Rozprawy doktorskie na temat „Soil microorganisms”

Cyanides enter the environment through both natural and man-made sources. Natural sources include cyanogenesis by bacteria, fungi and plants. A number of cyanide catabolising microorganisms have also been reported in literature. This is the first reported instance of cyanide catabolism in Trichoderma harzianum. Four strains of T. harzianum, one of T. pseudokoningii were evaluated. An investigation was made into the occurrence and distribution of the cyanide catabolising enzymes. Three enzymes, cyanide hydratase, beta-cyanoalanine synthase and rhodanese, were studied. All the strains showed a high capacity to degrade cyanide via both the cyanide hydratase and rhodanese pathways, beta-cyanoalanine synthase activity, however, was not detected in any of the selected strains. In the studies on the kinetic characterization of the rhodanese enzyme, a broad pH optimum of 8.5 - 10.5 was obtained for all the strains and a broad temperature optimum of 35 - 55 °C was also observed. The KmCN and Vmax values ranged from 7-16 mM and from 0.069 - 0.093 betamoles. Min-1. mg protein-1, respectively, between the selected strains of Trichoderma. Strong evidence of cyanide biodegradation and co-metabolism emerged from studies with flask cultures where glucose was provided as a co-substrate. The rate of degradation of 2000 ppm CIST was enhanced almost three times in the presence of glucose. Plant microcosm studies carried out using pea and wheat seeds too gave further corroboration of the cyanide degrading and plant growth promotion capabilities of Trichoderma. Microcosms set-up with cyanide at 50 or 100 ppm CN, in the presence of Trichoderma, showed germination of both pea and wheat seeds. There was no seed germination in any of the controls in the absence of Trichoderma inoculation.

Soils are complex and highly variable ecosystems within which a multitude of diverse microbial populations can be found. Here the effects of co-correlating environmental variables and spatial separation upon the diversity and community structure of two microbial kingdoms is investigated using several molecular based community assessment methods. Using an optimised nucleic acid extraction procedure, suitable for landscape scale surveys of microbial biogeography, large scale bacterial and fungal targeted terminal restriction fragment length polymorphism (t-RFLP) analysis was undertaken on soils collected as part of the Countryside Survey 2007 to show that populations of both kingdoms are structured, in part, by variability in environmental and edaphic conditions as well as spatial separation. In the case of soil bacteria, pH was identified as the most important environmental variable, although computed models suggest that many other environmental variables also play strong roles. Fungal systems have weaker relationships with environmental variability and stronger spatial relationships, although dominant plant species and soil pH were shown to significantly affect community structure. However, comparing results generated from different genes with different taxonomic resolutions hinders accurate comparisons between divergent microbial kingdoms. Pyrosequencing analysis was undertaken on 15 geographically isolated soil samples forming a natural pH gradient to address the changes in bacterial and fungal populations at great sequencing depth and at taxonomic resolutions closer to the species level. Again, in both cases, members of these kingdoms responded to differences in soil pH and the above ground plant community. A number of bacterial and fungal taxa were found to be responsible for the changes in community structure and diversity noted in the t-RFLP based experiments and are proposed as candidates for indicators of soil pH. In summary soil bacterial and fungal populations are structured according to complex laws relating to co-correlating environmental and spatial variables. This leads to the hypothesis that microbial communities are structured in similar ways to terrestrial macro organisms, and thus ecological theories derived from observations of larger animals may be investigated within the microbial world.

Made available in DSpace on 2014-06-11T19:28:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2003Bitstream added on 2014-06-13T19:16:46Z : No. of bitstreams: 1 cruz_jcs_me_botfca.pdf: 261941 bytes, checksum: 429c0454a3c1d34be05ecfd232f3ad29 (MD5)
A técnica de solarização vem sendo utilizada em pequenas propriedades como uma alternativa de substituição de defensivos agrícolas no controle de fitopatógenos, insetos, plantas daninhas e nematóides de solo. Desta forma, instalou-se um experimento em condições de campo, numa área da Fazenda Experimental Lageado, campus da UNESP no município de Botucatu – SP (latitude 22°51’S e longitude 48°26’W) para se avaliar o impacto desta técnica sobre a comunidade microbiana de um solo caracterizado como Latossolo Vermelho Distrófico, textura média. Inicialmente, incorporou-se uma fonte de matéria orgânica ao solo (couve Brassica oleraceae var. acephala L. fresca e triturada) na quantidade de 4kg.m-2. Posteriormente, umedeceu-se o mesmo e cobriu-se com filme plástico transparente de polietileno aditivado com 150mm de espessura. Fez-se vedação lateral de cada parcela, para se evitar a dispersão de gases e aumentar-se o efeito térmico natural. O experimento obedeceu a delineamento fatorial 2x2x4 (solo solarizado e não solarizado x com e sem incorporação de couve x épocas de coleta). Os tratamentos foram: a)adição de couve sem solarização; b)solarização e adição couve; c)testemunha, sem adição de couve e sem solarização; d)solarização sem adição de couve, com três repetições cada tratamento... .
The soil solarization technique has been used in small properties as an alternative to substitute chemical defensives for phytopathogens, insects, damage causing plants and soil nematode control. A field condition experiment was carried out in an area of Faculdade de Ciências Agronômicas - Botucatu - SP - Brazil (latitude 22°51’S and longitude 48° 26’ W) in order to evaluate the technique impact on the microbial community of soil characterized as Distrofic Red Latosoil, medium texture. Initially, a source of organic material was incorporated to the soil (kale- Brassica oleraceae var acephala L. fresh and ground) in the amount of 4 kg.m-2. After that, it was moisturized a covered with transparent additivated polyethylene plastic film 150mm tick. Lateral sealing of each alloment was made, in order to avoid gas dispersal and to increase natural thermal effect. The experiment followed a 2x2x4 factorial outline (solarized and non solarized soil x with and without kale incorporaton x four times of harvest). The treatments were: a) addition of kale incorporation; b) solarization and addition of kale; c) witness, without addition of kale and without solarization; d) solarization without addition of kale; with three repetitions of each treatment. Samples composed of soil from each allotment were collected from 0-10cm deep, with the first collecting performed seven days after the experiment implantation in the field, and the further ones as intervals of 14 days, from January to March 2001, being afterwards taken to the area of Departamento de Produção Vegetal, (Defesa Fitossanitária) for microbiological analysis... (Complete abstract, click electronic address below).

