JOURNALOF FERMENTATIONAND BIOENGINEERING

Vol. 86, No. 1, 1-14. 1998

REVIEW
Microbial Conversion of D-Xylose to Xylitol
ELEONORA

AND

WINKELHAUSEN*

Faculty of Technology

and Metallurgy,

SLOBODANKA

Rudjer Boskovic

Received 25 February 199WAccepted

KUZMANOVA

16, 91000 Skopje,

Macedonia

11March 1998

Xylitol, a five carbon sugar alcohol, occurs widely in nature but it is also a normal intermediate in human
metabolism. As an alternative sweetener, it is recommended for diabetics and for the prevention of dental
caries. Xylitol is currently produced chemically on a large scale. Microbial production is lately becoming more
attractive since the downstream processing is expected to be cheaper. Among microorganisms, yeasts are the
best xylitol producers, particularly those belonging to the genus Candida. The key enzymes for xylitol
production in yeasts are o-xylose reductase which, using either NADH or NADPH, reduces o-xylose to xylitol,
and predominantly,
NAD-linked
xylitol dehydrogenase which reoxidizes xylitol to n-xylulose. Xylitol accumulation in yeasts is sensitive to environmental conditions such as nutrition, temperature, pH, inoculum,
substrate and aeration, with the last two being critical for yeast growth and fermentation. Hemicellulosic
hydrolysates derived from hardwood and particularly from agricultural residues, such as sugar cane bagasse,
corn cobs, wheat and rice straw, are used as feedstock for xylitol production. Due to the presence of inhibitory
components, some of the hydrolysates have to be treated prior to microbial utilization. The most investigated
types of processes have been batch ones, although fed-batch and immobilized systems have been characterized
by the highest yields and productivities. Apart from the naturally occurring yeasts, recombinant strains of
Saccharomyces cerevisiue in free and immobilized form were also investigated for xylitol production.
[Key words:

D-xylose fermentation,

xylitol, yeasts, D-xylose reductase, xylitol dehydrogenase]

While biomass has served as a substrate in microbial

processes for the production
of alcoholic beverages for
thousands of years, it is only recently that broader applications of this material
have been envisaged.
Thus,
biotechnologists
are now developing efficient systems for
the production
of liquid fuels, pharmaceuticals,
foods
and chemical feedstocks from waste organic materials
(1).
Lignocellulosics
are organic materials which are abundant and renewable. Their major components,
cellulose,
hemicellulose
and lignin, vary with plant species. The
pentose fraction,
composed
of D-xylose (usually not
less than 95%) and L-arabinose is much higher in hardwoods (19 to 33%) than in softwoods (10 to 12%). High
amounts of pentosans,
up to 40%, are also present in
agricultural residues (2). The fact that xylan is more easily
hydrolyzed than cellulose offers the technical possibility
of D-xylose and xylitol production (3).
Recently, considerable
efforts have been focused on
the microbial production
of xylitol from o-xylose. The
microbial production
of xylitol has been always closely
connected with that of ethanol and for years it was considered only as a by-product
in ethanol
fermentation
processes from n-xylose (4-10). Obviously,
this stems
from the fact that ethanol production
from n-xylose
was the process that first attracted the attention of researchers. However, for a considerable period of time, views
on the ability of yeasts to produce ethanol from aldopentoses were contradictory
(11). The matter was resolved in
the beginning of the eighties when a number of different
laboratories
independently
demonstrated
the direct conversion of D-xylose to ethanol (12-16) as well as ethanol
production
from D-xylulose (17, 18). This discovery
* Corresponding author.

opened the possibility not only for ethanol, but also for
polyol production,
primarily, xylitol. The first significant
papers on microbial production of xylitol refer to screening for suitable microorganisms
(19, 20), but the real
scientific interest began in the last few years, when
xylitol, due to its unique properties
as an alternative
sweetener, began to be more frequently
used (21-25),
and when awareness of protection
of the environment
grew.
This review attempts to examine the present literature
on microbial
production
of xylitol regarding,
first,
xylitol properties, and then, microorganisms
involved in
this process, as well as xylose metabolism,
the effect of
the process variables on xylitol production
and some
aspects of the process strategies.
THE OCCURRENCE,
APPLICATION

PROPERTIES
OF XYLITOL

AND

Xylitol, a pentahydroxy
sugar alcohol, occurs widely
in nature, in many fruits, and vegetables, among which
the yellow plum has the highest content, almost 1% on a
dry solid basis (26). Xylitol is also a normal metabolic
intermediate
in mammalian
carbohydrate
metabolism
with an endogenous production and further utilization of
some 5-15 g daily in the average adult human. Slow adsorption and entry into metabolic pathways independently of insulin and without rapid fluctuation
of blood
glucose levels support the use of xylitol as a diabetic
sweetener (27, 28). In this respect, it is comparable
to
sorbitol and other slowly absorbed carbohydrates.
Extensive clinical and laboratory
experience has shown that
the only side effect of xylitol is the possibility
of an
unpleasant but harmless osmotic diarrhea after relatively
large initial oral doses (20-30g) in unadapted
subjects

WINKELHAUSEN

J. FERMENT. BIOENG.,

AND KUZMANOVA

(27, 29).
Primary
interest in xylitol therefore
centers on its
properties and potential uses as an alternative sweetener.
In contrast to other alternative
noncaloric
sweeteners,
such as saccharine, xylitol has many properties similar to
those of sucrose. It dissolves readily in water, it is as
sweet as sucrose and hence approximately
twice as sweet
as sorbitol and nearly three times as sweet as mannitol.
Its caloric content
is the same as that of sucrose,
17 kJ/kg. In addition, it gives a pleasant cool and fresh
sensation due to its high negative heat of solution (23,
26).
Perhaps the most significant characteristic
of xylitol,
however, is the fact that it is not utilized by the acidproducing,
cariogenic bacteria of the human oral cavity
and therefore inhibits demineralization
of tooth enamel
(21, 30). A number of long-term field trials in different
countries and hence in different nutritional,
social and
economic environments
demonstrated
that the consumption of even relatively small amounts of xylitol can significantly reduce the formation of new dental caries (21,
22, 24, 31). In the light of the scientific evidence currently available, it may be regarded as the best of all alternative sweeteners with respect to caries prevention (24, 28).
Xylitol finds a broad application
either as the sole
sweetener or in conjunction
with other sweeteners in the
preparation
of a wide variety of full- and reduced-energy
sugarless confectionery
products suitable for infants and
diabetics
(32). Bakery products,
spices and relishes,
jams, jellies, marmalades
and desserts represent other
potential applications
of xylitol in food products (29). It
can also be used in pharmaceuticals
and oral hygiene
products (23).
CONVENTIONAL

PRODUCTION

OF XYLITOL

Although xylitol occurs in many fruits and vegetables,

it would be very uneconomical
to extract it from such
sources due to their high cost and relatively low xylitol
content, On a large-scale, xylitol is currently produced
by chemical reduction
of xylose derived mainly from
wood hydrolysates.
The conventional
process of xylitol
production
includes four main steps: acid hydrolysis of
plant material, purification of the hydrolysate to either a
pure xylose solution or a pure crystalline xylose, hydrogenation of the xylose to xylitol, and crystallization
of
the xylitol (26).
The critical step in this process is the purification
of
the xylose from the acid hydrolysate. Ion exchange chromatography
is employed to remove salts and charged
degradation
products, and activated carbon is used to
remove color (33, 34). Ion exchange chromatography,
however,
does not remove or separate
the various
hemicellulosic
sugars. This is a problem because acid
hydrolysis releases appreciable amounts of D-galactose,
D-mannose
and L-arabinose
in addition
to D-xylose.
The exact proportions
of the various sugars depend on
the nature of the feedstock and the manner in which it is
hydrolyzed.
These contaminating
sugars can complicate
crystallization
and purification
of xylose (Leleu, J-B,
Duflot, P., and Caboche, J-J., U.S. patent 5,096,820,
1992). The yield of xylitol from the xylan fraction is
about SO-60:6 or S-1596 of the raw material employed
(35).
The existing drawbacks of conventional
xylitol production methods motivated researchers to seek alternative

ways for its production.

One of the most attractive
procedures, today, is microbial production.
To a certain
extent, another motivation
was the high pollution levels
and waste-treatment
concerns.
XYLITOL-PRODUCING

MICROORGANISMS

Microorganisms
more readily assimilate and ferment
glucose than xylose. However, although in small numbers, there are bacterias, yeasts and fungi capable of
assimilating and fermenting xylose to xylitol, ethanol and
other compounds (36).
A few bacteria such as Cor_vnebacteriurn sp. (37),
Enterobacter
liquefaciens (38, 39), and Mycobacterium
smegmatis (40) have been reported to produce xylitol.
For the first two bacteria, D-xylose was mainly used as a
substrate while for the last one, the substrate was D-xylulose or D-xylose isomerized by commercially
immobilized D-xylose isomerase. However, due to the relatively
small quantities
of xylitol formed,
xylitol-producing
bacteria do not presently attract researchers interest.
Regarding the fungi, there is only one significant report
regarding
Petromyces
albertensis
(41). This fungus
accumulated 39.8 g/l of xylitol when cultured for 10 d on
100 g/l D-XylOSe. Nevertheless, after initial studies regarding the effects of environmental
conditions
on xylitol
production
by this fungus, no further reports were published.
In general, among microorganisms,
the yeasts are considered to be the best xylitol producers and therefore,
the majority of publications
deal with them. Some of
the yeasts screened for xylitol production
are presented
in Table 1. While considering
Table 1, it should be
noted that: (i) The xylitol concentrations
indicated are
those obtained
during the screening process, that is
before any optimization
of the cultural conditions,
(ii)
different media and culture conditions have been applied
TABLE

1.

Screening

Yeast

of yeasts for xylitol production

Xylitol
k/0

2.9
Candida boidinii NRRL Y-17213
C. guilliermondii FTI-20037
16.0
5.7
C. intermedia RJ-248
31.0
C. mogii ATCC 18364
20.0
C. parapsilosis ATCC 34078
4.3
C. pseudotropicalis IZ-43 1
2.1
C. tropicalis
4.8
C. tropicalis HXP 2
17.0
C. tropicalis 1004
20.0
C. tropicalis ATCC 7349
5.5
C. tropicalis ATCC 20240
1.8
C. utilis ATCC 22023
3.0
C. utilis C-40
0.8
Debarvomvces hansenii C-98 M-21
6.1
Hansenula anomala IZ-1420
4.6
Kluvveromvces franilis FTI-20066
6.1
K. karxiar&
Ii-1821
Pichia (Hansenuia) anomala NRRL
2.0
Y-366
Pachysolen tannophilus NRRL
2.2
Y-2460
0.7
Saccharomyces SC- 13
2.3
Saccharom.vces SC-37
Schizosaccharomyces pombe 16919
0.2
d n.d., Nor detected.
h n.r., Not reported.

Ethanol
k/A

from o-xylose
Reference

3.9
n.d.a
3.6
n.r.h
n.r.
3.0
n.r.
n.r.
n.d.
n.r.
n.r.
n.r.
n.r.
n.d.
n.d.
3.5
0.6

42
20
20
43
43
20
19
19
20
43
43
43
44
42
20
20
20

n.d.

42

5.2
n.r.
n.r.
n.r.

