U.S. patent application number 13/297413 was filed with the patent office on 2012-05-24 for continuously fed biomass pretreatment process for a packed bed reactor.
This patent application is currently assigned to E. I. DU PONT DE NEMOURS AND COMPANY. Invention is credited to JEFFREY DAVID COHEN.
Application Number | 20120125548 13/297413 |
Document ID | / |
Family ID | 45094804 |
Filed Date | 2012-05-24 |
United States Patent
Application |
20120125548 |
Kind Code |
A1 |
COHEN; JEFFREY DAVID |
May 24, 2012 |
CONTINUOUSLY FED BIOMASS PRETREATMENT PROCESS FOR A PACKED BED
REACTOR
Abstract
Biomass pretreatment using anhydrous ammonia is effective in a
static reactor vessel when the ammonia can penetrate through the
biomass particles or pieces in vapor state, and when biomass is
continuously fed and moved through the reactor. To achieve this
condition, total system moisture content is kept below 40 weight %
based on total mass in the system. The pretreated biomass product
is effectively saccharified to produce fermentable sugars for
biocatalyst production of a product.
Inventors: |
COHEN; JEFFREY DAVID;
(Kennett Square, PA) |
Assignee: |
E. I. DU PONT DE NEMOURS AND
COMPANY
Wilmington
DE
|
Family ID: |
45094804 |
Appl. No.: |
13/297413 |
Filed: |
November 16, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61416484 |
Nov 23, 2010 |
|
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|
Current U.S.
Class: |
162/17 |
Current CPC
Class: |
C12P 19/02 20130101;
C12P 19/14 20130101; D21C 3/024 20130101; C12P 2201/00 20130101;
Y02P 20/582 20151101; D21C 1/06 20130101 |
Class at
Publication: |
162/17 |
International
Class: |
D21C 3/26 20060101
D21C003/26 |
Claims
1. A process for treating biomass to produce a pretreated biomass
product comprising: a) providing biomass with dry matter content of
at least about 60%; b) charging a static pretreatment vessel with
the biomass of (a) by continuous feeding whereby the charged
biomass in the vessel moves through the vessel; c) contacting the
charged biomass of (b) as it moves through the pretreatment vessel
with at least about 4% anhydrous ammonia measured as a function of
dry weight of the biomass in the vessel, under conditions whereby
the total moisture content in the vessel remains below 40 weight %
measured as a function of total mass in the vessel; whereby a
majority of the biomass is permeated by ammonia vapor; and d)
exiting the biomass from the pretreatment vessel; wherein the
biomass exiting the vessel is pretreated biomass product.
2. The process of claim 1 wherein after exiting of (d), vapors are
separated from the pretreated biomass product.
3. The process of claim 2 wherein vapors comprise ammonia vapor
which is recovered and recycled to the pretreatment vessel or to an
ammonia vapor handling system.
4. The process of claim 1 wherein after step (d) the pretreated
biomass product is recovered.
5. The process of claim 1 wherein at step (c) the charged biomass
moves through the pretreatment vessel with a residence time of
between about 10 minutes and about 5 hours.
6. The process of claim 1 wherein where the anhydrous ammonia is in
contact with the biomass for less than about 5 hours.
7. The process of claim 1 wherein the temperature of the biomass in
the vessel is at least about 70.degree. C.
8. The process of claim 1 wherein the temperature of the vessel is
raised to at least about 70.degree. C. prior to step (b).
9. The process of claim 8 wherein the temperature prior to step (b)
is between about 70.degree. C. and about 190.degree. C.
10. The process of claim 8 wherein the temperature is raised
directly by injecting hot gas or indirectly by application of heat
to the vessel.
11. The process of claim 1 wherein the concentration of anhydrous
ammonia at step (c) is less than about 20% measured as a function
of dry weight of the biomass in the vessel.
12. The process of claim 1 wherein pressure in the vessel is
between about 0 and 20 atmosphere gauge over steps (b) through
(d).
11. The process of claim 1 wherein the biomass of (a) is
mechanically size-reduced biomass.
12. The process of claim 1 wherein the biomass is cellulosic
biomass comprising cellulose, hemicellulose and lignin.
13. The process of claim 12 wherein the biomass is selected from
the group consisting of corn cobs, corn husks, corn stover,
grasses, wheat straw, barley straw, oat straw, canola straw, hay,
rice straw, switchgrass, miscanthus, cord grass, reed canary grass,
waste paper, sugar cane bagasse, sorghum bagasse or stover, soybean
stover, components obtained from milling of grains, trees,
branches, roots, leaves, wood chips, sawdust, shrubs and bushes,
vegetables, fruits, flowers and animal manure.
Description
[0001] This application claims the benefit of U.S. Provisional
Application 61/416,484, filed Nov. 23, 2010 which is incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to processes for treating biomass to
obtain fermentable sugars. Specifically, a continuously fed
pretreatment process for biomass in a packed bed reactor using
anhydrous ammonia for production of readily saccharifiable material
is provided.
BACKGROUND OF THE INVENTION
[0003] 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 chemicals, plastics,
fuels and feeds. Lignocellulosic feedstocks and wastes containing
the carbohydrate polymers cellulose and hemicellulose, as well as
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, or polysaccharides, of lignocellulosic biomass more
readily accessible to cellulolytic enzymes used in
saccharification. Various pretreatment methods are known, including
ammonia pretreatment of biomass. Typically, ammonia has been used
in aqueous state while treating biomass in preparation for
saccharification.
[0005] For example, commonly owned U.S. Pat. No. 7,932,063
discloses methods for pretreating biomass under conditions of high
solids and low aqueous ammonia concentration. The concentration of
ammonia used is minimally a concentration that is sufficient to
maintain the pH of the biomass-aqueous ammonia mixture alkaline and
maximally less than about 12 weight percent relative to dry weight
of biomass. The dry weight of biomass is at an initial
concentration of at least about 15% up to about 80% of the weight
of the biomass-aqueous ammonia mixture.
