APPuED MICROBIOLOGY, Dec. 1969, p.

1031-1035
Copyright ( 1969 American Society for Microbiology

Vol. 18, No. 6

Printed in" U.S.A.

Microbial Production of Xylitol

from Glucose'
HIROSHI ONISHI AND TOSHIYUKI SUZUKI
Noda Institute for Scientific Research, Noda-shi, Chiba-ken, Japan

Received for publication 22 July 1969

A microbiological method is described for the production of xylitol, which is

used as a sugar substitute for diabetics. A sequential fermentation process yielded
9.0 g of xylitol from 77.5 g of glucose via D-arabitol and D-xylulose. Candida guilliermondii var. soya (ATCC 20216) consumed 5.1 g of D-xylulose and produced
2.8 g of xylitol per 100 ml. Pentitol production from D-xylulose by yeasts was
divided into three types: I, yeast-produced xylitol; II, yeast-produced D-arabitol;
and III, yeast-produced xylitol and D-arabitol. D-Xylulose, but not glucose, was
dissimilated to xylitol by yeasts under aerobic conditions.
rouxii, 6 strains of S. rouxii var. halomembranis, 16
strains of Debaryomyces hansenii, 3 strains of Pichia
farinosa, P. hyalospora, 3 strains of P. membranaefaciens, P. polymorpha, P. pseudopolymorpha, P.
quercuwn, P. rhodanensis, P. robertsdi, P. scolyti, P.
wickerhamli, Hansenulda anomala, H. anomala var.
productiva, H. saturnus, H. schneggii, H. suaveolens,
H. subpelliculosa, 4 strains of Hansenula species
isolated from miso paste, Endomycopsis chodatii,
Candida albicans, 2 strains of C. guilliermondii, C.
guilliermondii var. soya, C. melibiosii, 2 strains of
C. parapsilosis, C. parapsilosis var. intermedia, C.
polymorpha, 5 strains of C. tropicalis, C. utilis, 3
strains of Candida species, 2 strains of Torulopsis
famata, Torulopsis halophila, Torulopsis sake, Torulopsis versatilis, Trichosporon cutanewn, Trigonopsis
variabilis, Cryptococcus neoformans, Cryptococcus
laurentil, and Rhodotorula rosa.
Media. The composition of the standard medium
used for screening of xylitol production from glucose
(medium A) or D-xylulose (medium B) was as follows.
Medium A: 10% glucose, 0.1% KH2PO4, 0.05%
MgSO4- 7H20, 0.01% CaCl2- 2{HaO, 0.01% NaCl,
0.4% vitamin-free Casamino Acids (Difco), 0.1%
Yeast Extract (Difco), pH 5.0. Medium B: 5%
D-xylulose was used instead of 10% glucose in medium A and added aseptically after sterilizing separately at 110 C for 5 min. Changes in the composition
of the medium are noted.
Fermentation conditions. To screen for xylitol production from glucose, one loopful of yeast from a
5-day koji-agar slant culture was inoculated into
large test tubes, each containing 8 ml of sterilized
medium A. The tubes were shaken on a reciprocal
Saccharomyces acidifaciens, 2 strains of S. acidi- shaker at 300 rev/min with a stroke of 2 cm at 30 C
faciens var. halomembranis, S. cerevisiae, S. fermen- for 2 to 5 days. The reduction of D-xylulose to xylitol
tati, S. fragilis, S. logos, S. mellis, 44 strains of S. and some factors affecting the fermentation were also
examined under the same conditions except that 10
I A preliminary report on this topic was presented at the annual
meeting of the Agricultural and Chemical Society of Japan, 1-4 ml of medium B was used. Fermentation temperature
of the latter was 30 C unless stated otherwise.
April 1969.
1031

A large variety of polyalcohols are produced

in good yields by aerobic dissimilation of various
pentoses and hexoses by yeasts (10-15, 20-22).
No specific commercial outlet for these polyalcohols has been found. Recently, however, it
has been shown that xylitol may be used as a
sugar substitute for diabetics (5, 7), and xylitol
is now commercially available for this purpose
in Japan.
Xylitol is readily produced chemically (6) or
biologically (13) by the reduction of D-xylose,
but the process is expensive because of the high
cost of the substrate (D-xylose).
It has been well demonstrated that glycerol,
erythritol, D-arabitol, and D-mannitol are produced by aerobic dissimilation of glucose by
yeasts (10, 11, 15, 20-22), but xylitol has not so
far been found to be a fermentation product of
glucose. We examined microbiological conversion of glucose to xylitol. Although we failed to
obtain xylitol from glucose by one fermentation
step, a microbiological method was developed
which converted glucose to xylitol via D-arabitol
and D-xylulose, in which three sequential steps
are involved.
MATERIALS AND METHODS
Yeast strains examined. A total of 128 yeast strains
were screened for their ability to produce xylitol from
glucose and D-xylulose. Most of them are from the
collection of our institute. They were: 2 strains of

