Appl Microbiol Biotechnol (1986)24:140--143
Microbiology
Short contribution
Wine-making by cell-recycle-batch fermentation process*
Gianfranco Rosini
Istituto di Biologia Vegetale Sezione di Microbiologia, University of Perugia, Borgo XX Giugno no. 74, 1-06100 Perugia, Italy
Summary. In order to check the overall validity
of more efficient fermentation systems to reduce
wine-making costs, we carried out an off-skins
fermentation of clarified Trebbiano toscano
grape-juice, making use of a non-conventional
"cell-recycle-batch fermentation" process. The re-
suits showed that the process causes a reduction
of the fermentation length as well as an improve-
ment in ethanol productivity and yield and can be
conveniently applied to the production of ordi-
nary table wines.
Introduction
Wine-making is generally carried out by coven-
tional batch fermentations. In fact, despite their
name, continuous wine-fermentors in Italy and in
other European countries are not used in order to
optimize fermentation kinetics but to increase the
extraction of pigments from red grape skins (Kun-
kee and Goswell 1977).
Since in conventional batch fermentations
ethanol productivity is rather low, various new
techniques of fermentation have been proposed in
order to maximize efficiency in the ethanol indus-
try as well as in the production of beer and other
alcoholic beverages (Jones et al. 1981; Maiorella
et al. 1981). Continuous systems are not em-
ployed, since they would require a complete re-
versal of traditional wine-making procedures.
More acceptable appears to be another non-
conventional technique, the "cell-recycle-batch
* This work was supported in part by a grant from the Italian
C.N.R. (Consiglio Nazionale delle Ricerche)
Offprint requests to: Gianfranco Rosini
Applied
Biotechnology
© Springer-Verlag 1986
fermentation" (CRBF), whose principal innova-
tion is the multiple successive use of the same
yeast starter in different batch fermentations
(Jones et al. 1981). Unlike continuous systems, the
C R B F does not require radical changes in the wi-
nery procedures nor does it imply capital invest-
ment in new equipment. In fact, yeast cells to be
recycled can be recovered through natural sedi-
mentation, or by filtration or centrifugation with
machines already present in most wineries. The
objective of this work was to examine the overall
effects of repeated cell reuse in an off-skin natural
grape must fermentation.
Materials and methods
Microorganism. A Saccharomyces cerevisiae strain selected for
wine-making and labelled for not producing hydrogen sul-
phide (H2S-) was used as fermentation starter.
Fermentation conditions. Green grape juice of the cultivar
Trebbiano toscano (sugar content 180 g / l ; p H = 2.9) was clari-
fied with a rehydrated commercial powder (micronized potas-
sium caseinate; food grade animal gelatine; bentonite 3 × ;
colloidal soil) and treated with 200 mg/1 sulphur dioxide.
After clarification (48 h at 8 ° C) it was stored at 6 ° C. A sche-
matic diagram of the cell-recycle batch culture is shown in Fig.
1.
The precultured S. cerevisiae (H2S-) was inoculated into
a 14-1 Chemap fermentor (working volume 10 1) to give an ini-
tial cell concentration of 0.32 g/1 (dry wt.). After the first batch
culture, the fermented medium was thoroughly discharged and
the cells were recovered by centrifugation (3000 rpm at 5°C
for 15 min), washed in sterile water and reused as fermenta-
tion starter.
All batch cultures were carried out at 27°C under gentle
agitation, and centrifuged wines were stored at 27 ° C up to the
end of fermentation.
Analyses. Cell concentration was measured as dry weight. For
dry weight determination the sample was filtered on pre-
weighed filters (millipore membrane; pore size 0.45 Ixm),
G. Rosini: Fast fermentation in wine-making 141

1/!, Fig. 1. Schematic diagram of the cell-recycle-batch cul-

ture system
1, grape juice reservoir; 2, feed pump; 3, fermentor; 4,
centrifugal apparatus; 5, centrifuged wine; 6, cell mass
reused as fermentation starter

washed with distilled water and dried at 100°--105°C for
24 h, to constant weight.
Sugar content was determined by the method of Dische
(1955), ethanol concentration by the technique of Rebelein
(1980). Other fermentation characteristics were evaluated ac-
cording to the indications of Fiechter (1981)•
The presence of wild yeasts was checked by replica-plat-
ing the colonies from an isolation culture on Malt-Agar onto
Difco Batco ABY (Acid Bismuth yeast agar medium -0636)
where labelled starter colonies are white and those of wild
strains are black• The frequency of white over total colonies
was evaluated after incubation at 25 °C for 48 h (Rosini 1984).
The presence of bacterial contamination was checked by mi-
croscopic observation.
The percentage of new cells (Xo/o)was determined by the
ratio between biomass produced and final cell mass at the end
of a single fermentation. Analytical characteristics of the wines
were determined according to the Italian Official Methods for
Wines and Vinegar (1958).
Results and discussion
Grape juice clarification treatment produced a
substrate without substances in suspension and
consequently an easy cell mass recovery was pos-
sible. Throughout all the fermentations carried
out in this work no wild yeast and no bacterial
contaminations were detected. In Fig. 2 A - - C the
concentrations of sugar, ethanol and the evolu-

