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The potential for biodegradation of contaminants in soil was assessed using an array of molecular methods, including terminal restriction fragment length polymorphism (TRFLP), quantitative polymerase chain reaction (qPCR), and traditional culturing techniques combined with sequencing of the 16S or ITS regions of the cultured bacteria and fungi. Soil was collected from the Santa Susana Field Laboratory (SSFL), which was the site of numerous liquid-propulsion rocket engine tests as well as nuclear energy research and development, which led to contamination of the soil with a wide variety of constituents. The contaminants of interest (COIs) at this site include polychlorinated biphenyls (PCBs), dioxins, polycyclic aromatic hydrocarbons (PAHs) and non-PAH petroleum hydrocarbons (PHCs). Various metals, most notably mercury and silver, are also present on the site. The purpose of this study was to determine if biodegradation is contributing to natural attenuation of contaminants in the soil, what organisms are likely causing biodegradation, and what rate(s) can be expected in the future. A literature review was conducted to investigate the chemical properties of theses COIs, their toxicity, and abiotic and biotic degradation. This research concluded that these COIs can be biodegraded if the right bacteria and/or fungi are present and active in the soil in sufficient numbers under the right conditions. Many known biodegraders of the COIs were identified in the literature review along with the most common pathways of biodegradation and degradation rates observed in field and laboratory studies. Soil was collected from 30 sample locations, with 3 sets of 10 samples containing high concentrations of one COI but low concentration of the others. PHCs and PAHs were found to be largely co-located, so 10 samples were selected for both of them. The remaining 20 samples were split evenly between PCBs and dioxins. DNA was extracted directly from all 30 soil samples and amplified using PCR for TRFLP analyses. Two soil samples were sent to Microbial Insights® for qPCR analysis. This analysis included 18 gene targets for the degradation of PHCs and PAHs, as well as the target gene for Dehalococcoides (an anaerobic dechlorinating bacteria). For each culturing a model chemical was selected to represent each COI and added to Bushnell-Haas agar plates containing no added carbon source other than the model compounds. The model chemicals were No. 2 diesel fuel for PHCs, naphthalene for PAHs, PCB #1 (monochloro) for PCBs, and dibenzofuran for dioxin. These plates were used to screen for biodegrading bacteria and fungi for each COI. Once cultured, 16S and ITS sequencing were used to identify these potential COI degraders and determine what TRFLP peak they would produce. The identity of isolated organisms was compared to information from the literature to assess the likelihood of COI biodegradation at SSFL. From the culturing experiments, 45 organisms were isolated, sequenced, and identified. The 45 included 14 unique bacteria and seven unique fungi. Of these, 10 different bacterial species and 5 different fungal species have been reported as COI biodegraders or belong to genera that contain reported COI biodegraders. TRFLP analysis revealed that the soil type has more effect on the microbial population than the presence of any of the COIs. There were no specific peaks that were significantly correlated to any specific COI. The peak distributions were fairly even, indicating a large amount of biodiversity in the microbial populations of the soil samples. The qPCR analysis revealed that SSFL soils contain significant populations of microbes that can degrade PHCs aerobically. Anaerobic PHC, anaerobic PAH, and aerobic PAH targets were not detected. A small amount of Dehalococcoides was detected in one of the samples. Collectively this study suggests that microbes present in SSFL soils are capable of biodegrading PHCs, and the genes for such biodegradation are actively being expressed. With the exception of a small population of Dehalococcoides, bacteria associated with the biodegradation of PAHs, PCBs, and/or dioxins were not detected. However, several strains of fungi were identified which have been reported to mediate cometabolic biodegradation of these compounds. Since these fungi do not require anaerobic conditions, they are more likely to contribute to natural attenuation than bacterial reductive dechlorination. Laboratory microcosm experiments are suggested for estimating rates of biodegradation at SSFL under natural attenuation conditions. Bioaugmentation and/or biostimulation methods should also be investigated in addition of natural attenuation. These microcosm experiments are currently underway in a companion study at Cal Poly by graduate student Mackenzie Billings.

