TITLE: Sugar Mixtures and Methods for Production and Use thereof
RELATED APPLICATIONS
In accord with the provisions of 35 U.S.C. §119(e) and §363, this application claims the
benefit of:
US 61/358,894 filed 26 June 2010 by Aharon EYAL and entitled "Fermentation
Feedstock Precursor and Methods for the Production Thereof; and
US 61/491,243 filed 30 May 2011 by Robert JANSEN et al. and entitled "Lignin
Compositions, Systems and Methods for Processing Lignin and/or HC1"; and
US 61/500,169 filed 23 June 2011 by Aharon EYAL et al. and entitled "Sugar Mixtures
and Methods for Production and Use thereof;
In accord with the provisions of 35 U.S.C. §119(a) and/or §365(b), this application
claims priority from:
prior Israeli application IL206896 filed on 8 July 2010 by Aharon EYAL and entitled
"Fermentation Feedstock Precursor and Methods for the Production Thereof: and
prior Israeli application IL207313 filed on 29 July 2010 by Aharon EYAL et al. and
entitled "Methods for the Production of a Fermentation Feedstock"; and
prior PCT application IL2011/000130 filed on 6 February 2011 by Aharon EYAL et al.
and entitled " Methods for the Separation of HC1 from a Carbohydrate and Compositions
Produced thereby" which corresponds to IL 210998 filed 1 February 2011.
Each of these priority documents is fully incorporated by reference.
This application is also related to the following co-pending applications which are each
fully incorporated herein by reference:
US 61/473,134 filed 7 April 201 1 by Aharon EYAL and entitled "Lignocellulose
Conversion Processes and Products"; and
US 61/483,663 filed 7 May 201 1 by Aharon EYAL and entitled "Lignocellulose
Conversion Processes and Products"
US 61/483,777 filed 9 May 201 1 by Robert JANSEN et al. and entitled "Hydrolysis
Systems and Methods"; and
US 61/487,319 filed 18 May 2011 by Robert JANSEN et al. and entitled "Hydrolysis
Systems and Methods"; and
prior Israeli application IL 211093 filed on 6 February 2011 by Aharon EYAL and
entitled "A Method for Processing a Lignocellulosic Material and for the Production of a
Carbohydrate Composition"
FIELD OF THE INVENTION
This invention relates to sugars and production and use thereof.
BACKGROUND OF THE INVENTION
The carbohydrate-conversion industry is large and rapidly increasing in size. Currently,
about 100 million tons of carbohydrates are fermented annually, primarily to provide fuel-grade
ethanol. This number is predicted to triple in the next decade.
Millions of tons of carbohydrates are also fermented every year to provide food and feed
products, such as citric acid and lysine. Also large and increasing is fermentation to produce
other products, such as monomers for the polymer industry, e.g. lactic acid for the production of
polylactide.
Fermentation media typically include, in addition to carbohydrates and/or another carbon
source, other nutrients and factors such as nitrogen sources, minerals, vitamins, and growth
factors. In some cases, fermentation media comprise well identified chemicals. In other cases,
various preparations (e.g. yeast extract or tryptone broth) are incorporated without fully
understanding the effect of each component in the preparation. Some of those preparations result
from natural sources, such as extracts. Some of those preparations are of relatively high cost.
With the advent of molecular biology techniques, a new generation of industrial
fermentation, also known as conversion, based upon genetically modified microorganisms has
emerged. In some cases these microorganisms rely upon inducible promoters for induction of a
specific gene. Some of the inducible promoters respond to specific sugars.
Although conversion of lignocellulosic material to carbohydrates via enzyme-catalyzed
and/or acid-catalyzed hydrolysis of polysaccharides and pyrolysis of lignocellulosic material
have been previously described, industrial scale application of the proposed technologies has
presented technical problems which remain to be overcome. Hydrolysis of hemicellulose is
relatively easy, but hydrolysis of cellulose (typically more than 50% of total polysaccharides) is
more difficult due to its partial crystalline structure.
This application refers to various solvents defined in terms of Hoy's cohesion parameter
Delta-P and/or Delta-H. By way of review:
Delta-P is the polarity related component of Hoy's cohesion parameter and delta-H is the
hydrogen bonding related component of Hoy's cohesion parameter.
The cohesion parameter, as referred to above or, solubility parameter, was defined by Hildebrand
as the square root of the cohesive energy densi
where DEn r and V are the energy or heat of vaporization and molar volume of the liquid,
respectively. Hansen extended the original Hildebrand parameter to a three-dimensional cohesion
parameter. According to this concept, the total solubility parameter, delta, is separated into three
different components, or, partial solubility parameters relating to the specific intermolecular
interactions:
in which delta-D, delta-P and delta-H are the dispersion, polarity, and Hydrogen bonding
components, respectively. Hoy proposed a system to estimate total and partial solubility
parameters. The unit used for those parameters is MPa1 2. A detailed explanation of that
parameter and its components can be found in "CRC Handbook of Solubility Parameters and
Other Cohesion Parameters", second edition, pages 122-138. That and other references provide
tables with the parameters for many compounds. In addition, methods for calculating those
parameters are provided.
SUMMARY OF THE INVENTION
One aspect of some embodiments of the invention relates to heterogeneous sugar
mixtures. The term "sugar" as used in this specification and the accompanying claims refers to
monosaccharides and oligosaccharides (disaccharides or greater) soluble in water at 25 degrees
centigrade. Throughout this application, the term disaccharide refers to sugar dimers and "higher
oligosaccharides" or "higher saccharide" refers to oligomers comprising three or more sugar
units. According to various exemplary embodiments of the invention dimers may be homodimers
and/or hetero-dimers. Alternatively or additionally, higher oligosaccharides may include
same and/or different sugar units.
In many exemplary embodiments of the invention, the sugar mixtures result from acid
hydrolysis of lignocellulosic or "woody" substrates. In some exemplary embodiments of the
invention, acid hydrolysis is conducted with HCl, optionally at a concentration of 37% W/W
HCl/ [HCl+water], optionally 39, 41, 43 or even 45 % HCl on the same basis. In some
exemplary embodiments of the invention, the hydrolysis is conducted at temperatures below 50
degrees centigrade, optionally below 40 degrees, optionally below 30 degrees, optionally at 25
degrees or less. In some exemplary embodiments of the invention, at least a portion of the
hydrolysis is conducted at 20 degrees or less, optionally 15 degrees or less.
According to various exemplary embodiments of the invention the sugar mixture is
provided as a syrup and/or liquid, optionally including residual acid from hydrolysis. Total sugar
concentrations in such a syrup/liquid can be 15, 20, 25, 30, 35, 40, 45 or 50% by weight or
intermediate or higher percentages. Optionally, the mixture is provided as dry crystals.
In some exemplary embodiments of the invention, a percentage of oligosaccharides to
total saccharides in the mixture is greater than 4, optionally 6, optionally 8, optionally 10,
optionally 15% or intervening or greater percentages.
In some exemplary embodiments of the invention, a percentage of disaccharides to total
saccharides in the mixture is greater than 3, optionally 5, optionally 7, optionally 10% or
intervening or greater percentages.
"Disaccharide" indicates two sugars connected by an alpha bond or by a beta bonds or
bonded via various hydroxyls on the molecule, and combinations thereof.
In some exemplary embodiments of the invention, a percentage of pentoses to total
saccharides in the mixture is greater than 3, optionally 5, optionally 7, optionally 10% or
intervening or greater percentages. Optionally, at least a portion of the pentose is present as part
of a disaccharide or longer oligosaccharide.
According to various exemplary embodiments of the invention the mixture includes at
least one alpha-bonded di-glucose and/or at least one beta-bonded di-glucose. Optionally, at least
a portion of the di-glucoses are present as parts of higher oligosaccharides.
In some exemplary embodiments of the invention, residual acid (e.g. HC1) may be
present in the mixture. Various exemplary embodiments of the invention are concerned with
ways to remove this residual acid. Optionally, such removal contributes to added value for use in
downstream processes (e.g. fermentation). In some exemplary embodiments of the invention,
removal of residual acid involves extraction with an extractant containing an alcohol. Optionally,
two or more extractions are conducted. In some exemplary embodiments of the invention, at
least one of the extractions employs a mixture of two solvent types.
Another aspect of some embodiments of the invention relates to hydrolyzing a
lignocellulosic substrate in HC1 to form a hydrolyzate comprising total saccharides to (total
saccharides + water) of at least 20%, optionally 25%, optionally 30% by weight and deacidifying
the hydrolyzate while increasing the sugar concentration. Optionally, the sugar
concentration is increased to 35, 40, 45 or 50% or greater or intermediate percentages. In some
exemplary embodiments of the invention, the disaccharides in the de-acidified hydrolyzate are at
least 5%, optionally 10%, optionally 20%, optionally 30% of the total saccharides or
intermediate or greater percentages. Optionally, a portion of the saccharides in the de-acidified
hydrolyzate can be enzymatically digested.
Another aspect of some embodiments of the invention relates to fermenting such a sugar
mixture in a fermentor to produce a desired fermentation product or "conversion product".
Optionally, the fermentation product can include one or more of alcohols, carboxylic acids,
amino acids, monomers for production of industrially important polymers and proteins. In some
exemplary embodiments of the invention, a fermentation product is produced and then converted
into the monomer (e.g. 3-hydroxy-propionic acid to be converted into acrylic acid, which is then
polymerized). In other exemplary embodiments of the invention, the monomer is produced
directly (e.g. lactic acid as a source of polylactide).
Optionally, the proteins are heterologous proteins produced by genetically modified
microorganisms. Such heterologous proteins include, but are not limited to, hormones, enzymes
(e.g. cellulases), growth factors, cytokines and antibodies. Optionally, the antibodies are fusion
proteins including a non-immunoglobulin domain.
