Methanotrophs are of great interest due to their distinguish ability of recovering value-added commodities such as methanol and lipids while mitigating methane. The enhancement of methanotrophs cultivation process conditions is a pivotal step to develop a feasible methanotrophic bioreactor. In this study, multiple batch tests have been performed to evaluate the aqueous growth medium elements including nitrogen, copper, and biomass density and the gaseous headspace composition influence on methanotrophs activity and the associated microbial community. It was found that increasing copper concentration to 20 mu M and nitrate concentration to 40 mM result in higher growth rate and yield. In contrast, increasing the biomass density resulted in a declination in the growth rate and yield. The 16S rRNA gene sequencing shows that the used culture was dominated by type I Methylomonas genus at relative abundance of 60.9% compare to 1% of the enriched population for type II and type III methanotrophs. Thereafter, the specific nitrate uptake rate has been determined to be ranging from 0.05 to 0.62 mgN-NO3/mgTSS/day based on the cultures OD600. The attained results would facilitate the continuous cultivation of type I methanotrophs dominated culture as a first step for any methanotrophic based biotechnology. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.