Enzyme and Microbial Technology 41 (2007) 576–582

Kefir-yeast technology: Industrial scale-up of alcoholic fermentation of

whey, promoted by raisin extracts, using kefir-yeast granular biomass
Athanasios A. Koutinas a,∗ , Ilias Athanasiadis b , Argyro Bekatorou a , Costas Psarianos a ,
Maria Kanellaki a , Nikolaos Agouridis a , Georgios Blekas b
a Food Biotechnology Group, Department of Chemistry, University of Patras, GR-26500 Patras, Greece
b Laboratory of Food Chemistry and Technology, Department of Chemistry,

Aristotle University, Thessaloniki 54006, Greece

Received 22 November 2006; received in revised form 20 April 2007; accepted 9 May 2007

Abstract
Industrial scale-up of whey fermentation, promoted by raisin extracts, using free kefir-yeast cells is reported. The fermented whey would be
exploited as raw material to produce kefir-like whey-based drinks, potable and fuel alcohol, as well as kefir-yeast biomass for use as baker’s yeast.
The scale-up process involved the development of a technology transfer scheme from lab-scale experiments to a successive series of increased
capacity bioreactors (100, 3000 and 11,000 L). The development of this technology was supported by the six-fold promotion of whey fermentation
by the addition of 1% black raisin extracts (optimum concentration found in this investigation), as well as by the formation of kefir-yeast in the
form of easily precipitated granular biomass that led to the avoidance of centrifugal separators, which are equipment of high cost. A cost analysis
showed a big reduction of the production cost of the aforementioned products in comparison with the market prices, attributed to the negligible
cost of raw materials (whey) and the granular biomass formation.
© 2007 Elsevier Inc. All rights reserved.

Keywords: Kefir; Whey; Alcoholic fermentation; Promotion; Scale-up; Raisin extracts

1. Introduction as suitable biocatalysts for lactose fermentation. Recombinant

Whey is the liquid effluent of the dairy industries and there-
fore it is produced in large capacities worldwide, creating
serious environmental pollution problems. However, intense
recent research attempts try to deal with the problem focus-
ing on the development of technologies that employ whey as
raw material to produce foods or chemicals of added value.
It is stressed that the production of a series of products is
necessary in order to manipulate effectively the world whey
capacity. Products such as SCP [1,2], lactic acid [3], citric acid
[4], enzymes [5], propionic acid [6], lactose and ethanol have
been proposed. In the case of ethanol production from lac-
tose, various research efforts have been undertaken the last two
decades. Yeasts like Kluyveromyces fragilis [7–9], and K. marx-
ianus [10,11], free or immobilized [12], have been proposed
∗ Corresponding author. Tel.: +30 2610 997104; fax: +30 2610 997105.
E-mail address: a.a.koutinas@upatras.gr (A.A. Koutinas).
DNA techniques [13,14] as well as processes employing co-
immobilized yeasts and enzymes, e.g. Saccharomyces cerevisiae
with beta-galactosidase [15], have been also examined. The
above efforts, showed the need for the selection of suitable bio-
catalysts and design of suitable large scale processes for the
effective utilization of lactose. In the frame of such efforts,
kefir-yeast culture, in granular biomass form, was recently found
suitable for alcoholic fermentation of whey [16]. Additionally,
the stability of a fermented kefir-cheese whey-milk mixture [17]
proved the possibility for kefir-like drink production based on
whey. These, in combination with the potential production of
potable and fuel alcohol from whey, makes the scale-up of alco-
holic fermentation of whey by kefir-yeast granular biomass very
attractive. Although there are numerous reports concerning alco-
hol production from whey in the literature, the scale-up of such
processes has not been reported. The results of Athanasiadis et
al. [16] and those of Paraskevopoulou et al. [17] encouraged
the industrial scale-up of alcoholic fermentation of whey using
kefir-yeast granular biomass, which is the aim of the current
investigation.

