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LWT - Food Science and Technology 43 (2010) 498–506

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LWT - Food Science and Technology


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

Application of a model using the phenomenological approach for prediction of


growth and xanthan gum production with sugar cane broth in a batch process
Sandra Faria, Patrı́cia Angélica Vieira, Miriam Maria Resende*, Eloı́zio Júlio Ribeiro, Vicelma Luiz Cardoso
Faculty of Chemical Engineering, Uberlândia Federal University, P.O. Box 593, 38400-902 Uberlândia, MG, Brazil

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

Article history: Production of xanthan gum was studied and modelled using unstructured kinetic models composed of
Received 23 January 2009 three differential equations, which considered the microbial biomass, carbon source, and xanthan
Received in revised form concentration. The fermentation process, using Xanthomonas campestris pv. campestris NRRL B-1459, was
9 September 2009
conducted under controlled conditions with diluted sugar cane broth at different initial sucrose
Accepted 18 September 2009
concentrations (15.0, 25.0, and 35.0 g L1). Unstructured kinetic models proposed in the literature for this
system were reviewed and applied. These models were tested against the experimental results, calcu-
Keywords:
lating the parameters by nonlinear regression. The kinetic models used in this study provided estima-
Unstructured kinetic model
Xanthan gum production tions of microbial growth, substrate consumption, and product formation, and, therefore, these
Sugar cane broth parameters were quantified in the fermentation experiments. Higher yield of xanthan per amount of
Model validation sucrose (0.58 g g1) and productivity (0.63 g L1 h1) were obtained using initial sucrose concentrations
of 25.0 and 35.0 g L1, respectively. The models were used to predict the kinetic parameters for a medium
containing an intermediate and a larger initial sucrose concentration (27.0 and 40.0 g L1). When tested
experimentally, the measured fermentation parameters were in close agreement with the values pre-
dicted by the model that presented the best adjustment, demonstrating its validity.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction rice, barley and corn flour, acid whey, sugar cane molasses, coconut
juice, sugar cane, etc., but glucose is still the best in terms of
Xanthan gum is a commercially important hetero-poly- product yield, supply, and product quality (El-Salam, Fadel, &
saccharide produced by strains of Xanthomonas campestris. Because Murad, 1994; Garcı́a-Ochoa, Santos, & Alcon, 2004; Kongruang,
of its rheological properties, xanthan is widely used as a suspen- 2005; Rosalam & England, 2006).
sion-thickening and stabilising agent as well as an emulsifier, The sizeable sugar cane industry in Brazil has the potential to
mainly in the food, cosmetics, and pharmaceutical industries explore sugar cane broth as an alternative raw material for xanthan
(Kennedy & Bradshaw, 1984; Rosalam & England, 2006). The gum production. Once that process has started, the investigation
X. campestris strain used for xanthan gum production is an aerobic will proceed along the most economically viable route, driving and
microorganism that can grow in complex and synthetic media redirecting the researchers in the search for new biotechnological
(Bradbury & Genus, 1984). Many variables, such as composition of processes. The main objective of this research was to use sugar cane
the culture medium, temperature, pH, and oxygen transfer rate broth as the carbon source for determining the kinetic parameters
affect the production of xanthan (Eto & Garcia-Cruz, 2007; Garcı́a- employed by a logistic model, adjusted for biosynthesis of xanthan
Ochoa, Santos, & Casas, 1999; Garcı́a-Ochoa, Santos, & Fritsch, 1992; gum (using X. campestris pv. campestris) in a batch process.
Garcı́a-Ochoa, Santos, Casas, & Gómez, 2000; Oliva Neto & Santos, Selection and development of the appropriate kinetic models,
2005; Rosalam & England, 2006; Scamparini & Rosato, 1990). particularly for certain microorganisms, should begin with an
Most commercial production methods for xanthan gum use understanding of the behaviour and habitat of the microorganisms.
glucose or invert sugars, and most industries prefer batch processes Two main options are available for kinetic processes, batch or
to continuous processes (Letisse, Chevallereau, & Simon, 2001). continuous operation. The time course of microbial growth differs
Other substrates have also been tested, such as sucrose, hydrolyzed in each type of operation.
Studies of unstructured kinetics models that describe the
synthesis of xanthan gum by X. campestris sp. in a batch process
* Corresponding author. Tel.: þ55 34 32394249; fax: þ55 34 32394188. have been undertaken by many authors (e.g., Cadmus, Knutson,
E-mail address: mresende@feq.ufu.br (M.M. Resende). Lagoda, Pittsley, & Burton, 1978; De Vuyst, Vermiere, Van Loo, &

0023-6438/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2009.09.018
S. Faria et al. / LWT - Food Science and Technology 43 (2010) 498–506 499

Nomenclature PO inorganic phosphate concentration (g L1)


