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Journal of Biotechnology 226 (2016) 3543

Contents lists available at ScienceDirect

Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Process optimization and kinetic modelling of cyclic (1 3,


1 6)--glucans production from Bradyrhizobium japonicum
MTCC120
Anju V. Nair, Sathyanarayana N. Gummadi, Mukesh Doble
Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India

a r t i c l e

i n f o

Article history:
Received 28 January 2016
Received in revised form 21 March 2016
Accepted 31 March 2016
Available online 1 April 2016
Keywords:
Bradyrhizobium japonicum
Cyclic (1 3, 1 6)--glucans
Response surface methodology
Kinetic modelling
Encapsulation

a b s t r a c t
Cyclic (1 3, 1 6)--glucans are water soluble, biocompatible polymers with potential applications
in food and pharmaceutical industries but have not yet been exploited due to their poor yield. In the
present study statistical experimental design methodology was employed to improve their production.
Initial screening indicated arabinose and peptone as best carbon and nitrogen source respectively, for
glucan production. Arabinose and osmolyte concentrations as well as pH signicantly contributed to
the glucan production. Central composite design indicated a signicant interaction between osmolyte
concentration and pH on glucan production. The maximum amount of cyclic glucan produced was 6.7 g/L
in a 2.5 L reactor in batch conditions. The logistic equation for cell growth and Luedeking-Piret equation for
glucan production could satisfactorily simulate the batch kinetics data. Cyclic -glucans could efciently
encapsulate a hydrophobic molecule, curcumin and increase its solubility in water, thus indicating that
these glucans have potential as drug delivery systems.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Glucans are polymers of glucose molecules linked by glycosidic
linkages. They are a diverse group of molecules differing in their
molecular weight, 3-dimensional structure, viscosity and solubility. They may be linear, branched or cyclic in nature, each with a
different set of biological activities (McIntosh et al., 2005). Cyclic
-glucans are molecules that are produced and localized within
the periplasmic region of the Rhizobiaceae family (McIntosh et al.,
2005; Venkatachalam et al., 2012). They constitute 520% of the
total cellular dry weight. Cyclic -glucans were rst reported in
the extracellular medium of Agrobacterium tumefaciens (Breedveld
and Miller, 1994). They are linked by -(1 2) glycosidic linkage.
Cyclic (1 2) -glucans are also produced by other fast growing Rhizobiaceae such as Rhizobium, Sinorhizobium, Mesorhizobium,
Brucella with the degree of polymerization ranging from 17 to
28 glucose units and substituted with phosphoglycerol, phosphoethanolamine, phosphocholine, succinate, methylmalonate, or
acetate (McIntosh et al., 2005). Slow growing Rhizobiaceae such as
Bradyrhizobium species, Rhizobium loti, Azorhizobium caulinodans
and Azospirillum brasilense produce cyclic glucans that are linked by

Corresponding author.
E-mail address: mukeshd@iitm.ac.in (M. Doble).
http://dx.doi.org/10.1016/j.jbiotec.2016.03.055
0168-1656/ 2016 Elsevier B.V. All rights reserved.

-(1 3) and -(1 6) linkage and are made of 10 to 13 glucose


molecules substituted with non sugar moieties such as phosphocholine (Rolin et al., 1992), acetate and succinate (Choma and
Komaniecka, 2011) at the C(O)6 position.
Since these molecules are water soluble and have an unusual
cyclic structure they nd applications in food and pharmaceutical industries similar to cyclodextrin, for example to form
inclusion complexes with hydrophobic drug molecules for drug
delivery applications and as immunomodulators. Among the glucans, soluble -(1 3) glucans with -(1 6) side chains have
been reported to have immunepotentiating activities (Laroche and
Michaud, 2007). Being biocompatible, they can also be used as
immune response modiers and as adjuvants in vaccines. They
have also been used as novel chiral additives for the enantiomeric
separation of some avanones such as eriodictyol, homoeriodictyol, hesperetin, naringenin, and isosakuranetin in capillary
electrophoresis (Kwon et al., 2007).
Though these molecules have potential applications, they have
still not been exploited fully unlike linear glucan, owing to their low
production. Generally, all the cultures produce milligram level per
litre of medium (Breedveld and Miller, 1994). The cellular concentrations of the cyclic (1 3, 1 6)--glucans within B. japonicum
culture is approximately 50 mg/g of cellular dry weight (Miller and
Gore, 1992). For cyclic (1 2)--glucans, reports indicate that their
production can be enhanced by modifying temperature, osmotic

36

A.V. Nair et al. / Journal of Biotechnology 226 (2016) 3543

strength, type of osmolyte and different media which leads to high


cell densities and in turn increase their production (Breedveld et al.,
1990, 1991; Venkatachalam et al., 2013). But systematic studies to
improve cyclic (1 3, 1 6)--glucans production have not been
reported so far and are needed to exploit their potential.
In the present study, the production of biomass and cyclic (1 3,
1 6)--glucans from Bradyrhizobium japonicum MTCC 120 were
enhanced in shake asks by optimizing the culture conditions using
response surface methodology. This is the rst study to the best of
our knowledge reported to improve the production of this biopolymer using the design of experiment strategy. The process was
then scaled up to a 2.5 L reactor and a kinetic model was developed to predict its production as a function of time. Experiments
were also performed to encapsulate curcumin, a poorly water soluble phytochemical using this biopolymer. Studies on use of cyclic
(1 3) (1 6)--glucan for inclusion complexation have not been
reported yet. This could be because of their low yield, which has
been addressed in our work. The preliminary study on ability of
cyclic (1 3) (1 6)--glucan as an encapsulating agent can pave
the way for its use as delivery systems for drugs or hydrophobic
molecules.

