U.S. patent application number 14/729756 was filed with the patent office on 2015-12-10 for multi-ligand metal complexes and methods of using same to perform oxidative catalytic pretreatment of lignocellulosic biomass.
The applicant listed for this patent is Board of Trustees of Michigan State University. Invention is credited to Namita Bansal, Aditya Bhalla, Eric L. Hegg, David B. Hodge, Zhenglun Li, Vaidyanathan Mathrubootham.
Application Number | 20150352540 14/729756 |
Document ID | / |
Family ID | 54768796 |
Filed Date | 2015-12-10 |
United States Patent
Application |
20150352540 |
Kind Code |
A1 |
Hodge; David B. ; et
al. |
December 10, 2015 |
MULTI-LIGAND METAL COMPLEXES AND METHODS OF USING SAME TO PERFORM
OXIDATIVE CATALYTIC PRETREATMENT OF LIGNOCELLULOSIC BIOMASS
Abstract
A homogeneous catalyst is provided comprising one or more
metals; and at least two metal coordinating ligands wherein the
homogeneous catalyst is a multi-ligand metal complex_adapted for
use with an oxidant in an oxidation reaction to catalytically
pretreat lignocellulosic biomass. In one embodiment, the homogenous
catalyst is copper (II) 2,2' bipyridine ethylenediamine
(Cu(bpy)en). Related methods are also disclosed.
Inventors: |
Hodge; David B.; (East
Lansing, MI) ; Hegg; Eric L.; (East Lansing, MI)
; Li; Zhenglun; (Lansing, MI) ; Mathrubootham;
Vaidyanathan; (Cary, NC) ; Bhalla; Aditya;
(Lansing, MI) ; Bansal; Namita; (Lansing,
MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Trustees of Michigan State University |
East Lansing |
MI |
US |
|
|
Family ID: |
54768796 |
Appl. No.: |
14/729756 |
Filed: |
June 3, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62007306 |
Jun 3, 2014 |
|
|
|
Current U.S.
Class: |
435/99 ; 127/34;
127/37; 546/12; 546/2; 556/110; 556/116; 556/45; 556/50 |
Current CPC
Class: |
B01J 31/1805 20130101;
C13K 1/02 20130101; D21B 1/021 20130101; B01J 2231/70 20130101;
C07F 13/005 20130101; B01J 31/183 20130101; C13K 13/002 20130101;
B01J 31/2243 20130101; B01J 2531/842 20130101; B01J 2531/72
20130101; C12P 2201/00 20130101; B01J 31/1815 20130101; B01J
2531/16 20130101; B01J 2531/845 20130101; C07F 1/08 20130101 |
International
Class: |
B01J 31/18 20060101
B01J031/18; C07F 13/00 20060101 C07F013/00; C13K 13/00 20060101
C13K013/00; C13K 1/02 20060101 C13K001/02; C12P 19/14 20060101
C12P019/14; C12P 19/02 20060101 C12P019/02; C07F 1/08 20060101
C07F001/08; D21B 1/02 20060101 D21B001/02 |
Goverment Interests
STATEMENT OF GOVERNMENT RIGHTS
[0002] This invention was made with government support under
DE-FC02-07ER64494 awarded by the U.S. Department of Energy. The
government has certain rights in the invention.
Claims
1. A homogeneous catalyst comprising: one or more metals; and at
least two metal coordinating ligands, wherein the homogeneous
catalyst is a multi-ligand metal complex adapted for use with an
oxidant in an oxidation reaction to catalytically pretreat
lignocellulosic biomass.
2. The homogeneous catalyst of claim 1 wherein the multi-ligand
metal complex is a multi-ligand copper complex.
3. The homogeneous catalyst of claim 2 wherein said metals capable
of interacting with the multi-ligand metal complex are selected
from aluminum, zinc, nickel, magnesium and combinations
thereof.
4. The homogenous catalyst of claim 3 wherein said metals are
selected from Fe(II), Fe(III)), Cu(I), Cu(II), Co(III), Co(VI)),
V(II), V(III), V(IV), V(V) and combinations thereof.
5. The homogeneous catalyst of claim 1 wherein the metal
coordinating ligand is selected from pyridine, 1,10-phenanthroline,
ethylenediamene, histidine, glycine and combinations thereof.
6. The homogeneous catalyst of claim 1 comprising copper (II) 2,2'
bipyridine ethylenediamine (Cu(bpy)en).
7. The homogeneous catalyst of claim 1 wherein the oxidant is
selected from air, oxygen, hydrogen peroxide, persulfate,
percarbonate and sodium peroxide and/or ozone.
8. The homogenous catalyst of claim 1 wherein the lignocellulosic
biomass contains more than trace amounts of at least one transition
metal.
9. The homogenous catalyst of claim 8 wherein the transition metal
is selected from iron, cupper and/or manganese.
10. The homogenous catalyst of claim 1 wherein said metals and said
metal coordinating ligands are in a state of interaction with each
other.
11. A method of pretreating plant biomass comprising catalytically
pretreating the plant biomass with a multi-ligand metal complex and
oxidant in an alkaline oxidative pretreatment to produce a
catalytically pretreated plant biomass.
12. The method of claim 11 wherein the plant biomass, the
multi-ligand metal complex and the oxidant form a solution having a
pH of at least 11.5.
13. The method of claim 12 wherein the oxidant is hydrogen peroxide
and the multi-ligand metal complex is a multi-ligand copper
complex.
14. The method of claim 13 wherein the copper complex is a
copper(II) 2,2'-bipyridine complex (Cu(bpy)) modified to contain at
least one additional metal-coordinating ligand.
15. The method of claim 14 wherein said additional metal
coordinating ligand is ethylenediamine.
16. The method of claim 11 wherein the oxidant is added at a
gradual rate.
17. The method of claim 16 wherein the gradual rate is equal to or
less than a rate of consumption of the oxidant by the plant biomass
and the multi-ligand metal complex.
18. The method of claim 11 wherein the method further comprises
extracting the lignocellulosic biomass prior to produce a solids
fraction and a liquid fraction, wherein the solids fraction is
catalytically pretreated.
19. The method of claim 11 wherein the method further comprises
recovering and reusing the multi ligand metal complex.
20. The method of claim 11 wherein the catalytic pretreating step
also produces a liquid phase and the method further comprises:
separating the catalytically pretreated biomass from the liquid
phase to produce separated catalytically pretreated biomass; and
hydrolyzing the separated catalytically pretreated biomass to
produce hydrolyzed catalytically pretreated biomass.
Description
[0001] This application claims the benefit under 35 U.S.C. 119 (e)
of U.S. Provisional Application Ser. No. 62/007,306 filed on Jun.
3, 2014.
BACKGROUND
[0003] Cellulosic biofuels offer enormous potential as sustainable,
low-carbon alternative liquid transportation fuels to
petroleum-derived fuels. The vast majority of carbon in the
terrestrial biosphere is contained in the cell walls of plants or
lignocellulose. This enormous reservoir of reduced carbon is
largely untapped for conversion to fuels, chemicals, and polymers
as consequence of the difficulty in deconstructing the biopolymers
contained in the plant cell wall matrix to suite a chemicals that
are amenable to conversion processes.
SUMMARY
[0004] The various embodiments described herein provide
multi-ligand metal complexes and methods of using same to perform
oxidative catalytic pretreatment of lignocellulosic biomass. In one
embodiment, the multi-ligand metal complex is a multi-ligand copper
complex. In one embodiment, the copper complex is a copper(II)
2,2'-bipyridine complex (Cu(bpy)) modified to contain at least one
additional metal-coordinating ligand. In a particular embodiment,
copper(II) 2,2' bipyridine ethylenediamine (Cu(bpy)en) is used.
[0005] In various embodiments, the oxidant is added gradually,
rather than in batch. In one embodiment, an integrated conversion
process for lignocellulosic biomass that produces bio-based
chemicals and fuels, is provided. In one embodiment, these
processes depend, in part, on particular chemical structures
contained in plant cell macromolecules. In one embodiment, plant
biomass having more than trace amounts of one or more transition
metals (e.g., Cu, Fe and/or Mn) is used.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0007] FIG. 1 is a graph showing the effect of metal catalyst
addition to a 24 hr AOP pretreatment of hybrid poplar according to
an embodiment.
[0008] FIG. 2 is a graph showing enzymatic digestibility of
pretreated poplar according to an embodiment.
[0009] FIG. 3 is a graph showing use of recovered catalyst in
catalytic AOP according to an embodiment.
[0010] FIG. 4A is a graph showing phenolic acid and aldehyde yields
during various pretreatments of hybrid poplar according to an
embodiment.
[0011] FIG. 4B is a graph showing p-hydroxybenzoate yield during
various pretreatments of hybrid poplar according to an
embodiment.
[0012] FIG. 5 is a graph showing the effect of a catalyst in PTL
for oxidative pretreatment on enzymatic digestibility of pretreated
biomass according to an embodiment.
[0013] FIG. 6 is a graph showing yields of glucose and xylose (with
copper recycling) under varying pretreatment conditions according
to various embodiments.
[0014] FIG. 7 is a graph showing glucose and xylose yield (with
extractives removal) under varying conditions according to various
embodiments.
[0015] FIG. 8 is a graph showing glucose and xylose yield (with
NaOH recycle) under varying conditions according to various
embodiments.
[0016] FIGS. 9A-9C are Transmission Electron Microscopy (TEM)
micrographs of cross sections of an untreated (A) and Alkaline
Oxidative Peroxide (AOP)-only pretreated (B, C) hybrid poplar cell
wall according to various embodiments.
[0017] FIGS. 10A-10D are TEM micrographs of a hybrid poplar cell
wall after copper(II) 2,2'-bipyridine complex (Cu(bpy))-catalyzed
AOP pretreatment showing delamination (A) and dislocations of cell
wall layers (B), together with accumulation of nanoparticles in
disrupted regions (C, D) according to various embodiments.
[0018] FIGS. 11A-11F are TEM micrographs of hybrid poplar cell wall
(A) and high resolution image of an electron-opaque aggregate (B)
together with acquired Energy Dispersive X-ray Spectroscopy (EDS)
spectra of select regions within this sample (C--F) according to
various embodiments.
[0019] FIG. 12 shows SEC elution profiles for plant cell wall
polymers solubilized during pretreatments referenced to elution
times for polystyrene standards according to various
embodiments.
[0020] FIGS. 13A-13F are partial 2D Heteronuclear Single-Quantum
Coherence (HSQC) Nuclear Magnetic Resonance (NMR) spectra of (A,B)
whole cell wall untreated poplar, (C,D) solubilized lignin, and
(E,F) residual poplar cell walls following Cu-catalyzed AOP
pretreatment showing polysaccharide correlations and colored
contours to match structures for aromatic components according to
various embodiments.
[0021] FIG. 14A-14L are representations of various known chemical
structures.
[0022] FIG. 15 is an Electron Energy Loss Spectroscopy (EELS)
spectrum of the Cu-containing nanoparticles showing the Cu
L.sub.2,3 edge providing evidence that the Cu in these particles is
primarily in the Cu(I) oxidation state with contributions by Cu(0)
according to various embodiments.
[0023] FIG. 16 is a graph comparing yields of xylose and glucose
when using catalytic AOP with a batch addition of hydrogen peroxide
versus a slow addition of hydrogen peroxide according to an
embodiment.
[0024] FIG. 17 is a graph comparing yields of xylose and glucose
yields when using catalytic AOP with a batch addition of hydrogen
peroxide versus a slow addition of hydrogen peroxide in combination
with alkali pre-extraction in a modified Cu-AOP pretreatment
according to an embodiment.
[0025] FIG. 18 is a graph showing the correlation between lignin
removal and glucose yields with different concentrations of slowly
added H.sub.2O.sub.2 in combination with alkali pre-extraction in a
modified Cu-AOP pretreatment according to an embodiment.
[0026] FIG. 19 is a graph showing glucose yields obtained with
different concentrations of enzymes at different loadings of
H.sub.2O.sub.2 on biomass utilizing a modified Cu-AOP pretreatment
according to an embodiment.
[0027] FIG. 20 is a graph showing glucose yields obtained with
different concentrations of bipyridine at different loadings of
H.sub.2O.sub.2 and enzymes on biomass utilizing modified Cu-AOP
pretreatment according to an embodiment.
[0028] FIG. 21 is an image of lignin after the biomass in which it
was located was subject to a Cu-AOP pretreatment according to an
embodiment.
[0029] FIGS. 22A-22C are graphs correlating sugar yields with
different cell wall properties according to an embodiment.
[0030] FIG. 23A is a graph showing metal content of various
genotypes of Populus trichocarpa species according to an
embodiment.
[0031] FIG. 23B is a graph showing lignin content vs guaiacyl and
syringyl (S/G) ratio according to an embodiment.
[0032] FIG. 24 is a graph showing glucose yields for various
chelated and non-chelated for four different genotypes of Populus
trichocarpa according to an embodiment.
[0033] FIG. 25 is a graph showing total number of cell wall redox
metal ions in various chelated and non-chelated for four different
genotypes of Populus trichocarpa according to an embodiment.
[0034] FIG. 26 is a graph showing glucose yields for various
hardwood species after undergoing different pretreatments according
to various embodiments.
[0035] FIG. 27 is a graph showing glucose yield to S/G ratio for
various types of hardwood species after undergoing various types of
pretreatment processes according to various embodiments.
[0036] FIG. 28 is a graph showing glucose yield correlation to cell
wall redox active metal content for Copper (Cu), Manganese (Mn) and
iron (Fe) after undergoing a pretreatment process according to
various embodiments.
[0037] FIG. 29A is a graph showing glucose yields to S/G ratio for
various hardwood species after undergoing an AOP-chelated and
AOP-non-chelated pretreatment according to various embodiments.
[0038] FIG. 29B is a graph showing glucose yields to S/G ratio for
various hardwood species after undergoing a Cu-AOP-chelated and
Cu-AOP-non-chelated pretreatment according to various
embodiments.
[0039] FIG. 30 is a graph showing glucose yields for various
hardwood species after undergoing different pretreatments according
to various embodiments.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0040] In the following detailed description, embodiments are
described in sufficient detail to enable those skilled in the art
to practice them, and it is to be understood that other embodiments
may be used and that chemical and procedural changes may be made
without departing from the spirit and scope of the present subject
matter. The following detailed description is, therefore, not to be
taken in a limiting sense, and the scope of embodiments of the
present invention is defined only by the appended claims.
[0041] The term "biomass" as used herein, refers in general to
organic matter harvested or collected from a renewable biological
resource as a source of energy and bioproducts. The renewable
biological resource can include plant materials, animal materials,
and/or materials produced biologically. The term "biomass" is not
considered to include fossil fuels, which are not renewable.
[0042] The term "plant biomass" or "ligno-cellulosic biomass (LCB)"
as used herein is intended to refer to virtually any plant-derived
organic matter containing cellulose and/or hemicellulose as its
primary carbohydrates (woody or non-woody) available for producing
energy on a renewable basis and bioproducts. Plant biomass can
include, but is not limited to, agricultural residues such as corn
stover, wheat straw, rice straw, sugar cane bagasse, sorghum and
the like. Plant biomass can also include agricultural residues and
forest residues that are dedicated for bioenergy purposes, such as
residues of grasses and trees. Plant biomass further includes, but
is not limited to, "woody biomass", i.e., woody energy crops, wood
wastes and residues such as trees, including fruit trees, such as
fruit-bearing trees, (e.g., apple trees, orange trees, and the
like), softwood forest thinnings, barky wastes, sawdust, paper and
pulp industry waste streams, wood fiber, and the like. Additionally
perennial grass crops, such as various prairie grasses, including
prairie cord grass, switchgrass, Miscanthus, big bluestem, little
bluestem, side oats grama, and the like, have potential to be
produced large-scale as additional plant biomass sources. For urban
areas, potential plant biomass feedstock includes yard waste (e.g.,
grass clippings, leaves, tree clippings, brush, etc.) and vegetable
processing waste. Plant biomass is known to be the most prevalent
form of carbohydrate available in nature and corn stover is
currently the largest source of readily available plant biomass in
the United States. When describing the various embodiments and used
without a qualifier, the term "biomass" is intended to refer to
"plant biomass," i.e., lignocellulosic biomass (LCB) containing
plant cell wall polysaccharides.
[0043] The term "biofuel" as used herein, refers to any renewable
solid, liquid or gaseous fuel produced biologically and/or
chemically, for example, those derived from biomass. Most biofuels
are originally derived from biological processes such as the
photosynthesis process and can therefore be considered a solar or
chemical energy source. Some types of biofuels, such as some types
of biodiesel, can be derived from animal fats. Other biofuels, such
as natural polymers (e.g., chitin or certain sources of microbial
cellulose), are not synthesized during photosynthesis, but can
nonetheless be considered a biofuel because they are biodegradable.
There are generally considered to be three types of biofuels
derived from biomass synthesized during photosynthesis, namely,
agricultural biofuels (defined below), municipal solid waste
biofuels (residential and light commercial garbage or refuse, with
most of the recyclable materials such as glass and metal removed)
and forestry biofuels (e.g., trees, waste or byproduct streams from
wood products, wood fiber, pulp and paper industries). Biofuels
produced from biomass not synthesized during photosynthesis
include, but are not limited to, those derived from chitin, which
is a chemically modified form of cellulose known as an N-acetyl
glucosamine polymer. Chitin is a significant component of the waste
produced by the aquaculture industry because it comprises the
shells of seafood.
[0044] The term "agricultural biofuel", as used herein, refers to a
biofuel derived from agricultural crops, lignocellulosic crop
residues, grain processing facility wastes (e.g., wheat/oat hulls,
corn/bean fines, out-of-specification materials, etc.), livestock
production facility waste (e.g., manure, carcasses, etc.),
livestock processing facility waste (e.g., undesirable parts,
cleansing streams, contaminated materials, etc.), food processing
facility waste (e.g., separated waste streams such as grease, fat,
stems, shells, intermediate process residue, rinse/cleansing
streams, etc.), value-added agricultural facility byproducts (e.g.,
distiller's wet grain (DWG) and syrup from ethanol production
facilities, etc.), and the like. Examples of livestock industries
include, but are not limited to, beef, pork, turkey, chicken, egg
and dairy facilities. Examples of agricultural crops include, but
are not limited to, any type of non-woody plant (e.g., cotton),
grains such as corn, wheat, soybeans, sorghum, barley, oats, rye,
and the like, herbs (e.g., peanuts), short rotation herbaceous
crops such as switchgrass, alfalfa, and so forth.
