Anne-Sophie ALDIGUIER

Transcription

1 n d ordre : 820 Thèse présentée devant L Institut National des Sciences Appliquées de Toulouse en vue de l obtention du DOCTORAT spécialité : SCIENCES ECOLOGIQUE, VETERINAIRE, AGRONOMIQUE ET BIOINGENIERIES filière : MICROBIOLOGIE ET BIOCATALYSE INDUSTRIELLES par Anne-Sophie ALDIGUIER Ingénieur INSA Toulouse Activité bio-catalytique en haute densité cellulaire de Saccharomyces cerevisiae pour l intensification de la production de bio-éthanol Soutenue le 20 janvier 2006 devant la commission d examen Rapporteurs Marc I. Directeur de Recherche CNRS, Nancy Sablayrolles J.M. Directeur de Recherche INRA, Montpellier Président Ghommidh C. Professeur, Polytech Montpellier Examinateurs Alfenore S. Maître de Conférences, INSA Toulouse Goma G. Professeur, INSA Toulouse Jouve-Molina C. Maître de Conférences, INSA Toulouse Poitrat E. Ingénieur ADEME, Paris Cette thèse a été préparée au Laboratoire de Biotechnologie-Bioprocédés UMR CNRS 5504, l INRA et du Département de Génie Biochimique et Alimentaire de l INSA dans le cadre de l école doctorale SEVAB.

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19 3B- 2, / 1 / B- 2, >, // / 1 / 1 DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD 15 3&B- 2,, // / 1 / 1 DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD 154 3%B,:*9;=/ 1 /DDDDDDDDDDDDDD B 9 #/ 6 ' (2 G&:;/ DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD 17 34B/ 1 /DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD B- >2 / DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD B - 2, 2 A DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD 17& &B- γ2 :/ 1 ; #1#DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD 17 &1B- ":/ 1 ;2 :/ 1 ; #1#/ DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD 17 &B- 2 / 1 / DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD 174 &3B-, 2:M/ 1 ;2 :'/ 1 ; =3E/ DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD 177 &&B- 0,η :6/;2 :'/ 1 ;/ DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD &%B- 2,ε G 2 :/ 1 ; 1 &B.η 2 2,ε G / DDDDDDD 3 &4B" > η 2 2,ε G /DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD & &5B" >2 DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD &7B- 6@":;2 :/ 1 ; 2 1'/ 1 /DDDDDDDDDDDDDDDDDDDDDDDDDDD 7 %B- 8:;2 :/ 1 ; 2 1'/ 1 / DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD 1 %1B- =τ:6;2 :/ 1 ; 2 1'/ 1 /DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD 11 %B- #0 #2 :/ 1 ;/ DDDDD 1 %3B- 2 L: 3 / / 1 ;2 :; 06@" '44/ DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD 1&

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34 % 4( I.1 Métabolismes de la levure,(, 0& 0( & ( 0,# (0,,(9$LE9KK::# &,.,(,0(= 0 =, 0 0 #G &.' /,,,(,#% (&, &,(, 0.(.' # I.1.1 Métabolisme oxydatif,(,.'.',, E(=00,( ''&' M( 9D : '.'0# $. =,(,# G.' & (& 0H, # 1 &0,,(93'/9K>::#%E 0,,E,'&E',' 'M(,# (,.,0,(0 % G 7 U7G U;7*U;7$*7%G U;7*U>@ G -1E',,$@D$@ & 'M(=,,(.',,,9!@9KJ::# '.'0 1E',$@D$@ $ U D$#(=.',,, #.,,,,(,,(,,# & =,,'*=$* * V=*,,(# 0,(,*,,.',,*PG#0*PG0 #" ( ( 9 & ;>

35 % 4( && &:000, 39;::# 0,( (,.'(#, 0.'0, (,&H C # 1 9MW#9K7:&*,#9KK::# I.1.2 Métabolisme fermentaire,(,, A,,,( (.',, #%, #,(,&.' ( # '' 9D : 0 '0# 0.'0, 0'0#$,(,,&,.'(#, 0 0 = ',, #, ' (,, 09!@9KJ:&3'/9KK::#( 0 % G 7 7 GU%G U*,R,0 &, +'1&&H * # 1 #%,,,1 9., '&T:&(,#&, =1KX,#-0&,(, & C # 1 (,=(,(,.'#,,(.'. ;> *,,(,.'& Y, '.'09MW/9K7::# ;H

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38 % 4( # # #; 7:%(.;!!('(!(",& - I.1.4 Effet glucose G,,(, 1& 0 E',0 &E', (, #GN O# 0E',(=,!, & =, 0 =, 0 E',#%A.,9+9KK::# I.1.5 Effet Pasteur *(,#G =, 0,,.' # *,E00( 9%((:9!@9KJ::# %(=E',,0 R '0&,(,,.'# E', '0 (.' 9*$%:& 0 0,& ( E','0' &.00.'09+9KK::# G 0,(& 1 A?, ' # ;

39 % 4( I.1.6 Les réserves en hydrates de carbone '.. 0 E 0 0 6= HX, 00,#,. ^19&:# ' ',& &,( α19&>:α19&7:0,#, ' = (#- &,,&,& & 0 & & (0 ',, (=0=(0 9@ #9KJ::&9*#9KKJ::# ' ( # $,,,,( & &(0&,A,. 9@*:,#, 0& 171*, 0,.#,?,. 9@ / 9KJ::# $,, (, = 0, 0, 9* 9KK;::# $ (,,(,#, 0 ( 0 ' 9* 9KK;::# & 6 B 0 # ',0( 9D8*9::# - & B..',,(,,1, 0 (,, 9,(,.': 0,. (, 9,(,.'1:# $ & 1A?,0 ' #".',=,1,#-&1= ;K

40 % 4(. T,, 0 0# >

41 % 4( I.2 Influence des paramètres environnementaux, 0# I.2.1 Exigences nutritionnelles 00.=, (#G(& 0,, &, E& & &,& 0, 1, ( = 0,# I L azote 0 E,& & (, 9&(_:#" E,,,,,,(#80,.,''*9)/ 9K:&`9K::# I Le phosphore 0 &,,,0&,,9`9K::#=' &'(,,,(9) /9K::# I Le soufre,,, 9 > #,,,&,9!@9KJ::# >

42 % 4( I Les oligo-éléments 1, =,1, = # B. 1,,( (0#0.# 1,1,M U &" U &% U &a U &D U1;U &" U &% 1,,0&=,"5 1,1,% U &4 U &% U &% U &% U & 1 &" U & U & 3.U,,0 &=Z"5 1 ( U & ;1 &@ U & U & U &G.U &*& 1 &,, 0 = Z"&,0# %,1,,1, 0 0 (! "!%2;!%"!*&'(!%=":",'>?' >2562@@- %!B M U U =('a U % U # "1, " U " U ', 0# " U # % U % U,,( = 0 0,# a U -, ''&0,,# $.M U.a U,# " U,'# D U &D ;U *.# U $0,=0#,# % 1 )B 0# "1, " U % U 4 U,(# >

43 % 4( I Les vitamines &=(&='.&,'1=,9!@ 9KJ::# 0,,1E',E',#,,(,0#*&,? 0, (&,'1 0 9` 9K::# % 0, 6 B,6 =,,,NO# - & 6 ( 0,, K& + >H, N1(O 0 0 >&, ; % (#, H X =,N1(O6# 2,0((0=0,9/9::# I.2.2 Effet de la température B,, R0 0,,, 9Z:&9I * :&,00(# I La vitesse spécifique de croissance µ 0,.,&Z,. &,& 0,, ;% 0 9+,9K:&@ #9K>:&#9:& # 9>::# = 0& 0,, A,,(,90 $9KJ:& #9>::# >;

44 % 4( I La vitesse spécifique de production d éthanol P 0 0,., 9I *,. : 0,=,=(0Z,.# 9+,9K::#,=0,.,I *,. # 9KJ: ( %,, #% A Y = 0,., '. 0 >%#",, ;%,, 9 /9>::# I Les rendements globaux 0,,E0 N1(O ( ' ( # /, (P(,! CP (P! *P (,,#,! CP /=,,,#*&,! *P,.,, 0/=9 /9>::#! "!%.;**&"!,(!%('%("'(&,'&(&%& >"&:%( >.44A@@- &,'./B 14B 11B 18B 15B 9C : 9 -: 2 &JK &7> &>K &> &; +C : + -: 2 &>; &>H &>J &>; # 9K>:,, 0&,,, & C00#((.,A,,,,,0&;%=>%# >>

45 % 4( I La viabilité,, R0 0, 0( #,,,R0,1,94'#9:: 9/9K::#2,"*/=,H%=0(HX,A,, =H%=0(>HX#*&,,,0# $,A,&/9K:,,.( KH:& / 9K:& # 9>::# & ( R/ # 1 &,(, 0(, ;# #, 1 = #, 1,,;%=>%9/9K::# * # 1 &'N1(O 0 & 0( X = HHX,,;%=;7%9 #9>::# *,, 90,:,,,1,# I.2.3 Ethanol & A ( ' 0&,,,# $0,&,,0,.&0 & 0,, 9+ ' 9KJK:& 9K>:& " 9KH:: 0( 9%' b 9K7:& G0 # 9KKK:& / 9:& / 9>::# $ &,(6B(,0 #,3,. 9329K::#,0,,(.,# 8,,,, K:& 329KH::# >H

46 % 4( -,&. &.,=,(, 9+' 9KJK::# *.&.,0,# 1 *, = ' # 1 *, = ' # 9+ ' & 9KJK:: ( 0 *, *, 0 J # 1 > # 1 #$,(,9 / 9:& / 9>::& 0, *, *, (0, # 1 > # 1 #%,06 0,,B,,,=# 0. =,(. (( 0#-&, 9 : 6,. 9. :# & 0? # 9K: 0 ( 6; = #, = ;'0&,,9+6 9K>:& $ / 9KH::# $,A,& ( = 0&,, # (.. 1,((#,,,( ', 0 9$% 9KK::# & &,,( E /, & KKJKK&,&(,,(,(# G&,,(,( 6 =,,,E',&=09329KH::# %E / &, &, 9,! KJK:&,,,9329K;::,(,,0 # G& =. & 0 >7

47 % 4( *1" U,,(, (0E',#-KJ&%b,E/& &.,&.,,#$&,,,0(*,,(# $,,,., 9, 4?9K>:&$% 9KK:&( #9KKH::,, =#$&0. 0,, 0 0( 9` 9K:& # 9:& / 9>::# I.2.4 Influence du ph $ 0 ; = J 9) / 9K::# 0,.,#*@1=0&,,#*@=&>=&7&, ( 9) / 9K::#- (&,00# &,@#* 0A.&,,*A 0, 0 H&E0# I.2.5 Influence du CO 2.' ( A,, ( 0,,1,0(.'&P,,,(, 0. (.''# >J

48 % %G,, E,, 7 :%( 1 ; *"%, N,,, O 9@ ( D ; &% 0 '%( "! (!( 0. %G; 1:& / 9 : & '!(! '.3 B - ' "%,,,!$% %' # 2 &H H %# 9) + %G, 0 E',, & &, & (,,, & =, %G &, %G, R 0 ' 0 ( ( P, / 0 0 &0 ( N ( 0 &,,, #,,, 77 >&H 0 & (, %4,&,, $, (! %( & (,, & %( % 9K ::# %G /, 0 # 0, 4( # & 0 #, ( O 0., ( 4, N ( 1( 0,, # O ,, >

49 % 4(, 0 # &,, R0 0;%&=@>,.' 9/9:& / 9>:& / 9>::# 0,,,, / 9:#% 0,, ((*,= # 1 & K # 1 0,,#. 1&, N1(O 0 0%477&,&,& E #-&1= 9*,1:&0 =6=.,0 9*,:[1,A,0('# &,,,1, &,,/0,,, ( P,# $ &,,,0, '0&S1=1,(,,1,#0,(, 00(#., 0 (( = ( # >K

50 % 4( I.3 Vers de hautes densités cellulaires (, 0 ; # 1 =; # 1,#(( ', 91: ( 9( =,,(, (1 :, 0#$&0,,, # I.3.1 Hautes densités cellulaires et modes de conduite de réacteur discontinu (Batch) et discontinu alimenté (Fedbatch) I Les réacteurs Batch "c#9:&,((& 7 # 1 H00M0%'" 9K;:J # 1 K0C002#,(,P (0,&; C # 1 &> C # 1 #*&0 (,&J # 1 # 1 #0(,(,( (#, (,(,, (,# 2,,6 =,.,,( ( 0 ( #* &,,&@&.&#0, (,(#, 1(& &,,,/ 0,9,,,,(. (:(,0(,(# H

51 % 4( I Les réacteurs Fed-Batch "E13 /9KK>:(,7 # 1 HJ, B = (, #,(,.B.00 =&H 1 #3@? /9:((0(, # 1 > B(&9L: #0(,(9&J # 1 # 1 :#, 9, (,.' : 0 = 0,.',(,#$&,0,,(,00,#, N1(O, 0 (, 0&; = >, ( ( V=,/,(,0#%B=0,, #, (,,, A,#",,/,(&, 'N1(O0A,,&,(&(0,9 0, E:# &,, (,, 1,(,& 1 0,& 1 A(0,# *&',(A# I.3.2 Les Bioréacteurs Continus et les Bioréacteurs à Recyclages Cellulaires (BRC) ',,#*9:((,J # 1. '&H 1 &H # 1, H

52 % 4( 0 0 # 0 (, # 1 # 1 # 0(,(( ' N1(O#% 0 (,,A,,,(#",,. '#. ',.,,( A = 0,.,,1, 0 0 #"A, ', (&',,= (,#*,0 0 &# &='.0 #*, 6 ', 6 (,& (0 1 & = =, &,,1,06=; # 1 9 9K::5 1 & =,,/ 0,,9,&0,&: (,,# $. ' &,,(&& # I Réacteurs à recyclage par floculation, immobilisation ou centrifugation * 0 9+ ' 9KJK:& +, 9KJ:&$, /9KKK:&+/9H::00 ;K # 1 0A,,,. #" 0 0,6,,,# (,#-& 1=. ' & 0# &,,( 990 9K:& / 9K;:&/9K;(:&"%'9K>:&/9KH::, H

53 % 4(,0 #0#9K>: ; # 1 #,',R0 (,#0,N1(O= &%'b? `9KJJ: # 1 D?/9KJ:>H # 1 (,#% ( # &.,.,,# $&,.' &6,,( # &,,(&,1, 0,( 9% "1 F 9K:&."/9KK>::#$,.'&,, # ',,,( 0=(,# I Les bioréacteurs à recyclage membranaire..'(=',,( 1,1,1 & 1.,, ',,# 1 0 6#( (,9L 4 ]# 1 :#G,NO#. ' 0# ( 9,:,,., 9$/9KK::# 1 0 #( (,9L 4 d# 1 :#.' 0 0,,# %,,..# H;

54 % 4( I Les bioréacteurs à recyclage cellulaire mono-étagés = ' =,,(.#,(,1 =,.D ># L C&&* 3 L * &*&C] L 4 C&&* 7:%(A;,!&)%(!%(D(#"!:""%"!(,, (!!(,!:- 2 & &)!",!>- 4 "!(!'%(&!'")!",!>:- '% '(!>:- + & & (,!>- & #,, # # 0,,,# ;# *,.9,( :# >#,,( 0, (,&,,,(# H#, 6 (, ( (# G,, 1 0,$9 1 :(,L 9# 1 :0,9:# F6 + F F * = = >2@.. 1 $.9 1 :( (,L 4 9# 1 : 0,9:# H>

55 % 4( F * = G. >.@ (,(,&,,&=0 = GF + IG. >1@ [ I = * G >A@ 1 0 9!: (, (,#,#L. =&(, '( (,#.'! *&(0 F F6 # = = >3@ F F I. # = >8@ F I Performances des bioréacteurs mono-étagés % 9KJ:((, # 1.'&;7 1 &.,H # 1 #,1,0 0 %% >H#,1 0, =,,(H?$7,e# / 9KK>: 9( (, : (, # 1 0, # 1.'&;H # (,1 0, & =,,(K,e# * &,, 0 (, S1=1 0 ( (,&`/9KK>:( (,J&J # 1 &.' 1. &7 1 &0,H # 1 #2 (,K # 1 *?/9KKK:J # 1 /9KK:. % / 9KK>:. ' HH

56 % 4( &; 1,> # 1 #",,(90(,:# 0# 0,0( /9KJ:&,,& ; # (JHX&= ', &0.'&H 1,H # 1 #-(/9:=.(,0 # 1 00( HX=JX&,,.# ( = ',,(,1, ( (, 0& 0 0' #"(,,/(,0,0# % 0 (,, &, # 0 # $ & ' =, 0(,1, 0,, # (,, ( = (,,,0(,# I Impacts des paramètres opératoires sur les bioréacteurs à recyclage membranaire monoétagé (,,9. '&,&.T:, (, NO 9 (,:,NO90( :,,# H7

57 % 4( % 9K7: %% >H,. ',,,,1 = ' #*.'&H 1, # 1 &(,0> # 1 #* 0.'&H 1 &(, ;H # 1 #-.&(,,,;&;0# &,& (,(,,,,. ',,,D H1# 7:%(3;,!&'%("!(!""%"!(->.A63AE14B 0A3!( (-, 2 A44@>!:>2568@@- +,$9K7:& 9K:?'/9:, (0#,.',0 (9&0,&:A,9`9K::,, ( 9" 9KH::# 0 (, 0&0(,9G,$9KKJ::#& HJ

58 % 4(. & 1 &0(JX.' &H 1 KX.'& 1 # "/9KJ:,.'0,1,00&=' #; # 1,.#('D 7# 7:%(8; *"%&%!%=&&"%#&(!%"$%> (!!(@>! ->256/@@- (,> # 1 &.' & 1 &0,1,&H 1 #*&.'& 1 &0, 6= 0 &; 1 # & 0,H# %,,,,,& (,&,. &, 0,& / 0(,9+,/9K:& 9K::# H

59 % 4(.,% 9K7:,,9####:,,#*. ' 1 & ( (, # 1, # 1 (, # 1 > # 1 9((%(G%(&%( (%! "-H:#,,,,.0,(#&,1,,# Y,,# 2,0(9"9KH::#,, A,#., N,P.'O,,(#.,,00;1> # 1 9"9KH::# $, / 9KH:, = ' #&0.,A,. '# *! & (,, (.#*! =E&',0 (, =,# $ & ' (,#*!]&(,(;&> # 1!]&KJ;7& # 1 9$,/9KH::# $>&. 0,1,# $ & 0, (, &. ' &,. ' =,( (,#&,. HK