An additional aspect of some embodiments of the invention relates to enzymatic
hydrolysis of a portion of the sugars in the mixture. For purposes of this specification and the
accompanying claims, the term "enzyme" indicates a single enzyme or a mixture including two
or more enzymes. Optionally, an enzyme is provided as a crude preparation (e.g. cell extract)
characterized by a type and/or level of activity, as opposed to a precise molecular definition.
According to various exemplary embodiments of the invention enzymes capable of hydrolyzing
alpha and/or beta bonds are used. Optionally, specificity for a desired bond type can be achieved
by appropriate enzyme selection and/or selection of suitable reaction conditions. In some
exemplary embodiments of the invention at least 10% of disaccharides in the mixture are
converted to monosaccharides by this enzymatic treatment. Alternatively or additionally, at least
10% of oligosaccharides in the mixture are enzymatically hydrolyzed to release additional
monosaccharides. In some exemplary embodiments of the invention, enzymes are immobilized.
Optionally, immobilization can be on beads and/or a membrane. In some exemplary
embodiments of the invention, immobilization contributes to an increase in yield of an enzymatic
hydrolysis product per unit of enzyme.
For purposes of this specification and the accompanying claims an "S1 solvent" or "S1" is
an organic solvent with a water solubility of less than 15% characterized by a polarity related
component of Hoy's cohesion parameter (delta-P) between 5 and 10 MPa1 2 and/or by a hydrogen
bonding related component of Hoy's cohesion parameter (delta-H) between 5 and 20 MPa1/2. In
some exemplary embodiments of the invention, HCl tends to selectively transfer to an SI solvent
upon contact therewith.
For purposes of this specification and the accompanying claims an "S2 solvent" or "S2" is
an organic solvent having a water solubility of at least 30% and characterized by a delta-P greater
than 8 MPa1 2 and/or a delta-H greater than 12 MPa1 2. In some exemplary embodiments of the
invention, HCl tends to selectively transfer to an extractant including both SI and S2 solvents
upon contact therewith.
For purposes of this specification and the accompanying claims "extract" and "extraction"
indicate bringing an extractant into contact with a substrate and then separating an extract from
an extracted substrate.
According to various exemplary embodiments of the invention an extraction may be on
an indicate stream or fraction per se or on a modified stream or fraction. Optional modifications
include, but are not limited to, dilution, concentration, mixing with another stream or fraction,
temperature adjustment, and filtration. Optionally, two or more modifications may be performed
prior to extraction.
"Woody materials" or "lignocellulosic materials" are an attractive and environmentfriendly
substrate for sugar production since they are obtained from renewable resources. Many
non-food lignocellulosic materials are potential sources of soluble carbohydrates. These
lignocellulosic materials include, but are not limited to, wood and by-products of wood
processing (e.g. chips, sawdust, and shavings) as well as residual plant material from agricultural
products and paper industry byproducts (e.g. cellulose containing residues and/or paper pulp)
Residual plant material from agricultural products includes processing by-products and
field remains.
Processing by-products include, but are not limited to, corn cobs, sugar cane bagasse,
sugar beet pulp, empty fruit bunches from palm oil production, straw (e.g. wheat or rice), soy
bean hulls, residual meals from the vegetable oil industry (e.g. soybean, peanut, corn or
rapeseed), wheat bran and fermentation residue from the beer and wine industries.
Field remains include, but are not limited to, corn stover, post harvest cotton plants, post
harvest soybean bushes and post harvest rapeseed plants.
Lignocellulosic materials also include "energy crops" such as switch grass and broom
grass which grow rapidly and generate low-cost biomass specifically as a source of
carbohydrates.
These lignocellulosic carbohydrate sources contain cellulose, hemicellulose and lignin as
their main components and also contain mineral salts (ashes) and lipophilic organic compounds,
such as tall oils. The degree and type of theses non-carbohydrate materials can create technical
problems in production of soluble carbohydrates.
Lignocellulosic materials typically contain 65-80% cellulose and hemicelluloses on a dry
matter basis. Cellulose and hemicellulose are polysaccharides which can release carbohydrates
suitable for fermentation and/or chemical conversion to products of interest if they are
hydrolyzed. Lignin is typically resistant to acid hydrolysis.
It will be appreciated that the various aspects described above relate to solution of
technical problems associated with obtaining specific ratios of disaccharides and/or higher
oligomers relative to total saccharides produced by acid hydrolysis of a lignocellulosic substrate.
Alternatively or additionally, it will be appreciated that the various aspects described
above relate to solution of technical problems associated with the need for defined mixtures of
sugars containing specific disaccharides and/or higher oligomers. In many cases this "need" is
defined by a specific downstream application.
In some exemplary embodiments of the invention, there is provided a sugar mixture
including: (i) monosaccharides; (ii) oligosaccharides in a ratio to total saccharides > 0.06; (iii)
disaccharides in a ratio to total saccharides >_0.05; (iv) pentose in a ratio to total saccharides >
0.05; (v) at least one alpha-bonded di-glucose; and (vi) at least one beta-bonded di-glucose.
Optionally, the mixture has a higher oligosaccharides in a ratio to total saccharides <_0.2.
Optionally, the mixture has a ratio of at least one of the alpha-bonded di-glucose and the
beta-bonded di-glucose relative to total saccharides is > 0.01.
Optionally, the mixture has a ratio of at least one of the alpha-bonded di-glucose and the
beta-bonded di-glucose relative to total saccharides is > 0.03.
Optionally, the alpha-bonded di-glucose includes at least one member of the group
consisting of maltose, isomaltose and trehalose.
Optionally, the beta-bonded di-glucose includes at least one member selected from the
group consisting of gentiobiose, sophorose and cellobiose.
In some exemplary embodiments of the invention, there is provided a method including
(a) hydrolyzing a lignocellulosic material in a medium containing HCl in a ratio to (HCl + water)
> 0.37 to form a hydrolyzate including total saccharides in a ratio to (total saccharides + water) >
0.20 by weight; (b) de-acidifying the hydrolyzate to form a de-acidified hydrolyzate including:
(i) total saccharides in a ratio to (total saccharides + water) >_0.35 and; (ii) total disaccharides in
a ratio to total saccharides >_0.05; and (c) adjusting a composition of the de-acidified hydrolyzate
to form a mixture according to any of claims 1 to 6.
In some exemplary embodiments of the invention, there is provided a method including
(a) hydrolyzing a lignocellulosic material in a medium containing HCl in a ratio to (HCl
+ water) > 0.37 by weight to form a hydrolyzate including total saccharides in a ratio to (total
saccharides + water) >_0.20 by weight; (b) de-acidifying the hydrolyzate to form a de-acidified
hydrolyzate including a mixture as described above.
Optionally, the hydrolyzing is conducted in a counter-current mode of operation.
Optionally, the hydrolyzing is conducted at a temperature of less than 25°C.
Optionally, the lignocellulosic material includes softwood, for example, pine.
Optionally, the de-acidifying includes selective extraction of HCl and water with an
extractant including alcohol.
Optionally, the de-acidifying is conducted at a temperature of less than 80°C.
In some exemplary embodiments of the invention, there is provided a method including: (i)
providing a preparation including HCl and a sugar mixture according to any of claims 1 to 6, and
(ii) de-acidifying the preparation to form a de-acidified preparation.
Optionally, the de-acidifying includes selective extraction of HCl with an extractant
including alcohol.
Optionally, the de-acidifying is conducted at a temperature of less than 80°C.
In some exemplary embodiments of the invention, there is provided a method including:
(a) providing a fermentor; and (b) fermenting a medium including a sugar mixture as described
above in the fermentor to produce a fermentation product.
In some exemplary embodiments of the invention, there is provided a method including:
(a) providing a fermentor; and (b) fermenting a medium including a de-acidified hydrolyzate
according as described above or a de-acidified preparation as described above to produce a
fermentation product.
Optionally, the fermentation product includes at least one member selected from the
group consisting of alcohols, carboxylic acids, amino acids, monomers for the polymer industry
and proteins.
Optionally, the method includes processing the fermentation product to produce a
consumer product selected from the group consisting of detergent, polyethylene-based products,
polypropylene-based products, polyolefin-based products, polylactic acid (polylactide)- based
products, polyhydroxyalkanoate-based products and polyacrylic-based products.
Optionally, the detergent includes a sugar-based surfactant, a fatty acid-based surfactant,
a fatty alcohol-based surfactant, or a cell-culture derived enzyme.
Optionally, the polyacrylic-based product is selected from plastics, floor polishes,
carpets, paints, coatings, adhesives, dispersions, flocculants, elastomers, acrylic glass, absorbent
articles, incontinence pads, sanitary napkins, feminine hygene products, and diapers.
Optionally, the polyolefin-based products are selected from milk jugs, detergent bottles,
margarine tubs, garbage containers, water pipes, absorbent articles, diapers, non wovens , HDPE
toys and HDPE detergent packagings.
Optionally, the polypropylene based products are selected from absorbent articles,
diapers and non wovens.
Optionally, the polylactic acid based products are selected from packaging of agriculture
products and of dairy products, plastic bottles, biodegradable products and disposables.
Optionally, the polyhydroxyalkanoate based products are selected from packaging of
agriculture products, plastic bottles, coated papers, molded or extruded articles, feminine hygiene
products, tampon applicators, absorbent articles, disposable nonwovens and wipes, medical
surgical garments, adhesives, elastometers, films, coatings, aqueous dispersants, fibers,
intermediates of pharmaceuticals and binders.
Optionally, the fermentation product includes at least one member of the group consisting
of ethanol, butanol, isobutanol, a fatty acid, a fatty acid ester, a fatty alcohol and biodiesel.
Optionally, the method includes processing of the fermentation product to produce at
least one product selected from the group consisting of an isobutene condensation product, jet
fuel, gasoline, gasohol, diesel fuel, drop-in fuel, diesel fuel additive, and a precursor thereof.