[0045] The term "pretreatment step" as used herein, refers to any
step intended to alter native biomass so it can be more efficiently
and economically converted to reactive intermediate chemical
compounds such as sugars, organic acids, etc., which can then be
further processed to a variety of end products such as ethanol,
isobutanol, long chain alkanes etc. Pretreatment can reduce the
degree of crystallinity of a polymeric substrate, reduce the
interference of lignin with biomass conversion, and hydrolyze some
of the structural carbohydrates, thus increasing their enzymatic
digestibility and accelerating the degradation of biomass to useful
products. Pretreatment methods can utilize acids of varying
concentrations, including dilute acid pretreatments, concentrated
acid pretreatments (using, for example, sulfuric acids,
hydrochloric acids, organic acids, and the like) and/or
pretreatments with alkali such as ammonia and/or ammonium hydroxide
and/or calcium hydroxide and/or sodium hydroxide and/or lime, and
the like, and/or oxidative pretreatments using oxidants such as
air, oxygen, hydrogen peroxide, organic peroxide, ozone, and the
like.
[0046] Pretreatment methods can additionally or alternatively
utilize hydrothermal treatments including water, heat, steam or
pressurized steam pretreatments, including, but not limited to,
hydro-thermolysis pretreatment and liquid hot water pretreatment,
further including, for example, acid catalyzed steam explosion
pretreatment (e.g., SO.sub.2 catalyzed). Pretreatment can occur or
be deployed in various types of containers, reactors (e.g., batch,
counter-current, and the like), pipes, flow through cells and the
like. Many pretreatment methods will cause the partial or full
solubilization and/or destabilization of lignin and/or hydrolysis
of hemicellulose to pentose sugars. Further examples of
pretreatment include, but are not limited wet oxidation, organosolv
pretreatment and mechanical extrusion.
[0047] The term "alkaline oxidative pretreatment" as used herein
refers to a pretreatment process in which plant biomass is
pretreated under alkaline conditions using oxidative chemicals,
which can include, but are not limited to, hydrogen peroxide,
oxygen, ozone, hydroperoxide anion, superoxide radical, hydroxyl
radical, and peroxy acids (e.g., peracetic acid, peroxymonosulfuric
acid, peroxyphosphoric acid, meta-chloroperoxybenzoic acid). See,
for example, Liu, et al., Coupling alkaline pre-extraction with
alkaline-oxidative post-treatment of corn stover to enhance
enzymatic hydrolysis and fermentability, Biotechnology for
Biofuels, 2014, 7:48, which describes example conditions for
alkaline oxidative pretreatment. An alkaline oxidative pretreatment
which uses hydrogen peroxide as the oxidant is to be distinguished
from a conventional "alkaline hydrogen peroxide (AHP)" pretreatment
which is a one-step catalytic pretreatment process which requires
much higher oxidant loadings. See, for example, Biotechnol Bioeng
1984, 26:46-52; Biotechnol Bioeng 1984, 26:628-631; Biotechnol
Bioeng 1985, 27:225-231; Science 1985, 230:820-822 and Biotechnol
Biofuels 2011, 4:16, which describe conventional AHP with much
higher oxidant loadings.
[0048] The term "metal-ligand complex" as used herein refers to a
metal complex containing one or more metal-coordinating ligands and
one or more metal atoms which are in a state of interaction with
each other. Such interactions include various types of forces and
bonds, which include, but are not limited to, ionic bonds, covalent
bonds, and van der Waals forces.
[0049] The term "metal-coordinating ligand" as used herein refers
to a ligand, such as an ion, a molecule, or the like, that is
capable of interacting with the metal portion of a metal-ligand
complex. When used without qualification, the term "ligand" is
intended to refer to a "metal-coordinating ligand."
[0050] The term "copper-coordinating ligand" as used herein refers
to a metal coordinating ligand capable of interacting with copper
atoms or copper ions.
[0051] The term "single-ligand metal complex" as used herein refers
to a metal-ligand complex containing only one ligand that
coordinates with, i.e., interacts with metal atoms or metal
ions.
[0052] The term "multi-ligand metal complex" as used herein refers
to a metal-ligand complex containing more than one ligand that
coordinates with, i.e., interacts with metal atom or metal
ions.
[0053] The term "toxicity" as used herein refers to ions, molecules
and metal-ligand complexes present in the process streams during
biomass conversion and cellulosic biofuel production that
negatively impact the yield of the products.
[0054] The term "slow add" as used herein refers to a gradual rate
of addition of a reagent to a reaction vessel. The gradual rate can
be continuous or discontinuous, i.e., can include intermittent
periods of no reagent being added. A "slow add" is in contrast to a
batch method of adding a reagent, in which all the desired reagent
is added to the reactive vessel at once.
[0055] The term "state of interaction" as used herein refers to an
interaction between a ligand and a metal or between a metal and
multiple ligands. Such an interaction can include various types of
forces and bonds, which include, but are not limited to, ionic
bonds, covalent bonds, and van der Waals forces.
[0056] Nearly all forms of lignocellulosic biomass, i.e., plant
biomass, such as monocots, comprise three primary chemical
fractions: hemicellulose, cellulose, and lignin. Lignin, which is a
polymer of phenolic molecules, provides structural integrity to
plants, and is difficult to hydrolyze. As such, after sugars in the
biomass have been fermented to a bioproduct, such as alcohol,
lignin remains as residual material, i.e., a non-easily digestible
portion.
[0057] Cellulosic biofuel production from lignocellulosic biomass
has gained considerable momentum due to both environmental and
social sustainability benefits. However, the technology is not yet
fully commercialized. One issue impeding cellulosic biofuel
production using the sugar platform is the hydrolysis-resistant
nature of certain components in the lignocellulosic biomass.
[0058] Cellulose and hemicelluloses in plant cell walls exist in
complex structures within the residual material. Hemicellulose is a
polymer of short, highly-branched chains of mostly five-carbon
pentose sugars (xylose and arabinose), and to a lesser extent
six-carbon hexose sugars (galactose, glucose and mannose). Because
of its branched structure, hemicellulose is amorphous and
relatively easy to hydrolyze into its individual constituent sugars
by enzyme or dilute acid treatment. Cellulose is a linear polymer
comprising of .beta.(1.fwdarw.4) linked D-glucose in plant cell
wall, much like starch with a linear/branched polymer comprising of
.alpha.(1.fwdarw.4) linked D-glucose, which is the primary
substrate of corn grain in dry grind and wet mill ethanol plants.
However, unlike starch, the glucose sugars of cellulose are strung
together by .beta.-glycosidic linkages which allow cellulose to
form closely-associated linear chains. Because of the high degree
of hydrogen bonding that can occur between cellulose chains,
cellulose forms a rigid crystalline structure that is highly stable
and much more resistant to hydrolysis by chemical or enzymatic
attack than starch or hemicellulose polymers. Although
hemicellulose sugars represent the "low-hanging" fruit for
conversion to a biofuel, the substantially higher content of
cellulose represents the greater potential for maximizing biofuel
yields, on a per ton basis of plant biomass.
[0059] Lignocellulose can also be characterized as a highly
heterogeneous composite material comprised of multiple cell wall
biopolymers (cellulose, heteropolysaccharides including
hemicelluloses and pectins, and lignins) associated primarily by
non-covalent interactions which are assembled into cell walls with
composition and properties varying by cell and tissue type. These
components are interconnected through a variety of covalent and
noncovalent interactions, giving rise to a highly organized network
which is assembled in a tightly controlled sequence during plant
growth. This heterogeneous higher order structure of the cell wall
impacts the cell wall's response to deconstruction and
conversion.
[0060] Plant cell walls exhibit substantial heterogeneity in both
content and distribution of the inorganic elements which also have
implications for biomass conversion processes. This includes
differences between content and distribution of inorganics in
disparate plant taxa, differences between related species, within a
single species as a function of its phenotype and environment, and
even between tissues in a single plant.
[0061] Inorganic elements in plants are known to be responsible for
diverse roles, including maintenance of ionic equilibrium in cells
(e.g., K) and storage (e.g., Fe in ferritin), which, despite being
localized in plastids, is water-extractable. A subset of the
inorganic elements in plants is strongly associated with the cell
wall. These elements are more resistant to aqueous extraction and
include inorganic elements that may have structural roles,
including, but not limited to, Ca and B ionic cross-links in pectic
polysaccharides, calcium oxalate raphide crystals in some grasses,
and Si in the cell walls of grasses which can comprise a
significant fraction of the mass of a plant. Another lass of role
of cell-wall associated inorganic elements are metal co-factors in
enzymes (e.g., Zn, Fe, Mn, Cu).
[0062] Redox-active metals, such as Cu, Mn, Fe, can exist in
multiple oxidation states in vivo and are often involved in
reactions involving electron transfer. Specifically, Fe in plants
is associated with Fe-heme proteins and iron-sulfur (Fe--S)
clusters in proteins, such as ferredoxins, which function as
electron carriers in the photosynthetic electron transport chain.
Cu in plants has diverse roles as a structural element in
regulatory proteins, in photosynthetic electron transport,
mitochondrial respiration, and Fe mobilization, among others.
Metals may also be associated with metallothioneins (MTs) and
phytochelatins (PCs), which are cysteine-rich polypeptides involved
in either ameliorating the toxicity or controlling homeostasis of
metals such as Fe, Ni, Cd, Zn, and Cu by coordination by thiols. In
addition to its involvement with enzymes associated with the
shikimic acid pathway and lignin biosysthesis, Mn is contained in a
metallo-oxo cluster containing 4 Mn ions at differing oxidation
states in the oxygen evolving complex of photosystem II.
[0063] There are differences in the strength and nature of
association of cell wall-associated metals. Specifically, Mn may be
strongly associated with the cell wall and be s present in "organic
chelates" or "bound to lignin." Alkali delignified hardwoods are
known to have differences in the extractability of cell
wall-associated Mn versus Fe using chelating compounds. Mg, which
is a component of chlorophyll, is useful for photosynthesis and
protein synthesis, although a portion of Mg may be bound to pectin
or precipitated as salts in the vacuole, while the remainder is
extractable with water. During either oxidative delignification or
biomass conversion processes where oxygen may be present, cell
wall-associated transition metals can catalyze the formation of
reactive oxygen species through Fenton chemistry. This catalytic
activation of oxygen by transition metals has been shown to
contribute to the oxidative scission of polysaccharides during
alkaline-oxidative bleaching or delignification using
H.sub.2O.sub.2 or O.sub.2. As a result, precautions are taken
during these processes through chelation and washing steps to
remove metals and addition Mg salts and silicates to complex
transition metals during these unit operations.
[0064] Therefore, a pretreatment process is typically used to alter
and open up the cell wall matrix, to hydrolyze the hemicelluloses,
and to alter the hemicelluloses. Pretreatment disrupts the
non-easily digestible portion of lignocellulosic biomass, e.g.,
cellulose and lignin, thus improving its accessibility. After
pretreatment, much of the biomass becomes easily digestible, while
a portion remains non-easily digestible. Ultimately, the
pretreatment process makes the cellulose more accessible (during a
subsequent hydrolysis process, such as with lytic enzymes) for
conversion of the lignocellulose polysaccharides (e.g., cellulose
and hemicellulose) to monomeric sugars, which can be transformed to
target products via catalytic conversion or microbial
fermentation.
[0065] However, enzymatic hydrolysis of lignocellulose
polysaccharides is usually hindered by the natural resistance of
plant cell wall against deconstruction. To overcome this
resistance, pretreatment processes of biomass feedstock have been
developed and employed. Biomass pretreatment modifies cell wall
structure and renders the biomass more digestible by enzymes.
[0066] A wide range of pretreatments are known, but few
pretreatment methods have been identified as effective for biomass
feedstocks, such as woody biomass, which are highly resistant to
enzymatic hydrolysis. For example, enzymatic hydrolysis of hybrid
poplar wood usually produces sugars at only 5 to 30% of the
theoretical maximum yield. As noted above, such resistance involves
the structural rigidity of the plant cell wall, the crystallinity
of cellulose, and the presence of lignin, which remains as a
residual material.
[0067] However, in the embodiments described herein, the alkaline
pretreatments not only solubilize the lignin, it is expected that
the lignin produced may closely resemble native lignin, such that
less than 35% of the .alpha.-carbon of the solubilized lignin is
oxidized from a hydroxyl to a carbonyl.
[0068] Lignin is known to be useful in a variety of applications
including, but not limited to, carbon fiber composites, bio-oil,
resins, adhesive binders and coating, plastics, paints, enriching
soil organic carbon, fertilizer, rubbers and elastomers, paints,
antimicrobial agents and slow nitrogen release fertilizer, and the
like, and can be a substitute for polymers produced using crude
oil.
[0069] One current source of lignin in the market is produced from
sulfite (or sulfonate) based paper/pulp mills, a kraft pulping
process, and the like. Most such mills currently burn the lignin to
recover energy, in an attempt to reduce the environmental impact of
discharge. Very few sulfite mills currently process the
lignosulfonates from sulfite spent liquors. Additionally, the
quality and quantity of lignin obtained via currently known methods
are inadequate for most applications. As such, methods to
fractionate and convert lignin into value-added products is
needed.
[0070] Known methods for pretreating plant biomass are typically
performed under elevated pressures and temperatures (above room
temperature). Such methods include hot water and steam treatments,
ammonia treatments and sulfite treatments.
[0071] Other pretreatment methods utilize an oxidant-based
pretreatment, such as the alkaline oxidative pretreatment process
defined herein or a conventional alkaline hydrogen peroxide (AOP)
known by those skilled in the art. Yet other methods include
catalytic processes. Catalytic approaches to plant cell wall
deconstruction and conversion of insoluble biomass rely on
homogeneous catalysts to allow the catalyst to diffuse through
nano-scale pores within the cell walls to perform the desired
reactions. Heterogeneous catalysis is known to be inefficient
unless the cell walls are solubilized in expensive solvents such as
ionic liquids. In one embodiment, homogeneous catalysts are used in
many applications, such as homogeneous copper catalysts used for
atom transfer radical polymerization where catalyst removal to
prevent contamination of the product adds cost to the process.
[0072] Use of a single ligand copper complex as a catalyst in
combination with an alkaline oxidative pretreatment (AOP) process
is known. See, for example, Li et al., Rapid and Effective
Oxidative Pretreatment of Woody Biomass at Mild Reaction Conditions
and Low Oxidant Loadings Biotechnol Biofuels 6(1), 119 (2013), and
Li, et al., Catalysis with Cu.sup.II(bpy) Improves Alkaline
Hydrogen Peroxide Pretreatment. Biotechnol Bioeng. 110(4):1078-1086
(2013), each of which is incorporated by reference in its entirety.
However, the amount of oxidant required in such processes is high,
such as at least 10% of the weight of the biomass to be treated.
Additionally, in order to achieve suitable pretreatment results,
the amount of metal utilized in a single-ligand copper complex is
high (e.g., more than 50 .mu.mol of metal complexes per gram of
biomass to be pretreated). Use of such high levels of a metal can
pose toxicity issues in subsequent processes (e.g., fermentation).
Moreover, use of such high amounts of metals and oxidants can be
cost prohibitive.
[0073] As such, the various embodiments described herein provide a
multi-ligand metal complex for use in an oxidative pretreatment
process, such as an alkaline oxidative (AOP) process, which not
only allows for a reduction in the amount of metals used in the
process, but also a reduction in the amount of oxidant. In one
embodiment, the multi-ligand metal complex is used with a
conventional alkaline hydrogen peroxide (AOP) process. In one
embodiment, the multi-ligand metal complex is a multi-ligand copper
complex. In one embodiment, the copper complex is a copper(II)
2,2'-bipyridine complex (Cu(bpy)) modified to contain at least one
additional metal-coordinating ligand, such as pyridine;
1,10-phenanthroline; ethylenediamene; histidine; and/or
glycine.
[0074] While not wishing to be bound by this proposed theory, both
the single- and multi-ligand metal complexes are thought to
function as suitable catalysts for lignocellulosic biomass (i.e.,
cause sufficient catalyst sorption into the biomass) due to the
ability of the cationic metal, such as copper, to interact with
(e.g., bond with) charged anionic groups, such as deprotonated
phenolic hydroxyls in lignin, carboxylate groups in lignin, and/or
uronic acids in pectins and hemicelluloses.
[0075] In one embodiment, the pH of the plant biomass being
pretreated is adjusted to increase the number of deprotonated
groups. In one embodiment, the pH of the pretreated biomass is, or
the biomass is pH adjusted to achieve, a neutral pH during the
pretreatment process. In one embodiment, the pH is adjusted to
achieve an alkaline pH to deprotonate the phenolic groups in lignin
and to increase lignin solubility. In one embodiment, the pH is
adjust to at least 11, such as at least 11.5, including any value
in between. In some embodiments, elevation of the pH is achieved
with bases such as ammonia and/or ammonia derivatives, such as
amines, in which copper is stabilized in solution in the form of a
complex ion. In one embodiment, the pH is adjusted via addition of
a base, which can react with lignin and cause depolymerization
and/or solubilization, i.e., helps the plant cell wall to become
degraded and/or destroyed, thus reducing resistance to subsequent
hydrolysis.
[0076] In one embodiment, oxidants useful in an oxidative
pretreatment process include, but are not limited to, air, oxygen,
hydrogen peroxide, ozone, persulfate, percarbonate and sodium
peroxide. In one embodiment, the metal-coordinating ligands include
2,2'-bipyridine and at least one of another metal-coordinating
ligand, including, but not limited to nitrogen-donating ligands
such as pyridine, 1,10-phenanthroline, and ethylenediamene, and
ligands containing both a nitrogen donor and a carboxylate group
such as the amino acids including histidine or glycine. In one
embodiment, the catalytic metal element(s) (i.e., metal or metals)
in the catalyst can include, but are not limited to, aluminum,
zinc, nickel, magnesium, manganese, iron, copper cobalt and/or
vanadium in various oxidation states. In one embodiment, the
elements include, but are not limited to, iron (e.g., Fe(II),
Fe(III)), copper (e.g., Cu(I), Cu(II)), cobalt (e.g., Co(III),
Co(VI)), and/or vanadium (e.g., V(II), V(III), V(IV), V(V)).
[0077] By substituting an amount of the 2,2'-bypyridine (bpy) with
other, lower costs ligands, substantial savings can be achieved. In
one embodiment, about 1 weight/weight (w/w)% up to about 99% or
higher, such as 100% of bpy is substituted, such as about 10 to
about 90%, such as about 20 to about 80%, such as about 35% to
about 60%, including any range there between. In the various
embodiments described herein, the multi-ligand metal complexes have
low production costs. In one embodiment, substitution of bpy with
other metal coordinating ligands provides a savings on the order of
10-fold or more, such as a savings of about 20 to 30 times the cost
of using bpy alone.
[0078] An additional benefit relates to reduced microbial toxicity.
Microbial toxicity is characterized by the final growth of yeast
cells during yeast fermentation, and/or the growth rate of yeast
during fermentation, and/or the length of the lag phase during
fermentation. Such toxicity is caused by metal ions and other
chemicals present in the processing stream, including the metals
present in the multi-ligand catalyst and metal elements present in
the plant biomass itself. Since the various embodiments allow for a
reduced amount of metal as compared to conventional processes, the
yeast used downstream is less adversely affected down to minimally
adversely affected. As such, in one embodiment, the multi-ligand
complexes have minimal microbial toxicity towards yeast
fermentation (i.e., less than 50% reduction in final growth of
yeast cells, as quantified with optical density).
