OA16926A - Processing biomass for use in fuel cells. - Google Patents

Abstract

Biomass (e.g., plant biomass, animal biomass, and municipal waste biomass) is processed to produce a carbohydrate solution that can be used in a fuel cell, e.g., a direct glucose fuel cell.

Description

PROCESSING BIOMASS FOR USE IN FUEL CELLS

RELATED APPLICATIONS

This application daims priority to U.S. Provisional Application Serial No.

61/579,568, filed December 22,2011, the complété disclosure thereln incorporated herein by reference.

BACKGROUND

Cellulosic and lignoceilulosic materials (e.g„ biomass materials) are produced, processcd, and used in large quantités in a number of applications. Often such materials are used once, and then discarded as waste, or are simply considered to be waste materials, e.g., sewage, bagasse, sawdust, and stover.

A typical biomass material contains cellulose, hemicellulose, and lignin plus lesser amounts of proteins, extractables and minerais. The complex carbohydrates contained in the cellulose and hemicellulose fractions can be processed into fermentable sugars by saccharification, using a cellulolytic enzyme, and the sugars can then bc used as an end product or intermediate, or converted b y further bioprocessing, e.g., fermentation, into a varicty of products, such as alcohols or organic acids. The product obtained dépends upon the microorganism utilized and the conditions under which the bioprocessing occurs.

SUMMARY

This invention relates to methods of processing (e.g., saccharifying) carbohydrate-containing materials (e.g., biomass materials or biomass-derived materials) to produce sugar (e.g., glucose) solutions that can be used in fuel cells such as direct sugar fuel cells. Indirect sugar fuel cells and biological fuel cells. The invention also relates to utilizing the carbohydrate-containing material derived sugars solutions in fuel cells.

ln some implémentations, the sugar solution is produced by the saccharification of a lignoceilulosic or cellulosic material, for example by contacting the material with an enzyme (e.g., a cellulose). In other implémentations the recalcitrancc of the lignoceilulosic or cellulosic material has been reduced relative to

that of the material ïn its native state prior to saccharification. In some cases, reducing the recalcitrance of the feedstock includes trcating the feedstock with a treatment. The treatment can be, for example, ionization radiation (e.g., électron beam radiation), sonication, pyrolysîs, oxidation, steam explosion, chemical treatment, various mechanical treatments and combinations of any of these treatments. The physical treatment can comprise any one or more of the treatments disclosed herein, applied alone or in any desired combination, and applied once or multiple times. When trcating with ionizing radiation, e.g., électron beam radiation, the dosage can be at least 10 Mrad and less than about 200 Mrad (eg., 50 Mrad to 150Mrad).

In some implémentations, the method can include using an additive in the fuel cell. For example additives can be acids, bases, buffers (e.g., phosphate buffers), minerais, colloïde, émulsions, emulsifiers, parti eu late s, nano- particles, cations, anions, métal ions (e.g., Fe^u,Fe^J*» Mn^I+, Cu²⁺, K⁺, Na*), vitamins, enzymes, peptones, extracts, surfactants, nutrients, gases (e.g., hydrogen, nitrogen, hélium, argon, carbon monoxide, carbon dioxide), chemicals, nitrogen sources (e.g., ammonia, urea), pigments, fragrances, anionic polymers, cationic polymers, non-ionic polymers, oligomers, lipids, fats, surfactants, dispersants, anti-foam agents, bacteriostatic agents, antimicrobial agents, microorganisms, viscosity modifiers, oxidizing agents (e.g., peroxides, chlorates), reducing agents, anti-scale agents, corrosion inhibitors, anti20 fouling agents, precipitating agents, coagulants added in any combination and sequence.

A lypical biomass resource contains cellulose, hemicellulose, and lïgnîn plus lesser amounts of proteins, cxtractablcs and minerais. In some implémentations, cellulosic or lignocellulosic material includes paper, paper products, paper waste, paper pulp, pigmented papers, loadcd papers, coated papers, filled papers, magazines, printed matter, printer paper, polycoated paper, card stock, cardboard, paperboard, offal, cotton. wood, particle board, forestry wastes, sawdust, aspen wood, wood chips, grasses, switchgrass, miscanthus, cord grass, reed canary grass, grain residues, rice hulls, oat hulls, wheat chaff, barlcy hulls, agriculture! waste, sïlage, canola straw, wheat straw, barlcy straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, com cobs, com stover, soybean stover, com fiber, alfalfa, hay, coconut haïr, sugar processing residues, bagasse, beet pulp, agave bagassc, algae, scaweed, manure, sewage, arracacha, bue kw heat, banana, barley, cassava, kudzu, oca, sago, sorghum.

potato, sweet potato, taro, yams, beans, favas, lentils, peas, and mixtures of any of these.

The cellulosic or lignocellulosîc material can be mechanlcally treated to reduce the bulk density of lhe cellulosic or lignocellulosîc material and/or increase its 5 surface area. !n some implémentations, the method includes mcchanically treating the fccdstock before and/or after reducing its recalcitrance. Mechanical treatments include, for example, cutting, milling, e.g., hammermilling, pressing, grinding, shearing and chopping. For example, comminuting lhe bîomass material can be effective treatment appiied to the biomass material. Mechanical treatment can reduce 10 the bulk density of the feedstock and/or increase lhe surface area of the feedstock. In some embodiments, after mechanical treatment the material has a bulk density of less than 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 0.025 g/cm³. Bulk density is determined using ASTM D1895B.

In one aspect, the invention features utilizing, a sugar solution derived from a cellulosic or lignocellulosîc material as described above in a fuel cell, e.g., a direct sugar fuel cell, an indirect sugar fuel cell and/or a biological fuel cell. Optionaily the fuel can be a sugar or an alcohol derived from the saccharification of lhe cellulosic or lignocellulosîc material.

2a Other features and advantages of the invention will be apparent from the following detailed description, and from the daims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram illustrating the enzymatic hydrolysis of cellulose to glucose.

FIG. 2 is a flow diagram illustrating conversion of a bîomass feedstock for use în a fuel ccll.

FIG. 3 îs a diagram showing various processes using saccharified feedstock in fuel cells.

FIG. 4 is a diagrammatic view of an example of a direct glucose fuel cell.

FIG. 5 is a diagrammatic view of an example of an indirect sugar fuel cell.

FIG. 6 is a diagrammatic view of a hal f-cell of a biological fuel cell.

DETAILED DESCRIPTION

Rcfcning to FIG. I, during saccharification, a cellulosic or lignocellulosic substrate is initially hydrolyzed by endoglucanases at random locations producing oligomeric intermediates. These intermediates are then substrates for exo-splitting glucanases such ns ccllobiohydrolase to produce cellobiosc from the ends of the cellulose polymer. Ccllobiose is a water-soluble 1,4-Iinked dimerof glucose. Final ly cellobiase cleaves cellobiose to yield glucose. The glucose, or other sugars, derived from saccharification can be utilized In a fuel cell as will be described in detail herein.

Rcfaring to FIG. 2, a process for manufacturing sugar solutions for incorporation Into a fuel cell system can include, for example, optionally mechanically treating a cellulosic and/or lignocellulosic feedstock (step 110). Before and/or after this treatment, the feedstock can optionally be treated with another physical treatment, for example irradiation, to reduce or further reduce its recalcitrance (step 112). A sugar solution is formed by saccharifying the feedstock (step 114) by, for example, the addition of one or more enzymes (step III). Optionally, the method may also include transportîng, e.g., by pipeline, railcar, truck or barge, the sugar solution (or the feedstock, enzyme and water, if saccharification is performed en route) to a manufacturing plant (step 116). For example, the methods of transportîng and processing biomass as discussed in U.S. Patent 8,318,453 filed J an

21,2009 can be used herein; the complété disclosure of which Is incorporated herein by référencé. If desired, the steps of measuring lignin content (step 118) and setting or adjusting process parameters based on this measurement (step 120) can be performed at various stages of the process, for example, as described in U.S. Application Number 12/704,519, filed on February 11,2011, the complète disclosure ofwhich is incorporated herein by référencé. The sugar solution is then incorporated into a fuel cell or fuel cell system (122). Optionally, products produced in the process can be further proccssed and/or modified, for exemple, if sugars from the process are fermented to products, the product can be purified, for example by distillation (124). The products produced in these processes can also be utilized in a fuel cell system.

