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

Abstract

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 pO 2 levels in the bioreactor were maintained at low levels of dissolved O 2 saturation (∼7%). Structural characterization of the isolated polysaccharides from the fermentation broths by 13 C 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 ( M w=5.3–14.7×10 4), root-mean square of the radius of gyration ( R g=24–33 nm) and polydispersity index ( M w/ M n=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.