Biochemical Engineering Journal 110 (2016) 7183

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Medium optimization and kinetics modeling for the fermentation of

hydrolyzed cheese whey permeate as a substrate for Saccharomyces
cerevisiae var. boulardii
D.E.G. Trigueros a, , M.L. Fiorese a , A.D. Kroumov b , C.L. Hinterholz a , B.L. Nadai a ,
G.M. Assunco a
a
Department of Chemical Engineering Postgraduate Program, West Paran State University UNIOESTE, Rua da Faculdade 645, Jd. Santa Maria, Toledo,
85903-000 PR, Brazil
b
Institute of Microbiology Stephan Angeloff Bulgarian Academy of Sciences, Department of Applied Microbiology, Acad. G. Bonchev str., Bl.26, Soa
1113, Bulgaria

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

Article history: This paper presents an efcient approach to culture medium optimization by a central composite rotat-
Received 28 August 2015 able design (CCRD) coupled to non-linear mathematical programming (NLP). This procedure was applied
Received in revised form 12 January 2016 for the investigation regarding the growth of Saccharomyces cerevisiae var. boulardii in cheese whey per-
Accepted 15 February 2016
meate (CWP), which is an attractive raw material for the production of bioproducts, mainly due to its
Available online 18 February 2016
high lactose content. The tests comprehended lactose hydrolysis followed by glucose-galactose anaerobic
fermentation in a batch system. A CCRD was proposed to evaluate the following factors: concentration
Keywords:
of hydrolyzed CWP, and N, Mg and K supplementation, where the system response was considered to be
Saccharomyces cerevisiae var. boulardii
Cheese whey permeate
cell growth. The cultivation media NLP model was empirically determined by a statistical methodology,
Medium optimization and was also based on the elemental composition and the stoichiometry of the yeast anaerobic culti-
Catabolite repression vation. The kinetic mathematical model of the yeast cultivation was proposed, where inhibition by the
substrates of fast-glucose and slow-galactose metabolization, inhibition by the product ethanol, catabo-
lite repression by glucose, and metabolic products formation were adequately considered. In addition,
the response surface provided by the kinetic modeling was also analyzed. The global optimum value of
biomass yield was equal to Yx/s = 0.50, where the cell biomass was estimated to be 8.20 g L1 by using
40 g L1 hydrolyzed CWP.
2016 Elsevier B.V. All rights reserved.

1. Introduction different residual waste from cheese and derivatives manufactur-

Cheese whey permeate emerges as a byproduct from the whey
protein concentrate fabrication during the membrane separation
process [1]. This agro-industrial byproduct is characterized by the
high content of lactose (7090%), presence of minerals (620%),
proteins (0.53%) and low levels of fats (<2%), besides signicant
concentrations of macro and micro elements, and some vitamins
B-Complex [1]. These characteristics point to various possibilities
for the use of the CWP by industries, such as the production of bev-
erage, soups, bakery products and others [2], or the obtainment of
lactitol and lactulose [3]. Appropriate and effective treatment of
dairy industry efuents are known [4], although the valuation of
Corresponding author.
E-mail address: estelita trigueros@yahoo.com.br (D.E.G. Trigueros).
ing has gained attention for its nutritional and functional content.
One alternative has been the use of dairy waste as a carbon source in
the production of various bioproducts by fermentation mainly due
to the high lactose content [5]. Some examples were shown dur-
ing the ethanol production [612], hydrogen obtainment [1315],
lactic acid production [16,17], polyhydroxyalkanoates production
[1820], organic acids production [21,22], and single cell protein
production [12,2325].
The single cell protein (SCP) production adds value and signi-
cance to the CWP, once the yeast biomass can be used in animal food
supplementation, and also in the human diet due to its favorable
and balanced composition, being constituted by proteins (4555%),
fats (513%), minerals (6.6%), besides B vitamins: thiamine (B1),
riboavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6),
biotin (B8), folic acid (B9), p-aminobenzoic acid (B10), associated
organic compounds, such as streptogenin and glutathione, and