20
44
44
19

VOL. 86, 1998

MICROBIAL

and (iii) the yeasts, for which no data regarding their

screening for xylitol production
were available, were not
included in the survey. Due to this, it is difficult here to
really compare the production
capacities
of different
yeasts. However it is obvious that the best xylitol producers belong to the genus Can&da.
Screening of more than 30 yeast strains, Ojamo (Ojamo,
H., Ph.D.
thesis University
of Technology,
Espoo,
Finland,
1994) also revealed that the yeasts from the
genus Candida, such as C. guilliermondii
VTT-C-71006,
C. tropicalis VTT-C-78086
and C. tropicalis VTT-C78087, were the best xylitol producers.
Unfortunately,
the results of the screening were not precisely quantified
and hence could not be included in Table 1.
CONVERSION

OF D-XYLOSE
YEASTS

TO XYLITOL

BY

The first step in the metabolism

Xylose transport
of D-xylose is the transport of the sugar across the cell
membrane. A number of investigations
have shown that
under aerobic and oxygen limited conditions
the rate
of transport can limit the utilization
of D-xylose in P.
stipitis CBS 7126 and C. shehatae ATCC 22984 (45-47).
Under anaerobic conditions, D-xylose metabolism has not
appeared to be transport-limited
either in C. shehatae
(45) or in P. stipitis (47). Instead, the limitation was in
the two initial steps of D-xylose metabolism,
reduction
of D-xylose
and
subsequent
oxidation
of xylitol.
Studying the oxygen requirement
for D-xylose uptake,
Skoog and Hahn-HPgerdal
(48) found
that oxygen
induces or activates a transport system in P. stipitis CBS
6054.
Starvation induced both proton symport and a facilitated xylose uptake diffusion system in C. shehatae CBS
2779 (49). In non-starved
cells, D-xylose was transported
by a facilitated diffusion system. Killian and van Uden
(46) reported on a low-affinity and a high-affinity xylose
proton symport operating simultaneously
in both starved
and non-starved
cells of P. stipitis IGC 4374. From the
differences between the kinetic parameters of C. shehatae
CBS 2779 (Km varies from 1 to 125 mM) and P. stipitis
IGC 4374 (K, varies from 0.06 to 2.26mM) stems the
observation that transport systems in the latter are more
efficient.
A typical xylitol-producing
yeast, the transport system
of which has been studied, is Candida mogii ATCC 18
364 (43). The o-xylose uptake rate in the yeast followed
Michaelis-Menten
kinetics which suggested a carriermediated facilitated diffusion transport system. A kinetic
analysis of i4C-xylose transport
in intact cells of C.
mogii supported this hypothesis. To our knowledge, the
transport
systems of no other xylitol-producing
yeasts
have been investigated to date.
Metabolic pathways
In 1960, Chiang and Knight
(50) found
that the filamentous
fungus
Penicillium
chrysogenum
carried out the conversion
of D-xylose to
D-xylulose through a two-step reduction and oxidation.
It possessed enzymes that differed from xylose isomerase
in bacteria. This finding, as well as some further investigations (51) led to the conclusion that the two-step conversion of D-xylose to D-xylulose is specific for yeasts
and fungi, whereas in bacteria the same conversion
is
catalyzed by xylose isomerase in a single step. The detection of xylose isomerase in the yeasts Rhodotorula
(52)
and C. boidinii no. 2201 (53) is one of the few excep-

CONVERSION

OF D-XYLOSE

TO XYLITOL

tions to this generalization.

Once inside the yeast cell, D-xylose is reduced to
xylitol by either NADH- or NADPH-dependent
xylose
reductase
(aldose reductase
EC 1.1.1.21).
Xylitol is
either secreted from the cell or oxidized to xylulose by
NAD- or NADP-dependent
xylitol dehydrogenase
(EC
1.1.1.9). The first two reactions are considered to be
limiting in D-xylose fermentation.
The phosphorylation
of xylulose to xylulose 5-phosphate
is catalyzed by
xylulokinase (EC 2.7.1.17) (54, 55).
The conversion of pentoses to xylulose-5-phosphate
is
a prerequisite for its utilization by the central catabolic
pathways (56). Xylulose-5-phosphate
can subsequently
enter the pentose phosphate pathway (Fig. 1). This pathway consists of an oxidative phase that converts hexose
phosphates
to pentose phosphates
providing
NADPH
needed in biosynthetic
pathways and a non-oxidative
phase in which the pentose phosphates
are converted
into hexose and triose phosphates (3). The pentose phosphate pathway also yields ribose-5-phosphate
used for
the synthesis of nucleic acids and histidine
and of
erythrose-4-phosphate
necessary
for the synthesis
of
aromatic amino acids.
Glyceraldehyde-3-phosphate
and fructose-6-phosphate
are products of the non-oxidative
phase of the pentose
phosphate pathway. Both of them can be converted to
pyruvate
in the Embden-Meyerhof-Parnas
pathway.
Pyruvate can either be decarboxylated
and reduced to
ethanol or can enter the tricarboxylic acid cycle. The conversion of xylulose-5-phosphate
into glyceraldehyde-3phosphate and acetyl phosphate by xylulose-5-phosphate
phosphoketolase
presents an alternative
route for the
utilization of xylulose-5-phosphate
(57). A detailed study
of the biochemistry
and physiology of the yeasts metabolizing xylose was recently published (58).
Xylose metabolism
in yeasts yields a variety of carbon-containing
products which include carbon dioxide,
ethanol, acetic acid and polysaccharides.
Product yields
are dependent
upon the regulation
of carbon
flow
through available metabolic routes (56). D-Xylose conver-

xylitol
k NAD(P)
+- NADLP)h
0 -xyl$o$
+-ADP

QyceraidehydL-3

-phosphate

ethanol

1
Embden-Meyerhoff-Parnas

pyruvate
Tricarboxylic
FIG.
yeasts.

1.

Schematic

Pathway
NADH

CO2
-

I
i
Acid

acetaldehyde

NAD
ethanol

Cycle

representation

of

D-xylose

metabolism

in

WINKELHAUSEN

J. FERMENT.BIOENG.,

AND KUZMANOVA

explains the higher n-xylose reductase activity in the former yeast (61). Some differences reported for the cofactor
requirements
of n-xylose reductase from C. guiliermondii NRC 5578 (Table 2) may stem from differences in
culture conditions.
The varying ratio of NADH- to NADPH-linked
Dxylose reductase activity with aeration conditions was first
found in Pachysolen tannophilus. This finding suggested
that there was probably more than one form of n-xylose
reductase in the yeast, which was indeed confirmed (68,
69). The same variations were observed in the yeasts C.
parapsilosis ATCC 28474 (61) and C. boidinii NRRL Y17213 (63).
It is noteworthy that C. boidinii under oxygen limitation, in contrast to all other D-xylose-fermenting
and
xylitol-producing
yeasts, (62, 65-67, 70) exhibits a NADH/
NADPH ratio higher than 1 (Table 2). Referring to
this, Vongsuvanlert
and Tani (53) speculated that in
C. boidinii xylitol could be formed by two metabolic
pathways. The first possibility was that n-xylose is directly reduced to xylitol and the second one is that n-xylose
is initially isomerized by D-xylose isomerase to D-xylulose that is subsequently
reduced to xylitol. In both
reductions,
NADPH was also active as a reductant but
with less efficiency.
The general characteristic
of most xylose-fermenting
yeasts is that their xylitol dehydrogenase
uses predominantly
NAD and very rarely the NADP cofactor
(55, 63, 64, 66, 71, 72). Regarding the ratio of NADHlinked D-xylose reductase and NAD-linked
xylitol dehydrogenase
activities, it has been noticed that oxygen
may lower it and consequently
minimize xylitol accumulation in n-xylose-fermenting
yeasts (56). This was also
observed
in C. boidinii NRRL Y-17213 (63). The
NADH/NAD
ratio decreased
%-fold with increasing
oxygen availability
in the investigated
range of oxygen
transfer rates from 10 to 30mmol/lh.
In addition
to aeration,
the activities of n-xylose
reductase and xylitol dehydrogenase
are also very sensitive to the substrate. Since the real substrates for xylitol
producing
microorganisms
are lignocellulosic
hydroly-

sion to xylitol in yeasts can not be separated from the

conversion of D-xylose to these products. The process of
xylitol formation can not be stopped after the first step,
when n-xylose is converted to xylitol. Cell growth depends on some of the above metabolic products and it is
also necessary that the cofactors be regenerated through
different steps in the metabolic pathway. Therefore, for
obtaining
good yields of xylitol, the amount of xylose
being converted to xylitol and the amount
of xylitol
which is available for further metabolism,
have to be
well balanced.
Coenzyme specificity
The first two enzymes, Dxylose reductase
and xylitol dehydrogenase
are key
enzymes in xylitol production by yeasts. They both require
pyridine nucleotide cofactors exhibiting different cofactor specificity in different yeasts. Under anaerobic
or
oxygen-limited
conditions,
the difference in the cofactor
requirements
of these enzymes causes a redox imbalance
which influences
xylitol accumulation
in yeasts (59).
Xylitol formation is favored under oxygen-limited
conditions, because of the NADH accumulation
and subsequent inhibition
of NAD-linked
xylitol dehydrogenase.
This phenomenon,
known as the Custer effect, results
from the incapability
of the yeasts to compensate
for
excess NADH as they have no transhydrogenase
activity
(60). Several studies showed the existence of a correlation between key enzyme activities, and the level of oxygenation
and xylitol
production
in yeasts (61-63).
However, due to the various presentation
of aeration,
when displaying the enzymes activities of D-xylose reductase and xylitol dehydrogenase
in Table 2, the aeration is
not given.
In most yeast cell-free extracts, n-xylose reductase has
a higher or even absolute preference for NADPH (Table
2). The xylose reductases
of C. parapsilosis
ATCC
28474, C. shehatae ATCC 22984 and CBS 5813, a
mutant strain of P. tannophilus U+U-27, and P. stipitis
CBS 5773 use both NADPH and NADH, the use of the
former being considerably
higher. The lower NADH/
NADPH ratio in C. guilliermondii NRC 5578 (0.1) compared to that in C. parapsilosis
ATCC 28474 (0.4)
TABLE

2.

Enzyme

activities

of o-xylose

reductase

and xylitol dehydrogenase

Soecific activitv

Microorganism

Candida boidinii (Kloeckera sp.) no. 2201

C. boidinii NRRL Y-17213
C. guilliermondii NRC 5578
C. guilliermondii NRC 5578
C. mogii ATCC 18364
C. parapsilosis ATCC 28474
C. shehatae ATCC 22984
C. shehatae CBS 5813
C. tropicalis IF0 06 18
C. utilis CBS 621
Debaryomyces hansenii DTIA-77
Pachysolen tannophilus CBS 4044
P. tannophilus NRRL Y-2460
P. tannophilus U-U-27 (mutant)
Pichia stipitis CBS 5773

o-Xylose

reductase

in various

yeasts with n-xylose

as a substrate3

(U/e orotein)b
Xylitol

dehydrogenase

NADPH

NADH

NAD

NADP

0.055
0.019
0.521
1.191
0.160
0.416
0.333
0.480
10.640
0.075
0.091
0.220
0.033
0.173
0.600

0.288
0.112
0.050
n.d.d
0.060
0.161
0.100
0.210
1.720
n.d.
n.r.
0.009
0.008
0.118
0.310

0.272
0.060
n.r.
n.r.
0.220
n.r.
0.240

0.096
0.003
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
0.120
n.d.
n.r.
0.070
n.d.
n.d.
0.075

2onko
0.280
0.047
0.910
0.049
0.160
0.720

a The activities were measured in crude extracts. When different levels of aeration were employed
presentation.
b Enzyme unit (U) is defined as mmol of oxidized or reduced coenzyme per minute.
c n.r., Not reported.
d n.d., Not detected.

the highest

activities

Reference

64
63
61
65
43
61
66
59
67
59
62
59
55
55
59
were selected for this

VOL. 86, 1998

TABLE 3.