Optionally, the gasahol is ethanol-enriched gasoline or butanol-enriched gasoline.
Optionally, the product is selected from the group consisting of diesel fuel, gasoline, jet
fuel and drop-in fuels.
In some exemplary embodiments of the invention, there is provided a consumer product,
a precursor of a consumer product, or an ingredient of a consumer product produced from a
fermentation product as described above.
In some exemplary embodiments of the invention, there is provided a consumer product,
a precursor of a consumer product, or an ingredient of a consumer product including at least one
fermentation product produced by a method as described above, wherein the fermentation
product is selected from carboxylic and fatty acids, dicarboxylic acids, hydroxylcarboxylic acids,
hydroxyl di-carboxylic acids, hydroxyl-fatty acids, methylglyoxal, mono-, di-, or poly-alcohols,
alkanes, alkenes, aromatics, aldehydes, ketones, esters, biopolymers, proteins, peptides, amino
acids, vitamins, antibiotics, and pharmaceuticals.
Optionally, the product is ethanol-enriched gasoline, jet fuel, or biodiesel.
In some exemplary embodiments of the invention, there is provided consumer product, a
precursor of a consumer product, or an ingredient of a consumer product according as described
above, wherein the consumer product has a ratio of carbon- 14 to carbon-12 of about 2.0 x 10 13
or greater.
Optionally, the consumer product including an ingredient according as described above
and an additional ingredient produced from a raw material other than lignocellulosic material.
Optionally, the ingredient and the additional ingredient produced from a raw material
other than lignocellulosic material are essentially of the same chemical composition.
Optionally, the consumable product includes a marker molecule at a concentration of at
least lOOppb.
Optionally, the marker molecule is selected from the group consisting of furfural,
hydroxy-methyl furfural, products of furfural or hydroxy-mathylfurfural condensation, color
compounds derived from sugar caramelization, levulinic acid, acetic acid, methanol, galcturonic
acid, and glycerol.
In some exemplary embodiments of the invention, there is provided a method including:
(a) de-acidifying an acid hydrolyzate including total saccharides in a ratio to (total
saccharides + water) > 0.20 by weight to produce a sugar mixture with total saccharides in a ratio
to (total saccharides + water) ratio > 0.35; the mixture including monosaccharides, the mixture
having disaccharides in a ratio to total saccharides > 0.05; and (b) enzymatically hydrolyzing the
mixture with an enzyme capable of catalyzing hydrolysis of alpha bonds in the mixture so that at
least 10% of the disaccharides are converted to monosaccharides; and (c) converting at least a
portion of the saccharides to a conversion product.
Optionally, the sugar mixture includes higher oligosaccharides.
Optionally, at least 10% of the higher oligosaccharides are hydrolyzed.
Optionally, the acid hydrolyzate is the result of counter-current hydrolysis.
Optionally, the acid hydrolyzate is the result of hydrolysis conducted at a temperature of
less than 25°C.
Optionally, the de-acidifying includes extraction with an extractant including an alcohol.
Optionally, the de-acidifying is conducted at a temperature of less than 80°C.
Optionally, the enzymatically hydrolyzing includes use of an enzyme capable of
catalyzing hydrolysis of beta bonds.
Optionally, the enzyme includes at least one enzyme selected from the group consisting
of amylases cellulases, hemicellulases, transglucosidases, glucoamylases, alpha-glucosidases and
pullulanases.
Optionally, the enzymatically hydrolyzing includes use of an immobilized enzyme.
Optionally, at least a portion of the converting is conducted simultaneously with the
enzymatically hydrolyzing.
Optionally, the total saccharides ratio to (total saccharides + water) is > 0.15 during the
enzymatically hydrolyzing.
Optionally, the enzymatically hydrolyzing includes incubation of the mixture with a
microorganism.
Optionally, the converting includes fermentation.
Optionally, the sugar mixture includes at least one pentose in a ratio to total saccharides >
0.05.
Optionally, the de-acidifying includes extracting the hydrolyzate, with a first extractant
including an SI solvent to form an HCl-carrying first extract and an HCl-depleted sugar solution.
Optionally, the de-acidifying includes including chromatographically separating the HCldepleted
sugar solution to produce a monosaccharide enriched monomer cut and an acid cut
enriched in disaccharides and higher oligosaccharides.
Optionally, the de-acidifying includes subsequently extracting the HCl-depleted sugar
solution with a second extractant including SI and a second solvent (S2).
Optionally, the SI of the extracting and the subsequently extracting each independently
include at least one member selected from the group consisting of alcohols, ketones and
aldehydes having at least 5 carbon atoms and combinations thereof.
Optionally, the second extractant is characterized by at least one of:
a delta-P greater than the delta-P of the first extractant by at least 0.2 MPa1 2; and
a delta-H greater than the delta-H of the first extractant by at least 0.2 MPa1 2.
Optionally, S2 includes at least one member selected from the group consisting of C 1-C4
mono- or poly-alcohols, aldehydes and ketones.
Optionally, a ratio of HC1 to total saccharides in the sugar mixture is < 0.03 by weight.
Unless otherwise defined, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which this invention
belongs. Although suitable methods and materials are described below, methods and materials
similar or equivalent to those described herein can be used in the practice of the present
invention. In case of conflict, the patent specification, including definitions, will control. All
materials, methods, and examples are illustrative only and are not intended to be limiting.
As used herein, the terms "comprising" and "including" or grammatical variants thereof
are to be taken as specifying inclusion of the stated features, integers, actions, ratios or
components without precluding the addition of one or more additional features, integers, actions,
ratios, components or groups thereof. This term is broader than, and includes the terms
"consisting of and "consisting essentially of as defined by the Manual of Patent Examination
Procedure of the United States Patent and Trademark Office.
The term "method" refers to manners, means, techniques and procedures for
accomplishing a given task including, but not limited to, those manners, means, techniques and
procedures either known to, or readily developed from known manners, means, techniques and
procedures by practitioners of architecture and/or computer science.
Percentages (%) and/or ratios of sugars (saccharides) to a total mixture, as well as ratios
of various sugars to one another (e.g. monosaccharides to disaccharides) are W/W (weight per
weight) unless otherwise indicated. Percentages ( ) and/or ratios of HC1 are also expressed as
W/W (weight per weight) unless otherwise indicate.
BRIEF DESCRIPTION OF THE DRAWINGS
In order to understand the invention and to see how it may be carried out in practice,
embodiments will now be described, by way of non-limiting example only, with reference to the
accompanying figures. In the figures, identical and similar structures, elements or parts thereof
that appear in more than one figure are generally labeled with the same or similar references in
the figures in which they appear. Dimensions of components and features shown in the figures
are chosen primarily for convenience and clarity of presentation and are not necessarily to scale.
The attached figures are:
Fig. 1 is a schematic overview of a system illustrating the industrial context of some
exemplary embodiments of the invention;
Figs. 2a and 2b are each simplified flow diagrams of methods according to exemplary
embodiments of the invention;
Fig. 3 is a simplified flow diagram of a method according to an exemplary embodiment
of the invention;
Figs. 4a and 4b are each simplified flow diagrams of methods according to exemplary
embodiments of the invention;
Fig. 5 is a simplified flow diagram of a method according to an exemplary embodiment
of the invention; and
Fig. 6 is a simplified flow diagram of a portion of the method depicted in Fig. 5 in greater
detail according to an exemplary embodiment of the invention.
DETAILED DESCRIPTION OF EMBODIMENTS
Embodiments of the invention relate to sugar mixtures, their preparation and use.
Specifically, some embodiments of the invention can be used as substrates for microbial
fermentation. Optionally, the specific microorganisms employed are selected to utilize one or
more sugars present in the mixture. Alternatively or additionally, some embodiments of the
invention relate to adjusting one or more component ratios within a mixture to render the mixture
more valuable for a specific downstream application.
The principles and operation of methods according to exemplary embodiments of the
invention may be better understood with reference to the drawings and accompanying
descriptions.
Before explaining at least one embodiment of the invention in detail, it is to be understood
that the invention is not limited in its application to the details set forth in the following
description or exemplified by the Examples. The invention is capable of other embodiments or of
being practiced or carried out in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description and should not be regarded as
limiting.
Overview of exemplary system
Fig. 1 is a simplified schematic diagram of a system for acid hydrolysis of a
lignocellulosic substrate indicated generally as 100. Depicted system 100 includes a main
hydrolysis reactor 110 adapted to receive a lignocellulosic substrate input 112. Optionally,
substrate 112 is provided as wood chips, although any "woody material" as described in the
background can be used instead of wood.
Substrate 112 is brought into contact with a concentrated HCl solution in reactor 110 and
hemicellulose and/or cellulose in the substrate are hydrolyzed to produce a mixture of soluble
sugars and residual lignin. These materials are collected separately as lignin stream 120 and sugar
mixture 130, each of which contains a large amount of HCl.
Since the acid acts as a catalyst, it is not consumed in the process. In addition, residual
acid content of the product and the co-products should be low in order to enable their use. Acid
recovery from the hydrolyzate should be conducted under conditions minimizing thermal
degradation.
Details of exemplary hydrolysis methods and systems are described in detail in co
pending US provisional applications 61/483,777 and 61/487,319, each of which is fully
incorporated herein by reference.
This application is primarily concerned with processing of sugar mixture 130. The
processing includes removal of HCl and/or adjustment of the mixture to achieve one or more
desired ratios of mixture components (e.g. disaccharides and/or monosaccharides). This
processing is conducted in a sugar refining module, designated here generically as 200.
Optionally, additional sugar mixture is recovered from lignin stream 120 as described in
co-pending US provisional application US 61/491,243 which is fully incorporated herein by
reference. In some exemplary embodiments of the invention, this additional sugar mixture is
routed to refining module 200. According to various exemplary embodiments of the invention
this additional sugar mixture increase a total sugar yield and/or changes a composition of the
mixture.
