useful for the present method include
cellulose-hydrolyzing glycosidases (for example, cellulases,
endoglucanases, exoglucanases, cellobiohydrolases, .beta.-glucosidases),
hemicellulose-hydrolyzing glycosidases (for example, xylanases,
endoxylanases, exoxylanases, .beta.-xylosidases, arabinoxylanases,
mannases, galactases, pectinases, glucuronidases), and starch-hydrolyzing
glycosidases (for example, amylases, .alpha.-amylases, .beta.-amylases,
glucoamylases, .alpha.-glucosidases, isoamylases). In addition, it may be
useful to add other activities to the saccharification enzyme consortium
such as peptidases (EC 3.4.x.y), lipases (EC 3.1.1.x and 3.1.4.x),
ligninases (EC 1.11.1.x), and feruloyl esterases (EC 3.1.1.73) to help
release polysaccharides from other components of the biomass. It is well
known in the art that microorganisms that produce
polysaccharide-hydrolyzing enzymes often exhibit an activity, such as
cellulose degradation, that is catalyzed by several enzymes or a group of
enzymes having different substrate specificities. Thus, a "cellulase"
from a microorganism may comprise a group of enzymes, all of which may
contribute to the cellulose-degrading activity. Commercial or
non-commercial enzyme preparations, such as cellulase, may comprise
numerous enzymes depending on the purification scheme utilized to obtain
the enzyme. Thus, the saccharification enzyme consortium of the present
method may comprise enzyme activity, such as "cellulase", however it is
recognized that this activity may be catalyzed by more than one enzyme.
[0079] Saccharification enzymes may be obtained commercially, such as
Spezyme.RTM. CP cellulase (Genencor International, Rochester, N.Y.) and
Multifect.RTM. xylanase (Genencor). In addition, saccharification enzymes
may be produced biologically, including using recombinant microorganisms.
[0080] Preferably the saccharification reaction is performed at or near
the temperature and pH optima for the saccharification enzymes. The
temperature optimum used with the saccharification enzyme consortium in
the present method may range from about 15.degree. C. to about
100.degree. C. The temperature optimum may range from about 20.degree. C.
to about 80.degree. C., or from about 30.degree. C. to about 60.degree.
C., or from about 45.degree. C. to about 55.degree. C. The pH optimum may
range from about 4 to about 6 or from about 4.5 to about 5.5 or from
about 4.5 to about 5.0.
[0081] The saccharification may be performed for a time of about several
minutes to about 168 hours, for example from about several minutes to
about 48 hours. The time for the reaction will depend on enzyme
concentration and specific activity, as well as the substrate used and
the environmental conditions, such as temperature and pH. One skilled in
the art can readily determine optimal conditions of temperature, pH and
time to be used with a particular substrate and saccharification
enzyme(s) consortium. These variables may be adjusted as necessary to
obtain an optimal saccharification product for use in fermentation.
[0082] The saccharification may be performed batch-wise or as a continuous
process. The saccharification may also be performed in one step, or in a
number of steps. For example, different enzymes required for
saccharification may exhibit different pH or temperature optima. A
primary treatment can be performed with enzyme(s) at one temperature and
pH, followed by secondary or tertiary (or more) treatments with different
enzyme(s) at different temperatures and/or pH. In addition, treatment
with different enzymes in sequential steps may be at the same pH and/or
temperature, or different pHs and temperatures, such as using
hemicellulases stable and more active at higher pHs and temperatures
followed by cellulases that are active at lower pHs and temperatures.
[0083] At least one additive may be present in the contacting of the
aqueous suspension of the solid fraction of the polysaccharide enriched
biomass with the saccharification enzyme consortium. The additive is
selected from the group consisting of polyethylene glycols, fatty acid
esters, fatty acid ethoxylates, nonionic surfactants derived from
polyethoxylated sorbitan and a fatty acid, sodium lauriminodipropionate,
sodium cocoamphoacetate, and sodium tridecyl ether sulfate, and a
combination of these. Examples of suitable additives include, but are not
limited to, polyethylene glycol having molecular weight from about 500 to
about 50,000 Daltons and commercial vegetable oil based surfactants such
as fatty acid esters. The fatty acid ester may be selected from the group
consisting of methyl esters of C.sub.12 to C.sub.30 fatty acid esters.
The fatty acid ethoxylate may be selected from the group consisting of
ethoxylates of C.sub.12 to C.sub.30 fatty acids. Also suitable are
nonionic surfactants derived from polyethoxylated sorbitan and a fatty
acid such as lauric acid or oleic acid, for example polysorbates. A
combination of surfactants may also be used. Examples of commercially
available additives useful in the present invention are shown in Table 9.
The concentration of the additive may be from about 0.1 weight percent to
about 5.0 weight percent or from about 0.25 weight percent to about 3.0
weight percent, based on the weight of the solid fraction of the
polysaccharide enriched biomass in the aqueous suspension.
[0084] In these methods, the use of an additive in conjunction with a low
enzyme loading enhances the saccharification yield, as compared to the
case where no additive is used. Use of an additive in conjunction with a
low enzyme loading also leads to higher saccharification enhancement as
compared to the case where higher loadings of enzyme are used with an
additive. The loading of the enzyme consortium is such that it is
sufficient for the additive to be effective. Reducing the loading of the
enzyme consortium reduces process cost and improves overall process
economics.
[0085] The saccharification product comprises sugars, wherein the sugars
comprise at least one sugar monomer selected from the group consisting of
glucose, arabinose, xylose, mannose, galactose, and a combination
thereof. The saccharification product may comprise: at least about 7
percent by weight sugars, based on the total weight of the
saccharification product, in 24 hours of contact with the
saccharification enzyme consortium; or at least about 18 percent by
weight sugars in 168 hours of contact with the saccharification enzyme
consortium. The concentration of the solid fraction of the polysaccharide
enriched biomass in the aqueous suspension for saccharification may be
from about 21 weight percent to about 30 weight percent, and the
saccharification product may comprise sugars corresponding to at least a
65% saccharification yield, based on the sum of glucan and xylan in the
polysaccharide enriched biomass.
