Studies were conducted to explore the interactions among rye grass, chlorophenols and microorganisms. The objectives were to examine some of the processes by which plants affect the fate of subsurface organic contaminants. The research was divided into three studies: interactions between live grasses and 2,4-dichlorophenol (DCP), 2,4,6-trichlorophenol (TCP), and pentachlorophenol (PCP); physico-chemical interactions between the three chlorophenols and root tissue; and effect of root exudates on biodegradation of TCP. To study the interactions between plants and organic contaminants, rye grass plants were grown in solutions containing DCP, TCP or PCP for one to three weeks. The grass removed substantial amounts of the chlorophenols throughout the incubation time. The majority of each chlorophenol removed from solution could not be recovered by non-destructive solvent extraction. The removal of the chlorophenols from solution and the unrecoverability of the removed compound followed different kinetics, indicating that the two are different processes. Both contaminant removal and unrecoverability were closely related to root surface area but not to transpiration. A qualitative model was developed to describe the uptake of organic contaminants by plants. The data demonstrate the importance of physico-chemical interactions between contaminants and roots and suggest that maximization of root surface area should be one consideration when selecting a plant species for phytoremediation. To study the physico-chemical interactions between plant roots and organic contaminants, the distribution of DCP, TCP and PCP within a three phase system was examined. The three phases were severed grass roots, water and an organic solvent, either hexane or ethyl acetate. The chlorophenol mass that partitioned into the solvent phase was inversely correlated with root mass and root surface area index. Partition coefficients calculated with respect to the organic liquid phase were inversely correlated with root mass and root surface area index. A similar partitioning experiment was conducted using PCP placed in a solution containing only the dissolved organic material released by roots. These resulting partition coefficients decreased with increasing organic carbon concentration. It appeared that the organic compounds released into solution by the roots affected the movement of the chlorophenol into the organic liquid phase. It is proposed that the presence of roots simultaneously promoted retention of the chlorphenols in the aqueous phase and provided a sorption site. The effect of grass root exudates and glucose on the lag time associated with 2,4,6-trichlorophenol (TCP) degradation by an unacclimated microbial inoculant and an acclimated microbial inoculant was investigated. The presence of an alternate organic carbon source reduced lag time for both the acclimated microbial inoculant and the inoculant that had not been previously exposed to chlorinated phenols. The lag time for acclimation of microbes to TCP mineralization was affected by the ratio of the alternate organic carbon source concentration to the biomass concentration. It is proposed that the presence of a readily available, alternate organic carbon source affected lag time through promotion of microbial population growth and provision of a preferred source of carbon and energy. The results indicate that rye grass may directly, through partitioning and uptake, and indirectly, through soil microbes, affect the fate of chlorophenols in the subsurface environment. / Ph. D.

Laboratory microcosm experiments were conducted to assess the potential for biostimulation and bioaugmentation as source reduction measures in support of a monitored natural attenuation remedial strategy at Naval Amphibious Base (NAB) Little Creek. Previous work with laboratory microcosms conducted under simulated natural (unamended) conditions has demonstrated that indigenous dehalorespirators were capable of partial dechlorination of tetrachloroethene (PCE) to cis-dichloroethene (cis-DCE). This study attempts to achieve complete reductive dechlorination with amendments to static microcosms to test the hypotheses that nutrient-limited or microorganism-limited conditions exist in aquifer sediments obtained from the site. The enhanced bioremediation experiments were comprised of nutrient-amended microcosms receiving additions of electron donors, mineral medium, or anaerobic digester supernatant, and dechlorinating culture-amended microcosms were inoculated with a culture capable of transforming PCE to ethene. Reductive dechlorination in the nutrient-amended microcosms proceeded to cis-DCE over a 260-day study period, at