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Bioresource Technology 101 (2010) 2837–2844

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Production of probiotic biomass (Lactobacillus casei) in goat

milk whey: Comparison of batch, continuous and fed-batch cultures
E.J. Aguirre-Ezkauriatza, J.M. Aguilar-Yáñez, A. Ramírez-Medrano, M.M. Alvarez *
Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Ave. Eugenio Garza Sada 2501 sur. Monterrey, N.L., C.P. 64849, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: This contribution examines the technical feasibility of producing high added value probiotic biomass
Received 13 August 2008 from deproteinized and non-supplemented milk whey. The kinetics of growth of Lactobacillus casei in
Received in revised form 15 October 2009 deproteinized goat milk whey was analyzed. Experiments in batch, continuous and fed-batch conditions
Accepted 21 October 2009
were conducted in a 3 L fully instrumented bioreactor. Final substrate and biomass concentrations, yields
Available online 29 December 2009
and productivities are reported for different culture strategies. A kinetic analysis was conducted to char-
acterize biomass production, product inhibition effects, and substrate consumption rates. Due to the
Keywords:
strong product inhibition, fed-batch cultures at high biomass concentration rendered higher productivity
Lactobacillus casei
Whey
(0.45 g L1 h1) than batch and continuous cultures (0.11 g L1 h1), complete lactose conversions (<1.0 g
Kinetics of lactose/L at the end of each fed-batch cycle), and a product with higher viable cell counts (2  1010 cell/
Fed-batch g of freeze-dried product). Based on our result, high-cell density fed-batch strategies are recommended
Probiotic for commercial production of probiotic L. casei biomass.
Ó 2009 Published by Elsevier Ltd.

1. Introduction
Milk whey, a byproduct of cheese making, is a rich substrate
that has been suggested for many applications including solid
enrichment in cheese manufacture, bacterial or yeast growth med-
ium to produce biomass, animal nutrients supplement, source of
added value proteins, among others. However, in many countries,
whey continues to be sub-utilized. In some instances, it is consid-
ered a serious ecological problem due to its high pollutant poten-
tial. In México, only 10% of the whey generated is recovered and
used, mainly for low value applications (animal feeding purposes).
A simple, robust, scalable, and low-cost process capable of using
non-supplemented whey to convert it into a high added value
product such as probiotics would be attractive for commercial
implementation. There is growing scientific evidence that specific
strains of probiotic microorganisms confer health benefits on the
host and are safe for human use (see for example recent reviews
by Reid and Hammond (2005), Meier and Steuerwald (2005),
Senok et al. (2005), Pham et al. (2008)).
Probiotics continue to find niches in both the food and pharma-
ceutical industries. In this context, an almost obvious high added
value venue for the use of milk whey is the production of probiotic
lactic acid bacteria. Whey has been used to culture lactic bacteria,
but mainly for lactic acid production rather than biomass genera-
tion (Lund et al., 1992; Youssef and Goma, 2005; Shahbazi et al.,
* Corresponding author. Tel.: +52 8183 28 4131; fax: +52 8183 28 4136.
E-mail address: Mario.Alvarez@itesm.mx (M.M. Alvarez).
2005; Altiok et al., 2006; Panesar et al., 2007; Agarwal et al.,
2008). In this contribution we present experimental results from
batch, continuous and fed-batch fermentation experiments where
non-supplemented, non-diluted, ultra-filtered whey was used for
the production of probiotic biomass of Lactobacillus casei.
L. casei strains are lactic acid bacteria with a remarkable pheno-
typic and genotypic variability (Cai et al., 2007, 2009) that colonize
diverse ecological niches, among them, the human gastrointestinal
tract (Kinoshita et al., 2007), and have found broad commercial
applications. L. casei has receive attention as a probiotic due to
evidence of antimicrobial (Ingrassia et al., 2005; Brink and Todo-
rov, 2006; Nemeth and Fajdiga, 2006); anti-diarrheal (Isolauri,
1991; Srinivasan and Meyer, 2006); and anti-mutagenic effects
(Wendakoon et al., 2007). In addition, some studies have pointed
to effects on cholesterol levels (Liong and Shah, 2006; Park and
Kim, 2008); on the immune system (Matsuzaki et al., 2007;
Ortiz-Andrellucchi and Sanchez-Villegas, 2008), and blood sugar
levels (Yadav et al., 2008). L. casei is highly tolerant to acidic con-
ditions (Yuki et al., 1999; Brink and Todorov, 2006), a characteris-
tic that might improve survival during passage through the gastric
environment, and consequently enhances its potential to act pos-
itively at the intestine level. Due to its high capacity to produce
lactic acid, bioreaction engineering research linked to L. casei has
almost exclusively focused on optimization/maximization of lactic
acid production (see for example Boudrant et al., 2005; John et al.,
2007; Panesar et al., 2007; Xu et al., 2007). Only a few reports of
culture of probiotic bacteria can be found in the literature. A
contribution by Schiraldi et al. (2003) documents a high density

