Process Biochemistry 40 (2005) 395400

Chitosan from Mucor rouxii: production andphysico-chemical characterization

S. Chatterjee, M. Adhya, A.K. Guha, B.P. ChatterjeeDepartment 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 alternativesource of chitosan is the cell wall of fungi. Fungal culture media and fermentation condition can be manipulated to provide chitosan ofmore consistent physico-chemical properties compared to that derived chemically from chitin. Chitosan has been isolated from Mucor rouxiicultured 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 and0.56 g/l for YPG respectively. Their physico-chemical properties such as ash, moisture, protein contents and specific rotation do not show muchdifference. However, variation has been observed in their polydispersed nature and crystallinity. Chitosan from MSM was less polydispersedand 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 de-rived by deacetylation of naturally occurring biopolymerchitin, which is present in the exoskeleton of crustacea suchas crab, shrimp, lobster, crawfish and insects, and is consid-ered to be the second most abundant polysaccharide in theworld after cellulose. Chitosan can also be found in the cellwall of certain groups of fungi, particularly zygomycetes. Itis a straight chain natural hydrophilic polysaccharide havinga three dimensional -helical configuration stabilized byintramolecular hydrogen bonding [1]. Chitosan being poly-cationic, nontoxic, biodegradable as well as antimicrobialfinds numerous applications especially in the agriculture,food and pharmaceutical industries, such as food preser-vation [27], fruit juice clarification [8] water purificationparticularly for removal of heavy metal ions [911]; sorp-tion for dyes and flocculating agent. Chitosan can alsobe 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).

properties [12], wound healing accelerator [13], and alsoin cosmetics industries. However, the efficiency of chitosandepends upon the molecular size [14], degree of deacetyla-tion, crystallinity, solubility and its derivatized products.

Shell waste from shrimp, crab and lobster processing in-dustries is the traditional source of chitin. However, commer-cial production of chitosan by deacetylation of crustaceanchitin with strong alkali appears to have limited potentialfor industrial acceptance because of seasonal and limitedsupply, difficulties in processing particularly with the largeamount of waste of concentrated alkaline solution causingenvironmental pollution and inconsistent physico-chemicalproperties. However, uniform deacetylation is a prerequisiteto specific industrial applications. With advances in fer-mentation technology chitosan preparation from fungal cellwalls becomes an alternative route for the production of thispolymer in an ecofriendly pathway [1517]. Adequate quan-tities of chitosan was found to be present in the hyphae ascell wall component in the dimorphic fungus Mucor rouxiias reported by White et al. [18] and Arcidiacono and Kaplan[19].

The present communication describes the production andphysico-chemical properties of chitosan obtained by fermen-tation of M. rouxii in three different media.

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

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

2. Materials and methods

2.1. Materials

M. rouxii (MTCC 386) used in this study was obtainedfrom Institute of Microbial Technology, Chandigarh, Indiaand maintained on potato dextrose agar slants. Chemicalsand biochemicals were purchased from E. Merck and mo-lasses was procured from local market.

2.2. Fermentation medium

The following three fermentation media were used tostudy the growth and production of chitosan from M. rouxii.

2.2.1. Molasses salt medium (MSM)This medium contains 0.2% NaNO3, 0.1% K2HPO4,

0.001% FeSO4, 0.001% MgSO4, 0.2% yeast extract andmolasses as carbon source. Molasses was added to the me-dia 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 eachmedium was added to a 250 ml Erlenmeyer flask and steril-ized by autoclaving at 121 C for 15 min.

2.3. Preparation of inoculum and fermentation

Inocula were prepared by growing the organism in potatodextrose agar (PDA) plates at 30 C for 3 days. Flasks con-taining the media were inoculated with one 5 mm diame-ter mycelium covered agar disk [20] containing 6.4 106spores/disk was used as inoculum and incubated at 30 Cunder submerged condition (120 rpm) for different periodsof time. At the end of the desired incubation period myceliawere harvested by filtration, dried by lyophilization andweighed. Mycelia and culture filtrates were stored at20 Cuntil use.

