JOURNAL OF FERMENTATION ANDBIOENGINEERING

Vol. 79, No. 5, 449-452. 1995

Factors Affecting the Ethanol Productivity of Yeast in Molasses

KAZUHIKO TAKESHIGE AND KOZO OUCHI*
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 3-6-6 Asahi-machi, Machida, Tokyo I94, Japan
Received 24 October 19941Accepted26 January 1995

The ethanol production by a laboratory yeast strain, X2180-lB, was less than half that by an alcohol yeast,
YOY655, in a molasses medium containing 30% sugars, although X2180-1B produced approximately the same
amount of ethanol as YOY655 in a nutrition medium with the same sugar content. The weak productivity of
X2180-1B in the molasses was ascribed to the limitation of sucrose hydrolysis in the molasses. The invertase
activity of X2180-1B was 0.019 (mmol sucrose/min/mg protein) in the nutrition medium, but substantially
zero in the molasses, while that of YOY655 was 1.75 in the nutrition medium and 1.15 even under the inhibitory
conditions in molasses. External addition of invertase greatly enhanced the ethanol productivity of only
X2180-1B. The inhibitory factors of invertase in molasses were heat-stable and dialyzable substances.

[Key words: Saccharomyces cerevisiae, ethanol production, molasses, invertase]

Molasses is widely used as a raw material for the pro- overnight at 30°C. The grown cells were concentrated 10
duction of ethanol for economic reasons, and strains of times and 0.2 ml of the cell suspension was inoculated
alcohol yeast have been selected for efficient ethanol pro- into 20ml of either YPS30 or Mo130 in a large test tube
duction. Laboratory strains of the yeast Saccharomyces (21 mm in diameter, 200 mm in height). The cultures
cerevisiae, however, have poor fermentation capacity in were incubated for ethanol fermentation at 30°C with
molasses, although they perform as well in nutrition rotation.
media as alcohol yeast does. One of the possible reasons Analyses of ethanol and sugars Three hundred
for such poor fermentation capacity of laboratory microliters of the culture was sampled and centrifuged,
strains is that they are more sensitive to certain inhibi- and the supernatant was stored at -20°C. For the as-
tory factors that probably exist in molasses. says of ethanol and sugars, the samples were diluted 20
Several factors, such as ethanol, salt and pH (l-3) or times with water and filtered through a 0.45-;lrn mem-
water activity (4, 5) have been reported to affect ethanol brane filter. The levels of ethanol and sugars were mea-
fermentation. Oderinde et al. (6) showed that the sured by gas chromatography, and by liquid chromatog-
removal of metal ions from molasses enhanced ethanol raphy using an HPLC column, Asahipak NH2P-50
production, but the reason remains to be clarified. Gaxiola (Shodex, Tokyo), respectively.
et al. (7) reported that overexpression of the HAL1 Assay of invertase activity The invertase activity of
gene in cells improved their growth under salt stress in a the yeasts was assayed according to Goldstein and Lam-
synthetic medium. However, they did not examine the pen (9). The enzyme reaction was initiated by adding
function of the HAL1 gene in ethanol fermentation. either Mo130 or YPS30 to yeast cells. The pH of Mo130
Praekelt and Meacock (8) reported that the Mall gene was adjusted to 5.0 with KOH and that of YPS30 was
was highly expressed in the early stationary stage of buffered to 5.0 with 0.1 M potassium acetate. The mix-
culture in molasses, but disruption of the Mall gene tures were incubated at 30°C for 20min and the reaction
revealed no remarkable phenotype. was stopped by boiling for 5 min. The quantity of glu-
In the present work, some factors affecting the ethanol cose liberated from sucrose was measured using a com-
productivity of yeast in molasses fermentation were mercial glucose kit (Kyowa Medix, Tokyo) and the inver-
examined. tase activity was determined based on the velocity of
glucose liberation (mmol glucose/min/mg protein).
Partition of molasses into dialyzable and undialyzable
MATERIALS AND METHODS
fractions For this purpose, 200ml of MollO was dia-
Yeast strains X2180-1B (MATa SUC2 ma1 mel lyzed twice against 1.2 I of distilled water. Since it was
gal2 CUPI) was purchased from the Yeast Genetic Stock necessary to remove sugars from the dialyzable fraction,
Center (California, USA). YOY655 (MATa) is a haploid 2 x 101’ cells of YOY655 were inoculated in this fraction
strain isolated in our laboratory from an industrial and allowed to consume sugars for 4 h. After centrifuga-
alcohol yeast. tion, the supernatant was heated at 90°C and concentrat-
Media YPD2 contained 1% yeast extract, 2% Poly- ed to 50 ml (dialyzable concentrate). The undialyzable
pepton and 2% glucose. YPS2 and YPSSO contained 1% fraction remaining in the dialysis bag was also concen-
yeast extract, 2% Polypepton and 2% or 30% sucrose, trated to 50 ml (undialyzable concentrate).
respectively. MollO and Mo130 contained 0.2% ammoni- Reagents Yeast invertase (,%fructosidase) was pur-
um sulfate and concentrated molasses to make the total chased from Wako Pure Chemical Industries (Tokyo).
sugar content either 10 or 30% (w/v), respectively.
Ethanol fermentation Yeast cells were cultured
RESULTS AND DISCUSSION
overnight at 30°C in 5 ml of YPD2. The culture was
seeded to YPS2 at the ratio of l/l000 and incubated Ethanol fermentation in molasses and nutrition me-
dium Ethanol production by X21 80-1B in Mo130
* Corresponding author. terminated after 30 h when 5% (v/v) of ethanol had accu-
449
450 TAKESHIGE AND OUCH1 J. FERMENT. BIOENG.,