0960-8524/$ - see front matter Ó 2009 Published by Elsevier Ltd.

doi:10.1016/j.biortech.2009.10.047
 Author's personal copy
2838 E.J. Aguirre-Ezkauriatza et al. / Bioresource Technology 101 (2010) 2837–2844
micro-aerophilic process to culture Lactobacillus delbrueckii var.
bulgaricus (also a probiotic strain) for both, the production of
lactic acid and biomass. To our knowledge, there is only one report
that suggests the culture of probiotic biomass from L. casei in bio-
reactors. Mondragón-Parada and Nájera-Martinez (2006) cultured
a L. casei strain in a batch bioreactor using a whey-based medium
supplemented with an ammonium salt and with low levels of
yeast extract (0.25 g/L). However, the authors provided only lim-
ited kinetic information, derived exclusively from batch experi-
ments at low lactose concentration. Here, unsupplemented whey,
deproteinized by ultrafiltration, was used as growth medium for
L. casei biomass production. We intended to validate that nutrients
present in the ultrafiltration permeate suffice, as culture medium,
to promote adequate biomass growth. In an effort to minimize fer-
mentation cost, an anaerobic process was preferred. Also with the
purpose of operative cost minimization, our experimental program
aimed to define an efficient, practical, and high productivity oper-
ation strategy. We compared the performance of batch, continu-
ous, and fed-batch culture strategies in terms of total biomass
production, biomass yields, productivities, reaction rates, and
product quality. We recommend basic operative conditions to
industrially implement this process.
2. Methods
2.1. Whey pretreatment
The general strategy followed in this experimental study is
illustrated in Fig. 1. Goat milk whey (referred to in the rest of the
document as GMW), kindly donated by CapricoÒ, N.L., México,
was processed in an ElecremÒ model No. 3, (France) to remove
fat and deproteinized by ultrafiltration using a QuixstandÒ unit
coupled to a peristaltic pump Watson-Marlow 313S, through an
ultrafiltration column UFP-5-C-4MA 5000 NMWC (surface area of
10% protein
90% lactose
0.76 Kg
1 Kg GMW
0.03% protein BRX
0.025% lactose UF
water
3.00
g biomasa/L
0.20 Kg water FD
85% protein
FD
15% lactose
0.010 kg
L. casei
0.025 kg
protein
Fig. 1. General scheme of a process to obtain freeze-dried protein and freeze-dried
probiotic biomass from goat milk whey (GMW). Quantities and percentages
presented correspond to typical values observed in our experiments. The nomen-
clature of the key unit operations in the process follows: (UF) ultrafiltration
column; (FD) freeze drying units; (BRX) fermentation bioreactor.
650 cm2), from AmershamÒ, Biosciences Corp. The filtrate was pas-
teurized at 80 °C for 20 min in a Yamato sterilizer, model SE510, Ja-
pan. No additional carbon or nitrogen source was used.
2.2. Inoculum
A food and pharmaceutical grade commercial strain of L. casei
BPG4 from SACCOÒ, Nuevo León, México was used as starter cul-
ture (identity confirmed by standard plate culture techniques
and PCR). For each fermentation experiment, 0.5–1.0 g of freeze
L. casei were suspended in 10 ml of GMW and added to 1800 ml
of whey.
2.3. Fermentation experiments
Batch, continuous and fed-batch experiments were conducted
in a 3 L fully instrumented ApplikonÒ model Ez-Control bioreac-
tor. Experiments were run at 37 °C, pH = 5.5 and 300 rpm. A so-
dium hydroxide solution (6 N) was used to control pH. In all
experiments, 25 ml liquid samples were taken from the bioreactor
to quantify biomass (L. casei), substrate (lactose), and product
(lactic acid) concentration. In order to control substrate inlet
and outlet flows during continuous and fed-batch experiments,
peristaltic pumps (MasterflexÒ, USA) were used. Results from
three batch experiments are reported at different inoculum (in
the range of 0.5–1.0 g/L) and initial substrate concentrations (in
the range of 35–50 g/L). One of these batch experiments, after
23 h of batch operation, was continued by a continuous cultiva-
tion period (inlet and outlet flowrate of 1.3 ml/min, operative vol-
ume = 1.8 L, for a residence time of 23.07 h, D = 0.043 h1). A
second batch experiment, after 20 h of operation, was followed
