Estudo da microfiltração de fluxo tangencial para a clarificação do caldo fermentado de Haemophilus influenzae tipo b

Abstract

Haemophilus influenzae type b (Hib) is a Gram-negative, facultative aerobic bacteria, responsible for respiratory infections, like pneumonia and meningitis, in children younger than 5 years. The main virulence factor of Hib is its capsular exopolysaccharide, Polyribosyl- Ribitol-Phosphate (PRP). Traditional PRP downstream processing comprises several steps of ethanol precipitation, centrifugation, ultracentrifugation, an also phenol extraction, resulting in a laborious, expensive and complex process. Therefore, a simpler and easily scalable process has been developed using tangential flow filtration (TFF) and ethanol precipitation steps. The aim of this work was to study the influence of the main variables during the microfiltration of the fermented broth for PRP recovery. Cell separation was performed at constant volume of 0.5 L, using a TFF system equipped with a Pellicon 2 Mini PVDF membrane (0.22 μm, 0.1 m2 ) and PBS pH 6.3 as dialysis buffer. Permeate flow was gradually increased during process, starting at 5 L.h-1.m-2 up to established values, in order to avoid membrane fouling. A full factorial experimental design was employed, in which three variables were combined at two levels, namely, TMP (15 and 30 psi), feed flow rate (400 and 500 L.h-1.m-2) and permeate flux (15 and 25 L.h-1.m-2), while temperature (22°C), PRP concentration (1 mg/mL), and broth volume/membrane area ratio (5 L/m2) remained constant. The effect of each variable was statistically evaluated by Analysis of Variance. The results indicate that TMP, feed flow, and permeate flow do not affect PRP retention by the membrane when evaluated independently. On the other hand, when combined on pairs or all simultaneously, these variables changed the PRP retention. The interaction between the higher experimental settings of TMP and feed flow or TMP and permeate flow increased PRP retention by around 8% and consequently reduced PRP recovery. Controlling these parameters is a promising strategy for the optimization of the broth clarification process, since the mean recovery observed in the trials was over 80%, in opposition to a mean recovery of around 60% obtained in former experiments.