Fine-scale microbial analysis on denitrifying bacteria for the improved control of wastewater treatment

The activated sludge process is the foundation of liquid waste management practice which removes soluble nutrients such as biochemical oxygen demand (BOD) and nutrients (e.g. nitrogen and phosphorus) from the wastewater. Nutrient removal efficiency depends on the bacterial composition and population. There are a number of operational parameters that may manipulate the microbial community and consequently change the process efficiency. Of these parameters, mixed liquor volatile suspended solids (MLVSS) is the quantitative operating parameter that represents the living bacterial biomass in full-scale water reclamation plants. However, it is only a crude estimation of the sum of bacterial biomass, and this simplification resulted in a poor indication of the actual treatment performance outcome. The advancement in molecular techniques allowed us to characterize the bacterial community within the MLVSS and to better understand the relationship between the bacterial composition and the treatment process. In this dissertation, I have used nitrate reduction as an example to demonstrate that microbial biomarkers are a better representation of biomass and correlate better between the wastewater operation and bacterial functionality.;The research started with developing effective biomarkers based on 16S rRNA genes and functional genes (dissimilatory nitrate reductases) that would be responsive to environmental variables and operational parameters. After validating the captured sequences from the water reclamation plants with the developed primers and probes, we evaluated the bacterial composition in two water reclamation plants. Bacterial composition varied seasonally and from one plant to another. The bacterial composition of the complete nitrifying plant was mainly denitrifying bacteria, but in the incomplete nitrifying plant it was mainly carbon oxidizing bacteria. Also, we applied the biomarkers to address the link between nitrate reduction and operational parameters such as temperature, solids retention time (SRT), pH and dissolved oxygen (DO) in the anoxic zone at both plants. The increase of temperature and SRT positively correlated with the number of targeted functional genes, but the pH and DO inversely correlated with the number of functional genes. narG has shown positive correlation with nitrate reduction in the complete nitrifying plant. Additionally, Paracoccus was also shown to be a better indicator of nitrate reduction in this plant. These findings provided a better understanding of microbial composition responding to the operational controls in activated sludge, and the biomarkers proved to be a useful indicator representing the biomass to project nutrient removal efficiency.