Microbial metabolism reacts quickly to environmental conditions. These reactions are dependent on the need for nutrients and respiration and can be measured using an assay of individual microbial enzyme activities (MEA’s). In this study, we measured MEA’s in the sediments of a stream in northeast Tennessee that had an approved fecal coliform Total Maximum Daily Load (TMDL). These values were compared to biochemical oxygen demand (BOD), phosphate concentration and nitrate concentration in the water column of this stream. Comparisons were grouped by season and land use. Stream sediments and water were collected monthly for one year and then quarterly for an additional two years at 14 sites located in agricultural, urban and forest regions. Dehydrogenase (DHA), a measure of microbial respiration, along with acid phosphatase (AcidPA), alkaline phosphatase (AlkPA), galactosidase (GalA) and glucosidase (GluA) activities were measured using colorimetric assays. BOD was determined using the standard 5-day BOD test (BOD5). Nitrate and phosphate concentrations were measured using colorimetric procedures. There were significant positive and negative correlations (p5, DHA vs. nitrate concentration, and DHA vs. phosphate concentration. Also in the fall months there were significant negative correlations between GalA and GluA vs. BOD5, and concentrations of nitrate and phosphate. There was also a negative correlation between AcidPA and BOD5. In the warmer months of spring and summer, there were positive correlations between AcidPA, AlkPA, GalA and GluA vs. the BOD5 ’s, and the concentrations of nitrate and phosphate. The only negative correlation in a warmer season was in the summer between AlkPA vs. BOD5 and phosphate concentration. No significant correlations were found by land use type. Results indicate that significant relationships may exist between MEA’s and other water quality measures (e.g. BOD5, nitrate concentration, and phosphate concentration) that could make it possible to use MEA’s as another tool for water quality assessment.

Four creeks within the Watauga River watershed in Northeast Tennessee are routinely monitored for water quality assessments. To identify sources and monitor remediation, Sinking Creek, Cash Hollow Creek, Buffalo Creek and Boones Creek are monitored for chemical and microbial parameters. These parameters include phosphates, nitrates, BOD and fecal coliforms. Sinking Creek is a tributary of the Watauga River with 10 miles of impaired water. Cash Hollow Creek enters the Watauga River at river mile 11.4 with 3.4 miles of impaired water. Boones Creek contains 18.6 impaired miles while the status of water quality in Buffalo Creek is not yet determined. Agricultural input is a major source of pollution for Sinking and Boones Creek. Cash Hollow Creek is impacted by a combination of sources of which urban runoff is the largest due to storm sewers and land development. Boones, Cash Hollow and Sinking Creeks are considered impaired and are on the state’s 303(d) list due to pathogen loading but only Sinking and Cash Hollow Creek have TMDLs. The seasonal and spatial patterns are more obvious for microbial than for chemical parameters. From 2002 - 2005, 14 stations on Sinking Creek were sampled quarterly. Fecal coliforms were elevated and always greater than 200 CFU/100ml for stations 1 – 5. Due to agricultural land use adjacent to stations 1 – 4, this would be expected. There was also a seasonal trend with higher concentrations found in the fall and spring. Cash Hollow Creek’s 9 stations were sampled monthly from 2002 - 2005. Although very high fecal coliforms concentrations were found, there were no obvious patterns. The 12 stations on Buffalo Creek were sampled quarterly from June 2004 to June 2005. Fecal coliform concentrations were high at station 8, which is adjacent to agricultural land. Boones Creek was sampled monthly from March 2005 to present and no obvious trends have been noted. The objective of this research is to compare patterns in these geographically similar creeks to identify any common patterns associated with various pollution sources. We will discuss the preliminary results and conclusions about the usefulness of these data to accomplish this objective.

Microbial enzyme activities (MEA’s) are measurements of microbial metabolism. These activities are dependent on the need for nutrients and respiration. This extended study evaluated four streams in the same watershed that had an approved fecal coliform Total Maximum Daily Load. Sediment and water samples were collected monthly for the first year of each specific stream study, and then quarterly to the end of 2006. Dehydrogenase, a measure of microbial respiration, along with acid phosphatase, alkaline phosphatase, galactosidase and glucosidase activities were measured using colorimetric assays. Biochemical oxygen demand (BOD) was determined using the standard 5-day test (BOD5). Nitrate and phosphate concentrations were measured using colorimetric procedures. Sediment MEA values were compared to the BOD, nitrate concentration and phosphate concentration in the overlying water. Seasonal means of each parameter were not significantly different (p5, nitrate concentration, and phosphate concentration). This suggests to us that MEA’s may be an alternative tool for water quality assessments.