FIG. 3 is a diagram showing processes using saccharified feedstock in fuel cells. The sugar solution from the saccharified feedstock can be used în a direct sugar fuel cell, an indirect sugar fuel ccll or a biological fuel cell. Glucose and xylose are often the most abundant sugars available from biomass and sugar solutions derived

from biomass can include a mixture of xylose and glucose in various ratios. For example, oniy glucose can be présent or only xylose can be présent, especially in cases when the sugars hâve been isolated and/or purified. When desired, other ratios cnn be utilized, for example as a percent of total glucose and xylose, the amount of glucose can be between 100% and 90%, 90% and 80%, 80% and 70%, 70% and 60%, 60% and 50%, 50% and 40%, 40% and 30%, 30% and 20%, 20% and 10%, 10% and 0%. Although, the glucose and xylose are often abundant biomass derived sugars, often providing more than 10 wt % of the sugar to biomass (e.g., more than 20 wL%, more than 30 wL%, more than 40 wt.%, more than 50 wL%, more than 60 wt.%, or even more than 70 wt.%) and arc useful in these different fuel cells, other sugars and polysaccharides can also be useful. For example, arabinose, mannose, galactose and rhamnose, cellulose, starch, xylan, glucuronoxylan, arabinoxylan, glucomannan and xylogulcan can be used. Mixtures of any of these sugars can be utilized. In addition the sugars described herein can be isomerized (e.g., to fructose) and then used in u fuel cell. These different fuel cells and their use are discusscd in more detail below. Direct sugar fuel cells, for example glucose fuel cells, generally include a cathode electrode, an anode electrode, and a separator (e.g., an anîon-cxchange membrane (AEM) or a diffusion layer.) The fuel cell may be acidic or alkaline. In the example shown in FIG. 4, an AEM is sandwiched between an anode electrode and 20 a cathode electrode, with flow fîelds being provided between each of the électrodes and the AEM. In some cases, thecell has a two-cylinder construction in which one electrode (e.g., the anode) is in the form of an inner cylinder and the other (e.g., the cathode) is in the form of an outer cylinder.

The active component of the anode may be, for example, PdNi or Pd-Pt, and the active component of the cathode may be, for example, a combined catalyst of cobalt porphyrin complex (CoTPP) and spinel (MnCoîCh) or other suitable catalyst.

In the embodiment of a direct sugar fuel cell shown in FIG. 4, a fuel solution containing glucose and, generally, potassium hydroxide (KOH), is fed into the anode flow channei, e.g., by a peristaltic pump (not shown), while oxygen is fed to the 30 cathode flow field. Glucose is oxidized at the anode and the reduced product flows away through an anode exit channei. The électrons flow from the anode and through a load. Oxygen is reduced at the cathode and exhaust gas is vented from the cathode flow field.

Direct sugar fuel cells may or may not completel y oxidize the sugar fuel to carbon dioxide and water while generating electricity. For example, the réaction for the total oxidation of glucose as shown here may occur.

Anode reaction: QHiîOe +24OH* > I8H2O + 6CO₂ + 18e5 Cathode reaction: 6Oj + 12H₂O + 24e- 24OH*

Overall reaction: CèHqOs + 6O₂ ·> 6CO2 + 6H₂O ΔΗ =-2830 KJ/mol

S in ce the total oxidation of glucose proceeds through many intermediates (e.g., gluconic acid, glucaric acid, gamma-gluconolactone, gomma-glucaro lactone, 2ketogluconic acid, arabinosc, trihydroxyglutaric acid, tartaric acid, hydroxyl malonic 10 acid and oxalic acid) any of these intermediates can also be used in a fuel cell. Any of these intermediates, if produced by some of the processed described herein, (e.g., saccharification, fermentation) can be useful in generating electricity in a fuel cell. Optionally, any sugar products not used in generating electricity in the direct sugar fuel cell can be further processed as shown in FIG. 2, for example lhey can be fermented to an alcohol and the alcohol isolated by distillation. In some cases the fuel cell can use one sugar, for example glucose, and does not use other sugars, for example xylose, and the second sugar can be used in subséquent processes. In some other instances, a process, for examplc fermentation, only uses one sugar (e.g., glucose) leaving other sugars which can be then used in a fuel cell. In many cases, only a partial oxidation of glucose occurs in a direct sugar fuel cell. For example, the oxidation of glucose to gluconic acid occurs quickly providing 2 électrons and releasing et most about 200 KJ/mol of energy (e.g., about 7% of the available energy). In terms of usable energy, the fuel cells convcrt at least 1% of the fuel to electric energy (e.g., at least 5%, at least 7%, at least 10%, at least 14%, at least 20%, at least

25%, at least 30 %, at least 40%, at least 50%, at least 60%, at least 70%, at least

80%, at least 90%). In some cases between about 1% and 90% of the fuel is converted to electric energy (e.g., between about 1% and 70%, between about 1% and 50%, between about 1% and 20 %, between about 1% and 10%, between about 5% and 90%, between about 5% and 70%, between about 5% and 50%, between about 5% and 25%, between about 5% and 10%, between about 10% and 90%, between about 10% and 70%, between about 10% and 50%, between about 10% and 30%, between about 10% and 20%, between about 20% and 70%).

In addition to sugars and products such as alcohols, the solutions derived from biomass by the processes described herein can include various solids or dissolved compounds and/or materials. For example the solutions can include enzymes (e.g.. parts oC enzymes, active enzymes, denatured enzymes), amino acids, nutrients, live 5 cells, dead cells, cellular débris (e.g., lysed cells, yeast extract), acids, bases, salis (e.g., ha!ides, sulfates, and phosphates, alkai, alkali earth, transition métal salts), partial hydrolysis products (e.g., cellulous and hemicellulose fragments), lignin, lignin residues, inorganic solids (c.g., siliceous materials, clays, carbon black, metals), remnants of saccharified biomass and combinations thereof.

Enzymes that can be présent can be the intact or denatured enzymes utilized in the processing, or derived from these enzymes (c.g„ proteins and amino acids). These can be dissolvcd/and or precipitated and suspended solids. For example, the sugar solutions can hâve up to about 10 wt.% enzymes (e.g., up to 9 wL %, up to 8 wt. %, up to 5 wl %, up to 2 wt %. up to 1 wt. %, between about 0.1 and 5 wt. %, between about 1 wt. % and 5 wt %, between about 2 wt. % and 5 wt. %, between about 0.1 wl % and 1 wL %, between about 0.01 wt.% and l wt.%, between about 0.001 wt.% and 0.1 wl%).

During saccharification of a biomass, an optimal pH can often be in the acidic région and therefore the solutions used, if used directly in a fuel cell system, can hâve 20 a pH between about 2 and 5 (e.g., between about 4 and 5). The pH can also be adjusled up or down after saccharificaion and or saccharification done at a higher or iower pH. In some embodiments the solution used In the fuel cell can therefore hâve pH values selected from a broad range. For examplc, the pH can be selected from a range of about 2 to about 10 (e.g., between about 2 and 5, between about 3 and 5, 25 between about 3 and 6, between about 4 and 6, between about 5 and 10, between about 6 and 10 between about 8 and 10).

