US009745604B2 (12 ) United States Patent ( 10) Patent No. : US 9 ,745 , 604 B2 Medoff (45 ) Date of Patent: * Aug. 29 , 2017 (54 ) PROCESSING BIOMASS B01D 39 / 18 (2013 . 01 ) ; C08H 8 /00 (2013 . 01 ) ; C08J 3 / 28 ( 2013 .01 ); C08L 1 /02 ( 2013 .01 ); (71 ) Applicant: Xyleco , Inc. , Wakefield , MA (US ) C08L 5 / 14 ( 2013 .01 ) ; C12P 7 /42 ( 2013. 01 ) ; (Continued ) ( 72 ) Inventor: Marshall Medoff, Brookline , MA (US ) (58 ) Field of Classification Search None ( 73 ) Assignee: Xyleco , Inc. , Wakefield ,MA (US ) See application file for complete search history. ( * ) Notice : Subject to any disclaimer, the term of this patent is extended or adjusted under 35 ( 56 ) References Cited U . S .C . 154 (b ) by 0 days . U . S . PATENT DOCUMENTS This patent is subject to a terminal dis 1 ,920 ,129 A * 7 / 1933 Munroe ...... B65G 3 / 00 claimer . 34 / 518 3 , 352 ,773 A 11/ 1967 Schwartz et al. (21 ) Appl. No. : 15/ 040 ,318 ( Continued ) ( 22 ) Filed : Feb . 10 , 2016 FOREIGN PATENT DOCUMENTS (65 ) Prior Publication Data CN 101041834 A 9 /2007 EP 2276795 A2 1 /2011 US 2016 /0165926 A1 Jun . 16 , 2016 (Continued ) Related U .S . Application Data OTHER PUBLICATIONS (63 ) Continuation of application No . 14 /813 ,898 , filed on Jul. 30 , 2015 , which is a continuation of application Adams, “ Corn Stover as Feed for Cattle ” , Penn State College of (Continued ) Agricultural Sciences, Penn State Extension , Feb . 1 , 2002 ( 2 pages) . ( Continued ) (51 ) Int. Ci. A23K 50 / 10 ( 2016 .01 ) Primary Examiner - Chris R Tate A23K 10 / 30 (2016 .01 ) Assistant Examiner — Aaron J Kosar (Continued ) (74 ) Attorney, Agent, or Firm — Wilmer Cutler Pickering (52 ) U . S . CI. Hale and Dorr LLP ??? ...... C12P 7 /62 ( 2013 .01 ) ; A23K 10 / 12 ( 2016 .05 ); A23K 10 / 30 ( 2016 .05 ) ; A23K 10 / 37 (57 ) ABSTRACT ( 2016 .05 ); A23K 20 / 163 ( 2016 . 05 ) ; A23K Biomass ( e. g . , plant biomass , animal biomass , microbial, 20 / 189 (2016 .05 ) ; A23K 40 / 10 (2016 .05 ) ; and municipal waste biomass) is processed to produce useful A23K 50 / 10 (2016 .05 ) ; A23K 50 / 80 ( 2016 .05 ); products , such as food products and amino acids . A23L 5/ 00 (2016 .08 ); A23L 5 /30 (2016 .08 ) ; A61K 8/ 97 (2013 .01 ); A61Q 19 /00 ( 2013 .01 ); 13 Claims, 54 Drawing Sheets
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Related U . S . Application Data 6 ,555 , 335 B14 / 2003 Saloheimo et al. 6 , 555 ,350 B2 4 /2003 Ahring et al. No . 14 /750 ,995 , filed on Jun . 25 , 2015 , which is a 6 , 620, 605 B2 9 /2003 Fowler et al . continuation of application No. 14 / 333 ,675 , filed on 6 , 808 , 600 B2 10 / 2004 Ross et al. Jul. 17 , 2014 , now Pat. No. 9 , 309, 545 , which is a 6, 942 ,973 B2 9 /2005 Yaver et al. continuation of application No. 13 / 902 ,246 , filed on 7 ,005 ,508 B2 2 / 2006 Benedini et al. May 24 , 2013 , now Pat . No. 8 , 877 , 472 , which is a 7 ,408 , 056 B2 8 / 2008 Medoff et al. 7 , 846, 295 B1 12 / 2010 Medoff continuation of application No. 12 /417 , 900 , filed on 7, 867, 358 B2 1 / 2011 Medoff Apr. 3 , 2009, now abandoned . 7 ,867 , 359 B2 1/ 2011 Medoff 7 , 900, 857 B2 3 / 2011 Medoff (60 ) Provisional application No . 61/ 139, 453, filed on Dec . 7 , 931, 784 B2 4 / 2011 Medoff 7 , 932 , 065 B2 4 / 2011 Medoff 19 , 2008, provisional application No . 61/ 073 , 674 , 7 , 935 , 219 B2 5 / 2011 Medoff filed on Jun . 18 , 2008 , provisional application No . 8 , 052 , 838 B2 11/ 2011 Medoff 61/ 049 ,405 , filed on Apr. 30 , 2008 . 8, 070 ,912 B2 12 / 2011 Medoff 8 , 083, 906 B2 12 / 2011 Medoff (51 ) Int. Ci. 8 , 142 , 620 B2 3 / 2012 Medoff C12P 7762 ( 2006 .01 ) 8 , 147 , 655 B2 4 / 2012 Medoff A61Q 19 /00 ( 2006 .01 ) 8 , 168 , 038 B2 5 / 2012 Medoff A61K 8 / 97 ( 2017 .01 ) 8 , 212, 087 B2 7 / 2012 Medoff BOID 39 / 18 ( 2006 . 01 ) 8 , 221 , 585 B2 7 / 2012 Medoff 8 , 236 , 535 B2 8 / 2012 Medoff et al . C08H 8 / 00 ( 2010 .01 ) 8 ,377 , 668 B2 2 / 2013 Medoff et al . C08J 3 / 28 ( 2006 . 01 ) 8 , 418 , 944 B2 4 / 2013 Medoff C12P 19 /44 ( 2006 .01 ) 8 , 454 , 803 B2 6 / 2013 Medoff C13K 1 /02 ( 2006 .01 ) 8 , 492, 128 B2 7 /2013 Medoff C08L 1 / 02 ( 2006 .01 ) 8 ,597 ,921 B2 12 / 2013 Medoff CO8L 5 / 14 ( 2006 .01 ) 8 ,603 , 787 B2 12 / 2013 Medoff C12P 13 /04 8 , 835 , 142 B2 9 /2014 Medoff ( 2006 .01 ) 8 , 841 , 101 B2 * 9 /2014 Medoff A61K 8 /97 D21C 5 / 00 ( 2006 .01 ) 162 / 50 D21C 9 /00 ( 2006 .01 ) 8 ,945 , 245 B2 2 /2015 Bals et al. A23K 10 / 12 ( 2016 . 01 ) 9 ,078 ,461 B2 7 / 2015 Medoff A23K 10 /37 ( 2016 .01 ) 9 , 278 , 896 B1 * 3 / 2016 Medoff B01J 19 /085 A23K 20 / 163 ( 2016 . 01 ) 9 , 309 , 545 B2 4 / 2016 Medoff A23K 20 / 189 (2016 .01 ) 9 , 328 ,393 B2 * 5 /2016 Medoff . .. C13K 1/ 06 A23K 50 /80 2004 / 0005674 A11 / 2004 Duck et al. ( 2016 .01 ) 2004 / 0231060 A1 11/ 2004 Burdette et al . A23L 5 /30 ( 2016 . 01 ) 2005 /0203291 A1 9 /2005 Svenson et al. C12P 7742 ( 2006 .01 ) 2006 / 0088922 AL 4 / 2006 Yang et al. A23L 5 /00 ( 2016 .01 ) 2006 / 0251764 A 11/ 2006 Abbas et al. A23K 40 / 10 ( 2016 .01 ) 2007/ 0015855 AL 1 /2007 Medoff et al . A23K 50 / 75 ( 2016 .01 ) 2009 / 0286295 AL 11 /2009 Medoff et al. A23K 50 / 30 ( 2016 .01 ) 2010 / 0087687 A1 4 / 2010 Medoff 2010 /0093241 A1 4 / 2010 Medoff A23K 50 /40 ( 2016 .01 ) 2010 /0105119 Al 4 / 2010 Medoff (52 ) U . S . CI. 2010 /0108567 A1 5 / 2010 Medoff ??? ...... C12P 13 /04 ( 2013 .01 ) ; C12P 19 /44 2010 /0124583 Al 5 / 2010 Medoff ( 2013 .01 ) ; C13K 1/ 02 ( 2013 . 01 ) ; D21C 5 / 005 2010 /0179315 A1 7 /2010 Medoff ( 2013 .01 ) ; D21C 9 /001 (2013 .01 ) ; A23K 50 / 30 2010 /0200806 A1 8 /2010 Medoff et al . ( 2016 .05 ); A23K 50 /40 (2016 . 05 ) ; A23K 50 / 75 2010 / 0203495 A1 8 / 2010 Medoff et al . ( 2016 . 05 ); C12P 2201/ 00 (2013 .01 ) ; C12P 2010 /0206501 A1 8 /2010 Medoff 2010 / 0297720 A111/ 2010 Medoff et al . 2203 /00 (2013 .01 ) ; YO2E 50 / 10 ( 2013 .01 ) ; 2010 /0304440 A112 / 2010 Medoff YO2E 50 / 16 (2013 . 01 ) ; YO2E 50 / 17 ( 2013 . 01 ) ; 2011/ 0011960 AL 1 / 2011 Medoff YO2E 50 / 30 (2013 . 01 ) ; YO2P 60 / 877 ( 2015 . 11 ) 2011/ 0027837 A1 2 / 2011 Medoff 2011/ 0039317 A1 2 /2011 Medoff (56 ) References Cited 2011/ 0081335 A1 4 / 2011 Medoff 2011 /0081336 A1 4 /2011 Medoff U . S . PATENT DOCUMENTS 2011 /0111456 AL 5 / 2011 Medoff 3 , 939 , 286 A 2 / 1976 Jelks 2011 /0139383 A1 6 / 2011 Medoff 4 , 268, 505 A 5 / 1981 Yoshikumi et al. 2011/ 0155559 A1 6 / 2011 Medoff 4 , 274 , 163 A 6 / 1981 Malcom et al . 2011/ 0262985 A1 10 / 2011 Medoff 4 , 275 , 163 A 6 / 1981 Gallo 2011/ 0265991 A1 11/ 2011 Medoff 4 , 321 ,328 A 3 / 1982 Hoge 2011 /0287498 AL 11/ 2011 Medoff et al. 4 , 769 ,082 A 9 / 1988 Kumakura et al . 2012 /0003704 A1 1 / 2012 Medoff 5 , 122 , 598 A 6 / 1992 della Valle et al. 2012 /0052536 A1 3 / 2012 Medoff et al . 5 , 221, 357 A 6 / 1993 Brink 5 ,314 ,978 A 5 / 1994 Kim et al. 2012 / 0074337 A1 3 /2012 Medoff 5 ,417 ,824 A 5 / 1995 Greenbaum 2012 /0077247 A1 3 / 2012 Medoff 5 , 538 , 730 A 7 / 1996 Romeo et al. 2012 /0094358 A14 /2012 Medoff 5 , 753 , 474 A 5 / 1998 Ramey 2012 /0100577 AL 4 /2012 Medoff et al. 6 ,011 ,008 A 1 / 2000 Domb et al. 2012 /0100586 A1 4 / 2012 Medoff et al. 6 , 334 , 347 B11 / 2002 Iscla 2012 /0142065 A1 6 / 2012 Medoff US 9 , 745 ,604 B2 Page 3
( 56 ) References Cited Ghosh , P. and Singh , A .“ Physicochemical and Biological Treat ments for Enzymatic /Microbial Conversion of Lignocellulosic Bio U . S . PATENT DOCUMENTS mass ” Advances in Applied Microbiology , 1993 , 39, pp . 295 -333 . 2012 /0142068 A1 6 / 2012 Medoff Gromov , A . V “ VI Inter- University Conference on Electron Accel 2012 /0315675 A1 * 12/ 2012 Medoff ...... C08H 8 /00 erators ” Atomic Energy , Aug . 1966 , 21 (2 ) , pp . 143 - 145 . 435 / 99 Han et al . ( 1983 ) “ Effect of Gamma- Ray Irradiation on Alcohol 2015/ 0342224 A1* 12 /2015 Medoff ...... A23K 1 / 1653 Production from Corn ” , Biotechnol. Bioeng . 25 : 2631 -2640 ( Abstract ) . 426 /63 Han et al. , “ Chemical and Physical Properties of Sugarcane Bagasse FOREIGN PATENT DOCUMENTS Irradiated with Gamma Rays, ” J. Agric . Food Chem . 1983 , 31 , 34 - 38 . 200200944 1 /2005 Ibrahim , M . N . M and Pearce , G . R . , “ Effects of Gamma Radiation on IN 200401988 8 /2006 the Composition and in Vitro Digestibility of Crop By -Products ," JP S5971700 A 4 / 1984 Agricultural Wastes , vol. 2 , 1980 , 253 - 259 . JP S59113856 A 6 / 1984 Ilan Levy et al ., “ Modification of Polysaccharides and Plant Cell KR 2002091479 12 / 2002 RU 2030155 Ci 3 / 1995 Wall by Endo - 1 , 4 ,Bet - Glucanase and Cellulose -Binding Binding SU 1262950 A1 5 / 1990 Domains, " Biomolecular Engineering, vol. 19 ( 1 ) , 2002 , pp . 17 - 30 . WO WO -01 / 15689 3 / 2001 Isono et al. , “ Ultrasonic Degradation of Waxy Rice Starch ” , Biosci WO WO -01 / 52620 7 / 2001 ence , Biotechnology , and Biochemistry , 58 ( 10 ): 1799 - 1802 , 1994 . WO WO - 0179241 10 / 2001 ISR — Corresponding PCT Application No . PCT/ US2009 /041963 , WO WO - 2006032282 A1 3 / 2006 Oct . 29 , 2009 by Kipo, 14 pages . WO WO - 2008073186 A2 6 / 2008 James M . Gibson ; Phillip S . Thomas; Joshua D . Thomas ; Jessica L . WO WO - 2009134791 A2 11/ 2009 Barker; Sunil S . Chandran ; Mason K . Harrup ; Karen M . Draths ; John W . Frost “ Benzene - Free Synthesis of Phenol” Angew . Chem . Int. Ed . 2001, 40 ( 1 ), pp . 1945 - 1948 . OTHER PUBLICATIONS Jessica L . Barker ; John W . Frost, “ Microbial Synthesis of Allen , Stephen Glen et al. , “ Fractionation of Sugar Cane with Hot , p - Hydroxybenzoic Acid from Glucose” Biotech . Bioeng . Dec . 2001, 76 ( 4 ), pp . 376 -390 . Compressed , Liquid Water” , Industrial & Engineering Chemistry Jian Shi, et al. , “ Impact of Mixed Feedstocks and Feedstock Research , vol. 35 , No . 8 , Jan . 1 , 1996 , pp . 2709 - 2715 . Densification on Ionic Liquid Pretreatment Efficiency” , Biofuels , Atchison , et al ., " Subcontractor Report : Innovative Methods for 4 ( 1) :63 - 72 , 2013 (10 pages ). Corn Stover Collecting, Handling, Storing and Transporting — Mar. Khan et al . ( 1986 ) “ Effect of Electron - Beam Irradiation Pretreat 2003” , National Renewable Energy Laboratory , NREL / SR -510 ment on the Enzymatic Hydrolysis of Softwood, ” Biotechnol. 33893, Apr. 2004 (63 pages ). Bioeng ., 28 : 1449 - 1453 Awafo et al. , “ Effect of Irradiation , as a Pretreatment, on Bioconver Kumakura et al. ( 1982 ) “ Radiation Degradation and the Subsequent sion of Corn Stover into Protein -Rich Mycelial Biomass of Enzymatic Hydrolysis of Waste Papers, " Biotechnol . Bioeng . Pluerotus Sajor- Caju ,” Radiation Physics and Chemistry, vol. 46 , 24 : 991- 997 . No. 4 -6 , pp . 1299 - 1302 , Sep . 12 , 1995 , XP004051286 . Leonhardt et al ., "Gamma and Electron Radiation Effects on Straw ," Bak , J . S . et al ., “ Improved Enzymatic Hydrolysis Yield of Rice Radiat . Phys . Chem ., vol. 21 , No. 4 , 1983 , pp . 397 - 400 . Straw Using Electron Beam Irradiation Pretreatment” Bioresource Madan , et al . , “ Cellulolytic Enzymes from an Edible Mushroom , Technology , 2009 ( online Oct. 17 , 2008 ) , 100 ( 3 ) , pp . 1285 - 1290 . Pleurotus Sajor -Caju ” , Biotechnology Letters, 5 (9 ): 601 -604 , Aug . Batrinou A . M . et al . “ Effect of Ionizing Radiation on the Quanti 1983 ( 4 pages) . fication of Genetically Modified Foods” , Food Biotechnology , Jul. Minea , R . et al . , " Accelerators Use for Irradiation of Fresh Medici Dec . 2008 , 22 ( 3 - 4 ) , pp . 338 -351 . nal Herbs” , Proceedings of EPAC 2004 , Lucerne, Switzerland , pp . Campbell, G .L . Au — Bedford , M .R ., “ Enzyme Applications for 2371- 2373 . Monogastric Feeds : A Review ” , Canadian Journal of Animal Sci Nemtanu , M . R . et al. , “ Microbial Safety Improvement of Sea Buckthorn by Electron Beam Irradiation ” , Sixth International Con ence, 72 ( 3 ) : 449 - 466 , Sep . 1 , 1992 . ference of the Balkan Physical Union , American Institute of Phys D . A . Teitge, G . L . Campbell, H . L . Classen , and P . A . Thacke, “ Heat ics, 2007, p . 792 . Pretreatment as a Means of Improving the Response to Dietary New Zealand Office Action , Corresponding Application No . Pentosanase in Chicks Fed Rye ” , Can . J. Anim . Sci. , 71 ( 2 ) : 507 -513 701322 , dated Sep . 7 , 2015 , 3 pages. ( Jun . 1991) . PTO - 14 - 2634 , English Translation of CN 101041834A , 11 pgs, Davis , “ International Research Team Seeks More Efficient Biomass published Sep . 26 , 2007 . Refinement Processes” , Virginia Tech News, Jul. 7 , 2015 (3 pages ). Saleh et al . ( 2004 ) “ Carbohydrases are Digested by Proteases DeKerf et al. ( 2001) “ Characterization and Disintegration Proper Present in Enzyme Preparations During in vitro Digestion , " J . ties of Irradiated Starch , ” Int. J . Pharmaceutics , 221 :69 - 76 . Poultry Sci. 41 : 229 -235 . Eichenberger , C . et al. , “ 7. 5 MeV High Average Power Linear Scharf , W . and Wieszczycka, W ., “ Electron Accelerators for Indus Acclerator System for Food Irradiation Applications” , Proceedings trial Processing — a Review ” , 15th International Conference on the of the International Symposium on the New Frontier of Irradiated Applications of Accelerators in Research and Industry , Denton , TX , Food and Non -Food Products and King Mongkut ' s Universityof USA , Nov . 4 - 7 , 1998 , pp . 949 -952 , archived reviewMay 1998 , 19 Technology Thonburi (KMUTT ), Sep . 2005 , 8 pages . pages . Elisashvili , et al. , “ Cellulase and Xylanase Activities in Higher Seiboth et al. (2011 ) “ Chap . 13 : Trichoderma reesei: A Fungal Basidiomycetes” , biochemistry (Mosc ) , 64 ( 6 ) : 718 -722 , Jun . Enzyme Producer for Cellulosic Biofuels ,” pp . 309 - 340 in : “ Biofuel 1999 — Abstract Only ( 2 pages) . Production — Recent Developments and Prospects , ” Aurelio ( ed . ) in European Search Report - Corresponding EP application No . Tech . 15171272. 6 , dated Sep . 15 , 2015 , 10 pages . Smith et al. , “ Irradiation Enhancement of Biomass Conversion ,” Extended European Search Report issued in EP15171272 .6 ; dated Radiation Physics and Chemistry , vol. 25, Nos. 1- 3 , pp . 27 -33 , Jan . Jan . 27 , 2016 ( 13 pages ) . 1 , 1985 , XP025752592 . FAO “ Cellulase Production ” FAO Agricultural Services Bulletin : Stipanovic , A . J . “ Electron Beam and X -Ray Irradiation of Lignocel Renewable Biological Systems for Alternative Sustainable Energy lulosic Biomass - Synergies with Biodelignification and Hemicel Production , Chapter 3 . 2 , 1997 , retrieved onlineURL :http : / /www . lulose Removal in Reducing Recalcitrance " USDA ( C . R . I . S . Acces fao .org / docrep /W7241E /W7241E00 . htm , 14 pages . sion No . 0211017 ) , Sep . 1 , 2007 , 6 pages. US 9, 745 ,604 B2 Page 4
( 56 ) References Cited Shin et al. , “ Improving enzymatic hydrolysis of industrial hemp ( Cannabis sativa L .) by electron beam irradiation ,” Radiation OTHER PUBLICATIONS Physics and Chemistry , Sep . 2008 ( epub May 29, 2008 ) , 77 ( 9 ) , pp . Taherzadeh et al. ( 2008 ) “ Pretreatment of Lignocellulosic Wastes to 103 - 1038 . Improve Ethanol and Biogas Production : A Review ” , Int. J . Mol. Srinivas et al. , “ Urea -molasses -mineral block licks supplementation Sci. 9 : 1621- 1651. for milk production in crossbred cows, " Asian Australas . J . Anim . Tewari et al. ( 1987 ) “ Role of Pretreatments on Enzymatic Hydro Sci, 1997 , v . 10 , No . 1 , pp . 47 -53 . lysis of Agricultural Residues for Reducing Sugar Production , ” J . Sun and Cheng , “ Hydrolysis of lignocellulosic material for ethanol Chem . Tech . Biotechnol. 38 : 153 - 165 . production : a review , ” Bioresource Technology , 83 , pp . 1 - 11 ( 2002 ) . Whitham , K . et al. “ 12 MW , 100 KW , 7000 Hour / Year Modulator Victorov and Menkin , “ Methodology and organization of for Titan Scan ” Pulsed Power Conference , 1995 , Digest of Tech zootechnical tests " 1991, Moscow , Agropromisdat, 112 , pp . 79 -82 nical Papers , Tenth IEEE International, Jul. 3 - 6 , 1995 , vol . 1 , pp . (cited in Russian Office Action for corresponding application No . 534 - 538 . RU 2015 127 786 ) . Xia et al. ( 1998 ) “ Saccharification of Corn Stover by Immobilized Wasikiewicz et al. , “ Degradation of chitosan and sodium alginate by Trichoderma reesei cells , ” Wei Shen Wu Xue Bao 38 :114 - 9 . gamma radiation , sonochemical and ultraviolet methods, ” Radiation Yang C ., et al ., “ Effect and Aftereffect of [Gamma ] -Radiation Physics and Chemistry , 2005 , 73 ( 5 ) , pp . 287 - 295 . Pretreatment on Enzymatic Hydrolysis of Wheat Straw ” , Xiong et al. , “ Xylanase production by Trichoderma reesi Rut C -30 Bioresource Technology , 2009 (online Jan . 14 , 2008 ), 99 ( 2 ), pp . grown on L - arabinose - rich plant hydrolysates, " Bioresource tech 6240 -6245 . nology , 2005 , v. 96 No. 7 , pp . 753 -759 . Chosdu et al. , “ Radiation and chemical pretreatment of cellulosic Ying et al . , “ Butyric acid production from acid hydrolysate of corn waste ," Radiation Physics and Chemistry, 1993 , v . 42 , No . 4 -6 , pp . fibre by Clostridium Tyrobutyricum in a fibrous -bed bioreactor, " 695 -698 . Process Biochemistry , 2002 , v . 38 , No . 5 , pp . 657 -666 . Dien et al. , “ Fermentation of sugar mixtures using Escherichia coli Zaldivar et al. , “ Fuel ethanol production from lignocellulose : a catabolite repression mutants engineered for production of - lactic challenge formetabolic engineering and process integration , ” Appl. acid ,” Journal of Industrial Microbiology & Biotechnology , 2002 , v. Microbiol. Biotechnol. , 2001, v. 56 , pp . 17 - 34 . 29, pp . 221 -227 . Zhao et al. , “ Effect of radiation pretreatment on enzymatic hydro Flachowsky et al ., " Studies on feeds from genetically modified lysis of rice straw with low concentration of alkali solution , " plants (GMP ) Contributions to nutritional and safety assessment, " Bioresource technology, 1993 , v .43 , No. 1 , pp . 13 - 17 . Animal Feed Science and Technology, 2007 , v . 133, pp . 2 -30 . Byrom , “ Polymer synthesis by microorganisms: technology and Gralak et al. , “ Rumen degradability of dry matter and crude fibre of economics” Trends in Biotechnology 5 (9 ) , pp . 246 - 250 ( 1987 ) . irradiated and sodium hydroxide treated straws, ” 1994 , Arch . Anim . Partial English translation of Office Action issued in corresponding Nutr. 47 , 63 -74 . Russian Patent Application No. 2015 127 760 , dated Feb . 8 , 2017 Gomes et al. , “ Production of cellulase and xylanase by a wild strain ( 3 pages) . of Trichoderma viride , ” Applied Microbiology and Biotechnology , Schafer et al ., “ Cattle , Corn , and Alternative Feeds” TheBeefSite 1992 , v . 36 , No. 5 , pp . 701 -707 . < thebeefsite .com / articles/ 840 /cattlecorn -and - alternative - feeds / > , 4 Huang et al . , “ Purification and characterization of an endoxylanase pages (Mar . 12 , 2007 ) . from Trichoderma koningii G -39 , " Biochemical Journal, 1991, v . Graham - Rowe, “ Electron Beams Could be Used to Irradiate Post, " 278 , No . 2 , pp . 329 - 333 . New Scientist , Oct. 24 , 2011 (1 page ). Kumakura and Kaetsu , “ Radiation - induced decomposition and I- AX Technologies Inc ., “ Hardware from I - AX Technologies Inc ., " enzymatic hydrolysis of cellulose, " Biotechnology and Bioengi
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No . 13 / 902 , 246 , filed May 24 , yeast , fungi, protists , e . g . , an algae , protozoa or a fungus - like 2013 , now U .S . Pat. No. 8 ,877 ,472 , issued on Nov . 4 , 2014 , 10 protist, e . g . , a slime mold ) , aquatic organisms ( e . g . , in which is a continuation of U . S . Ser. No. 12/ 417, 900 , filed aquaculture ), and /or plants and trees ( e . g . , in agriculture , Apr. 3 , 2009 , now abandoned , which claimed priority to hydroponics and silvaculture ) . U . S . Provisional Application 61/ 139 , 453 filed Dec . 19 , In another aspect, the present invention provides methods 2008 , U . S . Provisional Application 61/ 073 ,674 filed Jun . 18 , 5 of improving the pharmaceutical profile of materials . These 2008 and U . S . Provisional Application 61 /049 , 405 filed Apr. 15 methods include obtaining a first material including biomass 30 , 2008 . The entirety of each of these applications is ( e . g ., plant biomass , animal biomass ,microbial , and munici incorporated herein by reference . pal waste biomass ) containing polysaccharides in the form of cellulose , hemicellulose , and /or starch , and modulating TECHNICAL FIELD 20 ( e. g ., increasing , decreasing , or maintaining ) the molecular This invention relates to processing biomass , to compo - structure of the polysaccharides of the first material to sitions including saccharide units arranged in a molecular produce a second material , where one of the results of the chain , to methods of producing amino acids or antibiotics , to methods is that the pharmaceutical profile of the second methods of producing edible or immunostimulatory mate material is better or improved when compared to the phar rial, and to products of such methods . 25 maceutical profile of the first material. In some instances, the methods include using first materials with little or no phar BACKGROUND maceutical profile prior to modulating the molecular struc ture of the first material. The second materials produced Biomass , particularly biomass waste , is abundantly avail - using the methods described herein are suitable for admin able . It would be useful to derive products from biomass . 30 istration to an animal. In a further aspect, the invention provides methods for SUMMARY obtaining a plant- derived pharmaceutical. These methods include processing a material including biomass ( e . g . , plant Exemplary products that can be produced using the meth biomass, animal biomass, microbial, and municipal waste ods provided herein include foodstuffs suitable for use in , 35 biomass ) containing polysaccharides in the form of cellu e . g ., ingestion by a human and / or animal, aquaculture , lose , hemicellulose , and / or starch containing one or more agriculture, hydroponics, pharmaceuticals , nutraceuticals , plant made pharmaceuticals , using any one or more of pharmaceutical delivery vehicles and dosage forms, phar - radiation , sonication , pyrolysis , and oxidation to obtain a maceutical excipients , pharmaceutical conjugates, cross plant- derived pharmaceutical. In some instances the plant linked matrixes such as hydrogels , absorbent materials , 40 derived made pharmaceutical can be isolated and / or puri fertilizers, and lignin products . Any product disclosed herein fied . or produced by the methods disclosed herein can be used In yet another aspect, the present invention provides as - is , or as a precursor or an intermediate in the production methods of preparing nutraceuticals for human and / or a of another product . non -human animal consumption . These methods include In many embodiments , products can be produced using 45 processing a material including biomass ( e . g ., plant biomass , Natural ForceTM Chemistry . Natural ForceTM Chemistry animal biomass , microbial, and municipal waste biomass ) methods use the controlled application and manipulation of containing polysaccharides in the form of cellulose , hemi physical forces , such as particle beams, gravity , light, etc . , to cellulose , and / or starch so as to change the molecular create intended structural and chemicalmolecular change . In structure of the polysaccharides of the material ( e . g . , preferred implementations, Natural ForceTM Chemistry 50 increase or decrease the molecular weight of the material) . methods alter molecular structure without chemicals or These methods can optionally also include administering the microorganisms. By applying the processes of Nature , new resulting materials to humans and non -human animals . useful matter can be created without harmful environmental In an alternative aspect, the invention provides methods interference . of preparing biological agents and / or pharmaceutical agents . In one aspect , the present invention includes methods of 55 These method include processing a material including bio preparing feed materials for animals ( e . g . , humans and mass containing polysaccharides in the form of cellulose , animals , including but not limited to food animals , pets , zoo hemicellulose , and /or starch , so as to change the molecular animals , etc . ) , and for plants ( e . g . , agricultural plants or structure of the polysaccharides of the material. The result crops or aquatic plants , in particular in a hydroponic solution ing materials can then be combined with one or more or in aquaculture ) , and aquatic organisms ( e . g . , fish , crus- 60 biological agents and / or one or more pharmaceutical agents , taceans, mollusks and the like ). which can be administered to a subject. These methods include obtaining a first material including Also provided in the present invention are methods of biomass (e . g. , plant biomass , animal biomass ,microbial , and making hydrogels . These methods include processing a municipal waste biomass ) containing polysaccharides in the material including biomass containing polysaccharides in form of cellulose, hemicellulose, and /or starch . The molecu - 65 the form of cellulose , hemicellulose , and /or starch , and lar structure of the polysaccharides of the first material is changing the molecular structure of the polysaccharides to then modulated ( e . g ., increased , decreased , or maintained ) to produce a material that includes cross - linked polymer US 9 ,745 ,604 B2 chains . The method can further include cross -linking poly - ( e . g ., algae such alveolates , chlorarachniophytes, crypto mer chains in processed material. monads, euglenids, glaucophytes, haptophytes, red algae , In yet another aspect, the present invention provides stramenopiles , and viridaeplantae )) , seaweed , giant sea methods of making an absorbent or adsorbent material. weed , water hyacinth , plankton ( e . g . ,macroplankton , meso These methods include processing a material including 5 plankton , microplankton , nanoplankton , picoplankton , and biomass containing polysaccharides in the form of cellulose , femptoplankton ) , phytoplankton , bacteria ( e . g ., gram posi hemicellulose , and / or starch , and changing the molecular structure of the polysaccharides to produce an absorbent tive bacteria , gram negative bacteria , and extremophiles) , material. These absorbent materials can be charged , e . g . , yeast and / or mixtures of these . In some instances , biomass positively or negatively charged , and can have lipophilic 10 can include unicellular or multicellular organisms obtained and / or hydrophilic properties. As such , the materials can be from the ocean , lakes, and bodies of water including salt used as animal litter or bedding , and/ or absorbentmaterial to water and fresh water . In some instances, biomass can bind materials in a solution , ( e . g . , pollutants ) . In some include organic waste materials such as animal waste or embodiments , these absorbent materials can be used to bind excrement or human waste or excrement ( e . g ., manure and biological materials in solutions of blood or plasma. sewage ) . In some instances, biomass can include any com In a further aspect , the present invention provides meth bination of any of these . Other biomass materials are ods ofmaking fertilizers. These methods include processing described herein . Still other materials that include cellulose a material including biomass containing polysaccharides in are described in the patents , patent applications and publi the form of cellulose , hemicellulose , and /or starch , and cations that have been incorporated by reference herein . In changing the molecular structure of the polysaccharides to 20 some instances, biomass can be , e . g ., in solution , dry , and produce a material that has a greater solubility than the frozen . starting material and which is useful as a fertilizer . If biomass is or includes microorganisms, these microor Each of these methods includes treating the biomass using ganisms will generally include carbohydrates, e .g ., cellu one or more of ( e . g . , one , two , three , or four of) size lose . These microorganisms can be in a solution , dry , frozen , reduction ( e . g ., mechanical size reduction of individual 25 active , and /or inactive state . In some embodiments, these pieces of biomass) , radiation , sonication , pyrolysis , and microorganisms can require additional processing prior to oxidation to modulate the materials . In some embodiments, being subjected to the methods described herein . For the methods use a radiation dose, e . g . , from 0 . 1 Mrad to 10 example , the microorganisms can be in a solution and can be Mrad . In some embodiments , the methods use a radiation removed from the solution , e . g. , by centrifugation and /or dose , e . g . , from greater than 10 Mrad to 1000 Mrad . 30 filtration . Alternatively or in addition , the microorganisms In some aspects , the present invention also provides can be subjected to the methods described herein without compositions made using any of the methods described these additional steps, e . g . , the microorganisms can be used herein . For example , the invention features a composition in the solution . In some instances , the biomass can be or can including saccharide units arranged in a molecular chain , include a natural or a synthetic material. wherein from about 1 out of every 2 to about 1 out of every 35 Irradiation can be , e . g . , performed utilizing an ionizing 250 saccharide units comprises a carboxylic acid group , or radiation , such as gamma rays , a beam of electrons , or an ester or a salt thereof , and the composition is suitable for ultraviolet C radiation having a wavelength of from about consumption as a feed material. 100 nm to about 280 nm . The ionizing radiation can include By “ suitable for consumption as a feed material, ” we electron beam radiation . For example , the radiation can be mean that the composition is not toxic , under conditions of 40 applied at a total dose of between about 10 Mrad and about its intended use , to the living being to which it is fed , and 150 Mrad , such as at a dose rate of about 0 . 5 to about 10 provides some nutritional value to the being , e . g ., energy Mrad / day, or 1 Mrad/ s to about 10 Mrad / s . In some embodi and / or nutrients. ments , irradiating includes applying two or more radiation In some embodiments , the biomass feedstock is pre - sources, such as gamma rays and a beam of electrons. treated . In some embodiments , the methods disclosed herein 45 In some embodiments , the biomass exhibits a first level of can include a pre - treatment to reduce one or more dimen - recalcitrance and the carbohydrate material exhibits a sec sions of individual pieces of biomass . For example , pretreat- ond level of recalcitrance that is lower that the first level of ment can include reducing one or more dimensions of recalcitrance . For example , the second level of recalcitrance individual pieces of biomass can include , e . g . , shearing , can be lower than the first level of recalcitrance by at least cutting , crushing , smashing, or grinding. 50 about 10 % ( e . g . , 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , Pressure can be utilized in all of the methods described 90 % 0 , 99 % , 100 % . In some embodiments , the level of herein . For example , at least one of the treating methods, recalcitrance can be reduced by 50 % - 90 % . e . g ., radiation , can be performed on the biomass under a The biomass can be prepared by shearing biomass ( e . g . , pressure of greater than about 2 . 5 atmospheres, such as a biomass fiber source ) to provide a fibrous material. For greater than 5 or 10 atmospheres . 55 example , the shearing can be performed with a rotary knife Examples of biomass (also referred to as " biomass feed - cutter . The fibers of the fibrous material can have , e .g . , an stock ' or ' feedstock ' ) include cellulosic or lignocellulosic average length - to - diameter ratio ( L / D ) of greater than 5 / 1 . materials such as paper, paper products, paper waste , wood , The fibrous material can have, e . g ., a BET surface area of particle board , sawdust , agricultural waste , sewage , silage , greater than 0 . 25 mº /g (e . g ., 0 .3 m²/ g , 0 .35 m²/ g , 0 .35 m²/ g , grasses, rice hulls , bagasse , cotton , jute , hemp, flax , bamboo , 60 0 . 4 m?lg , 0 . 5 m?lg , 1 m?lg , 1 . 5 m?lg , 2 m?lg , 3 m?lg , 10 sisal, abaca , straw , corn cobs , corn stover, switchgrass , m²/ g , 25 m²/ g , or greater than 25 m²/ g ) . alfalfa , hay , rice hulls , coconut hair , cotton , cassava , and In some embodiments , the carbohydrate can include one synthetic celluloses and /or mixtures of these. In some or more B - 1, 4 - linkages and have a number average molecu instances , biomass can include unicellular and/ or multicel - lar weight between about 3 , 000 and 50, 000 daltons. lular organisms. Exemplary organisms include , but are not 65 In some examples, the pretreated biomass material can limited to , e . g . , protists ( e . g ., animal ( e . g ., protozoa such as further include a buffer , such as sodium bicarbonate or flagellates, amoeboids, ciliates, and sporozoa ) and plant ammonium chloride, an electrolyte , such as potassium chlo US 9 , 745 ,604 B2 ride or sodium chloride a growth factor , such as biotin and /or The term “ screen ," as used herein , means a member a base pair such as uracil , a surfactant, a mineral, or a capable of sieving material according to size . Examples of chelating agent. screens include a perforated plate , cylinder or the like , or a To aid in the reduction of the molecular weight of the wire mesh or cloth fabric . cellulose , an enzyme, e . g ., a cellulolytic enzyme, and/ or a 5 The term " pyrolysis , " as used herein , means to break swelling agent, can be utilized with any method described bonds in a material by the application of heat energy . herein . Pyrolysis can occur while the subject material is under When a microorganism is utilized , it can be a natural vacuum , or immersed in a gaseous material, such as an microorganism or an engineered microorganism ( e . g ., a oxidizing gas , e . g ., air or oxygen , or a reducing gas, such as genetically modified microorganism (GMM ) ) . For example , 10 hydrogen . the microorganism can be a bacterium , e. g ., a cellulolytic Oxygen content is measured by elemental analysis by bacterium , a fungus , e . g . , a yeast, a plant or a protist, e . g ., an pyrolyzing a sample in a furnace operating at 1300° C . or algae , a protozoa or a fungus - like protist , e . g ., a slime mold , above . protists ( e . g ., animal ( e . g . , protozoa such as flagellates , For the purposes of this disclosure , carbohydrates are amoeboids, ciliates , and sporozoa ) and plant ( e . g ., algae 15 materials that are composed entirely of one or more saccha such alveolates , chlorarachniophytes, cryptomonads, ride units or that include one or more saccharide units. The euglenids , glaucophytes, haptophytes, red algae , strameno saccharide units can be functionalized about the ring with piles , and viridaeplantae ) ), seaweed , plankton ( e . g ., mac one or more functional groups , such as carboxylic acid roplankton , mesoplankton , microplankton , nanoplankton , groups, amino groups, nitro groups, nitroso groups or nitrile picoplankton , and femptoplankton ), phytoplankton , and /or 20 groups and still be considered carbohydrates . Carbohydrates mixtures of these . In some embodiments , the microorganism can be polymeric ( e . g ., equal to or greater than 10 -mer , is white rot fungus . In some instances, the microorganism 100 -mer , 1 , 000 -mer , 10 , 000 -mer , or 100 ,000 -mer ) , oligo can include unicellular and /or multicellular organisms, e .g ., meric (e . g ., equal to or greater than a 4 -mer , 5 -mer , 6 -mer , the ocean , lakes, and bodies of water including salt water 7 -mer , 8 -mer , 9 -mer or 10 -mer ), trimeric , dimeric , or mono and fresh water . When the organisms are compatible , mix - 25 meric . When the carbohydrates are formed of more than a tures can be utilized single repeat unit, each repeat unit can be the same or Generally , various microorganisms can produce a number different. of useful products by operating on , converting , bioconvert Examples of polymeric carbohydrates include cellulose , ing , or fermenting the materials . For example , alcohols , xylan , pectin , and starch , while cellobiose and lactose are organic acids , hydrocarbons, hydrogen , proteins, carbohy - 30 examples of dimeric carbohydrates . Examples of mono drates, fats/ oils /lipids , amino acids, vitamins , or mixtures of meric carbohydrates include glucose and xylose . any of these materials can be produced by fermentation or Carbohydrates can be part of a supramolecular structure , other processes . e . g . , covalently bonded into the structure . Examples of such Examples of products that can be produced include mono - materials include lignocellulosic materials , such as that and polyfunctional C1 - C6 alkyl alcohols , mono - and poly - 35 found in wood . functional carboxylic acids, C1 - C6 hydrocarbons , and com - A starchy material is one that is or includes significant binations thereof. Specific examples of suitable alcohols amounts of starch or a starch derivative , such as greater than include methanol , ethanol, propanol, isopropanol, butanol, about 5 percent by weight starch or starch derivative . For ethylene glycol, propylene glycol, 1 , 4 -butane diol, glycerin , purposes of this disclosure, a starch is a material that is or and combinations thereof. Specific example of suitable 40 includes an amylose , an amylopectin , or a physical and / or carboxylic acids include formic acid , acetic acid , propionic chemical mixture thereof, e . g ., a 20 : 80 or 30 : 70 percent by acid , butyric acid , valeric acid , caproic acid , palmitic acid , weight mixture of amylose to amylopectin . For example , stearic acid , oxalic acid ,malonic acid , succinic acid , glutaric rice , corn , and mixtures thereof are starchy materials . Starch acid , oleic acid , linoleic acid , glycolic acid , lactic acid , derivatives include , e . g . , maltodextrin , acid -modified starch , y - hydroxybutyric acid , and combinations thereof. Examples 45 base -modified starch , bleached starch , oxidized starch , of suitable hydrocarbons include methane , ethane , propane , acetylated starch , acetylated and oxidized starch , phosphate pentane , n -hexane , and combinations thereof. modified starch , genetically -modified starch and starch that Another aspect of the invention features a method that is resistant to digestion . includes converting a low molecular weight sugar , or a For purposes of this disclosure , a low molecular weight material that includes a low molecular weight sugar , in a 50 sugar is a carbohydrate or a derivative thereof that has a mixture with a biomass , a microorganism , and a solvent or formula weight ( excluding moisture ) that is less than about a solvent system , e . g . , water or a mixture of water and an 2 ,000 , e . g ., less than about 1 , 800 , 1 ,600 , less than about organic solvent, to any product described herein . Without 1 ,000 , less than about 500 , less than about 350 or less than wishing to be bound by any particular theory , it is believed about 250 . For example , the low molecular weight sugar can that having a solid present, such as a high surface area and / or 55 be a monosaccharide , e . g . , glucose or xylose , a disaccharide , high porosity solid , can increase reaction rates by increasing e . g ., cellobiose or sucrose , or a trisaccharide . the effective concentration of solutes and providing a sub - Swelling agents as used herein are materials that cause a strate on which reactions can occur. Additional details about discernable swelling, e . g ., a 2 . 5 percent increase in volume such a conversion are described in U . S . patent application over an unswollen state of biomass materials , when applied Ser. No. 12 /417 , 840 , filed Apr. 3 , 2009 , the entire contents 60 to such materials as a solution , e .g ., a water solution . of which is hereby incorporated by reference herein in its Examples include alkaline substances , such as sodium entirety . hydroxide , potassium hydroxide , lithium hydroxide and The term “ fibrous material, ” as used herein , is a material ammonium hydroxides, acidifying agents , such as mineral that includes numerous loose , discrete and separable fibers . acids ( e . g . , sulfuric acid , hydrochloric acid and phosphoric For example , a fibrous material can be prepared from a 65 acid ) , salts, such as zinc chloride , calcium carbonate , sodium bleached Kraft paper fiber source by shearing , e . g . , with a carbonate, benzyltrimethylammonium sulfate , and basic rotary knife cutter. organic amines , such as ethylene diamine . US 9 ,745 ,604 B2 In some embodiments of the methods described herein , no App . Pub . No. 2010 /0093241 A1 (by Medoff ) , U . S . Pat . No . chemicals , e . g . , no swelling agents , are added to the bio 8 ,212 ,087 B2 ( to Medoff) , U . S . Pat. App . Pub . No. 2009/ mass , e . g ., none prior to irradiation . For example , alkaline 0312537 A1 (by Medoff ), U . S . Pat. No. 8 ,025 ,098 B2 ( to substances ( such as sodium hydroxide , potassium hydrox Medoff ) , and U . S . Pat. No . 7 , 867 ,359 B2 ( to Medoff ) . ide , lithium hydroxide and ammonium hydroxides ), acidi- 5 Any carbohydrate material described herein can be uti fying agents ( such as mineral acids ( e . g . , sulfuric acid , lized in any application or process described in any patent or hydrochloric acid and phosphoric acid ) ) , salts , such as zinc chloride , calcium carbonate , sodium carbonate , benzyltrim patent application incorporated by reference herein . ethylammonium sulfate, or basic organic amines , such as In any of the methods disclosed herein , radiation may be ethylene diamine , is added prior to irradiation or other 10 applied" from a device that is in a vault . processing . In some cases, no additional water is added . For Other features and advantages of the invention will be example , the biomass prior to processing can have less than apparent from the following detailed description , and from 0 . 