0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2007.05.013
 A.A. Koutinas et al. / Enzyme and Microbial Technology 41 (2007) 576–582
2. Materials and methods
2.1. Kefir-yeast culture and raw materials
The kefir-yeast culture, lactose synthetic media composition, and growth
process were recently reported [18]. Cheese whey was an effluent of the Greek
Dairy Company MEVGAL SA. It contained 51 g/L lactose. Sugar beet molasses
was a product of the sugar refinery located in Plati of Thessaloniki. Raisin
extracts were produced by extraction of Corinthiaki black currants (dried black
grapes, Corinth region, Greece) or golden Sultanina raisins (dried white grapes,
Achaia region, Greece). White Sultanina raisins and black currants contained
65 and 72.5% invertsugar, respectively, 13% dry matter (sugar excluded), var-
ious minerals, vitamins, amino acids, fibre, free fatty acids, lipids, phenolic
compounds, etc. The extraction was carried out at 70 ◦ C by mixing exactly
500 g of raisins with 1 L of hot tap water, until a raisin extract was obtained
having a density of 12◦ Be (specific gravity degrees of Baume hydrometer
scale).
2.2. Effect of raisin extracts on alcoholic fermentation of whey
Amounts of 1, 2, 3, 4 or 5% (v/v) black raisin extracts were added in 500 mL
glass bioreactors, containing 250 mL of whey and 6 g of suspended granular
kefir-yeast biomass. The pH was not adjusted, and the bioreactors were incu-
bated at 30 ◦ C. Three repeated batch fermentations were carried out under the
same conditions, by recovery of the precipitated granular biomass, and reuse
in the next fermentation batch, after removal of the excess amount to keep
the biomass concentration constant. The above experiments were performed to
estimate the effect of various amounts of black raisin extracts on the rate of
whey fermentation. A similar set of experiments was carried out using Sultan-
ina extracts. The effect of pH on the rate of whey fermentation was examined
in the presence of the optimum raisin extract concentration (1%). Five repeated
batch fermentations were carried out as described above, at various pH values
(3.5, 4.5, 5.5, 6.0 and 6.5). The effect of temperature was also examined, and fer-
mentations were also carried out at 30, 25, 20, 15, 10, 5 ◦ C in the same manner.
Finally, the operational stability of the biocatalyst was evaluated by conducting
35 repeated batch fermentations of whey at 30 ◦ C and pH 5.5 in the presence
of 1% of black raisin extracts. In all cases, fermentation times were monitored
to calculate productivity and yield, and samples were analyzed for ethanol and
residual lactose.
2.3. Pilot plant operations
2.3.1. Scale-up
The scale-up of the process was encouraged by the promising results obtained
in previous studies including lab-scale experiments concerning the alcoholic
fermentation of whey using kefir-yeast granular biomass [16], and the effect of
raisin extracts on the promotion of whey fermentation. The strategy adopted for
scale-up included (i) whey fermentation by kefir-yeast granules, (ii) addition of
1% black raisin extracts as promoter and (iii) successive scale-up of the system
in 100, 3000 and 11,000 L bioreactors.
2.3.2. Kefir-yeast granular biomass production
The preparation of kefir-yeast inoculates and granular biomass production
needed to pitch each of the 100, 3000 and 11,000 L bioreactors, were performed
as described in a previous paper [18].
2.3.3. Batch fermentations of whey by kefir-yeast granules promoted by
raisin extracts in the 100 L bioreactor
The 100 L bioreactor is shown in Fig. 1 and its construction is described in a
recent investigation [18]. The bioreactor was fed with 40 L of whey through
a peristaltic pump and then 0.4 L of 12◦ Be black raisin extract was added.
Subsequently, the temperature was adjusted at 30 ◦ C by wetting the bioreac-
tor externally with warm or cold tap water, and then the mixture was inoculated
with 1.5 kg of kefir-yeast granular biomass. The whole was allowed to ferment,
at constant temperature of 30 ◦ C, adjusted continuously by cooling or heating
with water. The pH was adjusted at 5.5 during fermentation, by the addition
577
Fig. 1. Bioreactors of 100 L (A) and 11,000 L (B) used for industrial scale-up
of the proposed technology.
of a sodium carbonate solution and the ◦ Be density was monitored at various
time intervals. After the end of fermentation the kefir-yeast granular biomass
was precipitated at the bottom of the bioreactor, and samples were collected for
ethanol and residual sugar determination. The precipitation of biomass required
about 1 h after the end of fermentation and then the supernatant fermented liquid
was decanted by one of the outlets near the bottom of the bioreactor. In order
to proceed to the next batch, 40 L of fresh whey were added and the process
was repeated again. A second feed of 40 L whey was added just after the end of
fermentation to increase the volume of the fermenting whey to 80 L.
2.3.4. Batch fermentations of whey by kefir-yeast granules promoted by
raisin extracts in the 3000 L bioreactor
The construction description and photo of the 3000 L bioreactor are available
in a recent investigation [18]. The bioreactor, which initially contained 30 kg
granular kefir-yeast biomass, was fed successively with 750, 1250, 1750 and
2250 L whey, in order to perform four repeated batch fermentations. In each
batch 1% (v/v) black raisin extracts of 12◦ Be were added. The temperature
was adjusted at 30 ◦ C by cooling or heating the supplied whey by a plate heat
exchanger. The system was allowed to ferment keeping the temperature constant
at 30 ◦ C by circulating the fermenting liquid through the plate heat exchanger for
cooling. The pH was also kept constant at 5.5 by addition of a sodium carbonate
solution. After the end of fermentation the fermented whey was allowed to stand
for at least 1 h for precipitation of the granular kefir-yeast biomass. Samples
were collected and analyzed for alcohol and residual sugar. Subsequently, the
fermented whey was decanted by an outlet at the base of the conical bottom
of the bioreactor. The granular kefir-yeast biomass was collected at an opening
of the top of the conical bottom of the bioreactor. To proceed to the second
fermentation batch and at the same time scale-up the process, 1250 L of whey
was pumped in the bioreactor and the fermentation was carried out as in the
first batch. In the same manner, repeated batch fermentations were successively
performed up to a final volume of 2250 L of whey.
2.3.5. Batch fermentations of whey by kefir-yeast granules promoted by
raisin extracts in the 11,000 L bioreactor
The 11,000 L bioreactor is shown in Fig. 1. It was constructed by De
Franceski Co. located at Halkida of Greece and installed in the factory of dairy
Co MEVGAL SA at Koufalia of Northern Greece. It was a cylindrical vessel of
3.5 m height and 2.0 m internal diameter, made from stainless steel AISI 304.
Its bottom was conical to collect the kefir-yeast granular biomass and it was fed
by an inlet at the base of the cone. A vertical glass tube fitted externally was
showing the whey level in the bioreactor. At the front, three glass windows and
three doors at various heights ensured observation of the fermentation at vari-
ous operating volumes. The bioreactor could be cooled or heated by externally
spraying with cold or warm water from a cyclic perforated pipe fitted at the top
of the bioreactor.
578 A.A. Koutinas et al. / Enzyme and Microbial Technology 41 (2007) 576–582