[0006] Disclosed in U.S. Pat. No. 4,064,276 is the use of anhydrous
ammonia for treatment of straw and other plant materials to improve
the nutritional value of the material. Straw having a dry matter
content of at least 60% by weight is treated with 15-40 kg of
anhydrous ammonia per ton of dry straw at ambient temperature and
atmospheric pressure for at least 10 days.
[0007] Disclosed in U.S. Pat. No. 7,915,017 is a process for the
pretreatment of biomass with anhydrous ammonia in liquid or vapor
state, and/or concentrated ammonia:water mixtures in the liquid or
vapor state, to obtain a mixture in which the ratio of ammonia to
dry biomass is between about 0.2 to 1 and 1.2 to 1, and the water
to dry biomass ratio is between about 0.2 to 1.0 and 1.5 to 1.
Processes are used to increase the fraction of the total ammonia
that is in the liquid phase. The temperature is maintained between
about 50.degree. C. and 140.degree. C. and the pressure is rapidly
released by releasing ammonia from the vessel to form a treated
biomass.
[0008] A packed bed flowthrough reactor is used in an
ammonia-recycled percolation process for pretreatment of biomass
(Yoon et al. (1995) Applied Biochemistry and Biotechnology
51/52:5-19). In this process aqueous ammonia is continuously
recycled through a bed of biomass.
[0009] There remains a need for a process that effectively
pretreats biomass with ammonia, producing a readily saccharifiable
material, that reduces reactor costs.
SUMMARY OF THE INVENTION
[0010] The invention provides processes for pretreating biomass in
a continuous manner with anhydrous ammonia in a static vessel to
produce a readily saccharifiable material.
[0011] Accordingly, the invention provides a process for treating
biomass to produce a pretreated biomass product comprising: [0012]
a) providing biomass with dry matter content of at least about 60%;
[0013] b) charging a static pretreatment vessel with the biomass of
(a) by continuous feeding whereby the charged biomass in the vessel
moves through the vessel; [0014] c) contacting the charged biomass
of (b) as it moves through the pretreatment vessel with at least
about 4% anhydrous ammonia measured as a function of dry weight of
the biomass in the vessel, under conditions whereby the total
moisture content in the vessel remains below 40 weight % measured
as a function of total mass in the vessel; whereby a majority of
the biomass is permeated by ammonia vapor; and [0015] d) exiting
the biomass from the pretreatment vessel; [0016] wherein the
biomass exiting the vessel is pretreated biomass product.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 illustrates a pretreatment system for the continuous
feeding of biomass into a pretreatment vessel.
DETAILED DESCRIPTION
[0018] When treating biomass with ammonia, contact of the ammonia
throughout the biomass is important for effective preparation of
the biomass for saccharification. Typically mechanical stirring is
used to mix ammonia and biomass to maximize contact. Under
conditions described herein, pretreatment may be performed in a
static reactor by continuous feeding of biomass to the reactor and
moving the biomass through the reactor, with no mechanical mixing,
thereby removing energy and maintenance costs associated with a
mixing reactor where ammonia is a reactant.
[0019] The following definitions and abbreviations are to be use
for the interpretation of the claims and the specification.
[0020] 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).
[0021] Also, the indefinite articles "a" and "an" preceding an
element or component of the invention are intended to be
nonrestrictive regarding the number of instances (i.e. occurrences)
of the element or component. Therefore "a" or "an" should be read
to include one or at least one, and the singular word form of the
element or component also includes the plural unless the number is
obviously meant to be singular.
[0022] The term "invention" or "present invention" as used herein
is a non-limiting term and is not intended to refer to any single
embodiment of the particular invention but encompasses all possible
embodiments as described in the specification and the claims.
[0023] 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. In one
embodiment, the term "about" means within 10% of the reported
numerical value, preferably within 5% of the reported numerical
value.
[0024] The term "fermentable sugar" refers to oligosaccharides and
monosaccharides that can be used as a carbon source by a
microorganism in a fermentation process.
[0025] The term "lignocellulosic" refers to a composition
comprising both lignin and cellulose. Lignocellulosic material may
also comprise hemicellulose.
[0026] The term "cellulosic" refers to a composition comprising
cellulose and additional components, including hemicellulose.
[0027] The term "saccharification" refers to the production of
fermentable sugars from polysaccharides.
[0028] The term "pretreated biomass" means biomass that has been
subjected to pretreatment prior to saccharification.
[0029] The term "butanol" refers to isobutanol, 1-butanol,
2-butanol, or combinations thereof.
[0030] The term "lignocellulosic biomass" refers to any
lignocellulosic material and includes materials comprising
cellulose, 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.
Lignocellulosic 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. Examples of biomass include, but are not limited
to, corn cobs, crop residues such as corn husks, corn stover,
grasses, wheat straw, barley straw, hay, rice straw, switchgrass,
waste paper, sugar cane bagasse, sorghum plant material, soybean
plant material, components obtained from milling of grains, trees,
branches, roots, leaves, wood chips, sawdust, shrubs and bushes,
vegetables, fruits, and flowers.
[0031] The term "dry matter content" refers to the amount by weight
of the subject material that exists after liquid content of the
material is removed.
[0032] The term "biomass hydrolysate" refers to the product
resulting from saccharification of biomass. The biomass may also be
pretreated or pre-processed prior to saccharification.
[0033] The term "biomass hydrolysate fermentation broth" is broth
containing product resulting from biocatalyst growth and production
in a medium comprising biomass hydrolysate. This broth includes
components of biomass hydrolysate that are not consumed by the
biocatalyst, as well as the biocatalyst itself and product made by
the biocatalyst.