J organic nitrogen concentration (g L1)
SRS sum of squares of residuals O2 concentration of dissolved oxygen (g L1)
t time (h) p dimensionless xanthan concentration (–)
YP/S yield of xanthan per amount of sucrose (g g1) s dimensionless carbon source concentration (–)
YX/P yield of biomass per amount of xanthan (g g1) x dimensionless biomass concentration (–)
YX/S yield of biomass per amount of sucrose (g g1) mO 2 dissolved oxygen consumption coefficient (g g1 s1)
YX/N yield of biomass per amount of nitrogen source (g g1) kLa oxygen mass transfer coefficient (s1)
YO2 =X yield of dissolved oxygen concentration per amount of k0S kinetic parameter (g L1)
biomass (g g1) k0P kinetic parameter (g L1)
YX/PO yield of biomass per amount of inorganic phosphate kx maximum specific growth rate (g1 g1 h1)
(g g1) kp maximum specific production rate (g1 g1 h1)
YX/NO yield of biomass per amount of nitrate salts K parameter of kinetic Contois model (g g1)
concentration (g g1) Ki saturation constant of component i in Monod-type
YX/J yield of biomass per amount of organic nitrogen (g g1) equations (g L1)
A parameter of Pinches and Pallent (1986) model
referred to oxygen consumption due to maintenance Greek symbols
(g g1 L1) b parameter that represents the carbon source
B parameter of Pinches and Pallent (1986) model consumption due to biomass (g g1 h1)
referred to oxygen consumption due to growth (g g1) a parameter that represents the carbon source
Ai nitrogen substrate consumption parameter ‘‘non- consumption due to growth (g g1)
associated’’ with growth (g g1 h1) mm maximum specific growth rate (h1)
yexp experimental values to xanthan, carbon source and mP kinetic parameter (g g1 h1)
biomass concentration (g L1) l parameter that represents the xanthan production due
ytheor calculated values to xanthan, carbon source and to biomass (g g1 h1)
biomass concentration (g L1) d biomass maintenance coefficient (g L1 h1)
Pr productivity [g L1 t1]
P xanthan concentration (g L1) subscripts
S carbon source concentration (g L1) m maximum value
X biomass concentration (g L1) A Dimensionless value
N ammonium concentration (g L1) 0 initial condition
NO nitrate salts concentration (g L1)

Vandamme, 1987; Garcia-Ochoa, Santos, & Alcon, 1995; Letisse, A more comprehensive kinetic model could also include the
Lindley, & Roux, 2003; Luong, Mulchandani, & Leduy, 1988; Mar- mass transfer limitation caused by increased viscosity, mechan-
garitis & Zajic, 1978; Moraine & Rogovin, 1971; Pinches & Pallent, ical design of equipment, and variation of adverse conditions
1986; Pons, Dussap, & Gros, 1990; Quinlan, 1986; Schweickart & with time, such as cell population density, which would
Quinlan, 1989; Souw & Demain, 1980; Weiss & Ollis, 1980). The contribute to increased cellular stress and endogenous rate.
models can be classified into two groups: (1) models that consider Garcia-Ochoa, Santos, and Alcon (1998) have proposed a meta-
growth and production to be dependent upon medium nutrients, bolic, structured kinetic model for xanthan gum production by
and (2) models that express growth and production only as X. campestris. Garcı́a-Ochoa, Santos, and Alcon (2004) proposed
a function of temporal changes in biomass. The only difference a structured kinetic model for xanthan gum production that
between them is in the type of kinetic equations used for the utilised the sources of carbon and nitrogen metabolism present
descriptions of growth and production. Such kinetic models tend to within the cell. This model includes eight lumped equations in its
be simpler, because the data is usually generated for a smaller structure (synthesis of amino acids, formation and non-forma-
number of process components. Some adjustments of the model tion of bases, nucleic acid RNA and DNA synthesis, xanthan gum
components are usually necessary so that the resulting information production, total sugar metabolism, oxidative phosphorylation,
corresponds to the established technical goals. These unstructured and maintenance energy). In these structured kinetic models, the
kinetics would include a balance on the cell mass (X), the product cells are described in more detail. For instance, intracellular
concentration (P), and the substrate concentration (S). They are components are included, allowing the model to describe the
simpler microbial modelling systems, because they describe the state of the cells and their adaptation to changes in the
microorganisms as biomass, which is considered a reagent or environment.
a component in the system being studied. The kinetic models employed in the literature in differential
Despite the simplicity of the unstructured models in relation form are summarised in Table 1.
to the structured models, they must describe, at a minimum, the In agreement with Table 1, the model proposed by Moraine and
evolution of biomass, the carbon source, the product, and often Rogovin (1971) uses Monod-type equations, Eqs. (1) and (3), for
another substrate, usually nitrogen (Garcı́a-Ochoa et al., 1995). biomass and xanthan production evolution. The nitrogen source is
Rosalam and England (2006), in their review of xanthan gum considered to be the limiting nutrient for growth Eq. (1) and the
production, also reported the characteristics of the unstructured carbon source for xanthan production Eq. (2). The evolution of the
and structured kinetics models, emphasising the behaviour of two substrates as a function of time is expressed in terms of stoi-
kinetics that have been described by the models of Garcı́a- chiometric coefficients Eqs. (2) and (4).
Ochoa et al. (1995), Luong et al. (1988), and Weiss and The model of Pinches and Pallent employs the same equations
Ollis (1980). for growth, xanthan production, and carbon source consumption
500
Table 1
Kinetic models for xanthan production in differential form (P is the xanthan concentration; S the carbon source concentration; X is the biomass concentration; N is the ammonium concentration; NO is the nitrate salts
concentration; PO is the inorganic phosphate concentration; J is the organic nitrogen concentration and O2 is the concentration of dissolved oxygen).