An amount of 300–500 L of fermented whey produced in the 3000 L biore- the bioreactor 3 was necessary. After the end of fermentation in the bioreactors
actor containing 100–120 kg kefir-yeast granular biomass was pumped in the 2 and 3, the granular biomass was allowed to precipitate and the supernatant
bioreactor of 11,000 L, and 2500 L of fresh whey and 25 L black raisin extracts fermented product was transferred to the tank 12.
were added. The temperature was adjusted at 30 ◦ C and the pH at 5.5, by addi-
tion of sodium carbonate solution, and was kept constant during fermentation.
2.5. Assays
The whey was allowed to ferment and samples were collected at various time
intervals, for determination of the ◦ Be density, to monitor the fermentation kinet-
Sugar and ethanol concentrations in all samples were determined by HPLC
ics. Before the end of fermentation 2000 L of whey containing 1% black raisin
as described in a recent paper [18]. Ethanol productivity expressed in g/L h and
extracts were pumped in and the fermentation was continued. In the same man-
ethanol yield factor was expressed as gram ethanol produced per gram of sugar
ner, another amount of 2000 L whey containing 1% black raisin extracts was
utilized (g/g). Conversion (%) was calculated by subtracting the residual from the
pumped in the bioreactor. After the end of each fermentation batch, samples
initial sugar concentration, dividing the result by the initial sugar concentration
were collected and analyzed for alcohol and residual sugar.
and multiplying by 100.

