|HAO WAKATI AKITUS009771603B2 A MAN UNION MANAT (12 ) United States Patent ( 10 ) Patent No. : US 9 ,771 , 603 B2 Smart et al. (45 ) Date of Patent: Sep . 26 , 2017 ( 54 ) FERMENTATION PROCESS OTHER PUBLICATIONS ( 71 ) Applicant: LanzaTech New Zealand Limited , PCT Search Report (PCT /US2014 /056846 ) dated Dec . 29 , 2014 . Skokie , IL (US ) Abrini, J. Naveau , H . & Nyns, E . J ., Archives of Microbiology , ( 1994 ) , 161 , 345 - 351 . (72 ) Inventors : Kathleen Frances Smart , Chicago , IL Hensirisak et al ., Scale -up of microbubble dispersion generator for (US ) ; Alexander PaulMueller , aerobic fermentation , Applied Biochemistry and Biotechnology , Oct. 2002 , vol. 101 , No . 3 . Chicago , IL ( US ) ; Michael James Klasson K . T . et al. , Bioconversion of synthesis gas into liquid or Harry Mawdsley , Skokie , IL (US ) ; gaseous fuels , Enzyme and Microbial Technology , ( 1992 ) , 14 , Christophe Daniel Mihalcea , Skokie , 602 -608 . IL (US ) Klasson K . 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C12P 7 / 18 (2013 .01 ) ; C12Y 202 /01006 Bioeng ., ( 1989 ), 34 . 6 . 785 - 793. ( 2013. 01 ) ; YO2P 20 / 52 ( 2015 . 11 ) Vega , J . L . , et al ., Study of gaseous substrate fermentations : Carbon ( 58 ) Field of Classification Search monoxide conversion to acetate . 1 . Batch culture , Biotechnology None and Bioengineering , ( 1989 ) , 34 . 6 . 774 - 784 . See application file for complete search history . Arfin et al ., Purification and Properties of the Acetohydroxy Acid Isomeroreductase of Salmonella typhimurium . The Journal of Bio (56 ) References Cited medical Chemistry , 1969 , vol . 244 , No . 5 , pp . 1118 - 1127 . Aulabaugh et al ., Oxalyl Hydroxamates as Reaction - Intermediate U . S . PATENT DOCUMENTS Analogues for Ketol - Acid Reductoisomerase , Biochemistry 1990 , 29 , pp . 2824 -2830 . 5 , 173 , 429 A 12 / 1992 Gaddy et al . 5 , 593 ,886 A 1 / 1997 Gaddy 5 , 807 , 722 A 9 / 1998 Gaddy Primary Examiner — Paul Holland 5 , 821, 111 A 10 / 1998 Grady et al. ( 74) Attorney , Agent, or Firm — Frank S Molinaro 6 , 136 , 577 A 10 / 2000 Gaddy 6 , 340 , 581 B1 1 / 2002 Gaddy (57 ) ABSTRACT 6 , 368 , 819 B1 4 / 2002 Gaddy et al. 6, 753 ,170 B2 6 / 2004 Gaddy et al . A methods for altering the metabolite profile of a fermen 8 , 143 ,037 B2 3 / 2012 Zahn et al. tation , by increasing flux through acetolactate . The methods comprises increasing production of one or more products FOREIGN PATENT DOCUMENTS derived from acetolactate . Further provided is a method for increasing the production of 2 , 3 -butandiol by microbial EP 117309 AL 9 / 1984 WO W098 /00558 A1 1 / 1998 fermentation of gaseous substrates, the method comprising WO WO00 /68407 AL 11 / 2000 providing a compound which inhibits one or more enzymes WO W002 /08438 A2 1 /2002 which convert acetolactate to branched chain amino acids to WO WO2007 / 117157 Al 10 /2007 the fermentation . The present invention further provides WO WO2008 /028055 A2 3 / 2008 methods for increasing the production of 2 , 3 -butandiol WO WO2008 / 115080 A1 9 /2008 WO WO2009 /022925 A1 2 / 2009 relative to other fermentation products such as ethanol and WO WO2009 /064200 A2 5 / 2009 acetic acid . WO WO2012 / 131627 A1 10 /2012 W0 2013076144 A2 5 / 2013 13 Claims, 7 Drawing Sheets atent Sep . 26 , 2017 Sheet 1 of 7 US 9 ,771 , 603 B2 IAcetate Ethanol Butanediol - Lactic acide Biomass Ethanol/ BDO Ratio HIBA - - * EtOH,HACConcentration(g/L) . **** ratioBiomass),Eth/BDO(gLHLACBDO **** * ** * ** * We O *** * * ** mm ***** o for someo is life for enterta i ning your stages . O 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Time (days ) FIG . 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - HIBAconcentration(g/L) 0 .00 0 .00 1 . 00 2 .00 3 .00 4 . 00 Time after HIBA addition (days ) FIG . 2 atent Sep . 26 , 2017 Sheet 2 of 7 US 9 ,771 , 603 B2 w Acetate Ethanol Butanediol Lactic acid waren Biomass Ethanol/ BDO Ratio 25 , . A : 14 . * * ** * * * * ** * ** ** BDO,HLACBiomass(g/L) ELOH,HACConcentrationVermont 0200 . Eth/BDOratio * * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * versin 2 ... * * * * * * * * * * * ** * * * * ** * * * * * ** ** * * * ** * * * ** * * * O animaciones 16 17 18 19 20 21 22 23 24 25 26 27 28 Time (days ) FIG . 