Fermentable sugars recovery from grape stalks for bioethanol production

DOI: h ps://doi.o g/10.1016/j. enene.2013.06.006
© 2013. This manusc ip e sion is made a ailable unde he CC-BY-NC-ND 4.0 license
h ps://c ea i ecommons.o g/licenses/by-nc-nd/4.0/(opens in new ab/window)
1
FERMENTABLE SUGARS RECOVERY FROM GRAPE STALKS FOR
BIOETHANOL PRODUCTION
I. Egüés a, L. Se ano a,*, D. Amendola b, D.M. De Fa e i b, G. Spigno b, J. Labidi a
a Chemical and En i onmen al Enginee ing Depa men , Uni e si y o he Basque
Coun y, Plaza. Eu opa 1, 20018, Donos ia-San Sebas ián, Spain
b Ins i u e o Oenology and Ag o-Food Enginee ing, Uni e si à Ca olica del Sac o
Cuo e, Via Emilia Pa mense, 84-29122 Piacenza, I aly
* Co esponding au ho . Tel.: +34-943017125; ax: +34-943017140.
E-mail ad ess: lui[email p o ec ed] (L. Se ano)
Abs ac
Th ee di e en p ocesses we e in es iga ed o he eco e y o e men able suga s
om g ape s alks: au ohyd olysis a 121 °C be o e and a e a p e-washing s ep and acid
hyd olysis (2% H2SO4 w/w) a e a p e-washing s ep. Mo eo e , op imal condi ions o a
cha coal-based pu i ica ion p ocess we e de e mined by expe imen al design. All
hyd olysa es, wi h hei co esponding syn he ic liquo s we e used as e men a ion
subs a es o he p oduc ion o me aboli es by he yeas : Deba yomyces nepalensis
NCYC 1026. The main e men a ion p oduc was e hanol, whe e a maximum p oduc ion
o 20.84 g/L, a con e sion yield o 0.35 g e hanol/g monome ic suga s and a p oduc i i y
o 0.453 g/Lh we e ob ained om non-pu i ied au ohyd olysa e liquo . In all cases,
e hanol p oduc ion and cell g ow h we e be e in non-pu i ied liquo s han in syn he ic
liquo s. These esul s could be in luenced by he p esence o o he suga s in he
hyd olysa es, wi h highe concen a ion in non-pu i ied ones.
Keywo ds: G ape s alks, hyd olysis, suga s, pu i ica ion, e men a ion, e hanol
2
1. In oduc ion
G ape is one o he mos impo an ui c ops and i icul u e is one o he mos
impo an ag icul u al ac i i ies. The p incipal solid esidues p oduced in g ape juice and
wine making p ocesses a e s alks and g ape ma c. G ape s alks a e he skele on o he
g ape bunch and consis in ligni ied issues [1]. I s composi ion is annins, lignin,
cellulose and hemicelluloses p incipally. The upg ading o his by-p oduc h ough he
use o i s componen s could en ail economical and en i onmen al imp o emen s o he
indus y.
In conc e e, hemicelluloses, which link he lignin and cellulose, a e no being
isola ed o indus ial use, bu hey ha e a high po en ial and hey could be used in many
applica ions. The hemicelluloses a e made up o pen oses (β-D-xylose, α-L-a abinose),
hexoses (β-D-mannose, β-D-glucose, α-D-galac ose) and/o u onic acids (α-D-
gluco onic, α-D-4-O-me hylgalac u onic and α-D-galac u onic acids) whe e he hyd oxyl
g oups o suga s can be pa ially subs i u ed wi h ace yl g oups [2]. Many s udies ha e
been ocused in he hemicelluloses hyd olysa es use o ob ain bio uels, especially
bioe hanol, [3-5] and chemicals like xyli ol [6], u u al [7] o lac ic acid [8]. The
composi ion and he concen a ion o hyd olysa es ich in hemicelluloses, depend on he
used aw ma e ial and he applied ex ac ion p ocess. En i onmen al- iendly
echnologies such as au ohyd olysis p ocess, which uses wa e as he only eagen , ha e
gained in e es o hemicelluloses suga s eco e y. Fo ha eason, in his s udy,
hyd olysis p ocess was s udied using wa e as p incipal eagen o g ape s alks
e men able suga s ex ac ion.
P elimina y expe imen s we e pe o med o de e mine au ohyd olysis op imum
condi ions in o de o eco e he maximum e men able suga s om g ape s alks.