2.3.6. Batch fermentations of mixtures of whey and molasses by kefir-yeast

granules in the 3000 L bioreactor
Five hundred liters of whey containing 1% black raisin extracts and 15 kg of
kefir-yeast granular biomass were pumped in the 3000 L bioreactor. The temper-
ature was adjusted at 30 ◦ C and the mixture was allowed to ferment. When the
fermentation was completed molasses was pumped in, through a pump suitable
for viscous liquids, to increase the density of the fermenting mixture by 2◦ Be.
After the completion of fermentation the product remained without agitation for
precipitation of the granular kefir-yeast biomass. The supernatant was decanted
and two more repeated batch fermentations were performed, in the same way,
using the same granular biomass. The products were analyzed for ethanol and
residual sugar.
2.4. Pilot plant operations
The process flow sheet of the pilot plant operations is shown in Fig. 2. The
granular biomass required to pitch the 11,000 L bioreactor was produced by aer-
obic fermentation in the 3000 L bioreactor. Scale-up was developed starting from
whey fermentations at lab-scale and successively in the 100, 3000 and 11,000 L
bioreactors numbered in the process flow sheet as 1, 2 and 3, respectively. Whey
contained in tank 4 was supplied to bioreactors using the pump 7 for both aero-
bic fermentation for biomass production and anaerobic bioconversion of whey.
The biomass needed for the alcoholic fermentation in the 100 L and the 3000 L
bioreactors was produced in the 100 L bioreactor. Aerobic fermentations were
performed supplying air through the sterile filter 5 using the air pump 6. Cooling
or heating the 3000 L bioreactor 2 was done using the plate heat exchanger 13.
The biomass produced in the bioreactor 2 could be pumped using the pump
9 directly in the bioreactor 3 (11,000 L) or be mixed with the raw material in
the whey-tank 4. The bioreactor 3 could be sterilized by the steam line 14 and
cooled or heated by the water line 15. The produced granular biomass could
be transferred from the bioreactor 3 to the kefir-tank 11 when sterilization of
3. Results and discussion
3.1. Promotion of fermentation by raisin extracts
After the observation that black raisin extracts promote the
alcoholic fermentation of whey, the effect of raisin extract con-
centration, temperature and pH were further examined. The
results are presented in Tables 1–3. Three repeated batch fer-
mentations of whey enriched with 1, 2, 3, 4 or 5% black raisin
extract were performed using kefir-yeast granules (Table 1).
The addition of black raisin extracts increased productivity by
more than six-fold, ethanol yield factor at least 10% and conver-
sion about 5% compared to results obtained in previous studies
involving kefir-yeast cells produced in synthetic media (not in
granular form) [16] or immobilized cells of K. marxianus [10].
These improvements were obtained by addition of only 1%
raisin extracts, while the increase of extract concentration did not
increase further the rate of fermentation. The promotion effect
of black raisin extracts was not obtained in the case of Sultanina
extracts where no substantial reduction of fermentation times
or improvement of other fermentation kinetics was obtained as
illustrated in Fig. 3. The effect of temperature on the fermenta-
tion of whey enriched with 1% black raisin extract is presented
in Table 2. Five repeated batch fermentations were carried out at
30, 25, 20, 15 and 10 ◦ C. The ethanol concentration, yield and
sugar conversion were not affected significantly in that range of
temperatures, while at lower temperatures (0–5 ◦ C) the fermen-
tation did not proceed. The effect of pH is reported in Table 3.

Fig. 2. Process flow sheet of the alcoholic fermentation of whey, promoted

by raisin extracts, using kefir-yeast granules. (1) Hundred liter bioreactor, (2)
3000 L bioreactor, (3) 11,000 L bioreactor, (4) whey tank, (5) sterile filter, (6)
air pump, (7) pump, (8) pump, (9) pump, (10) molasses tank, (11) kefir-yeast
tank, (12) fermented product tank, (13) plate heat exchanger, (14) steam line, Fig. 3. Effect of black raisin and golden Sultanina extracts on the kinetics of
and (15) water line. alcoholic fermentation of whey, using kefir-yeast granules.
 A.A. Koutinas et al. / Enzyme and Microbial Technology 41 (2007) 576–582 579

Table 1
Effect of black raisin extract addition on the kinetic parameters of repeated batch alcoholic fermentations of whey using kefir-yeast granules
Raisin extract Batch Initial density Fermentation Residual Ethanol (%, v/v) Ethanol productivity Yield Conversion
(%, v/v) (◦ Be) time (h) sugar (g/L) (g/L h) (g/g) (%)

