VTechWorks staff will be away for the Thanksgiving holiday beginning at noon on Wednesday, November 27, through Friday, November 29. We will resume normal operations on Monday, December 2. Thank you for your patience.

Browsing by Author "Hagedorn, Charles III"

Now showing 1 - 20 of 47
  • Thumbnail Image
    Antimicrobial Producing Bacteria as Agents of Microbial Population Dynamics
    Tanner, Justin Rogers (Virginia Tech, 2010-10-27)
    The need for new antibiotics has been highlighted recently with the increasing pace of emergence of drug resistant pathogens (MRSA, XDR-TB, etc.). Modification of existing antibiotics with the additions of side chains or other chemical groups and genomics based drug targeting have been the preferred method of drug development at the corporate level in recent years. These approaches have yielded few viable antibiotics and natural products are once again becoming an area of interest for drug discovery. We examined the antimicrobial "Red Soils" of the Hashemite Kingdom of Jordan that have historically been used to prevent infection and cure rashes by the native peoples. Antimicrobial producing bacteria were present in these soils and found to be the reason for their antibiotic activity. After isolation, these bacteria were found to excrete their antimicrobials into the liquid culture media which we could then attempt to isolate for further study. Adsorbent resins were employed to capture the antimicrobial compounds and then elute them in a more concentrated solution. As part of a drug discovery program, we sought a way to quickly characterize other soils for potential antibiotic producing bacteria. The community level physiologic profile was examined to determine if this approach would allow for a rapid categorizing of soils based on their probability of containing antimicrobial producing microorganisms. This method proved to have a high level of variability that could not be overcome even after mixing using a commercial blender. The role of these antimicrobial producing bacteria within their natural microbial community has largely been confined to microbe-plant interactions. The role of antimicrobial-producing microorganisms in driving the diversity of their community has not been a focus of considerable study. The potential of an antimicrobial-producing bacterium to act as a driver of diversity was examined using an artificial microbial community based in a sand microcosm. The changes in the microbial assemblage indicate that antimicrobial-producing bacteria may act in an allelopathic manner rather than in a predatory role.
  • Thumbnail Image
    Associations between Fecal Indicator Bacteria Prevalence and Demographic Data in Private Water Supplies in Virginia
    Smith, Tamara L. (Virginia Tech, 2013-06-12)
    Over 1.7 million Virginians rely on private water systems to supply household water. The heaviest reliance on these systems occurs in rural areas, which are often underserved in terms of financial resources and access to environmental health education. As the Safe Drinking Water Act (SDWA) does not regulate private water systems, it is the sole responsibility of the homeowner to maintain and monitor these systems. Previous limited studies indicate that microbial contamination of drinking water from private wells and springs is far from uncommon, ranging from 10% to 68%, depending on type of organism and geological region. With the exception of one thirty-year old government study on rural water supplies, there have been no documented investigations of links between private system water contamination and household demographic characteristics, making the design of effective public health interventions, very difficult. The goal of the present study is to identify potential associations between concentrations of fecal indicator bacteria (e.g. coliforms, E. coli) in 831 samples collected at the point-of-use in homes with private water supply systems and homeowner-provided demographic data (e.g. homeowner age, household income, education, water quality perception). Household income and the education of the perceived head of household were determined to have an association with bacteria concentrations. However, when a model was developed to evaluate strong associations between total coliform presence and potential predictors, no demographic parameters were deemed significant enough to be included in the final model. Of the 831 samples tested, 349 (42%) of samples tested positive for total coliform and 55 (6.6%) tested positive for E. coli contamination. Chemical and microbial source tracking efforts using fluorometry and qPCR suggested possible E. coli contamination from human septage in 21 cases.  The findings of this research can ultimately aid in determining effective strategies for public health intervention and gain a better understanding of interactions between demographic data and private system water quality.
  • Thumbnail Image
    Biological and Chemical Renovation of Wastewater with a Soil Infiltrator Low-Pressure Distribution System
    DiPaola, Tracey Stickley (Virginia Tech, 1998-06-23)
    An alternative on-site wastewater treatment and disposal system (OSWTDS) consisting of a soil infiltrator with low pressure distribution was evaluated in a soil that was unsuitable for a conventional OSWTDS under current Commonwealth of Virginia Sewage Handling and Disposal Regulations, due to a shallow seasonally perched water table and low hydraulic conductivity. The absorption field consisted of two subsystems numbered as 1 and 2 with effluent design loading rates of 5.1 and 10.2 Lpd/m2, respectively (actual loading rates of 2.4 and 4.9 Lpd/m2, respectively). Soil matric potentials compared seasonally for each subsystem and indicated that both provided similar hydraulic performance. Background water quality was generally improved by subsurface movement through the absorption fields. A bacterial tracer was found in shallow (45.7 cm) and deep (213.4 cm) sampling wells within 24 h in the two subsystems (but in low numbers) over both summer and winter sampling periods. A viral tracer was detected within 48 h in both shallow and deep wells, but only in subsystem 2 in the winter. In evaluating denitrification potential, the addition of glucose to soil core samples did increase quantitatively, although not significantly, nitrous oxide production in each subsystem, at each depth, during each season. Overall, the performance of both subsystems was very similar. The soil infiltrator functioned very well, as designed for the site and soil limitations. It appears to be a potential alternative OSWTDS for use in problem soils.
