Appl Microbiol Biotechnol (1994) 41:62-72

App//ed
Microbiology
Biotechnology
Springer-Verlag 1994
An interlaboratory comparison of the performance
of ethanol-producing micro-organisms
in a xylose-rich acid hydrolysate
B. Ha hn- H~ i g e r da l 1, H . J e p p s s o n 1, L. Ol s s o n 1, A . Mo h a g h e g h i 2
1 Department of Applied Microbiology, Lund Institute of Technology/University of Lund, P.O. Box 124, S-22100 Lund, Sweden
2 Bioprocess and Fuels Engineering Research Branch, National Renewable Energy Laboratory, 1617 Cole Boulevard, Golden,
CO 80401, USA
Received: 30 June 1993/Received revision: 29 September 1993/Accepted: 30 September 1993
Ab s t r a c t . A xylose-rich, di l ut e-aci d-pret reat ed corn-
cob hydrol ysat e was f er ment ed by Escherichia coli
ATCC 11303, recombi nant (rec) E. coli B ( pLOI 297
and KOl l ) , Pichia stipitis (CBS 5773, 6054 and R),
Saccharomyces cerev&iae isolate 3 in combi nat i on with
xylose isomerase, rec S. cerev&&e (T J1, H550 and
H477) and Fusarium oxysporum VTT-D-80134 in an
i nt erl aborat ory comparison. The micro-organisms
were studied according t o t hree different options: (A)
ferment at i on under consistent conditions, (B) fermen-
t at i on under optimal conditions for t he organism, and
(C) ferment at i on under optimal conditions for the or-
ganism with detoxification of t he hydrolysate. The
highest yields of ethanol, 0.24 g/g (A), 0.36 g/g (B) and
0.54 g/g (C), were obt ai ned from rec E. coli B, KOl l .
P, stipit& and F. oxysporum were sensitive t o the inhi-
bitors present in the hydrol ysat e and pr oduced a maxi-
mum yield of 0.34 g/g (C) and 0.04 g/g (B), respective-
ly. The analysis of the corn-cob hydrol ysat e and as-
pects of process economy of t he different ferment at i on
options (pH, sterilization, nut ri ent suppl ement at i on,
adaptation, detoxification) are discussed.
I n t r o d u c t i o n
As a renewabl e energy resource, fuel et hanol has re-
ceived consi derabl e scientific interest since the oil cri-
sis in the mid 1970s. Fuel et hanol can be pr oduced
from different sources, e.g. fast-growing wood, agricul-
tural or forest ry plant residues, and appropri at e muni-
cipal wast e (Kosari c et al. 1983; Magee and Kosaric
1985; Parisi 1989; Hahn-H/ i gerdal et al. 1991; Olsson
and Hahn-Hfi gerdal 1993). The raw material, lignocel-
lulose, has to be degr aded t o a ferment abl e hydroly-
sate from which et hanol can be produced. Fuel et hanol
is a bul k pr oduct with a worl d mar ket price of 0. 35-
Correspondence to: B. Hahn-H~igerdal
0.45 US dollars (May 1993), which requires an efficient
ferment at i on process to be economically feasible. Fur-
t hermore, envi ronment al benefits and political deci-
sions are of great i mport ance for the ext ent of fuel
et hanol appliCations (Kei m and Venkat asubramani an
1989; Lynd et al. 1991). Xylitol, which is of higher eco-
nomi c value than ethanol, can also be pr oduced by fer-
ment at i on of lignocellulose hydrolysates. It is widely
used in t he f ood industry as a sweet ener in the Nordi c
countries. Xylitol is anticariogenic and suitable as a su-
gar substitute for diabetics (M/~kinen 1979; Hyv6nen et
al. 1982). A ferment at i on process for xylitol product i on
could be advant ageous compared to the chemical proc-
ess used t oday (Melaja and H/~m/~l~iinen 1977) because
the downst ream processing is expect ed to be cheaper
(Oj amo et al. 1989).
Dependi ng on the raw material and pret reat ment ,
the lignocellulose hydrolysates contain many different
and variable amount s of monosacchari des (e.g. glu-
cose, galactose, mannose, xylose and arabinose), disac-
charides (e.g. cellobiose) and polysaccharides (e.g. xy-
lans and mannans) (Magee and Kosaric 1985). Special
at t ent i on has been given to the ferment at i on of xylose
as it is t he most difficult to ferment (Jeffries 1983;
Schneider 1989; Hahn-H/~gerdal et al. 1993). The pre-
t reat ment and hydrolysis of lignocellulose result in the
format i on of a number of degradat i on product s and ex-
tracts. These compounds, e.g. furfural, 5-hydroxyme-
thylfurfural, levulinic acid, vanillin, syringaldehyde and
acetic acid are known to inhibit micro-organisms
(Clark and Macki e 1984; Ando et al. 1986; Tran and
Chambers 1986; Nishikawa et al. 1988; Sanchez and
Bautista 1988; van Zyl et al. 1991). Moreover, the
product s of t he ferment at i on, et hanol and organic
acids, are also inhibitory t o micro-organisms (Maiorel-
la et al. 1983; Jones 1989). At t empt s have been made
to increase t he ferment abi l i t y of the hydrolysates (i.e.
to detoxify the hydrol ysat e) using different pret reat -
merits, e.g. ion-exchange (Clark and Macki e 1984; Fei n
et al. 1984; Frazer and McCaskey 1989), calcium hy-
droxide t reat ment (Strickland and Beck 1985; van Zyl
et al. 1988), mol ecul ar sieves (Tran and Chambers
63
1986), s t e a m s t r i ppi ng ( Yu et al. 1987) a n d i on excl u-
s i on ( Bu c h e r t et al. 1990).
An e c o n o mi c f uel e t h a n o l p r o d u c t i o n pr oc e s s r e-
qui r es a mi c r o - o r g a n i s m wi t h t he f ol l owi ng qual i t i es:
ef f i ci ent ut i l i zat i on o f t h e r a w ma t e r i a l ( hi gh yi el d) ,
hi gh f i nal e t h a n o l c o n c e n t r a t i o n ( mi n i mu m di s t i l l at i on
cost s) , a n d f as t e t h a n o l f e r me n t a t i o n r a t e ( hi gh p r o -
duct i vi t y) . I n a ddi t i on, t he s e d e ma n d s s h o u l d be ful -
f i l l ed wi t h o u t t h e n e e d f or e xt r a c he mi c a l s a n d pr oc e s s
st eps, s uc h as c o mp l e x n u t r i e n t s u p p l e me n t s , det oxi f i -
c a t i on c he mi c a l s , de t oxi f i c a t i on p r o c e d u r e s a nd st eri l i -
zat i on. Ge n e r a l l y , a hi gh yi el d a n d a hi gh e t h a n o l c on-
c e n t r a t i o n ar e c o n s i d e r e d t o h a v e t he gr e a t e s t i mp a c t
o n pr oc e s s e c o n o my , b u t b e c a u s e t he e t h a n o l p r o d u c -
t i on pr oc e s s is so c ompl e x, t h e pr oc e s s n e e d s de t a i l e d
i nve s t i ga t i on t o a c hi e ve a me a n i n g f u l e c o n o mi c e va l ua -
t i on ( Wr i g h t 1988; Za c c h i et al. 1988; Hi n ma n et al.