0 1–3 3.3 50.0 ± 2.0 3.31 ± 0.25 2.00 ± 0.10 0.32 ± 0.02 0.34 ± 0.02 93.4 ± 0.5
1 1–3 3.4 10.0 ± 1.0 1.10 ± 0.17 2.50 ± 0.00 1.99 ± 0.20 0.39 ± 0.00 97.9 ± 0.3
2 1–3 3.6 10.3 ± 1.5 1.88 ± 0.27 2.43 ± 0.06 1.89 ± 0.28 0.37 ± 0.01 96.5 ± 0.5
3 1–3 3.7 9.3 ± 1.2 2.35 ± 0.82 2.27 ± 0.15 1.95 ± 0.38 0.33 ± 0.02 95.8 ± 1.5
4 1–3 3.8 11.3 ± 1.5 1.78 ± 0.81 2.40 ± 0.10 1.72 ± 0.23 0.34 ± 0.01 96.9 ± 1.4
5 1–3 4.0 9.7 ± 0.6 1.58 ± 0.57 2.50 ± 0.10 2.05 ± 0.07 0.34 ± 0.01 97.4 ± 1.0

Table 2
Effect of temperature on the kinetic parameters of batch alcoholic fermentations of whey containing 1% black raisin extract using kefir-yeast granules
Temperature Initial density Batch Fermentation Residual Ethanol (%, v/v) Ethanol productivity Yield Conversion
(◦ C) (◦ Be) time (h) sugar (g/L) (g/L h) (g/g) (%)

30 3.4 1–5 9.8 ± 0.8 1.27 ± 0.28 2.54 ± 0.05 2.07 ± 0.19 0.40 ± 0.01 97.6 ± 0.5
25 3.4 1–5 21.0 ± 2.5 5.29 ± 0.64 2.28 ± 0.13 0.87 ± 0.12 0.39 ± 0.02 89.8 ± 1.2
20 3.4 1–5 40.8 ± 3.7 5.77 ± 1.05 2.30 ± 0.10 0.45 ± 0.05 0.40 ± 0.02 88.9 ± 2.0
15 3.4 1–5 70.8 ± 4.2 5.18 ± 1.57 2.14 ± 0.21 0.24 ± 0.03 0.36 ± 0.03 90.0 ± 3.0
10 3.4 1–5 119.6 ± 9.4 2.90 ± 0.51 2.30 ± 0.07 0.15 ± 0.01 0.37 ± 0.01 94.4 ± 1.0

Five repeated batch fermentations of whey containing 1% black
raisin extract were carried out at each pH value of 6.5, 6.0, 5.5,
4.5 and 4.0, showing that the optimum pH for whey conversion
by kefir-yeast granular biomass in the presence of raisin extracts
is 5.5. At that pH, the lowest fermentation time, and the highest
productivity, yield and conversion were observed.
Finally, the operational stability of the process was shown by
35 successive batch fermentations of whey enriched with 1%
black raisin extract at 30 ◦ C and the results are summarized in
Table 4. It was found that the system required about 10 fer-
mentation batches until its behaviour was stabilized, and after
that point, the fermentation kinetics remained constant for a long
period of time recycling the same granular biomass, without any
loss of activity.
3.2. Scale-up at industrial scale
The promising results obtained by the study of the effect of
raisin extracts on whey fermentation at lab-scale, encouraged
the industrial scale-up of the process. In each of the 100 and
3000 L bioreactors, four repeated batch fermentations of whey
containing 1% black raisin extract were performed at 30 and
20 ◦ C, while in the 11,000 L bioreactor, fed-batch fermentation
was carried out. The results are presented in Table 5. In the case
of the 100 L bioreactor, the results verified the observations at
lab-scale encouraging experiments at higher scale. Comparing
the kinetic parameters recorded at the two temperatures, it is
obvious that better results were obtained at 30 ◦ C. In the 3000 L
bioreactor batch fermentations of 750, 1250, 1750 and 2250 L
of whey containing 1% black raisin extract were carried out suc-
cessively. The ethanol concentrations obtained were similar to
those of lab-scale experiments, while conversion was higher than
75% and yield higher than 0.40 g/g, showing potential industrial
application. The increased fermentation times, though, may be
attributed to the lower biomass concentrations, since the biomass
needed for the first fermentation batch was produced aerobically
into the bioreactor, but from that point on, the biomass of each
anaerobic fermentation was used for the next fermentation batch.
Likewise, three fed-batch fermentations were carried out in the
industrial-scale bioreactor of 11,000 L. After the end of fermen-
tation, very small amounts of residual glucose and galactose
were found, while residual lactose concentrations were about
10 g/L, therefore conversion was not higher than 80%. Fermen-
tation times were about similar, while ethanol concentrations
(∼2%, v/v) were a little lower than those obtained in the 100 L
bioreactor, but equal to those recorded in the 3000 L bioreactor.
The above results, demonstrate that kefir-yeast granules are effi-
cient biocatalysts for alcoholic fermentation of whey, in batch as
well as fed-batch operation, with satisfactory fermentation times
and conversions even at large capacities. In Fig. 4, a compari-