http://dx.doi.org/10.1016/j.bej.2016.02.014
1369-703X/ 2016 Elsevier B.V. All rights reserved.
72 D.E.G. Trigueros et al. / Biochemical Engineering Journal 110 (2016) 7183
some essential amino acids: lysine, tryptophan, leucine and threo-
nine [26].
The use of the sugar present in the CWP by the yeast occurs by
direct fermentation of lactose or by fermentation of glucose and
galactose obtained during enzymatic hydrolysis of -galactosidase
[27]. Three yeast species are well known and used due to their abil-
ity to metabolize lactose: Kluyveromyces lactis [28], Kluyveromyces
fragilis [29,30], Kluyveromyces marxianus [6,7,11,12,3133].
In SCP production, the Saccharomyces genus is the most used for
presenting food safety for human consumption, according to Food
Drugs Administration. The metabolism of Saccharomyces cerevisiae
is well known and represents one of the most studied regulatory
systems [3436]. This specie is able to assimilate a great variety
of carbon sources, such as glucose, fructose and mannose [37,38].
However, S. cerevisiae is not able to metabolize lactose due to the
absence of -galactosidase (encoded by the LAC4 gene) and lactose
permease (encoded by the LAC12 gene) [39]. The diauxic growth
[4042] is observed when the cultivation medium for S. cerevisiae
has glucose and another carbon source, such as galactose [43]. This
phenomenon was observed in ethanol production by S. cerevisiae
in hydrolyzed CWP, where glucose was rstly and much faster con-
sumed than galactose [44]. According to Nielsen and Villadsen [45],
the glucose transportation system is constitutive, and therefore,
the cell is always ready to metabolize glucose. On the other hand,
the ability of the cell to grow on other carbon sources depends
on its energy state, which is associated to the glucose concentra-
tion. This means that the enzymatic system synthesis, necessary
for the absorption of the second carbon source, is controlled by glu-
cose concentration in the medium, resulting in different regulation
mechanisms such as the catabolite repression and the catabolite
inactivation [37,46,47].
The GAL genes allow S. cerevisiae to use galactose as a carbon
source, whose metabolization continues via Leloir [4851]. The
galactose control and regulation mechanism tolerates repression
by glucose, induction by galactose and catabolite inactivation by
galactose permease [43] which is responsible for galactose ow for
the interior of the cell. The GAL genes induction is determined by
the interaction between the top three specic regulatory proteins:
a transcription activator (Gal4p), a repressor (Gal80p) and a binder
sensor (Gal3p) [48,49,52]. The main carbon and energy source for
most yeasts is glucose, but overcoming the issues of the glucose
effects on cell growth has been the aim of researches that intend
to identify S. cerevisiae strains with new and efcient fermentative
activity [39,48,50,53].
S. cerevisiae Hansen CBS 5926 is a specic specie of S. cerevisiae,
better known as Saccharomyces boulardii [54,55]. Its difference
regarding the other S. cerevisiae strain is the presence of a sin-
gle and specic allele (sequence CAG 9 at locus 4) [56], and thus
researches indicate that the yeast must be considered as S. cerevisiae
var. boulardii [57]. S. boulardii is highly regarded for its probi-
otic potential, property able to maintain balanced the microbial
gastrointestinal ora [54,58]. This property can increase the bene-
ts of the consumption of SCP from the S. cerevisiae var. boulardii
yeast. The action of probiotics in animals promotes weight gain,
improve the immune system and recovery after microbial infec-
tions. In humans, the benets are associated with stimulation of
the immune system and preventing the formation of carcinogenic
substances in the intestine, as well as the reduction of lactose intol-
erance and reduction of serum cholesterol [59,60].
Besides the ideal conditions involved in the sugars metabolism,
the use of yeast for efcient production of SCP requires knowl-
edge of its nutritional demand for cell growth: carbon and nitrogen
sources, besides inorganics and some vitamins. The optimization
of the composition of the medium aiming maximum concentra-
tion of biomass is a difcult task for solving. In general, empirical
approaches trial and error procedure from a large number of
experiments are adopted, starting with the investigation of the
carbon source, then the nitrogen source, and nally, additional
sources of nutrients, until all components combinations supply
a culture medium with optimized composition. However, this
method can disregard the interactive effects between the vari-
ables. The response surface methodology (RSM) is a combination
of mathematical and statistical techniques which aims to estimate
the effects of different individual and conjugated independent vari-
ables on the system response [33], by means of an empiric model
which is, in general, characterized by a second order polynomial
equation adjusted in a region predened by the independent vari-
ables [6,29,33,61].
This paper presents a strategy for cell biomass maximization
based on RSM with non-linear mathematical programming (NLP)
that allows the optimization of the components concentrations that
are fundamental in the culture medium. The developed regres-
sion model takes into account all possible combinations of effects
over the microbial response, according to a CCRD design of experi-
ments which evaluates the concentrations of nitrogen, magnesium
and potassium over the cultivation of S. cerevisiae var. boulardii in
anaerobic batch system. The experimental study also evaluates the
initial pH and the inuence of the ratio of carbon and nitrogen
concentrations (C:N) on the specic growth rate. The kinetic math-
ematical model of the anaerobic batch cultivation of S. cerevisiae
var. boulardii was proposed considering the consumption of the
substrates of faster (glucose) and slower (galactose) metaboliza-
tion, the ethanol and CO2 production. The developed kinetic model
takes into account terms of inhibition by the substrates, inhibition
by the product, catabolic repression and the formation of metabolic
products.
2. Materials and methods
2.1. Yeast and cell activation
The lyophilized used S. boulardii17 yeast was obtained from
the commercial medicine Floratil 200 mg (Merk S/A). Each 200 mg
lyophilized capsule contains about 1 109 S. boulardii17 cells and
excipients lactose and magnesium stearate.
S. boulardii cells were activated in 250 mL YEPD (yeast extract
peptone dextrose) culture medium with the following compo-
sition: 5 g L1 yeast extract, 10 g L1 meat peptone and 20 g L1
glucose. The medium was sterilized in autoclave at 121 C for
15 min.
2.2. Maintenance culture
The yeast was inoculated in the YEPD activation medium, and
transferred to a shaker incubator (Solab Cientca SL 222/CFR)
with 100 rpm agitation and controlled temperature of 30 C for
24 h. Further, 25 mL medium with yeast was transferred to another
Erlenmeyer ask with sterilized YEPD medium, followed by incu-
bations under the same conditions. The yeast was kept in YEPD
medium with 20 g L1 agar-agar, in tubes kept in oven at 30 C for
48 h. After that, the tubes were stored in refrigerator at 4 C.
2.3. Cheese whey permeate
The CWP powder used as substrate for S. cerevisiae var. boulardii
was ceded by an industry (Sooro Concentrado Indstria de Produ-
tos Lcteos Ltda) located in the west region of Paran state, Brazil.
The composition of the powder permeate sample includes lactose
(90%), phosphorus (0.48%), potassium (0.04%), magnesium (0.09%),
sodium (0.50%), calcium (0.50%) and protein (3%). The pH range of
prepared 10% solution was from 6 up to 6.7.
 D.E.G. Trigueros et al. / Biochemical Engineering Journal 110 (2016) 7183 73

Table 1
Levels and real values of the variables considered in the CCRD design.
Factors Unit Level
2 1
x1 : (NH4 )2 SO4 (g L1 ) 3 4.5
x2 : MgSO4 (g L1 ) 0 1.25
x3 : K2 PO4 (g L1 ) 0 1.5
x4 : Hydrolyzed CWP (g L1 ) 55 85
2.4. Central composite rotatable design
A central composite rotatable design (CCRD) was proposed in
order to analyze the cultivation performance for S. cerevisiae var.
boulardii, as a function of chosen factors on the response surface of
the investigated region. Based on four factors (k), 28 experiments
(2k + 2k + 4) were performed, being 24 of them in unique conditions
and 4 central points (see Table 1). The real values of the levels of
each independent variable (xi ) were chosen from preliminary tests.
The ammonium sulfate concentration (x1 ), magnesium sulfate con-
centration (x2 ), monobasic potassium phosphate concentration
(x3 ) and hydrolyzed CWP concentration (x4 ) were investigated dur-
ing the cultivation of S. cerevisiae var. boulardii at 30 C, pH 5.5 and
100 rpm. The yeast cultivation performance, which was evaluated
by the dependent variable concentration of nal biomass reached
after 17 h of fermentation, was determined by using the response
surface methodology (RSM). The experimental data of CCRD design
were modeled and analyzed by means of the software Statistica
version 10 (Statsoft, Inc.), according to the analysis of variance
(ANOVA). The statistical signicance of the model was evaluated
by the F test with 5% signicance level. The quality of the polyno-
mial adjustment was evaluated by the determination coefcient
(r2 ). The polynomial equation (Eq. (1)) represents the dependent
variable concentration of biomass as a function of the effect of the
linear and quadratic terms of the independent variables, accord-
ing to the 1i and 2i coefcients, and their interactions effects,
according to the ij .
tion of NaOH and HCl solutions. The medium was pasteurized at
65 C for 30 min. Further, 30 mL of the pre-inoculum were trans-
ferred to the Erlenmeyer ask containing 270 mL inoculum. The
inoculum was kept in shaker (Solab Cientca SL 222/CFR) under
0 1 2
stirring of 100 rpm at 30 C for 12 h.
6 7.5 9
2.5 3.75 5
3 4.5 6 2.7. Cultivation of S. cerevisiae var. boulardii
115 145 175
The cultivations were performed in glass containers with 3 L
working volume. In each container 1820 mL cultivation medium
were prepared, according to the CCRD design. For further anal-
ysis, 20 mL of the cultivation medium were withdrawn. After
that, 200 mL of inoculum were transferred to 1800 mL cultivation
medium, which was kept in shaker incubator (Solab Cientca SL
222/CFR) at 100 rpm and 30 C for 30 h. The transferred inocu-
lum was properly diluted in order to keep the initial number of
S. cerevisiae var. boulardii cells constant. Samples of 15 mL were
taken every 3 h, of which 3 mL were used for absorbance reading
in spectrophotometer (600 nm), and 12 mL were stored in 1.5 mL
micro-tubes. The micro-tubes were centrifuged at 14000 rpm for
7 min. Further, the supernatant and the precipitated were sepa-
rated and stored in freezer at 10 C for further sugar analysis.
2.8. Analytical methods
The biomass concentration was obtained by a correla-
tion curve between both spectrophotometry and dry weight
(1OD600nm = 0.45 g L1 ) methods. The optical density of the cells
suspension was measured in triplicate in a spectrophotometer
UVvis (brand Shimadzu, model UV-1800). The total reducing sugar
(TRS) was quantied in triplicate by the 35 dinitrosalicylic acid
(DNS) method. The glucose concentration was determined in tripli-
cate by the enzymatic-colorimetric method by using the PP glucose
kit (brand Gold Analisa). The pH values were measured in a prop-
erly calibrated bench pH-meter (brand MS Tecnopon, model MPA
210).


