ARTICLES Determination and Kinetics of Producing Glucosamine Using Fungi Jui-Wei Hsieh, † Ho-Shing Wu,* ,†,‡ Yu-Hong Wei, ‡ and Shaw S. Wang †,‡,§ Department of Chemical Engineering and Materials Science and the Graduate School of Biotechnology and Bioinformatics, Yuan Ze University, Taoyuan, 32003, Taiwan, and Department of Chemical and Biochemical Engineering, Rutgers University, New Jersey This work used three fungi, Rhizopus oligosorus BCRC 31996, Monascus pilosus BCRC31527, and Aspergillus sp. BCRC31742, to produce glucosamine by using submerged fermentation and flask cultures. The reaction of glucosamine with 1-naphthyl isothiocyanate as derivatizing agent was carried out in pyridine at 50 °C for 1 h. The derivative was accurately analyzed and quantified by using high performance liquid chromatography. The relative standard deviation of glucosamine determined between experimental and real values were less than 2%. The kinetic and strategy of producing glucosamine in a flask culture was investigated to achieve an optimum yield of glucosamine under different conditions including three kinds of fungi, medium, and pH values. The descending ability of producing glucosamine for the three fungi was Aspergillus sp. BCRC31742 > Monascus pilosus BCRC31527 > Rhizopus oligosorus BCRC 31996 under the conditions studied. The experimental result shows that the glucosamine concentration had an optimum value and was 3430 mg/L by using Aspergillus sp. BCRC31742 culture in glucose and peptone (GP) medium, the yield of which was the best amount using wild-type microorganisms in the past. The generation culture of fungi and the pH control played important roles in enhancing the yield of glucosamine. The specific growth rate of the microorganism and the biomass, content, yield, and productivity of glucosamine were calculated as well. 1. Introduction Glucosamine is an amino-monosaccharide, which is a com- ponent of chitin and chitosan, whose chemical constitution is 2-amino-2-deoxy-D-glucose. The glucosamine provides a good regeneration for human joints cartilage. Glucosamine is readily soluble in water and hydrophilic solvents like methyl alcohol. In industry, the process for glucosamine production is from chitin hydrolysis using acid or enzymes by a chemical method in which the raw material is shrimp and crab shell powder. However, whether the raw material from the shrimp and crab shell powder is polluted or not must be taken into consideration, since the pretreatment is detailed and complicated in the production of glucosamine. In view of the literature, the yield from BTR company (American) patent (1) by using recombinant Escherichia coli can reach 17 g/L and hope to advance to 50 g/L. The yield by using wild-type bacteria to produce glu- cosamine only can reach 264 mg/L using Monascus pilosus culture in Rice bran solution (2). The research study of producing glucosamine using wild-type bacteria received little attention; hence, this work aims to study the mass production and the kinetics of submerged fermentation using wild-type bacteria to enhance the production of glucosamine. Three kinds of wild-type bacteria taken from literature are Rhizopus oli- gosorus (3), Monascus pilosus (2), and Aspergillus sp. (4). They all can produce glucosamine but the yield is low. In general, the quantitative methods of glucosamine that are commonly used are colorimetric assay (5), radioactivity (6), gas chromatography (7), capillary electrophoresis (8), and high performance liquid chromatograph (HPLC). Direct analysis does not work well as the glucosamine structure does not have a strong chromophore. To enhance glucosamine sensitivity and reduce noise, the glucosamine is derivatized and the derivatizing agents popularly used are phenyl isothiocyanate (9), 1-phenyl- 3-methyl-5-pyrazolone (10), anthranilic acid (11), aminopyrazine (12), p-aminobenzoic ethyl ether (13), 9-fluorenylmethoxycar- bonyl hydrazide (14), 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (15), 5-dimethylaminoaphthalene-1-sulfonyl chloride (16), and p-dimethylaminobenzaldehyde (17). Also, a new amino acid analysis system was developed that uses 1-naphthyl isothiocyanate (Edman’s reagent) and phenylisothiocyanate (PITC) to derivatize the amino acids in protein and peptide hydrolyzates prior to their separation by reverse phase liquid chromatography. The method is quantitative for primary and secondary amino acids, exhibits linear response over at least 2 orders of magnitude, and is extremely sensitive with detection limits being less than one picomole. Hence, this work analyzes glucosamine with 1-naphthyl isothiocyanate as a derivatizing agent (18) which is of high sensitivity. The derivatization time, concentration, and temperature of reacting glucosamine with * To whom all correspondence should be addressed. E-mail: cehswu@ saturn.yzu.edu.tw. Fax: (+886)-3-4559373. Tel: (+886)-3-4638800-2564. † Department of Chemical Engineering and Materials Science, Yuan Ze University. ‡ Graduate School of Biotechnology and Bioinformatics, Yuan Ze University. § Rutgers University. 1009 Biotechnol. Prog. 2007, 23, 1009-1016 10.1021/bp070037o CCC: $37.00 © 2007 American Chemical Society and American Institute of Chemical Engineers Published on Web 09/19/2007