A Novel Method for Screening of Fast-Growing Methanotrophs Using the Chemostat Principle

Abstract

Methane (CH4) is a potent greenhouse gas and, at the same time, is a promising resource as energy source and feedstock for chemical industry. Methanotrophs, the group of bacteria utilizing CH4 as their sole carbon and energy source, have recently gained interest as the biocatalysts for mitigation of greenhouse gas emissions and for conversion of CH4 to value-added products. Nonetheless, their slow growth rates have barred prompt genetic modifications and biomass generation. In this study, we have adopted the chemostat principle to isolate fast-growing methanotrophs by screening the methanotrophs by their growth rates (i.e., the dilution rate of the chemostat reactor). Soil from the northern bank of Gapcheon Stream, Daejeon, Korea was collected and 10 g of the soil sample was enriched in sealed batch culture before inoculation into a continuously stirred tank reactor (CSTR) fed with continuous stream of 20% CH4 (in air) in the headspace and fresh NMS medium in the aqueous phase. After initial fed-batch incubation in the CSTR, the dilution rate was gradually increased from 0.1 h-1 to 0.35 h-1(above which complete washout was observed) with 0.05 h-1 increments. The sample collected at the highest dilution rate was used for isolation. The shift in microbial population and enrichment of fast-growing methanotrophs at each step were monitored with 16S rRNA gene amplicon sequencing targeting the V6-V8 region. Diverse methanotrophs initially constituting <0.09% of the microbial population in the soil sample were enriched in initial batch incubation of 10 g soil, including Methylomonas spp. (0.0644%), Methylocystis spp. (0.0603%), Methylobacter spp. (0.5128%), Methylococcus spp. (0.0137%), and Methylocaldum spp. (0.0014%). After chemostat operation, the OTU grouped with Methylomonas sp. LW13 was exclusively enriched in the reactor among these methanotrophs. The isolated strain exhibited exponential growth rate >0.3 h-1, the highest growth rate ever reported for any methanotoph. The composition of non-methanotrophic microbial community (<30% of the total microbial community) shifted with the increase in the dilution rate, as the relative abundance of Methyloversatilis spp. decreased and that of Methylobacillus spp. increased. Also notably, Legionella spp., a pathogen previously known to be extremely difficult to enrich in a defined medium, was enriched at high dilution rate. The novel isolation method successfully screened for fast-growing methanotrophs with great industrial potential and significantly shortened the time for isolation.