B i o t e c h n o l o g y Letters Vol 12 No 1

R e c e i v e d as revised 14th N o v e m b e r

73-78

(1990)

Alcoholic F e r m e n t a t i o n of beet molasses :

study on stillage recycle.
E. Wolniewicz, J.J. Essia Ngang, F. Letourn~au, P. Villa
Laboratoire de Chimie Organique et Cin(~tique,
U.F.R. des Sciences Fondamentales et Appliqu@es,
33 rue Saint-Leu, 80039 Amiens, France.

SUMMARY
Stillage recycle in beet molasses alcoholic fermentation can be lower the production costs by
a decrease of energy requirements for waste water treatment but it becomes necessary to optimise,
separately, the sugar and non sugar contents of the wort. It is shown that the increase of the wort
osmolality, linked to stillage recycle disturbs yeast metabolism above 1,5 osmol. The observed
inhibition which is dependent on both sugar and non sugar concentrations leads to an apparent link
between yeast behaviour and the dry matter percent of the worts.
INTRODUCTION
Two ways are offered to industrial distillers to optimize alcohol production. The first
is to obtain wines with a high alcoholic degree and to balance low productivities by gains in
distillation energy requirements. This solution is not suitable for beet molasses fermentation if
sufficient growth is required (Letourneau and Villa, 1987).
The second one is to favour fermentation rates at the expense of the alcoholic degree and in this
case, stillage recycle in fermentation tanks allows a decrease in the energy needed for water
evaporation. One consequence of the stillage supply is the increase of the dry matter content in
the worts which can have two different effects :
-potential toxicity to microorganisms linked to accumulation of inhibitory substances
(Eddy,1982 ; Essia Ngang et aL, 1989 ; Maiorella et aL, 1983 ; Maiorella et aL, 1984) ;
-difficulties for yeasts to grow in hypertonic media (Brown et al., 1981; D'Amore et aL, 1988;
Jones and Greenfield, 1986 ; Kenyon et aL, 1986 ; Prior, 1979).
These two effects generally lead to a decrease of yield and ethanol productivity (Navarro, 1980
; Navarro and Durand, 1978 ; Novak et aL, 1981).
In a previous work (Letourneau and Villa, 1987), we have shown that yeast growth on beet
molasses was affected if initial sugar concentration was greater than 120 g.1-1 (i.e. about 200
g.1-1 dry matter in the wort). On the other hand, the alcohol inhibition was strictly dependent
on substrate concentration. With regard to stillage recycle, the aim of this work is to
distinguish between the effects of sugar and non sugar concentrations on the growth and
fermentative capacities of the yeast.

MATERIALS AND METHODS

Beet molasses (50 % sucrose and 30 % w/w non sugar) was diluted to obtain sucrose
concentrations between 20 to 120 g.I"1. Concentrated stillage (50 % non sugar w/w) was used to bring
non sugar concentrations to the desired value (up to 20 % w/w). Worts were adjusted to pH 5,2
with N HCI. Fermentations were carried out in duplicate at 33~ C in 250 ml closed conical flasks
containing 100 ml medium. Agitation was 120 r.p.m.. A strain of Saccharomyces cerevisiae, used in
previous studies in our laboratory, was inoculated, after a two days preculture on beet molasses media
73

at an initial concentration of 2.106 cells/ml.

The evolution of biomass was followed by O.D. measurements of the cell suspension at 630 nm. Ethanol
formation was measured by G.L.C. on a GIRDEL 3000 Chromatograph with a flame ionisation detector.
Sucrose and reducing sugars concentrations were determined by H.P.L.C. using a HPX-87C.BIORAD
; .
.
.
.
!
column. In,tlal osmolalmes were obtained by cryosopic measurements with reference to a standard
curve.
RESULTS
The variation of relative fermentation rates, expressed as CO 2 release per unit of time
(g.1-1 .h -1 ), with sugar and non sugar concentrations in the worts is shown on figure 1.
V

%
!