1989).
Th e p r e s e n t s t udy was u n d e r t a k e n as a c ol l a bor a -
t i ve p r o j e c t b e t we e n di f f e r e nt g r o u p s i n v o l v e d wi t h
f uel e t h a n o l r e s e a r c h ( Ta bl e 1), i n o r d e r t o c o mp a r e
t he r es ul t s o b t a i n e d f r o m f e r me n t i n g a l t e r na t i ve p r e p a -
r a t i ons of o n e pa r t i c ul a r l i gnocel l ul os e h y d r o l y s a t e
s ubs t r a t e wi t h Escheri chi a coli, r e c o mb i n a n t ( r ec) E.
coli, Sacchar omyces cerevisiae + xyl os e i s o me r a s e
( XI ) , r e c S. cerevisiae, Pi chi a stipitis a n d Fus ar i um ox-
ys por um. F o r t hi s s t udy a xyl os e - r i c h h y d r o l y s a t e de-
r i ve d f r o m c o r n c obs af t er di l ut e aci d p r e t r e a t me n t was
c h o s e n s i nce t he f e r me n t a t i o n o f t he xyl os e f r a c t i on
p r o d u c e s mo s t p r o b l e ms . Th e h y d r o l y s a t e was di st r i -
b u t e d t o t he pa r t i c i pa nt s at p H 1.5 t o as s ur e t h a t n o
me t a b o l i c act i vi t y o c c u r r e d dur i ng t r a n s p o r t a t i o n . Th e
i nve s t i ga t i on was di vi de d i nt o t h r e e e x p e r i me n t a l cat e-
gor i es. Fi r st , t h e f e r me n t a t i o n p e r f o r ma n c e o f t h e i n-
ve s t i ga t e d mi c r o - o r g a n i s ms was c o mp a r e d u n d e r c on-
s i s t ent c ondi t i ons c o n c e r n i n g p H, o x y g e n a t i o n , t e m-
p e r a t u r e a n d nut r i e nt s . Thi s was c o n s i d e r e d as t he ba -
sic e x p e r i me n t b e c a u s e it a l l owe d t h e c o mp a r i s o n o f a
b r o a d r a n g e o f o r g a n i s ms u n d e r i dent i cal condi t i ons .
Se c o n d , t he o r g a n i s ms we r e c o mp a r e d u n d e r wh a t was
c o n s i d e r e d t o b e t he o p t i ma l c ondi t i ons f o r e a c h i ndi -
Table 1. Participants in the interlaboratory collaboration
Name Affiliation
S. Amartey/T. W. Jeffries
D. Beall/L. O. Ingram
B. L. Boynton/J. D. McMillan
B. Hahn-Hggerdal/H. Jeppsson/T.
Linddn/N. Meinander/L. Olsson
H. G. Lawford/J. Rousseau
J. Pourquie
T. Seki/T. Yoshida
M.-L. Suihko
Forest Products Laborato-
ry, Madison, USA
University of Florida,
USA
National Renewable En-
ergy Laboratory, Gold-
en, USA
Lund Institute of Tech-
nologyAJniversity of
Luud, Sweden
University of Toronto,
Canada
Ministdre de l'Agricul-
ture, INA, Paris, France
Osaka University, Japan
VTT, Espoo, Finland
vi dua l o r g a n i s m wi t h r e s p e c t t o o x y g e n a t i o n a nd nu-
t r i e nt s u p p l e me n t a t i o n . Thi r d, t he o r g a n i s ms we r e
c o mp a r e d i n a s ubs t r a t e wh e r e de t oxi f i c a t i on was u s e d
t o r e d u c e t he c o n t e n t o f a n y i nhi bi t or s a nd i n s o me
cas es i n c o mb i n a t i o n wi t h a d a p t a t i o n o f t he mi c r o - o r -
g a n i s m t o t he hydr ol ys a t e . Th e bas i c e x p e r i me n t r e p r e -
s ent s t he f e r me n t a t i o n a l t e r na t i ve r e qui r i ng a mi ni -
mu m o f a ddi t i ona l cos t s wh e r e a s t he t wo o t h e r ge ne r -
al l y r e p r e s e n t mo r e c os t l y f e r me n t a t i o n al t er nat i ves . I n
a ddi t i on, t he a c c u r a c y o f t he anal ys i s o f t he c o r n - c o b
h y d r o l y s a t e was e va l ua t e d.
Materi al s and me t hods
Hydrolysate and pretreatments. The hydrolysate was prepared by
dilute acid pretreatment of corn cobs, obtained locally at the Na-
tional Renewable Energy Laboratory in Golden, Colo., USA.
The dry corn material was knife-milled to pass through a 2-mm
rejection screen. The corn-cob particles were pretreated with di-
lute (0.83%, w/w) sulphuric acid solutions (yielding a hydrolysis
pH of ].35-1.4) in a 2-gallon stainless-steel Parr reactor (Parr,
Moline, Ill., USA). A low-solid slurry (10%) of biomass in deion-
ized water was heated to 160 C, the acid was then injected and,
after being heated at 160C for 10 min, the pretreated material
was cooled to 90 C. The mixture was then immediately filtered
through a Buchner funnel to recover the hydrolysate at a final
pH of approximately 1.5.
Experimental design. Experiment category A - the basic experi-
ment: with minor deviations the following procedure was used.
The hydrolysate was supplemented with 2.5 g/1 of yeast extract,
0.25 g/1 of (NH4)zHPO4, 0.025 g/1 of MgSO4"7HzO and 0.1 M so-
dium phosphate. The pH was adjusted to 5.5 for the yeasts and
7.0 for the bacteria. The yeast fermentations were performed un-
der Oz-limited conditions, in half-filled (50/100, v/v) flasks at
30C and 150 rpm. The bacterial fermentations were performed
under anoxic conditions at 30 C. The fermentations were inocu-
lated to give a final cell mass concentration of approximately 5 g/1
(dry weight). Samples were taken regularly and the pH was ad-
justed each time a sample was taken.
Experiment categories B and C were performed under opti-
mal conditions for each type of micro-organism without and with
detoxification, respectively. The conditions are reported in the
Results.
Micro-organisms and cell mass production. Each type of micro-
organism was grown under the conditions given below and har-
vested in the log phase by centrifugation. The cells were washed
twice with physiological sodium chloride solution (9 g/l), if not
otherwise stated, and were then used as the inoculum for the fer-
mentation of the corn-cob hydrolysate under the conditions re-
ferred to as categories A, B and C.
E. coli ATCC 11303 was grown in a medium containing 10 g/1
of bactotryptone, 5 g/1 of yeast extract, 5 g/1 of sodium chloride,
10 g/1 of glucose, 10 g/1 of xylose and 10 g/1 of galactose, with the
pH adjusted to 7.5. The medium was inoculated with a 2.0% (v/v)
inoculum of an overnight culture. One-litre baffled erlenmeyer
flasks containing 200-ml aliquots of this culture were incubated in
a rotary shaking water bath at 37 C.
