Microaeration pretreatment can enhance lignin degradation of lignocellulosic biomass and subsequent biogas production during anaerobic digestion (AD). However, the reported values of effective aeration dose, aeration contact time, and aeration frequency differ with substrates riddling the selection of a suitable combination. A serial approach was adopted to identify the suitable combination for the degradation of lignin in the buffalo manure and improvement of the biogas production. Single and Multiple injections of three aeration doses of 5.3, 7.3, and 9.3 mL O₂/g VS were tested at 5 different contact times– 6, 12, 24, 36, and 47 h. Results for acid-soluble lignin (% ASL) indicated the suitability of 12 h contact time with a single injection of 7.3 mL O₂/g VS for lignin degradation and biogas enhancement for AD of buffalo manure. This combination achieved 78% higher ASL content and a 32% increase in average daily biogas yield than the control. Finally, the kinetic parameters obtained through the modified Gompertz model accurately predicted the biogas potential of the process (R² > 0.93). The results of this study can be referenced for improving the yield of similar biomass to bioenergy initiatives by employing a low-cost pretreatment strategy of in-situ microaeration.