Production of polyhydroxybutyrate by coupled saccharification-fermentation of inulin

Abstract

Inulin is a fructose-based polysaccharide that can be found in several plant species, from grass and onions to chicory roots; thus, it has the potential to be an excellent renewable source of fructose for several industrial applications. Among them, inulin hydrolysis can be coupled to a fermentation operation to produce polyhydroxybutyrate (PHB) using Cupriavidus necator H16. This work reports the PHB production process involving chicory root inulin hydrolysis using inulinase Novozym 960 followed by a C. necator fermentation. It was found that the maximum saccharification (95% wt.) was reached at 269 U/g_inulin after 90 min. The hydrolysates obtained were then inoculated with C. necator, leading to a biomass concentration of 4 g/L with 30% (w/w) polymer accumulation. Although PHB production was low, during the first hours, the cell growth and polymer accumulation detected did not coincide with a fructose concentration decrease, suggesting a simultaneous saccharification and fermentation process, potentially alleviating the product inhibition inherent to the inulinase-fructose system. The characterization of the obtained PHB showed a polymer with more homogeneous values of M_w, and better thermal stability than PHB produced using pure fructose as a fermentation substrate. The results obtained demonstrate a viable alternative carbon substrate for PHB production, opening the possibility for inulin-rich renewable feedstock valorization.

Keywords: Bioplastics; Cupriavidus necator; Enzymatic hydrolysis; Inulin; Polyhydroxybutyrate.

Fig. 1

Effect of the enzyme concentration on the hydrolysis of chicory root inulin; using Novozym 960 inulinase at 88 (squares), 180 (circles), 269 (down-pointing triangle), 359 (up-pointing triangle), and 449 U/g_inulin (diamonds); at pH 5.0 50 °C and 5 g/L of inulin

Fig. 2

Evaluation of the initial substrate concentration effect on the hydrolysis yield. a Reducing sugar concentration after (black squares) 20, (gray triangles) 30, and (open circles) 40 min at different initial inulin concentrations. b Hydrolysis product yields obtained at different inulin concentrations (20 min, black; 30 min, gray; 40 min, white). c Effect of Initial fructose concentration on the product yield for the enzymatic hydrolysis reaction. All reactions were carried out at pH 5.0 50 °C

Fig. 3

C. necator cell growth kinetics and polymer accumulation using an enzymatic inulin hydrolysate derived medium (solid symbols), and a fructose-based defined medium (open symbols); at 250 rpm at 30 °C. Squares, fructose concentration (g/L); circles, biomass concentration (g/L); triangles, PHB concentration (g/L)

Fig. 4

Inulin conversion under fermentation conditions at different times (light gray) and residual activity after incubation (dark gray), at 30 °C, pH 6.8 during 72 h

Fig. 5

DSC curves obtained from, A the cooling process, and (B) the second heating scan of PHB samples obtained from 72 h C. necator cultures grown in, (Fa) a synthetic medium using fructose, and (Hb) an enzymatic hydrolysate medium. Changes on the melting and crystallization temperatures (C) and enthalpies (D) measured through eight DSC heating and cooling cycles; obtained for PHB samples coming from a fructose-based medium, Fa (open squares); and a hydrolysate-based medium, Hb (closed squares). Solid line: heating scan (melting parameters). Dotted line: cooling scan (crystallization parameters)