  • Thumbnail Image
    A Comparative Study of Three Bacterial Source Tracking Methods and the Fate of Fecal Indicator Bacteria in Marine Waters and Sediments
    Irvin, Renee Danielle (Virginia Tech, 2010-11-30)
    E. coli and Enterococcus were used to determine the fate and survival of fecal indicator bacteria (FIB) in sand and sediments. The microbial source tracking (MST) methods antibiotic resistance analysis (ARA), Bacteroides human-specific primer test, and fluorometry were compared against the FIBs to determine how reliable each method was in detecting the presence of human fecal contamination. Two phases (Summer 2009 and 2010) were evaluated based on the type of contamination event. A combined sewage overflow (CSO) event was simulated in Phase I, where large amounts of influent were added to sand and bay water columns over 1 to 4 days. In 2010, a low volume sewage leak was simulated in which smaller doses of influent were added to sand and bay water columns over a period of 5 to 15 days. Within each of the phases, both non- and re-circulated columns were also evaluated. Evaluation of FIB survival indicated that Enterococcus was able to stabilize and re-grow in the water and at the sediment/water interface within the Phase I non-circulated columns. E. coli was unable to re-grow and/or stabilize within any environment. Comparisons between the ARA and the FIBs revealed a large majority of isolates identified as coming from either bird or wildlife sources. Human sources were identified but at much lower concentrations than expected. Bacteroides results indicated strong relationships between the increase of FIB concentrations and the presence of the human-specific Bacteroides. Fluorometry results did not indicate any relationship with the FIBs. Unexpectedly, fluorometry readings increased as time progressed indicating that another compound was present that fluoresced at the same wavelength as optical brighteners (OBs). This project was one of the first to study the differences related to two different pollution events (CSO vs. sewage leak) while also evaluating what happens to pollution as it settles into the sediment. It was also unique because it compared bacterial (ARA), molecular (Bacteroides), and chemical (fluorometry) MST methods.
  • Thumbnail Image
    Comparison of Enterococcus Species Diversity in Marine Water and Wastewater Using Enterolert and EPA Method 1600
    Ferguson, Donna M.; Griffith, John F.; McGee, Charles D.; Weisberg, Stephen B.; Hagedorn, Charles III (Hindawi, 2013-06-10)
    EPA Method 1600 and Enterolert are used interchangeably to measure Enterococcus for fecal contamination of public beaches, but the methods occasionally produce different results. Here we assess whether these differences are attributable to the selectivity for certain species within the Enterococcus group. Both methods were used to obtain 1279 isolates from 17 environmental samples, including influent and effluent of four wastewater treatment plants, ambient marine water from seven different beaches, and freshwater urban runoff from two stream systems. The isolates were identified to species level. Detection of non-Enterococcus species was slightly higher using Enterolert (8.4%) than for EPA Method 1600 (5.1%). E. faecalis and E. faecium, commonly associated with human fecal waste, were predominant in wastewater; however, Enterolert had greater selectivity for E. faecalis, which was also shown using a laboratory-created sample. The same species selectivity was not observed for most beach water and urban runoff samples. These samples had relatively higher proportions of plant associated species, E. casseliflavus (18.5%) and E. mundtii (5.7%), compared to wastewater, suggesting environmental inputs to beaches and runoff. The potential for species selectivity among water testing methods should be considered when assessing the sanitary quality of beaches so that public health warnings are based on indicators representative of fecal sources.
  • Thumbnail Image
    Control of Volatile Organic Compound (VOC) Air Pollutants
    Hunter, Paige Holt (Virginia Tech, 2000-06-01)
    A variety of methods exist to remove volatile organic compound (VOC) air pollutants from contaminated gas streams. As regulatory and public opinion pressures increase, companies are searching for more effective methods to control these emissions. This document is intended as a guide to help determine if existing systems are adequate and to provide additional information to improve the efficiency of the systems. It explores conventional methods of controlling VOC emissions, as well as innovative technologies including membrane separation, plasma destruction, and ozone catalytic oxidation. The conventional technologies covered include condensation, adsorption, absorption (or scrubbing), thermal incineration, flaring, catalytic incineration, and biofiltration. Each chapter includes a description of the technology, a discussion of the types of systems available, notes on the design of the system, economic estimates, an explanation of potential problems, and a list of considerations for installation and maintenance concerns. The final chapter is dedicated to the preparation and characterization of metal catalysts which were developed to improve the reaction rate of VOCs using ozone as an oxidant.