5 percent by weight added chemicals , e . g . , less than 0 . 4 , the claims. 0 .25 , 0 . 15 , or 0 . 1 percent by weight added chemicals . In some instances , the biomass has no more than a trace , e . g ., 15 DESCRIPTION OF DRAWINGS less than 0 . 05 percent by weight added chemicals , prior to irradiation . In other instances, the biomass prior to irradia FIG . 1 is a block diagram illustrating conversion of tion has substantially no added chemicals or swelling agents . biomass into products and co - products . Avoiding the use of such chemicals can also be extendedled FIG . 2 is block diagram illustrating conversion of a fiber throughout, e . g . , at all times prior to fermentation , or at all 20 source into a first and second fibrous material. times. FIG . 3 is a cross - sectional view of a rotary knife cutter. The term " edible , " as used herein , means fit to be eaten as FIG . 4 is block diagram illustrating conversion of a fiber food . source into a first , second and third fibrous material. A " sheared material, ” as used herein , is a material that FIG . 5 is block diagram illustrating densification of a includes discrete fibers in which at least about 50 % of the 25 material . discrete fibers , have a length /diameter (L / D ) ratio of at least FIG . 6 is a perspective view of a pellet mill . about 5 , and that has an uncompressed bulk density of less FIG . 7A is a densified fibrous material in pellet form . than about 0 . 6 g /cm3 . FIG . 7B is a transverse cross - section of a hollow pellet in In some embodiments, changing a molecular structure of which a center of the hollow is in - line with a center of the biomass , as used herein , means to change the chemical 30 pellet . bonding arrangement, such as the type and quantity of FIG . 7C is a transverse cross -section of a hollow pellet in functional groups or conformation of the structure . For which a center of the hollow is out of line with the center of example , the change in the molecular structure can include the pellet. changing the recalcitrance level of the material, changing the FIG . 7D is a transverse cross - section of a tri -lobal pellet . supramolecular structure of the material, oxidation of the 35 FIG . 8 is a block diagram illustrating a treatment sequence material , changing an average molecular weight, changing for processing feedstock . an average crystallinity , changing a surface area , changing a FIG . 9 is a perspective , cut -away view of a gamma degree of polymerization , changing a porosity , changing a irradiator housed in a concrete vault . degree of branching, grafting on other materials, changing a FIG . 10 is an enlarged perspective view of region R of crystalline domain size , or an changing an overall domain 40 FIG . 9 . size . FIG . 11 is a block diagram illustrating an electron beam Unless otherwise defined , all technical and scientific irradiation feedstock pretreatment sequence . terms used herein have the same meaning as commonly FIG . 11A is a schematic representation of biomass being understood by one of ordinary skill in the art to which this ionized , and then oxidized or quenched . invention belongs . Although methods and materials similar 45 FIG . 11B is a schematic side view of a system for or equivalent to those described herein can be used in the irradiating a low bulk density material, while FIG . 11C is practice or testing of the present invention , suitable methods cross -sectional of the system taken along 11C - 11C . and materials are described below . All publications, patent FIG . 11D is a schematic cross -sectional view of a fluid applications, patents , and other references mentioned herein ized bed system for irradiating a low bulk density material. are incorporated by reference in their entirety . In case of 50 FIG . 11E is a schematic side - view of another system for conflict , the present specification , including definitions, will irradiating a low bulk density material . control. In addition , the materials , methods, and examples FIG . 12 is a schematic view of a system for sonicating a are illustrative only and not intended to be limiting . process stream of cellulosic material in a liquid medium . As used herein , the term “ subject” is used throughout the FIG . 13 is a schematic view of a sonicator having two specification to describe an animal, human , or non -human . 55 transducers coupled to a single horn . The term includes , but is not limited to , birds, reptiles , fish , FIG . 14 is a block diagram illustrating a pyrolytic feed plants, amphibians , and mammals, e . g ., humans , other pri - stock pretreatment system . mates , pigs, rodents such as mice and rats , rabbits , guinea FIG . 15 is a cross -sectional side view of a pyrolysis pigs, hamsters , cows, horses , cats , dogs , sheep and goats . chamber. The full disclosure ofWO2008 /073186 is incorporated by 60 FIG . 16 is a cross- sectional side view of a pyrolysis reference herein in its entirety . The full disclosures of each chamber . of the following U . S . patents and published Patent Appli - FIG . 17 is a cross -sectional side view of a pyrolyzer that cations, which are being filed concurrently herewith , are includes a heated filament. hereby incorporated by reference herein : U .S . Pat. No . FIG . 18 is a schematic cross -sectional side view of a 7 , 867 , 358 B2 ( to Medoff ) , U . S . Pat. No . 7 , 846 ,295 B1 ( to 65 Curie - Point pyrolyzer. Medoff ) , U . S . Pat. No . 7 , 931 ,784 B2 ( to Medoff ), U . S . Pat. FIG . 19 is a schematic cross -sectional side view of a No. 8 , 236 ,535 B2 ( to Medoff and Masterman ) , U . S . Pat . furnace pyrolyzer. US 9 ,745 ,604 B2 10 FIG . 20 is a schematic cross- sectional top view of a laser FIG . 44 is a schematic diagram showing a variable pyrolysis apparatus . volume fed -batch fermentation process. FIG . 21 is a schematic cross- sectional top view of a FIG . 45 is a schematic diagram showing a fixed volume tungsten filament flash pyrolyzer. fed -batch fermentation process . FIG . 22 is a block diagram illustrating an oxidative 5 FIG . 46 is a schematic diagram showing the processing feedstock pretreatment system . steps required for the production of products 1, 2 , and 3 . Star FIG . 23 is block diagram illustrating a general overview indicates a step is optional. Black arrow indicates that an of the process of converting a fiber source into a product , optional densification step can be performed . FIG . 47 is a diagram illustrating the biosynthesis of e . g . , ethanol. explosion 10 aromatic amino acids such as tryptophan , phenylalanine, and FIG . 24 is a cross- sectional view of a steam explosion tyrosine through the shikimic acid pathway . apparatus . FIGS . 48A through 48F are diagrams illustrating antibi FIG . 25 is a schematic cross - sectional side view of a otics and other natural products that can be generated by hybrid electron beam / sonication device . microorganisms. FIG . 26 is a scanning electron micrograph of a fibrous 15 FIG . 49 is a diagram illustrating the biosynthesis of material produced from polycoated paper at 25x magnifi streptomycin , starting from D - glucose . cation . The fibrous material was produced on a rotary knife FIG . 50 is a diagram illustrating the biosynthesis of cutter utilizing a screen with 1/ 8 inch openings . vancomycin , starting from a glucose derivative . FIG . 27 is a scanning electron micrograph of a fibrous FIG . 51 is a diagram illustrating the biosynthesis of material produced from bleached Kraft board paper at 25x 20 ansamycin , starting from a glucose derivative . magnification . The fibrous material was produced on a rotary knife cutter utilizing a screen with 1 /8 inch openings . DETAILED DESCRIPTION FIG . 28 is a scanning electron micrograph of a fibrous material produced from bleached Kraft board paper at 25x Biomass ( e . g . , plant biomass, animal biomass, microbial magnification . The fibrous material was twice sheared on a 25 biomass , and municipal waste biomass ) can be processed rotary knife cutter utilizing a screen with 1 / 16 inch openings using the methods disclosed herein to produce useful prod during each shearing . ucts such as food products . In addition , functionalized FIG . 29 is a scanning electron micrograph of a fibrous materials having desired types and amounts of functionality , material produced from bleached Kraft board paper at 25x such as carboxylic acid groups, aldehyde groups, ketone magnification . The fibrous material was thrice sheared on a 30 groups, nitrile groups, nitro groups, or nitroso groups, can be rotary knife cutter. During the first shearing , a 1/ 8 inch screen prepared using the methods described herein . Such func was used ; during the second shearing , a 1 / 16 inch screen was tionalized materials can be , e . g . , more soluble , easier to used , and during the third shearing a 1 /32 inch screen was utilize by various microorganisms or can be more stable over used . the long term , e .g ., less prone to oxidation . Systems and FIG . 30 is a schematic side view of a sonication apparatus , 35 processes are described below herein that can use various while FIG . 31 is a cross -sectional view through the process - biomass materials , e . g . , cellulosic materials , lignocellulosic ing cell of FIG . 30 . materials , starchy materials, or materials that are or that FIG . 32 is a scanning electron micrograph at 1000x include low molecular weight sugars , as feedstock materials . magnification of a fibrous material produced from shearing Biomass materials are often readily available, can be diffi switchgrass on a rotary knife cutter , and then passing the 40 cult to process , e . g ., by fermentation , or can give sub sheared material through a 1/ 32 inch screen . optimal yields at a slow rate , for example , by fermentation . FIGS . 33 and 34 are scanning electron micrographs of the Biomass materials can be first pretreated , often by size fibrous material of FIG . 32 after irradiation with 10 Mrad reduction of raw feedstock materials . Pretreated biomass can and 100 Mrad gamma rays , respectively , at 1000x magni- then be treated using at least one of: radiation (under fication . 45 controlled thermal conditions) , sonication , oxidation , FIG . 35 is a scanning electron micrographs of the fibrous pyrolysis , and steam explosion . The various pretreatment material of FIG . 32 after irradiation with 10 Mrad and systems and methods can be used in combinations of two , sonication at 1000x magnification . three , or even four of these technologies. FIG . 36 is a scanning electron micrographs of the fibrous Alternatively or in addition , the present invention is material of FIG . 32 after irradiation with 100 Mrad and 50 based , at least in part , on the observation that the methods sonication at 1000x magnification . described herein can be used to convert biomass into non FIG . 37 is an infrared spectrum of Kraft board paper energy materials and compositions. Such materials and sheared on a rotary knife cutter . compositions include, but are not limited to , foodstuffs ( e . g . , FIG . 38 is an infrared spectrum of the Kraft paper of FIG . suitable for consumption by humans and /or animals ) , phar 37 after irradiation with 100 Mrad of gamma radiation . 55 maceuticals , nutraceuticals , pharmaceutical delivery FIG . 39 is a schematic view of a process for biomass vehicles and dosage forms, pharmaceutical excipients , phar conversion . maceutical conjugates , cross - linked matrixes such as hydro FIG . 40 is schematic view of another process for biomass gels , absorbent materials , fertilizers , and lignin products. conversion . Types of Biomass FIG . 41 is a schematic diagram of a truck -based mobile 60 Generally , any biomass material that is or includes car biomass processing facility . bohydrates composed entirely of one or more saccharide FIG . 42 is a schematic diagram of a train - based mobile units or that include one or more saccharide units can be biomass processing facility . processed by any of the methods described herein . As used FIGS . 43A and 43B are schematic diagrams showing the herein , biomass includes , cellulosic , lignocellulosic , hemi processing steps for generating products and co -products 65 cellulosic , starch , and lignin -containing materials . For from biomass ( A ) and for generating products using a example , the biomass material can be cellulosic or ligno bioconversion step . cellulosic materials , or starchy materials , such as kernels of US 9 , 745 ,604 B2 11 12 corn , grains of rice or other foods, or materials that are or pulp industry waste streams, wood fiber, and the like . that include one or more low molecular weight sugars, such Additionally grass crops , such as switchgrass and the like as sucrose or cellobiose . have potential to be produced on a large -scale as another For example , such materials can include paper , paper plant biomass source . For urban areas, the plant biomass products , wood , wood -related materials, particle board , 5 feedstock includes yard waste ( e . g . , grass clippings, leaves, grasses , rice hulls , bagasse , cotton , jute, hemp, flax , bamboo , tree clippings , and brush ) and vegetable processing waste . sisal, abaca , straw , corn cobs , rice hulls , coconut hair , algae , In some embodiments , biomass can include lignocellu seaweed ( e . g . , giant seaweed ), water hyacinth , cassava , coffee beans, coffee bean grounds (used coffee bean losic feedstock can be plant biomass such as , but not limited grounds ) , cotton , synthetic celluloses , or mixtures of any of 10 to , non -woody plant biomass , cultivated crops , such as, but these . not limited to , grasses, for example , but not limited to , C4 Fiber sources include cellulosic fiber sources , including grasses, such as switchgrass , cord grass , rye grass , miscan paper and paper products ( e . g . , polycoated paper and Kraft thus, reed canary grass , or a combination thereof, or sugar paper) , and lignocellulosic fiber sources, including wood , processing residues such as bagasse , or beet pulp , agricul and wood - related materials, e . g ., particle board . Other suit - 15 tural residues, for example , soybean stover , corn stover, rice straw , rice hulls , barley straw , corn cobs , wheat straw , canola able fiber sources include natural fiber sources , e. g. , grasses , straw , rice straw , oat straw , oat hulls , corn fiber , recycled rice hulls , bagasse, cotton , jute , hemp, flax , bamboo , sisal , wood pulp fiber , sawdust, hardwood , for example aspen abaca , straw , corn cobs, rice hulls , coconut hair ; fiber wood and sawdust, softwood , or a combination thereof. sources high in a -cellulose content, e . g ., cotton ; and syn Further , the lignocellulosic feedstock can include cellulosic thetic fiber sources , e . g ., extruded yarn ( oriented yarn or 20 waste material such as , but not limited to , newsprint, card un -oriented yarn ) . Natural or synthetic fiber sources can be board , sawdust, and the like . Lignocellulosic feedstock can obtained from virgin scrap textile materials , e . g . , remnants include one species of fiber or alternatively , lignocellulosic or they can be post consumer waste , e . g . , rags. When paper feedstock can include a mixture of fibers that originate from products are used as fiber sources, they can be virgin different lignocellulosic feedstocks . Furthermore , the ligno materials , e .g . , scrap virgin materials , or they can be post - 25 cellulosic feedstock can comprise fresh lignocellulosic feed consumer waste . Aside from virgin raw materials , post stock , partially dried lignocellulosic feedstock , fully dried consumer, industrial (e . g ., offal) , and processing waste ( e . g . , lignocellulosic feedstock , or a combination thereof. effluent from paper processing) can also be used as fiber Microbial biomass includes biomass derived from natu sourcesfrom human. Also ,(eg the .. fibersewage source ) , animal can ,be or obtained plant waste or derived. Addi- 30 rally occurring or genetically modified unicellular organ tional fiber sources have been described in the art , for isms and /or multicellular organisms, e . g ., organisms from example , see U .S . Pat. Nos . 6 ,448 , 307 , 6 ,258 ,876 , 6 , 207 , the ocean , lakes , bodies of water , e . g . , salt water or fresh 729 , 5 ,973 , 035 and 5 , 952 ,105 . water , or on land , and that contains a source of carbohydrate Microbial sources include , but are not limited to , any ( e . g . , cellulose ) . Microbial biomass can include , but is not naturally occurring or genetically modified microorganism 35 limited to , for example protists ( e . g . , animal ( e . g . , protozoa or organism that contains or are capable of providing a such as flagellates, amoeboids, ciliates, and sporozoa ) and source of carbohydrates (e . g. , cellulose ), for example , pro plant ( e . g ., algae such alveolates, chlorarachniophytes , cryp tists ( e . g ., animal ( e . g . , protozoa such as flagellates, amoe tomonads , euglenids, glaucophytes , haptophytes , red algae , boids, ciliates , and sporozoa ) and plant ( e . g ., algae such stramenopiles , and viridaeplantae )) , seaweed , plankton ( e. g ., alveolates, chlorarachniophytes , cryptomonadsiptomonads, euglenidseuglenids, 40 macroplankton , mesoplankton , microplankton , nanoplank glaucophytes, haptophytes, red algae , stramenopiles, and ton , picoplankton , and femptoplankton ), phytoplankton , viridaeplantae ) ) , seaweed , plankton ( e . g . , macroplankton , bacteria ( e . g . , gram positive bacteria , gram negative bacte mesoplankton , microplankton , nanoplankton , picoplankton , ria , and extremophiles ), yeast and / or mixtures of these . In and femptoplankton ), phytoplankton , bacteria ( e . g . , gram some instances, microbial biomass can be obtained from positive bacteria , gram negative bacteria , and extremo- 45s natural sources , e . g ., the ocean , lakes, bodies of water , e . g ., philes) , yeast and / or mixtures of these . In some instances , salt water or fresh water, or on land . Alternatively or in microbial biomass can be obtained from natural sources , addition , microbial biomass can be obtained from culture e . g ., the ocean , lakes , bodies of water, e . g ., salt water or systems, e . g . , large scale dry and wet culture systems. fresh water, or on land . Alternatively or in addition , micro Animal biomass includes any organic waste material such bial biomass can be obtained from culture systems, e . g ., 50so as animal- derived waste material or excrement or human large scale dry and wet culture systems. waste material or excrement ( e . g . , manure and sewage ) . Examples of biomass include renewable , organic matter, In some embodiments , the carbohydrate is or includes a such as plant biomass , microbial biomass , animal biomass material having one or more B - 1 , 4 - linkages and having a ( e . g . , any animalby - product, animal waste , etc . ) and munici number average molecular weightbetween about 3 ,000 and pal waste biomass including any and all combinations of 55 50 ,000 . Such a carbohydrate is or includes cellulose (I ) , these biomass materials . which is derived from ( B - glucose 1 ) through condensation of Plant biomass and lignocellulosic biomass includelude B 1( 4 ) - glycosidic bonds. This linkage contrasts itself with organic matter (woody or non -woody ) derived from plants , that for a (1 4 ) - glycosidic bonds present in starch and other especially matter available on a sustainable basis . Examples carbohydrates. include biomass from agricultural or food crops ( e . g ., sug - 60 arcane , sugar beets or corn kernels ) or an extract therefrom ( e . g . , sugar from sugarcane and corn starch from corn ) , HO agricultural crop wastes and residues such as corn stover, wheat straw , rice straw , sugar cane bagasse , and the like . LOH Plant biomass further includes, but is not limited to , trees , 65 HO HO- woody energy crops, wood wastes and residues such as OH softwood forest thinnings, barky wastes , sawdust, paper and US 9 ,745 ,604 B2 13 14 -continued - continued OH JOHJOH HOC HO ulo ?? OH - 5 ?? " HO HOY OH HOW OH - OH ÕH 2 -keto - gluconic acid glucose Starchy materials include starch itself , e . g . , corn starch , HO wheat starch , potato starch or rice starch , a derivative of starch , or a material that includes starch , such as an edible OH food product or a crop . For example , the starchy material can be arracacha , buckwheat , banana , barley, cassava , kudzu , oca , sago , sorghum , regular household potatoes, sweet HO potato , taro , yams, or one or more beans, such as favas, HOC lentils or peas. Blends of any one or more starchy material is also a starchy material. In particular embodiments , the starchy material is derived from corn . Various corn starches 2 **MOH and derivatives are known in the art, see, e . g ., “ Corn Starch ,” Corn Refiners Association ( 11th Edition , 2006 ) . OH Biomass materials that include low molecular weight 6 -MAS sugars can , e . g . , include at least about 0 . 5 percent by weight 6 -monoacid of sucrose of the low molecular sugar, e . g ., at least about 2 , 3 , 4 , 5 , 6 , . OH 7 , 8 , 9 , 10 , 12 . 5 , 25 , 35 , 50 , 60 , 70 , 80 , 90 or even at least HOC 0 WOH about 95 percent by weight of the low molecular weight ** 001 sugar . In some instances, the biomass is composed substan HOIA . tially of the low molecular weight sugar , e . g . , greater than 95 HOY POHOH percent by weight, such as 96 , 97 , 98 , 99 or substantially 100 20 percent by weight of the low molecular weight sugar. 30 OH HO OH Biomass materials that include low molecular weight glucuronic acid fructose sugars can be agricultural products or food products , such as HO . sugarcane and sugar beets or an extract therefrom , e . g ., juice from sugarcane , or juice from sugar beets . Biomass mate - 2 IIIIIII rials that include low molecular weight sugars can be ?? substantially pure extracts , such as raw or crystallized table sugar ( sucrose ). Low molecular weight sugars include sugar HO derivatives . For example , the low molecular weight sugars can be oligomeric ( e . g . , equal to or greater than a 4 -mer , 10 HOHO 5 -mer , 6 -mer , 7 -mer , 8 -mer , 9 -mer or 10 -mer ), trimeric , dimeric , or monomeric . When the carbohydrates are formed "" NIH of more than a single repeat unit , each repeat unit can be the Y OH same or different. OH Specific examples of low molecular weight sugars include sucrose cellobiose , lactose , sucrose , glucose and xylose, along with 45 derivatives thereof. In some instances, sugar derivatives are more rapidly dissolved in solution or utilized by microbes to Combinations ( e . g . , by themselves or in combination of provide a useful material. Several such sugars and sugar any biomass material, component , product, and / or co - prod derivatives are shown below . uct generated using the methods described herein ) of any 50 biomass materials described herein can be utilized for mak ing any of the products described herein . For example , HO blends of cellulosic materials and starchy materials can be utilized for making any product described herein . Systems for Treating Biomass OH 55 FIG . 1 shows a system 100 for converting biomass , particularly biomass with significant cellulosic and ligno cellulosic components and / or starchy components , into use HOC ful products and co - products . System 100 includes a feed O OIOH preparation subsystem 110 , a pretreatment subsystem 114 , a HO V 60 primary process subsystem 118 , and a post- processing sub system 122 . Feed preparation subsystem 110 receives bio HOY inNOH mass in its raw form , physically prepares the biomass for use as feedstock by downstream processes ( e . g ., reduces the size OH of and homogenizes the biomass ) , and stores the biomass 1 ' -MAS 65 both in its raw and feedstock forms. ( 1 '- monoacid of sucrose ) Biomass feedstock with significant cellulosic and /or lignocellulosic components , or starchy components can have US 9 ,745 ,604 B2 15 16 a high average molecular weight and crystallinity that can Size Reduction make processing the feedstock into useful products ( e . g ., In some embodiments , the biomass is in the form of a fermenting the feedstock to produce ethanol) difficult . fibrousmaterial that includes fibers provided by shearing the Accordingly it is useful to treat biomass feedstock , e .g ., biomass . For example , the shearing can be performed with using the treatment methods described herein . As described 5 a rotaryFor example knife cutter , and . by reference to FIG . 2 , a biomass fiber herein , in some embodiments , the treatment ofbiomass does source 210 is sheared , e . g . , in a rotary knife cutter , to provide not use acids, bases and / or enzymes to process biomass, or a first fibrous material 212 . The first fibrous material 212 is only uses such treatments in small or catalytic amounts . passed through a first screen 214 having an average opening Treatment subsystem 114 receives biomass feedstock size of 1 .59 mm or less ( 1/ 16 inch , 0 . 0625 inch ) to provide a from the feed preparation subsystem 110 and prepares the second fibrous material 216 . If desired , fiber source can be feedstock for use in primary production processes by, for cut prior to the shearing , e. g ., with a shredder. For example , example , reducing the average molecular weight and crys - when a paper is used as the fiber source , the paper can be first tallinity of the feedstock . Primary process subsystem 118 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 receives treated feedstock from treatment subsystem 114 and 15 those manufactured by Munson (Utica , N . Y . ) . As an alter produces useful products ( e . g . , ethanol, other alcohols , phar native to shredding , the paper can be reduced in size by maceuticals , and /or food products ) . In some cases , the cutting to a desired size using a guillotine cutter . For output of primary process subsystem 118 is directly useful example , the guillotine cutter can be used to cut the paper but, in other cases, requires further processing provided by into sheets that are , e . g . , 10 inches wide by 12 inches long . post -processing subsystem 122 . Post -processing subsystem 20 In some embodiments , the shearing of fiber source and the 122 provides further processing to product streams from passing of the resulting first fibrous material through first primary process system 118 which require it ( e . g ., distilla - screen are performed concurrently . The shearing and the tion and denaturation of ethanol) as well as treatment for passing can also be performed in a batch - type process . waste streams from the other subsystems. In some cases, the For example , a rotary knife cutter can be used to concur co - products of subsystems 114 , 118 , 122 can also be directly 25 rently shear the fiber source and screen the first fibrous or indirectly useful as secondary products and /or in increas - material. Referring to FIG . 3 , a rotary knife cutter 220 ing the overall efficiency of system 100 . For example , includes a hopper 222 that can be loaded with a shredded post - processing subsystem 122 can produce treated water to fiber source 224 prepared by standard methods. Shredded fiber source is sheared between stationary blades 230 and be recycled for use as process water in other subsystemsas fue 30 rotating blades 232 to provide a first fibrous material 240 . and/ or can produce burnable waste which can be used as fuel 30 First fibrous material 240 passes through screen 242 , and the for boilers producing steam and /or electricity . resulting second fibrous material 244 is captured in bin 250 . The optimum size for biomass conversion plants is To aid in the collection of the second fibrous material, the affected by factors including economies of scale and the type bin can have a pressure below nominal atmospheric pres and availability of biomass used as feedstock . Increasing1 35 sure, e . g. , at least 10 percent below nominal atmospheric plant size tends to increase economies of scale associated pressure , e .g ., at least 25 percent below nominal atmospheric with plant processes. However, increasing plant size also pressure , at least 50 percent below nominal atmospheric tends to increase the costs (e . g. , transportation costs ) per unit pressure , or at least 75 percent below nominal atmospheric of feedstock . Studies analyzing these factors suggest that the pressure . In some embodiments, a vacuum source 252 is appropriate size for biomass conversion plants can range 40 utilized to maintain the bin below nominal atmospheric from 100 to 1 ,000 or more, e . g . , 10 ,000 or more dried tons pressure . of feedstock per day depending at least in part on the type Shearing can be advantageous for “ opening up” and of feedstock used . The type of biomass feedstock can also “ stressing " the fibrous materials, making the cellulose of the impact plant storage requirements with plants designed materials more susceptible to chain scission and /or reduction primarily for processing feedstock whose availability varies 45 of crystallinity . The open materials can also be more sus seasonally (e . g ., corn stover ) requiring more on - or off -site ceptible to oxidation when irradiated . feedstock storage than plants designed to process feedstock In some embodiments , shearing can be advantageous for whose availability is relatively steady ( e . g ., waste paper ). " opening up ” and “ stressing " the fibrous materials , making the cellulose of the materials more susceptible to ruminant Biomass Pretreatment begin 50 digestion and absorption . In some cases, pretreatment methods of processing begin 30 di The fiber source can be sheared in a dry state , a hydrated with a physical preparation of the biomass , e . g . , size reduc state ( e .g ., having up to ten percent by weight absorbed tion of raw biomass feedstock materials , such as by cutting , water ) , or in a wet state , e .g ., having between about 10 grinding , crushing , smashing , shearing or chopping . In some percent and about 75 percent by weight water. The fiber embodiments , methods (e . g. , mechanical methods ) are used source can even be sheared while partially or fully sub to reduce the size and /or dimensions of individual pieces of merged under a liquid , such as water, ethanol, or isopropa biomass . In some cases, loose feedstock ( e . g . , recycled paperpaper nol. or switchgrass ) is pretreated by shearing or shredding . The fiber source can also be sheared in under a gas (such Screens and /or magnets can be used to remove oversized or as a stream or atmosphere of gas other than air ), e . g ., oxygen undesirable objects such as , for example , rocks or nails from 60 or nitrogen , or in steam . the feed stream . Other methods of making the fibrous materials include , Feed pretreatment systems can be configured to produce e . g . , stone grinding , mechanical ripping or tearing , pin feed streams with specific characteristics such as , for grinding , and /or air attrition milling . example , specific maximum sizes , specific length -to -width , If desired , the fibrous materials can be separated , e . g . , or specific surface areas ratios. As a part of feed pretreat - 65 continuously or in batches , into fractions according to their ment, the bulk density of feedstocks can be controlled ( e . g . , length , width , density , material type, or some combination of increased ) . these attributes . US 9 ,745 ,604 B2 18 For example , ferrousmaterials can be separated from any The average length - to -diameter ratio of the second fibrous of the fibrous materials by passing a fibrous material that material 14 can be greater than 5 / 1 , e . g . , greater than 8 / 1 , includes a ferrous material past a magnet, e . g ., an electro - e . g ., greater than 10 / 1 , greater than 15 / 1 , greater than 20 / 1 , magnet, and then passing the resulting fibrous material greater than 25 / 1 , or greater than 50 / 1 . An average length of through a series of screens, each screen having different 5 the second fibrous material 14 can be , e . g . , between about sized apertures . 0 . 5 mm and 2 . 5 mm , e . g . , between about 0 .75 mm and 1 . 0 The fibrous materials can also be separated , e .g ., by using mm , and an average width (e . g ., diameter ) of the second a high velocity gas, e .g ., air. In such an approach , the fibrous fibrous material 14 can be, e . g . , between about 5 um and 50 um , e . g . , between about 10 um and 30 um . materials are separated by drawing off different fractions, 10 In some embodiments , a standard deviation of the length which can be characterized photonically , if desired . Such a of the second fibrous material 14 is less than 60 percent of separation apparatus is discussed in Lindsey et al, U . S . Pat . an average length of the second fibrous material 14 , e . g . , less No . 6 , 883 , 667 . than 50 percent of the average length , less than 40 percent The fibrous materials can be pre - treated immediately of the average length , less than 25 percent of the average following their preparation , or they can be dried , e . g ., at 15 length less than 10 percent of the average length less than approximately 105° C . for 4 - 18 hours , so that the moisture 5 percent of the average length , or even less than 1 percent content is , e . g ., less than about 0 . 5 % before use . of the average length . If desired , lignin can be removed from any of the fibrous In some embodiments , a BET surface area of the second materials that include lignin . Also , to aid in the breakdown fibrous material is greater than 0 . 1 m²/ g , e . g . , greater than of the materials that include the cellulose , the material can 20 0 . 25 m²/ g , greater than 0 .5 m?lg , greater than 1 . 0 m?lg , be treated prior to irradiation with heat, a chemical (e . g . , greater than 1 .5 m²/ g , greater than 1 . 75 m²/ g , greater than mineral acid , base or a strong oxidizer such as sodium 5 .0 mº/ g , greater than 10 mº/ g , greater than 25 m²/ g , greater hypochlorite ) and / or an enzyme. than 35 m²/ g , greater than 50 m²/ g , greater than 60 m²/ g , In some embodiments , the average opening size of the greater than 75 m²/ g , greater than 100 m - / g , greater than 150 first screen is less than 0 . 79 mm ( 1 /32 inch , 0 . 03125 inch ) , 25 m?lg , greater than 200 m?lg , or even greater than 250 m?/ g . e . g ., less than 0 .51 mm (1 / so inch , 0 .02000 inch ) , less than A porosity of the second fibrous material 14 can be , e . g . , 0 .40 mm ( 1/ 64 inch , 0 . 015625 inch ), less than 0 . 23 mm (0 .009 greater than 20 percent, greater than 25 percent, greater than inch ), less than 0 .20 mm ( 1 /128 inch , 0 .0078125 inch ), less 35 percent, greater than 50 percent, greater than 60 percent , than 0 . 18 mm (0 .007 inch ) , less than 0 . 13 mm (0 .005 inch ) , greater than 70 percent, e . g ., greater than 80 percent, greater or even less than less than 0 . 10 mm (1 / 256 inch , 0 .00390625 30 than 85 percent, greater than 90 percent, greater than 92 inch ) . The screen is prepared by interweaving monofila - percent, greater than 94 percent, greater than 95 percent, ments having an appropriate diameter to give the desired greater than 97 . 5 percent, greater than 99 percent, or even opening size . For example , the monofilaments can be made greater than 99 .5 percent. of a metal, e . g ., stainless steel . As the opening sizes get In some embodiments , a ratio of the average length - to smaller, structural demands on the monofilaments can 35 diameter ratio of the first fibrous material to the average become greater. For example , for opening sizes less than length - to - diameter ratio of the second fibrous material is , 0 .40 mm , it can be advantageous to make the screens from e .g . , less than 1 . 5 , e . g . , less than 1 . 4 , less than 1 . 25 , less than monofilaments made from a material other than stainless 1 . 1 , less than 1 .075 , less than 1 .05 , less than 1 .025 , or even steel , e . g. , titanium , titanium alloys, amorphous metals , substantially equal to 1 . nickel, tungsten , rhodium , rhenium , ceramics, or glass . In 40 In particular embodiments , the second fibrous material is some embodiments , the screen is made from a plate , e . g . , a sheared again and the resulting fibrous material passed metal plate , having apertures, e . g . , cut into the plate using a through a second screen having an average opening size less laser. In some embodiments , the open area of the mesh is than the first screen to provide a third fibrous material. In less than 52 % , e . g . , less than 41 % , less than 36 % , less than such instances, a ratio of the average length - to - diameter 31 % , less than 30 % . 45 ratio of the second fibrous material to the average length In some embodiments , the second fibrous is sheared and to - diameter ratio of the third fibrous material can be , e. g . , passed through the first screen , or a different sized screen . In less than 1. 5 , e . g . , less than 1 . 4 , less than 1 .25 , or even less some embodiments , the second fibrous material is passed than 1 . 1 . through a second screen having an average opening size In some embodiments , the third fibrous material is passed equal to or less than that of first screen . 50 through a third screen to produce a fourth fibrous material. Referring to FIG . 4 , a third fibrous material 220 can be The fourth fibrous material can be , e . g ., passed through a prepared from the second fibrous material 216 by shearing fourth screen to produce a fifth material. Similar screening the second fibrous material 216 and passing the resulting processes can be repeated as many times as desired to material through a second screen 222 having an average produce the desired fibrous material having the desired opening size less than the first screen 214 . 55 properties . Generally , the fibers of the fibrous materials can have a Densification relatively large average length - to - diameter ratio ( e. g ., As used herein , densification refers to increasing the bulk greater than 20 - to - 1 ) , even if they have been sheared more density of a material. Densified materials can be processed , than once . In addition , the fibers of the fibrous materials or any processed materials can be densified , by any of the described herein can have a relatively narrow length and / or 60 methods described herein . length - to -diameter ratio distribution . A material, e . g ., a fibrous material, having a low bulk As used herein , average fiber widths ( e . g . , diameters ) are density can be densified to a product having a higher bulk those determined optically by randomly selecting approxi- density . For example , a material composition having a bulk mately 5 ,000 fibers . Average fiber lengths are corrected density of 0 . 05 g /cm can be densified by sealing the fibrous length -weighted lengths. BET (Brunauer , Emmet and Teller) 65 material in a relatively gas impermeable structure , e. g ., a bag surface areas are multi - point surface areas, and porosities are made of polyethylene or a bag made of alternating layers of those determined by mercury porosimetry . polyethylene and a nylon , and then evacuating the entrapped US 9 ,745 ,604 B2 19 20 gas, e . g . , air, from the structure . After evacuation of the air polyethylene glycol (PEG ) , polyethylene oxide , e . g . , from the structure , the fibrous material can have, e .g . , a bulk POLYOX® , copolymers of ethylene oxide and propylene density of greater than 0 . 3 g / cm " , e . g ., 0 . 5 g / cm°, 0 . 6 g / cm " , oxide , polyacrylic acid (PAA ) , polyacrylamide, polypep 0 . 7 g / cm or more , e . g ., 0 . 85 g /cmº . After densification , the tides , polyethylenimine , polyvinylpyridine , poly (sodium - 4 product can pre - treated by any of the methods described 5 styrenesulfonate ) and poly ( 2 - acrylamido -methyl - 1 -propane herein , e . g . , irradiated , e . g ., with gamma radiation . This can sulfonic acid ) . be advantageous when it is desirable to transport the mate In some embodiments , the binder includes a polymer that rial to another location , e . g ., a remote manufacturing plant, has a glass transition temperature less 25° C . Examples of where the fibrous material composition can be added to a such polymers include thermoplastic elastomers ( TPEs ) . solution , e . g . , to produce ethanol. After piercing the sub - 10 Examples of TPEs include polyether block amides , such as stantially gas impermeable structure , the densified fibrous those available under the tradename PEBAX® , polyester material can revert to nearly its initial bulk density , e .g . , to elastomers, such as those available under the tradename at least 60 percent of its initial bulk density , e . g . , 70 percent , HYTREL® , and styrenic block copolymers , such as those 80 percent, 85 percent ormore , e . g . , 95 percent of its initial available under the tradename KRATON® . Other suitable bulk density . To reduce static electricity in the fibrous 15 polymers having a glass transition temperature less 25° C . material, an anti -static agent can be added to the material. include ethylene vinyl acetate copolymer (EVA ) , polyole In some embodiments , the structure , e . g ., a carrier such as fins, e .g ., polyethylene , polypropylene , ethylene -propylene a bag , is formed of a material that dissolves in a liquid , such copolymers , and copolymers of ethylene and alpha olefins, as water. For example , the structure can be formed from a e. g ., 1 -octene , such as those available under the tradename polyvinyl alcohol so that it dissolves when in contact with a 20 ENGAGE® . In some embodiments , e . g ., when the material water - based solution . Such embodiments allow densified is a fiberized polycoated paper, the material is densified structures to be added directly to solutions that include a without the addition of a separate low glass transition microorganism , without first releasing the contents of the temperature polymer . structure , e . g . , by cutting. In a particular embodiment, the binder is a lignin , e . g ., a Referring to FIG . 