Process Biochemistry 40 (2005) 395400

Chitosan from Mucor rouxii: production and

physico-chemical characterization
S. Chatterjee, M. Adhya, A.K. Guha, B.P. Chatterjee
Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India
Received 30 June 2003; received in revised form 29 November 2003; accepted 15 January 2004

Abstract
Chitosan is obtained by chemical conversion of chitin, which is a constituent of the exoskeleton of crustacea and insects. An alternative
source of chitosan is the cell wall of fungi. Fungal culture media and fermentation condition can be manipulated to provide chitosan of
more consistent physico-chemical properties compared to that derived chemically from chitin. Chitosan has been isolated from Mucor rouxii
cultured in three different media, viz., molasses salt medium (MSM), potato dextrose broth (PDB) and yeast extract peptone glucose (YPG)
medium under submerged condition and their yield has been found to be the almost same, being 0.61 g/l for MSM, 0.51 g/l for PDB and
0.56 g/l for YPG respectively. Their physico-chemical properties such as ash, moisture, protein contents and specific rotation do not show much
difference. However, variation has been observed in their polydispersed nature and crystallinity. Chitosan from MSM was less polydispersed
and more crystalline compared to those from YPG and PDB.
2004 Elsevier Ltd. All rights reserved.
Keywords: Chitosan; Mucor rouxii; Fermentation; Polydispersity; X-ray diffraction

1. Introduction
Chitosan, a linear polymer of -1,4-glucosamine, is derived by deacetylation of naturally occurring biopolymer
chitin, which is present in the exoskeleton of crustacea such
as crab, shrimp, lobster, crawfish and insects, and is considered to be the second most abundant polysaccharide in the
world after cellulose. Chitosan can also be found in the cell
wall of certain groups of fungi, particularly zygomycetes. It
is a straight chain natural hydrophilic polysaccharide having
a three dimensional -helical configuration stabilized by
intramolecular hydrogen bonding [1]. Chitosan being polycationic, nontoxic, biodegradable as well as antimicrobial
finds numerous applications especially in the agriculture,
food and pharmaceutical industries, such as food preservation [27], fruit juice clarification [8] water purification
particularly for removal of heavy metal ions [911]; sorption for dyes and flocculating agent. Chitosan can also
be used as a biological adhesive for its hydrogel-forming

Corresponding author. Tel.: +91-33-2473-5904/4971/3372x321;

fax: +91-33-2473-2805.
E-mail address: bcbpc@mahendra.iacs.res.in (B.P. Chatterjee).

0032-9592/$ see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2004.01.025

properties [12], wound healing accelerator [13], and also

in cosmetics industries. However, the efficiency of chitosan
depends upon the molecular size [14], degree of deacetylation, crystallinity, solubility and its derivatized products.
Shell waste from shrimp, crab and lobster processing industries is the traditional source of chitin. However, commercial production of chitosan by deacetylation of crustacean
chitin with strong alkali appears to have limited potential
for industrial acceptance because of seasonal and limited
supply, difficulties in processing particularly with the large
amount of waste of concentrated alkaline solution causing
environmental pollution and inconsistent physico-chemical
properties. However, uniform deacetylation is a prerequisite
to specific industrial applications. With advances in fermentation technology chitosan preparation from fungal cell
walls becomes an alternative route for the production of this
polymer in an ecofriendly pathway [1517]. Adequate quantities of chitosan was found to be present in the hyphae as
cell wall component in the dimorphic fungus Mucor rouxii
as reported by White et al. [18] and Arcidiacono and Kaplan
[19].
The present communication describes the production and
physico-chemical properties of chitosan obtained by fermentation of M. rouxii in three different media.

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S. Chatterjee et al. / Process Biochemistry 40 (2005) 395400

2. Materials and methods

2.5. Estimation of sucrose

2.1. Materials

Sucrose was estimated by a phenol sulphuric acid method

[21].

M. rouxii (MTCC 386) used in this study was obtained

from Institute of Microbial Technology, Chandigarh, India
and maintained on potato dextrose agar slants. Chemicals
and biochemicals were purchased from E. Merck and molasses was procured from local market.
2.2. Fermentation medium
The following three fermentation media were used to
study the growth and production of chitosan from M. rouxii.
2.2.1. Molasses salt medium (MSM)
This medium contains 0.2% NaNO3 , 0.1% K2 HPO4 ,
0.001% FeSO4 , 0.001% MgSO4 , 0.2% yeast extract and
molasses as carbon source. Molasses was added to the media to obtain sucrose concentrations varying from 2 to 5%.
2.2.2. Yeast peptone glucose medium (YPG)
YPG was made with yeast extract 0.3%, peptone 1% and
glucose 2%.
2.2.3. Potato dextrose broth (PDB)
PDB contains potato extract 20% and dextrose 2%. The
pH of all media was adjusted to 5.0 and 50 ml of each
medium was added to a 250 ml Erlenmeyer flask and sterilized by autoclaving at 121 C for 15 min.
2.3. Preparation of inoculum and fermentation
Inocula were prepared by growing the organism in potato
dextrose agar (PDA) plates at 30 C for 3 days. Flasks containing the media were inoculated with one 5 mm diameter mycelium covered agar disk [20] containing 6.4 106
spores/disk was used as inoculum and incubated at 30 C
under submerged condition (120 rpm) for different periods
of time. At the end of the desired incubation period mycelia
were harvested by filtration, dried by lyophilization and
weighed. Mycelia and culture filtrates were stored at 20 C
until use.

