Environmental matrix and moisture are key determinants of microbial phenotypes expressed in a reduced complexity soil-analog

Abstract Soil moisture and porosity regulate microbial metabolism by influencing factors such as redox conditions, substrate availability, and soil connectivity. However, the inherent biological, chemical, and physical heterogeneity of soil complicates laboratory investigations into microbial phenotypes that mediate community metabolism. This difficulty arises from challenges in accurately representing the soil environment and in establishing a tractable microbial community that limits confounding variables. To address these challenges in our investigation of community metabolism, we use a reduced-complexity microbial consortium grown in a soil analog using a glass-bead matrix amended with chitin. Long-read and short-read metagenomes, metatranscriptomes, metaproteomes, and metabolomes were analyzed to test the effects of soil structure and moisture on chitin degradation. Our soil structure analog system greatly altered microbial expression profiles compared to the liquid-only incubations, emphasizing the importance of incorporating environmental parameters, like pores and surfaces, for understanding microbial phenotypes relevant to soil ecosystems. These changes were mainly driven by differences in overall expression of chitin-degrading Streptomyces species and stress-tolerant Ensifer . Our findings suggest that the success of Ensifer in a structured environment is likely related to its ability to repurpose carbon via the glyoxylate shunt while potentially using polyhydroxyalkanoate granules as a C source. We also identified traits like motility, stress resistance, and biofilm formation that underlie the degradation of chitin across our treatments and inform how they may ultimately alter carbon use efficiency. Together our results demonstrate that community functions like decomposition are sensitive to environmental conditions and more complex than the multi-enzyme pathways involved in depolymerization. Importance Soil moisture and porosity are critical mediators of microbial metabolism by influencing factors such as redox conditions, substrate availability, and soil connectivity. However, identifying how microbial community metabolism shifts in response to varying levels of moisture and porosity remains a challenging frontier. This difficulty arises from challenges in accurately representing the soil environment and in establishing tractable microbial communities that limit confounding variables. Moreover, inferring phenotypes based on “key” genes often fails to predict complex phenotypes that arise from cellular interactions. Here, we establish a tractably complex microbial community in a soil analog system amended with chitin and leverage it to understand how microorganisms respond to changes in porosity and moisture. By using genome-resolved metagenomics, metatranscriptomics, and metaproteomics, we report on the microbial lifestyle strategies that underpin changes in community expression like carbon conservation, biofilm production, and stress response.