Valorization of Wheat Bran for PHBV Copolymer Production by Bacillus subtilis SMI3: Optimization, Structural Characterization, and Biodegradability

Abstract

Background: Selecting an appropriate microbial host is as critical as choosing a suitable feedstock. Gram-negative bacteria such as Cupriavidus necator are known for their high polyhydroxybutyrate (PHB) yields; however, the presence of lipopolysaccharide endotoxins complicates downstream purification, particularly for applications in food and medicine. Aims: The present study evaluates the potential of wheat bran, an abundant agricultural by‑product, as a low‑cost substrate for polyhydroxyalkanoate (PHA) production using a newly isolated Bacillus subtilis strain SMI3, and to optimize fermentation conditions, characterize the polymer, and assess its biodegradability. Study Design: Laboratory‑scale experimental study for bioprocess optimization and polymer characterization. Place and Duration of Study: Department of Microbiology, Faculty of Life Sciences, Bayero University, Kano, Nigeria; between March 2024 and February 2025. Methodology: Bacillus subtilis SMI3 was isolated from fadama soil, screened for intracellular PHA accumulation using Sudan Black B and Nile Blue A, and identified by 16S rRNA gene sequencing. Wheat bran was hydrolyzed by dilute acid (5% H₂SO₄, 121°C, 30 min) to obtain fermentable sugars. One‑factor‑at‑a‑time (OFAT) screening evaluated incubation time (24–120 h), pH (6.5–8.5), temperature (25–45°C), substrate concentration (1–5% w/v), and inoculum size (0.5–4 McFarland). Response surface methodology (RSM) with a four‑factor central composite design (CCD) was then applied. The polymer was extracted and characterized by Fourier transform infrared (FTIR) spectroscopy and gas chromatography–mass spectrometry (GC‑MS). Biodegradability was tested by soil burial over 60 days. Results: OFAT identified baseline conditions: 72 h, pH 7.0, 35°C, 3 g/L wheat bran, and 3.0 McFarland inoculum. The RSM model was highly significant (R² = 0.9706, p < 0.0001), predicting a maximum yield of 660.00 mg/L, which was experimentally validated. Substrate concentration and inoculum size showed the strongest interactive effect (p = 0.0037). FTIR spectroscopy confirmed the PHA ester carbonyl peak at 1737 cm⁻¹, characteristic of the polyhydroxyalkanoate backbone. GC‑MS revealed a PHBV copolymer containing 28.18% 3‑hydroxybutyrate and 19.66% 3‑hydroxyvalerate – produced without adding any precursor. Soil burial caused 72.5% weight loss after 60 days. Conclusion: Wheat bran hydrolysate is an effective, low‑cost feedstock for PHBV production by Bacillus subtilis SMI3. The high yield, novel precursor‑free 3HV incorporation, and rapid biodegradability make this process a promising sustainable route for bioplastics within a circular bioeconomy.