Adsorption and biodegradation of the azo dye methyl orange using Ralstonia pickettii immobilized in polyvinyl alcohol (PVA)-alginate-hectorite beads (BHec-RP)

Abstract

Biological methods are widely used to treat dye waste, particularly methyl orange (MO) dye. The importance of MO degradation stems from its classification as a toxic dye. Within the scope of this research, successful bio-decolorization of MO was achieved through the use of Ralstonia pickettii bacteria immobilized in a PVA-alginate-hectorite matrix (BHec-RP). The optimum conditions for the degradation were observed at a composition of PVA (10%), hectorite (1%), static incubation, 40 °C, and pH 7. Subsequently, the adsorption kinetics of BHec-RP (dead cells) as well as the degradation kinetics of BHec-RP (live cells) and MO using free R. pickettii cells were evaluated. The decolorization of MO using BHec-RP (dead cells) is an adsorption process following pseudo-first-order kinetics (0.6918 mg g^-1 beads) and occurs in a monolayer or physical process. Meanwhile, the adoption of BHec-RP (live cells) and free R. pickettii cells shows a degradation process under pseudo-first-order kinetics, with the highest rates at an initial MO concentration of 50 mg L^-1 being 0.025 mg L^-1 h^-1 and 0.015 mg L^-1 h^-1, respectively. These results show that the immobilization system is superior compared to free R. pickettii cells. Furthermore, the degradation process shows the inclusion of several enzymes, such as azoreductase, NADH-DCIP reductase, and laccase, presumed to be included in the fragmentation of molecules. This results in five fragments based on LC-QTOF/MS analysis, with m/z values of 267.12; 189.09; 179.07; 169.09; and 165.05.

Fig. 1. Illustrative of preparation methodology for BHec-RP.

Fig. 2. Micrographs: (a) SEM of BHec, (b) SEM of BHec-RP, (c) SEM of Hec-40, and (d) TEM of Hec-40 (after calcination).

Fig. 3. SEM and EDS images: (a) BHec-RP (before); (b) BHec-RP (after); (c) BHec-RP (dead cell).

Fig. 4. TGA (a) and DTG (b) curves of Hec, BHec, and BHec-RP.

Fig. 5. N₂ adsorption–desorption (a) and PSD (b) curves of Hec and BHec-RP.

Fig. 6. Decolorization MO using BHec-RP (live and dead cells) and free cells (100 mg L⁻¹, static, pH 7, temperature 40 °C).

Fig. 7. MO adsorption capacity at different initial concentrations (PVA: 10% (w/w); alginate: 1% (w/w); hectorite: 1% (w/w); bead mass 10%, volume 25 mL).

Fig. 8. Non-linear sorption isotherm on BHec-RP (dead cells).

Fig. 9. The influence of initial MO concentrations on (a) BHec-RP (Live cells) and (b) Free R. pickettii cells.

Fig. 10. Effect of: (a) PVA concentration; (b) hectorite concentration; (c) pH; (d) temperature.

Fig. 11. Shape of beads at various PVA concentrations: (a) 2.5%; (b) 5%; (c) 7.5%; (d) 10%; (e) 15%.

Fig. 12. Bead morphology: (a) before degradation (BHec-RP; live cells); (b) after degradation at pH 7 (BHec-RP; live cells); (c) after degradation at pH 5 (BHec-RP; live cells); (d) after degradation at pH 9 (BHec-RP; live cells); (e) after decolorization at pH 7 (BHec-RP; dead cells).

Fig. 13. MO degradation cycles and residual viable cell.

Fig. 14. Chromatogram of MO control (blue line) and degradation results.

Fig. 15. Prediction of MO biodegradation pathway.