60 % 4(,,0,1,( (=(,9%'b?`9KJJ:&% 9K7::#$&,(,' 0,1,,( 9G,$9KKJ::#,, ', (,00(#$1 ' #- 0(,,,&,,,# I Réacteur à recyclage cellulaire multi-étagé $,,1 0 1,1 0 (, 9b? $ 9KKJ::# +, =,, 9!":0(, 5 1 (=',,(9b?$9KKJ::5 1 ', = ',,(9+/9KK:&Mb/9::# 0,,(,00,(0.,# b?$9kkj:,',a,1 (, =,1 # - & (,,,( 0.#(,0 (0# $ (1 &,, =.,=9b?9KKJ::[,1 7

61 % 4(, #% 0 09/9::#,=,.,,(, (=' =,, 9Mb/9:&+/9KK::&.(= ',,( 9Mb / 9:& 9b? / 9KKK::!" 0 (=',,(9Mb/9:#0,,,=,9Mb/9::#-& 0,,(0 (0# b?9kkj:b?$9kkk:,',,.(=,,(# Mb/9:.'((1 0,1, %% 7; ( = ' 0!"&.( =,,(# $, & (, (!" K # 1 7 # 1,#0(HJ # 1 # 1.=',,(J # 1 # 1 0 ( =,,( 0!"# ( = ' =!" 0( 0 0,# &,,(A.(=' # + / 9KK:. ( = '!" # $.,& 0., # &,(&>> # 1 0>&K # 1 # 1 0 (,J7 # 1 #$,, (&;7J # 1 0H& # 1 # 1 (,J # 1 #%&,(, V= 0,,(#, ; # 1 0,H7 # 1 #% (,1,# 7

62 % 4(!"(=' 0 Mb /9:(0 0#,0,& 1=.',0 A#*0,&Mb/ 9:0 (.,, 0.,# =',,(,.,( (0(,&,/0, B, &, (,, R0,(,,[0#&, &(,,9( &. '&,T:# ((1 0 0(,,(,#,&, 0 # - &. ' 0 A #, 0,,,(# =, 9% / 9KK>::# #, ' A.(# (;00 0' (# 7

63 % 4(! "!%1;!!:''&'(!%('(,!&H!&(&*('(!' ""%"!('>!: $%'!!:' ',, "'! 1*0. ' 1*,0,. 1*(,,.' 1,,((, 1$,, 1, 0,, 7"%"! (*%:! 1*(,,, 1*, 1 (. ( 1D,, 12(,0 1,. 1D( 1 *(,, 0 1 $, R0,,, 1@ '' 1*Y.,.,R0 &%" =( &%" ( 1D' 1@ 1!P0 f 0 1( 1* 1-, 1, 1@ 1!, fp0,,(p0, 14, 1,.' 1$= 1%,,,( 1, 1!, 1%,,,( 1*.( 1,P0 f I.3.3 Les phénomènes limitants,, ( = ' = 0,A,,1,& = 0, P = (, #- &,,,0,0( # $,A,&, 0, 9, (T: 0, 0( #* & ' 7;

64 % 4( =,,0,, #,,,,# I Limites physiologiques I Modification morphologique de la levure,=, (,(=,# 9K:, V=,#*(,J # 1 & (0 ;HX,,'>&7H&HZ, ;XH&>7Z,#*&(,; # 1 & 0 >7X,,',>&7H&H Z,JXH&>7Z,# $ & 0, ' # +V=,,2(/ 9KH:&+,$9K7: /9KJ:,, J # 1 =; # 1 =, H P,=&J P, # % 0 X# %, A.,,9:&,09 9K::# I Diminution de l activité spécifique du microorganisme, 0, 0,1, 9" / 9KJ::,,, ((%(G%(&%((%! "-7# 0(, A.& = & ',# 1 $, ( =, ( 0#%,,,(=.'(,&,(,0&= #-.,, ( 7>

65 % 4( 0,1,9#D 7((%(G %(&%((%! "-:# 1 $,,='#- &, 0,(,( E', 94?! 9KJ::# 0 (,(,# 1 $,, ( 0, 0# (,,& 0,,,9D J:#,(, '1, ( 0,1, 9 9K::,,,,#,.,. ( 0 A,. '#"&, &. A,.,. # I Limites physiques et physico-chimiques,,,,,, #%,,(# I La composition du milieu extracellulaire M / 9KK>:, 0,,,.,.,#-&,,, & & 0,#"9KKJ:,0 0 (, 0,.,,. 0#%0,,, A, (,,,(# &.,,, 7H

66 % 4(,,( 9 9K:: =, ( '.,,=# I Comportement rhéologique du milieu 2,(,.,# G,,, &.. 1,(, 5 1,,,# I Rhéologie des suspensions cellulaires,,,(. 1 0η *#5 1 00η &, η η = >/@ η [ η 0,.,1,*##!β / 9KJK:&" 9KK: " " 9: 0 0 (, 0'#,, 0 =, 0 9D J:,, b#% 0 6= H # 1!β/9KJK:& # 1 "9KK: # 1 " " 9:#* & 0,(& 9K:"b?/9KJ:,, b 6= JH # 1 # = 77

67 % 4( &,,,#(0& 0, 0, 0 '&..,,& ',,,#,,,, #,, A,0 τ = M 9γ: >6@ [?,, γ 0 1! b]?]η 2, (,,,,,?9+/9KKK::# "b? / 9KJ:, 0 0,, ' Gb11`& 0& 0 1 &, η =9 G + : >5@ [ &(&,,(,# 09b: =0, ε C,!β /9KJK:# 0, 0, 0, #.,(., 0 0 (,& 0,#!β /9KJK:&0,=(, # 0 0 0,,., (,,, #, (,=,009!β/ 7J

68 % 4( 9KJK:""9::#,,η &00A= 0,#%,,,,,,#,000 0,,, = &,(9L,/KK:#%,=,#- &, 0, 0 A,0,#,(,, 0,,,,., #%,,,,(,0, ' 0#,, 0,5 ( ' =,,(,# I Impact de la concentration cellulaire et de la modification de comportement rhéologique du milieu de fermentation sur la filtration (, 0, 0,,, 9,, (:# & G, $ 9KKJ:,,, 9 ( 0,:0(,# (,,,,1, 1= 0 0 0,,,.,,, D J# G, $ 9KKJ:,,, (, = # 1 0,1, =>%#,,,,,(#$,,,,,,(0# 7

69 % 4( &.,,. ',.,#,, ( =, ( #,,',,=0,.#&,,,&,, & ( &,,(,,,,(# 7K

70

71 % 4( "%';(%&I,(, 0 0, 0 # (, &, (#-,(,.'1&,( ( # #. &., 0 (.A,,0# (,(60.,,.,.,(,,.#,, (0 (,, 0, & ' 90 &,(, &,. T:& ( 9, & 0 &,T:#,,,,((=(,& 0 (, 0( 0, (# - & = &(,(=&=0 )%&% ":$% 1 L,(Q 1 L(,,(, 00Q 1 $ 0,&,,,,(0(,Q J

72 % 4( 1 %,,, 0..A, & (,,( 0Q 1 L,, 9,( 1.:,9. :,1,Q )%&%&'(!'*(' *',,(# 1 L,,0%G,,,.,,&,(Q *',,# % (,,( # 0.,((=,., ',,,( '#,0,.,((# -,&,( 0, 0&, 0 0# (0' ( (' & & 0 0,, # 0. (,, (.,,( & 9K: (& ' (,&,., 0 0,=6&=0;; # 1 #*& (, =( 0(0( # (,1,,,, b? $ 9KKJ: b? $ 9KK: (, 0( 0. (,# J

73 % 4( =0.(80&(,,.,,0, #, #, &!&*!('("!(!& (''! 0 B,(&#, # 1 &01=, (, 0 # # &!&,,,&!%:,( "! (!,!''!",,(,.,&&&(, 9 0,,1,,,/.,,,(:& 0 0 (. 0#,!( "!! " ")! &',((:!',' ' 0,, 960,,(= = # 1 : 0, 9(, (:# 0, ',0 N( O 0,!0![,.0,& 0 (, 0( 0.0&, R0#,!("! '''*$%& (&%&)!"&',( (:!','%"",8= 0 H # 1 ( # J;

74 % 4(,'("!%=&&"% #&(!%"$%,/.,,,(5,. ' 0,,& &, 0,,# %, 0 7:%(6;,!&% (!%(D,, (!!:D(#"!:""%"!(-J; '"& &,"%'!"- 2 2C. '"& &((%"!( L ( # 1 L P (!!# 1 L 0 (0,# 1 L 4 ( (,# 1 L` (# 1 L * (,# 1 L (,# 1 %( E,(= 0(! # $0 0.& &,!(,,',,,&0,,&.,,# L L " L L 0 L b ) +E L P C * + C * + ##1 L P +E L 4 ## L > L ;P L P; L ;!2 L * J>

75

76

77 % "" II Matériel et Méthodes (&% (6,,.,.,R0#, 0 =,,,,1,&,.(# $., &, ' # =., A'# JH

78 % "" II.1 Matériel de fermentation II.1.1 Souche %477,, % 4 0,, &JK G &7> &7 9 #9>::# II.1.2 Milieux de culture II Milieu de conservation 0=>% 4#, 40# 149$ g : 1%9*( g : 1+9*( g : ?$ g : H ?$ g : H,(0, #, 0, = % ( # II Milieu de culture 0 = > % 4,, F-*$#,,F-*$0# 149$ g : 1+9*( g : 1%9*( g : K ?$ g : J7

79 % "",(0, #, 0, = % ( # II Milieux de fermentation $.,. 1,, 9, (:& 1,4,# ",,(, ( =,,(#,,#* & 1,8=,6S= # 1 (,9/9::#,,0#. 1M@ *G > ;& 19@ > : G > ;& 1" G > &J@ G &H > &@ G ;& %,,E 1+,, & G 1, 1aG > &J@ G >&# 1 1%G >& H@ G K&# 1> 1"G > &@ G ;&# 1; 1@ ; 4G ; ;&# 1; 1%% &7@ G H&# 1> 1 "G > &@ G 7&# 1H 1%% &@ G &;# 1 19@ > : D9G > : 7 &7@ G &;# 1 3, 1 H&# 1; JJ

80 % "" 1, H&# 1; 1 H&# 1; 1*'. H&# 1; 1" H&# 1; 11,(E_ &# 1; 14 &# 1H,(0, #,,., 0,=%(#%,,, 1,0,# 4 % 1,,, "&Z,9!G!2 g :#@6=# 0=>%# 7&H& S,(, = S. (# (, 9 H C C:# " &Z,9!G!2 :0=>%# "4, 4&,, (&,,, ( ((,,,,., =, (,,, 9-9K::#,,40#. > >H& 19@ > : G > K&; 1" G > &J@ G J&> > &@ G J G 1, 1aG > &J@ G ;&; 1%G >& H@ G &> J

81 % "" 1"G > G & ; 4G ; & 1%% &7@ G & 1 "G > &@ G &; 1%% &@ G &; 19@ > : D9G > : 7 &7@ G ;&;,(0, #$(H#@,4 (=6@ ; *G ; #(H0 ;H,=%(# 4 0,&,, &,S9C:=@7&H&S,(,', ( 0 C# "&Z,9!G!2 :0=>%# (0,6(0# 9,':& =,( &. 1, *!G4G g &0,+" g,,"-!%m g # II.1.3 Bioréacteur à membrane II Principe de fonctionnement ( (1 0 ' ( ( 4 4,,(,., & 0&,,,(# (,1, 0(& ' 0 &,. 9Y,,&(:#,. # JK

82 % "" 1 2, 9!: #,(,1,' 0(= # A,7 # 1 1,.,1,, &(5 1, ( 0, 9`9K::# 1 2., 9!:,,,# = ', 0 6=;; # 1,09 9K::#2 0, B = &. # 0(0,1,!0!# II Description du pilote,,((1 0' 1 9D K:# L L " L L 0 L b ) +E C * + C * + ##1 L P L P +E L 4 ## L > L ;P L P; L ;!2 L * [ 7:%(5;,!&% (!%(D,, (!!:D(#"!:""%"!(- L " (,4# 1 L ( # 1 L 0 (0,# 1 L` (# 1 L (,# 1 L P (!! # 1 L P (!!# 1 L 4 ( (,# 1 L * (,# 1 #

83 % "",., 4# 4 4 %"D%, 0, 7 0,!!&0,.=>&H&H&.,,#$.,0!0,0,.=&H#0, K&H&>00, ',R0(9.,,( :# # ( '!#,,.'(,, 6, & & & & & E & = 0,& 6 (,#,. V = (0#$.,, (=&(#,,(,,(,&M-!-*9G&!:', &=J.,'7,,& J,,&,.H,,&H?$#,,( &HH, #, 0, 0,,.( 0 =,,(# 20=,,(,&,&,.,,(#,, 0!,# 2'=.,,(( (%G (#, ',,( 0 A,#2' 0'#,,(,8 =0 0 X9bP0:X9bP0:#,, # $ 8 = ' # ' &,,(06=(@J#

84 % "" 9,! = H # 1 &0,,5!0!&!0!,:, 1 ".,JH;1!&,JH1;H,0!&,JH1HJ.5 1,,"K,0,0!& (,0!#,,(,0A, ( > 0#., ',0( # (,,,.,& # (,,,.,,& ;# (,,,,#! "!%A;!,,&!"&&','>7:!,,,!=,!"&)%"'!&"!,*:!,,,,!"&)%"'!&"!,@- " A, +,,0 - + " *,!0! 7 &H=H# 1 D > &H=&H# 1 > &H=&H# 1 D ; &>=&># 1 H &=&# 1 D 7 &H=H# 1 H &=&# 1 D > &H=&H# 1 * (, ; &>=&># 1 D,,,(, 0,*%"'(,.,H, ; # 1 #$.(,,=%94?&",:,,(,((#(

85 % "",,,,. (,, E 9& 4?:# 2,( = 1& &,,(&,,',,,# ((0,0 B, # E,., 0' 0 %, *+E&0# %,, ',,, 0 (, 4 ( 4 14 ##### 0,,, 1, (,&, (,, E&,&@&.' &,0,&, E,5 1,,,,!0! 0.,0,!!5 1,# 2, B, 09D D :# ;

86 % "" FIR LICRA po2icr LICRA po2icr phicr TICR phicr TICR TIC SICR M ph SICR M PIR ER Air ER ER P TIC Pompe type Moineau FICRLA PIRHA M 7:%(24; (*!&(K">,,!&&%":"&)!$%'@&% (!%(!:D(#"!: ""%"!(- >

87 % "" 7:%(22;+:(!&"H'!""!- II.1.4 Préparation de l'inoculum.,;9(&0&0:# 2-,',H,,F-*$,= 4#%(1= ;% 9#, 1 :# -,', -,' H,, ( >,, ',, = H # 1 & 0,, 90:#0(1=;% 9#, 1 : =,-,'(;,7,, H

88 % "" ', = H # 1,, 0,,#%(1=;% 9#, 1 :#>,0(0=,.(=.X90P0:0,1>#0, , 0, =,,=X90P0:# II.1.5 Mise en œuvre des bioréacteurs,,# S,,,&,, 1,#;&H&H, 0,!!=094&%&;,:#0,,60,0, # =000#,# (, 0 H&H, # &K &K = H # 1 6 0,!!# ',,, ',= # 1 #0, 6,# S = 0,# $ 60,(=(, (, (#, E ', E9##7:#, X = > 0,,=>X90P0:# 4-1 =S9=H # 1 :# &, 0,&,,9%&,&(:# 1 0, = # 7

89 % "" II.2 Méthodes analytiques II.2.1 Caractérisation de la biomasse II Mesure de la densité optique DO S0,, = 7, 21: 0,, 6 # 8=(,&H &J((# II Détermination de la matière sèche totale (, &., " # 1,, 0,# 20,=SS,=0,,( ',&>HZ,9!G!2 g :(,#&,,(=S0=7%09,,@ :>& #,0,,(,,,9 " # 1 :# 0(,&0,( #& 9H,&; :0,,, V = 6. (- (, #( A0#=S0=7%09,,@ :>#, 0,,, 9 " # 1 :#,',(,,,;X# J

90 % "" II Détermination de la concentration cellulaire totale et de la viabilité cellulaire II Cellule de 00, 90( :#%, >:# 7:%(2.;""%"&,!-, =,G',%@19,>:# 0,,#,, # 1 2,,, ( ( #%(,# 1 ( & =,,&, #$&,#,(,A,;, = X 9* 9K7K::#* & 0 &, H.#,= 0

91 % "",(#, 1,(, = ffh>24@,(., (,'#%,,9%b'9K7:: $, (,',S5 6,'5,,("&Z,9!G!2 g :&,=,0S# =, 0,(,'0 0,,=,@G"= ;,1,#, H, =,,(&, U@G"#,,#00,(# 0(,( 00,(,,, D ; 0# %, %00 7:%(21;=,"&""%"!,(!(&, (,!""%"&,!%"'!&% "%&,#"L- II Comptage sur boîte (,0(,,(/*#Z( 0 94? $ :, 1' H # 1 &H # 1 # 1 # 1 K

92 % "" *0,&&H #%,=%(0(/ *# *,90,:& 80' #(/,, = 0 ( #, (>=>(=;%# II Détermination de l eau intracellulaire 0,,, 2(/9KH:(,0 #*& = ")7 = #,.=%,#,0, H,= > % = ; # #, 6, 0, ], f9ph:# 2, &,&H &= # D >1, # a * 7:%(2A;=,"&%( '&$%'&'!:- K

93 % "" ( 00,,,,,#,((0 ( # 0 (=E 1;#* & 0 0, ',, #,9= 0 :,.# II.2.2 Dosage des substance de réserves, ' 0 ( *D9KKJ:#1# II Le tréhalose 20,=;, (, HZ %G ; =&H"#(; =KH%# 6HZ"7Z,&"=@ H&&0HZ.&6Z,,9+": =2#, 1 #G(>=;J%# = ; H,& (,.'# II Glycogène 20,=;, (, HZ %G ; =&H"#(; =KH%# 6HZ"7Z,&"=@ H&&0HZ.&6Zα1,' =2#, 1 # G(>=H7%# = ; H,& (,.'# K

