NZ748522B2 - A method for producing carboxylic acid, or a salt thereof - Google Patents
A method for producing carboxylic acid, or a salt thereof Download PDFInfo
- Publication number
- NZ748522B2 NZ748522B2 NZ748522A NZ74852212A NZ748522B2 NZ 748522 B2 NZ748522 B2 NZ 748522B2 NZ 748522 A NZ748522 A NZ 748522A NZ 74852212 A NZ74852212 A NZ 74852212A NZ 748522 B2 NZ748522 B2 NZ 748522B2
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- NZ
- New Zealand
- Prior art keywords
- biomass
- fructose
- glucose
- treatment
- paper
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Abstract
Disclosed is a method of producing a carboxylic acid (e.g., butyric acid) comprising: saccharifying a cellulosic or lignocellulosic biomass with an enzyme to produce a solution comprising one or more sugars; contacting the solution with an isomerization agent to increase the concentration of fructose in the solution; and converting the fructose to a carboxylic acid, or a salt thereof, with a microorganism. se in the solution; and converting the fructose to a carboxylic acid, or a salt thereof, with a microorganism.
Description
A METHOD FOR PRODUCING CARBOXYLIC ACID, OR A SALT
THEREOF
by Marshall Medoff, Thomas Craig Masterman, l W. Finn
CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No.
61/579,559, filed on December 22, 2011. The entire sure of the above
application is incorporated herein by reference.
FIELD OF THE INVENTION
The invention pertains to methods of converting s to useful
products. In particular, the inventions relates to the production of ts, such as
butanol from sugars, such as fructose.
BACKGROUND
As demand for petroleum increases, so too does interest in ble
feedstocks for manufacturing biofuels and biochemicals. The use of lignocellulosic
biomass as a feedstock for such manufacturing processes has been d since the
1970s. Lignocellulosic biomass is attractive because it is abundant, renewable,
domestically produced, and does not compete with food industry uses.
Many potential lignocellulosic feedstocks are available today, including
agricultural residues, woody biomass, municipal waste, ds/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
cellulose fibrils embedded in a hemicellulose matrix, surrounded by lignin. This
compact matrix is difficult to access by s and other chemical, mical and
biological processes. Cellulosic 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, naturally-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
composition of ose, llulose and lignin.
While a number of methods have been tried to extract ural carbohydrates from
lignocellulosic biomass, they are either are too expensive, produce too low a yield, leave
undesirable chemicals in the resulting 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 developed that will make these
monosaccharides available in large quantities and at acceptable purities and prices.
SUMMARY OF THE ION
Provided 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.
In one aspect, the invention features a method for producing a product, the method
comprising: producing fructose by saccharifying a biomass and contacting the saccharifed
biomass with an ization agent, and converting the se to a product with a
microorganism and/or an enzyme.
] In some implementations, the biomass comprises a cellulosic or lignocellulosic
al. The cellulosic or lignocellulosic biomass is treated to reduce its recalcitrance to
saccharif1cation, for example using a treatment method selected from the group consisting of:
bombardment with electrons, sonication, oxidation, pyrolysis, steam explosion, chemical
treatment, mechanical treatment, freeze grinding and combination thereof
The ization agent may be, for example, an isomerase, e.g., xylose isomerase.
In some implementations, the cellulosic or lignocellulosic biomass is selected from
the group consisting of: paper, paper products, paper waste, paper pulp, pigmented papers,
loaded , coated papers, filled papers, magazines, printed matter, printer paper, polycoated
paper, card stock, cardboard, paperboard, cotton, wood, particle board, ry wastes, t,
aspen wood, wood chips, grasses, switchgrass, miscanthus, cord grass, reed canary grass, grain
residues, rice hulls, oat hulls, wheat chaff, barley hulls, agricultural waste, silage, canola straw,
WO 96703 2012/071097
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 processing residues,
bagasse, beet pulp, agave bagasse, algae, seaweed, manure, sewage, arracacha, buckwheat,
banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans,
favas, lentils, peas, industrial waste, and mixtures of any of these.
In some cases, the microorganism ses a strain of Clostridium spp. For
example, the microorganism may be C. roperbutylacetonicum, e.g., C.
saccharoperbutylacetonicum strain ATCC 27021 or C. saccharoperbutylacetonicum strain ATCC
27022.
The product may comprise a solvent, e.g., an alcohol such as isobutanol or n-butanol.
] In some embodiments described herein, while it is generally preferred that ts
such as butanol be produced from sugars, such as fructose, that is derived from a cellulosic or
lignocellulosic material, se from other sources may be used.
It should be understood that this invention is not limited to the embodiments
disclosed in this Summary, and it is intended 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 nce characters refer to the same parts throughout the different views. The drawings are
not necessarily to scale, emphasis instead being placed upon rating embodiments of the
present invention.
is a m illustrating enzymatic hydrolysis of cellulose to e.
Cellulosic substrate (A) is converted by endocellulase (i) to cellulose (B), which is converted by
exocellulase (ii) to cellobiose (C), which is converted to glucose (D) by cellobiase (beta-
glucosidase) (iii).
is a flow diagram illustrating conversion of a biomass feedstock to one or
more products. Feedstock is physically pretreated (e.g., to reduce its size) (200), optionally
treated to reduce its recalcitrance (210), saccharified to form a sugar solution (220), the solution
is transported (230) to a manufacturing plant (e.g., by pipeline, railcar) (or if saccharif1cation is
performed en route, the ock, enzyme and water is transported), the saccharified feedstock
is bio-processed to produce a d product (e.g., alcohol) (240), and the product can be
processed further, e.g., by lation, to produce a final product (250). Treatment for
recalcitrance can be d by measuring lignin content (201) and g or adjusting process
parameters (205). Saccharifying the feedstock (220) can be modified by mixing the feedstock
with medium and the enzyme (221).
is a diagram showing the preparatory phase of the metabolism of glucose and
fructose.
is a diagram showing a metabolic pathway for the formation of cerides
during metabolism of fructose.
is a diagram showing a fermentative pathway for a butanol-producing
organism.
DETAILED DESCRIPTION
] This invention relates to methods of processing s materials (e.g., biomass
materials or biomass-derived materials) to obtain sugars such as fructose that can then be utilized
to produce a product. For example, sugars, e.g. fructose, can be fermented to produce a solvent,
such as an alcohol, for example butanol, e.g., isobutanol or n-butanol. Butyric acid can also be
produced. The inventors have found that in some cases a fructose solution can be fermented to
an l more rapidly, and with better yields, than a glucose on.
Without being bound to any particular theory, it is believed that products, such as
solvents (e.g., butanol), are toxic to solvent-producing organisms, and that metabolism with
some sugars such as fructose produce protective substrates (e.g., triglycerides), to a greater
degree or faster than glucose metabolism. A suggested effect of solvents is that they interact
with cell membranes ting membrane fluidity. Solvents, such as butanol are also attributed
to have a chaotropic effect on the membrane. Chaotropic agents ere with stabilizing
intramolecular interactions mediated by valent forces. Due to these effects, solvents can
inhibit active nutrient transport, the activity of membrane-bound enzymes, and e uptake.
Solvents can also partially or completely abolish the ne pH gradient, lower intracellular
pH and ATP concentrations. In response to increasing solvents, the cells may attempt to adjust
lipid composition to maintain fluidity (Christopher A. Tomas, J. Bacteriol. 186:2006-2018
(2004)). Fructose metabolism can tate the increase of lipids such as triglycerides.
Without being bound to any particular theory, it is filrther believed that the benefit of
sugars such as fructose for solvent production may be related to regulation of glycolysis. The
purpose of regulation is to control the growth and health of the sm. It is believed that
since some sugars such as se are not as naturally nt in the world as glucose is, the
regulation mechanism for suppressing its glycolysis is not as well developed. This can allow a
higher intake and metabolism of those sugars such as fructose by an organism.