2. Materials and method

with pure commercial arabinose. The utilization of nitrogen during the fermentation was estimated by Kjeldahl Method (Khanna
and Srivastava, 2005). All the chemicals were obtained from Sigma
Aldrich (Bangalore, India), Himedia Laboratories Pvt. Ltd., (Mumbai,
India) and SRL (Mumbai, India).
2.2. Extraction of glucan
Cells pellets collected after centrifugation were washed with
1% NaCl, followed by distilled water. The cell-associated oligosaccharides were extracted by modied Bligh and Dyer extraction
procedure as described by Miller and Gore (1992). Glucan in the
upper aqueous methanol phase was then rotary evaporated and
lyophilized. For the extraction of the extracellular glucan, the cell
free supernatant was concentrated and three volumes of cold
ethanol were added to precipitate out the high molecular weight
exo-polysaccharides. The precipitate was removed and the supernatant was rotary evaporated. Ten volumes of ethanol was added
to this concentrate and incubated at 20 C, overnight. The precipitate was collected and lyophilized. The sugar content in the glucan
was determined by phenol-sulphuric acid method (Bhagwat et al.,
1996).
2.3. Experimental design

2.1. Microorganism and culture conditions


The strain B. japonicum MTCC 120 from Microbial Type Culture Collection, Chandigharh, India was used in the present study.
It was maintained in Yeast Mannitol (YM) agar medium (0.4 g
yeast extract, 10 g mannitol, 0.1 g NaCl, 0.2 g MgSO4 7H2 0, and
0.5 g K2 HPO4 per litre). The initial pH was adjusted to pH 7.0. The
inoculum was prepared in YM medium and incubated at 30 C and
180 rpm for 48 h (Miller et al., 1990). For glucan production, 1 mL
of the inoculum was added into 250 mL conical asks containing
100 mL of production medium, The production medium contains
2 g arabinose, 1 g yeast extract, 0.015 g CaCl2 , 0.16 g NH4 Cl, 0.125 g
Na2 SO4 , 0.125 g Na2 HPO4 , 0.18 g MgSO4 7H2 0, 0.016 g sodium ferric EDTA and 1.3 g HEPES buffer in 1 L of distilled water. The initial
pH of the medium was adjusted to pH 7.0. The asks were incubated for 120 h at 30 C and 180 rpm. The kinetics of the process was
monitored by withdrawing samples every 6 h. The biomass was collected by centrifugation at 8000 rpm for 10 min and the pellets were
dried and weighed. The utilization of substrate (arabinose) was
estimated by HPLC (Agilent 1100; BIORAD Aminex Column) with
5 mM H2 SO4 as the mobile phase. A standard curve was prepared

The initial experiments were performed o identify the best carbon and nitrogen sources. This was followed by full factorial and
central composite designs in shake ask level for process optimization. This was followed by scale-up in 2.5 L fermenter.
2.3.1. Screening for the best carbon and nitrogen source
Slow-growing rhizobia such as Bradyrhizobium have been
reported to use pentoses, hexoses, sugar alcohols as well as aromatic and hydroaromatic compounds for their growth, unlike the
fast growing rhizobia which utilize disaccharides, trisaccharides,
and organic acids (Stowers, 1985). The best carbon and nitrogen
source was identied by carrying out experiments with 16 different
combinations of carbon and nitrogen sources (Table 1).
2.3.2. Full factorial design
Following the selection of best carbon and nitrogen sources,
the effect of four factors (arabinose, peptone, osmolyte and pH)
on biomass and glucan production was determined using two level
full factorial design (FFD), which consisted of sixteen experiments
performed in duplicate (Table A.1a in Supplementary material).

Table 1
Biomass and glucan production on different carbon (10 g/L) and nitrogen (1 g/L) sources at the end of 120 h.
Carbon source

Nitrogen source

Biomass (g/L)

Arabinose
Mannitol
Glycerol
Glutamate

Yeast extract
Yeast extract
Yeast extract
Yeast extract

0.60
0.55
0.712
0.65

0.12
0.08
0.21
0.028

0.105
0.0751
0.0792
0.0417

0.028
0.07
0.015
0.014

0.175
0.138
0.11
0.063

0.084
0.033
0.021
0.004

Arabinose
Mannitol
Glycerol
Glutamate

Glutamate
Glutamate
Glutamate
Glutamate

0.8
0.35
0.65
0.55

0.014
0.04
0.39
0.026

0.113
0.0887
0.0707
0.0708

0.38
0.002
0.028
0.011

0.141
0.253
0.106
0.127

0.05
0.037
0.034
0.026

Arabinose
Mannitol
Glycerol
Glutamate

Peptone
Peptone
Peptone
Peptone

0.7 0.014
0.7 0.015
0.45 0.041
0.95 0.04

0.205 0.007
0.0965 0.012
0.0922 0.008
0.157 0.024

0.292
0.136
0.204
0.165

0.004
0.015
0.002
0.032

Arabinose
Mannitol
Glycerol
Glutamate

Malt extract
Malt extract
Malt extract
Malt extract

0.2
0.15
0.15
0.4

0.097
0.127
0.263
0.262

0.035
0.028
0.197
0.071

0.028
0.011
0.015
0.028

Total glucan (g/L)

0.02
0.019
0.038
0.104

0.01
0.023
0.025
0.02

Yield (g of glucan per g of biomass)