slightly higher rates than in experiments conducted with aquifer sediments from the same location under natural conditions. Inoculation of aquifer sediments with a small amount of dechlorinating culture initiated rapid transformation of PCE to vinyl chloride (VC) by day 18 of the study. Zero-order rates of PCE dechlorination in unamended, propionate-, formate-, mineral medium-, digester supernatant-, and dechlorinating culture-amended microcosms were 0.24, 0.750, 1.30, 0.339, 0.177, and 1.75 µM/day, respectively. The results of this study suggest that an engineered biostimulation approach alone may not be as beneficial for PCE source reduction at NAB Little Creek, than bioaugmentation with competent dehalorespirators, along with the inclusion of supplemental nutrients which would be available to stimulate dechlorination activity of both indigenous and introduced microorganisms. / Master of Science

Methyl tertiary-butyl ether (MTBE) and its degraded form tertiary-butyl alcohol (TBA) are both known carcinogens that have contaminated groundwater aquifers across the United States. MTBE is a synthesized compound, once widely used as an additive in gasoline to increase oxygenation. Because of its popularity, MTBE was released into the environment primarily through fuel combustion and leaking underground storage tanks. These two compounds are known to be recalcitrant to most conventional physico-chemical treatment methods. Previous studies have suggested that bioremediation is effective at degrading MTBE and TBA in contaminated groundwater. Bioremediation involves the injection of oxygen, nutrients, and pre-adapted bacterial cultures into contaminated groundwater to increase the rate of natural biodegradation. In this study, a historically documented spill in Cambria, CA was modeled employing the Groundwater Modeling System software (GMS) to compare the effectiveness of the baseline treatment approach to that of in-situ bioremediation. MODFLOW was used to simulate groundwater flow, while MT3DMS was used to simulate dispersal and biodegradation of MTBE. Well data from public records was used as comparative values for hydraulic head and MTBE concentrations. Additional information from cleanup reports provided data for the physical properties of the aquifer. This included bedrock elevation, soil types, and storativity. Conductance, recharge rate, and hydraulic conductivity were calibrated using Parameter Estimation Software (PEST). The constants applied in MT3DMS simulations, such as dispersivity values, molecular diffusion coefficients, and retardation factors, were calculated manually using available, semi-empirical approaches. The model was first run emulating bioremediation using a high first order biodegradation rate estimated to be 8.6 day-1. This was compared to an instance of natural attenuation, with a first order biodegradation rate of 0.0074 day-1. The case study investigated herein primarily implemented a pump and treat system relying on granular activated carbon and a series of trickling filters and clarifiers. Pump and treat operations began in 2000 and officially ended by the start of 2015. Even though treatment was terminated, the preliminary remedial goal for MTBE was not achieved. In the model created for this project, the bioremediation simulation predicted attainment of this treatment goal by 2010 after starting treatment in 2002. This increase in predicted removal rate over conventional approaches suggests bioremediation may be a viable and effective treatment technique when removing MTBE from groundwater. This predicted rate of removal suggests that bioremediation is more effective than the techniques used during the Cambria cleanup. It is important to note, there were many assumptions and simplifications made during the creation of the model. This includes the calibrated parameter values obtained from PEST iterations along with calculated parameter estimates regarding MTBE fate and transport. During set up, it was assumed that soil type consisted solely of silty clay and the bedrock layer was at a constant 45 ft below ground level. Additionally, the modeled in-situ bioremediation scenario assumes a best-case scenario, with the high first order biodegradation rate. For future modeling improvements, it is recommended to conduct onsite field testing to obtain degradation rates that more closely reflect rates found in the modeled region. A more complete mapping of the aquifer would also provide the model with increased reliability. Future models should also evaluate additional MTBE spill events and how differing terrains impact the effectiveness of in-situ bioremediation of MTBE.

No description available.