] As shown in for example, during saccharification a osic substrate (A) is
lly hydrolyzed by endoglucanases (i) at random locations producing oligomeric
ediates (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 iase (iii)
cleaves cellobiose (C) to yield glucose (D). Therefore, the endoglucanases are particularly
effective in attacking 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 substrate, the amount and type of the three ent s may need to be modified.
A process for manufacturing an alcohol, e.g., butanol, is shown in A process
for manufacturing an alcohol can include, for example, optionally mechanically treating a
feedstock, e.g., to reduce its size (200), before and/or after this treatment, optionally ng the
feedstock with another physical treatment to further reduce its recalcitrance (210), then
saccharifying the feedstock, using the enzyme complex, to form a sugar solution (220).
Optionally, the method may also include transporting, e.g. , by pipeline, railcar, truck or barge,
the solution (or the ock, enzyme and water, if saccharification is med en route) to a
manufacturing plant (230). In some cases the saccharified feedstock is r bioprocessed
(e. g., fermented) to produce a desired product e.g., alcohol (240). This resulting product may in
some entations be processed fiarther, e.g., by distillation (250), to produce a final product.
One method of reducing the recalcitrance of the feedstock is by electron bombardment of the
feedstock. If desired, the steps of measuring lignin content of the feedstock (201) and setting or
adjusting process parameters based on this measurement (205) can be performed at various
stages of the process, as described in US. Pat. App. Pub. 2010/0203495 A1 by Medoff and
Masterman, published August 12, 2010, the complete disclosure of which is incorporated herein
by nce. Saccharifying the ock (220) can also be modified by mixing the ock
with medium and the enzyme (221).
The method steps discussed above with reference to will now be discussed in
fiarther detail, followed by a discussion of the als used in the process.
FERMENTATION OF FRUCTOSE TO USEFUL PRODUCTS
The fructose solution produced by saccharification or saccharif1cation followed by
ization can be fermented to produce an alcohol, e.g., butanol, or butyric acid.
shows the preparatory phase of glycolysis for both fructose and glucose.
Fermentation includes a multiphase glycolysis reaction, the preparatory phase of which produces
glyceraldehyde 3-phosphate. As shown in and discussed in detail below, production of
aldehyde phate from fructose involves fewer ons than production from
glucose, which may contribute to the greater efficiency observed with fructose fermentation as
compared to glucose fermentation.
] Referring to the glucose pathway in glucose is converted to glucose 6-
ate by the action of hexokinase with ATP. Glucose 6-phosphate is then isomerized to
fructose 6-phosphate by phosphohexoisomerase and then is r converted to fructose 1,6-
phosphate by the action of phophofructokinase and ATP. At this point, the diphosphated sugar is
split by fructose bisphosphate aldolase into dihydroxyacetone phosphate and glyceraldehyde 3-
phosphate. The dihydroxyacetone phosphate is isomerized to glyceraldehyde 3-phosphate by the
action of triose phosphate isomerase.
Referring again to there are several paths for glycolysis of fructose. While
hexokinase reacts strongly with glucose, its affinity for fructose is low. Therefore, although
fructose can be phosphorylated to glucose 6-phosphate by hexokinase and ATP, it is ed
that the contribution to glycolysis by this pathway is quite low. The more likely path starts with
the phosphorylation of fructose by the action of fructokinase and ATP, giving fructose 1-
phosphate. se 1-phosphate is then split into dihydroxyacetone phosphate and D-
glyceraldehyde by fructose 1-phosphate aldolase. As in the glucose pathway, dihydroxyacetone
phosphate is isomerized to glyceraldehyde 3-phosphate by triose phosphate isomerase. The D-
glyceraldehyde is ted to the glyceraldehyde 3-phosphate by trioskinase and ATP.
The microorganism used in fermentation is preferably selected to produce butanol
e.g., isobutanol or n-butanol. Suitable microorganisms include those discussed in the Materials
section, below. Many butanol-producing organisms are obligate anaerobes.
Fructose can drive the production of trigylcerides as a by-product of glycolysis, as
shown in FIG 4. The final step shown in for the formation of triglycerides involves an
esterification between glycerol 3-phosphate and fatty acids. Fatty acids are formed from
aldehyde 3-phosphate, the ion of which has been described above, with multiple
intermediates not shown here. The formation of glycerol 3-phosphate is shown in and can
occur from the action of glycerol 3-phosphate dehydrogenase on dihydroxyacetone phosphate.
This can also occur through the action of glycerol dehydrogenase on D-glyceraldehyde, forming
glycerol, which is then phosphorylated with okinase and ATP to glycerol 3-phosphate.
Although the formation of glycerol 3-phosphate is possible from glucose through the
dihydroxyacetone phosphate intermediate, the additional pathway through D-glycerolaldehyde,
which is only available through fructose can produce more of this intermediate. The
triglycerides ed by esterification of the glycerol 3-phosphate may help in the production
of butanol by protecting the butanol-producing organism from the toxic effects of butanol.
shows a fermentative pathway for a butanol-producing organism (Clostrz'dz'um
acetobutylicz'um). In a typical fermentation, after an induction period, the cells enter an
exponential growth phase. In the growth phase, butyrate and acetate are first ed, along
with ATP needed for cell growth. This phase is also called the acidogenesis phase.
ching, and in the nary phase, the e undergoes a metabolic shift towards the
formation of acetone, butanol and ethanol as main solvent products. This stage is also known as
the solventogenic phase. During and after the togenic phase the cells will become
vegetative, die and/or sporulate. In reactions are represented by bold arrows and d
by symbols from R1 to R19. The acidogenic reactions are R9 and R18 (catalyzed by PTA-AK
and , respectively), generating e and butyrate respectively. The two acids are
reassimilated h R7 and R17 (the reverse paths of R9 and R18), or directly converted to
acetyl-CoA and butyryl-CoA through R8 and R15 yzed by CoAT). The solventogenic
reactions are R11, R16 and R19 (catalyzed by AAD, AADC and BDH, respectively), generating
ethanol, acetate and butanol respectively. R14 is a lumped reaction consisting of ons
catalyzed by BHBD, CR0 and BCD (http://www.biomedcentral.com/1752-0509/5/S 1/S 12 “An
improved kinetic model for the acetone-butanol-ethanol pathways of Clostridium acetobutylz’cum
and based perturbation analysis”).
The optimum pH for fermentation is from about pH 4 to 7. Typical fermentation
times are about 24 to 168 hours with temperatures in the range of 20°C to 40°C, however
thermophilic microorganisms prefer higher temperatures. For anaerobic organisms it is
preferable to t the fermentation in the absence of oxygen e.g., under a blanket of an inert
gas such as N2, Ar, He, C02 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.
Jet mixing or other agitation may be used during fermentation, and in some cases
rification and fermentation are performed in the same tank. In some embodiments the
fermentation is done without any mechanical mixing.
Nutrients may be added during rification and/or fermentation, for example the
food-based nutrient packages described in USSN 61/365,493 and US 6,358,717 the complete
disclosure of which is incorporated herein by reference.
] Mobile tors can be utilized, as bed in US. Serial No. 12/374,549 and
International Application No. WC 2008/01 1598. Similarly, the saccharification equipment can
be mobile. Further, saccharification and/or fermentation may be performed in part or entirely
during t.
PREFERRED 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, e.g., a cellulolytic bacterium, a fungus, e.g., a yeast, a plant or a protist, e.g., an algae,
a protozoa or a fungus-like protist, e.g. a slime mold. When the sms are compatible,
mixtures of sms can be utilized.
Suitable fermenting microorganisms have the ability to convert fructose and
preferably also other sugars, such as glucose, xylose, arabinose, mannose, galactose,
oligosaccharides or polysaccharides, into an alcohol, e.g., butanol or a butanol derivative.
