Valorization of wood waste for enhanced polyhydroxybutyrate production by Klebsiella sp. MK3

Abstract

The valorization of wood waste as a sustainable bacterial feedstock for the production of Polyhydroxybutyrate (PHB) is explored in this study, aiming to provide an environmentally friendly alternative to conventional plastics. Wood waste, treated with 4% sulfuric acid, served as the carbon source for isolating bacteria from Jalandhar waste streams, with the strain Klebsiella sp. MK3 identified as the most effective in PHB production after 16s rRNA sequencing. Analytical methods including the Molisch test, DNS, and sugar utilization tests confirmed sugar presence and consumption by the bacterial isolate. Media optimization using Design Expert 12.0 utilized a quadratic model, achieving a robust fit with an R² value of 98.6%. Optimization via Plackett-Burman design and response surface methodology enhanced PHB yield to 4.37 mg/mL, a significant increase over previous benchmarks. This yield was achieved under optimal conditions of 1.7% carbon concentration, 0.105% nitrogen concentration, and a constant temperature of 37 °C. Qualitative analysis of PHB by UV-Vis spectroscopy, FTIR, and NMR confirmed its purity and composition. The study highlights the potential of wood waste and wastewater as substrates for cost-effective PHB production, with significant applications in packaging, agriculture, medicine, and more, thus promoting reduced reliance on non-renewable resources and advancing sustainability goals.

Keywords: Klebsiella sp. MK3; Plackett-Burman design (PBD); Polyhydroxybutyrate (PHB); Response surface methodology (RSM); Statistical experimental design.; Wood waste valorization.

Fig. 1

Phylogenetic tree showing sample 3 is Klebsiella sp. MK3 prepared using MEGA11 software (version 11).

Fig. 2

Standard graph of glucose using DNS test for reducing sugar.

Fig. 3

Pareto showing factors that are most affecting production rate.

Fig. 4

Showing predicted and actual value of PHB in graphical form.

Fig. 5

Illustrating contour plots and 3D-Surface response prepared using Design Expert (version 12.0) by StatEase: interactive effects of (A) and (B) varied the intensity of the carbon and nitrogen at temperature 34; (C) and (D) varied the intensity of the carbon and Temperature at nitrogen 0.105; (E) and (F) varied the intensity of the nitrogen and Temperature at carbon 1.7.

Fig. 6

Displays the uniformly distributed form of the polyhydroxybutyrate (PHB) film following the evaporation of chloroform.

Fig. 7

UV spectroscopy analysis of PHA (orange line) and crotonic acid (black line).

Fig. 8

FTIR peaks representing the presence of PHB extracted from Klebsiella sp. MK3.

Fig. 9

(a) 13C-NMR spectrum of the PHB extracted using chloroform. (b) 1H-NMR spectrum of the PHB extracted using chloroform.