The sugar solutions derived from the processes described herein and used in fuei cells Systems can include non-sugar suspended or dissolved solids présent at concentrations up to about 50 wt.%, for example between about 1 and 50 wL%, 2 and 30 40 wL %, 3 and 25 wt.%, 5 and 25 wL%, 40 and 50 wL%, 30 and 40 wt.%. 10 and 20 wt.%, 1 and 5 wl%, 10 and 40 wt.%, less than about 50 wt.%, less than about 40 wt.%, less than about 30 wt.%, less than about 20 wt%, less than about 10 wt.%, less than about 5 wt.%, less than about 1 wt.%, less than about 0.5 wl%, less than about

0.01 wt.%. These solutions can hâve high turbidit y, for example at least about 5 nephelometric turbidit y unils (NTU) (e.g., at least aboutlO NTU, at least about 50 NTU, at least abouti 00 NTU, at least about 100 NTU, at least about 200 NTU, at least about 300 NTU, at least about 400 NTU and even greater than about 500 NTU). In some cases the solids are completely or partially removed prior to being use in the fuel cells. For example the solids can be removed by filtration, centrifuging, settling, floatation and combinations of these. In some cases lhe solids are derived from a previously soluble material that has been precipitated, for example an enzyme thaï has been denatured. After removing lhe solids lhe turbidity of the solutions can be reduced by up to about 500 NTU (e.g., reduced by up to about 100 NTU, reduced by up to about 50 NTU, reduced by up to about 5 NTU).

In addition to being turbid, the sugar solutions produced from lhe processes described herein can be colored due to colored impurilies. For example some métal ions and polyphenols and lignin derived products produced or released during lhe processîng of a lignocellulosic biomass can be highly colored. The solutions can be used directly in lhe fuel cell Systems described herein or can be partially or completely decolorized prior to being used. For example lhe colored impurities can be filtered oui of lhe solution, destroyed (e.g., by chemical décomposition) and/or precipitated out ofthe solution.

The ionic strength of the biomass derived sugar solutions can be high due to the source of lhe biomass as well as lhe processîng ofthe biomass as described herein. The solutions can be used directly or fully, sclectively or partially de-ionized prior to being used in the fuel cell Systems described herein.

In some embodiments the fuel cell can include biomass (e.g., lignocellulosic bîomass, cellulosic biomass, starch) as well as a saccharifying enzyme. For example, sugar can be utilized in a fuel cell system while it is being produced by lhe action of a saccharifying enzyme on a biomass material.

In yet other embodiments the sugar solutions used in lhe fuel cells herein described can include an additive. Such additives can modify properties of the solutions such as the pH, viscosity, chemical potential, surface tension, thermal properties, color, odor, opacily, ionic strength, conductivity, stcrility and/or nutrient value. For example additives can be acids, bases, buffers (e.g., phosphate buffers), minerais, colloids, émulsions, emulsifîers, particulates, nano-particles, cations, anions,

métal ions (e.g., Fe²*· Fe³*, Mn²*, Ci?*, K*, Na*), vitamins, enzymes, peptones, extracts, surfactants, nutrients, gases (e.g., hydrogen, nitrogen, hélium, argon, carbon monoxlde, carbon dioxlde), chemicals, nitrogen sources (e.g., ammonia, urea), pigments, fragrances, anionic polymers, cationic polymers, non-ionic polymers, oligomers, lipîds, fats, surfactants, dispersants, antl-foam agents, bacteriostatic agents, nntimicrobial agents, microorganisms, viscosity modifiers, oxidizing agents (e.g., peroxides, chlorates), reducing agents, anti-scale agents, corrosion inhibitors, antifouling agents, precîpitating agents, coagulants added in any combination and sequence. Additives can be added in ranges from a few parts per million to several percents. For example 1 to 1000 ppm, 5 to 500 ppm, 5 to 100 ppm, 50 to 100 ppm,

100 to 1000 ppm. 1 to 10 wt.%, 2 to 10 wt%, 5 to 10 wt.%, 2 to 5 wt.%). In some embodiments including cations, anions, métal anions the amounts are between 1 to lOOOppm. In some embodiments where acids, bases or buffers are added, the final pH after addition of tlie additive can be chosen to be between pH 2 and 10, (e.g., between 15 about 4 and 8, between about 5 and 7, between about 6 and 8, between about 4 and 5, between about 7 and 8, between about 8 and 10 and between about 2 and 4).

Additives can also be metered and added in amounts between about 1 uM to 5 M amounts (e.g., between about 1 mM and 1 M, between about 5 mM and 100 mM, between about 100 mM and 1 molar, between about 10 mM and lOOmM).

FIG. 5 is a diagrammatic view of an example of an indirect sugar fuel cell.

Generally, the indirect sugar fuel cell uses a biological process to convert a primary fuel to a secondary fuel and the secondary fuel generates a current using a fuel cell. The primary fuel (I) is brought into contact with a bio-component (2) where it produces a secondary fuel (3) and waste (4). The secondary fuel is brought into the fuel cell and cornes into contact with the anode (5) where it is oxidized, producing a reduced product (8), releasing an électron to an external circuit, and providîng a proton. The proton travels ïn the fuel cell through an ïon sélective membrane (6) to the cathode (7). Oxygen is supplied to the cathode where it is reduced by électrons supplied from the external circuit and combines with the proton producing water. In another possible design, the bio-component résides within the fuel cell, so that the production of product and electricity ail occur within the fuel cell. In some cases, the Ion sélective membrane is also not required. In other cases the oxidant may be ιο oxidants other than dioxygen (e.g., hydrogen peroxide, organic peroxides and inorganic peroxides).

The primary fuel used in the indirect fuel cell can be sugars (e.g., glucose and xylose) as well as polysaccharides that can be produced through the saccharification of biomass as previously discussed. The secondary fuel can be a fermentation product of the primary fuel. For example, the secondary fuel can be an alcohol or other fermentation product (e.g., éthanol, methanol, butanol, polyols, acetic acid, lactic acid and H₂ ). Generally, the primary and secondary fuels can be selected from the intermediates and products discussed below. The bio-component can be a microbïal material including but are not limited to, any naturally occurring or genetically modified microorganism or organisai, for example, protists, e.g., animal protists (e.g., protozoa such as flagellâtes, amoeboîds, ciliates, and sporozoa) and plant protists (e.g., algaesuch alveolates, chlorarachniophytes, cryptomonads, cuglenids, glaucophytes, haptophytes, red algae, stramenopiles, and viridaeplantae). Other examples include seaweed, plankton (e.g., macro plank ton, mesoplankton, micro plankton, nanoplankton, picoplankton, and fempto plank ton), phytoplankton, bacteria (e.g., gram positive bacteria, gram négative bacteria, and extremophiles), human cells, mammalian cells, yeast and/or mixtures of these. There may bc several bio-components, for example there may be several bacteria specialized to generate different products useful for producing a current from different or the same components of the fuel. For example, fermentation methods and fermenting organisms discussed herein can be utilized to produce the secondary product

Some species of microorganisms that can be used to produce the secondary fuel in the indirect sugar fuel cell are: Clostridium saccharobutylacetonicum, Clostridium saccharoperbutylacetonicum, Clostridium saccharobutylicum. Clostridium ptmiceum, Clostridium beijemckii. Clostridium acetobutylicum, Clostridium aurartlibutyricum. Clostridiumfelsineum. Clostridium butyricum, Geobacter species, strates of the genus Sacchromyces spp. e.g., Sacchrontyces cerevislae (baker’s yeast), Sacdtaromyces distaticus, Saccharomyces uvarttm, strates of the genus Kluyveromyces, e.g., species Kluyveromyces marxianus, Kluyveromyces fragiüs, strates of the genus Candida, e.g., Candlda pseudotropicalis, and Candida brassicae, Pichia stipttis (a relative of Candida sheltatae), strains of the genus Clavispora, e.g., species Clavispora lusitaniae and Clavispora opiintiae, strains of the

II gcnus Pachysolen, e.g., species Pachysolen tannophilus, and strains of the genus Brefannomyces, e.g., species Brefannomyces clausenli (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Ulilization, Wyman, C.E., ed., Taylor & Francis, Washington, DC, 179-212).