94 % "" II Dosage par la méthode de la glucose oxydase,1,e',.' 9+":0 % G 7 U@ GUG U@ G U19: 1.'9,: = # 2,, = = # 1 # =,,,1 9 Z: Z E', 1#,((;,=;J%,=>K,= -(ZZ# II.2.3 Composition élémentaire CHON de la biomasse,,,(, 8 90$ S (%B-%-9*&:#20, 9H,&>%&:'DH,# A 0# '(0=7%09,,@ :H7#,,,, (,#,,%&@&G&, *# %,,(, ',,(,=-%-,((,# E 9%G &@ G& G : ', E.' & E& ' (# * = &,,(,,0 8 90$ S & X8 f = X >22@ X9f 0 = Xf7 >2.@ K

95 % "" X$ f S = Xf> >21@ % γ=, γ = > + 0 ; S >2A@ %,,,,"9 #%, 1 :,0 " = > S >23@ II.2.4 Dosage du glucose extracellulaire,',fj 9F-G`*!+!2"- :#S.' (,.'.,,(#,, # 1 #SSX#,0 9; &;,:# 0,,S91&H # 1 :# II.2.5 Dosage des métabolites,(&& '&& 0 A ' # %@*,A, #, (6 9(H:#,,, #1 %, 9`-! & 7K: S, 9`-! >:&23=(9`-! KK7:S S,9",;#: %,.@*C1J@9;,,.J&,,:5, H%5 K;

96 % "" $S' ;,5 G > H,"5 $(,( &H,#, 1 5 3,S6 Z#! "!%3;'&',''$%!*! "'!(0+,'&((('&!'>,%'@-,' "%' & %$% "#(" &!$%!", 9,: & >& 7& &> 7&, 0 S =, = ',, 0(# 8 0 9; &;,:# 0SA '#,,S,#S,(,,,(=S V=,#,,(X# II.2.6 Dosage du CO 2, O 2, N 2, E,, E# 0# %, 9-!" g &+%"4:S,# %,*G!*ML&,,Hh#, ;J%&,# 6 ;J%& ;J%& E0 ' # 60.0# 3,S6 Z# $' E%*+H,# S S, %G &G & #S K>

97 % "" %G &G E.(,, 0. #%& ', S'& S S = &K;XSS.' /,.' #% %G #$ 0AS. %G &G & # X9 X$ X9 = >28@ α + 9α : X$ β X9 α X$ β X9 = >2/@ α + 9α : X$ β X$ = >26@ α + 9α : X$ β X 9 & X $ &X9.'(&E.',# α PE 9α ] &K7J: β.' 9β ]&K;KJ:# 2, # & = E., ( & S,(,0 %G &G & = ( E.=# KH

98 % "" II.2.7 Caractérisation rhéologique du milieu de culture $,0=,33HH 0,,. #,, 0 0 = # $,( A,'# 0,=,00,('1',,,('B#,,,, ( &=, ;%# II.2.8 Caractérisation de la pression osmotique $,, =,1,,,@,!( 'P$!9"?&4&+,':# %'S,,, #S(, = S,,# S = %# 2 0,,?S9,#M :=1&H%# G G S( ( S, V = #,,0,1;,,#M #A,;,0#*,&0Z (-#%(.A, (,, #,., 0# 0 1& ',, (-0 #,0iUj#-0,# G K7

99 % "" II.3 Traitements des données '0# II.3.1 Hypothèses.,, # 0,,,# ; *,.9,( :# >,,( 0, (,& H,,(',#, 6 (, ( (# 7 % E00 4 ( ',, 9, =0.(0:#,&0.,.9((.':. ', ( 9,,,:# (.,# 1 # *,( II.3.2 Conservation des débits F = F 6 + F >25@ 1*! F P = F + FP >.4@ 1*!9,U,: II.3.3 Temps de séjour F P = FP + F 6 + F >.2@,6 6 &[0(9(,:6,&.,.' 1 #,6(, C6 KJ

100 % "" G =. PFP >..@ 1 =. P9 FP + F : >.1@ G 1 G = : PF >.A@ 1. * = P >.3@ G, >.8@ G 1 =. P9 FP + F + F : >./@ 6 = :P9 F + F : >.6@ 1.' II.3.4 Bilans matières 6 * = P@ >.5@ (,.,,0,,# (=,0 1 9, # 1 :,! C 1 9, # 1 :,! C 1 0(,! X0( 1 0(,! X0( 1 (,9L :&9L P L P : 9L 4 L * :.,# 1 1 0,! 93 :! 93 :., 1! 9 :! 9 :., # 1 1! 9* :! 9* :., # 1 1 '! 9+ :! 9+ :., # 1 K

101 % "" 1! 9 :! 9 :., # 1 1! 9 :! 9 :., # 1 II Bilans matières sur le réacteur R1 en régime permanent 1 "!'%("!,!'' = G F G F + I G. >14@ P P Z 9 1 :0,1,0= (, # 0 0 = (, 0( 9Z 0 : = I I = >12@ X 1 "!'%(":"%' = F + F F, G. >1.@ P P 9 # 1 C # 1 :0,, #0,,=(,0( =,, = >11@ X 1 "!'%(")!" = +ν G. >1A@ 6FP 6F P 6 ν * 9 * # 1 C # 1 : 0 # 0 =(,0( = 6 ν 6 = >13@ X 1 "!'%(":"#(" ν KK

102 % "" = G. >18@ + FP + FP +, # 1 C # 1 :0 '#0 '=(,0( =,, + + = >1/@ X 1 "!%(")!&!$% = G. >16@ ) FP ) FP +,) 9 # 1 C # 1 :0#0 =(,0( =,, ) ) = >15@ X 1 "!%(")!&'%$% = F F +, G. >A4@ P P 9 # 1 C # 1 :0# 0=(,0( =,, = >A2@ X II Bilans matières sur le deuxième réacteur R2 en régime permanent 1 "!'%("!,!'' = G + I G. >A.@ F P G 9F + P F : Z 9 1 :0,1,#0 (,0(9Z 0 : =

103 % "" I I = >A1@ X 1 "!'%(":"%' =, G. >AA@ FP 9FP + F + F6: 9 # 1 C # 1 :0,, #0,,=(,0( =,, = >A3@ X 1 "!'%(")!" = ν G. >A8@ 6 FP 6 9FP + F + F6: + 6 ν * 9 * # 1 C # 1 : 0 # 0 =(,0( = ν 6 ν 6 = >A/@ X 1 "!'%(":"#(" = G. >A6@ + FP + 9FP + F + F6: +, # 1 C # 1 :0 '#0 '=(,0( =,, + + = >A5@ X 1 "!%(")!&!$% = G. >34@ ) FP )9FP + F + F6: +,) 9 # 1 C # 1 :0#0 =(,0( =,, ) ) = >32@ X

104 % "" 1 "!%(")!&'%$% =, G. >3.@ FP 9FP + F + F6: + 9 # 1 C # 1 :0# 0=(,0( =,, = >31@ X II Bilans gaz sur les deux réacteurs ( E..9!!:, 0%G 9 %G,# 1 :,,G 9 G,# 1 :# *& 0D#4%(97:90. #4#4#:#(1 E ( E9D J:#,0,,.' G E (%G &,0#

105 % "" " X X k!! XG XG k!! X%G k!! %*+ %*+ (!(2 L L! 9: %! L P &+E9 Gk! &T: L &+E9G! &T: (!(. L L! 9: %! X X k!! X XG XG k!! X%G k! X%G! " L &L +E9G! L 4 &T:! 9: L &+E9G! &T: L P &+E9 Gk! &T: "B L! L L! 9:! "B (>!(@ (>!(@ %, X XG X%G 7:%(23;'&% (!%(D(#"!:""%"!(!:%("!"%"&''''& ',,!&)=#:L&(&%&&=#&&!( - II Azote E E(, #,,00 E E,=%G =,,G,(,#2(E&( E=&,==&,( E= #*&0 0 9L #X :!6 $( L!6 = >3A@ X k!6 [ 6, L!6 ( E,# 1 L!6 (# 1 *&(.,,# 1 L L #!6!6 ;

106 Chapitre II : Matériel et Méthodes II Oxygène II Réacteur 1 : Le bilan matière sur l oxygène dans le réacteur R1 s écrit de la manière suivante : d(po ((O sat 2 2 _ R1.O dt *p sat 2 O2_R2.V.Q L_R1 2/1 %O d ) V + ) - (O sat 2.p O2_R1 dt.q. 2_ R1 V sortie G _ R1 mol 1/2 )) = -ro 2_R1 + ((Q entrée R1.%O entrée 2 ) - (Q sortie R1.%O 2_ R1 )) + (55) sat où O 2 représente la concentration en oxygène dissous en mol.l -1 dans le milieu à saturation po 2_Rj est la quantité d oxygène dissous mesurée dans le fermenteur par la sonde à oxygène en pourcentage par rapport à la saturation V L_Rj représente le volume de liquide dans le réacteur 1 ou 2 V G_Rj représente le volume de gaz dans le réacteur 1 ou 2 %O 2_Rj représente le pourcentage d oxygène dans les gaz de sortie du réacteur 1 ou 2 ro 2_Rj représente la vitesse de consommation d O2 dans le réacteur 1 ou 2 En régime permanent, on considère que le volume liquide V L_Rj, le volume de gaz V G_Rj, po 2_Rj et %O 2_Rj sont constants. ro ((O 2_R1 sat 2 V = *p O _R2 2 L_R1.Q.d(pO _ 2/1 2 dt ) - (O R1 sat 2.O.p sat 2 O _R1 2 V ).Q G _ R 1 1/2 )) %O. d V dt 2_ R1 sortie mol + ((Q entrée R1.%O entrée 2 ) - (Q sortie R1.%O 2_ R1 )) + (56) II Réacteur 2 : Le bilan matière sur l oxygène sur le réacteur R2 s écrit de la manière suivante : ro ((O 2_R2 sat 2 %O2_ R2 V. sat G _ R 2 d V.d(pO _.O ) sortie V L_R2 2 R2 2 mol entrée entrée sortie = + ((Q R2.%O2 ) -(Q R2.%O2_ R2)) + (57) dt dt sat *po _R1.Q1/2) -(O2.pO _R2.(Q2/1 + QP + QB))

107 Chapitre II : Matériel et Méthodes II Dioxyde de carbone - La phase liquide est saturée en CO 2. Il peut se dissocier en ions HCO 3 dont l accumulation peut sous estimer le bilan sur le CO 2. Toutefois, au ph auxquels nos cultures sont réalisées (ph 4), la quantité de HCO - 3 dissous est négligeable et représente moins de 0,1% du CO 2 dissous (Guillou (1996)). Nous avons considéré le bilan suivant pour chacun des réacteurs: %CO 2_ d sortie Vmol dt Rj. V G _ Rj = -rco 2_Rj + ((Q entrée Rj.%CO entrée 2 ) - (Q sortie Rj.%CO 2_ Rj )) (58) D où en régime permanent : rco 2_Rj = ((Q entrée Rj.%CO entrée 2 ) - (Q sortie Rj.%CO 2_ Rj %CO 2, Rj V G _ Rj. d sortie V mol )) - (59) dt II Algorithme de calcul Voici décrit succinctement l algorithme de calcul pour les gaz. 1. Acquisition en ligne de la composition des gaz en sortie des deux étages, de la concentration en oxygène dissous po2 et des débits d air en fonction du temps. 2. Lissage de l évolution de %O 2, %CO 2, po 2_R1, po 2_R2 en fonction du temps par une fonction polynomiale et calcul des dérivées en chaque point. 3. Calcul du volume molaire des gaz en entrée, en sortie et du débit d air d entrée en mol.h Calcul du débit de gaz de sortie en mol.h Calcul des vitesses spécifiques ro 2 et rco 2 en fonction du temps pour chacun des réacteurs. II.3.5 Logiciel de réconciliation des données Un logiciel de réconciliation de données a été développé par F. Ben Chaabane afin : - de réajuster statistiquement les données brutes pour boucler les bilans élémentaires (bilans carbone et oxydoréductif) lorsque les mesures contiennent de faible erreurs aléatoires ; 105

108 % "" 1,',,# $&,.,, 1,. (,, (,,5 1 0, = 0,,&.,%G 5 1,,,, (,. 9K:,,' HX,#% X= H # 1 # =0(,, ( 7#., (,& 6=X(,0# ( = 0, E ( X#! "!%8;!%"!*&'((%('!**'D!$%!(! " 3( - lcm&l*m X % # 1 lm X $(# 1 L &L P &L P &L 4 &L * X =,,,, 0 ( (, 9(.: = &&. U = && #. 0 ( #%,, 0(,.,# 00(C &0 %G,,G (#0=,,,, ((.'#0, 0 (9(0,:# 7

109 % "" 0,.0., 0 ', # 0, = 0 ' (0.,# II.3.6 Les rendements! "!%/;'(&,''=(,':-: 2 -!,,!!,,! $, D, $, D,! CP Z P! CP Z P! *P ν * P! *P ν * P J

110

111

112

113 % % III Caractérisation du pilote (&% ( (1 = ' & H, 6&(60., #.' (, M # #,,(,,.=,,(# ;#, '1, (,(, # ># (.,.,(# K

114 % % III.1 Phénomènes de transferts %, B,6.',(, 0&.'.!!, M # III.1.1 Transfert d oxygène III Mesure du K l a : Rappel $,M ==;%.!!, ', #, = 0,.' 9.,= : 0 = ( 0 E # $,,&E,6=X.' #,E(,, #,.' ( ) ( 9 9 9: ) = C >84@ 0G 9:G 9:] 69 9: 9 & 9.' =#, &,, # 9 9 : 9 (,,, = ',,.' -#,,9,7;X.' ':=0 &H#, =00M (9M :#, & ( ( ( #

115 % % III Mesures du K l a sur le premier réacteur R1 $, &, 0.' 9+=1G/9::#*(#, 1 & (M >; 1 H#, 1 1 H#, 1 #&M,>#*.= H#, 1 &M ((,=&H#, 1 #- & (.' = 0 0 E93 &,# 1 :&0 9& 1 :09η&*#:., β γ δ C = α. $ η >82@ [α,β,γ,δ # M ((9D 7:#M.,,., (;H 1 H#, 1 &H#, 1 # M 1 #, 1 $(#, 1 7:%(28;"%&% "!> 2 &"H!:!(-, 2 -!%(2-

116 % % "`9KKH:+=1G/9:.,M 6*,0,3, &7 β 6 δ C = α #. # # η >8.@. ""9K7:=., 0 &>H ; # 6 $ &H7 F 6 = α >81@ [,(,(!, L( E, ; # 1 α *.,,(&,(&,0,ρ 6 = 6 ρ >8A@ H ; $ $ $ * =79!An:#* &,&ρ&l3 #M = 0 = &K# D J 0M 0 9: &K # M =69`9KKH::( &H#, 1 #*&(&JH#, 1 & M 6#G(0 = &K =#%,M = # 0(, = 1, 9 E. : 9` 9KKH::# G E#* &, ',.' ( E#0M (#*( E =&H#, 1 &(,,,#

117 % % ;H ; H H M 1 &H#,1 1 &JH#,1 1 #,1 H &K # 1 H H H ; ;H 7:%(2/;"%&% "!> %''!&*!((!(D"!''&)!:!>(-' III Mesures du K l a sur le deuxième réacteur R2 A,M.,,, ( ( (!,.' # III Impact de l agitation de la turbine Rushton 0.,&,M 0 (!#0, #2M 77 1,,,, ((&H, ; # 1 &(H#, 1 ( (!#0M #, 1 #*& (!.,/;X.' M # %. 1 (!, ( 9? / 9KK::# * &, 9 : M, 9+ / 9:: (0 7ε = >83@ ;

118 % % [ (,(, ε 0, E0, E 0,# 1, 0 0,,,M # III Impact de la boucle de recirculation sur le deuxième réacteur R2 (0'(,M &H#, 1 &. ( (#! "!%6;'%('& "!> 2 > @&' & '& %"(''!'D!(!'!>.3-, :!(-, 2 %", 1-2 "! 2 & K &H &J ;H & ; & KJ &H &J,((&=&J, ; # 1 0,M ;7X #, 1 X #, 1 #"1=&H, ; # 1 &M =HX (, & 0 #* &M (00 #, 1 (. (00 #, 1 # %.,,M #, M,,, ####%,,,&., [.' ( E.A0# 1, 9 E.%G,: >

119 % %,%G,9&, (:(0,1,#, 0,., =,.' #0(, (B((#%&( (0,,,#.',M,, (0M,0,, 1, 9 E., : 0. #*&,&,, ( (, 1,.' (#((& 1.'(&(, (#,, (.. ',#,,( 8,#,. =,,(# III.1.2 Transferts membranaires 0.=,,(,'((0&H=;, ; D # &,,,(# H

120 % % K&-1H &-1H J&-1H 7&-1H H&-1H >&-1H ;&-1H &-1H ), ; #, 1 # 1 &H &H & ; $(, ; # 1 '];&K>-1H.! ]K&K7-1 &-1H &-U *" ( &H &H &H 7:%(26;"%&"!&'&*"%=?>, 1 -,. -' (!',, (!!(,#+>!(@DE14B-., ) 9, ; #, 1 # 1 :,,(,'*" 9(: #,,(,.& 0$!%F9$/9KK::,, [ 6@" L = f η >88@ ).,.,, ; #, 1 # 1,(,,(,=,..,, *",,(,'.,* η0',.,*# -,,(!, 9$/9KK::&>88@ ' 6@" L = η # #,,(=& = &>7, 1 #-.,&,,(&JJ(,,(&HH,e#*&(,.,,;H# 1.,,(,# 7

121 % % III.2 Optimisation de la phase de démarrage : stress thermique et stress mécanique, ((1 =',,(,,0&,(,,,, 94 %(& 0. :#, (, # 1 0,, # G,(, = # 1 # % A,,1,,@&,#.,, &,,,,( 0#, & 0, ( (.'(=KHX# * &. ( 0 ( #, ( 0 0 &,,(0(# III.2.1 Phase batch avec recirculation entre les deux réacteurs (Expérience 1),.0 1,.' * G nx5 1 0,&H!!5 =>.0@ ; >X0P05 1, =;%5 1 L P ]L P ]# 1,,.5 1 ((L P; ]&H, ; # ,0,/9:5 1,,(,'&H(# J

122 % % III Les cinétiques.,#,, H,#, & 0(,JX=0K1X#(, H&; # 1. H, 9D K:# (HJ # 1 ' # 1 #&. 0 ( L P L P. = # 1,,. 0# % # 1 J 7 H > ; 0 > 7 > 7 7:%(25;$%'&(&%&,!''>(%:@:"#(">('@&)!">M!%@& ',,!&:"%'> "%@"!! ">(@%("'&%=(!%('-+!'! 64880A14B,"%,(!"'!''!'",!&)=#:L-'(&'(('" (!%(2"'(!:"'"(!%(.- " # 1 3(X, 7 H > ; & &7 &> & III Vitesse spécifique de croissance,(& 0,.,,1,&7 1 #%0(,. ((,Z,. &>H 1 9M/9KKK:: 0'0&>> 1 % #9::#( 0#%&(. (9\; # 1 :,6,9 /9::#*'0A,=,