WO 96703
Exemplary microorganisms include, but are not d to, the following strains of
Clostridium:
Table l. Exemplary strains of Clostridium
XYLOSE ISOMERASE
Xylose isomerase (ES 5.3.1.5) is an enzyme the catalyzes the chemical reaction
back and forth between D-xylose and D-xylulose. It is also known systematically as glucose
isomerase and se aldose-ketose isomerase, and belongs to a family of isomerases,
WO 96703
specifically those intramolecular oxidoreductases interconverting aldoses and ketoses. Other
names in common use e se isomerase, D-Xylose ketoisomerase, and D-Xylose ketol-
isomerase. The enzyme ipates in pentose and glucuronate interconversions and se
and mannose metabolism. It is used industrially to convert glucose to fructose in the
manufacture of ructose corn syrup. It is sometimes referred to as “glucose isomerase.”
“Xylose isomerase” and “glucose isomerase” are used interchangeably herein. In vitro, glucose
isomerase zes the interconversion of glucose and fructose. In vivo, it catalyzes the
interconversion of xylose and xylulose.
Several types of enzymes are ered xylose isomerases. The first kind is
produced from Pseudomonas hydrophila. This enzyme has 160 times lower affinity to e
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 ichia 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 ed from Bacillus
megaterium AI and is NAD linked and is specific to glucose. Another glucose isomerase having
similar activity is isolated from Parac0l0bacterium aerogenoides. Glucose isomerases produced
by heterolactic acid bacteria require xylose as an inducer and are relatively unstable at high
temperature. The xylose isomerase (EC 5.3.1.5) is the most useful for commercial applications
as it does not e expensive cofactors such as NAD+ or ATP and it is relatively heat stable.
The e isomerases are usually produced intercellularly but reports of
ellular secretion of glucose isomerases are known. The enzyme used can be isolated from
many bacteria including but not limited to: Actinomyces 0liv0cinereus, Actinomyces
phaeochromogenes, Actinoplanes missouriensis, Aerobacter aer0genes,Aer0bacter cl0acae,
Aerobacter cum, Arthrobacter spp., Bacillus stearothermophilus, Bacillus megabacterium,
Bacillus ans, Bificlobacterium spp., Brevibacterium incertum, Brevibacterium
pentosoaminoacidicum, Chainia spp., Corynebacterium spp., C0rt0bacterium helv0lum,
Escherichiafreundii, Escherichia intermedia, Escherichia c0li, Flavobacterium arborescens,
Flavobacterium devorans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillusfermenti,
Lactobacillus mannitopoeus, Lactobacillus gayonii, Lactobacillus plantarum, acillus
lycopersici, Lactobacillus pentosus, Leuc0n0st0c mesenteroides, Microbispora r0sea,
Microellobosporiaflavea, Micr0m0n0sp0ra c0erula, Mycobacterium spp., N0cardia asteroides,
Nocarclz'a coralll'a, Nocarclz'a dassonvz'llez', Paracolobacterz'um aerogenoz'des, Pseudonocarclz'a
spp., Pseudomonas hydrophz'la, Sarcz'na spp., Staphylococcus bibila, Staphylococcusflavovz'rens,
Staphylococcus eclu'natus, Streptococcus ogenes, Streptococcus phaeochromogenes,
Streptococcusfraclz'ae, Streptococcus hromogenes, Streptococcus olivaceus,
Streptococcus calz'fornl'cos, Streptococcus venuceus, Streptococcus virginial, Streptomyces
hromogenes, Streptococcus venezaelz'e, Streptococcus wedmorensis, Streptococcus
griseolus, Streptococcus scens, Streptococcus bikiniensz's, Streptococcus rubigz'nosus,
Streptococcus achinatus, Streptococcus cinnamonensis, Streptococcusfraclz'ae, Streptococcus
albus, Streptococcus griseus, Streptococcus hivens, Streptococcus matensis, Streptococcus
murz'nus, Streptococcus nivens, Streptococcus platensz's, osporangl'um album,
Streptosporangl'um oulgare, Thermopolyspora spp., s spp., Xanthomonas spp. and
Zymononas mobilis.
Glucose isomerase can be used free in solution or immobilized on a support to
convert glucose to fructose. Whole cells or cell free s can be immobilized. The t
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 polystyrenes. 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 t. The solvent can be removed
by any suitable means, for example filtration or ation or spray drying. As another
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 process. For e a glucose isomerase producing bacteria can be co-cultured in
the process with an ethanol fermenting bacteria. Alternatively, the glucose-isomerase-producing
bacteria can be first contacted with the substrate, followed by inoculating with an ethanol-
ing substrate.
] Glucose isomerase can also be present within or ed from a cell also capable
of a further useful transformation of sugars. For example a e fermenting species can be
genetically modified to contain and express the gene for production of glucose isomerase.
ISOLATION OF SOLVENTS
After fermentation, the ing fluids can be purified using any useful method. For
example, some useful methods are distillation, tion, liquid-liquid extraction, perstraction,
reverse osmosis, oration and gas stripping (see, e.g., J. Ind. Microbiol. Biotechnol. (2009)
7-1138).
BIOMASS MATERIALS
As used herein, the term “biomass materials” includes lignocellulosic, cellulosic,
y, and microbial als.
Lignocellulosic materials include, but are not limited to, wood, particle board,
forestry wastes (e.g., sawdust, aspen wood, wood chips), grasses, (e.g., switchgrass, miscanthus,
cord grass, reed canary grass), grain residues, (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 processing residues (e.g., bagasse, beet pulp, agave bagasse), ,
algae, d, 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 vely uniform ess for irradiation,
and after irradiation are easy to se in the medium for fiarther processing. To facilitate
harvest and tion, 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, no additional nutrients (other than a nitrogen source, e.g., urea or
a) are required during fermentation of comcobs or cellulosic or ellulosic materials
containing significant amounts of s.
Comcobs, before and after comminution, are also easier to convey and disperse, and
have a lesser tendency to form explosive mixtures in air than other cellulosic or lignocellulosic
materials such as hay and grasses.
Cellulosic 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, labels, calendars, greeting cards, brochures, prospectuses,
newsprint), printer paper, polycoated paper, card stock, cardboard, paperboard, materials having
a high lulose 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 ry 14, 2012), the fill disclosure of which is incorporated herein by nce.
osic materials can also include ellulosic materials which have been de-
lignif1ed.
Starchy materials include starch itself, e.g., corn starch, wheat starch, potato starch or
rice , 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 es, sweet potato, taro, yams,
or one or more beans, such as favas, lentils or peas. Blends of any two or more starchy materials
are also starchy materials. Mixtures of starchy, cellulosic and or lignocellulosic als can
also be used. For example, a biomass can be an entire plant, a part of a plant or different 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 s described .
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, ts, e.g., animal protists (e.g.,
protozoa such as flagellates, amoeboids, ciliates, and sporozoa) and plant protists (e.g., algae
such alveolates, chlorarachniophytes, cryptomonads, euglenids, glaucophytes, haptophytes, red
algae, stramenopiles, and eplantae). Other examples include seaweed, plankton (e.g.,
macroplankton, mesoplankton, microplankton, nanoplankton, picoplankton, and
femptoplankton), phytoplankton, bacteria (e.g., gram positive bacteria, gram negative bacteria,
and extremophiles), yeast and/or mixtures of these. In some instances, microbial biomass can be
obtained from l sources, 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 e
systems, e.g., large scale dry and wet culture and fermentation systems.
The biomass material can also include offal, and similar sources of material.
In other embodiments, the biomass materials, such as cellulosic, starchy and
ellulosic 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 selection and ng to obtain desired traits in a plant.
Furthermore, the plants can have had genetic material d, d, silenced and/or added
with respect to the wild type variety. For example, genetically modified plants can be produced
by recombinant DNA methods, where genetic ations include introducing or modifying
specific genes from parental varieties, or, for example, by using transgenic breeding wherein a
specific gene or genes are uced to a plant from a ent species of plant and/or bacteria.
Another way to create genetic variation is through mutation breeding wherein new alleles are
ially created from endogenous genes. The artificial genes can be created by a variety of
ways including treating the plant or seeds with, for example, chemical ns (e.g., using
alkylating , epoxides, alkaloids, peroxides, formaldehyde), irradiation (e.g., X-rays,
gamma rays, neutrons, beta particles, alpha particles, protons, deuterons, UV radiation) and
temperature shocking or other external stressing and subsequent selection techniques. Other
methods of providing modified genes is h error prone PCR and DNA ng followed
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, biolistics, calcium phosphate precipitation, electroporation, gene splicing, gene silencing,
lipofection, microinjection and viral carriers. Additional genetically modified als have
been described in US. Application 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.