by a series of fed-batch periods, where two different strategies
were used: agitation was stopped and sedimentation of biomass
allowed for 5 h, removal of 900 ml of supernatant (half of the
reactor volume), and fresh GMW addition of the same volume,
and removal of 450 ml of fermented medium (one quarter of
the reactor volume) followed by immediate addition of 450 ml
of fresh GMW.
2.4. Analytical techniques
Biomass was estimated from samples taken at different fermen-
tation times by absorbance measurement at 560 nm. Absorbance
values were correlated to dry weight biomass values estimated
from 15 ml freeze-dried samples. Lactose, lactic acid, glucose, and
galactose concentrations were measured by High Pressure Liquid
Chromatography (HPLC) using a WatersÒ chromatographer (model
W1515) coupled with a refraction index detector from WatersÒ
(model W2414). An Aminex HPX 87H (BioradÒ) column and a
5 mM sulfuric acid mobile phase were used with a flow rate of
0.6 ml/min, and temperatures of 50 °C and 60 °C in the detector
and oven, respectively. Alternatively, lactose concentration was
evaluated by an enzymatic method. Tablets of commercial lactase
(LactaidÒ, one tablet equivalent to 9000 FCC Units of lactase) were
used. About 25-mL samples taken from the bioreactor, were centri-
fuged. One millilitre of the supernatant was mixed and incubated
for 15 min at 30 °C with 9 mL of lactase stock solution (1 LactaidÒ
tablet dissolved in 100 mL of water). After incubation, glucose,
product from lactose hydrolysis was measured using a commercial
blood glucose concentration tester (OptiumÓ, México). Glucose
readings were calibrated using monohydrate lactose dilutions in
the range of 1–50 g/L, relevant to the fermentation experiments.
Protein was determined using the Kjeldahl method AOAC 955.04
(AOAC, 2007).
 Author's personal copy
E.J. Aguirre-Ezkauriatza et al. / Bioresource Technology 101 (2010) 2837–2844
2.5. Kinetics analysis
The biomass and lactose concentration profiles observed in
batch experiments were analyzed to determine the experimental
biomass production rate (rx = d[X]/dt) and the lactose consumption
rate (rs = d[S]/dt). A first order growth kinetics with respect to bio-
mass concentration was assumed:
r x ¼ d½X=dt ¼ l½X
Here, [X] is the biomass concentration at any given time, t is time,
and l is the specific growth rate. To calculate the value of l at each
experimental point, the biomass concentration profiles in time were
fitted to a polynomial Eq. (2), where a, b, v, and d are fitting
constants.
½X ¼ a þ b tv t 2 þ d t 3
Differentiating:
r x d½X=dt ¼ d½a þ bt þ vt 2 þ dt3 =dtb þ 2vt þ 3dt2
Consequently, l can be calculated at each particular time from Eq.
(4):
l ¼ rx ½X ¼ d½X=dt=½X ¼ ðb þ 2vt þ 3dt2 Þ=½X
Regression analysis was conducted using KaleidagraphÒ of Synergy,
USA.
Similarly, a strategy of fitting substrate profiles in time to a
polynomial function and differentiation of such expression was
used to calculate rs. Polynomials providing fittings with correlation
index values of r2 > 0.98 were used.
2.6. Biomass recovery and freeze drying
Produced biomass was recovered by centrifugation using equip-
ment by DupontÒ, model Survall RC5C at 4000g for 15 min. Sam-
ples were frozen at 80 °C, and freeze-dried in a VirtisÒ
lyophilizer model Freezemobile 25EL, USA, operated at 80 °C
(condenser temperature) and vacuum between 0 and 50 militorr.
2.7. Microbiology techniques
Purity and viability of the final product was evaluated by stan-
dard plate count on MRS medium (FulkaÒ, USA). Samples of 1.0 g of
freeze-dried biomass from batch, continuous and fed-batch cul-
tures were suspended in 100 ml of MRS liquid medium. Dilutions
from 101 to 108 were prepared from 1 mL of this suspension.
One millilitre of each dilution was seeded in MRS agar and incu-
bated in a CO2 atmosphere for 48 h. Biomass purity was confirmed
by visual inspection of the colonies grown at plates corresponding
to different dilutions. Biomass identity was validated by PCR (Poly-
merase Chain Reaction) from colonies taken from MRS agar culture
plates. Specific primers for L. casei were used (Haarman and Knol,
2006): CTA TAA GTA AGC TTT GAT CCG GAG ATT T (forward pri-
mer) and CTT CCT GCG GGT ACT GAG ATG T (reverse primer). Each