A.V. Nair et al. / Journal of Biotechnology 226 (2016) 3543

Osmolarity of the medium was changed using non-ionic solute


mannitol (Choma and Komaniecka, 2011; Miller and Gore, 1992).
Generally, sodium chloride is used to increase osmolarity of the
media. Since Bradyrhizobia are found to be sensitive to NaCl, even
at low concentrations (Miller et al., 1990), mannitol was used here.
2.3.3. Central composite design (CCD)
CCD was employed to optimize the three most signicant factors (arabinose and osmolyte concentrations and pH) short listed
from FFD, to further enhance the glucan production. These variables were studied at ve different levels (1.68, 1, 0, +1 and
+1.68) (Table A.1b in the Supplementary material). Second order
regression models were developed for the production of biomass
and total glucan as a function of these parameters.
2.3.4. Statistical analysis
Minitab software version 1.4 (Minitab Inc., PA, USA) was used for
the generation of experimental design, data analysis, development
of regression models, Paretto plots, contour plots, ANOVA and two
sample t-test.
2.4. Fermentation in a batch reactor
The process was scaled up in a 2.5 L batch fermenter (Infors,
Switzerland), equipped with a 6-bladed disc impeller, a DO probe
and a pH probe. The working volume was 1 L which was maintained
at 30 C and 200 rpm. The pH was maintained at 6.8 using 2 N HCl
and 2NaOH. Foaming during the fermentation was controlled using
silicone oil, an antifoaming agent. Samples were withdrawn every
day and analysis was carried out as described above.

Integrating the above equation gives a sigmoidal behaviour for


X as a function of t,
1

(3)

2.5. Development of kinetic model for cyclic glucan production


The Logistic model, a substrate independent model, has been
reported for a number of fermentation processes especially for
polysaccharide fermentation (Weiss and Ollis, 1980). Here, the cell
growth rate, dX/dt (g/h) is dened as,

X
dX
= m 1
Xm
dt


X

(4)

where, max is the specic growth rate (h1 ) and Xm is the maximum cell growth (g/L).

(5)

(6)

X0 em t

P (t) = X0

 X0 

Xm

(1 em t )

 1

Xm
X0
ln 1
(1 em t )
m
Xm


(7)

Modied Luedeking-Piret equation was used for the substrate


(Arabinose) consumption kinetics. Arabinose is required for cell
components, product formation and cell maintenance and was
expressed as,
dA
=

dt

1
Yx

dX
+
dt

1
YP

dP
+ ms X
dt

(8)

where, Y x is the cell yield coefcient for arabinose, Y P is the product


s

yield coefcient for arabinose and ms is the maintenance coefcient.


Combining Eqs. (6) and (8) gives,

1
+
Yx
YP
s

dX
+
dt

+ ms
YP

(9)

dA
dX
=
+ X
dt
dt

where =

1
Yx

(10)

+ /Y P and =
S

YP

+ ms

Integrating Eq. (10), assuming, A = A0 at t = 0, gives,


A (t) = A0 X0


em t

 X0 
Xm

(1 em t )

 1

Xm
X0
ln 1
(1 em t )
m
Xm

(11)

(2)

where, C* = saturated oxygen concentration (7 mg/L) at


25 C, CL = Oxygen concentration in the liquid medium as a
function of time. The volumetric mass transfer coefcient was
determined by plotting ln (C* CL ) against time (t).The oxygen
transfer rate OTR was calculated using the equation:

where and are the product formation constants. Assuming the


initial concentration of the product to be zero and integrating the
equation gives,

OTR = KL a(C CL )

(1 em t )

Xm

dP
dX
=
+ X
dt
dt

KL a = 0.14N0.88 (VL /VO )0.8

dCL /dt = KL a(C CL )

 X0 0

where X0 is the cell concentration at time t = 0.


Luedeking-Piret equation (Luedeking and Piret, 1959) was used
to describe the product formation kinetics. According to the model,
the rate of product formation depends on both instantaneous cell
concentration (X) and growth rate (dX/dt) in a linear fashion,

dA
=

dt

where VL /VO is the ratio of volume of liquid to ask volume and N


is the incubator shaker speed.
The volumetric oxygen mass transfer coefcient (KL a) in the
bioreactor was measured at 30 C, in a cell-free medium by dynamic
gassing-out method (Kshirsagar et al., 2012). The variation in dissolved oxygen (DO) concentration with time, t, was obtained, using
the oxygen balance equation:

X em t

X (t) =

2.4.1. KL measurement
The volumetric oxygen mass transfer coefcient (KL a) in the
shake ask was estimated using the following equation
(1)

37

Nitrogen is required for the formation of cell components and


cell maintenance. Thus, utilization of nitrogen is given by the equation:

dN
=
dt

1
Yx

dX
+ ms X
dt

(12)

Estimation of the parameters was carried out by minimizing the


residual sum of squares of errors (RSS)
RSS =

n


(yi yi )

(13)

i=0

where n is the number of experimental points, yi is the experimental value and yi is the predicted value. The data from the batch
fermentation was used to develop the models. The minimization
was performed using the MATLAB function lsqnonlin and the system of differential equations (Eqs. (4),(6),(10),(12)) were solved
using the 4th order Runge Kutta Method (ode 45 function) in MATLAB program.

