US010508291B2

United States Patent ( 10 ) Patent No.: US 10,508,291 B2 Medoff (45 ) Date of Patent : * Dec . 17 , 2019

( 54 ) PROCESSING C12P 19/02 (2006.01 ) C12P 19/14 ( 2006.01) (71 ) Applicant: Xyleco, Inc. , Wakefield , MA (US ) C12P 710 (2006.01 ) C12N 9/00 (2006.01 ) ( 72 ) Inventor: Marshall Medoff , Wakefield , MA (US ) A23K 50/75 ( 2016.01) A23K 50/30 ( 2016.01) ( 73 ) Assignee : XYLECO , INC . , Wakefield , MA (US ) A23K 50/40 (2016.01 ) (52 ) U.S. CI. ( * ) Notice : Subject to any disclaimer , the term of this CPC C12P 7762 (2013.01 ) ; A23K 10/00 patent is extended or adjusted under 35 ( 2016.05 ); A23K 10/12 (2016.05 ); A23K 10/30 U.S.C. 154 (b ) by 0 days . ( 2016.05 ) ; A23K 10/37 ( 2016.05 ) ; A23K 20/163 (2016.05 ) ; A23K 20/189 ( 2016.05 ) ; This patent is subject to a terminal dis A23K 40/10 ( 2016.05 ); A23K 50/10 (2016.05 ) ; claimer . A23K 50/80 ( 2016.05) ; A23L 5/00 (2016.08 ) ; A23L 5/30 ( 2016.08 ) ; A61K 8/97 ( 2013.01 ) ; ( 21) Appl. No .: 15 /657,013 A61Q 19/00 (2013.01 ); B01D 39/18 ( 2013.01) ; C08H 8/00 ( 2013.01) ; C08J 3/28 (22 ) Filed : Jul, 21 , 2017 (2013.01 ) ; C08L 1/02 ( 2013.01 ) ; C08L 5/14 (2013.01 ) ; C12P 7/10 ( 2013.01) ; C12P 7/42 (65 ) Prior Publication Data ( 2013.01 ); C12P 13/04 ( 2013.01 ) ; C12P 19/02 US 2017/0321233 A1 ( 2013.01) ; C12P 19/14 ( 2013.01) ; C12P 19/44 Nov. 9 , 2017 ( 2013.01 ); C13K 1/02 ( 2013.01 ); D21C 5/005 (2013.01 ) ; D21C 9/001 (2013.01 ) ; A23K 50/30 Related U.S. Application Data (2016.05 ); A23K 50/40 (2016.05 ); A23K 50/75 (2016.05 ); A23V 2002/00 (2013.01 ); C12N (60 ) Division of application No. 15 /040,318 , filed on Feb. 9/00 (2013.01 ) ; C12P 2201/00 (2013.01 ) ; 10 , 2016 , now Pat. No. 9,745,604, which is a C12P 2203/00 (2013.01 ) ; YO2E 50/10 continuation of application No. 14 /813,898 , filed on ( 2013.01) ; YO2E 50/16 (2013.01 ) ; YO2E 50/17 Jul. 30 , 2015 , now Pat. No. 9,803,222 , which is a (2013.01 ) ; YO2E 50/30 (2013.01 ) ; YO2P continuation of application No. 14 /750,995 , filed on 60/877 (2015.11 ) Jun . 25 , 2015 , now Pat. No. 9,745,609 , which is a (58 ) Field of Classification Search continuation of application No. 14 / 333,675 , filed on CPC .... COSH 8/00 ; C12P 2201/00 ; C12P 2203/00 ; Jul. 17 , 2014 , now Pat . No. 9,309,545 , which is a C13K 1/02 , A23K 1/001 ; A23K 1/1643 ; continuation of application No. 13 /902,246 , filed on A23K 1/1813 ; A23K 1/188 ; A23L May 24 , 2013 , now Pat. No. 8,877,472 , which is a 1/0257 ; A61K 8/97 ; D21C 5/005 continuation of application No. 12/ 417,900 , filed on See application file for complete search history . Apr. 3 , 2009 , now abandoned . (Continued ) (56 ) References Cited (51 ) Int. Cl. U.S. PATENT DOCUMENTS A23L 5/30 ( 2016.01) 1,920,129 A 7/1933 Munroe et al. A23L 5/00 (2016.01 ) 3,352,773 A 11/1967 Schwartz et al . A23K 40/10 ( 2016.01 ) (Continued ) A61K 8/97 ( 2017.01 ) B01D 39/18 ( 2006.01 ) FOREIGN PATENT DOCUMENTS C08H 800 (2010.01 ) C08J 3/28 (2006.01 ) CA 2809329 6/2008 C12P 19/44 (2006.01 ) CN 101041834 A 9/2007 CO8L 1/02 ( 2006.01 ) ( Continued ) C08L 5/14 ( 2006.01 ) D21C 5/00 (2006.01 ) OTHER PUBLICATIONS D21C 9/00 ( 2006.01) A23K 10/12 ( 2016.01 ) Adams, “ Corn as Feed for Cattle, ” Penn State College of A23K 10/30 (2016.01 ) Agricultural Sciences, Penn State Extension , Feb. 1, 2002 ( 2 pages ) . A23K 10/37 ( 2016.01) (Continued ) A23K 20/163 ( 2016.01 ) A23K 20/189 ( 2016.01 ) Primary Examiner Aaron J Kosar C12P 7762 ( 2006.01) (74 ) Attorney , Agent, or Firm -Lando & Anastasi, LLP A61Q 19/00 ( 2006.01 ) C12P 13/04 (2006.01 ) (57 ) ABSTRACT A23K 50/10 (2016.01 ) Biomass ( e.g., plant biomass , animal biomass, microbial, A23K 50/80 ( 2016.01 ) and municipal waste biomass ) is processed to produce useful C13K 1/02 ( 2006.01 ) products , such as food products and amino acids . C12P 742 ( 2006.01 ) A23K 10/00 ( 2016.01 ) 15 Claims, 54 Drawing Sheets US 10,508,291 B2 Page 2

Related U.S. Application Data 2009/0286295 Al 11/2009 Medoff et al . 2010/0087687 A1 4/2010 Medoff 2010/0093241 A1 4/2010 Medoff (60 ) Provisional application No.61 / 139,453 , filed on Dec. 2010/0105119 A1 4/2010 Medoff 19, 2008 , provisional application No. 61/ 073,674 , 2010/0108567 Al 5/2010 Medoff filed on Jun . 18 , 2008, provisional application No. 2010/0124583 A1 5/2010 Medoff 61 /049,405 , filed on Apr. 30 , 2008 . 2010/0179315 A1 7/2010 Medoff 2010/0200806 A1 8/2010 Medoff et al. References Cited 2010/0203495 Al 8/2010 Medoff et al . ( 56 ) 2010/0206501 A1 8/2010 Medoff 2010/0297720 A1 11/2010 Medoff et al. U.S. PATENT DOCUMENTS 2010/0304440 A1 12/2010 Medoff 3,939,286 A 2/1976 Jelks 2011/0011960 A1 1/2011 Medoff 4,268,505 A 5/1981 Yoshikumi et al. 2011/0027837 A1 2/2011 Medoff 4,274,163 A 6/1981 Malcom et al. 2011/0039317 Al 2/2011 Medoff 4,275,163 A 6/1981 Gallo 2011/0081335 A1 4/2011 Medoff 4,321,328 A 3/1982 Hoge 2011/0081336 A1 4/2011 Medoff 4,431,675 A 2/1984 Schroeder et al. 2011/0111456 A1 5/2011 Medoff 4,769,082 A 9/1988 Kumakura et al . 2011/0139383 Al 6/2011 Medoff 5,122,598 A 6/1992 della Valle et al. 2011/0155559 Al 6/2011 Medoff 5,221,357 A 6/1993 Brink 2011/0262985 Al 10/2011 Medoff 5,314,978 A 5/1994 Kim et al. 2011/0265991 A1 11/2011 Medoff 5,417,824 A 5/1995 Greenbaum 2011/0287498 A1 11/2011 Medoff et al. 5,538,730 A 7/1996 Romeo et al. 2012/0003704 Al 1/2012 Medoff 5,753,474 A 5/1998 Ramey 2012/0052536 A1 3/2012 Medoff et al . 6,011,008 A 1/2000 Domb et al. 2012/0074337 A1 3/2012 Medoff 6,334,347 B1 1/2002 Iscla 2012/0077247 Al 3/2012 Medoff 6,344,347 B1 2/2002 Kino et al. 2012/0094358 A1 4/2012 Medoff 6,534,104 B1 3/2003 DeRouchey et al. 2012/0100577 A1 4/2012 Medoff et al . 6,555,335 B1 4/2003 Saloheimo et al . 2012/0100586 A1 4/2012 Medoff et al . 6,555,350 B2 4/2003 Ahring et al. 2012/0142065 A1 6/2012 Medoff 6,620,605 B2 9/2003 Fowler et al. 2012/0142068 A1 6/2012 Medoff 6,808,600 B2 10/2004 Ross et al. 2012/0315675 Al 12/2012 Medoff et al . 6,942,973 B2 9/2005 Yaver et al . 2013/0295624 Al 11/2013 Medoff et al. 7,005,508 B2 2/2006 Benedini et al. 2013/0302839 Al 11/2013 Harenberg et al . 7,408,056 B2 8/2008 Medoff et al. 2015/0342224 Al 12/2015 Medoff 7,846,295 B1 12/2010 Medoff 2015/0353974 A1 12/2015 Medoff 7,867,358 B2 1/2011 Medoff 7,867,359 B2 1/2011 Medoff FOREIGN PATENT DOCUMENTS 7,900,857 B2 3/2011 Medoff 7,931,784 B2 4/2011 Medoff EP 2276795 1/2011 7,932,065 B2 4/2011 Medoff IN 200200944 1/2005 7,935,219 B2 5/2011 Medoff IN 200401988 8/2006 8,003,368 B2 8/2011 Marchenko et al . JP S5971700 A 4/1984 8,052,838 B2 11/2011 Medoff JP S59113856 A 6/1984 8,070,912 B2 12/2011 Medoff KR 2002091479 12/2002 8,083,906 B2 12/2011 Medoff RU 2030155 C1 3/1995 8,142,620 B2 3/2012 Medoff SU 1262950 5/1990 8,147,655 B2 4/2012 Medoff WO WO -01 / 15689 3/2001 8,168,038 B2 5/2012 Medoff WO WO - 2001052620 7/2001 8,212,087 B2 7/2012 Medoff WO WO - 2001079241 10/2001 8,221,585 B2 7/2012 Medoff WO WO - 2006032282 3/2006 8,236,535 B2 8/2012 Medoff et al. WO WO - 2008073186 6/2008 8,377,668 B2 2/2013 Medoff et al. WO WO - 2009134791 11/2009 8,418,944 B2 4/2013 Medoff 8,454,803 B2 6/2013 Medoff 8,492,128 B2 7/2013 Medoff OTHER PUBLICATIONS 8,597,921 B2 12/2013 Medoff 8,603,787 B2 12/2013 Medoff Allen et al. , “ Fractionation of Sugar Cane with Hot , Compressed , 8,709,771 B2 4/2014 Medoff et al. Liquid Water, ” Industrial & Engineering Chemistry Research , 35 ( 8 ) , 8,835,142 B2 * 9/2014 Medoff A61K 8/97 pp . 2709-2715 ( 1996 ) . 435/171 8,841,101 B2 9/2014 Medoff Atchison et al ., " Subcontractor Report : Innovative Methods for 8,852,905 B2 10/2014 Medoff Collecting, Handling, Storing and Transporting — Mar. 8,877,472 B2 11/2014 Medoff 2003, ” National Renewable Energy Laboratory , NREL /SR - 510 8,945,245 B2 2/2015 Bals et al . 33893 , 63 pages (2004 ) . 9,078,461 B2 * 7/2015 Medoff A61K 8/97 Awafo et al. , “ Effect of Irradiation , as a Pretreatment, on Bioconver 9,278,896 B1 * 3/2016 Medoff BO1J 19/085 sion of Corn Stover into Protein -Rich Mycelial Biomass of Pluerotus 9,309,545 B2 4/2016 Medoff Sajor- Caju ,” Radiation Physics and Chemistry, 46 (4-6 ) pp . 1299 9,328,393 B2 5/2016 Medoff et al . 9,745,604 B2 * 8/2017 Medoff A61Q 19/00 1302 ( 1995 ). 9,745,609 B2 8/2017 Medoff Bak et al. , “ Improved Enzymatic Hydrolysis Yield of 9,803,222 B2 10/2017 Medoff A61Q 19/00 Using Electron Beam Irradiation Pretreatment, ” Bioresource Tech 2004/0005674 Al 1/2004 Duck et al. nology, 100 ( 3 ) , pp . 1285-1290 ( 2009 , online Oct. 17, 2008) . 2004/0231060 A1 11/2004 Burdette et al. Barker et al ., “Microbial Synthesis of p -Hydroxybenzoic Acid from 2005/0203291 A1 9/2005 Svenson et al. Glucose, ” Biotech . Bioeng. 76 ( 4 ) , pp . 376-390 (2001 ) . 2006/0088922 A1 4/2006 Yang et al . Batrinou et al. , “ Effect of Ionizing Radiation on the Quantification 2006/0251764 Al 11/2006 Abbas et al. of Genetically Modified Foods, ” Food Biotechnology , 22 ( 3-4 ) , pp . 2007/0015855 Al 1/2007 Medoff et al. 338-351 (2008 ) . US 10,508,291 B2 Page 3

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122 PRODUCTS/ POST PROCESSING PRODUCTS-CO 118 PRODUCTS/ IPRIMARY PROCESS PRODUCTSCO- FIG.1 114 PRETREATMENT 110 BIOMASS FEED PREPERATION U.S. Patent Dec. 17 , 2019 Sheet 2 of 54 US 10,508,291 B2

210 -212 FIRST 216 SCREEN SHEARING FIRST 214 SECOND SOURCE FIBROUS FIBROUS 200 MATERIAL MATERIAL

FIG . 2 U.S. Patent Dec. 17 , 2019 Sheet 3 of 54 US 10,508,291 B2

220

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PE VACUUM FIG . 3 SOURCE 252 U.S. Patent Dec. 17 , 2019 Sheet 4 of 54 US 10,508,291 B2

220 FIBROUS MATERIAL SECOND SCREEN 222 SECOND FIBROUS MATERIAL 216 FIRST SCREEN 214 212 FIRST FIBROUS MATERIAL FIG.4

210 FIBER SOURCE U.S. Patent Dec. 17 , 2019 Sheet 5 of 54 US 10,508,291 B2

ADDITIVES

LOW BULK DENSIFICATION HIGH BULK DENSITY DENSITY MATERIAL MATERIAL

BINDER

FIG . 5 U.S. Patent Dec. 17 , 2019 Sheet 6 of 54 US 10,508,291 B2

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TREATMENT

COMBINE WITH FIRST SECOND MICRO - ORGANISM COMPOSITE ( 4 ) MATERIAL MATERIAL PRODUCT ( 5 ) ( 2 ) OPTIONALLY ( 3 ) OR COMBINE UNDER WITH RESIN Thiny OXIDIZING TMng Ton CONDITIONS TC2 TOA TO2

FIG . 8 U.S. Patent Dec. 17 , 2019 Sheet 9 of 54 US 10,508,291 B2

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3052 Advance Carrying Medium To Electron Beam Irradiation Source #i Filter 3065 3053 Expose liquid 3054 i + 14 - ?

3060 yes MechanicalSeparation 3055 solid 3070 Advance To Next Processing FIG . 11 step U.S. Patent Dec. 17 , 2019 Sheet 11 of 54 US 10,508,291 B2

OH OH sto HO SANDRAMUKA HO HO OXIDATION QUENCH wshtainult BIOMASS INCLUDINGRADICALS .11AFIG PRETREATMENT IONIZINGRADIATION BIOMASS U.S. Patent Dec. 17 , 2019 Sheet 12 of 54 US 10,508,291 B2

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w the WU W w w ANS ** 11140 *** *** *** ** *** *** co ming 11142 COOLING ***

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1216 1252 1226 1250 1230 1222 1216 1226 1248 FIG.12 1244 ??????1218 1248 1224 1214 1236 1232 -1242 1216 1218 1210 1220 1234 1238 1212 1240 U.S. Patent Dec. 17 , 2019 Sheet 15 of 54 US 10,508,291 B2

82 POWER SUPPLY 92 94

86 84 90

60 62 TRANSDUCER

72 70 COUPLING HORN 76 78 000000 HORN 80 FIG . 13 U.S. Patent Dec. 17 , 2019 Sheet 16 of 54 US 10,508,291 B2

Dry Feedstock 6010 6000 6020 Mechanical 6220 Processing Dry Feedstock 6030 6260 Moisture Adjustment Water 6230 6040 Wet Feedstock Pre - Heating FIG . 14 6210 6050 Gases Pyrolysis

6260 6250 6270 Water Quenching Further Processing 6170 6060 Gas Separation Steam Gas

6130 6140 6070 Slurry De -watering Gas Blower Filtering 6150 6120 6080 6110 Compression & Mechanical Solids Drying Separation Storage

Liquids Condense and Purified Liquids Purify 6200 6190 6090 6100 Solvents 6180 U.S. Patent Dec. 17, 2019 Sheet 17 of 54 US 10,508,291 B2

6510 600 } {}

? ???? ???? ???? ???? 3 52 ) ( 2 ) 658 655 652 ) 6520 AAA {} / 35 { 566 10 ?????

?? Enrnrawe { FG . 15 7

{} 6700 6750 674 IC

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6572 FG . 16 U.S. Patent Dec. 17 , 2019 Sheet 18 of 54 US 10,508,291 B2

1712

t

GAS IN L 1715 men te 2w N AIKKEAKOCKA

1713

GAS OUT

FIG . 17 U.S. Patent Dec. 17 , 2019 Sheet 19 of 54 US 10,508,291 B2

1820 . GAS IN 1824 1825 1821 OSISSIMO 1823 1822

GAS OUT

FIG . 18 U.S. Patent Dec. 17 , 2019 Sheet 20 of 54 US 10,508,291 B2

130

GAS IN Eldi 134 137

136

131 135 132

GAS OUT

FIG . 19 U.S. Patent Dec. 17, 2019 Sheet 21 of 54 US 10,508,291 B2

142 144 143 193 LASER

146 141 145 FIG . 20 U.S. Patent Dec. 17 , 2019 Sheet 22 of 54 US 10,508,291 B2

151

FIG . 21 U.S. Patent Dec. 17 , 2019 Sheet 23 of 54 US 10,508,291 B2

5010 Dry Feedstock 5000 5020 Mechanical Processing 5150 5030 Processed Dry Water Feedstock 5170 5040 Fiber Feedstock Protecting Mixing Additive 5210 5050 5200 Waste Aqueous Extraction Feedstock Basic Stream Slurry Agents 5180 5130 Oxidation 5060 Oxidizing Preprocessing Further Processing Agents of Feedstock Slurry 5110 5160 5220 Thermal 5070 Drying Treatment Distillation , Waste Oxidation of Feedstock Extraction , Stream 5190 Slurry Separation 5080 5090 Mechanical Liquid Separation Phase 5100 Drying 5120 Solid Phase Stages

Further FIG . 22 Processing U.S. Patent Dec. 17 , 2019 Sheet 24 of 54 US 10,508,291 B2

402 400 SHEARED FIBER OPTIONAL OPTIONAL SHEARING SCREENING ADDITIONAL FIBER SOURCE SHEARING SOURCE

404 OPTIONAL STEAM STEAM LIQUOR FIBER EXPLOSION RECOVERY EXPLOSION

STEAM OPTIONAL OPTIONAL OPTIONAL EXPLODED FIBER REVERTIBLE BULK FIBER ADDITIONAL DENSIFICATION SOURCE RECOVERY SHEARING

OPTIONAL SHEARING 401 SCREENING FIBROUS MATERIAL FREEZE -DRIED YEAST , BACTERIA , AND /OR ENZYME OPTIONAL OVERLMINGI OPTIONAL REVERTIBLE BULK CELLULASES ENZYME DENSIFICATION HYDROLYSIS 414 TRANSPORT ANDI OR STORAGE 412 FERMENTABLE SUGARS

E.COLI FERMENTATION FIG . 23 K011 PRODUCT ( E.G., ETHANOL) U.S. Patent Dec. 17 , 2019 Sheet 25 of 54 US 10,508,291 B2

476 464 VENT TO ATMOSPHERE 465

470 462

472 467

funnenen 466 STEAM FROM BOILER -468 469 474

WWE 479 478 FIG . 24 U.S. Patent Dec. 17 , 2019 Sheet 26 of 54 US 10,508,291 B2

2500

2510

2540 2520 2520

2530 2545 2530 WWWWWWWWWWWW

2535 2535 2550

FIG . 25 U.S. Patent Dec. 17 , 2019 Sheet 27 of 54 US 10,508,291 B2

X25 1mm FIG . 26 U.S. Patent Dec. 17 , 2019 Sheet 28 of 54 US 10,508,291 B2

X25 1 mm FIG . 27 U.S. Patent Dec. 17 , 2019 Sheet 29 of 54 US 10,508,291 B2

2008 X25 FIG . 28 U.S. Patent Dec. 17 , 2019 Sheet 30 of 54 US 10,508,291 B2

X25 1mm FIG . 29 U.S. Patent Dec. 17 , 2019 Sheet 31 of 54 US 10,508,291 B2

500 502

504 506 508 512 517 516 WWW.MM.520

FIG . 30

506

510 508 512

FIG . 31 U.S. Patent Dec. 17 , 2019 Sheet 32 of 54 US 10,508,291 B2

FIG . 32

FIG . 33 U.S. Patent Dec. 17 , 2019 Sheet 33 of 54 US 10,508,291 B2

FIG . 34

FIG . 35 U.S. Patent Dec. 17, 2019 Sheet 34 of 54 US 10.508,291 B2

??