S. Faria et al. / LWT - Food Science and Technology 43 (2010) 498–506


Reference X S P N O2 PO NO J
Moraine and
Rogovin (1971) dX m NX dS 1 dP dP kp S dN 1 dX
¼ N (1)  ¼ (2) ¼ X (3)  ¼ (4)
dt KN þ N dt YP=S dt dt ðKS þ SÞ dt YX=N dt
Weiss and
 
Ollis (1980) dX X dS dX dP dX
¼ mm X 1  (5)  ¼ a þ dX (6) ¼ l þ gX (7)
dt Xm dt dt dt dt
Pinches and
 
Pallent (1986) dX X dS dX dP dX dN 1 dX dO2 dX
¼ mm X 1  (8)  ¼ a þ dX (9) ¼ l þ gX (10) ¼  (11) ¼ B þ AX (12)
dt Xm dt dt dt dt dt YX=N dt dt dt
Luong et al. (1988)
 
dX S X dS 1 dP dP dN 1 dX
¼ mm X 1 (13)  ¼ (14) ¼ kp SX (15) ¼  (16)
dt KS þ S Xm dt YP=S dt dt dt YX=N dt
Quinlan (1986)
and Schweickart dX dS 1 dP dP dN 1 dX
¼ kX NX (17)  ¼ (18) ¼ kP SX (19) ¼  (20)
and Quinlan (1989) dt dt YP=S dt dt dt YX=N dt
Letisse et al. (2003)
dX dS dX dP dX dN 1 dX dPO dNO dJ
¼ mðS; PO; J; N; NOÞX (21)  ¼ a þ dX (22) ¼ l þ gX (23) ¼ þ A1 X (24)
dt dt dt dt dt dt YX=N dt dt dt dt
1 dX 1 dX 1 dX
¼ ¼ ¼ þ A4 X (27)
YX=PO dt YX=NO dt YX=J dt
þA2 X (25) þA3 X (26)
Garcı́a-Ochoa
!  
et al. (1995) dX X0 dS dX dP dN 1 dX dO2
¼ kX þ N0 X  ¼ a þ dX (29) ¼ kP SX (30) ¼  (31) ¼ kLa O*2  O2
dt YX=N dt dt dt ds YX=N dt dt !
! 1 dX
X  mO2 X þ (32)
1 (28) YO2 =X dt
X0 þ YX=N N0
S. Faria et al. / LWT - Food Science and Technology 43 (2010) 498–506 501