2.6. Determination of degree of deacetylation

Degree of deacetylation of chitosan was measured by
first derivative UV spectroscopic method [22] using Shimadzu [option program/interface OPI-4] UV-Vis recording
spectrophotometer UV 240 graphtcord [scan speed-fast, slit
width 2 nm, scanning range 190240 nm].
2.7. Determination of weight average molecular weight
Chitosan solutions of varying concentrations ranging from
0.0625 to 1.00% were prepared with 2% AcOH. The viscosity of the solutions was measured at a particular shear
rate at 25 C by Haake Rheometer (Rotovisco model RT20,
Con/plate sensor C60/1 , and Haake software version V3).
The reduced viscosity (red ), which is specific viscosity
(sp )/concentration (C), as tabulated,
(red = sp /C) where (sp = relative 1),
viscosity of solution
relative =
viscosity of solvent

and

of solutions were plotted against different concentrations of

chitosan solution. By extrapolating of the curve to Y-axis
intrinsic viscosity (in ) was obtained. Using this value in
double logarithmic plot of the intrinsic viscosity ([in ], dl/g)
w ) of chitosan at
and weight average molecular weight (M
w chitosan was determined.
25 C [23], M
2.8. Estimation of protein
Chitosan (50 mg) was incubated with urea solution (1 ml,
5 M) for 30 min at 95 C with periodic vortexing. To the
mixture cooled to room temperature water was added followed by centrifugation at 5000 rpm for 2 min. The supernatant (400 l) was diluted with equal volume of water.
The protein content of the solution was determined by the
Bradford method [24].

2.4. Isolation of chitosan

2.9. Co-infrared spectroscopy
121 C

Mycelia (biomass) were autoclaved at

for 15 min
after homogenizing in a blender with 1N NaOH (1:40, w/v).
The alkali insoluble mass was washed thoroughly with water
followed by ethanol and refluxed with 100 volumes of 2%
AcOH (v/v) for 24 h at 95 C. The slurry was centrifuged
at 12,000 rpm in a Sorvall RC 5B refrigerated centrifuge
for 45 min at 4 C. Chitosan was precipitated out from the
supernatant by adjusting the pH to 8.5 with 1N NaOH;
washed several times with chilled water and triturated with
acetone.

Approximately 23 mg of chitosan was mixed with

100 mg of potassium bromide. Forty milligrams of the mixture was used to prepare KBr pellet. The pellets were subjected to Co-IR in Shimadzu FTIR-8300 spectrophotometer.
2.10. Dynamic light scattering with chitosan
Chitosan (1 g) was dissolved in 2% AcOH (1 l) and filtered
through Millipore (0.22 m). Light scattering experiment

S. Chatterjee et al. / Process Biochemistry 40 (2005) 395400

397

was performed in Photal DLS-700 Otsuka electronics, Japan

using HeNe laser at 632.8 nm at 28 C and at an angle 90 .
2.11. X-ray diffraction
Powder X-ray diffraction patterns were obtained using
a Seifert C 3000 instrument with the following operating
conditions 40 kV and 30 mA with a Cu/Ni radiation at =
1.5406. The relative intensity was recorded in a scattering
range (2) of 10100 .
Ash, moisture contents and specific rotation were measured by the standard (AOAC) method [25].

3. Results and discussion

Growth of M. rouxii under submerged fermentation condition in three different media is presented in the Fig. 1.
Sucrose concentration of MSM was standardized and maximum growth was obtained at 4%, further increase in sucrose
concentration did not improve the growth (data not shown).
Eighteen percent increase in biomass of M. rouxii was obtained when YPG was used as culture media compared to
MSM and PDB. However the time required to attain this was
almost double than that required with MSM. Alkali insoluble mass (AIM) represents around 39% of the total biomass
and this was not influenced by the composition of the growth

Fig. 1. Growth of M. rouxii in three different media under submerged

condition. () YPG; () MSM; () PDB.

media of the fungus. However, production of chitosan has

been found to be influenced by composition of the growth
medium, as the highest amount was obtained with MSM.
Chitosan obtained from 100 gm of dry biomass was varied
from 6.0 to 7.7% depending upon the growth media. These
values are higher than those reported by Tan et al. [26].
Co FT IR spectra of chitosan prepared using different
culture media along with commercial chitosan (Sigma) are
shown in Fig. 2. All chitosans showed bands at 2900 cm1

Fig. 2. Co FT-IR spectra of different fungal chitosans along with chitosan from Sigma. (I) Chitosan procured from Sigma; (II) chitosan obtained from
YPG; (III) chitosan from MSM; (IV) chitosan from PDB.

