In order to protect natural resources and ecosystems at Eglin Air Force Base (EAFB), a strategy must be developed for the containment and/or treatment of explosive contaminants on testing and training ranges under continuous use. Phytoremediation is the direct use of living plants for in situ (in place) remediation of contaminated soil, sludges, sediments, and groundwater through contaminant removal, degradation, or containment. Due to its ability to continuously treat large areas at low cost with low impact to the site, phytoremediation was implemented through a field study at EAFB in order to increase range sustainability.

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Perchlorate is a known inhibitor of the human thyroid gland. Perchlorate is destroyed by ubiquitous perchlorate-reducing bacteria. The bacteria often lack sufficient electron donor. Research was undertaken to evaluate the relationship between plants and perchlorate-reducing bacteria. To what degree can plant-produced electron donors stimulate perchlorate reduction in low organic carbon (LOC) and high organic carbon (HOC) soil? A complication is that plants have been shown to influence redox conditions which may inhibit perchlorate reduction. The removal of perchlorate in a flow-through reactor was monitored with variables of soil organic carbon, hybrid poplar trees, and bioaugmentation. The biodiversity was monitored using denaturing gradient gel electrophoresis. Low oxidation-reduction potential (ORP) was shown to indicate the capacity for greater perchlorate removal in soil. However, in planted LOC soil systems, evidence suggests that perchlorate reduction may also be possible at higher bulk redox conditions than previously observed. Increased hydraulic retention time was shown to both lower bulk ORP and increase perchlorate removal. Radiolabeled perchlorate was used to find that in planted systems as much as 11.7% of the influent perchlorate mass was taken up into the tree and 82% of the perchlorate taken up was accumulated in the leaves. The plant contribution to total perchlorate removal in nonbioaugmented LOC soil was 39%, with the balance of the removal being attributed to microbial reduction. In bioaugmented soil the microbial contribution to perchlorate removal was increased. Just planting poplar trees decreased the diversity of perchlorate reducers in the soil. However, when LOC soil was both planted and bioaugmented, the diversity of perchlorate reducers was not decreased. In HOC soil, the presence of an indigenous population of microorganisms competed with perchlorate reducers. At the increased ORP observed in planted HOC soil, the non-perchlorate-reducing bacteria appear to outcompete the perchlorate reducers and perchlorate removal is decreased. Engineering implications of this research are that perchlorate remediation in HOC soil does not benefit from planting hybrid poplar trees but that remediation in LOC soil is stimulated by planting and bioaugmentation.

Light, medium and heavy crude oils were studied at three concentrations and with two different sediments in experimental microcosm settings to determine the ability of Spartina alterniflora and associated microbes to breakdown total extractable hydrocarbons (TEH) in the water. It was a baseline experiment designed to quantify the rates of biodegradation under relatively quiescent conditions from different crude oils at moderate doses ranging from 0-150 mg/kg soil. Upon the completion of the experiment there were several key findings: (1) The lethal dosage for Spartina alterniflora was not reached within the 90 day experiment at these dosages, and all plants survived; (2) More than 97% of the total extractable hydrocarbons (TEH) were shown to be degraded by plants and rhizosphere microorganisms within the 90- day experiment; (3) The dose of oil introduced as a slick (simulated spill) on day zero did not significantly affect the results for TEH degradation within the range of dosages from 50-150 mg/g -- these dosages could be degraded by the marsh cord grass system; (4) A sediment type which was acclimated to oil for several months and one which was non-acclimated did not show significantly different results for TEH degradation in the microcosms -- both sediment systems resulted in TEH degradation over the 90-day experiment; and (5) A mathematical model was developed which simulated experiment results quite closely including TEH diffusion from the crude oil slick into the water and subsequent biodegradation.

