Utilization of toluene by Pseudomonas putida as its sole carbon and energy source affects morphology, outer membrane protein composition, and glycolipid production. Two strains of P. putida were found to utilize toluene and to coexist in continuous and batch culture. The two strains were designated translucent and opaque, based upon their readily identifiable coloration when grown on Luria agar. The translucent strain was the dominant strain in continuous culture conditions. The outer membrane proteins of P. putida were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. When toluene is the carbon and energy source, the trend in protein composition was towards a general increase in concentration of lower molecular weight proteins (wt). A similar decrease occurred in the concentration of higher molecular weight proteins in the range of 70X104-9X104 mol wt. P. putida produces glycolipids when grown on toluene as a sole carbon and energy source. Three glycolipids have been isolated from chemostat and batch culture spent media, using thin layer chromatography on silica gel GF254· The glycolipids are believed to be previously reported mono- and di-rhamnolipids that function as biosurfactants. The release of glycolipid into the media is believed to function to emulsify toluene, aiding in toluene uptake.

Natural organic matter (NOM), a complex mixture of organic compounds, influences drinking water quality and water treatment processes. The presence of NOM is unaesthetic in terms of colour, taste and odour, and may lead to the production of potentially carcinogenic disinfection by-products (DBPs), as well as biofilm formation in drinking water distribution systems. Some NOM removal processes such as coagulation, magnetic ion exchange resin (MIEXTM) and membrane filtration produce sludge and residuals. These concentrated NOM-containing sludges from alum precipitation, membrane treatment plants and MIEX regeneration must therefore be treated prior to disposal. The white-rot fungi possess a non-specific extracellular oxidative enzyme system composed of lignin peroxidase (LiP), manganese-dependent peroxidase (MnP) and laccase (Lac) that allows these organisms to mineralise lignin and a broad range of intractable aromatic xenobiotics. Rojek (2003) has shown the capabi lity of Phanerochaete chrysosporium ATCC 34541 to remove 40-50% NOM from solution, however, this was found to be mainly due to adsorption and to be a partially metabolically linked activity. Consequently, the bioremediation of NOM wastes by selected white-rot fungi was further investigated in the present study. The P. chrysosporium seemed to preferentially remove the very hydrophobic acid (VHA) fraction, and so was most effective for a NOM preparation with a high proportion of hydrophobic content (and so high in colour and specific UV absorbance (SUVA)). The extent of NOM decolourisation by P. chrysosporium in three growth media with different C:N ratios followed the trends: Waksman (C:N = 6) > Fahy (C:N = 76) > Fujita medium (C:N = 114), such that the lower the C:N ratio, the greater NOM removal. This was consistent with the findings of Rojek (2003), who used a different NOM preparation and demonstrated that the removal of NOM increased with decreased C:N ratio (1.58-15.81). As removals of NOM with P. c hrysosporium ATCC 34541 were low, and little biodegradation occurred, this organism was compared with P. chrysosporium strain ATCC 24725, Trametes versicolor ATCC 7731, and three strains of yeast (Saccharomyces species arbitrarily denoted 1, 2 and 3). T. versicolor gave the greatest removal (59%) which was attributed largely to degradation, whereas the NOM removal by the two strains of P. chrysosporium (37%) and the yeast was predominantly due to adsorption as indicated by the deep brown colouration of the biomass. Saccharomyces sp. 1, 2 and 3 removed 12%, 61% and 23% of the colour, respectively. Although Saccharomyces sp. 2 had similar high colour reduction to T. versicolor, the specific removal values differed markedly: 0.055 compared to 0.089 mg NOM/mg biomass, respectively. The low level of the ligninolytic enzymes secreted by both strains of P. chrysosporium corresponded with the low degree of NOM removal by biodegradation as shown by high performance size exclusion chromatography (HPSEC). The high NOM removal attained by T. versicolor was attributed to the activities of the ligninolytic enzymes, especially laccase. The NOM removal was attributed to the breakdown of the high molecular weight compounds to form a pool of low molecular weight materials, which were then most likely utilised by the T. versicolor. Growth of T. versicolor cultures at 36oC caused inhibition or denaturation of the activity of the phenoloxidase enzymes compared to those grown at 30oC. The low activity of LiP in both cultures suggested that this enzyme may not play much of a role in NOM removal. The higher levels of MnP and Lac activities at 30oC were responsible for the greater NOM removal (73% vs. 59%) and thus the cleavage of aromatic rings, conjugated and C-Cβ αbonds in phenolic moieties, as well as catalysing alkyl-aryl cleavage in the NOM structures. T. versicolor cultured in Waksman medium with higher initial glucose (5 g/L cf. 2 g/L) led to lower ligninolytic enzyme activities and a lower degree of NOM removal (25% less colour reduction), probably due to preferential use of glucose over NOM as carbon source. NOM removal (mg removed) increased linearly with NOM concentration up to 600 mg C/L (62 mg (A446); 31 mg (A254)), above which removal decreased markedly. This trend coincided with increasing total ligninolytic enzyme activity, where the level of Lac increased up to 600 mg C/L NOM although MnP decreased gradually across the range while LiP was only detected for 100 and 300 mg C/L NOM. Hence, the removal of NOM from solution by T. versicolor was associated with high oxidative enzyme activity, particularly of laccase. Laccase was the major extracellular enzyme secreted by T. versicolor and by deduction, played a major role in NOM removal. The optimum temperature for Lac activity secreted by T. versicolor cultured in Waksman medium supplemented with 4.5 g/L wheat bran plus 0.5% Tween 80 was determined to be 50oC. The optimum pH for the Lac activity for guaiacol and NOM was identified as pH 4.0-4.5. Although the optimum enzyme activity occurred at 50oC, 30oC was recommended for enzymatic removal of NOM as the phenoloxidase enzyme activity may be denatured if the NOM removal process were considered to run for long period at high temperature. Although agitation led to apparent enzyme denaturation, fermentations with continuous agitation promoted enzyme activity faster than those with occasional agitation (agitated every 6 hours for 30 minutes at 130 rpm and 30oC) as it provides better mass transfer. However, it seemed that continuous agitation had an adverse effect on the fungal growth and enzyme production over extended fermentation periods. Addition of 4.5 g/L wheat bran to modified Waksman medium in the absence of NOM led to high production of Lac activity compared with LiP and MnP activities, showing its great potential as a laccase inducer. Addition of Tween 80 alone to the cultures led to a small improvement in Lac activity; however, with the presence of wheat bran it caused marked increases in LiP, MnP and Lac activit ies. When NOM was added to cultures of T. versicolor with the two supplements, it led to markedly reduced Lac activity, but increased LiP and MnP activities, and no improvement in NOM removal compared with the cultures in the absence of supplements (12 mg (or 61%) cf. 15 mg (or 73%) for 100 mg C/L after corrected for colour from and adsorption by wheat bran).