as those of the model of Weiss and Ollis (1980) but also includes time is expressed in term of stoichiometric coefficients (Eqs. (14)
Eq. (11) for nitrogen evolution and adds an equation Eq. (12) for and (16)), where YP/S is the product yield coefficient based on
oxygen consumption, which also depends on biomass and growth. substrate and YX/N is the biomass yield coefficient based on
The Quinlan (1986) and Schweickart and Quinlan (1989) model nitrogen. Unfortunately, for the biomass in xanthan production,
also describes nutrient consumption by means of Eqs. (18) and the rate is not of the Monod type, so the equation proposed by
(20); however, biomass and xanthan production rates are not of these authors is not suitable to represent the synthesis of xanthan
the Monod type, but are instead like those described in Eqs. (17) gum.
and (19). In agreement with Letisse et al. (2003), the changes in biomass
Weiss and Ollis (1980) and Pinches and Pallent each proposed concentration and xanthan production are expressed by Eqs. (21)
models that are only dependent on biomass concentration and its and (23). The evolution of the five substrates with time is defined
evolution with time. These authors have expressed growth rate by Eqs. (22), (24)–(27). The growth rate (m) is expressed according
as a function of biomass using the logistic equation, also known to the consumption of the carbon substrate, the consumption of the
as the Verlhurst–Pearl or the autonomous equation Eq. (5). This various nitrogen sources (organic nitrogen, ammonium, nitrate
equation presents a similar tendency to the microorganism salts), and the consumption of phosphate. Models of the Monod
growth curve. Xanthan production and carbon source consump- type were used systematically to describe the consumption of the
tion are given by means of Luedeking–Piret equations [Eqs. (6) various substrates. This equation takes into account the various
and (7)]. These authors do not take into account the nitrogen nutritional events observed during the experiments, and it reflects
source. the successive consumption of the nitrogen sources. The xanthan
The Garcı́a-Ochoa et al. (1995) kinetic model takes into production rate (Eq. (23)) and the five substrate consumption rates
account dependencies of growth and production on nutrients, Eqs. (22), (24)–(27) can be described by the model of Luedeking–
including an expression for describing the evolution of another Piret.
nutrient in this system: dissolved oxygen. This equation Eq. (28) The model of Weiss and Ollis (1980) is the most widely used, in
takes into account both growth and nitrogen source consump- spite of the fact that the combination of Eqs. (5)–(7) predict infinite
tion. Carbon source consumption is expressed by noting that the production of xanthan even though no sucrose remains in the
carbon source is used both for maintenance and for growth, broth. In agreement with the model characteristics of the produc-
including xanthan production in ‘‘growth’’, since xanthan is tion of xanthan, the aim of this work was to apply our experimental
essentially the bacterial capsule. Xanthan production is data to the Weiss and Ollis model presented in Table 1, to the kinetic
expressed by means of Eq. (30), as in the model of Quinlan (1986) models of Contois and Chen and Hashimoto (Chen & Hashimoto,
and Schweickart and Quinlan (1989) and dissolved oxygen 1980), and to the logistic model of Weiss and Ollis (1980), while
evolution is described by Eq. (32). considering a variation in the equations of the model of Luedeking–
Luong et al. (1988) proposed a combination of Monod and Piret to xanthan production using X. campestris NRRL B-1459. For
logistic or modified logistic equations to describe the xanthan gum this procedure, the previous kinetic models given in the literature
production (Eq. (13)). The evolution of the two substrates with were taken into account.

Table 2
Parameter values with different sucrose concentrations to the logistic model of Weiss and Ollis (1980), while considering a variation in the equations of the model of Lue-
deking–Piret to xanthan production; the logistic model of Weiss and Ollis (1980) and kinetic models of Contois (Chen & Hashimoto, 1980).

Initial sucrose concentration [g L1]

Dimensionless 15.0 25.0 35.0


Parameters (this study)
mmA [h1] 11.646  0.749 0.485 0.485 0.485
XmAx [g L1] 1.008  2.381  102 2.364 2.611 2.313
aA [gS (gX)1] 0.411  6.148  102 2.629 3.967 6.267
bA [gS (gP)1] 0.609  8.252  102 2.139 1.074 1.412
lA [gP (gX)1] 6.180  102  2.430  103 0.112 0.338 0.407
mPA [gS (gX h)1] 4.098  7.073  101 0.311 0.935 1.123
k0S [gS L1] 0.183  4.386  102 2.745 4.575 6.405
k0PA [gP L1] 0.275  7.433  102 1.176 3.904 4.157
SRS 5.171  101

Parameters (Weiss & Ollis, 1980 model)


mmA [h1] 11.343  0.742 0.473 0.473 0.473
XmAx [g L1] 1.013  2.477  102 2.375 2.624 2.325
1
aA [gS (gX) ] 0.374  6.384  102 2.392 3.610 5.704
dA [gS (gX h)1] 0.832  0.108 0.222 0.335 0.529
lA [gP (gX)1] 6.032  105  1.303  106 1.098  104 3.302  104 3.968  104
gA [gP (gX h)1] 1.286  0.105 0.0976 0.293 0.353
SRS 5.646  101 – – –

Parameters (Chen & Hashimoto, 1980 kinetic model)


mmA [h1] 11.630  0.212 0.484 0.484 0.484
B [gP (gX)1] 3.238  9.648  102 20.712 31.254 49.381
aA [gS (gX)1] 0.459  6.148  102 2.936 4.430 7.0
bA [gS (gP)1] 0.618  0.126 2.186 1.089 1.432
lA [gP (gX)1] 0.116  2.492  102 0.211 0.635 0.763
1
mPA [gS (gX h) ] 2.302  2.415  101 0.175 0.525 0.631
k0S [gS L1] 5.244  102  0.134  102 0.787 1.311 1.835
k0PA [gP L1] 9.618  102  0.323  102 0.411 1.364 1.452
SRS 9.927  101
502 S. Faria et al. / LWT - Food Science and Technology 43 (2010) 498–506

1.2 1.2 20 6

Xanthan Concentration - P [dimensionless]


Sucrose Concentration - S [dimensionless]
Biomass Concentration - X [dimensionless]

1.2 2.5
18

Xanthan Concentration - P [g/L]


Sucrose Concentration - S[g/L]
1.0 1.0 5

Biomass Concentration - X[g/L]