[0034] The term "slurry" refers to a mixture of insoluble material
and a liquid. A slurry may also contain a high level of dissolved
solids. Examples of slurries include a saccharification broth, a
fermentation broth, and a stillage.
[0035] The term "target product" refers to any product that is
produced by a microbial production host cell in a fermentation.
Target products may be the result of genetically engineered
enzymatic pathways in host cells or may be produced by endogenous
pathways. Typical target products include but are not limited to
acids, alcohols, alkanes, alkenes, aromatics, aldehydes, ketones,
biopolymers, proteins, peptides, amino acids, vitamins,
antibiotics, and pharmaceuticals.
[0036] The term "static vessel" refers to a vessel that includes no
means for mixing of charged materials. A packed bed reactor employs
a static vessel.
[0037] The term "static pretreatment vessel" refers to a static
vessel that may be used for pretreatment. The vessel is built to
withstand the charged materials, such as ammonia, which is used in
the present process.
[0038] The term "venting" refers to allowing enclosed gases to
escape without an explosive effect.
[0039] The term "gas" refers to both gas and vapor, including a
condensable, low-density phase below its critical temperature, for
example, steam.
[0040] The term "total mass in the vessel" refers to the combined
masses of all of the components added to the vessel.
[0041] The term "a majority" refers to greater than 50%. Majority
may refer to any integer above 50%.
Continuous Pretreatment of Biomass in a Static Vessel
[0042] In the present method, biomass is fed continuously into a
static vessel for pretreatment to produce pretreated biomass
product. One embodiment of a continuously fed static vessel
pretreatment process is shown in FIG. 1. Aspects of the process
described below are exemplified in FIG. 1, with numbering below as
represented in FIG. 1.
[0043] Biomass is fed into a first end, or inlet (10), of the
vessel and moves through the vessel (11) to a second, opposite end,
or outlet (12), of the vessel. The biomass is fed to the vessel
using continuous feeding equipment (13) such as an extruder,
industrial baler, or concrete pump. Biomass may move into a feed
bin for charging the continuous feeding equipment by gravity flow,
or by an energy input method such as with an auger that drives the
feed into the continuous feeding equipment.
[0044] Biomass that is fed continuously into the static
pretreatment vessel moves from the inlet of the vessel to the
outlet of the vessel as a result of the force applied by the
feeding equipment. The vessel has greater dimension of the
inlet-outlet axis than of the width; for example the vessel may be
a cylinder or pipeline (as in FIG. 1). Downstream of the biomass
feed entrance point, a vapor seal may be used to prevent
pretreatment agents, including ammonia and steam, from escaping
upstream out of the inlet. This can be accomplished, for example,
by achieving sufficient swelling of the biomass between the biomass
entry point and the ammonia/and steam injection points to block
vapor escape from the inlet. In one embodiment, two different
biomass feeds are added in series, i.e. primarily switchgrass or
cob, with alternating zones of milled corn grain or starch that
swell and seal upon receiving hot moisture.
[0045] A pipeline vessel may contain one or more flared, expansion
sections (14) to allow for increased pipe diameter, beyond that of
the inlet end diameter, to reduce the overall vessel length while
providing adequate residence time for pretreatment. The increased
diameters will decrease the total frictional force versus a longer,
constant diameter pipeline. The increased diameters will also limit
the reverse movement of fed biomass toward the feed inlet.
[0046] One or more turns (15) may be included in the vessel to
reduce the overall dimension of the process. The vessel may have
increasing elevation (16) over all or a portion of its length to
elevate the biomass, such that the pretreated biomass product exits
at or near the entry point of a downstream receiving vessel (17)
which may be a saccharifier (18). This obviates the need for
buckets, augers, elevators or other equipment to load the
pretreated biomass product into the saccharifier or other receiving
vessel.
[0047] At one or more locations along the vessel length, inlets
(19) are used to feed pretreatment agents, including anhydrous
ammonia and steam. As indicated above, these inlets are downstream
of the biomass feeding inlet at a distance which reduces escape of
agents through the biomass feeding inlet. The pretreatment agents
are fed into the vessel, in communication with the biomass
undergoing pretreatment, in proportions sufficient to pretreat the
charged biomass as described below. In addition, heating is
provided to the biomass by direct injection of steam into the
vessel, or by heating the vessel using a jacket surrounding the
vessel containing a heating medium, such as steam or heated
oil.
[0048] The end section of the vessel (20) may be non-insulated, or
actively cooled such as by using a reactor jacket, to enable a
temperature decrease prior to biomass exiting the vessel. Sterile
water may be injected directly into the process to hasten
cooling.
[0049] The pretreated biomass product exits the vessel at the
outlet. The outlet may be an exit valve such as a hinged plate
(21), as is used in a swing-type, check valve. A controlled,
applied force opposing the flow may be used to control discharge
pressure.
[0050] Excess reagent, such as ammonia, may be captured and
recycled in the pretreatment process. Ammonia and steam vapors may
be vented from the pretreatment vessel, although typically these
vapors exit together with the pretreated biomass into a receiving
vessel (17) or hopper. In the receiving vessel the vapors may
expand and evaporate to become separated from the pretreated
biomass, and are typically vented (22) and channeled for recycling
into the pretreatment process.
[0051] Alternatively, the ammonia added may be limited to the
requisite stoichiometric ammonia, plus allowable ammonia residual,
to avoid the need to capture and recycle excess reagent.