[0086] The saccharification reaction may be performed in any suitable
vessel, such as a batch reactor a continuous reactor. The suitable vessel
may be equipped with a means, such as impellers, for agitating the
biomass/acid mixture. Reactor design is discussed in Lin, K.-H., and Van
Ness, H. C. (in Perry, R. H. and Chilton, C. H. (eds), Chemical
Engineer's Handbook, 5.sup.th Edition (1973) Chapter 4, McGraw-Hill, NY).
[0087] The degree of solubilization of sugars from biomass following
saccharification may be monitored by measuring the release of
monosaccharides and oligosaccharides. Methods to measure monosaccharides
and oligosaccharides are well known in the art. For example, the
concentration of reducing sugars can be determined using the
1,3-dinitrosalicylic (DNS) acid assay (Miller, G. L., Anal. Chem. (1959)
31:426-428). Alternatively, sugars can be measured by HPLC using an
appropriate column as described herein in the General Methods section.
Fermentation to Target Products
[0088] The readily saccharifiable biomass produced by the present methods
may be hydrolyzed by enzymes as described above to produce fermentable
sugars which then may be fermented into a target product. "Fermentation"
refers to any fermentation process or any process comprising a
fermentation step. Target products include, without limitation alcohols
(e.g., arabinitol, butanol, ethanol, glycerol, methanol, 1,3-propanediol,
sorbitol, and xylitol); organic acids (e.g., acetic acid, acetonic acid,
adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid,
formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid,
glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic
acid, malonic acid, oxalic acid, propionic acid, succinic acid, and
xylonic acid);
[0089] ketones (e.g., acetone); amino acids (e.g., aspartic acid, glutamic
acid, glycine, lysine, serine, and threonine); gases (e.g., methane,
hydrogen (H.sub.2), carbon dioxide (CO.sub.2), and carbon monoxide (CO)).
[0090] Fermentation processes also include processes used in the
consumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,
fermented dairy products), leather industry, and tobacco industry.
[0091] Further to the above, the sugars produced from saccharifying the
pretreated biomass as described herein may be used to produce in general,
organic products, chemicals, fuels, commodity and specialty chemicals
such as xylose, acetone, acetate, glycine, lysine, organic acids (e.g.,
lactic acid), 1,3-propanediol, butanediol, glycerol, ethylene glycol,
furfural, polyhydroxyalkanoates, cis, cis-muconic acid, and animal feed
(Lynd, L. R., Wyman, C. E., and Gerngross, T. U., Biocommodity
Engineering, Biotechnol. Prog., 15: 777-793, 1999; and Philippidis, G.
P., Cellulose bioconversion technology, in Handbook on Bioethanol:
Production and Utilization, Wyman, C. E., ed., Taylor & Francis,
Washington, D.C., 179-212, 1996; and Ryu, D. D. Y., and Mandels, M.,
Cellulases: biosynthesis and applications, Enz. Microb. Technol., 2:
91-102, 1980).
[0092] Potential coproducts may also be produced, such as multiple organic
products from fermentable carbohydrate. Lignin-rich residues remaining
after pretreatment and fermentation can be converted to lignin-derived
chemicals, chemical building blocks or used for power production.
[0093] Conventional methods of fermentation and/or saccharification are
known in the art including, but not limited to, saccharification,
fermentation, separate hydrolysis and fermentation (SHF), simultaneous
saccharification and fermentation (SSF), simultaneous saccharification
and cofermentation (SSCF), hybrid hydrolysis and fermentation (HHF), and
direct microbial conversion (DMC).
[0094] SHF uses separate process steps to first enzymatically hydrolyze
cellulose to sugars such as glucose and xylose and then ferment the
sugars to ethanol. In SSF, the enzymatic hydrolysis of cellulose and the
fermentation of glucose to ethanol is combined in one step (Philippidis,
G. P., in Handbook on Bioethanol: Production and Utilization, Wyman, C.
E., ed., Taylor & Francis, Washington, D.C., 179-212, 1996). SSCF
includes the cofermentation of multiple sugars (Sheehan, J., and Himmel,
M., Bioethanol, Biotechnol. Prog. 15: 817-827, 1999). HHF includes two
separate steps carried out in the same reactor but at different
temperatures, i.e., high temperature enzymatic saccharification followed
by SSF at a lower temperature that the fermentation strain can tolerate.
DMC combines all three processes (cellulase production, cellulose
hydrolysis, and fermentation) in one step (Lynd, L. R., Weimer, P. J.,
van Zyl, W. H., and Pretorius, I. S., Microbiol. Mol. Biol. Reviews, 66:
506-577, 2002).
[0095] These processes may be used to produce target products from the
readily saccharifiable biomass produced by the pretreatment methods
described herein.
EXAMPLES
[0096] The methods described herein are illustrated in the following
examples. From the above discussion and these examples, one skilled in
the art can ascertain the essential characteristics of this invention,
and without departing from the spirit and scope thereof, can make various
changes and modifications of the invention to adapt it to various uses
and conditions.
[0097] The following materials were used in the examples. All commercial
reagents were used as received. Sulfuric acid, ammonium hydroxide, acetic
acid, acetamide, yeast extract, glucose, xylose, sorbitol, MgSO4.7H2O,
phosphoric acid and citric acid were obtained from Sigma-Aldrich (St.
Louis, Mo.). The additives listed in Table 9 were obtained from Stepan
Company (Northfield, Ill.).