<p>The mass transfer of ethanol and toluene from air stream to liquid phase, and bioremediation of contaminated air streams containing either ethanol or toluene have been investigated using a stirred tank bioreactor. This investigation was conducted in six phases: </p> 1) mass transfer experiments involving the transport of toluene and ethanol from contaminated air streams into the liquid phase,</p> 2) study of air stripping effects of ethanol and toluene out of the liquid phase,</p> 3) batch growth experiments to determine growth kinetic models and model parameters,</p> 4) bioremediation of ethanol or toluene as the sole substrate to determine the capacity of Pseudomonas putida (P. putida) (ATCC 23973) growth on these substrates,</p> 5) toluene removal from contaminated air streams using ethanol and benzyl alcohol as co-substrates, and</p> 6) modelling the above studies using metabolic pathways to better understand the bioremediation process.</p> <p>Preliminary oxygen mass transfer studies showed that the presence of ethanol in the liquid phase enhances the overall oxygen mass transfer coefficients. Increasing the ethanol concentration from 0 to 8 g/L caused the oxygen mass transfer coefficients to increase from 0.015 to 0.049 s-1, and from 0.017 to 0.076 s-1, for impeller speeds of 450 and 600 rpm, respectively. Mass transfer studies using ethanol vapor in the air stream demonstrated complete absorption into the aqueous phase of the bioreactor at all operating conditions investigated (air flowrates up to 2.0 L/min and inlet concentrations up to 95.0 mg/L) and therefore mass transfer coefficients for ethanol absorption could not be determined. On the other hand, toluene mass transfer coefficients could be measured and were found to be 8.3x10-4, 8.8x10-4 and 1.0x10-3 s-1 at agitation speeds of 300, 450 and 600 rpm, respectively. The ethanol air stripping parameters (b values) were determined (at initial ethanol liquid concentration of 8.6 g/L) to be 0.002 and 0.007 h-1 for air flow rates of 0.4 L/min (0.3 vvm) and 1.4 L/min (1 vvm), respectively. The toluene air stripping rates, at initial liquid toluene concentration of 440 mg/L, were found to be 1.9, 5.3, 10.4, and 12.6 h-1 for air flow rates of 0.4, 0.9, 1.4, 2.1 L/min, respectively, which is much higher than those of ethanol at the same air flow rates and stirring speed of 450 rpm. It was also observed that benzyl alcohol was not stripped to any detectable level at any of the operating conditions used in this study.</p> <p>The growth of <i>P. putida</i> using toluene as sole substrate was carried out at several operating conditions by varying the dilution rates (D) from 0.01 to 0.1 h-1, the toluene air inlet concentration from 4.5 to 23.0 mg/L and air flow rates of 0.25 to 0.37 L/min (resulting in inlet toluene loadings from 70 to 386 mg/L-h). Steady state operation could not be achieved with toluene as the sole substrate. Ethanol and benzyl alcohol were therefore used as co-substrates for the toluene removal process. In order to understand the kinetics of P. putida growing on ethanol or benzyl alcohol, batch growth experiments were carried out at different initial substrate concentrations. The specific growth rates determined from the batch runs showed that ethanol had no inhibition effect on the growth of P. putida. The growth on ethanol followed the Monod equation with the maximum growth rate of 0.56 h-1 and yield of 0.59. The results from the batch growth experiments on benzyl alcohol showed that benzyl alcohol inhibits the growth of P. putida when the initial concentration of benzyl alcohol in the growth media is increased. The maximum growth rate was 0.42 h-1 in the inhibition model and the yield value was 0.45. </p><p>By operating the bioreactor in continuous mode using a pure strain of <i>P. putida</i>, it was possible to continuously convert ethanol into biomass without any losses to the gas phase or accumulation in the bioreactor at inlet ethanol concentrations of 15.9 and 19.5 mg/L. With ethanol as a co-substrate, toluene was efficiently captured in the bioreactor and readily degraded by the same strain of P. putida. A toluene removal efficiency of 89% was achieved with an ethanol inlet concentration of 15.9 mg/L and a toluene inlet concentration of 4.5 mg/L. With the introduction of benzyl alcohol as co-substrate at a feed rate of 0.12 g/h, the toluene removal efficiency reached 97% at toluene inlet concentrations up to 5.7 mg/L. All the experimental results at steady state were obtained when the bioreactor operated in a continuous mode at a dilution rate of 0.1 h-1, an air flowrate of 0.4 L/min, an agitation speed of 450 rpm and a reactor temperature of 25.0oC. The results of this study indicate that the well-mixed bioreactor is a suitable technology for the removal of VOCs with both high and low water solubility from polluted air streams. The results were achieved at higher inlet pollutant concentrations compared to existing biofilter treatments.