JOURNALOF FERMENTATIONAND BIOENGINEERING

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

REVIEW
Microbial Conversion of D-Xylose to Xylitol
ELEONORA WINKELHAUSEN* AND SLOBODANKA KUZMANOVA
Faculty of Technology and Metallurgy, Rudjer Boskovic 16, 91000 Skopje, Macedonia

Received 25 February 199WAccepted 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 ac-
cumulation 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 opened the possibility not only for ethanol, but also for
processes for the production of alcoholic beverages for polyol production, primarily, xylitol. The first significant
thousands of years, it is only recently that broader appli- papers on microbial production of xylitol refer to screen-
cations of this material have been envisaged. Thus, ing for suitable microorganisms (19, 20), but the real
biotechnologists are now developing efficient systems for scientific interest began in the last few years, when
the production of liquid fuels, pharmaceuticals, foods xylitol, due to its unique properties as an alternative
and chemical feedstocks from “waste” organic materials sweetener, began to be more frequently used (21-25),
(1). and when awareness of protection of the environment
Lignocellulosics are organic materials which are abun- grew.
dant and renewable. Their major components, cellulose, This review attempts to examine the present literature
hemicellulose and lignin, vary with plant species. The on microbial production of xylitol regarding, first,
pentose fraction, composed of D-xylose (usually not xylitol properties, and then, microorganisms involved in
less than 95%) and L-arabinose is much higher in hard- this process, as well as xylose metabolism, the effect of
woods (19 to 33%) than in softwoods (10 to 12%). High the process variables on xylitol production and some
amounts of pentosans, up to 40%, are also present in aspects of the process strategies.
agricultural residues (2). The fact that xylan is more easily
hydrolyzed than cellulose offers the technical possibility
THE OCCURRENCE, PROPERTIES AND
of D-xylose and xylitol production (3).
APPLICATION OF XYLITOL
Recently, considerable efforts have been focused on
the microbial production of xylitol from o-xylose. The Xylitol, a pentahydroxy sugar alcohol, occurs widely
microbial production of xylitol has been always closely in nature, in many fruits, and vegetables, among which
connected with that of ethanol and for years it was consi- the yellow plum has the highest content, almost 1% on a
dered only as a by-product in ethanol fermentation dry solid basis (26). Xylitol is also a normal metabolic
processes from n-xylose (4-10). Obviously, this stems intermediate in mammalian carbohydrate metabolism
from the fact that ethanol production from n-xylose with an endogenous production and further utilization of
was the process that first attracted the attention of resear- some 5-15 g daily in the average adult human. Slow ad-
chers. However, for a considerable period of time, views sorption and entry into metabolic pathways independent-
on the ability of yeasts to produce ethanol from aldopen- ly of insulin and without rapid fluctuation of blood
toses were contradictory (11). The matter was resolved in glucose levels support the use of xylitol as a diabetic
the beginning of the eighties when a number of different sweetener (27, 28). In this respect, it is comparable to
laboratories independently demonstrated the direct con- sorbitol and other slowly absorbed carbohydrates. Exten-
version of D-xylose to ethanol (12-16) as well as ethanol sive clinical and laboratory experience has shown that
production from D-xylulose (17, 18). This discovery the only side effect of xylitol is the possibility of an
unpleasant but harmless osmotic diarrhea after relatively
* Corresponding author. large initial oral doses (20-30g) in unadapted subjects
2 WINKELHAUSEN AND KUZMANOVA J. FERMENT. BIOENG.,

(27, 29). ways for its production. One of the most attractive
Primary interest in xylitol therefore centers on its procedures, today, is microbial production. To a certain
properties and potential uses as an alternative sweetener. extent, another motivation was the high pollution levels
In contrast to other alternative noncaloric sweeteners, and waste-treatment concerns.
such as saccharine, xylitol has many properties similar to
those of sucrose. It dissolves readily in water, it is as
XYLITOL-PRODUCING MICROORGANISMS
sweet as sucrose and hence approximately twice as sweet
as sorbitol and nearly three times as sweet as mannitol. Microorganisms more readily assimilate and ferment
Its caloric content is the same as that of sucrose, glucose than xylose. However, although in small num-
17 kJ/kg. In addition, it gives a pleasant cool and fresh bers, there are bacterias, yeasts and fungi capable of
sensation due to its high negative heat of solution (23, assimilating and fermenting xylose to xylitol, ethanol and
26). other compounds (36).
Perhaps the most significant characteristic of xylitol, A few bacteria such as Cor_vnebacteriurn sp. (37),
however, is the fact that it is not utilized by the acid- Enterobacter liquefaciens (38, 39), and Mycobacterium
producing, cariogenic bacteria of the human oral cavity smegmatis (40) have been reported to produce xylitol.
and therefore inhibits demineralization of tooth enamel For the first two bacteria, D-xylose was mainly used as a
(21, 30). A number of long-term field trials in different substrate while for the last one, the substrate was D-xylu-
countries and hence in different nutritional, social and lose or D-xylose isomerized by commercially immobi-
economic environments demonstrated that the consump- lized D-xylose isomerase. However, due to the relatively
tion of even relatively small amounts of xylitol can sig- small quantities of xylitol formed, xylitol-producing
nificantly reduce the formation of new dental caries (21, bacteria do not presently attract researchers’ interest.
22, 24, 31). In the light of the scientific evidence current- Regarding the fungi, there is only one significant report
ly available, it may be regarded as the best of all alterna- regarding Petromyces albertensis (41). This fungus
tive sweeteners with respect to caries prevention (24, 28). accumulated 39.8 g/l of xylitol when cultured for 10 d on
Xylitol finds a broad application either as the sole 100 g/l D-XylOSe. Nevertheless, after initial studies regard-
sweetener or in conjunction with other sweeteners in the ing the effects of environmental conditions on xylitol
preparation of a wide variety of full- and reduced-energy production by this fungus, no further reports were pub-
sugarless confectionery products suitable for infants and lished.
diabetics (32). Bakery products, spices and relishes, In general, among microorganisms, the yeasts are con-
jams, jellies, marmalades and desserts represent other sidered to be the best xylitol producers and therefore,
potential applications of xylitol in food products (29). It the majority of publications deal with them. Some of
can also be used in pharmaceuticals and oral hygiene the yeasts screened for xylitol production are presented
products (23). 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
CONVENTIONAL PRODUCTION OF XYLITOL
before any optimization of the cultural conditions, (ii)
Although xylitol occurs in many fruits and vegetables, different media and culture conditions have been applied
it would be very uneconomical to extract it from such
sources due to their high cost and relatively low xylitol TABLE 1. Screening of yeasts for xylitol production from o-xylose
content, On a large-scale, xylitol is currently produced
by chemical reduction of xylose derived mainly from Xylitol Ethanol
Yeast Reference
wood hydrolysates. The conventional process of xylitol k/0 k/A
production includes four main steps: acid hydrolysis of Candida boidinii NRRL Y-17213 2.9 3.9 42
plant material, purification of the hydrolysate to either a C. guilliermondii FTI-20037 16.0 n.d.a 20
pure xylose solution or a pure crystalline xylose, hydro- C. intermedia RJ-248 5.7 3.6 20
C. mogii ATCC 18364 31.0 n.r.h 43
genation of the xylose to xylitol, and crystallization of
C. parapsilosis ATCC 34078 20.0 n.r. 43
the xylitol (26). C. pseudotropicalis IZ-43 1 4.3 3.0 20
The critical step in this process is the purification of C. tropicalis 2.1 n.r. 19
the xylose from the acid hydrolysate. Ion exchange chro- C. tropicalis HXP 2 4.8 n.r. 19
matography is employed to remove salts and charged C. tropicalis 1004 17.0 n.d. 20
degradation products, and activated carbon is used to C. tropicalis ATCC 7349 20.0 n.r. 43
remove color (33, 34). Ion exchange chromatography, C. tropicalis ATCC 20240 5.5 n.r. 43
however, does not remove or separate the various C. utilis ATCC 22023 1.8 n.r. 43
hemicellulosic sugars. This is a problem because acid C. utilis C-40 3.0 n.r. 44
Debarvomvces hansenii C-98 M-21 0.8 n.d. 42
hydrolysis releases appreciable amounts of D-galactose,
Hansenula anomala IZ-1420 6.1 n.d. 20
D-mannose and L-arabinose in addition to D-xylose. Kluvveromvces franilis FTI-20066 4.6 3.5 20
The exact proportions of the various sugars depend on K. karxiar& Ii-1821 6.1 0.6 20
the nature of the feedstock and the manner in which it is Pichia (Hansenuia) anomala NRRL
hydrolyzed. These contaminating sugars can complicate Y-366 2.0 n.d. 42
crystallization and purification of xylose (Leleu, J-B, Pachysolen tannophilus NRRL
Duflot, P., and Caboche, J-J., U.S. patent 5,096,820, Y-2460 2.2 5.2 20
1992). The yield of xylitol from the xylan fraction is Saccharomyces SC- 13 0.7 n.r. 44
about SO-60:6 or S-1596 of the raw material employed Saccharom.vces SC-37 2.3 n.r. 44
Schizosaccharomyces pombe 16919 0.2 n.r. 19
(35).
The existing drawbacks of conventional xylitol produc- d n.d., Nor detected.
tion methods motivated researchers to seek alternative h n.r., Not reported.
VOL. 86, 1998 MICROBIAL CONVERSION OF D-XYLOSE TO XYLITOL 3

