VTechWorks Repository :: Browsing by Author "Benoit, Robert E."

Browsing by Author "Benoit, Robert E."

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The 16S rRNA characterization of a novel "microaerophilic" Pseudomonas sp. from the oligotrophic deep subsurface environment
Lampe, Robert Carl III (Virginia Tech, 1996-11-07)
A gram negative microaerophilic bacterium, designated Pseudomonas sp. strain MR 100, was isolated from a depth of 463 meters at the Savannah River DOE site and identified using 16S rDNA sequencing and DNA-DNA reassociation. Micro aerophiles from the Middendorf formation were isolated by use of a semi-solid agar assay, and constituted 10% of the plateable microorganisms. Genetic identification involved the isolation of genomic DNA and amplification of the gene encoding 16S rRNA by PCR, using universal primers. The amplified DNA was sequenced and compared to 16S rRNA sequences in Genbank. High sequence similarity (98.5%) was observed with the Pseudomonas mendocina type strain, indicating a similarity to the (Group I) pseudomonads. DNA-DNA reassociation was performed between Pseudomonas sp. strain MR 100 and 11 representative p seudomonads using the S 1 nuclease method. Strain MR 100 was found to be 20% homologous to the Pseudomonas mendocina type strain, 10% homologous to Pseudomonas alcaligenes, and 5% homologous to Pseudomonas aeruginosa. Data from biochemical tests confirm the hypothesis that strain MR 100 is a novel species of Pseudomonas
An assessment of factors controlling the biodegradation of benzene in the subsurface environment
Poor, Noreen D. (Virginia Tech, 1996)
The objectives of this research were to correlate benzene biodegradation with soil physical, chemical and biological properties, to determine if biodegradation could be predicted based on measured or observed soil properties, and to investigate the role of nutrients on benzene biodegradation in soil. Benzene disappearance over time was measured in aerobic active and control (autoclaved) microcosms prepared with previously-uncontaminated subsurface soils. Soil microcosm experiments were prepared with initial benzene concentrations of 1, 10 and 50 mg/L. For each soil, logistic, zero-, first-, and 3/2- order kinetic models were fit to benzene disappearance versus time data by regression analysis. The logistic and 3/2-order models fit the data better than zero- and first-order models for experiments prepared with initial benzene concentrations of 1 and 10 mg/L. For an initial benzene concentration of 50 mg/L, experimental data were often better fit by zero- or first-order kinetic models. To obtain predictive equations, logistic kinetic model rate constants were related to soil properties using multiple linear regression (MLR). The “best” MLR models and their regression coefficient estimates were statistically significant at p<0.05. For experiments prepared with an initial benzene concentration of 1 mg/L, the resulting predictive equation contained soil phosphorus concentration and cation exchange capacity (CEC). For experiments prepared with an initial benzene concentration of 10 mg/L, the predictive equation contained soil copper, nitrate-N and phosphorus concentrations, CEC, and % sand. A comparison was made between benzene biodegradation in unamended soil microcosms and soil microcosms amended with ammonium and potassium phosphates (11 mM nitrogen, 6 mM phosphorus and 0.6 mM potassium). Benzene disappearance over time in soil microcosms was stimulated by nutrient addition in one (11%), 6 (50%), and 5 (45%) soils at initial benzene concentrations of 1, 10 and 50 mg/L, respectively. In general, nutrient addition had the greatest affect on benzene biodegradation in low pH soils.
Bacterial extracellular polymers and flocculation of activated sludges
Kajornatiyudh, Sittiporn (Virginia Polytechnic Institute and State University, 1986)
The extracellular polymers produced by bacteria play an important role in bacterial aggregation or bacterial flocculation in secondary waste treatment. The mechanisms responsible for this floc formation are thought to be polymer induced adsorption and interparticle bridging among bacterial cells or between bacterial cells and inorganic colloids. The efficiency of the processes following flocculation in the treatment line such as sedimentation, sludge thickening, and sludge dewatering depends on the extent of this bacterial flocculation. In this research, sludge samples from under various substrate conditions were examined for type, molecular weight, physical characteristics„ and quantity of extracellular polymers so that the general characteristics of the various polymers could be established. An attempt was made to determine if a relationship exists between the state of bacterial aggregation and the polymer characteristics. This research also investigated the sludge physical properties. The effect of various parameters such as pH, divalent cation (mixture and concentration), and mixing (period and intensity) on dewatering properties were studied. A major goal of this study was to develop a flocculation model for activated sludge. This model could be used to determine if plants can increase the efficiency of waste treatment and sludge thickening and sludge dewatering processes.