k 
k 
k 
k 2.9. Mathematical modeling of the anaerobic batch cultivation
R = 0 + 1i xi + 2i x2i + ij Xi Xj (1) process of cerevisiae var. boulardii
i=1 i=1 i=1 j =
/ 1
The development of the mathematical model describing the
2.5. Enzymatic hydrolysis of the permeate cultivation of S. cerevisiae var. boulardii was performed from the
concepts of material balances for batch reactor. The kinetic math-
Twenty eight hydrolyzed samples were prepared according to ematical model proposed in this paper describes the dynamics of
the CCRD design. Solutions of 0.06; 0.10; 0.14; 0.18 and 0.22% (p/v) cell growth (X), substrate consumption (S) and product formation
permeate were prepared by adjusting the pH to 6.5 and adding (P), according to Eqs. (2)(4), respectively.
0.125% (p/v) -galactosidase enzyme ((Lactozym Pure 6500 L, dX
Novozymes). The enzymatic reaction occurred under incubation = x X (2)
dt
in shaker (Solab Cientca SL 222/CFR) stirred at 100 rpm and
dS 1 dX 1 dP
controlled temperature of 30 C for 3 h. Further, the enzyme was = (3)
inactivated by heating the solution in thermostatic bath at 100 C dt YX/S dt YP/S dt
for 5 min. dP dS
= p X = YP/S (4)
dt dt
2.6. Development of the inoculum
The specic rates of cell growth (Eq. (5)) and product formation
(Eq. (6)) were based on the models from Phisalaphong et al. [62].
In the pre-inoculum preparation, two loop full of the S. cere-
These models consider inhibition terms by the substrate [63] and
visiae var. boulardii yeast grown in agar medium were transferred
product [64].
aseptically to an Erlenmeyer ask containing 250 mL YEPD medium
 
sterilized in autoclave brand Phoenix model AV75 at 121 C for  
S P
15 min. The ask was kept in shaker under stirring of 100 rpm x = m 2
1 (5)
at 30 C for 24 h. For the inoculum preparation, ammonium S+Ks+ SKi P1m
sulfate(NH4 )2 SO4 , magnesium sulfateMgSO4 and monobasic  
potassium phosphateKH2 PO4 were added to the hydrolyzed CWP S
 P

under the conditions dened by the CCRD design. The pH of the p = Pm 2
1 (6)
S
S+KsP + Ki P2m
solution decreased to 5.5, and this value was controlled by the addi- P
74 D.E.G. Trigueros et al. / Biochemical Engineering Journal 110 (2016) 7183

where m is the maximum specic growth rate and m P is the max-

imum specic rate for the formation of the fermentation product.
Ks, Ki and P1m are respectively the saturation constant, the inhi-
bition by the substrate constant and the inhibition by the product
constant, all regarding cell growth. KsP , KiP and P2m are respectively
the saturation constant, inhibition by the substrate constant and
the inhibition by the product constant, all regarding the production
of the fermentation product.

2.10. Model parameters identication procedure

The model parameters were estimated by using the PSO opti-

mization method [65] implemented on software Maple 2015, as
described by Trigueros et al. [66]. The solution of the system of
ordinary differential equations was performed by the Rosenbrocks
method. The objective function was based on the least square sta-
tistical method (Eq. (7)), by assuming normal distribution of the
experimental variances and constant errors in all experimental
conditions [67].

NP   2

NV 
yij yij
FO = (7)
ymax
i
i=1 j=1

where yij is the value calculated by the model; yij is the experi-
mental value for the variable i and experimental data j. All the
computer simulations were performed in a microcomputer Intel
Core i7, 8 GB RAM memory.

3. Results and discussion

3.1. Preliminary cultivations

Three preliminary runs were performed in order to compare the

cultivation performance of S. cerevisiae var. boulardii in an anaerobic
batch system at 30 C, 100 rpm over 30 h of fermentation by using
pure CWP (medium 1), CWP supplemented with salts (medium
2) and glucose (medium 3), respectively. The composition of the
medium 1 was: 150 g L1 CWP [containing 90% lactose, of which
93% was hydrolyzed; 0.09% Mg; 0.04% K and 0.48% P]. The compo-
sition of the medium 2 was the same concentration of the medium 1
added supplementation: 3 g L1 (NH4 )2 SO4 ; 1.2 g L1 MgSO4 7H2 O
and 3 g L1 KH2 PO4 . The medium 3 was composed of 63 g L1 glu-
cose; 2 g L1 meat peptone; 2 g L1 yeast extract; 0.36 g L1 urea;
0.12 g L1 (NH4 )2 SO4 ; 2.4 g L1 MgSO4 7H2 O and 0.6 g L1 KH2 PO4 .
The results presented in Fig. 1 shows the potential of the CWP
utilization as a carbon source. The yeast growth in CWP pure or
supplemented was shown to be similar, which stimulated investi-
gations regarding the supplementation of the culture medium. On
the other hand, by using the same concentration of carbon source Fig. 1. Cultivation of S. cerevisiae var. boulardii on pure CWP (), supplemented
consumed on both tests (about 60 g L1 glucose), the growth of S. CWP () and sinthetic standard medium (): (A) biomass growth (ln N/N0); (B) TRS
consumption and (C) evolution of the medium pH as a function of time (h).
cerevisiae var. boulardii reached its maximum value shortly after
15 h of fermentation, followed by cell death. This result points out
that the standard medium is not ideal for yeast growth, what justify 180 5. These values were used to dene the real values of the lev-
the search for an optimized culture medium. els of the hydrolyzed CWP concentration (x4 ) dened during the
CCRD design.
3.2. Enzymatic hydrolysis of the cheese whey permeate
3.3. Experimental CCRD design and cell growth response
The enzymatic hydrolysis experiments from the -galactosidase
enzyme for lactose conversion in glucose and galactose were per- The real values for the independent variables and the respec-
fomed for 60, 100, 140, 180 and 220 g L1 CWP concentrations. tive experimental CCRD results obtained for the yeast cultivation
The reactions were performed at 30 C, pH 6.5 and 0.125% enzyme at 30 C, initial pH 5.5 and 100 rpm are presented in Table 2. The
over 180 min. Admiting 8892% lactose present in the powder analytical triplicate average error for all points of all runs were
whey permeate, the tests provided average conversions greater 0.85%. The pH 5.5 was chosen for the planning of runs because it
than 90%. The concentrations of the glucose and galactose mix- is the value that is closest to the pH of the cheese whey. The 30 C
ture resulted in (g L1 ): 50 5; 90 5.1; 120 5.9; 150 9.3 and temperature is considered optimal for culturing and growth of Sac-
 D.E.G. Trigueros et al. / Biochemical Engineering Journal 110 (2016) 7183 75

Table 2
Real values of the CCRD design and data experimental and predicted (in g L1 ) by multiple linear regression model response.

