PROGRAM | Chemical Engineering

By: Amalie Tuerk Levy Chair: Kelvin Lee

ABSTRACT

The metabolic capabilities of environmental microbes, which enable them to thrive in niche environments and survive harsh conditions, can inspire novel solutions to challenging problems. One such problem is the enormous amount of waste elemental sulfur produced as a byproduct of crude oil and natural gas refining. The supply of waste sulfur greatly exceeds demand, and the ~7 million tons produced annually is landfilled or stored in open piles at refineries. As sulfur oxidizing bacteria use elemental sulfur (S(0)) and other reduced sulfur compounds as substrates for growth, waste sulfur represents an untapped resource for fueling useful microbial processes.
The phototrophic sulfur oxidizing bacterium Chlorobaculum tepidum uses electrons obtained from the oxidation of reduced sulfur compounds for carbon dioxide fixation. C. tepidum oxidizes sulfide as the preferred electron donor, depositing S(0) into insoluble extracellular globules; once sulfide is depleted, C. tepidum oxidizes S(0) to sulfate. However, the mechanisms of S(0) production and degradation are poorly understood, as are the interactions that mediate cell-S(0) attachment. Thus the aim of this work was to improve understanding of S(0) metabolism in C. tepidum to facilitate alternative uses of waste sulfur via synthetic biology approaches.
Challenges with growth variability and a lack of reliable biomass quantitation methods in C. tepidum were initial obstacles to deriving meaningful information from quantitative systems-based studies. To address these issues, Design of Experiments methodology was used to evaluate the growth of C. tepidum across a 3×3 factorial space of S(0)-producing and S(0)-degrading states and a range of light fluxes: the ‘energy landscape’ of sulfur oxidation. Protein measurements collected across the landscape were calibrated against amino acid analysis quantitation, providing improved measurement methods for assessing biomass growth. Unexpectedly, these results also revealed adaptations that increase C. tepidum fitness in low-energy environments. Comprehensive measurements of the various intermediates of sulfur metabolism across this factorial space revealed that electron donor, but not light flux, affected growth yields on an electron equivalent basis, providing insight into pathways of energy conservation in C. tepidum. This work also revealed the presence of soluble intermediates of S(0) production and degradation, providing a mechanism to explain time-lapse microscopic observations of S(0) globules growing and degrading at a distance from cells.
Towards characterizing the surface properties of S(0)to better understand cell-S(0) interactions, analysis of the S(0) ‘proteome’ revealed intriguing similarities with the proteomes of the outer membrane vesicles of other gram negative bacteria. Of particular interest was the observation of proteins involved in spatial localization of import/export machinery and cell division, the envelope stress response, and inorganic ion transport and metabolism, along with uncharacterized outer membrane proteins. Together, these data suggest a new model for S(0) generation in C. tepidum, and provide new protein targets for characterization in the context of S(0) metabolism and cell-S(0) interactions.
The comprehensive ‘landscape’ approach used in this work enabled new insights into S(0) metabolism and low-energy adaptations in C. tepidum that would not have been observed by single-factor experiments. Furthermore, these efforts form a basis for future quantitative systems-based studies, and this approach is generalizable to preparing for these types of studies in other systems. The specific insights into S(0) metabolism obtained from this work could be applied to improved waste sulfur management and the low-energy adaptations have implications for optimizing the efficiency of “designer microbes” used in industrial processing.

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