NZ739947B2 - Methods for saccharifying biomass - Google Patents
Methods for saccharifying biomass Download PDFInfo
- Publication number
- NZ739947B2 NZ739947B2 NZ739947A NZ73994712A NZ739947B2 NZ 739947 B2 NZ739947 B2 NZ 739947B2 NZ 739947 A NZ739947 A NZ 739947A NZ 73994712 A NZ73994712 A NZ 73994712A NZ 739947 B2 NZ739947 B2 NZ 739947B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- biomass
- xylose
- glucose
- window
- sugar
- Prior art date
Links
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- 230000002588 toxic Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- JFALSRSLKYAFGM-UHFFFAOYSA-N uranium(0) Chemical compound [U] JFALSRSLKYAFGM-UHFFFAOYSA-N 0.000 description 1
- 229940005605 valeric acid Drugs 0.000 description 1
- 230000003612 virological Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamins Natural products 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
- 238000001238 wet grinding Methods 0.000 description 1
- 229910052724 xenon Inorganic materials 0.000 description 1
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon(0) Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 1
- 101700016253 xynX Proteins 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- SJZRECIVHVDYJC-UHFFFAOYSA-N γ-Hydroxybutyric acid Chemical compound OCCCC(O)=O SJZRECIVHVDYJC-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
- C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/16—Butanols
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
Disclosed is a method of saccharifying biomass, the method comprising: saccharifying recalcitrance-reduced lignocellulosic biomass with a cellulase in the presence of an isomerization agent (a strong acid) to obtain saccharified biomass, the isomerization agent reducing at least some feedback inhibition of the cellulase during saccharification, and wherein the recalcitrance-reduced lignocellulosic biomass has been pre-treated with a method of pre-treatment comprising bombardment with electrons. The method includes a way of avoiding feedback inhibition during the production of useful products. In a particular embodiment, the isomerization agent is polystyrene sulfonic acid or xylose isomerase. tion of the cellulase during saccharification, and wherein the recalcitrance-reduced lignocellulosic biomass has been pre-treated with a method of pre-treatment comprising bombardment with electrons. The method includes a way of avoiding feedback inhibition during the production of useful products. In a particular embodiment, the isomerization agent is polystyrene sulfonic acid or xylose isomerase.
Description
METHODS FOR SACCHARIFYING BIOMASS
by Marshall , Thomas Craig man, Michael W. Finn
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application Nos.
61/579,552 and 61/579,559, both filed on December 22, 2011. The entire disclosures
of the above applications are incorporated herein by reference.
FIELD OF THE INVENTION
The invention pertains to efficiencies useful in the processing of biomass
materials. For e, the invention relates to processes that circumvent negative
feedback of tic reactions.
BACKGROUND
As demand for petroleum increases, so too does interest in renewable
feedstocks for manufacturing biofuels and biochemicals. The use of lignocellulosic
biomass as a feedstock for such manufacturing processes has been studied since the
1970s. Lignocellulosic biomass is attractive because it is abundant, renewable,
ically produced, and does not compete with food industry uses.
Many potential lignocellulosic feedstocks are available today, including
agricultural residues, woody biomass, pal waste, oilseeds/cakes and sea weeds,
to name a few. At present these materials are either used as animal feed, biocompost
materials, are burned in a cogeneration facility or are landfilled.
Lignocellulosic biomass is recalcitrant to degradation as the plant cell
walls have a structure that is rigid and compact. The structure comprises crystalline
ose fibrils ed in a hemicellulose matrix, surrounded by lignin. This
t matrix is difficult to access by enzymes and other chemical, biochemical and
biological processes. osic biomass materials (e.g., biomass material from which
substantially all the lignin has been removed) can be more accessible to enzymes and
other conversion processes, but even so,
lly-occurring cellulosic materials often have low yields (relative to theoretical yields)
when contacted with hydrolyzing enzymes. Lignocellulosic biomass is even more recalcitrant to
enzyme attack. Furthermore, each type of lignocellulosic biomass has its own specific
ition of cellulose, hemicellulose and lignin.
While a number of methods have been tried to extract structural carbohydrates from
lignocellulosic biomass, they are either are too expensive, produce too low a yield, leave
undesirable chemicals in the ing product, or simply degrade the sugars.
Monosaccharides from renewable biomass sources could become the basis of
chemical and fuels industries by replacing, supplementing or substituting petroleum and other
fossil feedstocks. However, techniques need to be ped that will make these
monosaccharides available in large quantities and at acceptable purities and .
SUMMARY OF THE INVENTION
ed herein are methods of increasing the efficiency of saccharification of
biomass. In particular, efficiencies can be achieved by avoiding negative feedback inhibition of
enzymatic reactions.
ed herein is a method of making a product, where the method includes:
saccharifying recalcitrance-reduced ellulosic biomass, and adding an ization agent
to the saccharified biomass. In some implementations, the saccharified biomass comprises a first
sugar and a second sugar and the isomerization agent is used to convert the second sugar to a
third sugar. The method may also include, in some cases, contacting the saccharified biomass
with a rganism to convert the first sugar and third sugar to one or more t(s).
Also provided herein is a method of making a t with a microorganism from a
first sugar and a second sugar, where the microorganism can convert the first sugar to the
t, but cannot metabolize the second sugar, and where the method includes: providing a
cellulosic or lignocellulosic biomass; saccharifying the biomass to make a saccharified biomass,
wherein the saccharified biomass comprises a first sugar and a second sugar; providing a
microorganism that is capable of converting the first sugar into a product, but wherein the
microorganism cannot metabolize the second sugar; combining the microorganism and the
saccharified biomass, thereby producing a microorganism-biomass combination; maintaining the
microorganism-biomass combination under conditions that enable the microorganism to convert
the first sugar to the product, producing a combination that comprises the t and
the second sugar; converting the second sugar to a third sugar, wherein the
microorganism is capable of converting the third sugar to the product; and
maintaining the microorganism under conditions that enable the rganism to
convert the third sugar to the product; thereby making a product with a
microorganism from the first sugar and the second sugar.
In another aspect, the ion features a method of increasing the amount
of a product made by a microorganism from a saccharified biomass, the method
comprising: providing a cellulosic or lignocellulosic biomass; saccharifying the
biomass to make a saccharified biomass, wherein the saccharified biomass comprises
a first sugar and a second sugar; ing a microorganism that is capable of
converting the first sugar into a product, but wherein the microorganism cannot
metabolize the second sugar; combining the microorganism and the saccharified
biomass, thereby producing a microorganism-biomass combination; maintaining the
microorganism-biomass combination under ions that enable the microorganism
to convert the first sugar to the t, producing a combination that comprises the
product and the second sugar; converting the second sugar to a third sugar, wherein
the microorganism is capable of converting the third sugar to the product; and
maintaining the microorganism under conditions that enable the microorganism to
convert the third sugar to the product; thereby increasing the amount of the product
made by the microorganism from the saccharified s.
[0011B] In another , the t invention provides method of producing
one or more products, the method comprising: saccharifying itrance-reduced
lignocellulosic biomass to produce a saccharified mixture comprising glucose and
xylose; adding an isomerization agent to the saccharified mixture to produce an
isomerized mixture comprising glucose, xylose and fructose; and contacting the
isomerized mixture with a microorganism that converts e, but not xylose, to
product.
In any of the methods ed herein, the lignocellulosic biomass can be
treated to reduce its recalcitrance to saccharification. The ent method is
selected from the group consisting of: bombardment with electrons, tion,
oxidation, pyrolysis, steam explosion, chemical treatment, mechanical treatment, or
freeze grinding. The treatment method can be bombardment with electrons.
In any of the methods, the conversion of the second sugar to the third
sugar can be done before maintaining the microorganism-biomass ation under
conditions that enable the microorganism to convert the first sugar to the product.
The conversion of the second sugar to the third sugar can be done immediately after
saccharification of the biomass, or it can be done during saccharification of the
biomass.
In the methods provided , the lignocellulosic biomass can be
selected from the group consisting of: wood, particle board, forestry wastes, sawdust,
aspen wood, wood chips,
[Text continued on page 4]
grasses, switchgrass, miscanthus, cord grass, reed canary grass, grain residues, rice hulls, oat
hulls, wheat chaff, barley hulls, agricultural waste, , canola straw, wheat straw, barley
straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, corn cobs, corn stover,
soybean stover, corn fiber, alfalfa, hay, t hair, sugar processing residues, bagasse, beet
pulp, agave bagasse, algae, seaweed, , sewage, offal, agricultural or industrial waste,
arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato,
taro, yams, beans, favas, s, peas, or mixtures of any of these. The lignocellulosic biomass
can be mechanically treated to reduce its bulk density and/or increase its surface area. For
instance, it can be uted, e.g., by dry milling, or by wet milling. The biomass can be
saccharif1ed with one or more cellulases.
In the methods provided herein, the isomerization agent can se an acid, e.g.,
polystyrene sulfonic acid.
In the methods provided , the microorganism-biomass combination can be
maintained at a pH of about 10 to about 14, or at a pH of about 11 to about 13. It can be
maintained at a temperature of about 10°C to about 30°C, or at a temperature of about 20°C. It
can also be maintained at a temperature of about 60°C to about 65°C. It can be maintained at a
pH of about 6.0 to about 7.5, or a pH of about 7.
In the methods, the second sugar can be glucose, and the third sugar can be fructose.
The isomerization agent can comprise an enzyme. Alternatively, the second sugar can be xylose,
and the third sugar can be se. The enzyme can be xylose isomerase.
The microorganism can be yeast. The product can be alcohol. The microorganism
can be Clostrz'dz'um spp., and the product can be ethanol, butanol, butyric acid, acetic acid, or
acetone. The microorganism can be Lactobacillus spp., and the product can be lactic acid.
It should be understood that this invention is not limited to the embodiments
disclosed in this Summary, and it is ed to cover ations that are within the spirit and
scope of the invention, as defined by the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing will be apparent from the following more particular description of
example embodiments of the invention, as illustrated in the accompanying drawings in which
like reference characters refer to the same parts throughout the ent views. The drawings are
not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the
present invention.
is a diagram illustrating the enzymatic hydrolysis of cellulose to glucose.
Cellulosic substrate (A) is converted by endocellulase (i) to cellulose (B), which is converted by
exocellulase (ii) to cellobiose (C), which is converted to e (D) by cellobiase (beta-
glucosidase) (iii).
is a flow diagram illustrating the action of cellulase on cellulose and cellulose
derivatives. Cellulose (200) is broken down to cellobiose (210) by endoglucanases and exo-
glucanases/cellobiohydrolases (205) (A), which is then broken down by beta-glucosidase (215)
to glucose (220) (B). Endoglucanases and exo-glucanases/cellobiohydrolases are directly
inhibited by cellobiose (210) (D) and glucose (E), and beta-glucosidase is ted by glucose
(C).
is a flow diagram illustrating the conversion of biomass (300) to a product
(340). The feedstock (300) is combined (A) with cellulase (305) and fluid to form a mixture
(310), which is then allowed to rify (B), producing sugars (320). As disclosed herein, an
additive (325) is combined (C) with the e of sugars (320) to make a mixture of sugars and
ve (330). The resulting sugars are then used (D) in ream processing to produce one
or more products (340), such as alcohol, lactic acid, or one or more of the sugars themselves.
DETAILED DESCRIPTION
Provided herein are s of increasing the efficiency of production of sugars
(and/or products made from the ) from saccharified biomass. The methods are especially
useful in cases where one or more sugars or products cause negative feedback, limiting the
amount of sugars or products that can be produced.
Typically, the methods begin with saccharifying a biomass. Sacchariflcation usually
produces a mixture of sugars. The mixture includes sugars that can be converted to a useful
product. However, the e of sugars can include sugars that cannot be lized by the
microorganism. As these non-utilizable sugars increase in concentration, they ent a
metabolic “dead-end.” Furthermore, some sugars may form the basis of feedback inhibition, and
limit the throughput of metabolic pathways that make desired sugars or other desired products.
Disclosed herein are methods for preventing such feedback inhibition, and increasing
the amount of sugars and other useful products from the saccharif1cation of biomass.
The e produced during saccharification can inhibit further production of
glucose. In one embodiment, ore, the invention encompasses the ive removal of
glucose by converting it to fructose (which does not inhibit rif1cation), thereby allowing
for the tion of additional glucose. e can be converted to fructose by the action of
enzymes (such as xylose isomerase), strong acids or chemicals (such as polystyrene sulfonic
acid). Likewise, xylose, which cannot be metabolized by many microorganisms, can be
converted by xylose isomerase into xylulose, which can be metabolized by many
microorganisms. In addition, xylulose often does not inhibit its own production, unlike glucose.
For instance, biomass can be saccharif1ed to produce a mixture of sugars, ing
e and xylose. Most yeast strains can metabolize glucose, e.g, to an alcohol, but not
xylose. Therefore, if the desired end product is alcohol, then increased saccharification, and
increased production of e, followed by fermentation, will produce more alcohol, but it will
also produce more xylose. While the xylose is not harmful, it can represent a metabolic “dead
end.” If the xylose is converted to xylulose, it can be fermented to alcohol, and tion
efficiency can be increased.
As shown in for example, during saccharification a cellulosic substrate (A) is
initially hydrolyzed by endoglucanases (i) at random ons producing oligomeric
intermediates (e.g., cellulose) (B). These intermediates are then substrates for exo-splitting
glucanases (ii) such as cellobiohydrolase to produce cellobiose from the ends of the cellulose
polymer. Cellobiose is a water-soluble l,4-linked dimer of glucose. Finally cellobiase (iii)
cleaves cellobiose (C) to yield glucose (D). Therefore, the endoglucanases are particularly
effective in ing the crystalline portions of cellulose and increasing the effectiveness of
exocellulases to produce cellobiose, which then requires the specificity of the cellobiose to
produce glucose. Therefore, it is evident that depending on the nature and structure of the
cellulosic ate, the amount and type of the three different enzymes may need to be ed.