Tropical ecosystems are some of the most diverse and productive ecosystems on the planet. These ecosystems are also some of the most threatened worldwide and this is largely driven by agricultural expansion. Predicting biotic responses to such forms of environmental change is a challenge that requires an increased understanding of the factors structuring these communities in both pristine environments as well as environments that are undergoing environmental change. Studying patterns in the spatial structure of communities can provide important insights into ecological and evolutionary processes structuring communities. Combining such approaches with analyses of the distribution of activity and the genomic content of communities can help us better understand relationships between community structure and function. I explore the topics of microbial spatial scaling, activity, and gene content in both pristine tropical rainforest environments as well as tropical regions undergoing agricultural conversion. I first pose a fundamental question in microbial spatial ecology, i.e. why do microorganisms tend to show weaker spatial patterns than macro-organisms? I show that trees and soil microorganisms differ in the rates at which their communities change over space. I test the hypothesis that low rates of spatial turnover in microbial communities are an artifact of how we assess the community structure of microbial communities and show that sampling extent is likely the main driver of these differences. Next, I examine a Central Africa ecosystem that is undergoing conversion to agriculture. I show that there are numerous indications of biotic homogenization in these soil microbial communities and that the active fraction of the community shows a more pronounced response to environmental change. Finally, I examine two microbial processes in the Amazon Basin that have been reported to change following conversion to agriculture: methane production and methane consumption. I investigate changes to the genes and taxa involved in these processes and propose a new conceptual framework for how these processes might be changing. Work in this thesis contributes to a broader understanding of the spatial and functional ecology of tropical microorganisms and offers perspectives useful for those interested in predicting and mitigating the impacts of environmental change on these communities.
2019-02-17

Mycorrhizal fungi play a significant role in primary mineral weathering and soil formation. Due to their direct access to solar energy through symbiotic plant partners, fungi are able to extend into soils, acting as biosensors for nutrients which they subsequently uptake and supply to their plant partners. Mycorrhizal fungi operate at the individual hypha scale, mechanically forcing and chemically altering minerals to extract nutrient elements. The hyphae acidify their local environment by exuding organic acids, which are also involved in mineral breakdown. To extend the work on mycorrhizal fungal biotite weathering completed as part of the Weathering Science Consortium the mechanisms and kinetics of biotite dissolution were investigated. This was done by characterising the biotite surface as a function of fluid composition and measuring dissolution rates. During contact with dilute solutions, the chemical composition of the biotite surface changed dramatically as a function of pH. The rapid release of elements during these experiments was not stoichiometric but was highly pH dependent. A combination of electrokinetic measurements and potentiometric titrations further highlighted the variable composition of the biotite surface by yielding two values for zero points of charge, separated by ~7 pH units. Abiotic dissolution of biotite progressed by the formation of a dissolution front depleted in K Mg, Fe and Al, the extent of which varies spatially and with pH. The presence of the organic ligands, citric acid, oxalic acid and DFOB (desferrioxamine B) slightly enhanced the overall biotite dissolution rate in lightly acidic and near neutral pH conditions. The growth rate of mycorrhizal fungi over the surface of biotite was quantified at two levels of atmospheric CO2, 350 ppm and 1500 ppm. Initial growth rate calculations in the 1500 ppm experiments revealed hyphae to grow at an average of 10 μm d-1. Finally, changes in the biochemistry of fungal hypha were observed using μ-FTIR. Results suggested that biochemical changes present could be related to changes in fungal functionality spatially in future work.

Survival of P. fluorescens 10586s FAC510 was studied in liquid culture and soil microcosms in the presence of the ciliate C. steinii and the flagellate Cercomonas sp. Both protozoa caused a reduction in bacterial viable cell concentration, however the ciliate caused a greater decrease in abundance. Starvation of the bacterial prey caused a reduction in grazing rates of both the ciliate and flagellate, indicating the lower nutrional quality of starved as opposed to non-starved cells. Manipulation of the physiological state of prey cells, by starvation, had much the same effect as observed when bacterial prey resources are reduced. Luminescence provided a valuable marker for monitoring P. fluorescens 10586s FAC510 in liquid culture and soil, since detection by luminometry provided a sensitive, rapid, and non-extractive technique for measurement of microbial activity. In the presence of C. steinii, bacterial activity increased, whilst predation by Cercomonas sp. caused a reduction. The different responses of the bacterial inoculum may reflect the divergent balance between bacterial turnover, leading to nutrient regeneration, and grazing strategies, which are selective of the two protozoa. The distribution of bacterial cells in the soil pore network was manipulated by adjustment of the antecedant matric potential prior to inoculation. The ability to predominantly place bacterial and protozan cells in specific pore size classes was confirmed by partial chloroform fumigation and resin impregnated sections of soil.

Made available in DSpace on 2014-06-11T19:25:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-09-13Bitstream added on 2014-06-13T20:33:13Z : No. of bitstreams: 1 palmieri_ct_me_rcla.pdf: 395782 bytes, checksum: daeb45b072eb0ac555536326213d1e7b (MD5)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Este trabalho constitui-se de uma pesquisa multidisciplinar envolvendo física, química, e meio ambiente. Nesta pesquisa foi feito um estudo visando avaliar a absorção de radiação infravermelha da produção de CO2 em solo incorporado com substrato (sementes de aveia amarela, capim-seda, milho, milheto e outros invasores mais inoculação microbiana), com a finalidade de quantificar as emissões deste tipo de solo e as diferenças destas emissões de CO2 com relação à esse mesmo tipo de solo sem a incorporação deste substrato. O material foi coletado na Fundação Mokiti Okada localizada no Município de Ipeúna, interior do Estado de São Paulo SP. O solo retirado foi caracterizado como latossolo vermelho-escuro, argiloso, bem compactado, pouco poroso e com grande capacidade de saturação de água . Foram coletados dois tipos de solos: o solo incorporado com substrato e o não compactado com substrato. Ambos, foram devidamente triturados, peneirados com diâmetro de 2,5 mm e secos na sombra por um período de 12 dias e acondicionados em 12 vasos de cerâmica. Seis desses vasos continham solos não tratados com substrato e seis solos tratados com o substrato acima mencionado. Em cada vaso foi colocado uma quantia de 5.000 mL de solo, onde os mesmos foram umedecidos um dia antes da coleta dos dados. Os vasos receberam 1.200 mL de água, valor este gerado em função da capacidade de campo. No interior de cada vaso foi fixado balde de plástico que através do processo de oxidação da matéria orgânica foi produzido CO2 onde o mesmo foi medido pela câmera respirométrica LI-820 a qual possui leitor ótico infravermelho capaz de medir o fluxo de CO2. Os dados coletados demonstraram que os seis vasos tratados com substratos produziram CO2 em uma proporção muito maior do que os vasos com solos não tratados.
Abstrct: This work is constituted of a research multidisciplinar involving physics, chemistry, and environment. In this research it was made a study seeking to evaluate the absorption of infrared radiation of the production of CO2 in incorporate soil with substratum (seeds of oats yellow, grass-silk, corn, milheto and other invaders more microbial inoculation), with the purpose of quantifying the emissions of this soil type and the differences of these emissions of CO2 regarding to that same soil type without the incorporation of this substratum. The material was collected in the Fundação Mokiti located Okada in the Municipal district of Ipeúna, interior of the State of São Paulo - SP. The solitary soil was characterized as latossolo red-darkness, loamy, well compacted, little porous and with great capacity of saturation of water. Two types of soils were collected: the incorporate soil with substratum and the no compacted with substratum. Both, they were triturated properly, drizzled with diameter of 2,5 mm and dry in the shadow for a period of 12 days and conditioned in 12 ceramic vases. Six of those vases contained soils no treated with substratum and six soils treated with the substratum above mentioned. In each vase an amount of 5.000 soil mL was put, where the same ones were moistened one day before the collection of the data. The vases received 1.200 mL of water, value this generated in function of the field capacity. Inside each vase it was fastened bucket of plastic that through the process of oxidation of the organic matter CO2 was produced where the same was measured by the camera respirométrica READ-820 which possesses infrared optic reader capable to measure the flow of CO2. The collected data demonstrated that the six vases treated with substrata produced CO2 in a much larger proportion than the vases with soils no treated.