123 % % 1., Y = ( 9,,,(:5 1.,Y=, # - & / 9>:,,;K%0Z,# III.2.2 Etude de l impact du point chaud dues à la pompe d ultrafiltration (Expérience 2) *, & 0, & = (,1,&.,.,A,,#!,(&( Y=,## 0 0 =.,A,.,(09"9KH::# III Les cinétiques microbiennes 0,(,& & 0(.& D #,, K,# (, # 1.00(X 9D :#KJ # 1 '& # 1 # K

124 % % % % #1 # 1 " "1 # 1 #1 K J 3(X 7 7 H > & &7 > ;, ; > H 7 J K &> & 7:%(.4;"%&"!(!,!''>(%:@:"%'> "%@:"#(">('@&")!" >M!%@%("'&%=(!%('*&%,'-+!'! A14B,"%,(!"'!'",!&)=#:L-'(&'(('"(!%(2"' (!:"'"(!%(.-.&.,#%,( K H #%. 0(,.# III Vitesses spécifiques et rendements 0,.,, &>J 1 &>> 1.,&,Z,. X!7X!=,.# 0.. #, (P(, 0.,. 9, A,:,. #G(0,;;X,9(K:# &,.&,(,1, 0 0(,.,. (0#

125 % %! '(!C,!''> '% '(!C!"> -.! -.! Z,. 1 &7 &>J &>>,. # # 1 >&7 >& >&7 ν,. # # 1 &;7 &; &>! *P &;JH &> &;! CP &JH & &KK. 0 #, A,,,,1,, ( = ',,(# ( & 0,!!.,( 0,6, Y =, =, ( #,,(,,1,, (# %.,, (&,(.#,# 00=.,A,.# (, 0 # 1 &,#

126 % % III.3 Reproductibilité des expériences en régime permanent : phase continue (Expériences 3 et 4) $..0, (0((,,,# 0 1,.'!9* G nx:,1! 9* G ]:5 1 0,&H!!5 =>.0,,>X90P0:5 1, =;%5 1 ((L P; ]&H, ; # 1 5 1,,(,'&H(# (,=, ( (1 = ' =,,( (,., H # 1 9,D#4%(90.::#! "!%24; '(!:"%'&!'")!",!,''"('&'=('1A,&% AE14B,"%'#$%'!'",!. %(2,(!(!%(.- $( # 1 -. ; -. > + L " L " 3, L 3 LP * (, L 4 *, L * LP, L l+m # 1, &K f &J &; & &; &H7 &H7 H f &K f &J &; & &; &H7 &H7 H f f$( 6,,H # 1 (,, (,!=7X#

127 % % III.3.1 Cinétiques III Réacteur R1!(..9.;>: D # 0.0.,,,=,A, # J 7 H > ; % # 1 # 1 1!!P-.; 3(X & & &7 &> &, ; > H 7 J 7 H > ; % (1!!P-.> #1 # 1 3(X & & &7 &> &, H H H ; 7:%(.2;$%'&(&%&,!''>(%:":("@&)!">M!%@& ',,!&:"%'> "%@! ">(@%("(!%(2*&%,' AE14B,"%,(!"'!'",!&)=#:L-' (&'(('"'&' (%'"'"'"'&'("'&%(!%(2- :,(,!,,,, ; &, 6 ',,, ( 9((,:D #% ;

128 % %,.(9.;(:,,#! "!%22;+!(!,L('$%'&")=(1%("(!%(2(:,(,!!"%(' ("' 64880AE14B,"%,(!"'!'",!. - -.; 3 -'!%(2 % # 1 3(X H C & & H& &> * J& &; + &> & &J &H &K7 &;! "!%2.;+!(!,L('$%'&")=(A%("(!%(2(:,(,!!"%(' ("' 64880AE14B,"%,(!"'!'",!. -!! -.> 3 -' % # 1 3(X C J&J7 &HH H& &H7 * ;&JJ & + &> &J &7 &7 &7 & (,& 0,& # 1 o&&h& # 1 o&>j& # 1 o&;. ;9#(:J&J7 # 1 o&h&h& # 1 o&h7;&jj # 1 o&.>,9!:#,,&0(! XoHX.;JXoX.>9#(:#,, '& &> # 1 o&&&j # 1 o&h&k7 # 1 o&;.; &K # 1 o&j&&7 # 1 o&7&7 # 1 o&.>#,( (..& 1 &,A,0((,& && & ',!#..1HX# >

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144 %3 G( -,,&(,&!;&H;o&7 # 1 &7K&H7o&>K # 1 &K&77o &77 # 1 0(;o>X&!H&H7o&JK # 1 &&o& # 1 &;&Ho&;7 # 1 0(o>X#$8,,,,& '&!9&Ko& # 1 &&Jo&> # 1 &>o& # 1 :!9&o& # 1 &&o&7 # 1 &7o&; # 1 :# '.0,.,, X.,# IV Les vitesses spécifiques globales en régime permanent -,,& 0. Z 9 1 :&,, 9 # 1 # 1 :& ν * 9 # 1 # 1 :& ' + 9 # 1 # 1 :& 9 # 1 # 1 : 9 # 1 # 1 :&.& =(,,(#! "!%.4;''''*$%':"!"'(:,(,!-!%(2=(3- -. H f 3 # 1 # 1!,,!,, Z ν * + &o&> &;o&> &;Jo& &Jo&; &Ko&K &o& &o&; &;Ho&JJ &>o& &>Jo& &Ho&H &Ko& f3,!&h! -. H f! "!%.2;''''*$%':"!"'(:,(,!-!%(.=(3-!,,!,, 3 # 1 # 1 Z ν * + &;o& &>Jo& &o& &o& &o&; &o& &o& &;o&j &J7o&> &o& &Ho& &o& f3,!&h!!&.(&o&> 1,,,&o&; 1.,,,# 0,,. 0# 0 >

145 %3 G( &;Jo& # 1 # 1 &>o& # 1 # 1,,# '0,, & '=X#*&',&X&.06=X 0# $!&. (.,,, 9Z ]& o & 1 :&,. 0,, #0 =&o& # 1 # 1 =&J7o&> # 1 # 1 0,,,#%0(=!# %Y=,. (0,=; # 1.,=7 # 1 # 00,, &>Jo& # 1 # 1 &;o&j # 1 # 1,,#'. X,, (,&,0%G, # '. 0 9& ':=X# IV Les rendements en régime permanent,'(#*!,,&,(p(,! CP.=&o&> # 1 &o&; # 1 &,(P! *P =&;JHo&H # 1 = &;Jo&K # 1 #,(P(,(P., (0 0,(,P.'9& # 1 &> # 1 :# >

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151 %3 G( (6.(,, 0(,H # 1 # 1 # 1!#,, (& 0 0.,,94%(97::>X& J&HX&>X0,,;&>H# IV Les vitesses spécifiques globales en régime permanent ( '#*!&. =&>7o&K 1,,,9,,;:&=&>o& 1 9,,>:=&;;o& 1 9,,H:#0,, &HH7o&> # # 1 &&>;o&k # 1 # 1 &>Ho&H> # 1 # 1,,;&>H&0 &Jo&7 # 1 # 1 &&;Ko&7 # 1 # 1 &Ko&K # 1 # 1,, ;& > H# '. 0 '& = X#,,. 0,,.,,,9,,; >:#*&'0,, & (,,, 0 X# % Y. ( 0 0 (,, [& (,9 # 1 :&, 0,, 9, 0, (!,,9(1:#! "!%.8;''''*$%':"!"'(:,(,!%("(!%(2-=( f 3 # 1 # 1 Z ν *!,,;!,,>!,,H + &>7o&K &HH7o&> &Jo&7 &J>o& &o& &Ho& &>o& &>;o&k &;Ko&7 &>;o& &Ho& &>o& &;;o& &>Ho&H> &Ko&K &;o& &o& &o& f3,!>&h!k&h >J

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154 %3 G( IV Résultats macrocinétiques (Expérience 7) M*; &&%=(:,' (,!' '%*'!% (!,!''&54:- 2 %'.34:- 2 &!'"(!%(.# 0, >&H! K&H!#, 09(K:#! "!%.5; ','&'M%((!:"%'&!'")!",!-=('/ AE14B,"%'#$%'!'",!. %(2,(!(!%(.- -. J f $(# 1!,,7!,,J, L +L D. ( # 1 # 1 "L " 3,L 3 L P L P * *, 4,L 4 L * ;&> &K ;& & &; & 7& & ;& ;&K & ;& f &H &; &H H& & ;&K C C C P C $ ( 1 -. J f!,,7!,,j &J> &J> &; &> & H&;; &> & f3,!>&h!k&h IV Les cinétiques ( ; ;,,. J 9D H:#. J (.,,#,,,9,, 7: ( >, # % =, 6 '# (,& ;K&o;& # 1 &>7&Jo7&K # 1 J&o&K # 1!J&>o;&> # 1 &&o&; # 1 >J&Jo&J # 1!#0(HJoK X!H7oJX!,,,D H# '& =&>o& # 1 &&7o& # 1 &7o& # 1!=&Jo&7 # 1 &o& # 1 &Ko& # 1!#,,0,NO&0, 0., #" (, 9>7, :&,, =,.#*,!=&;J# 1 &7# 1 9',( H

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157 %3 G( IV Les vitesses spécifiques globales en régime permanent (; 0 0 '#*!&,,,9,,7:(=.&Ho& 1 '>HX&,,9,,J:=&; 1 #0,, &J;o&;K # 1 # 1 'HX&&; # 1 # 1 & 0&7o&; # 1 # 1 &H # 1 # 1,, 7 J#,, 0 & # 1 # 1 '& # 11 # 1 #.,,,9,,J: 0 '&, # -. J f! "!%1.;''''*$%':"!"'(:,(,!-!%(2=(/- 3 # 1 # 1 Z ν *!,,7!,,J + &Ho& &J;o&;K &7o&; &>o& &;o&7 &o&h &; &; &H & & & f3,!>&h!k&h (;; ' 0 0 '#*!&,,,9,,7:(=.&;o& 1 ';;X,,9,,J:=&H 1 #0,, &;HKo&> # 1 # 1 'HX&&;HK # 1 # 1 & 0&Ho&J # 1 # 1 &>J # 1 # 1,,7J#,,0 '& &H # 1 # 1.,,,9,,J:,# H;

158 %3 G(! "!%11;''''*$%':"!"'(:,(,!-!%(.=(/- -. J f 3 # 1 # 1 Z ν *!,,7!,,J + &;o& &;HKo&> &Ho&J &Ho& &Ho& &Ho& &H &;HK &>J & & & f3,!>&h!k&h IV Les rendements en régime permanent (;> ' 0,. J# *,,,9,,7:.&, (P(,,A,.#-&!! CP &Jo&;H # 1!! CP &7o&> # 1 #, (P,&;o&;7 # 1!&>o&> # 1!#'X0,# *.,,, 9,, J:&, (P(,, & &K # 1 & =.,&;K # 1 #,(P&;> &>0,!!#! "!%1A;&,''% '(!C,!'' 9C :-: 2 '% '(!C!" +C :-: 2 (:, (,!%(2%(.=(/- -. J f!, # 1! CP! *P! CP! *P!,,7!,,J &Jo&;H &;o&;7 &7o&> &>Jo&> &K &;> &;K &> f3,!>&h!k&h IV Résultats macrocinétiques (Expérience 8) M*;'! "'(%(!,!''&")(&(&234:- 2 '%("&%=L,(!%(!&(%(&%!"&)( A4:- 2-2 D%(!!"&84:- 2-0,>&H!K&H!#, &,0, 1 ( &HH# 1 & 1 (L P &;# 1 & H>

159 %3 G( 1 (L P H&H;# 1 & 1 (,;&># 1 & 1 ( (,L 4 &K# 1 # %(&=. &,(>H,0,.( ;H#! "!%13; ','&'M%((!:"%'&!'")!",!=(6%("!!'% AE14B,"%'#$%'!'",!. %(2,(!(!%(.- -.# f $(# 1!,,, D. ( * *, +L L # 1 # 1 "L " 3,L 3 L P L P 4,L 4 L * H&JK & 7& & &; >& K&JK &K H&J C C C P C $ ( 1 -.# f!,, &>7 &K7 & &> f3,!>&h!k&h IV Les cinétiques,=&j# 1 (>, =&H# 1 (J=&J# 1 =K#"&(,&,,=,.# 0.,0= # 1, # 1 # (.=&H# 1 #%, =,, 90,:(,#,, &, (, # > & =0,&H# 1 =&# 1 # HH

160 %3 G( K J 7 H > ; 7 > 7 > & &7 &> & & &7 &> & % # 1 ; > H 7 J % # 1 $(, # 1,, 3(X, ( 3(X, ; > H 7 J, ; > H 7 J K & & &7 &> & & & &7 &> & %(2,(!!%(.0AE14B 6488,"%,(!"-(!,!''>(%:@(!:"%'('&%" > "%@(!!">M!%@! ">(@-"%&"!':&"!,&)!M%& '% '(!>@- :,'(,!' H7

161 %3 G( 0'(;7(;J#,,>&,6'&( (,0 E,/(#,,(9,,: (,7&;o&H # 1 7>&H;o& # 1.,#0(> # 1 # 1 # = # 1 0( > X# (,&!&,,&HK&o;&K # 1 &JH&o;&J # 1 J&Ho&H # 1 # '&,.#-&77o&H # 1 &&;Ho &7 # 1 &Ho&; # 1!>&o&K # 1 &&>Ko& # 1 &Ko&;J # 1!# (6=0&= =(,#(0 (,HX=,#! "!%18;(!',!''!":"%'!":"#("!&!$%!&'%$%! "(:,(,!!%(2=(6- -.# f % 3( # 1 C * +!, HK&o;&; JH&o;&J H&Ho&H &77o&H &>o&; &;>o&7 &>;o&, f3,!>&h!k&h! "!%1/;(!',!''!":"%'!":"#("!&!$%!&'%$%! "(:,(,!!%(.=(6- -.# fff % # 1 C * + 3(!, 7&;o&H &o&>j 7>&H;o& >&o& &Ko&;J &>o& &;Ko&J, f3,!>&h!k&h IV Les vitesses spécifiques globales en régime permanent (; ' 0 0 '#*!&,,9,,:(=.&>;o&7 1 ';JX#0 HJ

162 %3 G(,, &>HJo&H # 1 # 1 ';X&0 &Ho& # 1 # 1 'X#,,& 0 & # 1 # 1 '&J # 1 # 1 #% 0 ' 1 (, (,,&,(,1,=,0#! "!%16;''''*$%'(:,(,!!%(2=(6- -.# f 3 # 1 # 1 Z!,, ν * + &>;o&7 &>HJo&H &Ho& &7Ko&7 &Ho& &o& f3,!>&h!k&h (;K00'# *!&,, 9,, : ( =. &H o & 1 ' X# 0,, =&>o&> # 1 # 1 'X& 0&JKo& # 1 # 1 'X#,,&0 ' & # 1 # 1 &; # 1 # 1 #! "!%15;''''*$%'(:,(,!!%(.=(6-3 # 1 # 1 Z ν * + -.# f!,, &Ho& &>o&> &JKo& &o& &o& &;o& f3,!>&h!k&h IV Les rendements,(p(,& # 1.0 '>X.#,(P&>;Ko&K # 1 &;;o&j> # 1 0,!!&, 0(#'., (P.X# H

163 %3 G(! "!%A4;&,''% '(!C,!'' 9C :-: 2 '% '(!C!" +C :-: 2 (:, (,!%(2%(.=(6- -.# f!, # 1! CP! *P! CP!,,! *P &KHo&; &;;o&j> &o&h &>;Ko&K f3,!>&h!k&h IV Application à la production d éthanol G.,, 0 0 &. A,6,= ((#6,,, (, 0, 0,., 0&,,,(#,,(>#,,(=,(0&,0#&/9:( 07&K # 1 # 1 0J7&K # 1,&>; ((=,,(# -( # 9:, 0 ( =,,( J 0 =K # 1 0;&> # 1 # 1 #0(,. # 1 &,,,(,(,#$ &(00(&.&,,# 2 ((1 =,,( + /9KK:#0=(&00,&H& 0&=0H;&> # 1 # 1 0H # 1 &, ( => # 1 #%(,(,0.,# HK

164 %3 G(! "!%A2;,!(!'&'(&%'!"%(&**(''#'L,'&*(,!! - ', 4 %, %,,( % # 1 *0 # 1 # 1 K > 7 ; 7 &J 7 ; J J7&7 H &7! "%' K7 4'? bh 4'? bh f 4'? bh +/ KJKf / KK;!',,( J7&K 7&K / 41 H H;&> +/KK 41!' 7> > 9H: f,,1 9H,:, R0 = ',,(&,,1 &,,, ( 0 ( 0#-&( 005&.,0,,# $&.(=(,, A 6 ( /(# 0., p. p, &,, 9,,, > :& (,7 # 1 7> # 1 # 0> # 1 # 1 # = # 1,&>> (# 7

165 %3 G( %,,,, 9#q#>##:&,(0H=. 0&,( 0Y 9 =+:&( (,0(P=7X0,# %0., (0 ( #, 0( 9, ;X.:9F#E1+E:,0,,00(,,# IV Synthèse $,, 0 ;=; # 1,(9(>:# ; # 1 &0=,/,,'1,'1,,, &%G =(,#,,.,,#! "!%A.;(!',!'':"%'('&%"!""!! "%(!%&' (!%('2.(:,(,! ff H f -. J ff -.# ff % # 1 C * 3( C * 3(!,, H&Ko&7J HH&Ko&H >&Jo&J &Ko&H ;J&KHo&HH 7&;o&H ;&o&;> &Ko&7!,, ;&H;o&7 7K&H7o&>K K&77o&77 &;o&> H&H7o&JK &o& ;&Ho&7K &o&;!,,; >&;o& HH&7o>&J J&o;&J &>Ho&; H7&HoH&J o H&7o>&H &>o&!,,> >&o;&h ;J&7o ;&Jo& &;o&; K&oJ&> o &o &;Ho&>!,,H H&>o&H >>&Ho;&J &o&h &o&7 >&o o K&o &7o&!,,7 ;Ko; H7&Jo7&K J&o&K &HJo&K J&>o;&> &o&; >J&Jo&J &H7o&J!,,J >J&K K&J J& &Ko& ;& &K 7&J &Ko&>!,, HK&o;&; JH&o;&J H&Ho&H &>;o& 7o&H &o&>j 7>&o& &;Ko&J f3,!&h!#ff3,!>&h!k&h# 7