Root symbioses with dark septate endophytic fungi (DSE) and arbuscular mycorrhizal fungi (AMF) provide plant tolerance to environmental stresses. This research answers several fundamental questions about the occurrence of these fungi in roots of prairie grasses. Traditional methods and current molecular techniques were combined in order to: 1) define the role and specificity of DSE in plant tolerance to drought; 2) assess the level of host specificity in DSE; 3) document AMF biodiversity and pattern of root colonization at different soil depths; 4) define the influence of soil depth and plant species on the distribution of DSE and AMF in roots and; 5) reveal how DSE and AMF interact in plant roots.<p> Under controlled conditions, DSE isolates showed host preference in colonizing roots and promoting plant growth. They colonized with more intensity the plant species from which they were isolated [Agropyron cristatum L. or Psathyrostachys juncea (Fisch) Nevski subsp. Juncea (Syn: Elymus junceus Fisch)]. Inoculation with five DSE isolates resulted in growth stimulation of the C3 grasses A. cristatum and P. juncea, and growth depression of the C4 grass Bouteloua gracillis (Willd. ex Kunth) Lag. ex Griffiths, under water stress. Plant C concentration suggested that DSE inoculation may have resulted in net C drain from B. gracillis.<p. In the field, soil depth influenced root colonization in A. cristatum, Panicum virgatum L., Nassella viridula Trin and Pascopyrum smithii (Rydb.) A. Löve., while AMF diversity was influenced by the interaction between soil depth and host plant species. Molecular analysis of roots serially sampled during one growing season from the A and B soil horizons, in stands of these grasses, revealed spatial and temporal changes in DSE and AMF community composition, and a significant correlation in DSE and AMF community structure.<p> These results suggest that DSE and AMF are adapted to specific environmental conditions and that root occupation by these fungi is a dynamic phenomenon. It is proposed that temporal variation in root occupation by DSE and AMF impacts plant and ecosystem processes at different times during the growing season.

Root symbioses with dark septate endophytic fungi (DSE) and arbuscular mycorrhizal fungi (AMF) provide plant tolerance to environmental stresses. This research answers several fundamental questions about the occurrence of these fungi in roots of prairie grasses. Traditional methods and current molecular techniques were combined in order to: 1) define the role and specificity of DSE in plant tolerance to drought; 2) assess the level of host specificity in DSE; 3) document AMF biodiversity and pattern of root colonization at different soil depths; 4) define the influence of soil depth and plant species on the distribution of DSE and AMF in roots and; 5) reveal how DSE and AMF interact in plant roots.<p> Under controlled conditions, DSE isolates showed host preference in colonizing roots and promoting plant growth. They colonized with more intensity the plant species from which they were isolated [Agropyron cristatum L. or Psathyrostachys juncea (Fisch) Nevski subsp. Juncea (Syn: Elymus junceus Fisch)]. Inoculation with five DSE isolates resulted in growth stimulation of the C3 grasses A. cristatum and P. juncea, and growth depression of the C4 grass Bouteloua gracillis (Willd. ex Kunth) Lag. ex Griffiths, under water stress. Plant C concentration suggested that DSE inoculation may have resulted in net C drain from B. gracillis.<p. In the field, soil depth influenced root colonization in A. cristatum, Panicum virgatum L., Nassella viridula Trin and Pascopyrum smithii (Rydb.) A. Löve., while AMF diversity was influenced by the interaction between soil depth and host plant species. Molecular analysis of roots serially sampled during one growing season from the A and B soil horizons, in stands of these grasses, revealed spatial and temporal changes in DSE and AMF community composition, and a significant correlation in DSE and AMF community structure.<p> These results suggest that DSE and AMF are adapted to specific environmental conditions and that root occupation by these fungi is a dynamic phenomenon. It is proposed that temporal variation in root occupation by DSE and AMF impacts plant and ecosystem processes at different times during the growing season.

