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Item Understanding Escherichia coli O157:H7 presence, pervasiveness, and persistence in constructed treatment wetland systems(Montana State University - Bozeman, College of Letters & Science, 2015) VanKempen-Fryling, Rachel Joy; Chairperson, Graduate Committee: Anne Camper; Otto R. Stein and Anne K. Camper were co-authors of the article, 'Presence and persistence of wastewater pathogen Escherichia coli O157:H7 in hydroponic reactors of treatment wetland species' in the journal 'Water science and technology' which is contained within this thesis.; Anne K. Camper was a co-author of the article, 'Escherichia coli O157:H7 attachment and persistence within root biofilm of common treatment wetlands plants' submitted to the journal 'Water research ' which is contained within this thesis.; Anne K. Camper was a co-author of the article, 'Using molecular and microscopic techniques to track the wastewater pathogen Escherichia coli O157:H7 within model treatment wetlands' submitted to the journal 'Applied and environmental microbiology' which is contained within this thesis.Treatment wetlands (TW) are a wastewater remediation technology that relies on the natural ability of wetland plant species and the associated microbial consortia to remove pollutants and improve water quality. Although there is substantial research on chemical pollutant remediation by TW, the removal of bacterial pathogens is much more varied and limited in scope. Escherichia coli O157:H7 is a bacterial pathogen that has caused numerous outbreaks and infections in the United States alone and is closely associated with improper water treatment. Understanding how E. coli O157:H7 could potentially persist and survive through a TW process is important in order to appropriately determine the efficacy of TW for treating water and protecting human health. This work used epifluorescent microscopy and qPCR relative DNA abundance to track E. coli O157:H7 tagged with a fluorescent DsRed protein in various environments pertaining to a TW. Two high performing wetland plant species, Carex utriculata and Schoenoplectus acutus, were used in hydroponic and simulated TW columns to better understand how the bacteria localize and persist. Teflon nylon strings (diameter 0.71-1.02 mm), cleaned and with established biofilm, were run hydroponically as control inert surfaces. Unplanted gravel columns were used as a nonplanted control for column experiments. E. coli O157:H7-DsRed were observed by microscopy on root surfaces both in hydroponic reactors and lab scale TW columns. The organisms persisted, forming microcolonies shortly after initial inoculation on both root and nylon surfaces. In the lab scale columns, cells persisted for three weeks, although strong biofilm formation was not observed. qPCR also provided evidence that E. coli O157:H7 was able to persist on the tested surfaces of plant roots, nylon inert surfaces, and gravel, showing higher abundance S. acutus roots than on the inert surface and gravel, however higher in unplanted gravel overall. For the plant types, C. utriculata was statistically lower for E. coli O157:H7 abundance than S. acutus over time. This work provides evidence that E. coli O157:H7 is able to colonize and persist in a TW environment, and plant surfaces may offer a higher inactivation than an inert matrix.Item Analysis of microbial biofilm community composition within constructed wetlands(Montana State University - Bozeman, College of Letters & Science, 2010) Faulwetter, Jennifer Lynn; Chairperson, Graduate Committee: Anne Camper; Vincent Gagnon, Carina Sundberg, Florent Chazarenc, Mark D. Burr, Jacques Brisson, Anne K. Camper and Otto R. Stein were co-authors of the article, 'Microbial processes influencing performance of treatment wetlands: a review' in the journal 'Ecological engineering' which is contained within this thesis.; Mark D. Burr, Otto R. Stein and Anne K. Camper were co-authors of the article, 'Characterization of sulfate reducing bacteria in constructed wetlands' in the journal 'Proceedings of the 11th international conference on wetland systems for water pollution control, Indore, India' which is contained within this thesis.; Mark D. Burr, Albert E. Parker, Otto R. Stein and Anne K. Camper were co-authors of the article, 'The effect of plant species and sample location on bacterial biofilm communities associated with constructed wetland microcosms' in the journal 'The international society for microbial ecology journal' which is contained within this thesis.; Mark D. Burr, Albert E. Parker, Otto R. Stein and Anne K. Camper were co-authors of the article, 'The influence of sulfate reducing bacteria and ammonia oxidizing bacteria on nutrient cycling in constructed wetland microcosms' in the journal 'Microbial ecology' which is contained within this thesis.; Mark D. Burr, Alfred B. Cunningham, Frank M. Stewart, Anne K. Camper and Otto R. Stein were co-authors of the article, 'Floating treatment wetlands for domestic wastewater treatment' in the journal 'Water science and technology' which is contained within this thesis.Constructed wetlands (CWs) are ecologically-based water treatment systems that provide cost-effective amelioration of waterborne pollutants. Fundamental understanding of removal mechanisms, especially microbial processes, limits greater usage of constructed wetlands as a wastewater treatment system. The influence of plant species selection, season, and organic load rate on pollutant removal was previously linked to the redox condition of the sub-surface wetland environment. The goal of this research was to determine which of these environmental variables (including spatial location within the CW) influenced the dominant microbial populations and/or the activity of various sub-populations. Once identified, a constructed wetland might be optimized for growth of microorganisms involved in removal of a specific pollutant. To assess environmental factors, microbial population samples were taken in six locations (effluent, 3 root and 2 gravel areas) within replicate unplanted microcosms and wetland microcosms planted with Deschampsia cespitosa or Leymus cinereus during the summer (24°C) and winter (4°C) seasons. Microcosms were fed a synthetic domestic wastewater in 20-day batches for at least 12 months prior to sampling. The most recent techniques in molecular biology including denaturing gradient gel electrophoresis (DGGE) and quantitative PCR were utilized and included treatment with and without propidium monoazide (PMA) to distinguish between "live" and "dead" microbial communities. Primer sets targeted the entire bacterial community (16S rDNA) and two functional groups, nitrifying bacteria (amoA gene) and sulfate reducing bacteria (dsrB gene). Results indicated that overall microbial community structure (16S rDNA) was affected by general location within the microcosm (effluent, root, gravel) as well the plant species present. Specific microbial groups appeared to be affected differently with relative gene quantities of sulfate reducing bacteria and nitrifying bacteria being influenced by a combined effect of plant species and season. For dsrB, D. cespitosa had the lowest relative gene quantities overall. Both genes were more abundant in the summer season, indicating seasonal importance. Location within the microcosms was also important, with anoxic environments (column bottom) being more important for dsrB presence and a diverse population of cultivated sulfate reducers. The roots were an important location for both microbial diversity and activity for all genes investigated.