and (iii) the yeasts, for which no data regarding their tions to this generalization.
screening for xylitol production were available, were not Once inside the yeast cell, D-xylose is reduced to
included in the survey. Due to this, it is difficult here to xylitol by either NADH- or NADPH-dependent xylose
really compare the production capacities of different reductase (aldose reductase EC 1.1.1.21). Xylitol is
yeasts. However it is obvious that the best xylitol pro- either secreted from the cell or oxidized to xylulose by
ducers belong to the genus Can&da. NAD- or NADP-dependent xylitol dehydrogenase (EC
Screening of more than 30 yeast strains, Ojamo (Ojamo, 1.1.1.9). The first two reactions are considered to be
H., Ph.D. thesis University of Technology, Espoo, limiting in D-xylose fermentation. The phosphorylation
Finland, 1994) also revealed that the yeasts from the of xylulose to xylulose 5-phosphate is catalyzed by
genus Candida, such as C. guilliermondii VTT-C-71006, xylulokinase (EC 2.7.1.17) (54, 55).
C. tropicalis VTT-C-78086 and C. tropicalis VTT-C- The conversion of pentoses to xylulose-5-phosphate is
78087, were the best xylitol producers. Unfortunately, a prerequisite for its utilization by the central catabolic
the results of the screening were not precisely quantified pathways (56). Xylulose-5-phosphate can subsequently
and hence could not be included in Table 1. enter the pentose phosphate pathway (Fig. 1). This path-
way consists of an oxidative phase that converts hexose
phosphates to pentose phosphates providing NADPH
CONVERSION OF D-XYLOSE TO XYLITOL BY
needed in biosynthetic pathways and a non-oxidative
YEASTS
phase in which the pentose phosphates are converted
Xylose transport The first step in the metabolism into hexose and triose phosphates (3). The pentose phos-
of D-xylose is the transport of the sugar across the cell phate pathway also yields ribose-5-phosphate used for
membrane. A number of investigations have shown that the synthesis of nucleic acids and histidine and of
under aerobic and oxygen limited conditions the rate erythrose-4-phosphate necessary for the synthesis of
of transport can limit the utilization of D-xylose in P. aromatic amino acids.
stipitis CBS 7126 and C. shehatae ATCC 22984 (45-47). Glyceraldehyde-3-phosphate and fructose-6-phosphate
Under anaerobic conditions, D-xylose metabolism has not are products of the non-oxidative phase of the pentose
appeared to be transport-limited either in C. shehatae phosphate pathway. Both of them can be converted to
(45) or in P. stipitis (47). Instead, the limitation was in pyruvate in the Embden-Meyerhof-Parnas pathway.
the two initial steps of D-xylose metabolism, reduction Pyruvate can either be decarboxylated and reduced to
of D-xylose and subsequent oxidation of xylitol. ethanol or can enter the tricarboxylic acid cycle. The con-
Studying the oxygen requirement for D-xylose uptake, version of xylulose-5-phosphate into glyceraldehyde-3-
Skoog and Hahn-HPgerdal (48) found that oxygen phosphate and acetyl phosphate by xylulose-5-phosphate
induces or activates a transport system in P. stipitis CBS phosphoketolase presents an alternative route for the
6054. utilization of xylulose-5-phosphate (57). A detailed study
Starvation induced both proton symport and a facili- of the biochemistry and physiology of the yeasts metab-
tated xylose uptake diffusion system in C. shehatae CBS olizing xylose was recently published (58).
2779 (49). In non-starved cells, D-xylose was transported Xylose metabolism in yeasts yields a variety of car-
by a facilitated diffusion system. Killian and van Uden bon-containing products which include carbon dioxide,
(46) reported on a low-affinity and a high-affinity xylose ethanol, acetic acid and polysaccharides. Product yields
proton symport operating simultaneously in both starved are dependent upon the regulation of carbon flow
and non-starved cells of P. stipitis IGC 4374. From the through available metabolic routes (56). D-Xylose conver-
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. xylitol
A typical xylitol-producing yeast, the transport system k NAD(P)
of which has been studied, is Candida mogii ATCC 18 +- NADLP)h
0 -xyl$o$
364 (43). The o-xylose uptake rate in the yeast followed
Michaelis-Menten kinetics which suggested a carrier- +-ADP
mediated 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 QyceraidehydL-3 -phosphate ethanol
(50) found that the filamentous fungus Penicillium 1
chrysogenum carried out the conversion of D-xylose to
D-xylulose through a two-step reduction and oxidation. Embden-Meyerhoff-Parnas Pathway
It possessed enzymes that differed from xylose isomerase
in bacteria. This finding, as well as some further investi- 1 CO2 NADH NAD

gations (51) led to the conclusion that the two-step con- pyruvate - acetaldehyde u ethanol
I
version of D-xylose to D-xylulose is specific for yeasts i
and fungi, whereas in bacteria the same conversion is Tricarboxylic Acid Cycle
catalyzed by xylose isomerase in a single step. The detec-
tion of xylose isomerase in the yeasts Rhodotorula (52) FIG. 1. Schematic representation of D-xylose metabolism in
and C. boidinii no. 2201 (53) is one of the few excep- yeasts.
4 WINKELHAUSEN AND KUZMANOVA J. FERMENT.BIOENG.,