166 %3 G(.,, =7 # 1 #0(=,,,A,.#G = 0, # 0( ( ( K X, ' (, 0,,# $.,&,,, (,,; # 1 &,.,, # 0(,. #, (, ((,0(0#, 6, =.,,( 0, 0( 9. H 7:# $ 0.,,(0#,,=; # 1, ( # $.,& =7 # 1 0( #%B,/..'# 1 0, (.,(,9:# *.&0,,,(9(>;:# 7

167 %3 G(! "!%A1;&,''% '(!C,!'''% '(!C!"%("'(!%('2.%(!%&'(:,'(,!' ff H f -. J ff -.# ff!,! # 1 CP! *P! CP! *P!,, &KJo&> &;JHo&H &H>o&; &>;7o&>>!,, &>o&; &;Jo&K &;Ho& &;>o&>!,,; &>o& &;;7o&7 &;Ko&H &>H;o&!,,> &KHo&; &;o&> &>Jo& &>o&>!,,h &o&hh &;o& &;o& &>Ho&H!,,7 &Jo&;H &;o&;7 &7o&> &>Jo&>!,,J &K &;> &;K &>!,, &KHo&; &;;o&j> &o&h &>;Ko&K f3,!&h!#ff3,!>&h!k&h#, (P(, 0, #(0 #.,&,(, 0,(,.' 9%(:0#,(,.,=& # 1 9M # 9K7::# *&,(P!,!# 0., #! "!%AA;''''*$%'(:,(,!%("'(!%(' ff H f -. J ff -.# ff 3 # 1 # 1 Z ν *!,,!,,!,,;!,,>!,,H!,,7!,,J!,, Z ν * &o&> &;o&> &;Jo& &;o& &>Jo& &o& &o&; &;Ho&JJ &>o& &o& &;o&j &J7o&> &>7o&K &HH7o&> &Jo&7 &o& &7Ho&H &o& &>o& &>;o&k &;Ko&7 &o& &7;o&J &7o&; &;;o& &>Ho&H> &Ko&K &7o& &Jo&; &o& &Ho& &J;o&;K &7o&; &;o& &;HKo&> &Ho&J &; &; &H &H &;HK &>J &>;o&7 &>HJo&H &Ho& &Ho& &>o&> &JKo& f3,!&h!#ff3,!>&h!k&h# 7;

168 %3 G( ' 0 (,#,000,'=,(0(,#.!,. 0; 1 1 H 1; 1 #. & &, 0# 0(&.&, 0 0 (!!# $ &, 6, &,.,, &J7 &,,1, 0, 0#- & E +EE 9>:, & 0 0,,;J # 1 &,60;0(#$,(0&# 1 0,!; #* &, 0, =0,0, &(&# 1 A#G(&=.&> 1 & (,,0.A,, ( #* &,, A 0 0,,((#,( 9!!:,(=,0. #%.,,,4%(,A,.,,(,(,#,&,&, 4%(&,( '0.,00(0,9,(&( =&=T:# *,&,', 0( =,. 7>

169 %3 G(.A,# - ' &,,,9&E1+EE:# -&(0((6, 0=(,0(,,,# IV.2.2 Etudes physiologiques ' = 0 ( 0 ( 0 9 ' :# $ & ',, (, #% ',, ',,1, 1,A,, = 0 F 11,,,Q IV Eau intracellulaire *,,&,, ( >H#, = (,# 0 (=,(#&'.Z,.&( =(,9lCmr1 # 1 :&=9l*m\; # 1 :& =, # 0, =(7&>Xo&HX# *,,(&,, =,., #!& 0 7 X = > X, X 9=0,.,;X=0:#*!07X=>X&,0X= 09(>H:# 7H

170 %3 G(! "!%A3;%('!%(!""%"!(N &!'"'(!%('2.(:,(,! H f J ff 7 ff! -.# ff D(!,,!,,!,,;!,,>!,,H!,,7!,,J!,, XPC!! 7&>Xo&HX C lcm # 1 9 o 1 H>&7> H>&H H&Ko&7J ;J&KHo&HH >&J H&;K ;&H;o&7 H&H7o&JK H&7 H7&7 >&;o& H7&HoH&J >7&K7 H&7; >&o;&h K&oJ&> >&H> H&J H&>o&H >&o H&K H&J ;Ko; J&>o;&> >J&7 >& >J&K ;& >&;; >K&;H HK&o;&; 7&;o&H f3,!&h!#ff3,!>&h!k&h# C IV Les réserves en hydrates de carbones : glycogène et tréhalose ' 0,, ( 0,1,.9':#*,,&(,&0 (,, ' #,0., 0,, (, # 0 =,(0,,#% ' 0 0,*/9KKK:&, ', 0. # ( >7 ' ',.,,# 77

171 %3 G(! "!%A8;%(':"#:L(!"'N,!''$%!((!(D"!,!'''L&!'"' (!%('2.&(:,(,! X"!! +' +' C lcm # 1 C H f J ff 7 ff! -.# ff D(!,,!,,!,,;!,,>!,,H!,,7!,,J!,, o o o o 9 o 1 &HJo&H &o&j &Ko&K ;&HHo&> H&Ko&7J ;J&KHo&HH o &o&7 &;o& &o&h ;&H;o&7 H&H7o&JK & &Ho& &o& &;o& >&;o& H7&HoH&J &;o& &Ho&H &>o&; ;&o& >&o;&h K&oJ&> &;;o& &>o& &Ho& >&o& H&>o&H >&o &>o&> &>>o&h> &Ko& &JJo&7K ;Ko; J&>o;&> &Jo&J &7o&7 &>o& >&Jo&7K >J&K ;& &7o&; &J>o& &>o&7 &H;o& HK&o;&; 7&;o&H f3,!&h!#ff3,!>&h!k&h#., =, =,,;#,.,,!,,J0&>X"&=!,.,,(,, 0&HJX"#'. X, 0 A, ; X&,,,,H!9(>7:# '.,,#,.,!,,J0>&JX"&!,,0&J>X"#$&',., = '.X,,,,!&, ',' X 9(>7:# IV La composition élémentaire,,%@g0& 9γ:,,,# 7J

172 %3 G( IV Le degré de réduction γ 9γ: (, =.' (,&,,9& & & : # ( >J ' ( # (,,(=0;&K#% 9;&K:,( 0 0 %4 77,,.',+9KK7:9>&;:#09&HX: 0,(.' ',(,9.'.':# 0 γ,,, 0 0(,9,,:#,( 0& γ,' >&J> o & 0,.,;X0.,# (, & ',, (,& #! "!%A/;:('&(&%γ %'%("'&**('(:,'(,!'&!'"'&%= (!%('2.- $ γ lcm # 1!!!! C C J ff! 7 ff H f D(!,,!,,!,,;!,,>!,,H!,,7!,,J ;&Ko&> ;&Ko&J 8 Ko - 10 >&o& >&o&7 H&Ko&7J ;J&KHo&HH >&;o& >&o& ;&H;o&7 H&H7o&JK >&7o& >&Ho& >&;o& H7&HoH&J >&o&h >&;o& >&o;&h K&oJ&> >&Jo& >&;o& H&>o&H >&o >&>o&; >&o&; ;Ko; J&>o;&> >&Ho& >&o&> >J&K ;& -.# ff!,, >&o& >&>o& HK&o;&; 7&;o&H f3,!&h!#ff3,!>&h!k&h 7

173 %3 G( IV La masse molaire M,,(,,!" &77 #%, 1!" 0;&H #%, 1 & 0>X.0,,,(>#%,, & 0 0 A, =,, ( + 9KK7: 0 0 %4 77& 7&J #%, 1 9,,.' :# ' ( =,. 0# *,5'(, ;X,,=&;X#,,,6=(,H # 1 0,'>&> #%, 1 o&7j0x= 09(>:# $LE/9KK;:,&6=(, J # 1 &,, (,,(,# 0 0 0#! "!%A6;!''',"!(':-," 2 %'%("'&**('(:,'(,!'&!'"'&%= (!%(' J ff 7 ff H f! -.# ff!,,!,,!,,;!,,>!,,h!,,7!,,j!,, "," #%, 1 lcm # 1!!!! C C D( &77o&> ;&Ho&; ;&Ko& ;&>Ko&H H&Ko&7J ;J&KHo&HH >&7o& ;&7Ko&; ;&H;o&7 H&H7o&JK ;&>Jo& >&>7o& >&;o& H7&HoH&J >&o&7 H&>o& >&o;&h K&oJ&> >&KKo&> >&JKo&K H&>o&H f3,!&h!#ff3,!>&h!k&h# 8 Ko - 10 >&o >&Ho&J >&;o& ;Ko; J&>o;&> >&>Ko&;H >&>Ko&K >J&K ;& >&>Jo&H >&K;o&H HK&o;&; 7&;o&H 7K

174 %3 G( IV Synthèse,, ' 0 0(,#*& (,=H # 1 &,,, #%,,, &&'0 0#$,&&, = # & '[,A,,(==(,,=&(0, P0,A,,.',, # * &, & =,,,'# 0 (', (,#,# ( '1,'1,(,,# IV.2.3 Résultats des études physico-chimiques/physicomécaniques &,0,,#,, 0,, (,#0,0 (,0,, (# IV La pression osmotique (>K(,,# 0,,(&o J

175 %3 G( H,,#M ' HX 0. #,,,,.7#*.&0!&,,H,,#M >H#0!&,J,,#M H#G(0>HX,,,;>,7X!JX!#,,,., =, # 0,(0.,#! "!%A5;'%('&"!(''',$%,',"-: 2 0.&!'"'&%=(!%('(:, (,!- *,,,#M lcm # 1!!!! C C! J ff 7 ff H f D( oh ohj!,,!,,!,,;!,,>!,,h!,,7!,,j 8 Ko - 10 K; H&Ko&7J ;J&KHo&HH J K ;&H;o&7 H&H7o&JK H J >&;o& H7&HoH&J 7; J >&o;&h K&oJ&> 7J H&>o&H >&o H H ;Ko; J&>o;&> ;7 J; >J&K ;&,f3,!&h!&ff3,!>&h!k&h# IV Etudes rhéologiques 0,,,,,#,,,. 9:&0η #0 & &',(η # J

176 Chapitre IV : Obtention des hautes densités cellulaires IV La viscosité du surnageant η 0 La viscosité du surnageant η 0 est exprimée en Pa.s. Elle a été déterminée pour chaque régime permanent dans les deux réacteurs. La Figure 27 représente les rhéogrammes des surnageants pour les réacteurs R1 (graphique a) et R2 (graphique b) lors de l expérience 6. Quel que soit le régime permanent, le comportement rhéologique de ce fluide n évolue pas, il reste newtonien sur la gamme d étude du gradient de vitesse (D en s -1 ). Il en est de même pour toutes les expériences réalisées. 3 τ en Pa a 2,5 RP5 RP4 2 1,5 RP3 Ref 1 0,5 0 D en s ,5 τ en Pa b 3 2,5 2 RP5 RP4 RP3 Ref 1,5 1 0,5 0 D en s Figure 27 : Evolution de la contrainte de cisaillement τ en Pa en fonction du gradient de vitesse D en s -1. Rhéogrammes des surnageants Expérience 6 Réacteur R1 (a) et Réacteur R2 (b) pour chacun des régimes permanents (RP) 3, 4 et 5 et référence (fin de la phase en mode batch). 172

177 Chapitre IV : Obtention des hautes densités cellulaires De plus, aucune modification de la viscosité du surnageant, quel que soit le réacteur et le régime permanent considérés, n est observée comme le montre le Tableau 50. La moyenne de cette viscosité est de 0,0013 Pa.s pour R1 et de 0,0014 Pa.s pour R2. Tableau 50 : Synthèse des viscosités des surnageants η 0 en Pa.s pour chacun des réacteurs R1 et R2 lors des différents régimes permanents. η 0 Pa.s [X] g.l -1 R1 R2 X t1 X t2 Ref Fin de batch 0,0012 0, ± 101 Expérience 5 * Expérience 6 ** Expérience 7 ** Régime permanent 1 0,0015 0, ,90 ± 0,67 37,95 ± 2,55 Régime permanent 2 0,0015 0, ,53 ± 0,62 115,56 ± 0,79 Régime permanent 3 0,0013 0, ,3 ± 2,2 56,5 ± 5,7 Régime permanent 4 0,0013 0, ,2 ± 3,5 129,2 ± 7,4 Régime permanent 5 0,0013 0, ,4 ± 0,85 204,8 ± 1 Régime permanent 6 0,0013 0, ± 3 87,4 ± 3,4 Régime permanent 7 0,0012 0, ,90 223,0 Exp. 8 ** Régime permanent 8 0,0013 0, ,22 ± 3,23 160,23 ± 8,58 * Volumes R1 1,5 L et R2 8 L. ** Volumes R1 4,5 L et R2 9,5 L. Suite à cette étude, le comportement rhéologique du milieu de fermentation (surnageant avec cellules) est analysé. IV La viscosité du milieu de culture : viscosité apparente η a La viscosité du milieu de culture contenant les cellules, viscosité apparente η a, est exprimée en Pa.s. Elle est déterminée pour chaque régime permanent pour les deux réacteurs. La Figure 28 représente les rhéogrammes des surnageants contenant les cellules pour les réacteurs R1 (graphique a) et R2 (graphique b) lors de l expérience 6. Les rhéogrammes ci-dessous montrent une corrélation linéaire entre la contrainte et le gradient de vitesse. Le fluide a donc un comportement linéaire traduisant un comportement newtonien. 173

178 Chapitre IV : Obtention des hautes densités cellulaires 4 τ en Pa a 3,5 3 2,5 2 RP5 RP4 RP3 Ref [X] = 58,4 g.l -1 [X] = 42,2 g.l -1 [X] = 24,3 g.l -1 1,5 1 [X] 0,5 0 D en s τ en Pa b RP5 RP4 RP3 Ref [X] = 204,8 g.l -1 [X] = 129,2g.L -1 [X] = 56,5 g.l -1 [X] 10 0 D en s Figure 28 : Evolution de la contrainte de cisaillement τ en Pa en fonction du gradient de vitesse D en s -1. Rhéogrammes des milieux de culture Expérience 6 Réacteurs R1 (a) et Réacteur R2 (b) en régime permanent (RP) 3, 4 et 5 par rapport à la référence (fin de la phase en mode batch). Dans le domaine de variation considéré du gradient de vitesse ( s -1 ), le comportement du milieu de fermentation est assimilé à un comportement newtonien. Les valeurs des viscosités apparentes du milieu de fermentation (milieu contenant les cellules) 174

179 Chapitre IV : Obtention des hautes densités cellulaires sont déterminées pour chacun des réacteurs et lors des différents régimes permanents. Elles sont synthétisées dans le Tableau 51. Les viscosités apparentes sont plus élevées dans le deuxième réacteur que dans le premier réacteur. Les gammes de variations sont de 0,0012 à 0,0448 Pa.s et de 0,0012 à 0,0022 Pa.s respectivement pour le deuxième et le premier réacteur. Ceci correspond à des variations de 3600 % pour le réacteur R2 et de 83 % pour le réacteur R1. Tableau 51 : Viscosité apparente du milieu de culture η a Pa.s dans les deux réacteurs R1 et R2 en régime permanent. η a Pa.s [X] g.l -1 R1 R2 X t1 X t2 Expérience Expérience Expérience 7 ** 6 ** 5 * Ref Fin de batch 0,0012 0, ± 10 1 Régime permanent 1 0,0024 0, ,90 ± 0,67 37,95 ± 2,55 Régime permanent 2 0,0022 0, ,53 ± 0,62 115,56 ± 0,79 Régime permanent 3 0,0015 0, ,3 ± 2,2 56,5 ± 5,7 Régime permanent 4 0,0016 0, ,2 ± 3,5 129,2 ± 7,4 Régime permanent 5 0,0016 0, ,4 ± 0,85 204,8 ± 1 Régime permanent 6 0,0017 0, ± 3 87,4 ± 3,4 Régime permanent 7 0,0016 0, ,90 223,0 Exp.8 ** Régime permanent 8 0,0022 0, ,22 ± 3,23 160,23 ± 8,58 * Volumes R1 1,5 L et R2 8 L. ** Volumes R1 4,5 L et R2 9,5 L. IV Synthèse L étude des paramètres physico-chimiques montre : - un comportement rhéologique newtonien pour le surnageant. Sa viscosité ne subit pas d évolution en fonction de l ensemble des conditions expérimentales explorées et sa valeur moyenne est de 0,0013 ± 0,0001 Pa.s. - un comportement rhéologique newtonien pour le milieu de culture dans les domaines de variation du gradient ( s -1 ) et de concentration en cellules (0-223 g.l -1 ) étudiés. La viscosité apparente du milieu biologique varie de 0,0012 à 0,0448 Pa.s respectivement pour une concentration en biomasse de 9 g.l -1 et de 223 g.l

180

181 %3 G( "%' -,&(( 0' & 0 0 %4 77 (, ;=; # 1 &0,,/ 9; >&,,:#, = 0 =7 # 1,,(0,(,# ',( '.0.,,# *,,(. (&, (., 0,#0,0( 0& 0 = (, 0(,( A #, (P(, (P, (0 0.,, 0., / $0,&0,0&,A,0 ' ( (,#,, # 2 0., (,,, #,,(,,, #(,&6=&,,,(, γ05,(0(&γ,'>&j>o& 0 0,., ;X 0#%,,,0(,,#,,0,, ' 0, '1, JJ

182 %3 G(,.#,,0,,b,0 0l1 1 m0; # 1 = ; # 1 #0,( 0&*#=&>>*# 0, =&;o&*# # $ 0& 0 0 0&,,,10,& ' (,# %( 3,(,0 0&0,,10,# $ 0& N,R0,O0 ( 0, J

183

184

185 %3, V Impacts des hautes densités cellulaires (&% (., ( =,, = (,,,; # 1 =; # 1 & & 3 ',,&, 0, (0 = # $, &, (,0( 00 ',& 0, '1,P'1,,10,# G ( &, '1,,,,,A,(&,,0,#%P&=& 1,&N,R0,O 0 ( 0,#