[0098] Corn cob was purchased from Independence Corn By Products (ICBP
Cob), Independence, Iowa. The seller stored the cob at 60.degree. C. and
milled and sieved the cob to 1/8''. The dry mass content of the cob was
about 92.5%. Another variety of cob referred to as MDO7 cob was obtained
from University of Wisconsin Farm, in Madison, Wis. and was milled to
assorted sizes. [0062]The following abbreviations are used: "HPLC" is
High Performance Liquid Chromatography, "C" is Centigrade, "kPa" is
kiloPascal, "m" is meter, "mm" is millimeter, "kW" is kilowatt, ".mu.m"
is micrometer, ".mu.L" is microliter, "mL" is milliliter, "L" is liter,
"min" is minute, "mM" is millimolar, "cm" is centimeter, "g" is gram(s),
"mg" is milligrams, "kg" is kilogram, "wt" is weight, "wt %" means weight
percent "h" is hour(s), "temp" or "T" is temperature, "theoret" is
theoretical, "pretreat" is pretreatment, "DWB" is dry weight of biomass,
"ASME" is the American Society of Mechanical Engineers, "s.s." is
stainless steel, "PEG" is polyethylene glycol.
[0099] Carbohydrate Analysis of Biomass
[0100] A modified version of the NREL LAP procedure "Determination of
Structural Carbohydrates and Lignin in Biomass" was used to determine the
weight percent glucan and xylan in the biomass. Sample preparation was
simplified by drying at 80.degree. C. under vacuum or at 105.degree. C.
under ambient pressure overnight. The samples were knife milled to pass
through a 20 mesh screen but were not sieved. The dry milled solids were
than subjected to the acid hydrolysis procedure at a 50 mg solids scale.
The solids were not first extracted with water or ethanol. HPLC analysis
of sugars was done on an Aminex HPX-87H column and no analysis of lignin
was attempted.
[0101] The soluble sugars glucose, cellobiose, and xylose in
saccharification liquor were measured by HPLC (Waters Alliance Model,
Milford, Mass.) using Bio-Rad HPX-87H column (Bio-Rad Laboratories,
Hercules, Calif.) with appropriate guard columns, using 0.01 N aqueous
sulfuric acid as the eluant. The sample pH was measured and adjusted to
5-6 with sulfuric acid if necessary. The sample was then passed through a
0.2 .mu.m syringe filter directly into an HPLC vial. The HPLC run
conditions were as follows: [0102] Biorad Aminex HPX-87H (for
carbohydrates): [0103] Injection volume: 10-50 .mu.L, dependent on
concentration and detector limits [0104] Mobile phase: 0.01 N aqueous
sulfuric acid, 0.2 micron filtered and degassed [0105] Flow rate: 0.6
mL/minute [0106] Column temperature: 50.degree. C., guard column
temperature <60.degree. C. [0107] Detector temperature: as close to
main column temperature as possible [0108] Detector: refractive index
[0109] Run time: 15 minute data collection After the run, concentrations
in the sample were determined from standard curves for each of the
compounds.
[0110] General Procedure for Delignification of Corn Cob
[0111] Corn cob was suspended in a specified volume of deionized water
containing a specified weight of nucleophilic base and then mixed with a
mechanical stirrer. The slurry was heated to the desired temperature for
a specified time. Following this the reaction mixture was cooled to
50.degree. C., vacuum filtered, and the solid residue was washed with
deionized water. The solid residue was dried at room temperature either
under ambient condition or laboratory vacuum (20 mm Hg). The dry mass
content of the solid residue was determined by weighing a known weight of
sample and heating to 99.degree. C. under nitrogen atmosphere until
constant weight was achieved.
Example 1
Production of Concentrated Sugar Solution from Corn Cob Delignification of
the Corn Cob
[0112] Corn cob (MDO7, 2.5 kg, moisture content 10%) was slowly added to a
stirred solution of 2% sodium hydroxide solution (10 L). The amount of
sodium hydroxide (NaOH) was 8.0 weight percent relative to the weight of
corn cob. The initial pH of the solution was 12.3. The mixture was heated
to reflux and maintained under reflux for 20 h. The reaction mixture was
allowed to cool to 60.degree. C. The reaction pH at this point was 9.80.
A portion of the mixture (200 g) was set aside. The remainder of the
mixture was transferred to a filter funnel with the aid of additional 1.9
kg water and filtered under laboratory vacuum. The filtrate was kept
separately for analysis. The solid residue from the filtration was washed
with water (4.times.5 L) and re-suspended in deionized water (10 L). The
pH of the suspension was maintained at 5.0 for 2 hours by one addition of
37% HCl (2.5 mL). The suspension was then filtered. After draining off
most of the liquid, the solid was collected and stored at room
temperature.
[0113] The weight of the solid recovered was 5.31 kg. A portion of the
solid was dried at 99.degree. C. under nitrogen atmosphere for 4 h to
determine the dry matter content of the cake, which was determined to be
25.9%. Further drying of the cake prior to saccharification was done
under atmospheric pressure and at ambient temperature for 4 days.
[0114] The glucan and xylan content of the cob before and after
delignification was determined by the NREL methods, well established in
the art, and were found to be as follows: [0115] Raw cob=39.2 wt %
glucan; 28 wt % xylan [0116] Delignified cob=51 wt % glucan, 38 wt %
xylan The weight ratio of glucan to xylan in the raw cob was 1.40. The
weight ratio of glucan to xylan in the delignified cob was 1.34.
Saccharification of the Delignified Cob Under High Solid Loading (22.6 wt
%)
[0117] The dried, delignified cob from the procedure above (208.9 g total,
moisture content 16.2 wt %) was used for saccharification. A portion of
the solid (155.4 g) was added to a solution of aqueous polyethylene
glycol (1.25 g PEG, molecular weight 2000), and the suspension was heated
to 50.degree. C. with good mixing. The pH of the slurry was adjusted to
5.0 by the addition of 2% aqueous sulfuric acid (10.6 g of solution)
followed by the addition of Accellerase.TM. 1000 cellulase (14.86 ml,
protein concentration 97 mg/mL) and Multifect.RTM. CX 12L enzyme (11.6
mL, protein concentration 56 mg/mL). The reaction was continued at this
temperature and pH (1.28 g of 2% aqueous sulfuric acid was added to
maintain this pH). After 3 h, the remainder of the delignified cob (53.5
g, moisture content 16.2 wt %) and additional Accellerase.TM. 1000
cellulase (5.14 mL) and Multifect.RTM. CX 12L enzyme (3.9 mL) were added,
followed by addition of a 0.512% aqueous solution of Penicillin G (0.5
mL, 2.5 ppm) and 2.1% aqueous solution of Virginiamycin (0.1 mL, 2 ppm).
