Bioresource Technology 98 (2007) 792–797

Optimization of nutrients for gellan gum production by Sphingomonas

paucimobilis ATCC-31461 in molasses based medium using
response surface methodology
a,*
R.M. Banik , A. Santhiagu a, S.N. Upadhyay b

a
School of Biochemical Engineering, Institute of Technology, Banaras Hindu University, Varanasi 221 005, India
b
Department of Chemical Engineering, Institute of Technology, Banaras Hindu University, Varanasi 221 005, India

Received 21 November 2005; received in revised form 13 March 2006; accepted 23 March 2006
Available online 16 May 2006

Abstract

A molasses based medium for the production of gellan by Sphingomonas paucimobilis ATCC-31461 was developed. Placket–Burman
design criterion was applied to study the effect of various nutrient supplements on gellan production using molasses. Among the 20 vari-
ables tested, molasses, tryptone, casaminoacid, disodium hydrogen orthophosphate and manganese chloride showed significant effect on
gellan production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained
from Placket–Burman design. Most suitable medium composition for production of gellan was (g/l): molasses—112.5; tryptone—1; cas-
aminoacid—1; disodium hydrogen orthophosphate—1; manganese chloride—0.947 and the optimum gellan production was 13.814 g/l.
Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: Gellan; Exopolysaccharide; Molasses; Sphingomonas paucimobilis; Central composite design; Response surface methodology

1. Introduction improve the fermentation process for reduction of its pro-

Gellan is an exopolysaccharide having various applica-
tions in food and pharmaceutical industry and produced
by fermentation using Sphingomonas paucimobilis (Banik
et al., 2000). Gellan forms thermoreversible gels which
are useful in food industry as thickening and binding
agents (Sutherland, 1998) and to maintain water capacity
during cooking and storage (Sanderson and Clark, 1983).
GELRITE, a commercial form of gellan is used for solidi-
fying microbial media, especially those used for the isola-
tion of thermophiles (Shangu et al., 1983; Lin and
Casida, 1984), also useful as vehicle for ophthalmic prepa-
rations (Calfors et al., 1998) and other sustained release
pharmaceutical preparations (Fattah et al., 1996). Gellan
is one of the expensive biogums and it is necessary to
*
Corresponding author. Tel.: +91 542 2367842; fax: +91 542 2368428.
E-mail address: rmbanik@rediffmail.com (R.M. Banik).
duction cost. Studies have been attempted to improve gel-
lan production using mutant strains (West, 2002) and whey
and corn starch as substrates (Dlamini and Peiris, 1997;
Madhavan et al., 2003) as well as optimization of culture
conditions (Kanari et al., 2002; Madhavan et al., 2003).
Molasses, a by-product of sugar industry, contains high
sugar concentration and other metals necessary for the fer-
mentation process and is inexpensive. Molasses has been
successfully used for fermentative production of polysac-
charides such as curdlan (Lee et al., 1997), xanthan (Kalo-
giannis et al., 2003) and dextran (Vedyashkina et al., 2005).
Response surface methodology (RSM) is a well-known
method applied in the optimization of medium constituents
and other critical variables responsible for the production
of biomolecules (Ya-Hong et al., 2004). Statistical experi-
mental designs can be employed at various phases of opti-
mization process, such as for screening experiments and for
finding optimum conditions for a desired response. Single
variable optimization methods are tedious and can also

0960-8524/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.03.012
 R.M. Banik et al. / Bioresource Technology 98 (2007) 792–797 793