E. coli B (ATCC 11303) carries the plasmid pLOI297, con-
taining the genes pdc and adhB coding for the enzymes pyruvate
decarboxylase and alcohol dehydrogenase II from Zymomonas
mobilis (Alterthum and Ingrain 1989). Cukures were stored
( - 10 C) in LB (Luria broth; 10 g/1 of tryptone, 5 g/1 of yeast ex-
tract, 5 g/1 of sodium chloride)/glycerol-citrate and were plated on
selective media (LB + 20 g/1 of agar + 10 mg/1 of tetracycline and
40 rag/1 of ampicillin). Cell mass was produced overnight in buff
64
fered LB cont ai ni ng ant i bi ot i cs (ampi ci l l i n and t et racycl i ne) and
30 g/1 of xylose as t he car bon source.
E. coli KO 11 carries t he genes pdc and adhB from Z. mobilis
i nt egr at ed i nt o t he chromosome. I n addi t i on, ot her genet i c
changes were carri ed out to mi ni mi ze by- pr oduct f or mat i on
( Oht a et al. 1991). The i nocul um was pr epar ed by t ransferri ng
cells from a single col ony to 125-ml flasks cont ai ni ng 50 ml fer-
ment at i on br ot h (LB + 50 g/1 of xylose). Cul t ures were i ncu-
bat ed overni ght at 30C wi t hout agitation.
F. oxysporum VTT- D- 80134 was grown i n a medi um consist-
i ng of 50 g/1 of xylose, 1.0 g/1 of yeast extract, 3.4 g/1 of NaNO3,
2.0 g/1 of KH2PO4, 0.4 g/1 of CaC12"2H20, 0.3 g/1 of
MgSO4"7H20 and 5.0 ml trace met al solution. The composi t i on
of t he t race met al sol ut i on was 2.0 g/1 of H3BO3, 0.2 g/1 of
COSO4"7H20 , 0.8 g/1 of CuSO4"5H20, 2.0 g/1 of ZnSO4" 7H20,
0.6 g/1 of MnSO4- H20, 0.2 g/1 of KI, 0.4 g/1 of Fe SO4. 7H20 and
0.6 g/1 of A12(SO4)3. The mycel i al mass was washed once with wa-
t er bef or e i nocul at i on. The p H was 5.0 and t emper at ur e 30 C.
P. stipitis CBS 5773 ( NRRL Y-7124) (a) was grown aerobi cal -
ly i n f our 1-1 DeLong cul t ure flasks cont ai ni ng 250 ml buf f er ed
yeast ni t r ogen base (YNB, Difco) suppl ement ed wi t h 2 g/1 of glu-
cose, 20 g/1 of xylose, 20 g/1 of arabi nose and 20 g/1 of galactose as
car bon sources. The YNB was buf f er ed at pH 5.5 usi ng KH2PO4
and Na2HPO4 at concent r at i ons of 10 g/1 and 0.5 g/l, respective-
l y .
P. stipitis CBS 5773 (/3) was obt ai ned from a cont i nuous cul-
t ur e grown aerobi cal l y at pH 4.5 on puri fi ed xylose (12 g/l) as t he
sole car bon source. The medi um composi t i on was not defi ned
furt her.
P. stipitis CBS 5773 (9,) was pr oduced at 30 C, pH 6.0, i n 500-
ml Sakaguchi flasks cont ai ni ng 50 ml medi um cont ai ni ng 20 g/1 of
xylose, 20 g/l of pol ypept one and 10 g/1 of yeast extract. The cells
were washed once wi t h 10 g/1 of sodi um chl ori de sol ut i on pri or to
i nocul at i on.
P. stipitis CBS 6054 (6) was grown i n a medi um cont ai ni ng
10 g/1 of xylose, 10 g/1 of glucose, 10 g/1 of galactose, 3 g/1 of yeast
extract, 3 g/l of mal t extract, 5 g/1 of bact opept one, 19 g/1 of
KH2PO4, 3 g/1 of (NH4)2HPO4 and 1.1 g/1 of MgSO4' 7H20. The
growt h medi um was i nocul at ed di rect l y f r om agar slants. One-
litre baffl ed er l enmeyer flasks cont ai ni ng 200-ml al i quot s of this
cul t ure were i ncubat ed i n a rot ary shaki ng wat er bat h at 30 C.
P. stipitis CBS 6054 (e) was grown i n a medi um cont ai ni ng
1.7 g/1 of yeast ni t r ogen base (wi t hout ami no acids and ammon-
i um sul phat e), 1.0 g/1 of casami no acids, 2.2 g/1 of urea, 40 g/1 of
glucose, 50 g/1 of xylose and 10 g/1 of arabi nose. The pH was ad-
j ust ed to 5.0. Cell mass was pr oduced at 30C and 150 r pm i n a
125-ml er l enmeyer flask filled to 50 ml.
P. stipitis R, a st rai n t hat has been adapt ed to l i gnocel l ul ose
hydrol ysat es (Parekh et al. 1986), was grown as for P. stipitis CBS
6054 (6).
S. cerevisiae Isol at e 3, a st rai n i sol at ed f r om a spent sul phi t e
l i quor f er ment at i on pl ant ( Li nd6n et al. 1992), was grown as for
P. stipitis CBS 6054 (3).
S. cerevisiae H477 is a st rai n carryi ng t he gene for t he enzyme
xylose reduct ase f r om P. stipitis CBS 6054. S. cerevisiae H477 was
const ruct ed as descri bed by Hal l bor n et al. (1991), al t hough tt/e
par ent st rai n was S. cerevisiae H308 (hi s3-del l , leu2-3, leu2-112,
trpl-289, ura3-52, cir +, Gal +) ( Hal l bor n, per sonal communi ca-
t i on). It was grown i n a medi um cont ai ni ng 6.7 g/1 of yeast ni t rog-
en base wi t hout ami no acids, 20 g/1 of glucose, 0.0135 g/1 of ade-
ni ne, 0.348 g/1 of argi ni ne, 0.266 g/1 of aspartic acid, 0.058 g/1 of
histidine, 0.036 g/1 of inositol, 0.525 g/1 of isoleucine, 0.091 g/1 of
lysine, 0.149 g/1 of met hi oni ne, 0.083 g/1 of phenyl al ani ne, 0.105
g/1 of seri ne, 0.119 g/ 1 of t hr eoni ne, 0.082 g/1 of t r ypt ophan,
0.018 g/1 of t yrosi ne, 0.022 g/1 of uraci l and 0.117 g/1 of val i ne. The
cells were grown i n two stages, first i n a 50-ml vol ume and t hen i n
a 500-ml vol ume at 30 C and 200 rpm.