BIOMASS MATERIAL PREPARATION -- MECHANICAL TREATMENTS
The biomass can be in a dry form, for example with less than about 35% re
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
cellulosic or lignocellulosic feedstocks that have been physically 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 ing cylinder ofknown volume
with a sample and obtaining a weight of the sample. The bulk y 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 nce.
In some cases, the pre-treatment processing includes screening of the biomass
material. ing can be through a mesh or ated plate with a d 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, 0.015625 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-
processed, for e by comminuting, or they can simply be removed from processing. In
another configuration material that is larger than the perforations is irradiated and the smaller
material is removed by the screening process or recycled. In this kind of a configuration, the
or itself (for example a part of the or) can be perforated or made with a mesh. For
example, in one particular embodiment the biomass material may be wet and the perforations or
mesh allow water to drain away from the biomass before irradiation.
Screening of material can also be by a manual method, for example by an or or
mechanoid (e.g., a robot equipped with a color, reflectivity or other sensor) that removes
ed 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 processing can include heating the material. For example a
portion of the conveyor can be sent through a heated zone. The heated zone can be created, for
e, by IR radiation, microwaves, combustion (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
. Heating can be, for example, for the purpose of drying the material. In the case of drying
the material, this can also be facilitated, with or without heating, by the nt of a gas (6.g.
air, , nitrogen, He, C02, Argon) over and/or through the biomass as it is being conveyed.
Optionally, pre-treatment processing can include cooling the material. g
material is described in US Pat. No. 7,900,857 to , 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 glycerol), 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 processing method can include adding a material to
the biomass. The additional material can be added by, for example, by showering, sprinkling
and or g the material onto the biomass as it is conveyed. Materials that can be added
include, for e, metals, cs and/or ions as described in US. Pat. App. Pub.
2010/0105119 Al (filed October 26, 2009) and US. Pat. App. Pub. 2010/0159569 Al (filed
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, tes), polymers, rizable monomers (e.g., containing
unsaturated bonds), water, catalysts, enzymes and/or organisms. Materials can be added, for
example, in pure form, as a on in a solvent (e.g., water or an organic solvent) and/or as a
solution. 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 m coating on
the biomass or be a homogeneous mixture of different components (e.g., s and additional
material). The added material can modulate the subsequent irradiation step by increasing the
efficiency of the irradiation, g the irradiation or changing 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 fithher downstream processing. The added material may help in
conveying the material, for example, by lowering dust levels.
Biomass can be delivered to the conveyor by a belt conveyor, a tic conveyor,
a screw conveyor, a hopper, a pipe, ly or by a combination of these. The biomass can, for
-l6-
example, be d, poured and/or placed onto the conveyor by any of these methods. In some
embodiments the material is delivered to the conveyor using an enclosed material distribution
system to help maintain a low oxygen atmosphere and/or l dust and fines. Lofted or air
suspended biomass fines and dust are rable because these can form an explosion hazard or
damage the window foils of an electron gun (if such a device is used for ng the material).
The material can be leveled to form a uniform thickness between about 0.0312 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,
0.150 --/- 0.025 inches, 0.200 --/- 0.025 inches, 0.250 --/- 0.025 inches, 0.300 --/- 0.025 inches,
0.350 --/- 0.025 inches, 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 , 0.900 --/- 0.025 .
Generally, it is preferred to convey the material as quickly as possible h 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
, at least 15 ft/min, 20, 25, 30, 35, 40, 45, 50 . The rate of conveying is d 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 ation dosage.
After the biomass material has been conveyed through the radiation zone, optional
post-treatment processing can be done. The optional post-treatment sing can, for example,
be a process described with t to the pre-irradiation processing. For example, the biomass
can be screened, heated, cooled, and/or combined with additives. Uniquely to post-irradiation,
quenching of the radicals can occur, for example, quenching of ls by the addition of fluids
or gases(e.g., oxygen, nitrous oxide, ammonia, liquids), using pressure, heat, and/or the addition
of radical scavengers. 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 groups. In one
embodiment the biomass is exposed during irradiation to the reactive gas or fluid. Quenching of
biomass that has been irradiated is described in US. Pat. No. 8,083,906 to Medoff, the entire
disclosure of which is incorporate herein by reference.
If d, one or more mechanical treatments can be used in addition to irradiation to
fiarther reduce the recalcitrance of the biomass al. These processes can be applied before,
during and or after irradiation.
In some cases, the ical treatment may include an initial ation of the
ock as received, e.g., size reduction of materials, such as by comminution, e.g, cutting,
ng, 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.
Mechanical treatment may reduce the bulk density of the biomass material, increase the surface
area of the biomass material and/or se one or more dimensions of the biomass material.
Alternatively, or in addition, the feedstock material can first be physically treated by
one or more of the other physical treatment methods, 6.g. chemical treatment, radiation,
sonication, oxidation, pyrolysis or steam explosion, and then mechanically treated. This
sequence can be advantageous since materials d by one or more of the other treatments,
6.g. irradiation or pyrolysis, tend to be more brittle and, therefore, it may be easier to further
change the ure of the material by mechanical treatment. For example, a ock al
can be conveyed through ionizing radiation using a conveyor as described herein and then
mechanically treated. 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 hydrolyzed and non-hydrolyzed
materials, for example with about 50% or more non-hydrolyzed material, with about 60% or
more non- hydrolyzed material, with about 70% or more drolyzed material, with about
80% or more non-hydrolyzed material or even with 90% or more non-hydrolyzed material.
In on to size reduction, which can be performed initially and/or later in
processing, mechanical treatment can also be advantageous for “opening up,3, “stressing,”
breaking or shattering the biomass materials, making the cellulose of the materials more
susceptible to chain scission and/or disruption of crystalline ure during the physical
treatment.
Methods of mechanically treating the biomass material include, for example, milling
or grinding. Milling may be med using, for e, a mill, ball mill, colloid mill, conical
or cone mill, disk mill, edge mill, Wiley mill, grist mill or other mill. Grinding may be
performed using, for example, a cutting/impact type grinder. Some exemplary grinders include
stone grinders, pin grinders, coffee grinders, and burr grinders. ng or g may be
provided, for e, by a reciprocating pin or other element, as is the case in a pin mill. Other
mechanical treatment methods include mechanical ripping, tearing, shearing or chopping, other
methods that apply pressure to the fibers, and air attrition milling. Suitable mechanical
treatments further include any other technique that continues the disruption of the internal
structure of the material that was initiated by the previous processing steps.
Mechanical feed preparation systems can be configured to produce streams with
specific characteristics such as, for example, specific maximum sizes, specific length-to-width,
or specific 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 accessible to processes and/or reagents, such as reagents in a
solution.
The bulk density of ocks can be controlled (e.g., increased). In some ions,
it can be desirable to prepare a low bulk density al, 6.g. the material (e.g.,
, by densifying
densif1cation 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 1ed, 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 densif1ed 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.
led materials can be processed by any of the methods described herein, or any material
processed by any of the s described herein can be subsequently densif1ed.
In some embodiments, the material to be sed is in the form of a fibrous material
that includes fibers provided by ng a fiber source. For example, the ng can be
performed with a rotary knife cutter.
2012/071097
For example, 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 shearing, 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. 1/4- to 1/2-inch wide, using
a shredder, e.g., a counter-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 cutting to a
desired size using a tine cutter. For example, the tine 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 ing
first fibrous material through a first screen are med concurrently. The shearing and the
passing can also be performed in a type s.
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 shredded fiber source prepared by shredding a fiber source. The shredded fiber source.