lead to misinterpretation of results because they overlook
the interaction between different factors involved (Francis
et al., 2003). In the present study, an attempt was made
to optimize the production of gellan gum using molasses
by S. paucimobilis ATCC-31461 using Placket and Burman
design and a central composite design.
2. Methods
2.1. Media and culture conditions
A strain of S. paucimobilis ATCC-31461 was used in this
study. It was maintained on YPG medium (glucose 2%,
yeast extract 0.3%, peptone 0.5%) and sub-cultured once
in two weeks. The strain was adapted with molasses (41%
w/w sugar content) for three successive transfers in slant
cultures prepared by replacing glucose with molasses. Gel-
lan fermentation was carried out in a medium containing
(per l): sucrose, 40 g; monosodium glutamate, 1.35 g; cas-
aminoacid, 1 g; KH2PO4, 3 g; Na2HPO4, 5 g; K2SO4, 1 g;
CaCl2 Æ 2H2O, 3 mM; MnSO4 Æ 7H2O, 1.5 mM; NaCl, 1 g;
(Kanari, 2004). Inoculum was developed by transferring
one loop full of the organism from the YPG slant culture
to the production medium in Erlenmeyer flasks. The flasks
were incubated in an orbital shaker at 30 ± 1 °C and
200 rev/min for 24 h for inoculum development.
gation at 10,000g for 45 min and dried at 80 °C and
weighed. Total sugar was determined by phenol sulphuric
acid method.
2.4. Response surface methodology
Response surface methodology was applied in two
stages, first to identify the significant nutrients for produc-
tion of gellan using Placket and Burman design criterion
and later the significant nutrients resulted from Placket
and Burman design were optimized by using a central com-
posite design. The experimental design and statistical anal-
ysis of the data were done by using Minitab statistical
software package.
2.4.1. Placket–Burman design
Each variable was tested at two levels namely a high
level denoted by (+) and a low level denoted by () as
listed in Table 1. Twenty variables were screened by con-
ducting 24 experiments and the experimental design is
given in Table 2. All experiments were conducted in dupli-
cate and the average value of gellan yield was used for sta-
tistical analysis. The variables, which were significant at 5%
level (P < 0.05) from the regression analysis were consid-
ered to have greater impact on gellan gum production
and were further optimized by a central composite design.

2.2. Production of gellan 2.4.2. Central composite design

A central composite design was applied to determine the
Initially the significance of 20 nutrients on the produc- optimum concentration of five significant nutrients
tion of gellan using Placket–Burman design criterion was screened from Placket–Burman design criterion. The effect
studied. Fermentations were carried out by inoculating of five nutrients (cane molasses, tryptone, casaminoacid,
(5% inoculum; 73.6 mg cell dry wt/ml) 50 ml medium in disodium hydrogen orthophosphate and manganese
250 ml Erlenmeyer flasks and incubating in an orbital sha- chloride) on the production of gellan was studied at five
ker at 30 ± 1 °C and 200 rev/min for 48 h. Optimization of
nutrients was done in a 5-l fermentor with a working vol- Table 1
ume of 3-l of production medium. The pH of the medium Level of nutrients used for the production of gellan by Sphingomonas
paucimobilis using Placket–Burman design criterion
was maintained at 6.5 by 2 M NaOH or 2 M H2SO4. Fer-
mentation was carried out with 5% inoculum and at Nutrient Nutrient High level Low level
code (+) (g/l) () (g/l)
30 ± 1 °C, 300 rpm with aeration at 3-l/min.
A Molasses 100 50
B Tryptone 0.5 0.25
2.3. Analytical methods
C Peptone 0.5 0.25
D Yeast extract 0.5 0.25
To isolate the polymer, the fermented broth was heated E Sodium nitrate 0.5 0.25
for 15 min in a boiling water bath, cooled and its pH was F Ammonium sulphate 0.5 0.25
adjusted to 10.0 by 2 M NaOH. Then the broth was heated G Ammonium chloride 0.5 0.25
H Ammonium nitrate 0.5 0.25
again for 10 min in a constant temperature water bath
I Potassium nitrate 0.5 0.25
(80 °C), cooled and pH was brought down to 7.0 by adding J Glycine 0.2 0.1
2 M H2SO4. This pretreatment killed the microorganisms, K Glutamine 0.2 0.1
deactivated the enzymes and enhanced the gelling proper- L Glutamic acid 0.2 0.1
ties of the exopolysaccharide (Kang et al., 1982). Then M Casaminoacid 0.2 0.1
N Potassium dihydrogen phosphate 0.1 0.05
the broth was centrifuged at 10,000g for 45 min to separate
O Disodium hydrogen phosphate 0.1 0.05
the cells. The cells were treated with dimethyl sulphoxide to P Manganese chloride 0.1 0.05
remove any adhering polymer and centrifuged (10,000g for Q Magnesium sulphate 0.1 0.05
45 min). The supernatant was added with twice the volume R Potassium sulphate 0.1 0.05
of isopropyl alcohol to precipitate the polymer and kept S Sodium chloride 0.1 0.05
T Calcium chloride 0.1 0.05
overnight at 4 °C. The polymer was separated by centrifu-
794 R.M. Banik et al. / Bioresource Technology 98 (2007) 792–797