As shown in hydrolysis of cellulose (200) to cellobiose (210) is a multi-step
process which includes initial breakdown at the solid-liquid interface via the synergistic action of
endoglucanases (EG) and ucanases/cellobiohydrolases (CBH) (205) (A). This initial
degradation is accompanied by fiarther liquid phase degradation, by hydrolysis of soluble
intermediate products such as oligosaccharides and cellobiose that are catalytically cleaved by
beta-glucosidase (BG; 215) (B) to glucose (220). However, cellobiose (210) directly inhibits (D)
both CBH and EG (205), and glucose (220) directly inhibits (C, E) not only BG (215), but also
CBH and EG (205). The invention as described herein reduces or avoids this tion.
shows a s for manufacturing a t (340) from a feedstock (300).
The feedstock can be pre-processed, such as by ion of the size and recalcitrance of the
feedstock. This can include, for example, optionally mechanically treating the feedstock and,
before and/or after this treatment, optionally treating the feedstock with r treatment, for
example, le bombardment, to further reduce its recalcitrance. The up-stream processed
feedstock (300) is then combined (A) with cellulase (305) and fluid to form a mixture (310),
which is then allowed to saccharify (B), producing sugars (320). As disclosed herein, an additive
(325) is combined (C) with the mixture of sugars (320) to make a mixture of sugars and additive
(330). The additive (325) increases the effectiveness of the cellulase during saccharification,
e.g., by reducing inhibition of the cellulase by iose and/or glucose. This increased
effectiveness of saccharif1cation results in increased levels of sugars, which are then used (D) in
downstream processing to produce one or more products (340), such as alcohol, lactic acid, or
one or more of the sugars themselves.
During saccharif1cation, the feedstock is treated with one or more cellulolytic
s, generally by combining the ock and the enzyme (305) in a fluid medium, e.g., an
aqueous solution. In some cases, the feedstock is boiled, steeped, or cooked in hot water prior to
saccharif1cation, as described in US. Pat. App. Pub. 100577 Al, filed October 18, 2011
and published April 26, 2012, the entire ts of which are incorporated herein by reference.
The additive can be added at the initiation of the saccharif1cation (B), for example,
with the biomass and cellulase. Alternatively, the additive can be added after some or all of the
saccharif1cation (B) has occurred. It can also be added at the start of producing a product.
The additive can be a chemical or an enzyme. es of suitable additives include
acids and bases. Bases can catalyze the Lobry-de-Bruyn-Alberda-van-Ekenstein ormation,
as described in more detail below. Acids can catalyze the hydrolysis of iose. Boronic
acids can be used to complex with the cis-diols of glucose. Xylose isomerase (a.k.a. glucose
isomerase) can be used to isomerize glucose to fructose.
The additive can be physically supported. Useful supports include but are not limited
to cationic polymeric supports, anionic polymeric supports, neutral polymeric supports, metal
oxide ts, metal carbonate supports, metal halide supports and/or es thereof. The
support can be added to the mixed sugars or can be stationary and the mixed sugars made to pass
through or over the ted additive.
The mixture containing the additive (330) can be returned to the biomass and
cellulase stage (310) to release more sugars before being r processed. This can include
returning the conditions to a state that preferably causes the saccharif1cation of cellulose rather
than conditions that favor the action of the additive. For example the pH can be optimized for
saccharif1cation in the acidic region (less than or equal to pH 7, less than or equal to pH 6, less
than or equal to pH 5) and greater than or equal to pH 2 (greater than or equal to pH 3, r
than or equal to pH 4). The temperature can be optimized for the action of cellulases, e.g., to
greater than or equal to 30°C (greater than or equal to 40°C, greater than or equal to 50°C,
greater than or equal to 60°C) and less than or equal to 90°C (less than or equal to 80°C, less
than or equal to 70°C, less than or equal to 60°C). Additional biomass, cellulase and additive
can optionally be added for increased production of sugars.
The sugar solution or suspension produced by saccharification can be ted to
downstream processing to obtain a desired t. For example, one or more of the sugars can
be isolated, and/or the solution can be fermented. When fermentation is utilized, the
tation product can be distilled. For example, sugars can be enated and sugar
alcohols isolated.
Without being bound by any particular theory, it is believed that this conversion
effectively removes glucose from the mix of sugars. As shown in this removal would
remove the inhibition steps C and E. This ses the overall saccharification of cellulose in
the biomass.
In many instances, the optimum temperature for using e isomerase ranges from
60 to 80°C. In the processes described herein, temperatures lower than the optimum may be
preferred because of cost and because the optimum temperature for other components of the
process can be different. For example cellulase ties are generally optimal between 30°C
and 65°C. A temperature range of about 60°C to about 65°C may therefore be preferred,
particularly if the glucose isomerase and cellulase are combined and used simultaneously. If
they are not used together, then optimal atures for each enzyme can be selected.
The optimum pH range for glucose isomerase ty is n pH 7 and 9. As
with the selection of the temperature range, in practicing this invention a lower pH can be
preferred because in some cases other components of the process may require a lower pH. For
example, cellulases are active over a range ofpH of about 3 to 7. The preferred pH for the
combined enzymes is ore generally at or below pH 7. If the glucose isomerase and
cellulase are not used together, then the optimal pH range for each enzyme can be selected.
Glucose isomerase can be added in any amount. For example, the concentration may
be below about 500 U/g of ose (lower than or equal to 100 U/g cellulose, lower than or
equal to 50 U/g cellulose, lower than or equal to 10 U/g cellulose, lower than or equal to 5 U/g
cellulose). The concentration can be at least about 0.1 U/g cellulose to about 500 U/g cellulose,
at least about 0.5 U/g ose to about 250 U/g cellulose, at least about 1 U/g ose to about
100 U/g cellulose, at least about 2 U/g cellulose to about 50 U/g cellulose.
In some cases, the addition of a glucose isomerase increases the amount of sugars
produced by at least 5 % (e.g., at least 10 %, at least 15 %, at least 20 %, at least 30, 40, 50, 60,
70, 80, 90, 100 %).
Another additive that can be used in the invention is, e.g., a chemical that increases
the activity of the saccharifying agent. The chemical can be, for example, a chemical that
facilitates the Lobry-de-Bruyn-van-Alberda-van-Ekenstein ormation (also called the
Lobry-de-Bruyn-van-Ekenstein transformation). This reaction forms an enol from an aldose
which can then form a ketose. For example, in the pH range of ll to 13 and at a temperature of
°C, alkali will catalyze the transformation of D-glucose into D-fructose and D-mannose.
Typically the reaction is base catalyzed, but it can also be acid catalyzed, or take place under
neutral conditions. As with the use of glucose isomerase, this on effectively removes
glucose.
As another example, an acid can be used to catalyze ysis of cellobiose. By
using chemical means to cleave cellobiose to glucose, rather than enzymatic or microbial means,
inhibition of these reactions by glucose does not occur.
In another example, the chemical can be one that reacts with glucose, such as a
c acid which binds preferentially to cis-diols.
The al can be on a t, for example, by polystyrene sulfonates (such as an
AmberlystTM) or polystyrene amines. The mixed sugars can be passed through the supported
chemical or flow over it. For example, the chemical can be a polystyrene ted boronic
acid. The glucose can be trapped as a borate by the polystyrene support and then ed at a
later stage, by addition of base for example.
XYLOSE ISOMERASE
Xylose isomerase (ES 5.3.1.5) is an enzyme the zes the chemical on back
and forth between D-xylose and D-xylulose. It is also known atically as glucose
isomerase and D-xylose aldose-ketose isomerase, and belongs to a family of isomerases,
specifically those intramolecular oxidoreductases onverting aldoses and ketoses. Other
names in common use include D-xylose isomerase, D-xylose ketoisomerase, and D-xylose ketol-
isomerase. The enzyme participates in pentose and glucuronate interconversions and fructose
and mannose metabolism. It is used rially to convert glucose to fructose in the
cture of high-fructose corn syrup. It is sometimes referred to as “glucose isomerase.”
“Xylose isomerase” and “glucose isomerase” are used interchangeably herein. In vitro, e
isomerase catalyzes the interconversion of glucose and fructose. In vivo, it catalyzes the
interconversion of xylose and xylulose.
Several types of enzymes are considered xylose isomerases. The first kind is
produced from Pseudomonas hydrophila. This enzyme has 160 times lower affinity to glucose
than xylose but nonetheless is useful for increasing the amount of fructose in the presence of
glucose. A second kind of enzyme is found in Escherichia intermedia. This enzyme is a
phophoglucose isomerase (EC 5.3.1.9) and can isomerize unphosphorylated sugar only in the
presence of arsenate. A glucose isomerase (EC 5.3.16) can be isolated from Bacillus
megaterium AI and is NAD linked and is specific to glucose. Another glucose isomerase having
similar activity is isolated from Paracolobacterium aerogenoides. Glucose isomerases produced
by heterolactic acid bacteria require xylose as an inducer and are vely unstable at high
temperature. The xylose isomerase (EC 5.3.1.5) is the most useful for commercial applications
as it does not require expensive cofactors such as NAD+ or ATP and it is relatively heat .
The glucose ases are usually produced intercellularly but reports of
extracellular secretion of glucose isomerases are known. The enzyme used can be isolated from
many bacteria including but not limited to: Actinomyces olivocinereus, Actinomyces
phaeochromogenes, Actinoplanes missouriensis, Aerobacter aerogenes,Aerobacter cloacae,
Aerobacter levanicum, Arthrobacter spp., Bacillus stearothermophilus, Bacillus megabacterium,
us coagulans, Bifidobacterium spp., Brevibacterium incertum, Brevibacterium
pentosoaminoacidicum, Chainia spp., Corynebacterium spp., Cortobacterium helvolum,
Escherichiafreundii, Escherichia intermedia, Escherichia coli, Flavobacterium arborescens,
Flavobacterium devorans, Lactobacillus brevis, Lactobacillus buchneri, acillusfermenti,
Lactobacillus opoeus, Lactobacillus gayonii, Lactobacillus plantarum, Lactobacillus
lycopersici, acillus pentosus, Leuconostoc mesenteroides, Microbispora rosea,
Microellobosporiaflavea, Micromonospora coerula, Mycobacterium spp., Nocardia asteroides,
Nocardia corallia, Nocardia dassonvillei, Paracolobacterium aerogenoides, Pseudonocardia
spp., Pseudomonas hydrophila, Sarcina spp., lococcus bibila, Staphylococcusflavovirens,
Staphylococcus echinatus, Streptococcus achromogenes, Streptococcus phaeochromogenes,
Streptococcusfracliae, Streptococcus roseochromogenes, Streptococcus olivaceus,
Streptococcus californicos, ococcus venuceus, Streptococcus virginial, Streptomyces
olivochromogenes, Streptococcus venezaelie, Streptococcus wedmorensis, ococcus
griseolus, Streptococcus glaucescens, Streptococcus bikiniensis, Streptococcus rubiginosus,
Streptococcus achinatus, Streptococcus cinnamonensis, ococcusfracliae, Streptococcus
albus, ococcus griseus, Streptococcus hivens, Streptococcus is, Streptococcus
murinus, Streptococcus nivens, Streptococcus sis, osporangium album,
Streptosporangium oulgare, Thermopolyspora spp., Thermus spp., monas spp. and
nas mobilis.
e isomerase can be used free in solution or immobilized on a support. Whole
cells or cell free enzymes can be immobilized. The support structure can be any insoluble
material. Support structures can be cationic, anionic or neutral materials, for example
diethylaminoethyl cellulose, metal oxides, metal chlorides, metal carbonates and yrenes.
Immobilization can be accomplished by any suitable means. For example immobilization can be
accomplished by contacting the support and the Whole cell or enzyme in a solvent such as water
and then removing the solvent. The solvent can be removed by any suitable means, for example
filtration or evaporation or spray drying. As r example, spray drying the Whole cells or
enzyme with a support can be effective.
Glucose isomerase can also be present in a living cell that produces the enzyme
during the s. For example a glucose isomerase producing bacteria can be tured in
the process with an ethanol fermenting bacteria. Alternatively, the glucose-isomerase-producing
bacteria can be first contacted with the substrate, followed by ating with an ethanol-
producing substrate.
Glucose isomerase can also be present within or ed from a cell also capable of a
fiarther useful transformation of sugars. For e a glucose fermenting species can be
genetically modified to n and express the gene for tion of glucose isomerase.
I. TREATMENT OF BIOMASS MATERIAL
A. PARTICLE BOMBARDMENT
One or more treatments with energetic particle bombardment can be used to process
raw feedstock from a wide y of different sources to extract useful substances from the
feedstock, and to provide partially degraded organic material which ns as input to further
processing steps and/or sequences. Particle bombardment can reduce the lar weight
and/or crystallinity of feedstock. In some embodiments, energy deposited in a material that
releases an electron from its atomic orbital can be used to treat the materials. The bombardment
may be provided by heavy charged particles (such as alpha particles or protons), electrons
(produced, for example, in beta decay or electron beam accelerators), or electromagnetic
radiation (for example, gamma rays, x rays, or ultraviolet rays). atively, radiation
produced by radioactive substances can be used to treat the feedstock. Any combination, in any
order, or concurrently of these treatments may be utilized. In another ch, electromagnetic
radiation (e.g., produced using electron beam emitters) can be used to treat the feedstock.
Each form of energy ionizes the biomass via particular interactions. Heavy charged
les primarily ionize matter via Coulomb scattering; fiarthermore, these interactions produce
energetic electrons that may further ionize matter. Alpha particles are cal to the nucleus of
a helium atom and are produced by the alpha decay of various radioactive nuclei, such as
isotopes of bismuth, polonium, astatine, radon, francium, radium, several actinides, such as
actinium, thorium, uranium, ium, curium, califomium, americium, and ium.
