Reactive Nitrogen (N) and phosphorus (P) pollution is responsible for a vast array of environmental problems, including eutrophication of nutrient limited water bodies, vast dead zones in the ocean margins, and ammonia toxicity to aquatic life. N pollution is also linked to the emission of the potent greenhouse gas nitrous oxide (N2O), which has a global warming potential 310 times that of CO2. In addition, P in fertilizers critical for global food production is derived primarily from phosphate rock, a geographically concentrated nonrenewable resource. P scarcity is an emerging global challenge in its own right, and there is increasing interest in reuse of P from wastewater.', 'Microbial bioprocesses at Water Resource Recovery Facilities (WRRFs) play a key role in preventing nutrient pollution. Unfortunately, current processes are energy intensive, costly, and characterized by emissions of N2O. Paradoxically, N2O is also a powerful potential energy source, as evidenced by its use in propulsion and automotive applications. Recently, a novel nutrient removal process, Coupled Aerobic-anoxic Nitrous Decomposition Operation (CANDO) was introduced to remove N from wastewater and generate N2O as a biofuel. Here, we developed a second generation of CANDO, termed CANDO+P, that combines N removal and energy recovery via microbial N2O generation with biological P removal and recovery. Simultaneous N and P removal by CANDO+P will have the chance to make this process more promising and fit a new vision of wastewater treatment targeting resource recovery in addition to environmental and public health protection. ', 'A proof-of-concept of CANDO+P was provided via long-term operation of a lab-scale bioreactor treating synthetic wastewater with biomass enriched in denitrifying polyphosphate accumulating organisms (DPAOs) for almost 1000 days. Over this period, stable denitrification performance with complete N and partial P removal coupled to high-rate and high-yield N2O production (>70% influent N) was achieved. Biomass aggregate structure shifted during operation from predominantly flocs to a hybrid mixture of flocs and dense microbial granules. A comprehensive study of both reactor kinetics and the underlying microbial community was conducted to understand the structure and function of the microbiome within CANDO+P, and to shed light on mechanisms of N2O production in this system. Based on high-throughput 16S rRNA gene amplicon sequencing, the reactor community rapidly shifted away from the inoculum under the selective pressures imposed mainly by high nitrite (NO2-, 40-50 mg-N/L) and phosphate (PO43-, 5-15 mg-P/L) in the synthetic wastewater feed. A denitrifying Enhanced Biological Phosphorus Removal (EBPR) enrichment dominated by DPAOs, denitrifying glycogen accumulating organisms (DGAOs) and other flanking organisms was selected. 41 near-complete draft genomes including two Candidatus Accumulibacter genomes (associated with clade IA and the first published genome associated with clade IC) were extracted through genome-resolved metagenomic sequencing to characterize genomic denitrification potential. To investigate kinetics of the selected microbial consortium, ex situ batch assays were performed to evaluate denitrification capabilities and denitrifying phosphate uptake with different nitrogen oxides (nitrate [NO3-], NO2- and N2O). Compared with aerobic EBPR reactors and other heterotrophic denitrifiers enrichments, the selected microbial consortium exhibited a strong preference for NO2- utilization, and the propensity to accumulate N2O in the presence of NO2-. ', 'To explain the mechanisms of N2O formation, three hypotheses were tested: (1) electron competition among denitrification enzymes, (2) the enrichment of Candidatus Accumulibacter (PAO) with truncated denitrification pathways, and (3) the selection of flanking organisms (non-PAOs) lacking nitrous oxide reductase (NOS), the terminal enzyme in the complete denitrification pathway. An observed imbalance of denitrification capabilities using different nitrogen oxides as electron acceptors suggested that electron competition was likely not the main driver of N2O formation in this microbial consortium. By screening denitrification genes within the 41 near-completed genomes, nitrous oxide reductase gene was discovered in the Accumulibacter genomes, but not in several flanking bacterial genomes. Taken together, our results suggest that the unusually high levels of N2O accumulation observed in this microbial consortium may be caused by a combination of different mechanisms, including the selection of flanking microorganisms with truncated denitrification pathways. These findings provide proof the feasibility of the CANDO+P bioprocess, and shed light on biological formation of N2O and P uptake by providing detailed information on the associated microbial community structure and function.

Last modified

• 10/21/2019

Creator

• Han Gao

DOI

• https://doi.org/10.21985/n2-bxh9-kt13

Subject

• Civil and Environmental Engineering

Keyword

• Environmental engineering

Date created

• 2018-01-01

Resource type

• Dissertation
• Dataset

Rights statement

• In Copyright