5 , a biomass material can be combined 25 natural or synthetically modified lignin . with any desired additives and a binder , and subsequently A suitable amount of binder added to the material, cal densified by application of pressure, e . g . , by passing the culated on a dry weight basis , is , e . g ., from about 0 . 01 material through a nip defined between counter - rotating percent to about 50 percent , e . g ., 0 . 03 percent, 0 . 05 percent, pressure rolls or by passing the material through a pellet 0 . 1 percent, 0 .25 percent, 0 . 5 percent, 1 . 0 percent, 5 percent, mill . During the application of pressure, heat can optionally 30 10 percent or more , e . g . , 25 percent , based on a total weight be applied to aid in the densification of the fibrous material. of the densified material. The binder can be added to the The densified material can then be irradiated . material as a neat, pure liquid , as a liquid having the binder In some embodiments , the material prior to densification dissolved therein , as a dry powder of the binder , or as pellets has a bulk density of less than 0 .25 g / cm ", e . g . , less than or of the binder. about 0 . 20 g /cm , 0 . 15 g/ cm°, 0 . 10 g /cm , 0 .05 g /cm3 or 35 The densified fibrous material can be made in a pellet less , e . g . , 0 . 025 g / cm ". Bulk density is determined using mill. Referring to FIG . 6 , a pellet mill 300 has a hopper 301 ASTM D1895B . Briefly , the method involves filling a for holding undensified material 310 that includes carbohy measuring cylinder of known volume with a sample and drate - containing materials , such as cellulose . The hopper obtaining a weight of the sample . The bulk density is communicates with an auger 312 that is driven by variable calculated by dividing the weight of the sample in grams by 40 speed motor 314 so that undensified material can be trans the known volume of the cylinder in cubic centimeters . ported to a conditioner 320 that stirs the undensified material The preferred binders include binders that are soluble in with paddles 322 that are rotated by conditioner motor 330 . water , swollen by water, or that have a glass transition Other ingredients , e . g . , any of the additives and / or fillers temperature of less 25° C ., as determined by differential described herein , can be added at inlet 332 . If desired , heat scanning calorimetry . Water - soluble binders have a solubil - 45 can be added while the fibrous material is in conditioner. ity of at least about 0 . 05 weight percent in water . Water After conditioned , the material passes from the conditioner swellable binders are binders that increase in volume by through a dump chute 340 , and to another auger 342 . The more than 0 . 5 percent upon exposure to water . dump chute, as controlled by actuator 344 , allows for In some embodiments , the binders that are soluble or unobstructed passage of the material from conditioner to swollen by water include a functional group that is capable 50 auger . Auger is rotated by motor 346 , and controls the of forming a bond , e . g . , a hydrogen bond , with the fibers of feeding of the fibrous material into die and roller assembly the fibrous material , e . g ., cellulosic fibrous material . For 350 . Specifically , the material is introduced into a hollow , example , the functional group can be a carboxylic acid cylindrical die 352 , which rotates about a horizontal axis and group , a carboxylate group, a carbonyl group , e. g. , of an which has radially extending die holes 250 . Die 352 is aldehyde or a ketone , a sulfonic acid group , a sulfonate 55 rotated about the axis by motor 360 , which includes a group , a phosphoric acid group , a phosphate group , an amide horsepower gauge , indicating total power consumed by the group , an amine group , a hydroxyl group , e . g . , of an alcohol, motor. Densified material 370 , e. g ., in the form of pellets , and combinations of these groups, e. g ., a carboxylic acid drops from chute 372 and are captured and processed , such group and a hydroxyl group . Specific monomeric examples as by irradiation . include glycerin , glyoxal , ascorbic acid , urea , glycine , pen - 60 The material , after densification , can be conveniently in taerythritol, a monosaccharide or a disaccharide, citric acid , the form of pellets or chips having a variety of shapes. The and tartaric acid . Suitable saccharides include glucose , pellets can then be irradiated . In some embodiments, the sucrose , lactose , ribose, fructose , mannose, arabinose and pellets or chips are cylindrical in shape , e . g ., having a erythrose . Polymeric examples include polyglycols , poly - maximum transverse dimension of, e . g ., 1 mm or more , e . g . , ethylene oxide , polycarboxylic acids, polyamides, 65 2 mm , 3 mm , 5 mm , 8 mm , 10 mm , 15 mm or more , e. g ., polyamines and polysulfonic acids polysulfonates . Specific 25 mm . Other convenient shapes include pellets or chips that polymeric examples include polypropylene glycol (PPG ), are plate - like in form , e . g ., having a thickness of 1 mm or US 9 , 745 ,604 B2 22 more , e . g ., 2 mm , 3 mm , 5 mm , 8 mm , 10 mm or more, e . g . , NREL /BR -510 -40742 , March , 2007 ) . For example , the 25 mm ; a width of, e . g ., 5 mm or more, e . g ., 10 mm , 15 mm , accessibility of polysaccharides ( e . g . , cellulose and hemi 25 mm , 30 mm or more, e .g ., 50 mm ; and a length of 5 mm cellulose ) in a first biomass material with a first recalcitrance ormore , e. g. , 10 mm , 15 mm , 25 mm , 30 mm ormore , e. g ., level will be lower than the accessibility of polysaccharides 50 mm . 5 ( e . g . , cellulose and hemicellulose ) in the same lignocellu Referring now FIG . 7A - 7D , pellets can be made so that losic material following treatment to reduce the recalcitrance they have a hollow inside . As shown , the hollow can be level of the material . In other words, the level of polysac generally in - line with the center of the pellet ( FIG . 7B ) , or charides available to polysaccharide degrading agents will out of line with the center of the pellet (FIG . 7C ) . Making be higher following treatment to reduce recalcitrance . the pellet hollow inside can increase the rate of dissolution 10 Assessing Recalcitrance Levels of Lignocellulosic Biomass in a liquid after irradiation . The recalcitrance level of a lignocellulosic material can be Referring now to FIG . 7D , the pellet can have , e .g . , a assessed using a number of art -recognized methods . transverse shape that is multi- lobal, e . g . , tri- lobal as shown , Examples of such methods include , but are not limited to , or tetra - lobal, penta - lobal, hexa - lobal or deca - lobal. Making surface characterization methods, enzymatic methods , and the pellets in such transverse shapes can also increase the 15 functional methods . rate of dissolution in a solution after irradiation . Exemplary surface characterization methods that can be Alternatively , the densified material can be in any other used to assess the recalcitrance level of lignocellulosic desired form , e . g . , the densified material can be in the form materials are known in the art ( for a review see Himmel et of a mat, roll or bale . al. , National Renewable Energy Laboratory (NREL ) Tech Examples of Densification 20 nical Report NREL / TP - 510 - 37902 , August , 2005 and Ding In one example , half - gallon juice cartons made of un - et al. , Microscopy and Microanalysis , 14 : 1494 - 1495 , 2004 ) . printed white Kraft board having a bulk density of 20 lb / ft For example , the recalcitrance level of lignocellulosic mate can be used as a feedstock . Cartons can be folded flat and rials can be assessed using microscopic and /or spectroscopic then fed into a shredder to produce a confetti - like material surface analysis methods ( e . g . , using one or more of the having a width of between 0 . 1 inch and 0 . 5 inch , a length of 25 surface analysis methods described below ) to identify , between 0 .25 inch and 1 inch and a thickness equivalent to assess, and /or quantify changes ( e . g ., structural changes ) in that of the starting material ( about 0 .075 inch ). The confetti - the lignocellulosic materials that indicate a reduction in the like material can be fed to a rotary knife cutter, which shears recalcitrance of thematerial . Exemplary changes that can be the confetti - like pieces, tearing the pieces apart and releasing used as indicia of a reduction in the recalcitrance of ligno fibrous material. 30 cellulosic materials include the appearance of pitting or In some cases , multiple shredder - shearer trains can be pores , and / or surface unwrapping of microfibrils . See, for arranged in series with output. In one embodiment, two example , Himmel et al. , National Renewable Energy Labo shredder - shearer trains can be arranged in series with output ratory (NREL ) Technical Report NREL / TP - 510 - 37902 , from the first shearer fed as input to the second shredder . In August, 2005 and Ding et al. , Microscopy and Microanaly another embodiment, three shredder -shearer trains can be 35 sis , 14 : 1494 - 1495, 2004 ), which describe the following arranged in series with output from the first shearer fed as methods : input to the second shredder and output from the second ( 1 ) Scanning election microscopy (SEM ) can be used to shearer fed as input to the third shredder. Multiple passes visualize the surface morphology of biological and non through shredder - shearer trains are anticipated to increase biological materials over a wide range of magnifications (as decrease particle size and increase overall surface area 40 high as 200 ,000x magnification ) and with high depth of field within the feedstream . ( see , e . g . , Gomez et al . , Biotechnology for Biofuels , 1 , Oct . In another example , fibrous material produced from 23 , 2008 ; Sivan et al. , Appl. Microbiol . Biotechnol ., 72 : 346 shredding and shearing juice cartons can be treated to 352, 2006 ). Typically , biological samples, such as lignocel increase its bulk density . In some cases , the fibrous material lulosic biomass samples, are coated with a thin layer of can be sprayed with water or a dilute stock solution of 45 electron dense material, such as carbon or atomized gold , POLYOXTM WSR N10 ( polyethylene oxide ) prepared in prior to analysis . For example , samples can be mounted in water. The wetted fibrous material can then be processed SEM stubs and coated with gold /palladium . These mounted through a pellet mill operating at room temperature . The specimens can then be observed using known methods and pelletmill can increase the bulk density of the feedstream by devices, e . g ., a JEOL JSM 6940LV SEM ( Jeol Ltd ., Tokyo , more than an order of magnitude . 50 Japan ) at an accelerating voltage of 5 kV . Treatment ( 2 ) More recently , methods have been developed for Pretreated biomass can be treated for use in primary analyzing samples containing natural moisture , a technique production processes by , for example , reducing the average referred to as environmentalmode SEM (ESEM ) , e . g. , using molecular weight , crystallinity , and /or increasing the surface the Quanta FEG 400 ESEM (FEI Company ) . The use of area and / or porosity of the biomass . In some embodiments , 55 ESEM in the analysis of yeast cells is described by Ren et the biomass can be treated to reduce the recalcitrance of the al. , Investigation of the morphology , viability and mechani biomass . Treatment processes can include at least one ( e . g ., cal properties of yeast cells in environmental SEM , Scan one, two , three , four, or five ) of irradiation , sonication , ning , published online Aug . 5 , 2008 ) . Such environmental oxidation , pyrolysis , and steam explosion . mode methods can be used to analyze lignocellulosic bio Recalcitrance is a term of art that, as used herein , broadly 60 mass containing moisture without the use of high electron refers to a biomass material' s resistance to the accessibility dense coatings . of polysaccharide degrading agents ( e . g . , microorganisms ( 3 ) Atomic force microscopy (AFM ) , e . g ., using DI- Veeco and / or enzymes ( e . g . , microbial enzymes ) ) to polysaccha - MultiMode PicoForce system ( see , e . g . , Stieg et al . , Rev. Sci. rides contained within biomass ( see , e . g ., Himmel et al. , Instrum . , 79 : 103701 , 2008 ) can also be used . AFM usefully National Renewable Energy Laboratory (NREL ) Technical 65 allows analysis of surface topography at very high magni Report NREL / TP - 510 -37902 , August , 2005 and National fication while also allowing analysis of the attractive and Renewable Energy Laboratory (NREL ) Technical Report repulsive forces between the scanning probe tip and the US 9 , 745 ,604 B2 23 24 sample surface , thus providing height and phase images. the activity of an enzyme ( e . g ., a cellulase ) in the same AFM is being increasingly applied to the analysis of bio sample following treatment to reduce the recalcitrance level logical samples due to its high atomic level resolution and its of the material. ease of use ( samples do not require extensive sample prepa Alternatively or in addition , a change (e .g . , decrease ) in ration ) . In addition , AFM can be used to observe dry and 5 the recalcitrance level of a lignocellulosic material can be hydrated surfaces directly using a tapping -probe . analyzed using functional methods. For example , a ligno ( 4 ) Transmission electron microscopy ( TEM ) , e . g . , using cellulosic material can be cultured in the presence of a sugar an FEI Tecnai F20 , allows the determination of the internal fermenting microorganism , e . g . , using the culture methods structures ofbiological and non - biological materials up to at disclosed herein , before and after treatment using the meth least 350 , 000x magnification . Typically , the determination 10 ods described herein . In some embodiments, an increase in of internal structures can be facilitated using shadowing the level of the one or more products generated by the techniques or staining with high contrast compounds . Com - microorganism indicates a change in the recalcitrant level of positional analysis of materials can also be performed by the material, e . g . , a decrease in the recalcitrance of the monitoring secondary X - rays produced by the electron material. specimen interaction using energy dispersive X - ray micro - 15 In some embodiments , the growth rate of a microorgan analysis . TEM - based methods for analyzing the recalci - ism and / or product generation by the microorganism in a trance levels of a lignocellulosic material are described in sample comprising a first lignocellulosic material with a first the art (see , e . g . , Rhoads et al ., Can . J . Microbiol. , 41: 592 recalcitrance level will be lower than the growth rate of the 600 , 1995 ) . microorganism and / or product generation by the microor ( 5 ) Near - field Scanning Optical Microscopy (NFSOM ) 20 ganism in the same sample following treatment to reduce the using , e . g ., a DI- Veeco Aurora - 3 NSOM (Nikon ) , permits recalcitrance level of the material. surfaces to be viewed with a long depth of field light In some embodiments , a change in the recalcitrance level microscope that is adapted to conduct secondary spectro - of a material can be expressed as ; ( 1 ) a ratio ( e . g . , a measure photometric analysis such as UV /VIS , fluorescence , and of the recalcitrance level of a material prior to treatment laser Raman . In some embodiments , NFSOM can be per - 25 versus a measure of the recalcitrance level or the material formed using an Olympus IX71 inverted microscope fitted post - treatment ) ; ( 2 ) a percent change ( e . g ., decrease ) in the with a DP70 high resolution CCD camera to perform single recalcitrance level of a material ; ( 3 ) a percent change ( e . g . , molecule microscopy . increase ) in the level of polysaccharide available to a poly (6 ) Confocal microscopy (CFM ) and confocal scanning saccharide degrading agent ( e . g ., an enzyme ) after treat laser microscopy ( CSLM ) ( see , e . g ., National Renewable 30 ment, as compared to before the treatment, per weight Energy Laboratory (NREL ) Technical Report NREL /BR - measure of the starting biomass material; or ( 4 ) a percent 510 - 40742, March , 2007 ) can be used to generate optical change ( e . g ., increase ) in the solubility of the material in a sections that can be used to build a three - dimensional image particular solvent. of a surface and internal structures. Typically, CFM and In some instances, the second material has cellulose that CSLM are performed in combination with labeling methods, 35 has a crystallinity ( " C ) that is lower than the crystallinity for example fluorescent stains (see , e. g ., Sole et al. , Microb . (FC ) of the cellulose of the first material . For example , Ecol. , Published online on Nov . 4 , 2008 ). ( " C ) can be lower than (™C ) by more than about 10 In some embodiments , the recalcitrance level of a ligno - percent, e . g . , 15 , 20 , 25 , 30 , 35 , 40 , or even more than about cellulosic material can be assessed using one or more 50 percent. methods known in the art, e . g . , methods described herein . 40 In some embodiments , the starting crystallinity index The same sample , or a portion thereof, can then be assessed (prior to irradiation ) is from about 40 to about 87 .5 percent, following treatment to observe a change ( e. g . , a structural e . g . , from about 50 to about 75 percent or from about 60 to change ) in the recalcitrance . In some embodiments , the about 70 percent , and the crystallinity index after irradiation appearance or observance of pitting or pores, and /or surface is from about 10 to about 50 percent, e . g ., from about 15 to unwrapping of microfibrils in or on a first lignocellulosic 45 about 45 percent or from about 20 to about 40 percent. material with a first recalcitrance level will be less than the However, in some embodiments , e . g ., after extensive irra appearance or observance of pitting or pores, and /or surface diation , it is possible to have a crystallinity index of lower unwrapping of microfibrils in the same sample following than 5 percent . In some embodiments , the material after treatment to reduce the recalcitrance level of the material. irradiation is substantially amorphous. Alternatively or in addition , a change ( e . g . , decrease ) in 50 In some embodiments , the starting number average the recalcitrance level of a lignocellulosic material can be molecular weight (prior to irradiation ) is from about 200 ,000 analyzed using enzymatic methods. For example , a ligno - to about 3 , 200 , 000 , e . g ., from about 250 , 000 to about cellulosic material can be incubated in the presence of one 1 ,000 , 000 or from about 250 , 000 to about 700 ,000 , and the or more cellulases , e . g . , before and after treatment using the number average molecular weight after irradiation is from methods described herein . In some embodiments , an 55 about 50 ,000 to about 200 ,000 , e . g ., from about 60 , 000 to increase in the break down of cellulose by the cellulase about 150 , 000 or from about 70 ,000 to about 125 , 000 . indicates a change in the recalcitrant level of the material, However, in some embodiments , e . g ., after extensive irra e . g . , a decrease in the recalcitrance of the material. In some diation , it is possible to have a number average molecular embodiments , the increase in the break down of cellulose by weight of less than about 10 ,000 or even less than about the cellulase causes an increase in the amount of monosac - 60 5 , 000 . charide and /or disaccharides in the sample. In some embodiments , the second material can have a In some embodiments , the amount ( e . g . , concentration ) of level of oxidation ( ' 02) that is higher than the level of monosaccharides and /or disaccharides resulting from the oxidation (" 0 ) of the first material . A higher level of activity of an enzyme ( e . g ., a cellulase ) in a sample com - oxidation of the material can aid in its dispersibility , prising a first lignocellulosic material with a first recalci- 65 swellability and / or solubility , further enhancing the materi trance level will be lower than the amount ( e . g . , concentra - al' s susceptibility to chemical, enzymatic , or biological tion ) ofmonosaccharide and / or disaccharides resulting from attack . In some embodiments, to increase the level of the US 9 ,745 ,604 B2 25 26 oxidation of the second material relative to the first material, growth factor, such as biotin and /or a base pair such as uracil the irradiation is performed under an oxidizing environment, or an equivalent thereof; a surfactant, such as Tween or e . g ., under a blanket of air or oxygen , producing a second polyethylene glycol; a mineral, such as such as calcium , material that is more oxidized than the first material. For chromium , copper, iodine , iron , selenium , or zinc ; or a example , the second material can have more hydroxyl 5 chelating agent, such as ethylene diamine, ethylene diamine groups , aldehyde groups, ketone groups , ester groups , or tetraacetic acid (EDTA ) (or its salt form , e . g . , sodium or carboxylic acid groups, which can increase its hydrophilic potassium EDTA ), or dimercaprol . ity . When radiation is utilized as or in the treatment, it can be Treatment Combinations In some embodiments , biomass can be treated by apply - 10 applied to any sample that is dry or wet, or even dispersed ing at least one ( e . g . , two , three , four, or five ) of the in a liquid , such as water . For example, irradiation can be treatmentmethods described herein , such as two or more of performed on biomass material in which less than about 25 radiation , sonication , oxidation , pyrolysis , and steam explo percent by weight of the biomass material has surfaces sion either with or without prior, intermediate , or subsequent wetted with a liquid , such as water. In some embodiments , biomass preparation as described herein . The treatment 15 irradiatingirradi is performed on biomass material in which sub methods can be applied in any order, in multiples ( e . g . , two stantially none of the biomass material is wetted with a ormore applications of a treatment method ) , or concurrently liquid , such as water . to the biomass , e . g . , a cellulosic and / or lignocellulosic In some embodiments , any processing described herein material. In other embodiments , materials that include a occurs after the biomass material remains dry as acquired or carbohydrate are prepared by applying three , four or more of 20 has been dried , e . g ., using heat and /or reduced pressure. For any of the processes described herein in any order or example , in some embodiments , the biomass material has concurrently ) . For example, a carbohydrate can be prepared less than about five percent by weight retained water , by applying radiation , sonication , oxidation , pyrolysis , and , measured at 25° C . and at fifty percent relative humidity . optionally , steam explosion to a cellulosic and / or lignocel- If desired , a swelling agent, as defined herein , can be lulosic material ( in any order or concurrently ) . The provided 25 utilized in any process described herein . In some embodi carbohydrate - containing material can then be converted by ments , when a biomass material is processed using radiation , one or more microorganisms, such as bacteria ( e . g . , gram less than about 25 percent by weight of the biomass material positive bacteria , gram negative bacteria , and extremo - is in a swollen state , the swollen state being characterized as philes ) , yeast , or mixtures of yeast and bacteria , to a number having a volume of more than about 2 . 5 percent higher than of desirable products, as described herein . Multiple pro - 30 an unswollen state , e . g . , more than 5 . 0 , 7 . 5 , 10 , or 15 percent cesses can provide materials that can be more readily higher than the unswollen state . In some embodiments , utilized by a variety of microorganisms because of their when radiation is utilized on a biomass material, substan lower molecular weight, lower crystallinity , and / or t ially none of the biomass material is in a swollen state . enhanced solubility . Multiple processes can provide syner - In specific embodiments when radiation is utilized , the gies and can reduce overall energy input required in com - 35 biomass material includes a swelling agent, and swollen parison to any single process . biomass material receives a dose of less than about 10 Mrad . For example , in some embodiments , biomass feedstocks When radiation is utilized in any process , it can be applied can be provided that include a carbohydrate that is produced while the biomass is exposed to air , oxygen - enriched air , or by a process that includes irradiating and sonicating in even oxygen itself , or blanketed by an inert gas such as either order or concurrently ) a biomass material, a process 40 nitrogen , argon , or helium . When maximum oxidation is that includes irradiating and oxidizing ( in either order or desired , an oxidizing environment is utilized , such as air or concurrently ) a biomass material, a process that includes oxygen . irradiating and pyrolyzing ( in either order or concurrently ) a When radiation is utilized , it can be applied to biomass biomass material , a treatment process that includes irradi- under a pressure of greater than about 2 . 5 atmospheres , such ating and pyrolyzing in either order or concurrently ) a 45 as greater than 5 , 10 , 15 , 20 or even greater than about 50 biomass material , or a process that includes irradiating and atmospheres . Irradiation can increase the solubility , steam -exploding ( in either order or concurrently ) a biomass swellability , or dispersibility of the biomass in a solvent. material. The provided biomass feedstock can then be con - In specific embodiments , the process includes irradiating tacted with a microorganism having the ability to convert at and sonicating and irradiating precedes sonicating . In other least a portion , e . g . , at least about 1 percent by weight , of the 50 specific embodiments , sonication precedes irradiating , or biomass to the product. irradiating and sonicating occur substantially concurrently . In some embodiments , the process does not include In some embodiments , the process includes irradiating hydrolyzing the biomass , such as with an acid , base , and /or and sonicating ( in either order or concurrently ) and further enzyme, e. g ., a mineral acid , such as hydrochloric or sulfuric includes oxidizing, pyrolyzing or steam exploding . acid . 55 When the process includes radiation , the irradiating can If desired , some or none of the biomass can include a be performed utilizing an ionizing radiation , such as gamma hydrolyzed material. For example , in some embodiments , at rays, X -rays , energetic ultraviolet radiation , such as ultra least about seventy percent by weight of the biomass is an violet C radiation having a wavelength of from about 100 unhydrolyzed material, e . g . , at least at 95 percent by weight nm to about 280 nm , a beam of particles, such as a beam of of the feedstock is an unhydrolyzed material. In some 60 electrons, slow neutrons or alpha particles . In some embodi embodiments , substantially all of the biomass is an unhy - ments , irradiating includes two or more radiation sources , drolyzed material. In some embodiments , 100 % of the such as gamma rays and a beam of electrons, which can be biomass is unhydrolyzed material. applied in either order or concurrently . Any feedstock or any reactor or fermentor charged with a In specific embodiments, sonicating can performed at a feedstock can include a buffer , such as sodium bicarbonate , 65 frequency of between about 15 kHz and about 25 kHz, such ammonium chloride or Tris ; an electrolyte , such as potas - as between about 18 kHz and 22 kHz utilizing a 1 KW or sium chloride, sodium chloride , or calcium chloride ; a larger horn , e. g ., a 2 , 3 , 4 , 5 , or even a 10 KW horn . US 9 ,745 ,604 B2 28 In some embodiments , the biomass has a first number material , and in some instances , a reduced crystallinity , the average molecular weight and the resulting carbohydrate second material is generally more dispersible , swellable includes a second cellulose having a second number average and / or soluble in a solution containing a microorganism . molecular weight lower than the first number average These properties make the second material 3 more suscep molecular weight . For example , the second number average 5 tible to chemical, enzymatic and / or biological attack ( e . g . , molecular weight is lower than the first number average by a microorganism ) relative to the first material 2 , which molecular weight by more than about twenty - five percent, can greatly improve the production rate and / or production e . g ., 2x , 3x , 5x , 7X , 10X , 25x , even 100x reduction . level of a desired product, e . g . , ethanol. Radiation can also In some embodiments , the first cellulose has a first sterilize the materials . crystallinity and the second cellulose has a second crystal- 10 In some embodiments , the second number average linity lower than the first crystallinity , such as lower than molecular weight (MN ) is lower than the first number about two , three , five , ten , fifteen or twenty - five percent average molecular weight ( My?) by more than about 10 lower. percent, e .g ., 15 , 20 , 25 , 30 , 35 , 40 , 50 percent, 60 percent, In some embodiments , the first cellulose has a first level or even more than about 75 percent. of oxidation and the second cellulose has a second level of 15 Ionizing Radiation oxidation higher than the first level of oxidation , such as Each form of radiation ionizes the biomass via particular two, three , four, five , ten or even twenty - five percent higher interactions, as determined by the energy of the radiation . In some embodiments , the first biomass has a first level of Heavy charged particles primarily ionize matter via Cou recalcitrance and the resulting biomass has a second level of lomb scattering ; furthermore , these interactions produce recalcitrance that is lower than the first level. 20 energetic electrons that can further ionize matter . Alpha Radiation Treatment particles are identical to the nucleus of a helium atom and One or more irradiation processing sequences can be used are produced by the alpha decay of various radioactive to process biomass from a wide variety of different sources nuclei, such as isotopes of bismuth , polonium , astatine, to extract useful substances from the feedstock , and to radon , francium , radium , several actinides , such as actinium , provide partially degraded organic material which functions 25 thorium , uranium , neptunium , curium , californium , ameri as input to further processing steps and / or sequences . Irra - cium , and plutonium . diation can reduce the recalcitrance, molecular weight and When particles are utilized , they can be neutral (un or crystallinity of feedstock . charged ), positively charged or negatively charged . When In some embodiments , energy deposited in a material that charged , the charged particles can bear a single positive or releases an electron from its atomic orbital is used to 30 negative charge , or multiple charges , e . g . , one , two , three or irradiate the materials . The radiation can be provided by 1 ) even four or more charges . In instances in which chain heavy charged particles, such as alpha particles or protons , scission is desired , positively charged particles can be desir 2 ) electrons , produced , for example , in beta decay or elec - able , in part , due to their acidic nature . When particles are tron beam accelerators, or 3 ) electromagnetic radiation , for utilized , the particles can have the mass of a resting electron , example , gamma rays , x rays, or ultraviolet rays . In one 35 or greater, e . g . , 500 , 1000 , 1500 , or 2000 or more times the approach , radiation produced by radioactive substances can mass of a resting electron . For example , the particles can be used to irradiate the feedstock . In some embodiments , have a mass of from about 1 atomic units to about 150 any combination in any order or concurrently of ( 1 ) through atomic units , e . g ., from about 1 atomic units to about 50 ( 3 ) can be utilized . In another approach , electromagnetic atomic units , or from about 1 to about 25 , e . g . , 1 , 2 , 3 , 4 , 5 , radiation ( e . g . , produced using electron beam emitters ) can 40 10 , 12 or 15 amu . Accelerators used to accelerate the be used to irradiate the feedstock . The doses applied depend particles can be electrostatic DC , electrodynamic DC , RF on the desired effect and the particular feedstock . For linear , magnetic induction linear or continuous wave . For example , high doses of radiation can break chemical bonds example , cyclotron type accelerators are available from within feedstock components and low doses of radiation can IBA , Belgium , such as the Rhodotron® system , while DC increase chemical bonding ( e . g . , cross - linking ) within feed - 45 type accelerators are available from RDI, now IBA Indus stock components. In some instances when chain scission is trial, such as the Dynamitron® . Ions and ion accelerators are desirable and / or polymer chain functionalization is desir - discussed in Introductory Nuclear Physics, Kenneth S . able, particles heavier than electrons, such as protons, Krane, John Wiley & Sons, Inc . ( 1988 ) , Krsto Prelec , helium nuclei, argon ions, silicon ions , neon ions, carbon FIZIKAB 6 (1997 ) 4 , 177 - 206 , Chu , William T . , “ Overview ions, phosphorus ions , oxygen ions or nitrogen ions can be 50 of Light- Ion Beam Therapy ” , Columbus -Ohio , ICRU - IAEA utilized . When ring - opening chain scission is desired , posi- Meeting , 18 - 20 Mar. 2006 , Iwata , Y . et al. , “ Alternating tively charged particles can be utilized for their Lewis acid Phase - Focused IH -DTL for Heavy -Ion Medical Accelera properties for enhanced ring -opening chain scission . tors ” , Proceedings of EPAC 2006 , Edinburgh , Scotland , and Referring to FIG . 8 , in one method , a first material 2 that Leitner , C . M . et al. , “ Status of the Superconducting ECR is or includes cellulose having a first number average 55 Ion Source Venus” , Proceedings of EPAC 2000 , Vienna , molecular weight ( ?My? ) is irradiated , e . g ., by treatment Austria . Typically, generators are housed in a vault, e . g ., of with ionizing radiation (e . g. , in the form of gamma radiation , lead or concrete . X -ray radiation , 100 nm to 280 nm ultraviolet (UV ) light, a Electrons interact via Coulomb scattering and beam of electrons or other charged particles ) to provide a bremsstrahlung radiation produced by changes in the veloc second material 3 that includes cellulose having a second 60 ity of electrons . Electrons can be produced by radioactive number average molecular weight ( Mys ) lower than the nuclei that undergo beta decay, such as isotopes of iodine , first number average molecular weight. The second material cesium , technetium , and iridium . Alternatively , an electron (or the first and second material) can be combined with a gun can be used as an electron source via thermionic microorganism ( e . g . , a bacterium or a yeast ) that can utilize emission . the second and / or first material to produce a product 5 . 65 Electromagnetic radiation interacts via three processes : Since the second material 3 has cellulose having a photoelectric absorption , Compton scattering , and pair pro reduced recalcitrance, molecular weight relative to the first duction . The dominating interaction is determined by the US 9 ,745 ,604 B2 29 30 energy of the incident radiation and the atomic number of pulsed accelerators. Electrons as an ionizing radiation the material. The summation of interactions contributing to source can be useful, e . g ., for relatively thin piles of mate the absorbed radiation in cellulosic material can be rials , e . g . , less than 0 . 5 inch , e . g . , less than 0 .4 inch , 0 . 3 inch , expressed by the mass absorption coefficient (see " Ioniza 0 .2 inch , or less than 0 . 1 inch . In some embodiments , the tion Radiation ” in PCT/ US2007 / 022719 ) . energy of each electron of the electron beam is from about Electromagnetic radiation is subclassified as gamma rays , 0 . 3 MeV to about 2 . 0 MeV (million electron volts ) , e . g . , x rays , ultraviolet rays , infrared rays , microwaves , or radio waves, depending on its wavelength . from about 0 . 5 MeV to about 1 . 5 MeV , or from about 0 . 7 For example , gamma radiation can be employed to irra MeV to about 1 .25 MeV . diate the materials . Referring to FIGS. 9 and 10 ( an enlarged 10 FIG . 11 shows a process flow diagram 3000 that includes view of region R ) , a gamma irradiator 10 includes gamma various steps in an electron beam irradiation feedstock radiation sources 408 , e . g ., 6o Co pellets, a working table 14 pretreatment sequence . In first step 3010 , a supply of dry for holding the materials to be irradiated and storage 16 , e . g . , feedstock is received from a feed source . As discussed made of a plurality of iron plates , all of which are housed in above , the dry feedstock from the feed source can be a concrete containment chamber ( vault ) 20 that includes a 15 pre - processed prior to delivery to the electron beam irradia maze entranceway 22 beyond a lead -lined door 26 . Storage tion devices . For example , if the feedstock is derived from 16 includes a plurality of channels 30 , e . g . , sixteen or more plant sources, certain portions of the plant material can be channels , allowing the gamma radiation sources to pass removed prior to collection of the plant material and /or through storage on their way proximate the working table . before the plant material is delivered by the feedstock In operation , the sample to be irradiated is placed on a 20 transport device. Alternatively , or in addition , as expressed working table . The irradiator is configured to deliver the in optional step 3020 , the biomass feedstock can be sub desired dose rate and monitoring equipment is connected to jected to mechanical processing ( e . g . , to reduce the average an experimental block 31 . The operator then leaves the length of fibers in the feedstock ) prior to delivery to the containment chamber, passing through the maze entrance - electron beam irradiation devices . way and through the lead - lined door. The operator mans a 25 In step 3030 , the dry feedstock is transferred to a feed control panel 32 , instructing a computer 33 to lift the stock transport device ( e . g . , a conveyor belt) and is distrib radiation sources 12 into working position using cylinder 36 uted over the cross - sectional width of the feedstock transport attached to a hydraulic pump 40 . device approximately uniformly by volume. This can be Gamma radiation has the advantage of a significant pen - accomplished , for example , manually or by inducing a etration depth into a variety of material in the sample . 30 localized vibration motion at some point in the feedstock Sources of gamma rays include radioactive nuclei , such as transport device prior to the electron beam irradiation pro isotopes of cobalt , calcium , technicium , chromium , gallium , cessing . indium , iodine , iron , krypton , samarium , selenium , sodium , In some embodiments , a mixing system introduces a thalium , and xenon . chemical agent 3045 into the feedstock in an optional Sources of x rays include electron beam collision with 35 process 3040 that produces a slurry . Combining water with metal targets , such as tungsten or molybdenum or alloys, or the processed feedstock in mixing step 3040 creates an compact light sources, such as those produced commercially aqueous feedstock slurry that can be transported through , for by Lyncean . Sources for ultraviolet radiation include deu example , piping rather than using , for example , a conveyor terium or cadmium lamps . Sources for infrared radiation belt . include sapphire , zinc , or selenide window ceramic lamps . 40 The next step 3050 is a loop that encompasses exposing Sources for microwaves include klystrons , Slevin type RF the feedstock ( in dry or slurry form ) to electron beam sources , or atom beam sources that employ hydrogen , oxy - radiation via one or more ( say , N ) electron beam irradiation gen , or nitrogen gases . devices. The feedstock slurry is moved through each of the Various other irradiating devices may be used in the N " showers” of electron beams at step 3052 . The movement methods disclosed herein , including field ionization sources , 45 can either be at a continuous speed through and between the electrostatic ion separators, field ionization generators , ther - showers , or there can be a pause through each shower, mionic emission sources , microwave discharge ion sources, followed by a sudden movement to the next shower. A small recirculating or static accelerators , dynamic linear accelera - slice of the feedstock slurry is exposed to each shower for tors , van de Graaff accelerators , and folded tandem accel- some predetermined exposure time at step 3053. erators . Such devices are disclosed , for example , in U . S . 