When particles are utilized, they can be neutral (uncharged), positively charged or
negatively charged. When charged, the charged particles can bear a single positive or negative
charge, or multiple charges, e.g., one, two, three or even four or more charges. In ces in
which chain scission is desired, positively charged particles may be desirable, in part, due to their
acidic nature. When particles are utilized, the particles can have the mass of a resting electron,
or greater, e.g., 500, 1000, 1500, or 2000 or more times the mass of a resting electron. For
example, the particles can have a mass of from about 1 atomic unit to about 150 atomic units,
e.g., from about 1 atomic unit to about 50 atomic units, or from about 1 to about 25, e.g., 1, 2, 3,
4, 5, 10, 12 or 15 atomic units. Accelerators used to accelerate the les can be electrostatic
DC, electrodynamic DC, RF linear, magnetic ion linear or continuous wave. For e,
cyclotron type accelerators are available from IBA (Ion Beam Accelerators, Louvain-la-Neuve,
Belgium), such as the RhodotronTM system, while DC type accelerators are available from RDI,
now IBA Industrial, such as the DynamitronTM. Ions and ion accelerators are discussed in
Introductory Nuclear Physics, Kenneth S. Krane, John Wiley & Sons, Inc. , Krsto Prelec,
FIZIKA B 6 (1997) 4, 177-206; Chu, William T., “Overview of Ion Beam Therapy”,
us-Ohio, ICRU-IAEA Meeting, 18-20 Mar. 2006; Iwata, Y. et al., “Altemating-Phase-
Focused IH-DTL for Heavy-Ion l Accelerators”, Proceedings of EPAC 2006, Edinburgh,
nd; and Leitner, C. M. et al., “Status of the Superconducting ECR Ion Source Venus”,
dings of EPAC 2000, Vienna, Austria.
The doses applied depend on the desired effect and the particular feedstock. For
example, high doses can break chemical bonds within feedstock ents and low doses can
increase chemical bonding (e.g., cross-linking) within feedstock components.
In some instances when chain scission is desirable and/or polymer chain
fianctionalization is desirable, particles heaVier than electrons, such as protons, helium nuclei,
argon ions, silicon ions, neon ions, carbon ions, phosphorus ions, oxygen ions or nitrogen ions
can be utilized. When ring-opening chain scission is desired, positively charged particles can be
utilized for their Lewis acid properties for ed ring-opening chain scission. For example,
when oxygen-containing fianctional groups are desired, treatment in the presence of oxygen or
even treatment with oxygen ions can be performed. For example, when nitrogen-containing
fianctional groups are desirable, treatment in the presence of nitrogen or even treatment with
nitrogen ions can be performed.
B. OTHER FORMS OF ENERGY
Electrons interact via Coulomb scattering and bremsstrahlung radiation produced by
changes in the ty of electrons. Electrons may be produced by radioactive nuclei that
undergo beta decay, such as es of iodine, cesium, technetium, and iridium. Alternatively,
an electron gun can be used as an electron source via thermionic emission.
Electromagnetic radiation interacts via three processes: photoelectric absorption,
Compton scattering, and pair production. The dominating ction is determined by the
energy of the incident radiation and the atomic number of the material. The summation of
interactions contributing to the absorbed radiation in cellulosic material can be expressed by the
mass absorption coefficient.
Electromagnetic radiation is subclassif1ed as gamma rays, x rays, ultraviolet rays,
infrared rays, microwaves, or radiowaves, depending on the wavelength.
For example, gamma radiation can be employed to treat the materials. Gamma
radiation has the age of a significant penetration depth into a y of material in the
. Sources of gamma rays include radioactive nuclei, such as isotopes of , calcium,
technetium, um, gallium, indium, iodine, iron, krypton, samarium, selenium, sodium,
m, and xenon.
Sources of x rays include on beam collision with metal targets, such as en
or molybdenum or alloys, or compact light sources, such as those produced commercially by
Lyncean.
Sources for ultraviolet radiation include deuterium or cadmium lamps.
s for infrared ion include sapphire, zinc, or selenide window ceramic
lamps.
Sources for microwaves include klystrons, Slevin type RF sources, or atom beam
sources that employ hydrogen, oxygen, or nitrogen gases.
Various other devices may be used in the methods disclosed herein, including field
ionization sources, electrostatic ion separators, field ionization generators, thermionic emission
sources, microwave discharge ion sources, recirculating or static rators, dynamic linear
accelerators, van de Graaff accelerators, and folded tandem accelerators. Such devices are
disclosed, for example, in US. Pat. No. 7,931,784 B2, the complete disclosure of which is
incorporated herein by reference.
C. ELECTRON BOMBARDMENT
1. Electron Beams
The feedstock may be treated with electron bombardment to modify its structure and
thereby reduce its recalcitrance. Such treatment may, for example, reduce the average molecular
weight of the feedstock, change the crystalline structure of the feedstock, and/or increase the
surface area and/or porosity of the feedstock.
Electron bombardment via an electron beam is generally preferred, because it
provides very high throughput and because the use of a vely low voltage/high power
electron beam device eliminates the need for expensive concrete vault shielding, as such devices
are “self-shielded” and provide a safe, efficient process. While the “self-shielded” devices do
include shielding (e.g. metal plate shielding), they do not require the construction of a te
vault, greatly reducing capital expenditure and often allowing an ng cturing facility
to be used without expensive modif1cation. Electron beam accelerators are available, for
example, from IBA (Ion Beam Applications, n-la-Neuve, Belgium), Titan Corporation
(San Diego, rnia, USA), and NHV Corporation n High Voltage, Japan).
Electron bombardment may be performed using an electron beam device that has a
nominal energy of less than 10 MeV, e.g., less than 7 MeV, less than 5 MeV, or less than 2 MeV,
e.g., from about 0.5 to 1.5 MeV, from about 0.8 to 1.8 MeV, from about 0.7 to 1 MeV, or from
about 1 to 3 MeV. In some implementations the l energy is about 500 to 800 keV.
The electron beam may have a relatively high total beam power (the combined beam
power of all rating heads, or, if multiple accelerators are used, of all rators and all
heads), e.g., at least 25 kW, e.g., at least 30, 40, 50, 60, 65, 70, 80, 100, 125, or 150 kW. In
some cases, the power is even as high as 500 kW, 750 kW, or even 1000 kW or more. In some
cases the electron beam has a beam power of 1200 kW or more.
This high total beam power is usually achieved by utilizing multiple accelerating
heads. For example, the electron beam device may include two, four, or more accelerating
heads. The use of multiple heads, each of which has a vely low beam power, prevents
excessive temperature rise in the material, thereby preventing burning of the material, and also
increases the uniformity of the dose through the thickness of the layer of material.
In some implementations, it is desirable to cool the material during on
bombardment. For example, the material can be cooled while it is being conveyed, for example
by a screw extruder or other conveying equipment.
To reduce the energy required by the recalcitrance-reducing s, it is desirable to
treat the material as quickly as possible. In general, it is preferred that treatment be performed at
a dose rate of greater than about 0.25 Mrad per second, e.g., greater than about 0.5, 0.75, l, 1.5,
2, 5, 7, 10, 12, 15, or even greater than about 20 Mrad per second, e.g., about 0.25 to 2 Mrad per
second. Higher dose rates generally require higher line speeds, to avoid thermal decomposition
of the material. In one implementation, the accelerator is set for 3 MeV, 50 mAmp beam
current, and the line speed is 24 feet/minute, for a sample thickness of about 20 mm (e.g.,
comminuted corn cob material with a bulk density of 0.5 g/cm3).
In some ments, electron bombardment is performed until the material receives
a total dose of at least 0.5 Mrad, e.g., at least 5, 10, 20, 30 or at least 40 Mrad. In some
embodiments, the treatment is performed until the material receives a dose of from about 0.5
Mrad to about 150 Mrad, about 1 Mrad to about 100 Mrad, about 2 Mrad to about 75 Mrad, 10
Mrad to about 50 Mrad, e.g., about 5 Mrad to about 50 Mrad, from about 20 Mrad to about 40
Mrad, about 10 Mrad to about 35 Mrad, or from about 25 Mrad to about 30 Mrad. In some
implementations, a total dose of 25 to 35 Mrad is preferred, d ideally over a couple of
seconds, e.g., at 5 Mrad/pass with each pass being applied for about one second. ng a
dose of greater than 7 to 8 Mrad/pass can in some cases cause l degradation of the
feedstock material.
Using multiple heads as discussed above, the material can be treated in multiple
passes, for example, two passes at 10 to 20 Mrad/pass, e.g., 12 to 18 Mrad/pass, separated by a
few seconds of cool-down, or three passes of 7 to 12 Mrad/pass, e.g., 9 to 11 ass. As
sed above, treating the material with several relatively low doses, rather than one high
dose, tends to prevent overheating of the material and also increases dose uniformity through the
thickness of the material. In some implementations, the material is stirred or otherwise mixed
during or after each pass and then smoothed into a uniform layer again before the next pass, to
r enhance treatment uniformity.
WO 96700
In some embodiments, electrons are accelerated to, for example, a speed of greater
than 75 percent of the speed of light, e.g., greater than 85, 90, 95, or 99 percent of the speed of
light.
In some embodiments, any sing described herein occurs on lignocellulosic
material that remains dry as acquired or that has been dried, e.g., using heat and/or reduced
re. For example, in some embodiments, the cellulosic and/or lignocellulosic material has
less than about five percent by weight retained water, measured at 25°C and at fifty percent
relative humidity.
Electron dment can be applied while the cellulosic and/or lignocellulosic
material is exposed to air, oxygen-enriched air, or even oxygen itself, or blanketed by an inert
gas such as nitrogen, argon, or helium. When maximum oxidation is desired, an oxidizing
nment is utilized, such as air or oxygen and the distance from the beam source is
zed to maximize reactive gas formation, e.g., ozone and/or oxides of nitrogen.
In some embodiments, two or more electron sources are used, such as two or more
ionizing sources. For example, samples can be treated, in any order, with a beam of electrons,
followed by gamma radiation and UV light having wavelengths from about 100 nm to about 280
nm. In some embodiments, samples are treated with three ionizing ion sources, such as a
beam of ons, gamma radiation, and energetic UV light. The biomass is conveyed through
the treatment zone where it can be bombarded with electrons. It is generally preferred that the
bed of biomass material has a relatively uniform thickness, as previously bed, while being
treated.
It may be advantageous to repeat the treatment to more thoroughly reduce the
recalcitrance of the s and/or filrther modify the biomass. In particular the process
parameters can be adjusted after a first (e.g, second, third, fourth or more) pass depending on the
recalcitrance of the al. In some embodiments, a conveyor can be used which includes a
circular system where the biomass is conveyed multiple times h the various processes
described above. In some other embodiments multiple treatment devices (e.g., electron beam
generators) are used to treat the biomass multiple (e.g., 2, 3, 4 or more) times. In yet other
embodiments, a single electron beam generator may be the source of le beams (e.g., 2, 3, 4
or more beams) that can be used for treatment of the biomass.
The effectiveness in changing the molecular/supermolecular structure and/or reducing
the recalcitrance of the biomass material depends on the electron energy used and the dose
applied, while exposure time depends on the power and dose.
In some embodiments, the treatment (with any electron source or a combination of
sources) is performed until the material receives a dose of at least about 0.05 Mrad, e.g., at least
about 0.1, 0.25, 0.5, 0.75, 1.0, 2.5, 5.0, 7.5, 10.0, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125,
150, 175, or 200 Mrad. In some embodiments, the treatment is performed until the material
es a dose of between 0.1-100 Mrad, 1-200, 5-200, 10-200, 5-150, 5-100, 5-50, 5-40, 10-50,
-75, 15-50, 20-35 Mrad.
In some embodiments, the treatment is performed at a dose rate of between 5.0 and
1500.0 kilorads/hour, e.g., n 10.0 and 750.0 kilorads/hour or between 50.0 and 350.0
kilorads/hours. In other embodiments the treatment is performed at a dose rate of between 10
and 10000 kilorads/hr, between 100 and 1000 kilorad/hr, or between 500 and 1000 kilorads/hr.
2. Electron Sources
Electrons interact via Coulomb scattering and bremsstrahlung radiation produced by
changes in the velocity of electrons. Electrons may be produced by radioactive nuclei that
undergo beta decay, such as isotopes of iodine, , technetium, and iridium. Alternatively,
an electron gun can be used as an electron source via onic emission and accelerated
through an accelerating potential. An on gun generates electrons, accelerates them through
a large potential (e.g., greater than about 500 thousand, greater than about 1million, greater than
about 2 million, greater than about 5 million, greater than about 6 million, greater than about 7
million, greater than about 8 million, greater than about 9 million, or even greater than 10 million
volts) and then scans them ically in the X-y plane, where the ons are initially
accelerated in the z direction down the tube and extracted through a foil window. Scanning the
on beam is useful for increasing the irradiation surface when irradiating als, e.g., a
biomass, that is ed through the scanned beam. Scanning the on beam also
distributes the thermal load homogenously on the window and helps reduce the foil window
e due to local heating by the electron beam. Window foil rupture is a cause of significant
ime due to subsequent necessary repairs and re-starting the electron gun.
Various other irradiating devices may be used in the methods disclosed herein,
including field ionization sources, electrostatic ion separators, field ionization generators,
thermionic emission sources, microwave discharge ion s, recirculating or static
accelerators, dynamic linear accelerators, van de Graaff accelerators, and folded tandem
accelerators. Such s are disclosed, for example, in US. Pat. No. 7,931,784 to Medoff, the
complete disclosure of which is orated herein by reference.
A beam of electrons can be used as the radiation source. A beam of electrons has the
memgmHfihghmmemmskgwL5¢newm10Mmdpflsmmmxhghmnmgmmfifls
containment, and less confinement ent. Electron beams can also have high electrical
efficiency (e.g., 80%), allowing for lower energy usage relative to other radiation methods,
which can translate into a lower cost of operation and lower greenhouse gas emissions
corresponding to the smaller amount of energy used. Electron beams can be ted, e.g., by
electrostatic generators, cascade generators, transformer tors, low energy accelerators with
a scanning , low energy accelerators with a linear cathode, linear accelerators, and pulsed
accelerators.