3 * Acetate w Ethanol w Butanediol * Lactic acid Biomass Ethanol /BDO Ratio 40 youare ? - ñ beone EtOH,HACConcentration(B/L) ó BDO,HLACBiomass(g/L)Ethratio .. to beim o wie A * * * * N " i meam * * * * * * * * * * ** * * * * o 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Time ( days ) FIG . 4 atent Sep . 26 , 2017 Sheet 3 of 7 US 9 ,771 , 603 B2 CO CO2 H2 7000 5000 3000 - 1000 - - CO/CO2Uptake(mmolLd) 1000 - ??o H2Uptake(mmol/Ld) - - 3000 . - - 5000 8 - 7000 ERRE - 9000 - 11000 16 17 18 19 20 Time21 (days 22 ) 23 24 25 26 27 FIG . 5 CO CO2 H2 8000 6000 4000 2000 CO/CO2Uptake(mmolLd) H2Uptake(mmol/Ld) - 2000 - 4000 - 6000 - 8000 +- - 10000 . YENE. - - - - - - - - - - - - - - - - - - I - - - 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Time (days ) FIG . 6 cocowatent wowSep . 26 , 2017 .Sheet. 4 of 7. .US .9 ,771 ., 603 . B2 - Acetate Ethanol Butanediol swipinas Lactic acid Biomass Ethanol/ BDO Ratio - % - % - % .:. Ethanol,AceticacidConc.(g/L) B . BDO,HLACBiomassConc.(g/L)Ethratio *** ** * + T - 8 - - h ?????????? ” oo Wono botercer cities. eest .. .. - 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 - Time ( d } FIG . 7 ** ?nawenye. 15147 Ethanol:2,3-BDOratio @ 15074 A 15093 . 15146 . o 0 0 .02 0 .04 0 . 06 0 .08 0 . 1 0 . 12 2 - HiBA concentration ( g / L ) FIG . 8 atent Sep . 26 , 2017 Sheet 5 of 7 US 9 ,771 , 603 B2 Acetate Ethanol W * Butanediol Biomass Ethanol BDO det *- Ethanol,AceticacidConc.Ig/L) Rritti BDO,BiomassConc.(OL) Eth/BDOratio 90 et wwwww . .. .. .. .. .. .. .. .. .. .. .. O 0 1 2 3 4 5 6 8 9 10 11 Time ( d FIG . 9 CO CO2 8000 6000 4000 watery 2000 MAKAN mmold]/LProduction(CO2Ço/ . p/1[ouwuomionpoldTH - 2000 -4000 -6000 . -8000 8 - 10000 - 12000 Super 0 1 2 3 4 5 6 7 8 9 10 11 Time ( d ] FIG . 10 wowU . S . Patent woSep . 26 , 2017- Sheet 6 of 7 US .9 ,771 , 603 . B2 Valine BCAAconcetration(mg/Lsample} Leucine Isoleucine Biomass(Bugunits X - Biomass år?nawatu www ouvid . , * * Y * * co OS0 . 5 1 1. 5 2 2. 5 Time (Hours FIGFIG .. 1111 Acetyl CoA - - - - Pyruvate Acetolactate Acetolactate synthase synthasease ( catabolic , AlsS ) (anabolic ) IlvBN 2 -Hydroxyisobutyric acid 3 ORF : 2389 , 2390 3 - isopropylmalate 3 dehydratase large 1 Acetolactate subunit hours 2 - isopropylmalate ORF : 1191 Ketol- acid syithase Acetolactate reductoisomerase ORF : 1190 decarboxylase IlyC ORF : 2387 ( BudA ) = 2 , 3 - dihydroxy - 3 2 - Oxoisovalerate Transaninasc isopropylmalatc 1 Acetoin methylbutanoatc Dihydrocyacid dehydratase dehydratasc ORF : 1193 2 ,3 -Butanediol ORF : 2388 dehydrogenasc 2 , 3 - Butanediol dehydrogenase Leucine ( 23Bdh ) (CSADH ) = = Valine and Isoleucine † 2 - 3 BDO Fig 1212 atent Sep . 26 , 2017 Sheet 7 of 7 US 9 ,771 , 603 B2 Acetate * Ethanol Biomass Butanediol . Ethanol/ BDO * WWWWW WWW www Ethanol,BiomassAceticacidConc.Ig/LI BDOConc.g/L), wwwwwwwwwwwwww Eth/BDOratio * wwwww wwww 1 2 13 Time [ d ] Fig . 13 Carbon distribution in metabolites 2 , 3 - BDO Ethanol Acetic acid Biomass Lactic acid Before HIBA addition 22 . 1 % 47 . 9 % 18 . 5 % 11 . 2 % 0 . 2 % After HIBA addition ARES41. 99 % 33 . 15 % 14 . 94 % 9 . 87 % 0 . 0 % Fig . 14 US 9 ,771 , 603 B2 FERMENTATION PROCESS where high temperatures are needed to generate desired steel qualities . For example, the steel industry in Australia is CROSS REFERENCE TO A RELATED reported to produce and release into the atmosphere over APPLICATION 500 ,000 tonnes of CO annually . Furthermore , CO is also a major component of syngas , This application claims priority from Provisional Appli where varying amounts of CO and H2 are generated by cation No . 61 /880 ,970 filed Sep . 22 , 2013 , the contents of gasification of a carbon - containing fuel . For example, syn which are hereby incorporated by reference . gas may be produced by cracking the organic biomass of waste woods and timber to generate precursors for the FIELD 10 production of fuels and more complex chemicals . The release of CO into the atmosphere may have signifi The present invention relates methods for altering the cant environmental impact.