Orientador: Marli Teixeira Almeida Minhoni
Resumo: A técnica de solarização vem sendo utilizada em pequenas propriedades como uma alternativa de substituição de defensivos agrícolas no controle de fitopatógenos, insetos, plantas daninhas e nematóides de solo. Desta forma, instalou-se um experimento em condições de campo, numa área da Fazenda Experimental Lageado, campus da UNESP no município de Botucatu - SP (latitude 22°51'S e longitude 48°26'W) para se avaliar o impacto desta técnica sobre a comunidade microbiana de um solo caracterizado como Latossolo Vermelho Distrófico, textura média. Inicialmente, incorporou-se uma fonte de matéria orgânica ao solo (couve Brassica oleraceae var. acephala L. fresca e triturada) na quantidade de 4kg.m-2. Posteriormente, umedeceu-se o mesmo e cobriu-se com filme plástico transparente de polietileno aditivado com 150mm de espessura. Fez-se vedação lateral de cada parcela, para se evitar a dispersão de gases e aumentar-se o efeito térmico natural. O experimento obedeceu a delineamento fatorial 2x2x4 (solo solarizado e não solarizado x com e sem incorporação de couve x épocas de coleta). Os tratamentos foram: a)adição de couve sem solarização; b)solarização e adição couve; c)testemunha, sem adição de couve e sem solarização; d)solarização sem adição de couve, com três repetições cada tratamento... (Resumo completo, clicar acesso eletrônico abaixo).
Abstract: The soil solarization technique has been used in small properties as an alternative to substitute chemical defensives for phytopathogens, insects, damage causing plants and soil nematode control. A field condition experiment was carried out in an area of Faculdade de Ciências Agronômicas - Botucatu - SP - Brazil (latitude 22°51'S and longitude 48° 26' W) in order to evaluate the technique impact on the microbial community of soil characterized as Distrofic Red Latosoil, medium texture. Initially, a source of organic material was incorporated to the soil (kale- Brassica oleraceae var acephala L. fresh and ground) in the amount of 4 kg.m-2. After that, it was moisturized a covered with transparent additivated polyethylene plastic film 150mm tick. Lateral sealing of each alloment was made, in order to avoid gas dispersal and to increase natural thermal effect. The experiment followed a 2x2x4 factorial outline (solarized and non solarized soil x with and without kale incorporaton x four times of harvest). The treatments were: a) addition of kale incorporation; b) solarization and addition of kale; c) witness, without addition of kale and without solarization; d) solarization without addition of kale; with three repetitions of each treatment. Samples composed of soil from each allotment were collected from 0-10cm deep, with the first collecting performed seven days after the experiment implantation in the field, and the further ones as intervals of 14 days, from January to March 2001, being afterwards taken to the area of Departamento de Produção Vegetal, (Defesa Fitossanitária) for microbiological analysis... (Complete abstract, click electronic address below).
Mestre

Thesis (Ph. D.)--Ohio State University, 2003.
Title from first page of PDF file. Document formatted into pages; contains xv, 160 p.; also includes graphics. Includes abstract and vita. Advisor: Warren A. Dick, Environmental Science Graduate Program. Includes bibliographical references (p. 135-160).

The abundance, distribution, and diversity of the soil microbial community from a seawater-irrigated, halophyte agroecosystem was examined. The abundance and trophic composition of decomposers responded to the biochemical composition of residue from three salt-tolerant plants, Salicornia bigelovii, Suaeda sp., and Batis maritima. Decomposers were also influenced by the decomposition environment as it interacted with residue resource quality. The microbial community responded to the availability of residues and the planting density of the halophyte Salicornia bigelovii (Chenopodiaceae) in a season-long study at a research farm in Sonora, Mexico. Soil microbial biomass, activity and efficiency, as well as nematode abundance and trophic composition, generally increased in association with soil-incorporated halophyte residues. However, soil moisture and salinity, which decreased with depth, may also have been influential. Microbial biomass and activity responded to plant density only after plants entered senescence. This result suggested that live-root exudations were not available to the microbial community possibly due to competition with plant roots for available nutrient resources. An increase in the abundance of plant-feeding, but not bacterial- or fungal-feeding, nematodes was associated with the presence of S. bigelovii. The functional diversity of a microbial community from an agronomic halophyte system was compared to natural and constructed halophyte salt marsh communities. Functional diversity of the microbial community at the agronomic sites was intermediate compared to the other sites; e.g., higher than the constructed marsh but lower than the natural salt marshes. Relationships among the study communities determined by microbial functional diversity reflected both habitat and geographical influences.

In this study bioluminescence-marker systems were used to investigate the fate of two genetically engineered degradative bacteria in soil under different conditions. These bacteria were chromosomally lux-marked Alcaligenes eutrophus H850 Lr with luxAB genes which was able to degrade polychlorinated biphenyls (PCBs), and Pseudomonas stutzeri P16. P. stutzeri P16 was genetically marked with the luxAB tet cassette by insertional mutagenesis using mini Tn5-transposons and was characterised for lux gene stability, phenanthrene degradability and sensitivity to aldehyde. Insertion of lux gene into genome of P. stutzeri P16 provided a novel strain with lower maximum specific growth rate and less sensitivity to high concentrations of phenanthrene in liquid culture. Survival and activity of P. stutzeri P16 luxAB4 was further investigated in soil amended with low and high concentrations of phenanthrene. Initially high concentrations of phenanthrene had a less toxic effect on viable cell concentrations and luminescence activity of genetically engineered P. stutzeri P16 comparing with its wild type showing that lux-marking the above degradative strain made the marked strain more suitable for environmental application in heavily polluted sites. However, resistant cells to high concentration of phenanthrene were not able to survive longer than those which was inoculated to microcosms with lower concentrations of phenanthrene. This indicates that toxicity of the target compound and its concentration have to be considered before releasing the GEMs. Degradation of phenanthrene was enhanced in both sterile and non-sterile soil inoculated with P. stutzeri P16 luxAB4. This study could be used as model for further investigated of degradation of other PAHs such as fluoranthene, benz[a]anthracene, benzo[a]pyrene, by degradative bacteria and also monitoring the survival and activity of those bacteria in the environment using bioluminescence-marker systems.