  • Thumbnail Image
    Denitrification in low pressure distribution onsite wastewater disposal systems
    Degen, Marcia J. (Virginia Tech, 1992-12-05)
    The effects of effluent type, effluent loading rate, dosing interval, and temperature on denitrification in low pressure distribution, on-site wastewater treatment and disposal systems (OSWTDS) were evaluated in this study. The treatments were surface and subsurface soil horizons; nitrified and non-nitrified wastewaters; 0.5, 1.0, and 1.5 times the Virginia Department of Health (VDH 1989) recommended wastewater loading rate; 24 and 48 hour dosing intervals; and summer and winter temperatures. Surface and subsurface soil cores were collected from a Groseclose silt loam soil (clayey, mixed, mesic Typic Hapludult) and subjected to the various treatments. The effects of the treatments on denitrification were evaluated based on analyses of leachate from the cores, soil chemical analyses, and microcosm studies to estimate actual denitrification activity. A model was developed from the study that estimated the mean N₂O production for each combination of experimental treatments. The results of the study and the model indicate that denitrification can be enhanced in OSWTDS by the application of non-nitrified wastewater at one-half the VDH recommended loading rate, or 1.25 cm/day, for surface soil horizons (30 min inch⁻¹ percolation rate) using a 48 hour dosing interval. A field study was conducted on a Lowell silt loam soil (fine, mixed, mesic Typic Hapludalf). Denitrification was measured at this site using acetylene blocking and the results compared to those predicted by the denitrification model developed from the laboratory data. The field measurements of denitrification based on N₂O concentration in the soil atmosphere were three orders of magnitude higher than that predicted by the model. It was concluded that the laboratory techniques can be used to determine optimum method of operation for denitrification in a low pressure distribution system, but it cannot be used to determine the field design loading rates.
  • Thumbnail Image
    Denitrification in onsite wastewater treatment and disposal systems
    Degen, Marcia B.; Reneau, Raymond B.; Hagedorn, Charles III; Martens, David C. (Virginia Water Resources Research Center, Virginia Polytechnic Institute and State University, 1991-11)
    The effects of effluent type, effluent loading rate, dosing interval, and temperature on denitrification in onsite wastewater treatment and disposal systems (OSWTDSs) were evaluated in this study. The variables were soil horizon, effluent type, effluent loading rate, dosing interval, and temperature. Surface and subsurface soil cores were collected from a Groseclose silt loam soil (clayey, mixed, mesic Typic Hapludult) and subjected to the following treatments: aerobic and anaerobic effluent, loading rates of 0.5, 1.0, and 1.5 times the Virginia Department of Health (VDH)-recommended levels, 24-hour and 48-hour dosing rates, and summer and winter temperatures. The effects of the treatments on denitrification were evaluated based on analyses of leachate from the cores, soil chemical analyses, and microcosm studies to estimate actual denitrification activity. From the study, a model was developed that predicted the mean nitrous oxide (N20) production for each combination of the experimental treatments. The results of the study and the model indicate that denitrification can be enhanced in OSWTDSs by the application of anaerobic effluent at the VDH-recommended effluent loading rate to surface soil horizons using a 48-hour dosing interval. A field study was conducted on a Lowell silt loam soil (fine, mixed, mesic Typic Hapludalf). Denitrification was measured at this site using acetylene blocking, and the results compared to those predicted by the denitrification model developed from the laboratory data. The field measurements of denitrification based on N20 concentration in the soil atmosphere were almost three orders of magnitude higher than that predicted by the model.
  • Thumbnail Image
    Determining fecal bacterial profiles of a human-habituated wild chimpanzee population in Mahale Mountains National Park, Tanzania
    Szekely, Brian (Virginia Tech, 2009-05-04)
    Intestinal flora of wild chimpanzee has not been studied. Fecal flora analyses currently give insight to this environment. We collected feces from twelve human-habituated wild chimpanzees in each of three age groups: four juveniles, four sub-adults, and four adults. We analyzed fecal samples using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) of amplified 16S rRNA genes to determine bacterial diversity present. Between 1 and 14 terminal-restriction fragments (T-RFs) were observed in each sample. A total of 26 unique T-RFs were produced from the samples and ranged in size from 92 to 837 base pairs (bps). Twenty-four of these T-RFs corresponded to five bacterial phyla: Actinobacteria, Bacteroidetes, Firmicutes, Mollicutes, and Proteobacteria, as well as uncultured and unidentified bacterial species. The remaining T-RFs corresponded solely to uncultured or unidentified bacteria. Firmicutes was the most common phylum, observed in 11 of the samples. Bacteroidetes was the second-most common phylum, detected in 8 of the samples. Principal Components Analysis (PCA) revealed a discrete clustering of 10 samples when looking at components one and two, and a clustering of 11 samples when looking at component three. These three components accounted for 72.5% of the variation within the data. Morisita indices were computed to compare T-RF profiles of two samples at a time, and were between 0 and 0.886. Results indicated that some fecal bacterial profiles were similar in the study group, but ultimately varied between samples when compared two at a time. Specific diet, physiology, and environmental reservoir exposure may play large roles in shaping such profiles.