50 Electron beam irradiation devices can be procured com Provisional Application Ser. No. 61/ 073 ,665 (which served mercially from Ion Beam Applications, Louvain - la -Neuve , as priority for U . S . Pat. No. 8 ,025 ,098 to Medoff ) , the Belgium or the Titan Corporation , San Diego , Calif. Typical complete disclosure of which is incorporated herein by electron energies can be 1 MeV , 2 MeV , 4 . 5 MeV , 7 . 5 MeV , reference . or 10 MeV . Typical electron beam irradiation device power Electron Beam 55 can be 1 kW , 5 kW , 10 kW , 20 kW , 50 kW , 100 kW , 250 kW , In some embodiments , a beam of electrons is used as the or 500 kW . Effectiveness of depolymerization of the feed radiation source . A beam of electrons has the advantages of stock slurry depends on the electron energy used and the high dose rates ( e . g . , 1 , 5 , or even 10 Mrad per second ) , high dose applied , while exposure time depends on the power and throughput, less containment, and less confinement equip - dose . Typical doses can take values of 1 kGy, 5 kGy, 10 kGy, ment. Electrons can also be more efficient at causing chain 60 20 kGy, 50 KGy, 100 kGy, or 200 kGy. scission . In addition , electrons having energies of 4 - 10 MeV Tradeoffs in considering electron beam irradiation device can have a penetration depth of 5 to 30 mm or more , such power specifications include cost to operate , capital costs , as 40 mm . depreciation , and device footprint. Tradeoffs in considering Electron beams can be generated , e . g . , by electrostatic exposure dose levels of electron beam irradiation would be generators , cascade generators , transformer generators , low 65 energy costs and environment, safety , and health (ESH ) energy accelerators with a scanning system , low energy concerns . Tradeoffs in considering electron energies include accelerators with a linear cathode , linear accelerators, and energy costs ; here , a lower electron energy can be advan US 9 ,745 ,604 B2 31 32 tageous in encouraging depolymerization of certain feed - until the material receives a dose of between 1 . 0 Mrad and stock slurry ( see, for example , Bouchard , et al, Cellulose 6 . 0 Mrad , e . g ., between 1. 5 Mrad and 4 .0 Mrad . ( 2006 ) 13 : 601 -610 ) . In some embodiments , the irradiating is performed at a It can be advantageous to provide a double -pass of dose rate of between 5 . 0 and 1500 . 0 kilorads/ hour , e . g . , electron beam irradiation in order to provide a more effective 5 between 10 . 0 and 750 . 0 kilorads/ hour or between 50 . 0 and depolymerization process . For example , the feedstock trans - 350 . 0 kilorads/ hours . port device could direct the feedstock ( in dry or slurry form ) In some embodiments , two or more radiation sources are underneath and in a reverse direction to its initial transport used , such as two or more ionizing radiations. For example , direction . Double - pass systems can allow thicker feedstock samples can be treated , in any order, with a beam of slurries to be processed and can provide a more uniform 10 electrons , followed by gamma radiation and UV light having depolymerization through the thickness of the feedstock wavelengths from about 100 nm to about 280 nm . In some slurry . embodiments , samples are treated with three ionizing radia The electron beam irradiation device can produce either a tion sources, such as a beam of electrons, gamma radiation , fixed beam or a scanning beam . A scanning beam can be and energetic UV light . advantageous with large scan sweep length and high scan 15 Alternatively , in another example , a fibrous biomass speeds, as this would effectively replace a large, fixed beam material that includes a cellulosic and/ or lignocellulosic width . Further, available sweep widths of 0 . 5 m , 1 m , 2 m material is irradiated and , optionally , treated with acoustic or more are available . One suitable device is referenced in energy , e . g . , ultrasound . Example 22 . In one example of the use of radiation as a treatment , Once a portion of feedstock slurry has been transported 20 half- gallon juice cartons made of un -printed polycoated through the N electron beam irradiation devices , it can be white Kraft board having a bulk density of 20 lb / ft are used necessary in some embodiments , as in step 3060 , to as a feedstock . Cartons are folded flat and then fed into a mechanically separate the liquid and solid components of sequence of three shredder- shearer trains arranged in series the feedstock slurry . In these embodiments , a liquid portion with output from the first shearer fed as input to the second of the feedstock slurry is filtered for residual solid particles 25 shredder , and output from the second shearer fed as input to and recycled back to the slurry preparation step 3040 . A the third shredder . The fibrous material produced by the can solid portion of the feedstock slurry is then advanced on to be sprayed with water and processed through a pellet mill the next processing step 3070 via the feedstock transport operating at room temperature . The densified pellets can be device . In other embodiments , the feedstock is maintained in placed in a glass ampoule which is evacuated under high slurry form for further processing. 30 vacuum and then back - filled with argon gas . The ampoule is Heavy Ion Particle Beams sealed under argon . The pellets in the ampoule are irradiated Particles heavier than electrons can be utilized to irradiate with gamma radiation for about 3 hours at a dose rate of carbohydrates or materials that include carbohydrates , e . g ., about 1 Mrad per hour to provide an irradiated material in cellulosic materials , lignocellulosic materials, starchy mate - which the cellulose has a lower molecular weight than the rials , or mixtures of any of these and others described herein . 35 starting material. For example , protons , helium nuclei , argon ions, silicon Quenching and Controlled Functionalization of Biomass ions , neon ions, carbon ions , phosphorus ions , oxygen ions After treatment with one or more ionizing radiations, such or nitrogen ions can be utilized . In some embodiments , as photonic radiation ( e . g . , X - rays or gamma- rays ) , e - beam particles heavier than electrons can induce higher amounts radiation or particles heavier than electrons that are posi of chain scission . In some instances , positively charged 40 tively or negatively charged ( e . g . , protons or carbon ions) , particles can induce higher amounts of chain scission than any of the carbohydrate - containing materials or mixtures negatively charged particles due to their acidity . described herein become ionized ; that is , they include radi Heavier particle beams can be generated , e . g . , using linear c als at levels that are detectable with an electron spin accelerators or cyclotrons. In some embodiments , the energy resonance spectrometer. The current limit of detection of the of each particle of the beam is from about 1 . 0 MeV / atomic 45 radicals is about 1014 spins at room temperature . After unit to about 6 ,000 MeV / atomic unit , e . g . , from about 3 ionization , any biomass material that has been ionized can MeV /atomic unit to about 4 ,800 MeV / atomic unit , or from be quenched to reduce the level of radicals in the ionized about 10 MeV /atomic unit to about 1 ,000 MeV / atomic unit . biomass , e . g . , such that the radicals are no longer detectable Electromagnetic Radiation with the electron spin resonance spectrometer . For example , In embodiments in which the irradiating is performed 50 the radicals can be quenched by the application of a suffi with electromagnetic radiation , the electromagnetic radia - cient pressure to the biomass and /or utilizing a fluid in tion can have , e . g . , energy per photon (in electron volts ) of contact with the ionized biomass , such as a gas or liquid , that greater than 10² eV, e. g. , greater than 10°, 104 , 10 ", 10° , or reacts with (quenches ) the radicals . Using a gas or liquid to even greater than 10 ' eV . In some embodiments , the elec - at least aid in the quenching of the radicals can be used to tromagnetic radiation has energy per photon of between 104 55 functionalize the ionized biomass with a desired amount and and 10 , e . g ., between 10 and 100 eV . The electromagnetic kinds of functional groups, such as carboxylic acid groups , radiation can have a frequency of, e. g. , greater than 1016 Hz, enol groups, aldehyde groups, nitro groups, nitrile groups, greater than 1017 Hz, 1018 , 1019 , 10², or even greater than amamino groups , alkyl amino groups, alkyl groups, chloroalkyl 1021 Hz. In some embodiments , the electromagnetic radia - groups or chlorofluoroalkyl groups . In some instances , such tion has a frequency of between 108 and 1022 Hz, e . g ., 60 quenching can improve the stability of some of the ionized between 1019 to 1021 Hz . biomass materials . For example , quenching can improve the Doses biomass ' s resistance to oxidation . Functionalization by In some embodiments , the irradiating (with any radiation quenching can also improve the solubility of any biomass source or a combination of sources ) is performed until the described herein , can improve its thermal stability , which material receives a dose of at least 0 . 25 Mrad , e . g . , at least 65 can improve material utilization by various microorganisms. 1. 0 Mrad , at least 2 . 5 Mrad , at least 5 . 0 Mrad , or at least 10 .0 For example, the functional groups imparted to the biomass Mrad . In some embodiments , the irradiating is performed material by the quenching can act as receptor sites for US 9 ,745 ,604 B2 33 34 attachment by microorganisms, e. g. , to enhance cellulose Other methods for quenching are possible . For example , hydrolysis by various microorganisms. any method for quenching radicals in polymeric materials FIG . 11A illustrates changing a molecular and /or a supra described in Muratoglu et al. , U . S . Patent Application Pub molecular structure of a biomass feedstock by pretreating lication No. 2008 /0067724 and Muratoglu et al. , U . S . Pat. the biomass feedstock with ionizing radiation , such as with 5 No . 7 , 166 ,650 , can be utilized for quenching any ionized electrons or ions of sufficient energy to ionize the biomass biomass material described herein . Furthermore any quench feedstock , to provide a first level of radicals . As shown in ing agent (described as a “ sensitizing agent” in the above FIG . 11A , if it ionized biomass remains in the atmosphere , it will be oxidized , such as to an extent that carboxylic acid noted Muratoglu disclosures) and /or any antioxidant groups are generated by reacting with the atmospheric 10 described in either Muratoglu reference can be utilized to oxygen . In some instances with some materials , such oxi quench any ionized biomass material. dation is desired because it can aid in the further breakdown Functionalization can be enhanced by utilizing heavy in molecular weight of the carbohydrate - containing bio charged ions , such as any of the heavier ions described mass, and the oxidation groups, e . g ., carboxylic acid groups herein . For example , if it is desired to enhance oxidation , can be helpful for solubility and microorganism utilization 15 charged oxygen ions can be utilized for the irradiation . If in some instances. However, since the radicals can “ live ” for nitrogen functional groups are desired , nitrogen ions or ions some time after irradiation , e. g ., longer than 1 day, 5 days , that includes nitrogen can be utilized . Likewise , if sulfur or 30 days, 3 months, 6 months or even longer than 1 year, phosphorus groups are desired , sulfur or phosphorus ions ma ties can continue to change over time, which can be used in the irradiation . in some instances, can be undesirable . Detecting radicals in 20 In some embodiments , after quenching any of the irradiated samples by electron spin resonance spectroscopy quenched ionized materials described herein can be further and radical lifetimes in such samples is discussed in Barto - treated with one or more of radiation , such as ionizing or lotta et al. , Physics in Medicine and Biology, 46 ( 2001) , non - ionizing radiation , sonication , pyrolysis , and oxidation 461 - 471 and in Bartolotta et al . , Radiation Protection for additional molecular and /or supramolecular structure Dosimetry, Vol. 84 , Nos. 1 - 4 , pp . 293 - 296 ( 1999 ). As shown 25 change . in FIG . 11A , the ionized biomass can be quenched to Particle Beam Exposure in Fluids functionalize and / or to stabilize the ionized biomass. At any In some cases , the cellulosic or lignocellulosic materials point, e . g. , when the material is “ alive” ( still has a substan can be exposed to a particle beam in the presence of one or tial quantity of reactive intermediates such as radicals ) , more additional fluids ( e . g . , gases and / or liquids ) . Exposure " partially alive ” or fully quenched , the treated biomass can 30 of a material to a particle beam in the presence of one or be converted into a product , e . g ., a food . more additional fluids can increase the efficiency of the In some embodiments, the quenching includes an appli - treatment. cation of pressure to the biomass , such as by mechanically In some embodiments , the material is exposed to a deforming the biomass, e . g ., directly mechanically com particle beam in the presence of a fluid such as air . Particles pressing the biomass in one , two , or three dimensions, or 35 accelerated in any one or more of the types of accelerators applying pressure to a fluid in which the biomass is disclosed herein ( or another type of accelerator ) are coupled immersed , e . g . , isostatic pressing . In such instances , the out of the accelerator via an output port ( e . g . , a thin deformation of the material itself brings radicals , which are membrane such as a metal foil) , pass through a volume of often trapped in crystalline domains, in close enough prox - space occupied by the fluid , and are then incident on the imity so that the radicals can recombine , or react with 40 material . In addition to directly treating the material, some another group . In some instances , the pressure is applied of the particles generate additional chemical species by together with the application of heat, such as a sufficient interacting with fluid particles ( e . g . , ions and /or radicals quantity of heat to elevate the temperature of the biomass to generated from various constituents of air , such as ozone and above a melting point or softening point of a component of oxides of nitrogen ) . These generated chemical species can the biomass , such as lignin , cellulose or hemicellulose . Heat 45 also interact with the material, and can act as initiators for can improve molecular mobility in the polymeric material, a variety of different chemical bond - breaking reactions in which can aid in the quenching of the radicals . When the material. For example , any oxidant produced can oxidize pressure is utilized to quench , the pressure can be greater the material, which can result in molecular weight reduction . than about 1000 psi, such as greater than about 1250 psi, In certain embodiments , additional fluids can be selec 1450 psi, 3625 psi , 5075 psi , 7250 psi, 10000 psi or even 50 tively introduced into the path of a particle beam before the greater than 15000 psi. beam is incident on the material. As discussed above , In some embodiments, quenching includes contacting the reactions between the particles of the beam and the particles biomass with a fluid , such as a liquid or gas, e . g . , a gas of the introduced fluids can generate additional chemical capable of reacting with the radicals , such as acetylene or a species , which react with the material and can assist in mixture of acetylene in nitrogen , ethylene , chlorinated eth - 55 functionalizing the material, and /or otherwise selectively ylenes or chlorofluoroethylenes , propylene or mixtures of altering certain properties of the material. The one or more these gases . In other particular embodiments , quenching additional fluids can be directed into the path of the beam includes contacting the biomass with a liquid , e . g. , a liquid from a supply tube, for example . The direction and flow rate soluble in , or at least capable of penetrating into the biomass of the fluid ( s ) that is /are introduced can be selected accord and reacting with the radicals , such as a diene , such as 60 ing to a desired exposure rate and / or direction to control the 1 , 5 - cyclooctadiene . In some specific embodiments , the efficiency of the overall treatment , including effects that quenching includes contacting the biomass with an antioxi- result from both particle -based treatment and effects that are dant, such as Vitamin E . If desired , the biomass feedstock due to the interaction of dynamically generated species from can include an antioxidant dispersed therein , and the the introduced fluid with the material. In addition to air, quenching can come from contacting the antioxidant dis - 65 exemplary fluids that can be introduced into the ion beam persed in the biomass feedstock with the radicals . Combi- include oxygen , nitrogen , one or more noble gases, one or nations of these and other quenching materials can be used more halogens, and hydrogen . US 9 ,745 ,604 B2 35 36 Irradiating Low Bulk Density Biomass Materials and Cool reduce the bulk density of the biomass feedstock as it is ing Irradiated Biomass being processed and can aid in the cooling of the biomass During treatment of biomass materials with ionizing feedstock . For example , the biomass can be conveyed from radiation , especially at high dose rates , such as at rates a first belt at a first height above the ground and then can be greater then 0 . 15 Mrad per second , e . g . , 0 . 25 Mrad / s , 0 . 35 5 captured by a second belt at a second level above the ground Mrad /s , 0 .5 Mrad /s , 0 .75 Mrad /s or even greater than 1 lower than the first level . For example , in some embodi Mrad /sec , biomass materials can retain significant quantities ments , the trailing edge of the first belt and the leading edge of heat so that the temperature of the biomass materials of the second belt defining a gap . Advantageously , the become elevated . While higher temperatures can , in some ionizing radiation , such as a beam of electrons , protons , or embodiments , be advantageous , e . g . , when a faster reaction 10 other ions , can be applied at the gap to prevent damage to the rate is desired , it is advantageous to control the heating of the biomass conveyance system . biomass to retain control over the chemical reactions initi - In the methods described herein , cooling of the biomass ated by the ionizing radiation , such as cross - linking, chain can include contacting the biomass with a fluid , such as a scission and /or grafting , e . g ., to maintain process control. gas , at a temperature below the first or second temperature , Low bulk density materials , such as those having a bulk 15 such as gaseous nitrogen at or about 77 K . Even water , such density of less than about 0 . 4 g / cm " , e . g ., less than about as water at a temperature below nominal room temperature 0 .35 , 0 .25 or less about 0 . 15 g /cm " , especially when com - ( e . g ., 25 degrees Celsius ) can be utilized . bined with materials that have thin cross -sections , such as The biomass feedstock can be treated at a first tempera fibers having small transverse dimensions, are generally ture with ionizing radiation for a sufficient time and / or a easier to cool. In addition , photons and particles can gener - 20 sufficient dose , such as from about 1 second to about 10 ally penetrate further into and through materials having a seconds at a dose rate of about 0 . 5 Mrad / s to about 5 Mrad /s , relatively low bulk density , which can allow for the pro - to elevate the biomass feedstock to a second temperature cessing of larger volumes of materials at higher rates , and higher than the first temperature . After applying the radia can allow for the use of photons and particles that having tion , the biomass can be cooled below the second tempera lower energies , e . g ., 0 .25 Mev , 0 . 5 MeV , 0 .75 MeV or 1 . 0 25 ture . The cooled treated biomass is treated with radiation , MeV , which can reduce safety shielding requirements . Many such as an ionizing radiation , and then the treated biomass of the biomass materials described herein can be processed is contacted with a microorganism having the ability to in one or more of the systems shown in FIGS . 11B , 11C , 11D convert at least a portion , e . g . , at least about 1 percent by and 11E , which are described below . The systems shown weight, of the biomass to the product. allow one or more types of ionizing radiation , such as 30 In some embodiments , a method of changing a molecular relativistic electrons or electrons in combination with and / or a supramolecular structure of a biomass feedstock X - rays , to be applied to low bulk density biomass materials includes optionally , pretreating the biomass feedstock by at highs dose rates , such as at a rate greater than 1 . 0 , 1 . 5 , 2 . 5 reducing one or more dimensions of individual pieces of the Mrad / s or even greater than about 5 . 0 Mrad / s , and then to biomass feedstock and applying ionizing radiation , such as allow for cooling of the biomass prior to applying radiation 35 photons , electrons or ions , to the biomass feedstock . In such for a second , third , fourth , fifth , sixth , seventh , eighth , ninth embodiments , the biomass feedstock to which the ionizing or even a tenth time . radiation is applied has a bulk density of less than about 0 .35 For example , in one method of changing a molecular g / cm " , such as less than about 0 . 3 , 0 .25 , 0 .20 , or less than and / or a supramolecular structure of a biomass feedstock , about 0 . 15 g /cm3 during the application of the ionizing the biomass is pretreated at a first temperature with ionizing 40 radiation . In such embodiments , the biomass feedstock can radiation , such as photons, electrons or ions ( e . g ., singularly be cooled , and then ionizing radiation can be applied to the or multiply charged cations or anions ), for a sufficient time cooled biomass. In some advantageous embodiments, the and / or a sufficient dose to elevate the biomass feedstock to biomass feedstock is or includes discrete fibers and / or a second temperature higher than the first temperature. The particles having a maximum dimension of not more than pretreated biomass is then cooled below the second tem - 45 about 0 . 5 mm , such as not more than about 0 . 25 mm , not perature . Finally, if desired , the cooled biomass can be more than about 0 . 1 mm , not more than about 0 .05 mm , or treated one or more times with radiation , e . g . , with ionizing not more than about 0 .025 mm . radiation . If desired , cooling can be applied to the biomass Referring particularly now to FIGS . 11B and 11C , which after and /or during each radiation treatment. shows a biomass material generating , treating , conveying , In some embodiments , the cooling of the biomass feed - 50 and irradiating device 1170 (shielding not illustrated in the stock is to an extent that, after cooling , the biomass is at a drawings) . In operation , paper sheet 1173 , e . g ., scrap third temperature below the first temperature . bleached Kraft paper sheet , is supplied from a roll 1172 and For example , and as will be explained in more detail delivered to a fiberizing apparatus 1174 , such as a rotary below , treating biomass feedstock with the ionizing radia - shearer. The sheet 1173 is converted into fibrous material tion can be performed as the biomass feedstock is being 55 1112 and is delivered to a fiber- loading zone 1180 by pneumatically conveyed in a fluid , such as a in a gas, such conveyer 1178 . If desired , the fibers of the fibrous material as nitrogen or air. To aid in molecular weight breakdown can be separated , e . g ., by screening , into fractions having and / or functionalization of the materials , the gas can be different L / D ratios. In some embodiments , the fibrous saturated with any swelling agent described herein and / or material 1112 of generally a low bulk density and advanta water vapor. For example , acidic water vapor can be utilized . 60 geously thin cross -sections , is delivered continuously to To aid in molecular weight breakdown , the water can be zone 1180 , and in other embodiments , the fibrous material is acidified with an organic acid , such as formic , or acetic acid , delivered in batches . A blower 1182 in loop 1184 is posi or a mineral acid , such as sulfuric or hydrochloric acid . tioned adjacent to the fiber- loading zone 1180 and is capable For example , and as will be explained in more detail of moving a fluid medium , e . g . , air , at a velocity and volume below , the treating biomass feedstock with the ionizing 65 sufficient to pneumatically circulate the fibrous material radiation can be performed as the biomass feedstock falls 1112 in a direction indicated by arrow 1188 through loop under the influence of gravity . This procedure can effectively 1184 . US 9 ,745 ,604 B2 37 38 In some embodiments , the velocity of air traveling in the combine to cause turbulence in the bed portion . Ionizing loop is sufficient to uniformly disperse and transport the radiation is applied to the fluidized bed portion as the fibrous fibrous material around the entire loop 1184 . In some material is conveyed through the bed portion . For example , embodiments , the velocity of flow is greater than 2 ,500 as shown , three beams of electrons from three Rhodotron® feet/ minute , e. g ., 5 , 000 feet/ minute , 6 ,000 feet/ minute or 5 machines 11135 , 11136 and 11137 can be utilized . more , e . g . , 7 ,500 feet/ minute or 8 ,500 feet/ minute . Advantageously, each beam can penetrate into the fluid The entrained fibrous material 1112 traversing the loop passes an application zone 1190 , which forms part of loop ized bed a different depth and /or each beam can emit 1184 . Here , any desired additives described herein are electrons of a different energy, such as 1, 3 , and 5 MeV . As applied , such as a liquid , such as water , such as acidified or 10 the irradiated fibrous material moves through the system , it water made basic . In operation , application zone 1190 cools by the action of gases , such as air , circulating at high applies an additive , such as a liquid solution 1196 to the speeds in the system and it is bathed in reactive gases, such circulating fibrous material via nozzles 98 , 99 and 11100 . as ozone and /or oxides of nitrogen , that are produced from When a liquid is applied , the nozzles produce an atomized the action of the ionizing radiation on the circulating gases, spray or mist of, which impacts the fibers as the fibers pass 15 such as air . If desired , the process can be repeated a desired in proximity to the nozzles . Valve 11102 is operated to number of times until the fibrous material has received a control the flow of liquid to the respective nozzles 1198 , desired dose . While the fluidized bed has been illustrated 1199, and 11100 . After a desired quantity of additive is such that its long axis is horizontal with the ground , in other applied , the valve 11102 is closed . implementations, the long axis of the bed is perpendicular to In some embodiments , the application zone 1190 is two to 20 the ground so that the fibrous material falls under the one hundred feet long or more , e . g . , 125 feet , 150 feet , 250 influence of gravity . feet long or more , e . g . , 500 feet long . Longer application Referring particularly now to FIG . 11E , which shows zones allow for application of over a longer period of time another fibrous material conveying and irradiating device during passage of fibrous material through application zone 11140 without shielding . Fibrous material 11144 is delivered 1190 . In some embodiments , the nozzles are spaced apart 25 from a bin 11142 to a first conveyer 11150 at a first level from about three to about four feet along the length of loop above the ground and then the material is transferred to a 1184 . second conveyer 11152 at a lower height than the first As the fibrous material moves in loop 1184 and through conveyer. The trailing edge 11160 of the first conveyer and the irradiating portion of the loop 11107 that includes a horn the leading edge 11161 of the second conveyer 11152 defines 11109 for delivering ionizing radiation , ionizing radiation is 30 a gap with a spacing S . For example , the spacing S can be applied to the fibrous material (shielding is not shown ) . between 4 inches and about 24 inches . Material 11144 has As the irradiated fibrous material moves around loop enough momentum to free fall under gravity and then to be 1184 , it cools by the action of gases , such as air , circulating captured by the second conveyer 11152 without falling into at high speeds in the loop and it is bathed in reactive gases , the gap . During the free fall, ionizing radiation is applied to such as ozone and / or oxides of nitrogen , that are produced 35 the material . This arrangement can be advantageous in that from the action of the ionizing radiation on the circulating the ionizing radiation is less likely to damage the conveying gases , such as air. After passing through the irradiating system because is not directly contacted by the radiation . portion 11107 , a cooling fluid , such as a liquid ( e . g . , water ) After passing through the irradiating portion , a cooling or a gas, such as liquid nitrogen at 77 K can be injected into fluid , such as a liquid (e . g. , water ) or a gas, such as liquid loop 1184 to aid in the cooling of the fibrous material. This 40 nitrogen at 77 K can be applied to the material to aid in the process can be repeated more than one time if desired , e . g . , cooling of the fibrous material. This process can be repeated 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 times or more , e . g . , 15 times, to more than one time if desired , e . g . , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 deliver the desired dose to the fibrous material . While , as times or more , e . g . , 15 times , to deliver the desired dose to shown , the long axis of the horn is along the direction of the fibrous material . While , as shown , the long axis of the flow , in some implementations , the long axis of the horn is 45 horn is transverse to the direction of the material flow , other transverse to the direction of the flow . In some implemen - beam arrangements are possible . In some implementations , tations , a beam of electrons is utilized as a principal ionizing a beam of electrons is utilized as a principal ionizing radiation source and X - rays as a secondary ionizing radia - radiation source and X - rays as a secondary ionizing radia tion source . X -rays can be generated by having a metal tion source . X -rays can be generated by having a metal target , such as a tantalum target 11111 , on the inside of loop 50 target , such as a tantalum target, in the gap on the opposite 1184 such that when electrons strike the target, X - rays are side of the material, such that as the electrons that pass emitted . through the material they strike the target, generating After a desired dose is delivered to the fibrous material, X - rays . the fibrous material can be removed from loop 1184 via a In one example of the use of radiation with oxidation as separator 11112 , which is selectively connected to loop 1184 55 a pretreatment, half - gallon juice cartons made of un -printed by section 11114 and gate valve 11116 . When valve 11116 is polycoated white Kraft board having a bulk density of 20 opened , another valve is also opened to allow air to enter the lb / ft are used as a feedstock . Cartons are folded flat and then loop 1184 to compensate for air exiting through separator fed into a sequence of three shredder - shearer trains arranged 11112 . in series with output from the first shearer fed as input to the Referring particularly now to FIG . 11D , which shows a 60 second shredder, and output from the second shearer fed as fluidized bed fibrous irradiating device 11121 with shielding input to the third shredder. The fibrous material produced by Fibrous material in a fluid , such as a gas, such as air under the can be sprayed with water and processed through a pellet pressure , is delivered to a shielded containment vessel 11123 mill operating at room temperature . The densified pellets can via piping 11125 and into a shielded fluidized bed portion be placed in a glass ampoule which is sealed under an 11127 . Counter -current streams 11131 of fluid , such as a gas, 65 atmosphere of air . The pellets in the ampoule are irradiated and transverse streams 11133 of fluid , such as a gas , that is with gamma radiation for about 3 hours at a dose rate of the same or different as a fluid delivered counter -currently , about 1 Mrad per hour to provide an irradiated material in US 9 , 745 ,604 B2 39 40 which the cellulose has a lower molecular weight than the cation , it is possible to have a number average molecular fibrous Kraft starting material. weight of less than about 10 ,000 or even less than about Sonication 5 ,000 . One or more sonication processing sequences can be used In some embodiments , the second material can have a to treat biomass from a wide variety of different sources to 5 level of oxidation (“ O2 ) that is higher than the level of oxidation ( “ O , ) of the first material. A higher level of extract useful substances from the feedstock , and to provide oxidation of the material can aid in its dispersibility, partially degraded organic material which functions as input swellability and /or solubility , further enhancing the materi to further processing steps and / or sequences. Sonication can als susceptibility to chemical, enzymatic or microbial attack . reduce the recalcitrance , molecular weight, and /or crystal 10 In some embodiments , to increase the level of the oxidation linity of feedstock , such as one or more of any of the of the second material relative to the first material, the biomass materials described herein , e . g ., one or more car sonication is performed in an oxidizing medium , producing bohydrate sources , such as cellulosic or lignocellulosic" a second material that is more oxidized than the first materials , or starchy materials . material . For example , the second material can have more Referring again to FIG . 8 , in onemethod , a first biomassomass 15 hydroxyl groups, aldehyde groups, ketone groups, ester material 2 that includes cellulose having a first number groups or carboxylic acid groups, which can increase its ??average molecular weight (“ My? ) is dispersed in a medium , hydrophilicity . such as water, and sonicated and / or otherwise cavitated , to In some embodiments , the sonication medium is an aque provide a second biomass material 3 that includes cellulose ous medium . If desired , the medium can include an oxidant, having a second number average molecular weight ( “ MN2 ) 20 such as a peroxide ( e . g . , hydrogen peroxide ) , a dispersing lower than the first number average molecular weight. The agent and /or a buffer. Examples of dispersing agents include second material (or the first and second material in certain ionic dispersing agents , e . g . , sodium lauryl sulfate , and embodiments ) can be combined with a microorganism ( e . g ., non - ionic dispersing agents , e . g . , poly ( ethylene glycol) . a bacterium or a yeast ) that can utilize the second and / or first In other embodiments , the sonication medium is non material to produce a product 5 . 25 aqueous. For example , the sonication can be performed in a Since the second material has cellulose having a reduced hydrocarbon , e . g ., toluene or heptane , an ether, e . g ., diethyl molecular weight relative to the first material, and in some ether or tetrahydrofuran , or even in a liquefied gas such as instances , a reduced crystallinity as well, the second material argon , xenon , or nitrogen . is generally more dispersible , swellable , and /or soluble in a Without wishing to be bound by any particular theory, it 30 is believed that sonication breaks bonds in the cellulose by solution containing the microorganism , e. g. , at a concentra creating bubbles in the medium containing the cellulose , tion of greater than 10 microorganisms/ mL . These proper which grow and then violently collapse . During the collapse ties make the second material 3 more susceptible to chemi of the bubble , which can take place in less than a nanosec cal, enzymatic , and /or microbial attack relative to the first ond , the implosive force raises the local temperature within material 2 , which can greatly improve the productionon rate 35 the bubble to about 5100 K ( even higher in some instance ; and /or production level of a desired product, e. g ., ethanol. see, e . g. , Suslick et al. , Nature 434 , 52 - 55 ) and generates Sonication can also sterilize the materials , but should not be pressures of from a few hundred atmospheres to over 1000 used while the microorganisms are supposed to be alive . atmospheres or more . It is these high temperatures and In some embodiments, the second number average pressures that break the bonds . In addition , without wishing molecular weight ( My2 ) is lower than the first number 40 to be bound by any particular theory , it is believed that average molecular weight ( “ Mni) by more than about 10 reduced crystallinity arises, at least in part , from the percent, e . g . , 15 , 20 , 25 , 30 , 35 , 40 , 50 percent, 60 percent, extremely high cooling rates during collapse of the bubbles, or even more than about 75 percent. which can be greater than about 1011 K / second . The high In some instances , the second material has cellulose that cooling rates generally do not allow the cellulose to organize has a crystallinity ( " C , ) that is lower than the crystallinity 45 and crystallize , resulting in materials that have reduced ( FC ) of the cellulose of the first material. For example , crystallinity . Ultrasonic systems and sonochemistry are dis ( "C2 ) can be lower than (™C ) by more than about 10 cussed in , e . g . , Olli et al ., U .S . Pat. No. 5 , 766 , 764 ; Roberts , percent, e . g ., 15 , 20 , 25 , 30 , 35 , 40 , or even more than about U . S . Pat. No . 5 ,828 , 156 ; Mason , Chemistry with Ultra 50 percent. sound , Elsevier, Oxford , ( 1990 ) ; Suslick ( editor ) , Ultra In some embodiments , the starting crystallinity index 50 sound : its Chemical, Physical and Biological Effects , VCH , (prior to sonication ) is from about 40 to about 87 . 5 percent, Weinheim , ( 1988 ) ; Price , “ Current Trends in Sonochemis e . g ., from about 50 to about 75 percent or from about 60 to try ” Royal Society of Chemistry , Cambridge , ( 1992 ) ; Sus about 70 percent, and the crystallinity index after sonication lick et al. , Ann . Rev . Mater . Sci. 29 , 295 , ( 1999 ) ; Suslick et is from about 10 to about 50 percent, e . g . , from about 15 to al. , Nature 353 , 414 ( 1991) ; Hiller et al ., Phys. Rev . Lett . 69 , about 45 percent or from about 20 to about 40 percent. 55 1182 ( 1992 ) ; Barber et al. , Nature , 352 , 414 ( 1991) ; Suslick However , in certain embodiments , e . g . , after extensive soni et al. , J . Am . Chem . Soc ., 108 , 5641 ( 1986 ) ; Tang et al. , cation , it is possible to have a crystallinity index of lower Chem . Comm . , 2119 ( 2000 ) ; Wang et al. , Advanced Mater. , than 5 percent. In some embodiments , the material after 12 , 1137 (2000 ) ; Landau et al ., J . of Catalysis , 201, 22 sonication is substantially amorphous. ( 2001 ) ; Perkas et al ., Chem . Comm . , 988 ( 2001) ; Nikitenko In some embodiments , the starting number average 60 et al. , Angew . Chem . Inter. Ed . (December 2001 ) ; Shafi et molecular weight (prior to sonication ) is from about 200 ,000 al. , J. Phys . Chem B 103, 3358 ( 1999 ); Avivi et al. , J. Amer. to about 3 , 200 ,000 , e . g . , from about 250 , 000 to about Chem . Soc . 121 , 4196 ( 1999 ) ; and Avivi et al. , J . Amer. 1 ,000 ,000 or from about 250 ,000 to about 700 ,000 , and the Chem . Soc . 122 , 4331 ( 2000 ) . number average molecular weight after sonication is from Sonication Systems about 50 , 000 to about 200 , 000 , e . g . , from about 60 ,000 to 65 FIG . 