Pr0ce55 parameter5 and env1r0nmenta1 fact0r5 affect1n9 D-xy105e fermentat10n 6y yea5t5

J. C. du Pree2

Department 0f M1cr0610109y and 810chem15try, Un1ver51ty 0f the 0ran9e Free 5tate, 810emf0nte1n, 50uth Afr1ca

Certa1n env1r0nmenta1 fact0r5 are 0f part1cu1ar 519n1f1cance 1n the fermentat10n 0f the hem1ce11u105e c0mp0nent 0f 119n0ce11u1051c 610ma55 6y yea5t5; the5e are d15cu55ed 1n re1at10n t0 pr0ce55 c0n51derat10n5 w1th a c0mparat1ve eva1uat10n 0f the re1evant fermentat10n parameter5. 7he c0mmerc1a1 exp101tat10n 0f the pent05e-ferment1n9 yea5t5 f0r ethan01pr0duct10n fr0m D-xy105e 15 re5tr1cted ma1n1y 6y the1r 10w ethan01 t01erance and 510w rate 0f fermentat10n, a5 we11 a5 6y the d1ff1cu1ty 1n c0ntr0111n9 the rate 0f 0xy9en 5upp1y at the 0pt1ma1 1eve1. Apart fr0m the5e 1ntr1n51c phy5101091ca1 c0n5tra1nt5, the u5e 0f 119n0ce11u1051c hydr01y2ate5 a5 5u65trate pre5ent5 add1t10na1 065tac1e5 t0 an eff1c1ent fermentat10n 6ecau5e 0f the pre5ence 0f 1nh161t0r5, e5pec1a11y acet1c ac1d, and 6ecau5e the fermentat10n 0f 5u9ar m1xture5 1ntr0duce5 further d1ff1cu1t1e5. Recent deve10pment5 1n the c0n5truct10n 0f rec0m61nant m1cr00r9an- 15m5 0ffer 900d pr05pect5 f0r the c0mmerc1a1 pr0duct10n 0f ethan01 fr0m renewa61e re50urce5.

Keyw0rd5: Hem1ce11u105e hydr01y2ate; 119n0ce11u105e; D-xy105e; pent05e 5u9ar5; ethan01; xy11t01; fermentat10n; ethan01; yea5t; Cand1da 5hehatae; P1ch1a 5t1p1t15; k1net1c parameter5

1ntr0duct10n

L19n0ce11u1051c 610ma55, c0mpr151n9 ce11u105e, hem1ce11u- 105e, and 119n1n, 15 0ne 0f the m05t a6undant renewa61e re50urce5 and may 6e c0n51dered a p0tent1a1 feed5t0ck f0r the pr0duct10n 0f ethan01 6y m1cr061a1 fermentat10n. Hem1- ce11u105e can c0n5t1tute up t0 35% 0f hardw00d 5pec1e5 and 0ther w00dy an9105perm5 6y dry we19ht, w1th the a1d0pent05e D-xy105e a5 the maj0r c0n5t1tuent 0fthe xy1an, am0unt1n9 up t0 25 % 0f the dry 610ma55.1,2 F0r the c0mmerc1a1 pr0duct10n 0f ethan01 fr0m 119n0ce11u1051c 610ma55, the fermentat10n 0f D-xy105e 15, theref0re, 0f cruc1a1 ec0n0m1c 1mp0rtance. H0wever, the c0mmerc1a1 exp101tat10n 0f the re1at1ve1y few 5pec1e5 0f pent05e-ferment1n9 yea5t5 f0r ethan01 pr0duc- t10n 15 c0n5tra1ned 6y the1r 510w rate5 0f fermentat10n, 10w ethan01 t01erance, p00r ethan01 y1e1d5 due t0 xy11t01 pr0duc- t10n, and the d1ff1cu1ty 1n c0ntr0111n9 certa1n env1r0nmenta1 parameter5 t0 0pt1m12e the fermentat10n.

A1th0u9h a num6er 0f exce11ent 9enera1 rev1ew5 0n pen- t05e fermentat10n have appeared 1n recent year5,3-7the

Addre55 repr1nt re4ue5t5 t0 Dr. du Pree2 at the Department 0f M1cr061- 0109y and 810chem15try, Un1ver51ty 0f the 0.F.5., P .0 .80x 339, 810em- f0nte1n 9300, 50uth Afr1ca 7h15 0verv1ew 15 6a5ed 0n a paper pre5ented 6y the auth0r 1n the 5e5510n 0n D-xy105e fermentat10n 6y yea5t5 dur1n9 the E19hth 1nternat10na1 5ymp0- 51um 0n Yea5t5 he1d 1n At1anta, 6e0r91a, Au9u5t 23-28, 1992 Rece1ved 14 5eptem6er 1993; accepted 29 March 1994

f0cu5 0f th15 rev1ew 15 0n the env1r0nmenta1 fact0r5 affect1n9 xy105e fermentat10n, a5 we11 a5 0n the pract1ca1 pr061em5 enc0untered 1n the fermentat10n 0f 119n0ce11u1051c hydr0- 1y2ate5. Furtherm0re, 1n th15 rev1ew the empha51515 ma1n1y 0n P1ch1a 5t1p1t15 and Cand1da 5hehatae, wh1ch are the m05t 5ucce55fu1 xy105e-ferment1n9 yea5t 5pec1e5 de5cr16ed t0 date, and c0mparat1ve ta6u1at10n5 0f the re1evant fermen- tat10n parameter5 are 91ven. 70 fac111tate c0mpar150n 0f the var10u5 yea5t 5tra1n5, the C85 cu1ture c011ect10n num6er5 are u5ed where app11ca61e (5ee Pr10r et a1. 3 f0r the e4u1va- 1ent cu1ture c011ect10n num6er5).

Env1r0nmenta1 fact0r5 affect1n9 xy105e fermentat10n

Nutr1t10na1 fact0r5, pH, and temperature

7he med1um c0mp051t10n and cu1ture c0nd1t10n5 exert a pr0f0und effect 0n D-xy105e fermentat10n5.7he fermenta- t10n 0f D-xy105e 6y C. 5hehatae 1n the a65ence 0f 610t1n and th1am1ne wa5 ne9119161e, 8 and Pachy501en tann0ph11u5 a150 re4u1red 610t1n and th1am1ne f0r9r0wth.9 p. 5t1p1t15 had n0

9 10 a6501ute v1tam1n re4u1rement,, a1th0u9h 610t1n and th1- am1ne dramat1ca11y 1ncrea5ed 60th ethan01 y1e1d and pr0duct1v1ty. 1° 8y c0ntra5t, 0ther5 f0und that 5upp1emen- tat10n 0f P. 5t1p1t15 C85 5773 w1th 610t1n and th1am1ne d1d n0t 1ncrea5e ethan01 y1e1d 0r pr0duct1v1ty. 11 1t ha5 6een rep0rted that m0re ethan01 accumu1ate51n a r1ch than 1n a def1ned med1um 12J3 and that yea5t extract 1ncrea5ed the

944 En2yme M1cr06.7echn01., 1994, v01.16, N0vem6er • 1994 8utterw0rth-He1nemann

Env1r0nmenta1 fact0r5 affect1n9 0-xy105e fermentat10n: J. C. du Pree2

9r0wth rate 0fP. 5t1p1t15. 9 0r9an1c n1tr09en 50urce5 5uch a5 urea 1mpr0ved the ethan01 y1e1d 06ta1ned w1th C. 5hehatae 1n c0mpar150n w1th 1n0r9an1c n1tr09en 50urce5, wherea5 0f the 1n0r9an1c n1tr09en 50urce5, amm0n1um 5u1fate 9ave the h19he5t ethan01 y1e1d and the h19he5t ethan01:xy11t01 rat10.14 7he add1t10n 0f re1at1ve1y 5ma11 am0unt5 0f D-91uc05e en- hanced ethan01 pr0duct10n fr0m D-xy105e 6y Pachy501en tann0ph11u5,15 5u99e5t1n9 that the pr0p0rt10n 0f pyruvate meta60112ed 6y the ethan01 pathway wa5 1ncrea5ed 6y 91u- c05e meta60115m. 4

W1th 60th C. 5hehatae C85 2779 andP. 5t1p1t15 C85 7126, the ethan01 y1e1d wa5 hard1y affected 6y pH va1ue5 6etween pH 2.5 and 6.5, wherea5 the fermentat10n rate wa5 0pt1ma1 6etween pH 4 and 5.5.16 A 6r0ader 0pt1ma1 pH ran9e f0r 9r0wth and f1na1 ethan01 c0ncentrat10n ha5 6een rep0rted f0r P. 5t1p1t15 C85 5773.17 0ther 0pt1ma1 va1ue5 a150 have 6een rep0rted, 13 6ut the5e d15crepanc1e5 may 6e due t0 d1fference5 1n exper1menta1 c0nd1t10n5 5uch a5 the u5e 0f med1a w1th0ut pH c0ntr01.

7he max1mum rate5 0f 9r0wth and xy105e fermentat10n w1th C. 5hehatae C85 2779 andP. 5t1p1t15 C85 7126 0ccurred at 30°C, w1th a 5harp decrea5e 1n the rate and y1e1d 0f eth- an01 pr0duct10n 6ut w1th an 1ncrea5ed xy11t01 y1e1d at h19her temperature5.16 8y c0ntra5t, the am0unt 0fxy11t01 pr0duced 6y P. tann0ph11u5 decrea5ed dramat1ca11y w1th n0 519n1f1cant chan9e 1n ethan01 pr0duct10n when the temperature wa5 1ncrea5ed fr0m 30 ° t0 37°C.18 1n 9en- era1, the 0pt1mum temperature f0r ethan01 pr0duct1v1ty 6y the5e yea5t 5pec1e515 1n the re910n 0f 30 ° t0 32°C, 5 6ut the 0pt1mum pr0ce55 temperature may depend 0n the 1nd1v1dua15tra1n, 5u65trate (xy105e 0r 91uc05e), and 5u9ar c0ncentrat10n. 17 7he dependence 0f the ethan01 11m1t perm1tt1n9 9r0wth 0n the 1ncu6at10n temperature ha5 6een dem0n5trated: w1th C. 5hehatae C85 2779 and P. 5t1p1t15 C85 7126, the max1mum ethan01 t01erance wa5 f0und 1n the re910n 0f 11 ° t0 22°C, w1th a 5harp decrea5e 1 19 •n ethan01 t01erance at h19her temperature5.

ce11 den51t1e5 reached at h19her 5u9ar 1eve15 rather than 6e1n9 a d1rect re5u1t 0f the xy105e c0ncentrat10n.16 A1terna- t1ve1y, the h19her ethan01 c0ncentrat10n c0u1d a150 re5u1t 1n 1ncrea5ed xy11t01 pr0duct10n. 7he xy11t01 y1e1d 0f P. 5t1p1t15 wa5 e5pec1a11y 5en51t1ve t0 the ethan01 c0ncentrat10n under anaer061c c0nd1t10n5, w1th a 11near 1ncrea5e 1n the xy11t01 y1e1d and a c0nc0m1tant decrea5e 1n the ethan01 y1e1d a5 the 1n1t1a1 ethan01 c0ncentrat10n wa51ncrea5ed. 24

Exper1menta1 data 5u99e5t that ethan01 t01erance d1ffer5 w1de1y 6etween 5tra1n5 and 15 a150 determ1ned 6y the cu1- ture c0nd1t10n5, e5pec1a11y 6y the temperature. 191n c0ntra5t t0 5acchar0myce5 cerev151ae, where aut09en0u51y pr0duced ethan0115 apparent1y m0re t0x1c than externa11y added eth- an01, n0 9reat d1fference 1n t0x1c1ty wa5 f0und w1th C. 5he- hatae C85 2779 and P. 5t1p1t15 C85 7126.19,25 W1th the5e yea5t5, 9r0wth cea5ed at ethan01 c0ncentrat10n5 0f 32 t0 35 91 - 1, wherea5 ethan01 pr0duct10n c0nt1nued up t0 44 t0 47 9 1-1. 25 7he max1mum ethan01 c0ncentrat10n a110w1n9 9r0wth 0fP. 5t1p1t6" C85 5773 at 25°C wa5 64 9 1-1.20 7he max1mum ethan01 c0ncentrat10n5 pr0duced rep0rted 6y 0ther5 ran9e fr0m 30 91 - 1 t0 57 91 - 1 f0r P. 5t1p1t1521,26 and w1th C. 5hehatae fr0m 25 91 - 1,26 t0 an a5t0und1n9 100 91 - 1 1n a wh01e 6ar1ey hydr01y2ate c0nta1n1n9 91uc05e and xy105e 1n a 70:30 rat10 w1th a 5tra1n der1ved fr0m C. 5hehatae A7CC 22984 adapted 6y recyc11n9 1n the hydr01y2ate. 27,28 1n 50me ca5e5, ethan01 t01erance depended 0n the nature 0f the car60n 50urce: P. tann0ph11u5 and a 5tra1n 0f C1av15p0ra pr0duced 519n1f1cant1y h19her c0ncentrat10n5 0f ethan01 fr0m 91uc05e than fr0m xy105e. 15,29 8y c0ntra5t, the ethan01 c0ncentrat10n511m1t1n9 9r0wth and fermentat10n 6y C. 5he- hatae C51R-Y981 and P. 5t1p1t15 C85 7126 were 51m11ar 1rre5pect1ve 0f whether 91uc05e 0r xy105e 5erved a5 car60n 50urce. 251t 5eem5 un11ke1y, theref0re, that the except10na11y h19h ethan01 c0ncentrat10n5 06ta1ned w1th the a60ve adapted 5tra1n der1ved fr0m C. 5hehatae A7CC 22984 c0u1d 6e due t0 the pre5ence 0f a 1ar9e am0unt 0f D-91uc05e 1n the hydr01y2ate.