[0079] In one embodiment, the final growth of yeast cells during
yeast fermentation is decreased by 50% or less as the result of the
presence of multi-ligand metal complexes, which indicates the low
toxicity of the multi-ligand metal complexes. In one embodiment,
the final growth of yeast cells is decreased by less than 40 to
50%, less than 30 to 50%, less than 20 to 50%, less than 10 to 50%,
less than 5 to 50%, including any range or value there between.
Additionally, the various embodiments provide for a pretreatment
method which produces pretreated biomass which is easily digestible
by commercial cellulase cocktails into fermentable sugars (glucose,
xylose, etc.)
[0080] Use of the multi-ligand metal complexes described herein
also reduces the amount of metal, such as copper, used in the
process as compared to a single ligand metal complex, such as a
single ligand copper complex, by at least 50%, or at least 40%, or
at least 30% or at least 20% or at least 10% or at least 5% or
lower, including any range therein. Use of a reduced amount of
metal not only reduces toxicity levels, but further reduces
costs.
[0081] Use of the multi-ligand metal complex may reduce the amount
of oxidant, such as hydrogen peroxide, used in the oxidative
pretreatment by at least 90%, by at least 80%, by at least 70%, by
at least 60%, by at least 50%, or at least 40%, or at least 30% or
at least 20% or at least 10% or at least 5% or lower, including any
range therein. Use of a reduced amount of oxidant further reduces
costs.
[0082] In one embodiment, one or more oxidants are combined with
the other reactants at a low weight percent (%) loading on biomass
(w/w), i.e., loading of no more than 15%. In one embodiment, the
oxidant loading is less than 10%, such as less than 5%. In one
embodiment, the oxidant loading ranges from about 1% to about 15%,
such as about 1% to about 10%, such as about 1% to 5% or less,
including any range there between. Such loadings are lower than
conventional loadings which can be as high as 200%. In one
embodiment, at least 0.1% to about 50 (w/w)%, such as greater than
31.5% oxidant is added, such as from about 31.51% to about 50
(w/w)%. In one embodiment, at least 2.5% to about 50 (w/w)%, such
as greater than 31.5% hydrogen peroxide is added, such as from
about 31.51% to about 50 (w/w)%.
[0083] In one embodiment, use of the multi-ligand metal complex as
a catalyst during an oxidative pretreatment may allow the
pretreatment process to proceed significantly faster (e.g., at
least two times as fast) as compared with an oxidative pretreatment
performed using a conventional single ligand metal complex as a
catalyst.
[0084] In one embodiment, the oxidant is added at a "slow add"
rate. In one embodiment, the "slow add" rate is equal to or less
than a rate of consumption of the oxidant by the other reactants.
Use of a slow rate, surprisingly, results in increased
effectiveness of the pretreatment, as evidenced by increased
downstream yields of various sugars.
[0085] Any suitable plant biomass can be used. In one embodiment,
the plant biomass contains transition metals. Use of a plant
biomass containing more than trace amounts of one or more
transition metals results in further cost savings, as a reduced
amount of catalyst is needed to affect the same or substantially
the same results. Examples of plant biomass containing more than
trace amounts of transition metals include, but are not limited to,
hardwoods of the genus Populus (e.g., various types of poplar
including hybrid poplar, hybrid aspen, western balsam poplar, and
the like), birch (including silver birch and the like), maple
(including sugar maple and the like), further including grasses
(including, but not limited to, corn, switchgrass, sorghum,
miscanthus) and gymnosperms, which are also referred to as conifers
and softwoods (including, but not limited to, the genus of Pinus,
such as Pinus resinosa, i.e., red pine).
[0086] In one embodiment, the plant biomass contains one or more
transition metals that a redox-active, including, but not limited
to, Fe, Mn, Cr, Co, Ni, Cu, Mo, Pd, Ru, Re, Pt, Pd, Os, Ir and
combinations thereof.
[0087] In one embodiment, prior to being subjected to a catalyzed
pretreatment process the plant biomass is first subjected to an
extraction step designed to facilitate oxidative pretreatment and
biomass hydrolysis.
[0088] In various embodiments, the biomass may be subjected to a
cycle of hydrolysis (e.g., enzymatic, acid, etc.) using any
conventional methods known in the art. In one embodiment, a reduced
amount of enzymes is used, as compared to hydrolysis of
conventionally catalyzed pretreated biomass.
[0089] In various embodiments, hydrolysis may optionally be
followed by or integrated with either fermentation or sugar
catalytic conversion of sugars to bioproducts, such as biofuels,
biochemicals and biopolymers. Use of the multi-ligand metal complex
as described herein provides improved downstream bioproduct yields,
such as sugar yields, as compared to yields obtained with a
single-ligand metal complexes, such as single-ligand copper
complexes. In one embodiment, such yields may be improved by at
least 5% or higher, such as at least 10%, at least 20%, at least
30%, at least 40% at least 50% or higher, up to two or three times
higher, including any range there between.
[0090] In one embodiment, the process can further include recovery
and reuse of the multi-ligand metal complex, including recovery of
the metal itself. Conventional technologies for metal removal
(e.g., copper) from wastewater streams are based on ion exchange,
precipitation/co-precipitation plus filtration, and membrane
separation. Additionally, lignocellulose such as waste biomass or
biomass fractions, such as lignin, have been proposed as biosorbant
materials in the treatment of wastewater to remove heavy metals,
including copper. Cationic metals can sorb to charged anionic
groups such as deprotonated phenolic hydroxyls in lignin or
carboxylate groups in lignin or uronic acids in pectins and
hemicellulose and are known to be strongly affected by pH with more
deprotonated groups at elevated pH.
[0091] In one embodiment, catalyst sorption to biomass is strongly
pH-dependent with near-complete catalyst adsorption to biomass at
alkaline pH and substantial desorption at neutral to acidic pH. In
one embodiment, pH is adjusted to recover the multi ligand metal
complex. In one embodiment, untreated plant biomass is used as an
adsorbent to both recover the catalyst and impregnate the catalyst
into the untreated plant biomass (such as woody biomass, including,
but not limited to, poplar, hybrid polar, and other trees).
[0092] In one embodiment, any conventional method is used to
recover the catalyst from either the unhydrolyzed pretreated
biomass (often referred to as "pretreatment liquor") and/or the
clarified (cell-free) stillage following fermentation and
distillation. Such methods include, but are not limited to
flocculation, precipitation, and filtration using a polyanionic
flocculant (e.g., Betz-Dearborn MR2405 or Ondeo-Nalco 8702) which
is commercially employed to remove heavy metals during wastewater
treatment. Such methods can further include recovery by adsorption
to a commercial ion exchange resin (e.g., Amberlyst.TM. 40Wet)
which is used industrially to recover and recycle copper catalyst
used in the production of adipic acid. In one embodiment, the
catalyst is recovered and recycled.
[0093] In embodiments which include a sugar conversion step,
recovery and reuse of the multi-ligand metal complex provides the
additional benefit of further reducing toxicity during subsequent
sugar conversion steps. Recovering and recycling the multi-ligand
metal complex further helps to reduce costs.
[0094] In one embodiment, the process may produce monomeric
aromatic compounds, such as, syringic acid, vanillin,
syringaldehyde acid, vanillic acid. Such aromatic compounds are
useful in a number of applications, such as food additives, polymer
precursors, and various types of chemicals.
[0095] In one embodiment, the process may produce aliphatic acids,
including, but not limited to formic acid, oxalic acid, acetic
acid, lactic acid, succinic acid, azaleic acid. Such aromatic
compounds are useful in a number of applications, such as food
additives, polymer precursors, and fine chemicals.
[0096] The various embodiments will be further described by
reference to the following examples, which are offered to further
illustrate various embodiments. It should be understood, however,
that many variations and modifications may be made while remaining
within the scope of the various embodiments.
Example 1
Screening with Metal Ligand Complexes
[0097] Screening of various metal ligand complexes (hereinafter
"catalysts") was performed via catalytic alkaline oxidative
pretreatment (AOP) pretreatment of alkali-extracted switchgrass
(AESG). To prepare AESG, 80 grams of untreated switchgrass (Panicum
virgatum, cv. Cave-In-Rock, hammermilled to pass a 5-mm screen, and
stored at a moisture content of approximately 5%) was soaked in an
aqueous solution of 5 g/L NaOH at 80.degree. C. for 2 hrs. The
weight-to-volume (w/v) solids loading of switchgrass during
alkaline extraction was 10% (e.g., 10 g of biomass in 100 mL NaOH
solution). After the alkaline extraction, the residual solid
biomass was recovered via filtration through a 200-mesh steel cloth
and washed with deionized water until the washing effluent had a
neutral pH. The washed solids was recovered as AESG and air-dried
for over 7 days.
[0098] Fe(III)-phthalocyanine (95%), Fe(III)-tetraphenylporphyrin
(.gtoreq.97%) and Fe(III)-tetrakis-pentafluorophenyl)-porphyrin
(.gtoreq.9%) catalysts were purchased from Sigma-Aldrich and used
without further purification.
[Al(III)(3,5-.sup.tBu.sub.2-salophen)Cl]
[3,5-.sup.tBu.sub.2-salophen=N,N'-bis(3,5-di-tert-butylsalicylidene)-1,2--
phenylenediamine], [Zn(II)(BPPA)Cl]Cl
[BPPA=N,N-bis(6-pivaloylamido-2-pyridylmethyl)-N-(2-pyridylmethyl)amine],
and [NEt.sub.4][Fe(III)(bpb)Cl.sub.2]
[H.sub.2(bpb)=N,N-bis(2-pyridinecarboxamide)-1,2-benzene] catalysts
were synthesized according to the methods described in (Gorski et
al. Analytica Chimica Acta 2010, 665, 39; Harata et al. Journal of
Coordination Chemistry 1998, 44, 311; Barnes et al. Journal of
Chemical & Engineering Data 1978, 23, 349; Afshar et al.
Inorganic Chemistry 2006, 45, 10347; Yang et al. Journal of
Molecular Catalysis A: Chemical 2007, 266, 284), which are hereby
incorporated by reference in their entireties, and verified by NMR
(Agilent Direct Drive 2, 500 MHz) and/or UV-Vis spectral analysis
(Hewlett Packard 8453 UV/Vis spectrophotometer). The Cu(bpy)
catalyst was prepared in an aqueous solution by mixing cupric
sulfate pentahydrate (Mallinckrodt, 99.8%) and 2,2'-bipyridine at a
ligand:metal molar ratio (L/M) of 3:1.
[0099] To study the effect of catalytic AOP pretreatment on AESG,
pretreatment was performed in a 100 g/L aqueous suspension of AESG
in the presence of each catalyst. The loading of hydrogen peroxide
(J.T. Baker, ACS reagent, as in an about 30 to about 32% w/v
aqueous solution) on biomass is approximately 10% by weight. The
concentration of each catalyst in the aqueous suspension of AESG is
shown in Table 1. The reaction mixture contained 0.04 M
Na.sub.2HPO.sub.4--NaOH buffer (prepared using disodium phosphate
and sodium hydroxide) to maintain the pH at 11.5 during
pretreatment. After 24 hrs of incubation at 30.degree. C. without
mixing, the pretreated switchgrass slurry was washed using
deionized water to remove the alkaline phosphate buffer.
[0100] The enzymatic digestibility of AOP-pretreated switchgrass
was estimated by the yield of glucose during the enzymatic
hydrolysis of AOP pretreated AESG. The percentage yield was
calculated on the basis of the glucan content in AESG prior to
catalytic AOP pretreatment. The theoretical maximum hydrolysis
yield was lower than 100% due to the loss of sugars in the washing
step between catalytic AOP pretreatment and enzymatic
hydrolysis.
[0101] For enzymatic hydrolysis, 500 .mu.L of 1 M Na-citrate buffer
(pH 5) and 40 .mu.L of 10 mM tetracycline (Sigma-Aldrich) were
added to AOP-AESG that was not dried after catalytic AOP
pretreatment. Accellerase 1500 (42 FPU/mL; Danisco-Genencor, Palo
Alto, Calif.) was added at 50 mg protein per gram of glucan in
AESG. The total volume of the mixture (solid and liquid) was
adjusted with deionized water to reach a 5% (w/v) solids
concentration, and the samples were incubated at 50.degree. C.
during hydrolysis.
[0102] The efficacy of catalysts on AOP pretreatment of AESG is
shown in Table 1. AOP pretreatment of AESG at pH 11.5 was not
improved via addition of metal catalysts, except for the case of Cu
(bpy) catalyst, which gave a very moderate improvement in the
enzymatic digestibility of AOP pretreated AESG. See Table 1
below.
TABLE-US-00001 TABLE 1 Efficacy of ligand-metal catalyst addition
to a 24 hour (hr) AOP Pretreatment of AESG Glucose yield of
enzymatic Catalyst hydrolysis (% of theoretical concentration
maximum) Catalyst (mM) pH 24 hrs 72 hrs No catalyst -- 3 30.06 .+-.
1.96 36.12 .+-. 2.33 9 32.37 .+-. 1.2 37.97 .+-. 0.57 10 31.25 .+-.
0.16 38.77 .+-. 0.28 11 37.74 .+-. 0.33 42.70 .+-. 0.59 13 36.82
.+-. 1.23 N.D. Fe(III)- 0.05 3 30.91 .+-. 0.24 36.80 .+-. 2.84
phthalocyanine 11 34.21 .+-. 0.23 38.75 .+-. 1.52 Fe(III)- 0.05 3
31.55 .+-. 1.6 42.13 .+-. 1.89 tetraphenylporphyrin 11 36.98 .+-.
1.3 42.14 .+-. 0.63 Fe(III)-tetrakis- 0.05 3 29.18 .+-. 0.33 35.38
.+-. 0.43 pentafluorophenyl)- 11 35.37 .+-. 0.45 40.25 .+-. 0.19
porphyrin [NEt.sub.4][Fe(III)(bpb) 2 9 27.07 .+-. 0.6 34.27 .+-.
0.21 Cl.sub.2] 10 25.54 .+-. 0.21 31.97 .+-. 0.13 11 27.44 .+-.
1.54 31.54 .+-. 0.23 [Al(III)(3,5-.sup.tBu.sub.2- 2 10 28.90 .+-.
0.17 N.D. salophen)Cl] 2 11 29.80 .+-. 0.25 N.D. 13 34.19 .+-. 0.41
N.D. 10 26.43 .+-. 0.31 N.D. [Zn.sup.II(BPPA)Cl]Cl 2 11 27.33 .+-.
0.62 N.D. 13 33.36 .+-. 0.3 N.D. 10 38.67 .+-. 0.3 42.98 .+-. 0.88
Cu(bpy), L/M = 3:1 5 11 41.65 .+-. 0.14 44.97 .+-. 0.42 13 41.56
.+-. 0.06 43.95 .+-. 0.17 (N.D.: not determined)
Example 2
Catalyst Screening with Multi-Ligand Metal Complexes
[0103] Metal complexes (hereinafter "metal catalysts") containing
multiple or single metal coordinating ligands were tested. The
metal catalysts tested included copper catalysts having a second
metal coordinating ligand substituted for at least a portion of the
2,2'-bipyridine. These catalysts were evaluated for their
performance in further enhancing the efficacy of AOP pretreatment
of hybrid poplar. Ligands involved in the screening included
histidine, glycine, di(2-picolyl)amine (dipica, 97%),
1,4,7-triazacyclononane (TACN, 95%), 2,2';6',2''-terpyridine
(Terpy, 98%), 1,10-phenanthroline (phen, .gtoreq.99%), and
tris(2-pyridylmethyl)amine (TPA, 98%, obtained from Sigma-Aldrich
and Acros Organics/0). An iron 2,2'-bipyridine complex was prepared
by mixing ferrous sulfate heptahydrate with 2,2'-bipyridine in an
aqueous solution, and this iron complex was also tested.
[0104] The hybrid poplar (Populus nigra var.
charkoviensis.times.caudina cv. NE-19) used in this testing was
grown at the University of Wisconsin Arlington Agricultural
Research Station. The hybrid poplar was harvested in 2011 and
debarked using methods known in the art at the University of
Wisconsin, Madison to produce heartwood and sap wood (different
parts of the wood stem which can be differentiated via chemical
testing).
[0105] The enzymatic digestibility of hybrid poplar after
pretreatment with AOP and metal complexes involving above mentioned
ligands was used to evaluate the performance of various
multi-ligand metal complexes (See FIG. 1). As these results show,
various types of multi-ligand metal complexes, as well as a
single-ligand iron complex are capable of enhancing oxidative
pretreatment of hybrid poplar. However, Cu(bpy) complexes
demonstrated superior performance among all candidates, and was
selected for further testing.
Example 3
Alkaline Extraction, Catalytic Pretreatment and Enzymatic
Hydrolysis of Hybrid Poplar
[0106] About 0.5 g of hybrid poplar, i.e., poplar (from the same
source as described in Example 2) was extracted in a temperature
controlled rotary shaker (New Brunswick Scientific Classic Series
C24KC, 210 rpm) at 30.degree. C. in a 5 mL aqueous solution
containing 270 mM NaOH (J.T. Baker, ACS reagent, 98.8%) for
approximately 1 hr to produce extracted wet poplar. The extracted
wet poplar was washed with 5 mL deionized water and centrifuged
(Thermo Scientific Sorvall ST-16R) at 1000 rpm for 1 min to
separate the solid and liquid phases. (The alkaline liquid from the
washing was used in later examples for the extraction step).
[0107] The separated solid phase of the extracted wet poplar was
then subjected to a catalytic AOP pretreatment in a 5 mL aqueous
solution containing 10.8 g/L NaOH, 10 g/L hydrogen peroxide and a
conventional single-ligand copper diimine catalyst at varying
concentrations of copper (0.5, 1, 2, 3, 4, and 5 mM) and varying
concentrations of 2,2'-bipyridine (1, 2, 4, 6, 8, 10, 15 and 20 mM)
to produce incubated catalyzed pretreated (solid phase) poplar
(hereinafter "solid poplar slurry"). The results presented herein
are for concentrations of 2 mM of copper and 4 mM of
2,2'-bipyridine.
[0108] Enzymatic hydrolysis was performed on the solid poplar
slurry at 50.degree. C. in the same rotary shaker used for the
extraction step. In preparation for hydrolysis, 20 .mu.L of 72%
(w/w) H.sub.2SO.sub.4 and 500 .mu.L of 1 M sodium citrate buffer
(pH 5) (prepared from sodium citrate and citric acid) were added to
the solid poplar slurry to adjust the pH to 5 (optimum pH for the
enzymes being used herein). Thereafter, 40 .mu.L of 10 mM
tetracycline (Sigma-Aldrich) stock solution was added to the solid
poplar slurry to inhibit microbial growth. This step was followed
by addition of a Cellic CTec2 and HTec2 (Novozymes A/S, Bagsv.ae
butted.rd, DK) enzyme cocktail at a loading of 30 mg protein/g
glucan in the biomass prior to extraction and catalytic
pretreatment, unless otherwise noted. The enzymatic reaction
(50.degree. C., 210 rpm, 72 hrs) was carried out at 5% solids
concentration by adjusting the volume of the poplar slurry to 10 mL
via the addition of deionized water.