38

A.V. Nair et al. / Journal of Biotechnology 226 (2016) 3543

2.6. Characterization of glucans


The functional groups and linkages present in the glucan
were determined with Fourier transform infrared spectrometer (FTIR; Jasco N4200, Tokyo, Japan), using KBr pellets. Proton
NMR and HSQC spectroscopic analysis of the sample dissolved
in D2 O was performed using Bruker Avance III 500 spectrometer (Bruker Instruments Inc., Billerica, Mass.). The mass spectrum
of the glucans were obtained using MALDI-TOF (Voyager-DETM
PRO BiospectrometryTM ), in the negative-ion mode using 2,5dihydroxybenozoic acid (DHB) as the matrix.
2.7. Encapsulation of curcumin
The encapsulation of curcumin by cyclic glucan was performed
by solvent evaporation method. 1 mL of 2 mM solution of curcumin
in acetone was slowly added to 1 mL of aqueous solution of 2 mM
glucan. The mixture was sonicated for 10 min and incubated in dark
for 24 h under stirring. The mixture was evaporated and centrifuged
to remove insoluble curcumin. The sample was later lyophilized. To
determine the amount of curcumin encapsulated, the complex was
dissolved in ethanol, ltered and absorbance measured at 428 nm
(Perkin Elmer Enspire multimode plate reader). The inclusion complex was analysed using FT IR spectroscopy (Jasco N4200, Tokyo,
Japan) and UVvis Spectroscopy (Perkin Elmer Enspire multimode
plate reader).

glucose from various carbon substrates, followed by glucose polymerization (Stowers, 1985). A number of studies have reported that
glutamate can be taken up rapidly and efciently by slow growing
B. japonicum strains than the fast growing Rhizobia (Stowers, 1985).
Arabinose is a good carbon source for the growth of B. japonicum.
Metabolism of arabinose is via arabinose dehydrogenase, nally
producing pyruvate (Duncan 1979). The metabolism of glycerol
proceeds via glycerol kinase and glycerolphosphate dehydrogenase, producing glycerol-3-Phosphate which is further metabolized
to pyruvate (Stowers, 1985). This could explain the slight lag in the
growth and glucan production when glycerol is used as the sole
carbon source (data not shown). The nitrogen source, peptone has
a signicant effect on biomass and glucan production. This could be
attributed to the type of nitrogen present in bacto-peptone which
is probably utilized by the bacterium more efciently when compared to the others.
The best combination of carbon and nitrogen source for biomass
production (0.95 g/L) is glutamate and peptone respectively. But
maximum glucan production (0.205 g/L) is obtained from arabinose and peptone with a biomass concentration of 0.70 g/L. Also,
in some conditions more glucan is produced in the culture medium
(extracellular) than in the periplasm of the cell (data not shown),
matching with ndings of others (Miller and Gore, 1992). Hence,
arabinose and peptone are chosen for further optimization studies.

3.3. Full factorial design


3. Results and discussion
The production of cyclic glucan by Gram negative B. japonicum
MTCC 120 was maximized in shake ask using statistical design of
experiments technique. Then, the process was scaled up to a 2.5 L
batch reactor and the kinetics was mathematically modelled.
3.1. Growth kinetics and glucan production
Growth of B. japonicum MTCC120 was carried out in arabinose
media as mentioned in Section 2.1. Doubling time for B. japonicum
is 1213 h and it reaches stationary phase in 84 h. The glucan
production is growth associated and continued even during the
stationary phase and reaches a maximum at 120 h (46.15 mg of
glucan per g of cellular biomass), similar to observations by Pfeffer
et al. (1994). They also observed that with B. japonicum USDA110,
glucan production continued beyond 96 h (the beginning of stationary phase) until 192 h. Maximum periplasmic cyclic (1 3)
(1 6)--glucans within B. japonicum USDA110 cultures that has
been reported is 50 mg/g of cellular dry weight (Breedveld and
Miller, 1994; Miller and Gore, 1992). Typically, all studies report
milligram quantities per litre. For their cost effective applications
in industries, it is very important to develop cultural conditions and
bacterial strains which overproduce these biopolymers.
3.2. Screening for carbon and nitrogen source
Various carbon and nitrogen sources were tested to determine
the best combination for maximum glucan production (Table 1).
Both the carbon and nitrogen sources used have a signicant effect
on glucan production (p = 0.091 and p = 0.033 respectively) whereas
only nitrogen source shows a signicant effect on biomass production (p = 0.006). It has been reported that Rhizobia are able
to use sugars, sugar alcohols and amino acids as carbon source
(Stowers, 1985). In the present study, we found the best carbon
source for biomass production is glutamate followed by arabinose
whereas glucan production is maximum with arabinose. Efcient
glucan production by the bacterium lies in its ability to synthesize

The inuence of four factors (arabinose, peptone and osmolyte


concentrations and pH) on biomass and glucan production was
determined using two level full factorial design (Table A.II in Supplementary material).The amount of biomass produced at the end
of 120 h varies from zero to 1.06 g/L biomass. The maximum glucan
production is 1.12 g/L in medium17 (see Table II in supplementary
material). Arabinose and osmolyte concentrations as well as pH
have a signicant inuence on both biomass and glucan production
(p < 0.1) (see Paretto plot in Fig. A.1 in supplementary material). The
p-value at the central point (curvature) is less than 0.1 indicating
that the curvature has a signicant effect and the optimum is well
within the factorial range. Variation in peptone concentration did
not have signicant effect (p > 0.1) (Table A.III in Supplementary
material). Arabinose concentration and pH are positively correlated with both the growth and glucan production. B. japonicum
cultures are known to grow on a wide range of pH. B. japonicum
USDA 110 can also grow at a pH as low as 4.7 (Puranamaneewiwat
et al., 2006). The acid tolerant property of Bradyrhizobium has relationship with wide range of bacterial metabolic pathways including
transport and metabolism of carbohydrate, amino acid and lipid
(Puranamaneewiwat et al., 2006). Osmolyte concentration has a
negative effect on both cell associated and extracellular glucan production. The glucan production is osmotically regulated similar to
that reported for other strains of Bradyrhizobium. Miller and Gore
(1992) observed that the amount of cyclic (1 3, 1 6)--glucans
synthesized by B. japonicum USDA 110 decreased considerably in
high osmolarity media containing 0.5 M sucrose or 0.5 M mannitol.