The reaction was continued with good mixing at 50.degree. C. and at pH
5.0 (6.42 g of 2% aqueous sulfuric acid was added to maintain the pH at
5.0). Samples were withdrawn at intervals of 24, 48, 72, 96, and 117 h
from the time of the last addition of solid. The sample was diluted 10
times (on a weight basis), filtered through 0.2 micron filter, and the
filtrate was analyzed by HPLC as described in the General Methods for
glucose, xylose, and cellobiose and compared to a standard aqueous
solution glucose (8.8 mg/g), xylose (8.9 mg/g), and cellobiose (8.8
mg/g). From this, the saccharification yield and sugar titer was
calculated. The results are shown in the following Tables.
TABLE-US-00001
TABLE 1
Cellobiose, glucose, and xylose concentrations at various
saccharification reaction times as determined by HPLC analysis.
CONCENTRATIONS (mg/g of reaction mixture)
Time (h)
Component 24 48 72 117
Cellobiose 0.0 0.0 0.0 0.0
Glucose 54.0 72.0 82.3 106.0
Xylose 56.3 62.5 63.1 71.8
Lactic Acid 0.0 0.0 0.0 0.2
TABLE-US-00002
TABLE 2
Total mass of sugar monomers produced
in the saccharification reaction with time.
TOTAL MASS (g)
Time (h)
24 48 72 117
Glucose 41.7 55.7 63.6 82.0
Xylose 43.5 48.3 48.8 55.5
Total Sugar 85.3 104.0 112.4 137.5
TABLE-US-00003
TABLE 3
Glucose, xylose, combined saccharification (total sugar)
percent yields, and percent sugar titer with time.
SACCHARIFICATION PERCENT YIELD
24 h 48 h 72 h 117 h
Glucose 42.1 56.2 64.2 82.7
Xylose 57.4 63.7 64.3 73.2
Total Sugar 48.8 59.5 64.3 78.6
Sugar Titer (%)
24 h 48 h 72 h 117 h
11.0 13.5 14.5 17.8
Example 2
Polysaccharide Enrichment and Delignification Of Corn Cob by Treatment
with Sodium Hydroxide at 5.1, 8.0 and 20.0% Wt. % (Relative to Weight of
Cob) and Comparison of Saccharification Performance
[0118] 5.1% Sodium hydroxide treatment (5.1 wt % NaOH relative to weight
of cob): Corn cob (ICBP, 100 g, milled to 2 mm) was suspended in 0.85%
aqueous sodium hydroxide (200 mL, pH 13.0) and heated to 110.degree. C.
for 18 h. When the pH was checked at this time, it was nearly neutral.
Another 200 mL of 0.85% aqueous sodium hydroxide and solid sodium
hydroxide (1.7 g) were added and the heating was continued with
occasional shaking of the flask. After 24 h, the hot solution was
filtered and extensively washed with water. Though brown color eluted out
with the filtrate, the solid material was brown colored indicating the
presence of lignin adsorbed to the material. Also, the corn cob pellets
retained their shape without as much chemical milling occurring as seen
in pretreatment with higher concentrations of NaOH solution. The residue
was suspended in water and the pH of the solution was adjusted to pH 5.0
with 20% aqueous citric acid. The residue was filtered and dried at room
temperature under laboratory vacuum for 24 h. Yield of solid was 70.3 g.
The sample was determined to have 6% moisture content.
8.0% Sodium Hydroxide Treatment (8.0 wt % NaOH Relative to Weight of Cob)
[0119] Corn cob (ICBP, 100 g, milled to 2 mm) was suspended in 2% aqueous
sodium hydroxide and heated to 110.degree. C. for 24 h. The solution was
filtered hot and the residue washed with water to neutral pH and dried
under laboratory vacuum for 48 h. The weight of pale yellow powder was
79.3 g. The moisture content of the solid was 20%.
[0120] A portion of the dried solid (42.0 g) was suspended in water (500
ml) and the pH (9.5) was lowered to 5.0 by the addition of 10% aqueous
citric acid solution. After 45 min at this pH the suspension was
filtered, washed with water and dried under laboratory vacuum. The
moisture content of this material was 7%.
20.0% Sodium Hydroxide Treatment (20.0 wt % NaOH Relative to Weight of
Cob)
[0121] Corn cob (ICBP, 1000 g, milled to 2 mm size) was suspended in 5%
aqueous sodium hydroxide (4000 mL) and heated to 110.degree. C. for 16 h.
The dark brown liquid was filtered hot and much of the liquid on the
solid was drained under laboratory vacuum. The solid residue on the
filter was washed with water until no more color eluted out. The solid
was dried under laboratory vacuum for 24 hours.
[0122] 100 gram of the above sample was suspended in water (700 mL) and
stirred. The pH of the solution was 11.2. Aqueous citric acid solution
(10%) was added to lower the pH to 5.0 and the suspension was stirred for
30 min. The solid was then filtered, washed with water and dried under
vacuum at room temperature for 24 hours. After drying, 86.2 g of
polysaccharide enriched biomass was obtained. The moisture content of
this material was 7.3 wt %.
[0123] Glucan/xylan ratios, glucan wt %, xylan wt %, lignin wt %, and the
percentage total carbohydrate content before and after sodium hydroxide
treatment, as determined by the NREL methods for carbohydrate analysis,
are presented in Table 4. The pretreatments with 5.1 and 8.0 weight
percent NaOH relative to the weight of the biomass used show
delignification of the biomass while maintaining a glucan/xylan weight
ratio within 15% of that for the untreated biomass.
