Fungal chitosan production and its characterization

Letters in Applied MicrobiologyVolume 35, Issue 1 p. 17-21 Free Access Fungal chitosan production and its characterization P. Pochanavanich, P. Pochanavanich Department of Microbiology, King Mongkut's University of Technology Thonburi, Bangkok, ThailandSearch for more papers by this authorW. Suntornsuk, W. Suntornsuk Department of Microbiology, King Mongkut's University of Technology Thonburi, Bangkok, ThailandSearch for more papers by this author P. Pochanavanich, P. Pochanavanich Department of Microbiology, King Mongkut's University of Technology Thonburi, Bangkok, ThailandSearch for more papers by this authorW. Suntornsuk, W. Suntornsuk Department of Microbiology, King Mongkut's University of Technology Thonburi, Bangkok, ThailandSearch for more papers by this author First published: 25 June 2002 https://doi.org/10.1046/j.1472-765X.2002.01118.xCitations: 152 Dr W. Suntornsuk, Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Tungkru, Bangkok 10140, Thailand (e-mail: worapot.sun@kmutt.ac.th). AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Aims: The objective of this investigation was to evaluate the chitosans produced by several species of fungi. Methods and Results: Representatives of four species of filamentous fungi, Aspergillus niger , Rhizopus oryzae , Lentinus edodes and Pleurotus sajo-caju , and two yeast strains, Zygosaccharomyces rouxii TISTR5058 and Candida albicans TISTR5239, were investigated for their ability to produce chitosan in complex media. Fungal chitosan was produced at 10–140 mg g –1 cell dry weight, had a degree of deacetylation of 84–90% and a molecular weight of 2·7 × 10 4 –1·9 × 10 5 Da with a viscosity of 3·1–6·2 centipoises (cP). Conclusions: Rhizopus oryzae TISTR3189 was found to be the producer of the highest amounts of chitosan. Significance and Impact of the Study: Commercial chitosan could be obtained from Rhizopus mycelia and would have potential applications for medical and agricultural uses. INTRODUCTION Chitosan (poly-N-acetylglucosamine) is a natural and biodegradable biopolymer. Chitosan and its derivatives can be variously used as a permeability control agent, an adhesive, a paper-sizing agent, a fining agent, flocculating and chelating agents, an antimicrobial compound and a chromatographic support (8; 2; 15). It is also used to immobilize enzymes or to deliver drugs to their target (4; 10). Chitosan is commercially produced from shrimp and crabshell chitin deacetylated by strong alkalis at high temperatures for long periods of time (7). However, supplies of raw materials are variable and seasonal and the process is laborious and costly (2). Furthermore, the chitosan derived from such a process is heterogeneous with respect to their physiochemical properties (2). Recent advances in fermentation technology suggest that the cultivation of selected fungi can provide an alternative source of chitosan. Fungal cell walls and septa of Ascomycetes, Zygomycetes, Basidiomycetes and Deuteromycetes contain mainly chitin, which is responsible for maintaining their shape, strength and integrity of cell structure (14; 6). These micro-organisms can be readily cultured in simple nutrients and cell wall chitosan easily recovered (19; 8; 16; 18; 20). The production and characterization of chitosan have been mostly studied in fungal species of Absidia and Mucor (19; 8; 9; 11). In this study, the production and properties of chitosan from representatives of four different filamentous fungi, including two yeasts, were investigated. MATERIALS AND METHODS Cultures The micro-organisms used in this study were Aspergillus niger TISTR3245, Rhizopus oryzae TISTR3189, Zygosaccharomyces rouxii TISTR5058 and Candida albicans TISTR5239 (Center of Culture Collection, Thailand Institute of Scientific and Technological Research, Bangkok, Thailand). Two strains of