Fu he mo e, p e ious s udies [9] showed he p esence o non-s uc u al ca bohyd a es,
3
which a e suga s de i ed om imp egna e g ape juice. Fo his eason, a aw ma e ial
washing s ep be o e au ohyd olysis p ocess was also s udied in o de o know he
con ibu ion o hese suga s.
Be o e e men a ion s udy, oxic compounds which a e ypically gene a ed du ing
he hyd olysis p ocess should be emo ed o imp o e he biocon e sion o hyd olysa es.
Adso p ion on ac i a ed cha coal could be an e ec i e and low cos echnique [10]
depending on he op imiza ion in he ea men a iables. The e o e, in his s udy, an
expe imen al design was applied o op imise he pu i ica ion p ocess wi h ac i a ed
cha coal, in es iga ing he in luence o empe a u e, ime, s i ing a e and pH on he
emo al a es o lignin deg ada ion p oduc s (LDP), colou (C) and suga s.
Finally, he hyd olysa es we e es ed as e men a ion subs a es. Fo indus ial
applica ions, i is e y impo an ha he mic oo ganism has he capaci y o me abolize
mos o he suga s p esen in he hyd olysa e, o ob ain an e icien biocon e sion o all
he suga s in o indus ial in e es me aboli es. Howe e , many wild- ype yeas s ains
canno u ilize de e mina e suga s o hei me abolism and a e easily inhibi ed by oxic
compounds gene a ed du ing he hemicelluloses hyd olysis. Fo his eason, in his wo k
he yeas Deba yomyces nepalensis NCYC 1026 was selec ed. In ac , his yeas ,
o iginally isola ed om o en apple, is known o i s abili y o me abolize bo h hexoses
and pen oses suga s simul aneously and o g ow also in non highly pu i ied media [11].
2. Ma e ials and Me hods
2.1 Raw ma e ial and au ohyd olysis condi ions
The aw ma e ial used in his wo k was a mix u e o g ape s alks om wo
di e en I alian ed cul i a s, Bona da and Ba be a, kindly supplied by a wine-making
ac o y in Piacenza (I aly) in he 2010 in age. The samples we e collec ed immedia ely
4
a e he ope a ion o p essing/des emming, o en d ied a 60 ºC o 24 h, inally g ound
in a mill and sie ed o ob ain he 1– 4 cm size ac ion.
Chemical composi ion o he aw ma e ial, gi en on an o en d y weigh basis, was
de e mined acco ding o TAPPI s anda ds [12] and bibliog aphic p ocedu es. Ashes
(T211 om-93), ho wa e (T264 cm-97) and 1% NaOH solubili y (T212 om-98), e hanol–
oluene ex ac i es (T204 cm-97), lignin (T222 om-98), holocellulose [13], cellulose and
hemicelluloses [14] con en s we e de e mined.
The hyd olysis ea men s we e ca ied ou using unwashed g ape s alks in a
e ical au ocla e, which suppo s a maximum empe a u e o 121 ºC and a maximum
p essu e o 198.67 kPa. The au ohyd olysis p ocess ime was a ied om 60 o 90, 105,
120 and 180 min, keeping cons an he au ohyd olysis empe a u e a 121 ºC and a
solid/liquid a io o 1:8 (w/ ) in all expe imen s o ind ou he bes condi ions o ge he
maximum concen a ion o e men able suga s.
Fo he aw ma e ial washing s ep, he g ape s alks we e suspended in dis illed
wa e (solid/liquid a io o 1:10/ w/ ) in an e lenmeye lask and main ained unde
agi a ion a oom empe a u e o 2 h. The solid phase was eco e ed by il a ion, o en
d ied a 50 ºC and used hen o hyd olysis p ocess a de e mined op imum condi ions.
Finally, he e ec o sulphu ic acid as au ohyd olysis p ocess ca alyse (2% w/ )
was s udied o obse e i s e ec on hemicellulosic s alks suga s ex ac ion a
au ohyd olysis op imum condi ions. All hese expe imen s (schema ised in Fig.1) allowed
ob aining di e en suga concen a ion liquo s, p o iding in o ma ion abou he e ec o
di e en hyd olysis condi ion had on di e en e men able suga s ac ions eco e y.
2.2 Hyd olysis liquo pu i ica ion
A 24 ull- ac o ial design wi h h ee coded le els leading 19 expe imen s was
made o s udy he e ec o ou di e en ac i a ed cha coal ea men a iables (wi h a

5
ixed cha coal:hyd olysa e a io 1:40): pH, s i ing a e, con ac ime, and empe a u e.