2.4. Isolation of chitosan

Mycelia (biomass) were autoclaved at 121 C for 15 minafter homogenizing in a blender with 1N NaOH (1:40, w/v).The alkali insoluble mass was washed thoroughly with waterfollowed by ethanol and refluxed with 100 volumes of 2%AcOH (v/v) for 24 h at 95 C. The slurry was centrifugedat 12,000 rpm in a Sorvall RC 5B refrigerated centrifugefor 45 min at 4 C. Chitosan was precipitated out from thesupernatant by adjusting the pH to 8.5 with 1N NaOH;washed several times with chilled water and triturated withacetone.

2.5. Estimation of sucrose

Sucrose was estimated by a phenol sulphuric acid method[21].

2.6. Determination of degree of deacetylation

Degree of deacetylation of chitosan was measured byfirst derivative UV spectroscopic method [22] using Shi-madzu [option program/interface OPI-4] UV-Vis recordingspectrophotometer UV 240 graphtcord [scan speed-fast, slitwidth 2 nm, scanning range 190240 nm].

2.7. Determination of weight average molecular weight

Chitosan solutions of varying concentrations ranging from0.0625 to 1.00% were prepared with 2% AcOH. The vis-cosity of the solutions was measured at a particular shearrate 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), andrelative = viscosity of solution

viscosity of solvent

of solutions were plotted against different concentrations ofchitosan solution. By extrapolating of the curve to Y-axisintrinsic viscosity (in) was obtained. Using this value indouble logarithmic plot of the intrinsic viscosity ([in], dl/g)and weight average molecular weight (Mw) of chitosan at25 C [23], Mw chitosan was determined.

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 themixture cooled to room temperature water was added fol-lowed by centrifugation at 5000 rpm for 2 min. The super-natant (400l) was diluted with equal volume of water.The protein content of the solution was determined by theBradford method [24].

2.9. Co-infrared spectroscopy

Approximately 23 mg of chitosan was mixed with100 mg of potassium bromide. Forty milligrams of the mix-ture was used to prepare KBr pellet. The pellets were sub-jected 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 filteredthrough Millipore (0.22m). Light scattering experiment

S. Chatterjee et al. / Process Biochemistry 40 (2005) 395400 397

was performed in Photal DLS-700 Otsuka electronics, Japanusing 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 usinga Seifert C 3000 instrument with the following operatingconditions40 kV and 30 mA with a Cu/Ni radiation at =1.5406. The relative intensity was recorded in a scatteringrange (2) of 10100.

Ash, moisture contents and specific rotation were mea-sured by the standard (AOAC) method [25].

3. Results and discussion

Growth of M. rouxii under submerged fermentation con-dition in three different media is presented in the Fig. 1.Sucrose concentration of MSM was standardized and maxi-mum growth was obtained at 4%, further increase in sucroseconcentration did not improve the growth (data not shown).Eighteen percent increase in biomass of M. rouxii was ob-tained when YPG was used as culture media compared toMSM and PDB. However the time required to attain this wasalmost double than that required with MSM. Alkali insolu-ble mass (AIM) represents around 39% of the total biomassand this was not influenced by the composition of the growth

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

Fig. 1. Growth of M. rouxii in three different media under submergedcondition. () YPG; () MSM; () PDB.

media of the fungus. However, production of chitosan hasbeen found to be influenced by composition of the growthmedium, as the highest amount was obtained with MSM.Chitosan obtained from 100 gm of dry biomass was variedfrom 6.0 to 7.7% depending upon the growth media. Thesevalues are higher than those reported by Tan et al. [26].