All societies are pressed to increase agricultural production – whether via enhancing crop yields in cultivated lands or via changing previous crop patterns into intensive agricultures, and the intensive agricultures are developing rapidly all over the world. Compared with the traditional agriculture, the intensive agriculture is under conditions of continuous production with high-intensity applications of fertilizers and other agrochemicals. Nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) is a commercial agrochemical applied to enhance nitrogen (N) fertilizer utilization efficiency. To maintain crop yield and quality, fungicide iprodione or herbicides atrazine and glyphosate are also applied into the agricultural soils, but their inhibitory or toxic effects are not constrained to hazardous fungi or weeds only. Once being applied into agricultural soils, the agrochemicals alone or with their degradation metabolites, may have negative effects on the entire soil microorganisms, and thereby soil biogeochemistry and quality. Apart from severe soil contaminations, the growing public concerns of agrochemicals are also associated with their impacts on soil environment and greenhouse gas emission. Previous studies have generally focused on single iprodione application, whereas in intensive agriculture production, iprodione is repeatedly applied. The DMPP and iprodione may be applied into agriculture soils simultaneously, but there are limited studies focusing on the interactive effects of DMPP and iprodione applications. For sugarcane farming, the herbicides different functions are essential to ensure sugarcane yields. However, few studies were conducted to reveal the impacts of DMPP and herbicides on soil N transformation rates and greenhouse gas emissions under different soil moisture conditions. Traditional researches about soil microbial population commonly utilized the culturing methods. However, the uncultured microorganisms far exceed their cultured counterparts, and approximately 80% of soil microorganisms are not discovered due to the shortages or costs of culturing methods. The superiorities of high-throughput sequencing technology enable detections of detailed microbial community changes that have not been reported with culturing methods, and it vastly enlarges our scientific horizon and understanding of bacterial and fungal responses to agrochemical applications at different taxonomic levels. Moreover, identifications of N flow and loss mechanisms are essential to enhance N utilization efficiencies and modify fertilizer and agrochemical management practices. External 15N isotope tracing method enables to accurately trace the transformations of applied N. In existing agricultural systems where N fertilizer or commercial agrochemical practices have already been established for decades, it is beneficial to apply the external 15N isotope tracing method to quantify effects of agrochemicals on the fluxes of applied N fertilizers...
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
Full Text

Organic matter amendments have long been known to improve native organic matter content, aggregation and structure of soils. In the laboratory, however, organic matter amendments to autoclaved soils have no such effect. This may explain the failure of many reclamation attempts on mine tailing wastes, which often proceed without regard for the microbiological processes necessary for soil formation and cycling of plant nutrients. In this study, incubation of tailing waste with soil microbes and a simple carbon source proved sufficient to increase the formation of water stable aggregates from tailing particles. Autoclaved control samples showed no change in aggregation. The incorporation of microbial cell mass into the mineral matrix of the tailing was observed using scanning electron microscopy. These results suggest that microbial activity is necessary in order to incorporate organic matter into the abiotic matrix of tailing, promoting aggregation and ultimately soil formation from this material.

European legislation has increased pressure on the use of land to represent the major disposal option for sewage sludge. Owing to their importance in regulating soil fertility, much research has been conducted into the effects of sewage sludge application on soil microorganisms. However, little knowledge is known about its effects on community dynamics of the beta-subgroup ammonia oxidising bacteria. This is despite the fact that nitrification activity of these bacteria plays an important role in soil nitrogen cycling. This investigation aimed to examine community dynamics of the beta-subgroup ammonia oxidising bacteria in sewage sludge amended soil by employing recently developed molecular based techniques. Firstly, a soil DNA extraction protocol was identified that allowed routine nested PCR amplification of 16S rDNA using beta-subgroup ammonia oxidiser directed primers. Reproducibility observed in denaturant gradient gel electrophoresis (DGGE) profiles suggested that 0.5 g samples used for DNA extraction allowed consistent detection of dominant beta-subgroup ammonia oxidiser community members. The effects of applying primary treatment sewage sludge to a variety of contrasting soils on (3-subgroup ammonia oxidiser community structure was unclear. This was partially hampered by lack of specificity of PCR primers for non ammonia oxidiser 16S rDNA sequences. Further, through measurement of net nitrification, there was no indication that sewage sludge addition stimulated the activity of nitrifier populations. Nevertheless, this study indicated the usefulness of DGGE for screening multiple environmental samples. A set of hierarchical oligonucleotide probes exhibiting specificity at the group, genus and cluster level were optimised using a non-radioactive system. These probes were used to assess the effects of application of aerobically digested sewage sludge to soil for 4 y on beta-subgroup ammonia oxidiser community structure. Despite significant changes in measurable soil parameters including net nitrification activity no changes were observed in beta-subgroup ammonia oxidiser community structure. This indicated resilience of these communities to change. A final study was conducted examining the effects of application of anaerobically digested sewage sludge to soil on beta-subgroup ammonia oxidiser community structure. Inhibition of nitrification by acetylene indicated the presence of a viable population of ammonia oxidising bacteria in continuously shaken samples of sludge. DGGE and oligonucleotide probing analysis provided evidence that sewage populations had the potential to outcompete indigenous soil populations of beta-subgroup ammonia oxidising bacteria. This was despite the fact that MPN enumeration suggested that soil populations of these bacteria were in some cases 10-fold greater than sludge populations. Evidence was also provided that suggested net nitrification to be an unreliable indicator of ammonia oxidiser activity in soil. It is possible to conclude that community structure of the beta-subgroup ammonia oxidising bacteria may be altered by application of sewage sludge to soil. The effects on community structure are likely to be influenced by both the type and level of sludge applied to soil.

The microbial life of sub-Antarctic islands plays a key role in the islands ecosystem, with microbial activities providing the majority of nutrients available for primary production. Knowledge of microbial diversity is still in its infancy and this is particularly true regarding the diversity of micro-organisms in the Antarctic and sub-Antarctic regions. One particularly important functional group of micro-organisms is the diazotrophs, or nitrogen-fixing bacteria and archaea. This group have not been well studied in the sub-Antarctic region, but play an important role in the nutrient cycling of the island. This thesis explored the diversity of nitrogen-fixing organisms in the soil of different ecological habitats on the sub-Antarctic Marion Island.

This study aims to investigate the link between genetic functioning and diversity, and to establish a relationship, if any, between that diversity and the stability of the community. Extreme transient perturbations were performed, using repeat chloroform fumigations, to reduce the community to its lowest genetic level (0.7% of the DNA of the control population). Severe reduction in community diversity and selection for bacilli was initially observed, the community subsequently recovered (6 months) to its previous diversity but was altered in community composition, bacilli were no longer dominant. Perturbations using benzene resulted in a reduced genetic diversity (16S rDNA and DGGE) which did not recover (after 4 weeks). However, full recovery was observed for culturable cell numbers, DNA concentration and community level physiological profile (Biolog)., suggesting high levels of genetic redundancy within soil. Investigation of the relationship between diversity and stability was performed by perturbing soils with naturally differing levels of diversity (Sourhope>Boyndie) to equivalent toxicity, using copper and benzene. Large decreases in biomass were observed for both soils after benzene perturbation. Assuming successful equivalent toxicity, the genetic diversity of the naturally more diverse soil was more resistant to benzene perturbation than the less diverse soil. The broad scale function of both benzene-treated soil communities was unaffected (14C-labelled wheat shoot mineralisation). Narrow niche function was impaired for both soils (14C-labelled 2,4-DCP mineralisation). The Sourhope soil recovered this function by the end of the experiment, but Boyndie did not. The genetic diversity of both soils was resistant to copper perturbation despite biomass decreases. Initial shifts in physiological profiles of both soils was observed but rapidly returned to that of the control. In conclusion, diversity-stability relationships may exist in soil communities, despite generally being an extremely robust system.