Biochemistry and genetics of the pathway for the anaerobic degradation of aromatic compounds by Eubacterium oxidoreducens
Haddock, John David (Virginia Tech, 1990-08-05)
The biochemical pathway for the anaerobic degradation of gallate, pyrogallol and phloroglucinol by Eubacterium oxidoreducens was investigated. Phloroglucinol reductase was purified 90-fold, from the soluble fraction of cell extract, to electrophoretic homogeneity. The enzyme was an Î±₂ homodimer with a native M_r of 78,000, did not contain metals or cofactors and was specific for phloroglucinol and NADPH with a K_m of 800 μM and 6.7 μM respectively at pH 6.8. The Km for phloroglucinol decreased with increasing pH. The enzyme catalyzed reaction was reversible and the equilibrium constant was 9.6. Dihydroresorcinol was a competitive inhibitor of the reverse reaction (K_i = 756 μM). Dihydrophloroglucinol produced in cell extract with H₂ as the reductant was identical to the compound produced by sodium borohydride reduction of phloroglucinol as shown by ¹H NMR spectroscopy. The ¹³C NMR spectrum was consistent with the structural assignment of dihydrophloroglucinol. The mechanism of the proposed enzymatically catalyzed reaction is proposed to involve transfer of a hydride equivalent from NADPH to the carbonyl carbon of the phloroglucinol dianion. Mutant strains of E. oxidoreducens that showed no gallate decarboxylase or dihydrophloroglucinol hydrolase activity were isolated after mutagenesis with ethylmethane sulfonate and emichment with ampicillin. The decarboxylase deficient mutants were unable to grow on gallate while pyrogallol and phloroglucinol supported growth. The hydrolase deficient mutants were unable to grow on any aromatic substrates and converted gallate to pyrogallol and dihydrophloroglucinol. The conversion of gallate to non-aromatic intermediates by cell extract of the wild-type stain was dependent on the presence of 1,2,3,5-benzenetetrol for the conversion of pyrogallol to phloroglucinol and on formate for the reduction of phloroglucinol to dihydrophloroglucinol. Transhydroxylase activity involved in the conversion of pyrogallol to phloroglucinol was induced by growth on aromatic substrates. The formate dehydrogenase was located in the soluble fraction of cell extract, and activity was protected from oxygen inactivation by sodium azide. The Km for formate and NADP was 290 μM and 140 μM respectively at pH 7.5. The pH optimum for activity was 7.5 and maximum activity was observed at a temperature of 50°C.
Biodegradation of methanol and tertiary butyl alcohol in previously uncontaminated subsurface systems
Goldsmith, Charles Douglas (Virginia Polytechnic Institute and State University, 1985)
The objective of this study was to determine the potential for biodegradation in subsurface soils and groundwater from sites in Williamsport, PA, Wayland, NY, and Dumfries, VA. These subsurface systems were characterized both physically, chemically and biologically. Bacterial populations were substantial in all systems and ranged from 10³ to 10⁸ colony forming units per gram. Soil sampling was done in a quality-controlled aseptic manner using conventional drilling end sampling equipment. A matrix of test-tube microcosms was used to determine biodegradation rates of methanol and t-butyl alcohol at concentrations ranging from 1 to 1000 mg/L. Methanol degraded readily at all sites ranging from 0.8 mg/L/day to 20.4 mg/L/day and rates were generally greater in the saturated zone. TBA biodegraded at all sites, but was refractory in nature. Biodegradation rates for TBA in anaerobic subsurface systems were found to increase directly with initial concentration from 10⁻⁴ mg/L/day for 1 mg/L to 10⁻¹ mg/L/day for 80 mg/L. TBA biodegradation in the aerobic system was essentially constant over all concentrations. Biokinetic coefficients were determined for methanol and TBA at each site based on plots of utilization rates versus substrate concentration and reciprocal plots of these values. The K values found suggest that aerobic subsurface systems can utilize alcohols at a greater rate than anoxic subsurface systems and can be used for comparative purposes. The K_s of anoxic subsurface systems were found to be large due to the low temperature (10°C) found in aquifers. The results indicate that methanol contamination in groundwater has much less associated risk to drinking water supplies due to the ease of biodegradation. However, TBA poses a much greater risk due to the very slow removal rates at low concentrations, which could result in a residual level for over a decade in some cases.