Run x1 x2 x3 x4 Response CCRD RSM

(NH4 )2 SO4 MgSO4 KH2 PO4 Hydrolyzed CWP Cell Biomass SD SE Predicted Value

1 4.5 1.25 1.5 85 6.05 0.04 0.03 6.09 0.44

2 7.5 1.25 1.5 85 5.50 0.11 0.06 5.60 0.44
3 4.5 3.75 1.5 85 6.58 0.03 0.02 6.40 0.44
4 7.5 3.75 1.5 85 5.52 0.04 0.03 5.57 0.44
5 4.5 1.25 4.5 85 5.97 0.08 0.05 5.95 0.44
6 7.5 1.25 4.5 85 5.09 0.12 0.07 5.46 0.44
7 4.5 3.75 4.5 85 5.98 0.12 0.08 6.75 0.44
8 7.5 3.75 4.5 85 6.25 0.04 0.02 5.94 0.44
9 4.5 1.25 1.5 145 8.44 0.10 0.05 8.40 0.44
10 7.5 1.25 1.5 145 10.00 0.04 0.03 9.24 0.44
11 4.5 3.75 1.5 145 8.84 0.17 0.09 8.48 0.44
12 7.5 3.75 1.5 145 9.33 0.14 0.08 8.99 0.44
13 4.5 1.25 4.5 145 8.08 0.17 0.09 8.04 0.44
14 7.5 1.25 4.5 145 9.06 0.14 0.08 8.89 0.44
15 4.5 3.75 4.5 145 9.08 0.31 0.02 8.62 0.44
16 7.5 3.75 4.5 145 9.17 0.17 0.09 9.14 0.44
17 3 2.5 3 115 7.93 0.11 0.06 7.90 0.44
18 9 2.5 3 115 7.56 0.09 0.05 7.93 0.44
19 6 0 3 115 7.73 0.13 0.08 7.82 0.44
20 6 5 3 115 8.12 0.11 0.06 8.38 0.44
21 6 2.5 0 115 6.21 0.06 0.04 6.78 0.44
22 6 2.5 6 115 7.02 0.09 0.05 6.80 0.44
23 6 2.5 3 55 4.39 0.06 0.04 3.81 0.44
24 6 2.5 3 175 8.40 0.14 0.09 9.33 0.44
25 6 2.5 3 115 7.22 0.08 0.06 6.71 0.29
26 6 2.5 3 115 6.78 0.14 0.08 6.71 0.29
27 6 2.5 3 115 6.76 0.08 0.05 6.71 0.29
28 6 2.5 3 115 6.09 0.08 0.06 6.71 0.29

SD: standard deviation; SE: standard error.

Table 3 ing to ANOVA (see Table 3), within the 95% condence interval
Effects values and ANOVA statistical data obtained by the CCRD experimental data.
(p-value < 0.05), the factors which show inuence on the growth
Effect SS df MS F-value p-value of S. cerevisiae var. boulardii are the concentrations of CWP, ammo-
x1 (Quadratic) 0.610 2.484 1 2.484 10.658 0.0034 nium sulfate, magnesium sulfate, and the interaction between
x2 (Quadratic) 0.700 3.271 1 3.271 14.033 0.0010 ammonium sulfate and CWP.
x4 (Linear) 2.756 45.590 1 45.590 195.550 0.0000 The greatest effect on the growth of S. cerevisiae var. boulardii
x1 :x4 (Linear) 0.668 1.783 1 1.783 7.650 0.0101 at 30 C, initial pH 5.5 and 100 rpm was assigned to the hydrolyzed
Error 5.362 23 0.233
CWP. The response surfaces generated from the statistical analysis
Total 57.680 27
of the CCRD results are presented in Fig. 2. It could be seen that
SS: Sum of Square; df: Degree of freedom; MS: Media Square.
the biomass reached greater levels when increasing CWP concen-
tration, according to its linear term positive effect (Table 3) (see
charomyces species, and the 100 rpm agitation speed did not limited Fig. 2ac). Although the effect of the ammonium sulfate concen-
the process during the preliminary tests. tration was low within the (NH4 )2 SO4 investigated concentration
The experimental data obtained by CCRD were used in the pre- range (39 g L1 ) (see Fig. 2a,e,f), the yeast cultivation using CWP as
diction of biomass concentration as a function of the concentrations a substrate requires supplementation with nitrogen source because
of hydrolyzed CWP and supplementaion by N, Mg and K from the of the importance of this element on the cell biomass synthe-
response surface methodology (RSM) according to multiple regres- sis [68]. The effect of the magnesium sulfate concentration was
sion performed on software Statistica version 10 (Statsoft, Inc.). positive, although low within the MgSO4 investigated concentra-
The ANOVA results for evaluation of the model validity are pre- tion range (05 g L1 ) (see Fig. 2b,d,f). Taking into account that
sented on Table 3. The model (Eq. (8)) considers only the statistically the CWP used as a substrate in the research constitutes of 0.09%
signicant regression coefcients and according to the F test (p- magnesium it is possible to use very low magnesium sulfate con-
value < 0.05) it describes adequately the experimental data, by the centration without undermining biomass growth, highlighting that
quadratic terms of the variables ammonium sulfate (x1 ) and mag- magnesium is the activator of the glucose 6-phosphate enzyme
nesium sulfate (x2 ), the linear term of the variable ammonium responsible for biomass production [38]. The effect of the potas-
sulfate (x1 ), and the linear interaction between ammonium sul- sium phosphate concentration was found to be negligible, as shown
fate and hydrolyzed CWP (x1 :x4 ). The Ftab value was obtained in Fig. 2ce. Therefore, not adding potassium sulfate does not harm
for 5% signicance (Ftab(5%,4.23) = 3.18). According to the value of the anaerobic growth of S. cerevisiae var. boulardii using CWP, since
the determination coefcient, the model is able to describe 92% this substrate constitutes of 0.04% potassium. Besides, according
(Radjusted = 0.84) of the responses as a function of the independent to Gancedo et al. [38] phosphate groups can promote competitive
variables in the region investigated by the CCRD design. inhibition with the substrate glucose 6-phosphate.

Cell biomass = 11 2.35x1 + 0.134x1 2 + 0.222x2 2

3.4. Effect of initial pH on specic growth rate
+ 0.00742x1 x4 (8)

The estimation of the variables effects and their interactions on the The pH of the cultivation medium plays an important role on
cell growth was obtained considering 5% signicance level. Accord- the activity of the enzyme glucose-6-phosphate-dehydrogenase
76 D.E.G. Trigueros et al. / Biochemical Engineering Journal 110 (2016) 7183

Fig. 2. Effects of the concentrations of ammonium sulfate, magnesium sulfate, potassium phosphate and hydrolyzed CWP on cell biomass efciency based on the CCRD and
RSM analysis, where: (a) x4 :x1 ; (b) x4 :x2 ; (c) x4 :x3 ; (d) x3 :x2 ; (e) x3 :x1 ; and (f) x2 :x1 .