  • Thumbnail Image
    Determining Sources of Fecal Contamination in Two Rivers of Northumberland County, Virginia
    Szeles, Cheryl Lynne (Virginia Tech, 2003-04-09)
    The goal of monitoring the water quality of shellfish beds is to provide protection against transmission of water-borne infectious diseases. The Coan River and the Little Wicomico River contain shellfish beds that are closed to harvest due to contamination with fecal bacteria. These two rivers are located in Northumberland County, Virginia, and empty into the lower Potomac River and the Chesapeake Bay. Bacterial source tracking (antibiotic resistance analysis of Eschericia coli) was used to determine the sources of fecal contamination that have caused shellfish harvest closures in these two rivers. A total of 1,248 Eschericia coli isolates were collected from known sources to build a regional library for the rivers. The Virginia Department of Shellfish Sanitation (DSS) and project cooperators collected known source samples from August 2001 to September 2002. The Average Rate of Correct Classification for the known source library was 71.9%, with a total of 930 isolates correctly classified. The categories (and rates of correct classification) were Birds (84.7%), Humans (74.8%), Livestock (72.4%), Pets (62.0%), and Wildlife (65.7%). The library was used to identify the sources of Eschericia coli isolated from DSS sampling stations along the Coan and Little Wicomico Rivers from August 2001 to September 2002. Some stations contained a substantial human signature, while wildlife and birds are also major contributors. The results will be used to decide the necessary changes that need to be addressed if the shellfish harvesting beds are to be reopened.
  • Thumbnail Image
    Determining Sources of Fecal Pollution in the Blackwater River Watershed, Franklin County, Virginia
    Bowman, Amy Marie (Virginia Tech, 2001-08-02)
    Antibiotic resistance analysis (ARA) was used to determine sources of fecal pollution in the Blackwater River in South-central Virginia. The Department of Environmental Quality designated six segments as impaired due to high fecal coliform concentrations with non-point source (NPS) agriculture the suspected source of impairment. The Blackwater River watershed encompasses 72,000 ha of dairy, beef, and intensive production agriculture, abundant wildlife populations and many homes with onsite septic systems. A library of antibiotic resistance profiles based on 30 concentrations of 9 antibiotics was developed for 1,451 enterococci isolates from human, cattle, chicken, horse, goat, sheep, deer, raccoon, muskrat, goose, duck, coyote, and wild turkey fecal samples. Each isolate was classified as human, wildlife or livestock. Correct classification rates were 82.3% for human, 86.2% for livestock and 87.4% for wildlife isolates when profiles were analyzed with discriminant analysis. Profiles were also determined for 48 isolates from 128 stream samples collected periodically from August 1999 thru April 2001 and compared to the known sources using discriminate analysis. A human signature was found at each site at least once during the year, ranging from 0.0% to 85.0% of the sample isolates. The livestock signature varied from 2.3% to 100% over sites and months, and the wildlife signature varied from 0.0% to 79.5%. The results indicate that both humans and wildlife contribute to fecal pollution in addition to the suspected source, livestock, and reducing fecal pollution will require consideration of all three sources. The results from this research are being used to develop a total maximum daily load (TMDL) project allocations for fecal coliforms in the Blackwater River. Isolates identified by ARA were also profiled using the Biolog metabolic identification system. A library of metabolic profiles was constructed from known source isolates. Stream isolates were identified by Biolog and the metabolic profile was compared to the Biolog library. Of ten stream isolates identified by ARA as human, the Biolog library identified one as human, four as livestock, and five as wildlife. Of ten isolates identified by ARA as livestock, the Biolog library identified seven as livestock and three as wildlife. Of ten isolates identified by ARA as wildlife, one was identified as human, three as livestock and six as wildlife. The overall correct classification of Blackwater isolates in the Biolog library was 14 of 30 isolates, or 47%. Although the Biolog library was constructed with some isolates from the Blackwater basin, there may not be enough isolates in the Biolog library to adequately represent the variability shown by the Blackwater isolates, resulting in lower than expected correct classifications. In spite of these results, Biolog remains promising as one of several tools with potential as a bacterial source tracking method.