Biodegradation of poorly water soluble hydrocarbons, such as n-alkanes and polycyclic aromatic hydrocarbons (PAHs) is often limited by the low availability of the pollutant to microbes. Adhesion of microorganisms to the oil-water interface can influence this availability. Our approach was to study a range of compounds and mechanisms to promote the adhesion of a hydrophilic PAH degrading bacterium, Pseudomonas fluorescens LP6a, to an oil-water interface and examine the effect on biodegradation of phenanthrene by the bacteria. The cationic surfactants cetylpyridinium chloride (CPC), poly-L-lysine and chlorhexidine gluconate (CHX) and the long chain alcohols 1-dodecanol, 2-dodecanol and farnesol increased the adhesion of P. fluorescens LP6a to n-hexadecane from ca. 30% to ca. 90% of suspended cells adhering. The alcohols also caused a dramatic change in the oil-water contact angle of the cell surface, increasing it from 24° to 104°, whereas the cationic compounds had little effect. In contrast, cationic compounds changed the electrophoretic mobility of the bacteria, reducing the mean zeta potential from –23 to –7 mV in 0.01M potassium phosphate buffer, but the alcohols had no effect on zeta potential. This results illustrate that alcohols acted through altering the cell surface hydrophobicity, whereas cationic surfactants changed the surface charge density. Phenanthrene was dissolved in heptamethylnonane and introduced to the aqueous growth medium, hence forming a two phase system. Introducing 1-dodecanol at concentrations of 217, 820 or 4100 mg/L resulted in comparable increases in phenanthrene biodegradation of about 30% after 120 h incubation with non-induced cultures. After 100 h of incubation with LP6a cultures induced with 2-aminobenzoate, 4.5% of the phenanthrene was mineralized by cultures versus more than 10% by the cultures containing initial 1-dodecanol or 2-dodecanol concentrations of 120 or 160 mg/L. The production and accumulation of metabolites in the aqueous phase responded similarly to the addition of 1-dodecanol. Further experiments showed that the positive influence of the alcohols could not be attributed to the changes in surface and interfacial tension or increase in biomass concentration. The results suggest that enhanced adhesion of bacterial cells to the oil-water interface was the main factor responsible for the observed increase in phenanthrene biodegradation by P. fluorescens LP6a. / Chemical Engineering