(G6PDH) responsible for biomass production. The initial effect respectively. Fig. 3a presents the experimental values of the maxi-
of pH was evaluated with the experimental data of the growth mum specic growth rate (max ) and the model tting x = f(pH).
of S. cerevisiae var. boulardii at 30 C, 100 rpm, 180 g L1 CWP, By analyzing Fig. 3a it can be seen that the cultivation of S. cerevisiae
7.5 g L1 ammonium sulfate, 1.25 g L1 magnesium sulfate, 1.5 g L1 var. boulardii at 30 C presents the greatest growth on the pH range
monobasic potassium phosphate. The pH values were 3.5; 4.5; 5.5; of 4.55.5.
6.5 e 7.5. The specic growth rate (x ) as a function of the medium
pH was determined according to Eq. (9) [69]. The model parameters
were estimated by PSO global search algorithm. The values of max ,
K1 and K2 parameters are 0.19 h1 , 15.104 g L1 and 1.107 g L1 , X = H+ max

K2
(9)
1+ K1
+ H+
 D.E.G. Trigueros et al. / Biochemical Engineering Journal 110 (2016) 7183
Fig. 3. Specic growth rate of S. cerevisiae var. boulardii at 30 C (A) effect of initial
pH (B) effect of initial C:N ratio at initial pH 5.5.
3.5. Effect of the initial C:N ratio on specic growth rate Subject to
The effect of the initial ratio between the carbon and nitro-
gen elements present on the cultivation medium was evaluated
by using as a criterion the maximum value of specic growth rate
(SGR) experimentally obtained. The cultivations without nitrogen
and cultivation with the C:N ratio equal to 2, 4, 7, 8, 12, 16, 20, 25,
30 and 35 were dened.
The model of Wu et al. [70], given by Eq. (10), was t to the
experimental data (see Fig. 3b). The model considers limiting and
inhibiting effects by the substrate (C:N ratio). The non-linearly cou-
pled kinetic model parameters of maximum specic growth rate x7 0.40x6 = 0
max , saturation constant Ks, inhibition constant Ki and n were x8 0.40x5 = 0
estimated by PSO search method.
max S 8 x8 20
X = S
n (10) M1 M1
Ks
1+ S
+ Ki
The experimental data obtained in the batch cultivation of S.
cerevisiae var. boulardii at 30 C and initial pH 5.5, resulted in the
following estimated kinetic parameters values: max = 0.19 h1 ; M2 =
Ks = 0.53 g L1 ; Ki = 1.1015 g L1 and n = 3.17, respectively (see Eq.
(10)). From the derivative of the function x = f(S), the estimated
parameters were used to calculate the critical initial ratio between
carbon and nitrogen present in the medium. The model prediction
presented in Fig. 3b shows that the anaerobic growth of S. cere-
visiae var. boulardii in batch system was limited by the substrate
where the initial C:N ratio was under 8. The inhibition by the sub-
77
strate occurred when the C:N ratio was over 20. The initial C:N
ratio between 8 and 20 provided the estimated value of maximum
specic growth rate max = 0.17 h1 , for the given experimental
conditions. If it is possible to compare, the growth of S. cerevisiae
var. boulardii at 30 C and initial pH 5.5 in the absence of nitrogen
was similar to the strong inhibition by the initial C:N ratio equal to
35.
3.6. Optimization of the nutrient medium
The strategy of cultivation medium optimization was based
on CCRD analysis methodology combined with mathematical pro-
gramming. Thus, a non-linear model (NLP) was proposed for
the optimization of medium components concentrations such as:
hydrolyzed CWP and (NH4 )2 SO4 , MgSO4 and KH2 PO4 . The objective
function was dened by statistical methods taken from the non-
linear multiple regression package coded in the software Statistica
version 10 (Statsoft, Inc.) (see Eq. (8)). The cultivation boundary
conditions were dened for the growth of the yeast Saccharomyces
using CWP as a substrate. Firstly the inequalities (Eqs. (11)(14))
were dened according to the elemental composition of the yeast
(4652% carbon, 59% nitrogen, 0.44.5% phosphorus; 0.22.5%
potassium and 0.10.5% magnesium). An equation was dened for
the concentration of glucose and galactose present in the lactose
(5050%) (Eq. (15)). The concentration of the glucose used for cell
growth was chosen based on the yield coefcient of biomass on this
substrate. Experimentally, this value (Yx/s = 0.35 0.55) (Eq. (16))
was obtained during the cultivation without aeration via oxide
reductive process. The carbon concentration regarding glucose
used for cell growth was also considered (Eq. (17)). The carbon con-
centration present in the medium was determined via the glucose
concentration (Eq. (18)). The values of C:N ratio was kept between
8 and 20 (Eq. (19)). The ratio between the molecular mass of the
nutrient source and its respective element was used as a conversion
factor (Eqs. (20)(22)), being all independent positive variables (Eq.
(23)).
maximizing z = 11 2.35x1 + 0.134x1 2 + 0.222x2 2 + 0.00742x1 x4
0.05M1 z x1 0.09M1 z (11)
0.001M2 z x2 0.005M2 z (12)
0.002M3 z x3 0.025M3 z (13)
0.46z x7 0.52z (14)
2x5 x4 = 0 (15)
x6 YX/S x5 = 0 (16)
(17)
(18)
x1 x1
(19)
MW(NH4 )2 SO4
M1 = (20)
MWN
MWMgSO4
(21)
MWMg
MWKH2 PO4
M3 = (22)
MWK
x1 ,x2 ,x3 ,x4 ,x5 ,x6 ,x7 ,x8 0 (23)
The model for the maximization of cell biomass at the end of
the anaerobic batch cultivation of S. cerevisiae var. boulardii on
78 D.E.G. Trigueros et al. / Biochemical Engineering Journal 110 (2016) 7183

Fig. 4. Dynamic proles of the anaerobic batch cultivation of S. cerevisiae var.

boulardii at 30 C, 100 rpm and initial pH 5.5: (A) experimental data for the biomass
growth (runs 8, 10, 23, 24 and 25 from the CCRD design, with 85, 145, 55, 175,
115 g L1 initial hydrolyzed CWP, respectively); (B) experimental data obtained
by the optimized run (x1 = 10 g L1 , x2 = 1.10 g L1 , x3 = 0.3 g L1 , x4 = 200 g L1 ) and
kinetics model simulations for the biomass growth, consumption of glucose and
galactose, and production of ethanol and carbon dioxide.