As will be explained in greater detail hereinbelow, refining module 200 employs a flow of
organic solvent 155 (solid arrows) to extract HCl 140 (dashed arrows) from sugar mixture 130.
De-acidified sugars 230 are the primary product of refining module 200. Module 200 also
produces a stream of HCl 140 mixed with solvent 155 (depicted as parallel dashed and solid
arrows respectively for clarity) which is routed to a solvent/HCl recovery module 150. Recovery
module 150 separates HCl 140 from solvent 155. In some exemplary embodiments of the
invention, separation is by distillation. HCl 140 is recycled to hydrolysis reactor 110 and solvent
155 is recycled to refining module 200.
De-acidified sugars 230 can be used in various industrial conversion processes as
described hereinbelow. Optionally, additional adjustments are made on de-acidified sugar 230
prior to these conversion processes as described below.
Exemplary sugar mixtures:
Some exemplary embodiments of the invention relate to sugar mixtures. In some
exemplary embodiments of the invention, the mixtures can be characterized as: containing
monosaccharides (e.g. hexoses such as glucose and/or galactose and/or mannose) and having an
oligosaccharides to total saccharides ratio >_0.06. Optionally, the mixture includes disaccharides
and a disaccharides to total saccharides ratio >_0.05. Optionally, the mixture includes a pentose
(e.g. xylose and/or arabinose and/or ribose and/or lyxose) and a pentose to total saccharides ratio
> 0.05. In some exemplary embodiments of the invention, the mixture includes at least one
alpha-bonded di-glucose (glucose bonded to glucose) and/or at least one beta-bonded di-glucose.
Optionally, the mixture is characterized by a higher oligosaccharides to total saccharide ratio <
0.2.
According to various exemplary embodiments of the invention the alpha-bonded diglucose
includes one or more of maltose, isomaltose and trehalose and the beta-bonded diglucose
includes one or more of gentiobiose, sophorose and cellobiose.
In some exemplary embodiments of the invention a ratio of at least one of said alphabonded
di-glucose and said beta-bonded di-glucose relative to the total saccharide content is >
0.01, optionally > 0.03.
In some exemplary embodiments of the invention, the alpha-bonded di-glucose includes
maltose and/or isomaltose and/or trehalose and/or kojibiose and/or nigerose. In some exemplary
embodiments of the invention, the beta-bonded di-glucose includes gentiobiose and/or sophorose
and/or cellobiose and/or laminaribiose and/or beta-trehalose.
In some exemplary embodiments of the invention, a proportion of the alpha-bonded diglucose
and/or the beta-bonded di-glucose is at least 0.01, optionally at least 0.02, optionally at
least 0.03 relative to total saccharides.
According to some exemplary embodiments, the mixture includes multiple alpha-bonded
di-glucoses and the combined proportion of these di-glucoses to total saccharides is at least 0.03.
According to still another embodiment, the mixture includes multiple beta-bonded di-glucoses
and the combined proportion to total saccharides of these beta-bonded di-glucoses is at least
0.03.
Exemplary methods to make such mixtures:
Fig. 2a is a simplified flow diagram depicting an exemplary method of making sugar
mixtures as described above indicated generally as 202. According to the depicted method a
lignocellulosic material is hydrolyzed 210 in HC1. In some exemplary embodiments of the
invention, hydrolysis 210 employs a medium containing a ratio of HC1 to (HC1 + water) > 0.37
by weight. Hydrolysis 210 yields a hydrolyzate 212 comprising total saccharides to (total
saccharides + water) >0.20 by weight. The depicted method also includes de-acidifying 220
hydrolyzate 212 to form a de-acidified hydrolyzate 222 comprising: (i) a ratio of total
saccharides to (total saccharides + water) >0.35 and; (ii) a ratio of total disaccharides to total
saccharides >0.05. Optionally, an increase in saccharide concentration resulting from deacidification
220 contributes to an increase in value.
Method 202 also includes adjusting 230 a composition of de-acidified hydrolyzate 222 to
form a sugar mixture as described above. According to various exemplary embodiments of the
invention adjusting 230 includes, but is not limited to, one or more of concentration, dilution,
polishing, active carbon treatment, ion exchange chromatography and enzymatic oligomerization
(e.g. to form dimers). Optionally, glycosyltransferases are employed.
Fig. 2b is a simplified flow diagram depicting an additional exemplary method of making
sugar mixtures as described above indicated generally as 204. Depicted method 204 includes
hydrolyzing 210 to produce hydrolyzate 212 as described for method 202.
However, in depicted method 204 de-acidifying 225 of hydrolyzate 212 yields a deacidified
hydrolyzate comprising a mixture 240 according to an exemplary embodiment of the
invention as described above.
With regards to methods 202 and/or 204 hydrolysis 210 is optionally conducted in a
counter-current mode of operation and/or at a temperature of less than 25°C. In some exemplary
embodiments of the invention, the lignocellulosic material hydrolyzed includes softwood,
optionally pine.
According to various exemplary embodiments of methods 202 and/or 204 de-acidifying
220 or 225 includes selective extraction of HC1 with an alcohol. In some exemplary
embodiments of the invention, the alcohol has a water solubility of less than 15%, e.g. an alcohol
with 5 to 8 carbon atoms. Optionally, some water is extracted with the HC1. Optionally; the deacidifying
is conducted at a temperature of less than 80°C.
Exemplary de-acidification methods
Fig. 3 is a simplified flow diagram depicting an exemplary method of de-acidifying a
sugar mixture as described above indicated generally as 300. According to the depicted method a
preparation including HC1 and a sugar mixture as described above is provided 310 and deacidified
320. In some exemplary embodiments of the invention, de-acidifying 320 includes
selective extraction of HC1 with an alcohol. Optionally, hexanol or 2-ethyl-l-hexanol is
employed for this extraction. Optionally, de-acidifying 320 is conducted at a temperature of less
than 80°C, optionally less than 70°C and optionally less than 60°C.
As used herein, "selective extraction" indicates that the HCl/carbohydrate ratio in the
extract is greater than that ratio in the acidic hydrolyzate, optionally at least 5 fold greater,
optionally at least 10 fold.
In some exemplary embodiments of the invention, de-acidifying is conducted according
to methods disclosed in co-pending Israeli patent application IL 206,152 which is fully
incorporated herein by reference.
Exemplary downstream processing of sugar mixtures
Fig. 4a is a simplified flow diagram depicting an exemplary method of producing a
fermentation product from a sugar mixture as described above indicated generally as 402.
According to the depicted method a fermentor is provided 410 and a media comprising a sugar
mixture as described above is fermented 420 to produce a fermentation product 430.
Fig. 4b is a simplified flow diagram depicting an exemplary method of producing a
fermentation product from a sugar mixture as described above indicated generally as 404.
According to the depicted method a fermentor is provided 410 and a media comprising a deacidified
hydrolyzate as described above or a de-acidified preparation as described above is
fermented 422 to produce a fermentation product 432.
According to various exemplary embodiments of the invention fermentation product 430
and/or 432 includes an alcohol and/or a carboxylic acid and/or an amino acid and/or a monomer
for the polymer industry and/or a protein.
One common fermentation product is ethanol, which may be useful, for example, as a
fuel. For example, ethanol may be added to gasoline to produce "gasohol" as is commonly done
in the United States, or used as a fuel itself as commonly done in Brazil.
Optionally, the fermentation product is a protein. In some exemplary embodiments of the
invention, the protein is a heterologous protein and a disaccharide in the sugar mixture triggers
an inducible regulatory element in a construct containing a sequence encoding the protein. In
some exemplary embodiments of the invention, increasing an amount and/or ratio of a specific
disaccharide in the sugar mixture contributes to an increased yield of fermentation product 430
and/or 432 per unit of sugar mixture in the media fermented 420 and/or 422.
Additional methods including enzymatic hydrolysis
Fig. 5 is a simplified flow diagram depicting an exemplary method for the production of
a conversion product from a sugar mixture as described above indicated generally as 500.
According to the depicted method, an acid hydrolyzate (as described above) including total
saccharides to (total saccharides + water) > 0.20 by weight is de-acidified 510 to produce a sugar
mixture 520 with a total saccharide to (total saccharide + water) ratio > 0.35 including
monosaccharides and having a disaccharides to total saccharides ratio > 0.05. This mixture is an
exemplary sugar mixture as described above.
Depicted exemplary method 500 also includes enzymatically hydrolyzing 522 a sugar
mixture with an enzyme capable of catalyzing hydrolysis of alpha bonds in the mixture so that at
least 10%, optionally at least 20%, optionally at least 30% of the disaccharides are converted to
monosaccharides. The phrase "capable of catalyzing hydrolysis of alpha bonds" indicates any
enzyme with at least 5% of the activity of alpha amylase with respect to alpha bonds. Optionally,
at least 10% of the di-saccharides and higher saccharides are hydrolyzed to release additional
monosaccharides. Optionally, higher saccharides release additional disaccharides.
In some exemplary embodiments of the invention, enzymatically hydrolyzing 522 is
performed without having to concentrate dilute saccharide solutions, i.e. the enzymatic
hydrolysis is performed at relatively high sugar concentration.
The depicted method also includes converting 530 at least a portion of the saccharides to
a conversion product 540.
According to various exemplary embodiments of the invention converting includes
fermentation and/or chemical conversion. Chemical conversion can be, for example, catalytic
conversion.
According to various exemplary embodiments of the invention, converting and/or
enzymatic hydrolysis may each be conducted in multiple stages in various sequences.
Optionally, one or more converting processes are conducted between enzymatic hydrolysis
stages. Alternatively or additionally, one or more enzymatic hydrolysis stages are conducted
between converting processes.
In some exemplary embodiments of the invention, the sugar mixture comprises
oligosaccharides with at least three sugar units.