  • Thumbnail Image
    Determining Sources of Fecal Pollution in Washington D.C. Waterways
    Porter, Kimberly Rae (Virginia Tech, 2003-11-19)
    Antibiotic resistance analysis (ARA) of Enterococci was used to determine sources of fecal contamination in three District of Columbia waterways: Rock Creek, the Anacostia River, and the Potomac River. These three waterways were identified as exceeding water quality standards set for fecal coliform levels and were designated by the District of Columbia to the Environmental Protection Agency's 303 (d) impaired waters list. A library profile of 1,806 enterococcus isolates from known sources was built based on antibiotic resistance patterns from thirty concentrations of nine antibiotics. These sources included human, cattle, chicken, horse, goat, sheep, deer, raccoon, muskrat, goose, seagull, coyote, duck, wild turkey, dog, and cat. Antibiotic profiles were characterized for 24 unknown enterococci isolates on each of 198 samples (38 samples from the Potomac River, 79 samples from the Anacostia River, and 81 samples from Rock Creek) collected periodically from July 2002 through April 2003. Two major storm events were also sampled during this period. These isolate profiles were compared to the known source library using logistic regression. Three dominant sources of fecal pollution were detected in the Potomac River: livestock (30%), human (29%), and wildlife (22%). Three dominant signatures were also detected in Rock Creek: horse (26%), human (26%), and wildlife (24%). Human was the only dominant source detected in the Anacostia River, averaging 43% over the sampling period. The results of this study indicate that human is a substantial contributor to the fecal contamination problems, especially in the Anacostia River, but there are significant agricultural and wildlife contributions as well. Significant and predictable seasonal variations were also detected, indicating the influence of precipitation on source distributions. The results of this study will aid the Metropolitan Washington D.C. Council of Governments in making important management decisions to help improve the water quality in and around the Washington D.C. area. Expanding the limits of ARA was also an integral part of this research. Three new and even controversial analytical techniques were run on the data collected from this project in an attempt to improve confidence and provide direction to the results of this study. The first was a comparison of the more commonly used statistical analysis model discriminate analysis (DA) with logistic regression (LR). No significant difference was found between the output of the two models for the known source libraries, therefore no suggestion could be made in favor of one model over the other. Another analytical test of the data was the introduction of a standard requiring isolates to meet a minimum of 80% similarity to the known source profiles where it was classified. With the 80% cutoff, between 41% and 44% of the isolates could not be classified to any source and were placed in an unknown category. Based on the remaining isolates, source distributions were recalculated and were not statistically different than those calculated with no restriction for isolate similarity for matching. The last major test of the data was the analysis of the library for representativeness via pulled sample cross validation and the exclusion of all duplicate patterns from the known source library. These analyses did not confirm the representativeness of the databases, but results were further analyzed based on the implications these analyses have on library based methods.
  • Thumbnail Image
    Determining Sources of Fecal Pollution in Water for a Rural Virginia Community
    Graves, Alexandria Kristen (Virginia Tech, 2000-08-01)
    This project involves developing and applying bacterial source tracking (BST) methodology to determine sources of fecal pollution in water for a rural community (Millwood, VA). Antibiotic resistance analysis (ARA) is the primary BST method for fecal source identification, followed by randomly amplified polymorphic DNA (RAPD) analysis for confirmation. Millwood consists of 66 homes, all served by individual septic systems, and a stream (Spout Run) passes through the center of the community. Spout Run drains a 5,800 ha karst topography watershed that includes large populations of livestock and wildlife. Stream and well samples were collected monthly and analyzed for fecal coliforms and fecal streptococci, starting in 5/99 and ending in 5/00. Twelve percent of the well samples and 92% of the stream samples were positive for fecal coliforms, and 26% of the stream samples exceeded the recreational water standard (1,000 fecal coliforms/100 ml). ARA of fecal streptococci recovered from the stream samples indicated that isolates of human origin appeared throughout the stream as the stream passed through Millwood with a yearly average of (approx. 10% human, 30% wildlife, and 63% livestock), and the percent human origin isolates declined downstream from Millwood. These results were obtained by comparing the antibiotic resistance profiles of stream isolates against a library of 1,174 known source isolates with correct classification rates of 94.6% for human isolates, 93.7% for livestock isolates, and 87.8% for wildlife isolates. There is a human signature in Spout Run, but it is small compared to the proportion of isolates from livestock and wildlife. The sporadic instances where well water samples were positive appeared primarily during very dry periods. Restricting livestock access to streams can dramatically lower fecal coliform counts during the unusually hot and dry periods. Reducing FC counts to below recreational water standards for Virginia (1000 per 100ml for any one sample) may be achievable, however to maintain streams below standards may prove to be difficult, as Spout Run is in an area where there are large populations of Canada geese, deer, and other wildlife, and will be hard to restrict these animals.
  • Thumbnail Image
    Development and evaluation of a colorimetric coliphage assay detection system
    Ijzerman, M. Marian; Hagedorn, Charles III; Reneau, Raymond B. (Virginia Water Resources Research Center, Virginia Polytechnic Institute and State University, 1994-03)
    A colorimetric coliphage assay detection system (CCADS), composed of a liquid colorimetric presence-absence (LCPA) method and a colorimetric agar-based (CAB) method, was developed to overcome the limitations imposed by the Standard Methods for the Examination of Water and Wastewater agar-based coli phage method (APHA method). Both CCADS methods are based on the induction of p-galactosidase in Escherichia coli and the release of the enzyme through a lytic cell infection. The released enzyme then cleaves a chromogenic substrate, which produces a colored reaction product. The CCADS was evaluated against the APHA method under laboratory conditions using a common sewage coliphage strain as a model (American Type Culture Collection-13706-B2), and under field conditions using water samples collected from four different sources. During thelaboratory evaluation, both the LCPA and CAB methods were found to be superior to the APHA method in coliphage detection because: 1) the LCPA and CAB methods were easier to read and interpret than the APHA method, 2) the LCPA and CAB methods were not subject to false positive results, 3) the ·LCPA method theoretically detected fewer coliphage particles than the APHA method, and 4) the CAB method detected roughly twice the number of coliphage particles detected with the APHA method. During the field evaluation, the results indicated: 1) the LCPA method was as reliable as either the CAB or APHA method in coliphage detection; 2) the LCPA and CAB methods were easier to read and interpret than the APHA method; 3) neither the LCPA method nor the CAB method were subject to false positive results; 4) the CAB method detected more coliphages than the APHA method under conditions of high fecal pollution, but both methods performed equally well in coliphage detection under conditions of low fecal contamination; and 5) the LCPA and CAB methods were equally as sensitive in coliphage detection as the APHA method. Finally, the coliphage group proved to be a useful indicator of fecal pollution in nonpotable water supplies exhibiting a high degree of fecal pollution, whereas they were not shown to be useful indicators in potable water supplies exhibiting low levels of fecal contamination. The lack of coliphage detection sensitivity under conditions of low fecal contamination does not appear to be method-limited, but the result of inefficiencies in processing environmental samples using the concentration methods currently available.
