ORIGINAL PAPER Enhanced pullulan production in a biofilm reactor by using response surface methodology Kuan-Chen Cheng • Ali Demirci • Jeffrey M. Catchmark Received: 13 January 2010 / Accepted: 22 February 2010 / Published online: 12 March 2010 Ó Society for Industrial Microbiology 2010 Abstract Pullulan is a linear homopolysaccharide that is composed of glucose units and often described as a-1, 6-linked maltotriose. In this study, response surface methodology using Box–Behnken design was employed to study the effects of sucrose and nitrogen concentrations on pullulan production. A total of 15 experimental runs were carried out in a plastic composite support biofilm reactor. Three-dimensional response surface was generated to evaluate the effects of the factors and to obtain the opti- mum condition of each factor for maximum pullulan pro- duction. After 7-day fermentation with optimum condition, the pullulan production reached 60.7 g/l, which was 1.8 times higher than the result from initial medium, and was the highest yield reported to date. The quality analysis demonstrated that the purity of produced pullulan was 95.2%, and its viscosity was 2.5 centipoise (cP), which is higher than the commercial pullulan and related to its molecular weight. Fourier transform infrared spectroscopy (FTIR) also suggested that the produced exopolysaccharide was pullulan. Keywords Pullulan  Aureobasidium pullulans  PCS Biofilm Reactor  Response surface methodology  Medium optimization Introduction Pullulan, an exopolysaccharide (EPS) synthesized by a yeast-like fungus Aureobasidium pullulans, is often described as an a-1, 6-linked maltotriose polymer [7]. With this unique linkage pattern, pullulan demonstrated dis- tinctive physical properties, such as adhesive ability, the capacity to form fibers, and the ability to be formed into thin and biodegradable films which are transparent and impermeable to oxygen. As a result, pullulan has been used for a wide range of applications in food, pharmaceutical, chemical, and environmental remediation applications [23]. At present, the number of patents related to pullulan has exceeded 300 [24] and has been produced commer- cially as products, such as edible packaging film, capsules, gelling agents, and emulsifiers [19, 20]. The effects of carbon source [27], nitrogen source [32], shear stress, agitation, aeration and dissolved oxygen levels [16], production enhancers such as zinc and iron [29], temperature [31], and cell morphology [30], have been studied to improve pullulan production. Each of the factors mentioned above will affect pullulan production and its materials property. A one-factor-at-a-time method has also been carried out for the cultivation parameters by Cheng et al. [9]. However, a more statistical method to determine the interaction among cultivation parameters for pullulan production is needed. Sucrose is a favorable carbon source for pullulan pro- duction by A. pullulans [17, 34]; however, some studies reported that excess sucrose (i.e., above 5%) will inhibit pullulan production and hence limit the application of a batch fermentation system [35]. Nitrogen source also plays a significant role on pullulan production. Catley [8] examined the nitrogen limitation effect when producing pullulan by A. pullulans. The results indicated that for K.-C. Cheng  A. Demirci (&)  J. M. Catchmark Department of Agricultural and Biological Engineering, The Pennsylvania State University, University Park, PA 16802, USA e-mail: demirci@psu.edu A. Demirci The Huck Institutes of Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA 123 J Ind Microbiol Biotechnol (2010) 37:587–594 DOI 10.1007/s10295-010-0705-x