28Background. The role of methane in global warming has become paramount to the environment 29 and the human society, especially in the past few decades. Methane cycling microbial 30 communities play an important role in the global methane cycle, which is why the 31 characterization of these communities is critical to understand and manipulate their behavior. 32 Methanotrophs are a major player in these communities and are able to oxidize methane as their 33 primary carbon source. 34Results. Lake Washington is a freshwater lake characterized by a methane-oxygen 35 countergradient that contains a methane cycling microbial community. The major microbial 36 members include methanotrophs such as Methylobacter Tundripaludum 37 21/22 and Methylomonas sp. LW13. In this work, these methanotrophs are studied via developing 38 highly curated genome-scale metabolic models. Each model was then integrated to form a 39 community model with a multi-level optimization framework. The metabolic interactions for the 40 community were also characterized. While both organisms are competitors for methane, 41 Methylobacter was found to display altruistic behavior in consuming formaldehyde produced 42 by Methylomonas that inhibits its growth. The community was next tested under carbon, oxygen, 43 and nitrogen limited conditions to observe the systematic shifts in the internal metabolic 44 pathways and extracellular metabolite exchanges. Each condition showed variable differences 45 within the methane oxidation pathway, serine cycle, pyruvate metabolism, and the TCA cycle as 46 well as the excretion of formaldehyde and carbon di-oxide from the community. Finally, the 47 community model was simulated under fixed ratios of these two members to reflect the opposing 48 behavior of the community in synthetic and natural communities. The simulated community 49 demonstrated a noticeable switch in intracellular carbon metabolism and formaldehyde transfer 50 between community members in natural vs. synthetic condition. 51 Conclusion. In this work, we attempted to reveal the response of a simplified methane recycling 52 microbial community from Lake Washington to varying environments and also provide an 53 insight into the difference of dynamics in natural community and synthetic co-cultures. Overall, 54this study lays the ground for in silico systems-level studies of freshwater lake ecosystems, 55 which can drive future efforts of understanding, engineering, and modifying these communities 56 for dealing with global warming issues. 57 58

Background The role of methane in global warming has become paramount to the environment and the human society, especially in the past few decades. Methane cycling microbial communities play an important role in the global methane cycle, which is why the characterization of these communities is critical to understand and manipulate their behavior. Methanotrophs are a major player in these communities and are able to oxidize methane as their primary carbon source. Results Lake Washington is a freshwater lake characterized by a methane-oxygen countergradient that contains a methane cycling microbial community. Methanotrophs are a major part of this community involved in assimilating methane from lake water. Two significant methanotrophic species in this community are Methylobacter and Methylomonas. In this work, these methanotrophs are computationally studied via developing highly curated genome-scale metabolic models. Each model was then integrated to form a community model with a multi-level optimization framework. The competitive and mutualistic metabolic interactions among Methylobacter and Methylomonas were also characterized. The community model was next tested under carbon, oxygen, and nitrogen limited conditions in addition to a nutrient-rich condition to observe the systematic shifts in the internal metabolic pathways and extracellular metabolite exchanges. Each condition showed variations in the methane oxidation pathway, pyruvate metabolism, and the TCA cycle as well as the excretion of formaldehyde and carbon di-oxide in the community. Finally, the community model was simulated under fixed ratios of these two members to reflect the opposing behavior in the two-member synthetic community and in sediment-incubated communities. The community simulations predicted a noticeable switch in intracellular carbon metabolism and formaldehyde transfer between community members in sediment-incubated vs. synthetic condition. Conclusion In this work, we attempted to predict the response of a simplified methane cycling microbial community from Lake Washington to varying environments and also provide an insight into the difference of dynamics in sediment-incubated microcosm community and synthetic co-cultures. Overall, this study lays the ground for in silico systems-level studies of freshwater lake ecosystems, which can drive future efforts of understanding, engineering, and modifying these communities for dealing with global warming issues.