16
1.0 2.0
14
0.8 0.8 4
0.8 1.5 12
0.6 0.6 10 3
0.6
1.0
8
0.4 0.4
0.4 2
0.5 6

0.2 0.2 0.2 4


0.0 1
2
0.0 0.0 0.0
-0.5 0 0
0.0 0.2 0.4 0.6 0.8 1.0
0 5 10 15 20 25
Time [dimensionless]
Time [h]
Fig. 1. Changes in the dimensionless concentration of microbial biomass (- 15.0 g L1,
Fig. 2. Changes in the concentration of microbial biomass (-), sucrose (D), xanthan
A 25.0 g L1,  35.0 g L1), sucrose (8  15.0 g L1,  25.0 g L1, X 35.0 g L1), xanthan
gum (>) when the initial sucrose concentration was 15.0 g L1. Symbols represent the
gum (c 15.0 g L1, – 25.0 g L1, > 35.0 g L1). Symbols represent the experimental
experimental results; lines represent the Verhulst–Pearl (V–P) and modified Luedek-
results; lines solids represent the Verlhurst–Pearl (V–P) and modified Luedeking–Piret
ing–Piret (L–P) kinetic model data (this study).
(L–P) kinetic model data; dash represent the Contois kinetic model and dot represent
the Weiss and Ollis model.

determine the possible presence of contaminants. All fermentation


2. Materials and methods runs were conducted in triplicate.

2.1. Organism and maintenance 2.3. Analytical determinations

The Tropical Collection Culture of the Fundação André Tosello, Sucrose disappearance was determined using a glucose-oxidase
Brazil supplied the X. campestris pv. campestris NRRL B-1459 used (GPO-PAP) kit on samples previously subjected to acid hydrolysis
for the studies. (Shu & Yang, 1990). For biomass determination, the fermented
X. campestris pv. campestris was grown and maintained in yeast broth samples were diluted at a 1:1 ratio with a NaCl solution
extract malt agar (YMA) (Nakajima, Funahashi, & Yoshida, 1990) (8.5 g/L) (De Vuyst & Vermeire, 1994), and the cells were separated
with the following composition (g L1): glucose 10.0, yeast extract by centrifugation at 18,900  g for 30 min. The cells were then
3.0, malt extract 3.0, peptone 5.0, and agar 15.0. The pH of the washed in a 8.5 g/L saline solution to remove any residual medium
medium was adjusted to 6.0 and the culture media were sterilised and metabolites. Cellular biomass was quantified by drying the
at 110  C for 20 min. After growing for 48 h at 30  1  C, the culture washed cell mass at 90  1  C until constant weight was achieved.
was maintained under sterile conditions at 5  1  C.
2.4. Recovery of xanthan gum

Fermented broth (samples of 50 mL each) was diluted 1:1 with


2.2. Fermentation experiments
deionised water and centrifuged in a Beckman Coulter Avanti J-25
centrifuge at 18,900  g for 30 min to remove the cells. The
The inoculum was prepared by overnight shake-flask cultiva-
supernatant was filtered and a saturated solution of KCl was added.
tion, agitated at 150 rpm and a temperature of 25  1  C, using
Xanthan gum was recovered by ethanol precipitation and vacuum
a medium containing (g L1): sucrose (from diluted sugar cane
broth) 20.0, yeast extract 3.0, NH4NO3 0.86, Na2HPO4 2.0, and
KH2PO4 2.0. The carbon source and the salt solution were sterilised 3.0 28 18
separately. The initial pH was adjusted to 7.0 with NaOH before
16
sterilisation. 24
2.5
Biomass Concentration - X[g/L]

Xanthan Concentration - P [g/L]

All the fermentation experiments were conducted in a 4.0-L


Sucrose Concentration - S[g/L]

14
bioreactor (Biostat-B, Sartorius, Germany). The production medium 20
2.0 12
was composed of sucrose (from sugar cane broth at 15.0, 25.0, 27.0,
16
35.0 or 40.0 g L1), yeast extract (3.0 g L1), NH4NO3 (0.86 g L1), 10
Na2HPO4 (2.5 g L1), KH2PO4 (2.5 g L1), and antifoam (0.5 mL L1). 1.5
12 8
The fermentation medium without the carbon source was sterilised
while in the fermentation vessel. The carbon source was sterilised 1.0 8 6
separately and then aseptically introduced into the vessel. During 4
4
fermentation runs, the dissolved oxygen concentration was main- 0.5
tained at 10–30% of saturation value by increasing the stirrer speed 2
0
as needed, while keeping a constant 0.5 vvm (air volume/medium 0.0 0
volume/minute) airflow rate. Temperature was maintained 0 5 10 15 20 25
constant at 28  1  C for 24 h. The pH of the medium was held at 7.5 Time [h]
by adding 2 mol equiv/L NaOH. During the process, the concentra-
Fig. 3. Changes in the concentration of microbial biomass (-), sucrose (D), xanthan
tions of the cells, sucrose, and xanthan gum were measured in the gum (>) when the initial sucrose concentration was 25.0 g L1. Symbols represent the
culture medium in nine samples of 50 mL each, and microscopic experimental results; lines represent the Verhulst–Pearl (V–P) and modified Luedek-
observations using the Gram method were carried out in order to ing–Piret (L–P) kinetic model data (this study).
S. Faria et al. / LWT - Food Science and Technology 43 (2010) 498–506 503

