Indian Journal of Biotechnology
Vol 12, July 2013, pp 429-431
Comparative studies for chitosan yield
and chelating ability of
Aspergillus niger and Rhizopus oryzae
S Bhuvaneshwari and V Sivasubramanian*
Department of Chemical Engineering,
National Institute of Technology, Calicut 673 601, India
Received 21 February 2012; revised 17 April 2012;
accepted 10 June 2012
Chitosan is a natural polysaccharide comprising copolymer of
glucosamine and N-acetyl glucosamine, and can be obtained by
the deacetylation of chitin. Due to its biodegradability,
biocompatibility and low toxicity, chitosan has received increased
attention as one of the promising renewable polymeric materials
for its various applications in the bioremediation, pharmaceutical,
chemical and biomedical industries. In the present study, chitosan
was extracted from cell walls of Aspergillus niger and Rhizopus
oryzae, and characterized for mol wt, viscosity and degree of
deacetylation. Further, effect of metal concentration, pH and
contact time on the adsorption of heavy metals from aqueous
solutions by extracted-chitosan was studied. The sorption
properties were also compared using live biomass and the
chitosan from A. niger and R. oryzae. It has been found that
A. niger gave good yield and the extracted chitosan also had high
sorption capacity compared to the chitosan derived from
R. oryzae. The maximum adsorption capacity for copper (79.65%)
and chromium (87.61%) was obtained using A. niger at optimized
conditions. Fourier transform infrared (FT-IR) analysis indicated
that hydroxyl and amine groups of chitosan structure were
involved in the mechanism of copper and chromium adsorption.
Keywords: Aspergillus niger, chitosan, chromium, copper,
Rhizopus oryzae
It has been well known that many biological materials
accumulate metals. There is an increasing interest in
investigating such biological adsorbents for a variety
of applications as the conventional methods have
disadvantages like incomplete removal, high energy
and reagent requirements1. Chitosan is a natural
polysaccharide comprising copolymer of glucosamine
and N-acetyl glucosamine. It is commercially
produced from shrimp and crab shell chitin via its
deacetylation by strong alkalis at high temperature for
long periods of time2. When chitosan is dissolved in
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*Author for correspondence:
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Mobile: +91-9446547421
E-mail: [email protected]
an acid solution, it becomes a cationic polymer due to
the protonation of free amino groups on the C-2
position of pyranose ring. The cationic properties in
acidic solution give it the ability to interact readily
with negatively charged molecules, such as, fats,
cholesterols, metal ions and proteins3. The chitosan
derived from different sources are heterogeneous with
respect to physiochemical properties4. Chitin
containing microbial biomass and in particular fungal
biomass is well suited for chitosan synthesis. Such
biomass usually has 10 to 20% chitin based on dry wt
of the biomass. In order to prepare the high quality
chitosan, it is desirable that the microbial biomass be
produced in a substantially controlled manner, having
relatively uniform temperature and nutrient levels
during the growth of the biomass. Fungi, in addition,
can be grown easily on any simple medium or
industrial byproducts, and this will also free the
environment from pollution concerns5. Moreover, in
order to sustain the marine biodiversity, it is also
advisable to use microorganism for the chitosan
production. In the present investigation, efforts were
made to extract chitosan from the Aspergillus niger
and Rhizopus oryzae for the adsorption of heavy
metals.
All the chemicals used were of analytical grade and
samples were prepared using double distilled water.
The freeze dried cultures of A. niger (strain no. 281)
and R. oryzae (strain no. 553) were collected from
Microbial Type Culture Collection (MTCC), Institute
of Microbial Technology, Chandigarh, India.
Subsequently, A. niger and R. oryzae were
sub-cultured in PDB (potato dextrose broth) and
Czapek medium for 3 and 4 d, respectively, and then
both were transferred on PDA (potato dextrose agar)
plates at room temperature and pH 7. PDA plates
were then kept under same conditions for about 4 d or
until the plates were fully grown. The grown fungus
(microbial biomass) was mass cultivated6. About
250 mL of sodium hydroxide solution (4% w/v) was
mixed with 250 g of microbial biomass of A. niger
and R. oryzae. A strong exothermic reaction occurred
when fungal biomass was dissolved into sodium
hydroxide solution. The flask containing the biomass
in the sodium hydroxide solution was placed in a
pre-heated oven at about 120°C for 30 min. After
INDIAN J BIOTECHNOL, JULY 2013
430
heating, the warm solution was filtered. The residuals
were completely washed with deionized water. This pretreated biomass containing chitosan-glucan complex was
rinsed with water many times until the pH of filtered
solution reached to less than pH 9. After rinsing, the
solids were transferred to a beaker and glacial acetic acid
was added till pH 3.5 was obtained. The mixture was
stirred for about 10 min to extract chitosan from the
glucan and then the mixture was centrifuged to6,7. The
crude chitosan was washed several times with distilled
water and air dried at 20°C to a constant wt.