(W w W
(a) I I
30 30

2
20
8
e
S
v) 10

0 0
0 30 60 90 0 30 60 90
/:- Time (h) Time (h)

0 30 60 90 - 0 30 60
(c) (d)
Time (h) Time (h)
FIG. 1. Ethanol production in Mo130 (a) and YPS30 (b) by
1

rjq&JJ;~
YOY655 (filled circles) and X2180-1B (open circles).

mulated, while that by YOY655 reached 12.5% in 60 h

(Fig. la). When YPS30 was fermented, however, X2180-
1B produced 14% of ethanol in 48 h (Fig. lb), an
amount which was comparable to the 15% produced by
YOY655. These results suggest that the poor fermenta-
tion of X2180-1B in Mo130 is not because of its weak
fermentation capacity per se, but arises from its suscep- 0 30 60 90 0 30 60 90
Time (h) Time (h)
tibility to some factor(s) in molasses other than sugars.
Changes in sugar concentrations during fermenta-
tion Sucrose, the major sugar in molasses, is hydro-
lyzed by yeast invertase to fructose and glucose, and
these monosaccharides are the direct substrates utilized
by yeast for fermentation.
The decrease of sucrose concentration in Mo130 was
much slower in X2180-1B than in YOY655 (Fig. 2a), the
rates of decrease being 4.6 and 0.52 mg sucrose/ml/h in
YOY655 and X2180-lB, respectively, as calculated from
the differences in the sucrose levels between 0 and 16 h.
On the other hand, the rates of decrease in YPS30 were
similar in the two strains (Fig. 2b) at 17 and 13 mg
sucrose/ml/h in YOY655 and X2180-lB, respectively. 0 30 60 90 0 30 60 90
The rapid sucrose hydrolysis was accompanied by tran- Tlme (h) Time (h)