Pretreatment Process Using Ammonia Vapor
[0052] In the present process continuously fed biomass charged to a
static vessel is treated with anhydrous ammonia in a relatively dry
system as it moves through the vessel. When the system is
relatively dry, ammonia may spread throughout the biomass in the
vapor state such that no mechanical mixing is required. Open
interstitial spaces between biomass particles, or pieces, for the
vaporous ammonia to pass through are maintained by keeping the
moisture content in the reactor low so that moisture present in the
reactor is associated with biomass, and does not fill interstitial
spaces. Thus the moisture content in the vessel is not constant,
since moisture is higher at the interface of biomass particles or
pieces, and lower in interstitial spaces between biomass particles
or pieces.
[0053] The overall total moisture content in the vessel remains
below 40 weight % measured as a function of total mass in the
vessel, which includes biomass, anhydrous ammonia, and any other
components added to the vessel such as steam. It is shown herein
that in the present process, with no mechanical mixing, ammonia
vapors are able to penetrate the biomass for effective pretreatment
to produce a readily saccharifiable biomass product, as determined
by sugar yields produced in subsequent saccharification. Samples
taken at different distances from an ammonia charge point were able
to produce high levels of sugars during saccharification. Sugars
produced in examples herein were in the range of 57-68% of
theoretical yield of monomer xylose and 80-90% of theoretical yield
of monomer glucose.
[0054] No mixing mechanism is needed in the vessel used for the
present pretreatment process; it is a static vessel. The vessel may
be any shape such as cylindrical, and oriented either horizontally
or vertically, or include horizontal and vertical portions. The
vessel has one or more ports for charging of biomass, ammonia, and
steam. The vessel may be built for running at pressures between
atmospheric (0 gauge) and 20 atmospheres gauge. The vessel may have
means to raise its temperature directly such as a heating jacket.
The vessel is of material designed to withstand ammonia vapor and
may be a packed bed type of reactor.
[0055] Biomass charged to the vessel is typically lignocellulosic
biomass, and has a dry matter content of at least about 60%. The
percent dry weight of the biomass may be about 60%, 65%, 70%, 75%,
80%, 85%, 90% or higher. For example, freshly harvested corn stover
is typically about 70% dry weight, whereas biomass may be dried,
such as air drying, to a higher percent dry matter. In addition,
biomass may be reduced in size prior to being charged in the
vessel. Typically prior to charging the biomass in the vessel, it
is size-reduced mechanically such as by chopping, cutting, or
grinding.
[0056] The biomass in the vessel is contacted with anhydrous
ammonia by charging anhydrous ammonia to the vessel containing
biomass and allowing it to penetrate the biomass. A majority of the
biomass in the vessel is contacted with anhydrous ammonia in the
present method. Typically greater than 50% of the biomass is
contacted with anhydrous ammonia. At least about 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, or 95% of the biomass is contacted
with anhydrous ammonia.
[0057] Anhydrous ammonia is added in an amount that is at least
about 4% based on dry weight of the biomass in the vessel. The
range of concentration of anhydrous ammonia used is between about
4% and about 20%. Using 6% ammonia increased yield of xylose from
saccharification of the resulting anhydrous ammonia pretreated
biomass, as shown in Example 3 herein, comparing to effects of 3%
anhydrous ammonia. Introduction of anhydrous ammonia raises the
temperature of the biomass in the contained vessel.
[0058] The temperature of the reactor and/or the biomass may be
increased prior to addition of anhydrous ammonia. Temperature may
be raised either indirectly or directly by any method, including by
application of heat to the vessel, such as using a heating jacket
or heating coils, or by introducing hot gas or vapor, such as
steam, into the reactor vessel. Steam may be injected as
superheated steam or dry steam to avoid introduction of moisture,
for maintaining the total moisture content in the vessel below 40
weight % of the total mass in the vessel. Moisture in the vessel
may come primarily from the biomass and added gases. When very dry
biomass is treated, such as biomass having a moisture content of
less than about 15%, then the steam does not need to be dry to
maintain the low 40% total moisture content. However, if biomass
has a higher moisture content such as about 35%, then dry steam is
used. The desired temperature is generally in the range of between
about 70.degree. C. and 190.degree. C. Typically the temperature is
between about 90.degree. C. and 150.degree. C.
[0059] Also, the pressure inside the vessel may be increased.
Injection of steam may be used to increase pressure. During
pretreatment, the pressure is maintained between 0 atmosphere gauge
and less than 20 atmospheres gauge. The residence time as the
biomass passes through the pretreatment vessel is between about 10
minutes and five hours. The residence time may include a first
segment after biomass charging where the biomass moves into the
vessel prior to encountering inlets for injection of pretreatment
agents such as steam and ammonia. Typically in a second segment of
residence time steam and ammonia are injected to contact the
biomass and pretreatment occurs. This second segment may be between
about 5 minutes and several hours. During this time pressure and
temperature are maintained within the vessel providing conditions
where ammonia continues to penetrate through interstitial spaces
among the biomass particles or pieces in a vapor state. There may
be a third segment of residence time as the biomass nears the
outlet where temperature is reduced as described above.
[0060] At the end of the desired residence time, the pretreated
biomass product exits the vessel through an outlet as it is pushed
out by force applied by the feeding equipment as described above.
The pretreated biomass product is in a dry state. It may be charged
to the next vessel by a mechanical process or by gravity.
[0061] Associated pretreatment agent vapors may be recycled as
noted above. It is preferred to recycle the ammonia for a
commercially viable process. Ammonia vapor may be recycled by
methods known to one skilled in the art, using an ammonia vapor
handling system. For example, ammonia vapor may be condensed and
recycled as aqueous ammonia. Alternatively ammonia vapor may be
recycled primarily in an anhydrous state.
