Methanotrophic based process can be the remedy to offset the wastewater treatment facilities increasing energy requirements due to methanotroph’s unique ability to integrate methane assimilation with multiple biotechnological applications like biological nitrogen removal and methanol production.Regardless of the methanotrophic process end product,the challenge to maintain stable microbial growth in the methanotrophs cultivation bioreactor at higher cell densities is one of the major obstacles facing the process upscaling.Therefore,a series of consecutive batch tests were performed to attentively investigate the biomass density influence on type Ⅰ methanotrophs bacterial growth.In addition,food to microorganisms (F/M),carbon to nitrogen (C/N) and nitrogen to microorganisms (N/M) ratio effect on the microbial activity was studied for the first time.It was clarified that the F/M ratio is the most influencing factor on the microbial growth at higher biomass densities rather than the biomass density increase,whereas C/N and N/M ratio change,while using nitrate as the nitrogen source,does not influence methanotrophs microbial growth.These study results would facilitate the scaling up of methanotrophic based biotechnology by identifying that F/M ratio as the key parameter that influences methanotrophs cultivation at high biomass densities.