The ange and le els (low -1, medium 0, high +1) o he a iables in es iga ed in his
s udy we e: pH 2-5-8, s i ing a e 150-200-250 pm; con ac ime 10-35-60 min;
empe a u e 25-35-50 ºC. The condi ions we e chosen in acco ding o p e ious s udies
and he wo k o Mussa o and Robe o [15]. Th ee assays in he cen e poin we e ca ied
ou o es ima e he andom e o o he expe imen al design. The emo al a es o lignin
deg ada ion p oduc s (LDP) and colou (C) we e aken as he esponses o he
expe imen al design.
All pu i ica ion ea men s we e ca ied ou in 250 ml E lenmeye laks ha
con ained 50 ml o unwashed g ape s alk hyd olysa e and we e agi a ed on an o bi al
shake du ing he pu i ica ion ea men . A e each ea men , he p ecipi a e was
emo ed by cen i uga ion a 5000 x g o 15 min and il e ed wi h glass ib e il e be o e
he HPLC analysis.
A e hyd olysa e pu i ica ion, he pH o all liquo s was adjus ed a pH ~ 5.4 wi h
NaOH 5 N and hen s e ilized in au ocla e a 121 ºC o 15 min o hei applica ion in
e men able p ocesses.
2.3 Fe men a ion condi ions
D. nepalensis NCYC 1026 used in his s udy was supplied as eeze-d ied s ain
by NCYC, Na ional Collec ion o Yeas Cul u es, UK. Acco ding o he gi en
ins uc ions, he yeas was ac i a ed in 10 ml mal ex ac (Oxoid), incuba ing o 5 days
in an o bi al shake (HT In o s AG CH-4103, Swi ze land) a 100 pm and a 25 ºC. The
ac i a ed yeas was hen di ided in o 3 aliquo s and 50 mL o esh mal ex ac was added
o each one. Fo h ee days, each day 20 ml o new esh mal ex ac we e added un il a
concen a ion o 1.07x108 cell/ml was ob ained. A e ha , he s ain was cen i uged,
washed and g own in wo di e en syn he ic media supplemen ed by simila suga s
6
composi ion o he hyd olysis liquo s ob ained in his s udy: 30 g/L o glucose + 30 g/L
o uc ose (wi h 6.5 g/L yeas ni ogen base, YNB om Di coTM) o L1, i s pu i ied L’1
liquo , L2 and i s pu i ied L’2 liquo , and 12 g/L glucose + 2.50 g/L uc ose (wi h 6.5 g/L
YNB) o L3 and i s pu i ied L’3 liquo . A e h ee days o g ow h a known olume o
he i s semi-syn he ic cul u e medium was ans e ed in o 50 ml o L1, L’1, L2, and L’2
expe imen al liquo s, co esponding o 1x106 cell/ml ini ial concen a ion, whe eas o he
second cul u e medium a known olume was ans e ed in o 50 ml o L3 and L’3 liquo s
always o ob ain a 1x106 cell/ml ini ial concen a ion. Addi ional syn he ic liquo s (B1, B2
and B3), wi h he same suga s concen a ion o L1, L2 and L3 hyd olysa es, we e also
p epa ed wi h s anda d suga s (Ca lo E ba, I aly) o compa e he e men a ion yield and
he g ow h in syn he ic liquo s wi h o iginal liquo s. All he inocula ed liquo s (in 250 ml
E lenmeye lasks) we e incuba ed in he o bi al shake a 140 pm and 25 ºC du ing he
expe imen s. Each ial was ca ied ou in duplica e.
Aliquo s o 1 mL we e pe iodically collec ed om di e en incuba ed liquo s o
obse e he cell g ow h, suga s consump ion and me aboli e p oduc ions. Fo his
objec i e, he aliquo s we e cen i uged a 8200 x g o 10 second o p ecipi a e he yeas
and o ob ain he supe na an .
2.4 Analy ical p ocedu es
To de e mine he lignin deg ada ion p oduc s, he samples we e dilu ed (1:1000)
in dis illed wa e and analyzed a 280 nm by a UV-1601 Shimadzu spec opho ome e .
Fo he colou de e mina ion, he samples we e dilu ed (1:50) and analyzed a 440 nm.