S. cerevisiae H550 is S. cerevisiae H308 t r ansf or med wi t h t he
genes for t he enzymes xylose reduct ase ( XR) and xylitol dehy-
drogenase ( XDH) from P. stipitis CBS 6054 on t he plasmids
pUA103 and pYEP- XYL2 ( Hal l bor n et al. 1992; Hal l bor n, per-
Tabl e 2. Def i ni t i on of cal cul at ed par amet er s
Par amet er s Cal cul at i ons Uni t s
Tot al et hanol yield Maxi mum et hanol con- g/g
cent rat i on/ t ot al i ni t i al
sugar concent r at i on
Et hanol yield f r om Maxi mum et hanol con- g/g
consumed sugars cent r at i on/ consumed
sugar concent r at i on
Maxi mum vol umet ri c The i ni t i al product i vi t y g/1 per hour
product i vi t y (fastest) (Fig. 1)
Maxi mum specific Maxi mum vol umet r i c g/g per hour
product i vi t y product i vi t y/ i ni t i al dry
weight (Fig. 1)
Concentration (g/t)
Maximum productivity Total productivity
Cell mass
Time (h)
Maximum ethanol concentration
Fig. 1. Schematic drawi ng for t he descri pt i on of product i vi t i es
sonal communi cat i on) . S. cerevisiae H550 was grown as for S. cer-
evisiae H477 wi t h uraci l excl uded from t he medi um.
S. cerevisiae TJ1, t r ansf or med wi t h XR and XDH genes from
P. stipitis CBS 5773 (Tant i rungki j et al. 1993), was grown as for P.
stipitis CBS 5773 (';/).
Xyl ose isomerase (XI). An i mmobi l i zed XI pr epar at i on deri ved
from Lactobacillus brevis (Li nd6n and Hahn-H~igerdal 1993).
Calculations. The f er ment at i ons were charact eri zed by t hei r t ot al
et hanol yield, et hanol yield based on consumed sugars, maxi mum
vol umet ri c product i vi t y and maxi mum specific product i vi t y (Ta-
bl e 2, Fig. 1). The cal cul at i ons were based on t he analysis results
r epor t ed by each part i ci pat i ng l aborat ory.
Analysis of sugars and products. Each part i ci pat i ng group ana-
lysed sugars and product s according to t hei r own rout i nes. The
met hods i ncl ude HPLC, gas chr omat ogr aphy (GC), glucostatic
and enzymat i c met hods (Tabl e 3).
R e s u l t s
An a l y s i s o f t he s ugar c o mp o s i t i o n o f t he h y d r o l y s a t e
T h e ma i n c o mp o n e n t s o f t h e c o r n - c o b h y d r o l y s a t e
we r e g l u c o s e , x y l o s e , a r a b i n o s e a n d a c e t i c a c i d. T h e
s a mp l e s we r e t a k e n at t h e s t a r t o f t h e f e r me n t a t i o n a n d
65


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addi t i on o f cell mas s and nutri ents accordi ng to opt i on
A caus ed a di l ut i on o f 2%. Compi l i ng t he analysis re-
sul ts o f t he hydrol ysat e f rom t he di fferent l aborat ori es
( Tabl e 3), t he me an val ue o f total amount o f sugar was
f ound to be 43. 6 g/1 wi t h a standard devi at i on of 5.2 g/1
and a concent rat i on range o f 31. %54. 7 g/1. For gl ucos e
a me an val ue of 6.2 g/1 wi t h a standard devi at i on o f
1.5 g/1 and a concent rat i on range o f 3. 6- 8. 1 g glucose/1
was f ound. Xyl os e gave a me an val ue of 32. 6 g/1 wi t h a
standard devi at i on of 4.0 g/1 and a concent rat i on range
of 23. 2- 40. 1 g xylose/1. For arabi nose a me an val ue of
4.8 g/1 wi t h a standard devi at i on o f 1.8 g/1 and a con-
cent rat i on range o f 3. 0- 7. 7 g arabinose/1 was f ound. A
me an val ue of 4.3 g/1 wi t h a standard devi at i on o f 0.6
g/1 and a concent rat i on range o f 3. 4- 4. 9 g/1 was ob-
t ai ned for aceti c acid. One report ed analysis resul t has
be e n omi t t ed becaus e o f unexpl ai nabl e l ow fi gures for
sugars, 3.0 g gl ucose/ l , 16.0 g xylose/1 and 2. 0 g arabi-
nose/1 ( bot h f rom Tabl e 3 and t he statistical compari -
sons) .
F er mentati ons
The f erment at i on perf ormance of t he bact eri um E.
coli, natural as wel l as recombi nant , natural l y occur-
ring yeast s (P. stipitis and S. cerevisiae), recombi nant
yeasts (S. cerevisiae) and a fungus (F. o x y s p o r u m) were
i nvest i gat ed.
Opt i on A
The f erment at i ons perf ormed under opt i on A were
consi st ent wi th respect t o nutri ent s uppl ement at i on,
pH, t emperat ure, and aerati on in order t o compare t he
perf ormance of di fferent mi cro- organi sms in t he s ame
hydrol ysate.
Natur al l y occurri ng mi cro- organi sms. E. col i ATCC
11303 pr oduced 2.9 g/1 o f bi omas s but no et hanol or
ot her product s ( Tabl es 4, 5 and 6). No n e of t he yeasts
P. stipitis CBS 5773 (oz), P. stipitis CBS 5773 ( ~) , P. sti -
pi ti s CBS 6054 (6) and P. sti pi ti s R produced any etha-
nol , bi omas s or by- product s ( Tabl es 4, 5, and 6). P. sti -
pi ti s CBS 5773 ( y) produced 8.4 g/1 o f et hanol wi t h a
yi el d of 0. 18 g/g and a maxi mum vol umet ri c product i v-
ity of 0. 20 g/l per hour, in addi t i on t o 0.19 g/g of xyl i t ol
( Tabl es 4, 5, and 6). P. sti pi ti s CBS 6054 (E) gave a
yi el d of 0.03 g/g, t he maxi mum vol umet ri c product i vi t y
was 0. 18 g/1 per hour and t he maxi mum speci fi c pro-
ducti vi ty was 0. 04 g/g per hour: xyl i t ol f ormat i on was
0. 02 g/g ( Tabl es 4, 5 and 6). S. cer evi si ae, bakers' yeast,
is not abl e t o uti l i se xyl os e, but can uti l i se t he i someri c
f orm o f xyl os e, xyl ul ose. The bacterial enzyme XI cata-
l yses t he i s omeri s at i on o f xyl os e t o xyl ul ose. Whe n us-
i ng S. cer evi si ae i sol at e 3 in combi nat i on wi t h XI, t he
yi el d was 0. 06 g/g, t he ma x i mum vol umet ri c producti vi -
ty 1.04 g/l per hour, t he ma x i mum speci fi c product i vi t y
0.31 g/g per hour and t he xyl i t ol f ormat i on was 0. 22 g/g
( Tabl es 4, 5 and 6).