12 shows a general system in which a biomass about 150 , 000 or from about 70 , 000 to about 125 ,000 . material stream 1210 is mixed with a water stream 1212 in However , in some embodiments , e . g ., after extensive soni a reservoir 1214 to form a process stream 1216 . A first pump US 9 , 745 ,604 B2 1218 draws process stream 1216 from reservoir 1214 and some embodiments , the flow rate of cooling fluid 1248 into toward a flow cell 1224 . Ultrasonic transducer 1226 trans heat exchanger 1246 is controlled to maintain an approxi mits ultrasonic energy into process stream 1216 as the mately constant temperature in reactor volume 1244 . In process stream flows through flow cell 1224 . A second pump addition or in the alternative , the temperature of cooling 1230 draws process stream 1216 from flow cell 1224 and 5 fluid 1248 flowing into heat exchanger 1246 is controlled to toward subsequent processing . maintain an approximately constant temperature in reactor Reservoir 1214 includes a first intake 1232 and a second volume 1244 . In some embodiments , the temperature of intake 1234 in fluid communication with a volume 1236 . A reactor volume 1244 is maintained at 20 to 50° C ., e . g ., 25 , conveyor ( not shown ) delivers biomass material stream 30 , 35 , 40 , or 45° C . Additionally or alternatively, heat 1210 to reservoir 1214 through first intake 1232 . Water 10 stream 1212 enters reservoir 1214 through second intake transferred to cooling fluid 1248 from reactor volume 1244 1234 . In some embodiments , water stream 1212 enters can be used in other parts of the overall process. volume 1236 along a tangent establishing a swirling flow An adapter section 1226 creates fluid communication within volume 1236 . In certain embodiments , biomass mate between reactor volume 1244 and a booster 1250 coupled rial stream 1210 and water stream 1212 are introduced into 15 ( e . g . , mechanically coupled using a flange ) to ultrasonic volume 1236 along opposing axes to enhance mixing within transducer 1226 . For example , adapter section 1226 can the volume. include a flange and O -ring assembly arranged to create a Valve 1238 controls the flow of water stream 1212 leak tight connection between reactor volume 1244 and through second intake 1232 to produce a desired ratio of booster 1250 . In some embodiments , ultrasonic transducer biomass material to water ( e . g . , approximately 10 % cellu - 20 1226 is a high -powered ultrasonic transducer made by losic material, weight by volume) . For example , 2000 tons / Hielscher Ultrasonics of Teltow , Germany. day of biomass can be combined with 1 million to 1 . 5 In operation , a generator 1252 delivers electricity to million gallons /day , e . g ., 1 .25 million gallons/ day , of water. ultrasonic transducer 1252 . Ultrasonic transducer 1226 Mixing of material biomass and water in reservoir 1214 is includes a piezoelectric element that converts the electrical controlled by the size of volume 1236 and the flow rates of 25 energy into sound in the ultrasonic range . In some embodi biomass and water into the volume. In some embodiments , ments , the materials are sonicated using sound having a volume 1236 is sized to create a minimum mixing residence frequency of from about 16 kHz to about 110 kHz, e . g ., from time for the biomass and water . For example , when 2000 about 18 kHz to about 75 kHz or from about 20 kHz to about tons /day of biomass and 1 .25 million gallons / day of water 40 kHz (e . g ., sound having a frequency of 20 kHz to 40 are flowing through reservoir 1214 , volume 1236 can be 30 kHz ) . about 32 ,000 gallons to produce a minimum mixing resi The ultrasonic energy is then delivered to the working dence time of about 15 minutes . medium through booster 1248 . Reservoir 1214 includes a mixer 1240 in fluid commu The ultrasonic energy traveling through booster 1248 in nication with volume 1236 .Mixer 1240 agitates the contents reactor volume 1244 creates a series of compressions and of volume 1236 to disperse biomass throughout the water in 35 rarefactions in process stream 1216 with an intensity sufii the volume. For example , mixer 1240 can be a rotating vane cient to create cavitation in process stream 1216 . Cavitation disposed in reservoir 1214 . In some embodiments , mixer disaggregates the cellulosic material dispersed in process 1240 disperses the biomass substantially uniformly through - stream 1216 . Cavitation also produces free radicals in the out the water. water of process stream 1216 . These free radicals act to Reservoir 1214 further includes an exit 1242 in fluid 40 further break down the cellulosic material in process stream communication with volume 1236 and process stream 1216 . 1216 . The mixture of biomass and water in volume 1236 flows out In general, 5 to 4000 MJ/mº , e . g. , 10 , 25 , 50 , 100 , 250 , of reservoir 1214 via exit 1242 . Exit 1242 is arranged near 500 , 750 , 1000 , 2000 , or 3000 MJ/ mº , of ultrasonic energy the bottom of reservoir 1214 to allow gravity to pull the is applied to process stream 16 flowing at a rate of about 0 . 2 mixture of biomass and water out of reservoir 1214 and into 45 m3/ s ( about 3200 gallons /min ). After exposure to ultrasonic process stream 1216 . energy in reactor volume 1244 , process stream 1216 exits First pump 1218 ( e . g . , any of several recessed impeller flow cell 1224 through outlet 1222 . Second pump 1230 vortex pumps made by Essco Pumps & Controls , Los moves process stream 1216 to subsequent processing ( e . g . , Angeles , Calif . ) moves the contents of process stream 1216 any of several recessed impeller vortex pumps made by toward flow cell 1224 . In some embodiments , first pump 50 Essco Pumps & Controls , Los Angeles, Calif. ) . 1218 agitates the contents of process stream 1216 such that While certain embodiments have been described , other the mixture of cellulosic material and water is substantially embodiments are possible . uniform at inlet 1220 of flow cell 1224 . For example , first A s an example , while process stream 1216 has been pump 1218 agitates process stream 1216 to create a turbu - described as a single flow path , other arrangements are lent flow that persists along the process stream between the 55 possible . In some embodiments for example , process stream first pump and inlet 1220 of flow cell 1224 . 1216 includes multiple parallel flow paths ( e . g . , flowing at Flow cell 1224 includes a reactor volume 1244 in fluid a rate of 10 gallon /min ) . In addition or in the alternative, the communication with inlet 1220 and outlet 1222 . In some multiple parallel flow paths of process stream 1216 flow into embodiments , reactor volume 1244 is a stainless steel tube separate flow cells and are sonicated in parallel ( e . g ., using capable of withstanding elevated pressures ( e . g . , 10 bars ) . In 60 a plurality of 16 kW ultrasonic transducers ) . addition or in the alternative , reactor volume 1244 includes As another example , while a single ultrasonic transducer a rectangular cross section . 1226 has been described as being coupled to flow cell 1224 , Flow cell 1224 further includes a heat exchanger 1246 in other arrangements are possible. In some embodiments, a thermal communication with at least a portion of reactor plurality of ultrasonic transducers 1226 are arranged in flow volume 1244 . Cooling fluid 1248 ( e . g . , water ) flows into 65 cell 1224 ( e . g . , ten ultrasonic transducers can be arranged in heat exchanger 1246 and absorbs heat generated when a flow cell 1224 ) . In some embodiments , the sound waves process stream 1216 is sonicated in reactor volume 1244 . In generated by each of the plurality of ultrasonic transducers US 9 ,745 ,604 B2 43 44 1226 are timed ( e . g ., synchronized out of phase with one 930 volt rms. The current flow through a transducer can vary another ) to enhance the cavitation acting upon process between zero and 3 . 5 ampere depending on the load imped stream 1216 . ance . At 930 volt rms the output motion is approximately 20 As another example , while a single flow cell 1224 has microns . The maximum difference in terminal voltage for been described , other arrangements are possible . In some 5 the same motional amplitude , therefore, can be 186 volt . embodiments , second pump 1230 moves process stream to Such a voltage difference can give rise to large circulating a second flow cell where a second booster and ultrasonic currents flowing between the transducers . The balancing transducer further sonicate process stream 1216 . As still another example , while reactor volume 1244 has unit 430 assures a balanced condition by providing equal been described as a closed volume, reactor volume 1244 is 10 current flow through the transducers , hence eliminating the open to ambient conditions in certain embodiments . In such possibility of circulating currents . The wire size of the embodiments , sonication pretreatment can be performed windings must be selected for the full load current noted substantially simultaneously with other pretreatment tech above and the maximum voltage appearing across a winding niques. For example , ultrasonic energy can be applied to input is 93 volt. process stream 1216 in reactor volume 1244 while electron 15 As an alternative to using ultrasonic energy , high - fre beams are simultaneously introduced into process stream quency, rotor- stator devices can be utilized . This type of 1216 . device produces high - shear , microcavitation forces , which As another example , while a flow through process has can disintegrate biomass in contact with such forces. Two been described , other arrangements are possible . In some commercially available high - frequency, rotor - stator disper embodiments , sonication can be performed in a batch pro - 20 sion devices are the SupratonTM devices manufactured by cess . For example , a volume can be filled with a 10 % Krupp Industrietechnik GmbH and marketed by Dorr -Oliver (weight by volume) mixture of biomass in water and Deutschland GmbH of Connecticut, and the DispaxTM exposed to sound with intensity from about 50 W / cm² to devices manufactured and marketed by Ika -Works , Inc. of about 600 W / cm , e . g . , from about 75 W / cm² to about 300 Cincinnati , Ohio . Operation of such a microcavitation W / cm2 or from about 95 W /cm² to about 200 W /cm² . 25 device is discussed in Stuart , U . S . Pat. No . 5 ,370 , 999 . Additionally or alternatively , the mixture in the volume can While ultrasonic transducer 1226 has been described as be sonicated from about 1 hour to about 24 hours, e . g . , from including one or more piezoelectric active elements to create about 1. 5 hours to about 12 hours, or from about 2 hours to ultrasonic energy, other arrangements are possible . In some about 10 hours. In certain embodiments , the material is embodiments, ultrasonic transducer 1226 includes active sonicated for a pre - determined time, and then allowed to 30 elements made of other types of magnetostrictive materials stand for a second pre - determined time before sonicating ( e . g . , ferrous metals ) . Design and operation of such a again . high -powered ultrasonic transducer is discussed in Hansen Referring now to FIG . 13 , in some embodiments , two et al ., U . S . Pat. No . 6 ,624 , 539 . In some embodiments , electro - acoustic transducers are mechanically coupled to a ultrasonic energy is transferred to process stream 16 through single horn . As shown , a pair of piezoelectric transducers 60 35 an electro -hydraulic system . and 62 is coupled to a slotted bar horn 64 by respective While ultrasonic transducer 1226 has been described as intermediate coupling horns 70 and 72 , the latter also being using the electromagnetic response of magnetorestrictive known as booster horns . The mechanical vibrations pro - materials to produce ultrasonic energy , other arrangements vided by the transducers , responsive to high frequency are possible . In some embodiments , acoustic energy in the electrical energy applied thereto , are transmitted to the 40 form of an intense shock wave can be applied directly to respective coupling horns, which can be constructed to process stream 16 using an underwater spark . In some provide a mechanical gain , such as a ratio of 1 to 1 . 2 . The embodiments , ultrasonic energy is transferred to process horns are provided with a respective mounting flange 74 and stream 16 through a thermo -hydraulic system . For example , 76 for supporting the transducer and horn assembly in a acoustic waves of high energy density can be produced by stationary housing . 45 applying power across an enclosed volume of electrolyte , The vibrations transmitted from the transducers through thereby heating the enclosed volume and producing a pres the coupling or booster horns are coupled to the input sure rise that is subsequently transmitted through a sound surface 78 of the horn and are transmitted through the horn propagation medium ( e . g . , process stream 1216 ) . Design and to the oppositely disposed output surface 80 , which , during operation of such a thermo -hydraulic transducer is discussed operation , is in forced engagement with a workpiece (not 50 in Hartmann et al. , U . S . Pat. No. 6 ,383 , 152 . shown ) to which the vibrations are applied . Pyrolysis The high frequency electrical energy provided by the One or more pyrolysis treatment sequences can be used to power supply 82 is fed to each of the transducers , electri- process biomass from a wide variety of different sources to cally connected in parallel , via a balancing transformer 84 extract useful substances from the biomass , and to provide and a respective series connected capacitor 86 and 90 , one 55 partially degraded organic material which functions as input capacitor connected in series with the electrical connection to further processing steps and /or sequences . to each of the transducers . The balancing transformer is Referring again to the general schematic in FIG . 8 , a first known also as “ balun ” standing for “ balancing unit. ” The biomass material 2 that includes having a first number balancing transformer includes a magnetic core 92 and a pair average molecular weight (" My ) is pyrolyzed , e .g . , by of identical windings 94 and 96 , also termed the primary 60 heating the first material in a tube furnace , to provide a winding and secondary winding , respectively . second material 3 that includes cellulose having a second In some embodiments , the transducers include commer number average molecular weight ( Myz ) lower than the cially available piezoelectric transducers, such as Branson first number average molecular weight. The second material Ultrasonics Corporation models 105 or 502 , each designed (or the first and second material in certain embodiments ) for operation at 20 kHz and a maximum power rating of 3 65 is /are combined with a microorganism ( e . g . , a bacterium or kW . The energizing voltage for providing maximum a yeast ) that can utilize the second and / or first material to motional excursion at the output surface of the transducer is produce a product 5 that. US 9 ,745 ,604 B2 45 46 Since the second biomass material has cellulose having a As described above , the dry biomass from the feed source reduced molecular weight relative to the first material, and can be pre -processed prior to delivery to the pyrolysis in some instances, a reduced crystallinity as well , the second chamber . For example , if the biomass is derived from plant material is generally more dispersible , swellable and /or sources, certain portions of the plant material can be soluble in a solution containing the microorganism , e . g . , at 5 removed prior to collection of the plant material and / or a concentration of greater than 100 microorganisms/ mL . before the plant material is delivered by the feedstock These properties make the second material 3 more suscep - transport device . Alternatively , or in addition , the biomass tible to chemical, enzymatic and /or microbial attack relative feedstock can be subjected to mechanical processing 6020 to the first material 2 , which can greatly improve the (e . g ., to reduce the average length of fibers in the feedstock ) production rate and /or production level of a desired product , 10 prior to delivery to the pyrolysis chamber. e . g ., ethanol. Pyrolysis can also sterilize the first and second Following mechanical processing, the biomass undergoes materials . a moisture adjustment step 6030 . The nature of the moisture In some embodiments , the second number average adjustment step depends upon the moisture content of the molecular weight ( “MN2 ) is lower than the first number mechanically processed biomass. Typically, pyrolysis of average molecular weight ( MN ) by more than about 10 15 biomass occurs most efficiently when the moisture content percent, e . g . , 15 , 20 , 25 , 30 , 35, 40 , 50 percent , 60 percent, of the feedstock is between about 10 % and about 30 % ( e . g . , or even more than about 75 percent. between 15 % and 25 % ) by weight of the feedstock . If the In some instances, the second material has cellulose that moisture content of the feedstock is larger than about 40 % has a crystallinity ( TC , ) that is lower than the crystallinity by weight, the extra thermal load presented by the water ( C ) of the cellulose of the first material. For example , 20 content of the biomass increases the energy consumption of (TC ) can be lower than ( " C ) by more than about 10 subsequent pyrolysis steps. percent, e . g . , 15 , 20 , 25 , 30 , 35 , 40 , or even more than about In some embodiments , if the biomass has a moisture 50 percent. content which is larger than about 30 % by weight, drier In some embodiments , the starting crystallinity (prior to biomass material 6220 , which has a low moisture content , pyrolysis ) is from about 40 to about 87 . 5 percent, e . g . , from 25 can be blended in , creating a feedstock mixture in step 6030 about 50 to about 75 percent or from about 60 to about 70 with an average moisture content that is within the limits percent, and the crystallinity index after pyrolysis is from discussed above . In certain embodiments , biomass with a about 10 to about 50 percent, e. g. , from about 15 to about 45 high moisture content can simply be dried by dispersing the percent or from about 20 to about 40 percent . However, in biomass material on a moving conveyor that cycles the certain embodiments , e . g . , after extensive pyrolysis , it is 30 biomass through an in -line heating unit . The heating unit possible to have a crystallinity index of lower than 5 percent. evaporates a portion of the water present in the feedstock . In some embodiments , the material after pyrolysis is sub - In some embodiments, if the biomass from step 6020 has stantially amorphous. a moisture content which is too low ( e . g ., lower than about In some embodiments , the starting number average 10 % by weight) , the mechanically processed biomass can be molecular weight (prior to pyrolysis ) is from about 200 ,000 35 combined with wetter feedstock material 6230 with a higher to about 3 , 200 , 000 , e . g . , from about 250 ,000 to about moisture content , such as sewage sludge . Alternatively , or in 1, 000 ,000 or from about 250 , 000 to about 700 , 000 , and the addition , water 6240 can be added to the dry biomass from number average molecular weight after pyrolysis is from step 6020 to increase its moisture content. about 50 ,000 to about 200 , 000 , e . g . , from about 60 ,000 to In step 6040 , the biomass — now with its moisture content about 150 , 000 or from about 70 , 000 to about 125 , 000 . 40 adjusted to fall within suitable limits can be preheated in However, in some embodiments, e . g ., after extensive an optional preheating step 6040. Treatment step 6040 can pyrolysis , it is possible to have a number average molecular be used to increase the temperature of the biomass to weight of less than about 10 ,000 or even less than about between 75º C . and 150° C . in preparation for subsequent 5 ,000 . pyrolysis of the biomass . Depending upon the nature of the In some embodiments, the second material can have a 45 biomass and the particular design of the pyrolysis chamber, level of oxidation ( " O , ) that is higher than the level of preheating the biomass can ensure that heat distribution oxidation (10 ) of the first material. A higher level of within the biomass feedstock remains more uniform during oxidation of the material can aid in its dispersibility, pyrolysis , and can reduce the thermal load on the pyrolysis swellability and / or solubility , further enhancing the materi- chamber . als susceptibility to chemical, enzymatic or microbial attack . 50 The feedstock is then transported to a pyrolysis chamber In some embodiments , to increase the level of the oxidation to undergo pyrolysis in step 6050 . In some embodiments , of the second material relative to the first material, the transport of the feedstock is assisted by adding one or more pyrolysis is performed in an oxidizing environment, pro - pressurized gases 6210 to the feedstock stream . The gases ducing a second material that is more oxidized than the first create a pressure gradient in a feedstock transport conduit, material. For example , the second material can have more 55 propelling the feedstock into the pyrolysis chamber ( and hydroxyl groups , aldehyde groups, ketone groups, ester even through the pyrolysis chamber ) . In certain embodi groups or carboxylic acid groups, which can increase its ments , transport of the feedstock occurs mechanically ; that hydrophilicity . is , a transport system that includes a conveyor such as an In some embodiments , the pyrolysis of the materials is auger transports the feedstock to the pyrolysis chamber. continuous . In other embodiments , the material is pyrolyzed 60 Other gases 6210 can also be added to the feedstock prior for a pre -determined time, and then allowed to cool for a to the pyrolysis chamber. In some embodiments , for second pre -determined time before pyrolyzing again . example , one or more catalyst gases can be added to the Pyrolysis Systems feedstock to assist decomposition of the feedstock during FIG . 14 shows a process flow diagram 6000 that includes pyrolysis . In certain embodiments , one or more scavenging various steps in a pyrolytic feedstock pretreatment system . 65 agents can be added to the feedstock to trap volatile mate In first step 6010 , a supply of dry feedstock is received from rials released during pyrolysis . For example , various sulfur a feed source . based compounds such as sulfides can be liberated during US 9 ,745 ,604 B2 48 pyrolysis , and an agent such as hydrogen gas can be added In some embodiments , pyrolysis of the feedstock occurs to the feedstock to cause desulfurization of the pyrolysis substantially in the absence of oxygen and other reactive products. Hydrogen combines with sulfides to form hydro - gases . Oxygen can be removed from the pyrolysis chamber gen sulfide gas , which can be removed from the pyrolyzed by periodic purging of the chamber with high pressure feedstock . 5 nitrogen ( e . g ., at nitrogen pressures of 2 bar or more ). Pyrolysis of the feedstock within the chamber can include Following purging of the chamber , a gas mixture present in heating the feedstock to relatively high temperatures to cause partial decomposition of the feedstock . Typically , the the pyrolysis chamber ( e . g . , during pyrolysis of the feed feedstock is heated to a temperature in a range from 150° C . stock ) can include less than 4 mole % oxygen ( e . g ., less than to 1100° C . The temperature to which the feedstock is heated 1011 mole % oxygen , and even less than 0 . 5 mole % oxygen ) . depends upon a number of factors , including the composi The absence of oxygen ensures that ignition of the feedstock tion of the feedstock , the feedstock average particle size , the does not occur at the elevated pyrolysis temperatures. moisture content, and the desired pyrolysis products . For In certain embodiments , relatively small amounts of oxy many types of biomass feedstock , for example , pyrolysis gen can be introduced into the feedstock and are present temperatures between 300° C . and 550° C . are used . 15 during pyrolysis . This technique is referred to as oxidative The residence time of the feedstock within the pyrolysis pyrolysis . Typically, oxidative pyrolysis occurs in multiple chamber generally depends upon a number of factors , heating stages . For example , in a first heating stage , the including the pyrolysis temperature , the composition of the feedstock is heated in the presence of oxygen to cause partial feedstock , the feedstock average particle size, the moisture oxidation of the feedstock . This stage consumes the avail content, and the desired pyrolysis products . In some embodi- 20 able oxygen in the pyrolysis chamber . Then , in subsequent ments , feedstock materials are pyrolyzed at a temperature heating stages , the feedstock temperature is further elevated . just above the decomposition temperature for the material in With all of the oxygen in the chamber consumed , however, an inert atmosphere , e . g . , from about 2° C . above to about feedstock combustion does not occur, and combustion - free 10° C . above the decomposition temperature or from about pyrolytic decomposition of the feedstock ( e . g ., to generate 3° C . above to about 7° C . above the decomposition tem - 25 hydrocarbon products ) occurs. In general, the process of perature . In such embodiments , the material is generally heating feedstock in the pyrolysis chamber to initiate kept at this temperature for greater than 0 .5 hours , e. g ., decomposition is endothermic . However, in oxidative greater than 1 . 0 hours or greater than about 2 . 0 hours . In other embodiments , the materials are pyrolyzed at a tem pyrolysis , formation of carbon dioxide by oxidation of the perature well above the decomposition temperature for the 30 feedstock is an exothermic process . The heat released from material in an inert atmosphere , e . g . , from about 75° C . carbon dioxide formation can assist further pyrolysis heating above to about 175° C . above the decomposition tempera stages , thereby lessening the thermal load presented by the ture or from about 85° C . above to about 150° C . above the feedstock . decomposition temperature. In such embodiments , the mate In some embodiments , pyrolysis occurs in an inert envi rial is generally kept at this temperature for less than 0 . 5 35 ronment , such as while feedstock materials are bathed in hour . e . g ., less 20 minutes, less than 10 minutes , less than 5 argon or nitrogen gas . In certain embodiments , pyrolysis can minutes or less than 2 minutes . In still other embodiments. occur in an oxidizing environment, such as in air or argon the materials are pyrolyzed at an extreme temperature , e . g . , enriched in air. In some embodiments , pyrolysis can take from about 200° C . above to about 500° C . above the place in a reducing environment, such as while feedstock decomposition temperature of the material in an inert envi- 40 materials are bathed in hydrogen gas. To aid pyrolysis, ronment or from about 250° C . above to about 400° C . above various chemical agents, such as oxidants , reductants , acids the decomposition temperature . In such embodiments , the or bases can be added to the material prior to or during material us generally kept at this temperature for less than 1 pyrolysis . For example , sulfuric acid can be added , or a minute , e . g . , less than 30 seconds, less than 15 seconds, less peroxide ( e . g . , benzoyl peroxide ) can be added . than 10 seconds , less than 5 seconds , less than 1 second or 45 As discussed above , a variety of different processing less than 500 ms. Such embodiments are typically referred conditions can be used , depending upon factors such as the to as flash pyrolysis . feedstock composition and the desired pyrolysis products . In some embodiments , the feedstock is heated relatively For example, for cellulose - containing feedstock material, rapidly to the selected pyrolysis temperature within the relatively mild pyrolysis conditions can be employed , chamber . For example, the chamber can be designed to heat 50 including flash pyrolysis temperatures between 375° C . and the feedstock at a rate of between 500° C ./ s and 11, 000° C ./ s . 450° C ., and residence times of less than 1 second . As Typical heating rates for biomass - derived feedstock material another example, for organic solid waste material such as are from 500° C ./ s to 1000° C ./ s , for example . sewage sludge, flash pyrolysis temperatures between 500° A turbulent flow of feedstock material within the pyroly . C . and 650° C . are typically used , with residence times of sis chamber is usually advantageous, as it ensures relatively 55 between 0 . 5 and 3 seconds. In general, many of the pyrolysis efficient heat transfer to the feedstock material from the process parameters , including residence time, pyrolysis tem heating sub - system . Turbulent flow can be achieved by perature , feedstock turbulence , moisture content, feedstock blowing the feedstock material through the chamber using composition , pyrolysis product composition , and additive one or more injected carrier gases 6210 , for example . In gas composition can be regulated automatically by a system general, the carrier gases are relatively inert towards the 60 of regulators and an automated control system . feedstock material, even at the high temperatures in the Following pyrolysis step 6050 , the pyrolysis products pyrolysis chamber. Exemplary carrier gases include, for undergo a quenching step 6250 to reduce the temperature of example , nitrogen , argon , methane , carbon monoxide , and the products prior to further processing . Typically , quench carbon dioxide . Alternatively, or in addition , mechanical ing step 6250 includes spraying the pyrolysis products with transport systems such as augers can transport and circulate 65 streams of cooling water 6260 . The cooling water also forms the feedstock within the pyrolysis chamber to create a a slurry that includes solid , undissolved product material and turbulent feedstock flow . various dissolved products. Also present in the product US 9 ,745 ,604 B2 49 50 stream is a mixture that includes various gases, including After separation in step 6080 , the solid product material product gases, carrier gases, and other types of process 6110 is optionally subjected to a drying step 6120 that can gases . include evaporation of water. Solid material 6110 can then The product stream is transported via in - line piping to a be stored for later use , or subjected to further processing gas separator that performs a gas separation step 6060, in 5 steps , as appropriate . which product gases and other gases are separated from the The pyrolysis process parameters discussed above are slurry formed by quenching the pyrolysis products . The exemplary. In general , values of these parameters can vary widely according to the nature of the feedstock and the separated gas mixture is optionally directed to a blower desired products . Moreover, a wide variety of different 6130 , which increases the gas pressure by blowing air into 10 pyrolysis techniques, including using heat sources such as the mixture . The gas mixture can be subjected to a filtration hydrocarbon flames and / or furnaces , infrared lasers, micro step 6140 , in which the gas mixture passes through one or wave heaters , induction heaters , resistive heaters , and other more filters ( e . g . , activated charcoal filters ) to remove par heating devices and configurations can be used . ticulates and other impurities . In a subsequent step 6150 , the A wide variety of different pyrolysis chambers can be filtered gas can be compressed and stored for further usese . 15 used to decompose the feedstock . In some embodiments , for Alternatively , the filtered gas can be subjected to further example , pyrolyzing feedstock can include heating themate processing steps 6160 . For example , in some embodiments , rial using a resistive heating member , such as a metal the filtered gas can be condensed to separate different filament or metal ribbon . The heating can occur by direct gaseous compounds within the gas mixture . The different contact between the resistive heating member and the mate compounds can include , for example , various hydrocarbon 20 rial . products ( e . g . , alcohols , alkanes , alkenes, alkynes , ethers ) In certain embodiments, pyrolyzing can include heating produced during pyrolysis . In certain embodiments, the the material by induction , such as by using a Currie -Point filtered gas containing a mixture of hydrocarbon compo - pyrolyzer. In some embodiments , pyrolyzing can include nents can be combined with steam gas 6170 ( e . g . , a mixture heating the material by the application of radiation , such as of water vapor and oxygen ) and subjected to a cracking 25 infrared radiation . The radiation can be generated by a laser, process to reduce molecular weights of the hydrocarbon such as an infrared laser. components . In certain embodiments , pyrolyzing can include heating In some embodiments , the pyrolysis chamber includes thematerial with a convective heat. The convective heat can heat sources that burn hydrocarbon gases such as methane , be generated by a flowing stream of heated gas . The heated propane , and / or butane to heat the feedstock . A portion 6270 30 gas can be maintained at a temperature of less than about of the separated gases can be recirculated into the pyrolysis 1200° C ., such as less than 1000° C . , less than 750° C ., less chamber for combustion , to generate process heat to sustain than 600° C . , less than 400° C . or even less than 300° C . The the pyrolysis process . heated gas can be maintained at a temperature of greater than In certain embodiments , the pyrolysis chamber can about 250° C . The convective heat can be generated by a hot receive process heat that can be used to increase the tem - 35 body surrounding the first material, such as in a furnace . perature of feedstock materials . For example , irradiating In some embodiments , pyrolyzing can include heating the feedstock with radiation (e . g . , gamma radiation , electron material with steam at a temperature above about 250° C . beam radiation , or other types of radiation ) can heat the An embodiment of a pyrolysis chamber is shown in FIG . feedstock materials to relatively high temperatures . The 15 . Chamber 6500 includes an insulated chamber wall 6510 heated feedstock materials can be cooled by a heat exchange 40 with a vent 6600 for exhaust gases , a plurality of burners system that removes some of the excess heat from the 6520 that generate heat for the pyrolysis process , a transport irradiated feedstock . The heat exchange system can be duct 6530 for transporting the feedstock through chamber configured to transport some of the heat energy to the 6500 , augers 6590 for moving the feedstock through duct pyrolysis chamber to heat (or pre -heat ) feedstock material , 6530 in a turbulent flow , and a quenching system 6540 that thereby reducing energy cost for the pyrolysis process . 45 includes an auger 6610 for moving the pyrolysis products , The slurry containing liquid and solid pyrolysis products water jets 6550 for spraying the pyrolysis products with can undergo an optional de- watering step 6070 , in which cooling water , and a gas separator for separating gaseous excess water can be removed from the slurry via processes products 6580 from a slurry 6570 containing solid and liquid such as mechanical pressing and evaporation . The excess products . water 6280 can be filtered and then recirculated for further 50 Another embodiment of a pyrolysis chamber is shown in use in quenching the pyrolysis decomposition products in FIG . 16 . Chamber 6700 includes an insulated chamber wall step 6250 . 6710 , a feedstock supply duct 6720 , a sloped inner chamber The de -watered slurry then undergoes a mechanical sepa - wall 6730 , burners 6740 that generate heat for the pyrolysis ration step 6080 , in which solid product material 6110 is process, a vent 6750 for exhaust gases, and a gas separator separated from liquid product material 6090 by a series of 55 6760 for separating gaseous products 6770 from liquid and increasingly fine filters . In step 6100 , the liquid product solid products 6780 . Chamber 6700 is configured to rotate in material 6090 can then be condensed ( e . g . , via evaporation ) the direction shown by arrow 6790 to ensure adequate to remove waste water 6190 , and purified by processes such mixing and turbulent flow of the feedstock within the as extraction . Extraction can include the addition of one or chamber . more organic solvents 6180 , for example , to separate prod - 60 A further embodiment of a pyrolysis chamber is shown in ucts such as oils from products such as alcohols . Suitable FIG . 17 . Filament pyrolyzer 1712 includes a sample holder organic solvents include , for example, various hydrocarbons 1713 with resistive heating element 1714 in the form of a and halo - hydrocarbons. The purified liquid products 6200 wire winding through the open space defined by the sample can then be subjected to further processing steps . Waste holder 1713 . Optionally , the heated element can be spun water 6190 can be filtered if necessary , and recirculated for 65 about axis 1715 ( as indicated by arrow 1716 ) to tumble the further use in quenching the pyrolysis decomposition prod material that includes the cellulosic material in sample ucts in step 6250 . holder 1713 . The space 1718 defined by enclosure 1719 is US 9 , 745 ,604 B2 52 maintained at a temperature above room temperature , e .g ., efficient heat transfer. The material can be heated by thermal 200 to 250° C . In a typical usage , a carrier gas, e . g ., an inert transfer from a hot metal or ceramic , such as glass beads or gas, or an oxidizing or reducing gas , traverses through the sand in the reactor, and the resulting pyrolysis liquid or oil sample holder 1713 while the resistive heating element is can be transported to a central production plant to manu rotated and heated to a desired temperature , e . g ., 325° C . 5 facture a product . After an appropriate time, e . g ., 5 to 10 minutes, the pyro - Oxidation lyzed material is emptied from the sample holder . The One or more oxidative processing sequences can be used system shown in FIG . 17 can be scaled and made continu - to process raw biomass feedstock from a wide variety of ous. For example , rather than a wire as the heating member, different sources to extract useful substances from the feed the heating member can be an auger screw . Material can 10 stock , and to provide partially degraded organic material continuously fall into the sample holder , striking a heated which functions as input to further processing steps and /or screw that pyrolizes the material. At the same time, the sequences. screw can push the pyrolyzed material out of the sample Referring again to FIG . 8 , a first biomass material 2 that holder to allow for the entry of fresh , unpyrolyzed material. includes cellulose having a first number average molecular Another embodiment of a pyrolysis chamber is shown in 15 weight ( My ) and having a first oxygen content ( 0 , ) is FIG . 18 , which features a Curie -Point pyrolyzer 1820 that oxidized , e . g ., by heating the first material in a tube furnace includes a sample chamber 1821 housing a ferromagnetic in stream of air or oxygen - enriched air , to provide a second foil 1822 . Surrounding the sample chamber 1821 is an RF material 3 that includes cellulose having a second number coil 1823 . The space 1824 defined by enclosure 1825 is average molecular weight ( Mys) and having a second maintained at a temperature above room temperature , e . g ., 20 oxygen content ( 0 ) higher than the first oxygen content 200 to 250° C . In a typical usage , a carrier gas traverses (40 . ) . The second material (or the first and second material through the sample chamber 1821 while the foil 1822 is in certain embodiments ) can be, e . g . , combined with a inductively heated by an applied RF field to pyrolize the material , such as a microorganism , to provide a composite 4 , material at a desired temperature . Yet another embodiment or another product 5 . Providing a higher level of oxidation of a pyrolysis chamber is shown in FIG . 19 . Furnace 25 can improve dispersibility of the oxidized material, e . g . , in pyrolyzer 130 includes a movable sample holder 131 and a a solvent. furnace 132 . In a typical usage , the sample is lowered ( as Such materials can also be combined with a solid and /or indicated by arrow 137 ) into a hot zone 135 of furnace 132 , a liquid . For example , the liquid can be in the form of a while a carrier gas fills the housing 136 and traverses solution and the solid can be particulate in form . The liquid through the sample holder 131 . The sample is heated to the 30 and / or solid can include a microorganism , e . g ., a bacterium , desired temperature for a desired time to provide a pyro - and /or an enzyme. For example , the bacterium and /or lyzed product . The pyrolyzed product is removed from the enzyme can work on the cellulosic or lignocellulosic mate pyrolyzer by raising the sample holder ( as indicated by rial to produce a product, such as a protein . Exemplary arrow 134 ). products are described in FIBROUS MATERIALS AND In certain embodiments , as shown in FIG . 20 , a cellulosic 35 COMPOSITES , ” U . S . Ser. No . 11/ 453 , 951, filed Jun . 15 , target 140 can be pyrolyzed by treating the target , which is 2006 . housed in a vacuum chamber 141, with laser light, e . g . , light In some embodiments , the second number average having a wavelength of from about 225 nm to about 1500 molecular weight is not more 97 percent lower than the first nm . For example , the target can be ablated at 266 nm , using number average molecular weight, e . g ., not more than 95 the fourth harmonic of a Nd - YAG laser (Spectra Physics , 40 percent, 90 , 85 , 80 , 75 , 70 , 65 , 60 , 55 , 50 , 45 , 40 , 30 , 20 , GCR170 , San Jose , Calif. ) . The optical configuration shown 12 . 5 , 10 . 0 , 7 . 5 , 5 . 0 , 4 . 0 , 3 . 0 , 2 . 5 , 2 . 0 or not more than 1 . 0 allows the nearly monochromatic light 143 generated by the percent lower than the first number average molecular laser 142 to be directed using mirrors 144 and 145 onto the weight. The amount of reduction of molecular weight will target after passing though a lens 146 in the vacuum cham - depend upon the application . ber 141 . Typically , the pressure in the vacuum chamber is 45 For example , in some embodiments the starting number maintained at less than about 10 - mm Hg. In some embodi- average molecular weight ( prior to oxidation ) is from about ments , infrared radiation is used , e .g ., 1. 