Commercîally available yeasts the may be used for the formation of secondary fuel include, for example, Red StarWLcsaffre Ethanol Red (available from Red Star/Lcsaffre, USA) FAL1® (available from Fleischmann's Yeast, a division of Bums Philip Food Inc., USA), SUPERSTART® (available from Alltech, now Lalemand), GERT STRAND* (available from Gcrt Strand AB, Sweden) and FERMOL® (available from DSM Specialties).

Biological fuel cells are devices capable of directly transforming achemical fuel to electrical energy via electrochemical reactions involving bîochemical pathways. Generally this involves enzymes or active parts of enzymes for catalysîs. The enzymes can be within a living organisai (e.g., microbial fuel cells) or can be 15 outside of a living cell (e.g., enzyme fuel cells). FIG. 6 shows a diagrammatic view of a generalized half-ccll for a biological fuel cell. A supplîed fuel is coniacted with a biological component which oxidizes the fuel and créâtes waste that is removed. The électrons released from the fuel are transferred from the biological component to a mediator which either diffuses to or is associated with the électrode where the 20 mediator is oxidized to its original state releasing an électron to an external circuit.

The oxidant side of the fuel cell is not shown. Some oxidants can be, for example, O₂, supplîed by air, or peroxides (e.g., hydrogen peroxidc, organic peroxides, inorganic perexides). Some biological fuel cells do not rcquîre a mediator; in such cells the électron is transferred directly from the biological component to the electrode. In some biological fuel cells the anode reaction with oxygen is catalyzed by a biological component. Some biological fuel cells hâve been described, for example by Derek Lovely in “The microbe electric: conversion of organic matter to electricity, Current Opinions in Biotechnology, 2008, Volume 19, pages 1-8, or in U.S. patent No. 8,283,076 filed May 18,2007; the enlire disclosure in these référencés are incorporated by reference herein.

The fuel used in the biological fuel cell can be the saccharification products from biomass as previously discussed. Especially in cases where organisais are utilized (e.g., microbial fuel cells) other nutrients and media components can be added to the fuels, for example Ions (e.g., sodium, potassium, iron, magnésium, manganèse, zinc, copper), phosphates, sulfates, ammonia, urea, amino acids, proteins, vitamîns, buffers, organic acids, inorganic acids, organic bases, inorganîc bases, nutrient rich extracts(e.g., yeast extracts, méat extracts and vegetable extracts). Additionally the température and pH can be controlled. For example températures between 10 and 70 °C can be used (e.g., between about 10 and 60 °C, between about 10 and 50 °C, between about 10 and 40 °C, between about 20 and 70 °C, between about 20 and 60 °C, between about 20 and 50 °C, between about 20 and 40 °C, between about 30 and 70 °C, between about 30 and 60 °C, between about 30 and 50 °C, between about 30 and 40 °C). The pH can be between about 3 and 10 (e.g., between about 3 and 9, between about 3 and 8, between about 3 and 7, between about 3 and 6, between about 3 and 5, between about 4 and 9, between about 4 and 8, between about 4 and 7, between about 5 and 9, between about 5 and 8, between about 5 and 7).

Examples of organisms that can be useful in biological fuel cells are species of gcobactcr, proteus vulgaris, Desulphovlbrio desulphuricans, E coli, Actinobacillus succinogenes, Desulphovibrio vulgaris, Shewanella putrefacîens and Rhodoferax ferrireducens.

Examples of enzymes that can be useful in biological fuel cells are glucose oxidase, alcoholdehydrogenase, aldéhyde dehydrogenase, formate dehydrogenase, oxidorcductase, diaphorase, flavor-oxido-rcductase, 1 accuse, microperoxidase, glucose dehydrogenase, hydrogenase, peroxidases, rcconstituted enzymes from this list and combinations thereof.

MECHANICAL TREATMENTS

Biomass feedstock (e.g., cellulosic and/or lignocellulosic material) can be mechanically treated prior to or after other treatments. Methods of mechonically treating the carbohydrate-containing material include, for example, milling or grinding. Milling may be performed using, for example, a hammer mill, bail mill, colloid mill, conical or cône mill, disk mill, edge mill, Wiley mill, grîst mill or other mills. Grinding may be performed using, for example, a cutting/impact type grinder. Some exemplary grinders include stone grinders, pin grinders, coffcc grinders, and burr grinders. Grinding or milling may be provided, for example, by a rcciprocating pin or other élément, as is the case in a pin mill. Other mechanical treatment methods include mechanical ripping or tcaring, other methods that apply pressure to the fibers, and air attrition milling. Suitable mechanical treatments further include any other technique that continues the disniption of the internai structure of the material that was initiated by the previous processing steps.

Mechanical feed préparation Systems can be configured to produce streams with spécifie characteristics such as, for example, spécifie maximum sizes, spécifie length-to-width, or spécifie surface areas ratios. Physical préparation can Increase the rate of reactions, improve the movement of material, 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 n solution.

The bulk density of feedstocks can be controlled (e.g., increased). In some situations, it can be désirable to préparé a low bulk density material, e.g., by denstfying the material (e.g., densification 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 densified, 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 0J g/cc, less than about 0.3 to more than about 0.9 g/cc, less than about 0.5 to more than about 0.9 g/cc, less than about 0.3 to more than about 0.8 g/cc, less than about 0.2 to more than about 0.5 g/cc). For example, the material can be densified by the methods and equipment disclosed in U.S. Pat. No. 7,932,065 to Medoff and International Publication No. WO 2008/073186 (which was filed October 26,2007, was published în English, and which designated the United States), the full disclosurts of which are incorporated herein by reference. Densified materials can be processed by any of the methods described herein, or any material processed by any of the methods described herein can be subsequently densified.

In some embodiments, the material to bc processed is in the form of a fibrous material thaï includes fibers provided by shearing a fiber source. For example, the shearing can be performed with n rotary knife cutter.

For exemple, a fiber source, e.g._t that is récalcitrant 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 eut prior to the shearing, e.g., with a shredder. For example, when n paper is used as the fiber source, the paper can be first cul into strips that are, e.g., 1/4- to 1/2-ïnch wide, using a shredder, e.g., a countcr-rotating screw shredder, such as those manufactured by Munson (Utîca, N.Y.). As an alternative to shredding, the paper can be redueed in size by cutting to a desired size using a guillotine cutter. For example, the guillotine cutter can be used to eut the paper into sheets that are, e.g._t 10 inches wide by 12 inches long.

ln some embodiments, the shearing of the fiber source and the passing of the resulting first fibrous material through a first screen are performed concurrently. The shearing and the passing can also be performed in a batch-type process.

For example, a rotary knife cutter can be used to concurrently shear the fiber source and screen the first fibrous material. A rotary knife cutter includes a hopper that can be loaded with a shredded fiber source prepared by shredding a fiber source.