sient increases in the fructose and glucose concentrations

FIG. 2. Changes in sugar concentrations during fermentation in
(Fig. 2d and 2f), indicating that the rates of sucrose Mo130 (a, c, e) and YPS30 (b, d, f). Yeast strains used for the fermen-
hydrolysis in YPS30 exceeded the rates of consumption tation were YOY655 (filled circles) and X2180-1B (open circles).
of the monosaccharides by yeast cells. A slight increase
in the fructose concentration was also observed when
Mo130 was fermented by YOY655 (Fig. 2c), and glucose the two strains were assayed in Mo130 and YPS30. The
was accumulated to some extent in the early stage of invertase activities measured in YPS30 were 1.745 and
the fermentation (Fig. 2e). These findings indicate that 0.019 (mmol sucrose/min/mg protein) for YOY655 and
the sucrose hydrolysis by YOY655 was fast enough to X2180-lB, respectively. When measured in Mo130, the
complete the fermentation in Mo130. When Mo130 was invertase activity of YOY655 decreased to 1.146 and that
fermented by X2180-lB, however, no increase in the of X2180-1B to 0.009. These data show that there was a
fructose level nor any accumulation of glucose occurred, lo-fold difference in invertase activity between the two
even though Mo130 had contained 6.2% of fructose and strains, and that the activity of each was suppressed in
4.7% of glucose at the beginning. The glucose level in Mo130 to nearly a half. These findings are substantially
X2180-1B simply decreased to nearly zero in 24 h and consistent with the observation that the decrease of su-
fructose was almost exhausted in 48 h, while most of the crose concentration was slower in Mo130 than in YPS30,
sucrose remained. These findings indicate that the rate and that it was slowest in the fermentation by X2180-1B
of sucrose hydrolysis by X2180-1B limited the fermenta- in Mo130. The suppression of invertase activity in Mo130
tion rate in Mo130. was not specific to these strains, since similar decreases
Invertase activities of YOY655 and X2180-1B in in the enzyme activity were also observed in other yeast
YPS30 and Mo130 The results outlined above suggest strains tested and in commercially available invertase
that invertase activity present in the periplasm of yeast (data not shown).
(10) is strongly suppressed in Mo130, and that X2180-1B Effect of external addition of invertase on ethanol
exhibits much lower invertase activity than YOY655 in production in Mo130 If the low invertase activity of
a molasses medium. Therefore, the enzyme activities of X2180-1B is the reason why this laboratory yeast per-
VOL. 79, 1995 FACTORS AFFECTING ETHANOL PRODUCTIVITY 451

TABLE 2. Analysis of metal ion contents in the dialyzable

concentrate

Metal ions (mg/O

Zn 401
Ni 3.88
Bi 33.4
Mn 57.2
Fe 231
cu 11.9
Al 462
Mg 2920
Na 841
Ca 5470
K 26300
Si 9.09

O- 60 60
Sn
V
18.6
86.8
Time (h)