In some exemplary embodiments of the invention, the initial acid hydrolyzate results
from counter-current hydrolysis. Optionally, the acid hydrolysis is conducted at a temperature of
less than 25°C.
In some exemplary embodiments of the invention, de-acidifying 510 includes extraction
with an extractant including an alcohol. Optionally, de-acidifying 510 is conducted at a
temperature of less than 80°C.
In some exemplary embodiments of the invention, enzymatically hydrolyzing 522
includes hydrolyzing beta bonds.
According to various exemplary embodiments of the invention enzymatically
hydrolyzing 522 includes use of at least one of one enzyme belonging to EC 3.2.1 according to
the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology
(NC-IUBMB) Enzyme Nomenclature Recommendations. This class of enzymes includes, but is
not limited to, amylases cellulases, hemicellulases, transglucosidases, glucoamylases, alphaglucosidases
and pullulanases. Optionally, an alpha amylase and/or a beta amylase and/or a 1-4
alpha glucosidase are employed.
Optionally, one or more of these enzymes may be provided as part of an enzyme cocktail
and/or a cellular extract including other undefined enzymes.
In some exemplary embodiments of the invention, an immobilized enzyme is used for
enzymatically hydrolyzing 522. Optionally, immobilization contributes to hydrolysis of a greater
number of bonds per enzyme molecule.
In some exemplary embodiments of the invention, at least a portion of converting 530 is
conducted simultaneously with enzymatically hydrolyzing 522. Optionally, this concurrent
processing prevents buildup of hydrolysis products which might adversely effect enzyme
hydrolysis kinetics. For purposes of this specification and the accompanying claims the term
"simultaneously" is used in its art accepted sense (i.e. simultaneous saccharification and
fermentation).
Optionally, a ratio of total saccharides to (total saccharides + water) > 0.15, optionally >
0.2 is maintained during enzymatic hydrolyzation.
According to some exemplary embodiments, enzymatically hydrolyzing 522 includes a
single reaction, or temporally and/or distinct reactions, which reactions differ, in the composition
of enzymes employed and/or in temperature and/or in pH.
In some exemplary embodiments of the invention, enzymatically hydrolyzing 522
includes fermentation with micro-organisms that produce the desired enzyme(s).
Alternatively or additionally, converting 530 comprises fermentation.
Optionally, sugar mixture 520 includes at least one pentose and a ratio between the at
least one pentose and total saccharides is > 0.05 by weight.
Exemplary de-acidification processes
Fig. 6 is a simplified flow diagram which illustrates some exemplary ways in which deacidification
510 of Fig. 5 can be accomplished.
In the depicted exemplary embodiments of the invention, de-acidifying 510 includes
extracting 511 the hydrolyzate with a first extractant including an SI solvent to form an HC1-
carrying first extract 612 and an HCl-depleted sugar solution 614. Optionally, the first extractant
includes 70, 80, 90, 95 or substantially 100% of the SI solvent. However, HC1 may be present in
sugar solution 614 at an unacceptable level. If that is the case, one or more additional separation
strategies can optionally be employed.
According to various exemplary embodiments of the invention residual HC1 in HCldepleted
sugar solution 614 is removed by chromatographic separation 620 and/or by a
subsequent extraction 630 with a second extractant including SI and a second solvent (S2).
Optionally, the total solvent concentration in the second extractant is 50m 60, 70, 80, 90, 95 or
substantially 100% or intermediate percentages.
In those exemplary embodiments of the invention which employ chromatographic
separation 620 sugar solution 614 is separated into an HC1 containing "acid cut" 624 which is
enriched in disaccharides and higher saccharides relative to total saccharides and a "monomer
cut" 622 enriched in monosaccharides relative to total saccharides. In some exemplary
embodiments of the invention, acid cut 624 is subject to further treatment to separate saccharides
from HC1 and/or to adjust a ration of monosaccharides to total saccharides. Exemplary
chromatographic separation techniques are disclosed in co-pending application IL 211093 which
is fully incorporated herein by reference.
In those exemplary embodiments of the invention which employ a subsequent extraction
630, there is a selective transfer of HC1 to the second extractant to form a second extract 634 and
the de-acidified hydrolyzate 520 depicted here as de-acidified sugar mixture 632. Exemplary
subsequent extraction techniques are disclosed in co-pending PCT application IL201 1/000 130
which is fully incorporated herein by reference.
Optionally, the SI solvent employed in the initial extracting 511 and the subsequently
extracting 630 includes a same solvent and/or a different solvent. SI solvents employed in
exemplary embodiments of the invention include, but are not limited to alcohols (e.g. hexanol
and 2-ethyl hexanol), ketones and aldehydes having at least 5 carbon atoms and combinations
thereof.
In some exemplary embodiments of the invention, the second extractant is characterized
by a delta-P greater than the delta-P of the first extractant by at least 0.2 MPa 2 and/or a delta-H
greater than the delta-H of the first extractant by at least 0.2 MPa1 2.
According to various exemplary embodiments of the invention, S2 includes at least one
member selected from the group consisting of -C4 mono- or poly-alcohols, aldehydes and
ketones.
In some exemplary embodiments of the invention, a ratio of HC1 to total saccharides in
the de-acidified hydrolyzate 520 and/or 632 is < 0.03 by weight.
Exemplary industrial contexts of downstream processing
Potential downstream applications of soluble carbohydrates include, but are not limited
to, production of bio-fuels (e.g. ethanol, butanol or hydrocarbons), use in the food industry (e.g.
fermentation to citric acid or xanthan gum and conversion of xylose to xylitol for use as an
artificial sweetener) and industrially useful monomers.
As new processes are developed for the production of alternative fuels such as fatty acid
esters and hydrocarbons (directly formed by fermentation or produced by conversion of
fermentation products), the demand for soluble carbohydrates is expected to increase.
By way of example, sugar mixtures according to various exemplary embodiments of the
invention are expected to be useful in fermentors which employ inducible promoters such as that
described in US 7,713,725 for example.
Alternatively or additionally, sugar mixtures according to various exemplary
embodiments of the invention are expected to be useful in production of fatty ester compositions
such as that described in, for example, US 2010/0071259.
Alternatively or additionally, sugar mixtures according to various exemplary
embodiments of the invention are expected to be useful in extractive fermentation such as
described in, for example, WO 2009/042950.
Alternatively or additionally, sugar mixtures according to various exemplary
embodiments of the invention are expected to be useful in production of fatty acid derivatives as
described in, for example, WO 2008/119082.
Alternatively or additionally, sugar mixtures according to various exemplary
embodiments of the invention are expected to be useful in production of peptides as described in,
for example, US 7595173.
In summary, direct downstream products of sugar mixtures according to various
exemplary embodiments of the invention resulting from fermentation and/or conversion (e.g.
alcohols, lactic acid, acrylic acid and antimicrobial peptide) are expected to give rise to a wide
variety of products resulting from further processing of these conversion products. Such products
include, but are not limited to, automobile fuel (e.g. for automobiles and/or airplanes), diapers,
plastic consumer products and paint.
Exemplary consumer products
Some exemplary embodiments of the invention, relate to consumer products and their
manufacture or preparation.
In some exemplary embodiments of the invention, a sugar mixture according to one
exemplary embodiment of the invention is converted to a fermentation product according to one
or more additional embodiments of the invention. Optionally, the fermentation product includes
at least one member selected from the group consisting of alcohols, carboxylic acids, amino
acids, monomers for the polymer industry and proteins.
In some exemplary embodiments of the invention, the fermentation product is processed
to produce a consumer product. Exemplary consumer products include but are not limited to
detergent, polyethylene-based products, polypropylene-based products, polyolefin-based
products, polylactic acid (polylactide)- based products, polyhydroxyalkanoate-based products
and polyacrylic-based products.
In some exemplary embodiments of the invention, the detergent includes a sugar-based
surfactant and/or a fatty acid-based surfactant and/or a fatty alcohol-based surfactant and/or a
cell-culture derived enzyme.
In some exemplary embodiments of the invention, the polyacrylic-based product includes
a plastic and/or a floor polish and/or a carpet and/or a paint and/or a coating and/or an adhesive
and/or a dispersion and/or a flocculants and/or an elastomer and/or acrylic glass and/or an
absorbent articles (e.g. incontinence pads, sanitary napkins, feminine hygene products, and
diapers).
Polyolefin-based products may be, for example, milk jugs, detergent bottles, margarine
tubs, garbage containers, water pipes, absorbent articles, diapers, non woven fabrics (e.g. premoistened
towellettes) , HDPE toys and HDPE detergent packagings.
In some exemplary embodiments of the invention, a polypropylene based product is
provided as an absorbent articles, optionally a diaper.
In other exemplary embodiments of the invention, a polypropylene based product is
provided as anon woven fabric item, optionally a pre-moistened towellette.
Polylactic acid based products may be, for example, packaging of agriculture or dairy
products, plastic bottles, biodegradable products and disposables.
Polyhydroxyalkanoate based products may be, for example, packaging of agriculture
products, plastic bottles, coated papers, molded or extruded articles, feminine hygiene products,
tampon applicators, absorbent articles, disposable nonwovens and wipes, medical surgical
garments, adhesives, elastometers, films, coatings, aqueous dispersants, fibers, intermediates of
pharmaceuticals and binders.
Optionally, the fermentation product includes at least one member of the group consisting
of ethanol, butanol, isobutanol, a fatty acid, a fatty acid ester, a fatty alcohol and biodiesel. In
some exemplary embodiments of the invention, the fermentation product is processed to produce
an isobutene condensation product and/or a jet fuel and/or gasoline and/or gasohol and/or diesel
fuel and/or drop-in fuel and/or a diesel fuel additive and/or a precursor thereof. According to
various exemplary embodiments of the invention gasahol is ethanol-enriched gasoline or
butanol-enriched gasoline.