In some implémentations, 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, chemical treatment, irradiation, sonication, oxidation, pyrolysls or steam explosion. Treatment methods can be used in combinations of two, three, four, or even ail of these technologies (in any order). When more than one treatment method is used, the methods can be applied at the same time or at different times. Other processes that change a molecular structure of q biomass feedstock may also be used, alone or in combination with the processes disclosed herein.

Mechanical trcQtments that may be used, and the characterislics of the mechanically treated feedstocks, are described in further detail in U.S. Serial No. 13/276,192, filed October 18,2011, the full disclosure of which is hereby incorporated herein by reference.

ln addition to this size réduction, which can be performed initlally and/or later during processing, mechanical treatment can also be advantageous for “opening up, stressing,” breakïng or shattering the feedstock materials, making the cellulose of the materials more susceptible to chain scission and/or disniption of crystalline structure during the structural modification treatment

The biomass can be in a dry form, for example with less than about 35% moisture content (e.g., less than about 20 %, less than about 15 %, less than

IS about 10 % less than about 5 %, less than about 4%, less than about 3 %, less than about 2 % or even less than about I %). 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 disciosed herein can utilize low bulk density materials, for example cellulosic or lignocellulosic feedstocks that hâve been physically pretreated to hâve 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.

io Bulk densityis determined usingASTM D1895B. Briefly, the method involves filling a measuring cylinder of known volume with a sample and obtaining a weight of the sample. The bulk density is calculated by divtding the weight of the sample in grains by the known volume of the cylinder in cubic centimeters. If desired, low bulk density materials can be densified, for example, by methods described in US. Pat No.

7,971,809 to Medoff, the full disclosure of which is hereby Incorporated by reference.

In some cases, the biomass can bc scrccned through a mesh or perforated plate with a desired opening size, for example, less than about 6.35 mm (¼ 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)).

Screening of biomass material can also be by a manual method, for example by an operator or mechanoid (e.g., a robot equipped with a color, reflectivity or other sensor) that removes unwanted material. Screening can also be by magnetic screening wherein a magnet is disposed near the conveyed material and the magnetic material Is removed magnetically.

Optionally, prior to further processing, the biomass material can be heated, for 30 example by IR radiation, microwaves, combustion (e.g., gas, coal. oil, biomass), résistive heating and/or inductive 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 movement of a gas (e.g., air, oxygen, nitrogen, Hc, COj, Argon) over and/or through the biomass.

Optionally, the biomass can be cooled prior to or after mechanical treatment Cooling material is described in US Pat No. 7,900.857 to Medoff, the disclosure of which in incorporated herein by rcfcrence.

RADIATION TREATMENT ln some cases, the feedstock may be irradiated to modify ils structure and thereby reduce ils recalcitrance. Irradiation may, for example, reduce the average molecular weight of the feedstock, change the crystalline structure of the feedstock, change the functionalization of the feedstock (e.g., by oxidation) and/or increase the surface area and/or pores ity of lhe feedstock

Various other irradialing devices may be used in the methods disclosed herein, including field ionization sources, electrostatic ion separators, field ionization generalors, thermionic émission sources, microwave discharge ion sources, recirculaiing or static accelerators, dynamic linear accelerators, van de Graaff accelerators, and folded tandem accelerators. Such devices are disclosed, for example, in U.S. PaL No. 7,931,784 to Medoff, the complété disclosure of which is incorporated herein by référencé.

A beam of électrons can be used as the radiation source. A beam of électrons has the advanlages of high dose rates (e.g., 1,5, or even 10 Mrad per second), high throughput, less containment, and less confinement equipment. Electron beams can also hâve high electrical efTiciency (e.g., 80%), allowing for lower energy usage relative to other radiation methods, which can translate into a lower cosl of operation and lower greenhouse gas émissions corresponding to the smaller amount of energy used. Electron beams can be generated, e.g., by electrostatic generators, cascade generators, transformer generalors, 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 structure of carbohydrate-containing materials, for example, by the mechanism of chain scission, ln addition, électrons having cnergies of 03-10 MeV can penetrate low density materials, such as the biomass materials described herein, e.g._t materials having u bulk density of less than 0.5 g/cm³, and a depth of 0.3-10 cm. Electrons as

an ionizing radiation source can bc useful, 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. ln some embodiments, the energy of each électron of the électron beam is from about 0.3 MeV to about 2.0 MeV (million électron volts), S e.g., from about 05 MeV to about 1.5 MeV, or from about 0.7 MeV to about 1.25 MeV. Methods of irradiating materials are discussed in U.S. Pat. App. Pub. 2012/01000577 Al, filed October 18,2011, the entîre disclosure of which is herein incorporated by reference.

Electron beam irradiation devices may be procured commercially from Ion

Beam Applications, Louvain-la-Neuve, Belgium or theTitan Corporation, San Diego, CA. Typical électron energies can bc 0.5 MeV, 1 MeV, 2 MeV, 4.5 MeV, 7.5 MeV, or 10 MeV. Typical électron 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 électron beam irradiation device power spécifications inciude cost to operate, capital cosis, dépréciation, and device footprint. Tradeoffs in considering exposure dose levels of électron beam irradiation would be energy costs and environment, safety, and health (ESH) conccms. Typically, generators are housed in a vault, e.g., of lead or concrète, cspeciajly for production from X-rays that are generated in the process. Tradeoffs in considering électron energies inciude energy costs.

The électron beam irradiation device can producc either a fixed beam or n scanning beam. A scanning beam may be advantageous with large scan sweep length 25 and high scan speeds, as this would effectively replace a large, fixed beam width.

Further, available sweep widths of 0.5 m, 1 m, 2 m or more are avaîlable.

ln some embodiments, two or more radiation sources are used, such as two or more ionizing radiation sources. For example, samples can be treated, in any order, with a beam of électrons, 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 électrons, gamma radiation, and energetlc UV light. The biomass is conveyed through the irradiation zone (354 in FIG. 3) where it can be irradîated, for example by électrons. Il is

generally preferred that the bed of bîomass material has a relatively uniform thîckncss, as previously described, while being irradiated.

Effectiveness of changing the molecular/supermolecular structure and/or reducing the recalcitrance of the carbohydrate-containing bîomass dépends on the électron energy used and the dose appiied, while exposure time dépends on the power and dose.

In some embodiments, lhe irradiating (with any radiation source or a combination of sources) is performed until the material receives a dose of at least about 0.05 Mrad, e.g., at least about 0.1,0.25,0.5,0.75,1.0,2.5,5.0,7.5,10.0,15,20, 10 25.30,40,50,60,70,80,90,100,125,150,175, or 200 Mrad. In some embodiments, the irradiating is performed until lhe material receives n dose of between 0.1 -100 Mrad, 1 - 200,5 - 200,10 - 200.5 -150,50 - 150,100 - 200,100 -150,80 - 120,5 - 100,5 - 50,5 - 40, 10 - 50,10 - 75,15 - 50,20 - 35 Mrad.

In some embodiments, the irradiating is performed at a dose rate of between

5.0 and 1500.0 kilorads/hour, e.g., between 10.0 and 750.0 kilorads/hour or between

50.0 and 350.0 kilorads/hours. In other embodiments the irradiation is performed at a dose rate of between 10 and 10000 kllorads/hr, between 100 and 1000 kilorad/hr, or between 500 and 1000 kilorads/hr.

In some implémentations, it is désirable to coo! the material during irradiation.

For example, the material can be cooled while it is being conveycd, for example by n screw extrader or other conveying equipment.

Radiation can be appiied while the cellulosic and/or lignocellulosîc material is exposed to air, oxygen-enriched air, or even oxygen itsel f, or blanketed by an inert gas such as nitrogen, argon, or hélium. When maximum oxidation is desired, an oxidizing 25 environment is utilized, such as air or oxygen and the distance from the radiation source is optimized to maximize réactivé gas formation, e.g., ozone and/or oxides of nitrogen.