Table 2
Nutrients for the production of gellan by Sphingomonas paucimobilis using Placket–Burman design criterion
Run Block A B C D E F G H I J K L M N O P Q R S T Gellan yield
(g/l)
1 1   +  +   + +   + +  +  + + + + 4.26
2 1   + +   + +  +  + + + + +     6.17
3 1 +     +  +   + +   + +  +  + 8.24
4 1  +  +   + +   + +  +  + + + + + 4.32
5 1 +   + +  +  + + + + +     +  + 4.21
6 1 +  + + + + +     +  +   + +   2.82
7 1 +  +  + + + + +     +  +   + + 4.20
8 1  +  + + + + +     +  +   + +  6.21
9 1  + +   + +  +  + + + + +     + 6.32
10 1 + + + +     +  +   + +   + +  6.18
11 1    +  +   + +   + +  +  + + + 4.16
12 1 +  +   + +   + +  +  + + + + +  8.30
13 1   + +  +  + + + + +     +  +  2.56
14 1  + + + + +     +  +   + +   + 6.19
15 1                     2.26
16 1 + +  +  + + + + +     +  +   + 6.23
17 1 +   + +   + +  +  + + + + +    8.27
18 1 + + + + +     +  +   + +   + + 8.19
19 1 + +   + +  +  + + + + +     +  6.20
20 1  + +  +  + + + + +     +  +   4.27
21 1 + + +     +  +   + +   + +  + 6.32
22 1  +   + +   + +  +  + + + + +   6.18
23 1 + +     +  +   + +   + +  +  8.21
24 1     +  +   + +   + +  +  + + 2.80

experimental levels: a, 1, 0, +1, +a, where a = 2n/4, here
n was the number of variables and 0 corresponded to the
central point. The experimental levels for these variables
were selected from our preliminary work, which indicated
that an optimum could be found within the level of
parameters studied. The levels of factors used for experi-
mental design are given in Table 3. The actual level of each
factor was calculated by the following equation (Paul et al.,
1992):
Actual level  ðhigh level þ low levelÞ=2
Coded value ¼
ðhigh level  low levelÞ=2
Gellan concentration was analyzed by using a second-
order polynomial equation and the data were fitted in to
the equation by multiple regression procedure. The model
equation for analysis is given below:
Y ¼ b0 þ b1 X 1 þ b2 X 2 þ b3 X 3 þ b4 X 4 þ b5 X 5 þ b11 X 21
þ b22 X 22 þ b33 X 23 þ b44 X 24 þ b55 X 25 þ b12 X 1 X 2
þ b13 X 1 X 3 þ b14 X 1 X 4 þ b15 X 1 X 5 þ b23 X 2 X 3
þ b24 X 2 X 4 þ b25 X 2 X 5 þ b34 X 3 X 4 þ b35 X 3 X 5
þ b45 X 4 X 5
where X1, X2, . . . , X5 are the levels of the factors and
b1, b2, . . ., b5 are linear coefficients, b11, b22, . . . b55 are
quadratic coefficients and b12, b13, . . . , b45 are interactive
coefficient estimates with b0 having a role of a scaling con-
stant. Analysis of variance (ANOVA), regression analysis
was done and contour plots were drawn by using Minitab
statistical software package.
3. Results and discussion
3.1. Screening of nutrient components using
Placket–Burman design criterion
In Placket and Burman design, among the inorganic
nitrogen sources tested, ammonium chloride showed some
significance at low level and others had no significance
on gellan production. Tryptone had greater influence on
ð1Þ gellan production than other organic nitrogen sources
studied. Casamino acid showed significant effect on
enhancement of gellan production as compared to other
amino acids tested. Among the metallic salts studied,
potassium dihydrogen phosphate increased gellan produc-
tion when used at low level but disodium hydrogen phos-
phate and manganese chloride showed greater impact on
gellan production. Out of the 20 nutrient supplements stud-
ied, five nutrients (cane molasses, tryptone, casaminoacid,
disodium hydrogen orthophosphate and manganese chlo-
ride) had significant influence on gellan production as evi-
denced by their P value (<0.05, significant at 5% level)
ð2Þ obtained from regression analysis. Gellan production
increased with increase in concentration of cane molasses,
tryptone, casaminoacid, disodium hydrogen orthophos-
phate and manganese chloride from low level to high level.
The coefficient of determination (R2) of the model was
0.905, which indicated the model could explain up to
90.05% variation of the data. Gellan yield obtained from
 R.M. Banik et al. / Bioresource Technology 98 (2007) 792–797 795