Northern peatlands are large repositories of carbon and little is known about the effect the microbial community has on carbon mineralization rates, and there is concern that a loss of microbial diversity due to environmental change may lead to reduced ecosystem functioning. Microbial communities vary among peatland types and abiotic variables such as temperature and pH have a large influence on carbon dioxide production, but distinguishing between abiotic controls and the role of microbial community structure has proved challenging. Microbial activity and community composition was characterized in three peatlands within the James Bay Lowlands, Ontario. Similar dominant microbial taxa were observed at all three peatlands despite differences in nutrient content and substrate quality and geographic location. In contrast, microbial activity differed among the sites, indicating that it is influenced by the quality of the peat substrate and the presence of microbial inhibitors. A series of reciprocal field and laboratory transplant experiments were conducted at a rich and poor fen near White River, Ontario to more explicitly distinguish between the abiotic and microbial controls on carbon mineralization. The effect of transplantation differed between the laboratory and field studies and when viewed individually could lead to different interpretations of the effect of substrate change. Surprisingly, intensive sampling within both fens was unable to reveal a difference between the rich and poor fen microbial community due to high within site temporal and spatial variation. Thus studies with small sampling effort will have a very incomplete understanding of microbial community structure and thus microbial ecology. A reciprocal sterilization transplant experiment was also conducted to examine how different microbial communities adapted to various peat substrates influenced C-mineralization patterns. Post-inoculation/incubation bacterial communities across peatlands converged towards a similar community structure, suggesting that abiotic variables are the dominant control on peatland microbial activity and community composition. The studies presented in this thesis collectively show that across a broad range of temperate and sub-arctic peatland types dominant members of the microbial community are generally similar, and decomposition rates can be predicted by broader controlling environmental factors rather than temporal niche or distributional constraints of the microbial community.

Northern peatlands are large repositories of carbon and little is known about the effect the microbial community has on carbon mineralization rates, and there is concern that a loss of microbial diversity due to environmental change may lead to reduced ecosystem functioning. Microbial communities vary among peatland types and abiotic variables such as temperature and pH have a large influence on carbon dioxide production, but distinguishing between abiotic controls and the role of microbial community structure has proved challenging. Microbial activity and community composition was characterized in three peatlands within the James Bay Lowlands, Ontario. Similar dominant microbial taxa were observed at all three peatlands despite differences in nutrient content and substrate quality and geographic location. In contrast, microbial activity differed among the sites, indicating that it is influenced by the quality of the peat substrate and the presence of microbial inhibitors. A series of reciprocal field and laboratory transplant experiments were conducted at a rich and poor fen near White River, Ontario to more explicitly distinguish between the abiotic and microbial controls on carbon mineralization. The effect of transplantation differed between the laboratory and field studies and when viewed individually could lead to different interpretations of the effect of substrate change. Surprisingly, intensive sampling within both fens was unable to reveal a difference between the rich and poor fen microbial community due to high within site temporal and spatial variation. Thus studies with small sampling effort will have a very incomplete understanding of microbial community structure and thus microbial ecology. A reciprocal sterilization transplant experiment was also conducted to examine how different microbial communities adapted to various peat substrates influenced C-mineralization patterns. Post-inoculation/incubation bacterial communities across peatlands converged towards a similar community structure, suggesting that abiotic variables are the dominant control on peatland microbial activity and community composition. The studies presented in this thesis collectively show that across a broad range of temperate and sub-arctic peatland types dominant members of the microbial community are generally similar, and decomposition rates can be predicted by broader controlling environmental factors rather than temporal niche or distributional constraints of the microbial community.