Optionally, the product is a diesel fuel. Optionally, the product is gasoline. Optionally,
the product is jet fuel. Optionally, the product is a drop-in fuel.
According to various exemplary embodiments of the invention a consumer product, a
precursor of a consumer product, or an ingredient of a consumer product is produced from a
fermentation product. Optionally, the consumer product, precursor of a consumer product, or
ingredient of a consumer product includes at least one fermentation product as described
hereinabove including one or more of carboxylic and fatty acids, dicarboxylic acids,
hydroxylcarboxylic acids, hydroxyl di-carboxylic acids, hydroxyl-fatty acids, methylglyoxal,
mono-, di-, or poly-alcohols, alkanes, alkenes, aromatics, aldehydes, ketones, esters,
biopolymers, proteins, peptides, amino acids, vitamins, antibiotics, and pharmaceuticals. In some
exemplary embodiments of the invention, the product is ethanol-enriched gasoline, jet fuel, or
biodiesel.
In some exemplary embodiments of the invention, a consumer product, or an ingredient
of a consumer product according as described above has a ratio of carbon-14 to carbon-12 of 1.8
x 10 13 , optionally 2.0 x 10~13 or greater.
In some exemplary embodiments of the invention, the consumer product includes an
ingredient as described above and an additional ingredient produced from a raw material other
than lignocellulosic material. Optionally, the ingredient and the additional ingredient are
essentially of the same chemical composition.
Some exemplary embodiments of the invention relate to a consumable product as
described above including a marker molecule at a concentration of at least lOOppb.
The marker molecule optionally includes one or more of furfural, hydroxy-methyl furfural,
products of furfural or hydroxy-mathylfurfural condensation, color compounds derived from
sugar caramelization, levulinic acid, acetic acid, methanol, galcturonic acid, and glycerol.
It is expected that during the life of this patent many EC 3.2.1 enzymes will be
characterized and the scope of the invention is intended to include all such new enzymes priori.
Although the invention has been described in conjunction with specific embodiments
thereof, it is evident that many alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications
and variations that fall within the spirit and broad scope of the appended claims.
Specifically, a variety of numerical indicators have been utilized. It should be understood
that these numerical indicators could vary even further based upon a variety of engineering
principles, materials, intended use and designs incorporated into the invention. Additionally,
components and/or actions ascribed to exemplary embodiments of the invention and depicted as
a single unit may be divided into subunits. Conversely, components and/or actions ascribed to
exemplary embodiments of the invention and depicted as sub-units/individual actions may be
combined into a single unit/action with the described/depicted function.
Alternatively, or additionally, features used to describe a method can be used to
characterize an apparatus and features used to describe an apparatus can be used to characterize a
method.
It should be further understood that the individual features described hereinabove can be
combined in all possible combinations and sub-combinations to produce additional embodiments
of the invention. The examples given above are exemplary in nature and are not intended to limit
the scope of the invention which is defined solely by the following claims. Specifically, the
invention has been described in the context of ethanol production but is widely applicable to any
fermentation or conversion process.
All publications, patents and patent applications mentioned in this specification are
herein incorporated in their entirety by reference into the specification, to the same extent as if
each individual publication, patent or patent application was specifically and individually
indicated to be incorporated herein by reference. In addition, citation or identification of any
reference in this application shall not be construed as an admission that such reference is
available as prior art to the present invention.
The terms "include", and "have" and their conjugates as used herein mean "including but
not necessarily limited to".
Additional objects, advantages, and novel features of various embodiments of the
invention will become apparent to one ordinarily skilled in the art upon examination of the
following examples, which are not intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated hereinabove and as claimed in the
claims section below finds experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above
descriptions, illustrate the invention in a non limiting fashion.
MA TERIALS AND METHODS
The following materials and methods are used in performance of experiments described in
examples hereinbelow:
Enzymes: Some enzymes (Examples 1-3) were from Novozymes (Novozymes A/S;
Denmark):
Spyrizyme Fuel HS: Glucoamylase with a 1-4 and 1-6 activity; 1425 AGU/g
declared activity; Light to dark-brown liquid.
Liquozyme SC DS: a-Amylase with 1-4 activity; 240 KNU-S/g declared activity;
Amber liquid.
Manufaturer's instructions are:
Spyrizyme Fuel HS : 0.025-0.035% (w/w) grain "as is"
Liquozyme SC DS : n.a.
Cellic CTec : 1.5%, 3.0%, and 15% w/w (g enzyme/g cellulose).
Cellic HTech : 0.1-0.5% (w/w TS).
Additional enzymes were obtained from Genencor (Danisco; Genencor, Beloit WI, USA):
Spezyme Alpha: <1% a-Amylase activity 13,775 (Alpha Amylase Units)/g (min.),
thermostable, dark-brown liquid, optimal pH 5.5-6.5.
Transglucosidase L-2000: 1-5% Active enzyme, having a 1-4 activity, yellow-brown
liquid, optimal pH 4.5-5.5.
The recommended usages of enzymes (A. Kelley, Genencor) are:
Spezyme Alpha: 0.025%
Transglucosidase L-2000: 0.01%
Accelerase BG: 8%
Accelerase DUET: 8%
Hydrolyzates: Hydrolyzates from Pinewood were employed as a substrate for enzymatic
analyses. The hydrolyzates were adjusted to contain a desired % TS (Total Suaccharides). All
experiments were conducted at pH 4.0-5.1 at various temperatures from 50 to 85°C. Loading
doses of the enzymes were also varied.
In all of the enzymatic hydrolysis experiments time 0 (hrs.) refers to the blank (without
enzyme).
Hydrolyzates from pinewood, sugarcane bagasse and eucalyptus wood were also subject
to detailed analyses of monosaccharide and disaccharide concentrations.
Control of reaction conditions: The hydrolyzate was diluted with deionized (DI) water
and buffer to a desired TS and pH. The enzyme solution was added and the flask was kept at a
selected temperature with stirring throughout incubation. Samples were withdrawn according to
an experimental schedule and the enzyme was denaturated by heating and filtered out.
Saccharide analyses:
Saccharride composition of the samples was analyzed by HPLC using a Varian Prostar®
and a Rezex RSO-Oligosaccharide Ag+, 10 x 200mm column and pre-column in the following
conditions:
Column Temp.: 80°C, Mobile Phase: Water (HPLC grade), Flow Rate: 0.3mL/min,
Injection: 5 - IOm (depending on sugars cone), Detector: RI, Detector Temp.: 40°C. The DP
groups HPLC results are given in area%, the x-axis in the graphs represents time (hrs.) and the yaxis
area%.
DP stands for degree of polymerization, so that DPI refers to all mono-saccharides, DP2
are all di-saccharides, DP3 are all tri-saccharides and DP>3 refers to oligosaccharides containing
more than three sugar units (DP groups given as %area).
Example 1:
Exemplary Enzymatic Hydrolysis of a
De-acidified Hydrolyzate with 30% TS using Spirizyme
In order to examine the influence of enzyme amounts on total monosaccharide yield,
enzyme amount was titrated and efficiency of enzymatic hydrolysis was measured.
In a first series of enzymatic hydrolysis experiment Spyrizyme Fuel HS and Liquozyme
SC DS were employed.
A pinewood hydrolyzate with 30% TS at pH 4.9 was hydrolyzed with 3mg enzyme
solution/lgr 100% sugar at a temperature of 62°C with stirring (enzyme was 0.3%). Results are
summarized in Table 1.
Table 1: Spirizyme Fuel HS (3mg enzyme solution/lgr 100% sugar)
In order to assess the impact of increasing the amount of enzyme, 33mg enzyme
solution/lgr 100% sugar were employed. Results are summarized in Table 2.
Table 2: Spirizyme Fuel HS (33mg enzyme solution/lgr 100% sugar)
Results summarized in Tables 1 and 2 demonstrate a trend towards increased proportion
of DPI and DP2 saccharides at the expense of DP3 and DP>3 saccharides. This trend increased
as a function of time.
In order to assess the impact of increasing the amount of enzyme even further, 65.6 mg
enzyme solution/lgr 100% sugar were employed. Results are summarized in Table 3.
Table 3: Spirizyme Fuel HS (65.6mg enzyme solution/lgr 100% sugar)
Results presented in Table 3 indicate that it is possible to increase the yield of
monosaccharides even further.
In order to assess the impact of increasing the amount of enzyme even further, 282.6 mg
enzyme solution/lgr 100% sugar were employed. Results are summarized in Table 4.
Table 4: Spirizyme Fuel HS (282.6mg enzyme solution/lgr 100% sugar)
Time (hr) DPI (%area) DP2 (%area) DP3 (%area) DP>3 (%area)
0 45.7 31.8 13.7 8.8
2.5 70.6 22.4 5.6 1.5
4.0 71.1 21.6 5.5 1.8
Results summarized in Tables 1 to 4 indicate that enzymes capable of hydrolyzing alpha
bonds can be employed to increase monosaccharide concentrations in acid hydrolyzates of
cellulose. The enzyme dose of 282.6 mg gave the highest yield of mono-saccharides.
However, other conditions may contribute to enzymatic hydrolysis results. Exemplary
other conditions include, but are not limited to, reaction temperature, exact nature of the sugars in
the hydrolyzate in terms of both bond type and oligomer length distribution.
EXAMPLE 2:
Influence of TS on Enzymatic Hydrolysis of a
De-acidified Hydrolyzate using Spirizyme
In order to examine the influence of TS in the substrate on total monosaccharide yield,
enzyme amount was titrated again against a hydrolyzate similar to that used in Example 1, but
with decreasing amounts of TS and efficiency of enzymatic hydrolysis was measured.
A pinewood hydrolyzate with 15% TS at pH 5.1 was hydrolyzed with 72.8 mg enzyme
solution/lgr 100% sugar at a temperature of 60°C with stirring (enzyme -243 of manufacturer's
recommendation). Results are summarized in Table 5. This experiment confirms results
presented in Table 2 of Example 1 and suggests that more dilute sugar solutions are more
amenable to enzymatic hydrolysis.