mushroom, Lentinus edodes no. 1 and Pleurotus sajo-caju no. 2, were also tested (Center of Mushroom Collection, Department of Agriculture, Ministry of Agriculture and Cooperation, Bangkok, Thailand). Cultivation media Potato Dextrose Broth medium (PDB; Merck, Darmstadt, Germany) was used for fungal and mushroom cultivation. Yeast Malt Extract Broth medium (YMB; Merck) was used for yeast cultivation. Cultivation conditions For fungal cultivation, 1 ml spore suspension (107 spores ml–1), prepared from a 7-d-old slant, was inoculated into 100 ml PDB in a 250-ml Erlenmeyer flask. The cultures were incubated at 30 °C, 180 rev min–1 for 15–21 d. For yeast cultivation, 1 ml yeast cell suspension (107 cells ml–1), prepared from a 7-d-old slant, was inoculated into 100 ml YMB in a 250-ml Erlenmeyer flask. The cultures were incubated at 30 °C, 180 rev min–1 for 6 d. Fermentation broth from each culture was sampled to monitor growth. Fungal dry weights were determined by filtration (GF/C glass microfibre; Whatman, Maidstone, UK), washing twice with distilled water and drying the biomass pellet at 85 °C to constant weight. All experiments were carried out in triplicate and the results are expressed as mean ± S.D. Chitosan isolation After cultivation, fungal mycelia were recovered by filtration (no. 1; Whatman), washed twice with distilled water until a clear filtrate was obtained and then dried at 65 °C to a constant weight. Yeast cells were harvested by centrifugation at 8000 g for 30 min, washed twice with distilled water and dried at 65 °C to a constant weight. Chitosan extraction was carried out by a modified method of 12) and 2). Dry fungal mycelia and dry yeast cells were finely ground, suspended with 1 mol l–1 NaOH solution (1 : 30 w/v) and autoclaved at 121 °C for 15 min. Alkali-insoluble fractions were collected after centrifugation at 12 000 g for 15 min, washed with distilled water and recentrifuged to a neutral pH (pH 7). The residues were further extracted using 2% acetic acid (1 : 40 w/v) at 95 °C for 8 h. The extracted slurry was centrifuged at 12 000 g for 15 min and the insoluble acid discarded. The pH of the supernatant fluids was adjusted to 10 with 2 mol l–1 NaOH, the solution centrifuged at 12 000 g for 15 min and the precipitated chitosan was washed with distilled water, 95% ethanol (1 : 20 w/v) and acetone (1 : 20 w/v), respectively and dried at 60 °C to a constant weight. Chitosan characterization Molecular weight. The molecular weights of fungal chitosan were determined by gel permeation chromatography (PL-GPC110) on an Ultralinear hydrogel column (103–2 × 107 Da) using pullulan as a standard. The calibration curve consisted of pullulan standards with molecular weights of 5·8 × 103–1·6 × 106 Da. Standards and samples (0·01 g suspended in 2·5 ml 0·5 mol l–1 acetic acid and 2·5 ml 0·5 mol l–1 sodium acetate) were introduced into the column at a flow rate of 0·6 ml min–1 using a mixture of 0·5 mol l–1 acetic acid and 0·5 mol l–1 sodium acetate (1 : 1 v/v) as eluent. The operation was carried out at 30 °C and the peaks monitored by a refractive index detector. Deacetylation. The extent of chitosan deacetylation was determined by titration with 0·01 mol l–1 NaOH (3). The method involved hydrolysing the acetyl groups in chitosan with a strong alkali and converting the salt to acetate, which was evaporated as an azeotrope with water and titrated. The acetyl percentage was determined from the equation: where V is the corrected volume of NaOH and w is the weight of the sample. The degree of deacetylation was calculated using the equation: Viscosity. The viscosity of 1% chitosan in 2% acetic acid solution was determined using an Ubbelohde viscometer (type/capillary no. 53233/111c) at 25 °C. RESULTS AND DISCUSSION The growth of four fungi, A. niger TISTR3245, R. oryzae TISTR3189, L. edodes no. 1 and P. sajo-caju no. 2, is shown in 1Fig. 1a–d. Aspergillus