Ethan01 and 5u65trate c0ncentrat10n

1n m05t 1n5tance5 the h19he5t ethan01 c0ncentrat10n5 have 6een rec0rded at 0-xy105e c0ncentrat10n5 0f 100 91 - 1 and 9reater, wherea5 the h19he5t y1e1d5 and 5pec1f1c rate5 0f ethan01 pr0duct10n have 6een f0und at c0n51dera61y 10wer xy105e c0ncentrat10n5. 3 7he xy105e c0ncentrat10n 6ecame 1nh161t0ry t0 the 9r0wth 0f P. 5t1p1t15 C85 5773 a60ve 40 9 1-1,20 wherea5 the ethan01 y1e1d 0f th15 5tra1n 0fP. 5t1p1t15, 6ut n0t C. 5hehatae A7CC 22984, rema1ned 5ta61e at a va1ue 0f a60ut 0.44, w1th n0 xy11t01 accumu1at10n up t0 an 1n1t1a1 xy105e c0ncentrat10n 0f 110 91 - 1.21 51m11ar1y, 0ther5 f0und that the v01umetr1c ethan01 pr0duct1v1ty 0f th15 P. 5t1p1t15 5tra1n wa51nh161ted at 1n1t1a1 xy105e c0ncentrat10n5 0f 6etween 76 and 99 9 1-1, wherea5 the ethan01 y1e1d wa5 affected 0n1y a60ve 145 9 1 - 1.22

A tendency f0r xy11t01 pr0duct10n t0 1ncrea5e w1th an 16 1ncrea5e 1n the x~105e c0ncentrat10n ha5 6een n0ted, even

w1th P. 5t1p1t15, 23 wh1ch u5ua11y pr0duce5 n0 detecta61e am0unt5 0f xy11t01 under 5em1aer061c c0nd1t10n5.7h15 c0r- re1at10n c0u1d, h0wever, 6e a c0n5e4uence 0f m0re 5evere 0xY9en-11m1ted 9r0wth c0nd1t10n5 a5 a re5u1t 0f the h19her

0xY9en 11m1tat10n

7he 0xY9en 5upp1y 15 0ne 0f the m05t 1mp0rtant env1r0n- menta1 fact0r5 1n xY105e fermentat10n 6y yea5t5, affect1n9 60th the rate and the y1e1d 0f ethan01 pr0duct10n a5 we11 a5 xY11t01 accumu1at10n dramat1ca11y. 7he aerat10n rate deter- m1ne5 the part1t10n1n9 0f the f1ux 0f car60n fr0m 0-xy105e 6etween 9r0wth and pr0duct f0rmat10n.4 Xy105e fermenta- t10n 15 9reat1y enhanced 6y a 11m1ted 0xY9en 5upp1y, pr06a- 61y due t0 the r01e 0f 0xy9en a5 term1na1 e1ectr0n aceept0r wh1ch 15 re4u1red t0 re11eve the part1a1 red0x 1m6a1ance 1n the 1n1t1a1 tw0 5tep5 0f 0-xy105e meta60115m. 3° At decrea5- 1n9 5pec1f1c 0xY9en uptake rate5, the e1ectr0n tran5fer 5y5- tem 15 una61e t0 re0x1d12e a11 0f the pr0duced NADH2 6y re5p1rat10n and/0r fermentat10n; a5 a c0n5e4uence the 1n- trace11u1ar NADH2 1eve1 1ncrea5e5, reduc1n9 the react10n rate 0f NAD-xy11t01 dehydr09ena5e, w1th the end re5u1t that xy11t01 accumu1ate5. 31,32 0xY9en 15 a150 re4u1red f0r 9r0wth 3° and 5eem5 t0 6e nece55ary f0r m1t0ch0ndr1a1 funct10n 33 and t0 9enerate ener9y f0r xy105e tran5p0rt. 34

7h1515 a150 the parameter that 15 the m05t d1ff1cu1t t0 4uan- t1fy and c0ntr01 dur1n9 a xy105e fermentat10n. C0n5e4uent1y, a 1ar9e num6er 0f paper5 have recent1y addre55ed th15 a5pect

En2yme M1cr06.7echn01., 1994, v01.16, N0vem6er 945

Rev1ew5

fr0m a phy5101091ca1 a5 we11 a5 a pr0ce55 v1ewp01nt. 0n1y the 1atter a5pect 15 further d15cu55ed 1n th15 rev1ew.

Under fu11y aer061c c0nd1t10n5, u5uaUy n0 ethan01 0r xy11t01 accumu1ate5,12,3°,35-4° a1th0u9h 1n 0ne 1n5tance a 5ma11 am0unt 0f ethan01 wa5 pr0duced under aer061c c0n- d1t10n5 6y C. 5hehatae A7CC 22984 and 1t wa5 5pecu1ated that th15 m19ht have 6een due t0 a nutr1t10na1 def1c1ency affect1n9 the aer061c meta60115m. 41 Apart fr0m 0xY9en 11m- 1tat10n, 1t 15 5u99e5ted that ethan01 accumu1at10n can a150 6e a550c1ated w1th a 11m1tat10n 6y 0ther nutr1ent5. 4

6r0wth and ethan01 pr0duct10n 0n xy105e are 5evere1y re5tr1cted 6y anaer061c c0nd1t10n5, and a11 yea5t5 1nve5t1- 9ated t0 date e1ther fa11 t0 9r0w 0r are capa61e 0f 0n1y a60ut 0ne d0u611n9 0f ce11 ma55. 2°,3°,38,42 7he 610chem1ca1 6a515 f0r the 1na6111ty 0f the5e yea5t5 t0 9r0w anaer061ca11y 0n D-xy105e 15, a5 yet, unkn0wn. 4 1n 9enera1, a5 the extent 0f 0xy9en 11m1tat10n 1ncrea5e5, the max1mum c0ncentrat10n 0f ethan01 1ncrea5e5 w1th a c0nc0m1tant decrea5e 1n the 9r0wth rate and ce11 y1e1d 6ut w1th an 1ncrea5e 1n the xY11t01 y1e1d. 23,43,44 1f the 0xy9en 11m1tat10n 15 t00 5evere, h0wever, the ethan01 pr0duct1v1ty and y1e1d may decrea5e, 50 that an 0pt1ma1 rate 0f 0xY9en 5upp1y ex15t5 where ethan01 pr0duc- t10n 15 max1m12ed. 11,45 Wherea5 xY11t0115 a 6y-pr0duct c0m- m0n1y a550c1ated w1th ethan01 pr0duct10n 6y C. 5hehatae and P. tann0ph11u5, P. 5t1p1t15 u5ua11y 0n1y pr0duce5 xY11t01 under c0nd1t10n5 0f anaer0610515 0r 5evere 0xY9en 11m1tat10n. 25,44,45 6r0wth and ethan01 y1e1d5 are 1nver5e1y re1ated, and 1t 15, theref0re, n0t p055161e t0 f1nd a rate 0f 0xY9en 5upp1y where 60th ce11 and ethan01 pr0duct10n are max1ma1.11,23 7he5e re1at10n5h1p5 a5 a funct10n 0f the rate 0f 0xY9en 5upp1y are we11111u5trated 6y F19ure 1.

7he ma1n pr061em 15 that the 0pt1ma1 d15501ved 0xY9en ten510n (D07) f0r ethan01 pr0duct10n fr0m D-xy105e 15 50 10w a5 t0 6e at the 5en51t1v1ty 11m1t 0f the d15501ved 0xY9en e1ectr0de5 5u1ta61e f0r u5e 1n m1cr061a1 fermentat10n5.7he 0pt1ma1 D 0 7 va1ue5 f0r ethan01 pr0duct10n 6y C. 5hehatae

7a61e 1 Fermentat10n parameter5 f0r ethan01 pr0duct10n fr0m D-xy105e

0.30 1

0241

0 1 8 r.t] E-, .<

-~ 0.12 r,,.

921

m 0 . 0 6

0.00

F19ure 1

0.5

0.4

0.1

0.0 0 15 30 45 60 75

0XY6EN 7RAN5FER C0EFF1C1EN7 [h-•]

Y1e1d c0eff1c1ent5 0n D-xy105e f0r ce11 ma55 (11), ethan01

1 0.3 =~

¢/}

0.2

(0), and xy11t01 (•) pr0duct10n and max1ma15pec1f1c rate5 0f 9r0wth ([]) and 0f ethan01 pr0duct10n (0)1n 6atch pr0ce55e5 w1th c0mputer- c0ntr011ed c0n5tant 0xY9en ma55 tran5fer c0eff1c1ent5 (kLa). (Re- drawn fr0m De11we9 et aL 47)

and P. 5t1p1t15 are apparent1y we11 6e10w 1% 0f 5aturat10n, and the yea5t5 can d1ffer 1n the1r 5en51t1v1ty t0ward5 the D07 . 25,46 P. 5t1p1t15 pr0duced n0 ethan01 a60ve 3 1xm01 0xY9en 1 - ] (e4u1va1ent t0 a D 0 7 0f appr0x1mate1y 1%). 23 7he 5aturat10n c0n5tant f0r 0xY9en (k0) 1n term5 0f the 9r0wth 0f P. 5t1p1t15 C85 5773 wa5 determ1ned t0 6e 1n the ran9e 0f 0.1 t0 0.2 m91 - 1,20 wh1ch 15 e4u1va1ent t0 a D 0 7 0f a60ut 1% t0 2% 0f 5aturat10n.

50me attempt5 t0 4uant1fy the 0xy9en re4u1rement 1n term5 0f the 0xy9en tran5fer rate (07R) 9ave 900d re5u1t5 (7a61e 1). 7h15 appr0ach 15, h0wever, pr06a61y 0n1y

07R 402 Xy105e Ethan01 Qp,max 4p,max D07 (mm01 (mm01

Yea5t (9 1-1) (9 1-1) Yp/5 Yx/5 Yxy1 (9 1-1 h-1) (9 9-1 h-1) (%) 1-1 h-1) 9-1 h-1) Ref.

P, 5t1p1t15 C85 6054 50 n5 0.48 n5 0 n5 0.20 0 < 1 n5 137 C85 5773 50 - 2 1 . 5 0.43 0.12 0 n5 n5 - 0 1.75 n5 44 C85 5773 15 a n5 - 0 . 4 n5 0 n5 0.24 0 0.7 - 0 . 3 45 C85 5773 50 n5 0.34 0.16 n5 0.50 0.41 n5 5 n5 11 C85 5773 50 -- 18 0.39 n5 0 n5 0.12 < 1 3.75 n5 23 C85 7126 50 21.5 0.43 0.12 0 0.86 0.30 n5 n5 n5 16 C85 5773 200 57 0.39 n5 0.02 n5 n5 n5 n5 n5 26

C. 5hehatae A7CC 22984 50 - 19.5 0.39 0.09 0.02 n5 n5 - 0 3.9 n5 44 C85 4705 50 24 0.45 n5 0.02 n5 n5 n5 n5 n5 26 C85 2779 50 18.5 0.37 0.09 0.08 0.96 0.48 n5 n5 n5 16 C85 2779 1296 44 0.34 0.07 0.18 0.99 0.34 0.7 n5 n5 25

P. tann0ph11u5 NNRL Y-2460 50 16 0.32 n5 0.14 n5 n5 n5 n5 n5 26

Yp/5, Ethan01 y1e1d, 9 9 - 1 5u9ar a551m11ated; Yx/5, ce11 y1e1d, 9 9 - 1 5u9ar a551m11ated; Yxy1, xy11t01 y1e1d, 9 9 - 1 5u9ar a551m11ated; 0p,max, max1mum v01umetr1c ethan01 pr0duct1v1ty; 4p,max, max1mum 5pec1f1c ethan01 pr0duct1v1ty; D07, d15501ved 0xy9en ten510n; 07R, 0xY9en tran5fer rate; 402, 5pec1f1c rate 0f 0xY9en uptake aP1u5 4 9 1-1 D-91uc05e 6Fed-6atch cu1t1vat10n w1th 50% (w/v) 0-xy105e feed n5, N0t 5tated

946 E n 2 y m e M 1 c r 0 6 . 7 e c h n 0 1 . , 1994, v 0 1 . 1 6 , N 0 v e m 6 e r

Env1r0nmenta1 fact0r5 affect1n9 0-xy105e fermentat10n: J. C. du Pree2

app11ca61e t0 a 91ven fermentat10n c0nd1t10n 6ecau5e the 0 7 R 1nd1cate5 the rate 0f 0xY9en 5upp1y t0 the cu1ture a5 a wh01e, wherea5 the 0xy9en ava11a6111ty exper1enced 6y the ce115 15 a150 determ1ned 6y the ce11 c0ncentrat10n. 7here- f0re, the D 0 7 and the 5pec1f1c rate 0f 0xy9en uptake 6y the ce115 are m0re mean1n9fu1 fermentat10n parameter5. R1221 et a1. 23 deemed the 5pec1f1c 0xy9en uptake rate a5 the m05t 1mp0rtant var1a61e affect1n9 the y1e1d5 and rate5 0f the5e fermentat10n5, and pr0p05ed that xY105e fermentat10n5 6e perf0rmed at a c0n5tant 5pec1f1c 0xY9en uptake rate t0 0p- t1m12e ethan01 pr0duct10n. 7h15 wa5 ach1eved 6y u51n9 c0m- puter12ed c0ntr01 6a5ed 0n the 0 7 R t09ether w1th ph0t0- metr1c 0n-11ne 610ma55 determ1nat10n a5 c0ntr01 parameter t0 ma1nta1n a c0n5tant 5pec1f1c rate 0f 0xY9en uptake, re- 5u1t1n9 1n a 519n1f1cant1y 1ncrea5ed ethan01 y1e1d 0f 0.44 9 9-1 at a 5pec1f1c 0xY9en uptake rate 0f0.3 mm019-1 h - 1.47

Xy11t01 pr0duct10n A1th0u9h xy11t0115 an unwanted 6y-pr0duct 1n ethan01 fer- mentat10n5, u5ua11y pr0duced at the c05t 0f the ethan01 y1e1d, 50me 1nve5t19at10n5 attempted t0 max1m12e xy11t01 pr0duct10n a5 the end pr0duct. 7h15 p01ya1c0h01 ha5 p0ten- t1a1 u5e a5 a natura1 f00d 5weetener, e5pec1a11y a5 a 5u9ar 5u65t1tute f0r d1a6et1c5, and 1t5 u5e reduce5 denta1 car1e5. ~ Xy11t0115 c0mmerc1a11y pr0duced 6y the chem1ca1 reduct10n 0f xy105e der1ved fr0m hem1ce11u1051c hydr01y2ate5. 49 7he exten51ve pur1f1cat10n 5tep5 re4u1red, h0wever, render xy11- t01 pr0duct10n 6y th15 r0ute re1at1ve1y expen51ve. 5°

7he 6e5t xy11t01-pr0duc1n9 yea5t5 (7a61e 2) have 6een f0und 1n the 5~ec1e5 C. tr0p1ca115 and e5pec1a11y Cand1da 9u1111erm0nd11. 5 A5 w1th ethan01 pr0duct10n fr0m D-xy105e, the 0xY9en 5upp1y ha5 t0 6e carefu11y re9u1ated t0 max1m12e xy105e reduct10n w1th m1n1ma1 1055 0f xY11t01 t0 further me- ta60115m. An 1ncrea51n9 0xY9en 11m1tat10n enhanced xY11t01 pr0duct10n and decrea5ed ethan01 pr0duct1v1ty w1th C. 9u1111erm0nd11. 51 Furtherm0re, the xY11t01 y1e1d 9enera11y 1n- crea5e5, up t0 a p01nt, w1th 1ncrea51n9 xy105e 5u65trate

r 50 c0ncent at10n. 50me 1mpre551ve xy11t01 y1e1d5 (m0re than 94% 0f the the0ret1ca1) 52 and c0ncentrat10n5 (up t0 221 9

1 - 1) have 6een rep0rted (7a61e2). Recent1y, a rec0m61nant 5. cerev151ae 5tra1n, tran5f0rmed w1th the 9ene enc0d1n9 xy105e reducta5e 0fP. 5t1p1t15 and re4u1r1n9 a c05u65trate f0r c0fact0r re9enerat10n, ma1ntenance, and 9r0wth due t0 the 1ack 0f xy11t01 dehydr09ena5e act1v1ty, 9ave a 95% c0nver- 510n 0f xy105e t0 xy11t01. 53 7he5e fermentat10n5 a150 tend t0 6e 510w, h0wever.

