Characterization of pullulan produced from beet molasses by Aureobasidium pullulans in a stirred tank reactor under varying agitation

The molecular characteristics of pullulan produced from beet molasses, following their pretreatment with sulfuric acid and activated carbon, by Aureobasidium pullulans P56 (a melanin-deficient strain) in a stirred tank fermentor under varying agitation conditions have been examined. A maximum pullulan concentration of 49 g l−1, biomass dry weight of 25 g l−1, pullulan yield of 50% and sugar utilization of 97% were achieved at an initial sugar concentration in the medium of 100 g l−1 and impeller speed of 700 rpm; under these conditions the pO2 levels in the bioreactor were maintained at low levels of dissolved O2 saturation (∼7%). Structural characterization of the isolated polysaccharides from the fermentation broths by 13C NMR spectroscopy and pullulanase digestion combined with size exclusion chromatography confirmed the identity of pullulan and the homogeneity (>94% d.b.) of the elaborated polysaccharides by the microorganism. The exopolysaccharide preparations were also free of contaminating proteins. Using multiangle laser light scattering (MALLS) and refractive index (RI) detectors, in conjunction with high-performance size-exclusion chromatography (HPSEC), molecular size distributions and estimates of the molecular weight (Mw=5.3–14.7×104), root-mean square of the radius of gyration (Rg=24–33 nm) and polydispersity index (Mw/Mn=1.7–2.6) were obtained. In most culture systems examined there was a reduction in the molecular size of the isolated polysaccharides as time of fermentation progressed. In the concentration range of 2.8–9.5% (w/v), the solution rheological behavior of the isolated pullulans was almost Newtonian (within 1 and 1200 s−1 at 20 °C). Overall, beet molasses were proven as an attractive fermentation medium for the production of pullulan by A. pullulans.