Electrons can also be more efficient at causing changes in the molecular structure of
biomass materials, for example, by the mechanism of chain scission. In addition, electrons
having energies of 0.5-10 MeV can penetrate low density materials, such as the biomass
materials described , e.g., materials having a bulk density of less than 0.5 g/cm3, and a
depth of 03-10 cm. Electrons as an ng radiation source can be , e.g., for relatively
thin piles, layers or beds of materials, e.g., less than about 0.5 inch, e.g., less than about 0.4 inch,
0.3 inch, 0.25 inch, or less than about 0.1 inch. In some embodiments, the energy of each
electron of the electron beam is from about 0.3 MeV to about 2.0 MeV (million electron volts),
e.g., from about 0.5 MeV to about 1.5 MeV, or from about 0.7 MeV to about 1.25 MeV.
Methods of irradiating materials are sed in US. Pat. App. Pub. 2012/0100577 Al, filed
October 18, 2011, the entire disclosure of which is herein incorporated by reference.
Electron beam irradiation devices may be procured commercially from Ion Beam
Applications (Louvain-la-Neuve, Belgium), the Titan Corporation (San Diego, California, USA),
and NHV Corporation (Nippon High Voltage, Japan). Typical electron energies can be 0.5
MeV, 1 MeV, 2 MeV, 4.5 MeV, 7.5 MeV, or 10 MeV. l electron beam irradiation device
power can be 1 KW, 5 KW, 10 KW, 20 KW, 50 KW, 60 KW, 70 KW, 80 KW, 90 KW, 100 KW,
2012/071093
125 KW, 150 KW, 175 KW, 200 KW, 250 KW, 300 KW, 350 KW, 400 KW, 450 KW, 500 KW,
600 KW, 700 KW, 800 KW, 900 KW or even 1000 KW.
Tradeoffs in considering electron beam ation device power specifications
include cost to operate, capital costs, iation, and device footprint. Tradeoffs in
considering exposure dose levels of electron beam irradiation would be energy costs and
nment, safety, and health (ESH) concerns. Typically, generators are housed in a vault,
e.g., of lead or concrete, especially for production from X-rays that are generated in the process.
Tradeoffs in ering electron energies include energy costs.
The electron beam irradiation device can produce either a fixed beam or a scanning
beam. A scanning beam may be advantageous with large scan sweep length and high scan
speeds, as this would effectively replace a large, fixed beam width. Further, available sweep
widths of 0.5 m, 1 m, 2 m or more are available. The scanning beam is preferred in most
embodiments describe herein because of the larger scan width and reduced possibility of local
heating and failure of the windows.
3. Electron Guns - Windows
When d with an electron gun, the biomass is irradiated as it passes under a
window, which is generally a metallic foil (6.g. , titanium, titanium alloy, aluminum and/or
silicon). The window is impermeable to gases, yet electrons can pass with low resistance while
being impermeable to gasses. The foil windows are preferably between about 10 and 100
microns thick (e.g., a window can be 10 microns thick, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 microns . Thin windows dissipate less
energy as an electron beam passes h them (e.g., the resistive heating is less since Power =
12R) which is advantageous with respect to ating the target material (e.g., biomass) with as
much energy as possible. Thin windows are also less mechanically strong and more likely to fail
which causes increased expense and more downtime for the equipment.
The foil window can be cooled by passing air or an inert gas over the . When
using an ure, it is generally preferred to mount the window to the enclosure and to cool the
window from the side outside of the enclosed conveying system to avoid lofting up any
particulates of the material being irradiated.
The system can include more than one window, e.g., a y window and a
secondary . The two windows may form the enclosure to contain the purging gases
and/or the cooling gases. The secondary window may serve a fianction as a “sacrificial” window,
to protect the primary window. The electron beam apparatus es a vacuum between the
electron source and the primary window, and breakage of the primary window is likely to cause
biomass material to be sucked up into the electron beam apparatus, resulting in damage, repair
costs, and equipment downtime.
The window can be r, ceramic, coated ceramic, composite or coated
composite. The secondary window can be, for instance, a continuous sheet/roll of polymer or
coated polymer, which can be advanced continuously or at intervals to provide a clean or new
section to serve as the secondary window.
The primary window and the ary window can be made from the same material,
or different materials. For instance, the primary window foil can be made from titanium,
scandium, vanadium, chromium, nickel, zirconium, niobium, molybdenum, ruthenium, rhodium,
palladium, hafnium, tantalum, tungsten, rhenium, platinum, iridium, or alloys or mixtures of any
of these. The secondary single-type window foil can be made from titanium, scandium,
vanadium, chromium, nickel, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium,
hafnium, tantalum, tungsten, rhenium, platinum, iridium, beryllium, aluminum, silicon, or alloys
or mixtures of any of these. The primary and secondary windows can be of the same material,
mixture of materials, or alloy, or different materials, mixtures of material or alloys. One or both
of the windows can be laminates of the same of different materials, mixtures of materials, or
alloys.
One ofmore of the windows can have a support ure across its face. The term
“single-type window”, as used herein, means a window with no support structure across its face.
The term “double-type ”, as used herein, means a window with a support structure across
its face, where the support structure ively divides the e of the window into two parts.
Such a double-type window is shown in US. Pat. No. 5,877,582 to Nishimura. Additional
support ures can also be used.
The y window foil and the secondary window foil can both be made from low
Z element. Alternatively, the primary window foil can be made from a high Z element, and the
ary window foil can be made from a low Z element.
WO 96700
The embodiments described herein do not preclude the inclusion of additional
windows, which may have a protective function, or may be included to modify the radiation
exposure.
The windows can be concave, flat or convex. It is generally preferred that the
window be slightly convex, in a direction away from the direction of the cooling fluid. This
curvature improves the mechanical strength of the window and increases the permitted
temperature levels as well as allowing a better flow path for the cooling fluid. On the side of the
scanning horn the curvature tends to be towards the vacuum (e.g., away from the cooling fluid)
due to the vacuum (e.g., about 10'5 to 10'10 torr, about 10'6 to 10'9 torr, about 10'7 to 10'8 torr).
The cooling of the window and/or concave shape of the window become especially
important for high beam currents, for example at least about 100 mA on gun currents (e.g. ,
at least about 110 mA, at least about 120 mA, at least about 130 mA, at least about 140 mA, at
least about 150 mA at least about 200 mA, at least about 500 mA, at least about 1000 mA)
because ive heating is approximately related to the square of the current as discussed above.
The windows can be any shape but typically are imately rectangular with a high aspect
ratio of the width to the length (where the width direction is the same as the width of the
conveying system perpendicular to the conveying direction, and the length is the same as the
direction of conveying). The distance of the window to the conveyed material can be less than
about 10 cm (e.g., less than about 5cm) and more than about 0.lcm (e.g, more than about lcm,
more than about 2 cm, more than about 3 cm, more than about 4 cm). It is also possible to use
le windows (e.g., 3, 4, 5, 6 or more) with different and varied shapes and configured in
different ways. For example a primary or secondary foil window can include one, two or more
windows in the same plane or layered and can include one or more support ures. For
example support structures can be a bar or a grid in the same plane and contacting the windows.
In some embodiments, the window that is d on the enclosed ing system
is a secondary foil window of a two foil window extraction system for a scanning electron beam.
In other embodiments there is no enclosure for conveying the biomass material, e.g., the biomass
is conveyed in air under the irradiation device.
A two-foil window tion system for a scanning electron beam has two windows,
a primary and a secondary window. lly the y window is closest to the electron
source, and is concave towards the top of the scanning horn due to the vacuum on that side of the
window. The secondary foil window tends to be flatter but is also concave in the same direction.
This curvature helps provide structural support to the window and is ically stronger than
a flat window. Alternatively the windows can be flat or curved in any direction. The window
foils are typically at least about 10 microns thick to about 30 microns thick (e.g., about 15-40
microns, about 20-30 microns, about 5-30 microns, about 8-25 microns, about 10-20 microns,
about 20-25 microns thick). The distance between the front surface of the primary window foil
and back surface of the secondary window foil is preferably less than 30 cm, more preferably
less than 20 cm, and most ably less than 10 cm. Sidewalls, in combination with the
y and secondary windows, can define an interior space. Electrons travel through both
windows to impinge on and penetrate the al (6.g. beneath. A first inlet
, biomass) disposed
can be included on one sidewall can be ed to allow a cooling fluid (e.g., a liquid or a gas)
to impinge on the y window foil. The cooling fluid can run along the window and then
reverse direction on meeting the far (opposite) wall and flow back generally through the center
of the interior space and then out h an exhaust port and or . A second inlet can be
included on the sidewall and can be arranged to allow cooling fluid to impinge on the secondary
window foil in a similar fashion. Optionally more inlets (e.g., 2, 3, 4, 5, 6 or more) can bring
cooling fluid to the primary and secondary window surfaces and multiple outlets (e.g., 2, 3, 4, 5,
6 or more) can allow the cooling fluid to exit the interior space. In some embodiments one or
more side walls can even be a mesh, screen or grate with many gs through which cooling
gas can flow while providing structural support to the windows.
Such window s are described in US. Provisional App. No. 61/711,801, by
Medoff et al., which was filed on October 10, 2012, the entire contents of which are incorporated
herein by reference. A variety of configurations for such a system will also be known to those of
ordinary skill in the art.
4. on Guns - Window Spacing
Although a large spacing between the windows can be advantageous, for example, for
the reasons described above, the large spacing poses some disadvantages. One disadvantage of a
large spacing n windows is that the electron beams will pass through a larger volume of
cooling gas which can cause energy losses. For example a 1MeV beam loses about 0.2 MeV/M
of energy, a 5 MeV beam loses about 0.23 MeV/M and a 10 MeV beam loses about 0.26
MeV/M. Therefore with a 1 MeV beam of electrons passing through 1 cm of air, the beam loses
only 0.2% of its energy, at 10 cm of air, the beam loses 2% of its energy, at 20 cm this is 4% of
its , while at 50 cm the energy loss is 10%. Since the electrons also have to travel from
the secondary foil window to the biomass through additional air, the gap between the windows
must be carefully controlled. Preferably, energy losses are less that about 20% (e.g., less than
%, less than 5% or even less than 1%). It is therefore advantageous to minimize the spacing
between the windows to decrease energy losses. Optimal g (e.g., e spacing)
between the windows (e.g., n the surface side of the electron window foil and the facing
surface of the secondary window foil) for the benefit of cooling as bed above and for the
benefit of ng energy loss are between about 2 and 20 cm (e.g., between about 3 and 20 cm,
between about 4 and 20 cm, between about 5 and 20 cm, between about 6 and 20 cm, between
about 7 and 20 cm, between about 8 and 20 cm, between about 3 and 15 cm, between about 4
and 15 cm, between about 5 and 15 cm, between about 6 and 15 cm, between about 7 and 15 cm,
between about 8 and 15 cm n about 3 and 10 cm, between about 4 and 10 cm, between
about 5 and 10 cm, between about 6 and 10 cm, between about 7 and 10 cm, between about 8
and 10 cm).
One of ordinary skill in the art will balance the advantages and disadvantages of
window spacing to suit their needs.
In some embodiments support structures for the windows can be used across the
windows, although these types of structures are less preferred because of energy losses that can
occur to the electron beam as it strikes these kinds of structures.
A large spacing between the windows can be advantageous because it defines a larger
volume between the windows and allows for rapid flowing of a large volume cooling gasses for
very nt cooling. The inlets and outlets are between 1mm and 120 mm in diameter (e.g.,
about 2 mm, about 5 mm about 10 mm, about 20 mm, about 50 mm or even about 100 mm).
The cooling gas flow can be at between about 500-2500 CFM (e.g., about 600 to 2500 CFM,
about 00 CFM, about 800 to 2500 CFM, about 1000 to 2500 CFM, about 600 to 2000
CFM, about 700-2000 CFM, about 800 to 2000 CFM, about 1000 to 2000 CFM, about 600 to
1500 CFM, about 700-1500 CFM, about 800 to 1500 CFM, about 1000 to 1500 CFM). In some
embodiments, about 50% of the gas is exchanged per about 60 seconds or less (e.g., in about 50
sec or less, in about 30 sec or less, in about 10 sec or less, in about 1 sec or less).
. Electron Guns - Cooling and Purging Gases
The cooling gas in the two foil window extraction system can be a purge gas or a
mixture, for example air, or a pure gas. In one embodiment the gas is an inert gas such as
nitrogen, argon, helium and or carbon dioxide. It is preferred to use a gas rather than a liquid
since energy losses to the electron beam are minimized. Mixtures of pure gas can also be used,
either pre-mixed or mixed in line prior to impinging on the windows or in the space between the
windows. The cooling gas can be cooled, for example, by using a heat ge system (e.g., a
chiller) and/or by using boil off from a condensed gas (e.g., liquid en, liquid helium).
When using an enclosure, the enclosed conveyor can also be purged with an inert gas
so as to maintain an atmosphere at a reduced oxygen level. Keeping oxygen levels low avoids
the formation of ozone which in some instances is undesirable due to its reactive and toxic
nature. For example the oxygen can be less than about 20% (e.g., less than about 10%, less than
about 1%, less than about 0.1%, less than about 0.01%, or even less than about 0.001% oxygen).
Purging can be done with an inert gas including, but not limited to, en, argon, helium or
carbon dioxide. This can be ed, for example, from a boil off of a liquid source (e.g., liquid
nitrogen or helium), generated or separated from air in situ, or supplied from tanks. The inert gas
can be recirculated and any residual oxygen can be removed using a st, such as a copper
catalyst bed. Alternatively, combinations of purging, recirculating and oxygen removal can be
done to keep the oxygen levels low.
The enclosure can also be purged with a reactive gas that can react with the biomass.
This can be done before, during or after the irradiation process. The reactive gas can be, but is
not limited to, nitrous oxide, a, oxygen, ozone, hydrocarbons, aromatic nds,
amides, peroxides, azides, halides, oxyhalides, phosphides, phosphines, arsines, sulfides, ,
boranes and/or hydrides. The reactive gas can be ted in the enclosure, e.g., by irradiation
(e.g., electron beam, UV irradiation, microwave irradiation, heating, IR radiation), so that it
reacts with the biomass. The biomass itself can be activated, for example by irradiation.
ably the biomass is activated by the electron beam, to produce radicals which then react
with the activated or unactivated ve gas, e.g., by radical coupling or quenching.