Diarrheal diseases are a global public health burden, killing 1.8 million people annually. Diarrhea disproportionately affects children and those in poverty. Most diarrheal cases can be prevented through safe drinking water, basic hygiene and/or sanitation measures, with drinking water interventions having the most impact on reducing diarrheal disease. There is no generally agreed-upon field method for determining biosand water filter effectiveness that is usable in low-resource communities. A pilot study was conducted of potential field use indicators, including the Colilert coliform presence/absence (P/A) test, hydrogen sulfide, alkalinity, hardness, pH, and fluorescently labeled latex microspheres. The study included both laboratory and field testing. The Colilert P/A test had the highest correlation to the United States Environmental Protection Agency standard method (IDEXX Quanti-trays), but more data are needed before making a recommendation. This study adds to understanding about evaluation of biosand water filters and provides preliminary data to address the need for a field use indicator for biosand water filters.

More than 270,000 km of rivers and streams are impaired due to fecal pathogens, creating an economic and public health burden. Fecal indicator organisms such as Escherichia coli are used to determine if surface waters are pathogen impaired, but they fail to identify human health risks, provide source information, or have unique fate and transport processes. Statistical and machine learning models can be used to overcome some of these weaknesses, including identifying ecological mechanisms influencing fecal pollution. In this study, canonical correlation analysis (CCorA) was performed to select parameters for the machine learning model, Maxent, to identify how chemical and microbial parameters can predict E. coli impairment and F+-somatic bacteriophage detections. Models were validated using a bootstrapping cross-validation. Three suites of models were developed; initial models using all parameters, models using parameters identified in CCorA, and optimized models after further sensitivity analysis. Canonical correlation analysis reduced the number of parameters needed to achieve the same degree of accuracy in the initial E. coli model (84.7%), and sensitivity analysis improved accuracy to 86.1%. Bacteriophage model accuracies were 79.2, 70.8, and 69.4% for the initial, CCorA, and optimized models, respectively; this suggests complex ecological interactions of bacteriophages are not captured by CCorA. Results indicate distinct ecological drivers of impairment depending on the fecal indicator organism used. Escherichia coli impairment is driven by increased hardness and microbial activity, whereas bacteriophage detection is inhibited by high levels of coliforms in sediment. Both indicators were influenced by organic pollution and phosphorus limitation.

Background.The leading cause of surface water impairment in United States’ rivers and streams is pathogen contamination. Although use of fecal indicators has reduced human health risk, current approaches to identify and reduce exposure can be improved. One important knowledge gap within exposure assessment is characterization of complex fate and transport processes of fecal pollution. Novel modeling processes can inform watershed decision-making to improve exposure assessment.

In June 2008, the Cedar River crested flooding more than 5,000 Cedar Rapids homes. Residents whose homes were flooded were invited to participate in this study. We characterized exposures and symptoms experienced by individuals inhabiting 73 flood-damaged homes. Exposures and questionnaire-based health assessments were compared at two levels of remediation, in-progress and completed. Homes with remediation in-progress (n=24), as compared to the completed homes (n=49), had significantly higher airborne concentrations of mold, bacteria, iPM, endotoxin and glucan. Residents of in-progress homes had a significantly higher prevalence of doctor diagnosed allergies (adjusted OR=3.08; 95%CI: 1.05-9.02) and all residents had elevated prevalence of self-reported wheeze (adjusted OR=3.77; 95%CI: 2.06-6.92) and prescription medication use for breathing problems (adjusted OR=1.38; 95%CI: 1.01-1.88) after the flood as compared to before. Proper post-flood remediation led to improved air quality and lower exposures among residents living in flooded homes. Recognition of endotoxin as a proinflammatory ligand for pattern recognition receptors has increased the demand for endotoxin assessment in studies of environmental lung disease. Measurements using the Limulus amebocyte lysate (LAL) assay of air and reservoir dust samples are routinely incorporated into epidemiologic studies. However, it is unknown if endotoxin reactivity in the LAL assay varies by its physical presentation as aggregates, as membrane components of whole bacteria or as shed membrane blebs or if this parallels differences in the inflammatory potency of endotoxin in vivo. Endotoxins as14C-labeled-lipooligosaccharide (14C-LOS) and 14C- labeled-lipopolysaccharide (14C-LPS) were produced from Neisseria meningitidis and Escherichia coli. The reactivity of the endotoxin presentations was assessed in the LAL assay and in vivo using a murine model. The LAL assay significantly underestimated the quantity of endotoxin in the whole bacteria form whereas there was no significant difference in detecting endotoxin in aggregate and bleb forms. The failure of the LAL assay to equally quantify endotoxin was not mirrored in vivo where all three presentations of endotoxin were equally inflammatory. The inability of the LAL assay to detect the full quantity of endotoxin presented in the whole bacteria form has troubling implications for exposure assessment studies. Various extraction methods were applied to samples of known endotoxin quantity to improve the detection ability of the LAL assay. Extraction using EDTA and Tris/EDTA significantly improved the detection of endotoxin compared to the reference method of extracting in pyrogen-free water. These extraction methods also significantly increased the quantity of endotoxin measured in house and barn dust samples. A higher quantity of endotoxin measured in the LAL assay corresponded to a higher neutrophilic response in vivo. A standardized methodology for endotoxin detection that mimics the in vivo response is necessary for accurate and consistent endotoxin analysis.