Xy105e fermentat10n u51n9 xy105e 150mera5e C0mmerc1a1D-xy105e 150mera5e (D-91uc05e 150mera5e) may 6e u5ed t0 c0nvert D-xy105e t0 the ket0pent05e, r)-xy1u105e, wh1ch 15 read11y fermenta61e 6y a num6er 0f yea5t5, 1nc1ud- 1n9 5acchar0myce5 cerev151ae and 5ch1205acchar0myce5 p0m6e (7a61e 3). 7h15 appr0ach thu5 ena61e5 the u5e 0f yea5t5 w1th a h19her fermentat1ve capac1ty, ethan01 t01er- ance, and temperature t01erance than the pent05e- ferment1n9 yea5t5. An0ther advanta9e 15 that w1th the5e yea5t5 anaer061c fermentat10n5 are p055161e, thu5 e11m1nat- 1n9 the pr061em 0f aer061c re5p1rat10n 0f ethan01 dur1n9 the fermentat10n. 54 5. p0m6e ha5 an 0ut5tand1n9 a6111ty f0r rap1d 9r0wth and eff1c1ent ethan01 pr0duct10n fr0m D-xy1u105e. 55-58 0ther5 have, h0wever, rep0rted 6etter re- 5u1t5 w1th 5. cerev151ae than w1th 5. p0rn6e. 59 A5 w1th d1rect xy105e fermentat10n5, xy11t01 pr0duct10n tend5 t0 decrea5e the ethan01 y1e1d. 54

7he e4u1116r1um 0f the 150mer12at10n react10n 151n fav0r 0f D-xy105e and y1e1d5 0n1y 10% t0 30% D-xy1u105e, depend- 1n9 0n temperature. 6°-62 7he e4u1116r1um can 6e 5h1fted 1n fav0r 0f D-xy1u105e 6y the add1t10n 0f tetra60rate, 62 6ut th15 pr0cedure a150 ha5 certa1n d15advanta9e5. 4,5 A1ternat1ve1y, a pr0ce55 1nv01v1n9 51mu1tane0u5 150mer12at10n and fer- mentat10n, there6y rem0v1n9 the xy1u105e a51t 15 f0rmed, 15 re4u1red f0r the eff1c1ent c0nver510n 0f xy105e t0 ethan01. 7he pr061em 15 c0mp0unded 6y the d1fferent 0pt1ma10per- at1n9 c0nd1t10n5 0f pH and temperature f0r the 150mer12a- t10n pr0ce55 (pH 6-8, 70°C) and ethan01 pr0duct10n (pH 4-6, 35°C); c0n5e4uent1y, a c0mpr0m15e 0f 5u60pt1ma1 c0n- d1t10n5 (pH 5.5-6, 35-39°C) ha5 t0 6e u5ed 1f the pr0ce5515 c0nducted 1n a 51n91e ve55e1. 54,63,64 De5p1te th15, an eth- an01 y1e1d and a pr0duct10n rate 5urpa551n9 50me 0f the

7a61e 2 Fermentat10n parameter5 f0r xy11t01 pr0duct10n

Xy105e Xy11t01 Fermentat10n Yea5t 5u65trate (9 1-1) (9 1-1) Yxy1 Yet0h t1me (h) Ref.

Cand1da 5p. 8-22 Pure xy105e 249 - 2 1 0 --0.84 n5 - 167 52 C. 601d1n11 Pure xy105e 100 - 4 0 n5 n5 - 120 138 C, 9u1111erm0nd11 Pure xy105e 50

NRC 5578 300 221 0.75 0.02 406 NRC 5578 110 68.7 0.60 0.03 238

C, 9u1111erm0nd11 Pure xy105e 104 77.2 0.74 10w 80 51 F71-20037

C, 9u1111errn0nd11 Hem1ce11u105e hydr01y2ate 76 -34.5 0.48 n5 N 130 139 F71-20037

Cand1da 5p. 8-22 Hem1ce11u105e hydr01y2ate 105.4 94.7 -0 .89 14.3 9 1-1 a 96 52 5. cerev151ae 6 Pure xy105e and 91uc05e 19 - 18 0.95 c n5 - 9 5 53

YxyJ, xy11t01 y1e1d, 9 9 -1 xy105e a551m11ated; Yet0h, ethan01 y1e1d, 9 9-1 xy105e a551m11ated aPr0duced fr0m 91uc05e, n0t xy105e 67ran5f0rmed w1th 9ene enc0d1n9 P. 5t1p1t15 xy105e reducta5e cA551m11ated dur1n9 c0ur5e 0f fermentat10n n5, N0t 5tated

En2yme M1cr06 .7echn01. , 1994, v01.16, N 0 v e m 6 e r 947

Rev1ew5

7a61e 3 Fermentat10n parameter5 f0r ethan01 pr0duct10n fr0m D-xy105e 6y yea5t51n c0njunct10n w1th D-xy105e 150mera5e

Q~p1 mha Xy11t01 Fermentat10n Xy105e Ethan01 Y1e1d a x 1) (9 4-P~nahx- 1) Yea5t 150mera5e (9 1-1) (9 1-1) (9 9-1) (9 (9 1-1) t1me (h) Ref.

5. CereV151ae 150mera5e and 75 31 0.41 1.6 n5 n5 (yea5t Cake) Na-tetra60rate

5. p0m6e 5Weet2yme Q 60 20.6 0.35 0.58 0.22 7.9 NRRL Y-164

5. CereV151ae 0pt15Weet-P 127 62 0.49 1.35 n5 7.5 (yea5t Cake) (Fed-6atCh

CU1tUre W1th 4.6 mM a21de)

24 62

48 54

46 65

E)p,max, MaX1mUm V01Umetr1C ethan01 pr0dUCt1V1ty; 4p,max, max1mUm 5peC1f1C ethan01 pr0dUCt1V1ty aEthan01 y1e1d, 9 9 -~ 5U9ar a551m11ated n5, N0t 5tated

6e5t va1ue5 06ta1ned 1n d1rect fermentat10n5 (w1th0ut 150mera5e) have 6een rep0rted.65 A1ternat1ve1y, 5eparate ve55e15 may 6e u5ed w1th med1um recyc11n9, 6ut th15 ha5 the d15advanta9e 0f c0nt1nua1 adju5tment5 0f tempera- ture and pH.

Fermentat10n 0f 119n0ce11u1051c hydr01y2ate5 A9r1cu1tura1 re51due5 c0n51dered p0tent1a1 feed5t0ck5 f0r ethan01 pr0duct10n 1nc1ude 5u9ar cane 6a9a55e, c0rn 5t0- ver, wheat 5traw, and 6ar1ey 5traw. 66,67 D-Xy105e and a 5ma11er 4uant1ty 0f L-ara61n05e re5u1t fr0m the hydr01y515 0f the pent05an c0mp0nent 0f hem1ce11u105e, wherea5 D-9a1act05e and 9-mann05e are der1ved fr0m 0ther hem1- ce11u105e c0n5t1tuent5 w1th D-91uc05e and D-ce1106105e 1ar9e1y 0r191nat1n9 fr0m the ce11u105e c0mp0nent. 4 50me 0f the5e 5u9ar5 may 6e 0-acety1ated. 68 5pent 5u1f1te 11- 4u0r 15 a150 re9arded a5 a p0tent1a1 feed5t0ck 6ecau5e 1t c0nta1n5 a h19h pr0p0rt10n 0f D-xy105e. 27,69 50me recent re5u1t5 0n the fermentat10n 0f 5uch feed5t0ck5 are 5um- mar12ed 1n 7a61e 4.

Xy105e 150mer12at10n ha5 a150 6een app11ed t0 the fer- mentat10n 0f 119n0ce11u1051c hydr01y2ate5, w1th re5u1t5 that c0mpare fav0ra61y w1th d1rect fermentat10n5 (7a61e 4). W1th 150mer12at10n-fermentat10n pr0ce55e5, the 5ta6111ty 0f the 150mera5e5 pre5ent5 a pr061em 1n that they are 9enera11y very 5en51t1ve t0 h19h 5a11n1ty 5uch a5 f0und 1n 5u1f1te wa5te 114u0r and ac1d-hydr01y2ed 119n0ce11u1051c5, 59 and 1t wa5 5u99e5ted that yea5t pr0te01yt1c en2yme5 1n 51mu1tane0u5 150mer12at10n and fermentat10n pr0ce55e5 may 1nact1vate the 150mera5e. 7° A further draw6ack 0f the 150mer12at10n- fermentat10n r0ute 15 the 10w rate 0f xy1u105e fermentat10n, and 1t wa5 5pecu1ated that the 0vera11 c05t may 6e h19her than that 0f the d1rect fermentat10n 0f xy105e.71

1n add1t10n t0 the 1ntr1n51c phy5101091ca1 c0n5tra1nt5 0f the pent05e-ferment1n9 yea5t5, the u5e 0f 119n0ce11u1051c hydr01y2ate5 a5 car60n 5u65trate5 pre5ent5 part1cu1ar 065ta- c1e5 t0 an eff1c1ent fermentat10n. 7he5e are de5cr16ed 6e10w.

1nh161t0r5

A num6er 0f meta6011c 1nh161t0r5 0ccur 1n m1nera1 ac1d hydr01y2ate5 0f 119n0ce11u1051c mater1a15, even where 0n1y the hem1ce11u105e c0mp0nent 15 extracted 6y m11d ac1d hy-

dr01y515 (7a61e 5). Acet1c ac1d 15 c0mm0n1y f0und 1n 5uch hydr01y2ate5, 0r191nat1n9 fr0m the acety1 9r0up5 0n the xy- 1an, and 15 0ne 0f the maj0r 1nh161t0r5 0f 9r0wth and ethan01 pr0duct10n 1n hem1ce11u105e hydr01y2ate5. P. 5t1p1t15 15 c0n- 51dera61y m0re 5en51t1ve t0 acet1c ac1d than 5. cerev151ae. 59 Wherea5 acet1c ac1d c0ncentrat10n5 0f a60ut 2 t0 5 91 - 1 can 6e 1nh161t0ry t0 P. 5t1p1t15 and P. tann0ph11u5, 72,73 far h19her c0ncentrat10n5 (up t0 10 9 1-1) can 0ccur 1n hydr01y2ate5. 4 8ecau5e the de9ree 0f t0x1c1ty 15 1ar9e1y pH dependent, 74 m05t fermentat10n5 0f ac1d-cata1y2ed hem1ceUu105e hydr0- 1y2ate5 have 6een c0nducted at pH va1ue5 a60ve the 0pt1- ma1 va1ue5 0f pH 4 t0 5.51n 0rder t0 m1n1m12e the 1nh161t0ry effect 0f acet1c ac1d, re5u1t1n91n 5u60pt1ma1 fermentat10n5.

7he pent05e-ferment1n9 yea5t5 vary 1n the1r t01erance t0- ward5 the5e 1nh161t0r5. 1n c0ntra5t t0 P. 5t1p1t15, P. tann0ph11u5 1n crude hem1ce11u105e hydr01y2ate fr0m wheat 5traw wa5 t0- ta11y 1nh161ted. 75,76 A num6er 0f rep0rt5 1nd1cated that re515- tance t01nh161t0r5 can 6e marked1y enhanced 6y acc11mat12a- t10n (1nv01v1n9 exp05ure t0 1ncrea51n9 c0ncentrat10n5 0f the part1cu1ar hydr01y2ate) 0r 6y u51n91501ate5 0fyea5t5 that have

27 28 77 6een recyc1ed 1n the hydr01y2ate (7a61e 4). , , Var10u5 pretreatment5 t0 m1n1m12e the effect5 0f the5e

1nh161t0r5 have 6een 1nve5t19ated (7a61e 6). 7he add1t10n 0f 50d1um 5u1f1te t0 50ftw00d ac1d hydr01y2ate 1mpr0ved 5u9ar ut1112at10n 6y an ethan0109en1c rec0m61nant 5tra1n 0f E5- cher1ch1a c011. 78 7he eff1cacy 0f the treatment apparent1y depended 0n the nature 0f the hydr01y2ate, and varY1n9 de9ree5 0f 5ucce55 a5 we11 a5 c0nf11ct1n9 re5u1t5 have 6een rep0rted. 7he hydr01y515 pr0cedure ha5 a 9reat 1mpact 0n the pre5ence 0f t0x1c c0mp0und5 1n the hydr01y2ate. F0r examp1e, a h19h temperature dur1n9 ac1d hydr01y515 0f 5u9ar cane 6a9a55e 1ncrea5e5 the furfura1 1eve1 dramat1ca11y, 79 wherea5 502-cata1y2ed 5team prehydr01y515 0f w00d ch1p5 f0110wed 6y en2ymat1c hydr01y515 w1th c0mmerc1a1 ce11u- 1a5e, hem1ce11u1a5e, and ce11061a5e y1e1ded a hydr01y2ate w1th n0 ev1dence 0f 1nh161t10n 0r t0x1c1ty 0f e1ther the en- 2yme5 0r P. 5t1p1t15. 80 7he c05t-effect1vene55 and fea516111ty 0f u51n9 the5e pr0cedure5 0n an 1ndu5tr1a15ca1e have yet t0 6e eva1uated.

5u9ar m1xture5

7he 91uc05e c0ntent 0f 119n0ce11u1051c hydr01y2ate5 can 6e 4u1te h19h, depend1n9 0n the de9ree t0 wh1ch the hydr01y515

948 En2yme M1cr06.7echn01., 1994, v01.16, N0vem6er

Env1r0nmenta1 fact0r5 affect1n9 0-xy105e fermentat10n: J. C, du Pree2

7a61e 4 Fermentat10n parameter5 f0r ethan01 pr0duct10n fr0m hem1ce11u1051c and 119n0ce11u1051c hydr01y2ate5

5u9ar c0nc (9 1 - 1 )

Ethan01 Y1e1d Fermentat10n Yea5t 7ype 0f hydr01y2ate Pretreatment 61uc05e Xy105e 70ta1 (9 1-1) (9 9-1) t1me (h) Ref.

Cand1da 5hehatae A7CC 22984 ••R•• Wh01e 6ar1ey - - 126 - 54 180 84 0.48 72 28

en2ym1c hydr01y2ate A7CC 22984 "R•• 5u1f1te wa5te 114u0r 5team 5tr1pp1n9 - 3 - 7 - 31 - 14 0.48 18 27 A7CC 22484 Hardw00d a c 1 d 0ver11m1n9 and - 9 - 4 4 65.4 16 0.24 120 140

hydr01y2ate f11trat10n P1ch1a 5t1p1t15

C85 5776 ••R•• P1ne, 502- - n5 n5 134 55 0.43 n5 77 prehydr01y2ed en2ym1c hydr01y2ate

C85 5776 ••R•• C0rn 5t0ver, - n5 n5 120 52 0.46 n5 77 502-prehydr01y2ed en2ym1c hydr01y2ate

C85 5773 Cane 6a9a55e Na0H and n5 n5 140 24 0.35 70 22 ac1d hydr01y2ate act1vated car60n

5. cerev151ae 5traw ac1d - - 1 0 ~27 - 4 1 16.8 0.406 51 59 hydr01y2ate c

5. cerev151ae 5u1f1te wa5te 114u0r c - - 1 0 - 5 - 4 1 16.8 0.416 45 59 5. p0m6e 5u1f1te wa5te 114u0r d - - 10 - 5 - 4 1 10.3 0.306 47.5 59

C85 352

aEthan01 y1e1d, 9 9-1 5u9ar a551m11ated, 0r 69 9 - 1 t0ta11n1t1a1 5u9ar5 c150mera5e (Maxa2yme) p1u5 4.6 mM a21da d150mera5e (0pt15weet-P) p1u5 4.6 mM a21de

7a61e 5 6r0wth 1nh161t0r5 1n hem1ce11u105e hydr01y2ate5

1nh161t0r Ref.