06 micron radiation 200 ,000 to about 3 ,200 ,000 , e. g ., from about 250 ,000 to from a Nd - YAG laser. In such embodiments , a infrared about 1, 000 ,000 or from about 250 ,000 to about 700 , 000 , sensitive dye can be combined with the cellulosic material to and the number average molecular weight after oxidation is produce a cellulosic target . The infrared dye can enhance the 50 from about 175 , 000 to about 3 , 000 , 000 , e . g . , from about heating of the cellulosic material. Laser ablation is described 200 , 000 to about 750 ,000 or from about 225 , 000 to about by Blanchet- Fincher et al. in U . S . Pat. No . 5 , 942 ,649 . 600 , 000 . Referring to FIG . 21, in some embodiments , a cellulosic Resins utilized can be thermosets or thermoplastics . material can be flash pyrolyzed by coating a tungsten Examples of thermoplastic resins include rigid and elasto filament 150 , such as a 5 to 25 mil tungsten filament, with 55 meric thermoplastics . Rigid thermoplastics include polyole the desired cellulosic material while the material is housed fins ( e . g ., polyethylene, polypropylene, or polyolefin copo in a vacuum chamber 151. To affect pyrolysis , current is lymers ) , polyesters ( e . g . , polyethylene terephthalate ) , passed through the filament, which causes a rapid heating of polyamides ( e . g ., nylon 6 , 6 / 12 or 6 / 10 ) , and polyethyl the filament for a desired time. Typically , the heating is eneimines. Examples of elastomeric thermoplastic resins continued for seconds before allowing the filament to cool. 60 include elastomeric styrenic copolymers (e . g ., styrene - eth In some embodiments , the heating is performed a number of ylene -butylene - styrene copolymers) , polyamide elastomers times to effect the desired amount of pyrolysis . (e . g. , polyether -polyamide copolymers ) and ethylene - vinyl In certain embodiments , carbohydrate -containing bio - acetate copolymer. mass material can be heated in an absence of oxygen in a In particular embodiments , lignin is utilized , e . g ., any fluidized bed reactor. If desired , the carbohydrate containing 65 lignin that is generated in any process described herein . biomass can have relatively thin cross - sections , and can In some embodiments , the thermoplastic resin has a melt include any of the fibrous materials described herein , for flow rate of between 10 g / 10 minutes to 60 g / 10 minutes , US 9 ,745 ,604 B2 53 54 e . g . , between 20 g / 10 minutes to 50 g / 10 minutes, or includes a polymer copolymerized with and /or grafted with between 30 g / 10 minutes to 45 g / 10 minutes , as measured maleic anhydride . Such materials are available from DuPont using ASTM 1238 . In certain embodiments , compatible under the trade name FUSABOND® . blends of any of the above thermoplastic resins can be used . Generally , oxidation of first material 200 occurs in an In some embodiments , the thermoplastic resin has a 5 oxidizing environment . For example , the oxidation can be polydispersity index (PDI ) , e . g . , a ratio of the weight aver effected or aided by pyrolysis in an oxidizing environment, age molecular weight to the number average molecular such as in air or argon enriched in air . To aid in the oxidation , weight, of greater than 1 . 5 , e . g . , greater than 2 . 0 , greater than various chemical agents , such as oxidants , acids or bases can 2 . 5 , greater than 5 . 0 , greater than 7 . 5 , or even greater than be added to the material prior to or during oxidation . For 10 . 0 . 10 example , a peroxide ( e . g . , benzoyl peroxide ) can be added In specific embodiments , polyolefins or blends of poly - prior to oxidation . olefins are utilized as the thermoplastic resin . Oxidation Systems Examples of thermosetting resins include natural rubber , FIG . 22 shows a process flow diagram 5000 that includes butadiene - rubber and polyurethanes . various steps in an oxidative feedstock pretreatment system . In some embodiments the starting number average 15 In first step 5010 , a supply of dry feedstock is received from molecular weight (prior to oxidation ) is from about 200 ,000 a feed source . The feed source can include , for example , a to about 3 , 200 , 000 , e . g . , from about 250 , 000 to about storage bed or container that is connected to an in - line 1 ,000 ,000 or from about 250 ,000 to about 700 ,000 , and the oxidation reactor via a conveyor belt or another feedstock number average molecular weight after oxidation is from transport device . about 50 , 000 to about 200 , 000 , e . g . , from about 60 ,000 to 20 As described above , the dry feedstock from the feed about 150 ,000 or from about 70 ,000 to about 125 , 000 . source can be pretreated prior to delivery to the oxidation However, in some embodiments , e . g ., after extensive oxi- reactor. For example , if the feedstock is derived from plant dation , it is possible to have a number average molecular sources, certain portions of the plant material can be weight of less than about 10 ,000 or even less than about removed prior to collection of the plant material and /or 5 ,000 . 25 before the plant material is delivered by the feedstock In some embodiments , the second oxygen content is at transport device . Alternatively , or in addition , the biomass least about five percent higher than the first oxygen content, feedstock can be subjected to mechanical processing ( e . g . , to e .g ., 7 .5 percent higher, 10 .0 percent higher, 12. 5 percent reduce the average length of fibers in the feedstock ) prior to higher , 15 .0 percent higher or 17 . 5 percent higher . In some deldelivery; to the oxidation reactor. preferred embodiments , the second oxygen content is at 30 Following mechanical processing 5020 , feedstock 5030 is least about 20 . 0 percent higher than the oxygen content of transported to a mixing system which introduces water 5150 the first material. Oxygen content is measured by elemental into the feedstock in a mechanical mixing process. Com analysis by pyrolyzing a sample in a furnace operating bining water with the processed feedstock in mixing step 1300° C . or higher . A suitable elemental analyzer is the 5040 creates an aqueous feedstock slurry 5050 , which can LECO CHNS- 932 analyzer with a VTF - 900 high tempera - 35 then be treated with one or more oxidizing agents . ture pyrolysis furnace . Typically , one liter of water is added to the mixture for In some embodiments , oxidation of first material 200 does every 0 .02 kg to 1 . 0 kg of dry feedstock . The ratio of not result in a substantial change in the crystallinity of the feedstock to water in the mixture depends upon the source cellulose. However , in some instances, e . g ., after extreme of the feedstock and the specific oxidizing agents used oxidation , the second material has cellulose that has as 40 further downstream in the overall process . For example , in crystallinity ( " C , ) that is lower than the crystallinity ( C ) of typical industrial processing sequences for lignocellulosic the cellulose of the first material. For example , ( C , ) can be biomass , aqueous feedstock slurry 5050 includes from about lower than (™C ) by more than about 5 percent, e . g. , 10 , 15, 0 .5 kg to about 1 .0 kg of dry biomass per liter of water . 20 , or even 25 percent. This can be desirable to enhance In some embodiments , one or more fiber- protecting addi solubility of the materials in a liquid , such as a liquid that 45 tives 5170 can also be added to the feedstock slurry in includes a bacterium and/ or an enzyme. feedstock mixing step 5040 . Fiber -protecting additives help In some embodiments, the starting crystallinity index to reduce degradation of certain types of biomass fibers ( e . g . , (prior to oxidation ) is from about 40 to about 87 .5 percent, cellulose fibers ) during oxidation of the feedstock . Fiber e . g ., from about 50 to about 75 percent or from about 60 to protecting additives can be used , for example , if a desired about 70 percent, and the crystallinity index after oxidation 50 product from processing a lignocellulosic feedstock includes is from about 30 to about 75 . 0 percent, e . g ., from about 35 .0 cellulose fibers . Exemplary fiber -protecting additives to about 70 . 0 percent or from about 37 . 5 to about 65 . 0 include magnesium compounds such as magnesium hydrox percent. However, in certain embodiments , e . g ., after exten - ide . Concentrations of fiber -protecting additives in feedstock sive oxidation , it is possible to have a crystallinity index of slurry 5050 can be from 0 . 1 % to 0 . 4 % of the dry weight of lower than 5 percent. In some embodiments , the material 55 the biomass feedstock , for example. after oxidation is substantially amorphous. In certain embodiments , aqueous feedstock slurry 5050 Without wishing to be bound by any particular theory , it can be subjected to an optional extraction 5180 with an is believed that oxidation increases the number of hydrogen - organic solvent to remove water- insoluble substances from bonding groups on the cellulose , such as hydroxyl groups, the slurry . For example, extraction of slurry 5050 with one aldehyde groups , ketone groups carboxylic acid groups or 60 or more organic solvents yields a purified slurry and an anhydride groups, which can increase its dispersibility and organic waste stream 5210 that includes water- insoluble or its solubility (e . g ., in a liquid ) . To further improve materials such as fats , oils , and other non - polar , hydrocar dispersibility in a resin , the resin can include a component bon - based substances . Suitable solvents for performing that includes hydrogen -bonding groups, such as one ormore extraction of slurry 5050 include various alcohols , hydro anhydride groups, carboxylic acid groups, hydroxyl groups, 65 carbons, and halo -hydrocarbons , for example . amide groups , amine groups or mixtures of any of these In some embodiments , aqueous feedstock slurry 5050 can groups . In some preferred embodiments , the component be subjected to an optional thermal treatment 5190 to further US 9 ,745 ,604 B2 55 56 prepare the feedstock for oxidation . An example of a thermal For example , in some embodiments , nascent oxygen can be treatment includes heating the feedstock slurry in the pres - produced from a reaction between NO and 0 , in a gas ence of pressurized steam . In fibrous biomass feedstock , the mixture or in solution . In certain embodiments, nascent pressurized steam swells the fibers , exposing a larger frac - oxygen can be produced from decomposition of HOCl in tion of fiber surfaces to the aqueous solvent and to oxidizing 5 solution . Other methods by which nascent oxygen can be agents that are introduced in subsequent processing steps. produced include via electrochemical generation in electro In certain embodiments , aqueous feedstock slurry 5050 lyte solution , for example . can be subjected to an optional treatment with basic agents In general, nascent oxygen is an efficient oxidizing agent 5200 . Treatment with one or more basic agents can help to due to the relatively high reactivity of the oxygen radical. separate lignin from cellulose in lignocellulosic biomass 10 However , nascent oxygen can also be a relatively selective feedstock , thereby improving subsequent oxidation of the oxidizing agent. For example , when lignocellulosic feed feedstock . Exemplary basic agents include alkali and alka stock is treated with nascent oxygen , selective oxidation of line earth hydroxides such as sodium hydroxide , potassium lignin occurs in preference to the other components of the hydroxide , and calcium hydroxide . In general, a variety of feedstock such as cellulose . As a result , oxidation of feed basic agents can be used , typically in concentrations from 15 stock with nascent oxygen provides a method for selective about 0 .01 % to about 0 . 5 % of the dry weight of the removal of the lignin fraction in certain feedstocks . Typi feedstock . cally , nascent oxygen concentrations ofbetween about 0 . 5 % Aqueous feedstock slurry 5050 is transported ( e . g ., by an and 5 % of the dry weight of the feedstock are used to effect in - line piping system ) to a chamber, which can be an efficient oxidation . oxidation preprocessing chamber or an oxidation reactor. In 20 Without wishing to be bound by theory , it is believed that oxidation preprocessing step 5060 , one or more oxidizing nascent oxygen reacts with lignocellulosic feedstock accord agents 5160 are added to feedstock slurry 5050 to form an ing to at least two different mechanisms. In a first mecha oxidizing medium . In some embodiments , for example , nism , nascent oxygen undergoes an addition reaction with oxidizing agents 5160 can include hydrogen peroxide . the lignin , resulting in partial oxidation of the lignin , which Hydrogen peroxide can be added to slurry 5050 as an 25 solubilizes the lignin in aqueous solution . As a result , the aqueous solution , and in proportions ranging from 3 % to solubilized lignin can be removed from the rest of the between 30 % and 35 % by weight of slurry 5050 . Hydrogen feedstock via washing . In a second mechanism , nascent peroxide has a number of advantages as an oxidizing agent. oxygen disrupts butane cross - links and / or opens aromatic For example , aqueous hydrogen peroxide solution is rela rings that are connected via the butane cross- links. As a tively inexpensive , is relatively chemically stable , is not 30 result , solubility of the lignin in aqueous solution increases , particularly hazardous relative to other oxidizing agents ( and and the lignin fraction can be separated from the remainder therefore does not require burdensome handling procedures of the feedstock via washing . and expensive safety equipment ) . Moreover, hydrogen per - In some embodiments , oxidizing agents 5160 include oxide decomposes to form water during oxidation of feed - ozone (03 ) . The use of ozone can introduce several chemi stock , so that waste stream cleanup is relatively straightfor - 35 cal- handling considerations in the oxidation processing ward and inexpensive . sequence . If heated too vigorously , an aqueous solution of In certain embodiments , oxidizing agents 5160 can ozone can decompose violently , with potentially adverse include oxygen ( e . g ., oxygen gas ) either alone , or in com - consequences for both human system operators and system bination with hydrogen peroxide. Oxygen gas can be equipment. Accordingly , ozone is typically generated in a bubbled into slurry 5050 in proportions ranging from 0 . 5 % 40 thermally isolated , thick -walled vessel separate from the to 10 % by weight of slurry 5050 . Alternatively , or in vessel that contains the feedstock slurry , and transported addition , oxygen gas can also be introduced into a gaseous thereto at the appropriate process stage . phase in equilibrium with slurry 5050 ( e . g . , a vapor head Without wishing to be bound by theory , it is believed that above slurry 5050 ) . The oxygen gas can be introduced into ozone decomposes into oxygen and oxygen radicals , and either an oxidation preprocessing chamber or into an oxi- 45 that the oxygen radicals ( e . g . , nascent oxygen ) are respon dation reactor (or into both ) , depending upon the configu - sible for the oxidizing properties of ozone in the manner ration of the oxidative processing system . Typically , for discussed above . Ozone typically preferentially oxidizes the example , the partial pressure of oxygen in the vapor above lignin fraction in lignocellulosic materials , leaving the cel slurry 5050 is larger than the ambient pressure of oxygen , lulose fraction relatively undisturbed . and ranges from 0 . 5 bar to 35 bar, depending upon the nature 50 Conditions for ozone -based oxidation of biomass feed of the feedstock . stock generally depend upon the nature of the biomass . For The oxygen gas can be introduced in pure form , or can be example , for cellulosic and / or lignocellulosic feedstocks, mixed with one or more carrier gases . For example , in some ozone concentrations of from 0 . 1 g /m² to 20 g /m3 of dry embodiments , high -pressure air provides the oxygen in the feedstock provide for efficient feedstock oxidation . Typi vapor. In certain embodiments , oxygen gas can be supplied 55 cally , the water content in slurry 5050 is between 10 % by continuously to the vapor phase to ensure that a concentra - weight and 80 % by weight ( e .g ., between 40 % by weight tion of oxygen in the vapor remains within certain prede and 60 % by weight) . During ozone - based oxidation , the termined limits during processing of the feedstock . In some temperature of slurry 5050 can be maintained between 0° C . embodiments , oxygen gas can be introduced initially in and 100° C . to avoid violent decomposition of the ozone . sufficient concentration to oxidize the feedstock , and then 60 In some embodiments , feedstock slurry 5050 can be the feedstock can be transported to a closed , pressurized treated with an aqueous , alkaline solution that includes one vessel ( e . g ., an oxidation reactor ) for processing. or more alkali and alkaline earth hydroxides such as sodium In certain embodiments , oxidizing agents 5160 can hydroxide , potassium hydroxide, and calcium hydroxide , include nascent oxygen (e . g ., oxygen radicals ) . Typically , and then treated thereafter with an ozone - containing gas in nascent oxygen is produced as needed in an oxidation 65 an oxidation reactor . This process has been observed to reactor or in a chamber in fluid communication with an significantly increase decomposition of the biomass in slurry oxidation reactor by one or more decomposition reactions. 5050 . Typically , for example , a concentration of hydroxide US 9 ,745 ,604 B2 57 58 ions in the alkaline solution is between 0. 001 % and 10 % by reactor , aliquots of the slurry can be extracted from the weight of slurry 5050 . After the feedstock has been wetted reactor at specific intervals and analyzed to determine con via contact with the alkaline solution , the ozone -containing centrations of particular products of interest such as complex gas is introduced into the oxidation reactor, where it contacts saccharides . Information about the increase in concentra and oxidizes the feedstock . tions of certain products in slurry 5050 as a function of time Oxidizing agents 5160 can also include other substances . can be used to determine residence times for particular In some embodiments , for example , halogen - based oxidiz classes of feedstock material. ing agents such as chlorine and oxychlorine agents ( e . g . In some embodiments. during oxidation of feedstock hypochlorite ) can be introduced into slurry 5050 . In certain embodiments, nitrogen - containing oxidizing substances can 10 hslurry 5050 , adjustment of the slurry pH can be performed be introduced into slurry 5050 . Exemplary nitrogen - contain by introducing one or more chemical agents into the oxida ing oxidizing substances include NO and NO2, for example . tion reactor. For example , in certain embodiments , oxidation Nitrogen - containing agents can also be combined with oxy occurs most efficiently in a pH range of about 9 - 11. To gen in slurry 5050 to create additional oxidizing agents . For maintain a pH in this range , agents such as alkali and example , NO and NO2 both combine with oxygen in slurry 15 alkaline. earth hydroxides , carbonates, ammonia , and alka 5050 to form nitrate compounds, which are effective oxi line buffer solutions can be introduced into the oxidation dizing agents for biomass feedstock . Halogen - and nitrogen reactor. based oxidizing agents can , in some embodiments , cause Circulation of slurry 5050 during oxidation can be impor bleaching of the biomass feedstock , depending upon the tant to ensure sufficient contact between oxidizing agents nature of the feedstock . The bleaching can be desirable for 20 5160 and the feedstock . Circulation of the slurry can be certain biomass -derived products that are extracted in sub achieved using a variety of techniques . For example , in sequent processing steps. some embodiments , a mechanical stirring apparatus that Other oxidizing agents can include , for example , various includes impeller blades or a paddle wheel can be imple peroxyacids , peroxyacetic acids, persulfates, percarbonates, mented in the oxidation reactor. In certain embodiments , the permanganates , osmium tetroxide , and chromium oxides . 25 oxidation reactor can be a loop reactor, in which the aqueous Following oxidation preprocessing step 5060 , feedstock solvent in which the feedstock is suspended is simultane slurry 5050 is oxidized in step 5070 . If oxidizing agents ously drained from the bottom of the reactor and recirculated 5160 were added to slurry 5050 in an oxidation reactor, then into the top of the reactor via pumping , thereby ensuring that oxidation proceeds in the same reactor. Alternatively, if the slurry is continually re -mixed and does not stagnate oxidizing agents 5160 were added to slurry 5050 in a 30 within the reactor. preprocessing chamber, then slurry 5050 is transported to an After oxidation of the feedstock is complete , the slurry is oxidation reactor via an in - line piping system . Once inside transported to a separation apparatus where a mechanical the oxidation reactor, oxidation of the biomass feedstock separation step 5080 occurs . Typically , mechanical separa proceeds under a controlled set of environmental conditions . tion step 5080 includes one or more stages of increasingly Typically , for example , the oxidation reactor is a cylindrical 35 fine filtering of the slurry to mechanically separate the solid vessel that is closed to the external environment and pres - and liquid constituents . surized . Both batch and continuous operation is possible , Liquid phase 5090 is separated from solid phase 5100 , although environmental conditions are typically easier to and the two phases are processed independently thereafter. control in in - line batch processing operations . Solid phase 5100 can optionally undergo a drying step 5120 Oxidation of feedstock slurry 5050 typically occurs at 40 in a drying apparatus , for example . Drying step 5120 can elevated temperatures in the oxidation reactor. For example , include , for example , mechanically dispersing the solid the temperature of slurry 5050 in the oxidation reactor is material onto a drying surface , and evaporating water from typically maintained above 100° C ., in a range from 120° C . solid phase 5100 by gentle heating of the solid material . to 240° C . For many types of biomass feedstock , oxidation Following drying step 5120 (or , alternatively , without under is particularly efficient if the temperature of slurry 5050 is 45 going drying step 5120 ) , solid phase 5100 is transported for maintained between 150° C . and 220° C . Slurry 5050 can be further processing steps 5140 . heating using a variety of thermal transfer devices. For L iquid phase 5090 can optionally undergo a drying step example, in some embodiments , the oxidation reactor con - 5110 to reduce the concentration of water in the liquid phase . tacts a heating bath that includes oil or molten salts . In In some embodiments, for example , drying step 5110 can certain embodiments , a series of heat exchange pipes sur- 50 include evaporation and/ or distillation and / or extraction of round and contact the oxidation reactor, and circulation of water from liquid phase 5090 by gentle heating of the liquid . hot fluid within the pipes heats slurry 5050 in the reactor. Alternatively , or in addition , one or more chemical drying Other heating devices that can be used to heat slurry 5050 agents can be used to remove water from liquid phase 5090 . include resistive heating elements, induction heaters, and Following drying step 5110 (or alternatively, without under microwave sources , for example . 55 going drying step 5110 ) , liquid phase 5090 is transported for The residence time of feedstock slurry 5050 in the oxi- further processing steps 5130 , which can include a variety of dation reactor can be varied as desired to process the chemical and biological treatment steps such as chemical feedstock . Typically , slurry 5050 spends from 1 minute to 60 and / or enzymatic hydrolysis . minutes undergoing oxidation in the reactor. For relatively Drying step 5110 creates waste stream 5220 , an aqueous soft biomass material such as lignocellulosic matter , the 60 solution that can include dissolved chemical agents such as residence time in the oxidation reactor can be from 5 acids and bases in relatively low concentrations . Treatment minutes to 30 minutes , for example , at an oxygen pressure of waste stream 5220 can include , for example , pH neutral of between 3 and 12 bars in the reactor, and at a slurry ization with one or more mineral acids or bases . Depending temperature of between 160° C . and 210° C . For other types upon the concentration of dissolved salts in waste stream of feedstock , however, residence times in the oxidation 65 5220 , the solution can be partially de - ionized ( e . g . , by reactor can be longer , e. g ., as long 48 hours. To determine passing the waste stream through an ion exchange system ) . appropriate residence times for slurry 5050 in the oxidation Then , the waste stream — which includes primarily water US 9 ,745 ,604 B2 59 60 can be re- circulated into the overall process ( e . g ., as water embodiments , theheating is performed a number of times to 5150 ), diverted to another process , or discharged . effect the desired amount of oxidation . Typically , for lignocellulosic biomass feedstocks follow - Referring again to FIG . 12 , in some embodiments , feed ing separation step 5070 , liquid phase 5090 includes a stock materials can be oxidized with the aid of sound and /or variety of soluble poly - and oligosaccharides , which can 5 cavitation . Generally , to effect oxidation , the materials are then be separated and /or reduced to smaller -chain saccha sonicated in an oxidizing environment, such as water satu rated with oxygen or another chemical oxidant, such as rides via further processing steps. Solid phase 5100 typically hydrogen peroxide . includes primarily cellulose, for example , with smaller Referring again to FIGS . 9 and 10 , in certain embodi amounts of hemicellulose - and lignin -derived products . 10 ments , ionizing radiation is used to aid in the oxidation of In some embodiments , oxidation can be carried out at feedstock materials . Generally , to effect oxidation , the mate elevated temperature in a reactor such as a pyrolysis cham rials are irradiated in an oxidizing environment, such as air ber. For example , referring again to FIG . 17, feedstock or oxygen . For example , gamma radiation and / or electron materials can be oxidized in filament pyrolyzer 1712 . In a beam radiation can be employed to irradiate the materials . typical usage, anan Oxidizingoxidizing carriercarrier gas , ec. g 8 .,. air orofan an 1515 Other Treatment ProcessesProc air /argon blend , traverses through the sample holder 1713 Steam explosion can be used alone without any of the while the resistive heating element is rotated and heated to processes described herein , or in combination with any of a desired temperature , e . g . , 325 oC . After an appropriate the processes described herein . time, e . g . , 5 to 10 minutes , the oxidized material is emptied FIG . 23 shows an overview of the entire process of from the sample holder. The system shown in FIG . 2 can be 20 converting a fiber source 400 into a product 450 , such as scaled and made continuous. For example , rather than a wire ethanol, by a process that includes shearing and steam as the heating member , the heating member can be an auger explosion to produce a fibrous material 401 , which is then screw . Material can continuously fall into the sample holder , hydrolyzed and converted , e . g . , fermented , to produce the striking a heated screw that pyrolizes the material. At the product. The fiber source can be transformed into the fibrous same time, the screw can push the oxidized material out of 25 material 401 through a number of possible methods , includ the sample holder to allow for the entry of fresh , unoxidized ing at least one shearing process and at least one steam material. explosion process. Referring again to FIG . 18 , feedstock materials can be For example , one option includes shearing the fiber oxidized in a Curie -Point pyrolyzer 1820 . In a typical usage , source , followed by optional screening step ( s ) and optional an oxidizing carrier gas traverses through the sample cham - 30 additional shearing step ( s ) to produce a sheared fiber source ber 1821 while the foil 1822 is inductively heated by an 402 , which can then be steam exploded to produce the applied RF field to oxidize the material at a desired tem - fibrous material 401. The steam explosion process is option perature . ally followed by a fiber recovery process to remove liquids Referring again to FIG . 19 , feedstock materials can be or the “ liquor ” 404 , resulting from the steam exploding oxidized in a furnace pyrolyzer 130 . In a typical usage , the 35 process . The material resulting from steam exploding the sample is lowered (as indicated by arrow 137 ) into a hot sheared fiber source can be further sheared by optional zone 135 of furnace 132 , while an oxidizing carrier gas fills additional shearing step ( s ) and /or optional screening step ( s ) . the housing 136 and traverses through the sample holder I n another method , the fibrous material 401 is first steam 131. The sample is heated to the desired temperature for a exploded to produce a steam exploded fiber source 410 . The desired time to provide an oxidized product. The oxidized 40 resulting steam exploded fiber source is then subjected to an product is removed from the pyrolyzer by raising the sample optional fiber recovery process to remove liquids , or the holder ( as indicated by arrow 134 ) . liquor. The resulting steam exploded fiber source can then be Referring again to FIG . 20 , feedstock materials can be sheared to produce the fibrous material. The steam exploded oxidized by forming a cellulosic target 140 , along with an fiber source can also be subject to one or more optional oxidant, such as a peroxide, and treating the target, which is 45 screening steps and / or one or more optional additional housed in a vacuum chamber 141, with laser light, e .g . , light shearing steps . The process of shearing and steam exploding having a wavelength of from about 225 nm to about 1600 the fiber source to produce the sheared and steam exploded nm . The optical configuration shown allows the monochro - fibrous material will be further discussed below . matic light 143 generated by the laser 142 to be directed The fiber source can be cut into pieces or strips of confetti using mirrors 144 and 145 onto the target after passing 50 material prior to shearing or steam explosion . The shearing though a lens 146 in the vacuum chamber 141 . Typically , the processes can take place in a dry ( e . g . , having less than 0 . 25 pressure in the vacuum chamber is maintained at less than percent by weight absorbed water ) , hydrated , or even while about 10 - ° mm Hg . In some embodiments , infrared radiation the material is partially or fully submerged in a liquid , such is used , e . g . , 1 . 06 micron radiation from a Nd- YAG laser. In as water or isopropanol. The process can also optimally such embodiments , a infrared sensitive dye can be combined 55 include steps of drying the output after steam exploding or with the cellulosic material to produce a cellulosic target. shearing to allow for additional steps of dry shearing or The infrared dye can enhance the heating of the cellulosic steam exploding . The steps of shearing , screening, and material. Laser treatment of polymers is described by steam explosion can take place with or without the presence Blanchet - Fincher et al. in U . S . Pat . No. 5 , 942 ,649 . of various chemicalchemie solutions . Referring again to FIG . 21 , feedstock materials can be 60 In a steam explosion process, the fiber source or the rapidly oxidized by coating a tungsten filament 150 , together sheared fiber source is contacted with steam under high with an oxidant, such as a peroxide , with the desired pressure , and the steam diffuses into the structures of the cellulosic materialwhile the material is housed in a vacuum fiber source ( e . g ., the lignocellulosic structures) . The steam chamber 151. To affect oxidation , current is passed through then condenses under high pressure thereby " wetting ” the the filament , which causes a rapid heating of the filament for 65 fiber source . The moisture in the fiber source can hydrolyze a desired time. Typically , the heating is continued for any acetyl groups in the fiber source ( e . g ., the acetyl groups seconds before allowing the filament to cool. In some in the hemicellulose fractions) , forming organic acids such US 9 ,745 ,604 B2 62 as acetic and uronic acids. The acids , in turn , can catalyze lowering the pH to about 5 by adding H2SO4. The overlimed the depolymerization of hemicellulose , releasing xylan and fibrous material can then be used as is without the removal limited amounts ofglucan . The “ wet” fiber source ( or of precipitates . As shown in FIG . 23 , the optional overliming sheared fiber source , etc . ) is then “ exploded ” when the step occurs just prior to the step ofhydrolysis of the sheared pressure is released . The condensed moisture instanta - 5 and steam exploded fibrous material, but it is also contem neously evaporates due to the sudden decrease in pressure plated to perform the overliming step after the hydrolysis and the expansion of the water vapor exerts a shear force step and prior to the fermenting step . upon the fiber source or sheared fiber source , etc . ) . A FIG . 24 depicts an example of a steam explosion appa sufficient shear force will cause the mechanical breakdown of the internal structures ( e . g ., the lignocellulosic structures ) 10 ratus 460 . The steam explosion apparatus 460 includes a of the fiber source . reaction chamber 462 , in which the fiber source and /or the The sheared and steam exploded fibrous material is then fibrous material placed through a fiber source inlet 464 . The converted into a useful product , such as ethanol. One reaction chamber is sealed by closing fiber source inlet valve method of converting the fibrous material is by hydrolysis to 465 . The reaction chamber further includes a pressurized produce fermentable sugars , 412, which are then fermented 15 steamstea inlet 466 that includes a steam valve 467 . The reaction to produce the product. Other known and unknown methods chamber further includes an explosive depressurization out of converting fibrous materials can also be used . let 468 that includes an outlet valve 469 in communication In some embodiments , prior to combining the microor with the cyclone 470 through the connecting pipe 472 . Once ganism , the sheared and steam exploded fibrous material the reaction chamber includes the fiber source and /or 401 is sterilized to kill any competing microorganisms that 20 sheared fiber source and is sealed by closing valves 465 , 467 can be on the fibrous material. For example , the fibrous and 469 , steam is delivered into the reaction chamber 462 by material can be sterilized by exposing the fibrous material to opening the steam inlet valve 467 allowing steam to travel radiation , such as infrared radiation , ultraviolet radiation , or through steam inlet 466 . Once the reaction chamber reaches an ionizing radiation , such as gamma radiation . The micro target temperature , which can take about 20 -60 seconds , the organisms can also be killed using chemical sterilants , such 25 holding time begins . The reaction temperature is held at the as bleach ( e . g ., sodium hypochlorite ), chlorhexidine , or target temperature for the desired holding time, which ethylene oxide . One method to hydrolyze the sheared and typically lasts from about 10 seconds to 5 minutes . At the steam exploded fibrous material is by the use of cellulases. end of the holding time period , outlet valve is open to allow Cellulases are a group of enzymes that act synergistically to for explosive depressurization to occur. The process of hydrolyze cellulose . Commercially available Accellerase® 30 explosive depressurization propels the contents of the reac 1000 enzyme complex , which contains a complex of tion chamber 462 out of the explosive depressurization enzymes that reduces lignocellulosic biomass into ferment- outlet 468 , through the connecting pipe 472 , and into the able sugars can also be used . cyclone 470 . The steam exploded fiber source or fibrous According to current understanding, the components of material then exits the cyclone in a sludge form into the cellulase include endoglucanases , exoglucanases ( cellobio - 35 collection bin 474 as much of the remaining steam exits the hydrolases ), and b - glucosidases ( cellobiases) . Synergism cyclone into the atmosphere through vent 476 . The steam between the cellulase components exists when hydrolysis by explosion apparatus further includes wash outlet 478 with a combination of two or more components exceeds the sum wash outlet valve 479 in communication with connecting of the activities expressed by the individual components. pipe 472 . The wash outlet valve 479 is closed during the use The generally accepted mechanism of a cellulase system 40 of the steam explosion apparatus 460 for steam explosion , (particularly of T . longibrachiahum ) on crystalline cellulose but opened during the washing of the reaction chamber 462 . is : endoglucanase hydrolyzes internal B - 1 , 4 - glycosidic The target temperature of the reaction chamber 462 is bonds of the amorphous regions , thereby increasing the preferably between 180 and 240 degrees Celsius or between number of exposed nonreducing ends. Exoglucanases then 200 and 220 degrees Celsius . The holding time is preferably cleave off cellobiose units from the nonreducing ends , which 45 between 10 seconds and 30 minutes , or between 30 seconds in turn are hydrolyzed to individual glucose units by b - glu - and 10 minutes , or between 1 minute and 5 minutes . cosidases . There are several configurations of both endo - Because the steam explosion process results in a sludge of and exo - glucanases differing in stereospecificities . In gen - steam exploded fibrous material, the steam exploded fibrous eral , the synergistic action of the components in various material can optionally include a fiber recovery process configurations is required for optimum cellulose hydrolysis . 50 where the “ liquor " is separated from the steam exploded Cellulases , however, are more inclined to hydrolyze the fibrous material. This fiber recovery step is helpful in that it amorphous regions of cellulose . A linear relationship enables further shearing and / or screening processes and can between crystallinity and hydrolysis rates exists whereby allow for the conversion of the fibrous material into a higher crystallinity indices correspond to slower enzyme product. The fiber recovery process occurs through the use hydrolysis rates . Amorphous regions of cellulose hydrolyze 55 of a mesh cloth to separate the fibers from the liquor. Further at twice the rate of crystalline regions. The hydrolysis of the drying processes can also be included to prepare the fibrous sheared and steam exploded fibrous material can be per - material or steam exploded fiber source for subsequent formed by any hydrolyzing biomass process . processing Steam explosion of biomass sometimes causes the for - Any processing technique described herein can be used at mation of by - products , e . g . , toxicants , that are inhibitory to 60 pressure above or below normal, earth - bound atmospheric microbial and enzymatic activities . The process of convert pressure . For example , any process that utilizes radiation , ing the sheared and steam exploded fibrous material into a sonication , oxidation , pyrolysis , steam explosion , or com product can therefore optionally include an overliming step binations of any of these processes to provide materials that prior to fermentation to precipitate some of the toxicants include a carbohydrate can be performed under high pres For example , the pH of the sheared and steam exploded 65 sure , which , can increase reaction rates. For example , any fibrous material can be raised to exceed the pH of 10 by process or combination of processes can be performed at a adding calcium hydroxide (Ca (OH ) 2 ) followed by a step of pressure greater than about greater than 25 MPa , e . g ., greater US 9 ,745 ,604 B2 63 64 than 50 MPa, 75 MPa , 100 MPa , 150 MPa, 200 MPa, 250 radiation emitted from , e . g . , 6ºCo pellets , can be combined MPa, 350 MPa , 500 MPa , 750 MPa, 1 ,000 MPa , or greater with an electron beam source and / or an ultrasonic wave than 1 ,500 MPa . source . Combinations of Irradiating, sonicating , and Oxidizing The radiation devices for pretreating biomass discussed Devices 5 above can also be combined with one or more devices that In some embodiments , it can be advantageous to combine perform one or more pyrolysis processing sequences. Such two or more separate irradiation , sonication , pyrolization , a combination can again have the advantage of higher throughput. Nevertheless, caution must be observed , as there and /or oxidation devices into a single hybrid machine . For can be conflicting requirements between some radiation such a hybrid machine, multiple processes can be performedde 10 processes and pyrolysis. For example , ultrasonic radiation in close juxtaposition or even simultaneously, with the devices can require the feedstock be immersed in a liquid benefit of increasing pretreatment throughput and potential oxidizing medium . On the other hand , as discussed previ cost savings. ously , it can be advantageous for a sample of feedstock For example , consider the electron beam irradiation and undergoing pyrolysis to be of a particular moisture content. sonication processes . Each separate process is effective in 15 In this case, the new systems automatically measure and lowering the mean molecular weight of cellulosic material monitor for a particular moisture content and regulate the by an order of magnitude or more , and by several orders of same Further, some or all of the above devices, especially magnitude when performed serially . the pyrolysis device , can be combined with an oxidation Both irradiation and sonication processes can be applied device as discussed previously . using a hybrid electron beam / sonication device as is illus- 20 Primary Processes (Processing Treated Biomass ) trated in FIG . 