Lignocellulosic Biomass
[0062] Biomass used in the present process is lignocellulosic,
which contains polysaccharides such as cellulose and hemicellulose,
and lignin. Polysaccharides of biomass may also be called glucans
and xylans. Types of biomass that may be used include, but are not
limited to, bioenergy crops, agricultural residues, municipal solid
waste, industrial solid waste, sludge from paper manufacture, yard
waste, wood and forestry waste. Examples of biomass include, but
are not limited to corn cobs, corn husks, corn stover, grasses,
wheat straw, barley straw, oat straw, canola straw, hay, rice
straw, switchgrass, miscanthus, cord grass, reed canary grass,
waste paper, sugar cane bagasse, sorghum bagasse or stover, soybean
stover, components obtained from milling of grains, trees,
branches, roots, leaves, wood chips, sawdust, shrubs and bushes,
vegetables, fruits, flowers and animal manure. Biomass may include
other crop residues, forestry wastes such as aspen wood, other
hardwoods, softwood and sawdust; and post-consumer waste paper
products; and fiber process residues such as corn fiber, beet pulp,
pulp mill fines and rejects; as well as other sufficiently abundant
lignocellulosic material.
[0063] Biomass that is particularly useful for the invention
includes biomass that has a relatively high carbohydrate content,
is relatively dense, and/or is relatively easy to collect,
transport, store and/or handle.
[0064] The lignocellulosic 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 stems or stalks and
leaves.
[0065] The biomass may be used directly as obtained from the
source, or may be subjected to some preprocessing, for example,
energy may be applied to the biomass to reduce size or moisture.
Size reduction may be performed using methods that produce coarse
size reduced material, where the obtained size is greater than 0.1
mm. Methods that may be used include mechanical methods such as
knife milling, crushing, shredding, chopping, disc refining, and
coarse hammer milling. This type of size reduction may be performed
before or after treatment with anhydrous ammonia, but is typically
before. Drying may be by any conventional means such as by using a
drying oven, rotary dryer, flash dryer, or superheated steam dryer.
In addition, air drying may be sufficient for reaching a desired
biomass moisture content that is less than about 40%. For use in
the present method it is desirable that the biomass has a dry
matter content of at least about 60, 65, 70, 75, 80, 85, 90, or 93
weight percent.
Pretreated Biomass Product
[0066] The pretreated biomass product resulting from the present
process is used in saccharification to produce sugars for
fermentation by a biocatalyst to produce a desired product.
Saccharification
[0067] Enzymatic saccharification typically makes use of at least
one saccharification enzyme and often an enzyme consortium for
breaking down cellulose and hemicellulose to produce a hydrolysate
containing sugars including glucose, xylose, and arabinose.
Saccharification enzymes are reviewed in Lynd, L. R., et al.
(Microbiol. Mol. Biol. Rev., 66:506-577, 2002).
[0068] The enzyme(s) generally include one or more glycosidases.
Glycosidases 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.,
223:1-5, 1994; Eur. J. Biochem., 232:1-6, 1995; Eur. J. Biochem.,
237:1-5, 1996; Eur. J. Biochem., 250:1-6, 1997; and Eur. J.
Biochem., 264:610-650 1999, 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, arabino-xylanases, 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.
[0069] Saccharification enzymes may be obtained commercially, such
as Spezyme.RTM. CP cellulase, Multifect.RTM. xylanase,
Accelerase.RTM. 1500, and Accellerase.RTM. DUET (Danisco U.S. Inc.,
Genencor International, Rochester, N.Y.). In addition,
saccharification enzymes may be unpurified and provided as a type
of cell extract or whole cell preparation. The enzymes may be
produced using recombinant microorganisms that have been engineered
to express multiple saccharifying enzymes.
[0070] Of particular value in the present invention are classes of
glycoside hydrolases (GH), such as the families GH3, GH39, GH43,
GH55, GH10, and GH11. GHs are a group of enzymes that hydrolyze the
glycosidic bond between two or more carbohydrates, or between a
carbohydrate and a noncarbohydrate moiety. Families of GHs have
been classified based on sequence similarity and are available in
the Carbohydrate-Active enzyme (CAZy) database (Cantarel et al.
(2009) Nucleic Acids Res. 37 (Database issue):D233-238). These
enzymes are able to act on a number of substrates and are effective
in the saccharification process. Glycoside hydrolase family 3
("GH3") enzymes have a number of known activities: -glucosidase
(EC:3.2.1.21); .beta.-xylosidase (EC:3.2.1.37);
N-acetyl-glucosaminidase (EC:3.2.1.52); glucan
.beta.-1,3-glucosidase (EC:3.2.1.58); cellodextrinase
(EC:3.2.1.74); exo-1,3-1,4-glucanase (EC:3.2.1); and
.beta.-galactosidase (EC 3.2.1.23). Glycoside hydrolase family 39
("GH39") enzymes have .alpha.-L-iduronidase (EC:3.2.1.76) or
.beta.-xylosidase (EC:3.2.1.37) activity. Glycoside hydrolase
family 43 ("GH43") enzymes have the following activities:
L-.alpha.-arabinofuranosidase (EC 3.2.1.55); -xylosidase (EC
3.2.1.37); endoarabinanase (EC 3.2.1.99); and galactan
1,3-1-galactosidase (EC 3.2.1.145). Glycoside hydrolase family 51
("GH51") enzymes have L-.alpha.-arabinofuranosidase (EC 3.2.1.55)
or endoglucanase (EC 3.2.1.4) activity. Glycoside hydrolase family
10 ("GH10") enzymes are more fully described in Schmidt et al.
(1999, Biochemistry 38:2403-2412) and Lo Leggio et al. (2001, FEBS
Lett 509: 303-308). The glycoside hydrolase family 11 ("GH11")
enzymes are more fully described in Hakouvainen et al. (1996,
Biochemistry 35:9617-24).