Biosorption studies for the recovery of copper and
chromium was carried out using potassium chromate
and copper sulphate solutions in distilled water.
Known amount of chitosan was introduced to metal
solution at different metal concentration and pH.
Intimate contact for biomass and metal solution was
provided by agitating it at low speed of 150 rpm in a
rotary shaker. At different contact time, the biomass
was separated by filtering and the filtrate was
analyzed using Chemito Atomic absorption
spectrophotometer (AA 203).
The chitosan content of fungi depends on fungal
strains, mycelium age, cultivation medium and
cultural conditions8. The extracted chitosan from
fungal cell walls of A. niger and R. oryzae was
analysed by FT-IR spectrophotometer (Thermo
Nicolet Model-Avater 320 model) and compared with
the spectrum of standard chitosan derived from crab
shell5,9. The peaks obtained from the two samples
were found similar to the peaks of standard chitosan
(Figs 1a-c). This clearly reveals that the extracted
fraction from the cell walls of A. niger and R. oryzae
was chitosan. The yield of fungal biomass and
chitosan as well as degree of deacetylation (DD),
viscosity and mol wt of chitosan from the both fungi
were determined10 and results are presented in Table 1.
The results clearly show that the amount of biomass
produced and the yield of extracted chitosan was
higher in case of A. niger in comparison to
R. oryzae. Moreover, the chitosan extracted from
A. niger had higher DD, viscosity and mol wt as
compared to chitosan from R. oryzae.
Biosorption of Cu and Cr ions by chitsan from
A. niger and R. oryzae showed only slight increase
with the increase in initial metal concentration from
Table 1—Physio-chemical characteristics of fungal chitosan
Chitosan
source
Biomass Chitosan Mol wt Degree of Viscosity
obtained produced (kDa) deacetylation
(cp)
(g/L)
(mg/g)
(%)
A. niger
266.7
724.9
331.436
85.90
5.49
R. oryzae
98.3
121.3
85.142
82.12
2.98
Fig. 1 (a-c) FTIR spectrum: a. Standard chitosan derived from
crab shell chitin; b. Chitosan derived from R. oryzae; & c.
Chitosan derived from A. niger
SHORT COMMUNICATIONS
Table 2—Effect of contact time on biosorption of metals by
fungal chitosan
Biosorption
(%)
A. niger
Forms
Chitosan
Live
biomass
rd
th
Metals 3 d 5 d
Cu 73.87 79.65
Cr 76.48 87.61
Cu
Cr
55.1 68.22
67.76 70.87
R. oryzae
th
rd
7 d
80.11
88.02
3 d 5th d 7th d
77.88 77.95 78.16
67.64 74.76 74.27
68.26
71.09
61.19 66.09 66.18
63.66 66.09 66.10
431
5th d for both chitosan and fungal biomass (Table 2).
However, no substantial increase in metal sorption
was observed beyond 5th d. Therefore, 5 d contact
time for biosorption was optimum for recovery of
Cr and Cu for both chitosan and fungal biomass.
Moreover, in case of both chitosan and live fungal
biomass, pH 5 was observed optimal for biosorption
of both the metal ions (Fig. 2). Overall, copper and
chromium recovery was recorded higher in A. niger
compared to R. oryzae. Thus, biosorption using
chitosan is a promising technology for metal sorption
because of its cost effectiveness and high efficiency.
Acknowledgement
The study was financially (Project No.:
SR/FTP/CS-68/2007) supported by the Department
of Science and Technology, Government of India,
New Delhi.
References
Fig. 2Effect of pH on % metal recovery by chitosan derived
from A. niger and R. orzyae, and their live biomass.
2 to 6 mM, whereas no increase in biosorption was
observed beyond 6 mM initial concentration
(original data not shown). Therefore, 6 mM was
considered as an optimal metal concentration for
further studies. Moreover, biosorption of Cu and Cr
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concentrations (2 to 10 mM) used. Further studies
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A. niger and R. oryzae, increase in contact time
increased the biosorption of Cu and Cr ions only up to
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