In some implementations, the feedstock is physically treated prior to saccharification
and/or fermentation. Physical treatment processes can include one or more of any of those
described herein, such as mechanical treatment, al treatment, irradiation, sonication,
oxidation, sis or steam explosion. Treatment methods can be used in combinations of two,
three, four, or even all of these logies (in any order). When more than one treatment
method is used, the methods can be applied at the same time or at ent 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 materials, are described in fithher detail in US. Pat. App. Pub. 2012/0100577
A1, filed October 18, 2011, the fill disclosure of which is hereby incorporated herein by
reference.
TREATMENT OF BIOMASS MATERIAL -- PARTICLE BOMBARDMENT
One or more treatments with energetic particle bombardment can be used to process
raw feedstock from a wide variety of different sources to extract useful substances from the
feedstock, and to provide partially degraded organic material which functions as input to fithher
processing steps and/or sequences. Particle bombardment can reduce the molecular weight
and/or crystallinity of feedstock. In some embodiments, energy deposited in a material that
es an electron from its atomic orbital can be used to treat the materials. The bombardment
may be provided by heavy d particles (such as alpha particles or protons), electrons
(produced, for example, in beta decay or on 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 approach, electromagnetic
radiation (e.g., produced using electron beam rs) can be used to treat the feedstock.
] Each form of energy ionizes the biomass via particular interactions. Heavy d
particles ily ionize matter via Coulomb scattering; fithhermore, these interactions produce
energetic electrons that may further ionize matter. Alpha particles are identical 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, neptunium, curium, califomium, americium, and ium.
When particles are utilized, they can be neutral (uncharged), positively charged or
vely 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 instances in
which chain scission is desired, positively charged particles may be ble, 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 g on. 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., l, 2, 3,
4, 5, 10, 12 or 15 atomic units. Accelerators used to accelerate the particles can be electrostatic
DC, electrodynamic DC, RF linear, magnetic induction linear or uous wave. For example,
cyclotron type accelerators are available from IBA (Ion Beam Accelerators, n-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 r Physics, Kenneth S. Krane, John Wiley & Sons, Inc. (1988), Krsto ,
FIZIKA B 6 (1997) 4, 177-206; Chu, William T., “Overview of Light-Ion Beam Therapy”,
Columbus-Ohio, ICRU-IAEA Meeting, 18-20 Mar. 2006; Iwata, Y. et al., ating-Phase-
d IH-DTL for Heavy-Ion Medical Accelerators”, Proceedings of EPAC 2006, Edinburgh,
Scotland; and Leitner, C. M. et al., “Status of the Superconducting ECR Ion Source Venus”,
Proceedings 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 components 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 ties for enhanced ring-opening chain scission. For example,
when oxygen-containing fianctional groups are d, 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 en or even treatment with
nitrogen ions can be performed.
OTHER FORMS OF ENERGY
ons interact Via Coulomb ring and trahlung radiation produced by
changes in the velocity of electrons. ons may be produced by radioactive nuclei that
undergo beta decay, such as isotopes of iodine, cesium, technetium, and m. Alternatively,
an electron gun can be used as an electron source via thermionic emission.
Electromagnetic radiation interacts via three ses: photoelectric absorption,
Compton scattering, and pair production. The dominating interaction 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 subclassified as gamma rays, x rays, ultraviolet rays,
infrared rays, microwaves, or radiowaves, depending on the wavelength.
For e, gamma radiation can be employed to treat the materials. Gamma
radiation has the advantage of a cant penetration depth into a y of material in the
sample. Sources of gamma rays include radioactive nuclei, such as isotopes of cobalt, calcium,
technetium, chromium, m, indium, iodine, iron, krypton, um, selenium, sodium,
thalium, and xenon.
Sources of x rays include electron beam collision with metal targets, such as tungsten
or molybdenum or alloys, or compact light sources, such as those produced commercially by
Lyncean.
s for ultraviolet radiation include deuterium or cadmium lamps.
] Sources for infrared radiation 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 s may be used in the methods disclosed herein, including field
tion sources, electrostatic ion separators, field ionization generators, thermionic emission
s, ave discharge ion sources, recirculating or static accelerators, c linear
accelerators, van de Graaff accelerators, and folded tandem accelerators. Such s are
disclosed, for example, in US. Pat. No. 7,931,784 B2, the complete disclosure of which is
incorporated herein by reference.
TREATMENT OF BIOMASS MATERIAL -- ELECTRON BOMBARDMENT
The feedstock may be treated with electron bombardment to modify its structure and
thereby reduce its recalcitrance. Such ent 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 hput and because the use of a relatively low voltage/high power
electron beam device eliminates the need for expensive concrete vault ing, 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 e the construction of a concrete
vault, greatly reducing capital expenditure and often allowing an existing manufacturing facility
to be used without expensive modification. Electron beam accelerators are available, for
example, from IBA (Ion Beam Applications, Louvain-la-Neuve, Belgium), Titan Corporation
(San Diego, California, USA), and NHV Corporation (Nippon 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 nominal energy is about 500 to 800 keV.
The electron beam may have a relatively high total beam power (the ed 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 le accelerating
heads. For example, the electron beam device may include two, four, or more rating
heads. The use of multiple heads, each of which has a relatively low beam power, prevents
excessive temperature rise in the material, thereby preventing burning of the material, and also
ses the uniformity of the dose through the ess of the layer of material.
In some implementations, it is desirable to cool the material during electron
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 process, it is ble to
treat the al 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, 1, 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 embodiments, 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, lO
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, applied 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 thermal degradation of the
feedstock material.
Using multiple heads as discussed above, the material can be treated in multiple
, 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 ll Mrad/pass. As
sed above, treating the al 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
fiarther enhance treatment uniformity.
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 t of the speed of
light.
In some ments, any sing described herein occurs on ellulosic
material that remains dry as acquired or that has been dried, e.g., using heat and/or reduced
pressure. 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 ty.
Electron bombardment can be applied while the cellulosic and/or lignocellulosic
material is exposed to air, oxygen-enriched air, or even oxygen , or blanketed by an inert
gas such as nitrogen, argon, or helium. When maximum oxidation is desired, an oxidizing
environment is utilized, such as air or oxygen and the ce 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 s. 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 radiation sources, such as a
beam of electrons, 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 s material has a relatively uniform thickness, as previously described, while being
treated.
It may be advantageous to repeat the treatment to more thoroughly reduce the
recalcitrance of the biomass and/or fiarther 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 material. In some embodiments, a conveyor can be used which es a
ar system where the biomass is conveyed multiple times through the s processes
bed above. In some other embodiments multiple treatment s (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 multiple 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 biomass 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 med until the al es 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 med until the material
receives 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 ments, the treatment is performed at a dose rate of between 5.0 and
1500.0 kilorads/hour, e.g., between 10.0 and 750.0 ds/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.
ELECTRON SOURCES
Electrons interact via Coulomb scattering and bremsstrahlung radiation produced by
changes in the ty of ons. Electrons may be produced by radioactive nuclei that
undergo beta decay, such as isotopes of iodine, cesium, technetium, and iridium. Alternatively,
an electron gun can be used as an on source via thermionic emission and accelerated
through an accelerating ial. An electron gun generates electrons, accelerates them through
a large potential (e.g., greater than about 500 thousand, greater than about lmillion, greater than
about 2 million, greater than about 5 million, greater than about 6 million, r 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 magnetically in the X-y plane, where the electrons are initially
accelerated in the z direction down the tube and extracted through a foil window. Scanning the
electron beam is useful for increasing the irradiation surface when irradiating als, e.g., a
biomass, that is conveyed through the scanned beam. Scanning the electron beam also
distributes the thermal load homogenously on the window and helps reduce the foil window
rupture due to local heating by the electron beam. Window foil rupture is a cause of significant
down-time due to subsequent necessary repairs and rting the electron gun.
9] s other irradiating devices may be used in the s disclosed herein,
including field ionization sources, electrostatic ion separators, field ionization generators,
thermionic emission sources, ave discharge ion sources, recirculating or static
accelerators, dynamic linear accelerators, van de Graaff accelerators, and folded tandem
accelerators. Such devices are disclosed, for example, in US. Pat. No. 7,931,784 to Medoff, the
complete disclosure of which is incorporated herein by reference.