Biodegradation of organic contaminants in subsurface systems: kinetic and metabolic considerations
Morris, Mark S. (Virginia Polytechnic Institute and State University, 1988)
Groundwater contaminated by organic chemicals from industrial spills, leaking underground gasoline storage tanks and landfills has caused concern about the future of a major source of drinking water. Compounds from industrial sources such as alcohols and phenols are frequently found as groundwater contaminants. These compounds are highly soluble in water and do not adsorb well to aquifer material. They also have the potential to migrate in the subsurface system achieving significant levels in drinking water supplies. In addition, they can serve as carriers for carcinogenic compounds such as benzene, toluene and xylene which are relatively insoluble in water, but are quite soluble in alcohol. A potential alternative to expensive groundwater reclamation projects is the use of the natural soil bacteria to degrade organic contaminants. Very little is known, however, about subsurface soil bacteria to man-made organic chemicals or the degradation rates of these compounds. Such information would be useful in planning cleanup or protection strategies for groundwater systems. This study was designed to measure the kinetic response of tertiary butyl alcohol (TBA), determine the biological degradation rates of methanol, ethanol, propanol, l-butanol, TBA, pentanol, phenol and 2,4-dichlorophenol; describe site specific conditions which enhance or inhibit degradation and compare biodegradation rates with thermodynamic predictions. Laboratory microcosms utilizing soil from two previously uncontaminated sites of widely varying conditions were constructed to simulate the subsurface environment. Nitrate was added to some microcosms to stimulate denitrification and metabolic inhibitors were added to others to define conditions at each site which favor biodegradation. Each of the test compounds except TBA was readily degraded in the Blacksburg soil. Inhibition of sulfate reduction by the addition of molybdate stimulated degradation of all compounds including TBA, whereas, inhibition of methanogenesis with BESA slowed the degradation rates. The addition of nitrate did not affect the biodegradation in Blacksburg soil. In the Newport News soil, all of the test compounds were biodegraded at substantially higher rates than was observed in the Blacksburg soil. The presence of the metabolic inhibitors did not affect degradation, however, the addition of nitrate increased the degradation rates of the alcohols but not the phenols. The degradation rates in each of the soils did not correlate with the bacterial population size or free energies of the reactions.
The biodegradation potential of methanol, benzene, and m-xylene in a saturated subsurface environment
Frago, Cathia H. (Virginia Tech, 1993-03-15)
The increased use of alcohols as gasoline additives, and possible substitutes, has prompted the investigation of the fate of gasoline/alcohol mixtures in the environment. In situ bioremediation is one technique that can successfully be applied to remove ground water contaminants particularly in situations where the adsorptive capacity of the soil plays a major role. Frequently, enhanced in situ bioremediation techniques rely on indigenous microorganisms to degrade ground water contaminants; this technique may sometimes include the addition of acclimated bacteria. In this study, soil microcosms were constructed in order to simulate the conditions found in a saturated aerobic aquifer. The biodegradation potential of methanol, benzene, and m-xylene was investigated. Uncontaminated soil from the surface, 12, 16.5, and 18 foot depths was utilized to observe the differences in microbial responses throughout the soil profile. The biodegradation potential of the indigenous microbiota was determined and compared to that of benzene acclimated bacteria, for all the compounds in the mixture. To observe the impact that chemical and physical soil characteristics may have on microbial responses, soils from each depth were classified on the basis of their particle size, moisture content and pH. Substantial methanol, benzene, and m-xylene biodegradation by the indigenous microorganisms occurred in all subsurface soils. While methanol was readily biodegradable over concentrations ranging from about 80 mg/L to about 200 mg/L, benzene inhibited methanol biodegradation at about 125 mg/L in all soil depths. The addition of benzene acclimated bacteria considerably increased the biodegradation rates of all compounds in the mixture. Such increases in biodegradation rates may be attributed to the activities of both groups, the indigenous microorganisms and the benzene acclimated bacteria. The results obtained by this study suggest that biodegradation of methanol, benzene, and m-xylene can readily occur in a saturated aerobic subsurface environment. The physical and chemical properties of a ground water aquifer seem to have a marked effect on microbial responses, and consequently on the biodegradation potential of water contaminants.
Changes in respiration rates and biomass attributes of epilithon due to extended exposure to zinc
Colwell, Frederic S. (Virginia Polytechnic Institute and State University, 1986)
The purpose of this research was to determine the influence of extended dosing of zinc on the carbon cycling and biomass characteristics of freshwater epilithon. Experiments were conducted in artificial streams continuously dosed with 0.00, 0.05, or 1.00 mg Zn liter⁻¹ for 20 to 30 days during summer and fall, 1984 and 1985. Repeated measurement of epilithon structure and function included estimates of ¹⁴C-glucose respiration, ¹⁴C-glutamate respiration, O₂ and CO₂ flux rates, ash-free dry weight (AFDW), protein, carbohydrate, and algal pigment concentrations, and total and zinc-tolerant colony forming units. An increase in epilithic glucose respiration per unit biomass consistently occurred 5 to 10 days after dosing with 1.0 mg Zn liter⁻¹ was started. At the same time significantly lower epilithon biomass occurred in the high dosed streams relative to controls in 3 out of 4 studies. Although algal pigment concentrations were lowest in the high dose streams at the midpoint of the studies, the chlorophyll a-to-pheophytin a ratio remained high, indicating that the minimal algal population was not senescing in situ. After 30 days, the epilithon dosed with 1.0 mg Zn liter⁻¹ had higher AFDW, protein, and carbohydrate concentrations than the other treatments. By 20 days, the high zinc treatment showed evidence of more total and zinc-tolerant colony forming units and lower rates of O₂ and CO₂ flux than epilithon from control streams. The high rates of glucose respiration were characteristic of epilithic communities stressed by 1.0 mg Zn liter⁻¹, and this response was not apparently due to in situ senescence of zinc-sensitive cells; the results suggested that epilithic biomass was washed out of the systems, not being degraded in situ. The development of unique epilithon communities that are acclimated to prolonged zinc exposure is evident in the eventual recolonization of the artificial surfaces, glucose respiration rates that are comparable to controls, and presence of zinc-tolerant heterotrophs.
