Illuminating dark matter: Light microscopy and Raman spectroscopy through transparent porous media for applications in soil and sediment microbial ecology

Soils offer habitats to an unparalleled abundance and diversity of microorganisms, whose activities are critical to agriculture, ecosystem health, and biogeochemical cycling. A major barrier to understanding soil microbes within their habitats is the opacity of natural soils. Despite a long history of endeavors to visualize life in the soil, and promising advancements in this field, non-destructive approaches that allow dynamic insights into microbial life in soils are particularly lacking. Chapter 1 reviews this field and outlines the history and potential of optically transparent porous media as model soil systems amenable to non-destructive imaging of soil microorganisms within three-dimensional soil-like matrices. In Chapter 2, I introduce the utility of single-cell Raman spectroscopy (SCRS) for non-destructive stable isotope probing over time, particularly for monitoring the uptake of 13C by bacteria from complex natural polysaccharides. This spatially resolved and non-destructive approach allows us to ask the question, “Do bacterial biofilms allow bacteria to stick together in numbers large enough to initiate cooperative decomposition of necromass?’ In Chapter 3, I assess the polymer Nafion and the crystal cryolite as substrates for optically transparent model soil systems called “transparent soil” (TS) microcosms. I find that both substrates are compatible with optical microscopy and enable growth, maintenance, and visualization of micron-sized bacteria in three-dimensional porous matrices over time. Both substrates are also compatible with SCRS, and enable stable isotope probing (SIP) using deuterium (D2O) as a non-destructive marker of microbial activity in situ, while cryolite-based microcosms also enable measurement of 13C label uptake in bacteria. I use D2O label tracing to show that bacterial cells attached to dead fungal hyphae within a Nafion matrix show more metabolic activity after a dry-wet cycle than cells far away from the fungal hyphae, corroborating the important role of fungi in facilitating survival of bacteria in the fluctuating conditions found in soils. In Chapter 4, I present a method for rapid and inexpensive manufacture of microfluidics devices that were used to construct TS microcosms at the lab bench. Chapter 5 summarizes the dissertation overall and offers suggestions for future research.Doctor of Philosoph