Table 4. Experimental conditions and resulting yields
Organism Option pH Temperature Initial dry Growth Total sugar Ethanol Yield (g/g) Yi el d (g/g)
( C) weight (g/l) (g/l) (g/l) (g/l) (total) (consumed)
Escherichia coli AT CC 11303 A 7.0 30 5.5 2.9 40.9
E. coli B, pLOI 297 A 7.0 30 4.0 2.5 40.6
E. coli B, p LOI 297 A( a ) " 7.0 30 3.0 3.5 41.6
E. coli B, p LOI 297 A( a) a 7.0 30 0.5 0.5 51.6
E. coli B, KOl l A 7.0 30 5.0 n.s. 39.3
Pichia stipitis CBS 5773 a A 5.5 30 5.0 0.0 31.7
P. stipitis CBS 5773/ 3 A 4.5 28 6.4 0.0 54.7
P. stipitis CBS 5773 y A 6.0 30 2.6 2.9 47.6
P. stipitis CBS 6054 8 A 5.5 30 4.8 0.0 41.3
P. stipitis CBS 6054 A 5.0 30 4.7 0.8 44.1
P. stipitis R A 5.5 30 4.6 0.0 41.7
Saccharomyces cerevisiae 3 + XI A 5.5 30 3.4 5.6 42.4
S. cerevisiae TJ1 ( XR + XDH) A 6.0 30 2.3 1.2 47.8
S. cerevisiae H550 ( XR + XDH) A 5.5 30 5.0 2.9 42.2
E. coli B, KOl l B 7.0 30 5.0 n.s. 39.3
P. stipitis CBS 5773 fi B(b) b 4.5 28 6.4 0.0 54.7
P. stipitis CBS 5773/ 3 B( c ) b 4.5 28 6.4 0.0 54.7
P. stipitis CBS 6054 B 5.0 30 5.6 0.9 44.8
S. cerevisiae H550 ( XR + XDH) B 5.5 30 5.0 3.2 39.8
Fusarium oxysporum VTT- D- 80134 B 5.0 30 5.3 0.0 21.0
E. coli B, p LOI 297 C 7.0 30 0.5 0.5 49.8
E. coli B, KOl l C 6.0 30 5.0 n.s. 39.3
P. stipitis CBS 5773/ 3 C c 4.5 28 6.4 2.0 24.8
P. stipitis CBS 6054 C 5.0 30 7.0 2.0 43.5
0.0 0.00 0.00
2.5 0.06 0.08
9.5 0.23 0.25
7.9 0.15 0.21
9.4 0.24 (96 h) n.s.
0.0 0.00 0.00
0.0 0.00 0.00
8.4 0.18 (72 h) 0.21 (72 h)
0.0 0.00 0.00
1.5 0.03 0.21
0.0 0.00 0.00
2.7 0.06 0.06
3.5 0.07 (72 h) 0.13 (72 h)
2.9 0.07 0.31
14.0 0.36 (96 h) n.s.
0.0 0.00 0.00
0.0 0.00 0.00
1.9 0.04 0.24
3.4 0.09 0.44
0.8 0.04 (92 h) 0.20 (92 h)
7.3 0.15 0.22
21.4 0.54 0.55
8.3 0.33 (44 h) 0.39 (44 h)
13.3 0.31 0.41
For options, see Materials and methods; XI, xyl ose
XR, xyl ose reductase; XDH, xylitol dehydrogen~ise
a No washing of i nocul um prior to inoculation
isomerase; b For details, see text
c Fed-batch, no detoxification of the hydrolysate
Tabl e 5. Resulting productivities
Organism Option Maximum volumetric Maximum specific
productivity (g/l/h) productivity (g/g/h)
67
E. coli ATCC 11303 A 0.00 0.00
E. coli B, pLOI 297 A 0.02 < 0.01
E. coli B, pLOI 297 A(a) a 0.15 0.05
E. coli B, pLOI 297 A(a) a 0.10 0.20
E. coli B, KOl l A 0.88 0.18
P. stipitis CBS 5773 a A 0.00 0.00
P. stipitis CBS 5773/3 A 0.00 0.00
P. stipitis CBS 5773 y A 0.20 0.08
P. stipitis CBS 6054 6 A 0.00 0.00
P. stipitis CBS 6054 A 0.18 0.04
P. stipitis R A 0.00 0.00
S. cerevisiae 3 +XI A 1.04 0.31
S. cerevisiae TJ1 (XR + XDH) A 0.05 0.02
S. cerevisiae H550 ( XR+XDH) A 0.76 0.15
E. coIi B, KOl l B 1.20 0.24
P. stipitis CBS 5773/3 B(b) b 0.00 0.00
P. stipitis CBS 5773/3 B(c) b 0.00 0.00
P. stipitis CBS 6054 B 0.20 0.04
S. cerevisiae H550 ( XR+XDH) B 0.83 0.17
F. oxysporum VTT-D-80134 B < 0.01 < 0.01
E. coli B, pLOI 297 C 0.11 0.22
E. coli B, KOl l C 1.32 0.26
P. stipitis CBS 5773/3 C n.s. n.s.
P. stipitis CBS 6054 C 0.32 0.05
~ See footnotes to Table 4
Table 6. By-products produced
Organism Option Lactate Lactate Xylitol Xylitol
(g/g total sugar) (g/g sugar consumed) (g/g total sugar) (g/g sugar consumed)
E. coli ATCC 11303 A
E. coli B, pLOI 297 A 0.23 0.30
E. coli B, pLOi 297 A(a) a 0.31 0.34
E. coli B, pLOI 297 A(a) a 0.18 0.25
E. coli B, KOl l A
P. stipitis CBS 5773 a A
P. stipitis CBS 5773/3 A
P. stipitis CBS 5773 3' A
P. stipitis CBS 6054 6 A
P. stipitis CBS 6054 e A
P. stipitis R A
S. cerevisiae 3 + XI A
S. cerevisiae TJ1 (XR + XDH) A
S. cerevisiae H550 (XR + XDH) A
E. coli B, KOl l B
P. stipitis CBS 5773/3 B(b) b
P. stipitis CBS 5773/3 B ( c ) b
P. stipitis CBS 6054 B
S. cerevisiae H550 (XR +. XDH) B
F. oxysporum VTT-D-80134 B
E. coli B, pLOI 297 C 0.17 0.25
E. coli B, KOl l C
P. stipitis CBS 5773/3 C c
P. stipitis CBS 6054 e C
0.00 0.00
0.00 0.00
0.19 0.22
0.00 0.00
0.02 0.11
0.00 0.00
0.22 0.22
0.23 0.41
0.04 0.21
0.00 0.00
0.00 0.00
0.02 0.11
0.04 0.19
0.03 0.03
a-~ See footnotes to Table 4
68
Recombinant micro-organisms. The washing of E. coli
B, pLOI 297 cells severely affected bot h yield and pro-
ductivity; washing prior to inoculation was omi t t ed un-
der option A (a). For E. coli B, pLOI 297 t he yield was
0. 06g/ g under option A and 0.23g/g under option
A (a) (Table 4). The maxi mum volumetric productivity
was 0.02 g/1 per hour and 0.15 g/1 per hour, respectively
(Table 5). The yield for E. coli B, KOl l was 0.24 g/g,
t he maxi mum volumetric productivity was 0.88 g/1 per
hour (Tables 4 and 5). For all ferment at i ons with E.
coli B, KOl l , t he medi um was filter-sterilized before
use. Bot h recombi nant S. cerevisiae strains gave a yield
of 0.07 g/g (Table 4). The maxi mum volumetric pro-
ductivity of et hanol was 0.05 g/1 per hour for S. cerevi-
siae TJ1 and 0.76 g/1 per hour for S. cerevisiae H550,
and t he format i on of xylitol was 0.23 g/g and 0.04 g/g,
respectively (Tables 5 and 6).