[0109] Following enzymatic hydrolysis, the resulting solid and
liquid phases were separated by centrifugation in same manner as in
Example 2, to produce separated hydrolyzed solid and liquid phases.
The amount of glucose and xylose released into the aqueous phase
was quantified by High Performance Liquid Chromatography (HPLC)
(Agilent 1260 Series equipped with an Aminex.RTM. HPX-87H column
operating at 65.degree. C., a mobile phase of 0.05 M
H.sub.2SO.sub.4, and a flow rate of 0.6 mL per minute, and
detection by refractive index). The yield of glucose and xylose
released was defined as the amount of solubilized monosaccharide
divided by the total sugar content of the poplar biomass prior to
incubation and catalytic pretreatment as determined by chemical
composition analysis.
[0110] After the enzymatic hydrolysis of the solid poplar slurry,
over 80% of the polysaccharides (by weight) in the poplar biomass
were converted to fermentable sugars. This sugar yield was
significantly higher than the sugar yield produced using untreated
raw hybrid poplar. (See FIG. 2).
Example 4
Recovery of Copper Catalyst, and Catalyst Impregnation During
Alkaline Extraction
[0111] About 40 g hybrid poplar (same source as Example 2) was
catalytically pretreated with 400 mL of aqueous solution containing
2 mM copper sulfate, 4 mM 2,2'-bipyridine, 270 mM NaOH and 10%
(w/w) hydrogen peroxide loading on biomass at 30.degree. C. for 24
hrs to produce catalytically pretreated poplar slurry (hereinafter
"poplar slurry"). Following the pretreatment, a copper removal step
was performed to remove part or all of the copper present in the
poplar slurry for use again in the process.
[0112] The copper removal step used in this testing was a
desorption step in which the pH of the poplar slurry was adjusted
to 5 with 1.6 mL of 72% sulfuric acid (prepared via dilution of 98%
sulfuric acid, J.T. Baker) to produce a pH-adjusted poplar slurry.
The pH-adjusted poplar slurry was then incubated for approximately
1 hr in the same rotary shaker as Example 2 at 30.degree. C., with
120 rpm shaking speed to produce pH-adjusted poplar slurry. After
incubation, the residual solid poplar was separated from the
aqueous phase via centrifugation in the same manner as described in
Example 3. The separated solids were then converted to sugars via
enzymatic hydrolysis following the procedures described in Example
3.
[0113] The separated aqueous phase or pretreatment liquor (PTL)
produced following centrifugation containing the copper catalyst
(containing about 0.9 mM of coordinated copper complexes) was
collected and used again in another catalytic AOP pretreatment. In
this testing, about 0.5 g of hybrid poplar (same source as in
Example 2) was incubated in the rotary shaker (120 rpm) with a
mixture of 4.73 mL of the previously collected aqueous phase and
270 .mu.L of 5 M NaOH (J.T. Baker, ACS reagent, 98.8%) at
30.degree. C. for about 1 hr to produce a catalyst-impregnated wet
poplar slurry. The catalyst-impregnated wet poplar slurry was
thereafter washed with 5 mL deionized water and centrifuged in the
same manner as Example 2 to separate the solid and liquid
phases.
[0114] After another centrifugal solid-liquid separation (performed
in same manner as described in Example 3), the separated solid
phase of the incubated wet poplar biomass was catalytically
pretreated with 5 mL aqueous solution containing 2 mM
2,2'-bipyridine, 270 mM NaOH and hydrogen peroxide (sources of
chemicals used are as described in Example 3) at 30.degree. C. for
approximately 24 hrs. After the catalytic pretreatment, the biomass
slurry was hydrolyzed by enzymes to obtain fermentable sugars at
high yields (.gtoreq.80% of the theoretical maximum). See FIG.
3.
Example 5
Production of Aromatic Chemicals Via Catalytic Oxidative
Pretreatment
[0115] About 0.5 g hybrid poplar (same source as Example 2) was
catalytically pretreated with 5 mL of an aqueous solution
containing 2 mM copper sulfate, 4 mM 2,2'-bipyridine, 270 mM NaOH
and 10% (w/w) hydrogen peroxide loading on biomass at 30.degree. C.
for approximately 24 hrs to produce a catalytically pretreated
poplar slurry (sources of chemicals used are as described in
Example 3). The solid and liquid phases were separated as described
in the above examples.
[0116] For LC-MS analysis of the monomeric aromatic compound
content, 10 .mu.L of undiluted pretreatment liquor sample were
injected into a XEVO G2SQTOF mass spectrometer in combination with
a Waters Acquity.RTM. Ultra Performance Liquid Chromatograph (UPLC)
system, which was equipped with an ESI interface capable of
operating in both positive- and negative-ion modes. Chromatographic
separation was carried out on a Thermo BetaBasic 100.times.2.1 mm
C18 column (Thermo Fisher Scientific, Waltham, Mass., USA)
maintained at 40.degree. C. The binary solvent gradient comprised
0.1% formic acid in water (solvent A) and 100% methanol (solvent B)
in the following gradient: 95% solvent A for the first 3 min, 50%
solvent A over the next 1 min, 30% solvent A over next 2 min, and
5% solvent A over the final 2 min. The column was then returned to
95% solvent A, and equilibrated for 2 min prior to the next
injection. A solvent delay of 2 min was used to prevent saturation
of the detector with the sample solvent. The negative-ion mode mass
spectrometry conditions were constant during all experiments with a
voltage of -2.25 kV and a desolvation temperature of 350.degree.
C.
[0117] MassLynx.TM. software (Waters) version 4.1 was used for
system control and data acquisition. The raw data was processed
using the TargetLynx.TM. application. Pure standards for vanillin,
vanillic acid, acetovanillone, syringaldehyde, syringic acid,
acetosyringone, and p-hydroxybenzoate (Sigma-Aldrich, St. Louis,
Mo., USA) were used to validate peak compound identification and
for quantitation.
[0118] For quantitation of p-hydroxybenzoate, samples were prepared
following the same procedure used for the LC-MS analysis, with the
samples instead analyzed via high-performance liquid chromatography
(HPLC) (Agilent 1260 LC equipped with an Agilent Poroshell 120
EC-C18 column (4.6.times.50 mm) and a diode array detector (DAD).
Integration of the p-hydroxybenzoate peak at 280 nm and comparison
to a standard curve was used for quantitation. A binary isocratic
solvent system was utilized consisting of 80:20 solvent C to
solvent D, where solvent C is acetonitrile with 0.1% water, and
solvent D is acetonitrile with 0.1% trifluoroacetic acid. The
results are shown in FIGS. 4A and 4B.
Example 6
Catalytic Pretreatment of Hybrid Poplar Biomass Using Copper (II)
2,2'-Bipyridine Ethylenediamine (Cu(bpy)en)
[0119] Hybrid poplar (0.5 g) (same source as Example 2) was
catalytically pretreated using 1 mM of a stock solution of
multi-ligand copper complex, namely copper (II) 2,2' bipyridine
ethylenediamine (Cu(bpy)en) containing 40 mM copper sulphate
pentahydrate, 40 mM bipyridine and 40 mM ethylenediamine in
deionized water, together with 270 mM NaOH and 10% H.sub.2O.sub.2
loading (w/w) on biomass in a total 5 mL volume made with deionized
water. Other components of the pretreatment catalyst include
tetraacetylethylenediamine (TAED), ethylenediaminetetraacetic acid
(EDTA), manganese sulfate, and magnesium sulfate. The catalytic
pretreatment was allowed to proceed for approximately 24 hrs at
30.degree. C.
[0120] Thereafter, the resulting poplar slurry was pH adjusted to 5
and hydrolyzed according to the process described in Example 3.
Following enzymatic hydrolysis, the solid and liquid phases were
separated by centrifugation as described in Example 3.
[0121] Among all catalyst systems tested, Cu (bpy)en yielded higher
glucose yields compared to other catalyst systems tested. Also, by
utilizing 1/2 the concentration of copper and 1/4 the concentration
of (bpy), as compared with a conventional Cu(bpy) catalyst, the
overall cost is reduced. See Table 2 below.
TABLE-US-00002 TABLE 2 Catalyst and Glucose Conversions Catalyst
Glucose Cu(bpy).sub.2 55.76 Cu(bpy)TAED 55.01 Cu(bpy)en (1 mM)
55.00 Cu(bpy)en (2 mM) 48.15 Cu(bpy)MnSO.sub.4 46.78 CuSO.sub.4
40.62 Mn(TAED).sub.2 38.65 Cu(en).sub.2 34.22 Cu(TAED).sub.2 30.72
MgSO.sub.4 25.17 Mg(bpy) 25.09 Mn(en).sub.2 24.08 Mn(EDTA).sub.2
22.28 Mn(bpy).sub.2 21.21 Control 11.34
Example 7
Copper Recovery, Recycling and Extractives Removal Following
Catalytic AOP Pretreatment
Catalytic AOP Pretreatment
[0122] In one set of tests, 40 g hybrid poplar was catalytically
pretreated at 10% biomass solids content (w/vol or "solids
loading") with a reaction mixture containing 2 mM copper sulfate
pentahydrate, 4 mM 2,2'-bipyridine, 270 mM NaOH and 10% (w/w)
H.sub.2O.sub.2 loading on biomass at 30.degree. C., pH 11.5 for 24
hrs to produce pretreated poplar. Desorption of adsorbed copper
from pre-treated poplar was carried out by reducing the pH of the
pretreated poplar biomass to a pH of 5 by adding 1.6 mL of 72%
H.sub.2SO.sub.4. The reaction mixture was catalytically pretreated
in the same rotary shaker described above at 30.degree. C., 120 rpm
for 1 hr. After catalytic pretreatment, the resulting pretreated
poplar slurry was centrifuged to separate the pretreated poplar
from the aqueous pretreatment liquor (PTL), i.e., the aqueous phase
collected following catalytic pretreatment. The PTL was collected
to use for subsequent pretreatment step. One (1) mL of sample was
analyzed with inductively coupled plasma mass spectrometry (ICP-MS;
Thermoscientific iCAP 6500 ICP) to determine the concentration of
copper in the PTL, which was found to be about 0.9 mM.
Pretreatment Using the PTL
[0123] PTL (4.33 mL) was mixed with 0.5 g hybrid poplar (10% solids
loading) with copper catalyst (Copper:bipyridine) of varying
ratios. NaOH (270 mM) and H.sub.2O.sub.2 (10% loading on biomass)
were added to the reaction mixture and the pretreatment lasts for
24 hrs at 30.degree. C. After incubation, the biomass slurry was
enzymatically hydrolyzed to yield monomeric sugars.
[0124] Due to the presence of 50% of originally added copper in the
PTL, the slurry was loaded with different copper (.ltoreq.1 mM) and
bipyridine (.ltoreq.2 mM) concentrations to match the
concentrations with conventional AHP pretreatment (Copper 2 mM and
bipyridine 4 mM). Results demonstrated that there were 50% less
yields as compared to an AOP pretreatment in which a single ligand
was used. The same yields were also obtained for different extra
loadings of Cu and Bpy as obtained with just PTL (FIGS. 4A and
4B).
[0125] These results suggest that inhibition is occurring, which
could be due to the presence of extra free copper during enzymatic
hydrolysis.
[0126] This testing shows that using the catalyst in PTL for
oxidative pretreatment improves the enzymatic digestibility of
pretreated biomass by about 5% (FIG. 5). As demonstrated in other
testing described in Example 8, supplementing more catalyst in
addition to PTL does not improve the efficacy of the catalytic
oxidative pretreatment.
Example 8
[0127] Other approaches beyond the approach described in the above
examples were also used to pretreat the poplar biomass utilizing
PTL, with the preferred approach determined on the basis of higher
sugar yields after enzymatic hydrolysis.
[0128] In a second approach, PTL was added to the 0.5 g hybrid
poplar biomass (10% solids loading on biomass by weight) with or
without 2 mM bipyridine. NaOH (270 mM) and H.sub.2O.sub.2 (10%
loading on biomass by weight) were added to the reaction mixture to
carry out a catalytic pretreatment for 24 hrs at 30.degree. C. to
produce a pretreated poplar slurry which was washed using 5 mL
distilled water. The washed pretreated poplar slurry was
centrifuged in same manner as described in earlier Examples to
separate the solid and liquid phases. The solid phase was
enzymatically hydrolyzed to obtain the sugar yields.
[0129] In a third approach, 0.5 g of hybrid poplar (10% solids
loading on biomass by weight) was catalytically pretreated with
4.73 mL of PTL and 270 mM NaOH at 30.degree. C., 120 rpm for 1 hr
to produce pretreated poplar slurry. The pretreated poplar slurry
was washed with 5 mL of distilled water and centrifuged to separate
the copper-containing aqueous phase from the solid phase. The solid
phase (containing an amount of adsorbed copper) was subject to an
additional incubation with 2 mM bipyridine, 270 mM NaOH and 10%
H.sub.2O.sub.2 at 30.degree. C., pH-11.5 for 24 hrs. Thereafter,
the incubated pretreated poplar slurry was enzymatically hydrolyzed
to yield monomeric sugars.
[0130] No additional copper was added in the pretreatment utilizing
these different approaches. Control experiments were run to ensure
the recovery and adsorption of copper into the new biomass.
Extractives Removal and Alkali Recovery Experiment
[0131] On the basis of results obtained from above defined
approaches, a two-step pre-treatment process was designed. As such,
0.5 g of hybrid poplar biomass (10% w/v insoluble solids
concentration) was pre-incubated with 270 mM NaOH at 30.degree. C.
for 1 hr. After incubation, contents of the reaction were washed
with 5 mL water and centrifuged to separate solid and liquid
phases. Solid phase, i.e., alkali pre-extracted biomass was
subjected to catalytic AOP pretreatment, while the liquid phase was
recovered in the same manner as described in previous examples. The
pH of all the pretreatment reactions were carefully monitored and
documented.
Enzymatic hydrolysis
[0132] Enzymatic hydrolysis was performed at 50.degree. C. in a
temperature controlled incubator shaker at 210 rpm for 72 hrs.
After pretreatment, 20 .mu.L of 72% (w/w) H.sub.2SO.sub.4 and 500
.mu.L of 1 M sodium citrate buffer (pH 5.0) were added to the
pretreated slurry to adjust the pH to 5 (optimum pH for enzymes).
Next, 40 .mu.L of 10 mM tetracycline(Sigma-Aldrich) stock solution
was added to inhibit microbial growth, followed by addition of the
enzyme cocktail consisting of Cellic CTec2 and HTec2(Novozymes A/S,
Bagsv.ae butted.rd, DK) at a loading of 30 mg protein/g glucan each
on the untreated biomass unless otherwise noted.
[0133] The enzymatic reaction was carried out at 5% solids loading
by adjusting the volume to 10 mL by the addition of deionized
water. Following enzymatic hydrolysis, the solid and liquid phases
were separated by centrifugation, and the amount of glucose and
xylose released into the aqueous phase was quantified by HPLC
(Agilent 1100 Series equipped with an Aminex.RTM. HPX-87H column
operating at 65.degree. C., a mobile phase of 0.05 M
H.sub.2SO.sub.4, a flow rate of 0.6 mL/min, and detection by
refractive index). The yield of glucose and xylose released was
defined as the amount of solubilized monosaccharide divided by the
total sugar content of the biomass prior to pretreatment as
determined by chemical composition analysis.
[0134] ICP-MS metal analysis results showed that PTL contained 0.9
mM copper which was approximately 50% of copper concentration (2
mM) originally added during the pretreatment.
Results
[0135] Results of the second approach demonstrated that there was
an increase in the glucose yields compared to the yields obtained
from the first approach described in the other examples. Addition
of extra bipyridine further increased the yields. There was 6 to 8%
increase in the glucose yield whereas lower xylose yield was
obtained. Low xylose yields could be explained as due to washing
step after pretreatment, hemicellulose was also removed from the
reaction mixture.
[0136] No extra copper was added in these experiments. Yields
obtained were purely from recycled copper.
[0137] These yields matched the yields achieved with conventional
AOP pretreatment, i.e., one-step catalytic AOP with no extraction,
incubation or impregnation beforehand. Increased yields were
obtained with addition of bipyridine in the form of a
bipyridine-copper complex.
[0138] The third approach (described above) produced the highest
sugar yields as compared to the other approaches. The glucose
yields obtained from this approach were approximately 28% higher
compared to conventional catalytic AOP. Addition of only PTL to
biomass without any addition of bipyridine yielded 7% higher
glucose yields compared to conventional catalytic AOP.
[0139] No extra copper was added in these experiments. Yields
obtained were purely from recycled copper.
[0140] These results show that extractives from PTL can inhibit the
performance of catalytic AOP by affecting the pH of the reaction
and/or by interfering with useful reactions. It is also possible
that enzymatic hydrolysis was inhibited due to the presence of
extractives.
[0141] Overall results demonstrated the ability to recycle the
copper and increase the glucose yields by about 82% to about 85%
and xylose yields by about 55% to about 58%.
Modification of Catalytic AOP
[0142] On the basis of results obtained from copper recycling and
extractives removal, a two-step process was developed, with the
first step removing extractives and alkali-extractable lignin and
xylan with NaOH and the second step consisting of catalytic
AOP.
[0143] Sugar yields obtained from modified catalytic AOP were about
28 to about 30% higher than sugar yields obtained using
conventional single-ligand catalytic AOP. Different control
experiments were also carried out to ensure that the yields
obtained are due to extractives removal, see FIG. 6.
[0144] See also improved sugar yields with pre-extraction step
shown in FIG. 7.
Example 9
Recycling of Base for Alkaline Extraction
[0145] Alkaline extraction was performed following the procedure
described in Example 3. Hybrid poplar (from the same source as in
previous examples) was extracted with 135 mM or 270 mM aqueous
solution of NaOH, and the extracted biomass was then washed with
deionized water. The liquid from washing was recovered. The washed
solids were pretreated with 10.8 g/L NaOH, 10 g/L H.sub.2O.sub.2, 2
mM CuSO.sub.4 and 4 mM 2,2'-bipyridine. The pretreated solids was
enzymatically hydrolyzed following the procedure described in
Example 3. Over 80% of the glucose was recovered after enzymatic
hydrolysis (FIG. 7).
[0146] The liquid from washing ("Recycled NaOH") was used for
alkaline extraction of hybrid poplar, following the procedure
described in Example 3. After 1 hour of extraction at 30.degree.