C. 5 U.S. Patent Dec. 17 , 2019 Sheet 35 of 54 US 10,508,291 B2

100

80 70 100 95 90 85 80 %Transmittance 70 REGIONA 65 60 55

4000 3500 3000 2500 2000 1500 1000 500 *Wavenumbers ( cm - 1 ) FIG . 37 U.S. Patent Dec. 17, 2019 Sheet 36 of 54 US 10,508,291 B2

00 80

100 20 %Transmittance { { }

REGION A ?? 10 4000 35 ){ 3000 { } {( 2000 100) 000 Wavenumbers (cm - 1 ) ?? ?? FC . 38 U.S. Patent Dec. 17 , 2019 Sheet 37 of 54 US 10,508,291 B2

114 -ALCOHOLS ELECTRICITY STEAM * 1 PRETREATMENT SONICATION DENATURANT LIVESTOCK BURNER/BOILER TURBOGENERATOR 118 DEHYDRATION P PROCESSINGPOST- IRRADIATIONby RESIDUE

with HEAT ELECTRICTY DISTILLATION LIGNIN SEPARATION WATER * GE ELECTRICITY FIG.39 SHREDDERSLICER ELECTRICTY SCREEN CENTRIFUGE YEAST PRIMARY PROCESSES 118 SWITCH GRASS FERMENTATION 1 ** + PREPARATION U.S. Patent Dec. 17 , 2019 Sheet 38 of 54 US 10,508,291 B2

4 * 1 ?? i oth 4 ** *** 4 3 3 WA ***

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50

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3 3 PRETREATMENT 3 3 & ittim 3 3 3 ww 3 ??? * am } w 40 ??? * WATER FIG.40 PREPARED FEEDSTOCK IRRADIATION SONICATION PRIMARY PROCESS U.S. Patent Dec. 17 , 2019 Sheet 39 of 54 US 10,508,291 B2

8014 8000 8012 8002 8016

8030 8028 8020 8010 8026 8042 8036 8022 8044 8018 8032 8034 8038 8024 LA 8020 8010 8042 8026 8036 8044 8022 8018 8024 8032 8034 8038 8020 14 8042 8010 8026 8036 8022 8044 8018 8024

* WANAE 8032 8034 8038 8020 8010 8042 8026 8036 8022 8044 8018 8024 8032 8034 8038 FIG . 41 U.S. Patent Dec. 17 , 2019 Sheet 40 of 54 US 10,508,291 B2

8508

8502 8516 8514

8518

8520

8522 8504

FIG . 42 8524 THI 8506

8526 8532 8528

8510 8530 8534

8512 U.S. Patent Dec. 17 , 2019 Sheet 41 of 54 US 10,508,291 B2

BIOMASS

TREATMENT ( reducing racalcitrance levels )

PRODUCTS FIG . 43A

BIOMASS

TREATMENT (reducing racalcitrance levels )

BIOCONVERSION

FIG . 43B PRODUCTS U.S. Patent Dec. 17 , 2019 Sheet 42 of 54 US 10,508,291 B2

ORGANISM + PROCESSED BIOMASS / CULTURE SOLUTION (CELL DENSITY A / VOLUME X )

FERMENTATION

CELL DENSITY C / VOLUME Y

PROCESSED FERMENTATION BIOMASS / CULTURE SOLUTION CELL DENSITY CI VOLUME Y

wwwwwwwwwww www

FERMENTATION

PRODUCT

FIG . 44 U.S. Patent Dec. 17 , 2019 Sheet 43 of 54 US 10,508,291 B2

ORGANISM + PROCESSED BIOMASS / CULTURE SOLUTION (CELL DENSITY A / VOLUME X )

FERMENTATION

CELL DENSITY BI VOLUME X

PROCESSED FERMENTATION BIOMASS / CULTURE SOLUTION CELL DENSITY C / VOLUME X

FERMENTATION

CELL DENSITY D VOLUME X - PRODUCT

PRODUCT

FIG . 45 U.S. Patent Dec. 17 , 2019 Sheet 44 of 54 US 10,508,291 B2

BIOMASS

PRE - TREATMENT PRODUCT 1 (SHRED / SHEAR )

TREATMENT (RADIATION / SONICATION / PRODUCT 2 PYROLYSIS OXIDATION )

PROCESSING PRODUCT 3

FIG . 46 U.S. Patent Dec. 17 , 2019 Sheet 45 of 54 US 10,508,291 B2

FIG . 47 Biosynthesis Pathway of Aromatic Amino Acids

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... hosphodnopyruvic acid ... from gyalyzis;

Og 3jupiespirate -Deoxy -o3rabinoheniuiosondacio

Shikiria

Spe Phosphoencepravkem Hi 3 -enolpyruvy sakanic aci stilrosaksi

.

Setia ? withthe

21.11 Freglenk acis U.S. Patent Dec. 17 , 2019 Sheet 46 of 54 US 10,508,291 B2

FIKI

OGI

|---Oleandrose tobidesMacroiliesandk 48AFIG. $ OAJI Oledy

*

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$ mi Meg4111[lryIII FI??? wwwthHon H0Piko 1 12-memberedmacrolides 14-Memberedmacrolides ETYK U.S. Patent Dec. 17 , 2019 Sheet 47 of 54 US 10,508,291 B2

7 Oleandrose KARYAARPIRRY*1 Avermect410

AwWwwWwwmmm

Isobutyrate 7 Mycerose 110.4 N1144 .488FIG Wildaam

JIMTAL Mycaminose Mycarose Rago

maxidesmenteret12,14-and16-Biosynthesisof Mamlitesoldesan RapN Ranxa 2 191011 andrelatedcompoundsKaparnycin RapJ an Sama 16-Memberedacrolides RapP U.S. Patent Dec. 17 , 2019 Sheet 48 of 54 US 10,508,291 B2

Rite

OH 0HO

2

FIG.480 43117 OITSH

. 1 10 ADRP3 Biosynthesisof813mycina OHH antibioticsAnsamycin OTUS" an17 U.S. Patent Dec. 17 , 2019 Sheet 49 of 54 US 10,508,291 B2

Busconi Bluesoon (139 ION TN TYUNURKR FR-Ucria

*

FIG.48D ????? Puursoon Mycosamine

????"; imatein 3 loudy ITERBOWY ???? 7

Z KOKTE macrolidesPolgjene Tüg.* ????????????????????????????????? 44 OSU U.S. Patent Dec. 17 , 2019 Sheet 50 of 54 US 10,508,291 B2

SOM YIMBIOS

** WW * ??????

48EFIG.

OH

Epok Cheer EPOF productsMyxobacterial V Epod writerter w U.S. Patent Dec. 17 , 2019 Sheet 51 of 54 US 10,508,291 B2

www Wynamal Mixabi MXBZ

ka Mx FIG.48F

OH

misadeSiska Mis1 WoToRE U.S. Patent Dec. 17 , 2019 Sheet 52 of 54 US 10,508,291 B2

FIG . 49 Biosynthesis of Streptomycin U.S. Patent Dec. 17 , 2019 Sheet 53 of 54 US 10,508,291 B2

LeuD-

NH NHL 2 UouUTVALET Spinomosanus HeptDeprideFauconyinlycone

opti IIDP pivaraosamine1.-e

Epianco$40 ?

4Ozovacowning GIDP-LTRUCOS HisptapepudeVRComprixeglycon

(Hpe FIG.50 phenyiglycine DP-3-691-2,3,6tudoky D-ABOMOTOBYI0a4Pose

0ancomycin phenylenoxyike Becopyrexos-4uosoDLythro Vancosamint naroza TDD-3nito OLCH

3,5-Dünydroxy IDP1-eswrwith(R) 5-19.tyl-5-020-2Hpen 1-Hydroxy 9o-no4kyucoxy

Eve.

DUSA 6-teoxyDglucos <-HydoryPersyuva d'DP4-oxo 42.1.46***** tynosineandPhenylalaning, BiosynthesisVancomycinof tytophanbiosyntesis metabolismPyrwhite TDPlose-D Nucleotidesuarametabolism U.S. Patent Dec. 17 , 2019 Sheet 54 of 54 US 10,508,291 B2