Conventional plastics have been used for decades in a diverse range of applications, however, many are resistant to degradation, leading to environmental pollution and their manufacture is dependent on non-renewable fossil fuels. Therefore, there has been an increasing need for eco-friendly biodegradable materials from renewable resources. Poly(lactic acid) (PLA) is a compostable polyester with a hydrolysable backbone that is susceptible to biodegradation and produced from renewable feedstocks. PLA has mechanical qualities comparable to non-biodegradable plastics, and currently is commercialized as food-packaging polymer for short shelf-life products. However, while PLA hydrolysis at elevated temperatures proceeds abiotically, ultimately releasing lactic acid and short chain oligomers, the role of microorganisms is unclear. Since PLA short-shelf life products are disposed after use, understanding the role of microorganisms and the effect of degradation on microbial populations in the environment is important. Therefore, the aims of this research was to (a) determine the relative importance of biotic and abiotic factors on PLA degradation; (b) to isolate putative fungal PLA degraders from the surface of PLA when buried in compost or soil and to test their ability to degrade PLA; (c) to assess the impact of PLA degradation on fungal communities when entering compost systems. The roles of abiotic and biotic factors in the degradation of high molecular weight PLA were investigated by comparing degradation rates in compost, soil and sterile water at temperatures of 25°, 37°, 45°, 50° and 55°C. Tensile strength loss and molecular weight decline of PLA in microorganism-rich compost and soil were greater than chemical hydrolysis in sterile water at elevated temperatures (above 45°C) indicating microorganisms can directly enhance PLA degradation. Since extensive fungal growth was observed on the surface of PLA when buried in compost and soil, putative fungal PLA degraders were isolated from PLA surface and their community profile on PLA surface was compared with the compost and soil community with a molecular method, terminal restriction fragment polymorphism (TRFLP). Among the identified fungi, Thermomyces lanuginosus was the dominant isolate recovered and shown to enhance PLA degradation in compost at 50°C. The fungal community profile on PLA surface was different than the fungal profile in compost and soil suggesting enrichment for PLA degraders on the surface of PLA. In order to determine the impact of PLA degradation on the fungal compost community, two different high molecular weight PLA sources, films and granules were buried in compost at 10%, 25% and 50% (w/w) concentration for 4 months at 25°C and 50°C and the community profile analysed by TRFLP and pyrosequencing. TRFLP revealed that when PLA did not degrade, the fungal community shifted back toward the initial compost community profile, however, when PLA degraded to its monomers releasing lactic acid at 50°C at a concentration of 50% (w/w) it changed the fungal community profile and decreased the fungal diversity. Pyrosequencing revealed that the presence of PLA enriched for Thermomyces in the compost population over time.

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Os sistemas de cultivo agrícola são essenciais à sociedade. A questão atual é saber como mantê-los produtivos sem afetar drasticamente os diferentes ecossistemas e ciclos biogeoquímicos. Sabe-se que a atividade biológica dos solos é de crucial importância à saúde dos mesmos e à produtividade. Deste modo, a implantação de técnicas ambientalmente corretas com monitoramentos eficientes, baseados na qualidade do solo, é fundamental para valorizar a sua conservação. Esta tem sido o foco de pesquisas nas últimas décadas, sendo que refletem ação antrópica, principalmente pela emissão dos gases do efeito estufa (GEEs). O uso de sistemas conservacionistas destaca-se pela viabilidade econômica e ambientalmente benéfica em relação ao manejo convencional. O sistema integração lavoura-pecuária tem sido utilizado para minimizar os impactos ambientais da exploração agrícola, a fim de preservar as características físicas, químicas e biológicas do solo, estendendo sua resiliência e aumentando a produtividade. Os microrganismos são responsáveis por diversos processos biológicos essenciais ao ambiente e algumas espécies participam produzindo ou oxidando o metano (CH4), um dos gases do efeito estufa. Estes são influenciados principalmente pelas mudanças de uso do solo, que quando relacionadas ao manejo inadequado podem alterar a qualidade do solo, a produtividade e a emissão de