Project description:This study investigates the role of carbon-to-phosphorus (C/P) ratios in shaping microbial community dynamics and polyhydroxyalkanoates (PHA) production in sequencing batch reactors (SBR) fed with volatile fatty acids. Three conditions, characterized by fixed organic loading rates but varying C/P ratios (Run 1 = 170 Cmol Pmol-1; Run 2 = 235 Cmol Pmol-1; Run 3 = 400 Cmol Pmol-1), were tested to explore their impact on PHA accumulation, biomass growth, and reactor stability. Results indicate that the moderate phosphorus limitation of Run 2 achieves the best overall performance, with a PHA volumetric productivity of 2.02 g PHA L-1 d-1 and process stability. Conversely, higher C/P ratio increased the storage yield but lowered the productivity (1.55 gPHA L-1 d-1), compromising sludge settleability and reactor stability, indicating impaired microbial functionality. Full-length 16S/18S rRNA gene sequencing using PacBio technology enabled high-resolution profiling of microbial communities, revealing ecological shifts across conditions. Run 2 exhibited the highest bacterial and eukaryotic diversity, featuring multiple PHA-accumulating bacteria (mainly Sphaerotilus, Leadbetterella, and uncultured Rhodobacteraceae) and a well-structured eukaryotic community dominated by K-strategist bacterivorous protists, e.g. Rhogostoma (Rhizaria) and Vorticella (Ciliophora, Oligohymenophorea) and predatory protozoa, e.g. Tokophrya (Ciliophora, Suctoria). In contrast, Sphaerotilus dominated under suboptimal conditions and may be linked to bulking. These results suggest that eukaryotic diversity may support system stability through selective predation. Transmission electron microscopy (TEM) further confirmed the presence of intracellular PHA granules and polyphosphate reserves, reinforcing the connection between nutrient limitation and adaptive microbial strategies. Overall, these findings highlight the critical role of the C/P ratio in shaping the performance of mixed microbial cultures, demonstrating that a well-balanced nutrient supply can enhance PHA production while maintaining microbial community stability. The results contribute to optimizing the selection process for mixed microbial cultures, offering valuable insights into the impact of carbon-to-nutrient ratios in the feeding strategy.

Project description:We developed a laboratory-scale model to improve our understanding and capacity to assess the biological risks of genetically engineered bacteria and their genetic elements in the natural environment. Our hypothetical scenario concerns an industrial bioreactor failure resulting in the introduction of genetically engineered bacteria to a downstream municipal wastewater treatment plant (MWWTP). As the first step towards developing a model for this scenario, we sampled microbial communities from the aeration basin of a MWWTP at three seasonal time points. Having established a baseline for community composition, we investigated how the community changed when propagated in the laboratory, including cell culture media conditions that could provide selective pressure in future studies. Specifically, using PhyloChip 16S rRNA gene-targeting microarrays, we compared the compositions of sampled communities to those of inoculates propagated in the laboratory in simulated wastewater conditionally amended with various carbon sources (glucose, chloroacetate, D-threonine) or the ionic liquid 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl). Proteobacteria, Bacteroidetes, and Actinobacteria were predominant in aeration basin and laboratory-cultured populations. Laboratory-cultured populations were enriched in Gammaproteobacteria. Enterobacteriaceae and Aeromonadaceae were enriched by glucose, Pseudomonadaceae by chloroacetate and D-threonine, and Burkholderiaceae by high (50 mM) concentrations of chloroacetate. Microbial populations cultured with chloroacetate and D-threonine were more similar to sampled populations than thoes cultured with glucose or [C2mim]Cl. Although observed relative richness in operational taxonomic units was lower for laboratory cultures than for sampled populations, both flask and reactor systems cultured phylogenetically diverse communities. These results importantly provide a foundation for laboratory models of industrial bioreactor failure scenarios. 46 samples, flask and reactor experiments were conducted in triplicate with two exceptions: [C2mim]Cl_flask and No-Carbon_flask treatments had only one sample (no replicates).

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