FIG.51

... oh Ýmbeta198cial

*

3 BiosynthesisAnsamycin US 10,508,291 B2 1 2 PROCESSING BIOMASS lar structure of the polysaccharides of the first material is then modulated ( e.g. , increased, decreased , or maintained ) to RELATED APPLICATIONS produce a second material with a greater nutrient (e.g. , protein , carbohydrate , fat , vitamin , and /or mineral) avail This application is a divisional of U.S. Ser . No. 15/040 , 5 ability than the first material. The methods can optionally 318 , filed Feb. 10 , 2016 , which is a continuation of U.S. Ser. include providing the second material to animals ( e.g., humans and / or non -human animals ) . No. 14 /813,898 , filed Jul. 30 , 2015 , which is a continuation In some embodiments , the methods described herein can of U.S. Ser. No. 14 / 750,995 , filed Jun . 25 , 2015 , which is a be used to generate materials suitable for use in maintaining nowcontinuation U.S. Pat .of No. U.S. 9,309,545 Ser. No., 14issued /333,675 on Apr. , filed 12 ,Jul 2016. 17 , , which2014 , 10 or promoting the growth of microorganisms (e.g. , bacteria , is a continuation of U.S. Ser. 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 , 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 , 15 In another aspect , the present invention provides methods 2008, U.S. Provisional Application 61 /073,674 filed Jun . 18 , of improving the pharmaceutical profile of materials . These 2008 and U.S. Provisional Application 61/ 049,405 filed Apr. 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 20 of cellulose , hemicellulose , and /or starch , and modulating TECHNICAL FIELD (e.g. , increasing , decreasing , or maintaining ) the molecular structure of the polysaccharides of the first material to This invention relates to processing biomass , to compo produce a second material, where one of the results of the sitions including saccharide units arranged in a molecular methods is that the pharmaceutical profile of the second chain , to methods of producing amino acids or antibiotics , to 25 material is better or improved when compared to the phar methods of producing edible or immunostimulatory mate maceutical profile of the first material. In some instances, the rial, and to products of such methods . methods include using first materials with little or no phar maceutical profile prior to modulating the molecular struc BACKGROUND ture of the first material. The second materials produced 30 using the methods described herein are suitable for admin Biomass , particularly biomass waste , is abundantly avail istration to an animal. able . It would be useful to derive products from biomass . In a further aspect , the invention provides methods for obtaining a plant- derived pharmaceutical. These methods SUMMARY include processing a material including biomass ( e.g., plant 35 biomass , animal biomass , microbial, and municipal waste Exemplary products that can be produced using themeth biomass) containing polysaccharides in the form of cellu ods provided herein include foodstuffs suitable for use in , lose , hemicellulose , and /or starch containing one or more e.g., ingestion by a human and / or animal, aquaculture , plant made pharmaceuticals , using any one or more of agriculture , hydroponics , pharmaceuticals , nutraceuticals , radiation , sonication , pyrolysis , and oxidation to obtain a pharmaceutical delivery vehicles and dosage forms, phar- 40 plant- derived pharmaceutical. In some instances the plant maceutical excipients , pharmaceutical conjugates , cross derived made pharmaceutical can be isolated and / or puri linked matrixes such as hydrogels , absorbent materials , fied . fertilizers , and lignin products . Any product disclosed herein In yet another aspect, the present invention provides or produced by the methods disclosed herein can be used methods of preparing nutraceuticals for human and /or a as- is , or as a precursor or an intermediate in the production 45 non -human animal consumption . These methods include of another product . processing a material including biomass ( e.g. , plant biomass , In many embodiments , products can be produced using animal biomass , microbial, and municipal waste biomass ) Natural ForceTM Chemistry. Natural ForceTM Chemistry containing polysaccharides in the form of cellulose , hemi methods use the controlled application and manipulation of cellulose , and /or starch so as to change the molecular physical forces, such as particle beams, gravity , light, etc. , to 50 structure of the polysaccharides of the material ( e.g. , create intended structural and chemicalmolecular change. In increase or decrease the molecular weight of the material ). preferred implementations, Natural ForceTM Chemistry These methods can optionally also include administering the methods alter molecular structure without chemicals or resulting materials to humans and non -human animals . microorganisms. By applying the processes of Nature, new In an alternative aspect, the invention provides methods usefulmatter can be created without harmful environmental 55 of preparing biological agents and / or pharmaceutical agents . interference . These method include processing a material including bio In one aspect, the present invention includes methods of mass containing polysaccharides in the form of cellulose , preparing feed materials for animals ( e.g. , humans and hemicellulose , and /or starch , so as to change the molecular animals , including but not limited to food animals , pets , zoo structure of the polysaccharides of the material. The result animals , etc. ), and for plants ( e.g., agricultural plants or 60 ing materials can then be combined with one or more crops or aquatic plants, in particular in a hydroponic solution biological agents and /or one or more pharmaceutical agents , or in aquaculture ), and aquatic organisms ( e.g., fish , crus which can be administered to a subject. taceans, mollusks and the like ) . Also provided in the present invention are methods of These methods include obtaining a firstmaterial including making hydrogels . These methods include processing a biomass ( e.g. , plant biomass , animal biomass ,microbial , and 65 material including biomass containing polysaccharides in municipal waste biomass ) containing polysaccharides in the the form of cellulose , hemicellulose , and /or starch , and form of cellulose, hemicellulose , and /or starch . The molecu changing the molecular structure of the polysaccharides to US 10,508,291 B2 3 4 produce a material that includes cross- linked polymer flagellates , amoeboids, ciliates, and sporozoa ) and plant 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. 5 weed , water hyacinth , plankton ( e.g., macroplankton , meso These methods include processing a material including 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 tive bacteria , gram negative bacteria , and extremophiles) , structure of the polysaccharides to produce an absorbent yeast and /or mixtures of these. In some instances , biomass material. These absorbent materials can be charged , e.g. , 10 can include unicellular or multicellular organisms obtained positively or negatively charged , and can have lipophilic from the ocean , lakes , and bodies of water including salt and / or hydrophilic properties. As such , the materials can be water and fresh water . In some instances, biomass can used as animal litter or bedding, and / or absorbent material to include organic waste materials such as animal waste or bind materials in a solution , ( e.g., pollutants ) . In some excrement or human waste or excrement ( e.g., manure and embodiments , these absorbent materials can be used to bind 15 sewage ). In some instances , biomass can include any com biological materials in solutions of blood or plasma. bination of any of these . Other biomass materials are In a further aspect, the present invention provides meth described herein . Still other materials that include cellulose ods of making fertilizers . These methods include processing are described in the patents , patent applications and publi a material including biomass containing polysaccharides in cations that have been incorporated by reference herein . In the form of cellulose , hemicellulose, and / or starch , and 20 some instances, biomass can be, e.g., in solution , dry , and changing the molecular structure of the polysaccharides to frozen . produce a material that has a greater solubility than the If biomass is or includes microorganisms, these microor starting material and which is useful as a fertilizer. ganisms will generally include carbohydrates , e.g., cellu Each of these methods includes treating the biomass using lose. These microorganisms can be in a solution , dry , frozen , one or more of ( e.g. , one, two , three , or four of) size 25 active, and / or inactive state . In some embodiments, these reduction (e.g. , mechanical size reduction of individual microorganisms can require additional processing prior to pieces of biomass) , radiation , sonication , pyrolysis , and being subjected to the methods described herein . For oxidation to modulate the materials . In some embodiments , example , themicroorganisms can be in a solution and can be the methods use a radiation dose, e.g. , from 0.1 Mrad to 10 removed from the solution, e.g., by centrifugation and / or Mrad . In some embodiments , the methods use a radiation 30 filtration . Alternatively or in addition , the microorganisms dose, e.g. , from greater than 10 Mrad to 1000 Mrad . can be subjected to the methods described herein without In some aspects , the present invention also provides these additional steps , e.g., the microorganisms can be used compositions made using any of the methods described in the solution . In some instances, the biomass can be or can herein . For example , the invention features a composition include a natural or a synthetic material . including saccharide units arranged in a molecular chain , 35 Irradiation can be, e.g. , performed utilizing an ionizing wherein from about 1 out of ever 2 to about 1 out of every radiation , such as gamma rays , a beam of electrons, or 250 saccharide units comprises a carboxylic acid group , or ultraviolet C radiation having a wavelength of from about an ester or a salt thereof, and the composition is suitable for 100 nm to about 280 nm . The ionizing radiation can include consumption as a feed material. electron beam radiation . For example , the radiation can be By “ suitable for consumption as a feed material, ” we 40 applied at a total dose of between about 10 Mrad and about mean that the composition is not toxic , under conditions of 150 Mrad , such as at a dose rate of about 0.5 to about 10 its intended use , to the living being to which it is fed , and Mrad /day , or 1 Mrad / s to about 10 Mrad / s . In some embodi provides some nutritional value to the being, e.g. , energy ments, irradiating includes applying two or more radiation and /or nutrients . sources, such as gamma rays and a beam of electrons. In some embodiments , the biomass feedstock is pre- 45 In some embodiments , the biomass exhibits a first level of treated . In some embodiments , the methods disclosed herein recalcitrance and the carbohydrate material exhibits a sec can include a pre -treatment to reduce one or more dimen ond level of recalcitrance that is lower that the first level of sions of individual pieces of biomass . For example , pretreat recalcitrance . For example , the second level of recalcitrance ment can include reducing one or more dimensions of can be lower than the first level of recalcitrance by at least individual pieces of biomass can include , e.g. , shearing , 50 about 10 % ( e.g., 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , cutting , crushing , smashing , or grinding . 90 % , 99 % , 100 % ) . In some embodiments , the level of Pressure can be utilized in all of the methods described recalcitrance can be reduced by 50 % -90 % . herein . For example , at least one of the treating methods, The biomass can be prepared by shearing biomass ( e.g. , e.g., radiation , can be performed on the biomass under a a biomass fiber source ) to provide a fibrous material. For pressure of greater than about 2.5 atmospheres , such as 55 example , the shearing can be performed with a rotary knife greater than 5 or 10 atmospheres . cutter. The fibers of the fibrous material can have , e.g., an Examples of biomass (also referred to as 'biomass feed average length - to -diameter ratio ( L / D ) of greater than 5/1 . stock ' or 'feedstock ') include cellulosic or lignocellulosic The fibrous material can have , e.g. , a BET surface area of materials such as paper, paper products , paper waste , wood , greater than 0.25 m < / g ( e.g. , 0.3 m²/ g , 0.35 m?lg , 0.35 m²/ g , particle board , sawdust , agricultural waste , sewage , silage, 60 0.4 m²/ g , 0.5 m²/ g , 1 m / g , 1.5 m / g , 2 m²/ g , 3 m´/ g , 10 grasses , rice hulls , bagasse , cotton , jute , hemp , flax, bamboo , m / g , 25 m /g , or greater than 25 m /g ). sisal, abaca, straw , corn cobs, corn stover, switchgrass , In some embodiments , the carbohydrate can include one alfalfa , hay, rice hulls , coconut hair , cotton , cassava , and or more B - 1,4 - linkages and have a number average molecu synthetic and /or mixtures of these . In some lar weight between about 3,000 and 50,000 daltons. instances, biomass can include unicellular and /or multicel- 65 In some examples, the pretreated biomass material can lular organisms. Exemplary organisms include , but are not further include a buffer , such as sodium bicarbonate or limited to , e.g., protists ( e.g., animal (e.g. , protozoa such as ammonium chloride, an electrolyte , such as potassium chlo US 10,508,291 B2 5 6 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 slimemold , 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 ofmore 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, ormixtures 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 ofproducts 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 10,508,291 B2 7 8 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 patent application incorporated by reference herein . chloride , calcium carbonate , sodium carbonate , benzyltrim 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 extended 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 . 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 of WO2008 /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 applica FIG . 17 is a cross -sectional side view of a pyrolyzer that tions , 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 10,508,291 B2 9 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 optional densification step can be performed . of the process of converting a fiber source into a product, FIG . 47 is a diagram illustrating the biosynthesis of e.g., ethanol. 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 materialwas 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 use 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 10,508,291 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 , losic feedstock can be plantbiomass such as, butnot limited coffee beans, coffee bean grounds (used coffee bean to , non -woody plant biomass , cultivated crops, such as , but grounds) , cotton, synthetic celluloses, or mixtures of any of 10 not limited to , grasses , for example , but not limited to , C4 these . grasses , such as switchgrass , cord grass , rye grass , miscan Fiber sources include cellulosic fiber sources , including thus, reed canary grass, or a combination thereof, or sugar paper and paper products (e.g. , polycoated paper and Kraft processing residues such as bagasse, or beet pulp , agricul paper ) , and lignocellulosic fiber sources , including wood , tural residues , for example , stover, corn stover , rice and wood -related materials , e.g. , particle board . Other suit- 15 straw , rice hulls , straw , corn cobs, wheat straw , canola able fiber sources include natural fiber sources , e.g., grasses , straw , rice straw , 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 sources. Also , the fiber source can be obtained or derived rally occurring or genetically modified unicellular organ from human ( e.g. , sewage ), animal, or plant waste . Addi- 30 isms and/ or multicellular organisms, e.g., organisms from tional fiber sources have been described in the art, for the ocean , lakes , bodies of water , e.g. , salt water or fresh example , see U.S. Pat. Nos. 6,448,307 , 6,258,876 , 6,207 , water , or on land, and that contains a source of carbohydrate 29 , 5,973,035 and 5,952,105 . ( e.g., cellulose ). Microbial biomass can include , but is not Microbial sources include , but are not limited to , any limited to , for example protists ( e.g. , animal ( e.g., protozoa naturally occurring or genetically modified microorganism 35 such as flagellates, amoeboids, ciliates , and sporozoa ) and or organism that contains or are capable of providing a plant ( e.g., algae such alveolates , chlorarachniophytes , cryp source of carbohydrates ( e.g., cellulose) , for example , pro tomonads, euglenids, glaucophytes , haptophytes , red algae , tists ( e.g., animal ( e.g. , protozoa such as flagellates, amoe stramenopiles, and viridaeplantae) ) , seaweed , plankton ( e.g ., boids , ciliates, and sporozoa ) and plant ( e.g., algae such macroplankton , mesoplankton , microplankton , nanoplank alveolates , chlorarachniophytes , cryptomonads, euglenids, 40 ton , picoplankton , and femptoplankton ), phytoplankton , glaucophytes, haptophytes , red algae , stramenopiles, and bacteria ( e.g., gram positive bacteria , gram negative bacte viridaeplantae ) ), seaweed , plankton ( e.g., macroplankton , ria , and extremophiles ), yeast and /or mixtures of these . In mesoplankton , microplankton , nanoplankton , picoplankton , some instances , microbial biomass can be obtained from and femptoplankton ) , phytoplankton , bacteria ( e.g., gram natural sources, e.g., the ocean , lakes, bodies of water , e.g. , positive bacteria , gram negative bacteria , and extremo- 45 salt water or fresh water , or on land . Alternatively or in philes ) , yeast and /or mixtures of these. In some instances, addition , microbial biomass can be obtained from culture microbial biomass can be obtained from natural sources , systems, e.g. , large scale dry and wet culture systems. e.g., the ocean , lakes , bodies of water, e.g. , salt water or Animal biomass includes any organic waste material such fresh water , or on land . Alternatively or in addition , micro as animal- derived waste material or excrement or human bial biomass can be obtained from culture systems, e.g., 50 waste material or excrement (e.g. , manure and sewage ) . large scale dry and wet culture systems. In some embodiments , the carbohydrate is or includes a Examples of biomass include renewable , organic matter, material having one or more B - 1,4 -linkages and having a such as plant biomass , microbial biomass, animal biomass number average molecular weightbetween about 3,000 and ( e.g., any animal by -product , animal waste , etc. ) and munici 50,000 . Such a carbohydrate is or includes cellulose ( I) , pal waste biomass including any and all combinations of 55 which is derived from (ß - glucose 1 ) through condensation of these biomass materials . B (14 ) -glycosidic bonds . This linkage contrasts itself with Plant biomass and lignocellulosic biomass include that for a (1-4 ) -glycosidic bonds present in starch and other organic matter (woody or non -woody ) derived from plants , carbohydrates. especially matter available on a sustainable basis . Examples 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 ), ?? , agricultural crop wastes and residues such as corn stover, wheat straw , rice straw , sugar cane bagasse , and the like. OH Plant biomass further includes , but is not limited to , trees , 65 HOHO woody energy crops, wood wastes and residues such as OH softwood forest thinnings, barky wastes , sawdust, paper and US 10,508,291 B2 13 14 -continued -continued I OH OH HO JULIOH HO2C HO . OH 5 HOW HOH HOW OH HO OH HO OH OH f glucose 2 -keto - gluconic acid HO Starchy materials include starch itself , e.g., corn starch , 10 wheat starch , starch or rice starch , a derivative of OH starch , or a material that includes starch , such as an edible food product or a crop . For example , the starchy material can HO be arracacha , buckwheat, banana , barley , cassava , kudzu , oca, sago , sorghum , regular household potatoes, sweet 15 HO2C OH potato , taro , yams, or one or more beans, such as favas, lentils or peas . Blends of any one or more starchy material is also a starchy material . In particular embodiments , the ????? "OH starchy material is derived from corn . Various corn starches 20 OH and derivatives are known in the art , see , e.g., “ Corn Starch , ” 6 -MAS Corn Refiners Association (11th Edition , 2006 ). 6 -monoacid of sucrose Biomass materials that include low molecular weight OH sugars can , e.g., include at least about 0.5 percent by weight HO2C IOH OH of the low molecular sugar , e.g., at least about 2 , 3 , 4 , 5 , 6 , 25 7 , 8 , 9 , 10 , 12.5 , 25, 35 , 50 , 60 , 70 , 80 , 90 or even at least about 95 percent by weight of the low molecular weight ?? " "OH ??!!! sugar. In some instances , the biomass is composed substan OH HOS OH tially of the low molecular weight sugar, e.g., greater than 95 glucuronic acid percent by weight, such as 96 , 97 , 98 , 99 or substantially 100 30 fructose percent by weight of the low molecular weight sugar . HO Biomass materials that include low molecular weight sugars can be agricultural products or food products , such as OH sugarcane and sugar beets or an extract therefrom , e.g. , juice from sugarcane , or juice from sugar beets . Biomass mate- 35 HO . rials that include low molecular weight sugars can be substantially pure extracts , such as raw or crystallized table OH 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 , 40 HOM.... " OH 5 -mer , 6 -mer , 7 -mer , 8 -mer , 9 -mer or 10 -mer ) , trimeric , dimeric , or monomeric . When the carbohydrates are formed OH ofmore than a single repeat unit , each repeat unit can be the sucrose same or different. Specific examples of low molecular weight sugars include 45 cellobiose , lactose, sucrose , glucose and xylose , along with Combinations ( e.g., by themselves or in combination of derivatives thereof. In some instances, sugar derivatives are any biomass material, component, product , and /or co -prod more rapidly dissolved in solution or utilized by microbes to uct generated using the methods described herein ) of any provide a useful material . Several such sugars and sugar biomass materials described herein can be utilized for mak derivatives are shown below . 50 ing any of the products described herein . For example , blends of cellulosic materials and starchy materials can be utilized for making any product described herein . ?? . Systems for Treating Biomass FIG . 1 shows a system 100 for converting biomass, OH 55 particularly biomass with significant cellulosic and ligno cellulosic components and /or starchy components , into use ful products and co -products . System 100 includes a feed HO2C preparation subsystem 110 , a pretreatment subsystem 114 , a OH primary process subsystem 118 , and a post- processing sub HO 60 system 122. Feed preparation subsystem 110 receives bio mass in its raw form , physically prepares the biomass for use HO ... MOH as feedstock by downstream processes (e.g. , reduces the size of and homogenizes the biomass ), and stores the biomass OH both in its raw and feedstock forms. 1' MAS- 65 Biomass feedstock with significant cellulosic and /or ( 1 ' -monoacid of sucrose ) lignocellulosic components , or starchy components can have a high average molecular weight and crystallinity that can US 10,508,291 B2 15 16 make processing the feedstock into useful products ( e.g. , Size Reduction fermenting the feedstock to produce ethanol) difficult . In some embodiments , the biomass is in the form of a Accordingly it is useful to treat biomass feedstock , e.g. , fibrous material that includes fibers provided by shearing the using the treatment methods described herein . As described biomass . For example, the shearing can be performed with herein , in some embodiments , the treatment of biomass does 5 a rotary knife cutter. not use acids, bases and /or enzymes to process biomass , or For example , and by reference to FIG . 2 , a biomass fiber only uses such treatments in small or catalytic amounts . source 210 is sheared , e.g., in a rotary knife cutter , to provide a first fibrous material 212. The first fibrous material 212 is Treatment subsystem 114 receives biomass feedstock passed through a first screen 214 having an average opening from the feed preparation subsystem 110 and prepares the 10 feedstock for use in primary production processes by, for size of 1.59 mm or less ( 1/16 inch , 0.0625 inch ) to provide a example , reducing the average molecular weight and crys second fibrous material 216. If desired , fiber source can be tallinity of the feedstock . Primary process subsystem 118 cut prior to the shearing, e.g. , with a shredder. For example , receives treated feedstock from treatment subsystem 114 and when a paper is used as the fiber source, the paper can be first produces useful products ( e.g., ethanol, other alcohols, phar- 15 cut into strips that are , e.g., 1/ 4- to 1/ 2 -inch wide , using a maceuticals , and/ or food products ). In some cases, the shredder, e.g., a counter - rotating screw shredder , such as output of primary process subsystem 118 is directly useful those manufactured by Munson (Utica , N.Y.) . As an alter but, in other cases, requires further processing provided by native to shredding, the paper can be reduced in size by post- processing subsystem 122. Post- processing subsystem cutting to a desired size using a guillotine cutter. For 122 provides further processing to product streams from 20 example , the guillotine cutter can be used to cut the paper primary process system 118 which require it ( e.g., distilla into sheets that are , e.g. , 10 inches wide by 12 inches long . tion and denaturation of ethanol) as well as treatment for In some embodiments , the shearing of fiber source and the waste streams from the other subsystems. In some cases, the passing of the resulting first fibrous material through first co -products of subsystems 114 , 118 , 122 can also be directly screen are performed concurrently . The shearing and the or indirectly useful as secondary products and / or in increas- 25 passing can also be performed in a batch -type process . ing the overall efficiency of system 100. For example , For example , a rotary knife cutter can be used to concur post -processing subsystem 122 can produce treated water to rently shear the fiber source and screen the first fibrous be recycled for use as process water in other subsystems material. Referring to FIG . 3 , a rotary knife cutter 220 and /or can produce burnable waste which can be used as fuel includes a hopper 222 that can be loaded with a shredded for boilers producing steam and / or electricity . 30 fiber source 224 prepared by standard methods. Shredded The optimum size for biomass conversion plants is fiber source is sheared between stationary blades 230 and affected by factors including economies of scale and the type rotating blades 232 to provide a first fibrous material 240 . and availability of biomass used as feedstock . Increasing First fibrousmaterial 240 passes through screen 242 , and the plant size tends to increase economies of scale associated 35 resulting second fibrousmaterial 244 is captured in bin 250 . with plant processes . However , increasing plant size also To aid in the collection of the second fibrous material, the tends to increase the costs ( e.g., transportation costs ) per unit bin can have a pressure below nominal atmospheric pres of feedstock . Studies analyzing these factors suggest that the sure, e.g., at least 10 percent below nominal atmospheric appropriate size for biomass conversion plants can range pressure , e.g. , at least 25 percent below nominal atmospheric from 100 to 1,000 or more, e.g., 10,000 or more dried tons 40 pressure , orat atleast least 50 75 percent percent below below nominalnominal atmosphericatmospheric of feedstock per day depending at least in part on the type pressure . In some embodiments , a vacuum source 252 is of feedstock used . The type of biomass feedstock can also utilized to maintain the bin below nominal atmospheric impact plant storage requirements with plants designed pressure . primarily for processing feedstock whose availability varies 45 Shearing can be advantageous for “ opening up ” and seasonally ( e.g., corn stover ) requiring more on- or off- site “ stressing " the fibrous materials, making the cellulose of the feedstock storage than plants designed to process feedstock materials more susceptible to chain scission and /or reduction whose availability is relatively steady ( e.g., waste paper) . of crystallinity . The open materials can also be more sus Biomass Pretreatment ceptible to oxidation when irradiated . In some cases, pretreatmentmethods of processing begin 50 In some embodiments , shearing can be advantageous for with a physical preparation of the biomass , e.g. , size reduc “ opening up ” and “ stressing” the fibrous materials , making tion of raw biomass feedstock materials , such as by cutting , the cellulose of the materials more susceptible to ruminant grinding , crushing , smashing , shearing or chopping . In some digestion and absorption . embodiments , methods ( e.g. , mechanical methods ) are used The fiber source can be sheared in a dry state , a hydrated to reduce the size and /or dimensions of individual pieces of 55 state (e.g. , having up to ten percent by weight absorbed biomass . In some cases , loose feedstock (e.g. , recycled paper water ) , or in a wet state , e.g., having between about 10 or switchgrass ) is pretreated by shearing or shredding . percent and about 75 percent by weight water . The fiber Screens and / or magnets can be used to remove oversized or source can even be sheared while partially or fully sub undesirable objects such as, for example , rocks or nails from 60 merged under a liquid , such as water, ethanol, or isopropa the feed stream . nol . Feed pretreatment systems can be configured to produce The fiber source can also be sheared in under a gas ( such feed streams with specific characteristics such as, for as a stream or atmosphere of gas other than air ), e.g., oxygen example, specific maximum sizes , specific length - to - width , or nitrogen , or in steam . or specific surface areas ratios. As a part of feed pretreat- 65 Other methods of making the fibrous materials include , ment, the bulk density of feedstocks can be controlled ( e.g., e.g. , stone grinding , mechanical ripping or tearing , pin increased ). grinding , and /or air attrition milling . US 10,508,291 B2 17 18 If desired , the fibrous materials can be separated , e.g., length - weighted lengths . BET (Brunauer , Emmet and Teller) continuously or in batches , into fractions according to their surface areas are multi -point surface areas , and porosities are length , width , density ,material type , or some combination of those determined by mercury porosimetry . these attributes . The average length - to - diameter ratio of the second fibrous For example , ferrous materials can be separated from any 5 material 14 can be greater than 5/1, e.g., greater than 8/1 , of the fibrous materials by passing a fibrous material that e.g., greater than 10/1 , greater than 15/1 , greater than 20/1 , includes a ferrous material past a magnet, e.g., an electro greater than 25/1 , or greater than 50/1. An average length of magnet , and then passing the resulting fibrous material the second fibrous material 14 can be , e.g. , between about through a series of screens, each screen having different 0.5 mm and 2.5 mm , e.g., between about 0.75 mm and 1.0 sized apertures . 10 mm , and an average width ( e.g., diameter ) of the second The fibrous materials can also be separated , e.g., by using fibrous material 14 can be , e.g., between about 5 um and 50 a high velocity gas, e.g., air . In such an approach , the fibrous um , e.g. , between about 10 um and 30 um . materials are separated by drawing off different fractions, 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. 15 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 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 . 20 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 0.25 m²/ g , greater than 0.5 m²/g , greater than 1.0 m²/ g , 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 25 5.0 m²/ g , greater than 10 m²/ g , greater than 25 m²/ g , greater hypochlorite ) and /or an enzyme. than 35 m´/ 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 m²/ g , greater than 150 first screen is less than 0.79 mm (1/32 inch , 0.03125 inch ), m²/ g , greater than 200 m´/ g , or even greater than 250 m²/ 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 30 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 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 35 greater than 97.5 percent, greater than 99 percent , or even opening size . For example , the monofilaments can bemade 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 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 40 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 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 45 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 % . 