3.4. Central composite design


Based on the results of the factorial design, three factors, namely
arabinose (A) and osmolyte (B) concentrations and pH (C) were
chosen for further optimization using CCD. The design and the
experimental results are listed in Table A.IV (in Supplementary
material). The second order regression models for biomass and
glucan production developed are:

A.V. Nair et al. / Journal of Biotechnology 226 (2016) 3543

39

Fig. 1. Contour (a) and surface (b) plots depicting the effect of various parameters on glucan production based on CCD.

Biomass = 0.985 + 0.008 A + 0.024 B + 0.083 C 0.12


A B 0.091 A C 0.005 B C + 0.010 A2
0.08 B2 0.081 C2

(14)

Glucanproduction = 1.175 0.044 A + 0.094 B + 0.0413


C 0.020 A B 0.019 A C + 0.135 B C 0.087
A2 0.036 B2 0.035 C2

(15)

F test and ANOVA indicates that both the models are statistically
signicant (Tables A.V in Supplementary material). The R2 values
for biomass and glucan production are 0.87 and 0.84 respectively.
The p-values of lack of t for the models are greater than 0.05 indicating that the models could adequately predict biomass and glucan
production.
The response surface and contour plots provide graphical representation of the interaction between two variables. A circular
contour plot indicates that the interaction between the two variables is negligible, whereas an elliptical or saddle contour plot

A.V. Nair et al. / Journal of Biotechnology 226 (2016) 3543

The process was then scaled up to a 2.5 L bioreactor with 1 L


reaction volume. The pH was maintained at 6.8 and aeration rate
was maintained at 2 vvm. The time course of biomass, substrate
utilization and glucan production are shown in Fig. 2b. Bradyrhizobium grows faster in the reactor and has a specic growth rate
of 0.047 h1 . Biomass production reaches a maximum of 4.5 g/L at
the end of 120 h. The amount of glucan at the end of 120 h is 6.7 g/L.
Biomass and glucan production is higher than that obtained in ask
studies (Fig. 2).
KL a of the shaker at these operating conditions
(N = 180 rpm, VL = 100 mL and V0 = 250 mL) is 0.0078 s1 . At
CL = 0, OTR is 0.0546 mg/L/s, whereas for the reactor the KL a is
0.015 s1 and OTR is 0.107 mg/L/s. The OTR in the reactor is twice
that of the ask and also the pH in the reactor is controlled. These
factors must have contributed to the increase in biomass and
glucan production in the reactor.
3.6. Kinetic modelling
Various models have been developed to describe the bacterial growth and product formations in many strains, the most
common being the substrate limitations models, Monod model
being the simplest of them all. Logistic and Luedeking-Piret equations which were originally developed for lactic acid fermentation
have been used to describe the growth and product formation
in many polysaccharide fermentations (Weiss and Ollis, 1980).
Logistic model is a substrate independent model where maximum
biomass is considered to be the limiting factor (Wang et al., 2006).
Hence accurate estimation of maximum biomass is required to
predict the parameters of the model. These models have been
found to be more suitable and accurate in describing biopolymer
productions. Gellan gum production from Sphingomonas paucimobilis ATCC3146 (Wang et al., 2006) and Xanthum gum production
(Garca-Ochoa et al., 1995) could be satisfactorily demonstrated
using these models.
The logistic equation is used here to develop a kinetic model for
bacterial growth both in the ask and the reactor. The model parameters obtained by tting the batch experimental data are listed in
Table 2. The growth parameters, initial biomass concentration (X0 ),
maximum biomass concentration (Xm ) and specic growth rate
(m ) in the reactor are 0.3 g/L, 4.64 g/L and 0.05 h1 respectively.

6
1
4

0.5

24

48

72

96

120

Arabinose Ulizaon(g/L)

Nitorgen ulizaon(g/L)

1.5

Time (hours)

b)
8

10

6
4
4
2

24

48

72

96

120

Biomass producon, Arabinose Ulizaon (g/L)

3.5. Reactor studies

10

a)
Biomass, glucan producon (g/L)

indicates that the interaction is signicant. A signicant interaction


between osmolyte concentration and pH on glucan production is
observed here (Fig. 1), while other interactions are not signicant.
There is a negative interaction between arabinose and osmolyte
concentration on biomass production. A negative interaction is also
seen between pH and arabinose on biomass production (Fig. A.2
Supplementary material). Increasing arabinose concentration also
increases the osmolarity of the medium. This is evident from the
negative interaction between arabinose and osmolyte concentration.
Using the model, the concentrations of the variables for maximum glucan production is predicted as 1.62 g/L at arabinose and
osmolyte concentrations of 9.63 g/L and 0.23 M respectively, and at
a pH of 6.8 which is not statistically different (p > 0.21) from three
experiments performed at these conditions (1.59 0.14 g/L). This
validation study further strengthened the predictive capability of
the model.
The glucan production has increased several folds from the basal
unoptimized literature reported media conditions. This is the rst
report on the optimization of cyclic (1 3) (1 6)--glucan produced by any B. japonicum. There are very few reports available on
the optimization of cyclic glucans from Rhizobiaceae.