PCR reaction was conducted in a 50 lL volume, using the Advan-
tage™ 2 PCR kit, from Clonetech, USA, in a P  2 Thermal Cycler
from Thermo Scientific, USA (Haarman and Knol, 2006). The PCR
product was analyzed by electrophoresis in 4% agarose (w/v) in
TAE (Tris–acetic acid–EDTA) (Sanbrook and Russell, 2001) with SY-
BER SafeÒ from Invitrogen, USA to a 1X final dilution. A Trackit™
50 bp and a Trackit™ 20 bp were used as DNA fragment markers.
A strain of L. casei var. rhamnosus (LiolactilÒ from IVAX™, commer-
cial freeze-dried product) was used as a positive control.
2839
3. Results and discussion
The process depicted in Fig. 1 leads to the production of differ-
ent added value products from GMW (i.e. high protein/carbohy-
drates ratio, milk fat, lactic acid, and probiotic freeze-dried
biomass, specifically, L. casei. Particularly, our discussion addresses
the production of L. casei from non-supplemented and deprotei-
nized GMW.
ð1Þ Fresh whey exhibited an original 6.32% solid content, with an
average of 36.7 g/L of lactose, 16.4 g/L of protein, and 2.20 g/L of
fat. After fat removal, the resulting GMW had a 5.6% solid content,
with 27.7 g/L of lactose, 23.0 g/L of protein, and 0.0 g/L fat. There-
fore, from 1000 kg of original whey, an average of 2.2 kg of fat
can be recovered.
ð2Þ 3.1. Ultrafiltration
The ultrafiltration process used exhibited consistent perfor-
mance. On average, from each kg of fat free GMW, 0.76 kg of per-
ð3Þ meate and 0.20 kg of protein concentrate were recovered. Typical
volume lost by membrane retention was 4%. This value should be
expected to decrease at larger scales, as the ratio of the process vol-
ume/surface area of the filter increases. Protein recovery at the re-
tained fraction was reproducible throughout all experimental runs
ð4Þ
(±3% variability). In general, it is possible to recover 1 g of freeze-
dried protein per 73 g of protein concentrate. This implies that,
from 1000 kg of original whey, 2.739 kg of protein can be recov-
ered (from 16.4 kg originally present).
3.2. Fermentation experiments: batch protocols
Fig. 2 shows typical biomass, lactose, and lactic acid concen-
tration profiles during batch culture of L. casei in whey. Features
should be remarked. In general, a long lag phase occurred, rang-
ing from 6 to 10 h (see also Figs. 4 and 5). These extended lag
phases are attributed to the time required for the cells to acti-
vate lactase production. Two observations support this state-
ment. The onset of galactose production coincides with the
starting point of exponential biomass growth. No growth oc-
curred by direct utilization of lactose. Also, in experiments with
lactase addition (not shown), or larger inoculum, shorter lag
phases were observed.
Lactic acid production occurs slightly after exponential biomass
growth starts, suggesting that its production is only partially cou-
pled to biomass production. In addition, galactose accumulates in
the culture medium, suggesting that mostly glucose was used to
support biomass growth and lactic acid production. A mass balance
on lactose, glucose, and galactose confirms that glucose acts as the
main substrate for biomass and lactic acid production of L. casei
and that only less than 20% of galactose produced during the first
40 h of the process was consumed.
It is known that Lactobacillus spp. growth is inhibited by lactic
acid (i.e. Altiok et al., 2006; Youssef and Goma, 2005) and the main
engineering challenge of biomass production by lactic bacteria is to
circumvent product inhibition. One of the main objectives of this
contribution is to characterize the product inhibition effects in or-
der to device a robust strategy to overcome it.
In a typical batch fermentation process, a general first order
model with respect to biomass is frequently assumed (Eq. (1)).
Accordingly, the observable biomass production rate (rx) is the re-
sult of the combined effects of the specific growth rate and the bio-
mass concentrations. However, under a product inhibition
scenario, l is expected to be a fraction of l0, the specific growth
rate observable in absence of product. Moreover, the multiplying
effect of the biomass concentration is at its minimum at the begin-
 Author's personal copy