sion to xylitol in yeasts can not be separated from the explains the higher n-xylose reductase activity in the for-
conversion of D-xylose to these products. The process of mer yeast (61). Some differences reported for the cofactor
xylitol formation can not be stopped after the first step, requirements of n-xylose reductase from C. guiliermon-
when n-xylose is converted to xylitol. Cell growth de- dii NRC 5578 (Table 2) may stem from differences in
pends on some of the above metabolic products and it is culture conditions.
also necessary that the cofactors be regenerated through The varying ratio of NADH- to NADPH-linked D-
different steps in the metabolic pathway. Therefore, for xylose reductase activity with aeration conditions was first
obtaining good yields of xylitol, the amount of xylose found in Pachysolen tannophilus. This finding suggested
being converted to xylitol and the amount of xylitol that there was probably more than one form of n-xylose
which is available for further metabolism, have to be reductase in the yeast, which was indeed confirmed (68,
well balanced. 69). The same variations were observed in the yeasts C.
Coenzyme specificity The first two enzymes, D- parapsilosis ATCC 28474 (61) and C. boidinii NRRL Y-
xylose reductase and xylitol dehydrogenase are key 17213 (63).
enzymes in xylitol production by yeasts. They both require It is noteworthy that C. boidinii under oxygen limita-
pyridine nucleotide cofactors exhibiting different cofac- tion, in contrast to all other D-xylose-fermenting and
tor specificity in different yeasts. Under anaerobic or xylitol-producing yeasts, (62, 65-67, 70) exhibits a NADH/
oxygen-limited conditions, the difference in the cofactor NADPH ratio higher than 1 (Table 2). Referring to
requirements of these enzymes causes a redox imbalance this, Vongsuvanlert and Tani (53) speculated that in
which influences xylitol accumulation in yeasts (59). C. boidinii xylitol could be formed by two metabolic
Xylitol formation is favored under oxygen-limited condi- pathways. The first possibility was that n-xylose is direct-
tions, because of the NADH accumulation and subse- ly reduced to xylitol and the second one is that n-xylose
quent inhibition of NAD-linked xylitol dehydrogenase. is initially isomerized by D-xylose isomerase to D-xylu-
This phenomenon, known as the Custer effect, results lose that is subsequently reduced to xylitol. In both
from the incapability of the yeasts to compensate for reductions, NADPH was also active as a reductant but
excess NADH as they have no transhydrogenase activity with less efficiency.
(60). Several studies showed the existence of a correla- The general characteristic of most xylose-fermenting
tion between key enzyme activities, and the level of oxy- yeasts is that their xylitol dehydrogenase uses pre-
genation and xylitol production in yeasts (61-63). dominantly NAD and very rarely the NADP cofactor
However, due to the various presentation of aeration, (55, 63, 64, 66, 71, 72). Regarding the ratio of NADH-
when displaying the enzymes activities of D-xylose reduc- linked D-xylose reductase and NAD-linked xylitol de-
tase and xylitol dehydrogenase in Table 2, the aeration is hydrogenase activities, it has been noticed that oxygen
not given. may lower it and consequently minimize xylitol accumu-
In most yeast cell-free extracts, n-xylose reductase has lation in n-xylose-fermenting yeasts (56). This was also
a higher or even absolute preference for NADPH (Table observed in C. boidinii NRRL Y-17213 (63). The
2). The xylose reductases of C. parapsilosis ATCC NADH/NAD ratio decreased %-fold with increasing
28474, C. shehatae ATCC 22984 and CBS 5813, a oxygen availability in the investigated range of oxygen
mutant strain of P. tannophilus U+U-27, and P. stipitis transfer rates from 10 to 30mmol/lh.
CBS 5773 use both NADPH and NADH, the use of the In addition to aeration, the activities of n-xylose
former being considerably higher. The lower NADH/ reductase and xylitol dehydrogenase are also very sensi-
NADPH ratio in C. guilliermondii NRC 5578 (0.1) com- tive to the substrate. Since the real substrates for xylitol
pared to that in C. parapsilosis ATCC 28474 (0.4) producing microorganisms are lignocellulosic hydroly-

TABLE 2. Enzyme activities of o-xylose reductase and xylitol dehydrogenase in various yeasts with n-xylose as a substrate3

Soecific activitv (U/e orotein)b

Microorganism o-Xylose reductase Xylitol dehydrogenase Reference
NADPH NADH NAD NADP
Candida boidinii (Kloeckera sp.) no. 2201 0.055 0.288 0.272 0.096 64
C. boidinii NRRL Y-17213 0.019 0.112 0.060 0.003 63
C. guilliermondii NRC 5578 0.521 0.050 n.r.’ n.r. 61
C. guilliermondii NRC 5578 1.191 n.d.d n.r. n.r. 65
C. mogii ATCC 18364 0.160 0.060 0.220 n.r. 43
C. parapsilosis ATCC 28474 0.416 0.161 n.r. n.r. 61
C. shehatae ATCC 22984 0.333 0.100 0.240 n.r. 66
C. shehatae CBS 5813 0.480 0.210 n.r. 59
C. tropicalis IF0 06 18 10.640 1.720 2onko 0.120 67
C. utilis CBS 621 0.075 n.d. 0.280 n.d. 59
Debaryomyces hansenii DTIA-77 0.091 n.r. 0.047 n.r. 62
Pachysolen tannophilus CBS 4044 0.220 0.009 0.910 0.070 59
P. tannophilus NRRL Y-2460 0.033 0.008 0.049 n.d. 55
P. tannophilus U-U-27 (mutant) 0.173 0.118 0.160 n.d. 55
Pichia stipitis CBS 5773 0.600 0.310 0.720 0.075 59

a The activities were measured in crude extracts. When different levels of aeration were employed the highest activities were selected for this
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.
VOL. 86, 1998 MICROBIAL CONVERSION OF D-XYLOSE TO XYLITOL 5

TABLE 3. Relative specific activities of aldose reductase and xylitol dehydrogenase induced in the presence of various carbon sources
Relative specific activity (%)
Microorganism Substrate (% w/v) Aldose reductase xylitol dehydrogenase Reference
NADPH NAD
Cundidu guilliermondii NRC 5578 D-Xylose (2%) 100 n.r.= 65
D-Glucose (2%) 1 n.r.
L-Arabinose (2%) 52 n.r.
D-Galactose (2%) 7 n.r.
D-Mannose (2%) 3 n.r.
Glycerol 11 n.r.
Candida guilliermondii NRC 5578 D-Xylose (4%) 100 n.r. 73
D-Glucose (4%) 13 n.r.
L-Arabinose (4%) 52 n.r.
D-Xylose (4%) + D-glucose (4%) 59 n.r.
D-Xylose (4%) + D-glucose (1%) 80 n.r.
D-Xylose (4%) + L-arabinose (4%) 75 n.r.
L-Arabinose (4%) + D-glucose (4,%) 8 n.r.
Candida guilliermondii NRC 5578 D-Xylose (2,%) 100 100 72
D-Glucose (2%) 0 1
r.-Arabinose (2%) 92 94
D-Galactose (2%) 2 0
D-Mannose (2%) 11 0
D-Fructose (206) 8 54
D-Xylose (1%) + D-glucose (1%) 5 23
D-Xylose (1%) + L-arabinose (1%) 93 107
D-Xylose (1 ,O&)+ D-galactose (1,%) 94 107
D-Xylose (l%)+D-mannose (1%) 32 45
Glycerol (2%) 3 0
Pachysolen tannophilus NRRL Y-2460 D-Xylose (4%) 100 100 74
D-Glucose (4,‘d) 15 0
D-Xylose (4%) + D-glucose (4%) 34 7
D-Xylose (4%) + L-arabinose (4%) 110 80
D-Xylose (40&+D-galactose (4%) 107 180
D-Xylose (4%)+D-mannose (4?& 30 4
Glycerol (2%) 32 20
Pichia stipitis NRRL Y-7124 D-Xylose (4%) 100 100 74
D-Glucose (4%) 5 13
D-Xylose (4%)+D-glucose (4.W 19 17
D-Xylose (4%)+1.-arabinose (4%) 120 110
D-Xylose (4%)+Dgalactose (4%) 160 90
D-Xylose (4%)+D-mannose (4Pd) 54 38
Glycerol (2%) 7 0
a n.r., Not reported.

sates which often contain a variety of sugars (o-xylose, tors. For the oxidation of polyols, the cell-free extract
o-glucose, o-mannose, o-galactose, L-arabinose etc), it preferably oxidized xylitol and D-sorbitol using NAD as
is very important to investigate their influence on the an oxidant rather than NADP (64). The highest enzyme
induction of D-xylose reductase and xylitol dehydrogenase activity in C. guilliermondii NRC 5578 cells was ob-
(Table 3). served with L-arabinose as a substrate for the enzymatic
o-Xylose was found to be the best inducer of both reaction (65).
aldose reductase, strictly dependent on NADPH, and Yokoyama et al. (75) purified and characterized three
xylitol dehydrogenase from C. guilliermondii NRC 5578. NADPH-dependent o-xylose reductases from C. tropica-
It was closely followed by L-arabinose, indicating that lis IF0 0618 and tested their activities for several sub-
pentoses can be effective substrates for the induction of strates. The activity for DL-glyceraldehyde was the
enzyme activities (Table 3). The level of induction of al- highest, followed by L-arabinose and D-xylose. The D-
dose reductase activity depended on the initial concentra- xylose reductases were the least active for o-glucose.
tion of D-xylose. Low activity was found in o-glucose-,
D-mannose- and o-galactose-grown cells, suggesting that
PROCESS VARIABLES
the mechanism leading to the expression of this enzyme
is under catabolite repression control, which is typical All published data on xylitol production by yeasts
for o-glucose or other rapidly metabolizable carbon have demonstrated that xylitol accumulation is
sources (65). influenced by a number of experimental conditions.
Various sugars and polyols can be used as substrates Studying the effect of these conditions is of particular
for the . enzymatic reactions. The cell-free extract of C. interest as a prerequisite for higher xylitol yields and
bordmrr no. 2201 reduced o-xylulose, o-xylose, D-fruc- productivities.
tose and L-sorbose, preferably using NADH as a Nutrition Although various media have been used
reductant rather than NADPH, but exhibited very low to culture xylitol-producing yeasts, a few generalizations
affinity for the reduction of D-glucose for both cofac- can be made: (i) For some yeasts, yeast extract is an
6 WINKELHAUSEN AND KUZMANOVA _I. FERMENT.BIOENG.,