Table 5: Spirizyme Fuel HS (72.8 mg enzyme solution/lgr 100% sugar)
A pinewood hydrolyzate with 5% TS at pH 5.0 was hydrolyzed with 67 mg enzyme
solution/lgr 100% sugar at a temperature of 62°C with stirring (enzyme -233 times
manufacturer's recommendation). Results are summarized in Table 6. This experiment confirms
results presented in Table 2 of Example 1 and suggests that more dilute sugar solutions are more
amenable to enzymatic hydrolysis.
Table 6: Spirizyme Fuel HS (67 mg enzyme solution/lgr 100% sugar)
Results presented in table 6 confirm that enzymes capable of hydrolyzing alpha bonds can
be employed to increase monosaccharide concentrations in acid hydrolyzates of cellulose.
Comparison of results presented in table 6 with those presented in Tables 2 and 5 suggests
that dilution of sugar solutions prior to enzymatic hydrolysis contributes to an increase in
monosaccharide yield.
Again, the exact nature of the sugars in the hydrolyzate in terms of both bond type and
oligomer length distribution may influence the total yield of monosaccharides.
EXAMPLE 3:
Enzymatic Hydrolysis of a
De-acidified Hydrolyzate with 30% TS
using Liquozyme SC DS
In order to determine whether the results presented above were enzyme specific an
additional experiment was conducted on a 30% TS hydrolyzate using Liquozyme. A pinewood
hydrolyzate with 30% TS at pH 6.0 was hydrolyzed with 282.6 mg enzyme solution/lgr 100%
sugar at a temperature of 85°C with stirring (enzyme -942 times manufacturer's
recommendation for Spirizyme). Results are summarized in Table 7. This experiment confirms
results presented in Table 2 of Example 1 and suggests that more dilute sugar solutions are more
amenable to enzymatic hydrolysis.
Table 7: Liquozyme SC DS (282.6 mg enzyme solution/lgr 100% sugar)
Results presented in table 7 indicate that the ability of enzymes capable of hydrolyzing
alpha bonds to increase monosaccharide concentrations in acid hydrolyzates of cellulose is not
specific to Spirizyme Fuel HS.
These results also suggest that Liquozyme SC DS provides a higher yield of mono¬
saccharides than Spirizyme Fuel HS at 282.6mg enzyme solution/lgr 100% sugar (see Table 4
for comparison). However, the desirability of one enzyme over another may also be influence by
other considerations including, but not limited to, availability, purity, bond specificity and price.
EXAMPLE 4:
Enzymatic Hydrolysis of a
De-acidified Sugarcane Bagasse Hydrolyzate with Various Enzymes
In order to examine the possibility of applying enzymatic hydrolysis to hydrolyzates from
non-wood substrates, a series of experiments was conducted on hydrolyzates prepared from sugar
cane bagasse. The hydrolyzates used contained different TS (5%, 20%), the experiments were
conducted at pH 4.5-5.1, and at various temperatures (50 °C and 60°C). The loading doses of the
enzymes were also varied.
A first experiment was conducted using Spezyme Alpha at a concentration of_34.2mg
enzyme solution/lgr 100% sugar, at a temperature of 60°C and at pH 5.1, applied to a bagasse
hydrolyzate with 20% TS. Results are summarized in Table 8.
Table 8: Spezyme Alpha applied to Baggase hydrolyzate TS 20% (34.2mg enzyme
An additional experiment was conducted using Transglucosidase L-2000 at a
concentration of 14.8mg enzyme /lgr 100% sugar at a temperature of 60°C at pH 5.0 applied to a
bagasse hydrolyzate with 20% TS. Results are summarized in Table 9.
Table 9: Transglucosidase L-2000 applied to Baggase hydrolyzate TS 20% (14.8mg
enz me; about 148 times manufacturer's recommendation)
Results summarized in Tables 8 and 9 indicate that Transglucosidase L-2000 provides an
increase in mono-saccharides in the bagasse hydrolyzate while Spezyme Alpha does not provide
such an increase.
The results from Transglucosidase L-2000 (Table 9) confirm that enzymes capable of
hydrolyzing alpha bonds can be used to increase the yield of monosaccharides.
The negative results from Spezyme Alpha presented in table 8 suggest that there may be
inhibitors present in the hydrolyzate. At this stage it is not clear whether these inhibitors are
specific to the substrate subject to the initial acid hydrolysis (e.g. sugar cane bagasse as opposed
to pine wood) or are specific to the enzyme.
EXAMPLE 5:
Saccharide Composition of Hydrolyzates of a First Pine Wood
In order to obtain a sugar mixture, dry first pinewood was introduced into a six stage
hydrolysis reactor series in a counter-current operation as described in co-pending US provisional
application 61/48377 filed May 9, 201 1 and entitled "Hydrolysis systems and methods". This
application is fully incorporated herein by reference.
Briefly, an aqueous solution of 42% HC1 was introduced continually at a temperature of
10-15°C for 24 hours. The hydrolyzate was collected, HC1 was removed by extraction and the
deacidified hydrolyzate was concentrated to give a sugar composition. The composition was
analyzed by HPLC, the sample's total sugars was 74.3%. Analysis results of monosaccharides
and disaccharides are summarized in Tables 10 and 11 respectively. The results are calculated as
% from sample's refractive total saccharides (%/RTS).
Table 10: Monosaccharides in hydrolyzate of the first pine wood
Table 11: Disaccharides in hydrolyzate of the first pine wood
Results summarized in table 10 illustrate the presence of pentoses such as Arabinose and
Xylose and Rhamnose (de-oxy pentose). Since these pentoses are prone to degradation under
harsh acidic conditions, their presence suggests that the counter current design of the hydrolysis
reactor contributes to a "pentose sparing" effect.
Results summarized in Table 11 illustrate the presence of alpha-bonded di-glucose such
as Trehalose and Isomaltose, as well as the presence of beta-bonded di-glucose such as
Gentiobiose and Sophorose.
EXAMPLE 6:
Saccharide Composition of Hydrolyzates
of a Second Pine Wood
Similarly, a second pine wood was hydrolyzed, deacidified and concentrated to 77% TS.
Analysis results of monosaccharides and disaccharides are presented in Tables 12 and 13
respectively. The results are calculated as % from sample's refractive total saccharides (%/RTS).
Table 12: monosaccharides in hydrolyzate of the second wood
Table 13: results of disaccharides in hydrolyzate of the second wood
The second pine wood also contained 16.7% higher oligosaccharides.
Results summarized in tables 12 and 13 confirm the presence of pentose, of alpha-bonded
di-glucose and of beta-bonded di-glucose. There are differences in the saccharide profiles of
hydrolyzates of the two pine sources.
EXAMPLE 7:
Saccharide Composition of Hydrolyzate Prepared
from Non-Pine WoodSubstrates
In order to examine the effect of substrate composition on hydrolyzate composition in
terms of specific sugars, deacidified hydrolyzates prepared from sugar cane bagasse and
Eucalyptus wood were analyzed as in Example 6 .
Analysis results of monosaccharides and disaccharides from the sugar cane bagasse
hydrolyzate are presented in Tables 14 and 15 respectively. The results are calculated as % from
sample's refractive total saccharides (%/RTS).
Table 14: results of monosaccharides in hydrolyzate of sugar cane bagasse
Arabinose Galactose Glucose Xylose Mannose Fructose Sum
2.2 7.2 48.7 4.9 4.8 2.4 70.2
Table 15: results of disaccharides in hydrolyzate of sugar cane bagasse
Analysis results of monosaccharides and disaccharides from the eucalyptus wood
hydrolyzate are presented in Tables 16 and 17 respectively. The results are calculated as % from
sample's refractive total saccharides (%/RTS).
Table 16: results of monosaccharides in hydrolyzate of Eucalyptus wood
Table 17: results of disaccharides in hydrolyzate of sugar Eucalyptus wood
Results presented in tables 14 to 17, taken together with results presented in tables 10 to
13 suggest that the substrate used for the initial acid hydrolysis can influence the saccharide
profile of the resultant de-acidified hydrolyzate.

CLAIMS:
1. A sugar mixture comprising:
(i) monosaccharides;
(ii) oligosaccharides in a ratio to total saccharides > 0.06;
(iii) disaccharides in a ratio to total saccharides >_0.05;
(iv) pentose in a ratio to total saccharides >_0.05;
(v) at least one alpha-bonded di-glucose; and
(vi) at least one beta-bonded di-glucose.
2. A mixture according to Claim 1, having higher oligosaccharides in a ratio to
total saccharides <_0.2.
3. A mixture according to Claim 1, wherein a ratio of at least one of said alphabonded
di-glucose and said beta-bonded di-glucose relative to total saccharides is >
0.01.
4. A mixture according to Claim 3, wherein a ratio of at least one of said alphabonded
di-glucose and said beta-bonded di-glucose relative to total saccharides is >
0.03.
5. A mixture according to Claim 1, wherein said alpha-bonded di-glucose includes
at least one member of the group consisting of maltose, isomaltose and trehalose.
6. A mixture according to Claim 1, wherein said beta-bonded di-glucose includes
at least one member selected from the group consisting of gentiobiose, sophorose and
cellobiose.
7. A method comprising:
(a) hydrolyzing a lignocellulosic material in a medium containing HCl in a
ratio to (HCl + water) > 0.37 to form a hydrolyzate comprising total saccharides in a
ratio to (total saccharides + water) >_0.20 by weight;
(b) de-acidifying said hydrolyzate to form a de-acidified hydrolyzate
comprising: (i) total saccharides in a ratio to (total saccharides + water) >_0.35 and; (ii)
total disaccharides in a ratio to total saccharides >_0.05; and
(c) adjusting a composition of said de-acidified hydrolyzate to form a
mixture according to any of claims 1 to 6.