2840 E.J. Aguirre-Ezkauriatza et al. / Bioresource Technology 101 (2010) 2837–2844

[Lactose], [Galactose], [Lactic acid] (g/L)

4.0 50 3.5 0.35

cummulative productivity (g L h )
(g biomass produced/L)
3.5 3.0
40 0.30
3.0 2.5
[X] (g biomass/L)

0.25
30
2.5
2.0
0.20
2.0
20 1.5
1.5 0.15
10 1.0

o
(a)

[X]-[X]
1.0

-1 -1
0.5 0.10
0.5 0
0 5 10 15 20 25 30 35 40
0.0 0.05
time (hr) 0 3 5 7 9 11 13
time (h)

0.30 2.5 Fig. 3. Biomass production and productivity profiles in time for L. casei culture in
non-supplemented whey. Biomass production is presented for batch experiments at
2.0 (b) different biomass and substrate initial concentrations: [X]0 = 0.50 g/L and
specific growth rate, µ (h-1)

0.25 [S]0 = 41.0 g/L (d); [X]0 = 1.00 g/L and [S]0 = 35.0 g/L ( ); and [X]0 = 0.50 g/L y
ln([X]/[X] )
o

1.5 [S]0 = 50.0 g/L (s). The calculation of the average cumulative biomass productivity
for these three batch experiments is also presented (j).
0.20 1.0

0.5 Fig. 3 presents the biomass production profiles for the three
0.15
batch experiments under consideration. Despite the fact that they
0.0
0.0 0.50 1.0 1.5 2.0 were conducted at different biomass and lactose initial concentra-
0.10 time (h)
(c) tions, when the produced biomass ([X]  [X]0) is plotted versus
time, again, all experimental trends collapse into a single curve,
0.05 demonstrating a consistent underlying kinetic behavior during
the exponential growth phase of the culture. This result demon-
strates that the kinetic behavior and presumably the value of rele-
0.00
0 2 4 6 8 10 12 14 16 vant kinetic parameters are not affected by the variation of
time (h) substrate or inoculum concentrations in the range of conditions ex-
plored ([S]0 from 35 to 50 g/L and [X]0 from 0.5 to 1.0 g/L). In a
Fig. 2. (a) Biomass (d), lactose (j), lactic acid (h) and galactose () concentration batch process, productivity is a function of time (although gener-
profiles for a typical batch fermentation experiment (b) during the first 2 h of the
ally only an average integrated value is reported). In our experi-
exponential phase of a batch experiment, when no lactic acid has been produced
yet, the specific growth rate is constant, and its value can be calculated by the slope ments, the cumulative biomass productivity, calculated for the
of the straight line of the plot ln[X]/[X]0 vs. time. (c) Time evolution of the specific biomass production master curve, follows a trend consistent with
growth rate (l) for three independent batch experiments, as lactic acid accumulates the time dependence of l. For the first two hours of exponential
in the fermentor.

3.5 50
ning of the process, being not significant unless a large inoculum
concentration is used. Therefore, it is expected that, once lactic acid 45
starts to be produced, the biomass production rate will be severely 3.0
diminished. This behavior is analyzed in Fig. 2b and c and 3. Fig. 2b (a) (b) 40
[S] (g lactose/L)

presents a graph of ln[X]/[X]0 versus time for the first 2 h of the

[X] (g biomass/L)