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 50 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 . 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 55 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 . properties . Generally , the fibers of the fibrous materials can have a Densification relatively large average length - to - diameter ratio (e.g. , 60 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 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 65 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 US 10,508,291 B2 19 20 material in a relatively gas impermeable structure , e.g., a bag ethylene oxide, polycarboxylic acids, polyamides , made of polyethylene or a bag made of alternating layers of polyamines and polysulfonic acids polysulfonates. Specific polyethylene and a nylon , and then evacuating the entrapped polymeric examples include polypropylene glycol (PPG ), 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 5 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/ cm3 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 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- 10 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 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 15 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 or more, e.g. , 95 percent of its initial available under the tradename KRATON® . Other suitable bulk density . To reduce static electricity in the fibrous 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 20 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 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 25 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 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 30 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 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 . 35 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 / cm² or The densified fibrous material can be made in a pellet less , e.g., 0.025 g / cmº. 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 40 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 gramsby 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 45 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 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 50 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 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 55 350. 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 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 , 60 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 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 , 65 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. , US 10,508,291 B2 21 22 2 mm , 3 mm , 5 mm , 8 mm , 10 mm , 15 mm or more, e.g., National Renewable Energy Laboratory (NREL ) Technical 25 mm . Other convenient shapes include pellets or chips that Report NRELUTP -510-37902 , August , 2005 and National are plate - like in form , e.g., having a thickness of 1 mm or Renewable Energy Laboratory (NREL ) Technical Report more , e.g., 2 mm , 3 mm , 5 mm , 8 mm , 10 mm or more , e.g., NREIJBR -510-40742 , March , 2007) . For example , the 25 mm ; a width of, e.g., 5 mm or more , e.g. , 10 mm , 15 mm , 5 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 or more , e.g., 10 mm , 15 mm , 25 mm , 30 mm or more , e.g. , level will be lower than the accessibility of polysaccharides 50 mm . ( 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 10 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 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 15 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 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 20 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 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 / ft3 25 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 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- 30 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 . 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 35 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 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 40 ( 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 ofmagnifications (as decrease particle size and increase overall surface area 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 45 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 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 50 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 pellet mill 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 . 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 55 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 , 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. , 60 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 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 65 ( 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 US 10,508,291 B2 23 24 allows analysis of surface topography at very high magni prising a first lignocellulosic material with a first recalci fication while also allowing analysis of the attractive and trance level will be lower than the amount (e.g. , concentra repulsive forces between the scanning probe tip and the tion ) of monosaccharide and /or disaccharides resulting from 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- 5 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 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 10 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 of biological and non -biological materials up to at disclosed herein , before and after treatment using the meth least 350,000x magnification . Typically , the determination 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- 15 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 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 20 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 ) 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 25 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 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 30 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 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 35 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, has a crystallinity ( C2) that is lower than the crystallinity for example fluorescent stains (see , e.g. , Sole et al ., Microb . (TC ) of the cellulose of the first material. For example , Ecol. , Published online on Nov. 4 , 2008 ) . 40 ( C2) 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 . 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 45 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 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 50 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 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- 55 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 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, 60 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 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 65 level of oxidation (TO ) that is higher than the level of monosaccharides and /or disaccharides resulting from the oxidation (10 ) 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, US 10,508,291 B2 25 26 swellability and /or solubility , further enhancing the materi Any feedstock or any reactor or fermentor charged with a al's susceptibility to chemical , enzymatic , or biological feedstock can include a buffer, such as sodium bicarbonate , attack . In some embodiments , to increase the level of the ammonium chloride or Tris ; an electrolyte , such as potas oxidation of the second material relative to the first material, sium chloride , sodium chloride , or calcium chloride; a the irradiation is performed under an oxidizing environment, 5 growth factor, such as biotin and /or a base pair such as uracil e.g. , under a blanket of air or oxygen , producing a second or an equivalent thereof; a surfactant, such as Tween® or material that is more oxidized than the first material. For polyethylene glycol; a mineral, such as such as calcium , example , the second material can have more hydroxyl chromium , copper , iodine, iron , selenium , or zinc; or a groups , aldehyde groups, ketone groups , ester groups, or chelating agent, such as ethylene diamine , ethylene diamine carboxylic acid groups, which can increase its hydrophilic 10 tetraacetic acid ( EDTA ) ( or its salt form , e.g., sodium or ity . potassium EDTA ), or dimercaprol. Treatment Combinations When radiation is utilized as or in the treatment, it can be In some embodiments , biomass can be treated by apply 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 15 in a liquid , such as water. For example , irradiation can be treatment methods 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 irradiating is performed on biomass material in which sub methods can be applied in any order , in multiples ( e.g., two 20 stantially none of the biomass material is wetted with a or more 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 has been dried , e.g., using heat and /or reduced pressure. For any of the processes described herein ( in any order or 25 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 utilized in any process described herein . In some embodi carbohydrate - containing material can then be converted by 30 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 ofmore than about 2.5 percent higher than of desirable products , as described herein . Multiple pro an unswollen state , e.g., more than 5.0 , 7.5 , 10 , or 15 percent cesses can provide materials that can be more readily 35 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 tially 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 biomass material includes a swelling agent, and swollen parison to any single process . 40 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 nitrogen , argon , or helium . When maximum oxidation is that includes irradiating and oxidizing ( in either order or 45 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 as greater than 5 , 10 , 15 , 20 or even greater than about 50 biomass material , or a process that includes irradiating and 50 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 specific embodiments , sonication precedes irradiating, or biomass to the product . 55 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 . When the process includes radiation , the irradiating can If desired, some or none of the biomass can include a 60 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 electrons, slow neutrons or alpha particles . In some embodi embodiments , substantially all of the biomass is an unhy- 65 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 . US 10,508,291 B2 27 28 In specific embodiments , sonicating can performed at a microorganism ( e.g., a bacterium or a yeast ) that can utilize frequency of between about 15 kHz and about 25 kHz, such the second and /or first material to produce a product 5 . as between about 18 kHz and 22 kHz utilizing a 1 KW or Since the second material 3 has cellulose having a larger horn , e.g., a 2 , 3 , 4 , 5 , or even a 10 KW horn . reduced recalcitrance, molecular weight relative to the first In some embodiments , the biomass has a first number 5 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 molecular weight is lower than the first number average 10 tible to chemical, enzymatic and / or biological attack ( e.g. , molecular weight by more than about twenty - five percent, by a microorganism ) relative to the first material 2 , which e.g., 2x , 3x , 5x , 7x , 10x, 25x , even 100x reduction . can greatly improve the production rate and/ or production In some embodiments , the first cellulose has a first level of a desired product , e.g., ethanol. Radiation can also crystallinity and the second cellulose has a second crystal sterilize the materials . linity lower than the first crystallinity , such as lower than 15 In some embodiments, the second number average about two, three , five , ten , fifteen or twenty - five percent molecular weight (MN2 ) is lower than the first number lower. average molecular weight ( "My? ) by more than about 10 In some embodiments , the first cellulose has a first level percent, e.g., 15 , 20 , 25 , 30 , 35, 40 , 50 percent, 60 percent, of oxidation and the second cellulose has a second level of or even more than about 75 percent. oxidation higher than the first level of oxidation , such as 20 Ionizing Radiation two, three , four, five , ten or even twenty - five percent higher. Each form of radiation ionizes the biomass via particular In some embodiments, the first biomass has a first level of interactions , as determined by the energy of the radiation . recalcitrance and the resulting biomass has a second level of Heavy charged particles primarily ionize matter via Cou recalcitrance that is lower than the first level . lomb scattering ; furthermore , these interactions produce Radiation Treatment 25 energetic electrons that can further ionize matter. Alpha One ormore irradiation processing sequences can be used particles are identical to the nucleus of a helium atom and to process biomass from a wide variety of different sources are produced by the alpha decay of various radioactive to extract useful substances from the feedstock , and to provide partially degraded organic material which functions nuclei, such as isotopes of bismuth , polonium , astatine , as input to further processing steps and / or sequences . Irra- 30 radon , francium , radium , several actinides, such as actinium , diation can reduce the recalcitrance , molecular weight and / thorium , uranium , neptunium , curium , californium , ameri or crystallinity of feedstock . cium , and plutonium . In some embodiments , energy deposited in a material that When particles are utilized , they can be neutral (un releases an electron from its atomic orbital is used to charged ), positively charged or negatively charged . When irradiate the materials . The radiation can be provided by 1 ) 35 charged , the charged particles can bear a single positive or heavy charged particles , such as alpha particles or protons, negative charge , or multiple charges , e.g., one, two , three or 2 ) electrons , produced , for example , in beta decay or elec even four or more charges . In instances in which chain tron beam accelerators , or 3 ) electromagnetic radiation , for scission is desired , positively charged particles can be desir example , gamma rays, x rays , or ultraviolet rays. In one able , in part , due to their acidic nature . When particles are approach , radiation produced by radioactive substances can 40 utilized , the particles can have the mass of a resting electron , be used to irradiate the feedstock . In some embodiments , or greater, e.g., 500 , 1000 , 1500 , or 2000 or more times the any combination in any order or concurrently of ( 1 ) through mass of a resting electron . For example , the particles can (3 ) can be utilized . In another approach , electromagnetic have a mass of from about 1 atomic units to about 150 radiation ( e.g., produced using electron beam emitters ) can atomic units , e.g. , from about 1 atomic units to about 50 be used to irradiate the feedstock . The doses applied depend 45 atomic units , or from about 1 to about 25 , e.g. , 1 , 2 , 3 , 4 , 5 , on the desired effect and the particular feedstock . For 10 , 12 or 15 amu . Accelerators used to accelerate the example, high doses of radiation can break chemical bonds particles can be electrostatic DC , electrodynamic DC , RF within feedstock components and low doses of radiation can linear, magnetic induction linear or continuous wave . For increase chemical bonding ( e.g. , cross - linking ) within feed example , cyclotron type accelerators are available from stock components . In some instances when chain scission is 50 IBA , Belgium , such as the Rhodotron® system , while DC desirable and /or polymer chain functionalization is desir type accelerators are available from RDI, now IBA Indus able , particles heavier than electrons , such as protons, trial , such as the Dynamitron® . Ions and ion accelerators are helium nuclei, argon ions, silicon ions , neon ions, carbon discussed in Introductory Nuclear Physics, Kenneth S. ions, phosphorus ions, oxygen ions or nitrogen ions can be Krane , John Wiley & Sons , Inc. ( 1988 ) , Krsto Prelec , utilized . When ring -opening chain scission is desired , posi- 55 FIZIKAB 6 ( 1997 ) 4 , 177-206 , Chu , William T., “ Overview tively charged particles can be utilized for their Lewis acid of Light- Ion Beam Therapy ” , Columbus - Ohio , ICRU - IAEA properties for enhanced ring -opening chain scission . Meeting , 18-20 Mar. 2006 , Iwata , Y. et al ., “ Alternating Referring to FIG . 8 , in one method , a first material 2 that Phase - Focused IH -DTL for Heavy - Ion Medical Accelera is or includes cellulose having a first number average tors” , Proceedings of EPAC 2006 , Edinburgh , Scotland, and molecular weight ( TMy?) is irradiated , e.g., by treatment 60 Leitner , C. M. et al. , “ Status of the Superconducting ECR with ionizing radiation ( e.g., in the form of gamma radiation , Ion Source Venus” , Proceedings of EPAC 2000 , Vienna, X - ray radiation , 100 nm to 280 nm ultraviolet (UV ) light, a Austria . Typically , generators are housed in a vault, e.g. , of beam of electrons or other charged particles ) to provide a lead or concrete . second material 3 that includes cellulose having a second Electrons interact via Coulomb scattering and number average molecular weight ( TMN2) lower than the 65 bremsstrahlung radiation produced by changes in the veloc first number average molecular weight. The second material ity of electrons . Electrons can be produced by radioactive (or the first and second material ) can be combined with a nuclei that undergo beta decay, such as isotopes of iodine , US 10,508,291 B2 29 30 cesium , technetium , and iridium . Alternatively , an electron scission . In addition , electrons having energies of 4-10 MeV gun can be used as an electron source via thermionic can have a penetration depth of 5 to 30 mm or more , such emission . as 40 mm . Electromagnetic radiation interacts via three processes: Electron beams can be generated , e.g. , by electrostatic photoelectric absorption , Compton scattering , and pair pro 5 generators , cascade generators , transformer generators , low duction. The dominating interaction is determined by the energy accelerators with a scanning system , low energy energy of the incident radiation and the atomic number of accelerators with a linear cathode , linear accelerators, and the material . The summation of interactions contributing to pulsed accelerators . Electrons as an ionizing radiation the absorbed radiation in cellulosic material can be source can be useful , e.g., for relatively thin piles of mate expressed by the mass absorption coefficient (see " Ioniza 10 rials , e.g., less than 0.5 inch , e.g. , less than 0.4 inch , 0.3 inch , tion Radiation ” in PCT/ US2007 / 022719 ) . 0.2 inch , or less than 0.1 inch . In some embodiments , the Electromagnetic radiation is subclassified as gamma rays, energy of each electron of the electron beam is from about x rays , ultraviolet rays , infrared rays, microwaves, or radio 0.3 MeV to about 2.0 MeV (million electron volts ) , e.g. , waves, depending on its wavelength . 15 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 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. , " Co pellets , a working table 14 pretreatment sequence. In first step 3010 , a supply of dry for holding thematerials to be irradiated and storage 16 , e.g. , 20 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 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 25 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 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 30 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 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- sectionalwidth of the feedstock transport attached to a hydraulic pump 40 . 35 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 . 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 , 40 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 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- 45 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 . 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 . 50 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, 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- 55 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. 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 60 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 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 65 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 20 kGy, 50 kGy, 100 kGy, or 200 kGy. US 10,508,291 B2 31 32 Tradeoffs in considering electron beam irradiation device 1021 Hz. In some embodiments , the electromagnetic radia power specifications include cost to operate , capital costs , tion has a frequency of between 1018 and 1022 Hz, e.g., depreciation , and device footprint. Tradeoffs in considering between 1019 to 1021 Hz. exposure dose levels of electron beam irradiation would be Doses energy costs and environment, safety , and health (ESH ) 5 In some embodiments , the irradiating (with any radiation concerns. Tradeoffs in considering electron energies include source or a combination of sources ) is performed until the material receives a dose of at least 0.25 Mrad , e.g., at least energy costs; here , a lower electron energy can be advan 1.0 Mrad , at least 2.5 Mrad , at least 5.0 Mrad , or at least 10.0 tageous in encouraging depolymerization of certain feed Mrad. In some embodiments , the irradiating is performed stock slurry ( see, for example , Bouchard , et al, Cellulose 10 until the material receives a dose of between 1.0 Mrad and ( 2006 ) 13 : 601-610 ) . 6.0 Mrad , e.g., between 1.5 Mrad and 4.0 Mrad . It can be advantageous to provide a double -pass of In some embodiments , the irradiating is performed at a electron beam irradiation in order to provide a more effective dose rate of between 5.0 and 1500.0 kilorads/ hour , e.g. , depolymerization process. For example, the feedstock trans between 10.0 and 750.0 kilorads/ hour or between 50.0 and port device could direct the feedstock ( in dry or slurry form ) 15 350.0 kilorads /hours . underneath and in a reverse direction to its initial transport In some embodiments , two or more radiation sources are direction . Double -pass systems can allow thicker feedstock used , such as two or more ionizing radiations. For example , slurries to be processed and can provide a more uniform samples can be treated , in any order, with a beam of depolymerization through the thickness of the feedstock electrons, followed by gamma radiation and UV light having slurry . 20 wavelengths from about 100 nm to about 280 nm . In some The electron beam irradiation device can produce either a embodiments , samples are treated with three ionizing radia fixed beam or a scanning beam . A scanning beam can be tion sources, such as a beam of electrons, gamma radiation , advantageous with large scan sweep length and high sca and energetic UV light. speeds, as this would effectively replace a large, fixed beam Alternatively , in another example , a fibrous biomass width . Further , available sweep widths of 0.5 m , 1 m , 2 m 25 material that includes a cellulosic and /or lignocellulosic or more are available. One suitable device is referenced in material is irradiated and , optionally , treated with acoustic Example 22. energy , e.g., ultrasound . In one example of the use of radiation as a treatment, Once a portion of feedstock slurry has been transported half -gallon juice cartons made of un -printed polycoated through the N electron beam irradiation devices , it can be 30 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 shredder , and output from the second shearer fed as input to and recycled back to the slurry preparation step 3040. A 35 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 . vacuum and then back - filled with argon gas . The ampoule is Heavy Ion Particle Beams 40 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 . starting material. For example , protons , helium nuclei, argon ions, silicon 45 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 tively or negatively charged ( e.g., protons or carbon ions) , particles can induce higher amounts of chain scission than 50 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 cals 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 radicals is about 1014 spins at room temperature . After unit to about 6,000 MeV /atomic unit , e.g. , from about 3 55 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 the radicals can be quenched by the application of a suffi with electromagnetic radiation , the electromagnetic radia- 60 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 103, 104, 10 % , 106, or reacts with ( quenches ) the radicals. Using a gas or liquid to even greater than 107 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 functionalize the ionized biomass with a desired amount and and 10 %, e.g., between 10 % and 10 % eV. The electromagnetic 65 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 , 1020, or even greater than amino groups, alkyl amino groups, alkyl groups, chloroalkyl US 10,508,291 B2 33 34 groups or chlorofluoroalkyl groups . In some instances , such soluble in , or at least capable of penetrating into the biomass quenching can improve the stability of some of the ionized and reacting with the radicals , such as a diene, such as biomass materials . For example , quenching can improve the 1,5 - cyclooctadiene . In some specific embodiments , the biomass's resistance to oxidation . Functionalization by quenching includes contacting the biomass with an antioxi quenching can also improve the solubility of any biomass 5 dant, such as Vitamin E. If desired , the biomass feedstock described herein , can improve its thermal stability , which can include an antioxidant dispersed therein , and the can improve material utilization by variousmicroorganisms . quenching can come from contacting the antioxidant dis For example , the functional groups imparted to the biomass persed in the biomass feedstock with the radicals . Combi material by the quenching can act as receptor sites for nations of these and other quenching materials can be used . attachment by microorganisms, e.g., to enhance cellulose 10 Other methods for quenching are possible . For example , hydrolysis by various microorganisms. any method for quenching radicals in polymeric materials FIG . 11 A 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 No. 7,166,650 , can be utilized for quenching any ionized electrons or ions of sufficient energy to ionize the biomass 15 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 , noted Muratoglu disclosures ) and/ or any antioxidant it will be oxidized , such as to an extent that carboxylic acid described in either Muratoglu reference can be utilized to groups are generated by reacting with the atmospheric quench any ionized biomass material. oxygen . In some instances with some materials , such oxi- 20 Functionalization can be enhanced by utilizing heavy dation is desired because it can aid in the further breakdown charged ions , such as any of the heavier ions described in molecular weight of the carbohydrate - containing bio herein . For example , if it is desired to enhance oxidation , mass , and the oxidation groups, e.g., carboxylic acid groups charged oxygen ions can be utilized for the irradiation . If can be helpful for solubility and microorganism utilization nitrogen functional groups are desired , nitrogen ions or ions in some instances. However , since the radicals can “ live ” for 25 that includes nitrogen can be utilized . Likewise , if sulfur or some time after irradiation , e.g., longer than 1 day, days, phosphorus groups are desired , sulfur or phosphorus ions 30 days , 3 months , 6 months or even longer than 1 year, can be used in the irradiation . materials properties can continue to change over time ,which In some embodiments , after quenching any of the in some instances, can be undesirable . Detecting radicals in quenched ionized materials described herein can be further irradiated samples by electron spin resonance spectroscopy 30 treated with one or more of radiation , such as ionizing or and radical lifetimes in such samples is discussed in Barto non - ionizing radiation , sonication , pyrolysis, and oxidation lotta et al. , Physics in Medicine and Biology , 46 ( 2001 ), for additional molecular and / or supramolecular structure 461-471 and in Bartolotta et al. , Radiation Protection change . Dosimetry , Vol. 84, Nos. 1-4 , pp. 293-296 ( 1999 ). As shown Particle Beam Exposure in Fluids in FIG . 11A , the ionized biomass can be quenched to 35 In some cases , the cellulosic or lignocellulosic materials functionalize and /or to stabilize the ionized biomass . At any can be exposed to a particle beam in the presence of one or point, e.g. , when the material is “ alive ” ( still has a substan more additional fluids ( e.g. , gases and/ or liquids) . Exposure tial quantity of reactive intermediates such as radicals ) , of a material to a particle beam in the presence of one or " partially alive” or fully quenched , the treated biomass can more additional fluids can increase the efficiency of the be converted into a product , e.g., a food . 40 treatment . In some embodiments , the quenching includes an appli In some embodiments , the material is exposed to a cation of pressure to the biomass , such as by mechanically particle beam in the presence of a fluid such as air . Particles deforming the biomass , e.g., directly mechanically com accelerated in any one or more of the types of accelerators pressing the biomass in one , two, or three dimensions, or disclosed herein (or another type of accelerator ) are coupled applying pressure to a fluid in which the biomass is 45 out of the accelerator via an output port ( e.g., a thin immersed , e.g., isostatic pressing . In such instances , the membrane such as a metal foil ), pass through a volume of deformation of the material itself brings radicals , which are space occupied by the fluid , and are then incident on the often trapped in crystalline domains, in close enough prox material. In addition to directly treating the material , some imity so that the radicals can recombine , or react with of the particles generate additional chemical species by another group . In some instances , the pressure is applied 50 interacting with fluid particles ( e.g. , ions and / or radicals together with the application of heat, such as a sufficient generated from various constituents of air , such as ozone and quantity of heat to elevate the temperature of the biomass to oxides of nitrogen ) . These generated chemical species can above a melting point or softening point of a component of also interact with the material, and can act as initiators for the biomass, such as lignin , cellulose or hemicellulose. Heat a variety of different chemical bond -breaking reactions in can improve molecular mobility in the polymeric material, 55 the material. For example , any oxidant produced can oxidize which can aid in the quenching of the radicals. When the material, which can result in molecular weight reduction . pressure is utilized to quench , the pressure can be greater In certain embodiments , additional fluids can be selec than about 1000 psi, such as greater than about 1250 psi , tively introduced into the path of a particle beam before the 1450 psi, 3625 psi , 5075 psi, 7250 psi, 10000 psi or even beam is incident on the material . As discussed above , greater than 15000 psi . 60 reactions between the particles of the beam and the particles In some embodiments , quenching includes contacting the of the introduced fluids can generate additional chemical biomass with a fluid , such as a liquid or gas , e.g. , a gas species , which react with the material and can assist in capable of reacting with the radicals , such as acetylene or a functionalizing the material, and /or otherwise selectively mixture of acetylene in nitrogen , ethylene, chlorinated eth altering certain properties of the material. The one or more ylenes or chlorofluoroethylenes , propylene or mixtures of 65 additional fluids can be directed into the path of the beam these gases . In other particular embodiments , quenching from a supply tube , for example . The direction and flow rate includes contacting the biomass with a liquid , e.g., a liquid of the fluid ( s ) that is /are introduced can be selected accord US 10,508,291 B2 35 36 ing to a desired exposure rate and /or direction to control the water vapor. For example , acidic water vapor can be utilized . efficiency of the overall treatment, including effects that To aid in molecular weight breakdown , the water can be result from both particle -based treatment and effects that are acidified with an organic acid , such as formic , or acetic acid , due to the interaction of dynamically generated species from or a mineral acid , such as sulfuric or hydrochloric acid . the introduced fluid with the material. In addition to air, 5 For example , and as will be explained in more detail exemplary fluids that can be introduced into the ion beam below , the treating biomass feedstock with the ionizing include oxygen , nitrogen , one or more noble gases, one or radiation can be performed as the biomass feedstock falls more halogens , and hydrogen . under the influence of gravity . This procedure can effectively Irradiating Low Bulk Density Biomass Materials and Cool reduce the bulk density of the biomass feedstock as it is ing Irradiated Biomass 10 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 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 15 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 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- 20 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 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- 25 ( 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 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- 30 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 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 35 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 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 40 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 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. 