Polychlorinated biphenyls (PCBs) are carcinogenic, persistent, and bioaccumulative contaminants that pose risks to human and environmental health. PCB biodegradation by indigenous microbial communities could be a cost-effective and an environmental-friendly bioremediation strategy for in situ PCB removal. A comprehensive understanding of the microbial PCB degradation at the contaminated site is required for the acceptance and optimization of using microbial PCB degradation as the site clean-up strategy. This thesis describes investigations of the aerobic and anaerobic microbial degradation of PCBs under both field and laboratory conditions. The microbial PCB degradation potential in sediments from Indiana Harbor and Ship Canal (IHSC), a site that was historically contaminated by PCBs, was explored by analyzing the PCB congener distributions and microbial communities in two core sediment samples. PCB congener analysis suggested the possibility of in situ dechlorination in deep sediments. Molecular analysis of biomarker genes revealed the potential of both aerobic and anaerobic PCB degradation in sediments. Microbial communities were characterized by the combination use of terminal restriction fragment length polymorphism (T-RFLP), clone library, and pyrosequencing. These methods elucidated the dominant role of Proteobacteria, especially Acidovorax and Acinetobacter in sediments. To improve the microbial PCB degradation, phytoremediation with switchgrass (Panicum vigratum) was employed under laboratory conditions. Congener analysis showed that both phytoextraction and microbial PCB degradation contributed to the enhanced PCB removal in the presence of switchgrass. Bioaugmentation with Burkholderia xenovorans LB400 was performed to further promote aerobic PCB degradation. The presence of LB400 was associated with improved degradation of PCB 52, but not PCB77 or PCB 153. Increased abundance of the biphenyl dioxygenase gene, which is indicative of aerobic PCB degradation, and its transcript were observed after bioaugmentation, suggesting active aerobic PCB degradation. To promote the anaerobic PCB degradation, redox cycling (alternating flooding and non-flooding) was performed. Redox cycling was found to improve the removal of PCB 153 in unplanted soils and to increase the dechlorinating Chloroflexi population. Characterization of the microbial community by T-RFLP and clone library revealed that Proteobacteria and Acidobacteria were dominant. Species that contain dechlorination potential were identified, including Geobacter and Clostridium, suggesting that their possible role in PCB dechlorination. The research described in this thesis provides scientific knowledge and evidence for the feasibility of employing bioremediation including natural attenuation, phytoremediation, and bioaugmentation to clean up PCB contamination. Such information will be critical in selecting and optimizing remediation strategies for PCB contaminated sites.

Phytoremediation is an innovative technology employing plants for reclamation of soils that have been polluted by industrial contaminants. While it is frequently slower than traditional physicochemical remediation techniques, there are, however, many significant advantages including simplicity, low cost, improvement of soil structure and microbiological fertility. While various hyperaccumulators have been reported from Europe and the USA, there is a need to identify Australian species. This study reports a number of metal accumulators following a soil and vegetation survey of two metal contaminated sites in New South Wales, the spoil (filtercake) heaps of the BHP (Broken Hill Propriety ) steelworks at Port Kembla and the abandoned mine at Sunny Corner. BHP is the largest manufacturer of steel in Australia. The Port Kembla is the largest single steel manufacturing site. Solid by-products from this site totalled 3.1 million tons in 1988. The Sunny Corner mine site was mainly a silver mine which operated over approximately 100 hectares 1184 to 1922 and produced over 100 tonnes of silver during that period. The soil and water in and around the site is heavily contaminated with metals and is distinctly acidic. It was concluded that Ricinus communis, Sonchus oleraceus, baeckea utilis, Poa labillardieri and Pinus radiata are options for heavy metal phytoremediation of contaminated Australian soils. Arbuscular Mycorrhizal infection (particular with Glomus and Gigaspora strains) should be promoted for optimum growth. / Doctor of Philosophy (PhD)