Mestrado em Engenharia do Ambiente - Instituto Superior de Agronomia
Abandoned mines are a major problem worldwide, with inherited impacts both upstream and downstream, whose dimension is more than just environmental, as they also influence sociological and economic spheres. Therefore, it is urgent to find viable alternatives to the recover affected soils. However, these extreme environments may also provide a panoply of microorganisms that may be useful in agricultural soils. The inoculation of soils with microorganisms is a technique increasingly addressed in the field of research. Plant growth assays using very specific bacteria – plant growth promoting bacteria (PGPB) – have been tested. In soils with small amounts of phosphorus, especially if rich in iron and aluminum oxides and hydroxides, most of the macronutrient is adsorbed and unavailable to plants. The same is true in alkaline soils with large amounts of calcium. Organic forms of phosphorus are also directly unavailable to plants. Calcium phytate, one of the most abundant forms of organic phosphorus, is insoluble. Soil microorganisms can help plant to acquire phosphorus from the soil by means of several mechanisms, and stimulate plant growth by improving the microbial-plant nutritional status and resistance to plant pathogens. Same bacteria may also tolerate high concentrations of trace elements. The hypothesis tested in this work was that isolates from contaminated mine soils would have a large tolerance to trace elements present in those soils. For this purpose, the tolerance of phosphorus-solubilizing bacteria isolated from the rhizosphere of plants from an abandoned Portuguese mine (S. Domingos mine) to four trace elements (arsenic, lead, copper and zinc) was tested and the ability to promote plant growth was evaluated using maize as the model plant, due to the economic importance of this crop in the national context and because its growth is rapid, which from the point of view of the laboratory tests becomes an asset. The total number of bacteria able to grow in rich media or in minimal media with the different sources of phosphorus was evaluated. The results showed that the greater value in rich media was obtained in the uncontaminated soil and the smallest value in the mine soil with the lowest pH and with large total contents of lead and arsenic. Isolates that formed a halo in calcium phosphate or calcium phytate were then tested in the presence of different levels of copper, zinc, lead and arsenic. The solubilization efficiency was diverse, and the tolerance to different concentrations of trace elements also varied between isolates, with better results obtained with arsenic, generally. The study in hydroponics showed the capacity of bacterial isolates to promote plant growth highlighting the importance of the interaction plant-bacteria. In conclusion, it was possible, under laboratory conditions, to obtain isolates with ability to tolerate different trace elements, in different concentrations, and able to promote plant growth. The results suggest that it may be possible to use some of the isolates under field conditions as inoculants for agricultural soils.

The entrainment of microbes from agricultural land into overland flow during rainfall events is recognised as an important source of pathogenic microbes to surface water bodies and yet this transport process is poorly understood. In this study, a method has been developed to separate bacteria into the forms in which they have been postulated to exist in overland flow. Then Escherichia coli was used as a model organism to investigate the transported state of bacteria eroded from cowpats and their subsequent transport in overland flow. Simulated rainfall experiments were used to generate runoff direct from cowpats. Concentrations of E. coli in the runoff direct from cowpats were found to be directly proportional to the concentration in the cowpat, regardless of the age of the cowpat. It was also observed that E. coli were predominantly eroded from cowpats as individual cells. The interactions between E. coli and soil particles in overland flow were then examined in a small laboratory scale model system and showed that E. coli attached to large (>45 [mu]m) soil particles were transported significantly less than unattached cells. However, in the runoff from the model system, E. coli were found to be attached mainly to clay particles that were similar in size to the bacterial cells. Furthermore, the transport of E. coli through the model system appeared to follow the transport of a conservative chemical tracer implying that (a) the cells were being transported as a solute with the bulk of the water flow, and (b) that E. coli attached to small clay particles were as mobile in the overland flow as unattached cells. These observations imply that E. coli predominantly interact with small clay particles that are also being carried along in the overland flow. The transport of E. coli at a larger scale was then investigated using 5-metre long, 1-metre wide buffer strips operated under saturation excess conditions. In buffer strips using intact soils and existing pasture cover, E. coli removal was very poor (26 % removal) at the low flow rate of 2 L min⁻� with no removal observed at the higher flow rates of 6 and 20 L min⁻�. E. coli removal rates were increased to 41 % removal at 2 L min⁻� by cultivating the soils, with the removal rate again decreasing with increasing flow rate. E. coli in the overland flow from the buffer strips did not form into large flocs or attach to large soil particles, but were transported in small neutrally buoyant particles that remain entrained in the overland flow. Under saturation excess runoff conditions, E. coli in overland flow were not effectively removed by buffer strips as the small particles are transported either over the soil surface or, through large pores in the soil. This Thesis has shown that E. coli is transported in overland flow in small particle sizes that are difficult to trap or remove from overland flow thereby explaining the high fluxes of faecal bacteria observed in overland flow from agricultural land.