TABLE-US-00004
TABLE 4
Results for Polysaccharide Enriched Biomass
Obtained by NaOH Pretreatments (Example 2).
% Total
Glucan/Xylan Carbohy-
weight Glucan Xylan Lignin drate in the
Sample Ratio (wt %) (wt %) (wt %) Biomass
Untreated 1.33 37.5 28.74 13.88 66
corn cob
5.1% NaOH 1.33 47.8 35.8 ND .sup.1 84
8.0% NaOH 1.35 52.96 39.11 3.33 92
20% NaOH 1.84 58.55 31.86 5.43 90
Note:
.sup.1 ND means "not determined"
Comparative Saccharification of Solid Fractions of Polysaccharide Enriched
Biomass Samples Obtained by Treatment With 5.1, 8.0, and 20% NaOH
Relative to the Weight of the Cob
[0124] Polysaccharide enriched biomass samples (1.0 g, moisture content
7-8%) obtained as solids from each of the 5.1 wt %, 8.0 wt %, and 20.0 wt
% sodium hydroxide treatment cases were separately suspended in 50 mM
citrate buffers (pH 5.0, 6.4 ml). Spezyme.RTM. (Genencor) cellulase
solutions (100 .mu.L, protein concentration 150 mg/mL) and Multifect.RTM.
CX 12L enzyme (100 .mu.L, protein concentration 45 mg/mL) were added to
each buffer and the suspensions kept at 55.degree. C. in a rotating oven.
Samples (100 .mu.L) were withdrawn at intervals of 24 h, 72 h, and 144
hours (6 days) and diluted to 1 ml with de-ionized water, filtered
through 0.2 micron filter and subjected to HPLC analysis. At the end of
the sixth day, each reaction mixture was filtered. The insoluble residue
was dried in an oven at 99.degree. C. for 24 h, where as the filtrate was
lyophilized to determine the solid content.
[0125] Results are presented in Table 5, Table 6, and FIG. 1, which is a
graphical representation of the data in Table 5. As evident from the
[0126] Tables and FIG. 1, biomass delignified with 8.0 weight percent and
20.0 weight percent NaOH, relative to the weight of the corn cob,
produced the maximum amount of soluble sugars xylose and glucose, whereas
the 5.1% NaOH treated material was more resistant to hydrolysis.
TABLE-US-00005
TABLE 5
Xylose, glucose, and cellobiose in mg/mL of the saccharification
mixture analyzed at 24, 90, and 144 hours of reaction
time from the polysaccharide enriched biomass samples
obtained by treatment with 5.1 wt %, 8.0 wt %, and 20
wt % NaOH relative to the weight of the cob.
Xylose Glucose Cellobiose
Sample Time (h) (mg/mL) (mg/mL) (mg/mL)
5.1 wt % 24 13.23 15.04 3.61
NaOH 90 16.30 20.26 2.62
144 19.63 20.37 2.24
8.0 wt % 24 25.88 29.63 2.61
NaOH 90 27.85 43.99 0.20
144 44.80 67.54 1.03
20.0 wt % 24 19.00 32.20 3.02
NaOH 90 21.19 44.16 2.90
144 32.18 65.96 3.77
TABLE-US-00006
TABLE 6
Weight in grams of insoluble residue left behind after 144 hours
of saccharification of polysaccharide enriched biomass obtained
by treatment with 5.1 wt %, 8.0 wt %, and 20.0 wt % NaOH.
Insoluble Residue
Sample from Pretreatment with Remaining (g)
5.1 wt % NaOH 0.61
8.0 wt % NaOH 0.07
20.0 wt % NaOH 0.14
Soluble Content in
Sample from Pretreatment with the Filtrate (g)
5.1 wt % NaOH 0.53
8.0 wt % NaOH 1.07
20.0 wt % NaOH 0.92
Also, weight in grams of solid content of the filtrate for each case.
Example 3
Comparison of Saccharification Efficiency Of 8% NaOH Delignified Corn Cob
with/and Without Removal Of Soluble Lignin, to Show Detrimental Effect of
Lignin on Saccharification Efficiency
[0127] Two vials, each containing 1 gram of raw cob (ICBP) in 4 mL of 2%
aqueous NaOH solution were refluxed at 110.degree. C. for 24 h. The
amount of NaOH used was 8 weight percent relative to the weight of the
corn cob. The liquid from vial 1 was pipetted out and washed with
deionized water (4.times.2 mL) and filtered (total volume of the filtrate
was 10.5 mL, against expected 12 mL, indicating the retention of 1.5 mL
in the solid). It was then suspended in citrate buffer (4.9 mL) and 20%
citric acid solution (0.040 mL) was added to get a pH of 5.0. To vial 2,
20% citric acid solution (0.4 mL) was added followed by the addition of
citrate buffer (2 mL) to get a pH of 5.0. Both solid residues were then
saccharified with an enzyme cocktail (180 .mu.L) containing Spezyme.RTM.
(9 mg), Multifect.RTM.CX 12L (2.7 mg) and Novozyme 188 (3 mg) at
50.degree. C. Samples were withdrawn at the end 1, 2, and 5 days and the
sugar monomer content was determined by HPLC as described above. As can
be seen from Table 7, the removal of lignin facilitated higher production
of sugars.
TABLE-US-00007
TABLE 7
Total fermentable sugars (glucose and xylose, mg) produced
in delignified cob sample containing no lignin (Sample 1
and 1-Duplicate) and lignin (Sample 2 and 2-Duplicate.