MICROBIAL CONVERSION OF D-XYLOSE TO XYLITOL

Relative specific activities of aldose reductase and xylitol dehydrogenase induced in the presence of various carbon sources
Relative specific activity (%)

Microorganism

Cundidu guilliermondii NRC 5578

Candida guilliermondii NRC 5578

Candida guilliermondii NRC 5578

Pachysolen tannophilus NRRL Y-2460

Pichia stipitis NRRL Y-7124

Substrate

(% w/v)

Aldose

D-Xylose (2%)
D-Glucose (2%)
L-Arabinose
(2%)
D-Galactose
(2%)
D-Mannose (2%)
Glycerol
D-Xylose (4%)
D-Glucose (4%)
L-Arabinose
(4%)
D-Xylose (4%) + D-glucose (4%)
D-Xylose (4%) + D-glucose (1%)
D-Xylose (4%) + L-arabinose
(4%)
L-Arabinose
(4%) + D-glucose (4,%)
D-Xylose (2,%)
D-Glucose (2%)
r.-Arabinose
(2%)
D-Galactose
(2%)
D-Mannose (2%)
D-Fructose (206)
D-Xylose (1%) + D-glucose (1%)
D-Xylose (1%) + L-arabinose
(1%)
D-Xylose (1 ,O&)+ D-galactose (1,%)
D-Xylose (l%)+D-mannose
(1%)
Glycerol (2%)
D-Xylose (4%)
D-Glucose (4,d)
D-Xylose (4%) + D-glucose (4%)
D-Xylose (4%) + L-arabinose
(4%)
D-Xylose (40&+D-galactose
(4%)
D-Xylose (4%)+D-mannose
(4?&
Glycerol (2%)
D-Xylose (4%)
D-Glucose (4%)
D-Xylose (4%)+D-glucose
(4.W
D-Xylose (4%)+1.-arabinose
(4%)
D-Xylose (4%)+Dgalactose
(4%)
D-Xylose (4%)+D-mannose
(4Pd)
Glycerol (2%)

reductase

xylitol dehydrogenase

NADPH

NAD

100
1

n.r.=
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
100

52
7
3
11
100
13
52
59
80
75
8
100
0
92
2
11
8
5
93
94
32
3
100
15
34
110
107
30
32
100
5
19
120
160
54
7

Reference

65

73

72

94
0
0
54
23
107
107
45
0
100
0

74

7
80
180
4
20
100

74

13
17
110

90
38
0

a n.r., Not reported.

sates which often contain
a variety of sugars (o-xylose,
o-glucose, o-mannose,
o-galactose,
L-arabinose etc), it
is very important
to investigate their influence on the
induction of D-xylose reductase and xylitol dehydrogenase
(Table 3).
o-Xylose was found to be the best inducer of both
aldose reductase,
strictly dependent
on NADPH,
and
xylitol dehydrogenase
from C. guilliermondii NRC 5578.
It was closely followed by L-arabinose,
indicating
that
pentoses can be effective substrates for the induction of
enzyme activities (Table 3). The level of induction of aldose reductase activity depended on the initial concentration of D-xylose. Low activity was found in o-glucose-,
D-mannose- and o-galactose-grown
cells, suggesting that
the mechanism leading to the expression of this enzyme
is under catabolite repression control, which is typical
for o-glucose
or other rapidly metabolizable
carbon
sources (65).
Various sugars and polyols can be used as substrates
for the . enzymatic reactions. The cell-free extract of C.
bordmrr no. 2201 reduced o-xylulose,
o-xylose, D-fructose and L-sorbose,
preferably
using NADH
as a
reductant rather than NADPH, but exhibited very low
affinity for the reduction
of D-glucose for both cofac-

tors. For the oxidation of polyols, the cell-free extract

preferably oxidized xylitol and D-sorbitol using NAD as
an oxidant rather than NADP (64). The highest enzyme
activity in C. guilliermondii
NRC 5578 cells was observed with L-arabinose as a substrate for the enzymatic
reaction (65).
Yokoyama et al. (75) purified and characterized three
NADPH-dependent
o-xylose reductases from C. tropicalis IF0 0618 and tested their activities for several substrates.
The activity
for DL-glyceraldehyde
was the
highest, followed by L-arabinose and D-xylose. The Dxylose reductases were the least active for o-glucose.
PROCESS

VARIABLES

All published
data on xylitol production
by yeasts
have
demonstrated
that
xylitol
accumulation
is
influenced
by a number
of experimental
conditions.
Studying the effect of these conditions
is of particular
interest as a prerequisite
for higher xylitol yields and
productivities.
Nutrition
Although various media have been used
to culture xylitol-producing
yeasts, a few generalizations
can be made: (i) For some yeasts, yeast extract is an

WINKELHAUSEN

AND KUZMANOVA

important nutrient for xylitol production.

(ii) For other
yeasts, sometimes also including
yeasts from the first
group, but reported by different researchers, yeast extract has no significant effect on xylitol formation.
These
yeasts prefer urea or urea and Casamino acids. (iii) For
kinetic studies, synthetic media are used which provide
all the necessary minerals and vitamins.
The culture media for C. parapsilosis ATCC 28474,
(76) C. boidinii no. 2201, (64) C. guilliermondii
NRC
5578 (77) and C. tropicalis IF0 0618 (67) contain yeast
extract in concentrations
ranging
from 10 to 2Og/l.
Yeast extract at a maximum concentration
of 1 g/l was
sufficient for C. tropicalis DSM 7524. Concentrations
higher than 15 g/l, blocked the conversion
of p-xylose
to xylitol (78). Increased concentrations
of yeast extract
of 5 and log/l increased the biomass production
of C.
guilliermondii
FTI 20037, but sharply decreased
its
xylitol productivity
(79). Similarly, the addition of yeast
extract and peptone to the defined medium for C. lnogii
ATCC 18364 enhanced cell growth markedly but had no
significant effect on the yield and specific productivity of
xylitol (43).
Xylitol formation in C. guilliermondii
FTl 20037 (20,
61), D. hansenii DTIA-77 (62, 80), Candida sp. (81), C.
parapsilosis ATCC 28474 (61) and C. boidinii NRRL Y17213 (42) was highest with urea as a substrate. In most
cases, the medium was supplemented
with Casamino
acids. In some yeasts, special supplements
improved
xylitol production.
Thus, on studying the effect of biotin, Lee et a/. (82) found that in high-biotin media, in P.
tannophilus
NRRL Y-2460 ethanol
production
was
favored over that of xylitol, while in C. guilliermondii FTI
20037 xylitol formation
was favored.
When the C.
boidinii no. 2201 medium was supplemented
with l?,j
(v/v) methanol,
xylitol production
increased 2.5-fold
(64). This was explained by the oxidation of methanol,
providing NADH to the medium. Mahler and Guebel
(83) found that at low Mg+2 levels, 1 mM, xylitol formation by P. stipitis NRRL Y-7124 was higher than that of
ethanol.
Temperature
and pH
The most suitable temperature for xylitol production
in yeasts was shown to be
30C. Small temperature
variations above this temperature, do not significantly affect xylitol production
in C.
tropicalis DSM 7524. The xylitol yield was, for the most
part, temperature-independent
when the yeast was cultured in a temperature
range between 30C and 37C
but above 37C the xylitol yield decreased sharply (78).
Xylitol formation
in C. guilliermondii
FTI 20037 was
the same at 30 and 35C, but decreased when the temperature increased to 40C (20). The conversion of D-xylose
to xylitol by Candida sp. B-22 was relatively constant
over the temperature range of 35-40C. At temperatures
of 45C and higher, the conversion was sharply reduced
(84). This was probably due to loss of the activities of
both NADPH and NADH-dependent
xylose reductase,
as the temperature increased (56).
When investigating
the effect of temperature
on
ethanol and xylitol production,
du Preez et al. (7) found
that at higher temperatures,
production
of xylitol is
favored over that of ethanol. Xylitol production
of C.
shehatae CSIR-Y492 increased &fold as the temperature
increased
from 22 to 36C. P. stipitis CSIR-Y633
produced xylitol at 36C but no detectable amounts at
lower temperatures.
If uncontrolled,
pH drops during the fermentation,

_I. FERMENT.BIOENG.,

and therefore under such conditions the initial pH values

have to be higher than under controlled conditions. The
optimum
initial pH value for best xylitol yield in C.
boidinii was 7 (42, 64), whereas under controlled conditions, a pH of 5.5 was better (63). Batch culture of C.
parapsilosis ATCC 28474 (76) was performed at pH 6,
while for continuous culture, a pH of 4.5 was used (85).
The yeasts are generally
cultivated
at pH values
between 4 and 6. C. parapsilosis ATCC 28474 (61) and C.
guifliermondii
NRC 5578 (61, 77) were grown at pH 6,
C. mogii ATCC (43) and P. stipitis NRRL Y-7124 at
pH 5 and 5.5, respectively, while pH 4 was optimum for
C. tropicalis IF0 0618 (67). In contrast, da Silva and
Afschar (78) reported that C. tropicalis DSM 7524 was
not very sensitive to pH and attained a maximum xylitol
yield at pH 2.5. Increasing the pH from 2.5 to 4 led to
an increase in xylitol productivity
but a decrease in
xylitol yield.
Inoculum
On studying the effect of initial cell concentration
of Candida sp. B-22 on xylitol production
from n-xylose, Cao et al. (84) found that the rate of
xylitol production
was linear and the fermentation
time
was dramatically
reduced over an initial concentration
range of 3.8 to 26g/l. With an initial yeast cell concentration of 26g/l,
21Og/l xylitol was produced
from
26Og/l o-xylose. A high initial concentration
was also
beneficial for xylitol production by C. boidinii NRRL Y17213. With an initial D-xylose concentration
of 5Og//,
the xylitol yield and specific productivity
doubled when
the inoculum level increased from 1.3 to 5.1 g/l (42).
The effect of inoculum size on the microbial production of xylitol from hemicellulose hydrolysates was also
investigated (86, 87). A high initial cell density did not
have a positive effect when C. guilliermondii FTI 20037
was grown on rice straw hemicellulose hydrolysate since
increasing the initial cell density from 0.67 g/l to 2.41 g/f
decreased
biomass
formation,
xylose utilization
and
xylitoi accumulation
(86). On the contrary, D. hansenii
NRRL Y-7426 grown on wood hydrolysate
produced
more xylitol at higher initial cell densities (87).
In addition to inoculum size, the culture age, which is
related to the metabolic activity of cells, was also studied. Varying the inoculum age of C. guilliermondii FTI
20037 from 15 to 70 h demonstrated
that 15-h-old cells
gave poor results, whereas 24-h-old and older cells had
similar effects and influenced only the productivity
of
xylitol but not its final concentration
and yield (79, 88).
Cultivation
of the inoculum
using different
carbon
sources (o-xylose, mixture of n-xylose and glucose in a
4 : 1 ratio and glucose) had only a minor influence on
the bioconversion
of D-xylose (88).
n-Xylose concentration
has been shown
Substrate
to be critical for yeast growth and fermentation.
In the
absence of D-xylose, xylitol formation
does not occur.
Together with aeration,
p-xylose concentration
affects
xylitol formation the most. D-Xylose is required for the
induction of xylose reductase and xylitol dehydrogenase
activities in yeasts (Table 3).
High n-xylose concentration
induces xylitol formation
in yeasts. Increased xylose concentration
favors xylitol
production at the expense of ethanol production,
resulting in an increase in the xylitol/ethanol
ratio, a decrease
in the ethanol yield and without exception, an increase
in the xylitol yield (42, 48, 77, SO). As the initial Dxylose concentration
increases, the specific growth rate
decreases, demonstrating
substrate
inhibition,
whereas

VOL. 86, 1998

MICROBIAL

TABLE

4.