niger TISTR3245 had the highest growth rate with a maximal mycelial dry weight of 9 g l–1 after 6 d of cultivation, while L. edodes no. 1 grew very slowly with a maximal biomass of 1·4 g l–1 after 9 d of cultivation. The growth of the yeasts, Z. rouxii TISTR5058 and C. albicans TISTR5239, is also shown in 1Fig. 1e–f. The highest biomass of Z. rouxii TISTR5058 was 4·4 g l–1 after 3 d of cultivation while the biomass of C. albicans TISTR5239 reached only 1·8 g l–1 after 2 d of cultivation. Figure 1Open in figure viewerPowerPoint Mycelial growth (•) and chitosan production (▮) of (a) Aspergillus niger TISTR3245; (b) Rhizopus oryzae TISTR3189; (c) Lentinus edodes no. 1; (d) Pleurotus sajo-caju no. 2; (e) Zygosaccharomyces rouxii TISTR5058 and (f) Candida albicans TISTR5239. Each point is the mean of triplicate determinations and error bars represent ± S . D . The chitosan accumulation of each strain was found at a different stage of cell growth, as shown in 1Fig. 1a–f. However, it has been previously reported that the late exponential phase produced the most extractable chitosan (17). Chitosan yield and content are shown in Table 1. Rhizopus oryzae TISTR3245 was shown to give a maximal yield of chitosan at 138 mg g–1 dry weight (14% chitosan). Chitosan yields from this study were, however, lower than those of other fungal strains, e.g. Absidia spp., Actinomucor spp., Circinella spp., Mucor spp., Phycomycete, Rhizopus spp. and Zygorhynchus spp. (16). The chitosan yield of L. edodes was reported to be 20–50 mg g–1 dry weight (2), that of R. oryzae was 270–700 mg l–1 (5 mg l–1 (16). The chitin content of the mycelia of A. niger was reported to be 42% (7). The results confirmed that the chitosan content of fungi depends on fungal strains, mycelial age, cultivation medium and conditions. The chitosan content of fungal mycelia also depends on the chitosan extraction method (19). Table 1. Amount of chitosan produced by different fungi Both strains of mushroom were found to grow slowly and to produce little chitosan in submerged cultivation. However, 2) reported that L. edodes grew well (3 g l–1) and produced 121 mg l–1 chitosan after 12 d of submerged cultivation in a synthetic medium and the yield of chitosan from L. edodes grown on wheat straw (solid state cultivation) was over 50 times higher than the chitosan yield by submerged cultivation. Therefore, the cultivation method is also an important factor for fungal chitosan production. The properties of fungal chitosan compared with those of a commercial chitosan derived from crab shells are shown in Table 2. The degree of deacetylation of fungal chitosan was 84–90%, relatively lower than that of crab chitosan. The results were slightly different from the reported percentage degrees of deacetylation of chitosan from fungal mycelia of 65–95% (16; 1; 9). The degree of deacetylation is an important parameter affecting the physicochemical properties of chitosan. Chitosan with a high degree of deacetylation has high positive charges and is more suitable for food applications as a coagulating or chelating agent, a clarifying agent or an antimicrobial agent (2). Table 2. Properties of fungal chitosans The viscosity of fungal chitosan was 3·1–6·2, centipoises (cP), considerably lower than the viscosity of crab chitosan (Table 2). The results were similar to those reported by 16). The molecular weight of fungal chitosan was 0·3–1·9 × 105 Da. It has previously been reported to be 1·0 × 105–1·4 × 106 Da (1; 9). The molecular weight was relatively lower than that of chitosan from crab shells. Thus, fungal chitosan could have potential medical and agricultural applications. In addition, chitosan with a low molecular weight was reported to reduce the tensile strength and elongation of the chitosan membrane but to increase its permeability (13). References 1 Arcidiacono, S. and Kaplan, D.L. (1992) Molecular weight distribution of chitosan isolated from Mucor rouxii under different culture and processing conditions. Biotechnology Bioengineering 39, 281– 286. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 2 Crestini, C., Kovac, B. and Giovannozzi-Sermanni, G. (1996) Production and isolation of chitosan by submerged and solid-state fermentation from Lentinus edodes. Biotechnology Bioengineering 50, 207– 210. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 3 Donald, H.D. and Hayes, E.R. (1988) Determination of degree of acetylation of chitin and chitosan. Methods in Enzymology 161, 442– 446. CrossrefWeb of Science®Google Scholar 4 Goosen, M.F.A. (1997) Applications of Chitin and Chitosan Lancaster: Technomic Publishing. Google Scholar 5 Hang, Y.D. (1990) Chitosan production from Rhizopus oryzae mycelia. Biotechnology Letters 12, 911– 912. CrossrefCASWeb of Science®Google Scholar 6 Hon, D.N.S. (1996) Chitin and chitosan: Medical applications. In Polysaccharides in Medicinal Application ed. Dumitri, S. pp. 631–649. New York: Marcel Dekker. Google Scholar 7 Knorr, D. (1991) Recovery and utilization of chitin and chitosan in food processing waste management. Food Technology 26, 114– 122. Google Scholar 8 McGahren, W.J., Perkinson, G.A., Growich, J.A., Leese, R.A. and Ellestad, G.A. (1984) Chitosan by fermentation. Process Biochemistry 19, 88– 90. CASWeb of Science®Google Scholar 9 Miyoshi, H., Shimura, K., Watanabe, K. and Kasuki, O. (1992) Characterization of some fungal chitosans. Bioscience Biotechnology and Biochemistry 56, 1901– 1905. CrossrefCASWeb of Science®Google Scholar 10 Muzzarelli, R.A.A. (1977) Chitin Oxford: Pergamon Press. Google Scholar 11 Rane, K.D. and Hoover, D.G. (1993a) Production of chitosan by fungi. Food Biotechnology 7, 11– 33. CrossrefCASWeb of Science®Google Scholar 12 Rane, K.D. and Hoover, D.G. (1993b) An evaluation of alkali and acid treatments for chitosan extraction from fungi. Process Biochemistry 28, 115– 118. CrossrefCASWeb of Science®Google Scholar 13 Rong, H.C. and Horng, D.H. (1996) Effect of molecular weight of chitosan with the same degree of deacetylation on the thermal, mechanical, and permeability properties of the prepared membrane. Carbohydrate Polymers 29, 353– 358. CrossrefGoogle Scholar 14 Ruiz-Herrera, J., Sentandreu, R.R. and Martinez, J.P. (1992) Chitin biosynthesis in fungi. In Handbook of Applied Mycology, Vol. 4. Fungal Biotechnology ed. Dilip, K.A., Richard, P.E. and Mukerji, K.G. pp. 281–312. New York: Marcel Dekker. Google Scholar 15 Shahidi, F., Arachchi, J.K.V. and You, J.J. (1999) Food applications of chitin and chitosan. Trends in Food Science and Technology 10, 37– 51. CrossrefCASWeb of Science®Google Scholar 16 Shimahara, K., Takiguchi, Y., Kobayashi, T., Uda, K. and Sannan, T. (1989) Screening of mucoraceae strains suitable for chitosan production. In Chitin and Chitosan ed. Skjak-Braek, G., Anthonsen, T. and Sanford, P. pp. 171–178. London: Elsevier Applied Science. Google Scholar 17 Su, C.T., Teck, K.T., Sek, M.W. and Khor, E. (1996) The chitosan yield of zygomycetes at their optimum harvesting time. Carbohydrate Polymer 30, 239– 242. CrossrefWeb of Science®Google Scholar 18 Synowiecki, J. and Al-Khateeb, N.A. (1997) Mycelia of Mucor rouxii as a source of chitin and chitosan. Food Chemistry 60, 605– 610. CrossrefCASWeb of Science®Google Scholar 19 White, S.A., Farina, P.R. and Fultong, L. (1979) Production and isolation of chitosan from Mucor rouxii. Applied and Environmental Microbiology 38, 323– 328.CASPubMedWeb of Science®Google Scholar 20 Yokoi, H., Aratake, T., Nishio, S., Hirose, J., Hayashi, S. and Takasaki, Y. (1998) Chitosan production from Shochu distillery wastewater by funguses. Journal of Fermentation and Bioengineering 85, 246– 249. CrossrefCASWeb of Science®Google Scholar Citing Literature Volume35, Issue1July 2002Pages 17-21 FiguresReferencesRelatedInformation