Dec0mp051t10n pr0duct5 0f car60hydrate5 141 Furfura1 142 Hydr0xymethy1furfura1 Acet1c ac1d

De9radat10n pr0duct5 0f 119n1n 143 5yr1n9a1dehyde Van1111n 0ther ar0mat1c m0n0mer5 and d1mer5

Heavy meta110n5 a 79,144,145 1r0n Chr0m1um N1cke1 C0pper

aDue t0 c0rr0510n 0f e4u1pment

pr0cedure 5acchar1f1e5 the ce11u105e c0mp0nent 1n add1t10n t0 the hem1ce11u105e (7a61e 4). 7he fermentat10n perf0r- mance 0f yea5t5 that ferment D-xy105e 4u1te eff1c1ent1y 1n med1a c0nta1n1n9 th15 pent05e a5 501e 5u9ar 15 u5ua11y much p00rer 1n hydr01y2ate5 c0nta1n1n9 a m1xture 0f 5u9ar5. AJ- th0u9h the 11terature c0nta1n5 c0nf11ct1n9 rep0rt5, 1t can 6e 9enera112ed that the pre5ence 0f D-91uc05e 1nh161t5 0r re- tard5 D-xy105e ut1112at10n 6y C. 5hehatae, P. 5t1p1t15, and P. tann0ph11u5, 10,75,81-84 6ut th15 d1aux1c pha5e 15 man1fe5ted 0n1y a60ve a certa1n thre5h01d c0ncentrat10n wh1ch appear5 t0 6e 1n a w1de ran9e 0f 2.3 t0 20 91 - 1.39,85,86 7he5e 065er- vat10n5 are 1n a9reement w1th f1nd1n95 that 0-91uc05e (and D-mann05e) part1a11y repre55 the pr0duct10n 0f xy105e

reducta5e and xy11t01 dehydr09ena5e 1n P. 5t1p1t15 C85 5773 and that 60th 5ymp0rt 5y5tem5 f0r D-xy105e 1n P. 5t1p1t15 are 1nh161ted, 6ut n0t repre55ed, 6y D-91uc05e. 82,87 1n C. 5hehatae C85 2779 the fac111tated d1ffu510n 5y5tem 15 c0mpet1t1ve1y 1nh161ted 6y 91uc05e and mann05e (6ut n0t repre55ed), and pr0t0n 5ymp0rt 15 n0nc0m- pet1t1ve1y 1nh161ted a5 we11 a5 repre55ed. 82,88 1n P. tann0- ph11u5 1t wa5 5h0wn that D-91uc05e rever5161y 1nact1vated the ex15t1n9 D-xy105e cata6011c en2yme5. 84 7he var10u5 pent05e-ferment1n9 yea5t5 appear t0 d1ffer 1n the1r 5en51t1v- 1ty t0 the re9u1at0ry effect 0f 91uc05e. 84 Under certa1n phy5- 101091ca1 c0nd1t10n5, h0wever, 51mu1tane0u5 ut1112at10n 0f xy105e and 91uc05e can 0ccur. F0r examp1e, a C. 5hehatae 1n0cu1um 9r0wn aer061ca11y 0n 91uc05e fermented 91uc05e and xy105e 5e4uent1a11y, 6ut 1f the ce115 were aer061ca11y c0nd1t10ned 0n D-xy105e, there wa5 n0 1a9 1n xy105e fermentat10n. 89

7yp1ca11y, the 5u9ar5 are a551m11ated 1n the 5e4uence D-91uc05e, D-mann05e, and D-xy105e, w1th L-ara61n05e ut1- 112ed 1a5t, wh11e D-9a1act05e may 6e ut1112ed c0ncurrent1y w1th, pr10r t0, 0r 5u65e4uent t0 D-xy105e. 10,45,69,90,91 L-Ara61n05e can c0n5t1tute up t0 9.3% 0f the t0ta1 hem1ce1- 1u105e 5u9ar5 0f ac1d-hydr01y2ed 5u9ar cane 6a9a55e, 92 6ut t0 date n0 yea5t ha5 6een f0und t0 pr0duce ethan01 fr0m

10 th15 pent05e. P. 511p#15 can ferment ce1106105e a5 we11 a5 1archw00d xy1an d1rect1y t0 ethan01. 93

Pr0ce55 c0n51derat10n5

F0r ethan01 pr0duct10n fr0m D-xy105e t0 6e c0mmerc1a11y v1a61e, 1t wa5 5u99e5ted that the yea5t 5h0u1d 6e capa61e 0f

E n 2 y m e M 1 c r 0 6 . 7 e c h n 0 1 . , 1994, v 0 1 . 1 6 , N 0 v e m 6 e r 9 4 9

Rev1ew5

7a61e 6 Pretreatment pr0cedure f0r hem1ce11u1051c and 119n0ce11u1051c hydr01y2ate5

Pr0cedure 7ype 0f hydr01y2ate Effect 0f treatment Ref.

5team 5tr1pp1n9 L19n0ce11u1051c Rem0va1 0f v01at11e5 (furfura1, phen015) 1nc1ud1n9 acet1c 22 ac1d 140

Hem1ce11u1051c Reduct10n 0f acet1c ac1d c0ncentrat10n 22

Hem1ce11u1051c

Neutra112at10n w1th Ca0, Na0H, K0H; act1vated car60n; f11trat10n

Neutra112at10n t0 pH 6.5 0r 0vert1trat1n9 t0 pH 10 w1th Ca(0H)2, Ca0, 0r K0H; rem0va1 0f prec1p1tate; H2504 t0 pH 6.5

10n exc1u510n chr0mat09raphy (cat10n exchan9e re51n)

Ether extract10n Evap0rat10n under vacuum Ethy1acetate extract10n M01ecu1ar 51eve M1xed 6ed 10n re51n5 An10n/cat10n exchan9e re51n5

Hem1ce11u1051c

Prec1p1tat10n 0f acetate, heavy meta15, furfura15 tann1n5, terpene5, phen011c5

Rem0va1 0f ar0mat1c m0n0mer5 and d1mer5

22 140 92

135 143

Hem1ce11u1051c Rem0va1 0f furfura1 75 Hem1ce11u1051c Rem0va1 0f acet1c ac1d 73 Hem1ce11u1051c Rem0va1 0f 119n1n-de9radat10n pr0duct5 73 Hem1ce11u1051c Part1a1 rem0va1 0f acet1c ac1d, furfura1, and 501u61e 119n1n 146 Hem1ce11u1051c Part1a1 rem0va1 0f acet1c ac1d, furfura1, and 501u61e 119n1n 146 Hem1ce11u1051c Part1a1 rem0va1 0f acet1c ac1d, rem0va1 0f meta110n5 79

pr0duc1n9 50 t0 60 9 1-1 ethan01 w1th1n 36 h w1th a y1e1d 0f at 1ea5t 0.4 9 9 - 1.2 7here 15 9enera1 c0n5en5u5 that C. 5he- hatae and P. 5t1p1t15 are 5uper10r t0 a110ther yea5t 5pec1e5 f0r ethan01 pr0duct10n fr0m D-xy105e and certa1n 5tra1n5, par- t1cu1ar1y 0f P. 5t1p1t15, can meet the prere4u151te 0f a h19h ethan01 y1e1d. 0 the r fav0ra61e character15t1c5 0f P. 5t1p1t15 are that 1t 15 n0t a6501ute1y dependent 0n added v1tam1n5 and u5ua11y pr0duce5 n0 0r 0n1y 5ma11 4uant1t1e5 0f xy11t01. 5tra1n 5e1ect10n 151mp0rtant 6ecau5e 1t ha5 6een 5h0wn that d1fference51n ethan01 pr0duct1v1ty ex15t 6etween 5pec1e5 0r even 6etween 5tra1n5 w1th1n a 5pec1e5.12,29,94-96 7h15 t0p1c 15 d15cu55ed 1n m0re deta111n the rev1ew 6y Jeffr1e5 and Kurt2- man 1n th15 155ue.

7he pr061em area5 are the unaccepta61y 10n9 fermenta- t10n t1me and the re1at1ve1y 10w ethan01 t01erance, wh1ch a150 tend5 t0 510w d0wn the fermentat10n a5 ethan01 accu- mu1ate5. Ethan01 t01erance can 6e 1mpr0ved 6y c0nduct1n9 the fermentat10n at temperature5 0f 25°C 0r 1e5517,19,97-99 due t0 the 10wer 501u6111ty 0f ethan011n the ce11 mem6rane 11p1d5,100 6ut th15 w0u1d re5u1t 1n a c0mpr0m15e 6etween the h19her ethan01 c0ncentrat10n and a550c1ated cheaper d15- t111at10n 0n the 0ne hand, and the 5u65tant1a11y 1ncrea5ed fermentat10n t1me and h19her c0011n9 c05t5 0n the 0ther. An attempt t0 c1rcumvent ethan01 t0x1c1ty 6y 9r0w1n9 C. 5he- hatae 1n1t1a11y 1n aer061c c0nt1nu0u5 cu1ture and then feed- 1n9 the ce115 t0 a 5ec0nd anaer061c fermenter 1ncrea5ed the v01umetr1c ethan01 pr0duct1v1ty 6ut fa11ed t0 1mpr0ve the ethan01 y1e1d 0r c0ncentrat10n. 97 7he u5e 0f 1mm061112ed P. 5t1p1t15 ce115 a150 d1d n0t 1mpr0ve 0n 6atch fermentat10n5.101 A h19h-ce11-den51ty fermentat10n w1th C. 5hehatae wa5 0n1y mar91na11y fa5ter than a 6atch fermentat10n u51n9 far 1e55 610ma55, pre5uma61y 6ecau5e 0f a 1055 0f ce11 v1a6111ty, and the fermentat10n pr0duct1v1ty 06ta1ned 1n an 0xY9en- 11m1ted c0nt1nu0u5 cu1ture wa5 10wer than that 0f a 6atch fermentat10n. 41

F0r the fermentat10n 0f 5u9ar m1xture5 c0nta1n1n9 a 1ar9e am0unt 0f 91uc05e, 1t 15 prefera61e t0 ferment the hex05e w1th an ethan01-t01erant m1cr00r9an15m 5uch a5 5. cerev151ae 0r 2ym0m0na5 m061115 that 91ve5 a h19h ethan01 y1e1d and pr0duct1v1ty. 21 7he fermentat10n 0f a 91uc05e/

xy105e m1xture d0e5 pre5ent certa1n d1ff1cu1t1e5 1n pract1ce, h0wever. 1n the ca5e 0f a 5e4uent1a1 fermentat10n where the 91uc05e 15 fermented f1r5t, the 10w ethan01 t01erance 0f the pent05e-ferment1n9 yea5t pre5ent5 a c0n5tra1nt, 50 that 1t w0u1d 6e prefera61e t0 rem0ve the ethan01 pr10r t0 the pent05e fermentat10n. 7h15 pr061em c0u1d 6e c1rcumvented 1f a 91uc05e-ne9at1ve mutant were f1r5t u5ed f0r the prefer- ent1a1 fermentat10n 0f the xy105e, f0110wed 6y 91uc05e fer- mentat10n w1th 5. cerev151ae 0r 2. m061115. 21 7h15 ha5 n0t yet 6een d0ne.

Var10u5 attempt5 t0 ferment 91uc05e/xy105e m1xture5 1n c01umn and a1r11ft react0r5 6y u51n9 f10ccu1ent 0r 1mm061- 112ed P. 5t1p1t15 ce115, a10ne 0r t09ether w1th free 5. cerev151ae ce115, 0r 1n a n0ve1 appr0ach where the5e tw0 yea5t5 were c01mm061112ed, 9ave 10w ethan01 y1e1d5 (up t0 0.35 9 9 - 1) and 1nc0mp1ete 5u9ar ut1112at10n. 1°2-1°4 Ce11 1mm061112a- t10n 0r the u5e 0f a f10ccu1ent 5tra1n 0f P. 5t1p1t15, wh1ch 15 a cheap a1ternat1ve t0 ce11 retent10n 6y 1mm061112at10n, 1°4 9ave n0 advanta9e 1n the fermentat10n 0f 91uc05e/xy105e m1xture5 (7a61e 7). 6r00tjen et a1.102 c0nc1uded that 1mm0- 61112ed P. 5t1p1t15 ce115 had a much 10wer 5pec1f1c 5u9ar c0n- ver510n rate, re5u1t1n91n a 10w 0vera11 c0nver510n rate. 51m- 11ar1y, 511n1n9er et a1.1°5 f0und that 0n1y 40% 0f entrapped P. tann0ph11u5 ce115 were act1ve 1n fermentat10n 6ecau5e 0f d1ffu510na1 11m1tat10n5 and that 1mm061112at10n d1d n0t 1m- pr0ve the ethan01 y1e1d. 0ther5 c1a1med 1mpr0ved ethan01 y1e1d5 and pr0duct10n rate5 w1th 1mm061112ed P. 5t1p1t15 1n c0mpar150n w1th the1r 6atch cu1ture va1ue5.1°1 7he u5e 0f a f10ccu1ent P. 5t1p1t15 5tra1n 1n a react0r w1th ce11 retent10n ha5 50me p0tent1a1 f0r the fermentat10n 0f med1a c0nta1n1n9 ma1n1y D-xy105e, 6ut ha5 n0t yet 6een eva1uated. U51n9 re- act0r51n 5er1e5 w1th a 5u5pen510n 0fP. 5t1p1t15 0r 1n c0njunc- t10n w1th a 5u5pen510n 0f 5. cerev151ae a150 fa11ed t0 1mpr0ve 0n the ethan01 y1e1d 0f the af0rement10ned 1mm061112ed

106 ce11 pr0ce55e5. 1n a c0nt1nu0u5 c0cu1ture 0f a f10ccu1ent 5tra1n 0f P. 5t1p1t15 w1th a f10ccu1ent re5p1rat0ry-def1c1ent mutant 0f 5. d1a5tat1cu5, h0wever, the ethan01 y1e1d wa5 1mpr0ved t0 0.45 9 9-1 (7a61e 7) w1th a 94% ut1112at10n 0f

107 the 91uc05e/xy105e m1xture. 1n th15 1n5tance the u5e 0f a re5p1rat0ry-def1c1ent 5. d1a5tat1cu5 mutant fac111tated a 1eve1

950 En2yme M1cr06.7echn01., 1994, v01.16, N0vem6er

Env1r0nmenta1 fact0r5 affect1n9 D-xy105e fermentat10n: J. C. du Pree2

"1•a61e 7 Fermentat10n parameter5 f0r ethan01 pr0duct10n fr0m 0-xy105e and D-91uc05e w1th 1mm061112ed 0r f10ccu1ent yea5t ce115

5u9ar c0nc (9 1-1)

Ethan01 Y1e1d a ••p 4p.max Yea5t Xy105e 61uc05e (9 1-1) (9 9-1) (9 17x1) (9 9-1 h-1) Ref.