Influence

boidinii
Y-17213

C. guiliiertnondii
NRC 5578

C. guilliermondii
NRC

C.

5578

mogii ATCC

18364

C. parapsilosis
ATCC

a n.r.,

on the fermentation

parameters

TO XYLITOL

for some xylitol-producing

28474

2201

50
100
150
50
100
150
200
10
50
150
300
50
100
200
300
10.1
28.9
53.3
50
100
200
300

64.0
81.8
51.0
11.6
85.6
74.7
22.2
100
98.3
100
100
100
100
100
100
100
100
100
100
100
100
I00

17.1
36.0
17.0
4.8
25.2
53.1
10.0
6.2
30.9
110.3
221.0
22.5
49
116
207
1.7
14.5
37.3
29.5
61.0
116.0
93.0

0.53
0.44
0.22
0.12
0.29
0.47
0.22
0.62
0.63
0.74
0.74
0.45
0.49
0.58
0.69
0.17
0.50
0.70
0.59
0.61
0.58
0.31

58.4
48.4
24.2
13.2
31.9
51.7
24.2
68.1
69.1
81.3
81.3
49.5
53.8
63.7
75.8
18.7
54.9
16.9
64.8
67.0
63.7
34.1

0.35
0.25
0.01
0.05
0.13
0.16
0.04
0.13
0.19
0.46
0.54
0.055
0.066
0.099
0.164
n.r.
n.r.
n.r.
0.111
0.123
0.115
0.050

n.r.a
n.r.
n.r.
6.4
11.6
15.1
9.0
0.4
0.9
3.0
6.0
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.

n.r.
n.r.
0%
0.11
0.07
0.09
0.31
0.09
0.04
0.02
0.036
0.014
0.002
0.004
n.r.
n.r.

n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
0.11
0.11
0.03
0.01
0.050
0.030
0.007
0.010
0.005
0.004
0.003
0.026
0.020
0.020
0.009

o%o

0.018
0.016
0.004

yeasts
Reference

(l/;h)

(g/l)

Candida boidinii
(Kloeckera sp.) no.
NRRL

concentration

OF D-XYLOSE

D-Xylose

Microorganism

C.

of initial substrate

CONVERSION

48
144
144
96
132
336
192
46
165
238
406
409
742
1172
1269
n.r.
n.r.
n.r.
266
477
1009
1860

64

42

77

61

43

61

Not reported.

the overall
xylitol
productivity
depends on the yeast
type. However, all yeasts need a relatively long time for
the conversion of D-xylose to xylitol (Table 4). For most
yeasts, the initial n-xylose concentrations
resulting in the
highest yields are between 100 and 2OOg/l, with C. guilliermondii
NRC 5578 being an exception
for which
300 g/l is the most suitable concentration.
Certain xylose
concentrations
inhibit xylitol formation,
and such inhibitory concentrations
differ with yeast type (Table 4).
Although for osmophilic
yeasts it is intrinsic that a
high substrate concentration
induces polyol formation,
according to Prior et ai. (57) the correlation
between
xylitol accumulation
and D-xylose concentration
could
be a consequence of more severe oxygen-limited
growth
conditions as a result of the higher cell densities reached
at higher substrate levels than an effect of the D-xylose
concentration
itself.
Regarding the effect of hexoses on D-xylose utilization, several reports stated that D-galactose, D-cellobiose
and L-arabinose are not inhibitory
to D-xylose assimilation, while D-mannose and particularly
D-glucose considerably slow down the utilization
of D-xylose (72-74,
78).
In mixtures of glucose and D-XylOSe,
the yeasts
first
consume the glucose and only after the glucose is depleted do they consume D-xylose (89, 90). When a mixture
of glucose and xylose was used as a substrate for the
fed-batch culture of C. boidinii NRRL Y-17213 with the
former being l/10 of the concentration
of the latter, glucose was consumed first, resulting in faster growth with
a maximum specific growth rate of 0.067 l/h, compared
to 0.023 l/h in the process using xylose alone. In contrast, xylitol accumulation,
and consequently
xylitol
yield, were lower; 39.4g/l
and 0.57 g/g, compared to
46.5 g/l and 0.64 g/g, respectively (90). In the presence
of glucose (15 g/l), C. guilliermondii FTI 2003 converted
D-XylOSe
(65 g/l) to xylitol with lower yields relative to
those obtained
in a medium without glucose. It was
assumed that in the presence of glucose, there was a
partial inhibition
of xylose reductase and consequently

a smaller amount of xylitol was produced (91). Higher

amounts of glucose relative to D-xylose reduced the yield
further (79).
D-mannose
or n-galactose
was
When
D-glucose,
present in the medium with D-xylose, C. guilliermondii
NRC 5578 exhibited a sequential pattern of utilization
with the hexoses being consumed before D-xylose (72).
In general, the assimilation
of D-xylose began when
some of the hexoses were still present in the medium,
which indicated the existence of a threshold above which
hexose repression occurred. The preferential
utilization
of D-galactose over D-xylose by C. guifliermondii NRC
5578 was surprising
since D-galactose did not repress
the induction
of D-xylose reductase
and xylitol dehydrogenase activities. When grown on a fructose-xylose
mixture, the yeast utilized both sugars simultaneously
at
similar rates.
The utilization of various single sugars, other than Dxylose, was studied in batch cultures of C. guilliermondii
NRC 5578. Meyrial et al. (77) found that D-glucose, Dmannose and D-galactose were rapidly fermented with
specific uptake rates being 2.2, 1.8 and 1.5 times higher
than for D-XylOSe, although the hexoses were utilized by
the strain only for growth and ethanol production;
their
corresponding
polyols were not detected. On studying
the same yeast, Lee et al. (72) found that D-glucose was
the most rapidly utilized, followed by D-mannose,
Dxylose, D-galactose and D-fructose. With D-glucose and
D-fructose, ethanol was the only fermentation
product,
whereas with D-mannose and D-galactose, mannitol and
galactitol were produced in addition to ethanol.
Aeration
An experimental condition that is critically important for determining
the extent to which xylitol
accumulates in cultures is aeration. Under fully aerobic
conditions, xylitol is not produced. Under anaerobic conditions, all the yeasts tested failed to grow on D-xylose
to any appreciable extent, reaching about one doubling
at best (11). It was found that fermentation
and growth
occur simultaneously
only under oxygen limitation (92).
Supply of oxygen to the culture is required for yeast

WINKELHAUSEN

J. FERMENT.BIOENG.,

AND KUZMANOVA

growth even under fermentation

for the synthesis of
unsaturated
fatty acids and ergosterol required for sugar
transport through the membrane (93).
As Laplace et al. (94) suggested, the dependence
of
the fermentative behavior of the xylose-fermenting
yeasts
on oxygen availability can be presented in three stages:
(i) Under anaerobic conditions
or at very low oxygen
transfer rates, the electron transport system of the yeasts
is unable to oxidize NADH completely.
As a consequence, the intracellular
NADH concentration
increases
and this imbalance between NADH and NAD concentrations leads to xylitol secretion. (ii) Increasing the oxygen
transfer rate permits the enhancement
of xylose fermentation. This is believed to be due to the role of oxygen as
a terminal electron acceptor, thus relieving the imbalance
of the two initial steps of anaerobic xylose metabolism.
This hypothesis is supported by the inverse relationship
between the degree of aeration and xylitol production
observed for some yeasts. Under these circumstances,
the main metabolic product is ethanol. (iii) When oxygen is
supplied in excess, a deviation in the pyruvate flow from
the fermentative
pathway to the tricarboxylic
acid cycle
is observed resulting in increase in cell mass.
A survey of xylitol production by various yeasts under
different oxygenation
levels is presented in Table 5. It is
very difficult, however, to compare data from different
studies because oxygenation
is measured and reported
differently. Yet, it is evident that yeasts producing xylitol
require very small amounts of oxygen, which appear to
be specific for each yeast strain. It seems that D. hanTABLE
Microorganism
Candida boidinii
NRRL Y-17213

C. guilliermondii
FTI 20037
C. guilliermondii
FTI 20037

C. guilliermondii
NRC 5578
C. parapsilosis
ATCC 28474
C. parapsilosis
ATCC 28474

C. parapsilosis
ATCC 28474
Debaryomyces hansenii
DTIA-77
Debaryomyees hansenii
DTIA-77
Pachysolen tannophilus
ATCC 32691
a
b
c
*

cs

(g/l)

130
130
130
130
40
40
40
65a
65a
6ja
65
300
300
30
100
100
20
10
10
10
10
50
50
50
90
50
50
50
50

5.

SC

Influence

(o/d) k/O

of aeration

senii DTIA-77 has the highest demand for oxygen compared to the other yeasts listed in Table 5.
Under aerobic conditions,
as D-xylose is depleted,
some yeasts, such as D. hansenii DTIA-77 (80), C.
tropicalis ATCC 32113 (96) and C. boidinii NRRL Y17213 (42) can reassimilate both xylitol and ethanol.
When optimizing
the xylitol production
rate of C.
tropicalis
IS0
0618 by employing
the Box-Wilson
method, Horitsu et al. (67) found that the interaction
between D-xylose concentration
and aeration rate was
related to cell concentration.
If the cell concentration
was
low, the dissolved oxygen concentration
was maintained
at a high level, resulting in low xylitol production.
This
finding
indicates
that the most relevant
parameter
defining the oxygenation
of a culture is the specific oxygen uptake rate. Despite this, not many of the xylitolproducing
yeasts have been investigated
with this in
mind.
The influence of specific oxygen uptake at a constant
D-xylose concentration
of 35 g/l under pseudo-steady
state conditions
was studied in C. mogii ATCC 18364
(43). Decreasing the specific oxygen uptake rate in the
range of 1.65 to 0.50mmoVgh
decreased the specific
growth rate (0.040 to 0.003 I/h), the specific o-xylose
uptake rate (0.25 to 0.08 g/gh) and xylitol formation
(0.12 to 0.05 g/gh) but increased the yield of xylitol
(0.48 to 0.63 g/g).
To determine the specific oxygen uptake rate at which
C. boidinii NRRL Y-17213 begins to produce xylitol, the
yeast was cultivated continuously
under oxygen-limited

on xylitol formation

in yeasts with o-xylose

y,/s yx,,
(g/g)

(0)

(h)

31.8
90.2
82.2
79.3
100
100
90.2
19.3
80.2
98.1
100
n.r.
n.r.
48.3
n.r.
n.r.
49.0
44.2
58.9
66.4
76.5
100
100
100
75

15.8
56.3
36.3
23.7
11.3
18.3
24.3
4.7
21.4
6.4
39.0
n.r.
n.r.
12.6
n.r.
n.r.
3.3
1.37
0.50
0.10
0.31
22.0
32.5
30.4
36.0

0.38
0.48
0.34
0.23
0.28
0.46
0.67
0.37
0.41
0.10
0.60
0.50
0.66
0.87
0.70
0.60
0.34
0.31
0.08
0.02
0.04
0.61
0.65
0.44
0.54

41.8
52.8
37.4
25.3
31.0
50.5
73.6
40.9
45.3
11.1
66.3
54.9
71.4
95.6
76.9
65.9
37.0
34.1
8.8
2.2
4.4
67.0
71.4
48.4
59.5

9.2
14.2
13.2
10.1
4.4
1.7
0.3
n.r.h
n.r.
n.r.
n.r.
n.r.
n.r.
0.8
nr.
n.r.
0
n.r.
n.r.
n.r.
n.r.
n.d.c
nd.
n.d.
9.0

11
10
7
6
75
75
75
72
72
72
72
n.r.
n.r.
20
n.r.
n.r.
20

15
24
100
100

7.5
7.5
13.5
11.0

1.00
0.13
0.27
0.22

109.9
14.3
29.7
24.2

0.65
0.31
10.5d
6.5d

30
33
189
69

reached

after

The medium also contains glucose, 15 g/l.

n.r., Not reported.
n.d., Not detected.
These are the maximum ethanol concentrations

(l?h)

(2)

(2)

Agitation
(rev/min)

as a substrate
AR
(vvm)

150

OTR
(mmol/n

kt.a
(I/h)

20

200

200
300
400
300

0.064
0.059
0.066
0.069
117
123
147
32

165 and 75 h

2000
.I

1500
/

2000
.