  • Thumbnail Image
    Development and evaluation of a colorimetric coliphage assay detection system
    Ijzerman, M. Marian (Virginia Tech, 1993-07-14)
    A Colorimetric Coliphage Assay Detection System (CCADS) that is composed of a Liquid Colorimetric Presence-Absence (LCPA) method and a Colorimetric Agar-Based (CAB) method was developed to overcome the limitations imposed by the Standard Methods for the Examination of Water and Wastewater agar-based coliphage method (APHA method). Both CCADS methods are based on the induction of β-galactosidase in Escherichia coli and the release of the enzyme through a lytic cell infection. The released enzyme then cleaves a chromogenic substrate which produces a colored reaction product. The CCADS was evaluated against the APHA method under laboratory conditions using a common sewage coliphage strain as a model (American Type Culture Collection- 13706-B2), and under field conditions using water samples collected from four different sources. During the laboratory evaluation, both the LCPA and CAB methods were found to be superior in coliphage detection to the APHA method because: 1) the LCPA and CAB methods were easier to read and interpret than the APHA method, 2) the LCPA and CAB methods were not subject to false positive results, 3) the LCPA method theoretically detected fewer coliphage particles than the APHA method, and 4) the CAB method detected roughly twice the number of coliphage particles than the APHA method. During the field evaluation, the results indicated: 1) the LCP A method was as reliable as either the CAB or APHA methods in coliphage detection, 2) the LCP A and CAB methods were easier to read and interpret than the APHA method, 3) neither the LCPA method nor the CAB method were subject to false positive results, 4) the CAB method detected more coliphages than the APHA method under conditions of high fecal pollution, but both methods performed equally well in coliphage detection under conditions of low fecal contamination, and 5) the LCPA and CAB methods were equally as sensitive in coliphage detection as the APHA method. Finally, the coliphage group proved to be a useful indicator of fecal pollution in nonpotable water supplies that exhibited a high degree of fecal pollution, whereas they were not shown to be useful indicators in potable water supplies that exhibited low levels of fecal contamination. The lack of coliphage detection sensitivity under conditions of low fecal contamination does not appear to be method limited, but rather the result of inefficiencies in processing environmental samples using the concentration methods currently available.
  • Thumbnail Image
    Development and evaluation of a computerized leafspot advisory program for effective use of cultivar resistance, fungicide, and spray adjuvant to control early leafspot of peanut
    Cu, Ramon M. (Virginia Tech, 1991-04-15)
    An advisory program to determine the timing of fungicide application for control of early leafspot of peanut, caused by Cercospora arachidicola, was developed based on growth responses of the pathogen to specific environmental conditions. The advisory program (89-ADV) assigned time-duration values to conditions conducive for infection (TDVi). Cumulative TDVi levels were used to determine when fungicide applications were needed. Various spray thresholds (TDVi=48, 72, 96, 120) of the 89-ADV program were compared to a 14-day spray schedule and an advisory program that was released to growers in 1981 (81-ADV). Leafspot incidence, area under the disease progress curve (AUDPC), spray number, yield and value were used to evaluate the performance of spray programs. Preliminary trials used Florigiant peanut and the fungicide chlorothalonil (1.26 kg/ha). The 89-ADV program with TDVi=48 performed exceptionally well for three consecutive years in field tests and in simulated disease environments reconstructed from historical weather data. Benefits of this program compared to the 81-ADV program included significant improvement of leafspot control, and improved crop yield and value. Based on performance, the 89-ADV program was delivered to growers as the on-line peanut leafspot advisory in 1989. Subsequent evaluations of the 89-ADV program included cultivars and fungicides in large multi-factorial experiments. Three classes of cultivars were identified: class I or highly susceptible, Florigiant and NC 9; class II or moderately susceptible, NC 7 and NC-V11; and class III or moderately resistant, NC 6. The efficiency of fungicide sprays was improved through effective leafspot control with about three fewer sprays per season than the 14-day spray schedule when chlorothalonil at 1.26 kg/ha, diniconazole at 140 g/ha or terbutrazole at 126 g/ha was applied on class I cultivars according to the TDVi=48 threshold of the 89-ADV program. The same efficiency was achieved when chlorothalonil or terbutrazole was applied on class II cultivars according to the TDVi=96. Cupric hydroxide at 1.79 kg/ha plus sulfur at 1.04 kg/ha or terbutrazole at 126 kg/ha with TDVi=96 as well as chlorothalonil at 1.26 kg/ha with TDVi=120 resulted in efficient control of disease on the class III cultivar. The spray adjuvant SoyOil 937® at 0.5% of spray volume consistently improved the performance of chlorothalonil, and allowed a reduction of application rate from 1.26 to 0.95 kg/ha without sacrificing disease control. The integrated use of cultivar resistance, fungicide, spray adjuvant, and TDVi thresholds of the 89-ADV program contributed to a reduction of fungicide input and improved disease control.