Characterization of DNA-repair potential in deep subsurface bacteria challenged by UV light, hydrogen peroxide, and gamma radiation
Arrage, Andrew Anthony (Virginia Tech, 1991-08-07)
Subsurface bacterial isolates obtained through the DOE Subsurface Science Program were tested for resistance to UV light, gamma radiation and H₂0₂. Some deep subsurface bacteria were resistant to UV light, demonstrating ≥1.0% survival at fluences which resulted in a 0.0001% survival level of E. coli B. The percentage of UV resistant aerobic subsurface bacteria and surface soil bacteria were similar; 30.8% and 25.8% respectively. All of the microaerophilic subsurface isolates were UV sensitive as defined in this work; however, subsurface isolates demonstrated UV resistance levels similar to reference bacterial strains of the same Gram reaction. These results were not in agreement with the hypothesis that the resistance of an organism to UV is correlated with the amount of solar radiation in its natural habitat. Evidence for photoreactivation and the presence of an SOS-like mechanism was also detected in subsurface bacteria. The presence of UV resistance and photoreactivation in subsurface bacteria that have been shielded from solar radiation for millions of years may point to a limited rate of evolution in the deep subsurface environment. In subsurface bacteria, there was a relatedness between UV resistance and resistance to gamma radiation and H₂0₂ UV-resistant aerobic subsurface isolates were also gamma and H₂0₂- resistant compared to the microaerophilic isolates tested. Due to the similarities of bacterial responses to UV, H₂0₂ , and gamma radiation, either UV or H₂0₂ may be utilized to model the effects of ionizing radiation on bacterial cultures used for the bioremediation of organic and radioactive waste-containing environments.
Cod removal, nitrification and denitrification kinetics and mathematical modeling of an integrated fixed film activated sludge (IFAS) system
Sen, Dipankar (Virginia Tech, 1995)
Biological nitrogen and phosphorus removal are being recommended at several wastewater treatment facilities in the U.S. to control eutrophication in water bodies receiving their effluent. In several instances, nitrogen removal is being recommended on a year round basis at plants located in the temperate climates. Concerns have been raised regarding the cost of additional reactors and clarifiers required for nitrogen removal in winter using activated sludge systems. Several facilities do not have the space to construct additional units. This research was undertaken to evaluate the potential for reducing reactor and clarifier requirements, maximize use of existing facilities, and economize costs for year round nitrification and nitrogen removal. The purpose was to develop Integrated Fixed Film Activated Sludge Systems in which biofilm support media was installed in the activated sludge basins to enhance nitrogen removal. The objectives were to evaluate the COD removal, nitrification and denitrification kinetics, which would help develop design and mathematical models for the process, and optimize the layout in terms of costs and media required for nitrogen removal. A bench scale pilot study was undertaken in conjunction with two full scale studies with two types of media: (i) free floating media and (ii) media fixed in frames. Amongst biofilm support media in the first category, sponge cuboids (Captor) were selected. For the second category, rope type Ringlace media was selected. Following preliminary screening for satisfactorily simulation of full scale conditions in a bench scale system, the IFAS-Sponge system was selected for kinetic coefficient evaluation and IF AS system model development.