1 day 2 days 5 days
Sample (mg) (mg) (mg)
1 342 411 446
1- Duplicate 337 407 436
2 250 307 402
2-Duplicate 245 295 398
Example 4
Enhancement of Yield by Commercial Additives in Saccharification of
Delignified Corn Cob with Enzyme Cocktail at High (25 Mg/G Dry Biomass
[DB]) & Low Enzyme (8.4 Mg/G DB) Loading Delignification of Corn Cob With
8 percent by Weight NaOH Relative to Cob Followed by Pressure Filtration
[0128] Corn cob (ICBP, 400 g, moisture content 5%) was suspended in 3.2%
sodium hydroxide solution (1000 mL) and refluxed for 24 hrs at
110.degree. C. The reaction mixture was cooled and the contents
transferred in three portions to a pressure filtration system. The slurry
was initially pressure filtered under air at 10 psi followed by final
dewatering at 220 psi. This afforded three cakes with the indicated wet
mass, dry mass, and percent solid in the wet cake (on a dry basis). No
attempt was made to further wash the cakes.
TABLE-US-00008
TABLE 8
Weight of the wet cake and the solid content of the delignified
corn cob obtained after pressure filtration of the reaction
slurry with no further water washing of the cakes
Cake Mass (g) %
Run Wet Dry Solids
1 175 73 41.5%
2 202 84 41.6%
3 212 81 38.3%
Total 589 238
Evaluation of Additive Effect on the Saccharification Efficiency of
Delignified Corn Cob
[0129] The following procedure was used to evaluate the effect of each of
the additives listed in Table 9 on the saccharification of the
delignified biomass. Delignified corn cob (2.40 g, moisture content
58.4%, dry mass equivalent 1 gram) was suspended in 50 mM sodium citrate
buffer (4.35 mL, pH 5.0). The sample pH was adjusted to pH 5.0 with 20%
aqueous citric acid (0.1 mL). An aqueous solution of the additive (5%,
0.1 mL) and the enzyme solution (0.295 mL) containing the desired amount
protein (see Table 10) was added and the solution was incubated at
50.degree. C. Samples were analyzed at the end 1, 2, and 4 days by HPLC
using an Aminex HPX-87H column and 0.01 N H.sub.2SO.sub.4 as the mobile
phase. Results are reported in Tables 11 and 12.
TABLE-US-00009
TABLE 9
List of additives used in saccharification of delignified biomass
Sam- % Ac-
ple Additive Description tives
1 Control [no additive]
2 Agent X-2776-45-1 Fatty acid methyl ester
3 AMPHOSOL .RTM. 160C-30 Sodium 37.5
lauriminodipropionate
4 AMPHOSOL .RTM. 1C Sodium Cocoamphoacetate 30
5 CEDEPAL .RTM. Sodium tridecyl ether 50
TD-403MFLD sulfate
6 NINEX .RTM. MT-603 Fatty acid ethoxylate, 100
POE-3
7 NINEX .RTM. MT-615 Fatty acid ethoxylate, 70
POE-15
8 STEOL .RTM. CS-270 Sodium laureth sulfate (2 28
EO)
9 STEOL .RTM. CS-330 Sodium laureth sulfate 100
10 STEPAN .RTM. 108 Caprylic/capric triglyceride 100
11 STEPAN .RTM. C-25 Methyl caprylate/caprate 100
12 STEPAN .RTM. C-40 Methyl laurate 100
13 STEPAN .RTM. C-65 Methyl palmitate/oleate 100
14 Stepanate AXS Ammonium xylene 42
sulfonate
15 STEPSPERSE .RTM. DF-100 Nonionic/lignosulfonate 100
blend
16 STEPSPERSE .RTM. DF-200 Anionic/lignosulfonate 100
blend
17 STEPSPERSE .RTM. DF-400 Nonionic/lignosulfonate 100
blend
18 STEPSPERSE .RTM. DF-500 Anionic/lignosulfonate 100
blend
19 STEPSPERSE .RTM. DF-600 Nonionic/lignosulfonate 100
blend
20 Stepanate SXS Sodium xylene sulfonate 41
21 STEPOSOL .RTM. ROE-W Canola oil, methyl ester 100
22 STEPOSOL .RTM. SB-W Soybean oil, methyl ester >99
23 PEG 2000 Polyethylene glycol
24 PEG 6000 Polyethylene glycol
25 PEG 8000 Polyethylene glycol
26 NINEX .RTM. MT-610 Fatty Acid Ethoxylate, 100
POE-10
27 NINEX .RTM. MT-630F Fatty Acid Ethoxylate, 100
POE-30
TABLE-US-00010
TABLE 10
Enzymes used for each enzyme loading (high and low)
Enzyme Used (mg/g delignified cob)
Multifect .RTM.
Enzyme Loading Accellerase .TM.1000 CX 12L Total
High 20.0 5.0 25.0
Low 6.7 1.7 8.4
TABLE-US-00011
TABLE 11
Observed Percentage increase in total sugar monomers
relative to control upon including additive (0.5% relative
to weight of corn cob) in enzymatic saccharification
of delignified biomass at high enzyme loading.