1oo

'o

,~

20

~,';~

SO :

2 O L I "i

, so. ,o,~-,

o so. ,o,,-,
9 so. ,o,~-,

I~

I
20

I
40

1
60

I
100

80

r
120

I
140

I
160

I
180

!
200

) 1LS, ( ( J . l " }

Figure 1 - Variations of relative fermentation rates versus sugar (So) and

non sugar (N.S.) concentrations in the worts.
To avoid effects due to ethanol inhibition, these values are calculated in the first stage of
fermentation (alcohol < 2 g.l-1). The maximum fermentation rate (V o max. = 100 %) is
obtained for an initial sugar concentration (So) of 100 g.1-1 and a non sugar concentration
(N.S.) of 80 g.1-1. The data show large variation of yeast fermentative capacities linked to the
initial compositions of the media. Irrespective of the initial sugar concentration, there is one
optimal non sugar concentration which is about 80 g.1-1. This value corresponds, for our
stillage , to
the nutritional requirements of the yeast for fermentation. Below this content,
there is a lack of substrate and above it, we can observe a decrease of the relative fermentation
rates.

Table 1 . Minimal non sugar (N.S.min.) concentrations needed to obtain 90 %

of maximal rate for each series of sugar (So).
20

So

40

60

80

100

q.1-1

V~x

22,72

50,68

69,90

89,32

20,45

45,61

62,91

80,39

30

42

50

59

67

30

40

50

60

70

100

0,9

Vmax
%

N' S-rain.

g.! -1

S
g.1-1

74

Table 1 summarizes the maximum relative fermentation rates obtained with each S o and
minimal value of non sugar concentration necessary to get 90 % V max. 9 In these conditions,
minimum value of N.S. respondsto the empirical relation 9
(relation 1)

N.S.min. = 1/2 S o + 20

When N.S. is present at the optimal concentration, fermentation rates are strictly
dependent on S o, as can be s e e n in figure 2. V o max. is obtained for S O = 100 g.1-1 . Stimulation

of fermentation by substrate shows an

where

V=I/2V

apparent

saturation

kinetic and S o

concentration

o max. is about 4 0 g . I -1.

V %
Vm a x ,
100 ' m~

. . . . . . . . . . . . . . . . . . . . .max.
....

BO .

60

40

-r ....

=;.---

l
100

i
120

/
O

'
CI

Figure

2 -

1
~.0

T
40

I
60

i
80

> S o (g.1-1)

Variation of relative fermentation rates versus sugar concentrations

(So) in the worts when N.S. = 8 0 g.1-1 .

When N.S. concentrations are greater than 8 0 g . 1 - 1 , the inhibition of fermentation

rates can be plotted on a semi logarithmic scale versus dry matter (figure 3). W e o b s e r v e a
break of the slope for a dry matter concentration in worts of 2 0 0 g.1-1 .
Log (V..~= % )

So :
S o =

40 ~-1

So=
SO :

6Og.1-1

,~.
T

40

C ~A

9
9

,t

-~

"~0 g.i-~

8 0 gJ-~

gJ-'
S o = 1 2 0 ~.1-~
S O :100

80

120

160

200

240

280

t ]= D . M .

320

(g.l-~)

Figure 3 - Variation of relative fermentation rates versus dry matter concentrations

(D.M.). (V = Vmax.=100 % for maximal rate obtained for each serie So.)
These particular data lack biological significance (there being no single dry matter
composition linked to independent S o and N.S. distribution in the worts) but can be reanalysed
versus worts osmolalities (figure 4).

75

Log( V---~. %)

X
9
o
9
9

0,3

0,6

So = 20 g.j'~
So = 40 g.I-I
Sow 609.1-I
S 0 = 8Q ~.i - I
S O : 100 gj-;
SO ~ 120 i1.1"I

0,9

1,2

1,5

1,8

2,1

2,4

2,7

3,0

Figure 4 - Variations of relative fermentation rates versus osmolality (~m) of the

worts (V = Vmax.--100 % for maximal rate obtained for each value of So).
The osmotic inhibition of fermentation rates is observed for ~m = 1,5 osmol and S o acts on
the observed inhibition. Plotting the different slopes for ~m > 1,5 osmol as a function of S o
(figure 5) shows that sugar concentrations increase in a linear way the osmotic inhibition. The
synergistic inhibition between initial sugar concentration and medium osmolality on
fermentation rates can be described by the kinetic relation :

Vm,,x

e-(0,06

+ 6 , 3 3 . 1 0 - 3 S o ) ( ~ m - t ,5)

(relation

2)

Where Vmax. = V o max.(So / (K s + So))

(Vo max. obtained with : S o = 100 g.I"1 and N.S. = 80 g.1-1).