SONICATION, PYROLYSIS, OXIDATION, STEAM EXPLOSION lf desired, one or more sonication, pyrolysis, oxidative, or steam explosion processes can be used in addition to or instead of irradiation to reduce the recalcitrance of the feedstock. These processes arc described in detail in U.S. Serial

No. 12/429,045, the full disclosure of which is incorporated herein by référencé.

SACCHARIFICATION

The treated biomass materials can be saccharified, generally by combining the material and a cellulase enzyme in a fluid medium, e.g., an aqueous solution. In some cases, the material is boiled, steeped, or cooked in hot water prior to saccharification, 5 as described in U.S. Pat. App. Pub. 2012/01000577 Al, filed October 18,2011.

The saccharification process can be partially or compietely performed in a tank (e.g., a tank having a volume of at least 4000,40,000, or 500,000 L) in a manufacturing plant, and/or can be partially or compietely performed in transit, e.g., in a rail car, tanker truck, or in a supertanker or the hold of a ship. The time required 10 for complété saccharification wi11 dépend on the process conditions and the carbohydrate-containing material and enzyme used. If saccharification is performed in a manufacturing plant under controlled conditions, the cellulose may be substantially entirely converted to sugar, e.g., glucose in about 12-96 hours. If saccharification is performed partially or compietely in transit, saccharification may 15 take longer.

It is generally preferred thaï the tank contents be mixed during saccharification, e.g., using jet mixing os described in International App. No. PCT/US2010/035331, filed May 18,2010, which was published in English as WO 2010/135380 and designated the United States, the full disclosure of which is incorporated by reference herein.

The addition of surfactants can erthance the rate of saccharification. Examples of surfactants include non-ionic surfactants, such as a Twecn® 20 or Tween® 80 polyethylene glycol surfactants, ionic surfactants, or amphoteric surfactants.

It is generally preferred that the concentration of the sugar solution resulting 25 from saccharification 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 évaporation, to increase the concentration of the sugar solution. This reduces the volume to be shippcd, and also inhibits mïcrobial growth in the solution.

Alternative! y, sugar solutions of lower concentrations may be used, in which 30 case it may be désirable to add an antimicrobial additive, e.g., a broad spectrum antibiotic, in a low concentration, e.g„ 50 to 150 ppm. Other suitable antibiotics include amphotericin B, ampicillin, chloramphcnicol, ciprofloxacîn, gcntamicin. hygromycin B, kanamycin, neomycin, penicillin, puromycin, strcptomycin.

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, on antibiotic can be included even if the sugar concentration is rclatively high. Alternatively, other S additives with anti-microbial or preservative properties may be used. Preferably the antimicrobial additive(s) are food-grade.

A rclatively high concentration solution can be obtained by limîting the amount of water added to the carbohydrate-conta in in g material with the enzyme. The concentration can be controlled, e.g., by controlling how much saccharification takes 10 place. For example, concentration can be increascd by adding more carbohydratccontaining material to the solution. In order to keep the sugar that is being produced in solution, a surfactant can be added, e.g., one of those discussed above. Solubility can also be increascd by increasing the température of the solution. For example, the solution can be maintained at a température of40-50’C. 60-80°C, or even higher.

SUGARS

In the processes described herein, for example after saccharification, sugars (e·#*, glucose and xylose) can be isolated. For example sugars can be isolated by précipitation, crystallization, chromatography (e.g., simulated movingbed chromatography, high pressure chromatography), centrifugation, extraction, any other isolation method known in the ait, and combinations thereof.

FERMENTATION

Yeast and Zymomonas bacteria, for example, can be used for fermentation or 25 conversion of sugar(s) to alcohol(s). Other microorganisms are discussed below. The optimum pH for fermentations is about pH 4 to 7. For example, the optimum pH for yeast is from about pH 4 to 5, while the optimum pH for Zymomonas is from about pH5 to 6. Typical fermentation times are about 24 to 168 hours (e.g., 24 to 96 hrs) with températures in the range of 20°C to 40°C (e.g., 26°C to 40°C), however thermophilic microorganisms prefer higher températures.

In some embodiments, e.g., when anaérobie organisms are used, at least a portion of the fermentation is conducted in the absence of oxygen, e.g., under a blanket of an inert gas such as N₂, Ar, He, CO₂ or mixtures thereof. Additionally, the

mixture may hâve a constant purge of an inert gas flowing through the tank during port of or ail of the fermentation. In some cases, anaérobie condition, can be achieved or maintained by carbon dioxide production during the fermentation and no additional inert gas is needed.

S In some embodiments, al! or a portion of the fermentation process can be interrupted before the low molecular weight sugar is completely converted to a product (e.g._t éthanol). The intermediate fermentation products include sugar and carbohydrates in high concentrations. The sugars and carbohydrates can be isolated via any means known in the art These intermediate fermentation products can be used in préparation of food for human or animal consumption. Additionally or altematively, the intermediate fermentation products can be ground to a fine partidc size in a stainless-steel laboratory mil! to producc a flour-like substance.

Jet mixîng 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 described in U.S. Pat App. Pub. 2012/0052536, filed July 15,2011, the complété disclosure of which is incorporated herein by reference.

Mobile fermentera can be utilized, as described in International App. No.

PCT/US2007/074028 (which was filed July 20,2007, was publishcd in English as

WO 2008/011598 and designated the United States), the contents of which is incorporated herein în its entirety. Similarly, the saccharification equipment can be mobile. Further, saccharification and/or fermentation may bc performed in part or entirely during transit.

DISTILLATION

After fermentation, the resulting fluids can be distilled using, for example, a “beer column to separate éthanol and other alcohols from the majority of water and residual solids. The vapor exiting the beer column can be, e.g., 35% by weight 30 éthanol and can be fed to a rectification column. A mixture of nearly ozcotropic (92.5%) éthanol and water from the rectification column can be purified to pure (99.5%) éthanol using vapor-phase molecular sieves. The beer column bottoms can be sent to the first effect of a three-effect evaporator. The rectification column reflux

condenser can 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 effluent can be recyded to fermentation and the rest sent to the second and third evaporator effects. Most of the evaporator condensate can be rctumed to the process as fairly ctean condensate with a small portion split off to wnste water treatment to prevent buitd-upof low-boiling compounds.

INTERMEDIATES AND PRODUCTS

Using the processes described herein, the biomass material can be converted to 10 one or more products, such ns energy, fuels, foods and materials. Spécifie examples of products include, but are not limited to, hydrogen, sugars (e.g., glucose, xylose, arabinosc, mannose, galactose, fructose, disaccharides, oligosaccharides and polysaccharides), alcohols (e.g., monohydric alcohols or dihydric alcohols, such as éthanol, n-propanol, isobutanol, sec-butanol, tert-butanol or n-butanol), hydrated or 1S hydrous alcohols (e.g., containing greater than 10%, 20%, 30% or even greater than 40% water), biodicsel, organic acids, hydrocarbons (e.g.. melhane, ethane, propane, isobutene, pcntanc, n-hexane, biodiesel, bio-gasolinc and mixtures thereof), coproduis (e.g., proteins, such as cellulolytic proteins (enzymes) or single cell proteins), and mixtures of any of these in any combination or relative concentration, 20 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), aldéhydes (e.g., acetaldehyde), alpha and beta unsaturated acids (e.g., acrylic acid) and olefins (e.g., ethylene). Other alcohols 25 and alcohol dérivatives include propanol, propylene glycol, 1,4-butanedîol, 1,3propancdiol, sugar alcohols (e.g., erythritol, glycol, glycerol, sorbitol threitol, arabitol, rîbitol, mannitol, dulcitol, fucitol, iditol, isomalt, maltitol, lactitol, xylitol and other polyols), and methyl or ethyl esters of any of these alcohols. Other products include methyl acrylate, melhylmelhacrylalc, lactic acid, citric acid, formic acid, acetic acid.

propionic acid, butyric acid, succinic acid, valeric acid, caproic acid, 3hydroxypropionic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, glutaric acid, olcic 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. Many of the products obtained, such as éthanol or n-butanol, can be utilized as a fuel for powering cars, trucks, trac tors, ships or trains, e.g., as an internai combustion fuel or as a fuel cell feedstock. Many of the products obtained can also be utilized to power aircraft, such as planes, e.g., having jet engines or helicopters. In

B addition, the products described herein can be utilized for electrical power génération, e.g., tn a conventional stcam generating plant or in a fuel cell plant.