1500
.

.,

..

2000
.

2000
.
.

1400
..
.

1600
I.
..

1000

100

6000
.

4000
.I

2000
.,

1600
.

250

5.3
10.6
41
10.6
1
2.2
3.2
0.4
1.0
3.2
10.1
26.6
70.0
102.8
4.8
16.8
35.4
112.8

0.46
.

0.15
0.60
1.50
2.00
0.08
0.30
0.90

200

250

Reference
63

10
14
24
30

1
1
0.08
0.90

91

95

95

85

76

80
62

163
253
4.8
35.4

76

VOL. 86, 1998

conditions
(97). Xylitol
secretion
was triggered
at
0.91 mmol OJgh. Xylitol was not produced at specific
oxygen uptake rates above this value. Upon a shift to
lower specific oxygen uptake rates, as expected, xylitol
production
rates and yield increased more rapidly than
those of ethanol.
Since xylitol formation
in yeasts is most sensitive to
substrate concentration
and aeration rate, it would be
best if both parameters were simultaneously
taken into
consideration
when optimizing
xylitol production.
By
varying the initial o-xylose concentration
between 60
and 12Og/l and the oxygen transfer
coefficient, kra,
between 0.24 to 1.88 l/min, Roseiro et al. (80) optimized
xylitol production
in D. hansenii DTIA-77. Applying an
experimental
design with 2 factors, they obtained xylitol
yield of 0.54g/g when the yeast was cultivated in 9Og/l
o-xylose and with kLa, of 1.88 I/min. Horitsu et al. (67)
studied the influence of culture conditions on xylitol formation by C. tropicalis IF0 0618 and optimized the volumetric xylitol production
rate using Box-Wilson method.
The initial D-xylose concentration
(120 to 180 g/l), yeast
extract concentration
(12 to 18g/Z) and kLa (236 to 381
l/h) were chosen as independent
factors in a 23-factorial
experimental
design. A maximum xylitol productivity
of
2.67 g/Ih was obtained when the initial o-xylose concentration was 172 g/I, the yeast extract concentration
was
21 g/l and the kLa was 452 l/h.
The general conclusion from all investigations
regarding oxygen influence on D-xylose metabolism in xylitolproducing
yeasts is that oxygen supply rate is a key
parameter
which determines
whether D-xylose will be
fermented or respired. It is very important, therefore, for
an effective process, to determine the oxygen flux that
will enable balanced
utilization
of carbon both for
growth and fermentation.
FERMENTATION OF HEMICELLULOSIC
HYDROLYSATES
The ultimate goal of all investigations
regarding Dxylose fermentation
is to acquire sufficient knowledge for
establishing
fermentation
processes using the pentose
fraction of the lignocellulosic
hydrolysates.
Although the hydrolysis can be performed enzymatically, most fermentation
studies have focused on hydrolysates derived from acid hydrolysis. Due to its heterogeneous structure and relatively low degree of polymerization, hemicellulose
is much easier to hydrolyze than the
crystalline cellulosic components
of biomass. In many
cases, even a simple steam treatment without the aid of
acid catalysis
has been found
to be effective (1).
However, for the decomposition
of hemicellulose,
mild
hydrolysis is the most suitable. Its advantages are that it
prevents the formation
of some degradation
products,
enhances the susceptibility
of cellulose to subsequent
enzymatic or acid hydrolysis,
reduces the requirement
for expensive corrosion-proof
equipment, and avoids the
environmental
problems
incurred
through
the use of
strong chemical treatments (1).
A critical feature of hydrolysates prepared using acid
catalysis
is the presence
of inhibitors
of microbial
metabolism
(11) which makes them substantially
more
difficult for microbial utilization than the corresponding
mixtures of pure sugars (3). During the acid hydrolysis
of the lignocellulosics,
different types of sugars (D-glucose, D-galactose,
o-mannose,
D-xylose, L-arabinose),

MICROBIAL

CONVERSION

OF D-XYLOSE

TO XYLITOL

and degradation products (furfural, 5-hydroxymethyl

furfural, acetic acid, syringic acid, p-hydroxybenzoic
acid,
vanillin etc.) are formed. Knowledge regarding inhibitors
and how to minimize their effects is of the utmost importance to achieve efficient fermentation
processes (98, 99).
One of the most common inhibitors,
particularly
in
ethanol fermentations,
is acetic acid, the degree of toxicity of which is pH-dependent
(57). However, regard to
xylitol production
in xylitol-producing
processes,
no
negative influence of acetic acid has been clearly identified. The concentration
of acetic acid decreased continuously until its complete depletion,
when D. hansenii
NRRL Y-1426 (87) and C. guilliermondii
FTI 20037
(100) were grown on hemicellulosic hydrolysates.
The data on xylitol production
from hemicellulosic
hydrolysates with yeasts are summarized in Table 6. C.
guilliermondii
FTI 20037 grown on rice straw hydrolysate exhibited the highest production
rate of 0.56g/Th,
whereas D. hansenii NRRL Y-7426 grown on chips of
Eucalyptus glob&us had in fact the highest yield, 0.73
g/g.
The presence of inhibitory
substances in hydrolysates
very often imposes the necessity of purification
of the
hydrolysates prior to their utilization and/or adaptation
of the microorganisms
to the sugar which will be used.
Thus, in the process of xylitol production by C. guilliermondii FTI 20037 from sugar cane bagasse hemicellulosic hydrolysate, the hydrolysate was treated in seven different ways (101). The best results were obtained following
overtitration
of the hydrolysate with Ca(OH)* and subsequent use of HzS04 (Table 6).
A single hydrolysis stage in Eucaliptus globulus led to
the formation of about 17 g/l o-xylose with a low concentration of inhibitors (87). However, the relatively low
substrate concentration
limited both the productivity and
yield of the subsequent fermentation
step. Therefore, the
D-xylose content was raised by vacuum evaporation.
In
the comparatively
high range of xylose concentrations
studied (57-78 g/l), a single charcoal treatment was unable to reduce the amount of inhibitors to a satisfactory
level and convert the hydrolysate to a suitable fermentation medium.
Because of this, the combined
strategy
involving
both charcoal
adsorption
of concentrated
hydrolysates and a high initial cell concentration
of up
to 80 g/l was applied.
When the yeasts C. guilliermondii
FTI 20037 (100,
101) and C. mogii ATCC 18364 (102) were grown on
hemicellulose
hydrolysates
for xylitol production,
a
sequential pattern of sugar consumption was observed. In
addition,
as a result of carbon source limitation,
both
yeasts started to consume the xylitol produced when Dxylose was almost exhausted.
Besides the yeasts presented in Table 6, there are other
xylose-fermenting
yeasts such as P. stipitis NRRL Y7124 (104) and C. shehatae NRRL Y-12858 (105) which
produced xylitol when cultivated
on wheat straw and
corn cob hemicellulosic
hydrolysate,
respectively.
In
these cases, however, xylitol was only a by-product
of
ethanol fermentation.
PROCESS STRATEGIES
Most of the papers published
thus far on xylitol
production
by yeasts refer to batch culture methods
(flasks or lab batch reactors) as the simplest and hence
the most used culture methods (20, 42, 61-64, 77, 80,

sugar
cane
bagasse

rice
straw

wheat
straw

C. guilliermondii

C. guilliermondii

C. mogii

Eucaliptus
globulus

Debaryomyces
hansertii
loOC, 11 h,
solid/liquid
ratio 818

3.5% HzSOJ,

100C

2-3% H$O,,

15 %,HSO
121C,30 gin

KS%,
per g
rice straw,
145C, 20 min
solid/liquid
ratio l/IO

0.07 g cont.

35 mM HzS04,
19OC, 5 min.
steam
explosion,
solid/liquid
ratio l/6

B Initial concentrations.
b Substrate consumed refers to D-xylose only.
c n.r., Not reported.
J Values inside brackets are amounts present initially.

NRRL Y-1426

sugar
cane
bagasse

18364

Candida sp. 11-2

ATCC

FTI 20037

FTI 20037

Substrate

Yeast

Hydrolysis
conditions

6.

vacuum
concentration,
CaC03, pH 6.5,
charcoal
treatment

73
78
57

46

cation-exchange
resins, CaO,
CaCOI, pH 4.5-6

58
43

pH 4.5-6

28

45

activated
charcoal, CaO,
CaCOJ, pH 4.5-6

CaCO,,

KOH, pH 10,
H>SOd, pH 6.5
vacuum
concentration
at 7OC,
NaOH, pH 10,
H?SOJ, pH 5.3
63

2
2
4

n.r.

n.r.

n.r.

14

16

18

68

KOH,

pH 6.5

19

65

CaO, pH 10,
H$Od, pH 6.5

15

D-Glucose

Released

4.2
4.2
4.5

n.r.

n.r.

n.r.

6.5

n.r.

n.r.

n.r.

n.r.

n.r.c

L-Arabinose

compoundsa

hydrolysates

5.3
6.1
4.5

n.r.

n.r.

n.r.

n.r.

8.5

n.r.

n.r.

n.r.

n.r.

n.r.

Acetic acid

(g/l)

by yeasts from hemicellulose

19

61

D-Xylose

production

68

pH 6.5

Xylitol

Ca(OH)2, pH 10,
HzSOa, pH 6.5

Ca(OH)2,

Hydrolysate
treatment

TABLE

6 (0.8)d
41 (2)d
39 (3)d

10.1

10.5

2.6

27

16

24

12.7
68.4
92.8

83.9

94.8

5.1

70

86

56

98

95

20

30

($)

(zO

0.088
0.500
1.060

0.195

0.205

0.053

0.130

0.560

0.128

0.192

0.240

(gyh)

0.57
0.73
0.68

0.26

0.26

0.88

0.31

0.69

0.48

0.36

0.48

($;)

0.20
0.25
0.12

n.r.

n.r.

n.r.

0.71

0.14

n.r.

n.r.

n.r.

n.r.