exponential growth phase of the batch experiment shown in 2.5 Valves 35

Steady
Fig. 2a. For this time interval, no lactic acid has been produced, state
and data can be adjusted to a straight line. Therefore, the value 30
of the specific growth rate (l) is constant, and can be calculated 2.0
from the slope of the linear trend (l = 1.22 h1). Fig. 2c presents 25
values for l versus time for the exponential phase of three batch
1.5 20
experiments conducted under equivalent operative conditions,
but different initial biomass and lactose concentrations. If properly
15
displaced along the time axis, so that the starting of the exponen-
1.0
tial growth phase defines time = 0, the three experimental behav- 10
iors coincide in a single curve. The specific growth rate 0 10 20 30 40 50 60
exponentially drops from the value calculated in the absence of time (h)
product (first 2 h) as the lactic acid concentration increases. Prod-
uct concentrations as low as 1.0 g/L severely affect l. At concentra- Fig. 4. Biomass (d) and lactose ( ) concentration profiles for (a) a batch
fermentation experiment (b) continued with a period of continuous operation.
tions of 5 g/L, the l value is practically at its minimum
The vertical dashed line indicates the point in time at which valves were activated.
(l = 0.02 h1). This strong lactic acid inhibition has an important Horizontal dashed lines indicate the values observed at the steady state corre-
effect of biomass productivity. sponding to D = 0.0433 h1.
 Author's personal copy
E.J. Aguirre-Ezkauriatza et al. / Bioresource Technology 101 (2010) 2837–2844
4.0
(a) batch (b) (c)
3.0
[X] (g biomass/L)
fed-batch 2
fed-batch 1
[X] (g/L)
2.0
1.0 [S] (g/L)
0.0
0 5 10 15 20 25 30 35
time(h)
Fig. 5. Biomass (d) and lactose ( ) concentration profiles for (a) a batch
fermentation experiment (b–d) continued with three periods of fed-batch opera-
tion. In a first fed-batch period (b), the bioreactor content is allowed to settle for one
hour, and half a volume of supernatant is removed from the stirred tank to be
substituted with fresh medium. In a second and third fed-batch periods (c) and (d),
one quarter of the volume content is substituted with fresh medium while stirring.
growth, biomass productivity grows until it peaks at a value of
0.34 g L1h1. Afterwards, as the lactic acid concentration in-
creases, the cumulative productivity during the exponential phase
drops to a value of 0.21 g L1h1. If the extent of the lag phases
were considered in the productivity calculation (consideration that
makes sense from a practical perspective), values of 0.11–
0.17 g L1h1can be estimated (an average of 0.14 g L1h1).
3.3. Fermentation experiments: continuous and fed-batch protocols
Fig. 4b shows biomass and lactose profiles for a continuous fer-
mentation, preceded by a batch period (Fig. 4a). A dilution rate
D = 0.0433 h1 (residence time RT = 22.73 h) was used. This condi-
tion was chosen to directly compare batch and continuous fermen-
tation protocols with similar overall residence times. After 28 h of
continuous operation, a steady state was achieved after 28 h. The
average steady state biomass and lactose concentration were
[X] = 2.6 g/L and [S] = 28.5 g lactose/L, respectively. These values
correspond to a specific growth rate l = D = 0.0433 h1, a biomass
production rate rx = 0.11 g biomass/(L h), and a biomass productiv-
ity D[X] = 0.113 g biomass/(L h).
Fed-batch protocols have been used before to culture L. casei,
mainly with the purpose of producing lactic acid (Ding and Tan,
2006). Here we demonstrate the use of fed-batch strategies in
the context of biomass production from GMW. Fig. 5b–d presents
the biomass concentration and substrate concentration profiles
for three consecutive fed-batch (Fig. 5a). During the first 20 h of
batch operation, a biomass concentration [X] = 3.7 g/L was
achieved, practically exhausting the available substrate. In a first
fed-batch cycle (Fig. 5b), the agitation was interrupted and the bio-
reactor content was allowed to settle for 4 h. Afterwards, half of the
bioreactor volume was removed from the top liquid layer (900 ml)
and the same volume was replaced with fresh culture medium
with a substrate concentration [S]0 = 34 g lactose/L. The initial bio-
mass and lactose concentrations for this cycle were [X]0 = 2.0 g/L
and [S]0 = 11 g lactose/L, respectively. Within a period of 5 h, fol-
lowing biomass production and substrate consumption rates sim-
ilar to those observed for batch operation at high biomass
concentration, the substrate was exhausted (rs batch at [X]high 
rs fed-batch). In a second fed-batch cycle (Fig. 5c), without a settling
2841
35 period, one quarter of the bioreactor volume was replaced with
(d) fresh culture medium (450 ml). The initial biomass and substrate
30 concentration for this second fed-batch cycle were [X]0 = 2.25 g/L
y [S]0 = 7.5 g lactose/L, respectively. After 5 h, following trends pre-
viously observed for biomass production and substrate concentra-
25
[S] (g lactose/L)
tion, lactose was exhausted again. In Fig. 5d, results from a replica
of the second fed-batch cycle are shown. The biomass and sub-
fed-batch 3
20
strate profiles were practically equivalent. Fig. 6 presents a more
detailed kinetic analysis of the fed-batch experiments. In Fig. 6a,
15 biomass (black squares) and substrate concentration profiles (grey
circles) observed in the second and third fed-batch cycles are pre-
10 sented. Lactose profiles were fit to linear trends to calculate the
substrate consumption rates (rs). The calculated rates are practi-
5 cally equivalent in the second and third fed-batch cycles, and
slightly lower than the values observed from batch experiments
0 (rs batch = 1.9 g L1h1 vs. rs fed-batch = 1.7 g L1h1). Although sub-
40 strate consumption rates were lower for the experimental condi-
tions used for our fed-batch experiments (experiments run at
higher lactic acid concentrations and lower lactose concentration),
overall biomass production rates were higher than those observed
in batch experiments. In Fig. 6a and b a polynomial trend was ad-
justed to the biomass profile. In Fig. 6d and e, from the resulting
trend equation, the biomass rates and specific growth rates were
derived for each experimental fed-batch point. Higher biomass
concentrations occurred in our fed-batch experiments, caused by
the biomass retention strategies implemented in the first fed-batch
cycle. Consequently, the increase in biomass concentration posi-
tively impacted process productivity. The average productivity
for the second and third fed-batch cycles was 0.45 g biomass
L1 h1, a value significantly higher to that observed from our
batch experiments and 9% superior to that observed by Altiok
et al., 2006 in batch experiments with supplemented and diluted
milk whey (see Table 1).
3.4. Comparison of fermentation protocols
Table 1 compares our experimental results from batch, contin-
uous, and fed-batch protocols in terms of relevant process indica-
tors. In addition, data from experimental reports found in literature
for batch systems (supplemented with yeast extract) are
presented.
The first and second columns in Table 1 present data from a
batch and a continuous experiment with a similar overall residence
time of 23 h. When exclusively considering the exponential growth
phase for calculation, the batch protocols exceeded the productiv-
ity of the continuous experiment. If the extended lag phase ob-
served in batch experiments is considered, then, for a similar
overall residence time of 23 h, the batch experiments yielded an
equivalent average productivity of 0.11 g biomass L1 h1 but were
superior in terms of a higher biomass production and substrate
consumption. Therefore, for an equivalent residence time, a contin-
uous operation mode does not yield a significant benefit over batch
protocols. In continuous systems, productivity and steady state
biomass and substrate concentrations are a function of dilution
rate. Continuous processes with shorter residence times (higher
dilution rates and higher specific growth rates) will result in higher
productivities, but at the expense of lower substrate consumption,
and consequently higher residual substrate concentrations.
Fed-batch protocols exhibited productivities that exceeded by
more than threefold those observed in batch and continuous
experiments. In addition, they assured substrate exhaustion. In
fed-batch protocols, was possible to take advantage of high bio-
mass concentrations to partially overcome the strong acid lactic
inhibition effect. Additionally, fed-batch protocols save time by
reducing the extended lag phase characteristic of low cell density
batch processes.
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2842 E.J. Aguirre-Ezkauriatza et al. / Bioresource Technology 101 (2010) 2837–2844