important nutrient for xylitol production. (ii) For other and therefore under such conditions the initial pH values
yeasts, sometimes also including yeasts from the first have to be higher than under controlled conditions. The
group, but reported by different researchers, yeast ex- optimum initial pH value for best xylitol yield in C.
tract has no significant effect on xylitol formation. These boidinii was 7 (42, 64), whereas under controlled condi-
yeasts prefer urea or urea and Casamino acids. (iii) For tions, a pH of 5.5 was better (63). Batch culture of C.
kinetic studies, synthetic media are used which provide parapsilosis ATCC 28474 (76) was performed at pH 6,
all the necessary minerals and vitamins. while for continuous culture, a pH of 4.5 was used (85).
The culture media for C. parapsilosis ATCC 28474, The yeasts are generally cultivated at pH values
(76) C. boidinii no. 2201, (64) C. guilliermondii NRC between 4 and 6. C. parapsilosis ATCC 28474 (61) and C.
5578 (77) and C. tropicalis IF0 0618 (67) contain yeast guifliermondii NRC 5578 (61, 77) were grown at pH 6,
extract in concentrations ranging from 10 to 2Og/l. C. mogii ATCC (43) and P. stipitis NRRL Y-7124 at
Yeast extract at a maximum concentration of 1 g/l was pH 5 and 5.5, respectively, while pH 4 was optimum for
sufficient for C. tropicalis DSM 7524. Concentrations C. tropicalis IF0 0618 (67). In contrast, da Silva and
higher than 15 g/l, blocked the conversion of p-xylose Afschar (78) reported that C. tropicalis DSM 7524 was
to xylitol (78). Increased concentrations of yeast extract not very sensitive to pH and attained a maximum xylitol
of 5 and log/l increased the biomass production of C. yield at pH 2.5. Increasing the pH from 2.5 to 4 led to
guilliermondii FTI 20037, but sharply decreased its an increase in xylitol productivity but a decrease in
xylitol productivity (79). Similarly, the addition of yeast xylitol yield.
extract and peptone to the defined medium for C. lnogii Inoculum On studying the effect of initial cell con-
ATCC 18364 enhanced cell growth markedly but had no centration of Candida sp. B-22 on xylitol production
significant effect on the yield and specific productivity of from n-xylose, Cao et al. (84) found that the rate of
xylitol (43). xylitol production was linear and the fermentation time
Xylitol formation in C. guilliermondii FTl 20037 (20, was dramatically reduced over an initial concentration
61), D. hansenii DTIA-77 (62, 80), Candida sp. (81), C. range of 3.8 to 26g/l. With an initial yeast cell concen-
parapsilosis ATCC 28474 (61) and C. boidinii NRRL Y- tration of 26g/l, 21Og/l xylitol was produced from
17213 (42) was highest with urea as a substrate. In most 26Og/l o-xylose. A high initial concentration was also
cases, the medium was supplemented with Casamino beneficial for xylitol production by C. boidinii NRRL Y-
acids. In some yeasts, special supplements improved 17213. With an initial D-xylose concentration of 5Og//,
xylitol production. Thus, on studying the effect of bio- the xylitol yield and specific productivity doubled when
tin, Lee et a/. (82) found that in high-biotin media, in P. the inoculum level increased from 1.3 to 5.1 g/l (42).
tannophilus NRRL Y-2460 ethanol production was The effect of inoculum size on the microbial produc-
favored over that of xylitol, while in C. guilliermondii FTI tion of xylitol from hemicellulose hydrolysates was also
20037 xylitol formation was favored. When the C. investigated (86, 87). A high initial cell density did not
boidinii no. 2201 medium was supplemented with l?,j have a positive effect when C. guilliermondii FTI 20037
(v/v) methanol, xylitol production increased 2.5-fold was grown on rice straw hemicellulose hydrolysate since
(64). This was explained by the oxidation of methanol, increasing the initial cell density from 0.67 g/l to 2.41 g/f
providing NADH to the medium. Mahler and Guebel decreased biomass formation, xylose utilization and
(83) found that at low Mg+2 levels, 1 mM, xylitol forma- xylitoi accumulation (86). On the contrary, D. hansenii
tion by P. stipitis NRRL Y-7124 was higher than that of NRRL Y-7426 grown on wood hydrolysate produced
ethanol. more xylitol at higher initial cell densities (87).
Temperature and pH The most suitable tempera- In addition to inoculum size, the culture age, which is
ture for xylitol production in yeasts was shown to be related to the metabolic activity of cells, was also stu-
30°C. Small temperature variations above this tempera- died. Varying the inoculum age of C. guilliermondii FTI
ture, do not significantly affect xylitol production in C. 20037 from 15 to 70 h demonstrated that 15-h-old cells
tropicalis DSM 7524. The xylitol yield was, for the most gave poor results, whereas 24-h-old and older cells had
part, temperature-independent when the yeast was cul- similar effects and influenced only the productivity of
tured in a temperature range between 30°C and 37°C xylitol but not its final concentration and yield (79, 88).
but above 37°C the xylitol yield decreased sharply (78). Cultivation of the inoculum using different carbon
Xylitol formation in C. guilliermondii FTI 20037 was sources (o-xylose, mixture of n-xylose and glucose in a
the same at 30 and 35”C, but decreased when the temper- 4 : 1 ratio and glucose) had only a minor influence on
ature increased to 40°C (20). The conversion of D-xylose the bioconversion of D-xylose (88).
to xylitol by Candida sp. B-22 was relatively constant Substrate n-Xylose concentration has been shown
over the temperature range of 35-40°C. At temperatures to be critical for yeast growth and fermentation. In the
of 45°C and higher, the conversion was sharply reduced absence of D-xylose, xylitol formation does not occur.
(84). This was probably due to loss of the activities of Together with aeration, p-xylose concentration affects
both NADPH and NADH-dependent xylose reductase, xylitol formation the most. D-Xylose is required for the
as the temperature increased (56). induction of xylose reductase and xylitol dehydrogenase
When investigating the effect of temperature on activities in yeasts (Table 3).
ethanol and xylitol production, du Preez et al. (7) found High n-xylose concentration induces xylitol formation
that at higher temperatures, production of xylitol is in yeasts. Increased xylose concentration favors xylitol
favored over that of ethanol. Xylitol production of C. production at the expense of ethanol production, result-
shehatae CSIR-Y492 increased &fold as the temperature ing in an increase in the xylitol/ethanol ratio, a decrease
increased from 22 to 36°C. P. stipitis CSIR-Y633 in the ethanol yield and without exception, an increase
produced xylitol at 36°C but no detectable amounts at in the xylitol yield (42, 48, 77, SO). As the initial D-
lower temperatures. xylose concentration increases, the specific growth rate
If uncontrolled, pH drops during the fermentation, decreases, demonstrating substrate inhibition, whereas
VOL. 86, 1998 MICROBIAL CONVERSION OF D-XYLOSE TO XYLITOL 7

TABLE 4. Influence of initial substrate concentration on the fermentation parameters for some xylitol-producing yeasts

D-Xylose
Microorganism Reference
(g/l) (l/;h)