A beam of electrons can be used as the radiation . A beam of electrons has the
advantages of high dose rates (e.g., l, 5, or even 10 Mrad per second), high throughput, less
containment, and less confinement equipment. 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. on beams can be ted, e.g., by
electrostatic tors, e generators, transformer generators, low energy accelerators with
a scanning system, low energy accelerators with a linear cathode, linear accelerators, and pulsed
accelerators.
Electrons can also be more efficient at causing changes in the molecular ure of
biomass materials, for example, by the mechanism of chain scission. In addition, ons
having energies of 0.5-10 MeV can penetrate low density als, such as the biomass
materials described herein, e.g., materials having a bulk density of less than 0.5 g/cm3, and a
depth of 03-10 cm. Electrons as an ionizing 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 discussed 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
ations (Louvain-la-Neuve, Belgium), the Titan Corporation (San Diego, California, USA),
and NHV Corporation (Nippon High Voltage, . Typical electron energies can be 0.5
MeV, 1 MeV, 2 MeV, 4.5 MeV, 7.5 MeV, or 10 MeV. Typical 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,
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 irradiation device power specifications
e cost to operate, capital costs, depreciation, and device footprint. Tradeoffs in
considering exposure dose levels of electron beam irradiation would be energy costs and
environment, 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 considering electron energies include energy costs.
The on 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 e 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 red in most
ments describe herein because of the larger scan width and reduced possibility of local
heating and failure of the windows.
TREATMENT OF BIOMASS MATERIAL -- SONICATION, PYROLYSIS, OXIDATION,
STEAM EXPLOSION
If d, one or more sonication, pyrolysis, oxidative, or steam explosion 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. 7,932,065 to Medoff, the
filll disclosure of which is orated herein by reference.
USE OF D 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
ediates and products such as organic 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 ses are described herein that can use as feedstock cellulosic and/or
lignocellulosic materials that are readily available, but often can be difficult to process, e.g.,
municipal waste streams and waste paper streams, such as streams 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
cturing plant, or a fuel plant, 6.g. , an ethanol manufacturing facility.
The ock can be hydrolyzed using an enzyme, e.g., by combining the materials
and the enzyme in a t, 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 portions of the biomass, contain or manufacture various
cellulolytic enzymes (cellulases), ligninases or s small molecule s-degrading
metabolites. These enzymes may be a x of enzymes that act synergistically to degrade
crystalline cellulose or the lignin portions of s. es of olytic enzymes include:
endoglucanases, cellobiohydrolases, and cellobiases (beta-glucosidases).
2012/071097
During saccharification a cellulosic substrate can be lly hydrolyzed by
endoglucanases at random locations producing oligomeric intermediates. These intermediates
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 material.
INTERMEDIATES AND PRODUCTS
The processes described herein are preferably used to produce butanol, e.g.,
isobutanol or n-butanol, and tives. However, the processes may be used to produce other
products, co-products and ediates, for example, the products described in US. Pat. App.
Pub. 2012/0100577 Al, filed October 18, 2011 and published April 26, 2012, the full disclosure
of which is incorporated herein by reference.
Using the processes described herein, the s material can be converted to one or
more products, such as energy, fuels, foods and materials. Specific examples of products
include, but are not limited to, hydrogen, sugars (e.g., glucose, xylose, arabinose, mannose,
galactose, fructose, disaccharides, oligosaccharides and polysaccharides), alcohols (e.g.,
dric ls or dihydric alcohols, such as ethanol, n-propanol, isobutanol, sec-butanol,
tert-butanol or n-butanol), hydrated or hydrous ls (e.g., containing greater than 10%, 20%,
% or even greater than 40% water), biodiesel, organic acids, hydrocarbons (e.g., methane,
ethane, propane, isobutene, pentane, n-hexane, sel, bio-gasoline and es thereof), co-
products (e.g., proteins, such as cellulolytic proteins (enzymes) or single cell proteins), and
mixtures of any of these in any combination 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), aldehydes (e.g.,
acetaldehyde), alpha and beta unsaturated acids (e.g., acrylic acid) and olefins (e.g., ethylene).
Other alcohols and alcohol derivatives e ol, propylene glycol, l,4-butanediol, 1,3-
propanediol, sugar ls and polyols (e.g., glycol, glycerol, erythritol, threitol, arabitol,
xylitol, ribitol, mannitol, sorbitol, galactitol, iditol, inositol, tol, isomalt, ol, lactitol,
maltotriitol, maltotetraitol, and polyglycitol and other polyols), and methyl or ethyl esters of any
of these alcohols. Other ts include methyl acrylate, methylmethacrylate, lactic acid, citric
acid, formic acid, acetic acid, propionic acid, butyric acid, succinic 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 mixtures thereof,
salts of any of these acids, mixtures of any of the acids and their respective salts.
Any combination 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 sanitized or
sterilized prior to selling the products, e.g., after purification or isolation or even after ing,
to neutralize one or more potentially undesirable contaminants that could be t in the
product(s). Such tion 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 generated from burning by-product s 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
sources hout the process. For example, anaerobic ion of ater 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
llulose 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 fuel
for powering cars, trucks, tractors, ships or trains, e.g., as an internal combustion filel or as a fuel
cell feedstock. Many of the products obtained can also be utilized to power aircraft, such as
, e.g., having jet engines or helicopters. In on, 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. 124583 A1, published May 20, 2010, to Medoff, the fill
disclosure of which is hereby incorporated by reference herein.
SACCHARIFICATION
To obtain a fructose solution from the reduced-relacitrance feedstock, the treated
biomass materials can be rif1ed and then ized and optionally ed, generally by
combining the material and a cellulase enzyme in a fluid medium, 6.g. , an aqueous solution. In
some cases, the material is , steeped, or cooked in hot water prior to saccharification, as
described in US. Pat. App. Pub. 2012/0100577 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 lly 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 required for complete saccharif1cation will depend on
the process conditions and the biomass material and enzyme used. If saccharification is
performed in a manufacturing plant under controlled conditions, the ose may be
substantially entirely converted to sugar, e.g., glucose in about 12-96 hours. If saccharif1cation is
performed partially or completely in transit, saccharif1cation may take longer.
It is lly preferred that the tank contents be mixed during saccharif1cation, e.g.,
using jet mixing as described in International App. No. ZOlO/O3533 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 .
The addition of surfactants can e the rate of saccharif1cation. Examples of
tants include non-ionic surfactants, such as a Tween® 20 or Tween® 80 polyethylene
glycol surfactants, ionic surfactants, or amphoteric surfactants.
It is generally preferred 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 ation, to increase the
concentration of the sugar solution. This reduces the volume to be shipped, and also ts
microbial growth in the solution.
The concentration of se solutions, for example after ization of a
saccharif1ed on, can be between about 1 an 40%. For example between about 5 and 40%,
between about 10 and 40%, between about 15 and 40%, between about 5 and 10%, between
about 10% and 30% and between about 30% and 40%.
Other sources of fructose can also be utilized. For example fructose can be obtained
from Molasses. Some examples of different kinds of molasses are Sugar Cane Molasses, Citrus
Molasses, Starch Molasses, Blackstrap Molasses and/or Sugar Beet Molasses. Glucose in
molasses can range between about 30% to 70% (e.g., 40% to 60%, eg., 45% to 55%) of the
glucose/fructose total, for example high fructose corn syrup is 55% fructose and 45% glucose.
ts from fruits can also be a source of high fructose products, for example agava extract can
have 90% fructose and 10% glucose. The isomerization of glucose solutions can increase the
concentrations of glucose solutions and is another source of fructose. Isomerization can be done
by an isomerase as discussed herein. Another source of fructose is the hydrolysis of sucrose, for
example using an enzyme (e.g., se), using and acid and/or using a base.
