Graduate College
Permanent URI for this community
The doctoral dissertation and master's thesis reflects the scholarly research in a graduate program as required for the completion of the degree at the University of Delaware.
Browse
Browsing Graduate College by Subject "13C"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
Item Case studies in coculture syntrophy and 13C metabolic flux analysis(University of Delaware, 2022) Dahle, Michael LynnEngineering microbial consortia for fermentation allows for pathway compartmentalization, broadened metabolic capability, and improved robustness. However, tools for modeling consortia are underdeveloped, limiting predictability compared to monocultures. Identifying and quantifying interspecies cross-feeding and its effect on each species’ metabolism is crucial to understanding cooperation and exploitation in consortia. In this work, we develop techniques for conducting 13C metabolic flux analysis on consortia using a range of model cocultures. ☐ In one model coculture, a methionine-auxotrophic E. coli cleaves lactose, providing glucose and galactose to S. enterica. In turn, S. enterica excretes excess methionine that is taken up by E. coli. Knowing only the biomass composition of each species and the combined biomass 13C-labeling, we used our recently developed methodology to simultaneously resolve the ratio of S. enterica to E. coli and each species’ full metabolic profile, both in isolation and in coculture. We discovered that S. enterica feeds on pyruvate produced from E. coli. Transwell 13C tracer experiments show flux of valine and leucine from E. coli to S. enterica, and transwell 13C-MFA shows that acetate syntrophy is favored over pyruvate syntrophy when species populations are separated. ☐ In another coculture system, a complementary pair of Keio collection E. coli were grown. One strain cannot consume glucose (del-ptsI del-glk) and the other strain cannot consume galactose (del-galK). When the lac+ operon was reintroduced to the galactose-consuming strain (del-ptsI del-glk lac+), the coculture’s growth was limited by the slower-growing strain’s ability to cleave lactose into glucose and galactose. In a pair of coculture transwell experiments with either [U-13C-galactose]-lactose or [U-13C-glucose]-lactose as the sole carbon source, we discovered widespread exchange of carbon via acetate, CO2, TCA cycle dicarboxylic acids, and amino acids. ☐ In the final project, we study cocultures of C. acetobutylicum and C. ljungdahlii. Coculturing with C. ljungdahlii has been shown to increase C. acetobutylicum’s carbon efficiency. In the process, we develop techniques to study metabolism in undefined medium with multiple carbon sources, including: a) calculations to differentiate biomass derived from yeast extract or derived from hexose; b) co-culture 13C-MFA to discretize monoculture metabolism into distinct phases; and c) isotopomer spectral analysis (ISA) on cell membrane lipids to calculate marginal growth during stationary phase. We show from ISA analysis of fatty acids that C. acetobutylicum has irreversible excretion of acetate. In C. ljungdahlii we use ISA on fatty acids to discern two distinct acetyl-CoA labeling phases, showing that sodium acetate buffer freely exchanges with intracellular metabolite pools. We found that 30% of C. acetobutylicum biomass was derived from glucose and the remainder was derived from yeast extract, limited only by the complete consumption of every yeast extract amino acid except alanine and glutamate. For C. acetobutylicum, most yeast extract components flowed directly into protein synthesis. Only aspartate and glycine entered central carbon metabolism. These new methods were then applied to the CO2-fixing bacterium C. ljungdahlii during mixotrophic growth. Using 13C-tracers, we discovered that methionine supplementation allows growth in defined medium. Despite the availability of fructose and asparagine, externally supplied CO2 was the primary carbon substrate for growth (47% of total carbon-moles consumed). Asparagine was a major carbon source via deamination to fumarate, or via cleavage of threonine to acetaldehyde and glycine to feed the Wood-Ljungdahl pathway. In both C. acetobutylicum and C. ljungdahlii, we discover significant alanine excretion. Coculture 13C-MFA of C. acetobutylicum and C. ljungdahlii confirms exchange of intracellular metabolite pools via acetate, and recapture of C. acetobutylicum’s waste CO2 via C. ljungdahlii’s autotrophic metabolism. We developed a simplified model for coculture metabolism which combines ISA of C16:0 labeling, extracellular solvents’ MIDs, and extracellular concentration changes to solvents and substrates to calculate species ratios, relative growth, quantitative contributions to extracellular carbon pools, and carbon exchange.