Graduation date: 2005

Trichloroethene (TCE) is the most frequently detected organic contaminant in groundwater, is classified as a probable human carcinogen, and exhibits toxicological effects on the human endocrine, immune, developmental, and reproductive systems. While significant research efforts have been devoted to the development of strategies for remediating TCE-contaminated groundwater, their advancement is currently hindered by limitations in current methodologies for measuring in situ reductive dechlorination rates, especially for sorbing solutes. This dissertation describes the development, evaluation, and demonstration of a method for measuring in situ reductive dechlorination rates that utilizes single-well, "push-pull" test technology. Initial field tests indicated that trichlorofluoroethene (TCFE) could be used as a surrogate for TCE in push-pull tests since (a) TCE and TCFE were transported similarly and (b) TCFE underwent reductive dechlorination by a pathway analogous to that of TCE while retaining the fluorine label. Because TCFE and TCE experienced sorption at the selected field site, a novel data analysis technique called "forced mass balance" (FMB) was developed to obtain in situ transformation rates of sorbing solutes from push-pull test data. The FMB technique was evaluated by quantifying errors in rates derived by applying FMB to push-pull test data generated by a numerical model. Results from simulated tests indicated that an example in situ rate for the reductive dechlorination of TCFE, which was obtained by applying FMB to field data, was underestimated relative to the true in situ rate by 10%. The utility of the rate-determination method presented in this dissertation was demonstrated by using it to evaluate the effectiveness of a chemical amendment, namely fumarate, at enhancing in situ reductive dechlorination rates in TCE-contaminated groundwater. Reductive dechlorination rates increased following three consecutive additions of fumarate in all five of the tested wells. The development of the rate-determination method described in this dissertation advances the state of bioremediation technology because methods for measuring in situ transformation rates are needed to both assess the potential for natural attenuation and to quantify the effects of bioremediation techniques in the field. / Graduation date: 2003

Mycobacterium vaccae JOB5 and Graphium sp. were studied to evaluate their ability to cometabolize methyl tert-butyl ether (MTBE) and its metabolites after growth on two different alkanes, propane and iso-pentane. Both cultures were capable of cometabolizing MTBE and the metabolites, tert-butyl formate (TBF) and tert-butyl alcohol (TBA). MTBE, TBF, and TBA did not support growth of either microbe. Higher degradation rates were obtained in the bacterial system when the cultures were grown on iso-pentane. Nonlinear least squares regression and direct linear plot methods were used to estimate kinetic coefficients and provided comparable results. The enzymes from Mycobacterium vaccae JOB5 and Graphium sp. that promote the cometabolism of MTBE and its metabolites exhibited similar kinetics and substrate inhibition. The presence of the substrate decreased the degradation rate of MTBE and TBA suggesting competitive inhibition and preference for the substrate. Blockage experiment with acetylene suggested the presence of an alkane monooxygenase for the metabolism of MTBE and TBA, and a hydrolytic enzyme for the degradation of TBF. The presence of a hydrolase enzyme was supported by the fact that TBF was degraded to TBA under either aerobic or anaerobic conditions and was not inhibited by the presence of acetylene, propane, or isopentane. Measured rates of abiotic hydrolysis of TBF were significantly less than biodegradation rates. Acetylene acted as a reversible inhibitor for both cultures when tested in the presence of the growth media and as an inactivator when tested in the presence of a phosphate solution for the bacterial system. Growth-batch reactor experiments were conducted to compare the degradation of iso-pentane and MTBE with the predicted degradation rates based upon kinetic constants determined from single and dual-compound experiments. Experimental data was modeled with Monod kinetics and STELLA�� software. Reasonable predictions of reactor performance were achieved when Monod maximum utilization rates were increased compared to single and dual-compound experiments. / Graduation date: 2002