[0071] These enzymes may be isolated from their natural host
organism, or expressed in an engineered host organism for
production. For example, a chimeric gene containing a promoter
active in a target expression host cell, a sequence encoding a GH
given above, and a termination signal is expressed from a plasmid
vector or is integrated in the genome of a target expression host
cell using standard methods known to one skilled in the art. A
coding sequence used may be codon optimized for the specific host
used for expression. Expression host cells typically used include
bacteria such as Escherichia, Bacillus, Lactobacillus, Pseudomonas
and Streptomyces, yeasts such as Saccharomyces,
Schizosaccharomyces, Candida, Hansenula, Pichia, Kluyveromyces, and
Phaffia, and filamentous fungi such as Acremonium, Aspergillus,
Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysoporium, Coprinus,
Coriolus, Corynascus, Chaertomium, Cryptococcus, Filobasidium,
Fusarium, Gibberella, Humicola, Magnaporthe, Mucor, Myceliophthora,
Mucor, Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Phanerochaete, Phlebia, Piromyces, Pleurotus,Scytaldium,
Schizophyllum, Sporotrichum, Talaromyces, Thermoascus, Thielavia,
Tolypocladium, Trametes, and Trichoderma.
[0072] One skilled in the art would know how to determine the
effective amount of enzymes to use in a consortium and adjust
conditions for optimal enzyme activity. One skilled in the art
would also know how to optimize the classes of enzyme activities
required within a consortium to obtain optimal saccharification of
a given pretreatment product under the selected conditions. An
example of saccharification is described in U.S. Pat. No.
7,932,063.
[0073] The saccharifying can be performed for a time of about
several minutes to about 200 hours, typically from about 24 hours
to about 72 hours. The time for the reaction will depend on the
enzyme concentration and the 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 consortium.
[0074] The saccharification can be performed in a single batch,
fed-batch or as a continuous process. The saccharification can 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.
[0075] Prior to fermentation the saccharification mixture may be
concentrated by evaporation, for example, to increase the
concentration of fermentable sugars. Optionally, liquid in the
saccharification product may be separated from solids in a batch or
continuous method. Optionally, the liquid or the entire
saccharification product may be sterilized prior to fermentation.
Depending on the biocatalyst(s) used during fermentation and the pH
used during saccharification, the pH may be adjusted to that
suitable for fermentation.
[0076] Biomass hydrolysate containing fermentable sugars is
included in fermentation medium typically as a percent of the
medium, providing all or a portion of the carbon source for
biocatalyst growth and product production. The hydrolysate in a
fermentation medium is typically about 40% to 90% of the
fermentation medium. Examples of hydrolysate used as 40% or 80% of
fermentation medium are given in Example 9 of U.S. Pat. No.
7,932,063. Depending on the fermentable sugars concentration in the
hydrolysate, additional sugars may be added to the medium. For
example, when a hydrolysate containing about 80 g/L glucose and
about 50 g/L xylose is included at 40% of the fermentation medium,
additional glucose and xylose may be added to the desired final
sugars concentrations. In addition to hydrolysate, fermentation
medium may contain other nutrients, salts and factors required for
growth and production by the specific biocatalyst to be used for
product production, as well known to one skilled in the art.
Supplements may include, for example, yeast extract, specific amino
acids, phosphate, nitrogen sources, salts, and trace elements.
Components required for production of a specific product made by a
specific biocatalyst may also be included, such as an antibiotic to
maintain a plasmid or a cofactor required in an enzyme catalyzed
reaction.
[0077] Alternatively to preparing hydrolysate, adding it to
fermentation medium, then carrying out the fermentation, a
simultaneous saccharification and fermentation (SSF) process may be
used to produce a biomass hydrolysate fermentation broth. In this
process sugars are produced from biomass as they are metabolized by
the production biocatalyst.
Biocatalyst Fermentation And Target Products
[0078] Fermentable sugars in the fermentation medium are
metabolized by suitable biocatalysts to produce target products.
The sugars are contacted with a biocatalyst in a fermentation
process where the biocatalyst is grown under conditions where a
target product made by the biocatalyst is produced. Temperature
and/or headspace gas may be adjusted for fermentation, depending on
conditions useful for the particular biocatalyst(s) in use.
Fermentation may be aerobic or anaerobic. These and other
conditions including temperature and pH are adjusted for the
particular biocatalyst used.
[0079] Examples of target products produced by biocatalysts include
1,3-propanediol, butanol (isobutanol, 2-butanol, and 1-butanol),
and ethanol. Disclosed in U.S. Pat. No. 7,504,250 are recombinant
microorganisms that produce 1,3-propanediol. Production of butanol
by genetically modified yeast is disclosed for example in U.S. Pat.
No. 7,851,188. Genetically modified strains of E. coli have also
been used as biocatalysts for ethanol production (Underwood et al.,
(2002) Appl. Environ. Microbiol. 68:6263-6272). Ethanol has been
produced by genetically modified Zymomonas in lignocellulosic
biomass hydrolysate fermentation media (U.S. Pat. No. 7,932,063).
Genetically modified strains of Zymomonas mobilis with improved
production of ethanol that may be used are described in U.S. Pat.
No. 7,223,575 and U.S. Pat. No. 7,998,722.
EXAMPLES
[0080] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. 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.
[0081] The meaning of abbreviations used is as follows: "s" is
second, "min" means minute(s), "h" or "hr" means hour(s), ".mu.L"
means microliter(s), "mL" means milliliter(s), "L" means liter(s),
"m" is meter, "nm" means nanometer(s), "mm" means millimeter(s),
"cm" means centimeter(s), ".mu.m" means micrometer(s), "mM" means
millimolar, "M" means molar, "mmol" means millimole(s), ".mu.mole"
means micromole(s), "g" means gram(s), ".mu.g" means microgram(s),
"mg" means milligram(s), "kg" is kilogram, "rpm" means revolutions
per minute, "C" is Centigrade, "ppm" means parts per million, "psi"
is pounds per square inch.