</p><p>A metabolic model has been developed to simulate the bioremediation of ethanol, benzyl alcohol and toluene. For continuous steady state operations, ethanol as a sole substrate required less maintenance for biomass growth (0.010 C-mol/C-mol-h) than bioremediations in the presence of toluene, as seen with the ethanol/toluene mixture (0.027 C-mol/C-mol-h), and the benzyl alcohol/toluene mixture (0.069 C-mol/C-mol-h).</p>

<p>The mass transfer of ethanol and toluene from air stream to liquid phase, and bioremediation of contaminated air streams containing either ethanol or toluene have been investigated using a stirred tank bioreactor. This investigation was conducted in six phases: </p> 1) mass transfer experiments involving the transport of toluene and ethanol from contaminated air streams into the liquid phase,</p> 2) study of air stripping effects of ethanol and toluene out of the liquid phase,</p> 3) batch growth experiments to determine growth kinetic models and model parameters,</p> 4) bioremediation of ethanol or toluene as the sole substrate to determine the capacity of Pseudomonas putida (P. putida) (ATCC 23973) growth on these substrates,</p> 5) toluene removal from contaminated air streams using ethanol and benzyl alcohol as co-substrates, and</p> 6) modelling the above studies using metabolic pathways to better understand the bioremediation process.</p> <p>Preliminary oxygen mass transfer studies showed that the presence of ethanol in the liquid phase enhances the overall oxygen mass transfer coefficients. Increasing the ethanol concentration from 0 to 8 g/L caused the oxygen mass transfer coefficients to increase from 0.015 to 0.049 s-1, and from 0.017 to 0.076 s-1, for impeller speeds of 450 and 600 rpm, respectively. Mass transfer studies using ethanol vapor in the air stream demonstrated complete absorption into the aqueous phase of the bioreactor at all operating conditions investigated (air flowrates up to 2.0 L/min and inlet concentrations up to 95.0 mg/L) and therefore mass transfer coefficients for ethanol absorption could not be determined. On the other hand, toluene mass transfer coefficients could be measured and were found to be 8.3x10-4, 8.8x10-4 and 1.0x10-3 s-1 at agitation speeds of 300, 450 and 600 rpm, respectively. The ethanol air stripping parameters (b values) were determined (at initial ethanol liquid concentration of 8.6 g/L) to be 0.002 and 0.007 h-1 for air flow rates of 0.4 L/min (0.3 vvm) and 1.4 L/min (1 vvm), respectively. The toluene air stripping rates, at initial liquid toluene concentration of 440 mg/L, were found to be 1.9, 5.3, 10.4, and 12.6 h-1 for air flow rates of 0.4, 0.9, 1.4, 2.1 L/min, respectively, which is much higher than those of ethanol at the same air flow rates and stirring speed of 450 rpm. It was also observed that benzyl alcohol was not stripped to any detectable level at any of the operating conditions used in this study.</p> <p>The growth of <i>P. putida</i> using toluene as sole substrate was carried out at several operating conditions by varying the dilution rates (D) from 0.01 to 0.1 h-1, the toluene air inlet concentration from 4.5 to 23.0 mg/L and air flow rates of 0.25 to 0.37 L/min (resulting in inlet toluene loadings from 70 to 386 mg/L-h). Steady state operation could not be achieved with toluene as the sole substrate. Ethanol and benzyl alcohol were therefore used as co-substrates for the toluene removal process. In order to understand the kinetics of P. putida growing on ethanol or benzyl alcohol, batch growth experiments were carried out at different initial substrate concentrations. The specific growth rates determined from the batch runs showed that ethanol had no inhibition effect on the growth of P. putida. The growth on ethanol followed the Monod equation with the maximum growth rate of 0.56 h-1 and