Candida boidinii 50 64.0 17.1 0.53 58.4 0.35 n.r.a n.r. n.r. 48 64
(Kloeckera sp.) no. 2201 100 81.8 36.0 0.44 48.4 0.25 n.r. n.r. n.r. 144
150 51.0 17.0 0.22 24.2 0.01 n.r. n.r. 144
C. boidinii 50 11.6 4.8 0.12 13.2 0.05 6.4 0% n.r. 96 42
NRRL Y-17213 100 85.6 25.2 0.29 31.9 0.13 11.6 0.11 n.r. 132
150 74.7 53.1 0.47 51.7 0.16 15.1 0.07 n.r. 336
200 22.2 10.0 0.22 24.2 0.04 9.0 0.09 n.r. 192
C. guiliiertnondii 10 100 6.2 0.62 68.1 0.13 0.4 0.31 0.11 46 77
NRC 5578 50 98.3 30.9 0.63 69.1 0.19 0.9 0.09 0.11 165
150 100 110.3 0.74 81.3 0.46 3.0 0.04 0.03 238
300 100 221.0 0.74 81.3 0.54 6.0 0.02 0.01 406
C. guilliermondii 50 100 22.5 0.45 49.5 0.055 n.r. 0.036 0.050 409 61
NRC 5578 100 100 49 0.49 53.8 0.066 n.r. 0.014 0.030 742
200 100 116 0.58 63.7 0.099 n.r. 0.002 0.007 1172
300 100 207 0.69 75.8 0.164 n.r. 0.004 0.010 1269
C. mogii ATCC 18364 10.1 100 1.7 0.17 18.7 n.r. n.r. n.r. 0.005 n.r. 43
28.9 100 14.5 0.50 54.9 n.r. n.r. n.r. 0.004 n.r.

C. parapsilosis
ATCC 28474
53.3
50
100
100
100
100
37.3
29.5
61.0
0.70
0.59
0.61
16.9
64.8
67.0
n.r.
0.111
0.123
n.r.
n.r.
n.r.
o%o
0.018
0.003
0.026
0.020
n.r.
266
477
61

200 100 116.0 0.58 63.7 0.115 n.r. 0.016 0.020 1009
300 I00 93.0 0.31 34.1 0.050 n.r. 0.004 0.009 1860

a n.r., Not reported.

the overall xylitol productivity depends on the yeast a smaller amount of xylitol was produced (91). Higher
type. However, all yeasts need a relatively long time for amounts of glucose relative to D-xylose reduced the yield
the conversion of D-xylose to xylitol (Table 4). For most further (79).
yeasts, the initial n-xylose concentrations resulting in the When D-glucose, D-mannose or n-galactose was
highest yields are between 100 and 2OOg/l, with C. guil- present in the medium with D-xylose, C. guilliermondii
liermondii NRC 5578 being an exception for which NRC 5578 exhibited a sequential pattern of utilization
300 g/l is the most suitable concentration. Certain xylose with the hexoses being consumed before D-xylose (72).
concentrations inhibit xylitol formation, and such inhibi- In general, the assimilation of D-xylose began when
tory concentrations differ with yeast type (Table 4). some of the hexoses were still present in the medium,
Although for osmophilic yeasts it is intrinsic that a which indicated the existence of a threshold above which
high substrate concentration induces polyol formation, hexose repression occurred. The preferential utilization
according to Prior et ai. (57) the correlation between of D-galactose over D-xylose by C. guifliermondii NRC
xylitol accumulation and D-xylose concentration could 5578 was surprising since D-galactose did not repress
be a consequence of more severe oxygen-limited growth the induction of D-xylose reductase and xylitol de-
conditions as a result of the higher cell densities reached hydrogenase activities. When grown on a fructose-xylose
at higher substrate levels than an effect of the D-xylose mixture, the yeast utilized both sugars simultaneously at
concentration itself. similar rates.
Regarding the effect of hexoses on D-xylose utiliza- The utilization of various single sugars, other than D-
tion, several reports stated that D-galactose, D-cellobiose xylose, was studied in batch cultures of C. guilliermondii
and L-arabinose are not inhibitory to D-xylose assimila- NRC 5578. Meyrial et al. (77) found that D-glucose, D-
tion, while D-mannose and particularly D-glucose con- mannose and D-galactose were rapidly fermented with
siderably slow down the utilization of D-xylose (72-74, specific uptake rates being 2.2, 1.8 and 1.5 times higher
78). than for D-XylOSe, although the hexoses were utilized by
In mixtures of glucose and D-XylOSe, the yeasts first the strain only for growth and ethanol production; their
consume the glucose and only after the glucose is deplet- corresponding polyols were not detected. On studying
ed do they consume D-xylose (89, 90). When a mixture the same yeast, Lee et al. (72) found that D-glucose was
of glucose and xylose was used as a substrate for the the most rapidly utilized, followed by D-mannose, D-
fed-batch culture of C. boidinii NRRL Y-17213 with the xylose, D-galactose and D-fructose. With D-glucose and
former being l/10 of the concentration of the latter, glu- D-fructose, ethanol was the only fermentation product,
cose was consumed first, resulting in faster growth with whereas with D-mannose and D-galactose, mannitol and
a maximum specific growth rate of 0.067 l/h, compared galactitol were produced in addition to ethanol.
to 0.023 l/h in the process using xylose alone. In con- Aeration An experimental condition that is critical-
trast, xylitol accumulation, and consequently xylitol ly important for determining the extent to which xylitol
yield, were lower; 39.4g/l and 0.57 g/g, compared to accumulates in cultures is aeration. Under fully aerobic
46.5 g/l and 0.64 g/g, respectively (90). In the presence conditions, xylitol is not produced. Under anaerobic con-
of glucose (15 g/l), C. guilliermondii FTI 2003 converted ditions, all the yeasts tested failed to grow on D-xylose
D-XylOSe (65 g/l) to xylitol with lower yields relative to to any appreciable extent, reaching about one doubling
those obtained in a medium without glucose. It was at best (11). It was found that fermentation and growth
assumed that in the presence of glucose, there was a occur simultaneously only under oxygen limitation (92).
partial inhibition of xylose reductase and consequently Supply of oxygen to the culture is required for yeast
8 WINKELHAUSEN AND KUZMANOVA J. FERMENT.BIOENG.,

growth even under fermentation for the synthesis of senii DTIA-77 has the highest demand for oxygen com-
unsaturated fatty acids and ergosterol required for sugar pared to the other yeasts listed in Table 5.
transport through the membrane (93). Under aerobic conditions, as D-xylose is depleted,
As Laplace et al. (94) suggested, the dependence of some yeasts, such as D. hansenii DTIA-77 (80), C.
the fermentative behavior of the xylose-fermenting yeasts tropicalis ATCC 32113 (96) and C. boidinii NRRL Y-
on oxygen availability can be presented in three stages: 17213 (42) can reassimilate both xylitol and ethanol.
(i) Under anaerobic conditions or at very low oxygen When optimizing the xylitol production rate of C.
transfer rates, the electron transport system of the yeasts tropicalis IS0 0618 by employing the Box-Wilson
is unable to oxidize NADH completely. As a conse- method, Horitsu et al. (67) found that the interaction
quence, the intracellular NADH concentration increases between D-xylose concentration and aeration rate was
and this imbalance between NADH and NAD concentra- related to cell concentration. If the cell concentration was
tions leads to xylitol secretion. (ii) Increasing the oxygen low, the dissolved oxygen concentration was maintained
transfer rate permits the enhancement of xylose fermenta- at a high level, resulting in low xylitol production. This
tion. This is believed to be due to the role of oxygen as finding indicates that the most relevant parameter
a terminal electron acceptor, thus relieving the imbalance defining the oxygenation of a culture is the specific oxy-
of the two initial steps of anaerobic xylose metabolism. gen uptake rate. Despite this, not many of the xylitol-
This hypothesis is supported by the inverse relationship producing yeasts have been investigated with this in
between the degree of aeration and xylitol production mind.
observed for some yeasts. Under these circumstances, The influence of specific oxygen uptake at a constant
the main metabolic product is ethanol. (iii) When oxygen is D-xylose concentration of 35 g/l under pseudo-steady
supplied in excess, a deviation in the pyruvate flow from state conditions was studied in C. mogii ATCC 18364
the fermentative pathway to the tricarboxylic acid cycle (43). Decreasing the specific oxygen uptake rate in the
is observed resulting in increase in cell mass. range of 1.65 to 0.50mmoVgh decreased the specific
A survey of xylitol production by various yeasts under growth rate (0.040 to 0.003 I/h), the specific o-xylose
different oxygenation levels is presented in Table 5. It is uptake rate (0.25 to 0.08 g/gh) and xylitol formation
very difficult, however, to compare data from different (0.12 to 0.05 g/gh) but increased the yield of xylitol
studies because oxygenation is measured and reported (0.48 to 0.63 g/g).
differently. Yet, it is evident that yeasts producing xylitol To determine the specific oxygen uptake rate at which
require very small amounts of oxygen, which appear to C. boidinii NRRL Y-17213 begins to produce xylitol, the
be specific for each yeast strain. It seems that D. han- yeast was cultivated continuously under oxygen-limited