Other intermediates and products, including food and pharmaceutical products, are described in U.S. App. No. 12/417,900 filed April 3,2009, the full disclosure of which is hereby incorporated by reference herein.

to

CARBOHYDRATE CONTAINING MATERIALS (BIOMASS MATERIALS) As used herein, the term biomass materials is used interchangeably with the term*’carbohydrate-containing materials’’, and includes lignocellulosic, cellulosic, starchy, and microbial materials. Any of the methods described herein can be practiced with mixtures of any biomass materials described herein.

Lignocellulosic materials include, but are not limited to, wood, particle board, forestry wastes (e.g., sawdust, aspen wood, wood chips), grasses, (e.g., switchgrass, mi scan thus, 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, i

barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, com cobs, com stover, soybcitn stover, com fiber, alfalfa, hay, coconut hair). sugar processîng residues (e.g., bagasse, beet pulp, agave bagasse),, algae, scawecd, manure, sewage, and mixtures of any of these.

In some cases, the lignocellulosic material includes comcobs. Ground or homme rmil lcd comcobs can be s prend in a loyer of relatively uniform thickness for irradiation, and after irradiation are easy to disperse in the medium for further processîng. To facilitate harvest and collection, in some cases the entire com plant is used, including the com stalk, com kernels, and in some cases even the root system of the plant.

Advantageously, no additional nutrients (other thon a nitrogen source, e.g., urea or ammonia) are required during fermentation of comcobs or cellulosic or lignocellulosic materials containing signifïcant amounts of comcobs.

Comcobs, before and after comminution, are also casier to convey and disperse, and hâve 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, fillcd papers, magazines, printed matter (e.g„ books, catalogs, manuals, labels, calendars, greeting caïds, brochures, prospectuscs, newsprint), primer paper, polycoated paper, card stock, cardboard, paperboard, materials having a high α-ccllulose content such ns cotton, and mixtures of any of these. For example paper products as described in U.S. App. No. 13/396,365 (Magazine Feedstocks’’ by Medoff et al., filed February 14,2012), the full disclosure of which is incorporated herein by reference.

Cellulosic materials can also include lignocellulosic materials which hâve been de-!ignified.

Starchy materials include starch itself, e.g„ com starch, wheat starch, potato starch or rice starch, a dérivative 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, régulai houschold potatoes, sweet potato, taro, yams, or one or more bcans, 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 materials 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 com plant, rice plant or a tree. The starchy materials can be treated by any of the methods described herein.

Microbial materials include, but are not limited to, any naturally occurring or genetically modified microorganism or organism that contains or is capable of providing a source of carbohydrates (e.g., cellulose), for example, protists, e.g„ animal protists (e.g., protozoa such as flagellâtes, amoeboids, ciliates, and sporozoa) and plant protists (e.g., algae such alveolates, chlorarachniophytes, cryptomonads, euglenids, glaucophytes, haptophytes, red algae, stramenopiles, and viridaeplantae). Other examples include seaweed, plankton (e.g., macroplankton, mesoplankton, microplankton, nanoplankton, picoplankton, and femptoplankton), phytoplankton, bactcria (e.g., gram positive bacteria, gram négative bacteria, and exlremophiles), yeast and/or mixtures of thèse. In some instances, microbial biomass can be obtained from naturel sources, e.g., the océan, lakes, bodies of water, e.g., sait water or fresh water, or on land. Altematively or in addition, microbial biomass can be obtained from culture Systems, e.g., large scale dry and wet culture and fermentation Systems.

In other embodiments, the biomass materials, such as cellulosic, starchy and lignocel lulosic fecdstock materials, can be obtained from transgenic microorgantsms and plants that hâve been modified with respect to a wild type variety. Such modifications may be, for example, through the itérative steps of sélection and breeding to obtain desired traits in n plant. Furthermore, the plants can hâve had genetic material removed, modified, 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 modifications include introducing or modifying spécifie genes from parental varieties, or, for example, by using transgenic breeding wherein a spécifie gene or genes are introduced to a plant from a different species of plant and/or bacteria. Another way to create genetic variation is through mutation breeding wherein new alleles are artificially created from endogenous genes. The artificial genes can be created by a variety of ways including treating the plant or seeds with, for example, chemical mutagens (e.g., using alkylating agents, epoxides, alkaloids, peroxides, formai dch y de), irradiation (e.g., X-rays, gamma rays, neutrons, beta particles, alpha particles, protons, deuterons, UV radiation) and température shocking or other extemal stressing and subséquent sélection techniques. Other methods of providing modified genes is through errer prône PCR and DNA shufflmg 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 précipitation, electroporation, gene splicing, gene silencing, lipofection, microinjection and viral carriers. Additional genetically modified materials hâve been described in U.S. Application Serial No 13/396,369 filed February 14,2012 the full disclosure of which is incorporated herein by référence.

SACCHARTFYING AGENTS

Suitable cellulolytic enzymes include cellulases from species in the généra Bacillus, Coprinus, Myceliophthora, Cephalosporium, Scytalidium, Pénicillium, Aspergillus, Pseudomonas, Humicola, Fusarium, Thlelavîa, Acremonium,

Chrysosporium and Trichoderma, especially those produced by a strain selected from the species Aspergillus (see, e.g., EP Pub. No. 0 458 162), Humlcola insolens (redassified as Scytalidinm thermophilum, see, e.g., U.S. Pat. No. 4,435307), Copriitus cinereus, Fusarium oxysporum, Mycetiophthora thermophlla, Meripilus gigant eus, Thielavia terrestris, Acremoninm sp. (including, but not limited to, A.

perslclnum,A. acremonium, A. bracliypeiùum.A. dicltromosporum, A. obclavatum,A. pînkertoniae, A. roseogriseum, A. incoloratum, and A. furatum). Preferred strains include Humlcola insolens DSM 1800, Fusarium oxysporum DSM 2672, Myceliophthora thermophila CBS 117.65, Cephalosporium sp. RYM-202,

Acremonium sp. CBS 478.94, Acremonlum sp. CBS 265.95, Acremonium persicinum CBS 169.65, Acremonium acremonium AHU 9519, Cephalosporium sp. CBS 535.71, Acremonium brachypenium CBS 866.73, Acremonium dichromosporum CBS 683.73, Acremonium obclavatum CBS 311.74, Acremonium pînkertoniae CBS 157.70, Acremonium roseogriseum CBS 134.56, Acremonium incoloratum CBS 146.62, and

Acremoniumfuratum CBS 299.70H. Cellulolytïc enzymes may also be obtained from

Chrysosporium, preferably a strain of Chrysosporium lucknowense. Additional strains that can be used include, but are not limited to, Trichoderma (particularly T. viride, T. reesei, and T. konlngli), alkalophilic Baclllus (see, for example, U.S. Pat. No. 3,844,890 and EP Pub. No. 0 458 162), and Streptomyces (see, e.g„ EP Pub. No.