8. A method comprising:
(a) hydrolyzing a lignocellulosic material in a medium containing HCl in a
ratio to (HCl + water) > 0.37 by weight to form a hydrolyzate comprising total
saccharides in a ratio to (total saccharides + water) >_0.20 by weight;
(b) de-acidifying said hydrolyzate to form a de-acidified hydrolyzate
comprising a mixture according to any of claims 1 to 6.
9. A method according to Claim 7 or 8, wherein said hydrolyzing is conducted in a
counter-current mode of operation.
10. A method according to Claim 7 or 8, wherein said hydrolyzing is conducted at a
temperature of less than 25°C.
11. A method according to Claim 7 or 8, wherein said lignocellulosic material
comprises softwood.
12. A method according to Claim 11, wherein said softwood comprises pine.
13. A method according to Claim 7 or 8, wherein said de-acidifying comprises
selective extraction of HCl and water with an extractant including alcohol.
14. A method according to Claim 7 or 8, wherein said de-acidifying is conducted at
a temperature of less than 80°C.
15. A method comprising:
(i) providing a preparation comprising HCl and a sugar mixture according
to any of claims 1 to 6, and
(ii) de-acidifying said preparation to form a de-acidified preparation.
16. A method according to Claim 15, wherein said de-acidifying comprises selective
extraction of HCl with an extractant including alcohol.
17. A method according to Claim 15, wherein said de-acidifying is conducted at a
temperature of less than 80°C.
18. A method comprising:
(a) providing a fermentor; and
(b) fermenting a medium comprising a sugar mixture according to any one
of claims 1 to 6 in said fermentor to produce a fermentation product.
19. A method comprising:
(a) providing a fermentor; and
(b) fermenting a medium comprising a de-acidified hydrolyzate according to
claim 7 or 8 or a de-acidified preparation according to claim 15 to produce a
fermentation product.
20. A method according to Claim 18 or 19, wherein said fermentation product
includes at least one member selected from the group consisting of alcohols, carboxylic
acids, amino acids, monomers for the polymer industry and proteins.
21. A method according to claim 18 or 19, comprising processing said fermentation
product to produce a consumer product selected from the group consisting of detergent,
polyethylene-based products, polypropylene-based products, polyolefin-based products,
polylactic acid (polylactide)- based products, polyhydroxyalkanoate-based products and
polyacrylic-based products.
22. A method according to claim 21, wherein said detergent comprises a sugarbased
surfactant, a fatty acid-based surfactant, a fatty alcohol-based surfactant, or a cellculture
derived enzyme.
23. A method according to claim 21, wherein said polyacrylic-based product is
selected from plastics, floor polishes, carpets, paints, coatings, adhesives, dispersions,
flocculants, elastomers, acrylic glass, absorbent articles, incontinence pads, sanitary
napkins, feminine hygene products, and diapers.
24. A method according to claim 21, wherein said polyolefin-based products are
selected from milk jugs, detergent bottles, margarine tubs, garbage containers, water
pipes, absorbent articles, diapers, non wovens , HDPE toys and HDPE detergent
packagings.
25. A method according to claim 21, wherein said polypropylene based products are
selected from absorbent articles, diapers and non wovens.
26. A method according to claim 21, wherein said polylactic acid based products are
selected from packaging of agriculture products and of dairy products, plastic bottles,
biodegradable products and disposables.
27. A method according to claim 21, wherein said polyhydroxyalkanoate based
products are selected from packaging of agriculture products, plastic bottles, coated
papers, molded or extruded articles, feminine hygiene products, tampon applicators,
absorbent articles, disposable nonwovens and wipes, medical surgical garments,
adhesives, elastometers, films, coatings, aqueous dispersants, fibers, intermediates of
pharmaceuticals and binders.
28. A method according to claim 18 or 19, wherein said fermentation product
includes at least one member of the group consisting of ethanol, butanol, isobutanol, a
fatty acid, a fatty acid ester, a fatty alcohol and biodiesel.
29. A method according to claim 28, comprising processing of said fermentation
product to produce at least one product selected from the group consisting of an
isobutene condensation product, jet fuel, gasoline, gasohol, diesel fuel, drop-in fuel,
diesel fuel additive, and a precursor thereof.
30. A method according to claim 29, wherein said gasahol is ethanol-enriched
gasoline or butanol-enriched gasoline.
31. A method according to claim 29, wherein said product is selected from the
group consisting of diesel fuel, gasoline, jet fuel and drop-in fuels.
32. A consumer product, a precursor of a consumer product, or an ingredient of a
consumer product produced from a fermentation product according to claim 18 or 19.
33. A consumer product, a precursor of a consumer product, or an ingredient of a
consumer product comprising at least one fermentation product produced by a method
according to claim 18 or 19, wherein said fermentation product is selected from
carboxylic and fatty acids, dicarboxylic acids, hydroxylcarboxylic acids, hydroxyl dicarboxylic
acids, hydroxyl-fatty acids, methylglyoxal, mono-, di-, or poly-alcohols,
alkanes, alkenes, aromatics, aldehydes, ketones, esters, biopolymers, proteins, peptides,
amino acids, vitamins, antibiotics, and pharmaceuticals.
34. A consumer product according to claim 33, wherein said product is ethanolenriched
gasoline, jet fuel, or biodiesel.
35. The consumer product, a precursor of a consumer product, or an ingredient of a
consumer product according to claim 33, wherein said consumer product has a ratio of
carbon-14 to carbon-12 of about 2.0 x 10 13 or greater.
36. A consumer product comprising an ingredient according to claim 32 and an
additional ingredient produced from a raw material other than lignocellulosic material.
37. The consumer product according to claim 36, wherein said ingredient and said
additional ingredient produced from a raw material other than lignocellulosic material
are essentially of the same chemical composition.
38. A consumer product according to claim 32, comprising a marker molecule at a
concentration of at least lOOppb.
39. A consumer product according to claim 38, wherein said marker molecule is
selected from the group consisting of furfural, hydroxy-methyl furfural, products of
furfural or hydroxy-mathylfurfural condensation, color compounds derived from sugar
caramelization, levulinic acid, acetic acid, methanol, galcturonic acid, and glycerol.
40. A method comprising:
(a) de-acidifying an acid hydrolyzate comprising total saccharides in a ratio to
(total saccharides + water) > 0.20 by weight to produce a sugar mixture with total
saccharides in a ratio to (total saccharides + water) ratio > 0.35; said mixture comprising
monosaccharides, said mixture having disaccharides in a ratio to total saccharides >
0.05; and
(b) enzymatically hydrolyzing said mixture with an enzyme capable of
catalyzing hydrolysis of alpha bonds in said mixture so that at least 10% of said
disaccharides are converted to monosaccharides; and
(c) converting at least a portion of said saccharides to a conversion product.
41. A method according to claim 40, wherein said sugar mixture comprises higher
oligosaccharides.
42. A method according to claim 41, wherein at least 10% of said higher
oligosaccharides are hydrolyzed.
43. A method according to Claim 40, wherein said acid hydrolyzate is the result of
counter-current hydrolysis.
44. A method according to Claim 40, wherein said acid hydrolyzate is the result of
hydrolysis conducted at a temperature of less than 25°C.
45. A method according to Claim 40, wherein said de-acidifying comprises
extraction with an extractant including an alcohol.
46. A method according to Claim 40, wherein said de-acidifying is conducted at a
temperature of less than 80°C.
47. A method according to Claim 40, wherein said enzymatically hydrolyzing
comprises use of an enzyme capable of catalyzing hydrolysis of beta bonds.
48. A method according to Claim 40, wherein enzyme includes at least one enzyme
selected from the group consisting of amylases, cellulases, hemicellulases,
transglucosidases, glucoamylases, alpha-glucosidases and pullulanases.
49. A method according to Claim 40, wherein said enzymatically hydrolyzing
comprises use of an immobilized enzyme.
50. The method according to Claim 40, wherein at least a portion of said converting
is conducted simultaneously with said enzymatically hydrolyzing.
51. A method according to Claim 40, wherein the total saccharides ratio to (total
saccharides + water) is > 0.15 during said enzymatically hydrolyzing.
52. A method according to Claim 40, wherein said enzymatically hydrolyzing
comprises incubation of said mixture with a microorganism.
53. A method according to Claim 40, wherein said converting comprises
fermentation.
54. A method according to Claim 40, wherein said sugar mixture comprises at least
one pentose in a ratio to total saccharides > 0.05.
55. A method according to Claim 40, wherein said de-acidifying comprises
extracting said hydrolyzate, with a first extractant comprising an SI solvent to form an
HCl-carrying first extract and an HCl-depleted sugar solution.
56. A method according to claim 55, comprising chromatographically separating
said HCl-depleted sugar solution to produce a monosaccharide enriched monomer cut
and an acid cut enriched in disaccharides and higher oligosaccharides.
57. A method according to claim 55, comprising subsequently extracting said HCldepleted
sugar solution with a second extractant comprising SI and a second solvent
(S2).
58. A method according to Claim 57, wherein the SI of said extracting and said
subsequently extracting each independently include at least one member selected from
the group consisting of alcohols, ketones and aldehydes having at least 5 carbon atoms
and combinations thereof.
59. A method according to Claim 57, wherein said second extractant is
characterized by at least one of:
a delta-P greater than the delta-P of said first extractant by at least 0.2 MPa 2; and
a delta-H greater than the delta-H of said first extractant by at least 0.2 MPa 2.
60. A method according to Claim 57, wherein S2 includes at least one member
selected from the group consisting of -C mono- or poly-alcohols, aldehydes and
ketones.
61. A method according to Claim 40, wherein a ratio of HC1 to total saccharides in
said sugar mixture is < 0.03 by weight.