C., the mixture was washed with deionized water, and the remaining
solids was pretreated with 10.8 g/L NaOH, 10 g/L H.sub.2O.sub.2, 2
mM CuSO.sub.4 and 4 mM 2,2'-bipyridine. The pretreated solids were
enzymatically hydrolyzed following the procedure described in
Example 3. More than 60% of the glucose was recovered after
enzymatic hydrolysis (FIG. 8).
[0147] Compared to conventional single-ligand catalytic AOP,
consumption of alkali was doubled in this modified catalytic AOP.
To be more cost effective, experiments were performed to recycle
the alkali from pre-extraction step or use less alkali in the
respective step.
Results
[0148] Sugar yields obtained using less alkali, i.e., 135 mM NaOH,
were comparable to the yields obtained from the 270 mM NaOH
pre-extraction step. The NaOH was also partially recycled. Yields
obtained from the recycled 270 mM NaOH reaction were about 66%
glucose and 85% xylose. These yields are higher than yields
obtained with the conventional single-ligand catalytic AOP process
(FIG. 8).
Example 10
Biomass, Pretreatment, and Hydrolysis
[0149] Hybrid poplar (Populus nigra var.
charkoviensis.times.caudina cv. NE-19) was grown at the University
of Wisconsin Arlington Agricultural Research Station. Prior to
pretreatment, a mixture of heartwood and sapwood of the 18-year-old
poplar was hammer-milled to pass a 5 mm screen. See, for example,
Li Z, Chen C H, Liu T, Mathrubootham V, Hegg E L, Hodge D B:
Catalysis with Cu.sup.II(bpy) improves alkaline hydrogen peroxide
pretreatment. Biotechnol Bioeng 2013, 110:1078-1086.37 and Li Z,
Chen C, Hegg E, Hodge D: Rapid and effective oxidative pretreatment
of woody biomass at mild reaction conditions and low oxidant
loadings. Biotechnol Biofuel 2013, 6:119, for additional details as
to compositional analysis and pretreatment specifics. For AOP-only
pretreatment, hybrid poplar (0.5 g) was pretreated in 5 mL aqueous
aliquots of 10 g/L H.sub.2O.sub.2 (10% w/w loading on biomass) and
10.8 g/L NaOH (final pH of approximately 11.7) at 30.degree. C. for
1 hr unless otherwise noted. During the pretreatment, the samples
were agitated at 180 rpm in an orbital shaker. For Cu-catalyzed AOP
pretreatments, 5 mM CuSO.sub.4 and 25 mM 2,2'-bipyridine were
included in the 5 mL aliquot during pretreatment. For alkali-only
(AOP-only) pretreatment, 0.5 g of hybrid poplar was pretreated in 5
mL aqueous aliquots of 10.8 g/L NaOH.
TEM Imaging and Elemental Profiling of Pretreated Cell Walls
[0150] Structural modification of hybrid poplar cell wall by
pretreatment was studied using transmission electron microscope
(TEM) combined with energy-dispersive X-ray spectroscopy (EDS) and
electron energy-loss spectroscopy (EELS). The conditions used for
pretreatment were identical to those used to prepare size exclusion
chromatography (SEC) samples. Cell wall samples of untreated hybrid
poplar and hybrid poplar treated with AOP and Cu-catalyzed AOP for
24 hr were air dried and fixed (i.e., embedded) in a resin
comprising 0.1 M pH 7 phosphate buffer containing 2.5% (w/w)
glutaraldehyde and 2.5% (w/w) paraformaldehyde. The fixed cell wall
samples were embedded in Spun epoxy resin (Poly/Bed 812,
Polysciences) and sectioned to 100 nm thickness using a PowerTome
XL ultramicrotome (Boeckeler Instruments, Tucson, Ariz., USA). Thin
sections were placed on 150 mesh gold grids with Formvar/carbon
support film (Electron Microscopy Sciences, PA) and stained in 1%
aqueous solution of KMnO.sub.4 for 60 sec. Samples were then rinsed
with deionized water to remove excess stain. Bright field TEM
micrographs and EELS spectra were acquired under a JEOL 2200FS 200
kV field emission TEM (Peabody, Mass., USA) fitted with a Gatan
(Warrendale, Pa., USA) digital multi-scan camera. EDS spectra were
acquired using an Oxford INCA system (Oxford Instruments, Abington,
UK) coupled with the TEM.
Analysis or Pretreatment Liquors
[0151] For analysis by SEC or LC-MS, pretreatment liquors at
alkaline pH were filtered through a 0.22 .mu.m mixed cellulose
ester membrane filter (EMD Millipore, Billerica, Mass.). SEC
analysis was performed using an Agilent 1100 HPLC equipped with an
Ultrahydrogel 250 column (Waters, Milford, Mass., USA) as described
in Stoklosa R J, Hodge D B: Extraction, recovery, and
characterization of hardwood and grass hemicelluloses for
integration into biorefining processes. Ind Eng Chem Res 2012,
51:11045-11053. Aqueous solutions of monodisperse sodium
polystyrene sulfonate (Sigma-Aldrich, St. Louis, Mo., USA) of known
molar mass (2000, 4300, 6800, 10000, 32000, and 77000 Da) were used
as calibration standards.
[0152] Samples for Liquid Chromatography-Mass Spectrometry (LC-MS)
analysis were prepared as described above for SEC analysis, except
that the concentrations of CuSO4 and 2,2'-bipyridine during
pretreatment were 2 mM and 4 mM, respectively. For LC-MS analysis,
10 .mu.L of undiluted pretreatment liquor sample were injected into
a XEVO G2SQTOF mass spectrometer in combination with a Waters
Acquity.RTM. UPLC system and equipped with an Electrospray
Ionization (ESI) interface capable of operating in both positive-
and negative-ion modes. Chromatographic separation was carried out
on a Thermo Beta Basic 100.times.2.1 mm C18 column (Thermo Fisher
Scientific, Waltham, Mass., USA) maintained at 40.degree. C. The
binary solvent gradient consisted of 0.1% formic acid in water
(solvent A) and 100% methanol (solvent B) in the following
gradient: 95% solvent A for the first 3 min, 50% solvent A over the
next 1 min, 30% solvent A over next 2 min, and 5% solvent A over
the final 2 min. The column was then returned to 95% solvent A, and
equilibrated for 2 min prior to the next injection. A solvent delay
of 2 min was used to prevent saturation of the detector with the
sample solvent.
[0153] The negative-ion mode mass spectrometry conditions were
constant during all experiments with a voltage of -2.25 kV and a
desolvation temperature of 350.degree. C. Mass Lynx software
(Waters) version 4.1 was used for system control and data
acquisition. The raw data acquired were processed using the Target
Lynx application. Pure standards for vanillin, vanillic acid,
acetovanillone, syringaldehyde, syringic acid, acetosyringone, and
p-hydroxybenzoate (Sigma-Aldrich, St. Louis, Mo., USA) were used to
validate peak compound identification and for quantitation.
[0154] For quantitation of p-hydroxybenzoate, samples were prepared
following the same procedure as that for the LC-MS analysis, but
the samples were then analyzed via high-performance liquid
chromatography (Agilent 1260 LC equipped with an Agilent Poroshell
120 EC-C18 column (4.6.times.50 mm) and a diode array detector
(DAD). Integration of the p-hydroxybenzoate peak at 280 nm and
comparison to a standard curve was used for quantitation. A binary
isocratic solvent system was utilized consisting of 80:20 solvent C
to solvent D, where solvent C is acetonitrile with 0.1% water, and
solvent D is acetonitrile with 0.1% trifluoroacetic acid.
NMR Analysis of Whole Cell Walls and Pretreatment-Solubilized
Lignin
[0155] Following the pretreatment, the aqueous phase was separated
from the solid phase (i.e., the insoluble portion of pretreated
poplar) via filtration, and the filtrate was acidified to pH 2.0
with 72% (w/w) sulfuric acid. The precipitate from the acidified
filtrate was recovered via centrifugation and washed with a large
volume of aqueous sulfuric acid (pH 2.0) followed by a final
washing step of re-suspending and decanting the lignin sample in
pH-neutral deionized water. The washed lignin precipitate was
lyophilized prior to NMR analyses. The 2D HSQC NMR spectra of three
types of samples (untreated hybrid poplar, recovered solubilized
lignins and the insoluble portion of pretreated poplar) were
acquired and analyzed according to the methods described in Kim H,
Ralph J, Akiyama T: Solution-state 2D NMR of ball-milled plant cell
wall gels in DMSO-d.sub.6. Bioenerg Res 2008, 1:56-66 (hereinafter
"Kim 2008") Untreated and pretreated samples were prepared for
gel-state NMR as y described in Kim (2008).
[0156] In brief, the dried sample was pre-ground for 1 min in a
Retsch MM400 mixer mill at 30 Hz, using zirconium dioxide (ZrO2)
vessels (10 mL) containing ZrO.sub.2 ball bearings (2.times.10 mm).
The ground material was extracted with distilled water (1 hr, 3
times) and 80% of ethanol (1 hr, 3 times) with ultrasonication. The
cell walls were dried and finely ball-milled using a PULVERISETTE 7
(Fritsch, Idar-Oberstein, Germany) mill at 600 rpm with ZrO.sub.2
vessels (50 mL) containing with ZrO.sub.2 ball bearings
(10.times.10 mm). Each sample (200 mg) was milled for 1 hr, 40 min
in 10 min intervals with 5 min interval breaks. The ball-milled
samples (50 mg of each) were transferred into 5-mm NMR tubes and
gels formed using DMSO-d.sub.6/pyridine-d.sub.5 (4:1, v/v, 0.5 mL)
with sonication (30 min). NMR spectra were acquired on a Bruker
BioSpin (Billerica, Mass., USA) AVANCE 700 MHz spectrometer
equipped with a cryogenically-cooled 5-mm triple-resonance
1H/13C/15N TXI gradient probe with inverse geometry (1H coils
closest to the sample).
[0157] The central DMSO solvent peak was used as an internal
reference (.delta..sub.C 39.5, .delta..sub.H 2.49 ppm). The
.sup.1H-.sup.13C correlation experiment was an adiabatic HSQC
experiment (Bruker standard pulse sequence `hsqcetgpsisp.2`;
phase-sensitive gradient-edited 2D HSQC using adiabatic pulses for
inversion and refocusing). HSQC experiments were carried out using
the following parameters: acquired from 9 to 1 ppm in F2 (1H) with
1,200 data points (acquisition time 200 ms), 160 to 10 ppm in F1
(13C) with 512 increments (F1 acquisition time 13.6 ms) of 32 scans
with a one-second interscan delay; the d24 delay was set to 0.86 ms
(1/8J, J=145 Hz).
[0158] Volume integration of contours in HSQC plots used Bruker's
TopSpin 3.1 (Mac) software. Assignments of peaks from NMR spectra
were based on Kim 2008 and Kim H, Ralph J: Solution-state 2D NMR of
ball-milled plant cell wall gels in DMSO-d6/pyridine-d5. Org Biomol
Chem 2010, 8:576-591. TEM imaging was used to characterize corn
stover cell wall structural changes associated with pretreatments
by dilute acid, and anhydrous ammonia, as well as acid chlorite
delignification. The TEM micrographs of untreated hybrid poplar
(FIG. 9A) and AOP-only pretreated hybrid poplar (FIGS. 9B and 9C)
showed identifiable features of the cell walls comprising wall
layers that include secondary cell wall layers (FIGS. 13A-13F and
FIG. 15) and the compound middle lamella (CML), as well as cell
corners (CC) and individual lumens. Following AOP-only pretreatment
(FIGS. 9A and 9B), the cell walls retained much of their structural
integrity, as seen by the similarities to those of untreated cell
walls. The only notable changes were dislocations which formed
between the middle lamellae and the primary cell walls, possibly
due to removal of some lignin and pectic polysaccharides during
pretreatment. The dark black stripes observed in the micrographs
are artifacts introduced during ultramicrotome sectioning and
KMnO.sub.4 staining.
[0159] Addition of Cu(bpy) complexes during AOP pretreatment of
hybrid poplar improved delignification and enzymatic hydrolysis
yields. TEM characterizations of hybrid poplar cell wall after
Cu-catalyzed AOP pretreatment are shown in FIGS. 10A-10D, with FIG.
10A showing delamination, FIG. 10B showing dislocations of cell
wall layers along, and FIGS. 10C and 10D showing the accumulation
of nanoparticles in disrupted regions. "S1" and "S2" indicate
different layers of the secondary cell wall; "CC" refers to a cell
corner and "CML" refers to the compound middle lamella. As FIGS.
10A-10D show, significant cell wall structural changes occurred as
a result of Cu(bpy)-catalyzed AOP pretreatment. See, for example,
the major dislocations in the cell wall in FIG. 10A, together with
formation of fractures in which the secondary cell wall layers were
perturbed. Fractures and disruptions were also observed in other
lignin-rich regions, including cell corners (FIG. 10A) and compound
middle lamellae (FIG. 10B), suggesting that the structural changes
may be caused by lignin modification and/or removal.
[0160] The testing described in this example was performed at
temperatures ranging from about 25 to about 30.degree. C., which is
well below the lignin glass transition (which is about 100 to about
170.degree. C.). As a consequence, the lignin removal which
occurred is primarily due to chemical modification and/or solvent
effects rather than thermal effects. Additionally, and as shown in
FIGS. 10C and 10D, X-ray-opaque particles with diameters in the
range of about 20 to about 100 nm were often co-localized with the
modified regions of cell walls. Interestingly, such particles were
not found in untreated hybrid poplar (FIG. 9A) or AOP-only
pretreated poplar (FIGS. 9B and 9C-). As such, the development of
such particles can be hypothesized to originate from the copper
catalyst, rather than as artifacts introduced during the TEM sample
preparation. This suggests that copper is involved in the observed
cell wall modification (e.g., via lignin oxidation).
Elemental Profiling of Pretreated Cell Walls
[0161] When combined with electron microscopy, in situ elemental
profiling using energy-dispersive X-ray spectroscopy (EDS) and
electron energy-loss spectroscopy (EELS) can provide chemical
characterization at a spatial resolution on the order of 100 nm. To
characterize the elemental composition of the nano-scale particles
observed in the TEM images, EDS spectra were acquired at different
locations in a TEM sample (FIG. 11A), including at a cell corner
(identified as area "i" in FIG. 11A), within a secondary cell wall
(area "ii"), and over the previously described particles (areas
"iii" and "iv").
[0162] TEM images at high magnification show that these particles
are aggregates with dendritic structures and diameters on the order
of 50 nm (FIG. 11B). A comparison of the EDS spectra reveals both
similarities and differences in elemental composition of the cell
wall regions (FIGS. 11C-11F). Mn peaks are apparent in all four
spectra and are a consequence of the permanganate staining, whereas
the Au peaks correspond to the X-ray emissions from the grid that
supports the TEM sample. The EDS spectrum from the cell corner
(area "i") shows a strong Ca L-edge peak indicating the presence of
Ca ions, which are known to complex with pectin. Ca K-edge peaks
(3.7 keV) are also present at a lower relative abundance in the
spectra of the other cell areas. In the areas of "ii" and "iii"
where X-ray-opaque particles were analyzed, the EDS spectra feature
characteristic peaks for Cu. The Cu L-edge and K-edge peaks are not
seen in the EDS spectra of either the cell corner (area "i") or the
secondary cell wall (area "ii"). EELS was used to identify the
oxidation state of the Cu-containing particles (FIG. 15), which
shows the spectrum of a Cu-containing particle with the pre-edge
background subtracted. The white-line intensity (i.e., the sharp
threshold peaks) of the Cu L2,3 edge indicates that the majority of
the Cu is in the Cu(I) oxidation state, while the relatively low
white-line intensity of L3 implies that Cu(0) is also present.
[0163] The identification of Cu-containing particles suggests that
the Cu catalyst is localized in the cell wall matrix at sites
corresponding to those with significant structural modification.
This result suggests that the soluble Cu catalysts diffuse into the
porous cell wall matrix during pretreatment and subsequently
catalyze the formation of localized reactive oxygen species that
may be involved in the oxidative delignification and structural
modification of the cell wall in their vicinity. Although the
active catalytic complexes have not yet been identified, one
possibility is that the oxidation reactions were accelerated by Cu
complexes that catalyzed the decomposition of H.sub.2O.sub.2 and/or
activated H.sub.2O.sub.2 via the formation of Cu-peroxide
complexes.
[0164] With respect to the timing of the formation of the
Cu-containing particles, it is possible that the solubility and
speciation of the Cu(bpy) complexes are a function of pH,
concentration, and ligand to metal ratio, with the Cu(bpy)
complexes being substantially more soluble at the alkaline pH where
pretreatment occurs. As a consequence, Cu-containing particles may
be precipitating at the neutral pH where sample fixation is
performed. Another possibility is that the observed Cu-containing
particles are providing indirect evidence of catalytic activity as
the soluble Cu(bpy) complexes are reduced from Cu(II) to the
observed Cu(I) and Cu(0) oxidation states during the pretreatment
process and are subsequently deposited as insoluble aggregate
particles. It is not yet known whether or not these particles are
catalytically active or inactive. Furthermore, the reduction of
Cu(II) by the incident electrons during TEM imaging also cannot yet
be ruled out.
Characterization of Solubilized Cell Wall Biopolymers and Phenolic
Monomers
[0165] Cell wall biopolymer structural changes associated with this
pretreatment were assessed using multiple analytical approaches. In
the first approach, the relative abundance and molecular weight
distributions of the lignin solubilized during pretreatment were
determined by size-exclusion chromatography (SEC) as a function of
pretreatment time (FIG. 12).
[0166] As can be seen in FIG. 12, a single large peak representing
phenolic monomers and oligomers eluted below an apparent molecular
weight of 1000 Da. As these elution profiles show, Cu-catalyzed AOP
released significantly more soluble lignin fragments than AOP-only
pretreatment. Additionally, as FIG. 12 shows, the molecular weight
distributions of the solubilized lignins were not noticeably
altered for either of the pretreatments over time, indicating that
the soluble lignins were neither undergoing substantial
depolymerization, nor repolymerization through condensation
reactions.
[0167] The distribution and abundance of the phenolic monomers
solubilized following pretreatment were quantitated by LC-MS and
HPLC. These monomers arise primarily through the cleavage of ether
bonds or by saponification of phenolic acids that acylate the
lignin polymer. The distribution of phenolic monomers was found to
be substantially different when the Cu catalyst was present, with
aldehyde products favored over acids as shown in FIG. 4A.
[0168] This quantitative difference between the release of phenolic
acids and aldehydes suggests that the Cu-catalyzed reaction may
utilize a different reaction mechanism that results in less
oxidation of the lignin polymer, yet yields more intra-lignin bond
cleavage and lignin solubilization than AOP-only treatment.
[0169] Additionally, phenolic monomer yields reached only 2 mg/g
lignin (i.e., mass of monomer to mass of cell wall lignin) for both
AOP-only and Cu-catalyzed AOP pretreatment (FIG. 4.), and only 0.5
mg/g lignin by alkali-only pretreatment (FIG. 4A). T Such low
phenolic monomer yields suggest that the cleavage of
.beta.-O-4-bonds is not complete.