2.5 20 3. Kinetic model


35
18
The unstructured kinetic models used in this investigation were

X a n t h a n C o n c e n tr a t io n - P [ g /L]
Biomass Concentration - X[g/L]

2.0 30 16
developed by Weiss and Ollis (1980) and Chen and Hashimoto

Sucrose Concentration - S[g/L]


14 (1980). The logistic models and Chen and Hashimoto used in this
25
1.5 12 study modified Luedeking–Piret equations (Luedeking & Piret,
20 1959), [Eqs. (35), (36), (38) and (39)]. Weiss and Ollis (1980) [Eqs.
10
15
(5)–(7)] (Table 1) also were studied.
1.0 8
This study:
10 6  
dx x
0.5 4 ¼ mmA x 1  (34)
5
ds xmA
2
0
0.0 0
0 5 10 15 20 25
ds dx dp
 ¼ aA þ bA (35)
Time [h] ds ds ds

Fig. 4. Changes in the concentration of microbial biomass (-), sucrose (D), xanthan dp dx mPA sp
gum (>) when the initial sucrose concentration was 35.0 g L1. Symbols represent the ¼ lA þ  0  x (36)
experimental results; lines represent the Verhulst–Pearl (V–P) and modified Luedek- ds ds kSA þ s k0PA þ p
ing–Piret (L–P) kinetic model data (this study).
Chen and Hashimoto (1980)

drying at 30  1  C (Ramı́rez, Fucikovsky, Garcı́a-Jiménez, Quintero, dx m xs


¼ mA (37)
& Galindo, 1988). ds KA x þ s

ds dx dp
 ¼ aA þ bA (38)
ds ds ds
2.5. Modelling of xanthan gum production: parameters and
constant identification dp dx mPA sp
¼ lA þ  0  x (39)
ds ds kSA þ s k0PA þ p
The experimental values of the various treatment parameters
were identified through the assays by using different initial sucrose Where:
levels. The results were generated by nonlinear regression, using
a multi-response algorithm (Marquardt, 1963). Integration of the X S P t X P
x¼ ; s¼ ; p¼ ; s ¼ ; aA ¼ a f ; bA ¼ b f ;
set of differential equations forming the models was completed Xm s0 pm tm s0 s0
0
using a fourth-order Runge–Kutta algorithm (Gill, 1951). The Xf Xf k k0
residuals between the experimental and calculated model values
lA ¼ l ; mmA ¼ mm tm ; mPA ¼ mP ; k0SA ¼ S ; k0PA ¼ P ;
Pf Pf S0 Pf
were obtained by the minimisation of the sum of squares of S0 Pf KS
residuals (SRS), as defined by Eq. (33). dA ¼ d ; gA ¼ g ; KA ¼
Xf tm Xf tm S0
N 
X 2 The parameters of these models were determined using
SRS ¼ yexp  ytheor (33) a nonlinear regression, multi-response technique applied to Eqs.
i¼1
(34)–(39), and (5)–(7), combining the three experimental data
points for the concentrations 15.0, 25.0, and 35.0 g L1.

24 4. Results and discussion


3.0 22
40
20 4.1. Determination of parameters
Xanthan Concentration - P[g/L]
Biomass Concentration - X[g/L]

Sucrose Concentration - S[g/L]

2.5 18
30 16 A fourth-order Runge–Kutta algorithm was coupled with the
2.0
14 nonlinear regression method to determine the solutions for the
12 differential equations (34)–(39), and (5)–(7). The parameters were
1.5
20 obtained for all dimensionless data groups (Table 2).
10
8 The sum of squares of the calculated residuals was relatively
1.0
6
low, suggesting good model fits, as indicated in Table 2 and Fig. 1.
10
Another indicator of good fit was the standard deviation obtained
0.5 4
for the calculated parameters (Table 2).
2
0.0
Table 2 shows the results obtained for the parameter values
0 0
0 5 10 15 20 25 proposed by Weiss and Ollis and those obtained by the kinetics
Time [h] model of Chen and Hashimoto (1980) as well as the logistic model
with modified Luedeking–Piret equations for the different runs
Fig. 5. Change in the concentration of microbial biomass (-), sucrose (), xanthan fitted. The parameters obtained for all runs were similar to those
gum (A) when the initial sucrose concentration was 40.0 g L1 and biomass (,),
sucrose (B), xanthan gum (>) when the initial sucrose concentration was 27 g L1.
proposed by Weiss and Ollis, except for the non-associated
Symbols represent the experimental results; lines represent the Verhulst–Pearl (V–P) parameter for the growth of xanthan production (g). As pointed out
and modified Luedeking–Piret (L–P) kinetic model data (this study). in the Introduction, the third kinetic model applied was the same as
504 S. Faria et al. / LWT - Food Science and Technology 43 (2010) 498–506

