AbstractImpacts of microbial diversity and macronutrients levels (expressed as C:N and C:P ratios) on the methane production from an untreated lignocellulosic feedstock were assessed. Next-generation sequencing technology revealed the bacterial diversity of a lignocellulolytic inoculum. This inoculum comprised 75 bacterial species that were well distributed in 14 phyla, 67% of which belonged to Firmicutes and Bacteroidetes. The families Ruminococcaceae, Clostridiaceae, Bacteroidaceae, Bacillaceae, and Fibrobacteraceae comprised 46% of the identified families and were associated with hydrolytic members. Nutrient adjustment reduced 40% of the length of the lag phase and doubled methane production rate compared with a control. The highest methane production of 0.197 m3 per kg of total volatile solids observed at C:N of 31:1 and C:P of 428:1, peaked 20 days earlier than in previous studies using untreated lignocellulosic feedstock. Interestingly, the highest hydrolytic activities and solids removal rates were observed at high nitrogen contents; however, the conditions (pH > 8.0) inhibited methanogenesis.