Table 3
Gellan production by Sphingomonas paucimobilis using significant nutrients based on central composite design criteria
Run order Molasses (g/l) Tryptone (g/l) Casaminoacid (g/l) Na2HPO4 (g/l) MnCl2 (g/l) Gellan (g/l)
Experimental Predicted
1 112.50 1.00 1.00 2.00 1.00 11.480 11.513
2 075.00 1.50 1.50 0.75 1.50 10.320 10.664
3 150.00 1.50 1.50 1.50 1.50 09.302 09.307
4 075.00 1.50 1.50 1.50 0.50 09.690 09.672
5 075.00 1.50 0.50 0.50 0.50 10.611 10.586
6 037.50 1.00 1.00 1.00 1.00 11.220 11.282
7 075.00 0.50 0.50 1.50 0.50 10.151 10.121
8 150.00 0.50 1.50 0.50 1.50 10.040 10.000
9 112.50 1.00 1.00 1.00 1.00 13.732 13.667
10 112.50 1.00 1.00 1.00 1.00 13.746 13.667
11 112.50 1.00 1.00 1.00 1.00 12.830 13.667
12 075.00 1.50 0.50 1.50 1.50 10.168 10.316
13 150.00 0.50 0.50 1.50 1.50 09.920 09.851
14 150.00 1.50 1.50 0.50 0.50 10.091 10.067
15 187.50 1.00 1.00 1.00 1.00 10.650 10.440
16 150.00 1.50 0.50 0.50 1.50 10.512 10.470
17 112.50 1.00 2.00 1.00 1.00 10.763 10.663
18 112.50 1.00 1.00 1.00 1.00 13.718 13.667
19 112.50 1.00 1.00 1.00 1.00 13.700 13.667
20 075.00 0.50 1.50 0.50 0.50 10.240 10.172
21 150.00 0.50 0.50 0.50 0.50 10.450 10.646
22 112.50 1.00 1.00 1.00 0.50 09.786 09.375
23 112.50 1.00 1.00 1.00 2.00 09.936 09.965
24 112.50 1.00 1.00 1.00 1.00 13.730 13.667
25 150.00 1.50 0.50 1.50 0.50 09.660 09.512
26 112.50 0.50 1.00 1.00 1.00 09.812 12.500
27 075.00 0.50 0.50 0.50 1.50 10.880 10.887
28 150.00 0.50 1.50 1.50 0.50 09.320 09.092
29 112.50 2.00 1.00 1.00 1.00 09.750 09.722
30 112.50 1.00 1.00 0.50 1.00 11.678 13.606
31 112.50 1.00 1.00 1.00 1.00 13.730 13.667
32 075.00 0.50 1.50 1.50 1.50 09.660 09.661