3.2 16 3.2 16

3.1 14 3.1 (b) 14

[X] (g biomass/L)

[S] (g lactose/L)
[X] (g biomass/L)
[S] (g lactose/L)
3.0 (a) 3.0
12
12
2.9 2.9
10 10
2.8 2.8
8 8
2.7 2.7
6 2.6 6
2.6

2.5 4 2.5 4
2 3 4 5 6 7 8 2 2.5 3 3.5 4 4.5 5 5.5 6
time (h) time (h)
1.0 0.6

(c) 0.5
Biomass growth rate, rx

0.80

specific growth rate, µ (h-1)

(g biomass L-1 h-1 )

0.4
0.60

0.3

0.40
0.2
(e)
(d)
0.20
0.1

0.0 0
5 10 15 20 25 30 35 40
Time (h)

Fig. 6. Kinetics of L. casei growth in fed-batch conditions. In (a) and (b), biomass (d) and lactose ( ) concentration profiles observed in the second and third fed-batch
protocols were fitted to polynomial equations. In (d) and (e), based on the derivative of the first trend equation, the biomass growth rate (rx, ) and the specific growth rate (l,
d) are calculated for the same experiments. (c) Estimations of biomass growth rate (rx, d) and specific growth rate (l, d) are presented for the first fed-batch experiment.

Using diluted whey (strategy to minimize inhibition by lactic This comparison suggests that low supplementation with yeast ex-
acid) and nitrogen supplementation (2.5 g/L of yeast extract addi- tract (2.5 g/L) has only a marginal effect on a batch process perfor-
tion), Mondragón-Parada and Nájera-Martinez (2006) observed pro- mance, at least in terms of biomass production indicators. For
ductivities of 0.067 g L1h1 (calculated from data reported from the higher supplementation scenarios, much higher biomass productiv-
authors) for a particular L. casei strain. Regarding biomass/lactose ity values have been documented. Altiok et al. (2006) conducted L.
yields (Yx/s), the authors observed values of 0.165 g biomasa/g lac- casei batch culture experiments at different whey dilutions (from 9
tose for the most productive strain they studied, but yields in the to 70 g of lactose/L) and higher yeast extract supplementation. From
range of 0.06–0.11 g biomass/g lactose for other five L. casei strains. this data, at 50 g lactose/L and 10 g/L of yeast extract, biomass/lac-