hydrolyzed CWP was solved by using the local solver CONOPT3

available on software GAMS version 2.8. The NLP model resulted in
15 equations and 9 continuous variables (8 independent variables
and 1 dependent variable). The simulation results obtained in opti-
mization procedure based on NLP model are presented in Table 4, in
comparison with the values obtained experimentally by the CCRD.
The cell biomass concentration determined by NLP provided
results greater than the ones obtained experimentally (see Table 4).
Moreover, the demand for nitrogen source increases linearly with
the increase of hydrolyzed CWP concentration. The C:N ratio cal-
culated for all tests were on the optimal range (820). However,
for the experimental run 24 it is suggested that the unbalanced Fig. 5. Specic growth rate: (A) as a function of the variables glucose and ethanol
during glucose consumption; (B) as a function of the variables galactose, glucose
medium (C:N very close to the inhibition limit) was the reason for
and xed ethanol during galactose consumption; (C) as a function of the variables
the low specic growth rate experimentally observed (see Fig. 4a), galactose and glucose (with and without the effect of repression by glucose) during
where the yeast growth on 175 g L1 hydrolyzed CWP was lower galactose consumption.
than expected. Fig. 4a presents the curves for cell growth obtained
experimentally by CCRD (runs: 8, 10, 23, 24 and 25). These exper-
imental data were chosen with the aim to present a physiological stant growth around 17 h of fermentation (see Fig. 4a) with total
characteristic of cell growth, with no sharp uctuations resulted glucose and partial galactose metabolization (experimental data
from any possible errors. not shown). It must be noticed, that during the rst hours of fer-
The CCRD tests presented a tendency similar to the biomass mentation, a simultaneous consumption of glucose and galactose
growth, in other words, they showed maximum level and con- was observed. At this point, it is suggested that the simultane-
 D.E.G. Trigueros et al. / Biochemical Engineering Journal 110 (2016) 7183 79

Table 4
Experimental values by CCRD and Optimized values by nonlinear programming.

Independent variable (g L1 ) Dependent variable (g L1 )

Hydrolyzed CWP (NH4 )2 SO4 MgSO4 KH2 PO4 Cell biomass(a) C:N

55 2.74 0.06 0.08 7.50 18.9 NLP (Yx/s = 0.55)

6 2.5 3 4.39 0.06 9.5 CCRD (run 23)
85 4.00 0.08 0.11 7.40 20 NLP (Yx/s = 0.50)
7.5 3.75 4.5 6.25 0.04 10.7 CCRD (run 8)
115 5.42 0.10 0.14 8.20 20 NLP (Yx/s = 0.45)
6 2.5 3 7.22 0.08 16.3 CCRD (run 25)
145 9.46 0.11 0.15 12.50 14.44 NLP (Yx/s = 0.40)
7.5 1.25 1.5 10.00 0.04 14.6 CCRD (run 10)
175 9.99 0.12 0.16 15.55 16.5 NLP (Yx/s = 0.35)
6 2.5 3 8.4 0.14 19.25 CCRD (run 24)

(a) Time of fermentation: 17 h. CWPcheese whey permeate; Ccarbon; Nnitrogen.

ous consumption is related to the 50%:50% ratio for the substrates Table 5
Parameters values of the kinetic model describing the anaerobic batch growth of S.
concentrations. The utilization of galactose is an inducible process
cerevisiae var. boulardii, estimated by the PSO global search algorithm.
which is genetically regulated and occurs when the glucose con-
centration reaches an appropriate value during the rst hours of Glucose (S1) Galactose (S2)
fermentation. The exhaustion of the rst carbon source occurred m1 0.21 h1
p
m1 0.80 h1 m2 0.028 h1
p
m2 0.50 h1
after about 17 h. Shortly after, galactose consumption ceased, sug- Ks1 30 g L1 Ks1P 2 g L1 Ks2 5 g L1 Ks2P 1 g L1
gesting that the cultivation medium proposed by the CCRD are Ki1 80 g L1 Ki1P 65 g L1 Ki2 110 g L1 Ki2P 200 g L1
P1m 150 g L1 P2mS1 300 g L1 YX/S2 0.20 g g1 P2mS2 25 g L1
not balanced and thus, the additional nutrients (N, Mg and K)
YX/S1 0.23 g g1 YXS1/CO2 1.9 gg1 YXS2/CO2 2.3 g g1 kd 75 105 h1
were completely used during glucose metabolization, making the KR 75 g L1 mS1 35 103 g L1 mS2 35 103 g L1
enzyme activation required for galactose metabolization impossi-
ble. On the other hand, if diauxic growth is involved, it is possible
that a long time was necessary for cell machinery and internal dP P

= GLU + PGAL X (27)

metabolite control to synthesize enzymes involved in the metabolic dt
pathway of galactose utilization in a second phase of cell growth. dCO2
Based on the results obtained by optimized model, the following = Kla(CO2 CO2 ) (28)
dt
values of nutrients for the execution of an independent experiment
where
were proposed: 10 g L1 (NH4 )2 SO4 , 1.10 g L1 MgSO4 and 0.3 g L1  
KH2 PO4 in 250 g L1 CWP with average composition of 90% lactose, m1 S1
 P

resulting in 7.45 g L1 and 10.35 g L1 cell biomass in 17 and 30 h of GLU = 1 (29)
S1 2 P1m
cultivation, respectively. S1 + KsS1 + KiS1

Pm1 S1
 P

3.7. Microbial growth kinetics PGLU = 1 (30)
S2 P2mS1
S1 + KsS1P + 1
KiS1P
The kinetics of microbial growth (X), consumption of the sub-

strate of fast metabolization (S1), consumption of the substrate of   
m2 S2
slow metabolization (S2), besides the kinetics of product (P) and GAL = 1 P KR
(31)
carbon dioxide (CO2 ) formation were dened by the rearrange- (S02 S2 ) 2
P1m KR + S1
S2 + KsS2 + KiS2
ment of the system of differential equations (see Eqs. (2)(4)) as
a function of cell growth rate (X ) and product formation rate (P ),
Pm2 S2
 P

considering the models proposed by Phisalaphong et al. (2006) [62]
PGAL = 1 (32)
(see Eqs. (5)(6)). In this paper, the mathematical modeling of S. (SO2 S2 ) 2
P2mS2
S2 + KsS2P + KiS2P
cerevisiae var. boulardii fermentation process was proposed accord-
ing to Eqs. (24)(32), taking into account the inhibition by the The system of differential equations included seven sto-
substrates [63], the inhibition by the product [64], and the catabolic ichiometric parameters (YX/S1 ,YX/S2 ,YP/S ,YXS1/CO2 ,YXS2/CO2 ,
repression by glucose concentration [71]. Further, the modeling m, kd) and sixteen kinetic parameters
procedure considered the formation of metabolic products [72], the (m1 ,m2 ,KsS1 ,KsS2 ,KiS1 ,KiS2 ,P1m ,Pm1 ,Pm2 ,KiS1P ,KiS2P ,KR ). All
rate of cell maintenance by the substrate (ms ) and the cell death rate parameters were estimated by applying the Particle Swarm Opti-
(kd ). The experimental kinetic data obtained from the independent mization (PSO) global search algorithm implemented on software
run (200 g L1 hydrolyzed CWP) were used for the validation of the Maple 2015 and are presented in Table 5. The parameters of the
mathematical model. PSO algorithm were chosen as follows: c1 = c2 = 1.5, initial = 0.9,
dX end = 0.4. The minimum value of the objective function (Eq.
= (GLU + GAL kd) X (24) (7)), chosen on the base of least square optimization procedure,
dt
resulted in 7.103 by using 1000 particles and 25 iterations.
 