Nitrogen Ulizaon, Glucan producon(g/L)

40

Time (hours)

Fig. 2. Time course of batch culture of Bradyrhizobium japonicum MTCC120 in (a)


shake ask and (b) reactor, under optimized conditions. Symbols:  biomass; 
glucan; arabinose; nitrogen represent he experimental data. The solid lines
represent model predictions.
Table 2
Estimation of model parameters estimated from experiments in ask and reactor.
Parameters

Flask (100 mL)

Reactor (1 L)

m (h1 )
X0 (g/L)
Xm (g/L)
(g glucan/g cell)
(g glucan/g cell/h)
(g arabinose/g cell)
(g arabinose/g cell/h)
YX/N (g nitrogen/g cell)
mN (g nitrogen/g cell/h)

0.09
0.01
1.26
0.65
0.009
5.01
0.0067
0.5
0.0002

0.05
0.3
4.64
0.549
0.012
1.59
0.0035
0.089
0.0003

The model prediction correlated well for bacterial growth with the
experimental data (R2 = 0.99) (Fig. 2a). Luedeking-Piret equation
was used to develop the model for cyclic glucan production. The
growth associated () and non-growth associated () parameters
are 0.549 g/g cell and 0.012 g/g cell/h respectively. The growth associated constant value indicates that the glucan production starts
almost simultaneously with the cell growth. The non-growth associated constant, , shows that the glucan production occurs even
during the stationary phase. Also, from Fig. 2, it is observed that
glucan is produced during the exponential as well as the stationary phases indicating a mixed growth process. Increase in biomass
is associated with decrease in arabinose concentration. The consumption of arabinose is described by modied Luedeking-Piret
equation. The values of and are 1.59 g arabinose/g cell and

A.V. Nair et al. / Journal of Biotechnology 226 (2016) 3543

0.0035 g arabinose/g cell/h respectively in the reactor whereas the


corresponding values in the ask are 5.01 g arabinose/g cell and
0.0067 g arabinose/g cell/h. This clearly shows that the yield coefcients for biomass and glucan production are 3 and 2.5 times more
respectively, in the reactor than in the ask. Comparison between
the model prediction and experimental values from the reactor
indicates that the models are suitable to predict glucan production
as well as arabinose and nitrogen utilization (correlation coefcients were 0.99, 0.98 and 0.98 respectively).

3.7. Structural characterization


Proton NMR spectrum (Fig. A.3b Supplementary material) of
glucan shows signal clusters at 4.86 and 4.56 ppm which indicates the presence of glucose units that are involved in the (1 3)
and (1 6) glycosidic linkages, respectively as reported by Rolin
et al. (1992) with coupling constant (J1,2 ) 8 Hz, conrming the anomeric conrmation. The resonances for protons at 4.51, 3.31,
3.48, 3.61, 3.84, 4.2 ppm are assigned to H-1, H-2, H-3, H-4, H-5, H6a and H-6b respectively for -(1 6) linked glucose residues. The
resonances for protons at 4.81, 3.53, 3.87, 3.48, 3.49, 3.89, 3.72 are
assigned to H-1, H-2, H-3, H-4, H-5, H-6a, H-6b respectively for (1 3) linked glucose residues (Rolin et al., 1992; Komaniecka and
Choma, 2003).The spectrum also reveals resonances characteristic
of phosphocholine (H 3.2 ppm), succinyl (H 2.69 and 2.50 ppm for
HOOC CH2 CH2 COO ) and acetyl (H 2.10 ppm for CH3 COO )
groups (Choma and Komaniecka, 2011). The absence of cross peaks
between 92 and 96 ppm for carbon and 5.1 ppm for proton in HSQC

41

Table 3
Substitution pattern in cyclic -glucan as analysed by MALDI-TOF.
Molecular weight observed [M-1] in Da

Components

1619.0
1780.9
1821.8
1880.4
1924.4
1942.3
1981.3
2022.6
2042.5
2083.8
2141.8
2184.7
2319.7
2383.4

Glc10
Glc11
Glc11 -Ac
Glc11 -Suc
Glc11 -Ac-Suc
Glc12
Glc12 -Ac
Glc11 -(Suc)2 -Ac
Glc12 -Suc
Glc12 -Ac-Suc
Glc12 -(Suc)2
Glc12 -(Suc)2 -Ac
Glu12 -PC-Ac
Glu12 -PC-Suc

spectrum (Fig. A.3c in Supplementary material) corresponding to


reducing glucose units, conrms cyclic nature of the glucan (Miller
et al., 1990; Rolin et al., 1992). The MALDI-TOF MS data shows the
degree of polymerization of the glucan to be from 10 to 13 (Fig. A
3d in Supplementary), majorly made of 11 and 12 glucose residues.
The mass of the molecular ion species are 18 Da less than for a linear glucan (M = 162 n + 18, where n is the number of glucose units
and 18 corresponds to the reducing end sugar), conrming that the
glucans are cyclic (Miller et al., 1990). The mass spectrum also conrms that the glucans obtained are also substituted with non-sugar
moieties phosphocholine, acetate and succinate groups (Table 3),
where succinate and acetate substitutions are high.

Fig. 3. (a) FTIR spectrum of the curcumin, glucan-curcumin complex and glucan. (b) FTIR spectrum showing the region between 2000 cm1 500 cm1 (c) Absorption spectrum
of pure curcumin and inclusion complex.