$7:;

60
78
34

51

51

49

46

48

125

125

125

125

125

(A)

87

103

102

100

101

Reference

5
2

%
z

kc
$

VOL. 86, 1998

82, 100, 101). Batch cultures are characterized

by high
initial substrate concentrations
and high product concentrations at the end of the process and relatively low productivity.
Although continuous
culture techniques often provide
better productivities
and yields for many microorganisms, the production
rates decrease with increasing dilution rates. High productivities
can be achieved only by
using low dilution
rates, that is high residence time,
which is very difficult to achieve in practice. Therefore,
some continuous
processes should be replaced by fedbatch ones (101). However, when studying yeast physiology, the most accurate data can be obtained from continuous cultures (67, 85, 97).
In fed-batch processes, the substrate concentration
can
be maintained
at a suitable level throughout
the entire
course of fermentation,
that is, a level sufficient to
induce xylitol formation but not to inhibit yeast growth.
In addition, these processes generally operate with high
initial cell density which normally leads to an increase in
volumetric productivity.
The yeast C. boidinii NRRL Y17213 gave much better results when cultivated
in a
fed-batch fermentor compared to other ways of cultivation.
The highest xylitol yield was 75% of the theoretical
yield, compared
to 53% in the batch culture.
The
productivity
of 0.46 g/Ih was twice as high as the highest
obtained under batch conditions (90).
Another way to improve the process parameters is the
use of immobilized
cells. To convert
D-xylose into
xylitol, cells of C. pelliculosa and Methanobacterium
sp.
HU were either separately immobilized
or co-immobilized in an agar gel, a calcium alginate gel, a h--carrageenen gel and other supports. The highest conversion rate
was observed when benzene-treated
cells were co-immobilized in the photo-crosslinkable
resin prepolymers
ENT
2000 and 4000 (107). Almost
100% of the n-xylose
(4.5 g/l) was converted into xylitol after 33 h of incubation when the volume ratio of immobilized
methanogen
to immobilized
C. pelliculosa was 1 : 2. In the co-immobilized cell system, the degree of conversion and the
conversion
rate of D-xylose were higher than those in
the separately immobilized cell system. To allow continuous xylitol production,
immobilized cells were packed in
a column reactor. Co-immobilized
cells were stable for
about 2 weeks with approximately
35% conversion.
De Silva and Afschar (78) immobilized
the cells of C.
tropicalis DSM 7524 on a porous glass and used them in
a fluidized bed reactor. The authors intended to reuse
the immobilized
cells several times by repeating
the
batch fermentation
with substrate shift. However, the
yeast degenerated after completion of the first cultivation
and addition of fresh medium. Under continuous
conditions, the immobilized
cells of C. tropicalis DSM 7524
converted D-xylose into xylitol with a high productivity
of 1.35 g/lb.
In another study, the aim of which was not xylitol
production,
xylitol was the only fermentation
product
observed. Lohmeier-Vogel
et al. (108) studied the glucose and D-xylose metabolism in agarose-immobilized
C.
tropicalis ATCC 32113 by nucleic magnetic resonance.
NMR studies showed that neither glucose nor xylose
metabolism
was enhanced by use of an immobilization
process.
Attempts
to improve
the rate of D-xylose
metabolism
by increasing
the oxygen delivery to the
entrapped cells were not successful.

MICROBIAL

CONVERSION

OF D-XYLOSE

TO XYLITOL

11

FUTUREPROSPECTS
Instead of the classical approach to the improvement
and optimization
of xylitol productivity
and yield by
changing the fermentation
variables, metabolic engineering offers opportunities
to change the genetic properties
of the microorganisms
themselves.
In the quest for a
microorganism
which will efficiently convert o-xylose to
xylitol, a strain of S. cerevisiae was genetically modified.
This recombinant
S. cerevisiae harbors the gene coding
for xylose reductase from P. stipitis CBS 6054 (109).
The yeast achieved a high xylitol yield approaching
the
theoretically
expected yield, since it did not possess any
significant
xylitol dehydrogenase
activity. The recombinant yeast required a cosubstrate for the generation of
reduction equivalents used in the reduction of xylose and
for maintenance
and growth. When glucose was used as
a cosubstrate
under anaerobic
conditions,
with supply
rates of 1 and 0.1 g/lb, the specific rate of xylitol formation was higher at the higher glucose supply rate, that is
0.78 compared with 0.39 mmol/gh (110).
In another study (ill), the formation of xylitol by the
same transformant
was investigated
by comparing
the
efficiency of different
cosubstrates
(glucose,
ethanol,
acetate, glycerol), oxygenation levels and different ratios
of substrate
and cosubstrate.
With both glucose and
ethanol, the conversion
yields were close to 1 g xylitol
per g of consumed
D-xylose. Decreased aeration
increased the xylitol yield and decreased the productivity.
When the xylose : cosubstrate ratio increased, the xylitol
yield based on consumed cosubstrate also increased.
In both of these studies (109, ill), the initial D-xylose
concentration
did not exceed 2Og/l. However, the Dxylose concentration
of industrial hemicellulose hydrolysates is usually higher since they are concentrated,
resulting in D-xylose concentrations
between 30 and 350 g/l
(112). Therefore, Meinander
et al. (112) applied a fedbatch process for xylitol production
by S. cerevisiae expressing XYLI gene with a total D-xylose concentration
corresponding
to that of industrial hemicellulose hydrolysate. When the total D-xylose concentration
was 95 g/l,
93% of the D-xylose was converted
to xylitol. The
average volumetric
productivity
was 1 g/Th, and the
specific productivity
0.04 g/gh. The xylitol ethanol yield
was about 1.1 g/g.
Continuous
xylitol production
with two different immobilized recombinant
strains of S. cerevisiae (H475 and
S641), expressing low and high xylose reductase activities, was investigated in a lab-scale packed bed reactor.
The cells were immobilized
by gel entrapment
using Ca
alginate as the support. The effect of hydraulic residence
time, substrate/cosubstrate
ratio, recycling ratio, and aeration rate were studied (113). The overall xylitol yield
was higher for the low xylose/glucose
ratio than for the
high ratio, 0.6Og/g
and 0.42 g/g, respectively.
The
highest xylitol concentration
of 15 g/l was reached at
hydraulic residence time of 8.5 h. The 20-fold higher Dxylose reductase activity of the strain with higher xylose
reductase activity did not result in a proportional
increase in xylitol concentration
compared with the strain
with lower xylose reductase activity. Under anaerobic
conditions, with a recycling ratio of 10, the highest volumetric productivities
of 3.44 and 5.80 g//h were obtained
with the strain with lower xylose reductase activity at
a residence time of 1.3 h and with the strain with higher
xylose reductase activity at a residence time of 2.6 h, re-

12

WINKELHAUSEN

spectively. These are the highest productivities

reported
to date.
Despite the very high yields and productivities
that
have been obtained with genetically modified strains of
S. cerevisiae, it still remains to be shown whether these
microorganisms
can remain sufficiently stable over a relatively long period of time and endure the operational
condition prevailing during the production of xylitol.
NOMENCLATURE
: aeration,
volume of air per volume of medium
per minute, vvm
: ethanol concentration,
g/f
C,
: xylitol concentration,
g/Z
CX
D
: dilution rate, l/h
: oxygen transfer coefficient, l/h
kra
OTR : oxygen transfer rate, mmol/lh
: volumetric xylitol production rate, g/lb
C$
: initial substrate (D-XylOSe) concentration,
g//
: D-xylose consumed, ,?i
SC
t
: time, h
v,
: culture volume, ml
V
: vessel volume, ml
Ycm/s : cell mass yield coefficient, g cell mass/g D-xylose
consumed
YX/S . xylitol yield coefficient, g xylitol/g D-xylose consumed
Yx/r f. percentage of the xylitol yield from the theoretical value, ,?d; theoretical value is considered as
0.91 g xylitol/g D-xylose consumed
: specific growth rate, l/h
I
AR

ACKNOWLEDGMENT
We acknowledge
istry of Science
(DAAD).

J. FERMENT. BIOENG.,

AND KUZMANOVA

the financial support

of the Macedonian
Minand Deutscher
Akademischer
Austauschdienst

Chem. Sot., 58, 651-657 (1993).

of pentoses to ethanol by yeasts and
11. Schneider, H.: Conversion
fungi. CRC. Crit. Rev. Biotechnol.,
9, I-40 (1989).
S. L.: Fermentation
of pentose sugars to ethanol
12. Rosenberg,
and
other
neutral
products
by microorganisms.
Enzyme
Microbial.
Technol.,
2, 185-189 (1980).
13. Gong, C. S., Chen, L. F., Flickinger, C., Chiang, L.-C., and
Tsao, G. T.: Conversion
of hemicellulose
carbohydrate.
Adv.
Biochem. Eng. Biotechnol.,
20, 93-118 (1981).
of xylose to ethanol under aerobic
14. Jeffries, T. W.: Conversion
conditions
by Cundida tropicalis. Biotechnol.
Lett., 3. 213-216
(1981).
15. Schneider, H., Wang, P. Y ., Chan, Y. K., and Maleszka. R.:
Conversion
of o-xylose
into ethanol
by yeast Pachysolen
tannophilus. Biotechnol. Lett., 3, 89-92 (1981).
16. Slininger, P. J., Bothast, R. J., van Cauwenherge,
J. E.. and
Kurtzman, C. P.: Conversion
of o-xylose
to e<hanol by the
yeast Puchysolen tannophilus. Biotechnol.
Bioene..
24. 371_I

384 (1982).
17

18

19

20.

21.
22.

23.
24.

REFERENCES
of hemicelluloses.
1. Magee, R. J. and Kosaric, N.: Bioconversion
Adv. Biochem. Eng. Biotechnol.,
32, 61-93 (1985).
Elsevi2. Singh, A. and Mishra, P.: Microbial pentose utilization.
er Science, Amsterdam-Tokyo
(1995).
of xylose by bacteria,
yeasts and
3. Jeffries, T. W.: Utilization
fungi. Adv. Biochem. Eng. Biotechnol.,
27, l-32 (1983).
of xylose and
4. Jeffries, T. W.: Effects of nitrate on fermentation
glucose by Pichia sfipitis. Biotechnology,
1, 503-506 (1983).
5. Dellweg, H., Rizzi, M., Methner, H., and Debus, D.: Xylose
fermentation
by yeasts 3. Comparison
of Pachysolen tannophi/us and Pichia stipitis. Biotechnol. Lett., 6, 395-400 (1984).
6. du Preez, J. C. and Prior, B.A.: A quantitative
screening of
some xylose fermenting
yeast isolates.
Biotechnol.
Lett., 7,
241-246 (1985).
7. du Preez, J. C., Bosch, M., and Prior, B. A.: Xylose fermentation by Candida shehatae and Pichia stipitis: effects of pH,
temnerature
and substrate
concentration.
Enzyme
Microb.
Technol.,
8, 360-364 (1986).
P. J.. Bothast.
R. J.. Ladish, M. R., and Okos.
8. Slininaer.
Comparative
evaluation
of ethanol
production
by
M.R.:
xylose fermenting
yeasts presented
high xylose concentrations.
Biotechnol.
Lett., 7, 431-436 (1985).
9. Slininger,
P. J., Bothast,
R. J., Ladish, M. R., and Okos,
M. R.: Optimum
pH and temperature
conditions
for xylose
fermentation
by Pichia stipitis. Biotechnol.
Bioeng., 35, 727731 (1990).
E., Georgievska,
I., and Kuzmanova.
S.: Batch
10. Vandeska,
fermentation
of xylose to ethanol by Candida shehatae. J. Serb.

25.

26.

27.

28.

29.
30.
31.

32.
33.