Alternatively, sugar solutions of lower concentrations may be used, in which case it
may be desirable to add an antimicrobial additive, eg., a broad spectrum otic, in a low
concentration, eg., 50 to 150 ppm. Other suitable antibiotics include ericin 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 tration is vely high. Alternatively, other additives with
anti-microbial of preservative properties may be used. Preferably the crobial additive(s)
are food-grade.
A relatively high concentration solution can be obtained by limiting the amount of
water added to the biomass material with the enzyme. The tration can be controlled, 6.g.
by controlling how much saccharification takes place. For example, concentration can be
sed by adding more biomass material to the solution. In order to keep the sugar that is
being ed in solution, a surfactant can be added, e.g., one of those discussed above.
Solubility can also be increased by increasing the temperature of the solution. For e, the
solution can be ined at a temperature of 40-50°C, C, or even higher.
By adding glucose isomerase to the ts of the tank, a high concentration of
fructose can be ed without saccharification being inhibited by the sugars in the tank.
Glucose isomerase can be added in any amount. For example, the concentration may be below
about 500 U/g of cellulose (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 is at least about 0.1 U/g cellulose (at least about 0.5 U/g cellulose, at least
about lU/g cellulose, at least about 2 U/g cellulose, at least about 3 U/g cellulose).
The addition of glucose isomerase increases the amount of sugars produced by at
least 5 % (at least 10 %, at least to 15 %, at least 20 %).
0] The concentration of sugars in the solution can also be enhanced by limiting the
amount of water added to the feedstock with the enzyme, and/or the concentration can be
increased by adding more feedstock to the on during saccharification. 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 increased by increasing the temperature of the solution. For
example, the solution can be maintained at a temperature of 40-50°C, 60-80°C, or even higher.
SACCHARIFYING AGENTS
le cellulolytic enzymes include cellulases from species in the genera Bacillus,
CaprinuS, Mycell'ophthora, Cephalosporz'um, z'dz'um, Penicillium, ASpergz'lluS,
Pseudomonas, Humicola, Fusarium, Thielavz'a, nium, 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), Humicola insolenS (reclassified as Scytalz'dz'um thermophilum, see, e.g., US. Pat.
No. 4,435,307), CaprinuS cinereus, um oxySporum, Mycelz'ophthora thermophila,
'luS giganteus, Thielavz'a triS, Acremonium Sp. (including, but not limited to, A.
perSl'cz'num, A. acremonium, A. brachypem'um, A. dichromosporum, A. obclavatum, A.
pinkertonz'ae, A. roseogriseum, A. incoloratum, and A. furaz‘um). Preferred strains include
Humicola insolenS DSM 1800, Fusarium oxySporum DSM 2672, Mycelz'ophthora thermophila
CBS 117.65, Cephalosporz'um Sp. RYM-202, Acremonium Sp. CBS 478.94, Acremonium Sp.
CBS , Acremonium perSicz'num CBS 169.65, Acremonium acremonium AHU 95 19,
Cephalosporz'um Sp. CBS 535.71, Acremonium brachypem'um CBS , Acremonium
dichromosporum CBS 683.73, Acremonium obclavatum CBS 311.74, Acremonium pinkertom'ae
CBS 157.70, Acremonium roseogrz'seum 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), philic Bacillus (see, for example, US. Pat. No. 3,844,890 and EP Pub. No. 0 458
162), and omyces (see, e.g., EP Pub. No. 0 458 162).
Many microorganisms that can be used to saccharify biomass material and produce
sugars can also be used to ferment and convert those sugars to useful products.
SUGARS
In the processes described herein, for example after rification, sugars (e.g.,
glucose and ) can be isolated. For example sugars can be isolated by precipitation,
crystallization, tography (6.g. , simulated moving bed chromatography, high re
tography), centrifilgation, extraction, any other isolation method known in the art, and
combinations thereof.
HYDROGENATION AND OTHER CHEMICAL ORMATIONS
The processes 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 catalysts
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., furfural and furfural-derived
products). Chemical transformations of sugar d ts 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.
FERMENTATION
ably, Clostrz'dz'um spp. are used to convert sugars (e.g., fructose) to butanol.
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 embodiments, e.g., when anaerobic organisms are used, at least a portion of
the fermentation is ted in the absence of oxygen, e.g., under a blanket of an inert gas such
as N2, Ar, He, C02 or mixtures thereof Additionally, the mixture may have a constant purge of
an inert gas flowing h the tank during part of or all of the fermentation. In some cases,
anaerobic condition, can be ed or maintained by carbon dioxide production during the
fermentation and no additional inert gas is needed.
In some embodiments, all or a n of the fermentation process can be upted
before the low molecular weight sugar is completely converted to a t (e.g., ethanol). The
intermediate fermentation ts include sugar and carbohydrates in high concentrations. The
sugars and carbohydrates can be isolated via any means known in the art. These intermediate
fermentation products 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 produce a flour-like substance.
Jet mixing may be used during fermentation, and in some cases saccharification and
fermentation are performed in the same tank.
Nutrients for the microorganisms may be added during saccharification and/or
fermentation, for example the food-based nutrient packages bed in US. Pat. App. Pub.
2012/0052536, filed July 15, 2011, the te disclosure of which is orated herein by
reference.
“Fermentation” includes the methods and products that are disclosed 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 bed 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 ts of which is incorporated herein in
its entirety. Similarly, the saccharif1cation equipment can be mobile. Further, saccharif1cation
and/or fermentation may be performed in part or entirely during transit.
OTHER FERMENTATION AGENTS
2] Although Clostrz'dz'am is preferred, other microorganisms can be used. For instance,
yeast and Zymomonas bacteria can be used for fermentation or sion of sugar(s) to other
alcohol(s). Other microorganisms are discussed below. They can be lly-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 fiangus, (including, but
not d 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 glucose, fructose, xylose, arabinose, mannose, galactose, oligosaccharides or polysaccharides
into fermentation products. Fermenting microorganisms include strains of the genus
romyces spp. ding, but not limited to, S. cerevisiae (baker’s yeast), S. z'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. tropicalz’s, and C. brassz'cae), Pichia
stz’pz’tz’s (a relative of a shehatae), the genus Clavz'spora (including, but not limited to, C.
lasitam'ae and C. opantz'ae), the genus Pachysolen (including, but not limited to, P. hz'las),
the genus nomyces (including, but not limited to, e.g., B. clausem'z' (Philippidis, G. P.,
1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and
Utilization, Wyman, C.E., ed., Taylor & Francis, gton, DC, l79-2l2)). Other suitable
microorganisms include, for example, Zymomonas mobilis, Clostrz'dz'am spp. ding, but not
limited to, C. thermocellum (Philippidis, l996, supra), C.saccharobutylacetonicum, C.
saccharobutylz’cum, C. Puniceum, C. beijemckiz’, and C. acetobutylz'cum), Mom'lz'ella pollinz's,
Mom'lz'ella iliensz's, Lactobacz'llus spp. Yarrowz'a lipolytl'ca, Aureobasidz'um 519.,
Trichosporonoz'des 519., Trigonopsz's variabilis, Trichosporon sp., Monilz'ellaacetoabutans sp.,
Typhula variabilis, a magnoliae, Ustilaginomycetes sp.,Pseudozyma tsukubaensis,yeast
s of genera Zygosaccharomyces, omyces, Hansenula and Pichia,and fungi of the
dematioid genus Torula.
4] For instance, Clostrz'dz'um spp. can be used to produce ethanol, l, butyric acid,
acetic acid, and acetone. Lactobacillus spp., can be used to produce lactic acid.
Many such microbial strains are publicly available, either commercially or through
depositories such as the ATCC (American Type Culture Collection, Manassas, Virginia, USA),
the NRRL (Agricultural Research Service Culture Collection, Peoria, Illinois, USA), or the
DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig,
Germany), to name a few.
Commercially available yeasts include, for example, Red Star®/Lesaffre Ethanol Red
(available from Red esaffre, USA), FALI® (available from Fleischmann’s Yeast, a division
of Burns Philip Food Inc., USA), SUPERSTART® (available from h, now Lalemand),
GERT STRAND® (available from Gert Strand AB, Sweden) and FERMOL® (available from
DSM Specialties).