42

A.V. Nair et al. / Journal of Biotechnology 226 (2016) 3543

3.8. Encapsulation of curcumin


The cyclic nature of these glucans with inner hydrophobic cavity
indicates that these oligosaccharides can form inclusion complexes
with hydrophobic guest molecules and thus increasing their solubility and stability in aqueous medium. Hence, they can nd
applications in drug and food industries. In our present study we
nd that cyclic glucan forms inclusion complex with curcumin.
This was ascertained by FTIR spectroscopy (Fig. 3a and b). The
FTIR spectrum of curcumin shows sharp peaks at 1604 cm1 (C C
stretching vibration of benzene ring), 1510 cm1 (C O stretching vibrations), 3510 cm1 (phenolic O H stretching vibrations),
1435 cm1 (olenic C H) and 857 cm1 (C O C stretching vibration). The FTIR spectrum of cyclic glucan exhibits characteristics
peaks at 3286 cm1 (O H stretching vibrations) and 2934 cm1
(C H stretching vibrations). The peaks at 1640 cm1 and 1023 cm1
corresponding to H O H and C O C glucose units respectively,
are also present. The band at 890 cm1 indicates -linkage. The
spectrum of the inclusion complex shows all the peaks corresponding to glucan but only a few characteristic peaks of curcumin are
visible. In the complex, the shift in the band from 3286 to 3397 cm
1 and 1640 to 1629 cm1 shows the interaction between enolic
OH group of curcumin and OH groups of CBG and the shoulder
peak appearing at 1600 cm1 corresponding to the C C stretching
of the aromatic ring. The shift from 1510 cm1 (C O stretching) to
1506 cm1 is a good indication for complex formation. The peak
at 1155 cm1 of pure curcumin corresponds to C O vibration. The
appearance of a shoulder peak at 1144 cm1 in this region is an indication of a complex formation. The interaction between the cyclic
glucan and curcumin appears to be between the aromatic ring and
the cavity of glucan. Similar observations have been reported for
curcumin encapsulation by -cyclodextrin (Mangolim et al., 2014;
Yallapu et al., 2010).
The inclusion complex was also analysed by UVvis spectroscopy (Fig. 3c). The aqueous solution of curcumin shows
absorption maxima at 410 nm. Cyclic glucan does not show any
absorbance in this region whereas the cyclic glucan-curcumin complex shows a red shift from 410 nm to 420 nm. This change is
attributed to the complex formation between cyclic glucan and
curcumin, during which the guest molecule is transferred from
the aqueous medium to relatively less polar cavity of he host
(Singh et al., 2010). The inclusion complex shows improvement
in the aqueous solubility of curcumin (Fig. A.4 in Supplementary). The water solubility of the inclusion complex was 0.5 mg/mL
when compared to the pure curcumin (6g/mL). An earlier study
with -cyclodextin (formed by solvent evaporation) reported up
to 0.4 mg/mL of curcumin solubility in water (Yadav et al., 2009).
The current result indicates that cyclic -glucan can be used for
encapsulation of hydrophobic compounds as an alternative to
cyclodextrins.

4. Conclusion
The cultural conditions for cyclic (1 3) (1 6)--glucan production from B. japonicum MTCC 120 was optimized using RSM. It
was observed that concentrations of arabinose and osmolyte and
pH signicantly affected the glucan production. Maximum glucan
production (6.7 g/L) was obtained in a 2.5 L reactor with the optimized conditions. This was several orders of magnitude higher
than the values reported in the literature so far. A simple kinetic
model based on logistic equation was developed for the bacterial growth. The model parameters were estimated from batch
experiments and could satisfactorily simulate the batch kinetics.
LuedekingPiret equation predicts the substrate consumption and
product formation well. These developed models can further help in

