(A) novel method for screening of fast-growing methanotrophs using the chemostat principle

Abstract

however, their slow growth rates have been the major bottleneck for their industrial applications, barring prompt genetic modifications and biomass generation. In this study, the chemostat principle was adopted to isolate fast-growing methanotrophs by screening the methanotrophs by their growth rates (i.e., the dilution rate of the chemostat reactor). Sediment from Gapcheon Stream and anaerobic digestion effluent from Daejeon WWTP were collected and enriched in sealed batch culture before inoculation into a continuously stirred tank reactor (CSTR) fed with continuous stream of 20% $CH_4$ (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 $h^{-1}$ with an increment of $0.05^{-1}$ until complete washout was observed. The samples collected at the highest dilution rates were used for isolation. The shift in microbial population and enrichment of fast-growing methanotrophs at each step were monitored with 16S rRNA amplicon sequencing. Diverse methanotrophs initially constituting minor populations in the environmental samples were enriched in batch incubation, including genera Methylosinus, Methylocaldum, Methylomonas, Methylococcus, Methylomicrobium, Methylocella, and Methylosarcina. After chemostat operation, the OTUs affiliated with Methylomonas sp. LW13 (SS) and Methylosarcina spp. (AD) were exclusively enriched. The isolated strain exhibited exponential growth rates higher than 0.3 $h^{-1}$, the highest growth rate ever reported for any methanotroph grown at 30$\circ C$. The novel isolation method successfully screened for fast-growing methanotrophs with great industrial potential and significantly shortened the time for isolation.

Methane ($CH_4$) is a potent greenhouse gas (GHG) but, 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 GHG emissions and for conversion of $CH_4$ to value-added products