The cell g ow h was e alua ed measu ing he op ical densi y o cul u e a 600 nm
(OD600), whe eas he ela ion be ween abso bance and cell concen a ion (e alua ed by
cell coun ing in Bu ke came a unde op ic mic oscope) was p e iously de e mined o
ob ain a calib a ion cu e.
7
The suga s consump ion was e alua ed om supe na an using Megazyme ki
assays o D-Glucose, D-F uc ose and D-Xylose de e mina ion, whe eas he by-p oduc s
and o he suga s componen s we e analyzed by High Pe o mance Liquid
Ch oma og aphy (HPLC) Jasco LC Ne II/ADC equipped wi h a e ac i e index de ec o
and a pho odiode a ay de ec o . A Phenomenex Rezex ROA HPLC column (300 mm x
7.8 mm) was used o he expe imen , and 0.005 N H2SO4 p epa ed wi h 100 % deionised
and degassed wa e was used as mobile phase (0.35 mL/min low, 40 ºC and injec ion
olume 40 µL). High pu i y s anda ds o a abinose, galac u onic acid, lac ic acid, xyli ol
and e hanol (supplied by Fluka, Sigma Ald ich and Pan eac), we e used o he calib a ion
cu es.
3. Resul s and Discussion
The composi ion o he g ape s alks (weigh pe cen age) used in his wo k was:
22.61±2.14% ex ac i es, 32.35±0.31% lignin, 12.19±0.52% cellulose, 26.43±0.42%
hemicelluloses (13.35% glucose and 13.08% uc ose) and 6.11± 0.43% ashes. This high
uc ose concen a ion in he aw ma e ial de i ed om g ape juice which imp egna ed
s alks du ing des emming ope a ions.
3.1 Au ohyd olysis
Unwashed g ape s alks we e used o ind ou he op imum condi ions o
maximum e men able suga s ex ac ion. Table 1 shows he esul s o suga s
concen a ions in he liquo s o he ials aimed o s udy he bes condi ions o
au ohyd olysis in o de o maximize he glucose and uc ose con en .
All he liquo s p esen ed an acid pH alue due o he o ganic acid o med in he
au ohyd olysis p ocess. The op imal condi ions chosen o use he au ohyd olysis liquo
as a e men a i e medium we e 121 ºC, 90 min, 1/8 because his expe imen p esen ed
he highes e men able suga concen a ion.
8
3.2 Au ohyd olysis liquo pu i ica ion expe imen al design
Au ohyd olysis liquo con ained, in addi ion o uc ose and glucose as he majo
suga s, se e al compounds ha a e oxic o yeas s, namely, ace ic acid, u u al,
hyd ome hyl u u al (HMF), and lignin deg ada ion p oduc s.
The esul s o he expe imen al design (Table 2) showed ha he emo al o colou
and lignin deg ada ion p oduc s was dependen on he condi ions employed in he
ea men o he unwashed g ape s alk hyd olysa e wi h ac i a ed cha coal.
The hyd olysa e colou is di ec ly ela ed o he p esence o lignin deg ada ion
p oduc s (phenolic compounds), so a loss o colou in he ea men wi h ac i a ed
cha coal is ob ained by emo ing hese compounds om he hyd olysa e.
Lignin deg ada ion p oduc s and colou emo al we e s ongly in luenced by he
empe a u e and pH. pH 2 p oduced be e esul s han pH 5 and 8, p obably as a esul o
he low o ma ion o phenola e ions a low pH and he ac ha hese ions a e poo ly
adso bed on ac i a ed cha coal [16]. High empe a u es p omo ed an inc ease in he
densi y o he packing o phenolic molecules in he ac i a ed cha coal po es [17]
p oducing a high emo al o phenolic compounds, so he colou in ensi y o he
hyd olysa e also dec eased.
The condi ions p esen ed in he expe imen 11 (pH=2, 250 pm, 10 min, 50 ºC)
we e selec ed as he bes condi ions o emo al he colou and lignin deg ada ion
p oduc s wi h he objec i e o he applica ion o hyd olysa e in e men a ion p ocesses
a e he pu i ica ion p ocess.
The selec ed pu i ica ion condi ions we e applied o he liquo s om hyd olysis
o g ape s alks, hyd olysis o washed g ape s alks and acid hyd olysis o washed g ape
s alks. The esul s a e p esen ed in Table 3.
15
Lis o ables
Table 1. Au ohyd olysis condi ions in au ocla e (all expe imen s a 121 ºC and 1/8 a io
solid/liquid) and suga concen a ion o ob ained ini ial liquo s om unwashed g ape
s alks.
