Reexamining the engineered nitrogen cycle : microbial diversity, community dynamics, and immigration in nitrifying activated sludge bioreactors
- Biological wastewater treatment is a multi-billion dollar industry—the largest application of biotechnology in the world. Nitrification systems in these wastewater treatment plants are critically important barriers to nitrogen pollution, thereby protecting natural systems from ammonium toxicity, excess emissions of the potent greenhouse gas nitrous oxide, nitrogenous oxygen demand, and N-stimulated eutrophication. Despite the environmental and economic importance of these processes, surprisingly little is known about the nature of the key biocatalysts (microbial communities) within nitrifying wastewater treatment bioreactors. The body of research presented in this dissertation targets three knowledge gaps in the microbial ecology of biological nutrient removal systems: microbial community dynamics and associated deterministic drivers; microbial diversity—namely, the 'core' and 'dispensable' microbiome in activated sludge and the relative importance therein of different groupings of ammonia-oxidizing microorganisms to nitrogen transformations; and the importance of microbial immigration in structuring engineered microbial communities. In Chapter 2, I investigate the diversity, population dynamics, and relative importance of two key types of microorganisms in nitrogen removal processes—ammonia-oxidizing bacteria (AOB) and newly-discovered ammonia-oxidizing archaea (AOA)—in a one-year time series of weekly activated sludge samples from a nitrifying wastewater treatment plant in California. I demonstrate that AOB predominate by 3 orders of magnitude over AOA in this system—an important result, given recent reports of natural environments dominated by AOA, and the first quantitative comparison of AOA and AOB in activated sludge. Moreover, my results reveal a predominance in the AOB community of a novel Nitrosomonas-like lineage and strong associations between AOB community dynamics and temperature, dissolved oxygen, influent nitrite concentrations, and primary influent chromium concentrations. In Chapter 3, I employ molecular fingerprinting analyses (T-RFLP) to characterize overall bacterial dynamics in activated sludge over the same one-year time period and to test a fundamental prediction of macroecological theory—the "Species-Time Curve"—in engineered microbial systems. My results reveal surprisingly strong long-term temporal dynamics in the activated sludge bacterial assemblage during a period of stable performance, with a gradual succession away from initial conditions likely linked to variations in dissolved oxygen, temperature, influent silver, biomass levels, and influent nitrite concentrations. I also provide significant support for a power-law taxa-time relationship (TTR) in activated sludge systems, as predicted by macroecological theory, with a power-law exponent (w=0.209) well in-line with those observed in macrobial systems. In Chapter 4, I detail an astonishingly high reservoir of overall microbial phylogenetic and functional diversity and unexpectedly large community dynamics in activated sludge via application of cutting-edge phylogenetic (PhyloChip) and functional gene (GeoChip) microarrays. While nearly 2,500 distinct microbial taxa distributed throughout 48 bacterial and archaeal phyla were observed in 12 monthly samples from a full-scale wastewater treatment bioreactor, the set of taxa that were present in all samples—the "core" microbiome—was limited to ~700 taxa. Dynamics in the ~1800 taxa present in only a subset of samples—the "dispensable" microbiome—were significantly associated with temperature and influent nitrite. Of 10,267 unique functional genes in 266 gene families that showed statistically significant hybridization signals to our functional gene microarray platform, only 66 unique functional genes were detected in all samples. In contrast, representatives from 63% of detected functional gene families were present in all samples. This core functional gene set encoded for resistance to several metals, specific organic degradation functions, cellulose degradation, nitrification, denitrification, and, surprisingly, sulfate reduction and methanogenesis. This first examination of the core and dispensable microbiome in an activated sludge bioreactor suggests that activated sludge microbial communities are functionally and phylogenetically highly diverse, but that only a fraction of this diversity constitutes a true core microbiome. In Chapter 5, I resolve a puzzling connection in Chapters 2-4 between microbial community dynamics and small levels of nitrite in the bioreactor influent by demonstrating intra-plant microbial immigration between coupled process units at a full-scale wastewater treatment plant. I provide converging lines of retrospective and prospective evidence that these microbial immigrants may be significant drivers of microbial community dynamics in engineered systems. Quantitative PCR (qPCR) analyses demonstrated accumulation of AOB in a BOD--removal trickling filter and significant immigration to a downstream activated sludge bioreactor. T-RFLP analyses corroborated by clone libraries showed that Nitrosomonas europaea dominated the trickling filter, while a 'Nitrosomonas-like' lineage dominated in activated sludge. N. europaea was previously shown to predominate in activated sludge during elevated bioreactor influent NO2- events, strongly suggesting that activated sludge AOB community dynamics are driven in part by immigration via sloughing from the upstream trickling filter. High-density phylogenetic microarray (PhyloChip) analyses revealed an overabundance of methanogens in the trickling filter relative to the activated sludge bioreactor and demonstrated transport of a diverse heterotrophic assemblage to the activated sludge via the trickling filter effluent. Our results indicate that immigration may play an unexpectedly significant role in the microbial community assembly process in activated sludge bioreactors, with potentially profound implications for design and operation of this widely-used, environmentally and economically important technology. Taken together, my research suggests the utility of coupling fundamental microbial ecology research to bioprocess engineering. I anticipate that, in the long term, the results of my work on bioreactor microbial ecology will lay the framework for enhanced "microbial resource management" strategies for wastewater treatment bioreactors, a critically important application of environmental biotechnology.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Wells, George Fraser
|Stanford University, Civil & Environmental Engineering Department
|McCarty, Perry L
|McCarty, Perry L
|Statement of responsibility
|George Fraser Wells.
|Submitted to the Department of Civil and Environmental Engineering.
|Thesis (Ph.D.)--Stanford University, 2011.
- © 2011 by George Fraser Wells
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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