45 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/ cm during the application of the ionizing the biomass is pretreated at a first temperature with ionizing 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 50 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 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 55 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 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 . 60 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 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 65 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 US 10,508,291 B2 37 38 geously thin cross -sections , is delivered continuously to Referring particularly now to FIG . 11D , which shows a zone 1180 , and in other embodiments , the fibrous material is fluidized bed fibrous irradiating device 11121 with shielding . delivered in batches . A blower 1182 in loop 1184 is posi Fibrous material in a fluid , such as a gas, such as air under tioned adjacent to the fiber - loading zone 1180 and is capable pressure, is delivered to a shielded containment vessel 11123 ofmoving a fluid medium , e.g., air, at a velocity and volume 5 via piping 11125 and into a shielded fluidized bed portion sufficient to pneumatically circulate the fibrous material 11127. Counter -current streams 11131 of fluid , such as a gas, 1112 in a direction indicated by arrow 1188 through loop and transverse streams 11133 of fluid , such as a gas , that is 1184 . the same or different as a fluid delivered counter- currently , 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 10 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 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 15 ized bed a different depth and / or each beam can emit passes an application zone 1190 , which forms part of loop electrons of a different energy , such as 1, 3 , and 5 MeV . As 1184. Here , any desired additives described herein are the irradiated fibrous materialmoves through the system , it applied , such as a liquid , such as water, such as acidified or cools by the action of gases , such as air, circulating at high water made basic . In operation , application zone 1190 speeds in the system and it is bathed in reactive gases , such applies an additive, such as a liquid solution 1196 to the 20 as ozone and / or oxides of nitrogen , that are produced from circulating fibrous material via nozzles 98 , 99 and 11100 . the action of the ionizing radiation on the circulating gases, When a liquid is applied , the nozzles produce an atomized such as air. If desired , the process can be repeated a desired spray or mist of, which impacts the fibers as the fibers pass number of times until the fibrous material has received a in proximity to the nozzles. Valve 11102 is operated to desired dose . While the fluidized bed has been illustrated control the flow of liquid to the respective nozzles 1198 , 25 such that its long axis is horizontal with the ground , in other 1199 , and 11100. After a desired quantity of additive is implementations, the long axis of the bed is perpendicular to applied , the valve 11102 is closed . the ground so that the fibrous material falls under the In some embodiments , the application zone 1190 is two to influence of gravity . one hundred feet long or more , e.g., 125 feet , 150 feet , 250 Referring particularly now to FIG . 11E , which shows feet long or more , e.g., 500 feet long. Longer application 30 another fibrous material conveying and irradiating device zones allow for application of over a longer period of time 11140 without shielding . Fibrousmaterial 11144 is delivered during passage of fibrous material through application zone from a bin 11142 to a first conveyer 11150 at a first level 1190. In some embodiments , the nozzles are spaced apart above the ground and then the material is transferred a from about three to about four feet along the length of loop second conveyer 11152 a lower height than the first 1184 . 35 conveyer. The trailing edge 11160 of the first conveyer and As the fibrous material moves in loop 1184 and through the leading edge 11161 of the second conveyer 11152 defines the irradiating portion of the loop 11107 that includes a horn a gap with a spacing S. For example, the spacing S can be 11109 for delivering ionizing radiation , ionizing radiation is between 4 inches and about 24 inches . Material 11144 has applied to the fibrous material ( shielding is not shown ). enough momentum to free fall under gravity and then to be As the irradiated fibrous material moves around loop 40 captured by the second conveyer 11152 without falling into 1184 , it cools by the action of gases , such as air, circulating the gap . During the free fall , ionizing radiation is applied to at high speeds in the loop and it is bathed in reactive gases, the material. This arrangement can be advantageous in that such as ozone and /or oxides of nitrogen , that are produced the ionizing radiation is less likely to damage the conveying from the action of the ionizing radiation on the circulating system because is not directly contacted by the radiation . gases , such as air. After passing through the irradiating 45 After passing through the irradiating portion , a cooling portion 11107 , a cooling fluid , such as a liquid ( e.g., water ) fluid , such as a liquid ( e.g., water ) or a gas , such as liquid or a gas , such as liquid nitrogen at 77 K can be injected into nitrogen at 77 K can be applied to the material to aid in the loop 1184 to aid in the cooling of the fibrous material . This cooling of the fibrous material. This process can be repeated process can be repeated more than one time if desired , e.g., more than one time if desired , e.g., 2 , 3 , 4 , 5 , 6 , 7, 8 , 9 , 10 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 times or more, e.g. , 15 times, to 50 times or more, e.g. , 15 times , to deliver the desired dose to deliver the desired dose to the fibrous material . While , as the fibrous material . While , as shown, the long axis of the shown , the long axis of the horn is along the direction of horn is transverse to the direction of the material flow , other flow , in some implementations, the long axis of the horn is beam arrangements are possible . In some implementations , transverse to the direction of the flow . In some implemen a beam of electrons is utilized as a principal ionizing tations, a beam of electrons is utilized as a principal ionizing 55 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, in the gap on the opposite target, such as a tantalum target 11111 , on the inside of loop side of the material, such that as the electrons that pass 1184 such that when electrons strike the target , X - rays are through the material they strike the target, generating emitted . 60 X -rays . After a desired dose is delivered to the fibrous material , In one example of the use of radiation with oxidation as the fibrous material can be removed from loop 1184 via a a pretreatment , half - gallon juice cartons made of un - printed separator 11112, which is selectively connected to loop 1184 polycoated white Kraft board having a bulk density of 20 by section 11114 and gate valve 11116. When valve 11116 is 1b / ft are used as a feedstock . Cartons are folded flat and then opened , another valve is also opened to allow air to enter the 65 fed into a sequence of three shredder - shearer trains arranged loop 1184 to compensate for air exiting through separator in series with output from the first shearer fed as input to the 11112 . second shredder, and output from the second shearer fed as US 10,508,291 B2 39 40 input to the third shredder. The fibrous material produced by 1,000,000 or from about 250,000 to about 700,000 , and the the can be sprayed with water and processed through a pellet number average molecular weight after sonication is from mill operating at room temperature . The densified pellets can about 50,000 to about 200,000 , e.g., from about 60,000 to be placed in a glass ampoule which is sealed under an about 150,000 or from about 70,000 to about 125,000 . atmosphere of air. The pellets in the ampoule are irradiated 5 However, in some embodiments , e.g., after extensive soni with gamma radiation for about 3 hours at a dose rate of cation , it is possible to have a number average molecular about 1 Mrad per hour to provide an irradiated material in weight of less than about 10,000 or even less than about which the cellulose has a lower molecular weight than the 5,000 . fibrous Kraft starting material . In some embodiments , the second material can have a Sonication 10 One ormore sonication processing sequences can be used level of oxidation (TO ) that is higher than the level of to treat biomass from a wide variety of different sources to oxidation (10 ) 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 15 als susceptibility to chemical, enzymatic or microbial attack . reduce the recalcitrance , molecular weight, and / or crystal 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 . 20 material. For example, the second material can have more Referring again to FIG . 8 , in one method , a first biomass 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 25 ous medium . If desired , the medium can include an oxidant, having a second number average molecular weight ( TMx2 ) 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 30 In other embodiments , the sonication medium is non material to produce a product 5 . 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 35 Without wishing to be bound by any particular theory , it solution containing the microorganism , e.g., at a concentra is believed that sonication breaks bonds in the cellulose by tion of greater than 10 microorganisms/ mL . These proper creating bubbles in the medium containing the cellulose , ties make the second material 3 more susceptible to chemi which grow and then violently collapse . During the collapse cal , enzymatic , and /or microbial attack relative to the first of the bubble , which can take place in less than a nanosec material 2 , which can greatly improve the production rate 40 ond , the implosive force raises the local temperature within and /or production level of a desired product , e.g. , ethanol . the bubble to about 5100 K (even higher in some instance ; Sonication can also sterilize the materials , but should not be see , e.g., Suslick et al ., Nature 434 , 52-55 ) and generates used while the microorganisms are supposed to be alive . In pressures of from a few hundred atmospheres to over 1000 some embodiments , the second number average molecular atmospheres or more . It is these high temperatures and weight ( +M72 ) is lower than the first number average 45 pressures that break the bonds . In addition , without wishing molecular weight ( TMyi) by more than about 10 percent, to be bound by any particular theory , it is believed that e.g., 15 , 20 , 25 , 30 , 35 , 40 , 50 percent, 60 percent, or even reduced crystallinity arises, at least in part, from the more than about 75 percent. extremely high cooling rates during collapse of the bubbles , In some instances , the second material has cellulose that which can be greater than about 1011 K / second . The high has a crystallinity (TC2 ) that is lower than the crystallinity 50 cooling rates generally do not allow the cellulose to organize (TC ) of the cellulose of the first material. For example , and crystallize , resulting in materials that have reduced (TC ) can be lower than ( C ) by more than about 10 crystallinity . Ultrasonic systems and sonochemistry are dis percent, e.g. , 15, 20 , 25 , 30 , 35 , 40 , or even more than about cussed in , e.g., Olli et al. , U.S. Pat . No. 5,766,764 ; Roberts , 50 percent. U.S. Pat . No. 5,828,156 ; Mason , Chemistry with Ultra In some embodiments , the starting crystallinity index 55 sound , Elsevier, Oxford , ( 1990 ) ; Suslick ( editor) , Ultra (prior to sonication ) is from about 40 to about 87.5 percent, sound : its Chemical, Physical and Biological Effects , VCH , e.g., from about 50 to about 75 percent or from about 60 to Weinheim , ( 1988 ) ; Price, “ Current Trends in Sonochemis about 70 percent, and the crystallinity index after sonication try ” Royal Society of Chemistry , Cambridge , ( 1992 ) ; Sus is from about 10 to about 50 percent, e.g., from about 15 to lick et al. , Ann . Rev.Mater . Sci. 29 , 295 , (1999 ); Suslick et about 45 percent or from about 20 to about 40 percent. 60 al. , Nature 353, 414 ( 1991) ; Hiller et al ., Phys . Rev. Lett. 69 , However , in certain embodiments , e.g. , after extensive soni 1182 ( 1992 ); Barber et al. , Nature , 352 , 414 ( 1991 ); Suslick cation , it is possible to have a crystallinity index of lower et al ., J. Am . Chem . Soc. , 108 , 5641 ( 1986 ) ; Tang et al. , than 5 percent. In some embodiments , the material after Chem . Comm ., 2119 (2000 ) ; Wang et al ., Advanced Mater. , sonication is substantially amorphous . 12 , 1137 ( 2000 ) ; Landau et al. , J. of Catalysis , 201, 22 In some embodiments , the starting number average 65 ( 2001 ) ; Perkas et al. , Chem . Comm ., 988 ( 2001 ) ; Nikitenko molecular weight ( prior to sonication ) is from about 200,000 et al. , Angew . Chem . Inter. Ed . ( December 2001) ; Shafi et to about 3,200,000 , e.g., from about 250,000 to about al. , J. Phys. Chem B 103 , 3358 ( 1999 ) ; Avivi et al. , J. Amer . US 10,508,291 B2 41 42 Chem . Soc . 121 , 4196 (1999 ) ; and Avivi et al ., J. Amer . capable of withstanding elevated pressures ( e.g., 10 bars ). In Chem . Soc . 122 , 4331 (2000 ) . addition or in the alternative, reactor volume 1244 includes Sonication Systems a rectangular cross section . FIG . 12 shows a general system in which a biomass Flow cell 1224 further includes a heat exchanger 1246 in material stream 1210 is mixed with a water stream 1212 in 5 thermal communication with at least a portion of reactor a reservoir 1214 to form a process stream 1216. A first pump volume 1244. Cooling fluid 1248 (e.g. , water ) flows into heat exchanger 1246 and absorbs heat generated when 1218 draws process stream 1216 from reservoir 1214 and process stream 1216 is sonicated in reactor volume 1244. In toward a flow cell 1224. Ultrasonic transducer 1226 trans some embodiments , the flow rate of cooling fluid 1248 into mits ultrasonic energy into process stream 1216 as the 10 heat exchanger 1246 is controlled to maintain an approxi process stream flows through flow cell 1224. A second pump mately constant temperature in reactor volume 1244. In 1230 draws process stream 1216 from flow cell 1224 and addition or in the alternative , the temperature of cooling toward subsequent processing . fluid 1248 flowing into heat exchanger 1246 is controlled to Reservoir 1214 includes a first intake 1232 and a second maintain an approximately constant temperature in reactor intake 1234 in fluid communication with a volume 1236. A 15 volume 1244. In some embodiments , the temperature of conveyor ( not shown ) delivers biomass material stream reactor volume 1244 is maintained at 20 to 50 ° C., e.g. , 25 , 1210 to reservoir 1214 through first intake 1232. Water 30, 35, 40 , or 45° C. Additionally or alternatively , heat 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 20 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 ( e.g., mechanically coupled using a flange ) to ultrasonic volume 1236 ong opposing es 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 25 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 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. 30 ultrasonicincludes a piezoelectrictransducer 1252.element Ultrasonic that converts transducer the electrical 1226 Mixing of material biomass and water in reservoir 1214 is energy into sound in the ultrasonic range . In some embodi controlled by the size of volume 1236 and the flow rates of ments , the materials are sonicated using sound having a biomass and water into the volume. In some embodiments , frequency of from about 16 kHz to about 110 kHz , e.g., from volume 1236 is sized to create a minimum mixing residence 35 about 18 kHz to about 75 kHz or from about 20 kHz to about time for the biomass and water. For example , when 2000 40 kHz ( e.g. , sound having a frequency of 20 kHz to 40 tons/ day of biomass and 1.25 million gallons/ day of water kHz) . are flowing through reservoir 1214 , volume 1236 can be The ultrasonic energy is then delivered to the working about 32,000 gallons to produce a minimum mixing resi medium through booster 1248 . dence time of about 15 minutes . 40 The ultrasonic energy traveling through booster 1248 in Reservoir 1214 includes a mixer 1240 in fluid commu reactor volume 1244 creates a series of compressions and nication with volume 1236.Mixer 1240 agitates the contents rarefactions in process stream 1216 with an intensity suffi of volume 1236 to disperse biomass throughout the water in cient to create cavitation in process stream 1216. Cavitation the volume. For example ,mixer 1240 can be a rotating vane disaggregates the cellulosic material dispersed in process disposed in reservoir 1214. In some embodiments , mixer 45 stream 1216. Cavitation also produces free radicals in the 1240 disperses the biomass substantially uniformly through water of process stream 1216. These free radicals act to out the water. further break down the cellulosic material in process stream Reservoir 1214 further includes an exit 1242 in fluid 1216 . communication with volume 1236 and process stream 1216 . In general, 5 to 4000 MJ/ m ” , e.g., 10 , 25 , 50 , 100 , 250 , The mixture of biomass and water in volume 1236 flows out 50 500 , 750 , 1000, 2000 , or 3000 MJ/ m , of ultrasonic energy of reservoir 1214 via exit 1242. Exit 1242 is arranged near is applied to process stream 16 flowing at a rate of about 0.2 the bottom of reservoir 1214 to allow gravity to pull the mö/ s (about 3200 gallons/ min ). After exposure to ultrasonic mixture of biomass and water out of reservoir 1214 and into energy in reactor volume 1244 , process stream 1216 exits process stream 1216 . flow cell 1224 through outlet 1222. Second pump 1230 First pump 1218 ( e.g., any of several recessed impeller 55 moves process stream 1216 to subsequent processing ( e.g., vortex pumps made by Essco Pumps & Controls , Los any of several recessed impeller vortex pumps made by Angeles , Calif. ) moves the contents of process stream 1216 Essco Pumps & Controls , Los Angeles, Calif. ) . toward flow cell 1224. In some embodiments , first pump While certain embodiments have been described , other 1218 agitates the contents of process stream 1216 such that embodiments are possible . the mixture of cellulosic material and water is substantially 60 As an example , while process stream 1216 has been uniform at inlet 1220 of flow cell 1224. For example , first described as a single flow path , other arrangements are pump 1218 agitates process stream 1216 to create a turbu possible . In some embodiments for example , process stream lent flow that persists along the process stream between the 1216 includes multiple parallel flow paths ( e.g., flowing at first pump and inlet 1220 of flow cell 1224 . a rate of 10 gallon /min ) . In addition or in the alternative , the Flow cell 1224 includes a reactor volume 1244 in fluid 65 multiple parallel flow paths of process stream 1216 flow into communication with inlet 1220 and outlet 1222. In some separate flow cells and are sonicated in parallel ( e.g., using embodiments , reactor volume 1244 is a stainless steel tube a plurality of 16 kW ultrasonic transducers ). US 10,508,291 B2 43 44 As another example , while a single ultrasonic transducer of identical windings 94 and 96 , also termed the primary 1226 has been described as being coupled to flow cell 1224 , winding and secondary winding , respectively . other arrangements are possible . In some embodiments , a In some embodiments , the transducers include commer plurality of ultrasonic transducers 1226 are arranged in flow cially available piezoelectric transducers, such as Branson cell 1224 ( e.g., ten ultrasonic transducers can be arranged in 5 Ultrasonics Corporation models 105 or 502 , each designed a flow cell 1224 ) . In some embodiments, the sound waves for operation at 20 kHz and a maximum power rating of 3 generated by each of the plurality of ultrasonic transducers kW . The energizing voltage for providing maximum motional excursion at the output surface of the transducer is 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 10 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 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 15 currents flowing between the transducers . The balancing transducer further sonicate process stream 1216 . unit 430 assures a balanced condition by providing equal As still another example , while reactor volume 1244 has current flow through the transducers , hence eliminating the been described as a closed volume, reactor volume 1244 is possibility of circulating currents . The wire size of the open to ambient conditions in certain embodiments . In such windings must be selected for the full load current noted embodiments , sonication pretreatment can be performed 20 above and the maximum voltage appearing across a winding substantially simultaneously with other pretreatment tech input is 93 volt. niques. For example , ultrasonic energy can be applied to As an alternative to using ultrasonic energy, high - fre process stream 1216 in reactor volume 1244 while electron quency , rotor- stator devices can be utilized . This type of beams are simultaneously introduced into process stream device produces high - shear , microcavitation forces, which 1216 . 25 can disintegrate biomass in contact with such forces. Two As another example , while a flow through process has commercially available high - frequency , rotor- stator disper been described , other arrangements are possible . In some sion devices are the SupratonTM devices manufactured by embodiments , sonication can be performed in a batch pro Krupp Industrietechnik GmbH and marketed by Dorr -Oliver cess . For example , a volume can be filled with a 10 % Deutschland GmbH of Connecticut, and the DispaxTM (weight by volume) mixture of biomass in water and 30 devices manufactured and marketed by Ika -Works , Inc. of exposed to sound with intensity from about 50 W / cm² to Cincinnati, Ohio . Operation of such a microcavitation about 600 W / cm², e.g., from about 75 W / cm2 to about 300 device is discussed in Stuart, U.S. Pat. No. 5,370,999 . W / cm2 or from about 95 W /cm2 to about 200 W / cm2. While ultrasonic transducer 1226 has been described as Additionally or alternatively , the mixture in the volume can including one or more piezoelectric active elements to create be sonicated from about 1 hour to about 24 hours, e.g., from 35 ultrasonic energy, other arrangements are possible . In some about 1.5 hours to about 12 hours, or from about 2 hours to embodiments , ultrasonic transducer 1226 includes active about 10 hours. In certain embodiments , the material is elements made of other types of magnetostrictive materials sonicated for a pre - determined time, and then allowed to ( e.g., ferrous metals ) . Design and operation of such a stand for a second pre -determined time before sonicating high -powered ultrasonic transducer is discussed in Hansen again . 40 et al. , U.S. Pat. No. 6,624,539 . In some embodiments , Referring now to FIG . 13 , in some embodiments , two ultrasonic energy is transferred to process stream 16 through electro -acoustic transducers are mechanically coupled to a an electro -hydraulic system . single horn .As shown , a pair of piezoelectric transducers 60 While ultrasonic transducer 1226 has been described as and 62 is coupled to a slotted bar horn 64 by respective using the electromagnetic response of magnetorestrictive intermediate coupling horns 70 and 72 , the latter also being 45 materials to produce ultrasonic energy, other arrangements known as booster horns. The mechanical vibrations pro are possible . In some embodiments , acoustic energy in the vided by the transducers , responsive to high frequency form of an intense shock wave can be applied directly to electrical energy applied thereto , are transmitted to the process stream 16 using an underwater spark . In some respective coupling horns, which can be constructed to embodiments , ultrasonic energy is transferred to process provide a mechanical gain , such as a ratio of 1 to 1.2. The 50 stream 16 through a thermo- hydraulic system . For example , horns are provided with a respective mounting flange 74 and acoustic waves of high energy density can be produced by 76 for supporting the transducer and horn assembly in a applying power across an enclosed volume of electrolyte , stationary housing . thereby heating the enclosed volume and producing a pres The vibrations transmitted from the transducers through sure rise that is subsequently transmitted through a sound the coupling or booster horns are coupled to the input 55 propagation medium ( e.g., process stream 1216 ) . Design and surface 78 of the horn and are transmitted through the horn operation of such a thermo- hydraulic transducer is discussed to the oppositely disposed output surface 80 , which , during in Hartmann et al. , U.S. Pat. No. 6,383,152 . operation , is in forced engagement with a workpiece ( not Pyrolysis shown ) to which the vibrations are applied . One or more pyrolysis treatment sequences can be used to The high frequency electrical energy provided by the 60 process biomass from a wide variety of different sources to power supply 82 is fed to each of the transducers , electri extract useful substances from the biomass , and to provide cally connected in parallel , via a balancing transformer 84 partially degraded organic material which functions as input and a respective series connected capacitor 86 and 90 , one to further processing steps and / or sequences. capacitor connected in series with the electrical connection Referring again to the general schematic in FIG . 8 , a first to each of the transducers. The balancing transformer is 65 biomass material 2 that includes having a first number known also as “ balun ” standing for “ balancing unit. ” The average molecular weight ( Myi) is pyrolyzed, e.g., by balancing transformer includes a magnetic core 92 and a pair heating the first material in a tube furnace, to provide a US 10,508,291 B2 45 46 second material 3 that includes cellulose having a second for a pre -determined time, and then allowed to cool for a number average molecular weight ( TMx2) lower than the second pre - determined time before pyrolyzing again . first number averagemolecular weight. The second material Pyrolysis Systems (or the first and second material in certain embodiments ) FIG . 14 shows a process flow diagram 6000 that includes is/ are combined with a microorganism ( e.g., a bacterium or 5 various steps in a pyrolytic feedstock pretreatment system . a yeast) that can utilize the second and / or first material to In first step 6010 , a supply of dry feedstock is received from produce a product 5 that. a feed source . 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 10 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 removed prior to collection of the plant material and /or a concentration of greater than 10 microorganisms/ mL . before the plant material is delivered by the feedstock tibleThese to propertieschemical, make enzymatic the second and /or material microbial 3 attackmore relativesuscep 15 transport device. Alternatively , or in addition , the biomass to the first material 2 , which can greatly improve the feedstock can be subjected to mechanical processing 6020 production rate and /or production level of a desired product, ( e.g., to reduce the average length of fibers in the feedstock ) e.g., ethanol. Pyrolysis can also sterilize the first and second prior to delivery to the pyrolysis chamber . materials . Following mechanical processing, the biomass undergoes In some embodiments , the second number average 20 a moisture adjustment step 6030. The nature of the moisture molecular weight ( My2) is lower than the first number adjustment step depends upon the moisture content of the average molecular weight ( TMy?) by more than about 10 mechanically processed biomass . Typically , pyrolysis of percent, e.g., 15 , 20 , 25 , 30 , 35 , 40 , 50 percent, 60 percent, biomass occurs most efficiently when the moisture content or even more than about 75 percent. of the feedstock is between about 10 % and about 30 % ( e.g., In some instances, the second material has cellulose that 25 between 15 % and 25 % ) by weight of the feedstock . If the has a crystallinity ( C2) that is lower than the crystallinity moisture content of the feedstock is larger than about 40 % ( " C ) of the cellulose of the first material. For example , by weight, the extra thermal load presented by the water ( TC ) can be lower than (FC ) by more than about 10 content of the biomass increases the energy consumption of percent , e.g. , 15 , 20 , 25 , 30 , 35 , 40 , or even more than about subsequent pyrolysis steps. 50 percent. 30 In some embodiments , if the biomass has a moisture In some embodiments , the starting crystallinity (prior to content which is larger than about 30 % by weight, drier pyrolysis ) is from about 40 to about 87.5 percent, e.g., from biomass material 6220 , which has a low moisture content , about 50 to about 75 percent or from about 60 about 70 can be blended in , creating a feedstock mixture in step 6030 percent, and the crystallinity index after pyrolysis is from with an average moisture content that is within the limits about 10 to about 50 percent, e.g. , from about 15 to about 45 35 discussed above . In certain embodiments , biomass with a percent or from about 20 to about 40 percent. However, in high moisture content can simply be dried by dispersing the certain embodiments , e.g. , after extensive pyrolysis , it is biomass material on a moving conveyor that cycles the possible to have a crystallinity index of lower than 5 percent. biomass through an in - line heating unit . The heating unit In some embodiments , the material after pyrolysis is sub evaporates a portion of the water present in the feedstock . stantially amorphous . 40 In some embodiments , if the biomass from step 6020 has In some embodiments , the starting number average a moisture content which is too low ( e.g. , lower than about molecular weight (prior to pyrolysis ) is from about 200,000 10 % by weight) , the mechanically processed biomass can be to about 3,200,000 , e.g. , from about 250,000 to about combined with wetter feedstock material 6230 with a higher 1,000,000 or from about 250,000 to about 700,000 , and the moisture content, such as sewage sludge . Alternatively , or in number average molecular weight after pyrolysis is from 45 addition , water 6240 can be added to the dry biomass from about 50,000 to about 200,000 , e.g. , from about 60,000 to step 6020 to increase its moisture content. about 150,000 or from about 70,000 to about 125,000 . In step 6040 , the biomass now with its moisture content However, in some embodiments , e.g., after extensive adjusted to fall within suitable limits can be preheated in pyrolysis , it is possible to have a number average molecular an optional preheating step 6040. Treatment step 6040 can weight of less than about 10,000 or even less than about 50 be used to increase the temperature of the biomass to 5,000 . between 75º C. and 150 ° C. in preparation for subsequent In some embodiments, the second material can have a pyrolysis of the biomass. Depending upon the nature of the level of oxidation (702 ) that is higher than the level of biomass and the particular design of the pyrolysis chamber, oxidation (10 ) of the first material . A higher level of preheating the biomass can ensure that heat distribution oxidation of the material can aid in its dispersibility , 55 within the biomass feedstock remains more uniform during swellability and / or solubility , further enhancing the materi pyrolysis , and can reduce the thermal load on the pyrolysis als susceptibility to chemical, enzymatic or microbial attack . chamber . In some embodiments , to increase the level of the oxidation The feedstock is then transported to a pyrolysis chamber of the second material relative to the first material, the to undergo pyrolysis in step 6050. In some embodiments , pyrolysis is performed in an oxidizing environment, pro- 60 transport of the feedstock is assisted by adding one or more ducing a second material that is more oxidized than the first pressurized gases 6210 to the feedstock stream . The gases material . For example , the second material can have more create a pressure gradient in a feedstock transport conduit , hydroxyl groups, aldehyde groups, ketone groups, ester propelling the feedstock into the pyrolysis chamber (and groups or carboxylic acid groups, which can increase its even through the pyrolysis chamber ) . In certain embodi hydrophilicity . 