The breakdown of contaminants in soil resulting from microbial activity that is enhanced in the presence of the plant root zone, rhizosphere, has been termed rhizoremediation. To date, Australian native plants have not been assessed for their hydrocarbon rhizoremediation potential. The use of native plants offers an economically feasible and environmentally sustainable cleanup option for the rehabilitation and restoration of hydrocarbon contaminated sites in Australia. The aim of the study was to evaluate the potential of Australian native grass species for the rhizoremediation of aliphatic hydrocarbon contaminated soil from a mine site. Candidate Australian native grass species Poaceae were selected following the development of essential and desirable growth criteria. Nine perennial Australian grasses were evaluated for seedling emergence in sandy loam soil sourced from a mine site which was artificially contaminated with a 60:40 diesel/oil mix at concentrations of 30 000 mg/kg, 10 000 mg/kg, 5 000 mg/kg and 0 mg/kg control. Seedling emergence was not adversely affected by the presence of hydrocarbon contamination at the exposed concentrations for eight of the nine species studied p > 0.05. Three promising species were assessed for relative growth performance in diesel/oil contaminated 10 000 mg/kg, 5 000 mg/kg and uncontaminated control soils in greenhouse studies to assess their tolerance of aliphatic hydrocarbon contaminated soil. Cymbopogon ambiguus Lemon Scented grass is a summer growing perennial with widespread distribution throughout Australia including the region where the mine site is situated. Brachiaria decumbens Signal grass – naturalised - is adapted to humid tropical areas of Australia and is native to the site and sourced from seed banks. Microlaena stipoides Weeping grass var. Griffin is a cool season grass, widely distributed throughout Australia in moister regions. The three evaluated species survived for 120 days in the diesel/oil contaminated soil at the exposed concentrations without adverse growth affect p > 0.05. In some instances e.g. C. ambiguus growth stimulation occurred in the presence of contamination producing significantly more root biomass compared with the control p < 0.0001. Most hydrocarbon degradation is believed to occur through microbial processes, and so the plant-associated microbial community was examined in the three tolerant species. The assessment of the influence of grass on the abundance and activity of microorganisms in the rhizosphere revealed species-specific plant-induced changes in the soil microbial community. Selective enrichment of hydrocarbon degrading microorganisms was demonstrated in the rhizosphere soil of the Australian grasses tested, to varying degrees. C. ambiguus appeared to have the greatest influence on stimulation of hydrocarbon degrading microorganisms, followed by the cool season grass M. stipoides. B. decumbens showed consistently lower numbers of hydrocarbon degrading microorganisms in rhizosphere soil over time compared to the other two species p < 0.01. The influence of grasses on microbial community structure - defined as community DNA fingerprint - in diesel/oil contaminated soil suggested no new microbial population was favoured by the grasses - qualitative shift - rather there were relative quantitative changes in existing members of the microbial population. Soil lipase activity did not appear to be an optimal bioindicator of rhizoremediation and may encompass total soil microbial activity not exclusively the hydrocarbon degrading microorganisms of interest. The assessment of biodegradation of hydrocarbons in soil is essential to characterise the effectiveness of plant species in rhizoremediation. Residual diesel and oil concentrations as total petroleum hydrocarbons, TPH were measured using Gas Chromatography. The presence of single species successfully enhanced the removal of hydrocarbons from soil for all species. All showed significantly lower residual hydrocarbon concentrations than those in unplanted soil after 100 days p < 0.01. Significantly, it was not necessary to add N and P to achieve up to 90% reduction in hydrocarbon concentrations in the soil. The relative performance of each grass species varied. In soil planted with C. ambiguus hydrocarbon concentrations were reduced faster and to a greater extent than the other species studied, from 10 000 mg/kg to approximately 1 100 mg/kg TPH, 88% removal. Similar endpoint success was recorded for M. stipoides which facilitated 80% reduction in hydrocarbon concentrations. Interestingly, B. decumbens, the only naturalised species, did not perform as well as the other species, although still significantly better compared to unplanted controls, with hydrocarbon concentrations reduced to approximately 4 500 mg/kg, 49%. Hydrocarbon concentrations in unplanted control soil were reduced by 45% through natural biodegradation processes. Plant root and shoot tissue was periodically assessed for hydrocarbon accumulation and was shown to be negligible. A multispecies planted trial using C. ambiguus plus B. decumbens had no additional influence on total TPH removal. The final TPH removal efficiency in the multispecies trial was not significantly different p > 0.05 from that of the best single species performer of the two i.e. C. ambiguus. In a field application the planting of multiple species may still be desirable in order to preserve site biodiversity and assist rehabilitation of the area. A strong relationship between abundance of hydrocarbon degrading microorganisms in the rhizosphere and hydrocarbon biodegradation was demonstrated for all species p < 0.01. Those species which showed greatest stimulation of the microbial population resulted in enhanced TPH removal from soil. These species were the summer grass C. ambiguus and the winter species M. stipoides. This may allow for broader application both seasonally and geographically across Australia. B. decumbens showed successful rhizoremediation to a lesser degree, but may still be an option in multiple planting strategies. This investigation identified three Australian grass species from the nine evaluated that are candidates for further investigation for in situ rhizoremediation potential at field scale.