dS1 1 1 Fig. 4b shows the kinetic simulations for the growth of S. cere-
= GLU + PGLU + mS1 X (25) visiae var. boulardii, consumption of glucose and galactose, t to the
dt YX/S1 YP/S1
respective experimental data, and the curves for ethanol and CO2
  formation. By analyzing the simulated biomass growth curve and
dS2 1 1
= GAL + PGAL + mS2 X (26) its corresponding experimental data, the catabolic repression phe-
dt YX/S2 YP/S2
nomenon was observed, according to the diauxic growth [40]. It is
80 D.E.G. Trigueros et al. / Biochemical Engineering Journal 110 (2016) 7183
Fig. 6. Ethanol specic production rate: (A) as a function of the variables glucose and ethanol concentrations and (B) as a function of the variables galactose and ethanol
concentrations.
characterized by the preferential consumption of one carbon source
present in the cultivation media over the other source. In some
cases, glucose was preferentially used in comparison to galactose
[9], showing its effect on the metabolic regulation mechanisms [47].
This effect was observed during the diauxic lag phase after glucose
exhaustion, followed by the very slow consumption of galactose
[44]. It must be note that in practice, for the researcher, it is impor-
tant to experimentally obtain more details about the simultaneous
usage of glucose and galactose, how this reects on the process
performance and which is the quantitative criterion for this eval-
uation. This can be studied separately case by case. Nevertheless,
in practice, there are only two important possibilities. First, both
substrates are simultaneously used by the cells with different spe-
cic growth rates. Second, one of the substrates is a repressor of the
utilization of the other one (metabolic control on the cell level). In
both cases the value of the ratio (in our case Glu:Gal) can be used as
a quantitative criterion which shows when the phenomenon may
occur.
The tendency of the experimental data regarding galactose uti-
lization is assigned to the metabolic products formation and were
observed in the run with optimized medium, as well as in all CCRD
runs. This phenomenon occurred in simultaneous yeast growth on
two substrates during the ethanol and CO2 product formation. Such
(34)
anaerobic metabolism allowed formation and excretion of glycerol
and organic acids, besides other compounds with less quantitative
signicance [73].
Theory suggested that the production of glycerol, the most
abundant of the secondary organic compounds in fermentation
is coupled to the cell redox equilibrium and to a response to the
osmotic stress that occurred in the presence of high sugars or salts
concentrations. Therefore, intrinsically to the production of ATP for
cell growth, it occurs the formation of metabolic products that are
related to the yeast survival, such as ethanol, glycerol, acetates,
succinates, organic acids and others [7,74], which can be used by
the yeast. According to Abramov et al. [68], yeasts synthetize all
amino acids and proteins from inorganic nitrogen, organic carbon
and intermediary products from the degradation of carbohydrates
formed during the fermentation and respiration.
The values of the estimated kinetic parameters (m1 and m2 )
agreed with the experimental data, since the evaluated maxi-
mum specic growth rate was greater when using glucose than
when using galactose [71]. This suggested that the genes respon-
sible for the catabolism of the sugars of slow fermentation were
repressed by glucose. During the rst step of cell growth, the
ethanol formation occurred as a result of the energetic metabolism
and therefore, as a product associated with cell growth. The evalu-
ated ethanol yield (YP/S ) was twice higher than the one of biomass
obtained during the phase of glucose (YX/S1 ) and galactose (YX/S2 )
metabolization, and it agreed with the stoichiometry of the yeast
fermentation on carbon and nitrogen source [30]. When tting the
developed mathematical model to the experimental data, it was
conrmed involvement of a breath-fermentation metabolism in an
oxide-reductive metabolism of S. cerevisiae var. boulardii, forming
cell biomass, CO2 and ethanol, with accumulation and oxidation
of metabolic products. Various researches reported slow fermenta-
tions when using initial lactose concentration over 100 or 200 g L1 ,
assigning the issue to osmotic pressure, low ethanol tolerance or
high salts concentration [10,11].
3.7.1. Response surface analysis
Considering the inhibitory effect of the glucose and galactose
substrates concentrations, the initial critic substrate concentration
(S* ), evaluated maximum specic growth rate (* ) and inhibi-
tion intensity (I* ) were determined by the derivative of Andrewss
model [63], according to Eqs. (33)(35).