1032

ONISHI AND SUZUKI

Analytical methods. After removal of yeast cells by

filtration or centrifugation, the clear fermented broth
was analyzed for sugar and polyalcohol by the method
of Neish (9) and for D-xylulose by the method of
Dische and Borenfreund (2) with crystalline D-xylulose p-bromophenylhydrazone as a standard. Paper
chromatographic determination was performed by
double ascending development on Whatman no. 1
filter paper in ethylacetate-pyridine-water (10:4:3,
v/v). Reducing sugar was detected with aniline
hydrogen phthalate reagent (17) and polyalcohol by
KI04-p, p'-tetramethyldiaminodiphenylmethane reagent (23). Spots of xylitol, D-arabitol, and ribitol were
distinctly separated on the paper showing RF = 1.14,
1.21, and 1.27, respectively, with glucose as standard.
Preparation of D-xylulose. A 1-ml amount of a
2-day culture of Acetobacter suboxydans (ATCC 621)
grown in a medium described below was inoculated
into 500-ml shake flasks each containing 50 ml of the
medium composed of 5% D-arabitol, 0.5% polypeptone, 0.1% Yeast Extract (Difco), and 0.1% KH2PO4
(pH 6.0). The flasks were shaken on a reciprocal
shaker (140 rev/min, a 7.5-cm stroke) at 30 C. After
2 days of incubation, D-arabitol was almost completely consumed and converted to D-xylulose (93%
yield). The clarified broth treated by the method of
Neish (9) was concentrated in vacuo at 45 C to a
syrup which was extracted with hot 99% ethyl alcohol.
After evaporation of ethyl alcohol under vacuum, the
resulting syrup was dissolved in water and decolorized
by passage through a column of activated carbon.
The D-xylulose fraction thus obtained was chromatographically pure, [Ca]20 - 33.7 (c = 8.99 in water),
and the product, D-xylulose, was identified as its
p-bromophenylhydrazone derivative. The melting
point (128.5 to 129.5 C) was identical with the value
given [128 to 129 C, (3)]. Elementary analysis gave
the following results: Cu1H1504N2Br; calculated: C,
41.39; H, 4.74; N, 8.78; Br, 25.04; found: C, 41.29;
H, 5.01; N, 8.83; Br, 25.20. D-Arabitol was prepared
by aerobic fermentation of glucose by yeasts (11).
Isolation of xylitol and D-arabitol. After clarification by the method of Neish (9), the cleared broth
was concentrated in vacuo at 50 C to dryness. The
dried material was extracted with hot 99% ethyl
alcohol. When necessary, the extract was treated with
Amberlite IRA-400 in the hydroxyl form (19) to
remove the remaining reducing sugar. The ethyl
alcohol extract gave a crystalline product, and pure
pentitol was obtained by recrystallization.

RESULTS
Screening for yeasts producing xylitol from
glucose. A total of 128 yeast strains were screened
for their xylitol-producing ability from glucose.
Arabitol was the only pentitol produced, but
neither xylitol nor ribitol was found.
Screening for yeasts producing xylitol from
D-xylulose. D-Arabitol has been reported to be
produced from glucose in 40 to 50% yields by
yeasts (10, 20-22) and to be converted almost
quantitatively to D-xylulose by Acetobacter (8,

APPL. MICROBIOL.