Table 3 4.1.1. Model validation


Parameter values for sucrose concentrations of 27.0 and 40.0 g L1. It is always advisable to check the coherence between the
Parameters Initial sucrose concentration [g L1] experiment and a model, within its valid range, before the model
Dimensionless 27.0 40.0
can be considered ‘‘valid’’. The established model was validated by
simulating a culture carried out under different initial conditions,
mmA [h1] 11.646  0.749 0.485 0.485
XmAx [g L1] 1.008  2.381  102 2.812 2.409 and by comparing the results of this simulation with those obtained
aA [gS (gX)1] 0.411  6.148  102 3.977 6.879 experimentally.
bA [gS (gP)1] 0.609  8.252  102 1.028 1.768 The conditions chosen used a medium in which the initial
lA [gP (gX)1] 6.180  102  2.430  103 0.354 0.356
sucrose concentration in the sugar cane broth was larger than those
mPA [gS (gX h)1] 4.098  7.073  101 0.979 0.985
k0SA [gS L1] 0.183  4.386  102 4.941 7.32
used initially (i.e., 40.0 g L1 and an intermediate 27 g L1). A good
k0PA [gP L1] 0.275  7.433  102 4.405 3.794 correlation between the experimental values and the values given
SRS 5.171  101 – – by the model was observed (Fig. 5), validating the model. The rela-
tive average error between the experimental and modelled values
remained lower than 10%, comparable with the level of analytical
the kinetic model from Weiss and Ollis, but it included expressions
precision inherent in such fermentation. The parameters calculated
modified for Luedeking–Piret equations (Eqs. (35) and (36)). The
for the 40.0 and 27.0 g L1 (initial concentrations of sucrose) that
parameters obtained for all runs were similar to those proposed by
were used in the validation of the proposed model, which presented
Weiss and Ollis and to the kinetic model of Chen and Hashimoto
small sum of residuals squares (this study) were presented in Table 3.
(1980) [Eqs. (37)–(39)].
Table 2 shows that maximum specific growth rate and
maximum biomass concentration were not influenced by the 4.2. Comparison with published values
values of sucrose concentration employed in this study. In this
table, it is observed that parameter (a) increases at a higher initial Kinetic parameters obtained experimentally were compared
sucrose concentration. These results show that sucrose consump- with those reported in the literature (Table 4); significant vari-
tion increased in relation to biomass and they present values with ability was observed between them. This could reflect differences
the same order of magnitude for the studied models. in microbial fermentation conditions and xanthan gum production.
The parameter related to associated-growth xanthan production Weiss and Ollis (1980) modelled experimental data obtained by
(l) was not strongly dependent on sucrose concentration, but did Moraine and Rogovin (1971) for X. campestris pv. campestris NRRL
increase when the initial sucrose concentration was higher, indi- B-1459 grown in a medium with 50 g L1 glucose and 0.6 g L1
cating that xanthan yield increased at higher sucrose concentration. nitrogen initial concentration. Operational conditions were
Parameter (d) is the maintenance coefficient (Table 2). Using this pH ¼ 7.1  0.1, T ¼ 28  1  C, and aeration at 1 air volume/medium
model, it was not strongly influenced by initial sucrose concen- volume/minute (vvm). Pinches and Pallent (1986) used the same
tration, indicating a small increase of sucrose for cellular mainte- Moraine and Rogovin (1971) strain in a production medium
nance in the higher initial sucrose concentration. composed of 22.5 g L1 glucose broth with nitrogen from sodium
1
Parameter (b) in Table 2 represents the inverse of xanthan yield L-glutamate (0.837, 1.526 and 2.856 g L ) or peptone (0.1, 0.2 and
per amount of sucrose. It indicates a maximum yield approximated 0.3 g L1), and operational conditions set at pH ¼ 7.0  0.5,
to the initial sucrose concentration of 25 g L1, and its value is T ¼ 30  0.5  C, and aeration at 0.4 vvm.
similar for either model. Serrano-Carreón, Corona, Sánchez, and Galindo (1998) evalu-
The xanthan production parameter non-associated with growth ated X. campestris pv. campestris NRRL B-1459 also, but employed
(g) in Table 2 increased with increases in initial concentration of 40.0 g L1 sucrose and NH4Cl 2.0 g L1 as substrates in
sucrose. The orders of magnitude also characterise well the product pH ¼ 7.0  0.1, T ¼ 29  C and 0.5 vvm. Garcı́a-Ochoa et al. (1995)
formation not associated with growth. used sucrose 40.0 g L1 as a carbon source, 1.144 g L1 NH4NO3 and
The best adjustment was done to the kinetic model of Weiss and the pH was not controlled, while Letisse et al. (2003) worked with
Ollis with modified Luedeking–Piret equations for substrate and X. campestris ATCC 13951 using 42 g L1 sucrose and 1.125 g L1
xanthan gum. Indeed, when experimental data were compared to NH4NO3 with operating conditions set at pH ¼ 7.0 and T ¼ 28  C.
the quantitative responses predicted by this model, there was Garcia-Cruz (1983) evaluated Xanthomonas manihotis and several
a high degree of correspondence, regardless of the initial sucrose glucose and sucrose concentrations. Their best results for the
concentrations (Figs. 2–4). xanthan gum production were with sucrose 40 g L1, without pH
The experimental results and the model predictions of the control. The substrate, the source of nitrogen, and the process
concentrations of cells, sucrose, and xanthan gum at different initial conditions in this work differed from those reported by Weiss and
sucrose concentrations (15.0, 25.0, and 35.0 g L1) show that the Ollis (1980), Moraine and Rogovin (1971), Serrano-Carreón et al.
Weiss and Ollis kinetic model with modified Luedeking–Piret (1998), Garcı́a-Ochoa et al. (1995) and Letisse et al. (2003).
equations describes in an accurate way the changes in concentra- In agreement with the literature (Lima, Aquarone, Borzani, &
tion during the fermentation process. Schmidell, 2001), many factors contribute to differences in the