S = KsKi (33)
m
= S
2 Ki
+1
Ks
I = (35)
Ki
In order to avoid inhibition by the substrate, which occurs possibly
because of the elevated osmotic pressure, the CWP concentration
must be controlled under 156 g L1 (sugar < 75 g L1 ) in batch sys-
tem fermentations, according to Ozmihci and Kargi [10]. In this
present study, the critical concentrations of glucose (SS1 = 49gL1 )
and galactose (SS2 = 23gL1 ) were determined on the base of the
estimated kinetic parameters. Therefore, the use of concentrations
of total reducing sugar over 72 g L1 resulted in inhibition by the
substrate concentration, value similar to the one found by Ozmihci
and Kargi [10] and Ghaly et al. [25].
The Fig. 5a and b shows the inhibition by glucose and galactose
concentration, and their respective values of * equal to 0.094 h1
and 0.020 h1 . In Fig. 5b, it can be seen that the inhibition by
galactose concentration was much lower (IS2 = 0.045) than the one
estimated for glucose (IS1 = 0.375). It can be seen that using con-
centrations of hydrolyzed CWP over 250 g L1 results in extremely
low specic growth rate GLI (see Fig. 5a) during the anaerobic
batch cultivation of S. cerevisiae var. boulardii. Moreover, by analyz-
ing the response surface (Fig. 5a) there was a linear decreasing of
GLI as the ethanol concentration was approaching the maximum
 D.E.G. Trigueros et al. / Biochemical Engineering Journal 110 (2016) 7183
value of P1m equal to 150 g L1 . The calculations show that GLI
decreased approximately 5% every 15 g L1 of ethanol produced.
The developed kinetic model was able to simulate the ethanol
production curve, whose stoichiometric parameter value YP/S was
estimated equal to 0.4. During the rst growth phase, the simulated
curve showed that the ethanol concentration increased linearly
until reaching the maximum value, close to 35 g L1 (see Fig. 4b).
By analyzing Fig. 5b, it was observed that when galactose con-
centration is four times higher than its critical value (SS2 * ), there
is a reduction of the GAL value in 25%. This clearly showed how
much galactose consumption can be slow, making cell growth
null in high concentrations of the sugar of slow metabolization.
Moreover, it was veried that the greater the ethanol concen-
tration produced during fermentation, the lower the specic
growth rate GAL . For example, by using the critical galactose
concentration (SS2 = 23gL1 ), the GAL value decreased by increas-
ing the ethanol concentration, according to estimated values:
0.020 h1 (Eth = 0 g L1 ); 0.0135 h1 (Eth = 50 g L1 ); 0.0060 h1
(Eth = 100 g L1 ) (see Fig. 5b). The galactose consumption did not
occur when 150 g L1 ethanol was produced. Thus, it was possi-
ble that the galactose was not completely metabolized due to the
ethanol production responsible for the ceasing the cell biomass
growth.
Fig. 5b also shows the inuence of the glucose concentration on
GAL for different ethanol concentrations (0, 50, 100 and 150 g L1 ).
The glucose repression effect was evaluated by the term in Eq. (31)
which incorporated the parameter KR in the specic growth rate by
galactose consumption GAL . In Fig. 5c, the GAL value decreased
when glucose concentration was higher, increasing the catabolic
repression. For 75 g L1 glucose the value of GAL decreased 50%.
In Fig. 6 it can be seen that the specic rate of ethanol production
P decreased linearly when the ethanol concentration increased,
demonstrated by the value of KiP parameter, and became null when
its concentration reached a critic value (P2m ). This value was esti-
mated for the glucose consumption phase P2mS1 = 300 g L1 . During
the rst growth phase, as the glucose was consumed, the specic
production rate P increased until reaching the maximum real
value (0.60 h1 ) (see Fig. 6a) which corresponded to the end of the
rst exponential growth phase. In other words, the process was
close to the exhaustion of glucose, giving about 35g L1 ethanol
(see Fig. 4b). Further, the ethanol concentration curve decreased
until reaching the concentration of 25 g L1 , an evidence that on
the second growth phase the yeast most likely consumed metabolic
products. According to Luong [75], the ethanol tolerance by a strain
is about 100 g L1 for cell growth and 200 g L1 for ethanol produc-
tion.
3.7.2. Estimation of the maximum biomass growth and the
maximum ethanol production
On the base of the developed kinetic model and the optimized
kinetic parameters it was possible to estimate the quantity of
biomass produced during the diauxic growth: on the rst phase
which represents the moment of glucose exhaustion, as well as
during the second phase, where GAL is extremely low characteriz-
ing constant cell growth. The results presented in Table 6 showed
the efciency of the proposed kinetic mathematical model for the
simulation and prediction of the growth kinetics in anaerobic batch
cultivation of S. cerevisiae var. boulardii. The experimental value of
biomass growth during 17 h of cultivation (CCRD: run 8, 10, 23, 24
and 25) was compared to the value obtained by NLP optimization
(Yx/s = 0.35; 0.40; 0.45; 0.50 and 0.55) and to the value obtained
from the developed mathematical model.
Results obtained by the NLP cultivation media optimization
model showed that the global optimum value of biomass yield
was equal to Yx/s = 0.50, where the cell biomass (with 50% car-
81
Table 6
Values of biomass concentration and ethanol production obtained by the simula-
tion of the anaerobic cultivation kinetic model for each presented hydrolyzed CWP
concentration.
Hydrolyzed CWP (g L1 ) Biomass growth (g L1 ) Ethanol (g L1 )
First phasea Second phaseb Maximum productionc
55 d
4.50 (11 h) 5.00 (15 h) 15
85 8.00 (12 h)d 9.00 (16 h) 20
115 8.00 (13 h)d 10.00 (20 h) 25
145 11.00 (12 h)e 15.00 (23 h) 30
175 6.00 (24 h)e 15.00 (80 h) 35
a
Exhaustion of glucose.
b
GAL very low.
c
Product associated to the rst growth phase.
d
Unique growth ( = m1 + m2).
e
Diauxic growth.
bon) was estimated equal to 8.20 g L1 by using 40 g L1 hydrolyzed
CWP and 1.93 g L1 (NH4 )2 SO4 , 0.04 g L1 MgSO4 and 0.06 g L1
KH2 PO4 . This result showed that the maximum concentration of
biomass obtained during the anaerobic fermentation of S. cere-
visiae var. boulardii can be reached by using low concentrations
of CWP, what required lower use of nitrogen sources and insigni-
cant values for additional nutrients (Mg and K) since the substrate
contained enough nutritional elements necessary for yeast unlim-
ited growth. It was highlighted the importance of the nitrogen
source and its ideal concentration in the cultivation media, since
nitrogen can minimize the catabolic repression by glucose, and a
balanced C:N ratio can prevent the ammonium effect, which can
be toxic for cell synthesis. The utilization of 40 g L1 hydrolyzed
CWP was under the critical concentration considered for inhibi-
tion by the substrate (about 70 g L1 ) and under the value where it
was observed the catabolite repression by glucose (about 75 g L1 ).
Thus, disregarding these two phenomena, by using the simulations
of the growth kinetic model with evaluated optimal initial con-
ditions (X0 = 0.8 g L1 , S1 + S2 = 40 g L1 ), resulted in the maximum
biomass value equal to 4 g L1 during 8 h cultivation of S. cerevisiae
var. boulardii. Moreover, the yeast growth was unique by show-
ing total metabolization of both substrates glucose and galactose.
The estimation of the process performance by using the developed
kinetic model clearly demonstrated that after 10 h cultivation, the
cell growth suffer strong inuence by the production of ethanol,
which can reach 12 g L1 .
4. Conclusion
This paper presents an efcient strategy of optimization of the
cultivation medium for fermentation of hydrolyzed cheese whey
permeate as a substrate for S. cerevisiae var. boulardii by using the
CCRD coupled to NLP procedure, which analyze levels of compo-
nents that are already known to be essential to the growth cell. The
statistical analysis results have shown that the greatest effect on
cell growth was assigned by hydrolyzed CWP, while the inuence of
supplemented K source was shown to be insignicant. By modeling
the specic growth rate as a function of pH it is shown that the opti-
mal range of pH is between 4.5 and 5.5. The initial C:N ratio between
8 and 20 provided the estimated value of maximum specic growth
rate max = 0.17 h1 . Moreover, the kinetics mathematical model-
ing of the anaerobic batch cultivation of S. cerevisiae var. boulardii
shows itself to be adequate for the simulation and prediction of
the cell growth, consumption of the substrate of fast-glucose and
slow-galactose metabolization, and production of ethanol and CO2 .
The critical concentrations of glucose and galactose were deter-
mined equal to 49 g L1 and 23 g L1 , respectively (about 70 g L1
hydrolyzed CWP). The RSM dened by kinetic parameters esti-
mated by the PSO algorithm shows that the main inuence on
82 D.E.G. Trigueros et al. / Biochemical Engineering Journal 110 (2016) 7183
the yeast cell growth comes both from the catabolite repression
by glucose concentration and high value of the ethanol concentra-
tion produced during fermentation. The global optimum value of
biomass yield was equal to Yx/s = 0.50, where the cell biomass was
estimated to be 8.20 g L1 by using 40 g L1 hydrolyzed CWP, which
was under the critical concentration considered for inhibition by
the substrate (about 70 g L1 ) and under the value in which it was
observed the catabolite repression by glucose (about 75 g L1 ). In
addition, this investigation shows the potential of utilization of the
cheese whey permeate as a substrate for the growth of S. cerevisiae
var. boulardii with the aim of SCP production with high added value
by the probiotic feature of the specic yeast specie Saccharomyces.
Acknowledgments
We gratefully acknowledge Capes, CNPq and Araucria Funda-
tion for nancing this study through the postgraduate program and
SOORO Ltd. for supplying the cheese whey powder.
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