25 . Hybrid electron beam / sonication device Fermentation 2500 is pictured above a shallow pool (depth ~ 3 - 5 cm ) of a Generally , various microorganisms can produce a number slurry of cellulosic material 2550 dispersed in an aqueous, of useful products by operating on , e . g. , fermenting treated oxidant medium , such as hydrogen peroxide or carbamide biomass materials . For example, alcohols , organic acids , peroxide . Hybrid device 2500 has an energy source 2510 , 25 hydrocarbons , hydrogen , proteins or mixtures of any of which powers both electron beam emitter 2540 and sonica - these materials can be produced by fermentation or other tion horns 2530 . processes . Electron beam emitter 2540 generates electron beams, The microorganism can be a natural microorganism or an which pass though an electron beam aiming device 2545 to engineered microorganism . For example , the microorganism The 30 can be a bacterium , e . g ., a cellulolytic bacterium , a fungus , impact the slurry 2550 containing cellulosic material. The 30 e . g ., a yeast , a plant or a protist , e . g . , an algae, a protozoa or electron beam aiming device can be a scanner that sweeps a a fungus- like protist, e . g . , a slime mold . When the organisms beam over a range of up to about 6 feet in a direction are compatible, mixtures of organisms can be utilized . approximately parallel to the surface of the slurry 2550 . To aid in the breakdown of the treated biomass materials On either side of the electron beam emitter 23402540 areare 35 that include cellulose , one or more enzymes, e. g. , a cel sonication horns 2530 , which deliver ultrasonic wave energy lulolyticlulol enzyme can be utilized . In some embodiments , to the slurry 2550 . The sonication horns 2530 end in a materials that include cellulose are first treated with the detachable endpiece 2535 that is in contact with the slurry enzyme, e . g . , by combining thematerials and the enzyme in 2550 . an aqueous solution . This material can then be combined The sonication horns 2530 are at risk of damage from 40 with the microorganism . In other embodiments , the materi long -term residual exposure to the electron beam radiation . als that include the cellulose, the one or more enzymes and Thus, the horns can be protected with a standard shield 2520 , the microorganism are combined concurrently , e .g ., by com e . g . , made of lead or a heavy -metal - containing alloy such as bining in an aqueous solution . Lipowitz metal , which is impervious to electron beam Also , to aid in the breakdown of the treated biomass radiation . Precautions must be taken , however, to ensure that 45 materials , the treated biomass materials can be further the ultrasonic energy is not affected by the presence of the treated ( e . g ., post irradiation ) with heat, a chemical ( e . g . , shield . The detachable endpieces 2535 , are constructed of mineral acid , base or a strong oxidizer such as sodium the same material and attached to the horns 2530 , are used hypochlorite ) , and / or an enzyme. to be in contact with the cellulosic material 2550 and are During fermentation , sugars released from cellulolytic expected to be damaged . Accordingly , the detachable end - 50 hydrolysis or saccharification , are fermented to , e . g . , etha pieces 2535 are constructed to be easily replaceable . nol, by a fermenting microorganism such as yeast. Suitable A further benefit of such a simultaneous electron beam fermenting microorganisms have the ability to convert car and ultrasound process is that the two processes have bohydrates , such as glucose , xylose , arabinose , mannose , complementary results . With electron beam irradiation galactose, oligosaccharides , or polysaccharides into fermen alone , an insufficient dose can result in cross - linking of some 55 tation products . Fermenting microorganisms include strains of the polymers in the cellulosic material, which lowers the of the genus Saccharomyces spp . e. g ., Saccharomyces cer efficiency of the overall depolymerization process. Lower e visiae ( baker ' s yeast ), Saccharomyces distaticus, and Sac doses of electron beam irradiation and / or ultrasound radia - charomyces uvarum ; the genus Kluyveromyces , e . g . , species tion can also be used to achieve a similar degree of depo - Kluyveromyces marxianus, and Kluyveromyces fragilis ; the lymerization as that achieved using electron beam irradia - 60 genus Candida , e . g . , Candida pseudotropicalis , and Can tion and sonication separately . dida brassicae ; the genus Clavispora , e . g ., species Clav An electron beam device can also be combined with one ispora lusitaniae and Clavispora opuntiae ; the genus Pachy or more of high - frequency, rotor- stator devices , which can solen , e . g . , species Pachysolen lannophilus; the genus be used as an alternative to ultrasonic energy devices , and Bretannomyces , e . g . , species Bretannomyces clausenii ; the performs a similar function . 65 genus Pichia , e. g. , species Pichia stipitis ; and the genus Further combinations of devices are also possible . For Saccharophagus , e . g . , species Saccharophagus degradans example , an ionizing radiation device that produces gamma (Philippidis , 1996 , “ Cellulose Bioconversion Technology ” , US 9 , 745 ,604 B2 65 66 in Handbook on Bioethanol: Production and Utilization , 478 . 94 , Acremonium sp . CBS 265 . 95 , Acremonium per Wyman , ed ., Taylor & Francis , Washington , D .C ., 179 - 212 ). sicimm CBS 169 .65 , Acremonium acremonium AHU 9519 , Commercially available yeast include, for example , Red Cephalosporium sp . CBS 535 .71 , Acremonium brachype Star® /Lesaffre Ethanol Red ( available from Red Star /Lesaf - nium CBS 866 .73 , Acremonium dichromosporum CBS fre , USA ) ; FALI? (available from Fleischmann 's Yeast , a 5 683 .73 , Acremonium obclavatum CBS 311 . 74 , Acremonium division of Burns Philip Food Inc ., USA ) ; SUPERSTART® pinkertoniae CBS 157. 70 , Acremonium roseogriseum CBS (available from Alltech , now Lallemand ) ; GERT 134 .56 , Acremonium incoloratum CBS 146 .62 , and Acre STRAND® (available from Gert Strand AB , Sweden ); and FERMOL® (available from DSM Specialties ). monium fiuratum CBS 299 .70H . Cellulolytic enzymes can Bacteria that can ferment biomass to ethanol and other 10 also be obtained from Chrysosporium , preferably a strain of products include, e . g ., Zymomonas mobilis and Clostridium Chrysosporium lucknowense . Additionally , Trichoderma thermocellum ( Philippidis , 1996 , supra ) . Leschine et al. (particularly Trichoderma viride, Trichoderma reesei, and ( International Journal of Systematic and Evolutionary Trichoderma koningii ) , alkalophilic Bacillus ( see , for Microbiology 2002 , 52 , 1155 - 1160 ) describe an anaerobic , example , U . S . Pat . No. 3 , 844 ,890 and EP 458162 ), and mesophilic . cellulolytic bacterium from forest soil. 15 Streptomyces (see , e . g . , EP 458162 ) can be used . Clostridium phytofermentans sp . nov. , which converts cel- Cellulolytic enzymes produced using recombinant tech lulose to ethanol. nology can also be used ( see, e. g. , WO 2007/ 071818 and Fermentation of biomass to ethanol and other products WO 2006 / 110891 ) . can be carried out using certain types of thermophilic or The cellulolytic enzymes used can be produced by fer genetically engineered microorganisms, such as Thermoan - 20 mentation of the above -noted microbial strains on a nutrient aerobacter species, including T .mathranii , and yeast species medium containing suitable carbon and nitrogen sources and such as Pichia species . An example of a strain of T. inorganic salts , using procedures known in the art ( see , e . g . , mathranii is A3M4 described in Sonne -Hansen et al. ( Ap - Bennettand LaSure ( eds . ), More Gene Manipulations in plied Microbiology and Biotechnology 1993 , 38 , 537 - 541) Fungi. Academic Press . CA 1991 ) . Suitable media are or Ahring et al. ( Arch . Microbiol. 1997 , 168, 114 - 119 ) . 25 available from commercial suppliers or can be prepared Yeast and Zymomonas bacteria can be used for fermentation according to published compositions ( e . g ., in catalogues of or conversion . The optimum pH for yeast is from about pH the American Type Culture Collection ) . Temperature ranges 4 to 5 , while the optimum pH for Zymomonas is from about and other conditions suitable for growth and cellulase pro pH 5 to 6 . Typical fermentation times are about 24 to 96 duction are known in the art (see , e . g . , Bailey and Ollis , hours with temperatures in the range of 26° C . to 40° C ., 30 Biochemical Engineering Fundamentals , McGraw - Hill however thermophilic microorganisms prefer higher tem - Book Company ,NY , 1986 ) . peratures. Treatment of cellulose with cellulase is usually carried out Enzymes that break down biomass , such as cellulose , to at temperatures between 30° C . and 65° C . Cellulases are lower molecular weight carbohydrate - containing materials , active over a range of pH of about 3 to 7 . A saccharification such as glucose , are referred to as cellulolytic enzymes or 35 step can last e . g ., up to 120 hours . The cellulase enzyme cellulase ; this process is referred to an “ saccharification ” . dosage achieves a sufficiently high level of cellulose con These enzymes can be a complex of enzymes that act version . For example , an appropriate cellulase dosage is synergistically to degrade crystalline cellulose . Examples of typically between 5 . 0 and 50 Filter Paper Units ( FPU or LU ) cellulolytic enzymes include : endoglucanases , cellobiohy - per gram of cellulose . The FPU is a standard measurement drolases , and cellobiases ( ß -glucosidases ). For example , 40 and is defined and measured according to Ghose (1987 , Pure cellulosic substrate is initially hydrolyzed by endogluca - and Appl. Chem . 59 : 257 - 268 ) . nases at random locations producing oligomeric intermedi In particular embodiments , ACCELERASETM 1000 ates. These intermediates are then substrates for exo -split - (GENENCOR ) is utilized as the enzyme system at a loading ting glucanases such as cellobiohydrolase to produce of 0 .25 mL per gram of substrate . ACCELLERASE? 1000 cellobiose from the ends of the cellulose polymer . Cellobi- 45 enzyme complex is a multiple enzyme cocktail with multiple ose is a water- soluble B - 1 , 4 - linked dimer of glucose . Finally activities , mainly exoglucanase , endoglucanase , hemicellu cellobiase cleaves cellobiose to yield glucose . lase and beta- glucosidase . The cocktail has a minimum A cellulase is capable of degrading biomass and can be of endoglucanase activity of 2500 CMC U / g and a minimum fungal or bacterial origin . Suitable enzymes include cellu - beta - glucosidase activity of 400 PNPG U / g . The pH of the lases from the genera Bacillus, Pseudomonas , Humicola , 50 cocktail is from about 4 . 8 to about 5 .2 . In other particular Fusarium , Thielavia , Acremonium , Chrysosporium and embodiments , the enzyme system utilized is a blend of Trichoderma , and include species of Humicola , Coprinus, CELLUCLAST® 1 . 5 L and Novozyme 188 . For example , Thielavia , Fusarium , Myceliophihora, Acremonium , Cepha - 0 . 5 mL of CELLUCLAST® 1. 5 L and 0 .1 mL of Novozyme losporium , Scytalidium , Penicillium or Aspergillus ( see , e . g ., 188 can be used for each gram of substrate . When a higher hsEP 458162 ) , especially those produced by a strain selected 55 hemicellulase ( xylanase ) activity is desired , OPTIMASHTM from the species Humicola insolens ( reclassified as Scyta - BG can be utilized . lidium thermophilum , see , e . g ., U . S . Pat . No. 4 ,435 , 307 ) , Gasification Coprinus cinereus, Fusarium oxysporum , Myceliophthora In addition to using pyrolysis for pre - treatment of feed thermophila , Meripilus giganteus , Thielavia terrestris , Acre - stock , pyrolysis can also be used to process pre - treated monium sp ., Acremonium persicinum , Acremonium acremo - 60 feedstock to extract usefulmaterials . In particular , a form of nium , Acremonium brachypenium , Acremonium dichromo - pyrolysis known as gasification can be employed to generate sporum , Acremonium obclavatum , Acremonium fuel gases along with various other gaseous, liquid , and solid pinkertoniae, Acremonium roseogriseum , Acremonium products . To perform gasification , the pre - treated feedstock incoloratum , and Acremonium furatum ; preferably from the is introduced into a pyrolysis chamber and heated to a high species Humicola insolens DSM 1800 , Fusarium oxysporum 65 temperature , typically 700° C . or more . The temperature DSM 2672 , Ayceliophthora thermophila CBS 117 .65 , used depends upon a number of factors , including the nature Cephalosporium sp . RYM -202 , Acremonium sp . CBS of the feedstock and the desired products . US 9 ,745 ,604 B2 67 68 Quantities of oxygen ( e . g ., as pure oxygen gas and /or as effects . Most of the evaporator condensate can be returned to air ) and steam (e . g ., superheated steam ) are also added to the the process as fairly clean condensate with a small portion pyrolysis chamber to facilitate gasification . These com - split off to waste water treatment to prevent build -up of pounds react with carbon -containing feedstock material in a low -boiling point compounds . multiple - step reaction to generate a gas mixture called 5 Waste Water Treatment synthesis gas (or " syngas ” ) . Essentially , during gasification , Wastewater treatment is used to minimize makeup water a limited amount of oxygen is introduced into the pyrolysis requirements of the plant by treating process water for reuse chamber to allow some feedstock material to combust to within the plant. Wastewater treatment can also produce fuel form carbon monoxide and generate process heat. The ( e . g . , sludge and biogas ) that can be used to improve the process heat can then be used to promote a second reaction 10 overall efficiency of the ethanol production process . For that converts additional feedstock material to hydrogen and example , as described in further detail below , sludge and carbon monoxide. In a first step of the overall reaction ,heating the feedstock biogas can be used to create steam and electricity that can be material produces a char that can include a wide variety of used in various plant processes . different hydrocarbon - based species . Certain volatile mate - 15 Wastewater is initially pumped through a screen ( e . g . , a rials can be produced ( e . g . , certain gaseous hydrocarbon bar screen ) to remove large particles , which are collected in materials ) , resulting in a reduction of the overall weight of a hopper. In some embodiments , the large particles are sent the feedstock material. Then , in a second step of the reaction to a landfill. Additionally or alternatively , the large particles some of the volatile material that is produced in the first step are burned to create steam and / or electricity as described in reacts with oxygen in a combustion reaction to produce both 20 further detail below . In general , the spacing on the bar screen carbon monoxide and carbon dioxide . The combustion reac - is between 1 /4 inch to 1 inch spacing ( e . g ., 1 /2 inch spacing ) . tion releases heat , which promotes the third step of the The wastewater then flows to an equalization tank , where reaction . In the third step , carbon dioxide and steam ( e . g ., the organic concentration of the wastewater is equalized water ) react with the char generated in the first step to form during a retention time. In general, the retention time is carbon monoxide and hydrogen gas . Carbon monoxide can 25 between 8 hours and 36 hours ( e . g . , 24 hours ) . A mixer is also react with steam , in a water gas shift reaction , to form disposed within the tank to stir the contents of the tank . In carbon dioxide and further hydrogen gas. some embodiments , a plurality of mixers disposed through Gasification can be used as a primary process to generate out the tank are used to stir the contents of the tank . In products directly from pre - treated feedstock for subsequent certain embodiments, the mixer substantially mixes the transport and / or sale , for example . Alternatively , or in addi- 30 contents of the equalization tank such that conditions ( e . g . , tion , gasification can be used as an auxiliary process for wastewater concentration and temperature ) throughout the generating fuel for an overall processing system . The hydro - tank are uniform . gen - rich syngas that is generated via the gasification process first pump moves water from the equalization tank can be burned , for example , to generate electricity and /or through a liquid - to - liquid heat exchanger . The heat process heat that can be directed for use at other locations in 35 exchanger is controlled ( e . g . , by controlling the flow rate of the processing system . As a result , the overall processing fluid through the heat exchanger ) such that wastewater system can be at least partially self- sustaining. A number of exiting the heat exchanger is at a desired temperature for other products , including pyrolysis oils and gaseous hydro anaerobic treatment. For example , the desired temperature carbon -based substances , can also be obtained during and / or for anaerobic treatment can be between 40° C . to 60° C . following gasification ; these can be separated and stored or 40 After exiting the heat exchanger, the wastewater enters transported as desired . one or more anaerobic reactors . In some embodiments, the A variety of different pyrolysis chambers are suitable for concentration of sludge in each anaerobic reactor is the same gasification of pre - treated feedstock , including the pyrolysis as the overall concentration of sludge in the wastewater. In chambers disclosed herein . In particular , fluidized bed reach other embodiments, the anaerobic reactor has a higher tor systems, in which the pre - treated feedstock is fluidized in 45 concentration of sludge than the overall concentration of steam and oxygen /air , provide relatively high throughput sludge in the wastewater. and straightforward recovery of products . Solid char that A nutrient solution containing nitrogen and phosphorus is remains following gasification in a fluidized bed system ( or metered into each anaerobic reactor containing wastewater. in other pyrolysis chambers ) can be burned to generate The nutrient solution reacts with the sludge in the anaerobic additional process heat to promote subsequent gasification 50 reactor to produce biogas which can contain 50 % methane reactions . and have a heating value of approximately 12 ,000 British Processing Treated Biomass thermal units , or Btu , per pound ) . The biogas exits each Distillation anaerobic reactor through a vent and flows into a manifold , After fermentation , the resulting fluids can be distilled where a plurality of biogas streams are combined into a using , for example , a " beer column " to separate ethanol and 55 single stream . A compressor moves the stream of biogas to other alcohols from the majority of water and residual solids . a boiler or a combustion engine as described in further detail The vapor exiting the beer column can be 35 % by weight below . In some embodiments , the compressor also moves ethanol and fed to a rectification column. A mixture of nearly the single stream of biogas through a desulphurization azeotropic (92 .5 % ) ethanol and water from the rectification catalyst. Additionally or alternatively , the compressor can column can be purified to pure ( 99 . 5 % ) ethanol using 60 move the single stream of biogas through a sediment trap . vapor- phase molecular sieves . The beer column bottoms can A second pump moves anaerobic effluent from the anaero be sent to the first effect of a three - effect evaporator. The bic reactors to one or more aerobic reactors ( e . g . , activated rectification column reflux condenser can provide heat for sludge reactors ) . An aerator is disposed within each aerobic this first effect. After the first effect, solids can be separated reactor to mix the anaerobic effluent, sludge , oxygen ( e . g ., using a centrifuge and dried in a rotary dryer . A portion 65 oxygen contained in air ) . Within each aerobic reactor, oxi ( 25 % ) of the centrifuge effluent can be recycled to fermen - dation of cellular material in the anaerobic effluent produces tation and the rest sent to the second and third evaporator carbon dioxide , water , and ammonia . US 9 , 745 ,604 B2 69 70 Aerobic effluent moves ( e . g . , via gravity ) to a separator , While certain embodiments have been described , other where sludge is separated from treated water. Some of the embodiments are possible . sludge is returned to the one or more aerobic reactors to As an example , while the biogas is described as being create an elevated sludge concentration in the aerobic reac - diverted to a combustion engine connected to an electric tors , thereby facilitating the aerobic breakdown of cellular 5 generator, in certain embodiments, the biogas or some portion thereof can also be passed through a fuel reformer to material in the wastewater. A conveyor removes excess produce hydrogen . The hydrogen is then converted to elec sludge from the separator. As described in further detail tricity through a fuel cell. below , the excess sludge is used as fuel to create steam As another example , while the biogas is described as and / or electricity . 10 being burned apart from the sludge and post -distillate solids, The treated water is pumped from the separator to a in certain embodiments , some or all of the waste by - products settling tank . Solids dispersed throughout the treated water can be burned together to produce steam . settle to the bottom of the settling tank and are subsequently Products/ Co -Products removed . After a settling period , treated water is pumped In some embodiments , the present invention provides from the settling tank through a fine filter to remove any 15 . mamaterials generated using the methods described herein . In additional solids remaining in the water . In some embodi some cases, such materials can be used in the absence of ments , chlorine is added to the treated water to kill patho - materials added to the biomass pre or post processing, e . g . , genic bacteria . In some embodiments , one or more physical materials that are not naturally present in biomass . In such chemical separation techniques are used to further purify the cases , the materials will contain naturally occurring mate treated water . For example , treated water can be pumped 20 rials , e . g ., derived from biomass. Alternatively or in addi through a carbon adsorption reactor. As another example , tion , the materials generated using the methods described treated water can pumped through a reverse osmosis reactor. herein can be combined with other natural and / or synthetic Waste Combustion materials , e . g . , materials that are not naturally present in The production of alcohol from biomass can result in the biomass . production of various by -product streams useful for gener - 25 As described above , in some embodiments , the methods ating steam and electricity to be used in other parts of the described herein can be used for converting ( e . g . , ferment plant. For example , steam generated from burning by - ing ) biomass to an energy product ( e . g . , an alcohol such as product streams can be used in the distillation process . As ethanol or a hydrocarbon ) and /or other products that result another example , electricity generated from burning by - from the conversion process (e . g ., organic acids ) . In such product streams can be used to power electron beam gen - 30 cases , the biomass will be exposed to conditions suitable for erators and ultrasonic transducers used in pretreatment. such a conversion . Exemplary conditions can include , e . g . , The by -products used to generate steam and electricity are at least biomass and one or more microorganisms capable of derived from a number of sources throughout the process . converting the biomass to energy (e . g . , an alcohol) in an For example , anaerobic digestion of wastewater produces a environment suitable for those organisms to function . This biogas high in methane and a small amount of waste biomass 35 conversion process can be allowed to proceed to a point ( sludge ) . As another example , post -distillate solids ( e . g ., where at least a portion of the biomass is converted to energy unconverted lignin , cellulose , and hemicellulose remaining ( e . g . , ethanol) and / or other products that result from the from the pretreatment and primary processes) can be used as conversion process (e . g ., as described below ) and /or to a a fuel . point where all ( e. g . , essentially all ) of the materials are The biogas is diverted to a combustion engine connected 40 converted to energy ( e . g ., ethanol) and/ or other products that to an electric generator to produce electricity . For example , result from the conversion process . For example, at least the biogas can be used as a fuel source for a spark -ignited about 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 95 , 98 , 99 , 99 . 5 , or natural gas engine . As another example , the biogas can be 100 % of the materials exposed to conditions suitable for used as a fuel source for a direct- injection natural gas fermentation is converted to energy ( e . g . , ethanol) and /or engine . As another example , the biogas can be used as a fuel 45 other products that result from the conversion process . source for a combustion turbine . Additionally or alterna - Alternatively or in addition , the methods described herein tively , the combustion engine can be configured in a cogen - can be used to modify biomass , e . g . , to modify ( e . g . , eration configuration . For example , waste heat from the increase , decrease , or maintain ) the solubility of the native combustion engines can be used to provide hot water or materials, to change the structure of, e . g . , to functionalize , steam throughout the plant . 50 the native materials, and / or alter ( e . g ., lower ) the molecular The sludge and post - distillate solids can be burned to heat weight and / or crystallinity relative to a native material. Such water flowing through a heat exchanger. In some embodi methods can be performed together or alone . For example , ments , the water flowing through the heat exchanger is the methods described herein can be used to convert a evaporated and superheated to steam . In certain embodi- portion of the biomass to energy . The methods described ments , the steam is used in the pretreatment rector and in 55 herein can also be used to modify ( e . g . , increase , decrease , heat exchange in the distillation and evaporation processes . or maintain the solubility , to change the structure, e . g . , Additionally or alternatively , the steam expands to power a functionalize , and/ or alter ( e . g ., lower ) the molecular weight multi - stage steam turbine connected to an electric generator . and / or crystallinity of the biomass , or vice versa . Steam exiting the steam turbine is condensed with cooling In some embodiments , the methods described herein can water and returned to the heat exchanger for reheating to 60 be used to obtain ( e . g . , extract, isolate , and / or increase the steam . In some embodiments , the flow rate of water through availability of, e . g . , as compared to unprocessed biomass the heat exchanger is controlled to obtain a target electricity materials ) one or more components contained in an unpro output from the steam turbine connected to an electric cessed biomass material ( e . g ., a raw material) . Exemplary generator. For example , water can be added to the heat components that can be obtained ( e . g . , extracted , isolated , exchanger to ensure that the steam turbine is operating 65 and / or increased in availability ( e . g ., compared to unpro above a threshold condition ( e . g . , the turbine is spinning fast cessed biomass materials ) ) include , but are not limited to enough to turn the electric generator) . sugars ( e . g ., 1 , 4 - diacids ( e . g ., succinic acids, fumaric acids , US 9 ,745 ,604 B2 71 and malic acids ) , 2 , 5 - furan dicarboxylic acids, 3 -hydroxy material that can be present in the biomass. Such materials propionic acid , aspartic acid , glucaric acid , glutamic acid , include, e . g ., pathogenic and non - pathogenic bacteria , itaconic acid , 3 -hydroxy propionic acid , aspartic acid , glu - viruses, fungus, parasites , and prions ( e . g . , infectious pro caric acid , glutamic acid , itaconic acid , levulinic acid , 3 -hy teins ). In some instances , the methods described herein can droxybutyrolactone, glycerol, sorbitol , and / or xylitol/ aribi- 5 be used to remove , inactivate , and /or neutralize toxins, e . g ., tol) , dextrins , cyclodextrins, amylase, amylopectin , germ , bacterial toxins and plant toxins. Alternatively or in addition , proteins, amino acids, peptides, nucleic acids, fats , lipids, fatty acids , gluten , sweeteners ( e . g . , glucose ) , sugar alcohols the methods described herein can be used to remove, inac ( e . g . , arabitol , Xylitol, ribitol , mannitol , sorbitol, isomalt, tivate , and / or neutralize materials that can be present in the maltitol, and lactitol) , oils ( e. g ., triglyceride vegetable oils 10 biomass that are not necessarily harmful, but are undesirable ( e . g . , soybean oil, palm oil, rapeseed oil , sunflower seed oil, in a material to be used in humans and /or animals or in peanut oil , cottonseed oil, palm kernel oil , olive oil ) , corn agriculture or aquaculture . Exemplary materials include, but oil, oat oil, nut oil, and palm oil ) , minerals , vitamins , toxins , are not limited to , bacterial and fungal spores , insects , and and other chemicals , ash , and flavenoids. Such components larvae. can be used in various application described below , e . g . , as 15 In some embodiments , the methods described herein can individual components, in combination with one or more be used to produce the products and co - products and bio additional components , in combination with processed and / conversion products described herein in challenging envi or unprocessed biomass, and / or in combination with one or ronments . Such environments can include environments that more additional components not obtained ( e . g ., extracted , present space limitations and / or extreme environmental con isolated , and /or increased in availability ) from biomass . 20 ditions , for example , locations with excessive heat or cold , Methods for obtaining one or more of these components are locations with excessive radiation , locations with excessive known in the art . pollutants , and / or locations with limited oxygen supply or In some embodiments , the methods described herein can sunlight. In some embodiments , such environments can be used to increase the availability of one or more compo - include , but are not limited to , for example , on board space nents contained in biomass ( e . g . , unprocessed and / or par - 25 craft , on board space stations ( e . g . , extraterrestrial loca tially processed biomass ). Components with increased avail - tions ), on board submarines ( e . g ., nuclear submarines ) and ability can be more readily obtained ( e . g . , extracted and/ or other marine vessels or barges or platforms designed to isolated ) , more readily used , and /or can be more readily remain at sea for extensive time periods, submarine loca amenable to an animal ( e .g ., digested or absorbed by an tions ( e . g ., civilian and /or military underwater facilities ) , animal) . Components with increased availability can 30 desert environments , polar environments , subzero environ include , for example , components that occur naturally in ments ( e . g . , permafrozen locations ) , elevated environments biomass and /or components that are generated using the (e .g ., where oxygen supplies can be limited and /or extreme methods described herein ( e . g . , cross -linked species , low temperatures are present) , and remote locations ( e . g . , self molecular weight species ) . Such components can increase contained locations ) . the value of biomass . For example , low molecular weight 35 In some embodiments , the products and / or co -products species are more readily hydrolyzed in the stomach than described herein , e . g . , resulting from the treatment of bio unprocessed biomass . Thus , biomass containing more read - mass using the methods described herein can be , e. g . , solids ily available low molecular weight species can be used as a ( e . g ., particulates ( e . g . , films) , granulates, and / or powders ) , more valuable food source , e . g . , for animals or insects , or for semi- solids, liquids, gasses, vapors , gels , and combinations use in agriculture, aquaculture , e . g . , the cultivation of fish , 40 thereof . aquatic microorganisms, aquatic plants , seaweed and algae . Alcohols In some embodiments , the methods described herein can Alcohols produced using the materials described herein be used to sterilize biomass to render the materials suitable can include, but are not limited to , a monohydroxy alcohol, for consumption by animals and / or humans ( e . g . , ingestion e . g ., ethanol, or a polyhydroxy alcohol, e . g ., ethylene glycol or implantation ) , by insects , or for use in agriculture , aqua - 45 or glycerin . Examples of alcohols that can be produced culture , e . g ., the cultivation of fish , aquatic microorganisms, include , but are not limited to , methanol, ethanol, propanol, aquatic plants , seaweed and algae . In some embodiments, isopropanol, butanol, e . g ., n - , sec - or t - butanol, ethylene irradiation treatment of cellulosic material will render the glycol, propylene glycol, 1 , 4 -butane diol, glycerin or mix biomass sterile and , therefore , suitable for consumption in tures of these alcohols . animals and / or humans ( e . g . , ingestion or implantation ) . The 50 In some embodiments , the alcohols produced using the irradiated cellulose can also be used in other products or treatment methods disclosed herein can be used in the co - products. production of a consumable beverage . In some embodiments , the methods described herein can Hydrocarbons be used to process biomass into a material intended for Hydrocarbons include aromatic hydrocarbons or arenes , consumption ( e . g . , ingestion or implantation ) in humans 55 alkanes, alkenes and alkynes. Exemplary hydrocarbons and / or non -human animals . Generally , such materials should include methane , ethane, propane , butane , isobutene , be essentially free of infectious material ( e. g. , pathogenic hexane , heptane , isobutene, octane, iso -octane , nonane , and / or non -pathogenic material) , toxins , and/ or other mate decordecane , benzene and toluene . rials ( e . g ., bacterial and fungal spores , insects, and larvae ) Organic Acids that can be harmful to the human and /or animal . Methods 60 The organic acids produced using the methods and mate known in the art and / or described herein can be used to rials described herein can include monocarboxylic acids or remove, inactivate , and / or neutralize infectious material polycarboxylic acids . Examples of organic acids include ( e . g . , pathogenic and / or non -pathogenic material) and / or formic acid , acetic acid , propionic acid , butyric acid , valeric toxins that can be harmful to humans and / or animals or that acid , caproic , palmitic acid , stearic acid , oxalic acid , malonic are generally undesirable in a material intended for use in 65 acid , succinic acid , glutaric acid , oleic acid , linoleic acid , humans and /or animals . For example , the methods can be glycolic acid , lactic acid , y - hydroxybutyric acid , or mixtures used to remove , inactivate , and /or neutralize infectious of these acids . US 9 ,745 ,604 B2 73 74 Foodstuffs calcium , phosphorus, magnesium , iron , zinc , copper, potas As described herein , the present invention provides meth - sium , selenium , and sodium ) , and oils when the biomass is ods useful for modifying biomass , e . g . , by modifying ( e . g ., ingested by an animal. increasing , decreasing , or maintaining ) the solubility of the In general, the methods described herein can be selected native materials , changing the structure of (e . g ., functional - 5 and /or optimized to select a method or combination of izing ) the native materials , and /or altering ( e .g . , lowering methods that result in the most readily soluble , absorbable , the molecular weight and/ or crystallinity relative to a native and /or digestible material , e . g . , with a desired nutrient material. The methods can be used to prepare materials with availability ( e . g ., a higher nutrient ( e . g . , protein , amino acid , properties that can be favorable for use as or in the produc - carbohydrate , mineral , vitamin , fat lipid , and oil availability tion of a foodstuff . For example , the methods can be used to 10 than native unprocessed material) that can be used in prepare a material with improved ( e. g ., increased or humans and /or animals as a foodstuff . Because the biomass decreased ) solubility , e . g . , compared to a native material, materials are readily available and cheap , the materials which can be used as a more easily absorbed foodstuff . resulting from such methods will provide an economical Increased solubility can be assessed , e . g ., by dispersing f oodstuff and reduce waste . ( e. g ., dissolving ) unprocessed and processed materials in a 15 In some embodiments , the materials and methods suitable solvent, removing undissolved material, detecting described herein can be used in the production of a foodstuff , the materials and/ or specific components of the materials e . g . , agricultural foodstuffs and foodstuffs suitable for inges ( e . g . , sugars ) , and comparing the levels of the detected tion by mammals , birds , and /or fish . Such animals include , materials in the processed and unprocessed materials . In but are not limited to food production animals , domestic some cases , the solvent containing the materials can be 20 animals, zoo animals , laboratory animals , and / or humans . modified , e . g ., by heating or by adjusting the pH . In some embodiments, materials produced by the methods Alternatively or in addition , the methods can be used to described herein that are intended for use as foodstuffs (e . g ., prepare a material with a higher nutritional value ( e .g ., in humans and /or animals ) can be additionally processed , higher energy ( e . g . , more digestively available food energy ) e . g . , hydrolyzed . Hydrolization methods are known in the art and / or nutrient availability , when the material is ingested by 25 and include , for example , the use of enzymes , acids , and / or an animal , e . g ., compared to a native material or unpro - bases to reduce the molecular weight of saccharides . In some cessed biomass . Such methods will not necessarily increase embodiments , foodstuffs resulting from the methods the total amount of energy or nutrients present in a set described herein can include enzymes ( e . g ., dried enzymes , amount ( e .g ., weight ) of a specific type of processed biomass active enzymes , and /or enzymes requiring activation ) . compared to the same amount and type of unprocessed 30 In some embodiments, materials produced by the methods biomass . Rather, the methods described herein can be used described herein that are intended for use as foodstuffs ( e . g . , to increase the nutritional value ( e . g ., the availability of in humans and/ or animals ) can be additionally processed to energy and /or one or more nutrients ) in a set amount ( e . g ., increase sterility of the materials and / or remove , inactivate , weight) of a specific type of processed biomass compared to and /or neutralize materials that can be present in the bio the same amount and type of unprocessed biomass . 