0,8

0,6

1,. S= (~.1 "L)

Figure 5 - Variation of inhibition linked to the osmolality of the medium (when

8m>1,5 osmol) versus initial sugar contents (So).
(The points correspond to differents slopes obtained on figure 4).
Figure 6 (a,b) represents respectively, final biomass and alcohol production obtained
in these experiments. If product formation (6b) is not altered to a great extent in our
experimental conditions, we can see, on the other hand, that final yeast populations (6a) is very
much affected by initial worts composition. The best final populations are obtained with S o
varying between 80 and 120 g.l "1 but there is no variation in specific growth rates which are
constant and maximum between S o -- 20 and 120 g.1-1 (1). We can also see, contrary to
relative rates of product formation, that N.S. = 80 g.1-1 is not the optimal concentration for
yeast growth.
76

Xt(~.l")

~
i i

i ~, i

9 so= 4o,j-,
o so= 8 o . - ,

~"%-

! i i !~"'~.

9 So. OO,,-.

so.~oo,,-,

9 so.,,~

1o

la

20

40

80

tJO

100

120

140

11s0

1~10

-'o

:aO

6a

'Q

'

;o

'

lea

1;~

,io

,~o

11o

.......

::o

(~j-'~

Figure 6 - Final productions of biomas Xf (6a) and ethanol P (6b) versus

sugar (So) and non sugar (N.S.) concentrations in the worts.

DISCUSSION
In the use of stillage recycle, the problem arises of how to get a sufficient growing
biomass on highly osmotic media. Our experimental data show that the limits of S o - N.S.
concentrations permitting optimal fermentation conditions are very narrow. The inhibition
threshold is observed for 200 g.1-1 dry matter in the wort or an osmolality of 1,5
osmol. For an osmolality of 3 osmols, no growth is observed, this value being comparable to
those obtain by other workers (Herman et al., 1980 ; Jones and Greenfield, 1986 ;
Koppensteiner and Windisch, 1971 ; Onishi, 1963).
When osmotic inhibition is present (1,5 < S m < 3 osmols), sugar concentrations have an
important impact but the best fermentation rates are obtained with the highest S O (see figure1).
For SO greater or equal to 80 g.1-1, the growth or the fermentative activity of the yeasts are
dependent only on wort osmolality, arising from the non sugar content of the medium. The
results presented are consistent with those previously obtained (Letourneau and Villa, 1987),
that is to say that a rapid filling of the substrate to the growth tank is favourable to the
fermentation process. Keeping in mind the critical point of 200 g.1-1 dry matter in the
medium, the leakage of a wort containing 80 g.I 1 sucrose to favour the growth, allows the
recycle of 50 g.1-1 of non sugar issued from stillage, which corresponds to an improvement in
water demand of about 30 to 50 %.

Research support from G#n6rale Sucri#re, Eppeville, France, is gratefully acknowkedged. The
authors would like to thank Dr. J. C. Slaughter for his advice.

SYMBOLS
Vo max. maximum fermentation rate observed, expressed as CO2 release by unit of time
(g.l-1 .h-1).
Vmax. maximum fermentation rate obtained with each S o (gco2.1 "1 .h-1).
V
fermentation rate of each fermentation test (gco2.1-1 .h "1).
initial sugar concentration (g.1-1).
So
N.S.
initial non sugar concentration (g.I"1).
D.M.
initial dry matter concentration (g.l "1).
initial osmolality of the media (osmol)
~m

77

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78