GENERAL METHODS
Saccharification Enzymes
[0082] Accellerase.RTM. 1500 (A1500) was obtained from Danisco U.S.
Inc., Genencor, International (Rochester, N.Y.).
Packed-Bed Reactor
[0083] A packed-bed pretreatment reactor was used composed of a
vertically-oriented, stainless-steel, filter-housing pressure
vessel with a removable lid. The pressure vessel housed a
stainless-steel, removable, cylindrical basket with an open-top,
solid walls, and a perforated base. The basket allowed for the
static-containment of a packed-bed of biomass and the passage of
top-entering, or bottom-entering liquid, vapor, and/or gas
throughout the interstitial volume between biomass particles.
[0084] The filter-housing had an inner-diameter of 8 inches (20.3
cm) and an interior length of 29 inches (73.7 cm). The cylindrical
basket had a diameter of 6.5 inches (16.5 cm) a depth of 29 inches
(73.7 cm), and a volume of approximately 15.8 liters.
[0085] Flexible copper tubing surrounded approximately 30% of the
filter housing, external surface area. The copper tubing, used for
heating the outside of the vessel, was connected to a 164.7 psia
(1135.6 kpascal) steam supply.
[0086] Tubing connections were made to the packed-bed reactor
allowing for the: [0087] Evacuation of air to create a full, or
partial, vacuum [0088] Addition of saturated steam from a steam
generator operating at 84.7 psia (584 Kpascal) [0089] Addition of
anhydrous ammonia [0090] Evacuation of post-reaction, process
vapors [0091] Addition of air, post-reaction, to break the
vacuum
Example 1
Packed-Bed Pretreatment of Biomass in Preparation for
Saccharification
[0092] 4.07 kilograms of corn cob were and classified to obtain a
cob fraction with particle diameter greater than 1/4'' (0.64 cm)
and less than 3/8'' (0.375 cm). The cob was placed into a basket
which was then placed into the pretreatment reactor described in
General Methods. The corn cob had a moisture content of 8.6% and a
bulk density of 227.5 g/L. The pre-reaction, packed bed depth
measured approximately 29 inches (73.7 cm).
[0093] Air was evacuated from the reactor and a partial vacuum of
0.1 bar (10 kpascal) was achieved. 8 wt % of anhydrous ammonia,
based on mass of bone-dry biomass,was added by charging 297.6 grams
of ammonia over an approximate 60 minute period. Saturated steam at
a pressure of 84.7 psia (584 kpascal) was then added to bring the
reactor temperature to approximately 70.degree. C. At this time,
the temperature of the reactor was allowed to increase from
70.degree. C. to 90.degree. C. over a hold time of 40 minutes. The
pressure in the reactor was approximately 85 psia (584 kpascal)
during the hold time. After the hold time the pressurized gas and
vapors were released and captured in a cooled, external-containment
vessel. When the reactor reached atmospheric pressure, the reactor
was then evacuated to approximately 0.1 bar (10 kpascal) using a
vacuum pump, and held at that pressure for five minutes. The
reactor was then vented to relieve the vacuum to atmospheric
pressure. The pretreatment reactor was opened after the process
reached atmospheric pressure.
[0094] The pretreated biomass was removed in quartile fractions
from the top to the bottom of the packed-bed, i.e. first (top)
quarter, second quarter, third quarter, fourth (bottom) quarter as
in Table 1. These fractions were analyzed for moisture content and
residual ammonia and results are in Table 2. Residual ammonia was
determined by extracting a known amount of pretreated solids in
water for one hour, then using titration with 0.1N HCl to determine
the equivalents of acid to reach pH 5.3. The equivalents were then
normalized to the amount of dry matter in the extraction system. In
addition, equal masses of each fraction were subsequently combined,
then analyzed, to determine a mixing-cup composition of the
pretreated biomass.
TABLE-US-00001 TABLE 1 Samples collected from four separate layers
in the 29-inch (73.7 cm) deep reactor bed. 168-1 Top of packed bed
168-2 Approximately 10 inches (25.4 cm) down from top 168-3
Approximately 20 inches (50.8 cm) down from top 168-4 Bottom of
packed bed 168-C Composite: packed bed thoroughly mixed
TABLE-US-00002 TABLE 2 Pretreated material analysis 168-1 168-2
168-3 168-4 168-C Moisture content wt % 21.42 5.82 6.07 28.78 9.54
Conc. of NH.sub.3 pH = 5.3 0.275 0.122 0.133 0.331 0.163 (g/100 g
DM*) *DM is dry matter
[0095] A sample of each pretreated biomass fraction was
enzymatically saccharified to determine monosaccharide and
oligosaccharide yields of glucan and xylan.
Saccharification Procedure
[0096] 0.56 grams of each fraction was weighed into individual 20
ml scintillation vials. Appropriate amounts of 50 mM pH 5 acetate
buffer were added to achieve 18.6% solids per vial. pH was adjusted
to 5 by addition of 1N H.sub.2SO.sub.4. The acetate buffer solution
contained 0.005% Sodium Azide to inhibit bacterial growth within
the feedstock during incubation.
[0097] Enzymatic saccharifications were performed using
Accelerase.RTM. 1500 combined with a cocktail of hemicellulases
(Xyn3, Fv3A, Fv51A, and Fv43D) at either 7.2 mg/g glucan+xylan, or
21.7 mg/g glucan+xylan. The enzymes were added to each vial,
followed by the addition of 1, 1/2'' (1.3 cm) steel ball to provide
adequate grinding of the feedstock during incubation.
[0098] Vials were tightly capped and allowed to saccharify at about
48.degree. C. for 72 hours on a rotary shaker at 180 rpm in a
rotary shaker.