186 %3, V.1 Impacts de hautes densités sur l activité du micro-organisme (, (!#, ',, #, 09Z:&9ν :,,(9 :#, 0 ( NO,1, 0 0 0( 9Z 0& ν 0 & 0 :&,( 0. 0 = (, 9Z & ν & :# V.1.1 Vitesse spécifique de croissance D K, 0. = (, = (, 0( (,# &7 &H &> &; & & & Z 1 Z Z0 7:%(.5;"%&F. >'#, ""@F. >'#, "&@> lcm # 1 H H H.(,& 0, =,. (,,,0+,/9K:&"/ 9KJ: 9K:#% 0. C # 1

187 %3,, # 9K:,,,A,,(,,# -, (, 0 # - 0,(,( E',# 2,0 ', ( 0 =,='9 9K::# -, 0 (,& 0 =(,0(9&H 1 : (,., # $,(,(/&,,0( 0&,0(,(,'1, 0 0( 5 (,, 0# % ' =, 0,, # V.1.2 Vitesses spécifiques de consommation de glucose et de production d éthanol,a,,,, (,0,, 9 0 : 9ν ν 0 :# D ; D ; 0, 0 0,, 0 (,# =, 0 #2,0=(, (0 =,, ( #%,=,&,&0 = 0 & 0 0( (. 0 N (O 0 (, #* (,,00(,, # ;

188 %3, &H & # 1 # 1 Z Z0 &H & &H lcm # 1 & H H H 7:%(14;"%&$ '. >'#, ""@$ '. >'#, "&@>:-: :"!"&,!''>:- C # 1 & & ν # 1 # 1 Z ν Z0 ν0 & &7 &> & lcm C # 1 # 1 & H H H 7:%(12;"%&ν. >'#, ""@ν. >'#, "&@>:-:- :"!"&,!''>:- # 1 ; # 1 &, (,00( (0# ',( A 9 9K::,,60.,0#,.,, (0 9K:& = 0 HX >X,,. 0. >

189 %3, 9KK:# & ', (, 0#,. & = & 0., 0 0(& 0 0( (,# (6 /, (,, 0( 0 # V.1.3 Influence du flux de glucose sur les paramètres spécifiques "9KH:&"#9KJ: 9K:&,.,. 0. ',#.,,,#$0.& =7 # 1.',, 0(,#&0 A,(,&.' &0.(#,. ( 0 0 &,,, 0( 0# 0 0 0(. D ;# H

190 %3, &K Z 0 1 & &J &7 &H &> &; & & D.( # 1 # 1 H H H ; ;H > 7:%(1.;"%&"!'''*$%! "&(''! 2 *&%*"%=&:"%':- 2-2 $,,&. (, 0 0( 9. ' : = 0,' & 1 # *,,.&,BB.(# D ;; 0 0 0(. # & ν0 # 1 # 1 & &7 &> & D.( # 1 # 1 > 7 7:%(11;"%&"!'''*$%&(&%! "&H!"$. :-:- 2 *&%*"%= &:"%':

191 %3, 00= (, 0(, =. 0(#*. (,&0(&; # 1 # 1 #%,,,,&.,,& 0,# 2 0, 0,,. (0D ;>#*.(,& 0,, (=&7 # 1 # 1 &.' # ; 0 1 &H &H &H D. # 1 # 1 > 7 7:%(1A;"%&"!'''*$%&',,!&:"%'$ '. :-:- 2 *&%*"%=& :"%':- 2-2 & (,,,,. B# - &,. & 9 9K: " / 9KJ:: ( 0,=(,0(P0 ( (#% 0 0,,, ',,1,# ((,,,',,1,.,,. ' # (. J

192 %3, &., &, 0 B0 = 7 # 1,,0# /&.(,,6B0 0(&,,, & & B, (=,,(# *&. B,&.,.&0((9;KX:(,1(9X:9#9>::= 7H # 1 # ", 0, & 0 ( 0, &HJo& # 1 # 1 ((1 =' &7K # 1 # 1,1(#%09.',.,,:, (,,0,,( 0,&0,,. # V.2 Impact de la concentration en biomasse sur la «morphologie» de la cellule et sa composition,(,,1,& &, # 2,, &,, 9%@G: (, 0 (' (,#*(, $GP" P 9,( P,:& 0 0, &(#

193 %3, V.2.1 par ml Corrélations DO/Masse sèche/nombre de cellules V Corrélation DO/Masse sèche $GP,,&,, [ 0, 0,, #%,, = D ;H&, 9 = ; # 1 (,:# 0$G.,,,,0,9 # 1 : 1!"9 # 1 :]&KK7f$G& 1!"9 # 1 :]&f$g# %0 9K:( ; # 1 &,0, (,= # 1 # ; " # 1 H ']&.! ]&KJK H H!! 9!: 9!: ']&KK7.! ]&KJ $2$G H H H ; 7:%(13;(("!("!&'$%>@D8.4,"!,!'''L:- 2 - %,& 9K:,,,, &(0.#(=0 (,#%(=, 9K:&,' K

194 %3,,0. 9K:(,NO 0,,(# ( $( 7:%(18; '(!!%,('&'"%('("''%("(!%(.-!+!'!!' %,!:(''%(*&(9A4>,('@- - & ' / ' =,=,,(#,& 00& ( ',,,# ', ( 9D ;7: 0, (, #%, A! [ (, 0,,!# - = $GP" (,( 9 (:&, (.& (,& = 0,., J X, # (.,,# * & " 9KH: 9K>: =,,,,= 9K:#"9KH:,, (, = # 1 = 0 C]f92$G: ( &(0&H=&KJ#%,, =0(0,=&&KHK0 K

195 %3, &K!5 ]&K; (]&K7 0 &K!#%,A,.0(&.# %,,0,, (,,. 0., # $ ',, ( 0,(,&,,, = (,.,,# V Corrélation Masse sèche/nombre de cellules par ml D ;J,0.,,,,,(,# H O9P:- 2 H H lcm(%# O9P ""-, 1. 2 ; &-U &-UK >&-UK 7&-UK &-UK &-U &-U &>-U &7-U &-U 7:%(1/;(!,!''=(,:- 2 &,!L('L"!(!, ( &""%"'!(,-,( 0,,,. (,.,,#* (, # 1 &(0,,( 0,#*&,0, (& #,, = 9# q #;#:# - & (0, (, K

196 %3,,,#%,P,(,,, 0,, (,&,, 9K:&, = ' $GP"# *, 0, (,&, 0, 0, 0, ',.&, ', # V.2.2 Impact des hautes densités cellulaires sur l eau intracellulaire et le volume des cellules D ;, 0 (,# J X XPC 7 H > ;!! lcm # 1 H H H 7:%(16;"%&%%(!:&H!%(!""%"!(*&"!(!,!'' 'L!"%(!%&'(!%('-,(, =(.,=HX!=>X9 =,.0(,! =7 # 1 :#%(0,0. 9K:(0,&>X=; # 1 (,& (=>X#,., A ( 0# $ K

197 %3,.& 0,, E 0 0, # % 0(=,&, 0,,('#,!9;X0 :&! 9H X 0 :# *, 0! A =,,!! 1,, 9# 3: 0 X! =!. & X.HHX.J# 1 $ &,,( (, # &,, A =.,# 1 -& 0 &,.,,(,9/9KK::# $ ',& =,,&,.&=,,., (,#,.,,,(,,., 0, # & ',, ' '1,,&,0A#$'[0, 0, 0,. 9 && T:>?? (, 9 9K::& X&,.,,(H # 1 #, ',,. 9( :& 0,HZ,&0&;? # 1, 7 X 0 92( / 9KH::&,.,, 7 # 1 # K;

198 %3, $.,&,., (, ; # 1 0 HX& 1,0,0&0, 1 0,0&,0,(# D ;K00,ε C,( 0,#0,0, 0,#,,(0,&,A,0,&>H,,A, 0,& ( A,(# 0,,(/(,,# &H &>H &> &;H &; &H & ε. &H & &H (%P, &-U &-UK >&-UK 7&-UK &-UK &-U &-U &>-U &7-U &-U 7:%(15;"%&"!*(!"%,$%*&"!(!,!'', (& ""%"'C, $,( (, &, =,(#%'A6=#-"9KK>:, &&0,,(&, #, = (,., 0,., (,=(,&, (, # - & ' 0,=,,# K>

199 %3, V.2.3 Impact des hautes densités cellulaires sur la composition élémentaire de la biomasse G, 0,, &, A = # 0,,,(,,,01=109,P ':5,.=, = N(,O# &, (,,,(,9%@G:& 9γ:&,, 9": (' & ' & ( 0 ' (,1,& # 00,,9 #%, 1 : (, (,,&.D >D >#,,(,.(,(# %, ' (& 0 γ,,, 0, 0 (,#,(0&,' γ,' >&J>o &&0,.,;X0#$LE #9KK;:,6=(,J # 1 & (,,(,&, # $ 0,, (0 #-, X 6= 0,' >&HKo&>H=H # 1 (,# &,,,, = (, =H # 1 #%(0,,, (, 0,, & &09#3:#(,,&, KH

200 %3, 9&:0,A,,.' 9:, # & ',,&, & =,# >&> >&; γ >& >& > ;&K ;& ;&J ;&7 ;&H >&K H 7 > ; ; ;K > > H7 H 7H J H K 7 H ; lcm # 1!,!!!! 7:%(A4;"%&%&:(&(&%γ*&"!(!,!''>:- &%=(!%('2. H&H " #%, 1 H& >&H >& ;&H ;& &H & &H & >&K H& 7 > ; ; ;K > > H7 H 7H J H K 7 H ; lcm # 1!,!!!! 7:%(A2;"%&"!,!'',"!(>:-," %("'&%=(!%('2.- K7

201 %3, V.2.4 Impact de la concentration en biomasse sur les réserves intracellulaires $, ( 0 ', (. #*,, 0 &, '',&00, D ># X" & 7 &7 > ' &> & & 7 &7 > &> & lcm # 1 H H H 7:%(A.;"%&%%(!:,!L('L&'('('*&"!(!,!'':- 2 - *= # 1 & 0 =;X" ' &>X"# 1= (,& 0, 6= 0>X" ' &>X"# % 0 0, ( 0 = (,#,&,., 1= # 1,,,,(,(,1,( 0#$',0A,,, = # 1,,',,# KJ

202 %3, V.3 Impact des hautes densités cellulaires sur les paramètres physico-chimiques du milieu de culture V.3.1 Evolution de la masse volumique de la suspension cellulaire,0,(,, (9,=;%:#-& = =,& =, 0,'#,,=,,0,,,,D >;# * ( (, 9 = ; # 1 :&, 0,&K;? # 1,6=0&H?# 1 #%0 =,KX,H # 1 # "9KK:&=&(00,0,? # 1 =&>H?# 1 0'0&H= H # 1 &,>X# $ &,, 0,.,(0"9KK:#.,.0',, &,(&(T:=;%&,,."9KK: ' 0 ' = %#, 0, (,,&,,,A,, 9( &,,&,(T:# K

203 %3, & ρq:- 2 & &K & &J &7 O9P:- 2 H H H 7:%(A1;"%&"!,!''"%,$%&%,"%&*(,!>Q:- (!,!''>,!'''L:- V.3.2 Evolution de la viscosité du milieu % 0,,,0=; # 1 0 = 1 # V Etude du comportement rhéologique du milieu de fermentation $ &,,,, b,, =!β / 9KJK:,00'#%& 9K:,& &,,,, ' Gb11`#,,..,,9 &&&,,&(:,0((# D >>, 0 0, & =,, 9,,.:& 0 (,,,,,# 0 ',,,,,6= # 1 (,'N-O H η = & + &># G >8/@ KK

204 %3, [C(,., # 1 # &H &>H η *# &> &;H &; &H & &H & ']&># 1H #lcmu&!e]&kh &H lcm # 1 H H H 7:%(AA;"%&"!''&#!,$%!!(η! >+!-'@*&"!(!,!''>,!L('L:- 1=(,&0,8. 0 # $ (0, G,$9KKJ:#, # 1 1= 0,0,.& / 00,(, (,, 0 =,,#-&00η 0 η η #%0,000.,,,#20,,0 0(,,# V Etude de la viscosité relative, 0 0 9( H: 0., η 4 0,ε 9 0,# (H, 0(,A,,#

205 %3,! "!%3.;'(''%(("("!''&''%'''&"%('*&"!*(! "%,$%- %%(' 0 9KJ: L, f 9KK: G $0 9KK:! 9KJK: #& '%''', $,HZ,, $,HZ, /0 0 $,HZ, / 9,HZ,: &Hε G η = η + ε G &H,!($%' ε G ε &H,. = &H ε = + G η η &7;J ε = &,. 7;J &HHε G η = η + ε G &7; η = η ( ( &7ε ) ) &H G ε G ε ε G ε &>,. = &7 &>,. = 0, ε C 0, 0,, #%,# 0,, V=&0,D >H# &7 &H &>H ε ε C &> &;H &; &H ']&.! ]&KKH> & &H & &H lcm # 1 & H H H 7:%(A3;"%&"!*(!"%,$%ε 9 *&"!(!,!'''L>:- 0,,=(, 0'

206 %3, G [ G ] ε = & >86@ [ ε C 0,9,: lcm(,9,: # 1 # 0,,., ( &>H (,; # 1 #!β/9kjk:(,, 0 &; 0 0 #!β / 9KJK: 000'#G&,, 0,0,#$&,,=,,.,,# 0,!β / 9KJK: A. 9&;&:# &, ( 0, #, 0 9 :,,.9 0:,, 9(H;: ε 29[ G ]: (,,&( G = 0,=,, ( η 29ε: 9D >7:# = 00,(,H1 # 1 =;(,; # 1 #%(, (,=,00,#

207 %3,! "!%31;!%"!*&"!''("!>η ε 9 D!$%(!,!'':- 2. lcm # 1 ε ε C η >&7 & &JJ >&> &> & H& &; & H&H & & H& &> & H&> &> & H&JH &> & & &H & H&K & &H>J >&; &H &H> ;&H; &J &HH; ;J&KH &H &; ;K& &7J &>;H >& & &; >J&K &H &;;; H7&H &; &KK H&> &; &; 7>&7H &J &7K J&> &H ;&7 H&H7 &; ;& K& & H& 7& &;7 & >& &>H &7J ;& &>>K K&7J (,&,G $0 9KK:,(0,,9D >J:# ;H ; H H η 09KJ: G $09KK:!9KJK: L,9KK: 00, H ε ε C & &H & &H & &H &; &;H &> &>H &H 7:%(A8;''("!η ( &''%'''&"%(*&"!*(!"%,$%ε 9 - ;

208 %3, (,G $09KK:&,, ' η ) ε G = η + ε G ε,. = ]&7 ε,. ] &7;J &7# ε G η = + ε G &7;J ;H η ; H " 00, H H & &H & &H & &H &; &;H &> &>H &H 7:%(A/;&"'!&"H"%&"!''("!η ( '%!",&L"('*& "!*(!"%,$%ε 9 -,0.,. ( 0 0&, = 0,, 0 0# 0000,,,,1#,& 0, = &H 9 (,=JH # 1 :&00 = 0#',(,# * 0,, &H &;& = (,,JH # 1 H # 1 &000,;=J# * 0, = &;& (,=H # 1 &000,, ε C ε >

209 %3,.&, 0,#,, 0, & 0,,,,. 9,:#+V=&00, 0,#%,, 000#, 0,,& '1, '1, 0,, # V.4 Impact des hautes densités sur la filtration tangentielle *,&.,., 9): 9`:&,(,,(& = 0A,# V.4.1 Impact de la concentration en biomasse sur la pression transmembranaire, sur la perte de charge le long de la membrane et sur la contrainte à la paroi, 0,,( 0, 0#$& ==,,(# * &,,,,(& 0A, & = # 0, 0 A # V Explication des calculs,0, H

210 %3, 0, $,,,(. L P; (, ; # 1,e ].,,(,e,e 3, 0,# 1 *-.,,(0,( *=,,( ( E L * *- (!E 7:%(A6;, (!&H%"(!*"(! V Calcul de la perte de charge ( 6 6 ) + 9S S = ρ >85@ (& ' 4 =,,.,,(# 8 = >/4@ 8 4.,,(=, N0O, 7

211 %3, J S ; P S f f = = $ F. 8 ρ ρ >/2@ ; P &7; = $ F 8 ρ >/.@.,,((( J&7K&JJ# &0E ( ]E ( E ; P S ; P &7; f : 9 = = $ F $ F S ρ ρ ρ >/1@ + = S ; P &7; : 9 $ F S ρ ρ >/A@ V Calcul de la pression transmembranaire moyenne PTM *,,(,'& 0,*,,(*-.,,(#! G. 4 E (& ',,,,( 8 S. 6 S. 6( + = + + e e ρ ρ ρ >/3@ ( ) S 8 S.. 6 6( ρ ρ + = e e >/8@ G'3n3 ( &(,0

212 %3, S 8 S. 6 6( ρ ρ e >//@ " ( ) S S = ρ >/6@,,( *.,,(# ( ) S S 6 S 8 S ( S 6 = = ρ ρ ρ e : 9 >/5@ 8 S. S 6 6 S 6 = e : 9 : 9 ρ ρ >64@ + = S ; P &7; : 9 e : 9 : 9 $ F S 6 6 S. S 6 6 S 6 ρ ρ ρ ρ >62@ G e : 9. S 6 6 ρ ρ α = >6.@ + = S ; P &7; : 9 $ F S 6 6 ρ ρ β >61@. * S S 6 β = α : 9 >6A@,,(,' *",',,(;>#&.