Opt i on B
Ferment at i ons under option B were per f or med under
optimal conditions for each t ype of micro-organism
chosen by each participating laboratory. The ferment a-
tions were per f or med without detoxification of t he hy-
drolysate. Nut ri ent suppl ement at i on and t he amount
of available 02 were changed. Under option B, t he nat-
urally occurring micro-organisms P. stipitis CBS 5773
(/3), P. stipitis CBS 6054 (e) and F. oxysporum VTT-
D-80134 were investigated, and among t he recombi-
nants E. coli KOl l and S. cerevisiae H550 were investi-
gated.
Naturally occurring micro-organisms. P. stipitis CBS
5773 (/3) was investigated under two conditions B (b)
and B (c). Options B (b) and B (c) did not result in any
i mpr ovement compared to option A, i.e. no ethanol,
biomass or by-products were produced. Under option
B (b) P. stipitis CBS 5773 (/3) f er ment ed a hydrol ysat e
t hat had been suppl ement ed with t he same chemicals
as under option A and t hereaft er autoclaved, and un-
der option B (c) t he hydrol ysat e was suppl ement ed
with IFP mineral medi um and t hereaft er autoclaved.
IFP mi neral medi um was obt ai ned by blending 5 ml so-
lution A + 5 ml solution B + 50 ml solution C + 1.3 g
yeast extract. Solution A cont ai ned 1.2 g/1 of CaO,
0.4 g/I of ZnO, 5.4 g/1 of i ron(III) chloride, 0.35 g/1 of
MgO, 0.25 g/1 of CuSO4" 5 H20, 0.24 g/1 of
CaC12" 5 HzO, 0.06 g/1 of ort hobori c acid and 1.3 ml/1 of
concent rat ed HC1. Solution B cont ai ned 10.1 g/1 of
MgO and 45 ml/1 of concent rat ed HC1, and solution C
cont ai ned 64 g/1 of urea, 12 g/1 of KHzPO4 and 1.8 g/1 of
disodium hydrogen phosphate. IFP mineral medi um
(5 ml) was mi xed with 45 ml autoclaved hydrolysate. P.
stipitis CBS 6054 (e) was f er ment ed in hydrol ysat e sup-
pl ement ed with 1.7 g/1 of YNB (without amino acids
and ammoni um sulphate), 1.0 g/1 of casamino acids and
2.2 g/1 of urea. This resulted in only marginal changes
in yield, maxi mum volumetric productivity, maxi mum
specific productivity and xylitol format i on compared to
option A (Tables 4, 5, and 6). F. oxysporum VTT-D-
80134 produced et hanol with a yield of 0.04 g/g and a
maxi mum specific productivity of less than 0.01 g/g per
hour when t he hydrol ysat e was suppl ement ed with t he
same nutrients as in t he cultivation medi a (Tables 4
and 5). In addition t he ferment at i on was per f or med
under an N2 at mosphere.
Recombinant micro-organisms. For E. coli B, KOl l
t he yield increased by 50% to 0.36 g/g, t he maxi mum
volumetric productivity and t he maxi mum specific pro-
ductivity increased by 35% to 1.20 g/1 per hour and to
0.24 g/g per hour, respectively (Tables 4 and 5) when
t he hydrol ysat e was suppl ement ed with 1 ml of corn
steep liquor/50 ml hydrolysate. Oz-limited conditions
(25 ml of t he hydrol ysat e in a 25 ml flask, sealed with a
rubber stopper supplied with a cannula for COz and
stirred gently with a magnet i c stirrer) did not improve
t he yield for recombi nant S. cerevisiae H550 (Table 4),
but t he maxi mum volumetric and specific productivity
increased by 34% to 1.02 g/1 per hour and 0.20 g/g per
hour (Table 5).
Opt i on C
Ferment at i ons under option C were per f or med with
detoxification of t he hydrol ysat e and/ or adaptation of
t he micro-organism to t he hydrol ysat e in addition to
t he optimal nut ri ent and oxygenat i on conditions pre-
sent ed under option B. The organisms investigated un-
der this option were P. stipitis CBS 5773 (~), P. stipitis
CBS 6054 (e), E. coli B, pLOI 297 and E. coli B, KOl l .
The detoxification met hod, over-liming with Ca(OH)z,
was chosen by each individual laboratory. In one case
a form of adaptation, ferment at i on of a diluted hydro-
lysate in fed-batch mode, was performed.
Naturally occurring micro-organisms. When t he fer-
ment at i on with P. stipitis CBS 5773 (13) was per f or med
as a fed-bat ch ferment at i on, started from 0.6 1 of pro-
duction culture cell mass and t hen adding 0.5 1 of auto-
claved hydrol ysat e in a fed-batch mode, t he yield was
0.33 g/g, which should be compared to t he two pre-
vious conditions where no et hanol was pr oduced at all
(Table 4). This pr ocedur e allowed P. stipitis CBS 5773
(/3) to f er ment a diluted hydrol ysat e t her eby avoiding
pr onounced inhibition. Fed-bat ch ferment at i ons may
be consi dered as a way of adapting t he micro-organ-
isms to t he hydrolysate. P. stipitis CBS 6054 (e) was
adapt ed to t he hydrol ysat e by successively subculturing
it in 25%, 50% and 75% hydrol ysat e for 48 h at 30 ~ C
before ferment i ng a hydrolysate detoxified by overlim-
ing. This resulted in an increased yield by almost a fac-
t or of t en [0.04 g/g to 0.31 g/g (Table 4], but only a mar-
ginal increase in t he maxi mum productivity (Table 5).
Recombinant micro-organisms. E. coli B, pLOI 297
and E. coli B, KOl l were f er ment ed in a detoxified
hydrol ysat e (over-limed). This resulted in a yield o f
0.15 g/g for E. coli B, pLOI 297 compared to 0.54 g/g
for E. coli B, KOl l (Table 4). The maxi mum vol umet -
ric productivities were 0.11 g/1 per hour and 1.32 g/1 per
hour, respectively, and t he maxi mum specific produc-
tivities were 0.22 g/g per hour and 0.26 g/g per hour,
respectively (Table 5). The initial cell mass concentra-
tion was 0.5 g/1 (E. coli B, pLOI 297) and 5 g/1 (E. coli
B, KOl l ) , which to some ext ent explains t he maxi mum
volumetric productivity difference bet ween t he two re-
combi nant E. coli. E. coli B, pLOI 297 produced 0.24 g
lactate/g sugar (mean value) under option A and A (a)
and 0.17 g/g under option C (Table 6), compared to no
report ed lactate product i on for E. coli B, KOl l . Clear-
ly, E. coli B, pLOI 297 has a different ferment at i on
pat t ern compared to E. coli B, KOl l . In addition, t he
ferment at i on conditions for t he two recombi nant E.
coli differed with respect to pH, nut ri ent Supplementa-
tion and initial cell dry weight (Beall and Ingrain 1992;
Lawford and Rousseau 1992).