Table 3
Effect of pH on the kinetic parameters of batch alcoholic fermentations of whey containing 1% black raisin extract using kefir-yeast granules
pH Initial density Batch Fermentation Residual Ethanol Ethanol productivity Yield (g/g) Conversion
(◦ Be) time (h) sugar (g/L) (%, v/v) (g/L h) (%)

6.5 3.4 1–5 35.4 ± 3.9 7.05 ± 0.95 2.12 ± 0.13 0.47 ± 0.08 0.37 ± 0.02 86.4 ± 1.8
6 3.4 1–5 24.8 ± 2.6 4.33 ± 0.80 2.14 ± 0.05 0.69 ± 0.09 0.36 ± 0.01 91.6 ± 1.6
5.5 3.4 1–5 9.8 ± 0.8 1.27 ± 0.28 2.54 ± 0.05 2.07 ± 0.19 0.40 ± 0.01 97.6 ± 0.5
4.5 3.4 1–5 14.3 ± 2.3 3.48 ± 1.86 2.22 ± 0.19 1.05 ± 0.47 0.36 ± 0.02 93.3 ± 3.6
3.5 3.4 1–5 17.6 ± 2.1 6.28 ± 1.99 2.08 ± 0.13 0.95 ± 0.12 0.36 ± 0.02 88.0 ± 3.8
580 A.A. Koutinas et al. / Enzyme and Microbial Technology 41 (2007) 576–582

Table 4
Kinetic parameters of the repeated batch alcoholic fermentations of whey containing 1% black raisin extract, using kefir-yeast granules (operational stability)
Batch Initial density Fermentation Residual Ethanol Ethanol productivity Yield Conversion
(◦ Be) time (h) sugar (g/L) (%, v/v) (g/L h) (g/g) (%)

1 3.4 18 9.81 1.5 0.66 0.28 81.1

5 3.4 15 8.92 1.9 1.00 0.35 82.8
Mean ± S.D. 17 ± 2 9.37 ± 0.63 1.7 ± 0.3 0.83 ± 0.24 0.32 ± 0.05 82.00 ± 1.2

10 3.4 8 4.60 2.5 1.65 0.43 91.2

15 3.4 8 2.54 2.5 2.48 0.40 95.1
20 3.4 8 2.61 2.6 2.57 0.42 95.0
25 3.4 9 2.12 2.6 2.29 0.41 95.9
30 3.4 9 1.85 2.5 2.20 0.39 96.4
35 3.4 10 1.76 2.6 2.06 0.41 96.6
Mean ± S.D. 9±1 2.58 ± 1.05 2.6 ± 0.1 2.21 ± 0.33 0.41 ± 0.01 95.0 ± 2.00

Table 5
Kinetic parameters during the scale-up of the alcoholic fermentations of whey containing 1% (v/v) raisin extract, using kefir-yeast granules
Bioreactor Temperature Whey Initial density Fermentation Residual Ethanol Ethanol productivity Yield Conversion
(process) (◦ C) (L) (◦ Be) time (h) sugar (g/L) (%, v/v) (g/L h) (g/g) (%)