Time (d)
Sample Surfactant 1 2 3 4
TOTAL SUGAR MONOMER MASS (mg) - HIGH LOADING
Average Control 440.1 557.6 591.3 625.0
% INCREASE IN TOTAL SUGAR
MONOMER MASS - HIGH LOADING
2 Agent X-2776-45-1 4.6 3.3 3.4 2.6
3 AMPHOSOL .RTM. 160C-30 5.7 5.0 3.3 4.0
4 AMPHOSOL .RTM. 1C 6.4 4.4 4.0 3.1
5 CEDEPAL .RTM. TD- 1.8 4.8 1.5 1.4
403MFLD
6 NINEX .RTM. MT-603 2.3 4.4 3.9 3.1
7 NINEX .RTM. MT-615 22.2 8.7 6.2 6.2
8 STEOL .RTM. CS-270 5.8 2.8 3.5 2.0
9 STEOL .RTM. CS-330 7.1 3.1 3.0 1.9
10 STEPAN .RTM. 108 6.1 3.6 6.3 2.3
11 STEPAN .RTM. C-25 5.7 3.7 4.0 4.1
12 STEPAN .RTM. C-40 7.3 3.0 3.8 1.0
13 STEPAN .RTM. C-65 5.6 3.7 2.5 3.1
14 Stepanate AXS 7.4 2.1 1.8 1.6
15 STEPSPERSE .RTM. 10.0 6.5 6.1 2.6
DF-100
16 STEPSPERSE .RTM. 8.4 6.3 2.7 2.2
DF-200
17 STEPSPERSE .RTM. 4.1 2.8 2.6 2.2
DF-400
18 STEPSPERSE .RTM. 3.1 2.5 1.6 1.0
DF-500
19 STEPSPERSE .RTM. 5.6 5.5 3.4 2.3
DF-600
20 Stepanate SXS 7.3 4.8 2.9 1.2
21 STEPOSOL .RTM. ROE-W 7.9 1.3 1.2 -1.2
22 STEPOSOL .RTM. SB-W 6.4 1.9 1.0 -2.0
23 PEG 2000 20.7 9.4 8.0 5.5
24 PEG 6000 17.5 7.5 6.4 4.0
25 PEG 8000 19.5 7.6 6.9 4.7
26 NINEX .RTM. MT-610 8.6 10.1 9.5 9.8
27 NINEX .RTM. MT-630F 20.4 17.2 12.1 8.5
Reaction mixture analyzed by HPLC: end of days 1-4
TABLE-US-00012
TABLE 12
Observed percentage increase in total sugar monomers relative
to control upon the inclusion of additives (0.5% relative
to weight of corn cob) in the enzymatic saccharification
of delignified biomass at low enzyme loading.
Time (d)
Sample Surfactant 1 2 3 4
TOTAL SUGAR MONOMER MASS (mg) - LOW LOADING
Average Control 232.2 314.0 367.3 411.8
% INCREASE IN TOTAL SUGAR
MONOMER MASS - LOW LOADING
2 Agent X-2776-45-1 2.7 3.0 1.1 -0.4
3 AMPHOSOL .RTM. 160C-30 8.7 12.2 9.1 9.5
4 AMPHOSOL .RTM. 1C -1.6 2.1 5.4 5.5
5 CEDEPAL .RTM. TD- -3.7 3.7 2.2 3.1
403MFLD
6 NINEX .RTM. MT-603 -1.7 4.4 5.7 4.5
7 NINEX .RTM. MT-615 9.8 16.2 15.5 9.2
8 STEOL .RTM. CS-270 -5.5 0.7 0.1 -1.2
9 STEOL .RTM. CS-330 -1.7 9.7 2.9 3.7
10 STEPAN .RTM. 108 -4.2 7.0 5.6 3.0
11 STEPAN .RTM. C-25 -4.0 2.1 4.2 3.8
12 STEPAN .RTM. C-40 -6.0 0.8 3.2 0.8
13 STEPAN .RTM. C-65 0.9 3.8 6.1 4.0
14 STEPAN .RTM. AXS 1.6 3.8 4.7 2.7
15 STEPSPERSE .RTM. 15.2 9.6 10.5 7.0
DF-100
16 STEPSPERSE .RTM. 5.6 6.3 5.2 5.4
DF-200
17 STEPSPERSE .RTM. 13.3 14.1 14.2 10.8
DF-400
18 STEPSPERSE .RTM. 1.7 -1.0 0.6 1.8
DF-500
19 STEPSPERSE .RTM. 14.4 10.0 8.3 1.5
DF-600
20 STEPSPERSE .RTM. SXS 0.6 0.5 2.6 0.8
21 STEPOSOL .RTM. ROE-W 1.0 0.5 3.0 1.0
22 STEPOSOL .RTM. SB-W 7.3 2.5 4.8 6.9
23 PEG 2000 36.1 25.8 27.3 21.6
24 PEG 6000 31.2 23.6 21.8 18.7
25 PEG 8000 30.4 21.6 19.3 15.8
26 NINEX .RTM. MT-610 16.8 13.5 10.0 7.3
27 NINEX .RTM. MT-630F 22.4 15.1 14.2 10.7
Reaction mixture was analyzed at the end of days 1, 2, 3, and 4 by HPLC.
Unexpectedly, the increment is much higher at low enzyme loading as
compared to the high enzyme loading.
[0130] The list of best performing (over 10% increase in saccharification
yield) additives under the conditions tested: [0131] STEPSPERSE.RTM.
DF-100 [0132] PEG 2000 [0133] PEG 6000 [0134] PEG 8000 [0135] NINEX.RTM.
MT-610 [0136] NINEX.RTM. MT-630F
[0137] Other additives which can also provide an increase in total sugar
monomers relative to an additive-free control in the enzymatic
saccharification of delignified biomass at low enzyme loadings include
Tween 20 and Tween 80 (also known as polysorbate 20 and polysorbate 80,
respectively). These additives are nonionic surfactants derived from
polyethoxylated sorbitan and a fatty acid, for example lauric acid, oleic
acid, or other C.sub.12 to C.sub.30 fatty acids. Additionally, additives
such as secondary alcohol ethoxylates, fatty alcohol ethoxylates,
nonylphenol ethoxylate, and tridecyl ethoxylates, as disclosed in U.S.
Pat. No. 7,354,743 may be used.
Example 5
Enhancement of Enzymatic Saccharification of Delignified, Water Washed
Corn Cob with Preferred Additives in Example 4 at Low Enzyme Loading
(Less than 12 mg Protein/Gram Dry Delignified Corn Cob) and Absence of
Additive Effect At High Enzyme Loading (Greater than About 25 mg
Protein/Gram Dry Delignified Corn Cob)
[0138] Corn cob (ICBP, 100 g) was delignified with 2% aqueous sodium
hydroxide solution as described in Example 1. Instead of isolating the
polysaccharide enriched biomass by vacuum filtration, the reaction
mixture was filtered under pressure (8 to 20 psi) to squeeze out the
liquid, followed by washing with deionized water and finally compacting
the residue at 220 psi air pressure in a filtration unit. This afforded
141.19 g of polysaccharide enriched, delignified biomass as a wet cake,
which had 57.5% moisture content. The sugar analyses of the untreated cob
and the solid fraction of the delignified polysaccharide enriched biomass
obtained after sodium hydroxide pretreatment are shown in Table 13.