65 ments , transport of the feedstock occurs mechanically ; that In some embodiments , the pyrolysis of the materials is is , a transport system that includes a conveyor such as an continuous. In other embodiments , the material is pyrolyzed auger transports the feedstock to the pyrolysis chamber. US 10,508,291 B2 47 48 Other gases 6210 can also be added to the feedstock prior general, the carrier gases are relatively inert towards the to the pyrolysis chamber . In some embodiments , for feedstock material, even at the high temperatures in the example , one or more catalyst gases can be added to the pyrolysis chamber . Exemplary carrier gases include , for feedstock to assist decomposition of the feedstock during example , nitrogen , argon , methane, carbon monoxide, and pyrolysis . In certain embodiments, one or more scavenging 5 carbon dioxide . Alternatively , or in addition , mechanical agents can be added to the feedstock to trap volatile mate transport systems such as augers can transport and circulate rials released during pyrolysis . For example , various sulfur based compounds such as sulfides can be liberated during the feedstock within the pyrolysis chamber to create a pyrolysis , and an agent such as hydrogen gas can be added turbulent feedstock flow . to the feedstock to cause desulfurization of the pyrolysis 10 In some embodiments , pyrolysis of the feedstock occurs products . Hydrogen combines with sulfides to form hydro substantially in the absence of oxygen and other reactive gen sulfide gas , which can be removed from the pyrolyzed gases . Oxygen can be removed from the pyrolysis chamber feedstock . by periodic purging of the chamber with high pressure Pyrolysis of the feedstock within the chamber can include nitrogen ( e.g., at nitrogen pressures of 2 bar or more ) . heating the feedstock to relatively high temperatures to 15 Following purging of the chamber, a gas mixture present in 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 1 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 20 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 . 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 , 25 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 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 30 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 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., 35 decomposition is endothermic . However , in oxidative greater than 1.0 hours or greater than about 2.0 hours . In pyrolysis , formation of carbon dioxide by oxidation of the other embodiments , the materials are pyrolyzed at a tem feedstock is an exothermic process . The heat released from perature well above the decomposition temperature for the carbon dioxide formation can assist further pyrolysis heating material in an inert atmosphere, e.g. , from about 75 ° C. stages , thereby lessening the thermal load presented by the above to about 175° C. above the decomposition tempera- 40 feedstock . ture or from about 85 ° C. above to about 150 ° C. above the In some embodiments , pyrolysis occurs in an inert envi decomposition temperature . In such embodiments , the mate ronment, such as while feedstock materials are bathed in rial is generally kept at this temperature for less than 0.5 argon or nitrogen gas. In certain embodiments , pyrolysis can hour , e.g. , less 20 minutes , less than 10 minutes, less than 5 occur in an oxidizing environment, such as in air or argon minutes or less than 2 minutes. In still other embodiments , 45 enriched in air. In some embodiments , pyrolysis can take the materials are pyrolyzed at an extreme temperature , e.g. , place in a reducing environment, such as while feedstock from about 200 ° C. above to about 500 ° C. above the materials are bathed in hydrogen gas . To aid pyrolysis , decomposition temperature of the material in an inert envi various chemical agents , such as oxidants , reductants , acids ronment or from about 250 ° C. above to about 400 ° C. above or bases can be added to the material prior to or during the decomposition temperature . In such embodiments , the 50 pyrolysis . For example , sulfuric acid can be added , or a material us generally kept at this temperature for less than 1 peroxide ( e.g. , benzoyl peroxide ) can be added . minute , e.g. , less than 30 seconds, less than 15 seconds, less As discussed above , a variety of different processing than 10 seconds , less than 5 seconds, less than 1 second or conditions can be used , depending upon factors such as the less than 500 ms. Such embodiments are typically referred feedstock composition and the desired pyrolysis products . to as flash pyrolysis . 55 For example , for cellulose - containing feedstock material , In some embodiments , the feedstock is heated relatively relatively mild pyrolysis conditions can be employed , rapidly to the selected pyrolysis temperature within the including flash pyrolysis temperatures between 375 ° C. and chamber. For example , the chamber can be designed to heat 450 ° C., and residence times of less than 1 second . As the feedstock at a rate ofbetween 500 ° C./s and 11,000 ° C./s . another example , for organic solid waste material such as Typical heating rates for biomass -derived feedstock material 60 sewage sludge , flash pyrolysis temperatures between 500 ° are from 500 ° C./s to 1000 ° C./s , for example . C. and 650 ° C. are typically used , with residence times of A turbulent flow of feedstock material within the pyroly between 0.5 and 3 seconds. In general, many of the pyrolysis sis chamber is usually advantageous, as it ensures relatively process parameters, including residence time, pyrolysis tem efficient heat transfer to the feedstock material from the perature , feedstock turbulence, moisture content, feedstock heating sub - system . Turbulent flow can be achieved by 65 composition , pyrolysis product composition , and additive blowing the feedstock material through the chamber using gas composition can be regulated automatically by a system one or more injected carrier gases 6210 , for example . In of regulators and an automated control system . US 10,508,291 B2 49 50 Following pyrolysis step 6050 , the pyrolysis products organic solvents include, for example , various hydrocarbons undergo a quenching step 6250 to reduce the temperature of and halo -hydrocarbons . The purified liquid products 6200 the products prior to further processing . Typically , quench can then be subjected to further processing steps. Waste ing step 6250 includes spraying the pyrolysis products with water 6190 can be filtered if necessary, and recirculated for streams of cooling water 6260. The cooling water also forms 5 further use in quenching the pyrolysis decomposition prod a slurry that includes solid , undissolved product material and ucts in step 6250 . various dissolved products . Also present in the product After separation in step 6080 , the solid product material stream is a mixture that includes various gases, including 6110 is optionally subjected to a drying step 6120 that can product gases, carrier gases, and other types of process include evaporation of water . Solid material 6110 can then gases . 10 be stored for later use, or subjected to further processing The product stream is transported via in - line piping to a steps, as appropriate . gas separator that performs a gas separation step 6060 , in The pyrolysis process parameters discussed above are which product gases and other gases are separated from the exemplary . In general, values of these parameters can vary slurry formed by quenching the pyrolysis products . The widely according to the nature of the feedstock and the separated gas mixture is optionally directed to a blower 15 desired products . Moreover , a wide variety of different 6130 , which increases the gas pressure by blowing air into 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 20 A wide variety of different pyrolysis chambers can be filtered gas can be compressed and stored for further use . used to decompose the feedstock . In some embodiments , for Alternatively , the filtered gas can be subjected to further example , pyrolyzing feedstock can include heating the mate 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 25 contact between the resistive heating member and the mate compounds can include, for example , various hydrocarbon 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 30 heating the material by the application of radiation , such as of water vapor and oxygen ) and subjected to a cracking 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 the material with a convective heat . The convective heat can heat sources that burn hydrocarbon gases such as methane, 35 be generated by a flowing stream of heated gas. The heated propane , and/ or butane to heat the feedstock . A portion 6270 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 40 about 250 ° C. The convective heat can be generated by a hot receive process heat that can be used to increase the tem 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 45 15. Chamber 6500 includes an insulated chamber wall 6510 heated feedstock materials can be cooled by a heat exchange 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, 50 6530 in a turbulent flow , and a quenching system 6540 that thereby reducing energy cost for the pyrolysis process . 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 55 products . water 6280 can be filtered and then recirculated for further 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 60 process , a vent 6750 for exhaust gases, and a gas separator separated from liquid product material 6090 by a series of 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 65 chamber . more organic solvents 6180 , for example , to separate prod 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 US 10,508,291 B2 51 52 1713 with resistive heating element 1714 in the form of a In some embodiments , the heating is performed a number of wire winding through the open space defined by the sample times to effect the desired amount of pyrolysis . holder 1713. Optionally , the heated element can be spun In certain embodiments , carbohydrate -containing bio about axis 1715 (as indicated by arrow 1716 ) to tumble the mass material can be heated in an absence of oxygen in a material that includes the cellulosic material in sample 5 fluidized bed reactor. If desired , the carbohydrate containing holder 1713. The space 1718 defined by enclosure 1719 is biomass can have relatively thin cross- sections, and can maintained at a temperature above room temperature , e.g., include any of the fibrous materials described herein , for 200 to 250 ° C. In a typical usage, a carrier gas, e.g., an inert efficient heat transfer . The material can be heated by thermal gas , or an oxidizing or reducing gas , traverses through the transfer from a hot metal or ceramic , such as glass beads or sample holder 1713 while the resistive heating element is 10 cansand bein transportedthe reactor , toand a thecentral resulting production pyrolysis plant liquid to manu or oil rotated and heated to a desired temperature , e.g. , 325 ° C. 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 15 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 stock , and to provide partially degraded organic material continuously fall into the sample holder , striking a heated which functions as input 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 20 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 weight ( "My? ) and having a first oxygen content (50 ) 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 25 material 3 that includes cellulose having a second number coil 1823. The space 1824 defined by enclosure 1825 is average molecular weight ( TMxz) and having a second maintained at a temperature above room temperature , e.g. , oxygen content (702 ) higher than the first oxygen content 200 to 250 ° C. In a typical usage , a carrier gas traverses (TO ) . 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 30 material , such as a microorganism , to provide a composite 4 , material at a desired temperature . or another product 5. Providing a higher level of oxidation Yet another embodiment of a pyrolysis chamber is shown can improve dispersibility of the oxidized material, e.g., in in FIG . 19. Furnace pyrolyzer 130 includes a movable a solvent. sample holder 131 and a furnace 132. In a typical usage , the Such materials can also be combined with a solid and / or sample is lowered ( as indicated by arrow 137 ) into a hot 35 a liquid . For example , the liquid can be in the form of a zone 135 of furnace 132 , while a carrier gas fills the housing solution and the solid can be particulate in form . The liquid 136 and traverses through the sample holder 131. The and /or solid can include a microorganism , e.g., a bacterium , sample is heated to the desired temperature for a desired and /or an enzyme. For example, the bacterium and / or time to provide a pyrolyzed product. The pyrolyzed product enzyme can work on the cellulosic or lignocellulosic mate is removed from the pyrolyzer by raising the sample holder 40 rial to produce a product , such as a protein . Exemplary ( as indicated by arrow 134 ) . products are described in FIBROUS MATERIALS AND In certain embodiments , as shown in FIG . 20 , a cellulosic 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 45 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 , 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 50 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 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 55 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 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 60 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 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 65 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. include elastomeric styrenic copolymers ( e.g. , styrene - eth US 10,508,291 B2 53 54 ylene- butylene - styrene copolymers ) , polyamide elastomers anhydride groups, which can increase its dispersibility and / ( e.g. , polyether- polyamide copolymers ) and ethylene -vinyl or its solubility ( e.g. , in a liquid ). To further improve acetate copolymer. dispersibility in a resin , the resin can include a component In particular embodiments , lignin is utilized , e.g., any that includes hydrogen -bonding groups, such as one or more lignin that is generated in any process described herein . 5 anhydride groups, carboxylic acid groups , hydroxyl groups, In some embodiments , the thermoplastic resin has a melt amide groups , amine groups or mixtures of any of these flow rate of between 10 g / 10 minutes to 60 g /10 minutes , groups. In some preferred embodiments , the component 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 10 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 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 15 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 . 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, 20 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 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 25 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 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 30 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 . 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 35 reduce the average length of fibers in the feedstock ) prior to higher , 15.0 percent higher or 17.5 percent higher. In some delivery to the oxidation reactor. preferred embodiments , the second oxygen content is at Followingmechanical 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 40 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 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 45 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 further downstream in the overall process. For example , in crystallinity (FC2 ) that is lower than the crystallinity (FC ) of typical industrial processing sequences for lignocellulosic the cellulose of the firstmaterial . For example , (TC , ) can be biomass , aqueous feedstock slurry 5050 includes from about lower than (FC ) by more than about 5 percent, e.g. , 10 , 15 , 50 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 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, 55 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 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- 60 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 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 ofhydrogen- 65 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 or more organic solvents yields a purified slurry and an US 10,508,291 B2 55 56 organic waste stream 5210 that includes water- insoluble the feedstock can be transported to a closed , pressurized materials such as fats , oils , and other non -polar , hydrocar vessel ( e.g., an oxidation reactor ) for processing . bon -based substances. Suitable solvents for performing In certain embodiments , oxidizing agents 5160 can extraction of slurry 5050 include various alcohols , hydro include nascent oxygen ( e.g., oxygen radicals ) . Typically , carbons, and halo -hydrocarbons , for example . 5 nascent oxygen is produced as needed in an oxidation In some embodiments , aqueous feedstock slurry 5050 can reactor or in a chamber in fluid communication with an be subjected to an optional thermal treatment5190 to further oxidation reactor by one or more decomposition reactions. 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 O2 in a gas pressurizedence of pressurized steam swellssteam .the In fibrousfibers , exposingbiomass feedstocka larger ,frac the 10 mixture or in solution. In certain embodiments, nascent tion of fiber surfaces to the aqueous solvent and to oxidizing oxygen can be produced from decomposition of HOC1 in agents that are introduced in subsequent processing steps . solution . Other methods by which nascent oxygen can be In certain embodiments , aqueous feedstock slurry 5050 produced include via electrochemical generation in electro can be subjected to an optional treatment with basic agents 15 lyte solution , for example . 5200. Treatment with one or more basic agents can help to In general , nascent oxygen is an efficient oxidizing agent separate lignin from cellulose in lignocellulosic biomass due to the relatively high reactivity of the oxygen radical . feedstock , thereby improving subsequent oxidation of the However, nascent oxygen can also be a relatively selective feedstock . Exemplary basic agents include alkali and alka oxidizing agent. For example , when lignocellulosic feed line earth hydroxides such as sodium hydroxide , potassium 20 stock is treated with nascent oxygen , selective oxidation of hydroxide , and calcium hydroxide. In general, a variety of lignin occurs in preference to the other components of the basic agents can be used , typically in concentrations from feedstock such as cellulose . As a result, oxidation of feed about 0.01 % to about 0.5 % of the dry weight of the stock with nascent oxygen provides a method for selective feedstock . removal of the lignin fraction in certain feedstocks. Typi Aqueous feedstock slurry 5050 is transported ( e.g., by an 25 cally , nascent oxygen concentrations ofbetween about 0.5 % in -line piping system ) to a chamber, which can be an and 5 % of the dry weight of the feedstock are used to effect oxidation preprocessing chamber or an oxidation reactor . In efficient oxidation . oxidation preprocessing step 5060 , one or more oxidizing Without wishing to be bound by theory , it is believed that agents 5160 are added to feedstock slurry 5050 to form an nascent oxygen reacts with lignocellulosic feedstock accord oxidizing medium . In some embodiments , for example , 30 ing to at least two different mechanisms. In a first mecha oxidizing agents 5160 can include hydrogen peroxide. nism , nascent oxygen undergoes an addition reaction with Hydrogen peroxide can be added to slurry 5050 as an the lignin , resulting in partial oxidation of the lignin , which aqueous solution , and in proportions ranging from 3 % to solubilizes the lignin in aqueous solution . As a ult , the between 30 % and 35 % by weight of slurry 5050. Hydrogen solubilized lignin can be removed from the rest of the peroxide has a number of advantages as an oxidizing agent. 35 feedstock via washing . In a second mechanism , nascent For example , aqueous hydrogen peroxide solution is rela oxygen disrupts butane cross- links and / or opens aromatic tively inexpensive , is relatively chemically stable , is not rings that are connected via the butane cross- links. As a particularly hazardous relative to other oxidizing agents (and result , solubility of the lignin in aqueous solution increases , therefore does not require burdensomehandling procedures and the lignin fraction can be separated from the remainder and expensive safety equipment) . Moreover , hydrogen per- 40 of the feedstock via washing. oxide decomposes to form water during oxidation of feed In some embodiments , oxidizing agents 5160 include stock , so that waste stream cleanup is relatively straightfor ozone (03 ) . The use of ozone can introduce several chemi ward and inexpensive . cal -handling considerations in the oxidation processing In certain embodiments , oxidizing agents 5160 can sequence . If heated too vigorously , an aqueous solution of include oxygen ( e.g., oxygen gas) either alone, or in com- 45 ozone can decompose violently , with potentially adverse bination with hydrogen peroxide. Oxygen gas can be consequences for both human system operators and system bubbled into slurry 5050 in proportions ranging from 0.5 % equipment. Accordingly, ozone is typically generated in a to 10 % by weight of slurry 5050. Alternatively, or in thermally isolated , thick -walled vessel separate from the addition , oxygen gas can also be introduced into a gaseous vessel that contains the feedstock slurry, and transported phase in equilibrium with slurry 5050 ( e.g. , a vapor head 50 thereto at the appropriate process stage . above slurry 5050 ) . The oxygen gas can be introduced into Without wishing to be bound by theory , it is believed that either an oxidation preprocessing chamber or into an oxi ozone decomposes into oxygen and oxygen radicals , and dation reactor (or into both ) , depending upon the configu that the oxygen radicals ( e.g., nascent oxygen ) are respon ration of the oxidative processing system . Typically , for sible for the oxidizing properties of ozone in the manner example , the partial pressure of oxygen in the vapor above 55 discussed above. Ozone typically preferentially oxidizes the slurry 5050 is larger than the ambient pressure of oxygen , lignin fraction in lignocellulosic materials , leaving the cel and ranges from 0.5 bar to 35 bar , depending upon the nature lulose fraction relatively undisturbed . of the feedstock . Conditions for ozone -based oxidation of biomass feed The oxygen gas can be introduced in pure form , or can be stock generally depend upon the nature of the biomass . For mixed with one or more carrier gases . For example , in some 60 example , for cellulosic and / or lignocellulosic feedstocks , embodiments , high -pressure air provides the oxygen in the ozone concentrations of from 0.1 g/ m² to 20 g /m of dry vapor. In certain embodiments , oxygen gas can be supplied feedstock provide for efficient feedstock oxidation . Typi continuously to the vapor phase to ensure that a concentra cally , the water content in slurry 5050 is between 10 % by tion of oxygen in the vapor remains within certain prede weight and 80 % by weight ( e.g. , between 40 % by weight termined limits during processing of the feedstock . In some 65 and 60 % by weight) . During ozone- based oxidation , the embodiments , oxygen gas can be introduced initially in temperature of slurry 5050 can bemaintained between 0 ° C. sufficient concentration to oxidize the feedstock , and then and 100 ° C. to avoid violent decomposition of the ozone . US 10,508,291 B2 57 58 In some embodiments , feedstock slurry 5050 can be soft biomass material such as lignocellulosic matter , the treated with an aqueous, alkaline solution that includes one residence time in the oxidation reactor can be from 5 or more alkali and alkaline earth hydroxides such as sodium minutes to 30 minutes , for example, at an oxygen pressure hydroxide , potassium hydroxide , and calcium hydroxide , of between 3 and 12 bars in the reactor, and at a slurry and then treated thereafter with an ozone -containing gas in 5 temperature of between 160 ° C. and 210 ° C. For other types an oxidation reactor. This process has been observed to of feedstock , however, residence times in the oxidation significantly increase decomposition of the biomass in slurry reactor can be longer, e.g. , as long 48 hours. To determine 5050. Typically , for example , a concentration of hydroxide appropriate residence times for slurry 5050 in the oxidation 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 10 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- 15 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 slurry 5050, adjustment of the slurry pH can be performed embodiments , nitrogen -containing oxidizing substances can by introducing one or more chemical agents into the oxida be introduced into slurry 5050. Exemplary nitrogen - contain tion reactor. For example , in certain embodiments , oxidation ing oxidizing substances include NO and NO2, for example . 20 occurs most efficiently in a pH range of about 9-11 . To Nitrogen - containing agents can also be combined with oxy maintain a pH in this range , agents such as alkali and gen in slurry 5050 to create additional oxidizing agents . For alkaline earth hydroxides, carbonates, ammonia , and alka example , NO and NO2 both combine with oxygen in slurry line buffer solutions can be introduced into the oxidation 5050 to form nitrate compounds, which are effective oxi reactor . dizing agents for biomass feedstock . Halogen- and nitrogen- 25 Circulation of slurry 5050 during oxidation can be impor based oxidizing agents can , in some embodiments , cause tant to ensure sufficient contact between oxidizing agents bleaching of the biomass feedstock , depending upon the 5160 and the feedstock . Circulation of the slurry can be nature of the feedstock . The bleaching can be desirable for achieved using a variety of techniques . For example , in certain biomass -derived products that are extracted in sub some embodiments , a mechanical stirring apparatus that sequent processing steps. 30 includes impeller blades or a paddle wheel can be imple Other oxidizing agents can include , for example , various mented in the oxidation reactor. In certain embodiments, the peroxyacids, peroxyacetic acids, persulfates, percarbonates , oxidation reactor can be a loop reactor, in which the aqueous permanganates , osmium tetroxide, and chromium oxides. solvent in which the feedstock is suspended is simultane Following oxidation preprocessing step 5060 , feedstock ously drained from the bottom of the reactor and recirculated slurry 5050 is oxidized in step 5070. If oxidizing agents 35 into the top of the reactor via pumping, thereby ensuring that 5160 were added to slurry 5050 in an oxidation reactor, then the slurry is continually re -mixed and does not stagnate oxidation proceeds in the same reactor. Alternatively , if within the reactor . oxidizing agents 5160 were added to slurry 5050 in a After oxidation of the feedstock is complete , the slurry is preprocessing chamber , then slurry 5050 is transported to an transported to a separation apparatus where a mechanical oxidation reactor via an in -line piping system . Once inside 40 separation step 5080 occurs . Typically , mechanical separa the oxidation reactor , oxidation of the biomass feedstock tion step 5080 includes one or more stages of increasingly proceeds under a controlled set of environmental conditions . fine filtering of the slurry to mechanically separate the solid Typically, for example , the oxidation reactor is a cylindrical and liquid constituents . vessel that is closed to the external environment and pres Liquid phase 5090 is separated from solid phase 5100 , surized . Both batch and continuous operation is possible , 45 and the two phases are processed independently thereafter . although environmental conditions are typically easier to Solid phase 5100 can optionally undergo a drying step 5120 control in in - line batch processing operations. in a drying apparatus , for example . Drying step 5120 can Oxidation of feedstock slurry 5050 typically occurs at include, for example , mechanically dispersing the solid elevated temperatures in the oxidation reactor. For example , material onto a drying surface , and evaporating water from the temperature of slurry 5050 in the oxidation reactor is 50 solid phase 5100 by gentle heating of the solid material. typically maintained above 100 ° C., in a range from 120 ° C. Following drying step 5120 (or , alternatively , without under to 240 ° C. For many types of biomass feedstock , oxidation going drying step 5120 ), solid phase 5100 is transported for is particularly efficient if the temperature of slurry 5050 is further processing steps 5140 . maintained between 150 ° C. and 220 ° C. Slurry 5050 can be Liquid phase 5090 can optionally undergo a drying step heating using a variety of thermal transfer devices . For 55 5110 to reduce the concentration of water in the liquid phase . example , in some embodiments , the oxidation reactor con In some embodiments , for example , drying step 5110 can tacts a heating bath that includes oil or molten salts . In include evaporation and /or distillation and / or extraction of certain embodiments , a series of heat exchange pipes sur water from liquid phase 5090 by gentle heating of the liquid . round and contact the oxidation reactor, and circulation of Alternatively , or in addition , one or more chemical drying hot fluid within the pipes heats slurry 5050 in the reactor. 60 agents can be used to remove water from liquid phase 5090 . Other heating devices that can be used to heat slurry 5050 Following drying step 5110 ( or alternatively , without under include resistive heating elements , induction heaters , and going drying step 5110 ), liquid phase 5090 is transported for microwave sources , for example . further processing steps 5130 , which can include a variety of The residence time of feedstock slurry 5050 in the oxi chemical and biological treatment steps such as chemical dation reactor can be varied as desired to process the 65 and/ or enzymatic hydrolysis. feedstock . Typically , slurry 5050 spends from 1 minute to 60 Drying step 5110 creates waste stream 5220 , an aqueous minutes undergoing oxidation in the reactor. For relatively solution that can include dissolved chemical agents such as US 10,508,291 B2 59 60 acids and bases in relatively low concentrations . Treatment with an oxidant, such as a peroxide , with the desired of waste stream 5220 can include, for example , pH neutral cellulosic material while the material is housed in a vacuum ization with one or more mineral acids or bases. Depending chamber 151. To affect oxidation , current is passed through upon the concentration of dissolved salts in waste stream the filament, which causes a rapid heating of the filament for 5220 , the solution can be partially de - ionized (e.g. , by 5 a desired time. Typically , the heating is continued for passing the waste stream through an ion exchange system ) . seconds before allowing the filament to cool. In some Then , the waste stream which includes primarily water embodiments , the heating is performed a number of times to can be re - circulated into the overall process ( e.g., as water effect the desired amount of oxidation . 5150 ), diverted to another process , or discharged . Referring again to FIG . 12, in some embodiments , feed Typically , for lignocellulosic biomass feedstocks follow- 10 stock materials can be oxidized with the aid of sound and /or ing separation step 5070 , liquid phase 5090 includes a cavitation . Generally , to effect oxidation , the materials are variety of soluble poly- and oligosaccharides, which can sonicated in an oxidizing environment, such as water satu then be separated and /or reduced to smaller -chain saccha 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 15 Referring again to FIGS . 