  • Thumbnail Image
    Effects of soil texture, soil depth, and treatment on septic tank effluent renovation
    Keeling, William G. (Virginia Tech, 1995-12-05)
    This study compared the effects of soil texture, soil depth, and additional treatment, by constructed wetland (CW) and recirculating sand filter (RSF) systems, on wastewater renovation in a texture group 2 (loam) and a texture group 3 (clay loam) soil. The soils with depths of 15, 30, and 45 cm were placed in the columns with a Gidding's hydraulic soil probe in order to retain the soils structure in situ. The texture group 2 columns were dosed with septic tank eflluent (STE), constructed wetland effluent (eWE), and recirculating sand filter effluent (RSFE) at 685 cm³ per day and the texture group 3 columns were dosed at 670 cm³ per day with the same effluents. These dosing events were split into 2 separate (12 h) events. The leachate from both groups of soil columns were analyzed for fecal coliforms (FC), biochemical oxygen demand (BODs), ammonium (NH₄-N), nitrate (NO₃-N), phosphate (PO₄-P), pH, total dissolved solids (TDS), electrical conductivity (EC), and chloride. In the texture group 2 soil the reduction in fecal coliform numbers was 88% and 99%, respectively, for the CW and the RSF and, in the texture group 3 soil was 91% and 99%, respectively, for the CW and the RSF. There was a reduction in BODs concentrations of 32% and 69% ,respectively, for the CW and the RSF. There was a 42% and 84% decrease in NH₄ concentrations, respectively, for the CW and the RSF. These reductions show that treatment of STE by CW or RSF can be substituted for soil depth in texture group 2 and 3 soils and will improve drainfield performance. Texture group 2 soils are less effective at STE renovation than texture group 3 soils at all soil depths. This was evidenced by higher FC counts and concentrations of BOD, N, and P in the leachates from these columns.
  • Thumbnail Image
    Egyptian Broomrape (Orobanche aegyptiaca Pers.) and Small Broomrape (Orobanche minor Sm.) Parasitism of Red Clover (Trifolium pratense L.) in Vitro
    Morozov, Ivan Vladimirovitch (Virginia Tech, 1998-05-13)
    Broomrapes, Orobanche spp., are holoparasites that affect the growth of a variety of broadleaf crops. One of the distinct characteristics of the family Orobanchaceae is the lack of chlorophyll, and hence inability to synthesize their own food. Broomrapes subsist on the roots of the host plant from which they derive the carbon, water, and nutrients needed for further growth. Parasitism as such leads to yield reductions, and in case of heavy infestations, complete crop failure. Among other plants parasitized by broomrapes are several legumes, some of which are also the world's most economically important crops. As part of their unique biology, legumes provide an ecological niche for diazotropic soil bacteria, which belong to the family Rhizobiaceae. In return, the host plant receives fixed nitrogen from the nodules, specialized structures produced on the roots of most legume plants upon inoculation with bacteria. Orobanche spp. germination depends on the presence of chemical stimulant in host root exudates. It has been reported that inoculation of some legumes resulted in greater infestation by parasitic weeds. In addition, bacterial nodules were assumed to provide a place for broomrape invasion of host legume. Furthermore, infestations were observed to be more intense in aerobic conditions when rhizobia are most active. It is possible that production of the stimulant could be correlated with the infection of roots with Rhizobium, and that the nodules formed on the roots could play a role in broomrape parasitism; however, others have not confirmed this. Studies were conducted to investigate the possibility of interaction between Orobanche spp. attack and Rhizobium nodulation in legumes. Seed germination, number of parasitic attachments, as well as the morphology of two broomrape species, small broomrape (Orobanche minor Sm.) and Egyptian broomrape (Orobanche aegyptiaca Pers.), were studied. O. minor showed a greater percent seed germination, and formed a greater number of attachments on red clover (Trifolium pratense L.) inoculated with Rhizobium leguminosarum bv. trifolii in comparison with non-inoculated plants. However, the addition of the inoculum did not appear to enhance O. aegyptiaca seed germination or the number of its attachments on the host roots compared with the controls. Morphological observations of O. minor attachments on red clover suggest that parasitic attachments were not situated over the bacterial nodules, but perhaps involve parasite-induced enzymatic degradation followed by mechanical protrusion of host plant root cortex, possibly utilizing host plant-rhizobacteria interactions as well.