A comparison of subsurface biodegradation rates of methanol and tertiary butanol in contaminated and uncontaminated sites
White, Kevin D. (Virginia Polytechnic Institute and State University, 1986)
The use of alcohols as inexpensive octane enhancers in gasoline has contributed to an increased concern about the potential contamination of groundwater. Being highly soluble in water, alcohols may easily separate from other, more insoluble gasoline components, and rapidly enter the groundwater flow system. The alcohols are relatively tasteless and odorless, and thus, may go undetected until potentially harmful concentrations are reached. This study was designed to determine the potential for alcohol biodegradation in a groundwater system that had been previously contaminated with gasoline containing tertiary butyl alcohol (TBA). Laboratory microcosms, utilizing actual aquifer material and groundwater, were constructed to determine the rate of alcohol biodegradation in a system closely resembling the subsurface environment. The only microorganisms used were those naturally present in subsurface soil obtained aseptically. Bacterial counts and degradation kinetics were evaluated at each of three subsurface depths (10, 26, and 45 feet) and results were compared to similar studies utilizing uncontaminated aquifer material. Significant bacterial populations were found to exist at all depths studied in the contaminated subsurface system. Bacterial plate counts ranged from 10 6 to 10 7 colony forming units per gram of soil (dry weight). Methanol was found to be a readily degradable substrate. Complete degradation of up to 1000 mg/L was degraded in a matter of months. The biodegradation of methanol in the contaminated system was similar to that observed at pristine sites, indicating that a similar degradation mechanism is involved. TBA biodegradation in the contaminated system occurred and was accompanied by microbial growth. Complete TBA degradation of up to 100 mg/L occurred in less than one year. In contrast, TBA biodegradation in the uncontaminated systems occurred at a very slow rate, which appeared to be constant over time, and thus zero order. However, the zero order rate was found to vary directly with initial substrate concentration. Several mechanisms may explain TBA biodegradation, including the presence of a non-specific exocellular enzyme system. Such a system would describe observed results and suggest that a widespread potential exists for the degradation of a large number of organic compounds.
Degradation of gasoline oxygenates in the subsurface
Yeh, Kuei-Jyum (Virginia Tech, 1992)
Tertiary butyl alcohol (TBA), methyl tertiary butyl ether (MTBE) and ethyl tertiary butyl ether (ETBE) are compounds with the potential for use as oxygenates in reformulated gasolines. Being relatively soluble in water, these organics, if accidentally discharged into the subsurface, may rapidly spread and pose threats to groundwater. The purpose of this work was to evaluate the biodegradation potential of these oxygenates in soils and to determine the influence of subsurface environments on their degradation. Biodegradation was evaluated in static soil/water microcosms. Aquifer material was collected from various depths at three sites with different soil characteristics. Potential electron acceptors including O₂ in the form of H₂O₂, nitrate or sulfate were added to induce the desired metabolism (aerobic respiration, denitrification, sulfate reduction, or methanogenesis). In each metabolic process, the influence of several subsurface environmental factors on biodegradation was investigated. The data show that biodegradation potential of MTBE, ETBE and TBA varied substantially with site and depth. TBA was the easiest compound to biodegrade, whereas MTBE was the most recalcitrant. Cleavage of the ether bond is the first and rate-limiting step in the degradation of ETBE and possibly MTBE. Addition of H₂O₂, caused chemical oxidation of MTBE and ETBE. The chemical oxidation was faster in the organically rich soils, but slower in the organic-poor soils. Soil microorganisms were able to catalyze the cleavage of the ether bond in ETBE but not MTBE. This biological reaction was not significant when chemical oxidation occurred. TBA, on the other hand, was aerobically biodegraded in all soils. Under denitrifying and anaerobic conditions TBA degradation occurred in all soils but the degradation of ETBE and MTBE was only observed at one of three sites. TBA degradation was enhanced by nutrient addition in the nutrient-poor soil but hindered by the presence of other easily-degraded organic compounds. Degradation of MTBE and ETBE occurred only in soils containing low organic matter with a pH around 5.5. No degradation of MTBE and ETBE was observed in the organic-rich soils and in the organically poor soils, the addition of ethanol inhibited MTBE and ETBE degradation.
Densities of indicator bacteria in urban and rural runoff
Cox, James Warren (Virginia Tech, 1974-08-05)
This study was designed to investigate the indicator bacterial densities in runoff from urban and rural sources and to compare these densities with the densities in both raw and secondary-treated, unchlorinated sewage effluents. Analysis was performed on a total of 42 samples, 22 of which were taken at the urban and the rural sites and correlated with storm flow. The remaining 20 samples were taken, 5 at each of the 4 sites, in order to establish respective baseline densities for total coliform, fecal coliform, and fecal streptococcus. Results indicated that mean values for total coliforms and fecal coliforms during dry flow were within Virginia State standards for urban runoff, but exceeded those two standards 100 percent and 91.9 percent of the time, respectively, during storm flow conditions. Mean values for total coliforms and fecal coliforms during dry flow at the rural site exceeded Virginia State standards 100 percent and 75 percent of the time, respectively. During storm flow the standards for both of these indicators were exceeded 100 percent of the time. It was also determined that the increase in densities typically lagged behind flow increases and maintained this lag while returning to baseline values. Finally, the FC/FS ratio as originally proposed by Geldreich was statistically supported as being valid for use in the identification of fecal pollution sources. Individual ratios were less than 0.7 at the urban watershed for 80% of the dry flow samples and 64.6 percent of the storm flow samples. For the rural site, FC/FS ratios were less than 0.7 for 25 percent of the dry flow samples and 81.8 percent of the wet flow samples. Samples of raw and secondary treated sewage exhibited ratios above 4.0, 40 percent, and 25 percent of the time respectively.