Purging gases supplied to an ed conveyor can also be cooled, for example
below about 25°C, below about 0°C, below about -40°C, below about -80°C, below about -
120°C. For example, the gas can be boiled off from a compressed gas such as liquid nitrogen or
sublimed from solid carbon dioxide. As an alternative e, the gas can be cooled by a
chiller or part of or the entire conveyor can be cooled.
6. Electron Guns - Beam Stops
In some embodiments the systems and methods include a beam stop (e.g., a shutter).
For example, the beam stop can be used to quickly stop or reduce the ation of material
t powering down the electron beam device. atively the beam stop can be used while
powering up the electron beam, e.g., the beam stop can stop the on beam until a beam
current of a desired level is achieved. The beam stop can be placed between the primary foil
window and secondary foil window. For example the beam stop can be mounted so that it is
movable, that is, so that it can be moved into and out of the beam path. Even partial coverage of
the beam can be used, for example, to control the dose of irradiation. The beam stop can be
mounted to the floor, to a conveyor for the biomass, to a wall, to the radiation device (e.g., at the
scan horn), or to any structural t. Preferably the beam stop is fixed in relation to the scan
horn so that the beam can be effectively controlled by the beam stop. The beam stop can
incorporate a hinge, a rail, wheels, slots, or other means allowing for its operation in moving into
and out of the beam. The beam stop can be made of any material that will stop at least 5% of the
electrons, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, at least 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even about 100% ofthe electrons.
The beam stop can be made of a metal including, but not limited to, stainless steel,
lead, iron, molybdenum, silver, gold, titanium, um, tin, or alloys of these, or laminates
(layered materials) made with such metals (e.g., metal-coated ceramic, metal-coated polymer,
coated composite, multilayered metal materials).
The beam stop can be cooled, for example, with a cooling fluid such as an aqueous
solution or a gas. The beam stop can be lly or completely hollow, for example with
cavities. Interior spaces of the beam stop can be used for cooling fluids and gases. The beam
stop can be of any shape, including flat, curved, round, oval, square, rectangular, beveled and
wedged shapes.
The beam stop can have ations so as to allow some electrons through, thus
controlling (e.g., reducing) the levels of radiation across the whole area of the window, or in
specific regions of the window. The beam stop can be a mesh formed, for example, from fibers
or wires. Multiple beam stops can be used, together or independently, to control the irradiation.
The beam stop can be ly controlled, e.g., by radio signal or hard wired to a motor for
moving the beam into or out of position.
D. TREATMENT OF BIOMASS MATERIAL -- SONICATION, PYROLYSIS,
OXIDATION, STEAM EXPLOSION
If desired, one or more sonication, sis, oxidative, or steam ion processes
can be used in addition to or instead of other treatments to further reduce the recalcitrance of the
biomass material. These processes can be applied before, during and or after another treatment
or treatments. These processes are described in detail in US. Pat. No. 065 to Medoff, the
filll disclosure of which is incorporated herein by reference.
11. BIOMASS MATERIALS
As used , the term “biomass materials” includes lignocellulosic, cellulosic,
starchy, and microbial materials.
Lignocellulosic materials e, but are not limited to, wood, particle board,
ry wastes (e.g., sawdust, aspen wood, wood chips), grasses, (e.g., switchgrass, miscanthus,
cord grass, reed canary grass), grain es, (e.g., rice hulls, oat hulls, wheat chaff, barley
hulls), agricultural waste (e.g., silage, canola straw, wheat straw, barley straw, oat straw, rice
straw, jute, hemp, flax, bamboo, sisal, abaca, corn cobs, corn stover, soybean stover, corn fiber,
alfalfa, hay, coconut hair), sugar sing residues (e.g., bagasse, beet pulp, agave bagasse), ,
algae, seaweed, manure, sewage, and mixtures of any of these.
In some cases, the lignocellulosic material includes comcobs. Ground or
hammermilled comcobs can be spread in a layer of relatively uniform thickness for irradiation,
and after irradiation are easy to disperse in the medium for filrther processing. To facilitate
harvest and collection, in some cases the entire corn plant is used, including the corn stalk, corn
kernels, and in some cases even the root system of the plant.
Advantageously, for l production, no additional nutrients (other than a nitrogen
source, e.g., urea or ammonia) are ed during fermentation of comcobs or cellulosic or
lignocellulosic materials containing significant s of comcobs. Other products may
require addition of trace metals, vitamins, or buffering capacity, but these adjustment are well
within the knowledge of those of ordinary skill in the art.
Comcobs, before and after comminution, are also easier to convey and disperse, and
have a lesser tendency to form ive mixtures in air than other cellulosic or lignocellulosic
materials such as hay and grasses.
osic materials include, for example, paper, paper products, paper waste, paper
pulp, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed matter
(e. g., books, catalogs, manuals, , calendars, ng cards, brochures, prospectuses,
newsprint), printer paper, polycoated paper, card stock, cardboard, paperboard, materials having
a high oc-cellulose content such as cotton, and mixtures of any of these. For example paper
products as described in US. App. No. 13/396,365 (“Magazine Feedstocks” by Medoff et al.,
filed February 14, 2012), the fill disclosure of which is incorporated herein by reference.
osic materials can also include lignocellulosic materials which have been de-
lignif1ed.
Starchy materials e starch itself, e.g., corn starch, wheat starch, potato starch or
rice starch, a derivative of starch, or a material that includes starch, such as an edible food
product or a crop. For example, the starchy material can be arracacha, buckwheat, banana,
barley, cassava, kudzu, oca, sago, sorghum, regular household potatoes, sweet potato, taro, yams,
or one or more beans, such as favas, lentils or peas. Blends of any two or more starchy als
are also starchy materials. Mixtures of starchy, cellulosic and or lignocellulosic materials can
also be used. For example, a biomass can be an entire plant, a part of a plant or ent parts of
a plant, e.g., a wheat plant, cotton plant, a corn plant, rice plant or a tree. The starchy materials
can be treated by any of the methods bed herein.
Microbial materials include, but are not limited to, any naturally occurring or
genetically modified microorganism or organism that contains or is capable of providing a
source of carbohydrates (e.g., cellulose), for example, protists, e.g., animal protists (e.g,
protozoa such as flagellates, amoeboids, ciliates, and sporozoa) and plant protists (e.g., algae
such alveolates, chlorarachniophytes, cryptomonads, ids, phytes, haptophytes, red
algae, stramenopiles, and viridaeplantae). Other es include seaweed, plankton (e.g.,
macroplankton, mesoplankton, microplankton, ankton, picoplankton, and
femptoplankton), lankton, bacteria (e.g., gram positive bacteria, gram negative bacteria,
and extremophiles), yeast and/or mixtures of these. In some instances, microbial biomass can be
ed from natural s, e.g., the ocean, lakes, bodies of water, e.g., salt water or fresh
water, or on land. Alternatively or in addition, microbial biomass can be obtained from culture
systems, e.g., large scale dry and wet culture and fermentation systems.
The biomass material can also include offal, and r sources of material.
In other embodiments, the biomass materials, such as cellulosic, starchy and
lignocellulosic feedstock materials, can be obtained from transgenic microorganisms and plants
that have been modified with respect to a wild type variety. Such modifications may be, for
example, through the iterative steps of ion and breeding to obtain d traits in a plant.
Furthermore, the plants can have had genetic material removed, modified, ed and/or added
with respect to the wild type variety. For e, genetically modified plants can be produced
by recombinant DNA methods, where genetic modifications include introducing or modifying
specific genes from parental varieties, or, for example, by using transgenic breeding wherein a
specific gene or genes are introduced to a plant from a different species of plant and/or bacteria.
Another way to create genetic ion is through mutation breeding wherein new alleles are
artificially created from endogenous genes. The artificial genes can be created by a variety of
ways including treating the plant or seeds with, for e, chemical mutagens (e.g., using
alkylating agents, epoxides, alkaloids, peroxides, formaldehyde), irradiation (e.g., ,
gamma rays, neutrons, beta particles, alpha particles, protons, deuterons, UV radiation) and
temperature shocking or other external stressing and subsequent ion techniques. Other
methods of providing modified genes is through error prone PCR and DNA shuffling ed
by insertion of the desired modified DNA into the desired plant or seed. Methods of introducing
the desired genetic variation in the seed or plant include, for example, the use of a bacterial
carrier, tics, calcium ate precipitation, electroporation, gene splicing, gene silencing,
lipofection, microinjection and viral carriers. Additional genetically modified materials have
been described in US. ation Serial No 13/396,369 filed February 14, 2012 the full
disclosure of which is incorporated herein by reference.
Any of the methods described herein can be practiced with mixtures of any biomass
materials described herein.
III. BIOMASS MATERIAL PREPARATION -- MECHANICAL TREATMENTS
The biomass can be in a dry form, for example with less than about 35% moisture
content (e.g., less than about 20 %, less than about 15 %, less than about 10 % less than about 5
%, less than about 4%, less than about 3 %, less than about 2 % or even less than about 1 %).
The biomass can also be delivered in a wet state, for example as a wet solid, a slurry or a
suspension with at least about 10 wt% solids (e.g., at least about 20 wt%, at least about 30 wt.
%, at least about 40 wt%, at least about 50 wt%, at least about 60 wt%, at least about 70
wt%).
The processes disclosed herein can utilize low bulk density materials, for example
osic or ellulosic feedstocks that have been ally pretreated to have a bulk
density of less than about 0.75 g/cm3, e.g., less than about 0.7, 0.65, 0.60, 0.50, 0.35, 0.25, 0.20,
0.15, 0.10, 0.05 or less, e.g., less than about 0.025 g/cm3.
Bulk density is determined using
ASTM D1895B. Briefly, the method involves filling a measuring cylinder ofknown volume
with a sample and obtaining a weight of the sample. The bulk density is calculated by dividing
the weight of the sample in grams by the known volume of the cylinder in cubic centimeters. If
desired, low bulk density materials can be densified, for example, by methods described in US.
Pat. No. 7,971,809 to Medoff, the full disclosure of which is hereby incorporated by reference.
In some cases, the pre-treatment sing includes screening of the biomass
material. ing can be through a mesh or perforated plate with a desired opening size, for
example, less than about 6.35 mm (1/4 inch, 0.25 inch), (e.g., less than about 3.18 mm (1/8 inch,
0.125 inch), less than about 1.59 mm (1/16 inch, 0.0625 inch), is less than about 0.79 mm (1/32
inch, 0.03125 inch), e.g., less than about 0.51 mm (1/50 inch, 0.02000 inch), less than about 0.40
mm (1/64 inch, 25 inch), less than about 0.23 mm (0.009 inch), less than about 0.20 mm
(1/ 128 inch, 0.0078125 inch), less than about 0.18 mm (0.007 inch), less than about 0.13 mm
(0.005 inch), or even less than about 0.10 mm (1/256 inch, 0.00390625 inch)). In one
configuration the desired biomass falls through the perforations or screen and thus biomass
larger than the perforations or screen are not irradiated. These larger materials can be re-
sed, for example by comminuting, or they can simply be removed from processing. In
r configuration material that is larger than the perforations is irradiated and the r
material is removed by the screening s or recycled. In this kind of a configuration, the
conveyor itself (for example a part of the conveyor) can be perforated or made with a mesh. For
example, in one particular embodiment the biomass material may be wet and the ations or
mesh allow water to drain away from the biomass before irradiation.
Screening of al can also be by a manual method, for example by an operator or
mechanoid (e.g., a robot equipped with a color, reflectivity or other sensor) that removes
unwanted material. Screening can also be by magnetic screening wherein a magnet is disposed
near the conveyed material and the magnetic material is removed ically.
Optional pre-treatment sing can e heating the material. For example a
portion of the conveyor can be sent through a heated zone. The heated zone can be created, for
example, by IR radiation, aves, tion (e.g., gas, coal, oil, biomass), resistive
heating and/or inductive coils. The heat can be applied from at least one side or more than one
side, can be continuous or periodic and can be for only a portion of the material or all the
material. For example, a portion of the conveying trough can be heated by use of a heating
jacket. Heating can be, for example, for the e of drying the material. In the case of drying
the material, this can also be facilitated, with or without heating, by the movement of a gas (6.g.
air, oxygen, nitrogen, He, C02, Argon) over and/or through the biomass as it is being ed.
Optionally, pre-treatment processing can include cooling the material. Cooling
material is described in US Pat. No. 7,900,857 to Medoff, the disclosure of which in incorporated
herein by reference. For example, cooling can be by supplying a cooling fluid, for example
water (6.g. with ol), or nitrogen (e.g. to the bottom of the conveying
, , liquid nitrogen)
trough. Alternatively, a cooling gas, for example, chilled nitrogen can be blown over the
biomass materials or under the conveying system.
Another optional pre-treatment sing method can include adding a material to
the s. The additional material can be added by, for example, by showering, sprinkling
and or pouring the material onto the biomass as it is conveyed. Materials that can be added
include, for example, metals, ceramics and/or ions as bed in US. Pat. App. Pub.
105119 A1 (flled r 26, 2009) and US. Pat. App. Pub. 2010/0159569 A1 (flled
December 16, 2009), the entire disclosures of which are incorporated herein by reference.
Optional materials that can be added include acids and bases. Other materials that can be added
are oxidants (e.g., peroxides, chlorates), polymers, polymerizable monomers (e.g., containing
unsaturated bonds), water, catalysts, enzymes and/or organisms. Materials can be added, for
example, in pure form, as a solution in a t (e.g., water or an organic solvent) and/or as a
on. In some cases the solvent is volatile and can be made to evaporate e.g, by heating
and/or blowing gas as previously described. The added material may form a uniform coating on
the biomass or be a homogeneous mixture of different ents (e.g., s and additional
material). The added material can modulate the subsequent irradiation step by increasing the
efficiency of the irradiation, g the ation or ng the effect of the irradiation
(e.g., from electron beams to X-rays or heat). The method may have no impact on the irradiation
but may be useful for fiarther downstream sing. The added material may help in
conveying the material, for example, by lowering dust levels.