‘Aigarchaeota’, a deeply branching lineage in the domain Archaea with no cultivated representatives, includes both thermophilic and hyperthermophilic microorganisms that reside in terrestrial and marine geothermal environments. The ‘Aigarchaeota’ consists of at least nine proposed genus-level groups that have been confirmed via 16S rRNA sequencing, with ‘Aigarchaeota’ Group 1 (AigG1) being the focus of this study. Based on cultivation-independent genomic data available from several AigG1 members in Great Boiling Spring (GBS), NV, and Yellowstone National Park, 22 different types of growth media were designed and tested for their ability to support growth of AigG1. One of these cultures, G1-10, was found to contain AigG1 at ~5% abundance, as well as other novel thermophilic microbial groups including a new species of the genus Pyrobaculum, members of the candidate phyla ‘Calescamentes’ and ‘Fervidibacteria’, and the novel archaeal lineage NAG1 (‘Geoarchaeota’). To attempt to obtain pure cultures of AigG1 and other novel thermophiles, a single-cell sorting system using an optical trap and a microfluidic device was constructed. The system was validated by sorting E. coli cells, which demonstrated that single, viable cells could be reliably obtained. Using this single cell sorting device on the G1-10 culture, a pure culture of a member of the genus Pyrobaculum was obtained, which was shown to represent a distinct species in this phylum by whole genome sequencing and in silico DNA-DNA hybridization. Additionally, a pure culture of the first representative of the candidate phylum ‘Fervidibacteria’ from an enrichment culture derived from G1-10. Additionally, to aid in morphology-based sorting of AigG1 and stable isotope labeling studies, fluorescence in situ hybridization (FISH) based on catalyzed reporter deposition (CARD-FISH) were developed and an AigG1-specific probe was tested. CARD-FISH was successfully used to detect AigG1 in both the G1-10 culture and in natural sediment samples from GBS. Stable isotope labeling incubations were performed with a variety of 13C-labeled substrates (bicarbonate, amino acids, sugars, and short chain fatty acids) on GBS sediments and G1-10 culture samples, and CARD-FISH was used to specifically detect AigG1 in the fixed samples. Nanometer-scale secondary-ion mass spectrometry (nano-SIMS) will then be used to determine whether AigG1 was capable of taking up the different carbon substrates tested. Overall, the results and accomplishments from this project and follow up nano-SIMS analysis will allow a better understanding of the metabolic potential of AigG1 and will aid future efforts to attempt to obtain pure cultures of this novel lineage.

The objective of this thesis was to investigate BiOWiSHTM-Aqua, a commercial dry solid formulation containing a consortium of bacteria and yeast, as a biopesticide for treatment of Botrytis cinerea, a gray mold that affects strawberries. BiOWiSHTM-Aqua was compared with another commercial product specifically used as a fungicide and bacteriocide, Serenade® Garden Disease Control Spray (concentrated Bacillus subtilis strain QST 713). Both laboratory tests as well as in vivo lab tests were conducted. BiOWiSHTM-Aqua results varied widely from plate to plate, regardless of experimental conditions. In some of these plates, inhibition zones were observed around colonies from BiOWiSHTM-Aqua, indicating efficacy. The organism responsible for the inhibition zones of B. cinerea growth was isolated from BiOWiSHTM-Aqua, and 16s rRNA analysis identified this culture as a strain of B. subtilis. This strain was designated as B. subtilis ssp. KLB. The B. subtilis KLB concentration required to completely inhibit B. cinerea was 9.1x104 CFU/mL when B. subtilis KLB was inoculated 48 hours before B. cinerea, 1.3x105 CFU/mL at 24 hours, and 3.2x106 CFU/mL when both were inoculated at the same time. Various preliminary experiments using B. subtilis KLB were also conducted to investigate its economic feasibility, to characterize the organism, and to test its post-harvest in vivo viability. B. subtilis KLB cell concentration was 1.6x109 CFU/mL in a bioreactor with LB at the end of the log growth phase. B. subtilis KLB achieved cell concentrations as high as 5x109 CFU/mL in shake flasks with food-grade tapioca as a carbon source. Inoculation of B. subtilis KLB on post-harvest strawberries did not have an effect on Botrytis infection rates compared to the negative control. These various experiments were the first step in research to potentially produce B. subtilis KLB on a commercial scale.

