Extractive Fermentation for Process integration and amplified pullulan production by A. pullulans in Aqueous Two Phase Systems

Abstract

Extractive fermentation technique or in situ product recovery process is a novel technique to segregate the desired product simultaneously in a fermentation process. For economic and high yield production of pullulan, Extractive fermentation process was applied fermentation process of A. pullulans. Aqueous Two Phase system (ATPS) systems were designed with various molecular mass of PEG (400, 600, 4000 and 6000) and dextran or mono/bi-sodium phosphate salts. Systems with short Tie Line length (TLL) 6.7 and 7.5% w/w for PEG-Salt and PEG-dextran respectively were chosen. Volume ratio for all the systems was kept constant at 1.0 and pH 7.0 for PEG-dextran and PEG-NaH₂PO₄ was maintained, whereas pH 9.0 was kept for PEG-Na₂HPO₄. A. pullulans, was found to be viable with PEG-NaH₂PO₄ and PEG-dextran systems. The biomass partitioned in the PEG rich top phase and the exopolysaccharide pullulan shown affinity towards the bottom phase. A maximum yield (36.47 g/L) was found with PEG 4000-Dextran 500 system of extractive fermentation process. The proposed process aptly integrates upstream and downstream process for continuous production and recovery of pullulan from the biomass, thus reducing the time quotient of the whole process.

Figure 1

A symbolic flow diagram for the process integration of pullulan production with extractive aqueous two phase fermentation process.

Figure 2

A. pullulans growth pattern on PEG-Monosodium phosphate salt (○) and PEG-Bisodium phosphate (□) aqueous two phase systems, the cell growth was determined measuring optical density at 540 nm. Both the systems were maintained at similar TLL 6.7% w/w, V_R 1.0 but varying pH 7 and 9 respectively.

Figure 3

Yield of pullulan was observed in Aqueous two phase systems, PEG 6000-Dextran (□) and PEG-Monosodium phosphate salt (▵) and compared with standard pullulan yield (○) via conventional fermentation process in a span of 10 days. The culture conditions for fermentative pullulan production were 6.5 of ph at 200 rpm and 28 °C of temperature whereas for PEG-dextran 7.5% w/w TLL, VR 1.0 and 7.0 pH.

Figure 4

The FTIR absorption spectrum of standard Pullulan (A) and pullulan sample (B) obtained from extractive fermentation systems of ATPS, for characterization and identification of major bonds and configuration.