P. 5t1p1t15 C85 57736 50 0 19.5 -0.39 0.56 n5 101 P. 5t1p1t15 C85 5773 10 40 12 0.27 0.84 0.05 104

and 5. cerev151ae c P. 5t1p1t15 C85 7507 d 10 40 13 0.35 0.65 0.05 104 P. 5t1p1t15 C85 5773 d 15 35 21.5 0.45 4.3 n5 107

and 5acchar0myce5 d1a5tat1cu5 a

aEthan01 y1e1d, 9 9 -1 5u9ar a551m11ated 61mm061112ed cC01mm061112ed dF10ccu1ent 0p,max, Max1mum v01umetr1c ethan01 pr0duct1v1ty; 4p, rnax, max1mum 5pec1f1c ethan01 pr0duct1v1ty

n5, N0t 5tated

0f d15501ved 0xY9en fav0ra61e f0r xy105e fermentat10n 6y P. 5t1p1t15, and the 10w re51dua1 91uc05e c0ncentrat10n 1n c0n- t1nu0u5 cu1ture a11ev1ated 91uc05e repre5510n 0f xy105e ut1- 112at10n. 7he ma1n 11m1t1n9 5tep 0f th15 c0cu1ture pr0ce55 wa5 the 10w ethan01 t01erance 0fP. 5t1p1t15. 107,108

An a1ternat1ve 501ut10n t0 the pr061em 0f ferment1n9 91uc05e/xy105e m1xture5 w0u1d 6e t0 u5e a hydr01y515 pr0ce- dure 9enerat1n9 5eparate hydr01y2ate 5tream5 c0nta1n1n9 pred0m1nant1y e1ther xy105e 0r 91uc05e. 1n th15 ca5e a tw0- 5ta9e c0nt1nu0u5 react0r w0u1d 6e m0re eff1c1ent when fer- ment1n9 the xy105e 5tream f1r5t, wh1ch w0u1d 5u65e4uent1y 6e fed t0 the 5ec0nd react0r t0 6e 61ended w1th the 91uc05e 5tream and fermented w1th 5. cerev151ae. 109 A c0mp11cat1n9 fact0r 15 the recent d15c0very that many 5tra1n5 0f P. 5t1p1t15 exh161t an 1nh161t0ry 0r a k111er act1v1ty a9a1n5t 5acchar0my- ce5 5pec1e5, 44,11° wh1ch 11m1t5 the1r u5e 1n c0cu1ture5. N0 1nc0mpat16111ty ha5, a5 yet, 6een detected 6etween 5tra1n5 0f 5. cerev151ae and C. 5hehatae. 110

Pr0v1d1n9 a xy105e fermentat10n w1th the 0pt1ma1 rate 0f 0xY9en tran5fer t0 max1m12e the rate and y1e1d 0f ethan01 pr0duct10n thr0u9h0ut the ent1re fermentat10n, a5 we11 a5 m1n1m121n9 cata60115m 0f ethan011n the pre5ence 0f 0xy9en dur1n9 0r at the end 0f the fermentat10n, 15 d1ff1cu1t 1n prac- t1ce. Accurate c0ntr010fthe D 0 7 1n a 9r0w1n9 cu1ture w1th a c0nt1nu0u51y chan91n9 0xY9en demand 15 techn1ca11y d1f- f1cu1t t0 ach1eve 6ecau5e 0f the very 10w va1ue 0f the cr1t1ca1 D07. 25,46 An attempt at u51n9 the red0x p0tent1a1 a5 c0n- tr0Ued var1a61e, wh1ch can under certa1n c0nd1t10n5 pr0v1de a 5en51t1ve 1nd1cat10n 0f very 10w D 0 7 1eve15, met w1th 11m1ted 5ucce551n 0pt1m121n9 a xY105e fermentat10n w1th C. 5hehatae and pr0ved 1neffectua1 w1th P. 5t1p1t15.111 A m0re 50ph15t1cated and 5ucce55fu1 501ut10n t0 th15 pr061em wa5 ach1eved 6y u51n9 c0mputer-c0ntr011ed 5t1rrer 5peed t0 91ve a c0n5tant 0 7 R 23 and e5pec1a11y 6y u51n9 the 0n-11ne e5t1mat10n 0f 610ma55 a5 parameter f0r c0mputer c0ntr01 0fthe 5pec1f1c 0xY9en uptake rate, 47 wh1ch appear5 t0 6e the m05t 1mp0rtant var1a61e t0 0pt1m12e the 0xY9en tran5fer rate. A5 an a1ternat1ve t0 the ph0t0metr1c meth0d u5ed 6y De11we9 et aL, 47 d1e1ectr1c mea5urement 15 a new and pr0m151n9 meth0d f0r 0n-11ne 610ma55 e5t1mat10n 1n 610react0r5.112

Certa1n 0ther 0perat1n9 c0nd1t10n5 may a150 1mpr0ve ethan01 pr0duct10n fr0m xy105e t0 varY1n9 de9ree5. 7he5e are de5cr16ed 6e10w.

Ce11 recyc11n9

1mpr0vement51n the fermentat10n w1th1n the th1rd recyc1e 0fP. tann0ph11u5 have 6een n0ted, n3 6ut w1th P. 5t1p1t15 n0 detecta61e 1mpr0vement wa5 f0und 0ver the c0ur5e 0f f0ur recyc1e5.22 7he d15c0very that adaptat10n 0f C. 5hehatae and P. 5t1p1t15 c0u1d 6e ach1eved 6y repeated recyc11n9 1n w00d hydr01y2ate, re5u1t1n91n 5tra1n5 capa61e 0f pr0duc1n9 much h19her ethan01 c0ncentrat10n5 than the parenta1 5tra1n5 (7a61e 4) 77 and a150 w1th an 1ncrea5ed re515tance t0 the 1nh161t0r51n hydr01y2ate5, 27 6r1n95 the c0mmerc1a1 app11ca- t10n 0f the5e pent05e-ferment1n9 yea5t5 c105e t0 rea112at10n. A 51m11ar1y acc11mat12ed Cand1da 5p. f0r xy11t01 pr0duct10n, wh1ch had an 1ncrea5ed t01erance t0 the 1nh161t0r5 1n un- treated cane 6a9a55e hydr01y2ate, ha5 6een rep0rted. 52

1n0cu1um a9e

Ce115 fr0m y0un9 (24 h) C. 5hehatae cu1ture5 pr0duced ethan01 at rate5 up t0 threef01d fa5ter than 72 h cu1ture5. 39

Feed1n9 re91men 0f car60n 50urce

1nterm1ttent feed1n9 0f ce11u105e hydr01y2ate t0 hardw00d hem1ce11u105e hydr01y2ate enhanced the ethan01 y1e1d 0fP. tann0ph11u5. 91 A 51m11ar effect wa5 06ta1ned 6y the 510w feed1n9 0f D-xy105e t0 a 6atch cu1ture 0f th15 yea5t 5pec1e5.114

Add1t10n 0f 11p1d5

7he add1t10n 0f 10w c0ncentrat10n5 0f er905ter01, 11n01e1c ac1d, and 7ween 80 519n1f1cant1y 1ncrea5ed the peak ethan01 c0ncentrat10n and y1e1d 1n cu1ture5 0fP. tann0ph11u5, wh1ch wa5 attr16uted t0 an enhanced ethan01 t01erance cau5ed 6y the 11p1d5.115

En2yme M1cr06.7echn01., 1994, v01.16, N0vem6er 951

Rev1ew5

Add1t10n 0 f meta6011c 1nh161t0r5

A21de can prevent c0ntam1nat10n, 610ck re5p1rat10n, and repre55 6y-pr0duct f0rmat10n. 7he u5e 0f th15 re5p1rat0ry 1nh161t0r can 91ve a 5ma111ncrea5e 1n the ethan01 y1e1d, 6ut 1t5 app11cat10n 1n 1ndu5tr1a1 pr0ce55e515 n0t fea5161e. 59 W1th C. tr0p1ca1•, a21de 1ncrea5ed the ethan01 y1e1dper un1t ce11 ma55 up t0 f0urf01d 6y repre551n9 xy11t01 pr0duct10n, 6ut 6y act1n9 a5 an unc0up1er 0f 0x1dat1ve ph05ph0ry1at10n, a21de cau5ed the 1055 0fup t0 60% 0fthe car60n 5u65trate a5 car60n d10x1de. 116 7he re5p1rat0ry 1nh161t0r ant1myc1n A 1mpr0ved the ethan01 y1e1d 0fP. 5t1p1t15,117 a1th0u9h th15 re5u1t wa5 pr06a61y 06ta1ned under c0nd1t10n5 0f exce551ve aerat10n.

1n 51tu ethan01 rem0va1

7he 1n 51tu rem0va1 0f ethan01 1n 6atch cu1ture5 0f P. tan- n0ph11u5 6y a650rpt10n 0n a hydr0ph061c 5111cate 1ncrea5ed the 9r0wth rate and ethan01 pr0duct1v1ty 6ut fa11ed t0 1m- pr0ve the ethan01 y1e1d. 118

Fermentat10n perf0rmance 0f 9enet1caUy m0d1f1ed m1cr00r9an15m5 Mutant5 0f Cand1da 119,120 and P. tann0ph11u5121-124 have 6een 06ta1ned w1th 1mpr0ved fermentat10n character15t1c5 1n c0mpar150n w1th the parenta1 5tra1n. 7he appr0ache5 t0 06ta1n1n9 the5e mutant5 are rev1ewed 1n 50me deta11 6y 5chne1der et aL 4 and 6y Jeffr1e5 and Kurt2man 1n th15155ue. 7he fermentat10n perf0rmance 0f the5e mutant5 wa5 5t111 1nfer10r t0 th05e 0f C. 5hehatae and P. 5t1p1t15, h0wever. Wherea5 the 9r0wth rate and ethan01 y1e1d 0f P. tann0- ph11u5 1ncrea5ed w1th 1ncrea51n9 p101dy, 1ncrea51n9 the DNA c0ntent 0f P. 5t1p1t15 0r C. 5hehatae 6y pr0t0p1a5t fu510n fa11ed t0 1mpr0ve the1r fermentat10n character15- t1c5 519n1f1cant1y. 96,125,126 Pr0t0p1a5t fu510n 0f P. tann0- ph11u5 w1th 5. cerev151ae re5u1ted 1n a fu5ant w1th a h19her ethan01 t01erance than P. tann0ph11u5, 6ut wh1ch pr0- duced 1e55 ethan01 fr0m D-xy105e. 127 5u65e4uent1y, 1t wa5 c1a1med that 51m11ar 1nter9ener1c hy6r1d5 exh161ted a 6et- ter perf0rmance than the parenta1 P. tann0ph11u5 0r C. 5hehatae. 128

5. cerev151ae tran5f0rmed w1th the 9ene5 fr0m P. 5t1p1t15 f0r xy105e reducta5e and xy11t01 dehydr09ena5e ut1112ed xy105e 510w1y, 1nc0mp1ete1y, and a1m05t ent1re1y 0x1- dat1ve1y. 129 1n the pre5ence 0f the re5p1rat0ry 1nh161t0r ant1myc1n A, th15 tran5f0rmant fa11ed t0 9r0w 0n xy105e 6ut d1d pr0duce ethan01 at a y1e1d 0f a60ut 0.179 9-1 w1th a60ut 50% 0f the xy105e 5ecreted a5 xy11t01.117,13° 0ther5 51m11ar1y rep0rted that the1r 5. cerev151ae tran5f0rmant, a1- th0u9h exh161t1n9 a h19h expre5510n 0f the xy105e reducta5e and xy11t01 dehydr09ena5e 9ene5, pr0duced ethan01 at a y1e1d 0f 0n1y a60ut 0.05 9 9-1 c0nc0m1tant w1th 5u65tant1a1 xy11t01 accumu1at10n. 13° 1t wa5 c0nc1uded that xy105e fer- mentat10n wa5 11m1ted 6y c0fact0r (NAD/NADH2) 1m- 6a1ance 117,13° and 6y an 1n5uff1c1ent capac1ty 0f xy1u105e c0nver510n 6y the pent05e ph05phate pathway. 117 7hu5 far, attempt5 at c0n5truct1n9 a xy105e-ut11121n9 yea5t 6y the ex- pre5510n 0f 6acter1a1 xy105e 150mera5e 9ene5 1n 5. cerev151ae were un5ucce55fu1, 1n that heter01090u5 pr0te1n5 w1th n0 0r a very 10w en2yme act1v1ty were pr0duced. 131,132 8etter re- 5u1t5 were 06ta1ned 6y c10n1n9 the xy105e 150mera5e 9ene fr0m E5cher1ch1a c0111nt0 5ch1205acchar0myce5 p0m6e, w1th the rec0m61nant yea5t 91v1n9 an ethan01 y1e1d 0f a60ut 0.35 9 9-1, 6ut at a 10w pr0duct1v1ty and w1th apprec1a61e xy11t01 pr0duct10n. 133

1n recent year5, pr0m151n9 re5u1t5 w1th rec0m61nant 6ac- ter1a have 6een rep0rted, 50me 0f wh1ch are 5h0wn 1n 7a61e 8. 7he u5e 0f 5uch m1cr00r9an15m5 f0r xy105e fermentat10n 0ffer5 the advanta9e5 0f h19h fermentat10n rate5, h19h eth- an01 y1e1d5, the a6111ty t0 ferment 5u9ar5 wh1ch are c0n5t1t- uent5 0f 119n0ce11u1051c5, and t01erat10n 0f a re1at1ve1y 6r0ad ran9e 0f env1r0nmenta1 c0nd1t10n5; an add1t10na1 advan- ta9e 15 that 5tr1ct re9u1at10n 0f the rate 0f 0xy9en 5upp1y 15 n0t an 1mp0rtant fact0r. 7M Furtherm0re, fermentat10n5 can 6e carr1ed 0ut at pH va1ue5 c105e t0 neutra1, wh1ch, 6e51de5 6e1n9 the 0pt1ma1 pH f0r 6acter1a1 fermentat10n5, m1n1m12e5 the t0x1c1ty 0f acet1c ac1d 1n hem1ce11u1051c hy- dr01y2ate5. 7he fermentat10n perf0rmance 0f a rec0m61- nant E. c011, tran5f0rmed w1th pymvate decar60xy1a5e and a1c0h01 dehydr09ena5e 9ene5 fr0m 2ym0m0na5 m061115, c0mpared very fav0ra61y w1th ethan01 pr0duct10n fr0m hem1ce11u105e hydr01y2ate5 6y pent05e-ut11121n9 yea5t5. 78,135

7a61e 8 Fermentat10n parameter5 f0r ethan01 pr0duct10n fr0m 0-xy105e 6y rec0m61nant 6acter1a

5u9ar c0nc (9 1-1)

Ethan01 Y1e1d 1Qp 8acter1um 61uc05e xy105e 70ta1 (9 1-1) (9 9-1) (9 1rn~ x -1 ) Fermentat10n t1me (h) Ref.

K1e651e11a 0xyt0ca 0 100 100 46 0.48 2.0 48 147 K. p1ant1c01a 0 - 42 - 42 17.8 0.42 0.58 80 148 E5cher1ch1a c011 0 140 140 56 0.45 1.4 120 134 E. c011 0 41.8 41.8 18.6 0.44 0.77 - 72 135 E. c011 0 64.5 64.5 28.0 0.43 0.33 - 72 135 E. c011 n5 31.0 a n5 14.9 0.48 0.65 n5 135 E. c011 20.0 15.0 73.66 35 0.46 > 1 48 78

aHardw00d (a5pen) hem1ce11u105e hydr01y2ate; c0ncentrated under vacuum, 0ver11med t0 pH 10, neutra112ed w1th 5u1fur1c ac1d 650ftw00d (p1ne) hem1ce11u105e hydr01y2ate; 0ver11med t0 pH 10-10.5 w1th ca1c1um 0x1de p1u5 50d1um 5u1f1te, heated and 5u65e4uent1y neutra112ed w1th 5u1fur1c ac1d Qp,max, Max1mum v01umetr1c ethan01 pr0duct1v1ty n5, N0t 5tated

9 5 2 E n 2 y m e M 1 c r 0 6 . 7 e c h n 0 1 . , 1994, v01, 16, N 0 v e m 6 e r

Env1r0nmenta1 fact0r5 affect1n9 D-xy105e fermentat10n: J. C. du Pree2

Further deve10pment5 1n 9enet1c en91neer1n9 a10n9 the5e 11ne5 0ffer exc1t1n9 p05516111t1e5 f0r c0mmerc1a1 pr0- ce55e5. A recent 5urvey 0f the fermentat1ve perf0rmance 0f 5evera1 yea5t5 and 6acter1a 0n 5u1f1te wa5te 114u0r and 0n hardw00d en2ymat1c hydr01y2ate 5u99e5ted that a rec0m- 61nant 5. cerev151ae 5tra1n m19ht 0ffer the 6e5t pr05pect5 f0r 1ndu5tr1a1 ethan01 pr0duct10n fr0m 119n0ce11u1051c hydr01y2ate5.136 Whether the recent advance51n c0n5truct- 1n9 1mpr0ved xy105e-ferment1n9 yea5t5 w111 u1t1mate1y y1e1d fermentat10n perf0rmance5 51m11ar t0 0r 6etter than th05e 0f the rec0m61nant 6acter1a rema1n5 t0 6e 5een.