Placket–Burman design experiments showed wide variation
(2.26–13.0 g/l), which indicated that further optimization
was necessary.
3.2. Optimization of significant nutrients using central
composite design
Thirty two experiments were carried out from the design
(Table 3), by applying multiple regression analysis on the
experimental data, the following second-order polynomial
equation was found to explain the gellan production by
S. paucimobilis:
Y ¼ 3:211 þ 0:109X 1 þ 7:766X 2 þ 4:549X 3
þ 2:771X 4 þ 8:037X 5  0:0005X 21  3:94X 22
 2:501X 23  1:566X 24  3:86X 25  0:002X 1 X 4
þ 0:1486X 2 X 3  0:039X 2 X 4  0:041X 3 X 4
 0:157X 3 X 5
Regression analysis of the experimental data showed
that molasses, tryptone, casaminoacid, disodium hydrogen
orthophosphate and manganese chloride had positive lin-
ear effect on gellan production (P < 0.05). Among the five
nutrients, manganese chloride had highest impact on gellan
production as given by highest linear coefficient (8.037),
followed by tryptone (7.766), casaminoacid (4.594), diso-
dium hydrogen orthophosphate (2.711) and molasses
(0.109). These nutrients also showed significant negative
quadratic effects on gellan production, indicating that gel-
lan production increased as the level of these factors
increased and decreased as the level of these parameters
increased above certain values. Interaction between these
parameters was also significant. The interactions bet-
ween molasses–disodium hydrogen phosphate, tryptone–
casaminoacid, tryptone–disodium hydrogen phosphate,
casaminoacid–disodium hydrogen phosphate, casamino-
acid–manganese chloride were significant as shown by
low P values (<0.05) for interactive terms. But the interac-
tions between molasses–tryptone, molasses–casaminoacid,
molasses–manganese chloride, tryptone–manganese chlo-
ride and disodium hydrogen phosphate were found to be
ð3Þ insignificant as given by P value above 0.05. Hence, these
terms were excluded from the regression Eq. (3) used for
this model. Analysis of variance for the gellan production
obtained from this design is given in Table 4. ANOVA
796 R.M. Banik et al. / Bioresource Technology 98 (2007) 792–797

Table 4 2.0
11
Analysis of variance for gellan production by Sphingomonas paucimobilis 9
10
using central composite design criterion
10
Source DF Seq SS Adj SS Adj MS F P 1.5 13

Casaminoacid (g/l)
Regression 20 71.501 71.501 3.575 17,269.94 0.000
Linear 5 4.488 17.609 3.522 17,011.45 0.000
Square 5 660,944 66.945 13.389 64,677.3 0.000 1.0
Interaction 10 0.069 0.069 0.007 33.21 0.000
Residual error 11 0.02 0.002 0.000
Lack of fit 6 0.001 0.001 0.001 0.74 0.643
0.5
Pure error 5 0.001 0.001 0.001
Total 31 71.504 11
10
12
0.0
50 75 100 125 150 175
gives the value of the model and can explain whether this
Molasses (g/l)
model adequately fits the variation observed in gellan pro-
duction with the designed nutrients level. If the F-test for Fig. 2. Contour plot for gellan production at varying concentration of
the model is significant at the 5% level (P < 0.05), then molasses and casaminoacid. Hold values: tryptone—1 g/l; disodium
hydrogen phosphate—1 g/l; manganese chloride—1 g/l.
the model is fit and can adequately explain the variation
observed. If the F-test for lack of fit is significant
(P < 0.05), then a more complicated model is required to
fit the data. The closure the value of R (multiple correlation 2.0
10
coefficient) to 1, the better the correlation between the 12

observed and predicted values. Here, the value of R

11
(0.9814) revealed that the model could explain up to 1.5
Disod. Hyd. Phosphate