Table 2. Expe imen al design o he colou emo al (CR) and lignin deg ada ion
p oduc s emo al (LDPR) in he ea men o unwashed g ape s alk hyd olysa es wi h
ac i a ed cha coal.
Table 3. Cha ac e iza ion o di e en concen a ed hyd olysa e liquo s be o e and a e
pu i ica ion p ocess a op imal condi ions: liquo s ob ained om g ape s alks
au ohyd olysis (L1 and pu i ied L’1), liquo s ob ained om washed g ape s alks
au ohyd olysis (L2 and pu i ied L’2) and liquo s ob ained om washed g ape s alks using
2% H2SO4 (w/w) acid hyd olysis p ocess (L3 y L’3).
Table 4. E hanol maximum p oduc ion yields and olume ic p oduc i i ies by D.
nepalensis in s udied concen a ed liquo s.

16
Table 1
Expe imen s
Glucose (g/L)
F uc ose (g/L)
pH
60 min
5.50±0.50
6.80±0.60
3.92±0.10
90 min
8.30±0.80
9.60±0.50
3.98±0.10
105 min
7.40±1.10
8.70±1.00
3.89±0.10
120 min
7.05±1.25
8.40±1.15
3.86±0.10
180 min
8.20±1.50
8.90±1.10
3.87±0.10
17
Table 2
Expe imen
aX
1
bX
2
cX
3
dX
4
CR (%)
LDPR (%)
1
-1
-1
-1
-1
65.5
91
2
+1
-1
-1
-1
43.7
87.4
3
-1
+1
-1
-1
51.5
89.9
4
+1
+1
-1
-1
53.7
92.4
5
-1
-1
+1
-1
44.8
87.3
6
+1
-1
+1
-1
50.8
87.9
7
-1
+1
+1
-1
54.9
87.4
8
+1
+1
+1
-1
44.1
80.5
9
-1
-1
-1
+1
75.1
74.2
10
+1
-1
-1
+1
60.3
80
11
-1
+1
-1
+1
83.4
100
12
+1
+1
-1
+1
43.6
73.2
13
-1
-1
+1
+1
80.6
100
14
+1
-1
+1
+1
71.3
95.8
15
-1
+1
+1
+1
74.9
100
16
+1
+1
+1
+1
66.1
95.7
17
0
0
0
0
59
80.4
18
0
0
0
0
53.2
81.1
19
0
0
0
0
45.3
80.6
aX1: pH no malized; bX2: s i ing a e no malized; cX3: con ac ime no malized; dX4:
empe a u e no malized; anges: pH 2-5-8, s i ing a e 150-200-250 pm; con ac ime
10-35-60 min; empe a u e 25-35-50 ºC.
18
Table 3
Liquo s
Abso bance Concen a ion (g/L)
Colou
LDP
Glucose F uc ose Xylose
L1 0.87 0.53
30.96±1.19
30.70±1.50
0
L’1 0.14 0.00
32.00±1.00
30.00±1.20
0
L2 0.75 0.53
6.01±0.10
5.96±0.28
0
L’2 0.07 0.09
6.05±0.13
6.15±0.20
0
L3 0.38 0.69
12.22±0.33
2.57±0.20
9.78±1.19
L’3 0.009 0.17
10.90±0.10
2.54±0.05
9.27±0.10
19
Table 4
Liquo
Suga s
concen a ion
Fe men a ion Yields
(g/L)
Max
e hanol
p oduc ion
(g/L)
YE/S
(g/g)
YE/S
(g/g)
Qp
(g/Lh)
YB/S
(109cell/g)
YE/B
(g/109cell)
B
1
60.00
13.14±1.50
0.22
-
0.07
8.03
0.03
L
1
61.66
20.84±1.25
0.34
0.14
0.45
10.57 0.03
L’
1
62.00
13.16±1.10
0.30
0.09
0.45
14.39 0.02
B
2
12.00
1.90±0.25
0.18
-
0.09
16.17
0.01
L
2
11.97
2.66±0.30
0.22
0.01
0.12
25.56
0.01
L’
2
12.20
2.38±0.17
0.19
0.01
0.11
28.36
0.01
B
3
24.50
3.75±0.20
0.15 (0.26)1
-
0.07
8.86 (14.96)a
0.02
L
3
24.57
5.20±0.35
0.21 (0.35)1
0.02
0.07
19.21 (31.91)
a
0.01
L’
3
22.71
1.86±0.35
0.08 (0.14)1
0.01
0.06
11.41 (19.27)
a
0.01
Y
E/S
: e hanol yield (g e hanol pe g monome ic suga consumed)
YE/S : e hanol yield (g e hanol pe g o d y hyd olysed s alks)
Qp: olume ic p oduc i i ies (g/L o e hanol pe hou )
YB/S: biomass yield (x109cell biomass pe g monome ic suga consumed)
YE/B: e hanol yield (g e hanol pe x109cell biomass)
a: Yield aking in o accoun he p oduc ion o e hanol om glucose and uc ose (wi hou xylose)
20
Lis o igu es:
Figu e 1. Di e en hyd olysis ea men s condi ions used o g ape s alks e men able
suga s eco e y a op imal condi ions: g ape s alks eed (S1), insoluble au ohyd olysis
ac ion (S’1), au ohyd olysa e (L1), washed g ape s alks eed (S2), insoluble washed
au ohyd olysis ac ion (S’2), washed g ape s alks au ohyd olysa e (L2), washed g ape
s alks eed (S3), washed acid hyd olysis insoluble ac ion (S’3), and acid hyd olysa e
(L3).