yield of 0.59. The results from the batch growth experiments on benzyl alcohol showed that benzyl alcohol inhibits the growth of P. putida when the initial concentration of benzyl alcohol in the growth media is increased. The maximum growth rate was 0.42 h-1 in the inhibition model and the yield value was 0.45. </p><p>By operating the bioreactor in continuous mode using a pure strain of <i>P. putida</i>, it was possible to continuously convert ethanol into biomass without any losses to the gas phase or accumulation in the bioreactor at inlet ethanol concentrations of 15.9 and 19.5 mg/L. With ethanol as a co-substrate, toluene was efficiently captured in the bioreactor and readily degraded by the same strain of P. putida. A toluene removal efficiency of 89% was achieved with an ethanol inlet concentration of 15.9 mg/L and a toluene inlet concentration of 4.5 mg/L. With the introduction of benzyl alcohol as co-substrate at a feed rate of 0.12 g/h, the toluene removal efficiency reached 97% at toluene inlet concentrations up to 5.7 mg/L. All the experimental results at steady state were obtained when the bioreactor operated in a continuous mode at a dilution rate of 0.1 h-1, an air flowrate of 0.4 L/min, an agitation speed of 450 rpm and a reactor temperature of 25.0oC. The results of this study indicate that the well-mixed bioreactor is a suitable technology for the removal of VOCs with both high and low water solubility from polluted air streams. The results were achieved at higher inlet pollutant concentrations compared to existing biofilter treatments.</p><p>A metabolic model has been developed to simulate the bioremediation of ethanol, benzyl alcohol and toluene. For continuous steady state operations, ethanol as a sole substrate required less maintenance for biomass growth (0.010 C-mol/C-mol-h) than bioremediations in the presence of toluene, as seen with the ethanol/toluene mixture (0.027 C-mol/C-mol-h), and the benzyl alcohol/toluene mixture (0.069 C-mol/C-mol-h).</p>

Contamination of soils and groundwater with nitroaromatic compounds such as 2,4,6-trinitrotoluene (TNT) and dinitrotoluenes (DNTs) has drawn considerable attention due to widely distributed contamination sites and substantial efforts for cleanup. Two isomers of DNT, specifically 2,6-dinitrotoluene (2,6-DNT) and 2,4-dinitrotoluene (2,4-DNT), occur as soil and groundwater contaminants at former TNT production sites. The discovery of bacteria that use DNT isomers as electron donors has encouraged bioremediation at contaminated sites. Current work is extending the existing engineered bioremediation to naturally occurring in situ biodegradation and focuses on the application of natural attenuation (NA) as a remediation strategy for residual DNT at contaminated sites. More specifically this research evaluated factors influencing in situ bioremediation of DNTs and TNT in surface soils, vadose zones, and saturated medium. Applications involving surface soils and vadose zones investigated the potential of water infiltration to promote in situ bioremediation. Studies in saturated media were more applicable to NA. Factors that were also considered in studies conduced included: 1) the presence and distribution of degrading microbes in field soils (Barksdale, WI); 2) the dissolution and bioavailability of contaminants in historically contaminated soils; and 3) the effect of mixtures of contaminants (i.e., DNTs and TNT) on biodegradation processes. This research provided information useful for practitioners considering an in situ bioremediation NA as a remedial solution for contaminated sites. Under the condition simulating downflow of surface waters or rainwater, the rapid rate of DNT degradation could be facilitated by the availability of oxygen in the soil gas without concern of toxicity (i.e., nitrite evolution and pH drop) and addition of nutrients. As a result, in situ bioremediation or NA should be strongly considered as a remedial option for Barksdale soils and similar sites where relatively low concentrations of DNT isomers are present as contaminants. At TNT contaminated sites TNT was not mineralized by indigenous microorganisms despite oxidative biotransformation, and mixed culture capable of growth on DNT also could not develop the mineralization of TNT during DNT degradation. This suggests that the mixtures of contamination did not improve the potential for in situ TNT bioremediation.