<p>Phytoremediation as a way to control and lessen nutrient concentrations in landfill leachate is a cheap and environmentally sustainable method. Accumulated nutrients in the plants can then be removed by harvesting and anaerobically digesting the biomass. This study presents two aquatic plants (L. minor (L.) and P. stratiotes (L.)) and one microalgae species (C. vulgaris (L.)), their capacities for growth and nutrient removal in leachate from Häradsudden landfill, Sweden, are investigated. The biogas potential of the two plants is determined via anaerobic digestion in a batch run, followed by a lab-scale reactor run for L. minor only. Results show that growth in leachate directly from the landfill is not possible for the selected species, but at a leachate dilution of 50% or more. Nutrients are removed in leachates with plants to a higher extent than in leachates without, yet the actual amounts do not differ notably between plant species. L. minor proves a better choice than P. stratiotes despite this as growth is superior for L. minor under the experimental conditions of this study. Considering biogas production, L. minor gives more methane than P. stratiotes according to the results from the batch run. The former is however not suitable for large-scale anaerobic digestion unless as an additional feedstock due to practical cultivation issues.</p>

As the third most important greenhouse gas, nitrous oxide (N2O) is a stable greenhouse gas and also plays a significant role in stratospheric ozone destruction. The primary anthropogenic source of N2O stems from the use of nitrogen in agriculture, with soils being the major contributors. Currently, the annual N2O emissions from this “soil–microbe-plant” system is more than 2.6 Tg (one Tg equals a million metric tons) of N2O-N globally. My doctoral studies aimed to explore innovative strategies for N2O mitigation, in the context of environmental microbiology’s potential contribution to alleviating global warming. The bacterial enzyme nitrous oxide reductase (N2OR), naturally found in some soils, is the only known enzyme capable of catalyzing the final step of the denitrification pathway, conversion of N2O to N2. Therefore, to “scrub” or reduce N2O emissions, bacterial N2OR was heterologously expressed inside the leaves and roots of transgenic plants. Others had previously shown that the functional assembly of the catalytic centres (CuZ) of N2OR is lacking when only nosZ is expressed in other bacterial hosts. There, coexpression of nosZ with nosD, nosF and nosY was found to be necessary for production of the catalytically active holoenzyme. I have generated transgenic tobacco plants expressing the nosZ gene, as well as tobacco plants in which the other four nos genes were coexpressed. More than 100 transgenic tobacco lines, expressing nosZ and nosFLZDY under the control of rolD promoter and d35S promoter, have been analyzed by PCR, RT-PCR and Western blot. The activity of N2OR expressed in transgenic plants, analyzed with the methyl viologen-linked enzyme assay, showed detectable N2O reducing activity. The N2O-reducing patterns observed were similar to that of the positive control purified bacterial N2OR. The data indicated that expressing bacterial N2OR heterologously in plants, without the expression of the accessory Nos proteins, could convert N2O into inert N2. This suggests that atmospheric phytoremediation of N2O by plants harbouring N2OR could be invaluable in efforts to reduce emissions from crop production fields.

Phytoremediation systems for petroleum hydrocarbons rely on a synergistic relationship between plants and their root-associated microbial communities. Plants exude organic compounds through their roots, which increase the density, diversity and activity of plant-associated microorganisms, which in turn degrade hydrocarbons. Understanding the mechanisms driving this relationship poses one of the more intriguing challenges in phytoremediation research. This study was designed to address that challenge. Plant-microbe interactions in a weathered-hydrocarbon contaminated soil were examined under controlled growth chamber, and field conditions. In both environments single-species grass treatments initially facilitated greater total petroleum hydrocarbon (TPH) degradation than <i> Medicago sativa </i> (alfalfa), mixed species, or control treatments. In growth chamber studies increased degradation was linked to increased aliphatic-hydrocarbon degrader populations within the rhizosphere. Under field conditions, specific recruitment of endophytic aliphatic-hydrocarbon degraders in response to high TPH levels may have facilitated increased degradation by the grass <i> Elymus angustus</i>(Altai wild rye, AWR). AWR stably maintained these communities during times of local drought, enabling them to act as subsequent source populations for rhizosphere communities. The broad phylogenetic diversity of AWR endophytes, compared to the <i> Pseudomonas</i>-dominated communities of other plants, contributed to the observed stability. The relative composition of exudates released by plants also impacted both degradation activity and potential. Alfalfa released higher concentrations of malonate, which hindered degradation by decreasing metabolic activity and concomitantly inhibiting catabolic plasmid transfer. In contrast, AWR exudates contained high levels of succinate, which was linked to increased catabolic gene expression and plasmid transfer. A reciprocal relationship between exudation patterns and endophytic community structure likely exists, and both parameters have a specific influence on rhizosphere degradation capacity. In this study, grasses were more successful in maintaining the specific balance of all parameters required for the transfer, preservation, and stimulation of hydrocarbon catabolic competency.