Bacteria-Polymer Composite Material for Glycerol Valorization

Abstract

Bacterial immobilization is regarded as an enabling technology to improve the stability and reusability of biocatalysts. Natural polymers are often used as immobilization matrices but present certain drawbacks, such as biocatalyst leakage and loss of physical integrity upon utilization in bioprocesses. Herein, we prepared a hybrid polymeric matrix that included silica nanoparticles for the unprecedented immobilization of the industrially relevant Gluconobacter frateurii (Gfr). This biocatalyst can valorize glycerol, an abundant by-product of the biodiesel industry, into glyceric acid (GA) and dihydroxyacetone (DHA). Different concentrations of siliceous nanosized materials, such as biomimetic Si nanoparticles (SiNps) and montmorillonite (MT), were added to alginate. These hybrid materials were significantly more resistant by texture analysis and presented a more compact structure as seen by scanning electron microscopy. The preparation including 4% alginate with 4% SiNps proved to be the most resistant material, with a homogeneous distribution of the biocatalyst in the beads as seen by confocal microscopy using a fluorescent mutant of Gfr. It produced the highest amounts of GA and DHA and could be reused for up to eight consecutive 24 h reactions with no loss of physical integrity and negligible bacterial leakage. Overall, our results indicate a new approach to generating biocatalysts using hybrid biopolymer supports.

Keywords: Gluconobacter; bacterial immobilization; biocatalysis; glycerol; hybrid polymers; nanomaterials.

Figure 1

Glycerol conversion with Gluconobacter frateurii (Gfr) immobilized in different matrixes (a) Glyceric acid (GA) production in 20 h. (b) Dihydroxyacetone (DHA) production in 20 h. Reactions were performed in 30 mL of 200 g/L glycerol in water using 20 mg Dry Cell Weight (DCW) immobilized in the different supports.

Scheme 1

Schematic representation of hybrid immobilized preparations combining alginate and nanomaterials. MT: montmorillonite, SiNp: Silica nanoparticle.

Figure 2

Gfr immobilization on hybrid materials. (1) Macroscopic appearance of (a) A4. (b) A4M1. (c) A4M4. (d) A4S1. (e) A4S4. (2) Scanning electron microscopy image (100×) of (a) A4. (b) A4M1. (c) A4M4. (d) A4S1. (e) A4S4. (3) Scanning electron microscopy image (1600×) of (a) A4. (b) A4M1. (c) A4M4. (d) A4S1. (e) A4S4.

Figure 3

Resistance analysis of immobilized preparations performed using a texture analyzer. **** = p-value < 0.0001.

Figure 4

Confocal analysis of immobilized preparation of a fluorescent Gox within (a) A4 and (b) A4S4. BF: Brightfield, mCherry: mCherry fluorescent protein. Scale bar = 20 µm.

Figure 5

Reusability evaluation of Gfr-based biocatalysts. (a) Repeated conversion of pure glycerol using resting cells. (b) Repeated conversion of pure glycerol using A4 immobilized preparations. (c) Repeated conversion of pure glycerol using A4S4 immobilized preparations. Residual GA production activity (red), residual DHA production activity (blue). Each use lasted 24 h.