TABLE 5. Influence of aeration on xylitol formation in yeasts with o-xylose as a substrate

Microorganism cs SC G y,/s yx,, Agitation AR OTR kt.a

(g/l) (o/d) k/O (g/g) (“0’) (h) (l?h) (2) (2) (rev/min) (vvm) (mmol/n (I/h) Reference
Candida boidinii 130 31.8 15.8 0.38 41.8 9.2 11 150 10 63
NRRL Y-17213 130 90.2 56.3 0.48 52.8 14.2 10 14
130 82.2 36.3 0.34 37.4 13.2 7 4‘ 24
130 79.3 23.7 0.23 25.3 10.1 6 30
C. guilliermondii 40 100 11.3 0.28 31.0 4.4 75 200 20
“
FTI 20037 40 100 18.3 0.46 50.5 1.7 75
40 90.2 24.3 0.67 73.6 0.3 75
C. guilliermondii 65a 19.3 4.7 0.37 40.9 n.r.h 72 200 0.46 5.3 91
FTI 20037 65a 80.2 21.4 0.41 45.3 n.r. 72 300 .‘ 10.6
6ja 98.1 6.4 0.10 11.1 n.r. 72 400 ” 41
65 100 39.0 0.60 66.3 n.r. 72 300 ” 10.6
C. guilliermondii 300 n.r. n.r. 0.50 54.9 n.r. n.r. 2000 1500 1 95
.I /‘
NRC 5578 300 n.r. n.r. 0.66 71.4 n.r. n.r. 2.2
30 48.3 12.6 0.87 95.6 0.8 20 3.2
C. parapsilosis 100 n.r. n.r. 0.70 76.9 nr. n.r. 2000 1500 0.4 95
.‘ .
ATCC 28474 100 n.r. n.r. 0.60 65.9 n.r. n.r. 1.0
., ..
20 49.0 3.3 0.34 37.0 0 20 3.2
C. parapsilosis 10 44.2 1.37 0.31 34.1 n.r. 0.064 2000 1400 0.15 10.1 85
ATCC 28474 10 58.9 0.50 0.08 8.8 n.r. 0.059 .‘ .. 0.60 26.6
10 66.4 0.10 0.02 2.2 n.r. 0.066 ” .’ 1.50 70.0
10 76.5 0.31 0.04 4.4 n.r. 0.069 “ ” 2.00 102.8
C. parapsilosis 50 100 22.0 0.61 67.0 n.d.c 117 2000 1600 250 0.08 4.8 76
.‘ I.
ATCC 28474 50 100 32.5 0.65 71.4 nd. 123 0.30 16.8
.‘ ..
50 100 30.4 0.44 48.4 n.d. 147 0.90 35.4
Debaryomyces hansenii 90 75 36.0 0.54 59.5 9.0 32 1000 100 200 112.8 80
DTIA-77
Debaryomyees hansenii 50 15 7.5 1.00 109.9 0.65 30 6000 4000 1 163 62
.‘ .I
DTIA-77 50 24 7.5 0.13 14.3 0.31 33 1 253
Pachysolen tannophilus 50 100 13.5 0.27 29.7 10.5d 189 2000 1600 250 0.08 4.8 76
., .‘
ATCC 32691 50 100 11.0 0.22 24.2 6.5d 69 0.90 35.4

a The medium also contains glucose, 15 g/l.

b n.r., Not reported.
c n.d., Not detected.
* These are the maximum ethanol concentrations reached after 165 and 75 h
VOL. 86, 1998 MICROBIAL CONVERSION OF D-XYLOSE TO XYLITOL 9

conditions (97). Xylitol secretion was triggered at and degradation products (furfural, 5-hydroxymethyl fur-
0.91 mmol OJgh. Xylitol was not produced at specific fural, acetic acid, syringic acid, p-hydroxybenzoic acid,
oxygen uptake rates above this value. Upon a shift to vanillin etc.) are formed. Knowledge regarding inhibitors
lower specific oxygen uptake rates, as expected, xylitol and how to minimize their effects is of the utmost impor-
production rates and yield increased more rapidly than tance to achieve efficient fermentation processes (98, 99).
those of ethanol. One of the most common inhibitors, particularly in
Since xylitol formation in yeasts is most sensitive to ethanol fermentations, is acetic acid, the degree of toxic-
substrate concentration and aeration rate, it would be ity of which is pH-dependent (57). However, regard to
best if both parameters were simultaneously taken into xylitol production in xylitol-producing processes, no
consideration when optimizing xylitol production. By negative influence of acetic acid has been clearly iden-
varying the initial o-xylose concentration between 60 tified. The concentration of acetic acid decreased continu-
and 12Og/l and the oxygen transfer coefficient, kra, ously until its complete depletion, when D. hansenii
between 0.24 to 1.88 l/min, Roseiro et al. (80) optimized NRRL Y-1426 (87) and C. guilliermondii FTI 20037
xylitol production in D. hansenii DTIA-77. Applying an (100) were grown on hemicellulosic hydrolysates.
experimental design with 2 factors, they obtained xylitol The data on xylitol production from hemicellulosic
yield of 0.54g/g when the yeast was cultivated in 9Og/l hydrolysates with yeasts are summarized in Table 6. C.
o-xylose and with kLa, of 1.88 I/min. Horitsu et al. (67) guilliermondii FTI 20037 grown on rice straw hydroly-
studied the influence of culture conditions on xylitol for- sate exhibited the highest production rate of 0.56g/Th,
mation by C. tropicalis IF0 0618 and optimized the volu- whereas D. hansenii NRRL Y-7426 grown on chips of
metric xylitol production rate using Box-Wilson method. Eucalyptus glob&us had in fact the highest yield, 0.73
The initial D-xylose concentration (120 to 180 g/l), yeast g/g.
extract concentration (12 to 18g/Z) and kLa (236 to 381 The presence of inhibitory substances in hydrolysates
l/h) were chosen as independent factors in a 23-factorial very often imposes the necessity of purification of the
experimental design. A maximum xylitol productivity of hydrolysates prior to their utilization and/or adaptation
2.67 g/Ih was obtained when the initial o-xylose concen- of the microorganisms to the sugar which will be used.
tration was 172 g/I, the yeast extract concentration was Thus, in the process of xylitol production by C. guillier-
21 g/l and the kLa was 452 l/h. mondii FTI 20037 from sugar cane bagasse hemicellulos-
The general conclusion from all investigations regard- ic hydrolysate, the hydrolysate was treated in seven differ-
ing oxygen influence on D-xylose metabolism in xylitol- ent ways (101). The best results were obtained following
producing yeasts is that oxygen supply rate is a key overtitration of the hydrolysate with Ca(OH)* and subse-
parameter which determines whether D-xylose will be quent use of HzS04 (Table 6).
fermented or respired. It is very important, therefore, for A single hydrolysis stage in Eucaliptus globulus led to
an effective process, to determine the oxygen flux that the formation of about 17 g/l o-xylose with a low con-
will enable balanced utilization of carbon both for centration of inhibitors (87). However, the relatively low
growth and fermentation. substrate concentration limited both the productivity and
yield of the subsequent fermentation step. Therefore, the
D-xylose content was raised by vacuum evaporation. In
FERMENTATION OF HEMICELLULOSIC
the comparatively high range of xylose concentrations
HYDROLYSATES
studied (57-78 g/l), a single charcoal treatment was una-
The ultimate goal of all investigations regarding D- ble to reduce the amount of inhibitors to a satisfactory
xylose fermentation is to acquire sufficient knowledge for level and convert the hydrolysate to a suitable fermenta-
establishing fermentation processes using the pentose tion medium. Because of this, the combined strategy
fraction of the lignocellulosic hydrolysates. involving both charcoal adsorption of concentrated
Although the hydrolysis can be performed enzymatical- hydrolysates and a high initial cell concentration of up
ly, most fermentation studies have focused on hydroly- to 80 g/l was applied.
sates derived from acid hydrolysis. Due to its heterogene- When the yeasts C. guilliermondii FTI 20037 (100,
ous structure and relatively low degree of polymeriza- 101) and C. mogii ATCC 18364 (102) were grown on
tion, hemicellulose is much easier to hydrolyze than the hemicellulose hydrolysates for xylitol production, a
crystalline cellulosic components of biomass. In many sequential pattern of sugar consumption was observed. In
cases, even a simple steam treatment without the aid of addition, as a result of carbon source limitation, both
acid catalysis has been found to be effective (1). yeasts started to consume the xylitol produced when D-
However, for the decomposition of hemicellulose, mild xylose was almost exhausted.
hydrolysis is the most suitable. Its advantages are that it Besides the yeasts presented in Table 6, there are other
prevents the formation of some degradation products, xylose-fermenting yeasts such as P. stipitis NRRL Y-
enhances the susceptibility of cellulose to subsequent 7124 (104) and C. shehatae NRRL Y-12858 (105) which
enzymatic or acid hydrolysis, reduces the requirement produced xylitol when cultivated on wheat straw and
for expensive corrosion-proof equipment, and avoids the corn cob hemicellulosic hydrolysate, respectively. In
environmental problems incurred through the use of these cases, however, xylitol was only a by-product of
strong chemical treatments (1). ethanol fermentation.
A critical feature of hydrolysates prepared using acid
catalysis is the presence of inhibitors of microbial
metabolism (11) which makes them substantially more PROCESS STRATEGIES
difficult for microbial utilization than the corresponding Most of the papers published thus far on xylitol
mixtures of pure sugars (3). During the acid hydrolysis production by yeasts refer to batch culture methods
of the lignocellulosics, different types of sugars (D-glu- (flasks or lab batch reactors) as the simplest and hence
cose, D-galactose, o-mannose, D-xylose, L-arabinose), the most used culture methods (20, 42, 61-64, 77, 80,
 +