A pilot scale demonstration of a biological permeable barrier was conducted in a pentachlorophenol-contaminated aquifer at a wood preserving facility. A permeable reactor was constructed to fit within a large diameter well. Arranged in series, a cylindrical reactor 24" x 36" (0.61 x 0.91m) (diameter x height) was partitioned to provide three biological treatment zones. Pentachlorophenol (PCP) biodegradation was evaluated under several environmental conditions using a mixed microbial consortium supported on ceramic saddles. Imitation vanilla flavoring (IVF), a mixture of propylene glycol, guaiacol, ethyl vanillin and sodium benzoate, served as the electron donor. In the absence of exogenous substrate, PCP was not degraded in the inoculated permeable barrier. Substrate addition under oxidizing conditions also failed to initiate PCP removal. Anaerobic conditions however, promoted in-situ PCP degradation. PCP reductive dechlorination resulted in the transient production of 3,4,5-trichlorophenol through sequential ortho dechlorinations. Continued carbon reduction at the meta and para positions resulted in 3,4-dichlorophenol and 3,5-dichlorophenol production. Complete removal of all intermediate degradation products was observed. Reactor operation was characterized through two independent laboratory and field companion studies. Experiments were conducted to evaluate (1) the effect of supplemental electron donor concentration (IVF) and (2) the effect of sulfate, a competitive electron acceptor on PCP reductive dechlorination. Results from laboratory and field conditions were consistent. (1) In the presence of an exogenous electron donor, PCP degradation was independent of supplemental donor concentration (10, 25, 50, 100 mg COD/L). However, a comparatively slower rate of PCP degradation was observed in the absence of electron donor. (2) The presence of sulfate was not inhibitory to PCP degradation. However, compared to systems evaluated in the absence of sulfate, slower rates of PCP transformation were observed. Passive operation and low energy requirements, coupled with potential contaminant mineralization suggest that the biological permeable barrier is a highly effective tool for subsurface restoration. / Graduation date: 2000