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.
Disinfectant Susceptibility of Mycobacterium avium
Taylor, Robert Henry (Virginia Tech, 1998-12-04)
Mycobacterium avium, an opportunistic human pathogen, infects between 25 and 50% of advanced-stage acquired immuno-deficiency syndrome (AIDS) patients in the United States. M. avium has been isolated from many environmental sources including: natural waters, soils, and aerosols. M. avium has also been recovered from within municipal and hospital drinking water systems. Rhesus macaques (Macaca mulatta) infected with the simian HIV analog, SIV, have been shown to acquire M. avium infections from potable water. Reduced-aggregate fractions (cell suspensions free of large aggregates) of Mycobacterium avium were exposed to chlorine, monochloramine, chlorine dioxide, and ozone and kinetics of disinfection measured. Chlorine disinfection kinetics was also measured in M. avium cultures grown in biofilms. M. avium exhibited a high resistance to chlorine compared to E. coli. M. avium CT99.9% (disinfectant concentration x time to 3 logs cell inactivation) values were between 571- and 2318 -times those of E. coli. Clinical isolates of M. avium showed 0.24 and 2.5-fold increase in resistance to chlorine compared to their pulsed-field-gel-electrophoresis- (PFGE) matched environmental isolates. M. avium strains exhibited a mixed response to exposure to monochloramine. The CT99.9% values of three strains (2 clinical, 1 environmental) were between 6.3- and 23.5- times that of E. coli. Two strains (1 clinical, 1 environmental) exhibited CT99.9% values approximately the same as E. coli, a difference from all the other disinfectants which were much less effective on M. avium than on E. coli. M. avium strains exhibited a high resistance to chlorine dioxide when compared to E.coli. M. avium CT99.9% values of between 133- and 706- times higher that that of E. coli. In the paired isolates tested, the clinical isolate was 5.3 times more resistant than the matched environmental isolate. M. avium exhibited a high resistance to ozone when compared to E. coli. M. avium strains exhibited a CT99.9% value of between 52 and 90 times higher that that of E. coli. In the paired isolates tested, the clinical isolate was nearly identical as judged by CT99.9% values. M. avium strain 5002 exhibited an unusual disinfection kinetics curve. Disinfection rate increased by a non-logarithmic factor, indicating that inactivation efficiency was increasing over time. M. avium strain 1060 showed between a 17% decrease to a 265% increase in CT99.9% value when grown as biofilms as opposed to suspension. Due to the large variance in biofilm density and and CT99.9% values, any conclusions based on these experiments should be considered tentative at best. M. avium's resistance to chlorine and chlorine dioxide approaches that of the protozoan cysts of Giardia muris and Entamoeba hystolytica. M. avium is much less resistant, relatively, to monochloramine possessing values similar to E. coli. Ozone resistance of M. avium is two orders of magnitude greater than E. coli and one order of magnitude of less than G. muris cysts. A critical concentration threshold level for chlorine dioxide was found. That is, there was no linear relationship between concentration of chlorine dioxide and cell inactivation. Initial experiments using a range of concentrations from 0.1 ppm to 0.5 ppm chlorine dioxide showed a biphasic curve with the inflection point (indicating the critical concentration) between 0.3 and 0.4 ppm chlorine dioxide.
The distribution of anaerobic bacteria along a soil drainage catena
Dolan, Rodney Martin (Virginia Tech, 1977-03-05)
Strict anaerobic culture techniques were used to enumerate the anaerobic bacteria present in three soil sites located along a drainage catena near Blacksburg, Virginia. An anaerobic cooked meat plus 0.5% glucose medium cultured the largest number of anaerobes from the poorly drained soil. The population of obligate anaerobes ranged from 10⁶/ g dry weight soil on the poorly drained soil (% moisture = 112.06) to 10⁵/g dry weight soil on the intermediate soil (% moisture = 34.51) to 10⁴/g dry weight soil on the well drained soil (% moisture 20.81). The population of organisms able to grow anaerobically (facultative plus obligate) ranged from 10⁶/g dry weight soil on the poorly drained site to 10⁵/g dry weight soil on the well drained site. This same population on the poorly drained site was relatively constant over a nine month period with the exception of a sharp rise in early spring. The clostridia constituted at least one third of the obligately anaerobic bacteria present on the poorly drained soil. A sizeable percentage of the obligate anaerobic isolates on this site were either clostridia which formed spores unable to germinate in the medium employed, clostridia which were very pleomorphic in cell shape and gram reaction, or nonsporeforming obligate anaerobes. These results indicate that strict anaerobes and possibly nonsporeforming strict anaerobes exist in soils of different drainage character even though facultative organisms appear to be more successful competitors on the more well drained sites.