2
TABLE 6. Xylitol production by yeasts from hemicellulose hydrolysates
g
Hydrolysis Hydrolysate Released compoundsa (g/l) kc
Yeast Substrate (zO ($) (gyh) ($;) $7:; (A) Reference $
conditions treatment D-Xylose D-Glucose L-Arabinose Acetic acid
C. guilliermondii sugar 35 mM HzS04, Ca(OH)2, pH 6.5 61 15 n.r.c n.r. 0 20 0 0 n.r. 125 101 %
z
FTI 20037 cane 19O”C, 5 min.
bagasse steam Ca(OH)2, pH 10, 68 19 n.r. n.r. 30 95 0.240 0.48 n.r. 125 5
explosion, HzSOa, pH 6.5 u
solid/liquid
ratio l/6 CaO, pH 10, 65 19 n.r. n.r. 24 98 0.192 0.36 n.r. 125
2
H$Od, pH 6.5 “3
KOH, pH 6.5 68 18 n.r. n.r. 0 0 0 0 0 125 2
KOH, pH 10, 63 16 n.r. n.r. 16 56 0.128 0.48 n.r. 125
2
H>SOd, pH 6.5
C. guilliermondii rice 0.07 g cont. vacuum 45 14 I 8.5 27 86 0.560 0.69 0.14 48 100
FTI 20037 straw KS%, per g concentration
rice straw, at 7O”C,
145”C, 20 min NaOH, pH 10,
solid/liquid H?SOJ, pH 5.3
ratio l/IO

C. mogii wheat 15 %,’HSO 28 3 6.5 n.r. 6 70 0.130 0.31 0.71 46 102

ATCC 18364 straw 121”C,30 gin

Candida sp. 11-2 sugar 2-3% H$O,, CaCO,, pH 4.5-6 58 n.r. n.r. n.r. 2.6 5.1 0.053 0.88 n.r. 49 103
cane 100°C
bagasse activated 43 n.r. n.r. n.r. 10.5 94.8 0.205 0.26 n.r. 51
charcoal, CaO,
CaCOJ, pH 4.5-6

cation-exchange 46 n.r. n.r. n.r. 10.1 83.9 0.195 0.26 n.r. 51

resins, CaO,
CaCOI, pH 4.5-6

Debaryomyces Eucaliptus 3.5% HzSOJ, vacuum 73 2 4.2 5.3 6 (0.8)d 12.7 0.088 0.57 0.20 60 87
hansertii globulus loO”C, 11 h, concentration, 78 2 4.2 6.1 41 (2)d 68.4 0.500 0.73 0.25 78
NRRL Y-1426 solid/liquid CaC03, pH 6.5, 57 4 4.5 4.5 39 (3)d 92.8 1.060 0.68 0.12 34
ratio 818 charcoal
treatment

B Initial concentrations.
b Substrate consumed refers to D-xylose only.
c n.r., Not reported.
J Values inside brackets are amounts present initially.
VOL. 86, 1998 MICROBIAL CONVERSION OF D-XYLOSE TO XYLITOL 11