  • Thumbnail Image
    Environmental and agronomic aspects of municipal solid waste heavy fraction used for turfgrass production
    Flanagan, Mark Steven (Virginia Tech, 1991-01-15)
    Disposal of municipal solid waste has become a tremendous problem in the United States. To reduce the volume of garbage that requires deposition in landfills, innovative methods of recycling need to be investigated. Several experiments were conducted in Blacksburg, Virginia, to evaluate the use of heavy fraction, a by-product of a solid waste separation process, as a soil amendment for production of turfgrass sod. In a field plot experiment using Kentucky bluegrass (Poa pratensis L.), measurements of sod strength taken 8.5 and 9.5 months after seeding were greater for sod grown in topsoil amended with heavy fraction than for turf grown in topsoil only. These results imply that the use of this by-product for turfgrass production may reduce the time required to produce a harvestable sod. In a container study, physical properties of a loam topsoil were altered 16 months after addition of heavy fraction. Bulk density and particle density were reduced and organic matter content increased by soil incorporation of this by-product. Total porosity and air porosity (macropore space) of the topsoil increased whereas water porosity (micropore space) decreased with increasing amount of applied heavy fraction. Soil fertility was enhanced and soil pH raised by addition of heavy fraction. Concentrations of extractable NH₄ -N, P, K, Ca, Mg, Mn, and Zn in soil were increased by the application of heavy fraction, as were concentrations of K, Ca, S, Mg, and Mn in leachate collected in lysimeter studies. Improved fertility resulted in greater aesthetic quality, clipping yields, and tissue N content for tall fescue (Festuca arundinacea Schreb.). Lysimeter studies indicated that the greatest environmental concern associated with the use of heavy fraction for turfgrass production appears to be the potential for leaching of N0₃-N during turf establishment. With loading rates of 414,747 kg ha⁻¹ or lower, however, the amount of N0₃-N leached from heavy fraction-amended topsoil was no greater than that leached from topsoil containing no heavy fraction. Concentrations of N0₃-N in leachate for all loading rates of heavy fraction decreased to levels well below 10.0 mg L ⁻¹ approximately two months after sodding the lysimeters with tall fescue. Leachate analyses indicated minimum potential for P or heavy metal contamination of groundwater from heavy fraction.
  • Thumbnail Image
    Environmental controls on the abundance, diversity, growth, and activity of  ammonia-oxidizing microorganisms in temperate forest soils
    Norman, Jeffrey Stancill (Virginia Tech, 2014-01-31)
    The goal of my dissertation research was to investigate the structure and function of ammonia-oxidizing microbial communities in temperate forest soils. Accomplishing this goal required a hybrid approach: I used modern molecular biology techniques alongside soil biogeochemical measurements and framed my research using ecological theory largely developed in plant systems. All of my field work was done at Coweeta Hydrologic Laboratory, a Forest Service Station and Long Term Ecological Research Site near Otto, NC. Watershed-level land use manipulations have been performed at Coweeta since the 1930s, including clear-cutting, fertilizing, liming, burning, grazing by cattle, and replanting entire watersheds in white pine. While these treatments were originally imposed to assess the effects of land use on water yield, they have resulted in changes in soil characteristics as well. Working at Coweeta has therefore allowed me to sample ammonia-oxidizer communities across a gradient of soil variables, such as pH and nitrogen (N) availability, within the geographically-constrained area of the Coweeta Basin. First, I used amplicon-based pyrosequencing to independently assess the diversity of ammonia-oxidizing archaea (AOA) and bacteria (AOB) at several sites within Coweeta. I found that AOA and AOB diversity were a function of both resource availability (i.e. N availability) and environmental harshness (i.e. soil pH) in line with general ecological theory developed for plant systems by Tilman and Grime, respectively. Next, I tested whether AOA and AOB were substrate or nutrient limited in this system by adding either N or a nutrient solution containing both potassium and phosphorus to soil incubations and assessing the growth response of AOA and AOB using quantitative polymerase chain reaction (qPCR). I found strong evidence for substrate limitation by AOB and a marginally-significant positive effect of nutrient addition on growth of AOA. Another intriguing finding from this study was that both AOA and AOB grew during unamended soil incubations. Unamended (buried-bag) incubations have been used to estimate in situ rates of nitrification for over 50 years. By measuring the growth of AOA and AOB alongside nitrification during buried-bag incubations, I discovered that AOA are the dominant ammonia-oxidizers in temperate forest soils. However, I found that AOA are much less efficient at using the energy from ammonia oxidation to create biomass than AOB in the forest soils I sampled. Overall, I found that temperate forest soils contain low abundances of AOA and AOB, with relatively low diversity in both groups. This is especially true for the diversity of AOA, where a single taxon dominated the community at every site. Soil pH and N availability seem to be major selective forces for forest soil ammonia oxidizers, though other nutrients such as potassium and phosphorus may regulate the activity of AOA as well. AOA are most-likely the dominant ammonia oxidizers in temperate forest systems, though this may change with increased disturbance. In a broader sense, I found that ecological theory developed for plant communities was applicable to chemoautotrophic microbes despite the large differences in life history between these groups of organisms.