0 458 162).

FERMENTATION AGENTS

The microorganism(s) used in fermentation can be natural ly-occurrin g micro organisme and/or engineered microorganisms. These fennentation agents can 25 be used, for example, to convert a primary fuel to a secondary fuel to bc used for energy génération in an indirect fuel cell. Of the fermentation agents can be used to convert sugars or intermediates not used in fuel cells described in the methods herein. Examples of microorganism can be a bacterium (including, but not limited to, e.g., a cellulolytïc bacterium), a fungus, (including, but not limited to, e.g., a yeast), a plant, 30 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 organîsms are compatible, mixtures of organisms can be utilized.

Suitabie fermenting microorganisms hâve the abiiity to convert carbohydrates, such as glucose, fructose, xylose, arabinose, mannose, galactose, oligosaccharides or polysaccharides into fermentation products. Fermenting microorganisms include strains of the genus Sacchromyces spp. (including, but not limited to, S. cerevisiae (baker’s yeast), S. distaticus, S. uvarum), the genus Kluyveromyces, (including, but not limited to, K. marxianus, K.fragilis), the genus Candida (including, but not limited to, C pseudotropicalis, and C. brassicae), Pichia stipitls (a relative of Candida shehatae), the genus Clavispora (including, but not limited to, C. lusitanlae and C. opuntiae), the genus Pachysolcn (including, but not limited to, P. tannophllus), the genus Bretannomyces (including, but not limited to, e.g., B. clausenti (Philippîdis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization. Wyman, C.E., ed., Taylor & Francis, Washington, DC, 179-212)). Other suitabie microorganisms include, for example, Zymomonas mobilis, Clostridium spp. (including, but not limited to. Clostridium thermoceilum (Philippîdis, 1996, supra). Clostridium saccharobutylacetonicum. Clostridium felsineum,Clostridium saccharabutylicum. Clostridium Puniceum, Clostridium beijemckii, Clostridium acetobutylicum, and Clostridium aurantibutylicum), Moniliella pollinis, Yarrowia lipolytica,Aureobasidium sp., Trichosporonoides sp., Trigonopsis variabilis, Trichosporon sp., Moniliellaacetoabutans sp., Typhula variabllls, Candida magiioliae, Ustilaginomycetes sp., Pseudozyma tsukubaensis, yeast species of généra Zygosaccharomyces, Debaryomyces, Hansenula and Pichia, and fungi of the dematioid genus Torula.

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 Scvice Culture Collection, Peoria, Illinois, USA), or the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany), to name a few.p

Commercially available yeasts include, for cxample, Red StarO/Lesaffrc Ethanol Red (available from Red Star/Lesaffre, USA), FAL1® (available from Fleischmann’s Yeast, a division of Bums Philip Food Inc., USA), SUPERSTART® (available from Alltcch, now Lalemand), GERT STRAND® (available from Gert Strand AB, Sweden) and FERMOL® (available from DS M Specialties).

Other than in the examples herein, or unless otherwise expressly specified, ail of the numerical ranges, amounts, values and percentages, such as those for amounts of materials, elemental contents, times and températures of réaction, ratios of amounts, and others, in the following portion of the spécification 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 contrury. the numerical parameters set forth in the following spécification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the présent invention. At the very least, and not as an attempt to limit the application of the doctrine of équivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the spécifie examples are reported as precisely as possible. Any numerical value, however. inhérent! y contains error necessarily resulting from the standard déviation found in its underlying respective testing measurements. Furthermore, when numerical ranges are set forth herein, these ranges are inclusive of the recited range end points (i.e., end points may be used). When percentages by weight are used herein, the numerical values reported are relative to the total weighL

Also, it should be understood that any numerical range rccited herein is intended to include ali sub-ranges subsumed therein. For example, a range of “1 to 10 is intended to include ail sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10. that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10. The terms one, a, or “an as used herein arc intended to include “at least one or “one or more, unless otherwise indicated.

Any patent, publication, or other disclosure material. in whole or in part, that is said to be incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not conflict with existing définitions, statements. or other disclosure material set forth in this disclosure. As such. and to the extent necessary, the disclosure as explicitly set forth herein supersedes any confiicting

material incorporated herein by reference. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing définitions, statements, or other disclosure material set forth herein will only be incorporated to lhe extent that no conflict anses between that incorporated material 6 and the existing disclosure materiai.

While th Is invention has been particularly shown and described with référencés to preferred embodiments thereof, it will be understood by those skilled in lhe art that various changes in form and details may be made therein without dcparting from the scope of the invention encompassed by the appended claims.

Claims (21)

1. A method comprising:

utîlizing, in a fuel cell, a sugar solution comprising saccharified cellulosic or lignocellulosic material.

2. The method of claim 1 wherein, the fuel cell is selected from the group consisting of direct sugar fuel cells, indirect sugar fuel cells and biological fuel cells.

3. The method of claim 2 wherein, prior to saccharification, the recalcitrance of the cellulosic or lignocellulosic material is reduced relative to that of the cellulosic or lignocellulosic material in its native state.

4. The method of claim 3 wherein the method of reducing the recalcitrance of the material is selected from the group consisting of mechanically treating, chemically treating, sonicating, pyrolyzing, irradiating, oxidizing, steam exploding and combinations thereof.

5. The method of claim 3 wherein the method of reducing recalcitrance of the material comprises irradiation the material with ionizing radiation.

6. The method of claim 5 wherein irradiation comprises irradiation with a dose of at least 10 Mrad.

7. The method of claim 5 wherein ionizing radiation comprises électron beam irradiation.

8. The method of claim 1 wherein the cellulosic or lignocellulosic biomass is selected from the group consisting of: paper, paper products, paper waste, paper pulp, pigmented papers, loaded papers, coated papers, fillcd papers, magazines, printed matter, printer paper, polycoated paper, card stock, cardboard, paperboard, offal, cotton, wood.

particle board, forestry wastes, sawdust, aspen wood, wood chips, grasses, switchgrass, miscanthus, cord grass, reed canary grass, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, agricultural waste, sîlage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, com cobs, com stover, soybean stover, com 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, and mixtures of any of these.

9. The method of claim 1, further comprising mechanically treating the cellulosic or lignocellulosic material, for example, to reduce the bulk density of the material and/or increase its surface area.

10. The method of claim 1 further comprising utilizing an additive in the fuel cell.

11. A fuel cell comprising:

an electrode and saccharified cellulosic or lignocellulosic biomass.

12. A method for producing electricity, the method comprising: provîding a fuel to a fuel cell, wherein the fuel is produced by saccharifying a treated lignocellulosic material.

13. The method of claim 12 wherein the treatment of the lignocellulosic material is selected from the group consisting of sonication, pyrolysîs, irradiation, oxidation, steam explosion and combinations thereof.

14. The method of claim 12 further comprising contacting the lignocellulosic material with an enzyme.

15. The method of claim 14 wherein the enzyme is a cellulase.

16. The method of claim 12 wherein the fuel comprises a sugar.

17. The method of claim 12 further comprising fermenting the saccharified treated lignoceilulosic material to produce the fuel.

18. The method of claim 17 wherein the fuel is an alcohol.

19. The method of claim 13 wherein treating the lignoceilulosic material comprise irradiation of the lignoceilulosic material with ionizing radiation.

20. The method of claim 19 wherein irradiation comprises irradiation of the material with a dose of at least 10 Mrad.

21. The method of claim 19 wherein the ionizing radiation comprises an électron

15 beam.