[0099] After incubation, samples were diluted with water before
filtration and analysis by HPLC. Samples were subjected to HPLC
analysis using an HPX-87H column (BioRad) run at 60.degree. C. with
0.01N H.sub.2SO.sub.4 as the mobile phase at a flow rate of 0.6
mL/min.
[0100] Total sugar values were obtained by acid hydrolysis, by
addition of sulfuric acid, autoclaving, filtration and analysis by
HPLC. Oligomer values were determined by subtracting the monomer
from the total sugar concentrations for each sample. Results are
given in Table 3.
TABLE-US-00003 TABLE 3 Sugars produced fron hydrolysis of treated
biomass quartile fractions Enzyme loading mg enzyme/gm % Yield %
Yield % Yield % Yield Sample glucose + Monomer Monomer Oligomer
Oligomer # xylose Xylose Glucose Xylose Glucose JV-168- 21.67 mg/gm
57.6 83.5 42.0 22.3 1 7.22 mg/gm 44.5 47.0 47.1 17.9 JV-168- 21.67
mg/gm 58.6 82.1 44.7 19.8 2 7.22 mg/gm 40.7 46.2 44.0 14.8 JV-168-
21.67 mg/gm 59.0 79.7 45.3 18.1 3 7.22 mg/gm 41.3 44.9 45.9 13.8
JV-168- 21.67 mg/gm 60.7 87.2 45.9 22.6 4 7.22 mg/gm 43.8 45.6 50.0
17.1 JV-168- 21.67 mg/gm 61.4 85.5 37.7 17.0 C 7.22 mg/gm 41.3 47.3
47.0 16.7
Example 2
Packed-Bed Pretreatment of Biomass with Short Time at Higher
Temperature in Preparation for Saccharification
[0101] 3.74 kilograms of milled corn cob, as in Example 1, were
placed into the basket of the reactor described in General Methods.
The basket was placed into the pretreatment reactor. The corn cob
had a moisture content of 8.6%, and a bulk density of 227.5 g/L.
The pre-reaction, packed bed depth measured approximately 29 inches
(73.7 cm).
[0102] Steam at 150 psig (1034.2 kpascal) was applied to the
pretreater copper coil. When the pretreater lid reached 145.degree.
C. air was evacuated until a partial vacuum of 0.6 psia was
achieved. Steam was directly injected into the process reaching a
pressure of 17 psia (4.14 kpascal). The pretreater was then vented
by opening the bottom valve to release any condensate formed in the
system. Steam was injected into the pretreater top until dry steam
was observed exiting the opened bottom valve to ensure that all
condensate was purged from the system. At this point the
temperature in the reactor was approximately 100.degree. C. The
steam flow was stopped and the drain valve was then closed. Due to
heat absorption into the biomass, the temperature of the reactor
dropped to 80.degree. C., which caused a partial vacuum of 8 psia.
At this point, 190.3 grams of anhydrous ammonia was added to the
process to achieve an ammonia loading of 6 wt % based on the dry
matter charged into the reactor. After the addition of ammonia,
steam was added to the process sufficient to reach a reaction
temperature of 150.degree. C. and a pressure of 70 psia (482.6
kpascal). After 30 minutes the pressurized gas and vapors were
released and captured in a cooled, external-containment vessel.
When the reactor reached atmospheric pressure, the reactor was then
evacuated to approximately 0.1 bar (10 kpascal) using a vacuum pump
to further reduce residual ammonia from the biomass, and held at
that pressure for five minutes. The reactor was then vented to
relieve the vacuum to atmospheric pressure. The pretreatment
reactor was opened after the process reached atmospheric
pressure.
[0103] The pretreated biomass was removed in quartile fractions
from the top to the bottom of the packed-bed as in Table 4. These
fractions were analyzed for moisture content, and residual ammonia,
with results in Table 5. In addition, equal masses of each fraction
were subsequently combined, then analyzed, to determine a
mixing-cup composition of the pretreated biomass.
TABLE-US-00004 TABLE 4 Samples collected from four separate layers
in the 30-inch deep reactor bed. 170-1 Top of packed bed 170-2
Approximately 10 inches down from top 170-3 Approximately 20 inches
down from top 170-4 Bottom of packed bed 170-C Composite: packed
bed thoroughly mixed
TABLE-US-00005 TABLE 5 Pretreated material analysis 170-1 170-2
170-3 170-4 170-C % dry matter % 54.96 83.68 93.68 92.30 71.29 of
PT sample Conc. of NH.sub.3 pH = 0.239 0.150 0.121 0.132 0.192
(g/100 g DM*) 5.3 *DM is dry matter
[0104] A sample of each pretreated biomass fraction was
enzymatically saccharified to determine the monosaccharide and
oligosaccharide yields of glucan and xylan, as described in Example
1.
TABLE-US-00006 TABLE 6 Sugars produced fron hydrolysis of treated
biomass quartile fractions Enzyme loading mg enzyme/gm % Yield %
Yield % Yield % Yield Sample glucose + Monomer Monomer Oligomer
Oligomer # xylose Xylose Glucose Xylose Glucose JV-170- 21.67 mg/gm
67.6 88.8 42.9 22.0 1 7.22 mg/gm 41.8 60.6 53.0 17.2 JV-170- 21.67
mg/gm 62.6 89.6 35.0 12.1 2 7.22 mg/gm 42.1 67.2 43.7 8.8 JV-170-
21.67 mg/gm 59.1 86.3 39.3 10.4 3 7.22 mg/gm 39.4 58.7 49.6 13.2
JV-170- 21.67 mg/gm 61.5 88.8 42.3 15.4 4 7.22 mg/gm 38.5 62.9 41.2
6.8 JV-170- 21.67 mg/gm 70.7 100.6 49.7 18.7 C 7.22 mg/gm 48.4 67.1
54.2 16.5
* * * * *