30 40 3.2 10 1.7 2.5 1.95 0.52 95.6

30 40 3.4 08 1.2 2.6 1.96 0.54 90.5
30 40 3.5 10 1.5 2.5 2.54 0.49 97.2
100 L
(batch) Mean ± S.D. 3.4 ± 0.2 9.3 ± 1.2 1.5 ± 0.3 2.5 ± 0.1 2.15 ± 0.34 0.52 ± 0.03 94.4 ± 3.5
20 40 2.9 36 19.8 1.2 0.28 0.52 48.3

30 750 3.6 25 11.2 2.0 0.64 0.42 77.1

30 1250 3.0 21 11.9 1.9 0.71 0.39 76.2
3000 L 30 1750 3.0 24 7.2 2.2 0.73 0.40 85.8
(batch) 30 2250 3.0 22 9.8 2.1 0.75 0.40 80.8
Mean ± S.D. 3.2 ± 0.3 23.0 ± 1.8 10.0 ± 2.1 2.1 ± 0.1 0.71 ± 0.05 0.40 ± 0.01 80.0 ± 4.4

30 3000 3.6 18 12.8 2.0 – – –

11000 L 30 5000 3.1 12 12.7 2.0 – – –
(fed- 30 7000 2.7 12 11.9 2.1 – – –
batch)
Mean ± S.D. 3.1 ± 0.5 14.0 ± 3.5 12.5 ± 0.5 2.0 ± 0.1 – – –

son of fermentation kinetics in the three bioreactors is attempted,

showing that fermentation is conducted faster in the 100 L biore-
actor, which may be attributed to the better distribution, of kefir
biomass in the fermenting liquid. Nevertheless, fermentation
times in the larger scale bioreactors did not exceed 20 h, which is
readily accepted by industrial practice. Bioreactor design for bet-
ter biomass distribution, stirring and substrate/product diffusion
is needed to further optimize these findings.

3.3. Enhancing alcohol production

Since the products of fermentation could be used for potable

or fuel alcohol production, the alcohol concentrations obtained
during the fermentations of whey were considered low. To
increase alcohol concentration, fermentations of whey by addi-
tion of molasses were proposed (to increase initial density by
2–4◦ Be), which were performed in the bioreactor of 3000 L.
The results, shown in Table 6, prove the possibility to increase Fig. 4. Effect of scale-up on the kinetics of alcoholic fermentation of whey
the final alcohol concentration to 5.0% (v/v). A similar prac- containing 1% (v/v) black raisin extracts, using kefir-yeast granules.
 A.A. Koutinas et al. / Enzyme and Microbial Technology 41 (2007) 576–582 581

Table 6
Kinetic parameters of the repeated batch alcoholic fermentations of mixtures of whey and molasses, using kefir-yeast granules
Bioreactor Temperature Whey Density after addition Fermentation Residual sugar Ethanol
(L) (◦ C) (L) of molasses (◦ Be) time (h) (g/L) (% v/v)