[0170] Plants within the family Salicaceae, including the genus
Populus, are known to have lignins with p-hydroxybenzoate groups
acylating the .gamma.-OH of syringyl subunits. As expected, the
most abundant phenolic monomer in all of the pretreatment liquors
was p-hydroxybenzoate (FIG. 44B), as these esters are easily
saponified during alkaline pretreatments. The results show that
alkali-only treatment results in the highest yields (14.8 mg/g
lignin) with AOP-only and Cu(bpy)-AOP, releasing roughly one third
or one half of this quantity. The lower yields following the
oxidative treatments are presumably due to the lower pHs during
these treatments (due to the acidic contribution of H.sub.2O.sub.2)
that may result in incomplete saponification of the
p-hydroxybenzoate, although oxidative degradation/modification may
also contribute to this difference.
2D HSQC NMR of Whole Cell Walls and Solubilized Lignins
[0171] 2D HSQC (heteronuclear single-quantum coherence) NMR
spectroscopy was used to analyze lignin after Cu-catalyzed AOP
pretreatment. NMR characterization was performed on various
components, including untreated whole cell wall material from
hybrid poplar (FIGS. 13A and 13B), the lignin that was solubilized
following one hour of catalytic pretreatment and recovered via acid
precipitation (FIGS. 13C and 13D), and the residual insoluble cell
wall material following pretreatment (FIGS. 13E and 13F). Several
important insights can be gained from these experiments. First, the
NMR data provide evidence for lignin oxidation. The aromatic region
(FIG. 13D) revealed a substantial increase in oxidized S and G
units to their benzylic ketone analogues S' and G' plus new
vanillate units (VA). The aliphatic region (FIG. 13C) further
supports these chemical changes. Contours are colored to match the
structures for aromatic components.
[0172] Although most of the correlations corresponding to
.beta.-ether units in the aliphatic region remained intact,
correlations for the corresponding oxidized analogues A' provide
further evidence of benzylic oxidation. It is not fully understood
how other lignin structures, such as .beta.-5-linked units
(phenylcoumaran), and .beta.-.beta.-linked (resinol) units (See
FIGS. 14I and 14J), react. However, such structures remained intact
in this fraction. It was also observed that cinnamaldehyde end
groups (X1) were completely absent from the oxidized lignin
samples, whereas benzaldehyde end groups (X2) remained. Monomeric
and oligomeric fragments with aryl-aldehyde and aryl-acid
structures are known to be present in milled wood lignins following
catalytic oxidation, and also in lignosulfonates (i.e., lignins
derived from acid sulfite pulping of wood). Although the aromatic
ring is inactivated toward oxidation due to carbonyl conjugation,
the aryl .alpha.-carbonyl is susceptible to nucleophilic attack by
hydroxyl groups followed by cleavage of the side-chain
C.sub..alpha.--C.sub..beta. bond. Such cleavage decreases the
molecular weight of polymeric lignin and creates hydrophilic lignin
fragments with benzoate and benzaldehyde end-groups, consistent
with the MS data (FIG. 4A).
[0173] It was also observed that lignin depolymerization was not
extensive for pretreatment by Cu-catalyzed AOP. Using known peak
integration methods, it was observed that the .beta.-O-4, .beta.-5
and .beta.-.beta. linkages were still present in both the
solubilized lignin (FIG. 13C) and in the residual
pretreatment-insoluble lignin (FIG. 13E) in approximately the same
ratio as in the native lignin. Together with the low yields of
aromatic monomers observed in LC-MS (FIG. 4A) and the SEC studies
performed in this example that revealed an increase in solubilized
lignins without a noticeable shift in the molecular weight
distributions (FIG. 12), these NMR results strongly support a
pathway in which Cu-catalyzed AOP pretreatment solubilizes and
removes a fraction of the cell wall-bound lignin with minimal
depolymerization and minimal oxidation of the residual lignin.
[0174] The solubilized lignins (FIGS. 13C and 13D) showed minimal
depolymerization, exhibited minor oxidative modification, and
contain soluble xylan oligosaccharides (not shown) unless these are
further hydrolyzed and converted. The residual,
pretreatment-insoluble lignin (FIGS. 13E and 13F) exhibited minimal
modification as a consequence of the pretreatment with structures
closely resembling native lignins (albeit requiring recovery of
bipyridine). Furthermore, by controlling the pretreatment time and
the oxidation stoichiometry, it might be possible to control the
molecular weight and chemical properties of solubilized lignins,
customizing them for the production of functional materials and
fine chemicals with targeted properties.
CONCLUSIONS
[0175] These results provide insights into the structural changes
that occur to the cell wall and cell wall biopolymers following
Cu-catalyzed AOP pretreatment of hybrid poplar. Specifically, the
catalyzed pretreatment resulted in disrupted cell walls manifested
by dislocations between individual cell walls, as well as
delaminations within cell walls. Additionally, copper-containing
nanoparticles are co-localized within these zones of
disruption.
[0176] It is hypothesized that sorption of catalyst into the cell
wall during pretreatment results in oxidation, solubilization, and
removal of lignin causing observable cell wall disruptions and
enhanced susceptibility to enzymatic hydrolysis. Consistent with
this hypothesis, both LC-MS and NMR characterization of the
solubilized lignins and the residual material following
Cu-catalyzed AOP pretreatment revealed the presence of oxidized
lignin fragments. Specifically, a fraction of the hydroxyl groups
at the .alpha.-carbon in .beta.-O-4-units were oxidized to
carbonyls, and end-groups characteristic of hydrolytic cleavage of
oxidized lignin side-chains were created, suggesting that
depolymerization results in lignin solubilization and removal
during the pretreatment. Whereas the pretreatment-solubilized
lignins exhibited a more than three-fold increase in the oxidation
of the benzylic alcohol relative to native lignin (with correlation
peak integrals increasing from 5% to 18%), the extent of lignin
oxidation was limited in the pretreatment-insoluble lignin, which
resembled native lignins.
[0177] Formation of the Cu-containing nanoparticles with oxidation
states of Cu(I) and Cu(0) lower may be attributed to reduction of
soluble Cu(bpy) complexes during pretreatment, although it is
possible that these particles were formed during sample
preparation. Additionally, relative to the lignins generated during
other pretreatments and/or delignification processes that were
performed at elevated temperatures, substantial lignin modification
was often the result. Furthermore, the mildly oxidized lignins
generated retained features closely resembling native lignins,
which may provide added value to an integrated biorefining
process.
Example 11
Biomass and Compositional Analysis
[0178] Hybrid poplar (Populus nigra var.
charkoviensis.times.caudina cv. NE-19) grown at the University of
Wisconsin Arlington Agricultural Research Station was milled to
pass through a 1 mm screen (Circ-U-Flow model 18-7-300,
Schutte-Buffalo Hammermill, LLC). The initial composition of
structural carbohydrates and acid-insoluble lignin (Klason lignin)
of biomass were determined using the NREL two-stage acidolysis
method (Sluiter et al. 2011).
Catalytic Copper-Catalyzed AOP Pretreatment (Cu-AOP)
[0179] Biomass 0.51 g (about 0.5 g dry basis; from about 3 to about
5% moisture content) was pretreated in a total of 5 mL aqueous
solution (10% solids loading). The standard Cu-AOP pretreatment
reaction was carried out by adding 4330 .mu.L of distilled water
followed by 270 .mu.L 5 M NaOH (100 mg/g biomass), 125 .mu.L of a
40 mM CuSO.sub.4 solution, and 125 .mu.L of a solution containing
both 40 mM CuSO.sub.4 and 160 mM 2,2-bipyridine (bpy) (2 mM
Cu.sup.2 and 4 mM bpy final concentration) and finally 150 .mu.L of
30% H.sub.2O.sub.2 (w/w) (100 mg/g biomass) to the biomass.
Reactants were briefly vortexed and the slurry was incubated with
orbital shaking at 180 rpm and 30.degree. C. for 24 hrs. The
initial pH for the Cu-AOP pretreatment reaction was approximately
11.5.
"Slow Addition of Hydrogen Peroxide" (SH) with Cu-AOP Pretreatment
(SH/Cu-AOP)
[0180] SH/Cu-AOP pretreatment was performed as described above for
standard Cu-AOP except that the 150 .mu.L 30% H.sub.2O.sub.2 (w/w)
(100 mg/g biomass) was added to the reaction mixture slowly over a
10 hour period. Specifically, each hour 15 .mu.L of 30%
H.sub.2O.sub.2 was added to the reaction mixture, followed by brief
mixing in a vortex mixer to ensure an even distribution. Following
the final addition of H.sub.2O.sub.2, the mixture was incubated as
described for an additional 14 hrs (24 hrs total reaction
time).
Alkali Pre-Extraction (AP) with Cu-AOP Pretreatment (AP/Cu-AOP)
[0181] Alkali pre-extracted hybrid poplar was prepared by
incubating 0.51 g (approximately 0.5 g dry basis) of biomass in 270
.mu.L of 5 M NaOH (100 mg/g biomass) at 30.degree. C. for 1 hr,
(10% solids loading). After 1 hour of incubation, the remaining
insoluble biomass was washed with one volume of deionized water and
subjected to 23 hrs of Cu-AOP pretreatment (24 hrs total reaction
time including the 1 hour pretreatment) as described above.
Combined AP and SH with Cu-AOP Pretreatment (AP-SH/Cu-AOP)
[0182] Alkali pre-extraction and slow H.sub.2O.sub.2 addition
strategies were combined in the modified Cu-AOP (AP-SH/Cu-AOP)
pretreatment. Alkali pre-extracted biomass (0.51 g or 0.5 g dry
weight) was subjected to SH/Cu-AOP as described above except that
the concentration of the Cu(bpy) complexes was reduced by 50% (i.e.
1 mM Cu and 2 mM bpy total final concentrations). Whenever a
different concentration of any of the reactants was used to probe
the effect on the pretreatment process, an appropriate amount of
distilled water was added to the reaction mixture to maintain a
final solid biomass loading of 10%.
Enzymatic Hydrolysis
[0183] Following pretreatment, 0.5 mL of 1 M citric acid buffer (pH
5) was added to the pretreatment mixture, and the slurry was slowly
titrated with 72% (w/w) H.sub.2SO.sub.4 to adjust the pH to 5 prior
to enzymatic hydrolysis. An enzyme cocktail consisting of Cellic
CTec3 and HTec3 (kindly provided by Novozymes A/S, Bagsv.ae
butted.rd, DK) at a loading of 30 mg protein/g glucan each unless
otherwise noted was added to the hydrolysis reaction. The protein
concentrations of the stock enzyme cocktails were quantified by
determining the total protein concentration (and subtracting the
non-protein nitrogen contribution) using the Kjeldahl nitrogen
analysis method (AOAC Method 2001.11, Dairy One Cooperative Inc.,
Ithaca, N.Y., USA). The total volume of the enzymatic hydrolysis
reaction was then adjusted to 10 mL by the addition of deionized
water, and the samples were incubated at 50.degree. C. for 72 hrs
with orbital shaking at 210 rpm. Following enzymatic hydrolysis,
the solid and liquid phases were separated by centrifugation, and
the amount of glucose and xylose released into the aqueous phase
was quantified by HPLC (Agilent 1260 Series equipped with a Agilent
1260 infinity refractive index detector) using an Aminex.RTM.
HPX-87H column operating at 65.degree. C., a mobile phase of 0.05 M
H.sub.2SO.sub.4, and a flow rate of 0.6 mL/min. Standard curves
using pure glucose and xylose were prepared prior to each analysis
to convert peak area to concentration of monomeric sugar. The yield
of glucose and xylose released was defined as the amount of
solubilized monosaccharide divided by the total sugar content of
the biomass prior to pretreatment as determined by chemical
composition analysis. The error bars in the figures represent the
standard deviation from three or more biological replicates.
Results and Discussion
Slow Addition of H.sub.2O.sub.7 (SH/Cu-AOP)
[0184] It was hypothesized that by lowering the effective
H.sub.2O.sub.2 concentration during AOP treatment, a decrease in
non-productive reactions would occur, thus enabling more of the
reactive oxygen species to react with the biomass. To test this
hypothesis, the
TABLE-US-00003 TABLE 3 Sugar yields obtained from different
pretreatment strategies Method Glucose yields (%) Xylose yields (%)
Cu-AOP 62 75 AP/Cu-AOP 86 95 SH/Cu-AOP 77 93 AP-SH/Cu-AOP 97 94
[0185] H.sub.2O.sub.2 was added in a "slow add" manner.
Specifically, the H.sub.2O.sub.2 was added over the course of 10
hrs, without changing either the total amount of peroxide utilized
or the pretreatment time (SH/Cu-AOP). Relative to Cu-AOP
conditions, this modification of the rate of addition of oxidant
resulted in a greater than 1 fold increase in sugar yields, namely
an approximately 1.2 fold increase (77% glucose and 93% xylose)
following enzymatic hydrolysis (See FIG. 16 and Table 3 below).
[0186] To ascertain how SH/Cu-AOP alters the cell wall of hybrid
poplar relative to standard Cu-AOP, compositional analysis of the
biomass was performed both before and after pretreatment. These
results showed that the major impact of slow addition of
H.sub.2O.sub.2 was an increase in the extent of lignin removal,
with approximately 44% of the original lignin being solubilized
during SH/Cu-AOP compared to 28% during Cu-AOP (FIG. 17 showing an
increase in the yields of glucose and xylose to 96% and 93%,
respectively, (Table 4). As discussed herein, lignin removal is a
significant factor in overcoming biomass recalcitrance and
improving sugar yields, by improving access to the cellulose. This
aspect of lignin removal may help explain why the more efficient
utilization of H.sub.2O.sub.2 in SH/Cu-AOP significantly increased
the efficacy of pretreatment and subsequently the final sugar
yields.
TABLE-US-00004 TABLE 4 Percent mass loss obtained different
pretreatments Pretreatments of Glucan Xylan Lignin hybrid poplar
loss (%) loss (%) loss (%) Untreated N/A N/A N/A Normal Cu-AOP 3
.+-. 0.03 23 .+-. 0.01 28 .+-. 0.4 AP 1 .+-. 0.9 5 .+-. 0.3 5 .+-.
0.4 AP/Cu-AOP 5 .+-. 1.2 32 .+-. 0.3 40 .+-. 1.4 SH/Cu-AOP 6 .+-.
0.8 23 .+-. 0.5 44 .+-. 0.00 AP-SH/Cu-AOP 6 .+-. 1.5 39 .+-. 0.4 56
.+-. 0.3 Lignin: Acid insoluble Klason lignin Untreated: Untreated
hybrid poplar AP: Alkali pre-extraction only AP/Cu-AOP: Cu-AOP
followed by alkali pre-extraction SH/Cu-AOP: Cu-AOP pretreatment
with slow addition of H.sub.2O.sub.2 AP-SH/Cu-AOP: Combining alkali
pre-extraction and slow addition of H.sub.2O.sub.2 with Cu-AOP
Alkali Pre-Extraction Followed by Cu-AOP (AP/Cu-AOP)
[0187] Seeking to increase further the efficiency of the
pretreatment and to improve the accessibility of the cellulases and
hemicellulases to the polymeric sugars, an alkali pre-extraction
step was incorporated prior to Cu-AOP (AP/Cu-AOP). By removing
easily extracted hemicellulose and lignin, it was thought that the
porosity of the biomass would increase, resulting in improved
penetration of the Cu(bpy) complexes into the plant cell wall.
This, in turn, may ead to efficient activation and utilization of
active radical species. In addition, such a step may also remove
extractives and lignin to reduce inhibition of enzymes during
enzymatic hydrolysis step.
[0188] Based on the final sugar yields, the alkaline pre-extraction
step had a large positive impact on enzymatic digestibility.
Compared to standard Cu-AOP pretreatment of hybrid poplar,
AP/Cu-AOP pretreatment resulted in improved glucose yields from 62%
to 86% (an approximately 1.4 fold increase), while xylose yields
also improved from 75% to 93% (an approximately 1.2 fold increase)
(Table 3). Compositional analysis following alkali pre-extraction
of hybrid poplar indicated that while approximately 5% by weight of
both the lignin and xylan was solubilized during pre-extraction
(FIG. 17, Table 4), substantially none or only trace amounts of the
glucose was released. Cu-AOP pretreatment of this alkali
pre-extracted poplar (AP/Cu-AOP) resulted in loss of an additional
approximately 35% lignin, which is about 1.4 times higher than
Cu-AOP alone. The removal of lignin is synergistic with AP/Cu-AOP,
removing nearly 10% more than the sum of AP and Cu-AOP alone,
consistent with the significant increase in sugar yields following
enzymatic hydrolysis of AP/Cu-AOP poplar (FIG. 2).
[0189] Hypothesizing that alkaline pre-extraction aids
pretreatment, in part, by preparing the biomass for pretreatment
i.e., swelling the biomass and increasing the surface area for
enzyme accessibility, the water retention value (WRV) was measured
both before and after pre-extraction. Results showed that the WRV
of hybrid poplar increased from 1.15 g water/g biomass (untreated)
to 1.5 g water/g biomass following alkali pre-extraction step,
consistent with the importance of WRV to the pretreatment
process.
[0190] To ascertain if alkali pre-extraction reduced enzyme
inhibition by removing extractives and lignin, the effect of
pre-extraction liquor on the hydrolysis of Avicel.RTM. was
evaluated. These results demonstrated that enzymatic hydrolysis of
Avicel.RTM. in the presence of alkali pre-extraction liquor
resulted in 6% lower glucose yields compared to hydrolysis
reactions that did not contain the pre-extraction liquor.
Hydrolysis reactions were also performed in the presence of
pretreatment liquors obtained from normal Cu-AHPAOP and
AP/Cu-AHPAOP. Interestingly, the inhibitory effect of the Cu-AHPAOP
pretreatment liquor was approximately equal to additive inhibitory
effect of the AP pre-extraction liquor plus the AP/Cu-AHPAOP
pretreatment liquor. Together, these results suggest that
pre-extraction of the hybrid poplar with NaOH increases sugar
yields by removing alkali extractable compounds that inhibit
enzymatic hydrolysis.
[0191] In summary, alkali pre-extraction prior to Cu-AHPAOP
pretreatment (AP/Cu-AHPAOP) results in increased glucose and xylose
yields relative to our standard Cu-AHPAOP process, presumably by
These data are consistent with results from Yuan et al. [30]
demonstrating efficient and synergistic lignin and hemicellulose
removal from poplar using a two-step alkali and ionic liquid
pretreatment. In addition, Liu et al. [26] recently reported
enhanced enzymatic hydrolysis of corn stover by coupling alkaline
pre-extraction with alkaline-oxidative post-treatment.