gases. Assim, a avaliação dos solos dos sistemas agrícolas, sob a interação rizosférica de diferentes culturas e a criação de um ambiente anóxico se faz necessário para entender o comportamento de tais comunidades. Os sistemas de integração lavoura-pecuária e a rotação de culturas foram investigados em relação a microbiota funcional do solo, em função de fatores como ao histórico da área e umidade. Foram avaliadas a abundância, por PCR em tempo real, e a estrutura das comunidades Archaea e Bacteria por TRFLP, e a potencialidade de atuação do solo como emissor e mitigador de CH4 através da quantificação dos microrganismos metanogênicos e metanotróficos de áreas agrícolas e reflorestamento do Alto Xingu, no município de Querência. Com elas foi possível observar que os microrganismos são estruturados, primariamente, em função do tipo de solo e consecutivo efeito rizosférico dos vegetais, de forma que os sistemas de integração lavoura-pecuária (ILP) apresentaram sutilmente maior estabilidade em relação ao sistema rotacionado de soja/milheto, devido ao sistema radicular da pastagem fornecer maior proteção e liberação de exsudatos. Pelas semelhanças existentes com os sistemas ILP, a área de reflorestamento se encontra em recuperação transitória, em uma média da similaridade entre as enzimas de restrição HhaI e MspI, de 85%; e 65% em relação à floresta, que se estruturou de maneira diferenciada das demais áreas. Outro fator de diferenciação das áreas agrícolas foi a forte influência da calagem o que eleva o pH e concomitantemente apresentou teores elevados de Ca e Mg. Já as comunidades de metanotróficas não apresentaram variação em função das metanogênicas, em que a saturação hídrica promoveu seu crescimento somente nos solos de floresta, onde ocorre maior incorporação de matéria orgânica / Agricultural faming systems are essential to society. The current question is how to keep them productive without drastically affecting different ecosystems and biogeochemical cycles. It is known that the soil biological activity is crucial to the health and productivity. Thus, the implementation of environmentally correct techniques with efficient monitoring, based on soil quality is critical to enhance their conservation. This has been the focus of research in recent decades, and reflects human action, mainly by the emission of greenhouse gases (GHGs). The use of conservation tillage systems distinguished by economic viability and environmentally beneficial compared to conventional tillage systems. The crop-livestock system has been used to minimize the environmental impacts of farming, in order to preserve the physical, chemical and biological soil properties, extending its resilience and increasing productivity. Microorganisms are responsible for many essential biological processes to the environment and some species participate in production or oxidation of methane (CH4), a greenhouse gas. These are mainly influenced by land use changes, that when related to inadequate management may alter soil quality, productivity and gases emissions. Thus, the evaluation of soils for agricultural systems under the rhizospheric interaction of different cultures and creating an anoxic environment is needed to understand the behavior of such communities. Crop-livestock systems and crop rotation were investigated in relation to functional soil microbiota, depending on factors such as the area history and moisture. Genes abundance were assessed by real-time PCR, while Archaea and Bacteria community structure by TRFLP, and the potential role of soil as releaser and mitigator of CH4 through the quantification of methanogens and methanotrophs in agricultural areas and reforestation of the Upper Xingu, at Querência city. Through the techniques of qPCR and TRFLP was observed that microorganisms are structured primarily on the type of soil, followed by rhizosphere effect of plants. In this way, crop-livestock integration systems (CLI) are subtly stable then rotational system of soybean/millet due to pasture root system to provide greater protection and root exudation. Alike CLI systems, the reforestation area is in transient recovery on average between the restriction enzymes HhaI and MspI, 85%; and 65% in relation to forest, this structures itself in a differentiated manner from the other areas. The farming areas present strong influence of liming, which leads to grow pH and concomitantly high Ca and Mg contents. The methanotrophic community did not vary due to the methanogenic community, and the water saturation promotes the growth of methanogenic communities only in forest soils where occurs greater organic matter incorporation