213 %3, 69 S : + 69S: S + S = = α β >63@ V Calcul de la contrainte à la paroi =.,,0 * τ = 8 >68@ > V.4.2 Comportement des paramètres physiques liés au module de filtration 0 &,,(,' =,(,# V Pression transmembranaire moyenne PTM D >K 0,,(,' (,9, # 1 :# &H *( *"9(: &H &H C 9 # 1 : lcm # 1 H H H ; "R0.,,,( 7:%(A5;"%&"!(''(!',, (!!(+>!(@*&"!(!,!''>:- 2 &!'"!,, (!- * (,, = H # 1 &,,(=0&H(#1=&,,.0(,## K

214 %3, V Perte de charge le long de la membrane H m 0,,(,=0,,(,,,D H# &H &H C lcm 9 # 1 : H H H ; "R0.,,,( 7:%(34;"%&"!(&!(: 0,>!(@*&"!(!,!''>:- %(%''&%*"%&'!&28,-' 2 &!'"!,, (!- =&;((,0 =H # 1 #1=&,. (0,(,# V Contrainte à la paroi τ 0 D H, 0 = (,#0==H *,,,=H # 1 &,,. (,# % 0,=,,(#

215 %3, ;H τ* ; H H H lcm # 1 H H H ; "R0.,,,( 7:%(32;"%&"!(!D"!!(τ>+!@*&"!(!,!''>:- %(%''&%*"%&&!'"!,, (!'!&28,-' 2 - (,,,, 0, 1 &,,(,' = 1(, # $ 1 (,, ( 0#- & 0,,(#,, =(=,,,(&= # 1 %0,=0,, =(,# 1 (, H # 1,( A 0 0,# 0,,0',,,(&, '',0A# C # 1

216 %3, V Détermination du nombre de Reynolds,(!'!,(,0. *! = η ρ >6/@ D H,(!' (,# K! J 7 H > ; C lcm # 1 # 1 H H H ; 7:%(3.;"%&%, (&#"&'*&"!(!,!''>:- $ 1&,(!', (,#* ( 1; # 1 &,(!' =,'(#* & # 1 &, ',# E (,&,&,(!'0>94 9KH::&(,0 # 1, # 1=H # 1 &,,,#%', 0(=,,,(&,, # & N,O 0 '',

217 %3,, &,.,(,,( H # 1 # 03,,'$ & 0,(!', 0 '1, P '1,,,#,,(!',,,, Y = 0 0 η, 0, ρ# &,(!', 1 0,& 1,0,,# G&,,,,, 9 3#;#:&,,0,,KX,., >,(!' 9!:#%,., =,, 0,,,# V.4.3 Impact de la biomasse sur la densité de flux de perméat D H;, 0 ',., 9):,,.,9, ; # 1 :#.,,00, 9), :# 2,HX.,(0H,#%,0. 9K:# ( (, 0,,,, =,,(&,,1#%,,,,(# ;

218 %3, &7-1H &>-1H 7"%=&(,!?, 1 -,. -' 2 &-1H &-1H &-17 7&-17 >&-17 &-17,' &-U & H& & H& & H& ;& 7:%(31;"%&"!&'&*"%=&(,!?>, 1 -,. -' (''(!',, (!!(,#+'!&4//!(-,(,9,:(=,.,9D H>:# C # 1 J 7 H > ; C O9P:- # 1 2?, 1 -,. -' 2 lcm D.,) &7-1H &>-1H &-1H &-1H &-17 7&-17 >&-17 $.,) &-17,' &-U ; > H 7 J K 7:%(3A;"%&"!&'&*"%=&(,!?>, 1 -,. -' 'LO9. P>:- &((%"!'!>.3, (!',, (!!(,#>+E4//!(@'!- 0 =,,(&,1, 0,,,(, 0 L 6@" = >66@ ηf # >

219 %3, [ ).,, ; #, 1 # 1 *",,(,'* η0,*#!,,,(, 1 # *,,(!,.,,0 6@" # = >65@ L fη D HH 0,,( (,9,:# &>-U;!,, 1 &-U; &-U; ']-U.U-U! ]&K7 &-U 7&-U >&-U &-U C # 1 lcm # 1 &-U ; > H 7 J 7:%(33;"%&"!(''!,, (!!(, >, >,!'''L:- * (,&,,( =,.=9#q###:#,,(,& 06=0 &H U;, 1 (,7H # 1 #,,(,H&JH# $,.=0=7H # 1 & 0,, 0.,.,# &,,,( 9D HH: =,1,0, # H

220 %3,,,&, (,,,(& B # %, 0 ( = ' ',((,=0,,# V.4.4 Stabilisation de la concentration en biomasse en régime permanent et évolution de la densité de flux de perméat pour des régimes permanents successifs. (0,.,,,,,;>D H7#,H, (,;=00,, %G,&,/( # H H O9P:- 2 C 9 # 1 :?, 1 -,. -' 2 &-1H &-1H &-1H &-1H H,>,;,H lcm D.,) &-17,' &-U H H H ;,(, $,8, G0.,,,( &-17 7&-17 >&-17 7:%(38;"%&"!&'&*"%=&(,!?>, 1 -,. -' 'LO9P>:-

221 %3,,(,9: 9(:&,(.,,,, H ;& (,,H9 # 1 :;97 # 1 :#,8, ',( = ( ',, ( #- & 0,8,&(0,.,0(# (0 (,0 E, 0,#%, ( =,, =,,(, ',#-&"9KKJ:,,'',,,,,,(#6 E,,((,(0,,, 0,#.,,,& (., A ( & 1,#% Y,,.,,,(,R0=,,1=# V Aspect énergétique V Rappels $=' &,, ;,9R : =,,,,,,R 9:,,,,R 9:# - = '.& (0,,,# J

222 %3, * * * * L * L P; * * L * 7:%(3/;('!&H%(&%D(#"!:,, `?`#, 1; & =,,, ;,& P = P ) + P ( 6 6 ) ρ F6# FP; # 8 = + 7 ;&7# # F6 η ) η 9"9KK::>54@ 0 η η % 0,,,, ]* 1* * *,0,&,,(,'*"*"]9* U* :Pp* # * * *"#$&* =,# * P* P -,,.=&J&,,, ;, = ρ 8 P F 6# 6@" + F # 7 ;&7# # F f&j P; = 8 6 >52@,,,, ( 0,, & &,.,& A # $ P ρfp; # 8 = >5.@ 7 ;&7# # F f&j 6

223 %3, V Résultats D H0,, (,9 # 1 :# 7 > 7 > `?`#, 1; lcm C # 1 # 1 H H H ; 7:%(36;"%&"H(:'*$%!"(!,!%, 1 &(,!>QR-, (!,!''>:- 0,, (,,=,,,(,'& &=0#*=H # 1 & 00,' &H ± &H?`#, 1; # 1= H # 1 &,,,., 0.,# %,,,,.,,,, D HK#GE,&1= &A,,# K

224 %3, $.,) 7 > 7 > `?`#, 1; ), ; #, 1 # 1 &-1H K&-17 &-17 J&-17 7&-17 H&-17 >&-17 ;&-17 &-17 C lcm # 1 &-17 # 1 &-U H H H -,,` 7:%(35;"%&"H(:'*$%!"(!,!%, 1 &(,!>QR-, *"%=&(,!?>, 1 -,. -' %,,.,,(!,(AH # 1,Y # V Evolution de la densité de flux de perméat en fonction du débit de la pompe et de la pression transmembranaire en concentration croissante en biomasse,,r0,,(, #&(,0=,,0,,,(.,,,,,D 7#

225 %3, $.,, ; #, 1 # 1 $(, ; # 1. 1> 7:%(84;"%&"!&'&*"%=&(,!?>, 1 -,. -' &((%"! >, 1 -' (!!(,#>!(@-, (, =,.,#2(,,0,,(,, 0,,(#"1=0&,#,(0, = & 0,, 0 R,=,A,,=.,,,(#,', '', (,0,(,&,,(9,:,(,( ',,(#"0,Y.&, (& ', ( 0#

226 %3, ", 0 6 =,.,#-&"?',9KK;:& (0,,''&= 0 & V., =,,(, ' 0#" (, = &, 0 0,,, 0 R, # %,, V0A=(0 9!, / 9KK:&? / 9KK>::#, V,,', # G, 0,,(# 2,,,(,.,,&1=0&,,A,,,,(#.,# B60.&(,,,(,, &,#

227 %3, "%' % 0 =,,,',,1,0,(,10,[0# &, R0 0 (& ( (1 = ' &, (,, &(,0,,/0'1,'1,# ' = 0. (0 (,.0.#. ',( = 0&,&=&6,,0.# 0 &,, = (, 0(&,, 0(,1,#&,. (, & 0(0&,,, 1( 9 /& >:#- & = 097H # 1 :&000( 9#7K # 1 # 1 :(1(= # 1 (, 0 0( X ((1 = ' =7 # 1 (,0(;KX# %,, ',1,# $.,&,0, 0. # & (., ( 0 > # 1 # 1 = + = #-,(, & 0,, ( & =,,# ;

228 %3, $0,&,,,, (, # % 0,, '&#$ (0& ' &(0.&#,,, ' (, = # 1 #%, = A,,,(,, =,,,#,X!=,,,![,;X(0# %. ',&,,,&0#,,,,,, 0,, ' b,,,91; # 1 :,, :#000,,,,1; # 1,,'G $0 9KK:#000.0, (,# =,&, &,,(,'& =,,( = &,#& =0,,#,(,(.,, ( 0 Y 9, :,, 9, (,:#%,=(,H # 1 # >

229

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231 %+*0 Conclusion Générale et Perspectives 0.,0,., 0 = ( = (,.,/,(# 20((,0,.& 0,(0.,&, A,6, (#., 9( ': ( ( =, 0,,.# -,,,00&0..'# 2- &",&)%'(%%( ":$%! ( %( ") &!%'&''""%"!('!,,&,!&)!"!"! (&%&)!"!( %(!-., 0 ((1 0 ' &,(&,( 1 (0,5 1 &0,0,., (5 1 0(,/ 9,,0,:'. 5 1,/ 0,, 6 ', 6 (,# ( ',0,( ', ( 0 # & H(0.&,((#.',,( (# (,,,# J

232 %+*0.- )!($%!!&%,(,,(!%&'""%"!( 0,, & 0,,&,',10,#,& ; # 1 =; # 1 &(,,&,, 0 0 #,&=; # 1, =7 # 1.,&0 E &, (#%= 0.(0,&,'1,#,&=0,(P(,(P&,, & S1=1, 0., # ((.,' NO = (, & = 0, ( (,,(# %& 8 & / 0 (,&,,(&= (,0(&0,#*&0,1, # $ 0,&., (,, 0& &, # 2 ' 0 (., #0,.,;X,X., # 0 ' (& & 0 0, 6= (, # 1 # 1= &,00,(0&=0, 0 ' #,, (,,," γ#%=0,'

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234 %+*0 *,.,,,0&0..,. (,, (0 0 & =, 0 0 0# 2- -(,0&,(, E#, & & [ 0 A &, 1 9 E p(, p,,: #- &0 E,,,1, 1,( 5 1,,,(& 0,,&,,,(&0 =.1#.- ( (, 0,,,(00&,& ( (,(0#((. 0,, 1, &(,HH # 1 ; # 1 0,5 1 & (, 0,7 # 1 7> # 1 &0> M #, 1; # 1 &(= # 1,(P&>># %& ;K X, (, 0#0 # # 0,,, ( ' 0,,(& A ( ',.& ', T 1,,,(5(.,0,.5 ;

235 %+*0 1,, 9&,: 9 &'(1'&1(:0,,# (# 9( B 0,:, ((1 0 ' #,,,,1,. #, A 0, 0,(# # %G,,0#%G,(,0,#,( (0 0 0 ( =, ( 0,=# # ;

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239 !4( Références bibliographiques ##&#&%,'C#&+,+#&2()##&+#"1 )0 %#& >& ',,' 0 ',(0(1&44'#- #&37&J1 # #&"1)0%#&+#-#&2()##&+,+#4(#&&,0 0(','0('0, ' 1(&#"(#4&37&771J# &%,'C&4(&4.%&2()&+,+&"1)0%& + -& >& ' 0',,' 0 1( & "( 4#& 3 7;& H& H;J1># ( &@'%-&) G4&KKH& b,,' 0 b (',#&F&3&&KH;1KHK# $#&%%#&"!#&" D&&M,b (,,&4,4#&3 K1K;&;H;17H# 4'? $#*# b `##& H&-, & (1, & (,& `# )# "(#4#&3&;1# ;H

240 !4( 4?"#)#&! ##& KJ&%0 '(' '# 0# 4,# - #4#&3;H&J1J# 4 +# #& KH& D, 1b, & $&3H;&1;&HJ1JK# 4%(D#7&0.&# 4'#&"*##+0*#&&%,, 0('0&#"(#4&3H7&H;1 H7# %b %*#&3E'D)#!@#&KJ&-0',1,,( * &, '(' 0 &)# +# "(#53;;&>&HJ17H# %' +#*#& b `#"#& K7&- '#%!0"(#& 3 ;&;&K1#!0b# "#&K&-,.',,(E &#"(#4#&3K&J1# '(',,(1( '&4#0#&3&>7J1>J# %b'#&k7&-&,, ##J7## %'"# ""##& K>&-,,(( ' (1%0 0&*4,#&3H& >1# ;7

241 !4( %'"#""##&K7&",((&%,&37&7J7p7# %'"#&""##& K;& 1,,,((,&4##&3H&#HK s H># %'b? +#!# `%#!#& KJJ&!, 0, '&4#4 #3K&H1>;# $, $#& %#1#&) # ` ##& KH&*,&?& ( E ', b '(',,(& #"(#4#&3#7K s JJ# $ +#& M0E -#& % "## *,, #4#& KH& (,,, '#4##&3J&H>pH>7# $ +#&! D#,*#& KK&,,(,&%,& 0 $# $E)#@#&2()##*.#&KK;&-, (, ' '.0 ' 1' S0&#"(#4#&3;K&7K17># $ LE )#@& KK& D,,1 S0.(,( =' & 7J&## $% #&KK&0b &#"(4&3;J&7>J17H# ;J

242 !4( $, #&,#.)##&&%, ' (&4#4 #&37&H&H>1HJ# - &#D&#&K&'"2+bF "#&)#+#"(#&3;&;7H1;7K# -()#"#&! M#$# %'"#& &-*,,(& # 4,#4#&3K1K;&;1K7# D8 )# * )##& &!0 (',(, ' 0&D-""(#!0b&3H&H1>H# D?@#&#&G?`#"?b@#&KJ&b,(? ',0 b('(&)#d,##&3h7&;h>1;7# +-#&*? `# # b #`#& & ' ((( E,,',(&4#4 #&37K&& ;1# +)#"#&KK&F((&"(#"#4#!0b& 37&;>1;7# +&%#5 &!#& KK&- -#F#3#;#&2M,,*&###HpHK# +1G D#& +,E%-# 3#-#& & G.'?.,&-E#"(#&3J&717K# ;

243 !4( + C#"#& a C#L# 4 D#`#& H& G, E ',&4#4 #&3K& H&H;pH;# + #M# ' #$#& KJK&!, ''# # * ( (,E, # 4# 4 #&CC&>1># +,+#$+#&K7&0,0,1, (( = & % # D# "(#&&J1>;# +, +# " "# *#& K& ", (,(#( #',#G0 (&b$&kjp# +%##& 0 M## ##& KKK&,( ' 1b,(& #"(# 4#& 3 H& ;1;H# +`#)#&???%#"#&`,!#@#&3!#+#)#"#'(M#%##"#& KK&M('(?',,&4- #&3&;K1>J# +6)#"#!#& K>& 0, &)#4 '&37&;&J>1J# +3#&KK7&-,,',,'0.'0.##>## ;K

244 #&,E*#`?#&KJ#@,' $#4#&E#)#"'%#)#&K>&,? (',C0#4## 37&&17#, #G## 4? #"#& K>&-,1,& 0#"(# *'#&3H&H;p;# $##& " ##&!( %#!#& " #& KH& - (' 1 ',,(E b1(,,( (& # "(#4#&3;&H1K# $##&, `#)#& ' $#*#&% ##&" ##!(%#!#& (,,(E -/ '& 4#4 #&3H&7H;17# $##& ' $#*#& % ##& " ##!( %#!#& K;& - ('0,,(Eb1(,,((& #-0#"(&3>7&&7>1J# )!#)#+*#D#&K&-(.' b,& -E',"(##&3>&1;# >

245 !4( )!#*#&*,,#+*#D#&P"'K&,(''1 0,0(&*4,#&>1>K# M G& K7&! (,(, 0 '#0"(*'#!0b&3&1K# M )#$# $? M##& KKK& %'(, b ',,' 0 ( &)# 4#& 3 J& 1;& H1;# M M#@#& KK>&%1b ( &",( &3J&HK1;# (' (, b1,,( 1' (& 4#4 #&3J;&&H1;># %#& K& D, ( =,,(& JJ# # %#&"b?)# +, +#& KJ&@ ',b'((',&4#&3 K&H&;>J1;H# )#"#&$ )#*#&"!#0)#"#&KK;&%,.'(''1,010 b.'1, '&)D,#4 #&3JH&;&J1# >

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254

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271 Bioprocess Biosyst Eng (2004) 26: DOI /s ORIGINAL PAPER A. S. Aldiguier Æ S. Alfenore Æ X. Cameleyre Æ G. Goma J. L. Uribelarrea Æ S. E. Guillouet Æ C. Molina-Jouve Synergistic temperature and ethanol effect on Saccharomyces cerevisiae dynamic behaviour in ethanol bio-fuel production Received: 11 July 2003 / Accepted: 30 January 2004 / Published online: 20 April 2004 Ó Springer-Verlag 2004 Abstract The impact of ethanol and temperature on the dynamic behaviour of Saccharomyces cerevisiae in ethanol biofuel production was studied using an isothermal fed-batch process at five different temperatures. Fermentation parameters and kinetics were quantified. The best performances were found at 30 and 33 C around 120 g l -1 ethanol produced in 30 h with a slight benefit for growth at 30 C and for ethanol production at 33 C. Glycerol formation, enhanced with increasing temperatures, was coupled with growth for all fermentations; whereas, a decoupling phenomenon occurred at 36 and 39 C pointing out a possible role of glycerol in yeast thermal protection. Keywords Ethanol fermentation Æ Saccharomyces cerevisiae Æ Temperature effect Æ Ethanol tolerance Æ Glycerol production Introduction A. S. Aldiguier Æ S. Alfenore Æ X. Cameleyre Æ G. Goma J. L. Uribelarrea Æ S. E. Guillouet (&) Æ C. Molina-Jouve Institut National des Sciences Appliquées, De partement de Ge nie Biochimique et Alimentaire, 135 Avenue de Rangueil, Toulouse Cedex, France [email protected] Tel.: Fax: Very high ethanol performances in fermentation using Saccharomyces cerevisiae are determined by several factors such as medium composition and operating parameters including substrate and vitamin feeding strategy, oxygen level and temperature [1, 2, 3, 4]. This latter parameter becomes crucial for optimising low-added product bioprocesses. One of the constraints in such bioprocesses is to find a good compromise between on one hand the optimisation of the fermentation temperature for productivity, yield, ethanol titre and cell viability, and on the other hand the minimization of energetic cost linked to fermentation temperature regulation. However, as mentioned by Torija et al. [5], very few investigations were reported on the impact of temperature on the dynamic behaviour of S. cerevisiae during fermentation processes. When temperature and ethanol induced synergistic effects on dynamic growth and production rates, the experimental evaluation of fermentation parameters and kinetics may provide the determination of the optimal temperature profile for bio-ethanol production in dynamic processes. Our approach consists in the complete analysis of isothermal alcoholic fermentation at five different levels of temperature, in fed-batch culture, under perfectly controlled operating conditions. Global fermentation parameters and kinetics were determined when growth and ethanol production occurred. Complementing numerous studies on heat shocks and added ethanol stress on yeast physiology, this paper will extend the knowledge of the temperature effect on ethanol fermentation by S. cerevisiae where the cells have to deal with growth and ethanol production. Our study applies to the context of intensive bio-fuel production supported by environmental and economical issues and in a quite different well-documented application field such as vinification. Materials and methods Microorganism, media and growth conditions The S. cerevisiae CBS8066 strain was supplied from the Centraalbureau voor Schimmelcultures (The Netherlands). The strain was maintained on YPD (yeast extract 1% (w/v), bactopeptone 2% (w/v) and glucose 2% (w/v)) agar medium at 4 C. Pre-culture of yeast cells was carried out in a 5 ml tube of YPD rich medium at 30 C for 16 h on a rotary shaker (100 rpm) containing NaCl 0.9% (w/v). The culture was transferred into a 500 ml erlenmeyer flask containing 150 ml of mineral medium (ph 4) prepared as follows (all compounds are expressed in