Biomass can be delivered to the or by a belt conveyor, a pneumatic conveyor,
a screw conveyor, a hopper, a pipe, manually or by a combination of these. The biomass can, for
example, be dropped, poured and/or placed onto the conveyor by any of these methods. In some
embodiments the al is delivered to the conveyor using an enclosed material distribution
system to help maintain a low oxygen atmosphere and/or control dust and fines. Lofted or air
suspended biomass fines and dust are undesirable e these can form an explosion hazard or
damage the window foils of an electron gun (if such a device is used for treating the material).
The al can be leveled to form a uniform thickness between about 0.03 12 and 5
inches (e.g., between about 0.0625 and 2.000 inches, between about 0. 125 and 1 inches, between
about 0. 125 and 0.5 inches, between about 0.3 and 0.9 inches, between about 0.2 and 0.5 inches
between about 0.25 and 1.0 inches, between about 0.25 and 0.5 inches, 0.100 +/- 0.025 inches,
0.l50 --/- 0.025 inches, 0.200 --/- 0.025 inches, 0.250 --/- 0.025 inches, 0.300 --/- 0.025 inches,
0.350 --/- 0.025 , 0.400 --/- 0.025 inches, 0.450 --/- 0.025 inches, 0.500 --/- 0.025 inches,
0.550 --/- 0.025 inches, 0.600 --/- 0.025 inches, 0.700 --/- 0.025 inches, 0.750 --/- 0.025 inches,
0.800 --/- 0.025 inches, 0.850 --/- 0.025 inches, 0.900 --/- 0.025 inches, 0.900 --/- 0.025 inches.
Generally, it is preferred to convey the al as quickly as possible through the
electron beam to maximize throughput. For example the material can be conveyed at rates of at
least 1 ft/min, e.g., at least 2 ft/min, at least 3 ft/min, at least 4 ft/min, at least 5 ft/min, at least 10
ft/min, at least 15 ft/min, 20, 25, 30, 35, 40, 45, 50 ft/min. The rate of conveying is related to the
beam current, for example, for a 14 inch thick biomass and 100 mA, the conveyor can move at
about 20 ft/min to provide a useful irradiation dosage, at 50 mA the conveyor can move at about
ft/min to provide approximately the same irradiation dosage.
After the biomass material has been ed through the radiation zone, optional
post-treatment processing can be done. The optional reatment processing can, for example,
be a process described with respect to the pre-irradiation processing. For example, the biomass
can be screened, heated, cooled, and/or ed with additives. Uniquely to post-irradiation,
quenching of the radicals can occur, for example, quenching of radicals by the addition of fluids
or gases (e.g., oxygen, nitrous oxide, ammonia, liquids), using pressure, heat, and/or the addition
of l gers. For example, the biomass can be conveyed out of the enclosed conveyor
and exposed to a gas (e.g., oxygen) Where it is quenched, forming caboxylated . In one
embodiment the biomass is exposed during irradiation to the reactive gas or fluid. Quenching of
biomass that has been irradiated is bed in US. Pat. No. 8,083,906 to Medoff, the entire
sure of which is incorporate herein by reference.
If desired, one or more mechanical treatments can be used in addition to irradiation to
fiarther reduce the recalcitrance of the biomass material. These processes can be applied before,
during and or after irradiation.
In some cases, the mechanical treatment may include an initial preparation of the
feedstock as received, e.g., size reduction of materials, such as by comminution, e.g., cutting,
grinding, shearing, pulverizing or chopping. For example, in some cases, loose feedstock (e.g.,
recycled paper, starchy materials, or switchgrass) is prepared by shearing or shredding.
ical treatment may reduce the bulk density of the biomass material, increase the surface
area of the biomass material and/or decrease one or more dimensions of the biomass material.
Alternatively, or in on, the feedstock material can first be physically treated by
one or more of the other physical treatment methods, 6.g. chemical ent, radiation,
sonication, oxidation, pyrolysis or steam explosion, and then mechanically treated. This
ce can be advantageous since materials treated by one or more of the other treatments,
6.g. irradiation or pyrolysis, tend to be more e and, therefore, it may be easier to further
change the structure of the material by mechanical treatment. For example, a ock material
can be conveyed through ionizing radiation using a conveyor as described herein and then
ically d. Chemical treatment can remove some or all of the lignin (for example
chemical pulping) and can partially or completely hydrolyze the material. The methods also can
be used with pre-hydrolyzed material. The methods also can be used with material that has not
been pre hydrolyzed The methods can be used with mixtures of yzed and non-hydrolyzed
materials, for e with about 50% or more non-hydrolyzed material, with about 60% or
more non- hydrolyzed material, with about 70% or more non-hydrolyzed material, with about
80% or more non-hydrolyzed material or even with 90% or more non-hydrolyzed material.
In addition to size reduction, which can be performed initially and/or later in
processing, mechanical treatment can also be ageous for “opening up,3, “stressing,”
ng or shattering the biomass als, making the cellulose of the materials more
susceptible to chain scission and/or disruption of crystalline structure during the physical
treatment.
Methods of mechanically treating the biomass material include, for example, milling
or grinding. Milling may be performed using, for example, a hammer mill, ball mill, colloid
mill, conical or cone mill, disk mill, edge mill, Wiley mill, grist mill or other mill. Grinding may
be med using, for example, a cutting/impact type grinder. Some exemplary grinders
include stone grinders, pin grinders, coffee grinders, and burr grinders. ng or milling may
be provided, for example, by a reciprocating pin or other element, as is the case in a pin mill.
Other mechanical treatment methods include ical ripping or tearing, other methods that
apply pressure to the fibers, and air attrition milling. Suitable mechanical treatments filrther
include any other technique that continues the disruption of the internal structure of the material
that was initiated by the previous sing steps.
Mechanical feed ation systems can be configured to produce streams with
specific characteristics such as, for example, specif1c maximum sizes, specif1c length-to-width,
or c surface areas ratios. Physical preparation can increase the rate of ons, improve
the movement of material on a conveyor, improve the irradiation profile of the material, improve
the radiation uniformity of the material, or reduce the processing time required by opening up the
materials and making them more ible to processes and/or reagents, such as reagents in a
The bulk density of ocks can be controlled (e.g., increased). In some situations,
it can be desirable to prepare a low bulk density material, 6.g. the material (e.g.,
, by densifying
1cation can make it easier and less costly to transport to another site) and then reverting the
material to a lower bulk density state (e.g., after transport). The material can be densif1ed, for
example from less than about 0.2 g/cc to more than about 0.9 g/cc (e.g., less than about 0.3 to
more than about 0.5 g/cc, less than about 0.3 to more than about 0.9 g/cc, less than about 0.5 to
more than about 0.9 g/cc, less than about 0.3 to more than about 0.8 g/cc, less than about 0.2 to
more than about 0.5 g/cc). For example, the material can be densified by the methods and
equipment disclosed in US. Pat. No. 7,932,065 to Medoff and International Publication No. WO
2008/073186 (which was filed October 26, 2007, was published in English, and which
designated the United States), the filll disclosures of which are incorporated herein by reference.
Densified materials can be processed by any of the methods bed herein, or any material
processed by any of the methods bed herein can be uently densified.
In some embodiments, the material to be sed is in the form of a fibrous material
that includes fibers provided by shearing a fiber source. For example, the shearing can be
performed with a rotary knife cutter.
For e, a fiber source, e.g., that is recalcitrant or that has had its recalcitrance
level reduced, can be sheared, e.g., in a rotary knife cutter, to provide a first fibrous material.
The first fibrous material is passed through a first screen, e.g., having an average opening size of
1.59 mm or less (1/16 inch, 0.0625 inch), provide a second fibrous material. If desired, the fiber
source can be cut prior to the ng, e.g., with a shredder. For example, when a paper is used
as the fiber source, the paper can be first cut into strips that are, e.g. l/4- to l/2-inch wide, using
a shredder, e.g., a r-rotating screw shredder, such as those manufactured by Munson
(Utica, N.Y.). As an alternative to shredding, the paper can be reduced in size by g to a
desired size using a guillotine cutter. For example, the guillotine cutter can be used to cut the
paper into sheets that are, e.g., 10 inches wide by 12 inches long.
In some embodiments, the shearing of the fiber source and the passing of the resulting
first fibrous material through a first screen are performed concurrently. The shearing and the
passing can also be performed in a batch-type process.
For example, a rotary knife cutter can be used to concurrently shear the fiber source
and screen the first fibrous material. A rotary knife cutter includes a hopper that can be loaded
with a ed fiber source prepared by shredding a fiber source. The shredded fiber source.
In some implementations, the ock is physically treated prior to saccharification
and/or tation. Physical treatment processes can e one or more of any of those
described herein, such as mechanical treatment, chemical treatment, irradiation, sonication,
oxidation, pyrolysis or steam explosion. Treatment methods can be used in combinations of two,
three, four, or even all of these technologies (in any order). When more than one treatment
method is used, the methods can be applied at the same time or at different times. Other
processes that change a molecular structure of a biomass feedstock may also be used, alone or in
combination with the processes disclosed herein.
Mechanical treatments that may be used, and the characteristics of the mechanically
treated biomass als, are described in r detail in US. Pat. App. Pub. 2012/0100577
A1, filed October 18, 2011, the fill disclosure of which is hereby incorporated herein by
reference.
IV. USE OF TREATED BIOMASS MATERIAL
Using the methods described herein, a starting biomass material (e.g., plant biomass,
animal biomass, paper, and municipal waste biomass) can be used as feedstock to produce useful
intermediates and products such as c acids, salts of organic acids, anhydrides, esters of
organic acids and fuels, e.g., fuels for internal combustion engines or feedstocks for fuel cells.
Systems and processes are described herein that can use as feedstock cellulosic and/or
lignocellulosic materials that are readily ble, but often can be difficult to s, e.g.,
municipal waste streams and waste paper streams, such as s that include newspaper, kraft
paper, corrugated paper or mixtures of these.
In order to convert the feedstock to a form that can be readily processed, the glucan-
or xylan-containing cellulose in the feedstock can be hydrolyzed to low molecular weight
carbohydrates, such as sugars, by a saccharifying agent, e.g., an enzyme or acid, a process
referred to as saccharif1cation. The low molecular weight carbohydrates can then be used, for
example, in an existing manufacturing plant, such as a single cell protein plant, an enzyme
manufacturing plant, or a fuel plant, 6.g. , an ethanol manufacturing facility.
The feedstock can be yzed using an , e.g., by combining the materials
and the enzyme in a solvent, e.g., in an aqueous solution.
Alternatively, the enzymes can be supplied by organisms that break down biomass,
such as the cellulose and/or the lignin ns of the biomass, contain or cture various
cellulolytic enzymes (cellulases), ligninases or various small molecule biomass-degrading
metabolites. These enzymes may be a x of enzymes that act synergistically to degrade
crystalline cellulose or the lignin portions of biomass. Examples of cellulolytic enzymes e:
endoglucanases, cellobiohydrolases, and cellobiases (beta-glucosidases).
During saccharif1cation a cellulosic substrate can be initially hydrolyzed by
endoglucanases at random locations producing oligomeric intermediates. These ediates
are then substrates for exo-splitting glucanases such as cellobiohydrolase to produce cellobiose
from the ends of the cellulose polymer. Cellobiose is a water-soluble l,4-linked dimer of
e. Finally, cellobiase cleaves cellobiose to yield glucose. The efficiency (e.g., time to
hydrolyze and/or completeness of hydrolysis) of this process depends on the recalcitrance of the
cellulosic al.
V. INTERMEDIATES AND PRODUCTS
Using the processes described herein, the biomass material can be converted to one or
more products, such as energy, fuels, foods and materials. Specific es of products
e, but are not limited to, hydrogen, sugars (e.g., e, xylose, arabinose, mannose,
galactose, fructose, disaccharides, oligosaccharides and polysaccharides), alcohols (e.g.,
monohydric alcohols or dihydric alcohols, such as ethanol, n-propanol, isobutanol, sec-butanol,
tert-butanol or n-butanol), hydrated or hydrous alcohols (e.g., containing greater than 10%, 20%,
% or even greater than 40% water), biodiesel, c acids, hydrocarbons (e.g., methane,
ethane, propane, isobutene, pentane, n-hexane, biodiesel, bio-gasoline and mixtures f), co-
products (e.g., proteins, such as cellulolytic proteins (enzymes) or single cell proteins), and
mixtures of any of these in any ation or relative concentration, and optionally in
combination with any additives (e.g., fuel additives). Other examples include carboxylic acids,
salts of a carboxylic acid, a mixture of carboxylic acids and salts of carboxylic acids and esters of
carboxylic acids (e.g., methyl, ethyl and n-propyl esters), ketones (e.g., acetone), des (e.g.,
acetaldehyde), alpha and beta unsaturated acids (e.g., acrylic acid) and olef1ns (e.g., ethylene).
Other alcohols and alcohol derivatives include propanol, propylene , l,4-butanediol, 1,3-
propanediol, sugar alcohols and polyols (e.g., glycol, glycerol, erythritol, threitol, arabitol,
xylitol, ribitol, ol, sorbitol, galactitol, iditol, ol, volemitol, isomalt, maltitol, lactitol,
maltotriitol, maltotetraitol, and polyglycitol and other polyols), and methyl or ethyl esters of any
of these alcohols. Other products include methyl te, methylmethacrylate, lactic acid, citric
acid, formic acid, acetic acid, nic acid, butyric acid, ic acid, valeric acid, caproic
acid, 3-hydroxypropionic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, glutaric
acid, oleic acid, linoleic acid, glycolic acid, gamma-hydroxybutyric acid, and es thereof,
salts of any of these acids, mixtures of any of the acids and their respective salts.
Any ation of the above products with each other, and/or of the above products
with other ts, which other products may be made by the processes described herein or
otherwise, may be packaged together and sold as products. The products may be combined, e.g.,
mixed, blended or co-dissolved, or may simply be packaged or sold together.