Graduation date: 1997

2,4,6-trinitrotoluene (TNT) is a toxic and recalcitrant pollutant contaminating soils and groundwater. Therefore, characterization of microbial populations of TNT-contaminated soils and isolation of bacteria degrading this pollutant are of primordial importance. Comparison of hybridizations of 16S rRNA derived from uncontaminated and TNT-contaminated soil samples required the development of a functional ANOVA model. Specifically, a statistical tool was necessary to compare dissociation curves obtained from thermal dissociation analysis of RNA hybridizations to DNA microarrays, and to determine if the dissociation curves significantly differed. To test and validate the model, we used dissociation curves from in vitro transcribed 16S rRNA amplified from two environmental samples hybridized to a phylogenetic microarray. Detection and rejection of outlier curves was important for appropriate discrimination between curves. The identification of significantly different curves was more efficient with the model than approaches relying on measurements at a single temperature. This functional ANOVA analysis was used to improve discrimination between hybridizations of two soil microbial communities. Following hybridization of in vitro transcribed 16S rRNA derived from an uncontaminated and a TNT-contaminated soil sample to an oligonucleotide microarray containing group- and species-specific perfect match (PM) probes and mismatch (MM) variants, thermal dissociation was used to analyze the nucleic acid bound to each PM-MM probe set. Functional ANOVA of the dissociation curves generally discriminated PM-MM probe sets when values of Td (temperature at 50% probe-target dissociation) could not. Maximum discrimination for many PM and MM probes often occurred at temperatures greater than Td. Comparison of signal intensities measured prior to dissociation analysis from hybridizations of the two soil samples revealed significant differences in domain-, group-, and species-specific probes. Functional ANOVA showed significantly different dissociation curves for 11 PM probes when hybridizations from the two soil samples were compared, even though initial signal intensities for 3 of the 11 did not vary. These differences in hybridizations between the two soil samples were likely the result from the presence of TNT. The effect of TNT on soil microbial communities was further investigated with additional uncontaminated and TNT-contaminated soil samples using 16S rRNA PCR-DGGE and cultivation-dependent techniques. In all contaminated soil samples, the amount of DNA extracted was lower than in the uncontaminated ones. Analysis of bacterial diversity by DGGE showed a predominance of Pseudomonadaceae and Xanthomonadaceae in the TNT-contaminated soil samples compared to the uncontaminated ones. Caulobacteraceae were also present in several contaminated soil samples. The culturable microflora of these soils was studied by plate counts on agar supplemented with dilute nutrient broth. The number of CFUs was lower in a TNT-contaminated soil inoculum than in an uncontaminated one. In the former, most of the CFUs belonged to Pseudomonadaceae, and to a lesser extent, to Caulobacteraceae. In addition to the above contaminated soil samples, a pristine soil was artificially contaminated with different concentrations of TNT and incubated for 4 months. The amount of DNA extracted decreased in the highly contaminated soil samples (1.4 and 28.5 g TNT/kg soil). After 7 days of incubation of these soil samples, there was a clear shift of their original flora to a population dominated by Pseudomonadaceae, Xanthomonadaceae, Comamonadaceae and Caulobacteraceae. When the TNT concentration was lower (140 mg TNT/kg soil), a moderate shift in the bacterial population was observed. These results indicate that TNT affects soil bacterial diversity and richness by selecting for a narrow range of bacterial species that belong mostly to Pseudomonadaceae and Xanthomonadaceae. TNT-contaminated soil samples probably contained TNT-degrading bacteria. In order to isolate bacteria that can denitrate TNT, enrichment cultures were carried out with TNT as sole nitrogen source and in the absence of oxygen. These cultures were established starting with an uncontaminated or a TNT-contaminated soil inoculum, in the presence or absence of ferrihydrite. A significant release of nitrite was observed in the liquid culture containing TNT, ferrihydrite and inoculum from a TNT-contaminated soil. Under these conditions, Pseudomonas aeruginosa was the predominant bacterium in the enrichment, leading to the isolation of P. aeruginosa ESA-5 as a pure strain. The isolate had TNT denitration capabilities as confirmed by nitrite release in oxygen-depleted cultures containing TNT and ferrihydrite. Concomitantly, TNT-reduced compounds were detected as well as unidentified polar metabolites. The concentration of nitrite released from TNT was proportional to the concentration of ferrihydrite in the medium. The release of nitrite was lower when the concentration of initially spiked TNT was reduced by one order of magnitude. Under these conditions, the concentration of nitrite peaked and then its concentration slowly decreased and production of ferrous ions was detected. A decrease of nitrite concentration and production of ferrous ion were observed when TNT was omitted and nitrite and ferrihydrite were provided. These results suggest that nitrite-reducing conditions were initially achieved, followed by iron-reducing conditions. When grown aerobically on a chemically defined medium, P. aeruginosa strain ESA-5 produced a greenish extracellular compound. This product was identified as phenazine-1-carboxylic acid (PCA). When purified PCA was incubated with TNT in the presence of NADH, nitrite was released. The concentration of nitrite released was dependent on the concentration of NADH and PCA. Denitration also occurred with two TNT-related molecules, 2,4,6-trinitrobenzaldehyde and 2,4,6-trinitrobenzyl alcohol. The release of nitrite was coupled with the formation of two polar metabolites and mass spectrometry analyses indicated that each of these compounds had lost two nitro groups from the trinitroaromatic parent molecule. The results obtained with the PCA mediated denitration of TNT in the presence of inhibitors of oxygen reactive species suggested the involvement of superoxide (O2.-). When exogenous PCA was added to a P. aeruginosa ESA-5 liquid culture containing TNT as sole nitrogen source, bacterial growth was significantly enhanced compared to cultures containing TNT without PCA.

It is recognized that microbial transformations are the primary mechanism of organic contaminant removal in natural and constructed wetland systems. However, not much is known about urban wetland microbial communities or their functional capacity to process contaminants. The objective of this research was to first characterize the physiological and phylogenetic diversity of microbial communities of different urban wetland types using the BIOLOG™ method and through DGGE of 16S rRNA sequences. The capacity of urban wetlands to attenuate model chlorinated aromatic compounds (2,4-D and 3-CBA) was assessed by UPLC biodegradation and 14C mineralization experiments. Toxicity tests were conducted to assess microbial tolerance to pollutant addition. In general, results indicate that urbanization has a homogenizing effect on microbial community structure and distribution within urban wetland systems, regardless of type. Urban wetlands also appear to have a limited capacity to remove chlorinated organic pollutants. Microbial community tolerance to chlorinated organic pollutants is relatively high, whereas heavy metal tolerance was found to coincide with history of contaminant exposure. / UOIT