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View U.S. Patent Application Publication No. US-2020-0017891 In 1111111111111111 IIIIII IIIII 1111111111 11111 11111 111111111111111 11111 111111111111111 11111111 US 20200017891Al c19) United States c12) Patent Application Publication c10) Pub. No.: US 2020/0017891 Al Donohue et al. (43) Pub. Date: Jan. 16, 2020 (54) MICROBIOMES AND METHODS FOR filed on Jul. 12, 2018, provisional application No. PRODUCING MEDIUM-CHAIN FATTY 62/696,677, filed on Jul. 11, 2018. ACIDS FROM ORGANIC SUBSTRATES Publication Classification (71) Applicant: WISCONSIN ALUMNI RESEARCH (51) Int. Cl. FOUNDATION, Madison, WI (US) C12P 7164 (2006.01) (52) U.S. Cl. (72) Inventors: Timothy James Donohue, Middleton, CPC ......... C12P 716409 (2013.01); Cl2P 2203/00 WI (US); Matthew Scarborough, (2013.01) Madison, WI (US); Daniel Noguera, Madison, WI (US) (57) ABSTRACT Microbiome compositions and uses thereof. The microbi­ (73) Assignee: WISCONSIN ALUMNI RESEARCH ome compositions include a set of microbes. The sets of FOUNDATION, Madison, WI (US) microbes contain members of Lactobacillaceae, Eubacteri­ aceae, Lachnospiraceae, and Coriobacteriaceae. The number of individual physical microbes in the set constitutes a (21) Appl. No.: 16/433,639 certain percentage of the total number of individual physical microbes in the microbiome composition. The microbiome (22) Filed: Jun. 6, 2019 compositions can be used for producing medium-chain fatty acids from organic substrates through anaerobic fermenta­ Related U.S. Application Data tion in a medium. The medium can include lignocellulosic (60) Provisional application No. 62/846,378, filed on May stillage. 10, 2019, provisional application No. 62/697,249, Specification includes a Sequence Listing. Dwra1ion (d) 26% Relative- Abundance 1% Patent Application Publication Jan. 16, 2020 Sheet 1 of 16 US 2020/0017891 Al m N u u W"> <.ri :r: 0.. q w '.l: a.. 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I) denovo107219 ), 0 Bl Ato{Cbium unclitured bac',eri~n (EU773553) z"i ---- 0/seoola profusa DSM139a9(NR116939.1) 0 liJ Al(lXJbiumvaginaeDSM15829(NR 117757.1) ()► "i Oi5€1]8//ager,~ C1 l,/-,'1278623) m ] denovo9132 ► Atw,:l)ium rinaeJCM10299 (NR 113CJ8.1) - Atop?bium parvuk.Jm DSM2D400 (NR 102936.1) 0.07 FIG. 4 Patent Application Publication Jan. 16, 2020 Sheet 8 of 16 US 2020/0017891 Al 25 !Ill• Xylose + Lactic Acid _ 20 s:E c: 15 0 -:0: ffi 10 4) c; 8 5 0 0 5 10 15 20 25 30 35 40 Time (hr) FIG. 5 ""O ~..... ('D =..... t "e.... ,:~,rum ~I (')- ~..... ,o;,i;),(){)O "'?'' t Xy.lose ITT Uncharartedzed Carbohydrf)tes r: Other Characterized COD II Uncharacterized COD .... 0= MT ""O -~ 35,0C·O .l = MG O".... ~ ~~ I MG (')- 0 JC,J}OO ·i- ~ U j ......... 1 0 1 MG ·~r l.SJ}-00.... + = ~ i MG Q·.. 20,t>OO ' ;' ?.... ~a-­ N 0 N 0 rJ'1 ('D=­ ('D..... 1,0 0 .... CR 12 1.8 M ~ ~ G Q ~ 00 ~ n 78 84 W % 102 108 114 120 a-- Days of Reactor Operation----------------------------- c rJ'1 N 0 N 0 FIG. 6A ---0 0.... -....J QO 1,0.... >.... ""O ~..... ('D =..... 2.S,000 .,,,, t "e.... i Acetic: Add (C2) I@ Butyric Add (C4) li Hexanoic Acid {C61 11 Oi:tanoic Add {CS) (')- ~......... MG 0 _ 10,000 tI MT MG = =""O O".... (')- MG ;; re MG ,: I ~G ~..... t,,o,JE ,, ~,,,,,,,,,,,, ,,,,,,: ,,,,,,, ,,,,,,, i i I Ir .... I 0 = -·C ~ 0 .... i -~ ;' ... ? C .... ~ C ~a-­ N 8 0 5,000 N 0 rJJ ('D=­ ('D ......... CR 12: 18 24 30 36 42 48 54 60 &6 72 78 84 90 96 102 108 .114 120 0 0 .... Days. of Reactor Operation---------------------------~ a-- c rJJ N 0 N 0 ---0 FIG. 68 0.... -....J QO 1,0.... >.... Patent Application Publication Jan. 16, 2020 Sheet 11 of 16 US 2020/0017891 Al ·tf') V li 5w ff&l~§&~J.z.~111'Jjj... ,.,.,., .. ,.. , ... ,.. ,.. ,.,.,.,.z.,.,.. ,.. ,.. ,.,.,.,.z.,.,.':f.«, .. ,., .. ,.z.,.,.z., .. ,.;~l ... \ "'w· ·~ :5 ikl f'l'l 5 b N .~ .,,.J !Hl ....-1 I;_) 5 (.) a (0 M ■ t;t:; C) 8 LL N N t;t:; 0 t... .J k:; ~ ix: 0 V ,::,:) Cl ,ey- .-I ~ :, C w 0 ·.p {J Its,_ V Q. 0,_ 0 ,I..) V I I ro I JN Q.1 ill .... 0::: 0 -<I) >.. I aro }:•. •.❖'.❖.❖.❖'.❖.❖.•'.❖'.❖'.❖ ~-: '.•'.❖'.❖'.❖'.❖'.❖'.❖'.❖'.❖'.❖'.~'.•.❖'.❖'.❖'.❖'.❖'.❖'.❖'.❖'.❖'.: ~'.•.❖.❖'.❖'.❖.•'.❖'.❖'.❖'.❖'.❖ .•:½:•.•.❖'.❖'.❖'.❖'.❖'.❖'.❖'.❖'.❖ .i::•.•.❖'.❖'.❖'.❖'.❖'.❖'.❖'.❖'. >-~.. Patent Application Publication Jan. 16, 2020 Sheet 12 of 16 US 2020/0017891 Al 70% ■ Relative Abundance I Relative Expression 60% 50% 40% 30% 20% 10% 0% . -rl M N m ~ 0:::: 0:::: 0:::: u 8 0 0 0 <:( ....I u u u ....I FIG. 7 Patent Application Publication Jan. 16, 2020 Sheet 13 of 16 US 2020/0017891 Al LaciobaciJJus rennin! 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