show Abstracthide AbstractConnecting genes to phenotypic traits in bacteria is often challenging because of a lack of environmental cues in laboratory settings. However, laboratory-based model ecosystems offer a means to better account for natural conditions compared to standard planktonic cultures, aiding in the linking of genotypes and phenotypes. Here, we present a simple, cost-effective, laboratory-based model ecosystem to study aerobic methane-oxidizing bacteria (methanotrophs). This system, referred to as the gradient syringe, is made by inoculating bacteria into semi-solid agarose held within a disposable syringe. Empty space at one end of the syringe is flushed with methane gas, while the other end is open to the atmosphere through a sterile filter. We show this system replicates the methane-oxygen counter gradient typically found in the natural soil environment of methanotrophs. Culturing the methanotroph Methylomonas sp. strain LW13 in this system produced a distinct horizontal band at the intersection of the counter gradient, which we discovered was due not to increased cell growth at this location but instead to an increased amount of extracellular polymeric substances (EPS). We also discovered that different methanotrophic taxa formed EPS bands with distinct locations and morphologies when grown in the methane-oxygen counter gradient. By comparing transcriptomic data from LW13 growing within and surrounding this EPS band, we identified genes implicated in cell growth and EPS formation within the gradient syringe, and validated the involvement of these genes with knockout strains. This work highlights the use of a laboratory-based model ecosystem that more closely mimics the natural environment to uncover methanotroph phenotypes missing from standard planktonic cultures, and link these phenotypes their genetic determinants. Overall design: To investigate the cellular functions underlying the extracellular polymeric substance (EPS) phenotype observed at the midpoint of the methane-oxygen counter gradient within a semi-solid agarose matrix, we compared transcriptomes of the aerobic methanotroph Methylomonas sp. LW13 isolated from increasing depths. Late exponential/early stationary phase gradient syringes inoculated with LW13 were divided into three 2 mL segments, with the middle segment centered around the EPS band. These segments were designated “above”, “band”, and “below”, representing oxygen-exposed, band-centered, and methane-flushed regions. For each of three independent replicates, RNA from eight LW13-inoculated syringes were pooled.