Reference5 1 5j65tr0m, E. W00d Chem15try. Fundamenta15 and App11cat10n5. Ac-

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810techn01. 1985, 3, 208-212 3 Pr10r, 8. A., K111an, 5. 6. and du Pree2, J. C. Fermentat10n 0f

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4 5chne1der, H. C0nver510n 0f pent05e t0 ethan016y yea5t5 and fun91. CRC Cr1t. Rev. 810techn01. 1989, 9, 1-40

5 5k009, K. and Hahn-H~19erda1, 8. Xy105e fermentat10n. En2yme M1cr06. 7echn01. 1988, 10, 66-80

6 511n1n9er, P. J., 801en, P. L. and Kurt2man, C. Pachy501en tann0- ph11u5:Pr0pert1e5 and pr0ce55 c0n51derat10n5 f0r ethan01 pr0duct10n fr0m 1~-xy105e. En2yme M1cr06. 7echn01. 1987, 9, 5-15

7 Va11ander, L. and Er1k550n, K. -E. L. Pr0duct10n 0f ethan01 fr0m 119n0ce11u1051c mater1a15: 5tate 0f the art. Adv. 810chem. En9. 810- techn01. 1990, 42, 63-95

8 du Pree2, J. C., K0ck, J. L. F., M0nte1r0, A. M. 7. and Pr10r, 8. A, 7he v1tam1n re4u1rement5 0f Cand1da 5hehatae f0r xy105e fermen- tat10n. FEM5 M1cr0610L Lea. 1985, 28, 271-275

9 De11we9, H., R1221, M., Methner, H. and De6u5, D. Xy105e fermen- tat10n 6y yea5t5. 810techn01. Lett. 1984, 6, 395-400

10 du Pree2, J. C., 805ch, M. and Pr10r, 8. A. 7he fermentat10n 0f hex05e and pent05e 5u9ar5 6y Cand1da 5hehatae and P1ch1a 5t1p1t15. App1. M1cr06101. 810techn01. 1986, 23, 228-233

11 6ue6e1, D. V., C0rden0n5, A., Nude1, 8. C. and 61u1ett1, A. M. 1nf1uence 0f 0xy9en tran5fer rate and med1a c0mp051t10n 0n fermen- tat10n 0f 0-xY105e 6y P1ch1a 5t1p1t15 NRRL Y-7124. J. 1nd. M1cr06101. 1991, 7, 287-292

12 Ma1e52ka, R. and 5chne1der, H. Fermentat10n 0f D-xY105e, xy11t01, and D-xy1u105e 6y yea5t5. Can. J. M1cr06101. 1982, 28, 360-363

13 Jeffr1e5, 7. W. Effect5 0f cu1ture c0nd1t10n5 0n the fermentat10n 0f xy105e t0 ethan01 6y Cand1da 5hehatae. 810techn01. 810en9. 5ymp. 1985, 15, 149-166

14 Pa1n1tkar, 5. and Lachke, A. Effect 0f n1tr09en 50urce5 0n 0x1- d0reduct1ve en2yme5 and ethan01 pr0duct10n dur1n9 D-xy105e fer- mentat10n 6y Cand1da 5hehatae. Can. J. M1cr0610L 1992, 38, 258-260

15 Jeffr1e5, 7. W., Fady, J. H. and L19htf00t, E. N. Effect 0f 91uc05e 5upp1ement5 0n the fermentat10n 0f xy105e 6y Pachy501en tann0- ph11u5. 810techn01. 810en9. 1985, 27, 171-176

16 du Pree2, J. C., 805ch, M. and Pr10r, 8. A. Xy105e fermentat10n 6y Cand1da 5hehatae and P1ch1a 5t1p1t15:Effect5 0f pH, temperature and 5u65trate c0ncentrat10n. En2yme M1cr06. 7echn01. 1986, 8, 360-364

17 511n1n9er, P. J. 80tha5t, R. J., Lad15ch, M. R. and 0k05, M. R. 0pt1mum pH and temperature c0nd1t10n5 f0r xy105e fermentat10n 6y P1ch1a 5t1p1t15. 810techn01. 810en9. 1990, 35, 727-731

18 8ar605a, M. de F. 5., Lee, H., 5chne1der, H. and F0r56er9, C. W. 7emperature med1ated chan9e5 0f 0-xy105e meta60115m 1n the yea5t Pachy501en tann0ph11u5. FEM5 M1cr06101. Lett. 1990, 72, 35-40

19 du Pree2, J. C., 805ch, M. and Pr10r, 8. A. 7emperature pr0f11e5 0f 9r0wth and ethan01 t01erance 0f the xy105e-ferment1n9 yea5t5 Can- d1da 5hehatae and P1ch1a 5t1p1t15. App1. M1cr06101. 810techn01. 1987, 25, 521-525

20 511n1n9er, P. J., 8ran5trat0r, L E., 80tha5t, R. J., 0k05, M. R. and Lad15ch, M. R. 6r0wth, death, and 0xY9en uptake k1net1c5 0fP1ch1a 5t1p1t15 0n xy105e. 810techn01. 810en9. 1991, 37, 973-980

21 Lap1ace, J. M., De19ene5, J. P., M01etta, R. and Navarr0, J. M. C0m61ned a1c0h011c fermentat10n 0f D-xy105e and D-91uc05e 6y f0ur 5e1ected m1cr061a15tra1n5:Pr0ce55 c0n51derat10n51n re1at10n t0 eth- an01 t01erance. 810techn01. Lett. 1991, 13, 445-450

22 R06ert0,1. C., Lac15, L. 5., 8ar605a, M. F, 5. and de Manch111a, 1. M. Ut1112at10n 0f 5u9ar cane 6a9a55e hem1ce11u1051c hydr01y5ate 6y P1- ch1a 5t1p1t15 f0r the pr0duct10n 0f ethan01. Pr0ce55810chem. 1991, 26, 15-21

23 R1221, M., K1e1n, C., 5chu12e, C., 8u1-7hanh, N. -A. and De11we9, H. Xy105e fermentat10n 6y yea5t5.5. U5e 0fA7P 6a1ance5 f0r m0de11n9 0xY9en-11m1ted 9r0wth and fermentat10n 0f yea5t P1ch1a 5t1p1t15 w1th xy105e a5 car60n 50urce. 810techn01. 810en9. 1989, 34, 509-514

24 De19ene5, J. P., M01etta, R. and Navarr0, J. M. Xy105e meta60115m 6y P1ch1a 5t1p1t15: 7he effect 0f ethan01. App1. M1cr06101. 810techn01. 1991, 35, 656-661

25 du Pree2, J. C., van Dr1e55e1, 8. and Pr10r, 8. A. Ethan01 t01erance 0f P1ch1a 5t1p1t15 and Cand1da 5hehatae 5tra1n51n fed-6atch cu1ture5 at c0ntr011ed 10w d15501ved 0xY9en 1eve15. App1. M1cr06101. 810techn01. 1989, 30, 53-58

26 511n1n9er, P. J., 80tha5t, R. J., 0k02, M. R. and Lad15ch, M. R. C0mparat1ve eva1uat10n 0f ethan01 pr0duct10n 6y xy105e-ferment1n9 yea5t5 pre5ented h19h xy105e c0ncentrat10n5. 810techn01. Lett. 1985, 7, 431-436

27 Yu, 5., Wayman, M. and Parekh, 5. K. Fermentat10n t0 ethan01 0f pent05e-c0nta1n1n9 5pent 5u1ph1te 114u0r. 810techn01. 810en9. 1987, 29, 1144-1150

28 Wayman, M. and Parekh, 5. Ethan01 and 5u9ar t01erance 0f Cand1da 5hehatae. 810techn01. Lett. 1985, 7, 909-912

29 N19am, J. N., Mar9ar1t15, A. and Lachance, M. A. Aer061c fermen- tat10n 0f 9-xy105e t0 ethan01 6y C1av15p0ra 5p.AppL Env1r0n. M1cr0- 610L 1985, 50, 763-766

30 8ru1nen6er9, P. M., de 80t, P. H. M., van D1jken, J. P. and 5cheffer5, W. A. NADH-11nked a1d05e reducta5e: 7he key t0 anaer061c a1c0- h011c fermentat10n 0f xy105e 6y yea5t5. App1. M1cr06101. 810techn01. 1984, 19, 256-260

31 R1221, M., Harwart, K., 8u1-7hanh, N. -A. and De11we9, H. A k1net1c 5tudy 0f the NAD + -xy11t01-dehydr09ena5e fr0m the yea5t P1ch1a 5t1p1t15. J. Ferment. 810en9. 1989, 67, 25-30

32 R1221, M., Harwart, K., Er1emann, P., 8u1-7hanh, N. -A. and De11we9, H. Pur1f1cat10n and pr0pert1e5 0f the NAD •-xy11t01-dehydr09ena5e fr0m the yea5t P1ch1a 5t1p1#5. J. Ferment. 810en9. 1989, 67, 20-24

33 L19the1m, M. E., Pr10r, 8. A. and du Pree2, J. C. 7he effect 0f re5p1rat0ry 1nh161t0r5 0n the fermentat1ve a6111ty 0f P1ch1a 5t1p1t15, Pachy501en tann0ph11u5 and 5acchar0myce5 cerev151ae under var10u5 c0nd1t10n5 0faer0610515.App1. M1cr06101. 810techn01. 1988, 29, 67-71

34 H6fer, M. and Na55ar, F. R, Aer061c and anaer061c uptake 0f 5u9ar5 1n 5ch1205acchar0myce5 p0m6e. J. 6en. M1cr06101. 1987, 133, 2163- 2172

35 du Pree2, J. C., van Dr1e55e1, 8. and Pr10r, 8. A. Effect 0f aer0610515 0n fermentat10n and key en2yme 1eve15 dur1n9 9r0wth 0f P1ch1a 5t1p1t15, Cand1da 5hehatae and Cand1da tenu15 0n D-xy105e. Arch. M1cr06101. 1989, 152, 143-147

36 511n1n9er, P. J., 80tha5t, R. J., van Cauwen6er9e, J. E. and Kurt2- man, C. P. C0nver510n 0f 0-xy105e t0 ethan016y the yea5t Pachy501en tann0ph11u5. 810techn01. 810en9. 1982, 24, 371-384

37 5chve5ter, P,, R061n50n, C. W. and M00-Y0un9, M. Xy105e fermen- tat10n t0 ethan01 6y Pachy501en tann0ph11u5. 810techn01. 810en9. 5ymp. 1983, 13, 131-152

38 du Pree2, J. C., Pr10r, 8. A. and M0nte1r0, A. M. 7. 7he effect 0f aerat10n 0n xy105e fermentat10n 6y Cand1da 5hehatae and Pachy- 501en tann0ph11u5. App1. M1cr0610L 810techn01. 1984, 19, 261-266

39 5reenath, H. K., Chapman, 7. W. and Jeffr1e5, 7. W. Ethan01 pr0- duct10n fr0m D-xy105e 1n 6atch fermentat10n5 w1th Cand1da 5heha- tae: Pr0ce55 var1a61e5.App1. M1cr06101. 810techn01. 1986, 24, 294-299

40 61r10, F. M., Pe1t0, M. A. and Amara1-C011ac0, M. 7. En2ymat1c and phy5101091ca1 5tudy 0f D-xy105e meta60115m 6y Cand1da 5hehatae. App1. M1cr06101. 810techn01. 1989, 32, 199-204

41 A1exander, M. A., Chapman, 7. W. and Jeffr1e5, 7. W. C0nt1nu0u5 ethan01 pr0duct10n fr0m 0-xy105e 6y Cand1da 5hehatae. 810techn0L 810en9. 1987, 30, 685-691

42 De19ene5, J. P., M01etta, R. and Navarr0, J. M. 7he effect 0f aerat10n 0n D-xy105e fermentat10n 6y Pachy501en tann0ph11u5, P1ch1a 5t1p1t15, K1uyver0myce5 marx1anu5 and Cand1da 5hehatae. 810techn01. Lett. 1986, 8, 897-900

En2yme M1cr06.7echn01., 1994, v01.16, N0vem6er 953

Rev1ew5

43 Wat50n, N. E., Pr10r, 8. A., du Pree2, J. C. and Late9an, P. M. 0xY9en re4u1rement5 f0r D-xY105e fermentat10n t0 ethan01 and p01y- 015 6y Pachy501en tann0ph11u5. En2yme M1cr06. 7echn01. 1984, 6, 447-450

44 Lap1ace, J. M., De19ene5, J. P., M01etta, R. and Navarr0, J. M. A1c0h011c fermentat10n 0f 91uc05e and xy105e 6y P1ch1a 5t1p1t15, Can- d1da 5hehatae, 5acchar0myce5 cerev151ae and 2yrn0rn0na5 m061115: 0xY9en re4u1rement a5 key fact0r.App1. M1cr06101. 810techn01. 1991, 36, 158-162

45 De19ene5, J. P., M01etta, R. and Navarr0, J. M. Fermentat10n 0f D-xy105e, D-91uc05e, L-ara61n05e m1xture 6y P1ch1a 5t1p1t15: Effect 0f the 0xy9en tran5fer rate 0n fermentat10n perf0rmance. 810techn0L 810en9. 1989, 34, 398-402

46 du Pree2, J. C., van Dr1e55e1, 8. and Pr10r, 8. A. D-Xy105e fermen- tat10n 6y Cand1da 5hehatae and P1ch1a 5t1p1t15 at 10w d15501ved 0xY9en 1eve151n fed-6atch cu1ture5. 810techn01. Lett. 1989, 11, 131-136

47 De11we9, H., R1221, M. and K1e1n, C. C0ntr011ed 11m1ted aerat10n and meta6011c re9u1at10n dur1n9 the pr0duct10n 0f ethan01 fr0m D-xY105e 6y P1ch1a 5t1p1t15. J. 810techn0L 1989, 12, 111-122

48 Cu16ert, 5. J. and Wan9, Y. -M. 0ra1xY11t011n Amer1can adu1t5. Nutr. Re5. 1986, 6, 913-922

49 Hyv6nen, L. and K01v15t01nen, P. F00d techn01091ca1 eva1uat10n 0f xy11t01. Adv. F00d Re5. 1983, 28, 273-403

50 Meyr1a1, V., De19ene5, J. P., M01etta, R. and Navarr0, J. M. Xy11t01 pr0duct10n fr0m D-xy105e 6y Cand1da 9u111erm0nd11: Fermentat10n 6ehav10ur. 810techn0L Lett. 1991, 13, 281-286

51 8ar605a, M. F. 5., de Mede1r05, M. 8., de Manc11ha, 1. M., 5chne1- der, H. and Lee, H. 5creen1n9 0fyea5t5 f0r pr0duct10n 0fxy11t01 fr0m 0-xy105e and 50me fact0r5 wh1ch affect xy11t01 y1e1d 1n Cand1da 9u11- 11erm0nd11. J. 1nd. M1cr0610L 1988, 3, 241-251