Xylitol product i on
Duri ng et hanol ferment at i on xylitol format i on is nor-
mally an unwant ed by-product but xylitol per se is used
in t he food industry as an anticariogenic sweet ener as
well as a sugar substitute for diabetics. Product i on by
naturally occurring xylose-utilising yeasts cannot be
used in t he food industry since t hey are not classified
as GRAS (General l y Recognized As Safe) organisms.
S. cerevisiae is a GRAS organism and t herefore a xyli-
tol-producing recombi nant S. cerevisiae may be mor e
easily accepted in t he food industry. The recombi nant
S. cerevisiae H477 harbouri ng t he gene coding for XR
from P. stipitis CBS 6054 is an organism constructed
for t he product i on of xylitol (Hallborn et al. 1991). The
xylitol yield for S. cerevisiae H477 was 0.08 g xylitol/g
xylose under option A (Table 7). Under option B, in
which glucose was added to t he ferment at i on in a fed-
batch pattern, t he yield was 0.72 g xylitol/g xylose.
Conversion of xylose to xylitol does not produce any
energy for t he cells, t herefore a co-substrate (glucose)
was needed for growth and for cofactor regenerat i on
(Hallborn et al., unpublished data). The xylitol yield in
grams per gram of xylose consumed was 0.79 g/g under
option A, and 0.99 g/g under option B.
Di scussi on
The sugar and product analysis data are of crucial im-
port ance for t he significance of t he report ed ferment a-
69
tion parameters. Since eight different laboratories ana-
lysed t he same hydrol ysat e (corn-cob hydrolysate, dis-
t ri but ed at pH 1.5 to avoid sugar loss due to metabolic
activity) this study gives an idea of t he problems and
t he accuracy when analysing complex substrates. The
main component s in t he corn-cob hydrolysate were
glucose, xylose, arabinose and acetic acid with a mean
value of 6.2 g/l, 32.6 g/l, 4.8 g/1 and 4.3 g/l, respectively.
For t he total amount of sugar t he concent rat i on range
was as much as 31.7-54.7 g/1 (Table 3). In a ferment a-
tion where 18.1 g/1 of ethanol is obtained, t he corre-
sponding yield ranges from 0.33 g/g to 0.57 g/g when
t he yields are recalculated using t he lowest and t he
highest value of total amount of sugar, respectively.
This indicates an uncert ai nt y in t he comparison of re-
sults from different investigations. Despite t he scatter
in analysis results, t he conclusions drawn from this
study are valid because t he ferment at i on results for dif-
ferent organisms and for different ferment at i on op-
tions differ enough to allow conclusions to be drawn.
Several participants pointed out t he difficulties in-
volved with analysing lignocellulose hydrolysates due
to t he complex matrix of t he hydrolysate, lack of suita-
ble analytical instruments and lack of t rai ned person-
nel for i nt erpret at i on of chromatograms. Linddn and
Hahn-H~igerdal (1989a) devel oped a separation met h-
od for t he analysis of lignocellulose hydrolysates using
spent sulphite liquor as t he model substrate. The sepa-
ration was per f or med using a precol umn and two
HPX-87H (Biorad) columns coupled in series to en-
hance the resolution. The use of a precol umn results in
a longer life-time of t he main columns and an in-
creased stability of t he analysis system. This separation
col umn system is suitable for separation of glucose, xy-
lose and arabinose, which were t he mai n sugar compo-
nents in t he corn-cob hydrolysate. When a hydrolysate
also contains galactose and mannose, common in soft-
wood hydrolysates, t he sugars must be separated on an
HPX-87P col umn (BioRad). Mor e selective analysis
met hods for sugar and product analysis in complex ma-
trices are present l y under devel opment (Marko-Varga
et al. 1990; Buttler et al. unpublished data). An addi-
tional control of t he accuracy of t he analytical proce-
dure would have been to carry out a compl et e carbon
balance of individual ferment at i ons (Skoog and Hahn-
H~igerdal 1990; Senac and Hahn-H~igerdal 1991; Ols-
son and Hahn-H~igerdal 1993). A carbon balance can
also provide i nformat i on about t he metabolic path-
ways. In t he present study it was not possible to make
carbon balances due to the incomplete analysis results
report ed.
Tabl e 7. Xylitol pr oduct i on by S. cerevisiae H477
Or gani sm Opt i on Ini t i al dry Gr owt h p H Te mpe r a t ur e Xyl i t ol yield Xyl i t ol yield
wei ght (g/l) (g/l) ( C) (g/g xylose) (g/g xylose consumed)
S. cerevisiae H477 ( XR) A 5 2.2 5.5 30 0.08 0.79
S. cerevisiae H477 ( XR) B 5 5.7 5.5 30 0.72 0.99
70
A micro-organism suitable for t he ferment at i on of
lignocellulose hydrol ysat es should have the following
qualities: (i) be abl e to ferment all sugar component s in
t he hydrol ysat e to et hanol at a reasonabl e rat e and (ii)
be tolerant of pot ent i al inhibitors in the hydrolysate.
The st udy was designed to test the investigated micro-
organisms under t hree consistent options: a basic ex-
peri ment al ferment at i on and two ferment at i ons where
additional medi a component s and detoxification of the
hydrol ysat e were used. The latter t wo opt i ons general-
ly represent mor e costly alternatives.
In the economi c eval uat i on of fuel et hanol produc-
tion, by far the most i mport ant fact or is t he total yield
as t he raw material cost is of prime i mport ance (Wright
1988; Hi nman et al. 1989). Yields should be compar ed
to t he theoretical yield, which is generally consi dered
to be 0.51 g/g, but ot her possible theoretical yields
have been discussed (Slininger et al. 1987).
The total vol umet ri c product i vi t y (Fig. 1) deter-
mines t he capital i nvest ment cost, i.e. t he size of fer-
mentors. Al most any vol umet ri c product i vi t y can be
achieved by increasing the concent rat i on of cell mass,
although at t he cost of carbon. The specific productivi-
ty (Fig. 1, Tabl e 2), on t he ot her hand, gives a measure
of t he efficiency of the ferment i ng micro-organism.
Productivities (volumetric and specific) are oft en used
in the literature as an indication of t he efficiency of dif-
ferent micro-organisms under given ferment at i on con-
ditions. The value of comparing productivities ob-
t ai ned in different studies is highly quest i onabl e since
t hey can be calculated differently especially when
bat ch processes are considered. It is particularly un-
clear when a bat ch process should be consi dered
ended. In or der t o compare productivities t he calcula-
tions have to be clearly defined. In the present st udy
no total product i vi t y figures are r epor t ed because not
enough dat a were r epor t ed and/ or the sampling points
were t oo i nfrequent t o identify the time at which max-
i mum et hanol concent rat i on was reached. Addi t i onal -
ly, in t he literature t he maxi mum product i vi t y is oft en
r epor t ed t oget her with the maxi mum et hanol concen-
tration, although in typical bat ch ferment at i ons t hese
values are sel dom connect ed (Fig. 1).