Combining Alkali Pre-Extraction and Slow Addition
(AP-SH/Cu-AOP)
[0192] Having demonstrated that the efficacy of Cu-AOP could be
improved dramatically by slowly adding the H.sub.2O.sub.2 over the
course of 10 hrs (SH/Cu-AOP), as well as by pre-extracting the
poplar with alkali prior to Cu-AOP (AP/Cu-AOP), a combination of
the two strategies (AP-SH/Cu-AOP). As shown in Table 3, these two
modifications to the Cu-AOP pretreatment of hybrid poplar procedure
resulted in the removal of nearly 40% of the xylan and over 55% of
the lignin during pretreatment. As expected, this increase in
lignin and xylan (hemicellulose) removal resulted in a significant
improvement in enzymatic digestibility (FIG. 17). In fact,
enzymatic hydrolysis resulted in high sugar yields of 97% and 94%
of the theoretical maximum for glucose and xylose,
respectively.
[0193] This AP-SH/Cu-AOP pretreatment strategy was performed at
atmospheric pressure near room temperature. As a result, the
AP-SH/Cu-AOP pretreatment can be performed using relatively simple
and inexpensive equipment.
Optimization of AP-SH/Cu-AOP Pretreatment
[0194] Hypothesizing that these two modifications to the Cu-AOP
pretreatment process might allow a reduction in delignification
costs by lowering oxidant loadings, a series of experiments were
performed with varying H.sub.2O.sub.2 loadings (FIG. 18). The
results demonstrated that the H.sub.2O.sub.2 loading can be reduced
by 25% to 75 mg/g poplar while still maintaining sugar yields of
over 90%. Even at H.sub.2O.sub.2 loadings as low as 50 mg
H.sub.2O.sub.2/g biomass, the sugar yields following enzymatic
hydrolysis were higher (78%) than those obtained with normal Cu-AOP
which utilizes 100 mg/g biomass (62%).
[0195] As expected, compositional analysis of the biomass treated
with different H.sub.2O.sub.2 loadings revealed a clear
relationship between the extent of lignin removal during
AP-SH/Cu-AOP and the sugar yields following enzymatic hydrolysis
(FIG. 18). Sugar yields increased rapidly with lignin removal until
approximately 40% of the lignin had been removed, at which point
additional lignin removal had only a modest effect on sugar yields.
This digestibility "threshold" value in between a Klason lignin
content of 10-15% may be the point where cell wall loses its enough
recalcitrance to allow enzyme accessibility.
[0196] The experiments described above were performed utilizing 60
mg protein (enzyme) per g glucan. Hypothesizing that the alkali
pre-extraction and the more efficient utilization of H.sub.2O.sub.2
may allow for a reduction in enzyme loading, a series of studies
were performed to correlate glucose yields following enzymatic
hydrolysis of pretreated hybrid poplar using different enzyme
loadings (FIG. 19). Not surprisingly, there was a strong
correlation between enzyme loading and sugar yield. As predicted,
however, AP-SH/Cu-AOP method resulted in improved sugar yields even
at much lower enzyme concentrations. For instance, at 100 mg
H.sub.2O.sub.2 per gram poplar (10% H.sub.2O.sub.2), enzyme
loadings were reduced to 15 mg/g glucan (a four-fold decrease) with
higher glucose yields following enzymatic hydrolysis (approximately
80% versus 60% for our "standard" Cu-AOP conditions). In addition,
when the peroxide loadings were increased during pretreatment to
125 mg H.sub.2O.sub.2/g biomass, glucose yields increased to
>90% while still utilizing only 15 mg enzyme/mg glucan. This
improved saccharification of poplar wood with such low enzyme
loadings can be attributed to the relatively high lignin removal
during the AP-SH/Cu-AOP pretreatment process.
[0197] As demonstrated in FIG. 19, AP-SH/Cu-AOP at H.sub.2O.sub.2
loadings of 100-150 mg/g biomass removed a substantial amount of
lignin from the biomass. This provided an insight of possibility to
decrease the concentrations of other costly components involved in
Cu-AOP pretreatment, such as bpy
[0198] Above discussed strategies for improving pretreatment were
performed using 2 mM concentration of bpy. Trials were carried out
to select minimum concentration of bpy but still having relatively
high sugar yields. FIG. 20 shows sugar yields obtained at different
concentrations of bpy, utilizing 125 and 150 mg H.sub.2O.sub.2.
Results revealed the potential of low concentrations of bpy at low
enzyme loadings of 15 and 30 mg/g glucan. A high glucose yields of
75% were obtained at 0.5 mM concentration of bpy with 30 mg/g
glucan enzyme and 150 mg H.sub.2O.sub.2. An increase in the yields
to 85% was obtained by increasing the bpy to 1 mM with 30 mg/g
glucan enzyme and 150 mg H.sub.2O.sub.2. These results shows the
potential of AP-SH/Cu-AOP at low bpy loadings. By performing cost
analysis of the data, we could figure out the best combinations of
the component concentration to lower the cost and maintaining high
sugar yields.
Lignin Stream
[0199] As shown in FIG. 19, AP-SH/Cu-AOP pretreatment solubilized a
significant fraction of the total lignin present in hybrid poplar.
Recognizing that this delignification process not only improves the
enzymatic digestibility of the biomass, but also provides a lignin
stream for potential valorization to chemicals and/or fuels, we
sought to characterize the solubilized lignin. The solubilized
lignin was precipitated at pH 2, washed with deionized water, and
lyophilized to dryness. The recovered product contained .gtoreq.90%
Klason lignin, with only 3% xylan and 2% ash content. (See FIG.
21).
Example 12
Study on Cell Wall Properties of Natural Populus Variants for
Enhanced Digestibility with Alkaline Hydrogen Peroxide
Pretreatment
[0200] In this study, the effect of cell wall redox metal ions,
lignin content and S/G ratio on the efficacy of alkaline
pretreatments was studied. The results obtained were correlated to
digestibility i.e., sugar release from biomass followed by
enzymatic hydrolysis.
Materials and Method
Biomass
[0201] Thirty-six Populus trichocarpa genotypes were provided by
Oak Ridge National Laboratory, Oak Ridge, Tenn., USA. Biomass was
milled (Wiley, MiniMill, Thomas Scientific, Swedesboro, N.J.) to
pass through 20 mesh size screen, air dried, and stored in airtight
bags prior to pretreatment studies.
Biomass Analysis
[0202] The initial composition of structural carbohydrates and
acid-insoluble lignin (Klason lignin) were determined using the
NREL two-stage acidolysis method (Sluiter et al. 2011) with
modifications as described in Li M Y, Foster C, Kelkar S, Pu Y Q,
Holmes D, Ragauskas A, Saffron C M, Hodge D B: Structural
characterization of alkaline hydrogen peroxide pretreated grasses
exhibiting diverse lignin phenotypes. Biotechnol Biofuels 2012,
5:38. The S/G ratio for all samples were determined by
thioacidolysis as described in Li et al., 2012.
[0203] Quantification of redox-active metals in different popular
species were determined by inductively coupled plasma mass
spectrometry (ICP-MS) performed at A & L Great Lakes
Laboratories.
Pretreatment
[0204] Each biomass 0.51 g (approximately 0.5 g dry basis; about 3
to about 5% moisture content) was pretreated with three
pretreatments viz alkali, alkaline hydrogen peroxide (AOP) and AOP
pretreatment aided with Cu (bpy) catalyst (Cu-AOP) in a total of 5
ml, aqueous solution (10% solids loading). For alkali-only
pretreatment, the solution contained 50 mg NaOH (100 mg NaOH/g
biomass), while AOP pretreatment also contained 150 .mu.L of 30%
H.sub.2O.sub.2 (100 mg H.sub.2O.sub.2/g biomass). The Cu-AOP
pretreatment solution was prepared as described above for AOP
except that 125 .mu.L of a solution containing 40 mM CuSO.sub.4 as
well as 125 .mu.L of a solution containing both 40 mM CuSO.sub.4
and 80 mM 2,2-bipyridine (bpy) were added to the biomass slurry
after the addition of NaOH (2 mM Cu.sup.2 and 4 mM bpy final
concentration) but prior to the addition of H.sub.2O.sub.2. The
final pH for the alkali-only pretreatment was 13.2, while it was
approximately 11.5 for AOP and Cu-AOP due to the addition of
H.sub.2O.sub.2. For all three pretreatments, the reactants were
vortexed and the slurry incubated with orbital shaking at 180 rpm
at 30.degree. C. Solutions containing only biomass and deionized
water acted as the control.
Enzymatic Hydrolysis
[0205] After pretreatment, 0.5 mL of 1 M citric acid buffer (pH
4.8) was added to the pretreated slurry, and the slurry was slowly
titrated with 72% (w/w) H.sub.2SO.sub.4 to adjust the pH to 5.0
prior to enzymatic hydrolysis. An enzyme cocktail consisting of
Cellic CTec3 and HTec3 (gift from Novozymes A/S, Bagsv.ae
butted.rd, DK) at a loading of 30 mg protein/g glucan each was
added to the hydrolysis reaction. (The protein concentrations of
the stock enzyme cocktails were quantified using the Bradford Assay
(Sigma-Aldrich)). The total volume was adjusted to 10 mL by the
addition of deionized water, and the samples were incubated at
50.degree. C. with orbital shaking at 210 rpm. Following enzymatic
hydrolysis, the solid and liquid phases were separated by
centrifugation, and the amount of glucose and xylose released into
the aqueous phase was quantified by HPLC (Agilent 1260 Series
equipped with an Aminex.RTM. HPX-87H column operating at 65.degree.
C., a mobile phase of 0.05 M H.sub.2SO.sub.4, a flow rate of 0.6
mL/min, and detection by refractive index). The yield of glucose
and xylose released was defined as the amount of solubilized
monosaccharide divided by the total sugar content of the biomass
prior to pretreatment as determined by chemical composition
analysis. Although xylan is solubilized during pretreatment, no
monomeric sugars were detected in the pretreatment liquor. The
error bars in the figures represent the standard deviation from
.gtoreq.three biological replicates.
Chelation of Inorganic Ions from Native Biomass
[0206] Biomass (3 g) was mixed with 30 mL of 0.2% (w/v) of the
chelator diethylenetriaminepentaacetic acid (DTPA). The pH of the
slurry was adjusted to 7.0 with 5M NaOH, and the solution was
incubated for 24 hr at 30.degree. C. The biomass was then washed
thoroughly with 10 volumes of distilled H.sub.2O to remove the
DTPA, dried at room temperature for 2 days, and stored in airtight
bags. Biomass incubated for 24 hr at 30.degree. C. with only
distilled water was used as a control.
[0207] Pretreatment reactions were performed either in the presence
of bpy plus Cu.sup.2+, Mn.sup.2+, or Fe.sup.2+ ([metal]:[bpy]=2 mM:
4 mM) or in the absence of added metal to ascertain the effect
these ions had on the pretreatment of chelated woody biomass.
[0208] Twenty (20) samples of a natural population of Populus
trichocarpa trees were obtained from Oak Ridge National Laboratory,
out of which 16 were separated into two fractions individually on
the basis of growth, i.e. early growth (EGW) and late growth (LGW)
wood samples, which made total number of samples to 32. Lignin
content, S/G ratio and cell wall redox metal ions content were
quantified to perform correlation studies. Correlations were
established between final sugar release after enzymatic hydrolysis
and different pretreatments among different poplar samples.
Results
[0209] Sixteen (16) samples out of 20 samples were separated into
two fractions on the basis of their growth i.e. early growth wood
and late growth wood to study how cell wall properties of different
ages of wood relates with wood digestibility. In total, 36 biomass
samples were individually pretreated utilizing alkali, alkaline
hydrogen peroxide (AOP), and copper catalyzed alkaline hydrogen
peroxide pretreatment (Cu-AOP). To establish digestibility
correlation among cell wall properties and different pretreatments,
pretreated samples were enzymatically hydrolyzed using commercially
available optimized cocktail of cellulases and xylanases. Samples
without any pretreatment were also enzymatically hydrolyzed and
analyzed.
[0210] To establish significant correlations, samples were analyzed
to determine their lignin content, S/G ratio and total cell wall
redox metal ions content. Estimated lignin content was in the range
of 18.74 to 24.55%, S/G ratio was 1.62 to 3.98 (FIG. 23B) and metal
content was in the range of 10 to 26 ppm (FIG. 23A).
[0211] Results demonstrated that for all 36 samples, Cu-AOP
pretreatment yielded maximum sugars, followed by yields obtained
with AOP, and untreated samples. Sugar yields with untreated
samples were in the range from 13 to 38%, 61 to 85% with alkali
pretreatment, about 53 to about 81% with AOP pretreatment and 74 to
94% for Cu-AOP pretreatment (FIG. 22).
[0212] Specifically, as FIG. 22 shows, there is a negative
correlation for lignin content in all pretreatments. Additionally,
the S/G ratio correlation strongest for AOP-only treatment
(p-value=0.055). The redox-active metal content was only
significant for AOP-only treatment.
[0213] As demonstrated by the results, addition of copper catalyst
led to substantial increase in the sugar yields, these surprising
results provided an insight into the role of cell wall redox active
metal ions in improving effectiveness of AOP only pretreatment.
[0214] A positive correlation was discovered between cell wall
redox metal ions and sugar yields when biomass was treated with AOP
only. Correlations were insignificant for cell wall metal ions when
the biomass was pretreated with alkali and Cu-AOP. The results
clearly demonstrated the importance of cell wall redox metal ions
in AOP only method. While not wishing to be bound by this proposed
hypothesis, it is thought that cell wall metal ions efficiently
activated the hydrogen peroxide which catalyzed the formation of
highly reactive radical species that aided in the pretreatment
process. Correlation between S/G ratio and sugar yields was found
to be strongest for AOP pretreated samples compared to alkali and
Cu-AOP.
Chelation of the Biomass
[0215] To validate the relationship between intracellular metal
content and efficacy of AOP pretreatment, each of the selected four
samples was incubated with the metal chelator DTPA to remove the
cell wall redox metal ions prior to pretreatment. Results revealed
decreased sugar yields with chelated biomass compared to
non-chelated biomass (FIG. 24). For example, glucose yields were
reduced from 66% to 50% for sample 105. Similar trend was observed
for the other biomass samples. To confirm the efficiency of
chelation, ICP-MS analysis of the chelated biomass was performed.
Results demonstrated reduced cell wall redox metal content compared
to non-chelated biomass (FIG. 25).
Discussion
[0216] To further understand the mechanism of AOP pretreatment on
woody biomass components, additional studies were carried out.
[0217] A total of 36 woody biomass samples were studied to
determine the correlation between cell wall properties and
fermentable sugar release. Analysis of results revealed that sugar
release was negatively correlated to lignin content of the biomass.
Lignin serves as a physical blockage on the surface of the biomass
and chemical blockage through lignin carbohydrate complex.
Adsorption of enzymes on lignin occurs through hydrophobic
interactions, electrostatic interactions, and/or hydrogen-bonding
interactions, which leads to non-productive binding making
cellulose inaccessible to enzymes.
[0218] In addition to lignin, lignin composition, i.e., the S/G
ratio, was also analyzed, with results confirming the absence of
any correlation of S/G rations with the sugar yields. This is in
contrast to other reported results, which may be explained by use
of a different pretreatment processes.
[0219] The effect of total cell wall redox metal ions (Cu, Mn, and
Fe) on the digestibility of biomass treated with AOP only.
Interestingly, positive correlation was observed between glucose
yields and number of metal ions for biomass treated with AOP only
pretreatment whereas no correlations were established for alkali,
Cu-AOP and untreated samples.
Example 13
Biomass
[0220] Hybrid poplar (Populus nigra var.
charkoviensis.times.caudina cv. NE-19) was provided by the Great
Lakes Bioenergy Research Center (GLBRC), sugar maple (Acer
saccharum) was obtained from Todd Smith (Devereur sawmill, Pewamo,
Mich.), silver birch (Betula pendula) was acquired from Curt
Lindstrom (Smurfit-Kappa Kraftlina AB, Pitea, Sweden) and aspen
(Populus tremula.times.Populous tremuloides) was obtained from Dr.
Raymond Miller (Michigan state University Extensions). Biomass was
milled using a Wiley MiniMill (Thomas Scientific, Swedesboro, N.J.)
to pass through a 20-mesh size screen, air dried, and stored in
airtight bags prior to pretreatment studies.
Compositional Analysis and S/G Ratios
[0221] Quantification of structural carbohydrates, acid-insoluble
lignin (Klason lignin), cell wall metal content, and S/G ratio were
performed as described in Example 12.
Pretreatment
[0222] Three different pretreatment strategies were tested on each
of the biomass samples: alkali only, alkaline hydrogen peroxide
(AOP), and copper-catalyzed AOP (Cu-AOP). In all cases, 0.51 g of
biomass (approximately 0.5 g dry basis; about 3 to about 5%
moisture content) was pretreated in a total of 5.0 mL aqueous
solution (10% solids loading). For alkali-only pretreatment, the
solution contained 50 mg NaOH (100 mg NaOH/g biomass), while AOP
pretreatment also contained 150 .mu.L of 30% H.sub.2O.sub.2 (100 mg
H.sub.2O.sub.2/g biomass). The Cu-AOP pretreatment solution was
prepared as described above for AOP except that 125 .mu.L of a
solution containing 40 mM CuSO.sub.4 as well as 125 .mu.L of a
solution containing both 40 mM CuSO.sub.4 and 160 mM 2,2-bipyridine
(bpy) were added to the biomass slurry after the addition of NaOH
(2 mM Cu.sup.2 and 4 mM bpy final concentration) but prior to the
addition of H.sub.2O.sub.2. The final pH for the alkali-only
pretreatment was 13.2, while it was approximately 11.5 for AOP and
Cu-AOP due to the addition of H.sub.2O.sub.2. For all three
pretreatments, the reactants were mixed in a vortex mixer and the
slurry incubated with orbital shaking at 180 rpm and 30.degree. C.
for 24 hrs. Solutions containing only biomass and deionized water
acted as controls.
Enzymatic Hydrolysis
[0223] Following pretreatment, 0.5 mL of 1 M citric acid buffer (pH
4.8) was added to the pretreated slurry, and the slurry was slowly
titrated with 72% (w/w) H.sub.2SO.sub.4 to adjust the pH to 5.0
prior to enzymatic hydrolysis. An enzyme cocktail consisting of
Cellic CTec3 and HTec3 (gift from Novozymes A/S, Bagsv.ae
butted.rd, DK) at a loading of 30 mg protein/g glucan for both
Ctec3 and HTec3 was added to the hydrolysis reaction. The protein
concentrations of the stock enzyme cocktails were quantified as y
described in X. Gao, R. Kumar, S. Singh, B. A. Simmons, V. Balan,
B. E. Dale and C. E. Wyman, Biotechnol Biofuels, 2014, 7, 71, using
the Kjeldahl nitrogen analysis method, as described in, for
example, R. Kumar and C. E. Wyman, Enzyme Microb Tech, 2008, 42,
426-433. The total volume of the pretreated biomass slurry was
adjusted to 10 mL by the addition of deionized water, and the
samples were incubated at 50.degree. C. for 72 hrs with orbital
shaking at 210 rpm.
