BME PhD Dissertation Defense: Daniel Hart

Title: "Development of the Atmostat as a Tool for Anaerobic Microbial Ecology and Evolution"

Advisory Committee: Mo Khalil, PhD - BME (Research Advisor) Mary Dunlop, PhD - BME (Chair) Seth Rakoff-Nahoum, PhD - Pediatrics, HMS Daniel Segré - Biology Ben Woolston - Chemical Engineering, Northeastern

Abstract: Anaerobic microbial physiology underpins many of earth's ecosystems. In these ecosystems, the inhibition of anaerobes by oxygen is often used to explain ecosystem structure and function. For example, in the gut microbiome, oxygen intolerance is used to explain the patterns of neonatal succession and IBD (inflammatory bowel disease). Full understanding of the ecology of oxygen tolerance may lead to improved treatments for developmental disease and IBD, and untangling the mechanisms of oxygen intolerance is important for environmental science and biomanufacturing. However, isolating the effect of oxygen on any one species, or the role of oxygen in an ecosystem, is difficult with current technology. To address this technical gap, I developed the atmostat, a general-purpose tool for automated atmospheric control on the benchtop. The atmotstat enables facile control of any gas concentration across independent channels via programmable routing and pressure regulation, and low-cost, modular fluidic resistors. I added the atmostat as a module on eVOLVER, a high-throughput continuous culture platform, for parallel, individual control of oxygen in microbial cultures. I applied atmostat-eVOLVER to measure the oxygen sensitivity of a panel of anaerobic gut bacteria, and was able to distinguish intermediate oxygen tolerance from the classical measure of aerotolerance. I also uncovered an unexpected capacity of many anaerobic bacteria to deplete oxygen, and confirmed oxygen depletion as a mechanism of oxygen tolerance. I demonstrated this mechanism in an in vitro ecological model, where oxygen depletion by an aerotolerant anaerobe facilitates the growth of a strict anaerobe. Finally, I adapted a selection of these anaerobes to increased oxygen tolerance in an experimental evolution campaign, which will help to distinguish the mechanisms of oxygen sensitivity and depletion in genetically intractable organisms. Ultimately, this work proves the functionality of atmostat-eVOLVER to explore new genetic and ecological mechanisms, and complicates the existing picture of anaerobes as uniquely vulnerable to oxygen.