98.14% variation of gellan production. The P value for lack

of fit (0.643) indicated that the experimental data obtained
fitted well with the model and explained the effect of molas-
(g/l)

1.0
ses, tryptone, casaminoacid, disodium hydrogen phosphate
and manganese chloride on gellan production by S. pauc-
imobilis. Figs. 1–4 show the contour plots of gellan produc- 0.5
tion for each pair of nutrient concentration by keeping the
other three nutrients constant at its middle level. Maximum
11 11
gellan was produced at middle level of each pair of nutri- 13
0.0
ents at a constant middle level of the other three nutrients. 50 75 100 125 150 175
Further increase in these factors above the middle level Molasses (g/l)
(cane molasses—112.5 g/l, tryptone—1 g/l, casamino-
Fig. 3. Contour plot for gellan production at varying concentration of
acid—1 g/l, disodium hydrogen orthophosphate—1 g/l
molasses and disodium hydrogen phosphate. Hold values: tryptone—1 g/l;
and manganese chloride—1 g/l) showed decrease in gel- casaminoacid—1 g/l; manganese chloride—1 g/l.
lan production. Fermentations carried out in laboratory

2.0
2.0
10 8 9 10
9 9
9 12
12
Manganese chloride

1.5 1.5
Tryptone (g/l)

(g/l)

1.0 1.0

13 13
0.5 0.5
10 10

9 11 9 9 11
8 9
8
0.0 0.0
50 75 100 125 150 175 50 75 100 125 150 175
Molasses (g/l) Molasses (g/l)

