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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
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).
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).
1.2 1.2 20 6
1.2 2.5
18
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
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
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
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)
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.
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 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
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