18). If a yeast grows well on D-xylulose and

produces xylitol in good yields, it could be expected that xylitol would be obtained efficiently
from glucose through the following sequential
fermentation process: glucose -* D-arabitol

D-xylulose -* xylitol.
Accordingly, production of pentitol from
D-xylulose by yeasts was examined. The yeast
strains which utilized D-xylulose fairly well and
produced good yields of pentitols are shown in
Table 1. On the basis of pentitol produced, yeast
strains were divided into three groups. Group 1
strains produce xylitol [a representative strain,
Candida guilliermondii var. soya (ATCC 20216)],
group Il produces D-arabitol [a representative
strain, Debaryomyces hansenii (ATCC 20212)],
and group III produces xylitol and D-arabitol (a
representative strain, Candida guilliermondii
3529). The products were identified from melting
points and infrared spectra after crystallization
from the broth of representative strains of each
group.
Effect of cultural conditions on xylitol production. Of group I, Candida guilliermondii var. soya
(ATCC 20216) showed the most rapid fermentation with yields of 25.0% of the D-xylulose consumed. The concentration of 6.1 % D-xylulose
gave the highest xylitol production (34.2 %,
based upon the sugar used). Of several nitrogen
sources tested, corn steep liquor was most effective (Table 2).
It has been previously reported that the C :N
ratio of medium significantly affects polyalcohol
production (10, 16, 20). The polyalcohol production by Pichia miso was markedly stimulated
in a medium containing 0.1% yeast extract,
whereas ethyl alcohol became the principal
product when the yeast was cultivated in a
medium of 4% yeast extract (16). When the
effect of corn steep liquor and Casamino Acids
on xylitol production was examined, it was
found that 4 to 8 % of corn steep liquor markedly
increased the xylitol production. The highest
yield was 55% of the sugar used. High concentrations of Casamino Acids (1 to 2%) were also
effective (Table 3). It has been shown that an
excess of inorganic phosphate has a detrimental
effect upon the polyalcohol yield in some yeasts
(10, 20). The effect of phosphate concentration
on xylitol yield was examined in the modified
medium B in which KH2PO4 content was varied
from 0 to 2%, but the xylitol yields were not
affected by any variation of level of the phosphate concentration. Sugar utilization was slower
and the xylitol yield was lower at 25 C (5.6%
yield), but the yields of xylitol at 30 and 37 C
were similar (29.9% yield). Within the range of

Voi..

18, 1969

1033

XYLITOL FROM GLUCOSE

pH 3.3 to 7.5, the initial pH 6.3 gave the best

yields of xylitol (26.0%).
Production of xylitol from glucose by a sequenfermentation process. Our results suggested
tial
that xylitol would be produced from glucose in

approximately

15

application of three fermentation processes without isolation and purification of the intermediate
products (D-arabitol and D-xylulose), which
would make this technique very attractive commercially. Debaryomyces hansenii ATCC 20212
to 20% yield by a sequential was employed for the first step (glucose>

TABLE 1. Survey

of pentitol productivity from

Strain

Fermenta-

D-xylulose by yeasts
D-Xylulose a

Pentitol

produced

tion time

Initial

Final

6.4
6.4
6.4
6.4
6.4
6.4
6.4
6.4
6.4
6.4

5.5
3.6
2.3

%~~~~

days

days

I. Yeasts which produce xylitol

Saccharomyces rouxii N28
S. rouxii E7

S. rouxii 3281
S. rouxii 3292
S. rouxii 3215
S. rouxii 3217
S. rouxii 3219
S. rouxii 3221
S. rouxii 3222
S. rouxii 3224
S. rouxii var. halomembranis A31
S. acidifaciens S9
S. acidifaciens var. halomembranis H3
S. mellis 3220
Debaryomyces hansenii (ATCC 20220)
Pichia farinosa (ATCC 20210)
P. farinosa (ATCC 20218)
Hansenula anomala
H. suaveolens
Endomycopsis chodatii
Candida tropicalis (ATCC 20215)
C. tropicalis 3540
C. guilliermondii var. soya (ATCC 20216)
C. melibiosii (IFO 961)
Candida sp. 3547
Candida sp. 3548

Cryptococcus neoformans

II. Yeasts which produce D-arabitol

Saccharomyces rouxii 3218

Debaryomyces hansenii (ATCC 20212)

D.hansenii3170
D. hansenli 3176
D.hansenii 3114
D.hansenii 3179
Hansenula subpelliculosa

Torulopsis famata (ATCC 20214)

T. halophila
T. versatilis (CBS 1752)
Candida polymorpha (ATCC 20213)
C. parapsilosis (IFO 708)
C. parapsilosis var. intermedia (IFO 1021)
Trigonopsis variabilis