9 and 10 , in certain embodi amounts of hemicellulose- and lignin -derived products . 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 20 beam radiation can be employed to irradiate the materials . typical usage , an oxidizing carrier gas, e.g. , air or an Other Treatment Processes 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 ° C. After an appropriate the processes described herein . time, e.g., 5 to 10 minutes , the oxidized material is emptied 25 FIG . 23 shows an overview of the entire process of from the sample holder. The system shown in FIG . 2 can be 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 30 product. The fiber source can be transformed into the fibrous same time, the screw can push the oxidized material out of 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, 35 source , followed by optional screening step ( s ) and optional an oxidizing carrier gas traverses through the sample cham 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 40 or the “ liquor” 404, resulting from the steam exploding oxidized in a furnace pyrolyzer 130. In a typical usage, the 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 In another method , the fibrous material 401 is first steam 131. The sample is heated to the desired temperature for a 45 exploded to produce a steam exploded fiber source 410. The desired time to provide an oxidized product . The oxidized 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 50 fiber source can also be subject to one or more optional oxidant, such as a peroxide , and treating the target , which is 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 55 The fiber source can be cut into pieces or strips of confetti using mirrors 144 and 145 onto the target after passing 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 60 as water or isopropanol. The process can also optimally such embodiments , a infrared sensitive dye can be combined 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 . 65 of various chemical solutions . Referring again to FIG . 21, feedstock materials can be 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 US 10,508,291 B2 61 62 pressure , and the steam diffuses into the structures of the ing the sheared and steam exploded fibrous material into a fiber source ( e.g. , the lignocellulosic structures) . The steam product can therefore optionally include an overliming step then condenses under high pressure thereby “ wetting ” the prior to fermentation to precipitate some of the toxicants . fiber source. The moisture in the fiber source can hydrolyze For example , the pH of the sheared and steam exploded any acetyl groups in the fiber source ( e.g. , the acetyl groups 5 fibrous material can be raised to exceed the pH of 10 by in the hemicellulose fractions) , forming organic acids such adding calcium hydroxide (Ca ( OH )2 ) followed by a step of as acetic and uronic acids . The acids, in turn , can catalyze lowering the pH to about 5 by adding H2SO4. The overlimed the depolymerization ofhemicellulose , releasing xylan and fibrous material can then be used as is without the removal limited amounts of glucan . The “ wet” fiber source (or of precipitates . As shown in FIG . 23 , the optional overliming sheared fiber source , etc. ) is then " exploded ” when the 10 step occurs just prior to the step of hydrolysis of the sheared pressure is released . The condensed moisture instanta 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 15 ratus 460. The steam explosion apparatus 460 includes a of the internal structures ( e.g., the lignocellulosic structures ) reaction chamber 462, in which the fiber source and /or the of the fiber source . fibrous material placed through a fiber source inlet 464. The The sheared and steam exploded fibrous material is then reaction chamber is sealed by closing fiber source inlet valve converted into a useful product, such as ethanol. One 465. The reaction chamber further includes a pressurized method of converting the fibrous material is by hydrolysis to 20 steam inlet 466 that includes a steam valve 467. The reaction produce fermentable sugars , 412 , which are then fermented chamber further includes an explosive depressurization out to produce the product . Other known and unknown methods let 468 that includes an outlet valve 469 in communication of converting fibrous materials can also be used . with the cyclone 470 through the connecting pipe 472. Once In some embodiments , prior to combining the microor the reaction chamber includes the fiber source and/ or ganism , the sheared and steam exploded fibrous material 25 sheared fiber source and is sealed by closing valves 465 , 467 401 is sterilized to kill any competing microorganisms that and 469 , steam is delivered into the reaction chamber 462 by can be on the fibrous material. For example , the fibrous opening the steam inlet valve 467 allowing steam to travel material can be sterilized by exposing the fibrous material to through steam inlet 466. Once the reaction chamber reaches radiation , such as infrared radiation , ultraviolet radiation , or target temperature, which can take about 20-60 seconds, the an ionizing radiation , such as gamma radiation . The micro- 30 holding time begins . The reaction temperature is held at the organisms can also be killed using chemical sterilants , such target temperature for the desired holding time, which as bleach (e.g. , sodium hypochlorite ), chlorhexidine , or typically lasts from about 10 seconds to 5 minutes. At the ethylene oxide. One method to hydrolyze the sheared and end of the holding time period , outlet valve is open to allow steam exploded fibrous material is by the use of cellulases . for explosive depressurization to occur. The process of Cellulases are a group of enzymes that act synergistically to 35 explosive depressurization propels the contents of the reac hydrolyze cellulose . Commercially available Accelleraser tion chamber 462 out of the explosive depressurization 1000 enzyme complex , which contains a complex of outlet 468 , through the connecting pipe 472 , and into the enzymes that reduces lignocellulosic biomass into ferment cyclone 470. The steam exploded fiber source or fibrous able sugars can also be used . material then exits the cyclone in a sludge form into the According to current understanding, the components of 40 collection bin 474 as much of the remaining steam exits the cellulase include endoglucanases , exoglucanases ( cellobio cyclone into the atmosphere through vent 476. The steam hydrolases) , and b -glucosidases (cellobiases ). Synergism explosion apparatus further includes wash outlet 478 with between the cellulase components exists when hydrolysis by wash outlet valve 479 in communication with connecting a combination of two or more components exceeds the sum pipe 472. The wash outlet valve 479 is closed during the use of the activities expressed by the individual components . 45 of the steam explosion apparatus 460 for steam explosion , The generally accepted mechanism of a cellulase system but opened during the washing of the reaction chamber 462 . (particularly of T longibrachiahum ) on crystalline cellulose The target temperature of the reaction chamber 462 is is : endoglucanase hydrolyzes internal B - 1,4 - glycosidic preferably between 180 and 240 degrees Celsius or between bonds of the amorphous regions, thereby increasing the 200 and 220 degrees Celsius. The holding time is preferably number of exposed non - reducing ends. Exoglucanases then 50 between 10 seconds and 30 minutes, or between 30 seconds cleave off cellobiose units from the nonreducing ends, which and 10 minutes , or between 1 minute and 5 minutes . in turn are hydrolyzed to individual glucose units by b - glu Because the steam explosion process results in a sludge of cosidases. There are several configurations of both endo steam exploded fibrous material , the steam exploded fibrous and exo - glucanases differing in stereospecificities . In gen material can optionally include a fiber recovery process eral , the synergistic action of the components in various 55 where the “ liquor” is separated from the steam exploded configurations is required for optimum cellulose hydrolysis. fibrous material . This fiber recovery step is helpful in that it Cellulases , however , are more inclined to hydrolyze the enables further shearing and /or screening processes and can amorphous regions of cellulose. A linear relationship allow for the conversion of the fibrous material into a between crystallinity and hydrolysis rates exists whereby product. The fiber recovery process occurs through the use higher crystallinity indices correspond to slower enzyme 60 of a mesh cloth to separate the fibers from the liquor. Further hydrolysis rates. Amorphous regions of cellulose hydrolyze drying processes can also be included to prepare the fibrous at twice the rate of crystalline regions. The hydrolysis of the material or steam exploded fiber source for subsequent sheared and steam exploded fibrous material can be per processing formed by any hydrolyzing biomass process. Any processing technique described herein can be used at Steam explosion of biomass sometimes causes the for- 65 pressure above or below normal , earth -bound atmospheric mation of by -products , e.g., toxicants , that are inhibitory to pressure . For example , any process that utilizes radiation , microbial and enzymatic activities . The process of convert sonication , oxidation , pyrolysis , steam explosion , or com US 10,508,291 B2 63 64 binations of any of these processes to provide materials that An electron beam device can also be combined with one include a carbohydrate can be performed under high pres or more of high - frequency, rotor- stator devices, which can sure , which , can increase reaction rates. For example , any be used as an alternative to ultrasonic energy devices , and process or combination of processes can be performed at a performs a similar function . pressure greater than about greater than 25 MPa, e.g. , greater 5 Further combinations of devices are also possible . For than 50 MPa , 75 MPa , 100 MPa , 150 MPa , 200 MPa, 250 example , an ionizing radiation device that produces gamma MPa , 350 MPa , 500 MPa , 750 MPa , 1,000 MPa , or greater radiation emitted from , e.g., 6 ° Co pellets, can be combined than 1,500 MPa . with an electron beam source and /or an ultrasonic wave Combinations of Irradiating , Sonicating , and Oxidizing source . 10 The radiation devices for pretreating biomass discussed Devices 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 and / or oxidation devices into a single hybrid machine. For throughput . Nevertheless, caution must be observed , as there such a hybrid machine, multiple processes can be performed 15 can be conflicting requirements between some radiation in close juxtaposition or even simultaneously , with the processes and pyrolysis . For example, ultrasonic radiation benefit of increasing pretreatment throughput and potential devices can require the feedstock be immersed in a liquid cost savings. oxidizing medium . On the other hand , as discussed previ For example , consider the electron beam irradiation and ously , it can be advantageous for a sample of feedstock sonication processes. Each separate process is effective in 20 undergoing pyrolysis to be of a particular moisture content . lowering the mean molecular weight of cellulosic material In this case, the new systems automatically measure and by an order of magnitude or more, and by several orders of monitor for a particular moisture content and regulate the agnitude when performed serially . same Further, some or all of the above devices , especially Both irradiation and sonication processes can be applied the pyrolysis device , can be combined with an oxidation using a hybrid electron beam / sonication device as is illus- 25 device as discussed previously . trated in FIG . 25. Hybrid electron beam / sonication device Primary Processes (Processing Treated Biomass ) 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 30 biomass materials . For example , alcohols , organic acids , peroxide. Hybrid device 2500 has an energy source 2510 , 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 35 engineered microorganism . For example , the microorganism impact the slurry 2550 containing cellulosic material . The can be a bacterium , e.g. , a cellulolytic bacterium , a fungus, electron beam aiming device can be a scanner that sweeps a e.g., a yeast , a plant or a protist, e.g., an algae , a protozoa or beam over a range of up to about 6 feet in a direction a fungus- like protist , e.g. , a slime mold . When the organisms approximately parallel to the surface of the slurry 2550 . are compatible , mixtures of organisms can be utilized . On either side of the electron beam emitter 2540 are 40 To aid in the breakdown of the treated biomass materials sonication horns 2530 , which deliver ultrasonic wave energy that include cellulose , one or more enzymes , e.g. , a cel to the slurry 2550. The sonication horns 2530 end in a lulolytic enzyme can be utilized . In some embodiments , detachable endpiece 2535 that is in contact with the slurry materials that include cellulose are first treated with the 2550 . enzyme, e.g. , by combining the materials and the enzyme in The sonication horns 2530 are at risk of damage from 45 an aqueous solution . This material can then be combined long - term residual exposure to the electron beam radiation . with the microorganism . In other embodiments , the materi Thus, the horns can be protected with a standard shield 2520, als that include the cellulose, the one or more enzymes and e.g., made of lead or a heavy -metal -containing alloy such as the microorganism are combined concurrently , e.g., by com Lipowitz metal , which is impervious to electron beam bining in an aqueous solution . radiation . Precautions must be taken , however , to ensure that 50 Also , to aid in the breakdown of the treated biomass the ultrasonic energy is not affected by the presence of the materials , the treated biomass materials can be further shield . The detachable endpieces 2535 , are constructed of treated ( e.g., post irradiation ) with heat , a chemical ( e.g., the same material and attached to the horns 2530 , are used mineral acid , base or a strong oxidizer such as sodium to be in contact with the cellulosic material 2550 and are hypochlorite ) , and / or an enzyme. 55 During fermentation , sugars released from cellulolytic expected to be damaged . Accordingly , the detachable end 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 microorganismshave 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 60 galactose , oligosaccharides , or polysaccharides into fermen alone , an insufficient dose can result in cross- linking of some 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 evisiae (baker's yeast) , Saccharomyces distaticus, and Sac doses of electron beam irradiation and / or ultrasound radia charomyces uvarum ; the genus Kluvveromyces , e.g., species tion can also be used to achieve a similar degree of depo- 65 Kluvveromyces marxianus, and Kluyveromyces fragilis; the lymerization as that achieved using electron beam irradia genus Candida , e.g., Candida pseudotropicalis , and Can tion and sonication separately . dida brassicae ; the genus Clavispora , e.g. , species Clav US 10,508,291 B2 65 66 ispora lusitaniae and Clavispora opuntiae ; the genus Pachy sporum , Acremonium obclavatum , Acremonium solen , e.g. , species Pachysolen lannophilus; the genus pinkertoniae, Acremonium roseogriseum , Acremonium Bretannomyces, e.g., species Bretannomyces clausenii; the incoloratum , and Acremonium furatum ; preferably from the genus Pichia , e.g. , species Pichia stipitis ; and the genus species Humicola insolens DSM 1800 , Fusarium oxysporum Saccharophagus, e.g., species Saccharophagus degradans 5 DSM 2672, Myceliophthora thermophila CBS 117.65 , ( Philippidis , 1996 , “ Cellulose Bioconversion Technology ” , Cephalosporium sp . RYM -202 , Acremonium sp . CBS in Handbook on Bioethanol: Production and Utilization , 478.94, Acremonium sp . CBS 265.95 , Acremonium persici Wyman , ed ., Taylor & Francis , Washington , D.C., 179-212 ) . num 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- 10 nium CBS 866.73 , Acremonium dichromosporum CBS fre , USA ) ; FALI® (available from Fleischmann's Yeast, a 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 monium furatum CBS 299.70H . Cellulolytic enzymes can FERMOL® , (available from DSM Specialties) . 15 also be obtained from Chrysosporium , preferably a strain of Bacteria that can ferment biomass to ethanol and other Chrysosporium lucknowense. Additionally , Trichoderma products include , e.g. , Zymomonas mobilis and Clostridium (particularly Trichoderma viride, Trichoderma reesei, and thermocellum (Philippidis , 1996 , supra ) . Leschine et al. Trichoderma koningii) , alkalophilic Bacillus (see , for ( International Journal of Systematic and Evolutionary example , U.S. Pat . No. 3,844,890 and EP 458162 ) , and Microbiology 2002 , 52, 1155-1160 ) describe an anaerobic , 20 Streptomyces (see , e.g. , EP 458162 ) can be used . mesophilic , cellulolytic bacterium from forest soil, Cellulolytic enzymes produced using recombinant tech Clostridium phytofermentans sp . nov. , which converts cel nology can also be used ( see, e.g. , WO 2007/071818 and lulose to ethanol. WO 2006/110891 ) . Fermentation of biomass to ethanol and other products The cellulolytic enzymes used can be produced by fer can be carried out using certain types of thermophilic or 25 mentation of the above -noted microbial strains on a nutrient genetically engineered microorganisms, such as Thermoan medium containing suitable carbon and nitrogen sources and aerobacter species , including T. mathranii, and yeast species inorganic salts , using procedures known in the art ( see , e.g., such as Pichia species. An example of a strain of T. Bennettand LaSure (eds . ) , More Gene Manipulations in mathranii is A3M4 described in Sonne- Hansen et al . ( Ap Fungi. Academic Press . CA 1991) . Suitable media are plied Microbiology and Biotechnology 1993 , 38 , 537-541 ) 30 available from commercial suppliers or can be prepared or Ahring et al . (Arch . Microbiol. 1997 , 168, 114-119 ) . according to published compositions ( e.g., in catalogues of Yeast and Zymomonas bacteria can be used for fermentation the American Type Culture Collection ). Temperature ranges or conversion . The optimum pH for yeast is from about pH and other conditions suitable for growth and cellulase pro 4 to 5 , while the optimum pH for Zymnomonas is from about duction are known in the art ( see , e.g., Bailey and Ollis , pH 5 to 6. Typical fermentation times are about 24 to 96 35 Biochemical Engineering Fundamentals , McGraw -Hill hours with temperatures in the range of 26 ° C. to 40 ° C., Book Company, NY, 1986 ). however thermophilic microorganisms prefer higher tem Treatment of cellulose with cellulase is usually carried out peratures . at temperatures between 30 ° C. and 65 ° C. Cellulases are Enzymes that break down biomass , such as cellulose , to active over a range of pH of about 3 to 7. A saccharification lower molecular weight carbohydrate - containing materials , 40 step can last e.g., up to 120 hours . The cellulase enzyme such as glucose , are referred to as cellulolytic enzymes or dosage achieves a sufficiently high level of cellulose con cellulase ; this process is referred to an “ saccharification ” . version . For example , an appropriate cellulase dosage is These enzymes can be a complex of enzymes that act typically between 5.0 and 50 Filter Paper Units (FPU or LU ) synergistically to degrade crystalline cellulose . Examples of per gram of cellulose. The FPU is a standard measurement cellulolytic enzymes include : endoglucanases , cellobiohy- 45 and is defined and measured according to Ghose ( 1987, Pure drolases , and cellobiases (ß - glucosidases ). For example , and Appl. Chem . 59 :257-268 ) . cellulosic substrate is initially hydrolyzed by endogluca In particular embodiments , ACCELERASETM 1000 nases at random locations producing oligomeric intermedi (GENENCOR ) is utilized as the enzyme system at a loading ates. These intermediates are then substrates for exo - split of 0.25 mL per gram of substrate . ACCELLERASE? 1000 ting glucanases such as cellobiohydrolase to produce 50 enzyme complex is a multiple enzyme cocktail with multiple cellobiose from the ends of the cellulose polymer. Cellobi activities, mainly exoglucanase , endoglucanase , hemicellu ose is a water- soluble B - 1,4 - linked dimer of glucose. Finally lase and beta -glucosidase . The cocktail has a minimum cellobiase cleaves cellobiose to yield glucose . endoglucanase activity of 2500 CMC U / g and a minimum A cellulase is capable of degrading biomass and can be of beta - glucosidase activity of 400 PNPG U / g . The pH of the fungal or bacterial origin . Suitable enzymes include cellu- 55 cocktail is from about 4.8 to about 5.2 . In other particular lases from the genera Bacillus, Pseudomonas, Humicola , embodiments , the enzyme system utilized is a blend of Fusarium , Thielavia , Acremonium , Chrysosporium and CELLUCLAST® 1.5 L and Novozyme 188. For example , Trichoderma, and include species of Humicola , Coprinus, 0.5 mL of CELLUCLAST® 1.5 L and 0.1 mL of Novozyme Thielavia , Fusarium , Myceliophihora , Acremonium , Cepha 188 can be used for each gram of substrate . When a higher losporium , Scytalidium , Penicillium or Aspergillus (see , e.g., 60 hemicellulase (xylanase ) activity is desired , OPTIMASHTM hsEP 458162 ), especially those produced by a strain selected BG can be utilized . from the species Humicola insolens (reclassified as Scyta Gasification lidium thermophilum , see , e.g. , U.S. Pat . No. 4,435,307) , In addition to using pyrolysis for pre - treatment of feed Coprinus cinereus, Fusarium oxysporum , Myceliophthora stock , pyrolysis can also be used to process pre- treated thermophila ,Meripilus giganteus, Thielavia terrestris, Acre- 65 feedstock to extract usefulmaterials . In particular, a form of monium sp ., Acremonium persicinum , Acremonium acremo pyrolysis known as gasification can be employed to generate nium , Acremonium brachypenium , Acremonium dichromo fuel gases along with various other gaseous, liquid , and solid US 10,508,291 B2 67 68 products . To perform gasification , the pre - treated feedstock rectification column reflux condenser can provide heat for is introduced into a pyrolysis chamber and heated to a high this first effect . After the first effect, solids can be separated temperature, typically 700 ° C. or more. The temperature using a centrifuge and dried in a rotary dryer . A portion used depends upon a number of factors , including the nature (25 % ) of the centrifuge effluent can be recycled to fermen of the feedstock and the desired products . 5 tation and the rest sent to the second and third evaporator 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 10 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 plantby 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 15 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 . biogas can be used to create steam and electricity that can be In a first step of the overall reaction , heating the feedstock used in various plant processes . material produces a char that can include a wide variety of Wastewater is initially pumped through a screen ( e.g. , a different hydrocarbon -based species . Certain volatile mate- 20 bar screen ) to remove large particles , which are collected in rials can be produced ( e.g., certain gaseous hydrocarbon a hopper . In some embodiments , the large particles are sent materials ) , resulting in a reduction of the overall weight of to a landfill. Additionally or alternatively , the large particles the feedstock material . Then , in a second step of the reaction , are burned to create steam and /or electricity as described in some of the volatile material that is produced in the first step further detail below . In general, the spacing on the bar screen reacts with oxygen in a combustion reaction to produce both 25 is between 1/4 inch to 1 inch spacing (e.g. , 1/2 inch spacing ). carbon monoxide and carbon dioxide . The combustion reac The wastewater then flows to an equalization tank , where tion releases heat, which promotes the third step of the the organic concentration of the wastewater is equalized reaction . In the third step , carbon dioxide and steam ( e.g. , during a retention time. In general, the retention time is water ) react with the char generated in the first step to form between 8 hours and 36 hours ( e.g. , 24 hours ). A mixer is carbon monoxide and hydrogen gas. Carbon monoxide can 30 disposed within the tank to stir the contents of the tank . In also react with steam , in a water gas shift reaction , to form some embodiments , a plurality ofmixers disposed through carbon dioxide and further hydrogen gas. out the tank are used to stir the contents of the tank . In Gasification can be used as a primary process to generate certain embodiments , the mixer substantially mixes the products directly from pre - treated feedstock for subsequent contents of the equalization tank such that conditions ( e.g., transport and /or sale , for example . Alternatively , or in addi- 35 wastewater concentration and temperature ) throughout the tion , gasification can be used as an auxiliary process for tank are uniform . generating fuel for an overall processing system . The hydro A first pump moves water from the equalization tank gen - rich syngas that is generated via the gasification process through a liquid - to - liquid heat exchanger . The heat can be burned , for example , to generate electricity and /or exchanger is controlled ( e.g., by controlling the flow rate of process heat that can be directed for use at other locations in 40 fluid through the heat exchanger) such that wastewater the processing system . As a result , the overall processing exiting the heat exchanger is at a desired temperature for system can be at least partially self- sustaining . A number of anaerobic treatment. For example , the desired temperature other products , including pyrolysis oils and gaseous hydro for anaerobic treatment can be between 40 ° C. to 60 ° C. carbon -based substances, can also be obtained during and /or After exiting the heat exchanger , the wastewater enters following gasification ; these can be separated and stored or 45 one or more anaerobic reactors . In some embodiments , the transported as desired . concentration of sludge in each anaerobic reactor is the same A variety of different pyrolysis chambers are suitable for as the overall concentration of sludge in the wastewater. In gasification of pre - treated feedstock , including the pyrolysis other embodiments , the anaerobic reactor has a higher chambers disclosed herein . In particular, fluidized bed reac concentration of sludge than the overall concentration of tor systems, in which the pre- treated feedstock is fluidized in 50 sludge in the wastewater . steam and oxygen /air , provide relatively high throughput A nutrient solution containing nitrogen and phosphorus is and straightforward recovery of products . Solid char that metered into each anaerobic reactor containing wastewater . remains following gasification in a fluidized bed system (or The nutrient solution reacts with the sludge in the anaerobic in other pyrolysis chambers ) can be burned to generate reactor to produce biogas which can contain 50 % methane additional process heat to promote subsequent gasification 55 and have a heating value of approximately 12,000 British reactions . thermal units , or Btu , per pound ). The biogas exits each Processing Treated Biomass anaerobic reactor through a vent and flows into a manifold , Distillation where a plurality of biogas streams are combined into a After fermentation , the resulting fluids can be distilled single stream . A compressor moves the stream of biogas to using , for example , a " beer column " to separate ethanol and 60 a boiler or a combustion engine as described in further detail other alcohols from themajority of water and residual solids . below . In some embodiments , the compressor also moves The vapor exiting the beer column can be 35 % by weight the single stream of biogas through a desulphurization ethanol and fed to a rectification column. A mixture of nearly catalyst. Additionally or alternatively , the compressor can azeotropic (92.5 % ) ethanol and water from the rectification move the single stream of biogas through a sediment trap . column can be purified to pure ( 99.5 % ) ethanol using 65 A second pumpmoves anaerobic effluent from the anaero vapor- phase molecular sieves. The beer column bottoms can bic reactors to one or more aerobic reactors ( e.g. , activated be sent to the first effect of a three -effect evaporator. The sludge reactors ). An aerator is disposed within each aerobic US 10,508,291 B2 69 70 reactor to mix the anaerobic effluent, sludge , oxygen (e.g. , exchanger to ensure that the steam turbine is operating oxygen contained in air ). Within each aerobic reactor, oxi above a threshold condition ( e.g. , the turbine is spinning fast dation of cellular material in the anaerobic effluent produces enough to turn the electric generator ). carbon dioxide , water, and ammonia . While certain embodiments have been described , other Aerobic effluent moves ( e.g. , via gravity ) to a separator, 5 embodiments are possible . where sludge is separated from treated water . Some of the As an example , while the biogas is described as being sludge is returned to the one or more aerobic reactors to diverted to a combustion engine connected to an electric create an elevated sludge concentration in the aerobic reac generator, in certain embodiments , the biogas or some tors, thereby facilitating the aerobic breakdown of cellular portion thereof can also be passed through a fuel reformer to material in the wastewater. A conveyor removes excess 10 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 . 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 15 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 materials 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- 20 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 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. 25 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 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- 30 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 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 35 at least biomass and one or more microorganismscapable 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 amountof waste biomass 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 40 ( 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 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 45 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 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- 50 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. 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- 55 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 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 60 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 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 65 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 , US 10,508,291 B2 71 72 and / or increased in availability ( e.g., compared to unpro are generally undesirable in a material intended for use in cessed biomass materials ) ) include, but are not limited to humans and /or animals . For example , the methods can be sugars (e.g. , 1,4 -diacids ( e.g., succinic acids, fumaric acids, used to remove, inactivate , and /or neutralize infectious 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 , 5 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 be used to remove, inactivate , and/ or neutralize toxins, e.g. , tol) , dextrins, cyclodextrins , amylase , amylopectin , germ , bacterialtoxins and plant toxins . Alternatively or in addition , proteins, amino acids, peptides, nucleic acids, fats , lipids, 10 the methods described herein can be used to remove, inac fatty acids, gluten , sweeteners ( e.g. , glucose ), sugar alcohols tivate , and / or neutralize materials that can be present in the ( e.g., arabitol, xylitol, ribitol, mannitol, sorbitol, isomalt, biomass that are not necessarily harmful, but are undesirable maltitol, and lactitol) , oils ( e.g., triglyceride vegetable oils in a material to be used in humans and / or animals or in (peanut e.g., soybean oil, cottonseed oil , palm oiloil, , palmrapeseed kernel oil , oilsunflower, olive oil seed) , cornoil, 15 agriculture or aquaculture .Exemplary materials include, but oil , oat oil, nut oil, and ) , 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 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 / 20 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 . 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 . 