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Table 1
Physico-chemical properties of chitosan isolated from different media
Origin of
preparation

Degree of
deacetylation (%)

Weight average
molecular weight
w ) (Da) 104
(M

Average molecular
diameter (nm)

Ash (%)

Protein
(%)

Moisture
(%)

Specific rotation
[]25 ( )

MSM
PDB
YPG
Crustaceana

87.2
89.8
82.8
89.7

2.48
4.58
5.59
100.0

0.5
2.1
3.8
1448.6

0.83
0.89
0.91
0.60

0.1
0.1
0.2
0.05

4.82
4.90
5.01
5.12

19
19
21
21

Prepared from lobster shell.

and 3000 cm1 (NH bond stretching) at 1650 cm1 (C=O

bond stretching) and 1557 cm1 (NH vibrational mode). It
appears from these spectra that the degree of deacetylation
of all the polymers are very close to one another. This
was confirmed by UV first derivative spectra. The degree
of deacetylation of chitosan from PDB (89.8%) (Table 1)
was similar to that produced from MSM (87.2%), whereas
chitosan from YPG (82.2%) was found to be slightly lower.
Other parameters such as ash, protein contents and specific
rotation, which reflect the quality of chitosan, were found
to be almost same (Table 1). Ash and moisture content of
the chitosan was less than that reported by McGarhen et al.
[14]. Dynamic light scattering (Figs. 35) shows the molecular distribution pattern of chitosan isolated from M. rouxii
grown in three different media. It was observed that chitosan
isolated from MSM is less polydispersed, polydispersity index being 2.225 101 . This indicates that this polymer is
of high quality, whereas those isolated from PDB and YPG
were more polydispersed, polydispersity index obtained
4.363 101 and 4.977 101 , respectively. It is assumed
that such difference in polydispersity may arise from differences in chitosanase activity of the fungus, which awaits
further study. The above findings were also confirmed from
molecular size distribution results. It was found that the
average molecular size distribution in chitosan from MSM

was 0.5 nm, which is smallest with respect to those obtained

from other two media PDB and YPG, being 2.1 and 3.8 nm
respectively. Variation of molecular size of chitosan obtained from M. rouxii grown in three media gave an insight
w ), which was in good
to an average molecular weight (M
agreement with the experimental results obtained. From the
above it may be concluded that the higher the molecular
weight, the higher will be the molecular size distribution.

Fig. 4. Dynamic light scattering pattern of fungal chitosan from PDB.

Fig. 3. Dynamic light scattering pattern of fungal chitosan from MSM.

Fig. 5. Dynamic light scattering pattern of fungal chitosan from YPG.

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399

Fig. 6. X-ray powder diffraction pattern of different fungal chitosan. (a) Chitosan from MSM; (b) chitosan from PDB; (c) chitosan from YPG.

X-ray diffraction is commonly used to determine the polymorphic forms of a compound having different crystalline
structures for which distinct powered X-ray diffraction patterns are obtained. These patterns are indicative of different
spacing of the crystal planes, which provide strong evidence
for polymorphic differences. In addition, it provides accurate
measurements of crystallinic contents, which greatly affects
physical and biological properties of the polymer. Fig. 6
shows the powder diffraction pattern of M. rouxii grown in
three media viz., MSM, PDB and YPG. Strong Bragg refrac-

tions were observed at an angle 31.8 2 (d = 2.81 ), 32.7

2 (d = 2.73 ) and 33.95 2 (d = 2.64 ), the first one
showing strongest intensity being chitosan from MSM. The
chitosan for fungus grown in PDB shows strongest Bragg at
19.85 2 (d = 4.46 ) which for the fungus grown in YPG,
the corresponding one is at 19.61 2 (d = 4.52 ). From
the above results it can be concluded that chitosan from
MSM is more crystalline than those from YPG and PDB.
From the present study it may be concluded that MSM
is the best as well as the cheap medium for the production

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S. Chatterjee et al. / Process Biochemistry 40 (2005) 395400

of chitosan from M. rouxii as the cost of ingredients for

the preparation of one thousand litre of each medium was
calculated and MSM was found to be the cheapest among
the other two. Moreover, the yield and quality of chitosan
have been found to be better than the other two media.
Acknowledgements
Authors acknowledge Department of Biotechnology, New
Delhi for their financial support and wish to thank Dr. Subhasis Banerjee for various help in the present investigation.

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