5
5
5
5
7
5
6
7
5
5
5
5
5

6.4
6.4
6.4

5.6
2.5
1.2
2.8

7
4

6.4
5.9

5.1
0.2

3
6
4
3
3
4
3

5.9
5.0
5.9
5.6
5.9

2.3
0.1
0.2

Pentitol yield
based on
D-xylulose
used

3
3

5.0
4.7
5.0
6.4

1.6
0.3
0.4
1.0
1.9

0.3
0.8
1.1
0.1
0.4
0.2
0.9
0.7
1.2
0.2
0.5
1.4
0.6
0.6
1.5
0.6
0.8
0.3
0.5
0.3
0.8
1.1
1.0
0.7

5.0

0.3

0.7

14.8

4
4

5.0
5.9

0.3
0

0.7
0.2

14.8
3.3

5
5
4
5
4
4
4
4
5
6
4
3
3

6.4
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
6.4
6.4

5.1
0
1.5
0.6
0
1.9

0.2
2.8
0.8
2.9
2.9
0.5

0.9

1.9
3.6
3.0
1.1
1.0
0.1

0.1

1.5
1.5
2.3
1.5
0.8
1.1
1.9

15.3
47.4
18.2
54.7
49.1
12.5
15.2
37.5
65.2
79.3
31.2
14.8
20.7
38.7

5.9

2.4

1.8

51.4

5.0

2.1
3.0
5.8
4.4
3.9
4.0

1.8

33.3
28.5
26.8
2.3
11.7
33.3
45.0
28.0
50.0
25.0
12.8
26.9
16.6
46.1
26.3
16.6
16.3
5.2
13.2
7.0
17.0
25.5
25.0
15.5

III. Yeasts which produce xylitol and D-arabitol

Candida guilliermondii 3529

a Expressed as grams per 100 ml.

1034

ONISHI AND SUZUKI

TABLE 2. Effect of nitrogen sources-

APPL. MICROBIOL.

TABLE 4. Production of xylitol from glucose by a

sequential fermentation processa
Xy~litol

Nitrogen source

Fer- DXloeb
D-Xy]ulose
men
tation
tion

yield
oln
D~~~-xylu-

Initial Final

sumned

based

lose

Fermentation step

Yield of
the
Yield of
Amount of product the
prodon the
or
sugars
t onpolyols in substrate inition
thc brothb consumed glnuctale
at eachglcs

step

Casamino Acids, 0.4% .......

Ammonium lactate, 0.4%....

Urea, 0.05% ...............

Corn steep liquor, 1.6%

..

..

hr
69
60
69
57

%
3.6
3.6
3.6
3.6

1.2
0.4
1.4
0.3

20.6
35.3
17.3
46.1

"Medium: 4% D-xylulose, 0.1% KH2PO4, 0.05% Mg54O

7H20, 0.01 % CaC2- 2H20, 0.01 % NaCI, and vitamins (biotin,
0.4 pg; calcium pantothenate, 80 pg; inositol, 400 pug; niacin,
80 jug; p-aminobenzoic acid, 40 sg; pyridoxine hydrochloride,
80 pg; thiamine hydrochloride, 80 pxg; and riboflavine, 40 p&g,
per 100 ml of medium). The media were adjusted to pH 6.0.
Total nitrogen content of corn steep liquor used was 4.08%.
b Expressed as grams per 100 ml.
TABLE 3. Effect of concentration of Casamino Acids
and corn steep liquor upon yield of xylitola
D-XYluloseb

Casamino Acids, 0.1%,

+ yeast extract, 0.1%
Casamino Acids, 0.4%,
+ yeast extract, 0.1%
Casamino Acids, 1.0%,
+ yeast extract, 0.1%
Casamino Acids, 2.0%,
+ yeast extract, 0.1%
Corn steep liquor, 0.4%
Corn steep liquor, 1.6%
Corn steep liquor, 4.0%
Corn steep liquor, 8.0%

based on

Initial

Final

D-xylulose
consumed

5.4

3.3

14.7

5.4

1.4

31.2

5.4

0.3

41.3

5.4

0.3

36.3

5.3
5.3
5.3
5.3

3.1
0.9
0.2
0.2

21.1
36.1
50.3
55.4

%;

5.3

34.2

34.2

5.0

94.3

32.3

1.8

41.8

11.6

15.5
(77.5 g/
500 ml)