52 Chen, L. -F. and 60n9, C. -5. Fermentat10n 0f 5u9arcane 6a9a55e hem1ce11u105e hydr01y5ate t0 xy11t01 6y a hydr01y5ate-acc11mat12ed yea5t. J. F00d 5c1. 1985, 50, 226-228

53 Ha1160rn, J., Wa1fr1d550n, M., A1rak51nen, U., 0jam0, H., Hahn- H~19erda1, 8., Pentt11a, M. and Ker~1nen, 5. Xy11t01 pr0duct10n 6y re- c0m61nant 5acchar0myce5 cerev151ae. 810techn0109y 1991, 9, 1090-1095

54 La5t1ck, 5. M., 7ucker, M. Y., 8eyette, J. R., N011, 6. R. and 6r0h- mann, K. 51mu1tane0u5 fermentat10n and 150mer12at10n 0f xy105e. AppL M1cr06101. 810techn0L 1989, 30, 574-579

55 Ch1an9, L. -C., H51a0, H. -Y., Uen9, P. P., Chen, L. -F. and 75a0, 6. 7. Ethan01 pr0duct10n fr0m xy105e 6y en2ymat1c 150mer12at10n and yea5t fermentat10n. 810techn0L 810en9. 5ymp. 1981, 11, 263-274

56 Uen9, P. P., Hunter, C. A., 60n9, C. -5. and 75a0, 6 .7 . D-Xy1u105e fermentat10n 1n yea5t5. 810techn0L Lett. 1982, 3, 315-320

57 Wan9, P. Y. and 5chne1der, H. 6r0wth 0f yea5t5 0n D-xy1u105e. Can. J. M1cr06101. 1980, 26, 1165-1168

58 H51a0, H. -Y., Ch1an9, L. -C., Uen9, P. P. and 75a0, 6.7.5e4uent1a1 ut1112at10n 0f m1xed m0n05acchar1de5 6y yea5t5. AppL Env1r0n. M1- cr06101. 1982, 43, 840-845

59 L1nd~n, 7. and Hahn-H/19erda1, 8. Fermentat10n 0f 119n0ce11u105e hydr01y5ate5 w1th yea5t5 and xy105e 150mera5e. En2yme M1cr06. 7ech- n01. 1989, 11, 583-589

60 R0man, 6. N., Jan5en, N. 8., H51a0, H. Y. and 75a0, 6 . 7 . K1net1c 5tud1e5 0f the en2ymat1c 150mer12at10n 0f xy105e. En2yme M1cr06. 7echn0L 1985, 7, 129-133

61 011v1er, 5. P. and du 701t, P. J. 5u9ar cane 6a9a55e a5 a p055161e 50urce 0f fermenta61e car60hydrate5.11.0pt1m12at10n 0f the xy105e 150mera5e react10n f0r 150mer12at10n 0f xy105e a5 we11 a5 5u9ar cane 6a9a55e hydr01y2ate t0 xY1u105e 1n 1a60rat0ry-5ca1e un1t5.810techn01. 810en9. 1986, 28, 684-699

62 H51a0, H. -Y., Ch1an9, L. -C., Chen, L. F. and 75a0, 6 .7 . Effect5 0f 60rate 0n 150mer12at10n and yea5t fermentat10n 0f h19h xy1u105e 501ut10n and ac1d hydr01y5ate 0f hem1ce11u105e. En2yme M1cr06. 7echn01. 1982, 4, 25-31

63 Ch1an9, L. -C., 60n9, C. -5., Chen, L. -F. and 75a0, 6 .7 . D-Xy1u105e fermentat10n t0 ethan01 6y 5acchar0myce5 cerev151ae. AppL Env1r0n. M1cr06101. 1981, 41, 284-289

64 60n9, C. -5., Chen, L. -F., F11ck1n9er, M. C., Ch1an9, L. -C. and 75a0, 6. 7. Pr0duct10n 0f ethan01 fr0m 9-xy105e 6y u51n9 9-xy105e 150mera5e and yea5t5.AppL Env1r0n. M1cr0610L 1981, 41, 430-436

65 Hahn-H~19erda1, 8., 8erner, 5. and 5k009, K. 1mpr0ved ethan01 pr0duct10n fr0m xy105e w1th 91uc05e 150mera5e and 5acchar0myce5 cerev151ae u51n9 the re5p1rat0ry 1nh161t0r a21de. App1. M1cr0610L 810- techn0L 1986, 24, 287-293

66 51tt0n, 0. C., F0utch, 6. L., 800k, L. N. and 6addy, J. L. Ethan01 fr0m a9r1cu1tura1 re51due5. Pr0ce55 810chem. 1979, 14 (9), 7-10

67 60rden, A. H., L0max, J. A. and Che550n, A. 61yc051d1c 11nka9e5 0f 1e9ume, 9ra55 and cerea15traw ce11 wa115 6ef0re and after exten51ve de9radat10n 6y rumen m1cr00r9an15m5. J. 5c1. F00dA9r1c. 1983, 34, 1341-1350

68 8urden, D. W. and Eve1e19h, D. E. Yea5t5--D1ver5e 5u65trate5 and pr0duct5.1n: Yea5t 7echn0109y (5pencer, J. F. 7. and 5pencer, D. M., ed5.). 5pr1n9er-Ver1a9, 8er11n, 1990, pp. 199-227

69 Ne1r1nck, L., Ma1e52ka, R. and 5chne1der, H. A1c0h01 pr0duct10n fr0m 5u9ar m1xture5 6y Pachy501en tann0ph11u5. 810techn01. 810en9. 5ymp. 1982, 12, 161-169

70 Wan9, P. Y., J0hn50n, 8. F. and 5chne1der, H. Fermentat10n 0f D-xy105e 6y yea5t5 u51n9 91uc05e 150mera5e 1n the med1um t0 c0nvert D-xy105e t0 D-xy1u105e. 810techn01. Lett. 1980, 2, 273-278

71 Jeffr1e5, 7. W. C0mpar150n 0f a1ternat1ve5 f0r the fermentat10n 0f pent05e5 t0 ethan01 6y yea5t5. 1n: Ener9y App11cat10n5 0f810ma55: Pr0ceed1n95, Nat10na1 Meet1n9 0n 810ma55 R • D f0r Ener9y App11- cat10n5 (L0wen5te1n, M. 2., ed.). E15ev1er App11ed 5c1ence Pu6115h- er5, New Y0rk, 1985, pp. 231-252

72 Lee, H. and McCa5key, 7. A. Hem1ce11u105e hydr01y515 and fermen- tat10n 0f re5u1t1n9 pent05e5 t0 ethan01. 7app1J. 1983, 66, 102-107

73 W1150n, J. J., De5chate1et5, L. and N15h1kawa, N. K. C0mparat1ve fermenta6111ty 0f en2ymat1c and ac1d hydr01y5ate5 0f 5team- pretreated a5penw00d hem1ce11u105e 6y P1ch1a 5t1p1t15 C85 5776. App1. M1cr06101. 810techn01. 1989, 31, 592-596

74 Van 2y1, C., Pr10r, 8. A. and du Pree2, J. C. Acet1c ac1d 1nh161t10n 0f D-xy105e fermentat10n 6y P1ch1a 5t1p1t15. En2yme M1cr06. 7echn01. 1991, 13, 82-86

75 De19ene5, J. P., M01etta, R. and Navarr0, J. M. Ac1d hydr01y515 01 wheat 5traw and pr0ce55 c0n51derat10n5 f0r ethan01 fermentat10n 6y P1ch1a 5t1p1t15 Y7124. Pr0ce55 810chem. 1990, 25, 132-135

76 Detr0y, R. W., Cunn1n9ham, R. L. and Herman, A. 1. Fermentat10n 0fwheat 5traw hem1ce11u105e5 t0 ethan016y Pachy501en tann0ph11u5. 810techn0L 810en9. 5ymp. 1982, 12, 81-89

77 Parekh, 5. R., Parekh, R. 5. and Wayman, M. Ethan01 and 6utan01 pr0duct10n 6y fermentat10n 0f en2ymat1ca11y 5acchar1f1ed 502-prehydr01y5ed 119n0ce11u1051c5. En2yme M1cr06. 7echn01. 1988, 10, 660-668

78 8ar605a, M. de F. 5., 8eck, M. J., Fe1n, J. E., P0tt5, D. and 1n9ram, L. 0. Eff1c1ent fermentat10n 0f P/nu5 5p. ac1d hydr01y5ate5 6y an ethan0109en1c 5tra1n 0f E5cher1ch1a c011. App1. Env1r0n. M1cr0610L 1992, 58, 1382-1384

79 Wat50n, N. E., Pr10r, 8. A. and Late9an, P. M. Fact0r5 1n ac1d treated 6a9a55e 1nh161t1n9 ethan01 pr0duct10n fr0m D-xy105e 6y Pachy501en tann0ph11u5. En2yme M1cr06. 7echn01. 1984, 6, 451- 456

80 Wayman, M., Parekh, 5., Ch0rnet, E. and 0verend, R. P. 502-cata1y5ed prehydr01y515 0fc0n1fer0u5 w00d f0r ethan01 pr0duc- t10n. 810techn0L Lett. 1986, 8, 749-752

81 Parekh, 5. R., Parekh, R. 5. and Wayman, M. Fermentat10n 0f w00d-der1ved ac1d hydr01y5ate5 1n a 6atch 610react0r and 1n a c0n- t1nu0u5 dynam1c 1mm061112ed ce11 610react0r 6y P1ch1a 5t1p1t15 R. Pr0ce55 810chem. 1987, 22, 85-91

82 81ch0, P. A., Runna15, P. L., Cunn1n9ham, J. D. and Lee, H. 1nduc- t10n 0f xy105e reducta5e and xy11t01 dehydr09ena5e act1v1t1e51n Pach- y501en tann0ph11u5 and P1ch1a 5t1p1t15 0n m1xed 5u9ar5.App1. Env1r0n. M1cr06101. 1988, 54, 50-54

83 Jeffr1e5, 7. W. and 5reenath, H. K. Fermentat10n 0f hem1ce11u1051c 5u9ar5 and 5u9ar m1xture5 6y Cand1da 5hehatae. 810techn01. 810en9. 1988, 31, 502-506

84 Lee, H. Rever5161e 1nact1vat10n 0f D-xy105e ut1112at10n 6y D-91uc05e 1n the pent05e-ferment1n9 yea5t Pachy501en tann0ph11u5. FEM5 M1- cr06101. Lett. 1992, 92, 1-4.

85 Pancha1, C. J., 8a5t, L., Ru55e1,1. and 5tewart, 6 . 6 . Repre5510n 0[ xy105e ut1112at10n 6y 91uc05e 1n xy105e-ferment1n9 yea5t5. Can. J. M1cr06101. 1988, 34, 1316-1320

86 6r00tjen, D. R. J., van der Lan5, R. 6. J. M. and Luy6en, K. Ch. A. M. C0nver510n 0f 91uc05e/xy105e m1xture5 6y P1ch1a 5t1p1t15 under 0xY9en-11m1ted c0nd1t10n5. En2yme M1cr06. 7echn01. 1991, 13, 648-654

87 K111an, 5.6. and van Uden, N. 7ran5p0rt 0fxy105e and 91uc05e 1n the xy105e-ferment1n9 yea5t P1ch1a 5t1p1t15. AppL M1cr0610L 810techn0L 1988, 27, 545-548

954 En2yme M1cr06.7echn01., 1994, v01.16, N0vem6er

Env1r0nmenta1 fact0r5 affect1n9 0-xy105e fermentat10n: J. C. du Pree2

88 Luca5, C. and van Uden, N. 7ran5p0rt 0f hem1ce11u105e m0n0mer5 1n the xy105e-ferment1n9 yea5t. Cand1da 5hehatae. App1. M1cr06101. 810techn01. 1986, 23, 491-495

89 Ka5tner, J. R. and R06ert5, R. 5. 51mu1tane0u5 fermentat10n 0f D-xy105e and 91uc05e 6y Cand1da 5hehatae. 810tec1m01. Lett. 1990, 12, 57-60

90 Devere11, K. F. Ethan01 pr0duct10n fr0m w00d hydr01y5ate5 u51n9. Pachy501en tann0ph11u5. 810techn01. Lett. 1983, 5, 475-480

91 8eck, M. J. Effect 0f 1nterm1ttent feed1n9 0f ce11u105e hydr01y2ate t0 hem1ceUu105e hydr01y2ate 0n ethan01 y1e1d 6y Pachy501en tann0- ph11u5. 810techn01. Lett. 1986, 8, 513-516

92 Van 2y1, C., Pr10r, 8. A. and du Pree2, J. C. Pr0duct10n 0f ethan01 fr0m 5u9ar cane 6a9a55e hem1ce11u105e hydr01y2ate 6y P1ch1a 5t1p1- t15. App1. 810chem. 810techn01. 1988, 17, 357-369

93 Lee, H., 81e1y, P. Latta, R. K., 8ar605a, M. F. 5. and 5chne1der, H. Ut1112at10n 0fxy1an 6y yea5t5 and 1t5 c0nver510n t0 ethan016yP1ch1a 5t1p1t15 5tra1n5. App1. Env1r0n. M1cr06101. 1986, 52, 320-324

94 701v01a, A., Yarr0w, D., van den 805ch, E., van D1jken, J. P. and 5cheffer5, W. A. A1c0h011c fermentat10n 0f D-xy105e 6yyea5t5.App1. Env1r0r~ M1cr06101. 1984, 47, 1221-1223

95 60n9, C. -5., C1ayp001, 7. A., McCracken, L. D., Maun, C. M., Uen9, P. P. and 75a0, 6. 7. C0nver510n 0f pent05e5 6y yea5t5. 810techn01. 810en9. 1983, 25, 85-102

96 du Pree2, J. C. and Pr10r, 8. A. A 4uant1tat1ve 5creen1n9 0f 50me xy105e-ferment1n9 yea5t 1501ate5. 810techn01. Lett. 1985, 7, 241- 246

97 A1exander, M. A., Chapman, 7. W. and Jeffr1e5, 7. W. C0nt1nu0u5 xy105e fermentat10n 6y Cand1da 5hehatae 1n a tw0-5ta9e react0r. App1. 810chem. 810techn01. 1988, 17, 221-229

98 A1exander, M. A., Chapman, 7. W. and Jeffr1e5, 7. W. Xy105e meta60115m 6y Cand1da 5hehatae 1n c0nt1nu0u5 cu1ture. App1. M1- cr06101. 810techn01. 1988, 28, 478-486

99 A1exander, M. A. Chapman, 7. W. and Jeffr1e5, 7. W. C0nt1nu0u5- cu1ture re5p0n5e5 0f Cand1da 5hehatae t0 5h1ft51n temperature and aerat10n: 1mp11cat10n5 f0r ethan011nh161t10n.App1. Env1r0n. M1cr0- 6101. 1989, 55, 2152-2154

100 1n9ram, L. 0. and 8uttke, 7. M. Effect5 0f a1c0h015 0n m1cr0- 0r9an15m5. Adv. M1cr06. Phy5101. 1984, 25, 253-300

101 L1nk0, Y. -Y., Kaut01a, H., U0t11a, 5. and L1nk0, P. A1c0h011c fer- mentat10n 0f D-xy105e 6y 1mm061112ed P1ch1a 5t1p1t15 yea5t. 810tech- n01. Lett. 1986, 8, 47-52