No description available.

Arsenic (As) is a toxic metalloid and the remediation of soils and waters from this contaminant as well as the prevention of future contamination are still pending tasks in Chile. There are bacteria able to live in environments polluted with arsenic, as they have tolerance mechanisms for this metalloid, or even can use it for energy metabolism. The potential tolerance mechanisms include the production of siderophores, metabolites with chelating activity that can decrease the toxicity of metals and metalloids. Although a correlation between siderophore production and metalloid tolerance has been described, the structure of arsenic-binding siderophores and their implications in tolerance have not been elucidated yet. In this work, it is proposed that bacteria isolated from contaminated environments produce arsenic-binding siderophores. The main aims of this work are to study the production of the siderophores by arsenic-tolerant bacteria, to characterize these compounds and to determine their relation with tolerance to arsenic. Fourteen arsenic-tolerant bacteria were isolated from contaminated water, From these, four strains belonging to the species Rhodococcus erythropolis, Arthrobacter oxydans and Kocuria rosea were selected, in addition to the previously isolated Rhodococcus erythropolis S43, for a more detailed study. The isolates were used to produce siderophore extracts, which were then evaluated for their iron- and arsenic-binding activity. To detect the latter, a new method (As-mCAS) was set up, based on the Chrome Azurol S (CAS) test, an assay to detect iron-chelating activity of siderophores. After testing the extracts, R. erythropolis S43 was selected as the strain with the best arsenic-binding activity. For the subsequent chemical characterization, siderophores were produced under control conditions (iron-free M9 medium) and under stress conditions with arsenic (iron-free M9 medium with sodium arsenite). HPLC analysis of the extracts for both culture conditions showed the presence of a single compound with both an iron-chelating and an arsenic-binding activity. Analyses by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) for both culture conditions suggested the main presence of the siderophore heterobactin B. In addition, the genome of strain S43 was sequenced. A cluster of ars-genes was predicted, probably responsible for the arsenic-tolerance of the strain. In addition, a complete gene cluster for heterobactin production was found. However, no significant difference was obtained in the expression of these determinants in the presence or absence of arsenic, suggesting that the production of this siderophore in strain S43 is not responsible for the tolerance to the metalloid.

The ability of Chromolaena odorata propagated by stem cuttings and grown for six weeks in the greenhouse to thrive in soil containing different concentrations of PCB congeners found in Aroclor and transformer oil, and to possibly remediate such soil was studied under greenhouse conditions. Chromolaena odorata plants were transplanted into soil containing 100, 200, and 500 ppm of Aroclor and transformer oil (T/O) in 1L pots. The experiments were watered daily at 70% moisture field capacity. Parameters such as mature leaves per plant, shoot length, leaf colour as well as the root length at harvest were measured. C. odorata growth was negatively affected by T/O in terms of shoot length and leaf numbers, but no growth inhibition was shown by Aroclor. At the end of six weeks of growth, Plants size was increased by 1.4 and 0.46%, but decreased at -1.0% in T/O, while increases of 45.9, 39.4 and 40.0% were observed in Aroclor treatments. Mean total PCB recoveries were 6.40, 11.7, and 55.8μg in plants tissues at Aroclor treated samples resulting in a percentage reduction of PCB from the soil to 2.10, 1.50, and 1.10 at 100, 200, and 500mg/kg Aroclor treatments respectively. There was no PCB recovery from plants in transformer oil treatments as a result of its inhibition to growth. Root uptake was found to be the probable means of remediation of PCB-contaminated soil by C. odorata, this was perhaps aided by microbes. This study has provided evidence on the ability of C. odorata to remediate PCB contaminated soil. However, the use of C. odorata for phytoremediation of PCB contaminated soil under field condition is therefore advised. / Environmental Sciences / M.Sc. (Environmental Science)