Any of the products or combinations of products described herein may be zed or
sterilized prior to selling the products, e.g., after ation or isolation or even after packaging,
to lize one or more ially undesirable contaminants that could be present in the
product(s). Such sanitation can be done with electron bombardment, for example, be at a dosage
of less than about 20 Mrad, e.g., from about 0.1 to 15 Mrad, from about 0.5 to 7 Mrad, or from
about 1 to 3 Mrad.
The processes described herein can produce various by-product streams useful for
generating steam and electricity to be used in other parts of the plant (co-generation) or sold on
the open . For example, steam ted from burning by-product streams can be used in
a distillation process. As another example, electricity generated from burning by-product
streams can be used to power electron beam generators used in pretreatment.
The by-products used to generate steam and electricity are derived from a number of
s throughout the process. For example, anaerobic digestion of wastewater can produce a
biogas high in methane and a small amount of waste biomass (sludge). As another example,
post-saccharification and/or post-distillate solids (e.g., unconverted lignin, cellulose, and
hemicellulose remaining from the pretreatment and primary processes) can be used, e.g., burned,
as a fuel.
Many of the products obtained, such as ethanol or n-butanol, can be utilized as a filel
for powering cars, trucks, tractors, ships or trains, e.g., as an internal tion filel or as a fuel
cell feedstock. Many of the products obtained can also be utilized to power aircraft, such as
planes, e.g., having jet engines or helicopters. In addition, the products described herein can be
utilized for electrical power generation, e.g., in a conventional steam generating plant or in a fuel
cell plant.
Other intermediates and products, including food and pharmaceutical products, are
described in US. Pat. App. Pub. 2010/01245 83 Al, published May 20, 2010, to Medoff, the full
disclosure of which is hereby incorporated by reference herein.
VI. PRODUCTION OF ENZYMES BY MICROORGANISMS
Filamentous fungi, or ia that produce cellulase, typically require a carbon
source and an inducer for production of cellulase.
Lignocellulosic materials comprise different combinations of cellulose, hemicellulose
and lignin. Cellulose is a linear polymer of glucose forming a fairly stiff linear ure without
significant coiling. Due to this structure and the disposition of hydroxyl groups that can
hydrogen bond, cellulose contains lline and non-crystalline portions. The crystalline
portions can also be of different types, noted as I(alpha) and I(beta) for example, depending on
the location of hydrogen bonds between s. The polymer lengths lves can vary
lending more variety to the form of the ose. Hemicellulose is any of several
heteropolymers, such as xylan, glucuronoxylan, arabinoxylans, and xyloglucan. The y
sugar monomer present is xylose, although other monomers such as mannose, galactose,
rhamnose, arabinose and glucose are present. Typically hemicellulose forms ed structures
with lower molecular weights than cellulose. llulose is therefore an amorphous material
that is generally susceptible to enzymatic ysis. Lignin is a complex high molecular weight
heteropolymer generally. Although all lignins show variation in their composition, they have
been described as an amorphous dendritic network polymer of phenyl e units. The
amounts of cellulose, hemicellulose and lignin in a specific biomaterial depends on the source of
the biomaterial. For example wood derived biomaterial can be about 38-49% cellulose, 7-26%
hemicellulose and 23-34% lignin ing on the type. Grasses typically are 33-38% cellulose,
24-32% hemicellulose and 17-22% lignin. Clearly lignocellulosic biomass tutes a large
class of substrates.
The diversity of biomass materials may be r increased by pretreatment, for
e, by changing the crystallinity and molecular weights of the polymers.
The cellulase producing organism when contacted with a biomass will tend to
produce enzymes that release molecules advantageous to the organism’s growth, such as e.
This is done through the phenomenon of enzyme induction as described above. Since there are a
2012/071093
variety of substrates in a particular biomaterial, there are a variety of ases, for example, the
endoglucanase, exoglucanase and cellobiase discussed previously. By selecting a particular
lignocellulosic material as the inducer the relative concentrations and/or actiVities of these
enzymes can be modulated so that the resulting enzyme complex will work efficiently on the
lignocellulosic material used as the inducer or a similar material. For example, a biomaterial
with a higher n of crystalline ose may induce a more effective or higher amount of
endoglucanase than a biomaterial with little crystalline ose.
One of ordinary skill in the art can ze the tion of enzymes by
microorganisms by adding yeast extract, corn steep, peptones, amino acids, ammonium salts,
phosphate salts, potassium salts, magnesium salts, calcium salts, iron salts, manganese salts, zinc
salts, cobalt salts, or other additives and/or nutrients and/or carbon sources. Various components
can be added and removed during the processing to optimize the desired production of useful
products.
Temperature, pH and other conditions optimal for growth of microorganisms and
tion of enzymes are generally known in the art.
VII. SACCHARIFICATION
The treated biomass materials can be saccharified, generally by combining the
material and a cellulase enzyme in a fluid medium, e.g., an aqueous solution. In some cases, the
material is boiled, steeped, or cooked in hot water prior to saccharification, as described in US.
Pat. App. Pub. 100577 A1 by Medoff and Masterman, published on April 26, 2012, the
entire contents of which are incorporated herein.
The saccharif1cation process can be partially or completely performed in a tank (6.g.
a tank haVing a volume of at least 4000, 40,000, or 500,000 L) in a manufacturing plant, and/or
can be partially or completely performed in transit, e.g., in a rail car, tanker truck, or in a
supertanker or the hold of a ship. The time ed for complete saccharif1cation will depend on
the process ions and the biomass material and enzyme used. If saccharification is
performed in a manufacturing plant under controlled ions, the cellulose may be
substantially entirely converted to sugar, e.g., glucose in about 12-96 hours. If saccharif1cation is
performed partially or completely in transit, rif1cation may take longer.
It is generally preferred that the tank contents be mixed during saccharif1cation, e.g.,
using jet mixing as described in International App. No. PCT/U82010/03533 l , filed May 18,
2010, which was published in English as WC 2010/135380 and designated the United States, the
filll disclosure of which is incorporated by reference herein.
The addition of surfactants can enhance the rate of saccharif1cation. Examples of
surfactants e non-ionic surfactants, such as a Tween® 20 or Tween® 80 polyethylene
glycol surfactants, ionic surfactants, or eric surfactants.
It is generally red that the concentration of the sugar solution resulting from
saccharif1cation be relatively high, e.g., greater than 40%, or greater than 50, 60, 70, 80, 90 or
even greater than 95% by weight. Water may be removed, e.g., by evaporation, to increase the
concentration of the sugar solution. This s the volume to be shipped, and also inhibits
microbial growth in the solution.
atively, sugar ons of lower concentrations may be used, in which case it
may be ble to add an antimicrobial additive, e.g., a broad spectrum antibiotic, in a low
concentration, e.g., 50 to 150 ppm. Other suitable antibiotics include amphotericin B, ampicillin,
chloramphenicol, ciprofloxacin, gentamicin, hygromycin B, kanamycin, neomycin, penicillin,
puromycin, streptomycin. Antibiotics will inhibit growth of microorganisms during transport
and storage, and can be used at appropriate concentrations, e.g., between 15 and 1000 ppm by
weight, e.g., between 25 and 500 ppm, or between 50 and 150 ppm. If desired, an antibiotic can
be included even if the sugar concentration is relatively high. Alternatively, other ves with
anti-microbial of preservative properties may be used. Preferably the antimicrobial additive(s)
are food-grade.
A vely high tration solution can be obtained by limiting the amount of
water added to the biomass material with the enzyme. The concentration can be controlled, 6.g.
by controlling how much rification takes place. For example, concentration can be
increased by adding more biomass material to the solution. In order to keep the sugar that is
being produced in solution, a surfactant can be added, e.g., one of those discussed above.
Solubility can also be sed by increasing the temperature of the solution. For e, the
solution can be maintained at a temperature of 40-50°C, 60-80°C, or even higher.
V111. SACCHARIFYING AGENTS
Suitable cellulolytic enzymes include cellulases from species in the genera Bacillus,
CaprinuS, Mycell'ophthora, Cephalosporium, Scytalz'dz'um, Penicillium, ASpergz'lluS,
monas, Humicola, Fusarium, Thielavz'a, Acremonium, ChrySOSporz'um and Trichoderma,
ally those produced by a strain selected from the species ASpergz'lluS (see, e.g., EP Pub.
No. 0 458 162), la insolenS (reclassified as Scytalz'dz'um thermophilum, see, e.g., US. Pat.
No. 4,435,307), CaprinuS cinereus, Fusarium oxySporum, Myceliophthora thermophila,
Merlpl'luS giganteus, vz'a terrestriS, Acremonium Sp. (including, but not limited to, A.
perSl'cz'num, A. acremonium, A. pem'um, A. dichromosporum, A. obclavatum, A.
pinkertonz'ae, A. roseogriseum, A. incoloratum, and A. furaz‘um). red strains include
la insolenS DSM 1800, Fusarium oxySporum DSM 2672, Myceliophthora thermophila
CBS 117.65, Cephalosporium Sp. RYM-202, Acremonium Sp. CBS 478.94, Acremonium Sp.
CBS , Acremonium perSicz'num CBS 169.65, Acremonium acremonium AHU 9519,
Cephalosporium Sp. CBS 535.71, Acremonium pem'um CBS 866.73, Acremonium
dichromosporum CBS 683.73, Acremonium obclavatum CBS , Acremonium pinkertom'ae
CBS 157.70, Acremonium roseogriseum CBS 134.56, Acremonium incoloratum CBS 146.62,
and Acremom’umfuratum CBS 299.70H. Cellulolytic enzymes may also be obtained from
ChrySOSporium, preferably a strain of ChrySOSporz'um lucknowense. Additional strains that can
be used include, but are not limited to, Trichoderma (particularly T. viride, T. reesez’, and T.
koningii), alkalophilic Bacillus (see, for e, US. Pat. No. 3,844,890 and EP Pub. No. 0 458
162), and Streptomyces (see, e.g., EP Pub. No. 0 458 162).
Many microorganisms that can be used to rify biomass material and produce
sugars can also be used to ferment and convert those sugars to useful products.
IX. SUGARS
In the processes bed herein, for example after saccharification, sugars (e.g.,
glucose and xylose) can be isolated. For example sugars can be isolated by precipitation,
crystallization, chromatography (6.g. , simulated moving bed chromatography, high pressure
chromatography), filgation, extraction, any other isolation method known in the art, and
combinations thereof.
X. HYDROGENATION AND OTHER CHEMICAL TRANSFORMATIONS
The ses described herein can include hydrogenation. For example glucose and
xylose can be hydrogenated to sorbitol and xylitol respectively. Hydrogenation can be
accomplished by use of a catalyst (e.g., Pt/gamma-A1203, Ru/C, Raney Nickel, or other sts
know in the art) in combination with H2 under high pressure (e.g., 10 to 12000 psi). Other types
of chemical transformation of the products from the processes described herein can be used, for
example production of organic sugar derived products such (e.g., al and al-derived
products). Chemical transformations of sugar derived products are described in US Prov. App.
No. 61/667,481, filed July 3, 2012, the disclosure of which is incorporated herein by reference in
its entirety.
XI. FERMENTATION
Yeast and Zymomonas bacteria, for example, can be used for fermentation or
conversion of sugar(s) to alcohol(s). Other microorganisms are discussed below. The optimum
pH for fermentations is about pH 4 to 7. For example, the optimum pH for yeast is from about
pH 4 to 5, while the optimum pH for Zymomonas is from about pH 5 to 6. Typical fermentation
times are about 24 to 168 hours (e.g., 24 to 96 hrs) with temperatures in the range of 20°C to
40°C (e.g., 26°C to 40°C), however thermophilic microorganisms prefer higher temperatures.
In some ments, e.g., when anaerobic organisms are used, at least a portion of
the tation is conducted in the e of oxygen, e.g., under a blanket of an inert gas such
as N2, Ar, He, CO2 or mixtures thereof Additionally, the mixture may have a constant purge of
an inert gas flowing through the tank during part of or all of the fermentation. In some cases,
anaerobic condition, can be achieved or ined by carbon dioxide production during the
fermentation and no additional inert gas is needed.
In some embodiments, all or a portion of the fermentation s can be interrupted
before the low molecular weight sugar is completely converted to a product (e.g., ethanol). The
intermediate fermentation products include sugar and carbohydrates in high concentrations. The
sugars and carbohydrates can be isolated via any means known in the art. These intermediate
fermentation ts can be used in preparation of food for human or animal consumption.
Additionally or alternatively, the intermediate fermentation products can be ground to a fine
particle size in a stainless-steel laboratory mill to e a flour-like substance.
2012/071093
Jet mixing may be used during fermentation, and in some cases saccharification and
fermentation are med in the same tank.
Nutrients for the rganisms may be added during saccharification and/or
fermentation, for example the food-based nutrient packages described in US. Pat. App. Pub.
2012/0052536, filed July 15, 2011, the complete disclosure of which is incorporated herein by
reference.
“Fermentation” includes the methods and products that are sed in US. Prov.
App. No. 61/579,559, filed December 22, 2012, and US. Prov. App. No. 61/579,576, filed
December 22, 2012, the contents of both of which are incorporated by reference herein in their
entirety.
Mobile fermenters can be utilized, as described in International App. No.
(which was filed July 20, 2007, was published in English as WO
2008/01 1598 and designated the United States), the contents of which is orated herein in
its entirety. Similarly, the saccharification equipment can be mobile. Further, rification
and/or fermentation may be performed in part or entirely during transit.
XII. FERMENTATION AGENTS
The microorganism(s) used in fermentation can be naturally-occurring
microorganisms and/or engineered microorganisms. For example, the microorganism can be a
bacterium (including, but not limited to, e.g., a cellulolytic bacterium), a filngus, ding, but
not limited to, e.g., a yeast), a plant, a protist, e.g. a protozoa or a fungus-like protest (including,
but not limited to, e.g., a slime mold), or an alga. When the organisms are compatible, mixtures
of organisms can be utilized.