3000 30 500 3.1 20 14.2 3.7

3000 30 500 4.2 24 11.4 4.6
3000 30 500 5.2 38 13.1 5.0

Mean ± S.D. 4.2 ± 1.1 27 ± 9 12.9 ± 1.4 4.4 ± 0.7

tice was adopted by Zakrzewski and Zmarlicki [19], who added
molasses after the end of whey fermentation (by K. marxianus)
to increase ethanol production by following fermentation with
S. cerevisiae. The use of both microorganisms for simultane-
ous fermentation of mixture of whey and molasses did not yield
good results.
3.4. Economic evaluation
Table 7 summarizes the production costs, investments and
added value within the European Union and Greece for com-
panies that would utilize whey to produce kefir drink, potable
and fuel-grade alcohol. The results clearly show a big differ-
ence between market prices and estimated production costs for
all products that could be produced via the proposed kefir-yeast
technology. The added value created in the EU in the case of
a kefir-like drink production is much higher, compared with
potable and fuel-grade alcohol. As the results show, the required
investments for such product could be depreciated within a
period of 1 year. The big differences between market prices and
production costs can be attributed to the low cost of whey, the
lower costs for labour and electricity and the reduction of the
investment due to the formation of biomass in granular form,
leading to avoidance of centrifugal separators (machineries of
high cost). The increase of production cost and investment in
the case of fuel-grade alcohol is attributed to the additional cost
needed to produce the anhydrous alcohol, which is necessary
for automobiles.
3.5. Extension and exploitation of the kefir-yeast-based
technology
A recent investigation [18] reported a kefir-yeast based tech-
nology for the production of kefir-yeast biomass for use as
baker’s yeast, protein livestock feed for animals, and food emul-
sifiers. The technology was based on the formation of kefir-yeast
in the form of granular biomass that reduced production cost.
This technology is further extended, in the frame of this inves-
tigation, by the production of a whey-based drink, simulating
the traditional kefir drink, and of potable and fuel-grade alco-
hol, whose production was also based on kefir-yeast granular
biomass. However, for cost effective production of the above
products, the promotion of alcoholic fermentation by the addi-
tion of 1% black raisin extracts was needed, as reported in more
detail in the frame of this work. This important finding, made
the extension of kefir-yeast technology feasible, as proved by
the good results of the scale-up of the process.
The scale-up of the process showed an increase of the fer-
mentation time as the volume increased from 100 to 3000 L
(Fig. 4). This can be attributed to mass transfer phenomena,
which could be avoided by better mixing and suitable bioreac-
tor design. The high operational stability of the system, proved
by repeated batch fermentations, as well as further reduction of
cost (estimated to 25%) that could be obtained by the use of
the discarded kefir-yeast biomass as alternative baker’s yeast,
would further facilitate industrial application and exploitation
of the proposed technology. Also, since an alcoholic degree of
2.5% (v/v) is considered low for alcohol distillation (to reduce
the energy demand in an alcohol production plant), there are two
options to enhance alcohol production from whey. The first is to
add molasses just after the end of whey fermentation and to pro-
ceed in a second fermentation using the same kefir-yeast granular
biomass. The addition of molasses follows the fermentation of
whey in order to avoid inhibition problems. The produced fer-
mented liquid containing 5.2% (v/v) alcohol could further be
mixed with fermented molasses (8%, v/v). The second option is
to conduct whey fermentation and then add molasses to the fer-
mented product and finally ferment with S. cerevisiae. Finally,
the highest added value between the six possible products of the
proposed kefir-yeast technology would be created by the kefir

Table 7
Products and estimation of costs as well as added value created in EU and Greece by the alcoholic fermentation of whey using kefir-yeasts
Product Market price Construction costa Production Annual production Added value in Added value in
(D /kg) in Greece (D ) cost (D /kg) capacity in Greece (m3 ) Greeceb (D /year) EU (D /year)

Potable alcohol 0.74 8,000,000 0.26 21,000 12.4 × 106 600 × 106
Kefir drink 0.70 8,000,000 0.25 700,000 500 × 106 25 × 106
Fuel-grade alcohol 0.80c 9,000,000 0.28 21,000 12.4 × 106 600 × 106
a Annual production capacity of whey in Greece estimated to 700,000 m3 .
b Estimated taking into account the production capacity of whey in Greece.
c Market price of gasoline in Greece.
582 A.A. Koutinas et al. / Enzyme and Microbial Technology 41 (2007) 576–582
whey-based drink, due to the high annual production capacity
of whey.
3.6. Technological consideration and conclusions
The proposed technology is environmentally significant due
to the fact that a very polluting liquid industrial waste is
employed to produce various products of added value, including
the use of discarded kefir-yeast biomass as alternative baker’s
yeast. The feasibility of this technology was enhanced by the
finding of the promoting effect of black raisin extracts on the
alcoholic fermentation of whey, validated by scale-up of the pro-
cess at industrial level. Moreover, the use of kefir-yeast biomass
in granular form would provide the possibility to eliminate the
use of centrifugal separators, which are traditionally used in
alcohol production plants and are equipment of high energy
demand, investment and labour costs. Therefore, the two key
points for industrial application of the proposed technology is
the promotion of fermentation by raisin extracts and the forma-
tion of granular biomass. However, for successful application of
the proposed kefir-yeast technology, other requirements, such as
resistance to contaminations, and good organoleptic properties
of the products are necessary, which were both met during lab
and industrial scale experiments. Finally, the big differences of
production costs from market prices are also of high importance
for the industrialization of the proposed kefir-yeast technology.
Acknowledgments
The authors acknowledge financial support of this work with
750,000 euros by Greece General Secretarial of Research and
Technology. Also, the authors acknowledge the Greek Dairy
Company MEVGAL S.A. (Grant participant) which provided
the industrial experience and facilities.
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