[ES] Las preocupaciones ambientales y de salud han estimulado el interés en estrategias alternativas para el manejo de las malas hierbas. En todo el mundo se están haciendo esfuerzos para reducir la gran dependencia de los herbicidas sintéticos que se utilizan como principal método para el control de las plantas arvenses. Los herbicidas naturales basados en sustancias alelopáticas, como los aceites esenciales (AEs) extraídos de plantas, se han sugerido como una de las posibles alternativas para lograr un manejo sostenible de las arvenses. Por un lado, los AEs han mostrado capacidad para inhibir la germinación y el crecimiento de semillas de malas hierbas, por otro lado, hay una falta de estudios sobre los efectos de tales sustancias sobre los microorganismos del suelo. Por lo tanto, en esta tesis se ha investigado la actividad fitotóxica y herbicida de los AEs extraídos de plantas mediterráneas para determinar su potencial como herbicidas naturales. Se han ensayado los efectos de AEs, así como de otros extractos de plantas como hidrolatos, extractos acuosos y hojas frescas obtenidas de plantas mediterráneas, sobre los microorganismos del suelo. Las especies donadoras de AEs fueron seleccionadas en base a conocimientos previos del grupo de investigación, y de acuerdo con la bibliografía existente sobre la actividad herbicida de metabolitos secundarios de estas especies o de especies que están taxonómicamente estrechamente relacionadas: Eucalyptus camaldulensis Dehnh., Eucalyptus occidentalis Endl., Eucalyptus globulus Labill., Eucalyptus torquata Luehm., Eucalyptus lesoufii Maiden, Thymbra capitata (L.) Cav., Mentha × piperita L. y Santolina chamaecyparissus L. Como malas hierbas objetivo se seleccionaron dos monocotiledóneas, Avena fatua L. y Echinochloa crus-galli (L.) P. Beauv. y dos dicotiledóneas, Portulaca oleracea L. y Amaranthus retroflexus L. La composición de los AEs se analizó mediante Cromatografía de gases (CG) y Cromatografía de gases-Espectrometría de masas (CG-EM). Los ensayos in vitro se realizaron en cámaras de germinación, para evaluar los efectos fitotóxicos de los AEs sobre la germinación y el crecimiento de las plántulas. Los ensayos in vivo se realizaron en condiciones de invernadero, los AEs se aplicaron emulsionados con Fitoil mediante riego. El estudio de los efectos sobre los microorganismos del suelo se realizó en el laboratorio en macetas donde los suelos se trataron con los AEs y extractos de hojas y luego se incubaron a temperatura ambiente (20-23 ° C). Los ensayos in vitro revelaron que todos los AEs mostraron efectividad contra las malas hierbas ensayadas. Entre ellos, T. capitata fue el más eficaz. A las dosis más bajas, bloqueó completamente la germinación de todas las semillas. Los ensayos en invernadero demostraron la actividad herbicida de T. capitata, M. piperita y S. chamaecyparissus, aumentando su fitotoxicidad con la dosis. T. capitata fue el AE más eficaz contra todas las malas hierbas a la dosis máxima y P. oleracea fue la especie más resistente. Los microorganismos del suelo, después de un período transitorio de agitación, generalmente recuperaron su función y biomasa iniciales. Solo el AE de T. capitata a la dosis más alta no permitió que los microorganismos del suelo recuperaran completamente su funcionalidad inicial. La aplicación de extractos de hojas al suelo proporcionó evidencia de que las hojas de eucalipto y sus extractos (AE, hidrolatos y extractos acuosos), afectaron a la comunidad microbiana del suelo de diferente modo, dependiéndose de la especie de Eucalyptus considerada. Hasta el momento, los resultados permiten sugerir la aplicación de los Aes como bioherbicidas en entornos controlados y en condiciones de invernadero. Se debe identificar la dosis óptima de aplicación para controlar las malas hierbas y simultáneamente, no afectar negativamente a los microorganismos del suelo.
[CA] Les preocupacions ambientals i de salut han estimulat l'interès per estratègies alternatives de gestió de les males herbes. A tot el món, s'estan fent esforços per reduir la gran dependència dels herbicides sintètics que s'utilitzen com a principal mètode per al control de les males herbes. Els herbicides naturals basats en substàncies al·lelopàtiques, com els olis essencials (OEs) extrets de plantes, s'han suggerit com una de les alternatives possibles per aconseguir una gestió sostenible de les males herbes. Els OEs han mostrat capacitat per inhibir la germinació i el creixement de llavors de males herbes, però per altra banda, falten estudis sobre els efectes d'aquestes substàncies sobre els microorganismes del sòl. En aquesta tesi es van investigar les activitats fitotòxiques i herbicides dels OE extrets d'herbes mediterrànies per a un ús potencial com a herbicides naturals. A més, es van provar els efectes d'aquests OE i d'altres extractes vegetals, com hidrolats, extractes aquosos i fulles fresques obtingudes d'herbes mediterrànies sobre els microorganismes del sòl. Les espècies donants d'OEs es van seleccionar bassant-se en la experiencia previa del grup d'investigació i en la bibliografía existent sobre les activitats biològiques dels metabòlits secundaris d'aquestes espècies: Eucalyptus camaldulensis Dehnh., Eucalyptus occidentalis Endl., Eucalyptus globulus Labill., Eucalyptus torquata Luehm., Eucalyptus lesoufii Maiden, Thymbra capitata (L.) Cav., Mentha × piperita L. i Santolina chamaecyparissus L. Les males herbes objectiu seleccionades van ser dos monocotiledóneas, Avena fatua L. i Echinochloa crus-galli (L.) P. Beauv. i dos dicotiledóneas, Portulaca oleracea L. i Amaranthus retroflexus L. La composició dels OEs es va analitzar mitjançant Cromatografia de gasos (CG) i Cromatografia de gasos-espectrometria de mases (CG-EM). Els assajos in vitro es van realitzar en cambres de germinació per avaluar els efectes dels OEs sobre la germinació i el creixement de les plàntules. Els assajos in vivo es van realitzar en condicions d'hivernacle, on es van aplicar per reg els OEs emulsionats amb Fitoil a les males herbes. L'estudi dels efectes dels OEs sobre els microorganismes del sòl es van realitzar en un experiment en tiestos en condicions de laboratori, en el qual els sòls van ser tractats amb els OEs i els extractes de fulles i després van ser incubats a temperatura ambient (20-23 ° C). Els resultats dels assaigs in vitro van revelar que tots els OE van mostrar efectivitat contra les males herbes objectiu. Entre tots ells, T. capitata va ser el més eficaç. A dosis més baixes, va bloquejar completament la germinació de tots les llavors. Els assaigs en hivernacle van demostrar l'activitat herbicida de T. capitata, M. piperita i S. chamaecyparissus, augmentant la seva fitotoxicitat amb la dosi. T. capitata va ser l'OE més eficaç contra totes les males herbes a la dosi màxima i P. oleracea va ser la mala herba més resistent. Els microorganismes del sòl, després d'un període de trastorn transitori, en general van recuperar la seva funció inicial i la seva biomassa. Només l'oli de T. capitata, a la dosi més alta no va permetre als microorganismes del sòl recuperar completament la seva funcionalitat inicial. Els resultats de l'aplicació d'extractes de fulles al sòl van demostrar que les fulles d'eucaliptus i els seus extractes (OEs, hidrolats i extractes aquosos), afecten la comunitat microbiana del sòl de diferents maneres, depenien de les espècies d'eucaliptus. Fins ara, els resultats obtinguts fan possible suggerir l'aplicació d'EOs com a bioherbicides en entorns controlats com l'horticultura i en condicions d'hivernacle, però s'ha d'identificar la dosi òptima d'aplicació per controlar les males herbes i simultàniament, no afectar negativament els microorganismes del sòl.
[EN] Environmental and health concerns caused for traditional crop protection systems have stimulated interest in alternative weed management strategies. Worldwide, efforts are being made to reduce the heavy reliance on synthetic herbicides that are used to control weeds. Natural herbicides based on allelopathic substances, such as volatile essential oils (EOs) extracted from plants, has been suggested to be one of the possible alternatives for achieving sustainable weed management. From one hand, EOs have shown ability to inhibit weeds seed germination and growth, on the other hand there is a lack of studies about the effects of such substances on soil microorganisms. Therefore, in this thesis the phytotoxic and herbicidal activities of EOs extracted from Mediterranean plants were investigated for their potential use as natural herbicides. The effects of EOs, as well as, of other plant extracts, such as hydrolates, aqueous extracts and fresh leaves obtained from Mediterranean plants, were tested on soil microorganisms. The donor species of EOs were selected based on previous experience of the research group and according to the current literature about the herbicidal activities of the secondary metabolites of these species or from species that are taxonomically closely related: Eucalyptus camaldulensis Dehnh., Eucalyptus occidentalis Endl., Eucalyptus globulus Labill., Eucalyptus torquata Luehm., Eucalyptus lesoufii Maiden, Thymbra capitata (L.) Cav., Mentha × piperita L. and Santolina chamaecyparissus L. The target weeds were two monocotyledons, Avena fatua L. and Echinochloa crus-galli (L.) P. Beauv, and two dicotyledons, Portulaca oleracea L. and Amaranthus retroflexus L. EOs composition was analyzed by means of Gas Chromatography (GC) and Gas Chromatography-Mass Spectrometry (GC-MS). The in vitro assays were performed in germination chambers, to assess the effects of EOs on weed seed germination and seedling growth. The in vivo trials were conducted in greenhouse conditions, where EOs emulsified by Fitoil were applied on weed species by watering. The study of EOs effects on soil microorganisms was carried out in a laboratory pot experiment, where soils were treated with EOs and leaf extracts and then incubated at room temperature (20-23°C). Results obtained from the in vitro bioassays revealed that all used EOs displayed of effectiveness against assayed weeds, controlling completely their germination process or reducing it and significantly inhibiting their seedling growth. Among them, T. capitata was the most effective. At lower doses, it blocked completely the seed germination of A. retroflexus, P. olecerea, A. fatua and E. crus-galli. Greenhouse trials demonstrated herbicidal activity of T. capitata, M. piperita and S. chamaecyparissus, increasing their phytotoxicity with the dose. T. capitata was the most effective against all weeds at the maximum dose and P. oleracea was the most resistant weed. Soil microorganisms, after a transient upheaval period, induced by the addition of EOs, generally recovered their initial function and biomass. Only T. capitata EO at the highest dose did not allow soil microorganisms to completely recover their initial functionality. Results of leaf extracts application on soil provided evidence that Eucalyptus leaves and their extracts (EOs, hydrolates and aqueous extracts), affected soil microbial community in different ways, and those effects were dependent on the Eucalyptus species. So far, the results obtained make feasible to suggest EOs application as bio-herbicides in controlled environments, such as horticulture and in greenhouse conditions. However, the optimum dose of application must be determined, to control weeds and simultaneously, not negatively affect soil microorganisms.
Jouini, A. (2020). Herbicidal activity of Mediterranean essential oils and their effects on soil bioindicators [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/159914
TESIS