Co FT IR spectra of chitosan prepared using differentculture media along with commercial chitosan (Sigma) areshown in Fig. 2. All chitosans showed bands at 2900 cm1

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

Origin ofpreparation

Degree ofdeacetylation (%)

Weight averagemolecular weight(Mw) (Da) 104

Average moleculardiameter (nm)

Ash (%) Protein(%)

Moisture(%)

Specific rotation[]25 ()

MSM 87.2 2.48 0.5 0.83 0.1 4.82 19PDB 89.8 4.58 2.1 0.89 0.1 4.90 19YPG 82.8 5.59 3.8 0.91 0.2 5.01 21Crustaceana 89.7 100.0 1448.6 0.60 0.05 5.12 21

a Prepared from lobster shell.

and 3000 cm1 (NH bond stretching) at 1650 cm1 (C=Obond stretching) and 1557 cm1 (NH vibrational mode). Itappears from these spectra that the degree of deacetylationof all the polymers are very close to one another. Thiswas confirmed by UV first derivative spectra. The degreeof deacetylation of chitosan from PDB (89.8%) (Table 1)was similar to that produced from MSM (87.2%), whereaschitosan from YPG (82.2%) was found to be slightly lower.Other parameters such as ash, protein contents and specificrotation, which reflect the quality of chitosan, were foundto be almost same (Table 1). Ash and moisture content ofthe chitosan was less than that reported by McGarhen et al.[14]. Dynamic light scattering (Figs. 35) shows the molec-ular distribution pattern of chitosan isolated from M. rouxiigrown in three different media. It was observed that chitosanisolated from MSM is less polydispersed, polydispersity in-dex being 2.225 101. This indicates that this polymer isof high quality, whereas those isolated from PDB and YPGwere more polydispersed, polydispersity index obtained4.363 101 and 4.977 101, respectively. It is assumedthat such difference in polydispersity may arise from dif-ferences in chitosanase activity of the fungus, which awaitsfurther study. The above findings were also confirmed frommolecular size distribution results. It was found that theaverage molecular size distribution in chitosan from MSM

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

was 0.5 nm, which is smallest with respect to those obtainedfrom other two media PDB and YPG, being 2.1 and 3.8 nmrespectively. Variation of molecular size of chitosan ob-tained from M. rouxii grown in three media gave an insightto an average molecular weight (Mw), which was in goodagreement with the experimental results obtained. From theabove it may be concluded that the higher the molecularweight, the higher will be the molecular size distribution.

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

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

S. Chatterjee et al. / Process Biochemistry 40 (2005) 395400 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 poly-morphic forms of a compound having different crystallinestructures for which distinct powered X-ray diffraction pat-terns are obtained. These patterns are indicative of differentspacing of the crystal planes, which provide strong evidencefor polymorphic differences. In addition, it provides accuratemeasurements of crystallinic contents, which greatly affectsphysical and biological properties of the polymer. Fig. 6shows the powder diffraction pattern of M. rouxii grown inthree media viz., MSM, PDB and YPG. Strong Bragg refrac-

tions were observed at an angle 31.8 2 (d = 2.81 ), 32.72 (d = 2.73 ) and 33.95 2 (d = 2.64 ), the first oneshowing strongest intensity being chitosan from MSM. Thechitosan for fungus grown in PDB shows strongest Bragg at19.85 2 (d = 4.46 ) which for the fungus grown in YPG,the corresponding one is at 19.61 2 (d = 4.52 ). Fromthe above results it can be concluded that chitosan fromMSM is more crystalline than those from YPG and PDB.

From the present study it may be concluded that MSMis the best as well as the cheap medium for the production

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

of chitosan from M. rouxii as the cost of ingredients forthe preparation of one thousand litre of each medium wascalculated and MSM was found to be the cheapest amongthe other two. Moreover, the yield and quality of chitosanhave been found to be better than the other two media.

Acknowledgements

Authors acknowledge Department of Biotechnology, NewDelhi for their financial support and wish to thank Dr. Sub-hasis Banerjee for various help in the present investigation.

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Chitosan from Mucor rouxii: production and physico-chemical characterizationIntroductionMaterials and methodsMaterialsFermentation mediumMolasses salt medium (MSM)Yeast peptone glucose medium (YPG)Potato dextrose broth (PDB)

Preparation of inoculum and fermentationIsolation of chitosanEstimation of sucroseDetermination of degree of deacetylationDetermination of weight average molecular weightEstimation of proteinCo-infrared spectroscopyDynamic light scattering with chitosanX-ray diffraction

Results and discussionAcknowledgementsReferences