understanding, controlling and optimizing the production of cyclic


glucan. This biopolymer could encapsulate hydrophobic molecule,
curcumin efciently paving the way for applications in pharmaceutical and food industries for encapsulating and solubilising drugs,
food and avours. Since -glucans are more biocompatible than
cyclodextrins, they could be better alternatives in drug delivery
applications. Use of cyclic (1 3) (1 6)--glucans, for inclusion
complexes has not been reported yet, because of its low production,
which has been addressed to an extent in the current work.
Acknowledgements
We thank Department of Biotechnology, Government of India
for nancial support. DST Unit of Nanoscience and Thematic Unit of
Excellence, Department of Chemistry and Sophisticated analytical
Instrument facility (SAIF), Indian Institute of Technology, Madras,
for their analytical instrument facilities. The authors also thank Mr.
Sneh Badle, Ms Maya Raman and Mr Aslam Basha for technical
inputs.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jbiotec.2016.03.
055.
References
Bhagwat, A.A., Gross, K.C., Tully, R.E., Keister, D.L., 1996. Beta-glucan synthesis in
Bradyrhizobium japonicum: characterization of a new locus (ndvC) inuencing
beta-(1,6) linkages. J. Bacteriol. 178, 46354642.
Breedveld, M.W., Miller, K.J., 1994. Cyclic beta-glucans of members of the family
Rhizobiaceae. Microbiol. Rev. 58, 145161.
Breedveld, M.W., Zevenhuizen, L.P.T.M., Zehnder, A.J.B., 1990. Excessive excretion
of cyclic beta-(1,2)-glucan by Rhizobium trifolii TA-1. Appl. Environ. Microbiol.
56, 20802086.
Breedveld, M.W., Zevenhuizen, L.P.T.M., Zehnder, A.J.B., 1991.
Osmotically-regulated trehalose accumulation and cyclic -(1,2)-glucan
excretion by Rhizobium leguminosarum biovar trifolii TA-1. Arch. Microbiol.
156, 501506.
Choma, A., Komaniecka, I., 2011. Characterization of cyclic -glucans of
Bradyrhizobium by MALDI-TOF mass spectrometry. Carbohydr. Res. 346,
19451950.
Duncan, M.J., 1979. L-arabinose metabolism in Rhizobia. J. Gen. Microbiol. 113,
177179.
Garca-Ochoa, F., Santos, V., Alcon, A., 1995. Xanthan gum production: an
unstructured kinetic model. Enzyme Microb. Technol. 17, 206217.
Khanna, S., Srivastava, A.K., 2005. Statistical media optimization studies for growth
and PHB production by Ralstonia eutropha. Process Biochem. 40, 21732182.
Komaniecka, I., Choma, A., 2003. Isolation and characterization of periplasmic
cyclic -glucans of Azorhizobium caulinodans. FEMS Microbiol. Lett. 227,
263269.
Kshirsagar, P., Suttar, R., Nilegaonkar, S., Kulkarni, S., Kanekar, P., 2012. Scale up
production of polyhydroxyalkanoate (PHA) at different aeration, agitation and
controlled dissolved oxygen levels in fermenter using Halomonas campisalis
MCM B-1027. J. Biochem. Technol. 4, 512517.
Kwon, C., Park, H., Jung, S., 2007. Enantioseparation of some chiral avanones using
microbial cyclic -(1->3),(1->6)-glucans as novel chiral additives in capillary
electrophoresis. Carbohydr. Res. 342, 762766.
Laroche, C., Michaud, P., 2007. New developments and prospective applications for
beta (1,3) glucans. Recent Pat. Biotechnol. 1, 5973.
Luedeking, R., Piret, E.L., 1959. A kinetic study of the lactic acid fermentation: batch
process at controlled pH. J. Biochem. Microbiol. Technol. Eng. 1, 393431.
Mangolim, C.S., Moriwaki, C., Nogueira, A.C., Sato, F., Baesso, M.L., Neto, A.M.,
Matioli, G., 2014. Curcuminb-cyclodextrin inclusion complex stability,
solubility, characterisation by FT-IR, FT-Raman, X-ray diffraction and
photoacoustic spectroscopy, and food application. Food Chem. 153, 361370.
McIntosh, M., Stone, B.A., Stanisich, V.A., 2005. Curdlan and other bacterial
(1,3)--d-glucans. Appl. Microbiol. Biotechnol. 68, 163173.
Miller, K.J., Gore, R.S., 1992. Cyclic beta-1,6 -1,3 glucans of Bradyrhizobium:
functional analogs of the cyclic beta-1,2-glucans of Rhizobium? Curr. Microbiol.
24, 101104.
Miller, K.J., Gore, R.S., Johnson, R., Benesi, A.J., Reinhold, V.N., 1990. Cell-associated
oligosaccharides of Bradyrhizobium spp. J. Bacteriol. 172, 136142.
Pfeffer, P.E., Becard, G., Rolin, D.B., Uknalis, J., Cooke, P., Tu, S., 1994. In vivo nuclear
magnetic resonance study of the osmoregulation of
phosphocholine-substituted beta-(1,3) (1,6) cyclic glucan and its associated

A.V. Nair et al. / Journal of Biotechnology 226 (2016) 3543


carbon metabolism in Bradyrhizobium japonicum USDA 110. Appl. Environ.
Microbiol. 60, 21372146.
Puranamaneewiwat, N., Tajima, S., Niamsup, H., 2006. Proteomic analysis of
Bradyrhizobium japonicum USDA110 in acidic condition. Chiang Mai J. Sci. 33,
335345.
Rolin, D.B., Pfeffer, P.E., Osman, S.F., Szwergold, B.S., Kappler, F., Benesi, A.J., 1992.
Structural studies of a phosphocholine substituted -(1,3) (1,6) macrocyclic
glucan from Bradyrhizobium japonicum USDA 110. BBA-Gen. Subj. 1116,
215225.
Singh, R., Bharati, N., Madan, J., Hiremath, S.N., 2010. Characterization of
cyclodextrin inclusion complexesa review. J Pharm. Sci. Technol. 2, 171183.
Stowers, M.D., 1985. Carbon metabolism in Rhizobium species. Annu. Rev.
Microbiol. 39, 89108.
Venkatachalam, G., Gummadi, S.N., Doble M., 2012. Cyclic -Glucans from
Microorganisms: Production, Properties and Applications, SpringerBriefs in
Microbiology, Berlin.

43

Venkatachalam, G., Srinivasan, D., Doble, M., 2013. Cyclic -(1,2)-glucan


production by Rhizobium meliloti MTCC 3402. Process Biochem. 48, 18481854.
Wang, X., Xu, P., Yuan, Y., Liu, C., Zhang, D., Yang, Z., Ma, C., 2006. Modeling for
gellan gum production by Sphingomonas paucimobilis ATCC 31461 in a
simplied medium. Appl. Microbiol. Biotechnol. 72, 33673374.
Weiss, R., Ollis, D., 1980. Extracellular microbial polysaccharides. I. Substrate,
biomass, and product kinetic equations for batch xanthan gum. Biotechnol.
Bioeng. 22, 859873.
Yadav, V.R., Suresh, S., Devi, K., Yadav, S., 2009. Effect of cyclodextrin complexation
of curcumin on its solubility and antiangiogenic and anti-inammatory
activity in rat colitis model. AAPS Pharm. Sci. Technol. 10, 752.
Yallapu, M.M., Jaggi, M., Chauhan, S.C., 2010. -Cyclodextrincurcumin self
assembly enhances curcumin delivery in prostate cancer cells. Colloid Surf. B.
79, 113125.