Akram Zamani, Lars Edebo, Björn Sjöström, Mohammad Taherzadeh. Extraction and precipitation of chitosan from cell wall of zygomycetes fungi by dilute sulfuric acid. Biomacromolecules 2007; 8 (12): 3786-3790.

A new method was developed in this work for extraction of chitosan from the zygomycetes cell wall. It is
based on the temperature-dependent solubility of chitosan in dilute sulfuric acid. Chitin is soluble in neither
cold nor hot dilute sulfuric acid. Similarly chitosan is not soluble at room temperature but is dissolved in
1% H2SO4 at 121 °C within 20 min. The new method was developed to measure the chitosan content of the
biomass and cell wall. The procedures were investigated by measuring phosphate, protein, ash, glucuronic
acid, and degree of acetylation. The cell wall derivatives of fungus Rhizomucor pusillus were then examined
by this new method. The results indicated 8% of the biomass as chitosan. After treatment with NaOH, the
alkali-insoluble material (AIM) contained 45.3% chitosan. Treatment of AIM with acetic acid resulted in
16.5% acetic-acid-soluble material (AcSM) and 79.0% alkali- and acid-insoluble material (AAIM). AcSM
is usually cited as pure chitosan, but the new method shows major impurities by, for example, phosphate.
Furthermore, AAIM is usually considered to be the chitosan-free fraction, whereas the new method shows
more than 76% of the chitosan present in AIM is found in AAIM. It might indicate the inability of acetic
acid to separate chitosan from the cell wall.