Wang, Y., Jonson, B. F., and Schneider, H.: Fermentation

of
o-xylose by yeasts using isomerase
in the medium to convert
o-xylose to o-xylulose.
Biotechnol.
Lett., 3, 273-278 (1980).
Gong, C. S.. Chen, L. F., Flickinger, C., Chiang, L.-C., and
Tsao, G. T.: Production
of ethanol from D-xylose by using Dxylose isomerase
and yeasts. Appl. Environ.
Microbial.,
41,
430-436 (1981).
Gong, C. S., Chen, L. F., and Tsao, G. T.: Quantitative
production
of xylitol from o-xylose by a high-xylitol
producing
yeast mutant
Candidu tropicalis HXP2. Biotechnol.
Lett., 3,
130-135 (1981).
Barbosa, M. F. S.. de Medeiros, M. B., de Mancilha, I. M.,
Schneider, H., and Lee, H.: Screening of yeasts for production
of xylitol from o-xylose and some factors which affect xylitol
yield in Candidu guilliermondii. .I. Ind. Microbial.,
3, 241-251
(1988).
Bar, A.: Caries prevention
with xylitol. A review of the scientific evidence. Wld. Rev. Ntr. Diet., 55, 183-209 (1988).
Isokangas,
P., Alanen, P., Tiekso, J., and Makinen, K. K.:
Xylitol chewing
gum in caries prevention:
a field study in
children. J. Am. Dent. Assoc., 117, 315-320 (1988).
Pepper, T. and Olinger, P. M.: Xylitol in sugar-free
confections. Food Technol.,
10, 98-106 (1988).
Makinen, K. K.: Latest dental studies on xylitol and mechanism of action of xylitol in caries limitation,
p. 331-362.
In
Greenby, T. H. (ed.), Progress in sweeteners, 2nd. edn. Elsevier
Applied Science, London,
N. Y. (1992).
Waler, S. M., Assev, S., and Rella, G.: Xylitol 5-p formation
the dental plaque after 12 weeks exposure to the xylitol/sorbitol containing
chewing gum. Stand. J. Dent. Res., 100, 319321 (1992).
Aminoff, C., Vanninen, E., and Doty, T. E.: The occurrence,
manufacture
and properties
of xylitol, p. 1-9. In Counsell,
J. N. (ed.),
Xylitol.
Applied
Science
Publishers,
London
(1978).
Bassler, K.-H.: Biochemistry
of xylitol, p. 35-41. In Counsell,
J. N. (ed.),
Xylitol.
Applied
Science
Publishers,
London
(1978).
Bar, A.: Xylitol, p. 349-379.
In Nabors,
L. 0. and Gelardi.
R. C. (ed.), Alternative
sweetener,
2nd. edn. Marcel Dekker,
N.Y.. Basel, Hong-Kong
(1991).
Emodi, A.: Xylitol: its properties
and food applications.
Food
Technol.,
January, 28-32 (1978).
Rella, G., Schele, A. A., and Assev, S.: Plaque formation
and
plaque inhibition.
Dtsch. Zahnarztl.,
Z 42, 39-41 (1987).
Kandelman,
D. and Gagnon,
G.: Clinical
results after 12
months from a study of the incidence and progression
of dental caries in relation to consumption
of chewing-gum
containing xylitol in school preventive
programs.
J. Dent. Res., 60,
1407-1411 (1987).
production.
Pepper, T.: The use of xylitol in confectionery
Confectionery
Prod., 3, 253-256 (1989).
Nikolaev, D. I., Chernikova,
L. P., Glazman, B. A., Kostyuk,
M. S., and Chivyaga.
A. A.: New ionI,. N., Rutskaya,

VOL. 86, 1998

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

41.

48.
49.

50.
51.

52.

53.

54.

55.

exchange
resins in xylitol
production.
Gidroliz.
Lesokhim.
Prom-St.,
2, 16-18 (1983).
Kind, V. B., Vyglazov,
V.V.,
and Kholkin,
Y. J.: Use of
cationic surfactants
for clarification
of pentose hydrolyzates
in
xylitol production.
Gidroliz.
Lesokhim.
Prom-St.,
3, 11-12
(1987).
Nigam, P. and Singh, D.: Processes
for fermentative
production of xylitol: a sugar substitute.
Process Biochem.,
30, 117124 (1995).
Barnett, J. A., Payne, R. W.. and Yarrow, D.: Yeasts: characteristics
and identification,
2nd edn. Cambridge
University
Press, New York (1990).
Yoshitake, J., Obiwa, H., and Shimamura, M.: Production
of
polyalcohol
by Corynebacterium
sp. 1. Production
of pentitol
from aldopentose.
Agric. Biol. Chem., 35, 905-911 (1971).
Yoshitake,
J., Ishizaki,
H., Shimamura,
M., and Imai, T.:
Xylitol production
by an Enterobacfer
species. Agric. Biol.
Chem., 37, 2261-2267 (1973).
M., Ishizaki,
H., and Irie, Y.:
Yoshitake,
J., Shimamura,
Xylitol production
by Enferobacter
liquefaciens.
Agric. Biol.
Chem., 40, 1493-1503 (1976).
Izumori, K. and Tuzaki, K.: Production
of xylitol from Dxylulose by Mycobacterium
smegmatis.
J. Ferment.
Technol.,
66, 33-36 (1988).
by Petromyces
albertensis
Dahiya, J. S.: Xylitol production
grown on medium containing
D-xylose. Can. J. Microbial.,
37,
14-18 (1991).
Vandeska,
E., Amartey,
S., Kuzmanova,
S., and Jeffries,
T. W.: Effects of environmental
conditions
on production
of
xylitol by Candida boidinii. W. J. Microbial.
Biotechnol.,
11,
213-218 (1995).
Sirisansaneeyakul,
S., Staniszewski,
M., and Rizzi, M.: Screening of yeasts for production
of xylitol from D-xylose.
J.
Ferment. Bioeng., 6, 564-570 (1995).
Dahiya, D. S., Kumari, P., and Dahiya, J. S.: Xylitol production from sugar cane bagasse by fermentation,
p. 292-303.
In
Gehlawat,
J. K. (ed.), Modernization
of Indian Sugar Industry.
Arnold Publishers,
New Delhi (1990).
Alexander,
M. A., Chapman,
T. W., and JetTries, T. W.:
Xylose metabolism
by Candida shehatae in continuous
culture.
Appl. Microbial.
Biotechnol.,
28, 478-486 (1988).
Kilian, S. G. and van Uden, N.: Transport
of xylose and glucose in the xylose fermenting
yeast Pichia stipitis.
Appl.
Microbial.
Biotechnol.,
27, 545-548 (1988).
Ligthelm, M. E., Prior, B.A., du Preez, J. C., and Brand& V.:
An investigation
of ~-(l-l~C) xylose metabolism
in Pichia stipitis under aerobic and anaerobic
conditions.
Appl. Microbial.
Biotechnol.,
28, 293-296 (1988).
Skoog,
K. and Hahn-Hlgerdal,
B.: Xylose
fermentation.
Enzyme Microb. Technol.,
10, 66-80 (1988).
Lucas,
C. and van Uden,
N.: Transport
of hemicellulose
monomers
in the xylose-fermenting
yeast Candida shehatae.
Appl. Microbial.
Biotechnol.,
23, 491-495 (1986).
Chiang, C. and Knight, S. G.: Metabolism
of D-xylose by
moulds. Nature, 188, 79-81 (1960).
Chakravorty,
M., Veiga, L. A., Bacila, M., and Horecker,
B. L.: Pentose metabolism
in Candida II. The diphosphopyridine nucleotide-specific
polyol dehydrogenase
of Candida utilis.
J. Biol. Chem., 237, 1014-1020 (1962).
Hiifer, M., Betz, A., and Kotyk, A.: Metabolism
of the obligatory aerobic
yeast Rhodotorufa
gracilis IV. Induction
of an
enzyme necessary
for D-xylose catabolism.
Biochim.
Biophys.
Acta, 252, l-12 (1971).
Vongsuvanlert,
V. and Tani, Y.: Purification and characterization of xylose isomerase of a methanol yeast, Candidu boidinii,
which is involved in sorbitol production
from glucose. Agric.
Biol. Chem., 52, 1817-1824 (1988).
Smiley, K. L. and Bolen, P. L.: Demonstration
of D-xylose
reductase and D-xylitol dehydrogenase
in Pachysofen
tannophi/us. Biotechnol.
Lett., 4, 607-610 (1982).
Lathe, A. H. and Jetfries, T. W.: Levels of the enzymes of the
pentose phosphate
pathway
in Pachysolen
tannophilus
Y-2460
and selected mutants.
Enzyme Microb.
Technol.,
8, 353-359

MICROBIAL

CONVERSION

OF D-XYLOSE

TO XYLITOL

13

(1986).
56. Slininger, P. J., Bolen, P. L., and Kurtzman, C. P.: Pachysolen tannophilus:
properties
and process
consideration
for
ethanol production
from D-xylose. Enzyme Microb. Technol.,
9, 5-15 (1987).
51. Prior, B. A., Killian, S. G., and du Preez, J. C.: Fermentation
of D-xylose by the yeasts Candida shehatae and Pichia stipitis.
Proc. Biochem.,
24, 21-32 (1989).
58. Hahn-Hiigerdal,
B., Jeppsson,
H., Skoog,
K., and Prior,
B. A.: Biochemistry
and physiology
of xylose fermentation
by
yeasts. Enzyme. Microbial.
Technol.,
16, 933-943 (1994).
59. Bruinenberg, P.M., de Bot, P. H. M., van Dijken, J. P., and
Scheffers, W. A.: NADH-linked
aldose reductase:
the key to
anaerobic
alcoholic
fermentation
of xylose by yeasts. Appl.
Microbial.
Biotechnol.,
19, 256-260 (1984).
60. van Dijken, J. P. and Scheffers, W. A.: Redox balances in the
metabolism
of sugars by yeasts. FEMS Microbiology
Reviews,
32, 199-224 (1986).
L., Delgenes, J. P., and Navarro,
61. Nolleau, V., Preziosi-Belloy,
J. M.: Xylitol production
from xylose by two yeast strains:
sugar tolerance.
Current Microbial.,
27, 191-197 (1993).
J. C., S&Machado,
P., Duarte-Reis,
62. Girio, F. M., Roseiro,
A. R., and Amaral-Collaqo,
M. T.: Effect of oxygen transfer
rate on levels of key enzymes
of xylose
metabolism
in
Debaryomyces
hansenii. Enzyme Microb. Technol.,
16. 10741078 (1994).
E., Kuzmanova,
S., and Jeffries,
T. W.: Xylitol
63. Vandeska,
formation
and key enzyme activities in Cundidu boidinii under
different oxygen transfer
rates. J. Ferment.
Bioeng., 80, 513516 (1995).
V. and Tani, Y.: Xylitol
production
by a
64. Vongsuvanlert,
methanol
yeast Candida boidinii (Kloeckera sp.) no. 2201. J.
Ferment. Bioeng., 67, 35-39 (1989).
of aldose reductase
65. Sugai, J. K. and Delgenes, J. P.: Induction
activity in Cundida guilliermondii
by pentose sugars. J. Ind.
Microbial.,
1, 46-51 (1995).
M. A., Yang, V. W., and Jeffries, T. W.: Levels of
66. Alexander,
pentose phosphate
pathways
enzymes from Candida shehatae
in continuous
culture. Appl. Microbial.
Biotechnol.,
29, 282288 (1988).
H., Yahashi,
Y., Takamizawa,
K., Kawai, K.,
67. Horitsu,
Suzuki, T., and Watanabe, N.: Production of xylitol from Dxylose by Candida tropicalis: optimization
of production
rate.
Biotechnol.
Bioeng., 40, 1085-109 (1992).
G., Kubicek-Pranz,
E. M., RGhr, M., and
68. Ditzelmiiller,
Kubicek, C. P.: NADPH-specific
and NADH-specific
xylose
reductase
is catalyzed by two separate enzymes in Pachysolen
tannophilus.
Appl. Microbial.
Biotechnol..
22. 297-299 (1985).
C., Frank.
J., van Dijken, j. P., and Scheffeis,
69. Verdiyn,
W. A.: Multiple forms of xylose reductase in Pachysolen
tannophihts
CBS 4044. FEMS Microbial.
Lett.,
30, 313-317
(1985).
70. du Preez, J. C., van Driessel, B., and Prior, B. A.: Effect of
aerobiosis
on fermentation
and key enzymes
levels during
growth of Pichia stipitis, Candida shehatae and Candida tenius
on D-xylose. Arch. Microbial.,
152, 143-147 (1989).
F. M., Peito,
M. A.,
and Amaral-Collaqo,
M. T.:
71. Girio,
Enzymatic
and physiological
study of o-xylose
metabolism
by Cundida shehatae. Appl. Microbial.
Biotechnol.,
32, 199-204
(1989).
of xylose
72. Lee, H., Sopher, C. R., and Yau, K. Y. F.: Induction
reductase
and xylitol dehydrogenase
activities on mixed sugars
in Candida guilfiermondii.
J. Chem. Technol. Biotechnol.,
66,
375-379 (1996).
73. Sugai, J. K. and Delgenes, J. P.: Catabolite
repression of induction of aldose
reductase
activity
and utilization
of mixed
hemicellulosic
sugars
in Candida
guilliermondii.
Current
Microbial.,
31, 239-244 (1995).
74. Bichio, P. A., Runnals, P. L., Cunningham,
J. D., and Lee,
H.: Induction
of xylose reductase and xylitol dehydrogenase
activities in Pachysolen
tannophilus
and Pichia stipitis on mixed
sugars. Appl. Environ. Microbial.,
54, 50-54 (1988).
S. I., Suzuki, T., Kawai, K., Horitsu, H., and
75. Yokoyama,