TABLE-US-00013
TABLE 13
Sugar Analyses (Example 5)
% % % Total
Sample Glucan Xylan Sugars
Raw Cob 37.5 28.7 66.2
Delignified Cob 52.3 39.8 92.1
[0139] A portion of the above cake (0.5 g) was suspended in 50 mM sodium
citrate buffer (pH 5.0, 3.125 mL) containing a mixture of
Accelerase.TM.1000 cellulase and Multifect.RTM. CX 12L enzyme (protein
ratio 3.94:1) mixture containing 4.20 mg, 5.85 mg or 12.50 mg of protein
(corresponding respectively to 8.4 mg, 11.7 mg or 25 mg of protein per
gram of dry delignified corn cob) in the presence or absence of additives
PEG 2000 (Sigma-Aldrich, St. Louis, Mo.) or NINEX.RTM. MT-610 (fatty acid
ethoxylate ester, Stepan Company, Northfield, Ill.). The additive
concentration relative to the weight of dry biomass was 0.0%, 0.5% and
1.0% for each enzyme loading used. Table 14 summarizes the reaction
conditions used for the saccharifications performed in Example 5.
[0140] The reaction samples taken at intervals of 24 h, 48 h, 72 h, and
168 h (7 days) were analyzed by HPLC for glucose and xylose content. The
results are reported as % yield in Table 15 and represented graphically
in FIG. 2. The yields were calculated on the basis of expected total
glucan and xylan present in the delignified corn cob. Table 16 shows the
percentage increase in total sugar monomer mass produced with time for
each sample.
[0141] These results show that the effect of the added additive to
increase sugar yield is greatest with the lower enzyme loadings, for
example at the loadings below about 25 mg/g.
TABLE-US-00014
TABLE 14
Reaction Conditions Used in Each Saccharification
Performed for Example 5
Enzyme Additive
Example #.sup.1 Loading.sup.2 Additive Used Loading.sup.3
5A 8.4 None 0
5B 8.4 PEG 2000 0.5
5C 8.4 PEG 2000 1.0
5D 8.4 NINEX .RTM. MT-610 0.5
5E 8.4 NINEX .RTM. MT-610 1.0
5F 11.7 None 0
5G 11.7 PEG 2000 0.5
5H 11.7 PEG 2000 1.0
5J 11.7 NINEX .RTM. MT-610 0.5
5K 11.7 NINEX .RTM. MT-610 1.0
5L 25 None 0
5M 25 PEG 2000 0.5
5N 25 PEG 2000 1.0
5P 25 NINEX .RTM. MT-610 0.5
5Q 25 NINEX .RTM. MT-610 1.0
Notes:
.sup.1Refers to saccharification runs of Example 5
.sup.2in mg of protein per gram of dry delignified corn cob
.sup.3additive concentration relative to the weight of dry delignified
corn cob
TABLE-US-00015
TABLE 15
Yields of Total Sugars with Time for Saccharification of
Polysaccharide Enriched, Delignified Biomass at Three Different
Enzyme Loadings and with Two Different Surfactant Loadings.
SACCHARIFICATION % YIELD
Enzyme Additive Time (d)
Loading Additive Loading (%) 1 2 3 7
8.4 mg/g Control 24.0 30.0 45.9 44.3
PEG 2000 0.5 33.7 43.1 47.4 64.5
1.0 33.5 43.1 48.8 65.4
NINEX .RTM. 0.5 32.8 44.0 54.3 57.7
MT-610 1.0 37.0 46.2 54.3 71.0
11.7 mg/g Control 30.2 35.7 42.7 58.9
PEG 2000 0.5 38.4 50.4 61.3 76.3
1.0 33.8 48.2 59.7 73.4
NINEX .RTM. 0.5 33.5 50.8 59.5 71.6
MT-610 1.0 36.2 53.6 62.3 76.2
25 mg/g Control 56.3 71.0 74.2 83.1
PEG 2000 0.5 58.3 72.9 75.4 83.9
1.0 61.6 74.8 80.3 84.7
NINEX .RTM. 0.5 62.3 74.6 80.3 84.8
MT-610 1.0 63.0 77.4 82.7 88.1
TABLE-US-00016
TABLE 16
Increase over control (absence of additives) in weight of total monomer
sugars (glucose + xylose) on days 1, 2, 3, and 7, as result of additives
PEG 2000 and NINEX .RTM. MT-610 on saccharification of delignified,
polysaccharide enriched biomass with Accelerase .TM.1000-Multifect .RTM.
CX 12L at 3 different enzyme loadings and 2 additive concentrations
% INCREASE IN TOTAL SUGAR MONOMER MASS PRODUCED
Additive
Enzyme Loading Time (days)
Loading Additive (wt %) 1 2 3 7
8.4 mg/g PEG 2000 0.5 40.6 43.9 3.4 45.6
1.0 39.9 43.7 6.3 47.5
NINEX .RTM. 0.5 36.9 46.8 18.4 30.1
MT-610 1.0 54.1 54.3 18.4 60.2
11.7 mg/g PEG 2000 0.5 27.0 41.3 43.5 29.5
1.0 12.0 35.2 39.6 24.6
NINEX .RTM. 0.5 10.8 42.3 39.2 21.5
MT-610 1.0 19.8 50.1 45.8 29.3
25 mg/g PEG 2000 0.5 3.6 2.6 1.7 1.0
1.0 9.4 5.3 8.2 2.0
NINEX .RTM. 0.5 10.6 5.0 8.3 2.1
MT-610 1.0 11.9 9.0 11.5 6.1
* * * * *
