Enhanced production of bacterial nanocellulose by Bacillus subtilis using combined mutagenesis and response surface...

In the present study, a native Bacillus subtilis isolate-8 was subjected to sequential mutagenesis using ultraviolet (UV) irradiation, nitrous acid (HNO₂) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) to obtain enhanced BNC production by following mutagenesis steps, with maximum yields of 4.3 g/L, 8.1 g/L and 9.8 g/L achieved by the superior UV mutant (BCUV-08), nitrous acid mutant (BCN-06), and NTG mutant (BCNT-03), respectively. The hyperproducing mutant BCNT-03 was further optimized using response surface methodology (RSM) based on a central composite rotatable design (CCRD). Four critical process variables, namely inoculum size (0.5-2.5%, v/v), incubation temperature (20-40 °C), incubation period (5-25 days), and flask-to-medium ratio (1:6-1:2, v/v), were evaluated under static fermentation conditions using modified Hestrin-Schramm (HS) medium containing molasses as a low-cost carbon source. The developed quadratic model was highly significant (F = 16.27, p < 0.001) and explained 93.44% of the variation in BNC production (R²=0.9344). The optimum conditions predicted by the model corresponded to an inoculum size of 1.5% (v/v), incubation temperature of 30 °C, incubation period of 15 days, and flask-to-medium ratio of 1:4, resulting in a maximum BNC yield of 15.92 g/L. The combined mutagenesis and process optimization strategy substantially enhanced BNC production. The findings demonstrate the potential of B. subtilis as an alternative BNC-producing organism.