102 6r00tjen, D. R. J,, Me1j11nk, L. H. H. M., van der Lan5, R. 6. J. M. and Luy6en, K. Ch. A. M. C0fermentat10n 0f 91uc05e and xy105e w1th 1mm061112ed P1ch1a 5t1p1t15 and 5acchar0myce5 cerev151ae. En- 2yrne M1cr06. 7echn01. 1990, 12, 860-864

103 6r00tjen, D. R. J., Me1j11nk, L. H. H. M., V1ee5en6eek, R., van der Lan5, R. 6. J. M, and Luy6en, K. Ch. A. M. C0fermentat10n 0f 91uc05e and xy105e w1th 1mm061112ed P1ch1a 5t1p1t15 1n c0m61nat10n w1th 5acchar0myce5 cerev151ae. En2yme M1cr06. 7echn01. 1991, 13, 530-536

104 6r00tjen, D. R. J., V1ee5en6eek, R., W1ndme1jer, M. 6. A., van der Lan5, R. 6. J. M. and Luy6en, K. Ch. A. M. A f10ccu1at1n9 5tra1n 0f P1ch1a 5t1p1t15 f0r the c0nver510n 0f 91uc05e/xy105e m1xture5. En2yme M1cr06. 7echn01. 1991, 13, 734-739

105 511n1n9er, P. J., 80tha5t, R. J., 81ack, L. 7. and Mc6hee, J. E. C0nt1nu0u5 c0nver510n 0f D-xy105e t0 ethan01 6y 1mm061112ed Pachy501en tann0ph11a5. 810techn01. 810en9. 1982, 24, 2241-2251

106 6r00tjen, D. R. J., Jan5en, M. L., van der Lan5, R. 6. J. M. and Luy6en, K. Ch. A. M. React0r5 1n 5er1e5 f0r the c0mp1ete c0nver- 510n 0f 91uc05e/xy105e m1xture5 6y 1~ch1a 5t1p1t15 and 5acchar0myce5 cerev151ae. En2yme M1cr06. 7echn01. 1991, 13, 828-833

107 Lap1ace, J. M., De19ene5, J. P., M01etta, R. and Navarr0, J. M. Ethan01 pr0duct10n fr0m 91uc05e and xy105e 6y 5eparated and c0- cu1ture pr0ce55e5 u51n9 h19h ce11 den51ty 5y5tem5. Pr0ce55 810chem. 1993, 28, 519-525

108 Lap1ace, J. M., De19ene5, J. P., M01etta, R. and Navarr0, J. M. Effect5 0f cu1ture c0nd1t10n5 0n the c0-fermentat10n 0f a 91uc05e and xy105e m1xture t0 ethan01 6y a mutant 0f 5acchar0myce5 d1a- 5tat1cu5 a550c1ated w1th P1ch1a 5t1p1t15. App1. M1cr06101. 810techn01. 1993, 39, 760-763

109 511n1n9er, P. J. and 80tha5t, R. J. C0nt1nu0u5 fermentat10n 0f feed 5tream5 c0nta1n1n9 D-91uc05e and D-xy105e 1n a tw0-5ta9e pr0ce55 ut11121n91mm061112ed 5acchar0myce5 cerev151ae and Pachy501en tan- n0ph11u5. 810techn01. 810en9. 1988, 32, 1104-1112

110 Lap1ace, J. M., De19ene5, J. P. M01etta, R. and Navarr0, J. M. A1c0h011c 91uc05e and xy105e fermentat10n5 6y the c0cu1ture pr0- ce55: C0mpat16111ty and typ1n9 0f a550c1ated 5tra1n5. Can. J. M1cr0- 6101, 1992, 38, 654-658

111 du Pree2, J. C., van Dr1e55e1, 8. and Pr10r, 8. A. 7he re1at10n 6etween red0x p0tent1a1 and D-xy105e fermentat10n 6y Cand1da 5hehatae and P1ch1a 5t1p1t15. 810techn01. Lett. 1988, 10, 901-906

112 M15h1ma, K., M1mura, A. and 7akahara, 7. 0n-11ne m0n1t0r1n9 0f ce11 c0ncentrat10n5 dur1n9 yea5t cu1t1vat10n 6y d1e1ectr1c mea5ure- ment5. 2 Ferment. 810en9. 1991, 72, 296-299

113 Ma1e52ka, R., Ve11ky, 1. A. and 5chne1der, H. Enhanced rate 0f ethan01 pr0duct10n fr0m D-xy105e u51n9 recyc1ed 0r 1mm061112ed ce115 0f Pachy501en tann0ph11u5. 810techn01. Lett. 1981, 3, 415-420

114 W00d5, M. A. and M11115, N. F. Effect 0f 510w feed1n9 0f xy105e 0n ethan01 y1e1d 6y Pachy501en tann0ph11u5. 810techn01. Lett. 1985, 7, 679-682

115 Dekker, R. F. H. L1p1d-enhanced ethan01 pr0duct10n fr0m xy105e 6y Pachy501en tann0ph11u5. 810techn01. 810en9. 1986, 28, 605-608

116 L0hme1er-V09e1, E., 5k009, K., V09e1, H. and Hahn-H/19erda1, 8. 31p nuc1ear ma9net1c re50nance 5tudy 0f the effect 0f a21de 0n xy105e fermentat10n 6y Cand1da tr0p1ca115. App1. Env1r0n. M1cr06101. 1989, 55, 1974-1980

117 K6tter, P. and C1r1acy, M. Xy105e fermentat10n 6y 5acchar0myce5 cerev151ae. App1. M1cr06101. 810techn01. 1993, 38, 776-783

118 Chun9, 1. 5. and Lee, Y. Y. Effect 0f/n 51tu ethan01 rem0va1 0n fermentat10n 0f D-xy105e 6y Pachy501en tann0ph11u5. En2yme M1- cr06. 7echn01. 1985, 7, 217-219

119 McCracken, L. D. and 60n9, C. -5. D-Xy105e meta60115m 6y mu- tant 5tra1n5 0f Cand1da 5p.Adv. 810chem. En9. 810techn0L 1983, 27, 33-55

120 60n9, C. -5., McCracken, L. D. and 75a0, 6 .7 . D1rect fermenta- t10n 0f D-xy105e t0 ethan01 6y a xy105e-ferment1n9 yea5t mutant, Cand1da 5p. XF 217. 810techn01. Lett. 1981, 3, 245-250

121 Jeffr1e5, 7. W. Mutant5 0f Pachy501en tann0ph11u5 5h0w1n9 en- hanced rate5 0f 9r0wth and ethan01 f0rmat10n fr0m D-xy105e. En- 2yme M1cr06. 7echn01. 1984, 6, 254-258

122 C1ark, 7., Wed10ck, N., Jame5, A. P., DevereU, K. and 7h0rnt0n, R. J. 5tra1n 1mpr0vement 0f the xy105e-ferment1n9 yea5t Pachy501en tann0ph11u5 6y hy6r1d15at10n 0ftw0 mutant 5tra1n5.810techn01. Lett. 1986, 8, 801-806

123 801en, P. L. and 511n1n9er, P. J, C0nt1nu0u5 cu1ture 5e1ect10n 0f mutant 5tra1n5 0f Pachy501en tann0ph11u5 capa61e 0f rap1d aer061c 9r0wth 0n 0-xy105e. Dev. 1nd. M1cr06101. 1984, 25, 449-457

124 Lee, H., Jame5, A. P., 2aha6, D. M., Mahm0ur1de5, 6., Ma1e52ka, R. and 5chne1der, H. Mutant5 0fPachy501en tann0ph11u5 w1th 1m- pr0ved pr0duct10n 0f ethan01 fr0m 0-xy105e.App1. Env1r0n. M1cr0- 6101. 1986, 51, 1252-1258

125 6upthar, A. 5. C0n5truct10n 0f a 5er1e5 0fP/ch/a 5t1p1t15 5tra1n5 w1th 1ncrea5ed DNA c0ntent5. Curr. 6enet. 1987, 12, 605-610

126 J0hann5en, E., Ea91e, L. and 8redenhann, 6. Pr0t0p1a5t fu510n u5ed f0r the c0n5truct10n 0f pre5umpt1ve p01yp101d5 0f the D-xy105e ferment1n9 yea5t Cand1da 5hehatae. Curr. 6enet. 1985, 9, 313-319

127 Wan9, 6.5. and Wan9, L. H. 1mpr0vement 0n ethan01-t01erance 0f xy105e-ferment1n9 yea5t 6y pr0t0p1a5t fu510n. Rept. 7a1wan 5u9ar Re5. 1n5t. 1989, 124, 29-37

128 He1uane, H., 5pencer, J. F., 5pencer, D. and de F19uer0a, L. Char- acter12at10n and 6ehav10ur 0f1nter9ener1c hy6r1d5 06ta1ned 6y pr0- t0p1a5t fu510n 6etween Pachy501en tann0ph11u5 and 5acchar0myce5 cerev151ae. A65tr., 16th 1nt. C0nf. 0n Yea5t 6enet1c5 and M01ecu1ar 810109Y, V1enna, Au9. 15-21, 1992. Yea5t 8 (5pec1a1155ue)

129 K6tter, P., Am0re, R., H011en6er9, C. P. and C1r1acy, M. 1501at10n and character12at10n 0f the P1ch1a 5t~01t15 xy11t01 dehydr09ena5e 9ene, XYL2, and c0n5truct10n 0f a xy105e-ut11121n9 5acchar0myce5 cerev151ae tran5f0rmant. Curr. 6enet. 1990, 18, 493-500

130 7ant1run9k1j, M., Naka5h1ma, N., 5ek1, 7. and Y05h1da, 7. C0n- 5truct10n 0f xy105e-a551m11at1n9 5acchar0myce5 cerev151ae. J. Fer- ment. 810en9. 1993, 75, 83-88

131 Am0re, R., W11he1m, M. and H011en6er9, C. P. 7he fermentat10n 0f xy105e--An ana1y515 0f the expre5510n 0f8ac111u5 andAct1n0p1ane5 xy105e 150mera5e 9ene51n yea5t5.App1. M1cr06101. 810techn01. 1989, 30, 351-357

132 5arthy, A. V., McC0nau9hy, 8. L., L060, 2., 5und5tr0m, J. A., Fur10n9, C. E. and Ha11, 8. D. Expre5510n 0f the E5cher1ch1a c011

En2yme M1cr06.7echn01., 1994, v01.16, N0vem6er 955

Rev1ew5

xy105e 150mera5e 9ene 1n 5acchar0myce5 cerev151ae. App1. Env1r0n. M1cr06101. 1987, 53, 1996-2000

133 Chan, E. -C., Uen9, P. P. and Chen, L. 0-Xy105e fermentat10n t0 ethan01 6y 5ch1205acchar0myce5 p0m6e c10ned w1th xy105e 150mera5e 9ene. 810techn01. Lett. 1986, 8, 231-234

134 8ea11, D. 5., 0hta, K. and 1n9ram, L. 0. Parametr1c 5tud1e5 0f ethan01 pr0duct10n fr0m xy105e and 0ther 5u9ar5 6y rec0m61nant E5cher1ch1a c011. 810techn01. 810en9. 1991, 38, 296-303

135 Lawf0rd, H. 6. and R0u55eau, J. D. Fue1 ethan01 fr0m hardw00d hem1ce11u105e hydr01y5ate 6y 9enet1ca11y en91neered E5chet1ch1a c011 8 carrY1n9 9ene5 fr0m 2ym0m0na5 m061115. 810techn0L Lett. 1991, 13, 191-196

136 01550n, L. and Hahn-H~19erda1, 8. Fermentat1ve perf0rmance 0f 6acter1a and yea5t51n 119n0ce11u105e hydr01y5ate5. Pr0ce55 810chem. 1993, 28, 249-257

137 5k009, K. and Hahn-H/19erda1, 8. Effect 0f 0xY9enat10n 0n xy105e fermentat10n 6y P1ch1a 5t1p1t15. App1. Env1r0n. M1cr0610L 1990, 56, 3389-3394

138 V0n95uvan1ert, V. and 7an1, Y. Xy11t01 pr0duct10n 6y a methan01 yea5t, Cand1da 601d1n11 (R10eckera 5p.) N0. 2201. J. Ferment. 810en9. 1989, 67, 35-39

139 R06ert0, 1. C., Fe11pe, M. 6. A., Lac15, L. 5., 511va, 5.5. and de Manc11ha, 1. M. Ut1112at10n 0f 5u9ar cane 6a9a55e hem1ce11u1051c hydr01y2ate 6y Cand1da 9u1U1erm0nd11 f0r xy11t01 pr0duct10n. 810re- 50urce 7echn01. 1991, 36, 271-275

140 Pere90, P., C0nvert1, A., Pa1a221, E., de1 80r9h1, M. and Ferra1010, 6. Fermentat10n 0f hardw00d hem1ce11u105e hydr01y5ate 6y Pach-

y501en tann0ph11u5, Cand1da 5hehatae and P1ch1a 5t1p1t15. J.1nd. M1- cr06101. 1990, 6, 157-164

141 Hajny, 6. J. 8101091ca1 ut1112at10n 0f w00d f0r pr0duct10n 0f chem- 1ca15 and f00d5tuff5. U5 Dept. A9r1c. F0r. 5erv. Pr0d. La6., Mad1- 50n, W1, Re5earch Paper FPL 385, 1981

142 60n9, C. 5. and 75a0, 6 . 7 . Pr0duct10n 0f ethan01 fr0m 610- ma55 hem1ce11u105e hydr01y2ate 6y yea5t5. Paper N0. 43, 5ymp. Amer1can 1n5t1tute 0f Chem1ca1 En91neer5, 0r1and0, F10r1da, 1982

143 8uchert, J., N1eme1~1, K., Pu15, J. and P0utanen, K. 1mpr0vement 1n the fermenta6111ty 0f 5teamed hem1ce11u105e hydr01y5ate 6y 10n exc1u510n. Pr0ce55 810chem. 1nt. 1990, 25, 176-180

144 Ma9ee, R. J. and K05ar1c, N. 810c0nver510n 0f hem1ce11u1051c5. Adv. 810chem. En9. 810techn01. 1985, 32, 61-93

145 Fe1n, J. E., 7a111m, 5. R. and Lawf0rd, F. R. Eva1uat10n 0f 0-xy105e ferment1n9 yea5t5 f0r ut1112at10n 0f a w00d-der1ved hem1ce11u105e hydr01y5ate. Can. J. M1cr06101. 1984, 30, 682-690

146 7ran, A. V. and Cham6er5, R. P. Ethan01 fermentat10n 0f red 0ak ac1d prehydr01y5ate 6y the yea5t P1ch1a 5t1p1t15 C85 5776. En2yme M1cr06. 7echn01. 1986, 8, 439-444

147 0hta, K., 8ea11, D. 5., Mej1a, J. P. and 5hanmu9am, K. 7. Meta6011c en91neer1n9 0fK1e651e11a 0xyt0ca M5A1 f0r ethan01 pr0duct10n fr0m xy105e and 91uc05e.App1. Env1r0n. M1cr06101. 1991, 57, 2810-2815

148 Fe1dmann, 5., 5pren9er, 6. A. and 8ahm, H. Ethan01 pr0duct10n fr0m xy105e w1th a pyruvate-f0rmate-1ya5e mutant 0f K1e651eUa p1ant1c01a carrY1n9 a pyruvate-decar60xy1a5e 9ene fr0m 2ym0m0- na5 m061115. App1. M1cr06101. 810techn01. 1989, 31, 152-157

956 En2yme M1cr06.7echn01., 1994, v01.16, N0vem6er