Not only yield and product i vi t y are of i mport ance,
but also chemicals and process steps that increase pro-
duct i on costs. A low medi a pH (Esser and Karsch
1984; Bj6rling and Li ndman 1989) and a fast produc-
tion time reduce t he cont ami nat i on risk and t herefore
cont ri but e to process economy, whereas the use of me-
di um sterilisation and sterile techniques add extra costs
to t he process. Det oxi fi cat i on with mol ecul ar sieves
and mi xed-bed ion resins have been r epor t ed to re-
move sugars from t he hydrol ysat e (Tran and Cham-
bers 1986), but cont radi ct ory results (detoxification
with calcium hydroxide, extraction with organic sol-
vents, t reat ment with ion-exchange and adsorpt i on re-
sins) have also been r epor t ed (Frazer and McCaskey
1989). The Ca(OH)2 detoxification t reat ment used in
the present st udy is not economically sound since bot h
chemicals and equi pment add costs. Det oxi fi cat i on of
t he hydrol ysat e costs 0.40 SEK in t he chemical cost
alone of Ca(OH)2 (based on the price for a consump-
tion of 3500 metric tons per year, Chemat ur, Sweden,
1993), which should be compared with the world mar-
ket price of ethanol, 2.6-3.4 SEK (1 SEK equals 0.13
US dollars). Det oxi fi cat i on met hods such as organic
solvent extraction and ion exchange have been consid-
ered t o be even mor e costly t han Ca(OH)2-pret reat -
merit (Frazer and McCaskey 1989).
Af t er adapt at i on and dilution of t he hydrolysate, P.
stipitis CBS 5773 (/3) gave a yield of 0.34 g/g, and after
detoxification in combi nat i on with adapt at i on P. stipi-
tis CBS 6054 (e) gave a yield of 0.31 g/g. Detoxification
and/or adapt at i on is a prerequi si t e for obtaining rea-
sonabl e yields with P. stipitis, indicating that this mi-
cro-organism is highly sensitive to inhibitors present in
t he corn-cob hydrolysate. The inhibitor sensitivity is in
agreement with earlier results with P. stipitis (van Zyl
et al. 1988; Wilson et al. 1989). None of the ot her P.
stipitis strains nor t he fungus F. oxysporum or the bac-
t eri um E. coli ATCC 11303 pr oduced mor e than 0.04 g
ethanol/g total sugars (Tabl e 4). The poor results ob-
t ai ned with these micro-organisms make t hem unsuita-
ble for fuel et hanol product i on according to this corn-
cob hydrol ysat e study. E. coli B, KOl l was the most
promising et hanol producer in t he corn-cob hydroly-
sate. This organism gave t he highest yield in all fer-
ment at i on options [0.24g/g (A), 0, 36g/g (B) and
0.54 g/g (C), Tabl e 4]. Recombi nant E. coli has t he ad-
vant age of giving high total volumetric productivities
even at low cell densities (0. 033-330mg dry cell
weight/l), at least in l aborat ory substrates (Beall et al.
1991). However , for maxi mum ferment at i on perform-
ance this organism needed detoxification of t he corn-
cob hydrolysate. E. coli B, pLOI 297 has given reason-
able yields for sQme hydrolysates wi t hout detoxifica-
tion (Lawford and Rousseau 1993). A disadvantage for
bact eri a in general is the requi rement for a ferment a-
tion pH of 6.0 to 7.0, which increases the contamina-
tion risk. In the present st udy the ferment at i ons with
rec E. coli B, KOl l were per f or med in sterilized media.
Yeast, on the ot her hand, can ferment well at low pH
(3.0-4.0) (Esser and Karsch 1984; Bj6rling and Lind-
man 1989). S. cerevisiae is well known to exhibit high
t ol erance t o inhibitors present in non-det oxi fi ed ligno-
cellulose hydrolysates (Lind6n and Hahn-H~igerdal
1989b; Olsson and Hahn-H~gerdal 1993) and t ol erance
to high et hanol concentrations (Brown et al. 1981). En-
zymatic conversion of xylose to xylulose with XI fol-
l owed by ferment at i on of xylulose by S. cerevisiae has
been successfully per f or med in bot h a l aborat ory sub-
strate and a lignocellulose hydrol ysat e (Gong et al.
1981; Lind6n et al. 1992). However , with the hydroly-
sate used in this study, S. cerevisiae isolate 3 in combi-
nation with XI did not per f or m well. This is present l y
under investigation. In the xylose ferment at i on using
the recombi nant S. cerevisiae carrying the gene for XR,
xylose was convert ed to xylitol with an efficiency of
0.99 g xylitol/g xylose consumed when a co-subst rat e
was added. The recombi nant S. cerevisiae strains carry-
ing t he genes for XR and XDH showed poor et hanol
product i on from xylose, which is in agreement with
71
ear l i er s t udi es ( Ta nt i r ungki j et al. 1993; K6 t t e r a n d
Ci r i acy 1993). Thi s i ndi cat es f u r t h e r bl oc ks i n t he me -
t a b o l i s m o f xyl os e b y S. cerevisiae n o t ear l i er ob-
s er ved.
A c o mp a r a b l e e v a l u a t i o n o f t he s ui t abi l i t y o f mi c r o-
or ga ni s ms f o r t he f e r me n t a t i o n o f a di l ut e - a c i d- pr e -
t r e a t e d h y d r o l y s a t e t h a t is r i ch i n xyl os e was p e r f o r m-
ed. Thi s wo r k ga ve r i se t o s ugge s t i ons f or f u t u r e wo r k
c o n c e r n i n g d e v e l o p me n t of s t a n d a r d anal ys i s me t h o d s
f o r l i gnocel l ul os e hydr ol ys a t e s , e v a l u a t i o n o f t he e c o-
n o mi c f eas i bi l i t y o f s t er i l i zat i on a n d di f f e r e nt det oxi f i -
c a t i on me t h o d s , a n d f u r t h e r c o mp a r i s o n o f mi c r o - o r -
ga ni s ms f e r me n t i n g o t h e r l i gnoc e l l ul os e hydr ol ys a t e s .
Acknowledgements. This study was performed within Interna-
tional Energy Agency (IEA), subgroup Biomass utilization (Task
X). The xylose fermentation research at the Department of Ap-
plied Microbiology, Lund Institute of Technology/University of
Lurid has been supported financially by The Swedish Natural
Science Research Council (NFR), Swedish National Board for
Industrial and Technical Development (NUTEK), Swedish Na-
tional Board for Technical Development (STU), National Swe-
dish Energy Administration (STEV), Swedish Ethanol Develop-
ment Foundation (SSEU) and The Nordic Fund for Technology
and Industrial Development (NIF). The participating groups
have contributed with experimental data as well as valuable dis-
cussions for which they are gratefully acknowledged.
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