STRUCTURAL AND METABOLIC MECHANISMS UNDERLYING THE BIOLOGICAL FATES OF TWO RESOURCE RECOVERY BIOPOLYMERS: POLYPHOSPHATE AND POLY(Β-HYDROXYBUTYRATE)
Resource recovery is an important aspect of environmental engineering for sustainability purposes, and polyphosphate (polyP) and poly(β-hydroxybutyrate) (PHB) are examples of biopolymers that are utilized for resource recovery.Given its structure composed of multiple phosphate groups linked by phosphoanhydride bonds, polyP is an important source of phosphorus (P) recovery from wastewaters. Polyphosphate accumulating organisms (PAOs) are an example of such a group of organisms since they are capable of storing P in the form of polyP when subjected to varying oxygen availability conditions. These organisms have been studied extensively to optimize the performance of enhanced biological P removal (EBPR) systems for wastewaters and previous studies have suggested the correlation between metal composition and EBPR performances. However, the underlying mechanism remains largely unknown. Therefore, in Chapter 1, we investigated the coordination of physiological metal cation (K+, Na+, Ca2+, and Mg2+) chelated by polyPs of different chain lengths (with 10 and 30 phosphate moieties) by employing molecular dynamics simulations and experimental techniques. The simulation results showed that the Mg-polyP and K-polyP complexes were the most and least thermodynamically stable, respectively. Additionally, to gain insight into the conformation of polyP-30, we conducted gel permeation chromatography and small-angle X-ray scattering analysis on Mg-polyP-30 solution samples. Results showed varying thermodynamic stability amongst the tested metal-polyP complexes and we proposed the differing roles of metal-polyP complexes in maintaining the physiological pool of bacterial polyP: while the more thermodynamically stable Mg-polyP complexes could contribute to the long-term storage of polyP, the less stable K-polyP with weaker chelating strength would allow and favor enzymatic hydrolysis of polyP. Chapter 2 focuses on PHB which, due to its similar physical properties to conventional, petroleum-based plastics, is an attractive source of bioplastic. Accumulation of PHB in various organisms under nutrient limitations has been well reported, and Comamonas testosteroni is also capable of accumulating PHB under nutrient deficiency. However, how nutrient limitation induces PHB accumulation in bacteria like C. testosteroni remains unclear. Therefore, to gain insight into the nutrient-dependent carbon metabolism that leads to PHB production by C. testosteroni, we first investigated the effect of varying nutrient conditions on biomass growth of C. testosteroni KF-1. Second, we employed a quantitative 13C-metabolic flux analysis (MFA) using ultra-high-performance liquid chromatography with high-resolution mass spectroscopy to understand the effect of nitrogen (N) availability on carbon metabolism. Third, we compared the metabolite levels and relative protein abundances for the different N and P conditions to gain insight into how reduced nutrient availability may have impacted the cell’s response. Our findings implied that C. testosteroni is less sensitive to P-deficiency than to N-deficiency and that there may be a threshold at which N depletion favors the production of PHB compared to the replete condition due to compromised cellular growth further imposing imbalances between the metabolic carbon flux and energetics.
Comamonas testosteroni KF-1; molecular dynamics simulations; nutrient dependent metabolism; polyhydroxybutyrate; polyphosphate; X-ray scattering
Biological and Environmental Engineering
M.S., Biological and Environmental Engineering
Master of Science
dissertation or thesis