Figu e 2. Suga consump ion and by-p oduc s ob ained om D. nepalensis g ow h in 30
g/L glucose and 30 g/L uc ose o syn he ic liquo B1 (2a), g ape s alks au ohyd olysa e
liquo L1 (2b), and he pu i ied liquo L’1 (2c).
Figu e 3. Suga consump ion and by-p oduc s ob ained om D. nepalensis g ow h in 6
g/L glucose and 6 g/L uc ose o syn he ic liquo B2 (3a), washed g ape s alks
au ohyd olysa e liquo L2 (3b), and he pu i ied liquo L’2 (3c).
Figu e 4. Suga consump ion and by-p oduc s ob ained om D. nepalensis g ow h in 12
g/L glucose, 2.5 g/L uc ose and 10 g/L xylose o syn he ic liquo B3 (4a), washed g ape
s alks acid au ohyd olysa e liquo L3 (4b), and he pu i ied liquo L’3 (4c).
Figu e 5. Cell g ow h o D. nepalensis in B1, L1, L’1, B2, L2, L’2 and B3, L3, L’3 liquo s
ep esen ed as cell/ml wi h monome ic suga s consump ion (C ) espec o he ini ial
suga concen a ion (C0).

21
Fig. 1
22
Fig. 2
a)
B1
0
5
10
15
20
25
30
35
0 20 40 60 80 100 120 140 160 180 200
h
g/L
Glucose
F uc ose
E hanol
Lac ic acid
b)
L
1
0
5
10
15
20
25
30
35
0 10 20 30 40 50
h
g/L
Glucose
F uc ose
E hanol
Galac u onic acid
Lac ic acid
A abinose
c)
L'1
0
5
10
15
20
25
30
35
0 10 20 30 40 50
h
g/L
Glucose
F uc ose
E hanol
Galac u onic acid
Lac ic acid
A abinose
23
Fig. 3
b)
L2
0
1
2
3
4
5
6
7
0 5 10 15 20 25
h
g/L
Glucose
F uc ose
E hanol
Galac u onic acid
Lac ic acid
A abinose
c)
L'2
0
1
2
3
4
5
6
7
0 5 10 15 20 25
h
g/L
Glucose
F uc ose
E hanol
Galac u onic acid
Lac ic acid
A abinose
a)
B2
0
1
2
3
4
5
6
7
0 5 10 15 20 25
h
g/L
Glucose
F uc ose
E hanol
Lac ic acid
24
Fig. 4
c)
L'
3
0
2
4
6
8
10
12
14
0 20 40 60 80 100
h
g/L
Glucose
F uc ose
E hanol
Galac u onic acid
Lac ic acid
Xylose
Xyli ol
A abinose
b)
L
3
0
2
4
6
8
10
12
14
0 20 40 60 80 100 120 140 160 180 200
h
g/L
Glucose
F uc ose
E hanol
Galac u onic acid
Lac ic acid
Xylose
Xyli ol
A abinose
a)
B
3
0
2
4
6
8
10
12
14
0 20 40 60 80 100 120 140 160 180 200
h
g/L
Glucose
F uc ose
E hanol
Lac ic acid
Xylose
Xyli ol