This research tested the hypotheses that (a) suboptimal root-zone temperatures (RZT) limit the soybean-Bradyrhizobium N$ sb2$-fixing symbiosis primarily through an inhibition of symbiosis establishment and (b) this inhibition is modified by the genotype of micro- or macrosymbiont. Controlled environment and field experiments were conducted utilizing two soybean genotypes and six B. japonicum strains. At 19$ sp circ$C RZT fixed nitrogen levels decreased by 30-40%, predominantly due to a restriction in the latter stages of nodule development. Reductions of 10% and 30% in specific nodule activity rates at 19$ sp circ$C and 15$ sp circ$C RZT respectively, indicated nodule function to be comparatively insensitive to low RZT. Soybean genotypes did not differ in seedling nodulation or N$ sb2$-fixation under cool-soil, field or controlled environment, conditions. At all temperatures, commercial B. japonicum strain 532C was more efficient, but not effective, than strains obtained from the cool-soils of Northern Japan. Under cool-soil field conditions, two of the latter strains increased seedling nodulation and N$ sb2$-fixation.

Made available in DSpace on 2014-06-11T19:31:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2002-05Bitstream added on 2014-06-13T21:02:53Z : No. of bitstreams: 1 rocha_mm_dr_botfca.pdf: 1150326 bytes, checksum: 893acd7e01d7f269b3eaa875876c771e (MD5)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O manejo agrícola altera, em muito, as características físicas, químicas e biológicas dos solos. A cultura e as condições climáticas, assim como o tipo de solo propriamente dito, afetam a decomposição da matéria orgânica do solo e, consequentemente, a biogeociclagem dos nutrientes. A matéria orgânica é uma característica importante em relação à fertilidade do solo, de modo que o impacto do uso do solo precisa ser avaliado principalmente em agroecossistemas. O conhecimento dos efeitos do uso da terra e das práticas agrícolas sobre a comunidade microbiana é de fundamental importância, em vista das importantes funções que os microorganismos desempenham no solo e que irão se refletir na produtividade agrícola. Neste sentido, uma avaliação da biomassa microbiana e de microorganismos do solo pode evidenciar diversas alterações no ecossistema do solo que estão associadas ao teor de argila e/ou sistemas de plantio. No presente estudo, caracterizou-se a camada superficial do perfil de solos sob plantio direto e plantio convencional em quatro fazendas no Brasil em relação a alguns de seus componentes microbiológicos. Para tanto, analisou-se, mensalmente, a respiração do solo, a biomassa microbiana e grupos de microorganismos. A avaliação da biomassa microbiana foi feita através da técnica da fumigação-incubação (FI), utilizando-se a Equação: [(C-CO2 liberado pelo solo fumigado, no período de 0-10 dias de incubação) - (C-CO2 liberado pelo solo não-fumigado, ao longo de 10-20 dias de incubação)]/0,45. Os cálculos indicaram um conteúdo de carbono da biomassa microbiana significativamente maior nos solos sob sistema de plantio direto em relação àqueles sob plantio convencional, na camada amostrada (0-10 cm de profundidade). Quantidades significativamente maiores...
The crop management changes greatly the physical, chemical and biological soil properties. Furthermore, the crop and soil types, and the climatic conditions would affect on soil organic matter decomposition and on nutrients biogeociclying. Soil organic matter is a important characteristic in relationship soil fertility. The knowledge about effects on soil using and agriculture practices on soil microbial communities is very important, due to the function that microorganisms have in soil and it was going to in soil fertility. In this sense, evaluation of the soil microbial biomass and micro-organisms greatly aids predictions several changes in the soil ecosystems are associated with reduced tillage as compared with conventional tillage. Surface soils from long-term no-till and conventional tillage plots at four Brazil farms were characterised for microbial components. Soil respiration, microbial biomass carbon and counts of microorganisms were measured at intervals monthly. The evaluation of microbial biomass carbon was done by fumigation-incubation technique (FI). For calculating the soil microbial biomass carbon, the equation used was: Equation = [(CO2-C evolved by fumigated soil, 0-10 days) - (CO2-C evolved by unfumigated soil, 10-20 days)]/0,45. Significantly greater amounts of CO2-C were released from no-till than from conventional tilled soils. qCO2 values were not significantly different between tillage systems. This observation confirms that the tillage affected biological activity in those soils, further that qCO2 values didn't have significantily different which two tillage systems, in studied soils. Number both fungi and bacteria were assayed by Most Probable Number (MPN) by the agar drop counting technique and the microorganisms groups were calculated using by traditional Most Probable Number (MPN) method...(Complete abstract, click electronic access below)