272 218 gl )1 ): KH 2 PO 4, 3.0; (NH 4 ) 2 SO 4, 3.0; Na 2 HPO 4, 12H 2 O, 3.0; sodium glutamate, 1.0; MgSO 4,7H 2 O, 0.5; ZnSO 4, 7H 2 O, 0.04; MnSO 4,H 2 O, ; CoCl 2,6H 2 O, ; CuSO 4,5H 2 O, ; Na 2 MoSO 4,2H 2 O, ; CaCl 2 ; 2H 2 O, 0.023; (NH 4 ) 2 Fe(SO 4 ) 6,6H 2 O, 0.023; H 3 BO 3, 0.003; pantothenate, 0.005; nicotinic acid, 0.005; mesoinositol, 0.125; thiamine, 0.005; pyridoxine, 0.005; paraaminobenzoic acid, and biotin, Glucose was added at a final concentration of 40 g l )1. After 10 h of growth at the desired temperature (27, 30, 33, 36 and 39 C) on a rotary shaker (100 rpm), the 150 ml culture was used to inoculate the 2-litre fermentor containing 1.3 l of mineral medium with vitamins as described above. Fermentations Fed-batch experiments were performed in a 2 l fermentor using a SETRIC fermenting system. The temperature was regulated at 27, 30, 33, 36 and 39 C as desired, and the ph at 4 with the addition of a 14% (v/v) NH 3 solution. The fermentor was flushed continuously with air through the sparger placed at the bottom of the reactor. The flow rate was 18 l h )1 corresponding to a maximum VVM of 0.2. The stirring rate was fixed at 400 rpm until the dissolved oxygen (D.O.) reached 20% of saturation, then increased in order to avoid any oxygen limitation in the cultures. Chemicals Chemical products (glucose, salts, oligo-elements, orthophosphoric acid and NH 3 ) were provided from Prolabo, the vitamins from Sigma and the sodium glutamate from Merck. All products were of the highest analytical grade available. Feeding strategy Vitamin feeding strategy All fermentations were performed with an exponential feeding of biotin and vitamins based on the growth profile [2]. Glucose feeding strategy Fermentation was started with a glucose concentration of 100 g l )1. Each time that the residual glucose concentration was lower than 20 g l )1,a glucose feeding (700 g l )1 ) was carried out to reach a glucose concentration of 100 g l )1. When the ethanol concentration was above 90 g l )1, the glucose feeding brought the concentration back to 50 g l )1 (cf. Fig. 1). Fig. 1 Change in the mass of glucose, ethanol, glycerol and biomass in fed-batch fermentation at 27, 30, 33, 36 and 39 C. Thick arrows represent the glucose feeding to a final concentration of 100 g l -1, and thin arrows the glucose feeding to a final concentration of 50 g l -1 c

273 219 Analytical methods Yeast growth was evaluated by spectrophotometric measurements at 620 nm in a spectrophotometer (Hitachi U-1100) and calibrated against cell dry weight measurements. The spectrophotometric measurements enable evaluation during the fermentation of the doubling of the population and the generation time for the exponential addition of the vitamins during the growth. Cells were harvested by filtration on 0.45 lmpore-size polyamide membranes and dried to a constant weight at 60 C under a partial vacuum (200 mm Hg (ca kpa)). A change of 1 unit at OD 620 was shown to be equivalent to 0.84 g of dry matter per litre. The determination of glucose from fermentation supernatants was performed during the fermentation with a glucose analyser (YSI model 27 A: Yellow Springs Instruments). Concentrations of ethanol and acetic acid in the medium were determined by gas chromatography using a Poraplot Q column (25 m by 0.53 mm) with nitrogen as carrier gas and flame ionisation detection (Hewlett Packard, 5890 A) and the following conditions: an injection temperature of 250 C, a detector temperature of 270 C, an initial oven temperature of 115 C to a final temperature of 235 C, at a rate of 12 C min )1 and an isotherm of 10 min, a flow rate of carrier gas of 20 ml min -1 and an injected volume of 50 ml. Determination of organic acids and glucose from fermentation supernatants was performed by HPLC (Waters Alliance) using an Aminex HPX-87H column (300 mm by 7.8 mm) and the following conditions: a temperature of 50 C, with 5 mm H 2 SO 4 as eluant (flow rate of 0.5 ml min )1 ) and a dual detection (refractometer and UV at 210 nm). The biomass formula was determined at ENSIACET (Toulouse, France) by elemental analysis of C, H, O, N and ashes. The biomass formula used to convert cell dry weights into molar carbon concentration was C 1 H 1.79 O 0.64 N Determination of cell viability The cellular viability was determined by the methylene blue technique (Postgate 1967) [6]. A 200 ll sterile solution of methylene blue (0.3 mm in 68 mm Na 3 citrate) was mixed with 200 ll of a yeast suspension diluted to reach an OD 620 nm of 0.4 to 0.7. The mixture was shaken and placed in a Thomas s counting chamber after 5 minutes incubation. The number of stained (nonactive cells) or unstained (active cells) and the number of buds were counted in 5 different fields with a total of at least 200 to 300 cells. The percentage of viable cells was the number of unstained cells (living cells) divided by the total number of cells (stained and unstained). In our conditions, the mean (m) of the viability is estimated with an accuracy of 10% [6, 7]. This means that the interval m±0.1m contains the true value of viability with a probability of 95%. Results and discussion The impact of ethanol and temperature on the dynamic behaviour of S. cerevisiae in ethanol biofuel production was studied using isothermal fed-batch process at five different levels 27, 30, 33, 36 and 39 C. Fermentations parameters and kinetics were quantified to assess the effects of temperature on growth, ethanol, by-products formation and yeast tolerance. Impact of temperature on fermentation parameters Experiments were performed at different temperatures until an uncoupling effect of growth and ethanol production occurred. Glucose mass was consumed, cell and product (ethanol and glycerol) masses were plotted versus time in Fig. 1, and Table 1 summarizes the fermentation performances. Direct comparison of metabolite concentrations was allowed because of similar final volumes for the fermentations, except for the one at 39 C. Thus, the dilution factor does not affect the interpretation of the variation of the different parameters. The fermentation carried out at 39 C had very poor performance in terms of growth and ethanol production. As a result, only 100 g of glucose were consumed in 27 h and no glucose was added thereafter, explaining the lower final volume. When the temperature increased, biomass reached a maximum value at 30 C (39 g in 30 h) and then drastically decreased down to 2 g at 39 C. The optimum temperature for ethanol production (around 260 g, i.e., 120 g l )1 ) was found at 30 and 33 C. The main by-products were glycerol, succinic acid and acetic acid. Within the temperature range C, glycerol production was lower at 30 and 33 C and higher at 36 C; succinic acid and acetic acid masses gradually decreased Table 1 Temperature effect on biomass, ethanol production and by-product formation of Saccharomyces cerevisiae CBS 8066 during the Fed-batch fermentations 27 C 30 C 33 C 36 C 39 C Fermentation time (h) Final volume (l) [Ethanol] (g l 1 ) l max (h 1 ) m pmax (g g 1 h 1 ) m l=0 p (g g 1 h 1 ) Y X/S (g g 1 ) Y Eth/S (g g 1 ) Y Gly/S (g g 1 ) Average ethanol productivity (g l 1 h 1 ) l max : maximum specific growth rate, m pmax : maximum specific ethanol production rate, m p l=0 : specific ethanol production rate when growth ceased, Y i/s : yield factors of constituant i on glucose (with i: X biomass, Eth ethanol, Gly glycerol)

274 220 with increasing temperature from 10 to 1.9 g and 8 to 1 g respectively. At 39 C, the fermentation was sluggish with very low biomass (2 g), ethanol (43 g) and by-products (0.7 and 0.5 g succinic and acetic acid respectively) production, except for the glycerol production (11 g) that was enhanced. Fermentations at temperature above 35 C were already found very restrictive for growth and ethanol production in yeast cultivated on white must in aerobic static bottles [5], in batch cultures on wheat mashes [1], and in continuous mode with total cell recycling [4]. Within the temperature range C, the apparent growth yield on glucose was favoured with decreasing temperature: it decreased by a factor of 2 from 27 to 36 C and by a factor of 6 at 39 C. There was little effect on apparent ethanol yield on glucose as temperature increased from 27 to 36 C with a slight variation between 0.43 and 0.47 g g )1, supporting previous observations in static bottles [5], in batch [8] and continuous cultures [9], and then it decreased to 0.35 g g )1 at 39 C. As mentioned above, glycerol production was strongly enhanced at 36 and 39 C as shown by the significant increase in the apparent glycerol yield on glucose (about 4-fold and 6-fold higher at 36 C and 39 C compared to 30 C). This observation complements previous reports where glycerol production increased with increasing temperature within C with S. cerevisiae cultivated in grape juice in flasks [10], within C with mixed yeast cultivation on white must in static bottle [5] or after heat shocks from 27 Cto C on shochu brewing yeast [11]. It has been previously shown that cell viability fell with increasing temperature [1, 5, 12, 13, 14]. Then cell viability was quantified during our five fermentations. As shown in Fig. 2, a significant effect was observed on the cell viability due to temperature and ethanol concentration. Over 90% of viable cells was measured until the ethanol concentration reached a threshold value. Fig. 2 Comparison of the percentage of methylene blue-stained cells (Viability %) as a function of ethanol concentration in fedbatch fermentations at 27, 30, 33, 36 and 39 C Above this value the cell viability dropped drastically. This ethanol threshold value was found to be a function of temperature: it was about 80 g l )1 at 27 C, around 100 g l )1 at 30 and 33 C, and 50 g l )1 at 36 C. At 39 C, we observed a quick drop in the cell viability right at the beginning of the fermentation. As a result of the temperature effect on growth and ethanol production and cell viability, the best average ethanol productivities remained at about a maximum constant value 4 g l )1 h )1 at 30 C and 33 C. Impact of temperature on kinetic parameters Maximum specific growth rate (l max ) and ethanol production rate (m pmax ) have a similar classical profile with increasing temperature but a different optimum value, i.e., an optimum value of 0.43 h )1 at 30 C for growth and of 2.3 g g )1 h )1 at 33 C for ethanol production. The difference of temperature (3 C) between optimum growth and ethanol production was found here lower than that reported in the literature (5 to 10 C) though it is known to be strain-dependent as mentioned by numerous authors [9, 15, 16, 17]. Synergistic effects of temperature and ethanol concentrations have been shown on yeast growth upon addition of exogenous ethanol [15, 18, 19]. In this way, the ratio of specific growth rate (l) over maximum specific growth rate (l max ) and the ratio of specific ethanol production rate (m p ) over maximum specific ethanol production rate (m pmax ) were plotted versus ethanol concentration for the five fermentations (Fig. 3). These representations may exhibit the heat-induced and ethanol-induced damages on isothermal growth and production compared to the maximum capacities of the yeast at each given fermentation temperature. The inhibitory effect of ethanol on growth was observed for all fermentations whatever the temperature. For any given ethanol concentration, this effect was strongly pronounced for the highest temperatures as reported in the literature [9, 15, 20, 21]. A growth rate with 50% inhibition was seen for 40 g l )1 ethanol at 30 C, compared to 70% at 27, 33 and 36 C and complete inhibition at 39 C. The critical ethanol concentration (P crit ), for which growth stopped, was also found dependent on the fermentation temperature. The P crit value was the highest around 115 g l )1 at 30 and 33 C and then dropped to 90 g l )1 at 27 C, 75 g l )1 at 36 C and 20 g l )1 at 39 C. Temperature also affected ethanol impact on its own production as the specific ethanol production rate decreased with increasing ethanol concentration. The inhibitory effect of ethanol on specific ethanol production rate showed different patterns depending on the ethanol concentration range: for concentrations below 60 g l )1 ethanol, a negative effect of ethanol on production was less severe at 27 C andat 30 C for concentrations above 60 g l )1. When yeast growth ceased, we observed that fermentative activity of the yeast is not entirely inhibited.

275 221 Fig. 3 Ratios of specific growth rate over maximum specific growth rate (l/l max ) and of specific ethanol production rate over maximum specific ethanol production rate (m p /m pmax ) vs. ethanol concentration in fed-batch fermentations at 27, 30, 33, 36 and 39 C The specific ethanol production rates, at the P crit values mentioned above, gradually increased with temperature (Table 1) from 0.19 g g )1 h )1 at 27 C to 0.35 g g )1 h )1 at 33 C and then decreased to 0.29 g g )1 h )1 at 36 C. The value obtained at 39 C as 0.99 g g )1 h )1 cannot be compared to the others because of premature cessation of yeast activities resulting in a sluggish fermentation and, consequently very low ethanol concentration performed (20 g l )1 ). As depicted in Fig. 4 where specific glycerol production rate was plotted against specific growth rate, fermentation temperature affected the main by-product formation. We observed the well-known coupling effect between growth and glycerol production rate, and glycerol-biomass apparent yield can be calculated from the slope. The apparent glycerol-biomass yield exponentially increased with increasing temperature within the range C. More interestingly, a decoupling Fig. 4 Specific glycerol production rate (mglycerol) vs. specific growth rate (l) in fed-batch fermentations at 27, 30, 33, 36 and 39 C

276 222 occurred between growth and glycerol production at 36 and 39 C. For these temperatures, glycerol kept being produced even after growth ceased. In this range of fermentation temperature, the specific glycerol production rate, when growth ceased, was increased 6-fold with temperature. Glycerol formation in yeast is well-known as a response to aeration conditions, due to its role as a redox sink, and to the medium osmotic pressure as reviewed by Wang [22]. Increasing glycerol production was also reported after submitting yeast cells to heat shock [11, 23]. The authors explained this observation by an overexpression of the glyceraldehyde-3-phosphate dehydrogenase. Moreover, the few studies reporting an increase in glycerol production with increasing temperature during isothermal cultures were obtained using S. cerevisiae cultivated in flasks. Because the oxygen solubility decreases with increasing temperature, these systems are not suitable to differentiate temperature and aeration effect. Our data presented here in non-oxygen limiting and isothermal conditions raise the question about a potential role of glycerol in yeast thermal protection. Conclusion The study presented here clearly showed the contribution of fermentation temperature combined with high ethanol concentration on the dynamic biomass, ethanol and glycerol production, and the cell viability. As a result of the impact of temperature and ethanol production dynamic on cell viability, biomass and ethanol production, temperatures of 30 and 33 C led to better performances with a slight benefit for growth at 30 C and for ethanol production at 33 C. These data are of primary interest for designing process strategies such as two-stage high cell density bioreactors where growth and intensive ethanol production may be monitored separately. References 1. Thomas KC, Hynes SH, Jones AM, Ingledew WM (1993) Production of fuel alcohol from wheat by VHG technology: effect of sugar concentration and fermentation temperature. Appl Biochem Biotechnol 43: Alfenore S, Molina-Jouve C, Guillouet SE, Uribelarrea J-L, Goma G, Benbadis L (2002) Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process. Appl Microbiol Biotechnol 60: Alfenore S, Cameleyre X, Benbadis L, Bideaux C, Uribelarrea J-L, Goma G, Molina-Jouve C, Guillouet SE (2003) Aeration strategy: a need for very high ethanol performance in Saccharomyces cerevisiae. Appl Microbiol Biotechnol (in press) 4. Atala DIP, Costa CA, Maciel R, Maugeri F (2001) Kinetics of ethanol fermentation with high biomass concentration considering the effect of temperature. Appl Biochem Biotechnol 91: Torija MJ, Rozes N, Poblet M, Guillamon JM, Mas A (2003) Effects of fermentation temperature on the strain population of Saccharomyces cerevisiae. Int J Food Microbiol 80: Postgate JP (1967) Viable counts and viability. In: Norris JR, Ribbons DW (eds) Methods in microbiology. Academic Press, London, pp Nielsen LK, Smyth GK, Greenfield PF (1991) Hematocytometer cell count distributions: implications of non-poisson behavior. Biotechnol Prog 7: Jones AM, Ingledew WM (1994) Fuel alcohol production: optimization of temperature for efficient very-high-gravity fermentation. Appl Environ Microbiol 60: Groot WJ, Waldram RH, Van der Lans RGJM, Luyben KChAM (1992) The effect of repeated temperature shock on baker s yeast. Appl Microbiol Biotechnol 37: Gardner N, Rodrigue N, Champagne CP (1993) Combined effects of sulfites, temperature, and agitation time on production of glycerol in grape juice by Saccharomyces cerevisiae. 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Process Biochem 16: Van Uden N (1971) Kinetics and energetics of yeast growth. In: Rose AH, Harrison JS (eds) The yeast, vol 2. Academic Press, London, pp Sa-Correia I, Van Uden N (1983) Temperature profiles of ethanol tolerance: effects of ethanol on the minimum and the maximum temperatures for growth of the yeasts Saccharomyces cerevisiae and Kluyveromyces fragilis. Biotechnol Bioeng 25: Van Uden N (1985) Ethanol toxicity and ethanol tolerance in yeasts. In: Annual reports on fermentation processes. Academic Press, London, 8: Brown SW, Oliver SG (1982) The effect of temperature on the ethanol tolerance of the yeast Saccharomyces uvarum. Biotechnol Lett 4: D amore T, Stewart GG (1987) Ethanol tolerance of yeast. Enzyme Microb Technol 9: Wang ZX, Zhuge J, Fang H, Prior BA (2001) Glycerol production by microbial fermentation: a review. 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