Intensification of hydrogen production by B. licheniformis using kitchen waste as substrate

Bacillus licheniformis AP1 isolated from dairy waste cheese whey water possess pyruvate formate lyase (PFL) and formate hydrogen lyase (FHL) enzyme gene which can hydrolyze pyruvate and formate to produce hydrogen under anaerobic conditions. Molecular characterization of this strain was done using 16rRNA gene sequencing. Phylogenetic tree was formed on the basis of neighbor-joining method using MEGA5 which showed that no significant change occurred in 16s rRNA during the course of evolution. Biohydrogen production using this laboratory isolate was performed using pre-treated kitchen waste as substrate at optimized pH 6.5 with yield of 12.29 ± 1.2 mmolH2/gCOD reduced. Effect of macronutrients and micronutrients were studied by varying concentrations on the hydrogen production. Hydrogen production substantially increased from 14.10 ± 1.4 mmolH2/gCOD, 17.027 ± 1.7 mmolH2/gCOD, 17.029 ± 1.7 mmolH2/gCOD to 17.62 ± 1.8 mmol/gCOD reduced kitchen waste by B. licheniformis at optimized concentrations of -different metals like magnesium (MgCl2) 0.59 g/L, nitrogen (NH4Cl) 7 g/L, nickel (NiCl2) 180 μg/L, and iron (II) (FeSO4) 67 μg/L respectively. The optimized temperature for this process was found to be 34 ± 2 °C with the maximal hydrogen yield of 17.62 ± 1.8 mmol/g COD reduced kitchen waste. The end fermentation metabolites detected were acetic acid, isobutyl acid, butyric acid, and pyruvic acid in the proportion of 3.75:1:1:1 under the optimized conditions in batch experiment (30 ml MGY media broth). These are the products of pyruvate-formate lyase enzyme complex that indicates the major electron flux towards formate during hydrogen production by B. licheniformis in hydrolyzed kitchen waste.