Table 4
Comparison of kinetic constant values from modeling and those in literature. Runs 1–4 represents the four experimental data points for the concentrations 15.0, 25.0, 35.0 and
40.0 g L1, respectively.

Parameters Run 1 Run 2 Run 3 Run 4 Weiss and Pinches and Garcı́a-Ochoa Serrano-Carreón Letisse Garcia-Cruz
Ollis (1980) Pallent (1986) et al. (1995) et al. (1998) et al. (2003) (1983) a
mm [h1] 0.485 0.485 0.485 0.485 0.152 0.29 0.67 0.25 0.38 –
Xm [g L1] 2.364 2.611 2.313 2.409 2.450 2.15 1.580 4.42 3.30 –
a [gS gX1] 2.629 3.967 6.267 6.879 2.00 1.24 5.22 3.03 3.23 30.44
l [gP (gX)1] 0.112 0.338 0.407 0.356 0.155 0.13 0.0 0.25 0.23 –
a
Calculated values from the experimental data for the best conditions of xanthan gum production presented in the author’s dissertation.
S. Faria et al. / LWT - Food Science and Technology 43 (2010) 498–506 505

Table 5 The best model studied in this paper adequately described


Yield (Y) and productivity (Pr) under different initial sucrose concentrations. microbial growth, xanthan gum production, and carbon source
Parameters Initial sucrose concentration [g L1] consumption, as is evident from Figs. 2–5. Operational conditions
15.0 25.0 35.0 40.0
defined in the second assay provided the best conversion of
substrate to product, and the three assay conditions provided the
Y [gP gS1]exp 0.28  0.02 0.58  0.03 0.43  0.02 0.35  0.03
Y [gP gS1]mod 0.31 0.61 0.47 0.38 best productivity. The model, therefore, was sufficient to predict
Pr [gP L1 h1] 0.18  0.04 0.59  0.03 0.63  0.04 0.57  0.03 the performance of the production system, given different initial
sucrose concentrations and the kinetic events occurring in
kinetic parameter values, for example, the strain selected, C/N response to the specific experimental conditions. Given these
relationship, nitrogen source, temperature, with or without control conditions, the parameters derived from nonlinear regression
pH in the medium, and essentially the use of complex mediums, analysis differed from the values for those parameters cited in the
such as the sugar cane broth. literature. The results of the simulation indicate that for sucrose
The works of Garcı́a-Ochoa et al. (1990, 1995); Serrano-Carreon concentrations of 27.0 and 40.0 g L1, a good compromise between
et al. (1998) and Letisse et al. (2003) show the influence of the yield and productivity should be obtained (Table 5). Thus, the
operational conditions, clearly including the relationship C/N in the model proposed in this work was able to describe growth, xanthan
results of the xanthan gum production and consequently in the production, and consumption of carbon source, as can be seen in
adjusted parameters. Garcia-Ochoa et al., 1990 and Serrano-Carreon Fig. 5.
et al., 1998 used sucrose 40 g L1 and X. campestris pv. campestris
NRRL B-1459, only differing in the source of nitrogen (Garcı́a-Ochoa References
used NH4NO3 and Serrano-Carreon, NH4Cl). Their results for the
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