82, 100, 101). Batch cultures are characterized by high

FUTUREPROSPECTS
initial substrate concentrations and high product concen-
trations at the end of the process and relatively low pro- Instead of the classical approach to the improvement
ductivity. and optimization of xylitol productivity and yield by
Although continuous culture techniques often provide changing the fermentation variables, metabolic engineer-
better productivities and yields for many microorgan- ing offers opportunities to change the genetic properties
isms, the production rates decrease with increasing dilu- of the microorganisms themselves. In the quest for a
tion rates. High productivities can be achieved only by microorganism which will efficiently convert o-xylose to
using low dilution rates, that is high residence time, xylitol, a strain of S. cerevisiae was genetically modified.
which is very difficult to achieve in practice. Therefore, This recombinant S. cerevisiae harbors the gene coding
some continuous processes should be replaced by fed- for xylose reductase from P. stipitis CBS 6054 (109).
batch ones (101). However, when studying yeast physiol- The yeast achieved a high xylitol yield approaching the
ogy, the most accurate data can be obtained from con- theoretically expected yield, since it did not possess any
tinuous cultures (67, 85, 97). significant xylitol dehydrogenase activity. The recom-
In fed-batch processes, the substrate concentration can binant yeast required a cosubstrate for the generation of
be maintained at a suitable level throughout the entire reduction equivalents used in the reduction of xylose and
course of fermentation, that is, a level sufficient to for maintenance and growth. When glucose was used as
induce xylitol formation but not to inhibit yeast growth. a cosubstrate under anaerobic conditions, with supply
In addition, these processes generally operate with high rates of 1 and 0.1 g/lb, the specific rate of xylitol forma-
initial cell density which normally leads to an increase in tion was higher at the higher glucose supply rate, that is
volumetric productivity. The yeast C. boidinii NRRL Y- 0.78 compared with 0.39 mmol/gh (110).
17213 gave much better results when cultivated in a In another study (ill), the formation of xylitol by the
fed-batch fermentor compared to other ways of cultivation. same transformant was investigated by comparing the
The highest xylitol yield was 75% of the theoretical efficiency of different cosubstrates (glucose, ethanol,
yield, compared to 53% in the batch culture. The acetate, glycerol), oxygenation levels and different ratios
productivity of 0.46 g/Ih was twice as high as the highest of substrate and cosubstrate. With both glucose and
obtained under batch conditions (90). ethanol, the conversion yields were close to 1 g xylitol
Another way to improve the process parameters is the per g of consumed D-xylose. Decreased aeration in-
use of immobilized cells. To convert D-xylose into creased the xylitol yield and decreased the productivity.
xylitol, cells of C. pelliculosa and Methanobacterium sp. When the xylose : cosubstrate ratio increased, the xylitol
HU were either separately immobilized or co-immobi- yield based on consumed cosubstrate also increased.
lized in an agar gel, a calcium alginate gel, a h--carragee- In both of these studies (109, ill), the initial D-xylose
nen gel and other supports. The highest conversion rate concentration did not exceed 2Og/l. However, the D-
was observed when benzene-treated cells were co-immobi- xylose concentration of industrial hemicellulose hydroly-
lized in the photo-crosslinkable resin prepolymers ENT sates is usually higher since they are concentrated, result-
2000 and 4000 (107). Almost 100% of the n-xylose ing in D-xylose concentrations between 30 and 350 g/l
(4.5 g/l) was converted into xylitol after 33 h of incuba- (112). Therefore, Meinander et al. (112) applied a fed-
tion when the volume ratio of immobilized methanogen batch process for xylitol production by S. cerevisiae ex-
to immobilized C. pelliculosa was 1 : 2. In the co-im- pressing XYLI gene with a total D-xylose concentration
mobilized cell system, the degree of conversion and the corresponding to that of industrial hemicellulose hydroly-
conversion rate of D-xylose were higher than those in sate. When the total D-xylose concentration was 95 g/l,
the separately immobilized cell system. To allow continu- 93% of the D-xylose was converted to xylitol. The
ous xylitol production, immobilized cells were packed in average volumetric productivity was 1 g/Th, and the
a column reactor. Co-immobilized cells were stable for specific productivity 0.04 g/gh. The xylitol ethanol yield
about 2 weeks with approximately 35% conversion. was about 1.1 g/g.
De Silva and Afschar (78) immobilized the cells of C. Continuous xylitol production with two different im-
tropicalis DSM 7524 on a porous glass and used them in mobilized recombinant strains of S. cerevisiae (H475 and
a fluidized bed reactor. The authors intended to reuse S641), expressing low and high xylose reductase activi-
the immobilized cells several times by repeating the ties, was investigated in a lab-scale packed bed reactor.
batch fermentation with substrate shift. However, the The cells were immobilized by gel entrapment using Ca
yeast degenerated after completion of the first cultivation alginate as the support. The effect of hydraulic residence
and addition of fresh medium. Under continuous condi- time, substrate/cosubstrate ratio, recycling ratio, and aer-
tions, the immobilized cells of C. tropicalis DSM 7524 ation rate were studied (113). The overall xylitol yield
converted D-xylose into xylitol with a high productivity was higher for the low xylose/glucose ratio than for the
of 1.35 g/lb. high ratio, 0.6Og/g and 0.42 g/g, respectively. The
In another study, the aim of which was not xylitol highest xylitol concentration of 15 g/l was reached at
production, xylitol was the only fermentation product hydraulic residence time of 8.5 h. The 20-fold higher D-
observed. Lohmeier-Vogel et al. (108) studied the glu- xylose reductase activity of the strain with higher xylose
cose and D-xylose metabolism in agarose-immobilized C. reductase activity did not result in a proportional in-
tropicalis ATCC 32113 by nucleic magnetic resonance. crease in xylitol concentration compared with the strain
NMR studies showed that neither glucose nor xylose with lower xylose reductase activity. Under anaerobic
metabolism was enhanced by use of an immobilization conditions, with a recycling ratio of 10, the highest volu-
process. Attempts to improve the rate of D-xylose metric productivities of 3.44 and 5.80 g//h were obtained
metabolism by increasing the oxygen delivery to the with the strain with lower xylose reductase activity at
entrapped cells were not successful. 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 AND KUZMANOVA J. FERMENT. BIOENG.,

spectively. These are the highest productivities reported Chem. Sot., 58, 651-657 (1993).
to date. 11. Schneider, H.: Conversion of pentoses to ethanol by yeasts and
Despite the very high yields and productivities that fungi. CRC. Crit. Rev. Biotechnol., 9, I-40 (1989).
have been obtained with genetically modified strains of 12. Rosenberg, S. L.: Fermentation of pentose sugars to ethanol
and other neutral products by microorganisms. Enzyme
S. cerevisiae, it still remains to be shown whether these
Microbial. Technol., 2, 185-189 (1980).
microorganisms can remain sufficiently stable over a rela- 13. Gong, C. S., Chen, L. F., Flickinger, C., Chiang, L.-C., and
tively long period of time and endure the operational Tsao, G. T.: Conversion of hemicellulose carbohydrate. Adv.
condition prevailing during the production of xylitol. Biochem. Eng. Biotechnol., 20, 93-118 (1981).
14. Jeffries, T. W.: Conversion of xylose to ethanol under aerobic
conditions by Cundida tropicalis. Biotechnol. Lett., 3. 213-216
NOMENCLATURE (1981).
AR : aeration, volume of air per volume of medium 15. Schneider, H., Wang, P. Y ., Chan, Y. K., and Maleszka. R.:
per minute, vvm Conversion of o-xylose into ethanol by yeast Pachysolen
: ethanol concentration, g/f tannophilus. Biotechnol. Lett., 3, 89-92 (1981).
C, 16. Slininger, P. J., Bothast, R. J., van Cauwenherge, J. E.. and
CX : xylitol concentration, g/Z Kurtzman, C. P.: Conversion of o-xylose to e<hanol by the
D : dilution rate, l/h yeast Puchysolen tannophilus. Biotechnol. Bioene.. 24. 371-
: oxygen transfer coefficient, l/h
_I

kra 384 (1982).

OTR : oxygen transfer rate, mmol/lh 17 Wang, Y., Jonson, B. F., and Schneider, H.: Fermentation of
C$ : volumetric xylitol production rate, g/lb o-xylose by yeasts using isomerase in the medium to convert
: initial substrate (D-XylOSe) concentration, g// o-xylose to o-xylulose. Biotechnol. Lett., 3, 273-278 (1980).
SC : D-xylose consumed, ,?i 18 Gong, C. S.. Chen, L. F., Flickinger, C., Chiang, L.-C., and
t : time, h Tsao, G. T.: Production of ethanol from D-xylose by using D-
v, : culture volume, ml xylose isomerase and yeasts. Appl. Environ. Microbial., 41,
430-436 (1981).
V” : vessel volume, ml
19 Gong, C. S., Chen, L. F., and Tsao, G. T.: Quantitative pro-
Ycm/s : cell mass yield coefficient, g cell mass/g D-xylose duction of xylitol from o-xylose by a high-xylitol producing
consumed yeast mutant Candidu tropicalis HXP2. Biotechnol. Lett., 3,
YX/S . xylitol yield coefficient, g xylitol/g D-xylose con- 130-135 (1981).
sumed 20. Barbosa, M. F. S.. de Medeiros, M. B., de Mancilha, I. M.,
Yx/r f. percentage of the xylitol yield from the theoreti- Schneider, H., and Lee, H.: Screening of yeasts for production
cal value, ,?d; theoretical value is considered as of xylitol from o-xylose and some factors which affect xylitol
0.91 g xylitol/g D-xylose consumed yield in Candidu guilliermondii. .I. Ind. Microbial., 3, 241-251
: specific growth rate, l/h (1988).
I’ 21. Bar, A.: Caries prevention with xylitol. A review of the scien-
tific evidence. Wld. Rev. Ntr. Diet., 55, 183-209 (1988).
ACKNOWLEDGMENT 22. Isokangas, P., Alanen, P., Tiekso, J., and Makinen, K. K.:
Xylitol chewing gum in caries prevention: a field study in
We acknowledge the financial support of the Macedonian Min- children. J. Am. Dent. Assoc., 117, 315-320 (1988).
istry of Science and Deutscher Akademischer Austauschdienst 23. Pepper, T. and Olinger, P. M.: Xylitol in sugar-free confec-
(DAAD). tions. Food Technol., 10, 98-106 (1988).
24. Makinen, K. K.: Latest dental studies on xylitol and mechan-
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