Suitable fermenting microorganisms have the ability to convert carbohydrates, such
as e, se, xylose, arabinose, mannose, galactose, oligosaccharides or polysaccharides
into fermentation products. Fermenting microorganisms include strains of the genus
Saccharomyces spp. (including, but not limited to, S. cerevisiae (baker’s yeast), S. distatz'cas, S.
avaram), the genus Klayveromyces, (including, but not limited to, K. marxz’anas, K. fragilis), the
genus Candida (including, but not limited to, C. pseudotropz'calis, and C. brassz'cae), Pichia
’tz’s (a relative of Candida shehatae), the genus Clavz'spora (including, but not limited to, C.
lasitam'ae and C. 'ae), the genus Pachysolen (including, but not limited to, P. tannophz'las),
2012/071093
the genus Bretannomyces (including, but not d to, e.g., B. clausem'z' (Philippidis, G. P.,
1996, Cellulose version technology, in Handbook on Bioethanol: Production and
Utilization, Wyman, C.E., ed., Taylor & Francis, Washington, DC, 179-212)). Other suitable
microorganisms include, for example, Zymomonas mobilis, Clostrz'dz'am spp. (including, but not
d to, C. thermocellam (Philippidis, 1996, , C.saccharobatylacetonicam, C.
saccharobaz‘ylz'cam, C. Paniceam, C. beijemckl’i, and C. acetobatylz'cam), Mom'lz'ella pollinz's,
Mom'lz'ella iliensz's, Lactobacz'llas spp. Yarrowz'a lipolytl'ca, Aareobasidiam 519.,
Trichosporonoides 519., Trigonopsz's variabilis, Trichosporon sp., Monilz'ellaacetoabatans sp.,
Typhala variabilis, Candida magnoliae, Ustilaginomycetes sp.,Pseadozyma tsakabaensis,yeast
species of genera Zygosaccharomyces, Debaryomyces, Hansenala and Pichia,and fungi of the
dematioid genus Torala.
For instance, Clostrz'dz'am spp. can be used to produce ethanol, butanol, butyric acid,
acetic acid, and acetone. Lactobacz'llas spp., can be used to produce lactice acid.
Many such microbial strains are publicly available, either cially or h
depositories such as the ATCC can Type Culture Collection, Manassas, Virginia, USA),
the NRRL (Agricultural Research Sevice Culture Collection, Peoria, Illinois, USA), or the
DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig,
Germany), to name a few.
cially available yeasts include, for example, Red Star®/Lesaffre Ethanol Red
(available from Red Star/Lesaffre, USA), FALI® able from Fleischmann’s Yeast, a division
of Burns Philip Food Inc., USA), SUPERSTART® (available from Alltech, now Lalemand),
GERT STRAND® (available from Gert Strand AB, Sweden) and FERMOL® (available from
DSM lties).
Many rganisms that can be used to saccharify biomass material and e
sugars can also be used to ferment and convert those sugars to useful products.
XIII. DISTILLATION
After fermentation, the resulting fluids can be distilled using, for example, a “beer
column” to separate ethanol and other alcohols from the majority of water and residual solids.
The vapor exiting the beer column can be, e.g., 35% by weight ethanol and can be fed to a
rectification column. A mixture of nearly azeotropic (92.5%) ethanol and water from the
rectification column can be purified to pure (99.5%) ethanol using vapor-phase molecular sieves.
The beer column bottoms can be sent to the first effect of a three-effect evaporator. The
rectification column reflux condenser can e heat for this first effect. After the first effect,
solids can be separated using a centrifuge and dried in a rotary dryer. A portion (25%) of the
centrifuge effluent can be recycled to fermentation and the rest sent to the second and third
evaporator effects. Most of the evaporator condensate can be returned to the process as fairly
clean condensate with a small portion split off to waste water treatment to prevent up of
low-boiling compounds.
EXAMPLES
Example 1. Effect of Exogenous Fructose on Saccharification
This example tests whether or not exogenous se inhibits saccharification
enzymes.
Three 225mL Erlenmeyer flasks were ed, each with 10g of treated corn cob
biomass (mesh size between 15 and 40, and ated to 35 Mrad with an electron beam) lOOmL
of water and 2.5mL of Duet AcceleraseTM (Danisco). To the first, second, and third flask were
added, respectively: 0g, 5g and 10g of fructose. The flasks were covered with aluminum foil and
set in an incubator shaker at 50°C and 200rpm for four days. The amount of xylose and glucose
was monitored by HPLC. The results of the saccharification are shown in the table below.
Table 1. Saccharification under varying levels of exogenous fructose.
Sample Glucose yield (g/L) Xylose yield (g/L) % Glucose
0g added fructose 17.9 13.8 100.0
5g added fructose 16.7 12.3 93.5
10g added fructose 18.1 12.6 101.3
Unlike glucose (a known tor of cellobiase), 5% or 10% added fructose does not
inhibit the rification of comcob.
Example 2. Effect of Xylose Isomerase on Saccharif1cation
Glucose is a known inhibitor of cellobiase. This e tests if the conversion of
glucose to the isomer fructose by xylose isomerase can increase sacchariflcation.
Four 225mL Erlenmeyer flasks were prepared, each with 10g of treated corn cob
biomass and 100mL of water. The biomass was treated as described in Example 1. To the first,
second, and third flask was added 2.5mL of Duet raseTM (Danisco). To the , third,
and fourth flasks were added, respectively: 1g, 0.1g and 0.1g of glucose isomerase
(SweetzymeTM, Aldrich). The flasks were d with aluminum foil and set in an incubator
shaker at 50°C and 200rpm for four days. The amount of xylose and glucose was monitored by
HPLC. The results of the sacchariflcation are shown in the table below.
Table 2. Effectiveness of cellulase with added xylose isomerase.
2.5mL Duet -- 1g GI 28.3 20.6 125.2 122.3
2.5mL Duet -- 0.1g GI 24.6 18.5 109.0 109.4
0.1g G1 1.6 Not detected 6.9 Not detected
The addition of glucose isomerase was observed to increase the effectiveness of the
cellulase enzyme, with flask 2 producing about 25% more sugars than flask 1.
Example 3. Use of a Strong Acid to Cleave Cellobiose
This example tests the use of a strong acid to cleave iose to glucose, to increase
rif1cation yield. The strong acid used was yst-15TM, a polystyrene ic acid.
This is a strongly acidic ic acid macroreticular polymeric resin that is based on crosslinked
styrene divinylbenzene copolymers. Published studies indicate that Amberlyst-15 can cleave the
dimer cellobiose to glucose.
Three 225mL Erlenmeyer flasks were prepared, each with 10g of treated corn cob
biomass, 100mL of water and 2.5mL Duet AcceleraseTM. The biomass was treated as described
in Example 1. In the second flask 1g of glucose isomerase (SweetzymeTM, Aldrich) was added;
and in the third 1 g of e isomerase and 0.1 g of polystyrene sulfonic acid (Amberlyst-15TM,
DOW) was added.
The flasks were covered with aluminum foil and set in an incubator shaker at 50°C
and 200rpm for four days. The amount of xylose and glucose was monitored by HPLC. The
results of the saccharification are shown in the table below.
Table 3. Effect of an Acid on Saccharification.
(g/L) (g/L) improved with G1
Duet alone 21.1 16.1 100 100 NA
Duet -- GI 26.5 19.2 125 119 NA
Duet -- G1 + 27.9 20.5 131 127 14
Amberlyst
The results show an improvement in the saccharification with the addition of glucose
ase. The experiment also shows an improvement in the saccharification with the addition
of polystyrene ic acid.
Example 4. Removal of Cellobiase
This example es saccharification where cellobiase has been removed, while
the endo- and exo-cellulases have been retained.
Chromatofocusing was used to separate the enzymes. Duet AcceleraseTM co)
was injected onto a Mono P column using an AKTA system. The nd llulases bound
to the column, while the cellobiase passed through and was removed. The exo- and endo-
cellulases were then eluted from the column by shifting the pH to 4.0. The resulting fractions
were combined and immediately applied to a saccharification reaction.
Table 4. Accumulation of Cellobiose and Sugars in the Absence of Cellobiase.
Sample Cellobiose Glucose Xylose Xylitol Lactose
AKTA 1.057 4.361 5.826 0.556
purified Duet
Duet 0.398 16.999 14.830 0.726
Comcob (no 0.673 0.550
enzymes)
Spun/Filtered 17.695 15.053 0.770 1.052
Duet
The expected result was that without cellobiase, there would be an accumulation of
cellobiose. Although the yield was low, the table below shows that a detectable amount of
cellobiose was indeed generated.
Other than in the es , or unless otherwise expressly specified, all of the
cal ranges, amounts, values and percentages, such as those for amounts of materials,
elemental contents, times and temperatures of reaction, ratios of amounts, and others, in the
following portion of the specification and attached claims may be read as if prefaced by the word
“about” even though the term ” may not expressly appear with the value, amount, or
range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the
following specification and attached claims are approximations that may vary depending upon
the desired properties sought to be obtained by the present invention. At the very least, and not
as an attempt to limit the application of the doctrine of equivalents to the scope of the claims,
each numerical ter should at least be construed in light of the number of reported
significant digits and by applying ordinary ng techniques.
Notwithstanding that the numerical ranges and ters setting forth the broad
scope of the invention are approximations, the numerical values set forth in the specific
examples are reported as precisely as possible. Any numerical value, however, inherently
contains error necessarily resulting from the standard deviation found in its underlying respective
testing measurements. Furthermore, when numerical ranges are set forth herein, these ranges are
inclusive of the recited range end points (2'.e., end points may be used). When percentages by
weight are used herein, the numerical values ed are relative to the total weight.
Also, it should be tood that any numerical range recited herein is intended to
include all sub-ranges subsumed therein. For e, a range of “1 to 10” is intended to
include all sub-ranges between (and including) the recited minimum value of 1 and the recited
maximum value of 10, that is, having a minimum value equal to or greater than 1 and a
m value of equal to or less than 10. The terms ) :4 a)
a or “an” as used herein are
intended to include “at least one” or “one or more,” unless otherwise indicated.
Any patent, publication, or other disclosure material, in whole or in part, that is said
to be incorporated by reference herein is incorporated herein only to the extent that the
incorporated material does not conflict with existing definitions, statements, or other disclosure
material set forth in this disclosure. As such, and to the extent necessary, the disclosure as
explicitly set forth herein edes any conflicting material incorporated herein by reference.
Any material, or portion thereof, that is said to be incorporated by nce herein, but which
conflicts with existing definitions, statements, or other disclosure material set forth herein will
only be orated to the extent that no conflict arises between that incorporated material and
the existing disclosure material.
While this invention has been particularly shown and described with references to
preferred embodiments thereof, it will be understood by those skilled in the art that various
changes in form and details may be made therein without departing from the scope of the
invention assed by the ed claims.
Claims (20)
1. A method of producing one or more products, the method comprising: saccharifying recalcitrance-reduced lignocellulosic biomass to produce a saccharified mixture comprising glucose and ; adding an isomerization agent to the saccharified mixture to produce an isomerized mixture comprising glucose, xylose and fructose; and contacting the isomerized mixture with a microorganism that converts glucose, but not xylose, to product.
2. The method of claim 1, wherein the recalcitrance-reduced biomass is produced by treating s with accelerated ons.
3. The method of any one of claim 1 or claim 2, wherein the lignocellulosic biomass is selected from the group consisting of: wood, particle board, forestry wastes, sawdust, aspen wood, wood chips, s, switchgrass, miscanthus, cord grass, reed canary grass, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, ltural waste, silage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, , sisal, abaca, corn cobs, corn stover, soybean stover, corn fiber, alfalfa, hay, coconut hair, sugar processing residues, e, beet pulp, agave bagasse, algae, seaweed, manure, sewage, offal, agricultural or rial waste, arracacha, eat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, peas, and mixtures of any of these.
4. The method of any one of claims 1-3, wherein one or more cellulases saccharify the recalcitrance-reduced biomass.
5. The method of claim 4, wherein the one or more cellulases is an endoglucanase, an exo-splitting glucanase, a cellobiase, or a combination thereof.
6. The method of any one of claims 1-5, wherein the isomerization agent comprises an enzyme.
7. The method of claim 6, wherein the enzyme is immobilized on a t.
8. The method of claim 6, wherein the enzyme is xylose ase.
9. The method of claim 8, wherein the xylose isomerase is produced from Pseudomonas hydrophila, Escherichia intermedia, Bacillus megaterium, Paracolobacterium aerogenoides, or a combination f.
10. The method of any one of claims 1-9, n the steps of saccharifying and adding are performed at a temperature of 60 degrees to 65 degrees Celsius.
11. The method of any one of claims 1-9, wherein the steps of saccharifying and adding are performed at a pH of 7 or below.
12. The method of any one of claims 1-11, wherein the saccharifying is performed during the addition of the isomerization agent.
13. The method of any one of claims 1-11, wherein the saccharifying is performed before the addition of the isomerizing agent.
14. The method of any one of claims 1-13, n the microorganism is from the genus Saccharomyces ssp.
15. The method of any one of claims 1-14, wherein the product comprises ethanol.
16. The method of any one of claims 1-15, further comprising isolating the product.
17. The method of any one of claims 1-16, wherein the product is isolated by distillation.
18. The method of any one of claims 1-17, r comprising isolating the xylose.
19. The method of claim 18, wherein the xylose is isolated by crystallization.
20. The method of any one of claims 1-18, further comprising isolating the xylose and isolating the product.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161579552P | 2011-12-22 | 2011-12-22 | |
US201161579559P | 2011-12-22 | 2011-12-22 | |
US61/579,559 | 2011-12-22 | ||
US61/579,552 | 2011-12-22 | ||
NZ724310A NZ724310B2 (en) | 2011-12-22 | 2012-12-20 | Processing Biomass |
Publications (2)
Publication Number | Publication Date |
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NZ739947A NZ739947A (en) | 2019-10-25 |
NZ739947B2 true NZ739947B2 (en) | 2020-01-28 |
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