Many microorganisms that can be used to saccharify biomass material and produce
sugars can also be used to ferment and t those sugars to useful products.
DISTILLATION
After fermentation, the ing 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 l and can be fed to a
rectif1cation 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
rectif1cation column reflux ser can provide 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 t 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 sate with a small portion split off to waste water treatment to prevent build-up of
low-boiling compounds.
Other than in the examples herein, or unless otherwise expressly specified, all of the
cal ranges, s, values and percentages, such as those for amounts of materials,
elemental contents, times and atures of reaction, ratios of amounts, and , in the
following portion of the specification and attached claims may be read as if prefaced by the word
“about” even though the term “about” 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 ne of equivalents to the scope of the claims,
each cal parameter should at least be construed in light of the number of reported
significant digits and by ng ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters 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
g ements. 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 reported are relative to the total .
Also, it should be understood that any numerical range recited herein is intended to
include all sub-ranges subsumed therein. For e, a range of “l to 10” is intended to
include all sub-ranges between (and including) the recited minimum value of l and the d
maximum value of 10, that is, having a minimum value equal to or r than 1 and a
maximum value of equal to or less than 10. The terms “one,a) :4 a)
a or “an” as used herein are
intended to include “at least one” or “one or more,” unless otherwise indicated.
EXAMPLES
e 1. Butanol Production on Glucose, Xylose and Fructose
A P2 based medium as described in US 6,358,717 was used for the following tests.
The medium was composed of the following separately prepared solutions (in grams per 100 ml
of distilled water, unless indicated otherwise): sugar (see below for types and amounts), 790 ml
of distilled water (solution I), 0.5 g of K2HPO4, 0.5 g of KH2P04, 2.2 g of CH3COONH4
(solution 11), 2.0 g ofMgSO4-7H20, 0.1 g oanSO4- H20, 0.1 g ofNaCl, 0.1 g ofFeSO4-7H20
(solution III), and 100 mg of p-aminobenzoic acid, 100 mg of thiamine, 1 mg of biotin (solution
IV). Solutions I and II were filter sterilized and subsequently mixed to form a sugar buffer
solution. Solutions III and IV were filter sterilized. Portions (10 and 1 ml) of solutions III and
IV, respectively, were added aseptically to the sugar-buffer solution. The final pH of the P2
medium was 6.6.
The amounts of sugars used were: for medium GXP2, 43g e and 24g xylose;
for medium FP2, 43g of fructose; for medium FGP2, 43g fructose and 43g glucose.
The solutions were d with argon for 45 min and then brought into an anaerobic
chamber. Solutions (lOmL) were measured into 21, 20mL previously aved serum vials.
The vials were sealed with a sealable septum and then brought out of the anaerobic box. Vials
were inoculated with 1 vol% Clostrz'dz'um saccharoperbutylacetom’cum (ATCC 27021) from an
aqueous glycerol stock prepared following the ATCC recommended protocol from the ATCC
provided pellet. The vials were grown at 30°C for 48 or 96 hours. The head space was analyzed
for l tion using GC. The results (in g/L) are shown in the table below.
Table 2. Results of butanol tion on three different carbon sources.
Sample ID Time Point Media n-Butanol
1 48hr GXP2 3.2
2 48hr GXP2 2.9
3 48hr GXP2 2.0
4 48hr FP2 11.5
48hr FP2 11.0
6 48hr FP2 11.7
7 48hr FGP2 3.1
8 48hr FGP2 3.0
9 48hr FGP2 3.5
96hr GXP2 2.6
11 96hr GXP2 2.9
12 96hr GXP2 3.0
Control-13 96hr GXP2 0.0
14 96hr FP2 11.1
96hr FP2 12.0
16 96hr FP2 11.4
Control-17 96hr FP2 0.0
18 96hr FGP2 2.3
19 96hr FGP2 2.7
96hr FGP2 3.4
Control-21 96hr FGP2 0.0
Example 2. Butanol tion on Fructose vs. Glucose & Xylose
Ten ml of P2 media containing either a e/xylose mixture of fructose alone (32
g/L) were incubated at 30°C with one of either Clostridium saccharoperbutylacetonium ATCC
strain 27021 or 27022. As in Example 1, the results presented in the table below show that more
butanol is generated when Clostridium is grown on fructose, as opposed to glucose or xylose.
Table 3. Clostridium Vial growth on fructose or glucose/xylose as the carbon source.
Strain Substrate Butanol Production (g/L) Timepoint (hrs)
ATCC 27021 Fructose 11.7 48
ATCC 27021 Glucose/Xylose 2.3 48
ATCC 27022 Fructose 11.6 96
ATCC 27022 Glucose/Xylose 4.0 96
Any patent, publication, or other disclosure al, 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 sure
material set forth in this disclosure. As such, and to the extent necessary, the sure as
explicitly set forth herein supersedes any conflicting material incorporated herein by reference.
Any material, or n thereof, that is said to be incorporated by reference herein, but which
ts with existing definitions, statements, or other disclosure material set forth herein will
only be incorporated to the extent that no conflict arises between that orated material and
the ng disclosure material.
While this invention has been particularly shown and described with references to
preferred embodiments f, 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 encompassed by the appended claims.
Claims (12)
1. A method for ing a carboxylic acid, or a salt thereof, the method comprising: rifying a cellulosic or lignocellulosic biomass with an enzyme to produce a solution comprising one or more sugars; contacting the solution with an isomerization agent to increase the concentration of fructose in the solution; and converting the fructose to a carboxylic acid, or a salt thereof, with a microorganism.
2. The method of claim 1, further comprising maintaining metabolic conditions that cause the microorganism to produce a protective substrate that protects the microorganism from a toxic effect of the carboxylic acid or a salt thereof, n the protective substrate comprises one or more lipids.
3. The method of claim 1 or 2, wherein the isomerization agent ses glucose isomerase or xylose isomerase.
4. The method of any one of the preceding claims, wherein the cellulosic or lignocellulosic s is treated to reduce its recalcitrance to saccharification.
5. The method of claim 4, wherein the treatment is ed from the group consisting of: bombardment with electrons, sonication, oxidation, pyrolysis, steam ion, chemical treatment, mechanical treatment, freeze grinding, and any combination thereof.
6. The method of claim 4 or 5, wherein the treatment comprises bombardment with electrons.
7. The method of any one of the preceding claims, wherein the cellulosic or ellulosic biomass is selected from the group consisting of: paper, paper products, paper waste, forestry wastes, s, grain residues, agricultural waste, silage, sugar processing es, manure, sewage, industrial waste, and mixtures of any of these.
8. The method of any one of the ing claims, wherein the cellulosic or lignocellulosic biomass is selected from the group consisting of: paper pulp, pigmented papers, loaded papers, coated papers, filled papers, magazines, d matter, printer paper, polycoated paper, card stock, cardboard, paperboard, cotton, wood, particle board, sawdust, aspen wood, wood chips, switchgrass, miscanthus, cord grass, reed canary grass, rice hulls, oat hulls, wheat chaff, barley hulls, 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, bagasse, beet pulp, agave bagasse, arracacha, buckwheat, banana, barley, a, kudzu, oca, sago, m, potato, sweet potato, taro, yams, beans, favas, s, peas, algae, seaweed, and mixtures of any of these.
9. The method of any one of the preceding claims, wherein the microorganism comprises a strain of Clostridium spp.
10. The method of claim 2, wherein the one or more lipids comprises a triglyceride.
11. The method of claim 10, wherein the triglyceride is produced by esterification of glycerol 3-phosphate through a D-glyceraldehyde pathway.
12. The method of any one of the preceding claims, wherein the carboxylic acid or a salt thereof comprises c acid, or a salt thereof WO 96703 SUBSTITUTE SHEET (RULE 26)
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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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 | ||
NZ73115812 | 2012-12-20 |
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NZ748522B2 true NZ748522B2 (en) | 2020-06-30 |
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