[0224] Following enzymatic hydrolysis, the solid and liquid phases
were separated by centrifugation, and the amount of glucose and
xylose released into the aqueous phase was quantified by HPLC
(Agilent 1260 Series equipped with an Aminex.RTM. HPX-87H column
operating at 65.degree. C., a mobile phase of 0.05 M
H.sub.2SO.sub.4, a flow rate of 0.6 mL/min, and detection using an
Agilent 1260 infinity refractive index detector).
[0225] The yield of glucose and xylose released was defined as the
amount of solubilized monosaccharide divided by the total sugar
content of the biomass prior to pretreatment as determined by
chemical composition analysis. Prior to each analysis, standard
curves were generated using pure solutions of glucose and xylose to
convert peak area to concentration of monomeric sugar. The error
bars in the figures represent the standard deviation from three or
more biological replicates.
Chelating Inorganic Ions from Native Biomass
[0226] Biomass (3 g) was mixed with 30 mL of 0.2% (w/v) of the
chelator diethylenetriaminepentaacetic acid (DTPA). The pH of the
slurry was adjusted to 7.0 with 5 M NaOH, and the solution was
incubated for 24 hr at 30.degree. C. The biomass was then washed
thoroughly with 10 volumes of distilled H.sub.2O to remove the
DTPA, dried at room temperature for 2 days, and stored in airtight
bags. Biomass incubated for 24 hr at 30.degree. C. with only
distilled water was used as a control.
[0227] Pretreatment reactions as described above were performed
either in the presence of 2,2'-bipyridine (bpy) plus Cu.sup.2+,
Mn.sup.2+, or Fe.sup.2+ ([metal]:[bpy]=2 mM:4 mM) or in the absence
of added bpy and metal to ascertain the effect these metal
complexes had on the pretreatment of chelated woody biomass. The
error bars represent the standard deviation from three or more
biological replicates.
Results and Discussion
[0228] Different woods are known to respond differently to
oxidative pretreatment..sup.32 To ascertain the key factors that
lead to these variations, we analyzed the enzymatic digestibility
of four hardwood samples (hybrid poplar, silver birch, aspen, and
sugar maple) following alkali-only, AOP, or Cu-AOP pretreatment.
Each of these biomass samples had differences in glucan and xylan
composition, lignin content and composition, and redox active metal
content, and these characteristics were compared with their
enzymatic digestibilities following pretreatment.
Cell Wall Composition Analysis
[0229] Cell wall composition is an important determinant in the
enzymatic digestibility of lignocellulosic biomass, and not
surprisingly, analysis of the biomass composition of the four
different hardwood samples showed a range of lignin, hemicellulose
and cellulose content (See Table 5 below). The amount of glucan
varied from 32% to 44%, with the lowest content found in aspen
(32%) and the highest in hybrid poplar (44%). Conversely, hybrid
poplar had the lowest xylan content at 17%. Importantly, silver
birch had the lowest lignin content (18%), while aspen, hybrid
poplar, and sugar maple contained roughly equal amounts of lignin
(25-26%).
Comparison of Different Alkaline Pretreatments
[0230] To ascertain the susceptibility of silver birch, aspen,
hybrid poplar, and sugar maple to different alkaline pretreatments,
each biomass was subjected to alkali-only, AOP, or Cu-AOP
pretreatment followed by enzymatic hydrolysis (FIG. 27 and Table
7). While silver birch and aspen both responded quite well to
alkali-only pretreatment, the enzymatic digestibility of hybrid
poplar and sugar maple rose much more modestly when only NaOH was
used for pretreatment. Intriguingly, while three of the four woody
biomass samples tested had very high levels of enzymatic
digestibilities following AOP pretreatment and exhibited only a
slight increase in digestibility when AOP was performed in the
presence of copper 2,2'-bipyridine complexes (Cu-AOP), hybrid
poplar behaved quite differently. In the case of hybrid poplar,
enzymatic digestibility following alkali-only pretreatment and AOP
pretreatment were nearly identical, while glucose yields more than
doubled following Cu-AOP pretreatment. Xylose yields demonstrated
very similar results (Table 7). Ultimately, silver birch exhibited
the highest sugar yields following Cu-AOP and enzymatic hydrolysis
(79%), followed by aspen (69%), hybrid poplar (60%), and sugar
maple (51%).
TABLE-US-00005 TABLE 7 Glucose and xylose yields (%) following
enzymatic hydrolysis of alkali, alkaline oxidative peroxide (AOP)
and copper catalyzed AOP (Cu-AOP) pretreatment for different
biomasses Biomass Alkali AOP Cu-AOP Glucose yields (%) Silver Birch
64.2 .+-. 3.4 74.0% .+-. 2.5.sup. 80.0 .+-. 1.1 Aspen 57.4 .+-. 5.0
63.3 .+-. 1.4 69.0 .+-. 2.3 Hybrid Poplar 32.5 .+-. 1.5 28.3 .+-.
0.2 60.6 .+-. 0.9 Sugar Maple 33.7 .+-. 0.3 48.7 .+-. 0.7 51.4 .+-.
0.8 Xylose hydrolysis yields (%) Silver Birch 75.6 .+-. 3.9 78.12
.+-. 1.9 79.5 .+-. 1.3 Aspen 63.7 .+-. 2.5 61.35 .+-. 0.6 62.9 .+-.
2.1 Hybrid Poplar 59.5 .+-. 0.3 48.29 .+-. 0.01 74.6 .+-. 1.4 Sugar
Maple 42.7 .+-. 0.1 56.43 .+-. 0.7 72.8 .+-. 0.4
[0231] Silver birch, which had the lowest lignin content (Table 5),
gave the highest sugar yields following enzymatic digestion (FIG.
26). However, the other three hardwoods all contained approximately
the same amount of lignin yet differed significantly their
enzymatic digestibility following pretreatment, indicating that
there must be other differences between these four biomass samples.
FIG. 26 shows glucose yields of various types of wood under various
treatment conditions according to various embodiments.
[0232] The S/G ratio, which is an important determinant in the
extent of lignin crosslinking, is also known to affect biomass
recalcitrance. S rich lignin has a less branched structure and
lower degree of polymerization than G rich lignin, resulting in S
rich lignin generally being more easily removed. In the present
study, four different hardwoods were compared, and a correlation
was found between the S/G ratio and biomass digestibility FIG. 28).
Silver birch, with the lowest lignin content and the highest S/G
ratio (2.7), showed highest glucose yields with all pretreatments
(Table 5, FIG. 30).
TABLE-US-00006 TABLE 5 Compositional analysis of hardwoods used in
this study.sup.a Glucan Xylan Klason S/G Biomass (%) (%) Lignin (%)
ratios Silver birch 39.0 .+-. 1.9 24.2 .+-. 0.1 17.8 .+-. 0.8 2.7
.+-. 0.1 Aspen 32.0 .+-. 1.7 20.1 .+-. 1.0 25.0 .+-. 0.9 1.3 .+-.
0.1 Hybrid poplar 44.0 .+-. 1.2 17.0 .+-. 0.2 24.6 .+-. 1.0 1.2
.+-. 0.1 Sugar maple 42.0 .+-. 2.0 20.0 .+-. 0.8 25.0 .+-. 2.5 1.7
.+-. 0.1 .sup.aErrors represent standard deviations from either 6
(compositional analysis) or 3 biological replicates.
[0233] However, the digestibility of silver birch was only slightly
higher than that of aspen even though aspen's S/G ratio was
significantly lower (1.3). In addition, sugar maple had the second
highest S/G ratio (1.7) and yet resulted in lower glucose yields
following enzymatic digestion compared to both silver birch and
aspen for all pretreatment tested. Together these results highlight
the fact that while lignin content and composition are clearly
important, other factors also impact how these four hardwoods
respond to different alkali pretreatments.
Redox-Active Metal Content of the Cell Wall
[0234] It was noted that the addition of copper 2,2'-bipyridine
complexes during AOP pretreatment (Cu-AOP) substantially increased
the digestibility of hybrid poplar but only slightly increased the
digestibility of the other hardwoods. It was hypothesized that AOP
requires the presence of metal ions to be an effective pretreatment
for hardwoods. It was further hypothesized that while the silver
birch, aspen, and sugar maple samples already contained sufficient
redox-active metal ions in their cell wall (thereby obviating the
need for the additional copper ions during pretreatment), the
hybrid poplar samples contained a relatively low level of
redox-active metal ions. In this scenario, the addition of Cu(bpy)
complexes during AOP pretreatment would compensate for the low
natural levels of metal ions in our hybrid poplar samples.
[0235] To test these hypotheses, ICP-MS was performed to quantify
cell wall redox-active metal ions in the four different hardwood
samples (Table 6).
TABLE-US-00007 TABLE 6 ICP-MS analysis for total redox-active
metals present in cell wall Cell Wall Redox Metals (ppm) Biomass
Manganese Iron Copper Total.sup.a Silver birch (control).sup.b 100
10 1 111 .+-. 5 Silver birch (non chelated).sup.c 98 16 1 115 .+-.
3 Silver birch (chelated).sup.d 4 5 1 11 .+-. 2 Aspen (control) 19
26 6 51 .+-. 5 Aspen (non chelated) 16 23 6 45 .+-. 2 Aspen
(chelated) 2 11 4 17 .+-. 3 Hybrid poplar (control) 1 5 1 7 .+-. 3
Hybrid poplar (non 1 5 1 7 .+-. 2 chelated) Hybrid poplar
(chelated) 1 2 1 4 .+-. 4 Sugar maple (control) 34 10 1 45 .+-. 3
Sugar maple (non chelated) 34 6 1 41 .+-. 4 Sugar maple (chelated)
3 2 1 6 .+-. 3 .sup.aErrors represent the standard deviation from 3
biological replicates .sup.bControl samples are untreated biomass.
.sup.cNon-chelated samples were incubated in pure deionized water
for 24 hrs. .sup.dChelated samples were treated with the chelator
DTPA for 24 hrs as described in the experimental section.
[0236] The values ranged from only 7 ppm found in hybrid poplar to
over 110 ppm in silver birch. As predicted, a strong positive
correlation was discovered between the redox-active metal content
of the woody biomass and enzymatic digestibility following AOP
pretreatment (FIG. 28). This same correlation was not observed
following Cu-AOP pretreatment. Analysis via ICP-MS of the cell wall
metal ion content in Cu-AOP pretreated biomass demonstrated that
all samples exhibited a very large increase in the amount of copper
relative to the untreated samples, with copper essentially
dominating the metal ratio (Table 8).
[0237] To corroborate the relationship between intracellular metal
content and efficacy of AOP pretreatment, each of the different
hard woods was incubated with the metal chelator DTPA prior to
pretreatment. CP-MS analysis of the chelated biomass revealed a
substantial decrease in metals, with DTPA treatment removing
approximately 96% of cell wall redox metals (Table 6).
TABLE-US-00008 TABLE 8 ICP-MS concentrations (ppm) for untreated
and Cu-AHP, Fe-AHP and Mn-AHP pretreated biomasses Cu-AHP Fe-AHP
Mn-AHP (Copper) (Iron) (Manganese) Biomass ppm ppm ppm Silver Birch
1481 .+-. 6 Aspen 1238 .+-. 9 Hybrid Poplar 1182 .+-. 8 619 249
Sugar Maple 1176 .+-. 9
[0238] The effect of chelation was dramatic for biomass that
initially contained a large amount of redox-active metal ions. For
example, the enzymatic digestibility of chelated silver birch was
significantly diminished following uncatalyzed AOP pretreatment
relative to biomass that had not been chelated, with glucose yields
reduced from 70% to only 50% (FIG. 29A). Likewise, chelated aspen
and sugar maple also exhibited lower enzymatic digestibility
following uncatalyzed AOP pretreatment relative to unchelated
samples. Conversely, the digestibility of hybrid poplar, which
naturally had very low cell wall metal content, was not affected by
incubation with DTPA.
[0239] To verify that the decreased efficacy of uncatalyzed AOP
following incubation with DTPA was due to the loss of the metal
ions, chelated biomass was subjected to metal-catalyzed AOP
pretreatment (FIG. 29B). As expected, chelation of the hardwoods
with DTPA prior to pretreatment did not significantly alter the
efficacy of Cu-AOP, presumably because the addition of Cu(bpy)
complexes obviated the need for naturally occurring intracellular
metal ions. Interestingly, the addition of Cu(bpy) complexes led to
significantly higher enzymatic digestibility than the addition of
either Mn(bpy) or Fe(bpy) complexes (i.e. Mn-AOP and Fe-AOP) (FIG.
30). Whether this difference is due to the superior reactivity of
Cu(bpy) complexes, the result of better penetration of Cu(bpy)
complexes into the plant cell wall (Table 8), or some other
property cannot be determined from this data.
Prophetic Examples
[0240] Testing with other ligands will be performed to determine
other candidates for use in the various embodiments described
herein.
[0241] Recycling of the novel catalysts described herein will also
be completed.
[0242] Optimization studies will be carried out to further reduce
catalyst loading, alkali loading and H.sub.2O.sub.2 loading.
[0243] Detailed study for lignin degradation products will be
conducted to identify lignin removal mechanism.
[0244] To determine sorption behavior of the catalyst for several
other biomass fractions. Specifically, we will utilize untreated
hybrid poplar as well as hybrid poplar that has been pretreated or
pretreated and hydrolyzed, a model cellulose (Avicel.RTM.), and a
model lignin generated in our lab from the alkali pulping of hybrid
poplar. These feedstocks will be used to determine the sorption
isotherms of both copper salt (e.g., as CuSO.sub.4) and
Cu(bpy)(ethylenediamine). The copper content of free liquid will be
determined using atomic adsorption spectroscopy in the Chemistry
department as employed previously by our group. Additionally, the
survival of ligand-metal complexes and their residual catalytic
activity following pretreatment will be evaluated using GC-MS,
LC-MS and UV/Vis spectrometry.
[0245] The recycling of copper catalyst from pretreatment liquors,
enzymatic hydrolysates, and clarified (cell-free) stillage
following fermentation and distillation using untreated biomass as
the bio-sorbent as a strategy to simultaneously detoxify liquors
prior to fermentation, to recover catalyst, and to impregnate
untreated biomass with catalyst will be further explored.
Considering that about 60 to about 70% of the original mass of the
biomass is solubilized by pretreatment and hydrolysis, even if the
residual biomass strongly adsorbs catalyst, there will be
substantially less present. Catalyst recovery will be assessed by
analyzing the amount and activity of recovered catalyst as
functions of operation parameters including pH, temperature,
impregnation time and solids loading.
[0246] The feasibility of utilizing polyanionic flocculants and
cationic ion exchange resins as a strategy to either remove
residual catalyst not recovered by adsorption to untreated biomass
or as a standalone recovery strategy will also be explored.
Catalyst recovery will be by quantification of copper removal as a
function of pH.
[0247] In one embodiment, a commercial process for the production
of lignocellulose derived sugars is provided. This process can, for
example, be converted by biological, chemical, or catalytic
conversion to renewable biofuels, biochemicals, and biopolymers.
This may function as a standalone pretreatment, or as a component
of a multi-stage pretreatment process (as in the case of oxidative
bleaching or delignification following alkaline pulping).
[0248] The various embodiments further include a homogeneous
catalyst (e.g., copper (II) 2,2' bipyridine ethylenediamine
(Cu(bpy)en) comprising one or more metals; and at least two metal
coordinating ligands, wherein the homogeneous catalyst is a
multi-ligand metal complex adapted for use with an oxidant (e.g.,
air, oxygen, hydrogen peroxide, persulfate, percarbonate and sodium
peroxide and/or ozone) in an oxidation reaction to catalytically
pretreat lignocellulosic biomass. In one embodiment, the
multi-ligand metal complex is a multi-ligand copper complex. In one
embodiment, the metals are selected from aluminum, zinc, nickel,
magnesium and combinations thereof. In one embodiment, said metals
and said metal coordinating ligands are in a state of interaction
with each other.
[0249] In one embodiment, said metals are selected from Fe(II),
Fe(III)), Cu(I), Cu(II), Co(III), Co(VI)), V(II), V(III), V(IV),
V(V) and combinations thereof.
[0250] In one embodiment, the metal coordinating ligand is selected
from pyridine, 1,10-phenanthroline, ethylenediamene, histidine,
glycine and combinations thereof.
[0251] In one embodiment, the lignocellulosic biomass contains more
than trace amounts of at least one transition metal, such as, but
not limited to, iron, cupper and/or manganese.
[0252] In one embodiment, a method of pretreating plant biomass is
provided, comprising catalytically pretreating the plant biomass
with a multi-ligand metal complex and oxidant in an alkaline
oxidative pretreatment to produce a catalytically pretreated plant
biomass.
[0253] In one embodiment, the plant biomass, the multi-ligand metal
complex and the oxidant form a solution having a pH of at least
11.5. In one embodiment, the oxidant is hydrogen peroxide and the
multi-ligand metal complex is a multi-ligand copper complex.
[0254] In one embodiment, the copper complex is a copper(II)
2,2'-bipyridine complex (Cu(bpy)) modified to contain at least one
additional metal-coordinating ligand, such as ethylenediamine.
[0255] In one embodiment, the oxidant is added at a gradual rate.
In one embodiment, the gradual rate is equal to or less than a rate
of consumption of the oxidant by the plant biomass and the
multi-ligand metal complex.
[0256] In various embodiments, the method can further comprise
extracting the lignocellulosic biomass prior to produce a solids
fraction and a liquid fraction, wherein the solids fraction is
catalytically pretreated and/or recovering and reusing the multi
ligand metal complex.
[0257] In one embodiment, the catalytic pretreating step also
produces a liquid phase and the method further comprises separating
the catalytically pretreated biomass from the liquid phase to
produce separated catalytically pretreated biomass; and hydrolyzing
the separated catalytically pretreated biomass to produce
hydrolyzed catalytically pretreated biomass.
[0258] All publications, patents and patent documents are
incorporated by reference herein, as though individually
incorporated by reference, each in their entirety, as though
individually incorporated by reference. In the case of any
inconsistencies, the present disclosure, including any definitions
therein, will prevail.
[0259] Although specific embodiments have been illustrated and
described herein, it will be appreciated by those of ordinary skill
in the art that any procedure that is calculated to achieve the
same purpose may be substituted for the specific embodiments shown.
For example, although the process has been discussed using
particular types of plant biomass, any type of plant biomass, such
as grasses, rice straw and the like, for example, may be used.
Additionally, although the process has been discussed using
primarily copper as the metal in the multi ligand metal catalyst,
other metals, such as iron, in various oxidation states, for
example, may be used. This application is intended to cover any
adaptations or variations of the present subject matter. Therefore,
it is manifestly intended that embodiments of this invention be
limited only by the claims and the equivalents thereof.
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