The effect of carbon addition, pH and Fe concentration of microbial sulfate reduction and the subsequent precipitation of Fe and Mn from acid mine drainage in wetland mesocosms
Duddleston, Khrystyne Noel (Virginia Tech, 1993-06-05)
A wetland was constructed near Norton, VA by Westmoreland Coal Company to treat acid mine drainage (AMD) from an inactive coal refuse pile. The AMD had an average inflow pH of 7.0, and average inflow concentrations measuring 4 mg/L total Fe, 3 mg/L total Mn and 450 mg/L dissolved sulfate. An 18 month field study of water quality improvement and sulfate-reducing bacterial (SRB) populations revealed that the wetland was effectively treating the AMD. Iron and Mn both met compliance standards set by the EPA requiring an instream Fe concentration of 3 mg/L and an instream Mn concentration of 2 mg/L. SRB averaged 8.7 x 10⁴ through the 18 month study period as determined by the Most Probable Number (MPN) method. The concentration of sulfate was decreased by an average of 360 mg/L as the AMD passed through the wetland. In a separate laboratory study, the effect of carbon addition, pH and Fe concentration on microbial sulfate reduction and the subsequent precipitation of Fe and Mn was determined in mesocosms built to simulate a wetland. Mesocosms were constructed with plexiglass sheets and measured 6" x 6" x 24". Each mesocosm was filled with a 4-inch layer of limestone gravel beneath 17 inches of weathered pine bark mulch. A perforated PVC pipe was installed within the limestone layer to act as an underground drain. With mulch as the only source of available carbon, a 15% decrease in total sulfate concentration occurred in AMD containing initial concentrations of 1500, 750 and 375 mg/L sulfate. The population of SRB averaged 10⁵/ g dry mulch. The addition of 300 mg/L carbon as lactate resulted in an a 3 log₁₀ increase in SRB population. Following the addition of carbon as lactate, the concentration of sulfate decreased 95%. Total Fe decreased 90% from inflow concentrations prior to the addition of lactate, and decreased 96% following the addition of lactate to the AMD. The effect of varying the influent pH of AMD was studied using wetland mesocosms, and a pH of 3.5 adversely affected microbial sulfate reduction and water quality improvement. Populations of SRB decreased by 3 log₁₀ from an initial population of 10⁸ SRB/g dry mulch. Iron and Mn concentrations decreased 70 and 37% respectively. Hydrogen ion concentration increased to 7.0 and above when inflow pH was 4.5 and 6.0, but increased to an average of 6.4 when inflow pH was 3.5. The effect of different concentrations of Fe within AMD was investigated using wetland mesocosms, and total inflow Fe concentrations of 155 and 301 mg/L resulted in a greater percent decrease in sulfate concentrations than at a lessor Fe concentration averaging 85 mg/L. Total Mn decreased 12% at an inflow Fe concentration of 85 mg/L, and decreased 43% at an inflow Fe concentration of 301 mg/L. The results generated from both the analysis of the Pine Branch wetland and the laboratory mesocosm experiments demonstrate that subsurface flow constructed wetlands are a viable form for treatment of AMD. 2197820b-4775-4425-b667-55393f34b513,"This thesis deals with the deliberate insertion of nonlinear elements in second-order linear control systems for the purpose of improving their transient response. The main body consists of a method of obtaining a desired step response by placing a nonlinear computer in the forward loop. This computer fixes the system trajectory in the phase plane by determining the required output velocity for the error present at any time. An inner control loop adjusts the output velocity to agree with the computed signal in an extremely short time, thus giving a very close agreement between actual and desired responses. Several examples are presented to show the application of this method, and experimental verification is obtained with an analog computer. Areas of future study and practical limitations are discussed in the final sections of the thesis.