14

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

WINKELHAUSEN

J. FERMENT. BIOENG.,

AND KUZMANOVA

Takamizawa,
K.: Purification,
characterization
and structure
analysis of NADP-dependent
D-xylose from Candida tropiculis. J. Ferment. Bioeng., 79, 217-223 (1995).
Furlan, S. A., Boutlloud,
and de Castro, H. F.: Influence of
oxygen on ethanol and xylitol production
by xylose fermenting
yeasts. Process Biochem.,
29, 657-662 (1994).
Meyrial, V., Delgenes, J. P., Moletta, R., and Navarro,
J. M.:
Xylitol production
from D-xylose by Candidu guiUiermondii:
fermentation
behaviour.
Biotechnol.
Lett., 11, 281-286 (1991).
de Silva, S. S. and Afschar, A. S.: Microbial production
of
xylitol
from o-xylose
using Candida
tropicalis.
Bioprocess
Eng., 11, 129-134 (1994).
Silva, S. S., Quesada-Chanto,
A., and Vitolo, M.: Upstream
parameters
affecting the cell growth and xylitol production
by
Candidu guilliermondii
FTI 20037. Z. Naturforsch.,
52c, 359363 (1997).
Roseiro, J. C., Peito, M. A., Girio, F. M., and Amaral-Collaqo,
M. T.: The effects of oxygen transfer
coefficient and substrate
concentration
on the xylose fermentation
by Debaryomyces
hansenii. Arch. Microbial.,
156, 484-490 (1991).
Lu, J., Tsai, L. B., Gong, C. S., and Tsao, G. T.: Effect of
nitrogen sources on xylitol production
from D-xylose by Candida sp. L-102. Biotechnol.
Lett., 17, 167-170 (1995).
Lee, H., Atkin, A. L., Barbosa, M. F. S., Dorscheid,
D.A.,
and Schneider, H.: Effect of biotin limitation
on the conversion of xylose to ethanol and xylitol by Puchysolen tannophilus
and Candida guilliermondii.
Enzyme
Microb.
Technol.,
10,
81-84 (1988).
Mahler, G. P. and Guebel, D. V.: Influence of magnesium
concentration
on growth, ethanol and xylitol production
by Pichia
stipitis NRRL Y-7124. Biotechnol.
Lett., 16, 407-412 (1994).
Cao, N-J., Tang, R., Gong, C. S., and Chen, L. F.: The effect
of cell density on the production
of xylitol from D-xylose by
yeast. Appl. Biochem. Biotechnol.,
45-46, 515-519 (1994).
Furlan, S. A., Boutlloud, P., Strehaiano, P., and Riba, J. P.:
Study of xylitol formation from xylose under oxygen limiting
conditions. Biotechnol. Lett., 13, 203-206 (1991).
Roberto, I. C., Sate, S., and de Mancilha, I. M.: Effect of inoculum level on xylitol production from rice straw hemicellulose hydrolysate by Cundida guilliermondii.
J. Ind. Microbial.,
16, 348-350 (1996).
Parajo, J. C., Dominguez,
H., and Dominguez, J. M.: Production of xylitol from concentrated
wood hydrolysates
by Debaryomyces
hansenii: effect of the initial cell concentration.
Biotechnol.
Lett., 18, 593-598 (1996).
Pfeifer, M. J., Silva, S. S., Felipe, M. G. A., Roberto, 1. C.,
and Mancilha, I. M.: Effect of culture conditions
on xylitol
production
by Candida
guiiliermondii
FTI 20037.
Appl.
Biochem. Biotechnol.,
57-58, 423-430 (1996).
Delgenes, J. P., Moletta, R., and Navarro, J. M.: Fermentation of D-xylose,
D-glucose,
L-arabinose
mixture
by Pichia
stipitis Y-7124: sugar tolerance.
Appl. Microbial.
Biotechnol.,
29, 155-161 (1988).
Vandeska,
E., Amartey,
S., Kuzmanova,
S., and Jeffries,
T. W.: Fed-batch
culture
for xylitol production
by Candida
boidinii. Process Biochem.,
31, 265-270 (1996).
Silva, S. S., Roberto, I. C., Felipe, M. G. A., and Mancilha.
I. M.: Batch fermentation
of xylose for xylitol production
in
stirred tank bioreactor.
Process Biochem.,
31, 549-553 (1996).
Alexander,
M. A., Chapman,
T. W., and Jeffries, T. W.: Continuous
culture
responses
of Candidu shehatae to shifts in
temperature
and aeration:
implications
for ethanol inhibition.
Appl. Environ. Microbial.,
55, 2152-2154 (1989).
Kuriyama, H. and Kobayashi, M.: Effect of oxygen supply on
yeast growth and metabolism
in continuous
fermentation.
.I.
Ferment. Bioeng., 75, 364-367 (1993).
Laplace, J. M., Delgenes, J. P., Moletta, R., and Navarro,
J. M.: Alcoholic fermentation of glucose and xylose by Pichia
stipitis, Candida shehatae, Saccharomyces
cerevisiae and Zynlomonas mobilis:
oxygen requirement
as a key factor.
Appl.
Microbial.
Biotechnol.,
36, 158-162 (1991).
Nolleau,
V., Preziosi-Belloy,
L., and Navarro,
J. M.: The
reduction
of xylose to xylitol by Candida guilliermondii
and

96.

97.

Cundida parapsilosis:
incidence
of oxygen
and pH. Biotechnol. Lett.. 17. 417-422 (1995).
Lohmeier-Vogel,
E. M., Hahn-Higerdal,
B., and Vogel, H. J.:
Phosphorus-31
and carbon-13
nuclear
magnetic
resonance
studies of glucose and xylose metabolism
in Cundidu tropicalis
cell suspension.
Appl. Environ.
Microbial..
61. 1414-1419
(1995).
Winkelhausen,
E., Pittman,
P., Kuzmanova,
S., and Jeffries,
T. W.: Xylitol formation
by Candida boidinii in oxygen limited
chemostat
culture. Biotechnol.
Lett., 18, 753-758-- (19961.
du Preez, J. C.: Process parameters
and environmental
factors
affecting
o-xylose
fermentation
by yeasts. Enzyme
Microb.
Technol.,
16, 944-956 (1994).
Olsson, L. and Hahn-Hiigerdal,
B.: Fermentation
of lignocellulosic hydrolysates
for ethanol production.
Enzyme Microb.
Technol.,
18, 312-331 (1996).
Roberto,
I. C., Mancilha,
I. M., de Souza, C. A., Felipe,
M. G. A., Sate, S., and de Castro, H. F.: Evaluation
of rice
straw hemicellulose
hydrolysate
in the production
of xylitol by
Candida
guilliermondii.
Biotechnol.
Lett..
16. 1211-1216
(1994).
Roberto,
1. C., Felipe, M. G. A., Lacis. L. S., Silva, S. S.,
and de Mancilha,
I. M.: Utilization
of sugar cane bagasse
hemicellulosic
hydrolyzate
by Candida
guilliermondii
for
xylitol production.
Bioresource
Technol.,
36, 271-275 (1991).
Rizzi,
M., Sirisansaneeyakul,
and Reuss,
M.: Microbial
production
of xylitol from wheat straw hydrolysates,
p. 541544. In DECHEMA
Biotechnology
Conference
5. VCH
Verlagsgemeinschaft,
Weinheim (1992).
Dominguez,
J. M., Gong, C. S., and Tsao, G. T.: Pretreatment of sugar cane bagasse
hemicellulose
hydrolysate
for
xylitol production
by yeast. Appl. Biochem. Biotechnol.,
575X. 49-56 (1996).
Delgenes,
J. P:, Moletta,
R., and Navarro,
J. M.: Acid
hydrolyzate
of wheat straw and process
consideration
for
ethanol
fermentation
by Pichia
stipitis
Y-7124.
Process
Biochem. Inter.. 25. 132-135 (19901.
Kuzmanova, S.; Vahdeska, E:, and Georgievska,
I.: Selection
of yeasts
for
fermentation
of corn
cob hemicellulosic
hydrolyzates.
Mikrobiologija,
30, 19-30 (1993).
Schiigerl, K.: Bioreaction
engineering,
vol. 1. John Willy &
Sons., N. Y. (1987).
Nishio, N.. Sugawa, K., Hayase, N., and Nagai, S.: Conversion of D-xylose into xylitol by immobilized
cells of Candidu
peliculosa
and Methanobacterium
sp. HU.
.I. Ferment.
Bioeng., 67, 356-360 (1989).
Lohmeier-Vogel,
E. M., Hahn-Htigerdal,
B., and Vogel,
Phosphorus-31
and
carbon-l 3 nuclear
magnetic
H. J.:
resonance
studies
of glucose
and xylose
metabolism
in
agarose-immobilized
Candidu
tropicalis.
Appl.
Environ.
Microbial.,
61, 1420-1425 (1995).
Hallborn, J., Walfridsoon,
M., Airaksinen,
U., Ojamo, H.,
Hahn-Hlgerdal,
B., Penttilal, M., and KerBnen. S.: Xvlitol
cerevisiae.
production
by
recombinant
Saccharomyces
Biotechnology,
9, 1090-1095 (1991).
The&up,
G; N. and Hahn-Htigerdal,
B.: Xylitol formation
and reduction
equivalent
generation
during anaerobic
conversion with glucose as cosubstrate
in recombinant
Saccharomyces cerevisiae expressing
the xyli gene. Appl.
Environ.
Microbial.,
61, 2043-2045 (1995).
Hallborn, J., Gorwa, M.-F., Meinander, N., B., Penttilii, M.,
Keriinen, S., and Hahn-Higerdal,
B.: The influence of cosubstrate and aeration on xylitol formation
by recombinant
Saccharomyces
cerevisiae
expressing
the XYLI
gene.
Appl.
Microbial.
Biotechnol.,
42, 326-333 (1994).
Meinander, N., Hahn-Hlgerdal,
B., Linko, M., Linko, P.,
and Ojamo, H.: Fed-batch
xylitol production
with recombinant
XYL-l-expressing
Saccharomyces
cerevisiae
using
ethanol
as a cosubstrate.
Appl. Microbial.
Biotechnol.,
42,
334-339 (1994).
Roca, E., Meinander, N., and Hahn-Higerdal,
B.: Xylitol
recombinant
Saccharomyces
production
by immobilized
cerevisiae in a continuous
packed-bed
bioreactor.
Biotechnol.
Bioeng., 51, 317-326 (1996).
\----I

98.

99.

100.

101.

102.

103.

104.

105.

106.
107.

108.

109.

110.

111.

112.

113.