The biodiversity of two distinct marine environments was observed to describe the biogeocomplexity of these extreme ecological systems. A shallow-water hydrothermal vent in Papua New Guinea served as a study of a thermophilic ecosystem influenced by arsenic rich vent fluids while a 60 m deep offshore primarily anoxic karst sink served as a study of an anaerobic sulfur-influenced habitat. Both environments support unique biological communities that are influenced by the physical and chemical pressures imposed on them by the harsh conditions of these systems. In Tutum Bay, Ambitle Isle, Papua New Guinea, a transect was created from a shallow hydrothermal vent that extended 120 m away from the vent. Previous studies have shown that the geochemistry of the system is heavily influenced by arsenic which is toxic to most organisms. In this study, macro- and meiofauna were collected and scored and combined with bacterial sequence data collected along the length of the transect. It was found that near vent sites harbored biological communities more similar than sites further from the vent. Many species were found only at sites near the hydrothermal vent. Near-vent communities were less diverse than those away from the vent, and biodiversity generally increased as distance from the vent increased. Distinct correlations between thermophilic organisms and temperature were observed. The metabolic repertoire of the microbial communities suggests that many strategies are used to obtain energy and carbon. The relative abundance of bacteria containing genes to reduce arsenic was comparable to those able to reduce sulfur compounds. Primary production appeared to be a mix of chemo- and phototrophy. Food webs and association analysis suggest a complex interplay between macrofaunal, meiofaunal and bacterial communities. While the system is heavily influenced by arsenic, no specific correlation between the relative abundance of arsenic metabolizing organisms and the amount of arsenic in the system could be drawn. This is likely due to the fact that most of the arsenic produced by the system is readily adsorbed onto iron oxyhydroxides, reducing the arsenic's bioavailability. The anoxic conditions at Jewfish sink provide a different hurdle than the hot arsenic conditions found in Papua New Guinea. The anoxic conditions are shared by other pit features found in karst geography, but the metabolic processes between Jewfish sink and these other karst habitats are different. The blue holes and black holes of the Bahamas are some of the most well-studied of these karstic pits. In these features, which are large circular pits with diameters of over 300 m, light and sulfur are used as a means of energy acquisition. Jewfish sink, having an opening only 6 m in diameter, is light restricted compared to these systems. As a result, the strategy of organisms dwelling in the anoxic conditions of the sink is different than those found at the well-studied holes in the Bahamas. Geochemical measurements were recorded over two time periods spanning a combined total of 6 years. The anoxic bottom waters of Jewfish sink remain stable and contained high levels of sulfide throughout most of the seasons studies. Sequence analysis of prokaryotes within the sink showed that sulfur reducers had the highest relative abundance compared to other functional guilds. To monitor the changes of the microbial communities within the sink, bacterial communities were examined at 4 depths within the sink at 9 different intervals over a period of 685 days. Denaturing Gradient Gel Electrophoresis (DGGE) was used to fingerprint 16s rRNA bacterial communities and dissimilatory sulfite reducing communities by targeting the 16s rRNA bacterial gene and the dsr gene associated with dissimilatory sulfite reducing bacteria and archaea. The lowest depth studied within the sink (40 m) remained stable chemically and biologically until a turnover event occurred within the second winter of the study. This turnover event disrupted the biological communities at 40 m and led to a reestablished community comprised of different species that those found prior to the event. Upper waters within the sink show that clines establish themselves seasonally and partition zones that confine bacterial communities that are more similar to each other within these zones while excluding bacterial communities that are outside of these zones. Oxygenated water was shown to not contain prokaryotes containing the dsr gene. As the oxycline changed seasonally, dissimilatory sulfite reducing prokaryotes containing the dsr gene remained in the anoxic zone and required time to reestablish themselves whenever oxygenated water displaced them.