FIG. 3. Effect of externally added invertase on ethanol produc- tivity was apparently inhibited in the assay media con-
tion in Mol30. After 16 h of fermentation, 20 units/ml of commercial taining the dialyzable concentrate, the activities mea-
invertase (filled symbols) or distilled water (open symbols) were added
sured in the assay media containing 10 and 20% of dialyz-
to the culture of YOY655 (circles) or X2180-1B (triangles).
able concentrate being 77 and 45% of the control, respec-
tively. The latter medium corresponds to Mo130 with
forms so poorly in ethanol production from Mo130, repect to dialyzable components other than sugar, and
external addition of invertase to the culture medium the inhibition rate (45%) measured in this medium was
should effectively enhance its ethanol production. This nearly equal to that in Mo130 (50%). These results indi-
was confirmed in the experiments shown in Fig. 3. A cate that the inhibitors in molasses are dialyzable molec-
commercially available invertase was added to the Mo130 ules stable to heating at 90°C.
culture at 20 units/ml after 16 h of fermentation. Upon Metal ions are possible candidates because molasses
the addition of invertase, the ethanol production by contains high concentrations of metal ions, as shown
X2180-1B began to increase rapidly and 12% of ethanol in the data on metal ions existing in the dialyzable con-
was finally attained in 60 h, which was more than two centrate (Table 2). Yamamoto et al. (11) reported that
times the final amount attained in the control. On the 0.1 mM Cu*+ inhibited almost all the activity of purified
contrary, external addition of invertase to the culture of invertase from Brevibacterium. The effects of several
YOY655 had no enhancing effect on its ethanol produc- metal ions on the invertase activity were thus examined
tion. These findings suggest that the invertase activity (Table 3). Metal ions were added to MollO on the basis
which X2180-1B exhibits by itself in Mo130 is far less of the analytical data, but the levels of the ions of inter-
than that required for the completion of fermentation. est were increased by 10 to 100 times over those in the
They also suggest that the invertase activity of YOY655 dialyzable concentrate in order that the inhibition could
is sufficient to support full fermentation in Mo130. be detected easily. Among the metal ions tested, copper,
Analysis of invertase inhibitors in molasses Since potassium, and calcium showed stronger inhibition. In
the molasses appeared to contain some inhibitor(s) of particular, the inhibition by copper ion was strong
invertase, it was partitioned by dialysis to determine
whether the dialyzable or undialyzable fraction would TABLE 3. Effects of metal ions on invertase activity
retain the inhibitory action. Dialyzable and undialyzable lnvertase activity (%)
concentrates with little fermentable sugars were prepared Additive
(mmol/min/me nrotein)
and separately added to MollO medium. MollO was none 44.99 (100)
employed as the basal assay medium because the suppres- l.OM KC1 33.69 ( 75)
sion of sucrose hydrolysis occurred only in molasses 2.OM KC1 21.79 ( 48)
media that contained more than 20% (w/v) sugars (data 0.1 M NaCl 42.36 ( 94)
not shown). The inhibition assay was performed using 0.2M NaCl 40.75 ( 91)
the commercial yeast invertase. The invertase activity 0.62 M CaC& 34.90 ( 74)
measured in the basal medium was 38 (mmol sucrose/ 1.24 M CaC12 24.01 ( 53)
min/mg protein), which was no difference from that 0.2M MgS04 40.95 ( 91)
measured in the assay medium containing 20% of un- 0.4M MgS04 39.14 ( 87)
0.02 M FeS04 44.79 (100)
dialyzable concentrate (Table 1). However, invertase ac-
0.04 M FeS04 43.78 ( 97)
0.02 M MnC& 46.00 (102)
TABLE 1. Effects of dialyzable and undialyzable fractions in 0.04 M MnCl, 46.00 (102)
molasses on invertase activity 0.02 M ZnSO, 40.35 ( 90)
0.04 M ZnSOl 36.31 ( 81)
Invertase activity (%)
Assay medium 0.02 M CuCl2 11.50 ( 26)
(mmol/min/ma -. protein)
0.04 M CuC12 7.46 ( 17)
MollO 38.28 (100)
MollO+ 10% dialyzable concentrate 29.25 ( 77) Metal ions were added to MollO medium so that final concentration
Mol10+20% dialyzable concentrate 17.05 ( 45) was 10 to 100 times higher than that of the corresponding ions in
Moll0+20% undialvzable concentrate 38.23 (100) Mol30 medium. Invertase activity was measured as described in
Materials and Methods.
452 TAKESHIGE AND OUCH1 J. FERMENT. BIOENG.,

50
itory condition, until1 the fermentation was completed.

40 8i
ACKNOWLEDGMENTS

L
b We are grateful to Ms. M. Umeda for her excellent technical
:g 30 assistance and Mr. N. Yokota, Technical Research Laboratories,
m Kyowa Hakko Kogyo Co. Ltd. for the analysis of molasses compo-
I nents. This work was supported by the “Research and Development
z? 20 Concerning Highly Efficient Ethanol Fermentation Techniques by
-c High Productivity Yeasts.” Project of the Ministry of International
Trade and Industry, Japan.
10

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