25 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 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 30 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 ) , imal) . Components with increased availability can 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 35 ( 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 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- 40 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 , thereof . aquatic microorganisms, aquatic plants , seaweed and algae . Alcohols In some embodiments , the methods described herein can 45 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 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 , 50 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 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 . 55 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 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 60 hexane, heptane, isobutene, octane, iso -octane , nonane, and / or non - pathogenic material) , toxins, and/ or other mate decane , 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 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 65 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 US 10,508,291 B2 73 74 acid , succinic acid , glutaric acid , oleic acid , linoleic acid , ( saturated , monounsaturated , and polyunsaturated ), choles glycolic acid , lactic acid , y -hydroxybutyric acid , or mixtures terol, dietary fiber, vitamins ( e.g., vitamin A , E , C , B6 , B12 , of these acids . carotene , thiamin , riboflavin , and niacin ), minerals ( e.g. , Foodstuffs calcium , phosphorus, magnesium , iron , zinc , copper , potas As described herein , the present invention provides meth- 5 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 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 10 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 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, 15 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 foodstuff and reduce waste . ( e.g., dissolving) unprocessed and processed materials in a 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 20 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 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 25 described herein that are intended for use as foodstuffs (e.g. , prepare 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 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 30 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 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 35 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 . mass , e.g., infectiousmaterial ( 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 40 bacterial and fungal spores , insects , and larvae) . In general , energy intake (MEI ) of that biomass . Methods for measuring 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 neutralization of materials 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 45 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 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 50 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 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 55 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 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 . 60 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 , 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 65 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 US 10,508,291 B2 75 76 embodiments , the methods described herein can be used to In some embodiments , the methods described herein can increase the nutrient availability of any of these materials , be used to process biomass to generate the materials e.g. , to maintain or improve the health of a specific type or described herein . These materials can include , but are not 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 5 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 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 , 10 100-200 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 5 , 4 , 3 , 2 , and 1 saccharide units . tation . For example, ruminants only digest about 30-50 In some embodiments , the methods produce disaccharides percent of the cellulose and hemicellulose . In some embodi ( e.g., sucrose, lactose, maltose, trehalose, and cellobiose ). In ments , the methods described herein can be used to increase 15 some embodiments , the methods produce monosaccharides the nutrient availability or nutritional value of any of these (e.g. , glucose (dextrose ), fructose , galactose , xylose , and materials , e.g., to maintain or improve the health of a ribose ) . These shorter chain molecules will be more easily specific type or species of animal, promote the growth of a absorbed by an animal and will thereby increase the nutrient target animal ( e.g., tissue gain ), and /or to promote food availability of biomass . Consequently , the methods and production . The methods described herein use reduced 20 materials described herein can be used as foodstuffs or in the amounts of foodstuffs , at a lower cost , and / or with less production of foodstuffs . waste . In some embodiments , the materials described herein can Generally, increasing the nutrient availability of an animal be used as a foodstuff e.g., agricultural foodstuffs and /or foodstuff will reduce the amount of feed required to be fed foodstuffs suitable for ingestion by mammals , birds, and / or to an animal for the animal to receive the same amount of 25 fish . Alternatively or in addition , the methods described energy . Consequently , the animal will require less foodstuff herein can be used to process a raw material suitable for use thus providing a more economical foodstuff . as or in an animal foodstuff . Various techniques have been attempted to increase the Materials that can be usefully processed using the meth nutrient availability of a foodstuff with limited success . Such ods described herein include cellulosic and lignocellulosic techniques include the use of enzymes, such as cellulosic 30 materials, e.g. , arable products , crops, grasses , plants and / or enzymes, to break down cellulosic material into shorter feed grains, for example including , but not limited to , plant chain oligosaccharides, which can be more readily digested . material ( e.g. , forage such as alfalfa meal, hay , Bermuda Although used in Europe and Australia , this practice is coastal grass hay, sweet grass, corn plant, and soybean hay ) , expensive and not widely used in developing countries . grains ( e.g., barley, corn ( including organic and genetically Other techniques include removing stover to prevent leaf 35 modified corn ), oats , rice , sorghum , and wheat ), plant pro loss , air removal , physically treating the material ( e.g., tein products ( e.g. , canola meal, cottonseed cakes and meals , compacting cellulosic material, reducing particle size, and safflower meal , and soybean ( including organic and geneti fine grinding ) , chemical treatment, and overfeeding . Addi cally modified soybean ) feed and meal) , processed grain tionally , foodstuffs composed largely from cellulosic mate by -products (e.g. , distillers products , brewers dried grains, rial are frequently supplemented with nutrient systems ( e.g. , 40 corn gluten , sorghum germ cake and meal , peanut skins , and premixes ) . These nutrient systems are typically designed to wheat bran ) , fruit and fruit by -products ( e.g., dried citrus provide the nutritional requirements of a target animal . pulp , apple pomace , and pectin pulp ), molasses ( e.g. , beet , Despite ensuring the animals receive the required nutrients , citrus , starch , and cane molasses ), almond hulls , ground such systems do not make efficient use of the cellulosic shells, buckwheat hulls , legumes and legume by -products , material . 45 and other crop by - products . Other raw materials include , but The methods described herein provide methods for are not limited to , alfalfa , barley , birdsfoot trefoil, brassicas improving the nutrient availability or nutritional value of ( e.g., chau moellier, kale , (canola ), rutabaga biomass ( e.g., by modifying ( e.g., increasing, decreasing, or ( swede ), and turnip ), clover (e.g. , alsike clover, red clover , maintaining ) the solubility of the biomass and /or changing subterranean clover , and white clover) , grass ( e.g. , false oat the structure ( e.g., functionalizing) of the native materials , 50 grass , fescue, Bermuda grass, brome, heath grass , meadow and / or altering (e.g. , lowering ) the molecular weight and /or grass , orchard grass , ryegrass , and Timothy grass ) , crystallinity ) , as described above, thereby producing a more (corn ), millet , oats , sorghum , and . In some valuable foodstuff . In some embodiments , the methods embodiments , the raw material can be animal waste ( e.g. , described herein can be used to increase the nutrient avail ruminant waste ) or human waste . ability of the biomass by breaking down cellulosic material 55 In some embodiments , the foodstuff contains only the ( e.g. , cellulose and /or hemicellulose ) into shorter chain materials produced using the methods described herein . saccharides and /or monosaccharides . By improving the Alternatively or in addition , the foodstuff contains additional nutrient availability of the biomass , these methods will raw materials (including raw materials not treated using the result in a more efficient foodstuff that can be used to methods described herein ) and additives. Such foodstuffs maintain or improve the health of a specific type or species 60 can be formulated to meet the specific requirements of a of animal, promote the growth of a target animal ( e.g. , tissue target animal, e.g., to maintain or improve the health of a gain ), and / or to promote food production . specific type or species of animal, to promote the growth of In some embodiments, a useful animal foodstuff can a target animal , tissue gain , and/ or to promote food produc include partially processed biomass , e.g. , biomass that has tion . In some cases , a foodstuff can be formulated to meet been sheared using the methods described herein . Such 65 the nutritional requirements of a target animal for the least partially processed biomass can be more readily hydrolyzed cost ( the “ least cost ration ” ) . Methods for determining the in the stomach of an animal. formulation of a foodstuff and the least cost ration are well US 10,508,291 B2 77 78 known to those ofskill in the art ( see, for example , Pesti and described herein can be provided to the foodstuff manufac Miller , Animal Feed Formulation : Economic and Computer turer in a form that is suitable for incorporation into a Applications (Plant and Animal Science ) , Springer Publish foodstuff. Alternatively or in addition , the materials ing , Feb. 28 , 1993 and world wide web address liveinfor described herein can be prepared from a raw material at the matics.com ). 5 site at which the foodstuff is prepared . Additional raw materials and additives that can be use In some embodiments , the materials described herein can fully combined with a material produced using the methods be distributed alone and ingested by an animal in the absence described herein include , but are not limited to , animal of any additional raw materials and / or additives . products ( e.g. , meatmeal , meat meal tankage , meat and bone In some embodiments , the materials will require post meal , poultry meal , animal by - product meal , dried animal 10 processing prior to use as food . For example , a dryer can be blood , blood meal, feather meal , egg - shell meal , hydrolyzed used to remove moisture from the intermediate fermentation whole poultry , hydrolyzed hair , and bone marrow ) , animal products to facilitate storage, handling, and shelf life . Addi waste , marine products and by -products (e.g. , krill , fish parts tionally or alternatively , thematerials can be ground to a fine and meal, fish residue meal, crab parts and meal, shrimp particle size in a stainless- steel mill to produce a flour- like parts and meal, fish oil, fish liver and glandular meal, and 15 substance . other fish by -products ), dairy products ( e.g., dried cow milk , Typically , biomass based foodstuffs are usefully fed only casein , whey products , and dried cheese ), fats and oils (e.g. , to ruminants that are capable of at least partially digesting animal fat , vegetable fat or oil, and hydrolyzed fats ), res cellulose . As the present disclosure provides materials in taurant food waste (e.g. , food waste from restaurants , bak which the cellulosic material has been broken down into eries , and cafeterias ), and contaminated / adulterated food 20 shorter chain sugars , these materials can also be used as a treated to destroy pathogens. viable foodstuff for animals that are incapable of cellulose or Other additives include antibiotics ( e.g. , tetracyclines, hemicellulose digestion . Therefore , foodstuffs prepared macrolides, fluoroquinolones, and streptogramins ), flavor using the materials and methods described herein can be ing , brewers oats , by -products of drug manufacture (e.g. , usefully fed to animals including, but not limited to , food spent mycelium and fermentation products ), minerals and 25 production animals , zoo animals , and laboratory animals , trace minerals ( e.g. , bone charcoal, calcium carbonate , chalk and /or domestic animals . The foodstuffs can also be used in rock , iron salts , magnesium salts , oyster shell flour, and agriculture and aquaculture . In addition , because foodstuffs sulphate ) , proteinated minerals ( e.g., proteinated selenium prepared using the materials described herein have a higher and chromium ) , vitamins ( e.g. , vitamin A , vitamin D , vita nutrient availability , less foodstuff will be required by the min B12, niacin , and betaine ), direct fed organisms/ probiot- 30 animal to receive the same amount of energy , which will ics ( e.g., Aspergillus niger , Baccillus subtillis , Bifidobacte reduce the overall cost of the foodstuff. Alternatively , ani rium animalis , B. bifidium , Enterococcus faecium , mals will be able to consume more energy , which will result Aspergillus oryzae, Lactobacillus acidophilus, L. bulgari in higher growth rates, tissue gain , milk production , and egg cus , L. planetarium , Streptococcus lactis , and Saccharomy production . ces cerevisiae ), prebiotics ( e.g. , mannan -oligosaccharides 35 In some embodiments , the materials described herein can (MOS ), fructo - oligosaccharides , and mixed oligo -dextran ) , be usefully fed to ruminants ( e.g. , cattle , goats , sheep , flavors ( e.g., aloe vera gel concentrate , ginger, capsicum , horses, elk , bison , deer , camels , alpacas , llamas, giraffes, and fennel ) , enzymes (e.g. , phytase , cellulase , lactase, yaks, water buffalo ,wildebeest , and antelope ), poultry , pigs, lipase , pepsin , catalase , xylanase , and pectinase ) , acetic acid , boars , birds , cats , dogs, and fish . sulfuric acid , aluminum salts , dextrans, glycerin , beeswax , 40 In some embodiments , distillers grains and solubles can sorbitol, riboflavin , preservatives ( e.g. , butylated hydroxy be converted into a valuable byproduct of the distillation anisole and sodium bisulfite ), nutraceuticals ( e.g., herbal and dehydration process . After the distillation - dehydration pro botanical products ) , amino acids, by pass protein , urea , cess, distillers grains and solubles can be dried to improve molasses , fatty acids, ( e.g., acetic , propionic, and butyric the ability to store and handle the material. The resulting acid ) and metabolic modifiers ( e.g. , somatotropins and 45 dried distillers grains (DDG ) and solubles is low in starch , adrenergic agonists ). In some cases , thematerials produced high in fat , high in protein , high in fiber , and high in using the methods described herein can be combined or phosphorous. Thus, for example , DDG can be valuable as a incorporated into a urea molasses 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 50 tives to meet specific dietary requirements of specific cat animal. In some cases the foodstuff can be a solid . Alterna 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, 55 DDG can be optimized to meet the needs of target animals . pellets , biscuit, and mixtures ofan unprocessed raw material 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, 60 the methods described herein can be used in animal feed . e.g., for local use and /or small scale distribution . In such 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. , 65 novel foodstuff for human consumption . In order for a mixed ) into a foodstuff by a foodstuff manufacturer , e.g. , for 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 US 10,508,291 B2 79 80 ity of the biomass would have to be increased by ( 1 ) In some embodiments , the materials described herein can be increasing the solubility of the biomass; (2 ) changing the combined with protein , fats , carbohydrates , minerals , phar structure ( e.g. , functionalizing ) of the native materials ; ( 3 ) maceuticals , and vitamins. altering ( e.g., lowering) the molecular weight and /or crys Proteins tallinity relative to a native material; and /or ( 4 ) breaking 5 In some embodiments , the methods described herein can down cellulosic material into smaller saccharides, for be used to obtain ( e.g., extract, isolate, and /or purify proteins example , saccharides with length of greater than 1000 (e.g. , polypeptides , peptides, and amino acids ) from bio saccharide units ; about 1000 sugar saccharide units ; about mass . Such proteins ( e.g., polypeptides, peptides , and amino 800-900 saccharide units ; about 700-800 saccharide units ; 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 bataua , 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 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 10,508,291 B2 81 82 Vitamins herbs with medicinal properties . Additionally or alterna In some embodiments , the methods described herein can tively , irradiation can stimulate bioactivity and / or bioavail be used to obtain ( e.g. , extract , isolate , and /or purify ) ability of the medicinal components of plants and /or herbs vitamins from biomass . Such vitamins can be used , e.g., with medicinal properties. alone or in combination with one or more of the materials 5 In some embodiments , the methods described herein can and biomass components obtained using the methods be used to increase the solubility of a plant and /or herbal described herein , e.g., in the food industry ( e.g. , as additives , material. Alternatively or in addition , the methods described and supplements ) , in the healthcare industry , in the cosmetic herein can be used to reduce the toxicity of a plant and / or industry ( e.g. , in the compounding of cosmetics ), and /or in herbalmaterial withoutreducing the medicinal properties of agriculture . 10 the plant and /or herb . In some embodiments , the methods Minerals described herein are useful for isolating and /or purifying In some embodiments , the methods described herein can pharmaceutical compounds from plant material (which be used to obtain ( e.g., extract, isolate , and /or purify ) without being bound by theory, is possible due to the more minerals from biomass. Such minerals can be used , e.g. , efficient break down of cellulosic material) as the methods alone or in combination with one or more of the materials 15 disrupt, alter, modify , or restructure cellulose , e.g., present in and biomass components obtained using the methods the leaves of plant material. Desired compounds released described herein , e.g., in the food industry ( e.g., as additives , using the methods described herein can then be isolated and supplements ), in the healthcare industry, in the cosmetic from the undesired material, whereas less efficient methods industry ( e.g., in the compounding of cosmetics ), and /or in can not allow the release of the desired material from agriculture. undesired material. Inevitably, therefore, less efficient meth Ash 20 ods will result in the carry over of undesired material, which In some embodiments , the methods described herein can can lower the efficacy of the desired (e.g. , pharmaceutical be used to obtain ( e.g., extract, isolate , and/ or purify ) ash compound ) and / or be associated with potentially toxic side from biomass. Such ash can be used , e.g., alone or in effects . Themethods described herein can , therefore , be used combination with one or more of the materials and biomass to generate highly purified forms of potentially pharmaceu components obtained using the methods described herein , 25 tical compounds, e.g. , free of undesirable plant material , that e.g., in the food industry ( e.g., as an additive, supplement, are not obtainable using current practices. These highly and /or filler ). purified compounds can be more efficacious then less puri Pharmaceuticals fied forms of the same compounds . In some embodiments , Over 120 currently available pharmaceutical products are the increased efficacy attainable using themethods described plant - derived . As themethods described herein are useful for 30 herein can allow reduced dosing . In turn , this reduction in processing cellulolytic material, these methods can be useful the amount of material administered to a subject can reduce in the isolation , purification , and /or production of plant associated toxicity . Alternatively or in addition , the removal based pharmaceuticals . of surplus or undesirable plant material can help reduce or In some embodiments , the materials described herein can eliminate any toxicity associated with a plant based com have medicinal properties. For example , the methods pound that has not been processed using the methods described herein can result in the production of a material 35 described herein . with novel medicinal properties ( e.g. , not present in the Examples of plants and / or plant material that can be native material) . Alternatively or in addition , the methods usefully treated using the methods described herein include , described herein can result in the production of a material for example , sonication and irradiation can be combined in with increased medicinal properties ( e.g., a greater medici the pretreatment of willow bark to stimulate the isolation , nal property than that of the native material ). 40 purification , and /or production of salicin . Alternatively or in In some embodiments , the methods described herein can addition , the methods described herein can be used to be used to modify ( e.g. , increase , decrease, or maintain ) the process plant material comprising comfrey plants to facili solubility of a material, e.g., a material with medicinal tate the isolation , purification , and /or production of allan properties . Such a material can be more easily administered toin . Alternatively or in addition , the methods described and / or absorbed , e.g., by a human and / or animal than a 45 herein can be used to facilitate the isolation , purification , native material. and /or production benzoin . Alternatively or in addition , the In some embodiments , the methods described herein can methods described herein can be used to process plant be used to functionalize ( e.g. , alter the structure , expose a material comprising camphor basil to facilitate the isolation , reactive side chain , and /or modify the charge ) a material purification , and/ or production of camphor. Alternatively or with medicinal properties . Such materials can have, e.g., 50 in addition , the methods described herein can be used to altered reactivity , altered charge, and /or altered solubility . process plant material comprising plants in the genus Ephe In some embodiments , the methods described herein can dra to facilitate the isolation , purification , and /or production be used to modify the molecular structure of a material , e.g. , of ephedrine . Alternatively or in addition , the methods a material with medicinal properties . Such materials can described herein can be used to process plant material have altered ( e.g., increased or decreased ) average molecu comprising Duboisia myoporoides R. Br. ( Australian cork lar weights , average crystallinity , surface area , and /or poros 55 tree ) to facilitate the isolation , purification , and /or produc ity . Such materials can have , e.g. , altered reactivity , altered tion of atropine . In some embodiments , the atropine charge, altered solubility . obtained using the methods described herein will have an In some embodiments , the methods described herein can increased anticholinergic effect. Alternatively or in addition , be used as high efficiency processing methods , e.g., to obtain the methods described herein can be used to process plant plant- based pharmaceuticals from a cellulosic raw material 60 material comprising Mucuna deeringiana (velvet bean ) to such as plants . In some embodiments , the methods described facilitate the isolation , purification , and /or production of herein can be used to increase the pharmaceutical activity of L -dopa . In some embodiments , the L - dopa obtained using a plant -based pharmaceutical. For example , in some the methods described herein will have an increased anti embodiments , the methods described herein can be applied parkinsonian effect . Alternatively or in addition, the methods to plants and / or herbs with medicinal properties. For 65 described herein can be used to process plant material example , sonication can stimulate bioactivity and /or bio comprising Physostigma venenosum Balf. ( ordeal bean ) to availability of the medicinal components of plants and /or facilitate the isolation , purification , and /or production of US 10,508,291 B2 83 84 physostigmine . In some embodiments , the physostigmine that are capable of promoting a healthy lifestyle . In such obtained using the methods described herein will have an cases , the materials described herein can either enhance the increased anticholinesterase effect. Examples of other plant activity of the one or more nutritional supplements and /or based pharmaceuticals in which the methods described enhance the solubility and /or pharmacokinetics of the one or herein can be used to process plant material to facilitate the 5 more nutritional supplements. Exemplary nutritional supple isolation , purification , and / or production of include , but are ments that can be combined with the materials described not limited to , bromelain , chymopapain , cocaine, deserpi herein include, but are not limited to , for example , silica , dine , emetine, hyoscyamine , kawaina , monocrotaline, oua silicon , boron , potassium , iodine, beta - carotene, lycopene , bain , papain , pilocarpine, quinidine, quinine , rescinnami, insoluble fiber , monosaturated fatty acids , omega - 3 fatty reserpine, scopolamine , tubocurarine , vinblastine, yohim- 10 acid , flavonols , sulforaphane, phenols (e.g. , caffeic acid and bine , caprylic - acid , cineole , citric acid , codeine , cresol, ferulic acid ), plant stanols and sterols ( including esters guaiacol, lecithin , menthol, phenol pseudephedrine , sorbitol , thereof) , polyols (e.g. , sugar alcohols ) , prebiotics and pro and tartaric acid . biotics ( e.g., Lactobacilli and bifidobacteria ) , phytoestro In some embodiments , the methods described herein can gens ( e.g., isoflavones such as daidzein and genistein ), be used to process herbs, e.g. , medicinal herbs, including , 15 proanthocyanidins, soy protein , sulfides and thiols ( e.g. , but not limited to , basil , lemon grass , parsley, peppermint, dithiolthiones ), vitamins ( e.g., vitamin A , vitamin B1, vita and celery. Additional medicinalherbs thatcan be processed min B2, vitamin B3, vitamin Bs, vitamin B6, vitamin B7, using the methods described herein can be found at world vitamin B12 , vitamin C , vitamin D , vitamin E , vitamin K , wide web address altnature.com . including combinations thereof) minerals ( e.g., iron , cal An emerging technology is the production of pharmaceu cium ,magnesium , manganese, phosphorus, potassium , zinc, ticals in plants . Pharmaceutical produced using plants , 20 trace minerals , chromium , selenium , including combina which are commonly referred to as plant made pharmaceu tions thereof) , and folic acid . ticals (PMPs ) , include pharmaceutical compounds and vac Pharmaceutical Dosage Forms and Drug Delivery Com cines . Typically , PMPs are expressed in the leaves of the positions respective plants . Clearly , therefore , the methods described Drug substances are seldom administered alone , but herein can be useful for processing plant material compris- 25 rather as part of a formulation in combination with one or ing PMPs to facilitate the isolation , purification , and /or more non -medical agents that serve varied and often spe production of the PMPs. cialized pharmaceutical functions. Pharmaceutics is the sci Additional exemplary medicinal plants that can be treated ence of dosage form design , e.g., formulating a drug into a using the methods described herein can be found e.g., at dosage form suitable for administration to a subject. These world wide web address nps.gov/plants/MEDICINAL/ 30 non -medical agents , referred to as pharmaceutic or pharma plants.htm . ceutical ingredients , can be formulated to solubilize , sus In some embodiments , material that has been processed pend , thicken , dilute , emulsify , stabilize , preserve , color, using the methods described herein can be combined with a flavor, and fashion medicinal agents into efficacious and pharmaceutical excipient, e.g., for administration to a sub appealing dosage forms. Such dosage forms can be unique ject. Exemplary excipients that can be used include buffers in their physical and pharmaceutical characteristics . The ( for example , citrate buffer, phosphate buffer , acetate buffer , 35 drug and pharmaceutic ingredients will typically be com and bicarbonate buffer ), amino acids, urea, alcohols , ascor patible with each other to produce a drug product that is bic acid , phospholipids , polypeptides ( for example , serum stable , efficacious, attractive, easy to administer, and safe . albumin ), EDTA , sodium chloride, liposomes, mannitol, The product should be manufactured under appropriate sorbitol , water , and glycerol. Dosage forms can be formu measures of quality control and packaged in containers that lated to be suitable for any standard route of administration . 40 contribute to promote stability . Methods describing the For example , administration can be parenteral, intravenous, preparation of specific dosage forms are well known in the subcutaneous , or oral or any route of administration art and can be found in , for example , Ansel el al. , Pharma approved by the Federal Drug Administration ( see world ceutical Dosage Forms and Drug Delivery Systems, Seventh wide web address fda.gov/cder/dsm/DRG/drg00301.htm ) . Edition , Lippincott , Williams, & Wilkins and , “ Remington's Nutriceuticals and Nutraceuticals 45 Pharmaceutical Sciences, 18th Ed ., Gennaro , ed ., Mack Foods with a medical health benefit, including the pre Publishing Co., Easton , Pa ., 1990 . vention and /or treatment of disease , are referred to as In some embodiments , the pretreated materials described nutraceuticals or nutriceuticals . For example , nutraceuticals herein can be used as pharmaceutic ingredients e.g., inactive and nutriceuticals are naturally occurring or artificially gen ingredients . For example , the materials described herein can erated nutritional supplements capable of promoting a 50 be used , e.g., formulated , to solubilize, suspend , thicken , healthy lifestyle , for example , by reducing disease related dilute , emulsify , stabilize , preserve , color, flavor , and fash symptoms, reducing the incidence or severity of disease , and ion medicinal agents into efficacious, palatable , and appeal promoting long- term health . ing dosage forms. In such cases, the materials described In some embodiments , the methods described herein can herein can be mixed with a drug and /or conjugated to a drug be used to generate combinations of monosaccharides , such that the solubility , concentration , viscosity , emulsion disaccharides, oligosaccharides , and / or polysaccharides that 55 stability , shelf life , color, and flavor of the drug is increased are capable of promoting a healthy lifestyle . In some or decreased . embodiments, the methods described herein can be used to In some embodiments , the methods described herein can generate a material that is useful for promoting weight loss be used to modify ( e.g., increase , decrease, or maintain ) the in a human . For example , the material can have low nutrient solubility of a material. Such materials can be used to availability with low digestibility , e.g., a fibrous material. 60 facilitate the administration of a drug to a subject. For Such materials could be used as a dietary supplement, e.g., example , certain of the new pretreated materials are excep to suppress hunger and / or to promote satiety . Consuming tionally soluble in liquids , such as water, and can be used , such materials would allow a subject to avoid consuming when mixed with active ingredients to form a pharmaceu high nutrient availability and/ or highly digestible foods and tical composition , to allow the inert ingredients to be easily thus would facilitate weight loss in the individual. 65 dissolved in liquids . In some embodiments , the materials described herein can Alternatively or in addition , the materials described be supplemented with one or more nutritional supplements herein can be used to delay , control, or modify the release of