Fig. 1. Contour plot for gellan production at varying concentration of Fig. 4. Contour plot for gellan production at varying concentration of
molasses and tryptone. Hold values: casaminoacid—1 g/l; disodium molasses and manganese chloride. Hold values: tryptone—1 g/l; disodium
hydrogen phosphate—1 g/l; manganese chloride—1 g/l. hydrogen phosphate—1 g/l; casaminoacid—1 g/l.
 R.M. Banik et al. / Bioresource Technology 98 (2007) 792–797
fermentor gave higher yield of gellan than shake flask cul-
tures due to pH control, better mixing, mass and oxygen
transfer in fermentor. The experimental data were fitted
in to Eq. (3) and the optimum values were found to be:
cane molasses—112.5 g/l, tryptone—1 g/l, casamino-
acid—1 g/l, disodium hydrogen orthophosphate—1 g/l
and manganese chloride—0.947 g/l. At these optimum level
of nutrients, gellan production of 13.814 g/l was obtained,
which was very close to the predicted value (13.71 g/l).
About 91% of sugar was consumed during the fermenta-
tion process, which indicated molasses as useful substrate
for gellan production.
4. Conclusions
To develop a low-cost fermentation medium for gellan
production by S. paucimobilis, 20 nutrients were tested by
using Placket and Burman design criterion and five nutrients
viz. molasses, tryptone casaminoacid, disodium hydrogen
orthophosphate and manganese chloride showed significant
effect on gellan production. The nutrients concentration was
optimized by applying central composite design and the
most suitable medium composition for gellan production
was (g/l) cane molasses—112.5, tryptone—1, casamino-
acid—1, disodium hydrogen orthophosphate—1 and man-
ganese chloride—0.947. This is the first report about
production of gellan by S. paucimobilis using molasses based
medium.
Acknowledgements
We are thankful to UGC, New Delhi, India, for finan-
cial support of the research work and Senior Research fel-
lowship awarded to Mr. A. Santhiagu.
References
Banik, R.M., Kanari, B., Upadhyay, S.N., 2000. Exopolysaccharides of
gellan family: prospects and potential. World J. Microbiol. Biotechnol.
16, 407–414.
Calfors, J., Edsman, K., Peterson, R., Journving, K., 1998. Rheological
evaluation of gelrite in situ for ophthalmic use. Eur. J. Pharm. Sci. 6,
113–119.
Dlamini, A.M., Peiris, P.S., 1997. Production of exopolysaccharide by
Pseudomonas sp. ATCC 31461 (Pseudomonas elodea) using whey as
fermentation substrate. Appl. Microbiol. Biotechnol. 47, 52–57.
797
Fattah, E.A., Grant, D.J., Gabe, K.E., Meshali, M.M., 1996. Physical
characteristics and release behaviour of salbutamol sulphate beads
prepared with different ionic polysaccharide. Drug Dev. Ind. Pharm.
24, 541–547.
Francis, F., Sabu, A., Madhavan, K.N., Sumitra, R., Sanjoy, G., George,
S., Pandey, A., 2003. Use of response surface methodology for
optimizing process parameters for the production of a-amylase by
Aspergillus oryzae. Biochem. Eng. J. 15, 107–115.
Kalogiannis, S., Iakovidou, G., Kyriakides, M.L., Kyriakides, D.,
Skaracis, N.G., 2003. Optimization of xanthan gum production by
Xanthomonas campestris grown in molasses. Proc. Biochem. 39, 249–
256.
Kanari, B., 2004. Studies on production, purification and properties of
gellan gum. Ph.D. Thesis, Banaras Hindu University, Varanasi, India.
Kanari, B., Banik, R.M., Upadhyay, S.N., 2002. Effect of environment
and carbohydrate on gellan gum production in batch mode. Appl.
Biochem. Biotechnol. 102, 129–140.
Kang, S.K., Veeder, T.G., Mirrasoul, J.P., Kaneko, T., Cottrell, W., 1982.
Agar-like polysaccharide produced by a Pseudomonas species: pro-
duction and basic properties. Appl. Environ. Microbiol. 43 (5), 1086–
1091.
Lee, I.Y., Seo, W.T., Kim, M.K., Park, C.S., Park, Y.H., 1997.
Production of curdlan using sucrose or sugar cane molasses by two-
step fed-batch cultivation of Agrobacterium species. J. Ind. Microbiol.
Biotechnol. 18, 255–259.
Lin, C.C., Casida, L.E., 1984. GELRITE as a gelling agent in media for
the growth of thermophilic microorganisms. Appl. Environ. Micro-
biol. 47, 427–429.
Madhavan, N.K., Reeta, R.S., Sabarinath, C., Pandey, A., 2003.
Fermentative production of gellan using Sphingomonas paucimobilis.
Proc. Biochem. 38, 1513–1519.
Paul, G.C., Kent, C.A., Thomas, C.R., 1992. Quantitative characteriza-
tion of vacuolization in Penicillium chyrsogenum using automatic
image analysis. Trans I. ChemE. 70, 13–20.
Sanderson, G.R., Clark, R.C., 1983. Laboratory-produced microbial
polysaccharide has many potential food applications as a gelling,
stabilizing and texturing agent. Food Technol. 37, 63–70.
Shangu, D., Valiant, M., Tutlane, V., Weinberg, E., Weissberger, B.,
Koupal, L., Gadebusch, H., Stapley, E., 1983. GELRITE as an agar
substitute in bacteriological media. Appl. Environ. Microbiol. 46, 840–
845.
Sutherland, I.W., 1998. Novel and established applications of microbial
polysaccharides. Trends Biotechnol. 16, 41–46.
Vedyashkina, T.A., Revin, V.V., Gogotov, I.N., 2005. Optimizing the
conditions of dextran synthesis by the bacterium Leuconostoc mesen-
teroides grown in a molasses-containing medium. Appl. Biochem.
Microbiol. 41 (4), 409–413.
West, T.P., 2002. Isolation of mutant strain of Pseudomonas sp. ATCC
31461 exhibiting elevated polysaccharide production. J. Ind. Micro-
biol. Biotechnol. 29, 185–188.
Ya-Hong, X., Jian-Zhong, L., Hai-Yan, S., Liang-Nian, J., 2004.
Enhanced production of extracellular ribonuclease from Aspergillus
niger by optimization of culture conditions using response surface
methodology. Biochem. Eng. J. 21, 27–32.