35 mass , e . g . , infectious material ( e . g . , pathogenic and / or non Increasing the availability of food energy of a particular pathogenic material) , toxins, and /or other materials ( e . g . , type of biomass can be used to increase the metabolizable bacterial and fungal spores , insects , and larvae ) . In general, energy intake (MEI ) of that biomass .Methods formeasuring the methods described herein can be selected and optimized food energy are known in the art . MEI is typically calculated in order to promote optimal removal, inactivation , and / or by multiplying the number of kilocalories or kilojoules 40 neutralization ofmaterials that may be undesirable . contained in a food item by 85 % . In some embodiments , the Animal Foodstuffs methods described herein can be used to increase the MEI of In excess of 600 million tons of animal foodstuff is biomass . produced annually around the world with an annual growth Methods for comparing the MEI of processed and unpro rate of about 2 % . Agriculture is one of the largest consumers cessed biomass can include , for example , feeding equal 45 of animal foodstuffs with farmers in the United States amounts of processed or unprocessed biomass to at least two spending in excess of $ 20 billion dollars per year on distinct groups of one or more animals, and measuring the agricultural foodstuffs for food producing animals . Other growth response of the animals . foodstuffs consumers include , for example , pet owners, Nutrient availability can be assessed by conducting a zoos , and laboratories that keep animals for research studies . digestion trial. Protocols for conducting digestion trials are 50 In general, an animal foodstuff should meet or exceed the known in the art . For example , total nutrient levels can be specific requirements of a target animal , e . g ., to maintain or determined in processed and /or unprocessed biomass . Equal improve the health of a specific type or species of animal, amounts of processed or unprocessed biomass can be fed to promote the growth of a target animal ( e . g ., tissue gain ) , at least two distinct groups of one or more animals . Fecal and/ or to promote food production . Improved animal food loss of one or more nutrients is then determined for a defined 55 stuffs ( e . g ., more soluble , absorbable , and /or digestible food period of time. Increased nutrient availability is defined as stuffs ) will promote or support these same effects using a lower amounts of one or more nutrients in the animal feces. smaller amount of foodstuff and /or for a lower cost . Alternatively or in addition , nutrient availability can be Currently used raw materials in commercially prepared assessed by measuring and comparing the levels of one or foodstuff include feed grains ( e . g ., corn , soybean , sorghum , more nutrients in the blood of animals fed processed and 60 oats , and barley ) . The feed industry is the largest purchaser unprocessed biomass . of U . S . corn , feed grains , and soybean meal. However, with In some embodiments , the nutritional value of biomass the escalating price of feed grains such as corn , cheaper can be increased by increasing the digestibility of one or alternatives are desired . The most abundantly available more of , food energy , carbohydrates , sugars , proteins, fats foodstuff is biomass , e . g . , cellulosic material. In some ( saturated , monounsaturated , and polyunsaturated ) , choles - 65 embodiments , the methods described herein can be used to terol, dietary fiber , vitamins ( e . g . , vitamin A , E , C , B6 , B12 , increase the nutrient availability of any of these materials , carotene, thiamin , riboflavin , and niacin ), minerals ( e. g ., e. g ., to maintain or improve the health of a specific type or US 9 ,745 ,604 B2 75 76 species of animal, promote the growth of a target animal limited to , e . g . , polysaccharides with a length of greater than ( e . g . , tissue gain ) , and / or to promote food production . The 1000 saccharide units ; about 1000 sugar saccharide units ; low nutrient availability of commonly used foodstuffs ( e . g . , about 800 - 900 saccharide units ; about 700 -800 saccharide hay and grasses ) is largely attributed to the high cellulose, units ; about 600 -700 saccharide units ; about 500 -600 sac hemicellulose, and lignin content of such material. Unlike 5 charide units ; about 400 -500 saccharide units ; about 300 humans, who cannot digest cellulose , herbivores, e . g . , rumi 400 saccharide units ; about 200 - 300 saccharide units ; about nants , are capable of digesting cellulose , at least partially, 100 - 200 saccharide units ; 100 , 90 , 80 , 70 , 60 , 50 , 45 , 40 , 35 , through a process known as rumination . This process , how - 30 . 25 . 20 . 19 . 18 . 17 . 16 . 15 . 14 . 13 . 12 . 11 . 10 . 9 , 8 , 7 , 6 , ever , is inefficient and requires multiple rounds of regurgi tation . For example, ruminants only digest about 30 -50 10 5 , 4 , 3 , 2 , and 1 saccharide units . percent of the cellulose and hemicellulose . In some embodi In some embodiments , the methods produce disaccharides ments , the methods described herein can be used to increase ( e . g ., sucrose, lactose , maltose , trehalose , and cellobiose ) . In the nutrient availability or nutritional value of any of these some embodiments, the methods produce monosaccharides materials , e . g . , to maintain or improve the health of a ( e . g ., glucose (dextrose ) , fructose , galactose , xylose , and specific type or species of animal, promote the growth of a 15 ribose ) . These shorter chain molecules will be more easily target animal ( e . g . , tissue gain ) , and / or to promote food absorbed by an animal and will thereby increase the nutrient production . The methods described herein use reduced availability of biomass . Consequently , the methods and amounts of foodstuffs , at a lower cost, and / or with less materials described herein can be used as foodstuffs or in the waste . production of foodstuffs . Generally , increasing the nutrient availability of an animal 20 In some embodiments , the materials described herein can foodstuff will reduce the amount of feed required to be fed be used as a foodstuff e . g ., agricultural foodstuffs and /or to an animal for the animal to receive the same amount of foodstuffs suitable for ingestion by mammals , birds, and/ or energy . Consequently , the animal will require less foodstuff fish . Alternatively or in addition , the methods described thus providing a more economical foodstuff . herein can be used to process a raw material suitable for use Various techniques have been attempted to increase the 25 as or in an animal foodstuff . nutrient availability of a foodstuff with limited success. Such Materials that can be usefully processed using the meth techniques include the use of enzymes , such as cellulosic o ds described herein include cellulosic and lignocellulosic enzymes , to break down cellulosic material into shorter m aterials , e . g . , arable products , crops , grasses , plants and / or chain oligosaccharides , which can be more readily digested . feed grains, for example including , but not limited to , plant Although used in Europe and Australia , this practice is 30 material ( e . g . , forage such as alfalfa meal, hay , Bermuda expensive and not widely used in developing countries . coastal grass hay, sweet grass , corn plant, and soybean hay ) , Other techniques include removing stover to prevent leaf grains (e . g. , barley, corn (including organic and genetically loss , air removal, physically treating the material ( e . g ., modified corn ) , oats, rice , sorghum , and wheat) , plant pro compacting cellulosic material , reducing particle size, and tein products ( e. g ., canolameal , cottonseed cakes and meals , fine grinding ) , chemical treatment, and overfeeding . Addi- 35 safflower meal, and soybean ( including organic and geneti tionally , foodstuffs composed largely from cellulosic mate - cally modified soybean ) feed and meal) , processed grain rial are frequently supplemented with nutrient systems ( e .g ., by -products (e . g. , distillers products , brewers dried grains, premixes ) . These nutrient systems are typically designed to corn gluten , sorghum germ cake and meal, peanut skins, and provide the nutritional requirements of a target animal. wheat bran ) , fruit and fruit by - products ( e . g . , dried citrus Despite ensuring the animals receive the required nutrients , 40 pulp , apple pomace , and pectin pulp ) , molasses (e .g ., beet, such systems do not make efficient use of the cellulosic citrus, starch , and cane molasses) , almond hulls , ground material . shells , buckwheat hulls , legumes and legume by -products , The methods described herein provide methods for and other crop by - products . Other raw materials include , but improving the nutrient availability or nutritional value of are not limited to , alfalfa , barley , birdsfoot trefoil , brassicas biomass ( e . g . , by modifying ( e . g . , increasing , decreasing, or 45 ( e . g . , chau moellier, kale , rapeseed ( canola ) , rutabaga maintaining ) the solubility of the biomass and /or changing ( swede ) , and turnip ), clover ( e . g ., alsike clover , red clover, the structure ( e . g . , functionalizing ) of the native materials , subterranean clover, and white clover ) , grass ( e . g . , false oat and / or altering ( e . g . , lowering ) the molecular weight and / or grass , fescue , Bermuda grass , brome, heath grass , meadow crystallinity ) , as described above , thereby producing a more grass , orchard grass, ryegrass , and Timothy grass ) , maize valuable foodstuff . In some embodiments , the methods 50 (corn ), millet , oats , sorghum , and soybeans . In some described herein can be used to increase the nutrient avail embodiments , the raw material can be animal waste (e . g. , ability of the biomass by breaking down cellulosic material ruminant waste ) or human wasteeste . ( e . g . , cellulose and / or hemicellulose ) into shorter chain In some embodiments , the foodstuff contains only the saccharides and /or monosaccharides . By improving the materials produced using the methods described herein . nutrient availability of the biomass , these methods will 55 Alternatively or in addition , the foodstuff contains additional result in a more efficient foodstuff that can be used to raw materials ( including raw materials not treated using the maintain or improve the health of a specific type or species methods described herein ) and additives. Such foodstuffs of animal, promote the growth of a target animal ( e . g . , tissue can be formulated to meet the specific requirements of a gain ) , and /or to promote food production . target animal , e . g. , to maintain or improve the health of a In some embodiments , a useful animal foodstuff can 60 specific type or species of animal, to promote the growth of include partially processed biomass , e . g ., biomass that has a target animal , tissue gain , and /or to promote food produc been sheared using the methods described herein . Such tion . In some cases, a foodstuff can be formulated to meet partially processed biomass can be more readily hydrolyzed the nutritional requirements of a target animal for the least in the stomach of an animal. cost ( the " least cost ration ” ) . Methods for determining the In some embodiments , the methods described herein can 65 formulation of a foodstuff and the least cost ration are well be used to process biomass to generate the materials known to those of skill in the art ( see , for example , Pesti and described herein . These materials can include , but are not Miller , Animal Feed Formulation : Economic and Computer US 9 ,745 ,604 B2 77 78 Applications (Plant and Animal Science ) , Springer Publish turer in a form that is suitable for incorporation into a ing , Feb . 28 , 1993 and world wide web address liveinfor foodstuff. Alternatively or in addition , the materials matics . com ). described herein can be prepared from a raw material at the Additional raw materials and additives that can be use - site at which the foodstuff is prepared . fully combined with a material produced using the methods 5 In some embodiments , the materials described herein can described herein include , but are not limited to , animal be distributed alone and ingested by an animal in the absence products ( e . g . ,meat meal, meatmeal tankage ,meat and bone of any additional raw materials and / or additives. meal, poultry meal, animal by - product meal, dried animal In some embodiments , the materials will require post blood , blood meal, feather meal, egg -shell meal , hydrolyzed processing prior to use as food . For example, a dryer can be whole poultry , hydrolyzed hair , and bone marrow ) , animal 10 used to remove moisture from the intermediate fermentation waste , marine products and by - products ( e . g . , krill , fish parts products to facilitate storage , handling , and shelf life . Addi and meal, fish residue meal, crab parts and meal , shrimp t ionally or alternatively , the materials can be ground to a fine parts and meal, fish oil, fish liver and glandular meal, and particle size in a stainless- steel mill to produce a flour- like other fish by -products ) , dairy products ( e . g . , dried cow milk , substance . casein , whey products , and dried cheese ) , fats and oils ( e . g ., 15 Typically , biomass based foodstuffs are usefully fed only animal fat, vegetable fat or oil , and hydrolyzed fats ) , res - to ruminants that are capable of at least partially digesting taurant food waste ( e . g . , food waste from restaurants , bak - cellulose . As the present disclosure provides materials in eries , and cafeterias ) , and contaminated /adulterated food which the cellulosic material has been broken down into treated to destroy pathogens. shorter chain sugars , these materials can also be used as a Other additives include antibiotics ( e .g . , tetracyclines , 20 viable foodstuff for animals that are incapable of cellulose or macrolides, fluoroquinolones, and streptogramins) , flavor- hemicellulose digestion . Therefore , foodstuffs prepared ing, brewers oats , by - products of drug manufacture ( e . g ., using the materials and methods described herein can be spent mycelium and fermentation products ) , minerals and usefully fed to animals including , but not limited to , food trace minerals ( e . g . , bone charcoal, calcium carbonate , chalk production animals , zoo animals , and laboratory animals , rock , iron salts , magnesium salts , oyster shell flour, and 25 and /or domestic animals . The foodstuffs can also be used in sulphate ) , proteinated minerals ( e . g . , proteinated selenium agriculture and aquaculture . In addition , because foodstuffs and chromium ) , vitamins (e . g ., vitamin A , vitamin D , vita - prepared using the materials described herein have a higher min B12, niacin , and betaine ), direct fed organisms/ probiot nutrient availability , less foodstuff will be required by the ics ( e . g . , Aspergillus niger, Baccillus subtillis , Bifidobacte - animal to receive the same amount of energy , which will rium animalis , Bifidobacterium . bifidum , Enterococcus 30 reduce the overall cost of the foodstuff . Alternatively , ani faecium , Aspergillus oryzae, Lactobacillus acidophilus , mals will be able to consume more energy , which will result Lactobacillus bulgaricus, Lactobacillus plantarum Strepto in higher growth rates, tissue gain , milk production , and egg coccus lactis , and Saccharomyces cerevisiae ) , prebiotics production . ( e . g . , mannan -oligosaccharides (MOS ) , fructo -oligosaccha In some embodiments , the materials described herein can rides , and mixed oligo - dextran ) , flavors ( e . g . , aloe vera gel 35 be usefully fed to ruminants ( e . g ., cattle , goats, sheep , concentrate , ginger , capsicum , and fennel ), enzymes ( e. g ., horses, elk , bison , deer , camels , alpacas, llamas, giraffes , phytase , cellulase , lactase, lipase, pepsin , catalase, xylanase , yaks , water buffalo , wildebeest , and antelope ), poultry , pigs, and pectinase ) , acetic acid , sulfuric acid , aluminum salts , boars, birds, cats , dogs, and fish . dextrans , glycerin , beeswax , sorbitol, riboflavin , preserva - In some embodiments, distillers grains and solubles can tives ( e . g . , butylated hydroxyanisole and sodium bisulfite ) , 40 be converted into a valuable byproduct of the distillation nutraceuticals ( e . g . , herbal and botanical products ), amino dehydration process. After the distillation - dehydration pro acids , by pass protein , urea , molasses, fatty acids, ( e . g ., cess , distillers grains and solubles can be dried to improve acetic , propionic , and butyric acid ) and metabolic modifiers the ability to store and handle the material. The resulting ( e. g ., somatotropins and adrenergic agonists ) . In some cases, dried distillers grains (DDG ) and solubles is low in starch , the materials produced using the methods described herein 45 high in fat, high in protein , high in fiber, and high in can be combined or incorporated into a urea molasses phosphorous. Thus , for example , DDG can be valuable as a mineral block (UMMB ) . source of animal feed ( e . g . , as a feed source for dairy cattle ). Foodstuffs prepared using the materials described herein DDG can be subsequently combined with nutritional addi can be in a form suitable for ingestion , e . g ., by a target tives to meet specific dietary requirements of specific cat animal. In some cases the foodstuff can be a solid . Alterna - 50 egories of animals ( e . g . , balancing digestible lysine and tively or in addition , the foodstuff can be in a liquid form , phosphorus for swine diets ) . Alternatively or in addition , e . g . , the foodstuff can be in a liquid suspension or solution biomass processed using the methods described herein can in a suitable solvent. Exemplary forms include, but are not be combined with DDG . The ratio of processed biomass to limited to solids such as powders , tablets , mineral blocks, DDG can be optimized to meet the needs of target animals . pellets , biscuit, and mixtures of an unprocessed raw material 55 In some embodiments, oils obtained from biomass using ( e . g . , grass ) and a material processed using the methods the methods described herein can be used in animal feed , described herein . e . g ., as a pet food additive . In some embodiments , the materials described herein can In some embodiments , as obtained from biomass using be incorporated ( e . g . , mixed ) into a foodstuff by a farmer , the methods described herein can be used in animal feed . e . g ., for local use and / or small scale distribution . In such 60 Human Foodstuffs cases, the materials described herein can be provided to the As described above , humans are typically less able to farmer in a packaged form , e . g . , in a form that is suitable for digest cellulose and cellulosic material. Biomass is an abun incorporation into a foodstuff . Alternatively or in addition , dantly available material , however , that could serve as a the materials described herein can be incorporated ( e . g ., novel foodstuff for human consumption . In order for a mixed ) into a foodstuff by a foodstuff manufacturer, e . g . , for 65 biomass material ( e . g . , a material containing cellulose ) to be large scale distribution . In such cases , the materials useful as a human foodstuff , however, the nutrient availabil described herein can be provided to the foodstuff manufac - ity of the biomass would have to be increased by ( 1 ) US 9 ,745 ,604 B2 79 80 increasing the solubility of the biomass ; ( 2 ) changing the In some embodiments , thematerials described herein can be structure ( e .g . , functionalizing ) of the native materials ; ( 3 ) combined with protein , fats , carbohydrates , minerals , phar altering ( e . g ., lowering) the molecular weight and / or crys - maceuticals , and vitamins. tallinity relative to a native material; and /or ( 4 ) breaking Proteins down cellulosic material into smaller saccharides, for 5 In some embodiments, the methods described herein can example , saccharides with a length of greater than 1000 be used to obtain ( e . g . , extract , isolate , and /or purify proteins saccharide units : about 1000 sugar saccharide units ; about ( e . g ., polypeptides , peptides , and amino acids ) from bio 800 - 900 saccharide units ; about 700 - 800 saccharide units ; mass . Such proteins ( e . g . , polypeptides , peptides , and amino acids ) can be used , e . g ., alone or in combination with one or about 600 - 700 saccharide units ; about 500 -600 saccharide 10 more of the materials and biomass components obtained units ; about 400 - 500 saccharide units ; about 300 - 400 sac using the methods described herein , in the food industry charide units ; about 200 - 300 saccharide units ; about 100 ( e. g ., as additives , supplements , and /or fillers ), in the cos 200 saccharide units ; 100 , 90 , 80 , 70 , 60 , 50 , 45 , 40 , 35 , 30 , metic industry ( e . g . , in the compounding of cosmetics ) , 25 , 20 , 19 , 18 , 17 , 16 , 15 , 14 , 13 , 12 , 11, 10 , 9 , 8 , 7 , 6 , 5 , and /or in agriculture ( e . g . , as foodstuffs or to feed or 4 , 3 , 2 , and 1 saccharide units . Such materials will have an 15 maintain crops ) or aquaculture . increased nutrient availability (as described above ), e. g . , in In some embodiments . the methods described herein can humans , and will be useful as a human foodstuff . In general, be used to obtain proteins ( e . g . , polypeptides , peptides , and a useful human foodstuff should , e. g . , provide a usable and amino acids ) from e . g ., okra seed , Lipimus mutabilis , nuts accessible energy and nutrient source to the human to , e . g ., ( e . g ., macadamia nuts ) , Jessenia batana , Oenocarpus, maintain or improve the health of a human , and /or promote 20 Stokesia laevis , Veronia galamensis , and Apodanthera undu the growth of a human ( e . g . , tissue gain ). The methods late . described herein can be used to produce such a useful human Fats , Oils , and Lipids foodstuff , e . g . , from a biomass -based material. Fats consist of a wide group of compounds that are In some embodiments , the materials will require process - generally soluble in organic solvents and largely insoluble in ing prior to use as food . For example , a dryer can be used to 25 water. Fats that are solid at room temperature . Fats that are remove moisture from the intermediate fermentation prod - liquid at room temperature are typically referred to as oils . ucts to facilitate storage, handling , and shelf life . Addition - The term lipids typically refers to solid and liquid fats . As ally or alternatively , the materials can be ground to a fine used herein , the terms fats , oils , and lipids include, but are particle size in a stainless -steel mill to produce a flour - like not limited to , edible oils , industrial oils, and those materials substance . 30 having an ester , e . g ., triglyceride and /or hydrocarbon . Such foodstuffs can include, but are not limited to , for In some embodiments , the methods described herein can example , energy supplements ( e . g ., powders and liquids) . be used to obtain ( e . g ., extract, isolate, and /or purify ) fats Alternatively or in addition , the materials described herein ( e . g ., lipids, and fatty acids ) from biomass . Such fats ( e . g . , can be combined with a first food to increase the nutritional lipids, and fatty acids ) can be used , e . g . , alone or in value of the first food . For example , the foodstuffs described 35 combination with one or more of the materials and biomass herein can be combined with a low energy food to increase components obtained using the methods described herein , in the energy of the food . the food industry ( e . g . , as additives, supplements, and / or Alternatively or in addition , the materials described fillers) , in the cosmetic industry ( e . g . , in the compounding of herein can be used to increase the sweetness of the food , e . g ., cosmetics ), and / or in agriculture ( e . g . , as foodstuffs) . as a sweetening agent , as well as the nutritional value of the 40 In some embodiments , the methods described herein can food . In such cases , it can be desirable to obtain one or more be used to obtain ( e . g . , extract, isolate , and / or purify ) oils specific sugars (e . g . , a monosaccharide , a disaccharide , a from biomass . Such oils can be used , e . g ., alone or in oligosaccharide, and /or a polysaccharide ) from the materi- combination with one or more of the materials and biomass als , e . g . , by isolating the one or more specific sugars from components obtained using the methods described herein , in the materials . Methods for isolating sugars are known in the 45 the food industry ( e . g . , as additives , supplements , and / or art . fillers ) , in the cosmetic industry ( e . g . , in the compounding of In some embodiments , the materials described herein can cosmetics ) , in agriculture ( e . g ., as foodstuffs ) , as biofuels , be used as a low cost material for food production . For drying oils ( e . g . , in paints ) , and pet food additives . example , the materials can be supplied to bakeries for use in In some embodiments , the methods described herein can bread and / or confectionery , and to food manufacturers to be 50 be used to obtain fats , oils , and / or lipids from e . g . , sun used as a filler, e . g ., to increase the volume and /or nutritional flower, okra seed , buffalo gourd ( Cucurbita foetidissima ) , value of a food . Lipinus mutabilis , nuts ( e . g . , macadamia nuts ) , Jessenia In some embodiments , the materials can further serve as bataua , Oenocarpus, Crambe abyssinica (Crambe ) , Mon a source of fiber for human consumption . In such cases, the oecious jojoba (jojoba ), Cruciferae sp . (e .g ., Brassica jun methods used to break down the cellulolytic material will be 55 cea , B . carinata , B . napas ( common rapeseed ) , and B . configured to provide a less complete reduction in molecular campestris ), Stokesia laevis , Veronia galamensis , and Apo weight, e . g ., the methods will result in materials containing danthera undulate . some cellulose and / or result in longer chain length polysac - Carbohydrates and Sugars charides that are not easily absorbed by humans . Such In some embodiments, the methods described herein can materials can be fed to a human in the form of a solid ( e . g ., 60 be used to obtain ( e . g ., extract, isolate , and/ or purify ) a tablet or a granular powder ) or a liquid ( e . g ., a solution , carbohydrates and /or sugars from biomass . Such carbohy gel, colloid , or suspension ) . drates and sugars can be used , e . g ., alone or in combination In some embodiments , the materials described herein can with one or more of the materials and biomass components be fed to a human alone or in the combination with a second obtained using the methods described herein , e . g ., in the food that is suitable for human consumption . Such foods 65 food industry ( e . g . , as additives, supplements , syrups, and /or include , but are not limited to , breads , dairy products, meats , fillers ) , in the cosmetic industry ( e . g . , in the compounding of fish , cereals , fruits , vegetables , beans ( e . g ., soy ) , and gums . cosmetics) , and / or in agriculture ( e . g ., as foodstuffs ) . US 9 , 745 ,604 B2 81 82 Vitamins to plants and / or herbs with medicinal properties . For In some embodiments , the methods described herein can example , sonication can stimulate bioactivity and /or bio be used to obtain ( e . g ., extract, isolate , and / or purify ) availability of the medicinal components of plants and /or vitamins from biomass . Such vitamins can be used , e . g . , herbs with medicinal properties. Additionally or alterna alone or in combination with one or more of the materials 5 tively , irradiation can stimulate bioactivity and / or bioavail and biomass components obtained using the methods ability of the medicinal components of plants and /or herbs described herein , e . g ., in the food industry ( e . g . , as additives, with medicinal properties . and supplements ) , in the healthcare industry , in the cosmetic In some embodiments, the methods described herein can industry ( e . g ., in the compounding of cosmetics) , and / or in be used to increase the solubility of a plant and / or herbal agriculture . 10 material . Alternatively or in addition , the methods described Minerals herein can be used to reduce the toxicity of a plant and / or In some embodiments , the methods described herein can herbal material without reducing the medicinal properties of be used to obtain ( e .g ., extract , isolate , and /or purify ) the plant and /or herb . In some embodiments , the methods minerals from biomass. Such minerals can be used , e . g ., described herein are useful for isolating and /or purifying alone or in combination with one or more of the materials 15 pharmaceutical compounds from plant material (which and biomass components obtained using the methods without being bound by theory , is possible due to the more described herein , e. g ., in the food industry (e . g ., as additives, efficient break down of cellulosic material) as the methods and supplements ) , in the healthcare industry , in the cosmetic disrupt , alter, modify , or restructure cellulose , e . g . , present in industry ( e . g . , in the compounding of cosmetics ), and /or in the leaves of plant material. Desired compounds released agriculture . 20 using the methods described herein can then be isolated Ash from the undesired material, whereas less efficient methods In some embodiments , the methods described herein can can not allow the release of the desired material from be used to obtain ( e . g ., extract, isolate , and /or purify ) ash undesired material. Inevitably , therefore , less efficient meth from biomass . Such ash can be used , e . g ., alone or in ods will result in the carry over of undesired material, which combination with one or more of the materials and biomass 25 can lower the efficacy of the desired ( e . g ., pharmaceutical components obtained using the methods described herein , compound ) and/ or be associated with potentially toxic side e . g ., in the food industry ( e . g ., as an additive, supplement, effects . The methods described herein can , therefore , be used and /or filler ). to generate highly purified forms of potentially pharmaceu Pharmaceuticals tical compounds , e . g . , free of undesirable plantmaterial , that Over 120 currently available pharmaceutical products are 30 are not obtainable using current practices. These highly plant- derived . As the methods described herein are useful for purified compounds can be more efficacious then less puri processing cellulolytic material, these methods can be useful fied forms of the same compounds. In some embodiments , in the isolation , purification , and / or production of plant - the increased efficacy attainable using the methods described based pharmaceuticals . herein can allow reduced dosing. In turn , this reduction in In some embodiments , the materials described herein can 35 the amount of material administered to a subject can reduce have medicinal properties . For example , the methods associated toxicity . Alternatively or in addition , the removal described herein can result in the production of a material of surplus or undesirable plant material can help reduce or with novel medicinal properties ( e . g . , not present in the eliminate any toxicity associated with a plant based com native material ) . Alternatively or in addition , the methods pound that has not been processed using the methods described herein can result in the production of a material 40 described herein . with increased medicinal properties ( e . g ., a greater medici Examples of plants and /or plant material that can be nal property than that of the native material) . usefully treated using the methods described herein include , In some embodiments , the methods described herein can for example , sonication and irradiation can be combined in be used to modify ( e . g . , increase , decrease , or maintain ) the the pretreatment of willow bark to stimulate the isolation , solubility of a material, e . g . , a material with medicinal 45 purification , and / or production of salicin . Alternatively or in properties . Such a material can be more easily administered addition , the methods described herein can be used to and / or absorbed , e . g . , by a human and/ or animal than a process plant material comprising comfrey plants to facili native material. tate the isolation , purification , and /or production of allan In some embodiments , the methods described herein can t oin . Alternatively or in addition , the methods described be used to functionalize ( e . g . , alter the structure , expose a 50 herein can be used to facilitate the isolation , purification , reactive side chain , and /or modify the charge ) a material and / or production benzoin . Alternatively or in addition , the with medicinal properties . Such materials can have , e . g . , methods described herein can be used to process plant altered reactivity , altered charge , and / or altered solubility . material comprising camphor basil to facilitate the isolation , In some embodiments , the methods described herein can purification , and / or production of camphor. Alternatively or be used to modify the molecular structure of a material, e . g . , 55 in addition , the methods described herein can be used to a material with medicinal properties . Such materials can process plant material comprising plants in the genus Ephe have altered ( e . g . , increased or decreased ) average molecu - dra to facilitate the isolation , purification , and / or production lar weights , average crystallinity , surface area , and / or poros - of ephedrine . Alternatively or in addition , the methods ity . Such materials can have , e . g . , altered reactivity , altered described herein can be used to process plant material charge , altered solubility . 60 comprising Duboisia myoporoides R . Br. (Australian cork In some embodiments , the methods described herein can tree ) to facilitate the isolation , purification , and / or produc be used as high efficiency processing methods, e . g . , to obtain tion of atropine. In some embodiments , the atropine plant- based pharmaceuticals from a cellulosic raw material obtained using the methods described herein will have an such as plants . In some embodiments , themethods described increased anticholinergic effect . Alternatively or in addition , herein can be used to increase the pharmaceutical activity of 65 the methods described herein can be used to process plant a plant- based pharmaceutical. For example , in some material comprising Mucuna deeringiana ( velvet bean ) to embodiments , the methods described herein can be applied facilitate the isolation , purification , and /or production of US 9 ,745 ,604 B2 83 84 L -dopa . In some embodiments , the L - dopa obtained using in a human . For example , the material can have low nutrient the methods described herein will have an increased anti - availability with low digestibility , e. g. , a fibrous material . parkinsonian effect. Alternatively or in addition , the methods Such materials could be used as a dietary supplement, e . g . , described herein can be used to process plant material to suppress hunger and /or to promote satiety . Consuming comprising Physostigma venenosum Balf (ordeal bean ) to 5 such materials would allow a subject to avoid consuming facilitate the isolation , purification , and /or production of high nutrient availability and /or highly digestible foods and physostigmine . In some embodiments , the physostigmine obtained using the methods described herein will have an thus would facilitate weight loss in the individual . increased anticholinesterase effect. Examples of other plant In some embodiments , the materials described herein can based pharmaceuticals in which the methods described 10 be supplemented with one or more nutritional supplements herein can be used to process plant material to facilitate the that are capable of promoting a healthy lifestyle . In such isolation , purification , and /or production of include , but are cases , the materials described herein can either enhance the not limited to , bromelain , chymopapain , cocaine, deserpi activity of the one or more nutritional supplements and / or dine, emetine, hyoscyamine , kawaina, monocrotaline, oua enhance the solubility and / or pharmacokinetics of the one or bain , papain , pilocarpine , quinidine, quinine , rescinnami, 1515 more nutritional supplements . Exemplary nutritional supple reserpine , scopolamine, tubocurarine , vinblastine, yohim ments that can be combined with the materials described bine. caprylic - acid , cineole . citric acid . codeine . cresol. herein include, but are not limited to , for example , silica , guaiacol, lecithin ,menthol , phenolpseudephedrine , sorbitol, silicon , boron , potassium , iodine, beta -carotene , lycopene, and tartaric acid . insoluble fiber, monosaturated fatty acids, omega - 3 fatty In some embodiments , the methods described herein can 20 acid , flavonols , sulforaphane , phenols ( e . g . , caffeic acid and be used to process herbs, e. g. , medicinal herbs, including, ferulic acid ), plant stanols and sterols ( including esters but not limited to , basil , lemon grass , parsley, peppermint, thereof ) , polyols ( e . g ., sugar alcohols ), prebiotics and pro and celery . Additional medicinal herbs that can be processed biotics ( e . g . , Lactobacilli and bifidobacteria ), phytoestro using the methods described herein can be found at world gens (e . g ., isoflavones such as daidzein and genistein ), wide web address altnature .com . 25 proanthocyanidins, soy protein , sulfides and thiols ( e . g . , An emerging technology is the production of pharmaceu - dithiolthiones )), , vitaminsvitamins (Teo e . g . , vitamin AA , vitamin B1B , vitavita ticals in plants . Pharmaceutical produced using plants, min B2, vitamin B2 , vitamin Bs, vitamin B , vitamin B , which are commonly referred to as plant made pharmaceu ticals ( PMPs ) , include pharmaceutical compounds and vac vitamin B12 , vitamin C , vitamin D , vitamin E , vitamin K , cines . Typically , PMPs are expressed in the leaves of the 30 including combinations thereof) minerals ( e . g . , iron , cal respective plants . Clearly , therefore, the methods described cium , magnesium , manganese , phosphorus, potassium , zinc , herein can be useful for processing plant material compris trace minerals , chromium , selenium , including combina ing PMPs to facilitate the isolation , purification , and / or tions thereof) , and folic acid . production of the PMPs. Pharmaceutical Dosage Forms and Drug Delivery Com Additional exemplary medicinal plants that can be treated 35 pos using the methods described herein can be found e . g ., at Drug substances are seldom administered alone, but world wide web address nps . govvirlanta/ plants MEDICINALIMEDICINAL/ ) rather as part of a formulation in combination with one or plants .htm . more non -medical agents that serve varied and often spe In some embodiments , material that has been processed cialized pharmaceutical functions . Pharmaceutics is the sci using the methods described herein can be combined with a 40 ence of dosage form design , e. g ., formulating a drug into a pharmaceutical excipient, e . g . , for administration to a sub - dosage form suitable for administration to a subject. These ject . Exemplary excipients that can be used include buffers non -medical agents , referred to as pharmaceutic or pharma ( for example, citrate buffer, phosphate buffer, acetate buffer, ceutical ingredients , can be formulated to solubilize , sus and bicarbonate buffer ) , amino acids, urea , alcohols , ascor pend , thicken , dilute , emulsify , stabilize , preserve, color, bic acid , phospholipids , polypeptides ( for example , serum 45 flavor, and fashion medicinal agents into efficacious and albumin ) , EDTA , sodium chloride , liposomes, mannitol, appealing dosage forms. Such dosage forms can be unique sorbitol, water, and glycerol. Dosage forms can be formu - in their physical and pharmaceutical characteristics . The lated to be suitable for any standard route of administration . drug and pharmaceutic ingredients will typically be com For example , administration can be parenteral, intravenous, patible with each other to produce a drug product that is subcutaneous , or oral or any route of administration 50 stable , efficacious , attractive , easy to administer , and safe . approved by the Federal Drug Administration ( see world The product should be manufactured under appropriate wide web address fda . gov / cder/ dsm /DRG / drg00301. htm ) . measures of quality control and packaged in containers that Nutriceuticals and Nutraceuticals contribute to promote stability . Methods describing the Foods with a medical health benefit, including the pre - preparation of specific dosage forms are well known in the vention and /or treatment of disease , are referred to as 55 art and can be found in , for example , Ansel et al ., Pharma nutraceuticals or nutriceuticals . For example , nutraceuticals ceutical Dosage Forms and Drug Delivery Systems, Seventh and nutriceuticals are naturally occurring or artificially gen - Edition , Lippincott , Williams, & Wilkins and , “ Remington ' s erated nutritional supplements capable of promoting a Pharmaceutical Sciences, 18th Ed ., Gennaro , ed . , Mack healthy lifestyle , for example , by reducing disease related Publishing Co ., Easton , Pa ., 1990 . symptoms, reducing the incidence or severity of disease , and 60 In some embodiments , the pretreated materials described promoting long -term health . herein can be used as pharmaceutic ingredients e . g . , inactive In some embodiments , the methods described herein can ingredients . For example , the materials described herein can be used to generate combinations of monosaccharides, be used , e . g . , formulated , to solubilize , suspend, thicken , disaccharides , oligosaccharides , and / or polysaccharides that dilute , emulsify , stabilize , preserve , color , flavor, and fash are capable of promoting a healthy lifestyle . In some 65 ion medicinal agents into efficacious, palatable , and appeal embodiments , the methods described herein can be used to ing dosage forms. In such cases , the materials described generate a material that is useful for promoting weight loss herein can be mixed with a drug and /or conjugated to a drug