200

13°
120

10 ~

i •
• ~J
~
, I
~
8
0 10 20 30 5 0 5
Culture time ( l)

60

40
Fig. 2 A - - D . Profile of wine
making by cell-recycle-batch fer-
mentation
A Typical batch culture; B 4th
5,o
B
batch culture; C 7th batch cul-
ture; D ethanol production by re-
cycled cells.
Symbols: • ethanol; • sugar; •
cell mass
0 20 40 60 80
Running time (h)
142 G. Rosini: Fast fermentation in wine-making

tion of cell mass in the 1st, 4th and 7th batch cul-
tures are indicated. In the 1st cultures (Fig. 2A)
(typical batch) the time required to produce 80 ml
ethanol was 31 h, due to the low cell mass concen-
tration (7.60 g/1 dry wt.). The cell concentration
increased progressively up to the 7th cycle, where
a final cell concentration of 28.40 g/1 dry wt. was
observed.
Fermentation length was consistently reduced
at higher cell concentration (Table 1, Fig. 2D);
after the 6th batch culture, the time required for
one operation was 7 h.
The rate of ethanol production was also
greatly improved under these conditions. In fact,
during the steady state of the operation, reached
after the 6th batch, alcohol productivity was
11.44 g/1 per hour, approximately five times the
rate of a traditional batch fermentation (lst cycle).
This improved ethanol yield with our CRBF sys-
tem may be due to a lower rate of new cells pro-
duction (Xo/o), a low cell biomass yield (Yx) and a
growth rate reduction (Ix).
Analyses carried out on wines after the end of
the fermentation showed (Table 2) the absence of
any detectable differences in alcohol content and
total acidity compared with wines produced by
traditional batch fermentation.
However, wine-making by "cell reuse" re-
suited in a reduction of SO2 content and in a
higher quantity of volatile acids, not caused by
bacterial contaminations.
The above results confirmed the possibility of
obtaining better fermentation kinetics by the use
of the cell-recycle batch fermentation process as
in off-skins wine-making. The rate of sugar trans-
formation and consequently the ethanol produc-
tivity are favourably affected. Fermentation costs
could be considerably decreased due to an etha-
nol productivity which is five times that of a batch
fermentation process. In fact, for the same work-
ing capacity a winery would reduce capital invest-
ment and probably also management costs as a
consequence of more rational utilization of wi-
nery equipment and labour. Thus the cell-recycle-

Table 1. Effect of cell-recycle-batch culture on different kinetics parameters of fermentation

Fermentation Fermentation cycles

parameters
1 2 3 4 5 6 7

Fermentation time (h) 31.5 13 11 9 8.5 7 7

Presence of labelled yeast (%) 100. 100 100 100 100 100 100
X0= Starting cell mass (g/l) 0.32 5.26 11.12 16.18 20.70 25.08 25.20
Xt = Final cell mass (g/l) 7.60 10.96 17.12 21.78 26.90 27.58 28.40
Xp = Cell mass produced (g/l) 7.27 5.70 6.00 5.60 6.20 2.50 3.20
X0/0=Percentage of new cells (%) 95.65 52.00 35.05 25.71 23.05 9.06 11.26
S = S u g a r utilized (g/l) 156.30 135.00 153.30 165.00 161.30 165.00 155.00
Eth= Ethanol produced (ml/l) 77.10 72.50 79.90 88.90 84.90 84.70 80.10
YE~h= Ethanol yield (ml/g) 0.49 0.54 0.52 0.54 0.53 0.51 0.52
QEth = Ethanol productivity
( m l / l h - 1) 2.45 5.57 7.26 9.87 9.98 12.10 11.44
# = Growht rate (h - ~) 0.077 0.061 0.042 0.040 0.045 0.016 0.018
(9--22) a (1--13) (0--5.5) (1--6) (1--3.5) (1--7) (1--7)

a Time period of/.t calculation

Table 2. Analytical characters of wines obtained by cell-recycle fermentation systems

Analytical Wines
characters
1 2 3 4 5 6 7

Ethanol (% vol) 8.5 9.2 9.3 9.1 9.0 9.2 9.1

Volatile acidity (g/l) 0.24 0.31 0.42 0.42 0.50 0.52 0.52
Total acidity (g/l) 9.37 9.30 9.00 9.16 9.22 9.37 9.20
Total SO2 (mg/1) 121.18 88.00 86.96 87.14 87.36 73.92 72.96
Free SO2 (mg/1) 3.10 2.88 2.56 2.54 2.56 2.24 1.60
pH 2.83 2.82 2.91 2.94 2.90 2.93 2.96
G. Rosini: Fast fermentation in wine-making
batch fermentation process could be a more con-
venient method for the production of ordinary ta-
ble wines a n d / o r wines for compulsory distilla-
tion, even if they require the use of highly clad-
fled musts.
Acknowledgements. We thank F. Maccarelli for his technical
assistance.
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Received September 6, 1985/Revised December 2, 1985