(9.0 g/

Xylitol

yield

Nitrogen source added to the

basal medium

Glucose
I. Debaryomyces
hansenii fermentation for 99 hr
D-Arabitol
II. Acetobacter suboxydans fermen1, tation for 48 hr
D-Xylulose
III. Candida guilliermondii var. soya
fermentation for
64 hr
Xylitol

Residual D-xylulose

500 ml)
0.7

a In all fermentation steps, 10 shake flasks, each

containing 50 ml of medium (total 500 ml), were
run at the same time. At the end of each step,
the broth of 10 flasks was mixed, supplemented
with corn steep liquor when necessary, adjusted
to pH 6.0, and made up to 500 ml. Glucose and Darabitol at the first and the second steps were exhausted during the respective fermentations.

b Expressed as grams per 100 ml.

a Standard medium B without Casamino Acids

and yeast extract was employed as the basal medium. The media were adjusted to pH 6.0. Fermentation time: 69 hr.
b Expressed as grams per 100 ml.

D-arabitol). A modified medium A was used containing 15% glucose and 4 % corn steep liquor.
After 4 days of incubation, 13.8 g of glucose was
consumed per 100 ml of the medium, and 5.9 g
of D-arabitol was accumulated per 100 ml of the
broth. The yield of D-arabitol was 42.9% of the
glucose consumed. When glucose was almost
completely exhausted and yielded D-arabitol, the
fermented broth was adjusted to pH 6.0 with
NaOH without removing yeast cells and autoclaved at 120 C for 15 min. The broth was thus
enriched with the nutrients favorable to the

growth of Acetobacter suboxydans, which oxidized the D-arabitol almost quantitatively to

D-xylulose in 2 days at 30 C in shake flasks. For
the reduction of D-xylulose to xylitol by C.
guilliermondii var. soya (ATCC 20216), further
addition of 4% corn steep liquor to the broth
fermented by A. suboxydans was necessary for
the best yield. The fortified broth was adjusted
to pH 6.0 with NaOH and autoclaved at 10 lb of
steam pressure for 5 min to prevent browning
reaction of the medium as much as possible. In
54 hr, 3.6 g of D-xylulose was consumed per 100
ml of the broth and 1.5 g of xylitol was produced
per 100 ml of the broth (41.1% yield).
Thus, a practical fermentation in shake flasks
was run in sequential three-step combination,
with optimal conditions for each step. A 9.0-g
amount of xylitol was produced from 77.5 g of
glucose with a final yield of 11.6% (Table 4).
Xylitol was isolated in pure crystalline form from
the broth and identified by melting point, elementary analysis, and infrared spectrum.

XYLITOL FROM GLUCOSE

VOL. 18, 1969

DISCUSSION
The data presented in this paper demonstrate
the practicability of xylitol production from glucose by means of three sequential steps (glucose

--

D-arabitol

-+

D-xylulose

--

xylitol).

The

xylitol formation was carried out without isolation and purification of the intermediates and
yielded 11% xylitol from glucose.
The type of pentitol produced from D-xylulose
depended on the yeasts which were used for
such a reduction. As a result, xylitol, D-arabitol,
or a mixture of both were formed. C. guilliermondii var. soya (ATCC 20216), a group I
strain (Table 1), produced xylitol (55% yields on
the sugar used); D. hansenii (ATCC 20212), a
group II strain, produced D-arabitol (50%
yields); and Candida guilliennondii 3529, a group
III strain, produced almost equal amounts of
xylitol and D-arabitol (50% yields of both pentitols).
Although xylitol was not found in earlier
experiments to be formed from glucose by yeasts
(10, 11, 20-22), D-arabitol was the only pentitol
detected and identified by us as the dissimilation
product of yeasts.
D. hansendi (ATCC 20212) and S. rouxii E7,
both of which are typical D-arabitol producers,
elaborated different pentitols, D-arabitol or
xylitol, respectively, from D-xylulose. It has been
suggested (1, 4) that D-arabitol formation from
glucose by S. rouxii proceeds through two different intermediates, namely, D-xylulose and D-ribulose. This system offers a suitable model for the
study of the complex formation of pentitol, especially since it is believed (20) that the intrinsic
factors of such formation will most likely be
found in the metabolic controls in the cells.
ACKNOWLEDGMENTS
We thank K. Arima and Y. Ikeda of the University of Tokyo
for their guidance. We also thank M. Mogi and N. Iguchi of our
Institute for their encouragement. The technical assistance of K.
Kouchi is gratefully acknowledged.
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