Table 1
Comparative summary of key L. casei production process indicators observed with different culture strategies.
a e f
Batch culture Continuous Fed-batch Batch culture Batch culture
culture b culture c
Indicator This report This report This report Altiok et al. (2006) Mondragón-Parada and Nájera-Martinez (2006)
Residence time (h) 23 23 6 h Cycles 14 45
Cumulative productivity (g biomass/(L h)) 0.11 ± 0.02 0.11 ± 0.01 0.45 ± 0.01 0.42 0.067
Biomass Production [X]final  [X]0 (g/L) 3.25 ± 0.3 2.6 ± 0.1 2.7 ± 0.2 6.0 3.0
Substrate consumption (g/L) 28.36 ± 1.7 20.0 ± 3.0 33.0 45 10.0
Yield Yx/s (g biomass/g substrate) 0.1145 ± 0.021 0.13 ± 0.01 0.1060 ± 0.01 0.15–0.17 0.16
Viable count d (CFU/g) 5.17  109 1.95  1010 2.43  1010
a
Values correspond to averages of three independent runs.
b
Values correspond to time averages during the steady state of one experiment.
c
Values correspond to the average of three fed-batch cycles.
d
Viable counts were estimated from consolidated samples corresponding to the final freeze-dried product from different experiments (batch and fed-batch) or different
samples taken at the steady state of the continuous fermentation.
e
Altiok et al. (2006): experiments in whey supplemented with 10 g of yeast extract/L.
f
Values calculated from Mondragón-Parada and Nájera-Martinez (2006): experiments in diluted whey supplemented with 2.5 g of yeast extract/L.
 Author's personal copy
E.J. Aguirre-Ezkauriatza et al. / Bioresource Technology 101 (2010) 2837–2844
tose yields within the range of 0.15–0.16 g/g and productivities of
0.42 g L1h1 can be calculated. Although much higher than those
observed in our non-supplemented batch experiments, these pro-
ductivity values are 10% lower than those obtained in non-supple-
mented fed-batch fermentations (Table 1).
3.5. Biomass characterization
Recent experimental evidence suggests that some of the benefi-
cial health effects of probiotics are not associated with live cells
(Rachmilewitz et al., 2004; Meier and Steuerwald, 2005). However,
most beneficial effects of probiotics have been clearly associated
with live cells and their metabolites (Wendakoon et al., 2007;
Pham et al., 2008). Therefore, the determination of the viable count
of beneficial bacteria present in a product continues to be the most
valid method to test for probiotic quality and processes that max-
imize viable count in the final product would be preferable. Once
the purity of the freeze-dried product from our fermentation
experiments was validated by standard microbiology plate tech-
niques, identity was confirmed by PCR using primers specific for
L. casei. Viable counts of the biomass produced in the fermentation
experiments were between 5  109 (LOG 10 = 9.7) and 2.5  1010
(LOG 10 = 9.7) viable microorganisms per g of freeze-dried biomass
(Table 1). Our results suggest that continuous and fed-batch proto-
cols render a higher quality probiotic. We selected our batch, fed-
batch and continuous fermentation protocols such that each litre
of culture media remained in the fermentor approximately for
the same process time (1.8 L/23 h). Therefore, these viability differ-
ences could not be attributed to differences in the average resi-
dence time of the biomass within the bioreactor, but to
differences in the ratios of lactic acid/biomass in the bioreactor.
Even for the case of the batch product, the observed viable counts
allow for the use of the product as a probiotic (viable count must
be higher than 1  106/g of product (Sanders, 2000; Szily et al.,
2005)). In average, the batch and the fed-batch products exhibited
500 fold and 2500 fold this accepted threshold value respectively.
Commercial equivalent products based on L. casei (i.e. LiolactilÒ,
from IVAX™, France) have a minimum viable cell count of
8.0  108/g. Therefore, the fed-batch product described here has
31-fold viable cells/g with respect to these products. Considering
that the final price to the public of the commercial equivalent is
$4 dollars/g, the proposed fed-batch process would potentially ren-
der, in product equivalent sales, $434,000 dlls/m3 of milk whey.
4. Conclusions
Our results suggest that non-supplemented and non-diluted
whey can be used to produce high added value products such as
high-quality/high solubility protein and probiotic biomass through
low-cost ultrafiltration and fermentation technology. Microbiolog-
ical analysis on samples from batch, continuous and fed-batch
experiments render viable counts on the range of 5.0  109–
2.5  1010 viable cells/g of freeze-dried biomass. Particularly, the
implementation of fed-batch protocols seems to be a cost effective
strategy to produce probiotic L. casei probiotic biomass from whey.
When compared to batch and continuous strategies, fed-batch pro-
tocols exhibited superior performance, specifically higher biomass
productivity, lower residual substrate concentrations, and higher
viable counts in the freeze-dried product.
Acknowledgements
We gratefully acknowledge the support and funding of Tec-
nológico de Monterrey (seed funding-CAT122) and CONSEJO NAC-
IONAL DE CIENCIA Y TECNOLOGÍA (CONACyT).
2843
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