The effect of influent organic compounds on the performance of biological nutrient removal systems
Abu-Ghararah, Ziad (Virginia Polytechnic Institute and State University, 1988)
The main objective of the research was to investigate the effect of influent organic compounds on the performance of biological nutrient removal system. To carry out the investigation, a pilot plant system was designed and constructed. The system was operated as a UCT process at an influent flow rate of 0.15 liters/minute and a sludge age of 13 days. The influent wastewater was domestic sewage. Excess biological phosphorus removal and steady-state conditions were established before making experimental measurements, or adding supplemental substrate. The effects of separate addition of formic, acetic, propionic, butyric, isobutyric, valeric, and isovaleric acid, plus glucose, addition on phosphorus release under anaerobic conditions, and phosphorus uptake under aerobic conditions, were studied. The effects of the organic acid additions on the removal of nitrogen and COD, and changes in SOUR, MLVSS, and metals such as iron, magnesium, calcium and potassium, were also studied. In all experiments, the specific substrate was added continuously to the first anaerobic reactor for three days at an influent concentration of 100 mg COD/liter. Samples were collected from each reactor at the end of the addition period and analyzed for orthophosphate, nitrate, nitrite, sulfate, volatile fatty acids, COD, MLVSS, pH and metals. All added substrates, except formic acid and dextrose, caused significant increases in phosphorus release in the anaerobic stage, and phosphorus uptake, in the aerobic stage, and consequently, an increase in phosphorus removal efficiency. The molar ratios of phosphorus release to volatile fatty acid added obtained for propionic acid, acetic acid, butyric acid, and valeric acid were 0.44, 0.77, 0.78, and 1.72 respectively. However, on a COD basis, the greatest ratios of mg phosphorus released to mg COD utilized was produced by the addition of acetic acid (0.37) and valeric acid (0.19). It was also found that the branched organic acids, isobutyric and isovaleric, caused more phosphorus release in the anaerobic stage and better phosphorus removal efficiencies as compared with the nonbranching forms of the same organic acids. The molar ratios of phosphorus release for these two acids were 0.8 and 2.3, respectively, and on a COD basis were 0.16 and 0.25. For engineering applications, it is suggested by this research that at least 20 mg COD equivalent of acetic acid is needed for the removal of I mg phosphorus. The results obtained by this investigation were consistent with the hypothesis proposed by Marais et al., 1983. The most recent biochemical models, proposed by Comeau et al., 1986 and Wentzel et al., 1986, were also tested using the data collected in the present investigation. Both models, in most cases, overestimated the ratios of phosphorus release to volatile fatty acid utilized. A speculative model for anaerobic metabolism by poly-p bacteria of volatile fatty acids which contain both odd and even numbers of carbon atoms was proposed. All added substrates produced no effect on both COD and TKN removals. Metal releases were found to correlate with the amount of phosphorus release.
The effect of water quality on the metabolism of the lactic streptococci
Benoit, Robert E. (Water Resources Research Center, Virginia Polytechnic Institute, 1968)
The effects of atrazine on nitrogen cycling in a freshwater wetland microcosm
Wilhite, Rhonda E. (Virginia Tech, 1996)
Atrazine and other pesticides may damage non-target ecosystems, such as wetlands, by destroying vegetation or by disrupting microbial communities and nutrient cycling. The addition of atrazine to a wetland microcosm will disrupt the nitrogen cycle by inhibiting nitrifying and denitrifying bacteria. The inhibition or absence of functional groups of bacteria will limit the ability of the wetland to remove nutrients and herbicides, thus increasing nitrogen levels in non-point source pollution. All tests were conducted in the laboratory using microcosms established from intact plant/sediment core subsamples collected from a natural freshwater wetland. Each microcosm received distilled water, atrazine, or acidified distilled water (pH 2) by groundwater seepage. Microcosms were dosed with 1.5 mg/L and 4.5 mg/L atrazine. Acid treatment of microcosms was done to provide a perturbation reference to the microcosm. Nitrite, nitrate, and ammonia levels were determined, as was dissolved oxygen, pH, and conductivity. The nitrifying bacteria and denitrifying bacteria were enumerated. Data were analyzed with one way analysis of variance (ANOVA) with block and treatment interaction and Tukey's Studentized Range test. Acid treatment of microcosms provided a positive control of microcosm disturbance. Acidification of the sediment disturbed the nitrogen cycle by suppressing ammonia dependent microbial processes. Atrazine (at 1.5 and 4.5 mg/L atrazine) did not significantly affect the number of nitrifying bacteria. The denitrifying or nitrate reducing bacteria were stimulated by exposure to atrazine and nitrate reduction became the dominant microbial process following exposure to atrazine. This shift toward denitrification and nitrate reduction processes caused increased levels of nitrite in the overflow waters. The continued accumulation of atrazine may cause nitrogen losses from an ecosystem which is already nitrogen limiting. Over time, predominant plant species may change, becoming primarily nitrogen fixing species. Changes in plant species would bring about changes in associated microbial populations. Excess nitrogen present in wetland runoff may cause algal blooms in receiving waters and pose health risks in contaminated drinking water. Accumulation of atrazine in wetlands may, ultimately, result in the loss of buffer zones.

Browsing by Author "Benoit, Robert E."

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