Strategies for the enhanced biological degradation of pesticides were examined in this research project. In one approach, the concept of the plant—microbial rhizosphere association was investigated as a laboratory model using the herbicide 2,4-D as a test compound. In another, an enhanced degradation of the recalcitrant herbicide atrazine was shown. Here, two microbial populations each capable of rapid atrazine metabolism were studied. The metabolism of 2,4-D by bacteria associated with the root system of wheat and canola seedlings was demonstrated in this study using a hydroponic system as well as a solid medium of sand and gravel. Significant and rapid 2,4-D metabolism (near 100% within 24h) was found in all hydroponic systems where the 2,4-D degrading microorganisms, Acinetobacter baumannii pJP4 transconjugant, Alcaligenes eutrophus and Azospirillum brasilense pIP4 transconjugant were associated with the roots. The metabolism of 2,4-D by Azospirillum brasilense pJP4 transconjugant associated with wheat was less rapid than associations with the other 2,4-D degrading bacteria. There was little difference in the rates of degradation between the hydroponic system and the sand/gravel mixture. The colonisation of the roots of seedlings by microbes was studied by both fluorescence and laser scanning confocal microscopy. Colonisation was often prolific without favoured areas of attachment on the root. A pre-treatment of seedlings with a synthetic auxin which formed para-nodular structures had little effect on the nature of colonisation. Counts of colony forming units, however, established that there was an increase of an order in magnitude of cells per root system when the plants were pretreated with this synthetic auxin. An average of 5.5 x 106 viable cells of 2,4-D degrading Acinetobacter baumannii were counted on para-modulated wheat root systems. It was demonstrated that the colonisation of the rhizosphere by suitable microbes could protect canola seedlings against phytotoxic effects of the applied herbicide. Whether this bio-safening effect will be seen in solid media or in field situations with these nonleguminous plants was not investigated. Significant rates of atrazine degradation either in the laboratory or in the field have rarely been reported. Attempts were therefore made to obtain microbes capable of such metabolism. These attempts had the ultimate goal of providing microbes for application in the model plant microbial rhizosphere association. Two microbial cultures, each capable of rapid atrazine metabolism, were obtained and studies of the metabolic processes were conducted. Rhodococcus sp. NI86/21 metabolised atrazine within l44h to two N—dealkylated products, desisopropylatrazine and desethylatrazine. Mineralisation of the ethyl-14C labelled sidechain to 14CO; was demonstrated, accounting for 25% of the total applied label in the broth culture. Desisopropylatrazine was shown to be the major metabolite. Desethylatrazine was shown as a terminal metabolite in the degradation of atrazine by Rhodococcus sp. N186/21, accumulating in the broth. In other studies using it as the substrate, no firrther metabolism was found. Desisopropylatrazine was also indicated to have been a terminal metabolite as it too accumulated in the broth. The metabolism of prometryn, a thio-ester (non-chlorinated) s-triazine was also investigated in these studies. Although Rhodococcus Sp. N186/21 did not rapidly degrade prometryn under similar conditions to those used with the atrazine assays, a mono-N-dealkylated metabolite was identified by mass-spectrometry. Possibly, prometryn was toxic to the Rhodococcus at the concentration used (lOug mL'l), as only minimal growth of the bacteria in the broth was observed by increased absorbance. Such a toxic effect was likely to have inhibited the N-dealkylation metabolism of the prometryn. In another approach, soil from a pesticide sullage site on a farm in northern NSW was assayed for its atrazine metabolising ability. Although there was no initial activity, after 30 months of perfirsion of the soil with a concentrated solution of the herbicide, it had acquired the ability to rapidly mineralise atrazine. A rapid conversion of the three carbons in the s—triazine ring to C02 was demonstrated using radiolabelled atrazine. Also, the labelled carbon in the ethyl sidechain of atrazine was rapidly metabolised to CO2. The sidechain 14C label was mineralised to 14C02 at a slower rate than the carbons in the ring. It was demonstrated that there was a likelihood of the sidechain carbon being incorporated into an unextractable intermediate metabolite, which was subsequently also less susceptible to attack by the microorganisms. There were no significant metabolites of atrazine accumulated in the broth. The likely presence of hydroxyatrazine was noted in the assay using uniformly ring—labelled [14C] atrazine. Hydroxyatrazine was also identified in the assay with ethyl-sidechain labelled [14C] atrazine, however, the amount detected was less. The presence of atrazine at 25pg mL-l inhibited nitrification reactions in the soil, however, at a saturating concentration of SOug mL-l there was some ammonia oxidation noted. Attempts to isolate single bacterial colonies capable of the metabolism of atrazine were unsuccessful. Although there was insufficient time to utilise these microbial cultures in the plant microbial rhizosphere associations, studies on the metabolism of atrazine have sufficiently characterised the nature of biodegradation to suggest that plant-microbial associations can be confidently tested in fiiture experiments.

A field study to determine the effects of a switchgrass buffer strip (SBS) on soil microorganisms near a field applied with endosulfan was carried out. Soil samples were taken from a SBS and bare soil area downslope from a field applied with endosulfan at different distances, days, and two seasons (wet and dry). Soil samples were analyzed for endosulfan, soil fungi, and bacteria. Analysis of endosulfan concentrations was done by reversed-phase liquid chromatography. No endosulfan runoff was detected by this method. Analysis of soil fungi and bacteria was done by fungal and bacterial enumeration by plate count method on rose bengal agar and tryptic soy agar, respectively. Soil fungi and bacteria were higher in the SBS than in the bare soil area. Also, soil bacteria was higher during the wet season than during the dry season. The opposite trend was observed for soil fungi.

This study aimed to assess the effect of heavy metals, especially Cu, Zn and Cd, on the ability of the white rot fungus (WRF) Bjerkandera sp. BOS55 and several soil bacterial isolates to degrade a selection of polycyclic aromatic hydrocarbons (PAHs). To investigate the effect of heavy metals on PAH degradation by Bjerkandera sp. BOS55, the fungus was grown in both a rich undefined medium and a defined medium containing the PAH pyrene and different concentrations of Cu or Cd. It was found that both metals reduced fungal growth and production of ligninolytic enzymes but that toxicity was modified by the growth medium. The fungus exhibited adaptation and tolerance to both metals in the defined medium containing up to 600 ppm metal but was inhibited in the rich medium containing as little as 50 ppm metal. Whilst WRF are capable of PAH oxidation, complete mineralisation to CO2 is often unachieved resulting in the production of polar and often toxic metabolites. In soil, these metabolites can be further oxidised by bacterial populations to non-toxic products. However, bacteria often exhibit a greater sensitivity to heavy metals than WRF. This sensitivity to heavy metals could block the detoxification process of the soil environment. The three PAH-oxidising bacterial isolates that were tested for metal tolerance were found to be highly susceptible to Cu, Zn and Cd at concentrations of 200 ppm each, resulting in a reduced degradation of phenanthrene and pyrene. It was therefore concluded that the presence of heavy metals in the environment, even at fairly low levels, could potentially lead to the incomplete degradation of PAHs, and possible accumulation of toxic metabolites. Finally, co-cultures of the metal-sensitive bacterial isolates and the fungus were used to establish if a combination of WRF and a bacterial isolate could be used to accelerate the degradation of PAHs. Anthracene and anthraquinone were used as model PAHs because anthraquinone is a dead-end metabolite produced during the oxidation of anthracene by Bjerkandera sp. BOS55. It was found that some of the fungal-bacterial co-cultures tested synergistically removed anthracene, resulting in significantly faster degradation than observed for each organism individually. Moreover, one co-culture removed anthraquinone, suggesting that a combination of WRF and bacteria are essential for the rapid mineralisation of PAHs in the environment.

The objective of this research was to evaluate the impact of bioaugmentation of soil with microorganisms harboring plasmid pJP4 on remediation, plasmid transfer, and plasmid dispersal. Divided into three sections, this research showed that use of microbial inocula harboring self-transmissible plasmids holds promise as an applicable bioremediation approach. In the first study, a pJP4 donor that could readily be counter-selected due to a lack of chromosomal genes necessary for 2,4-dichlorophenoxyacetic acid (2,4-D) mineralization was generated to allow detection of transconjugants in soil. Plasmid pJP4 was introduced into Escherichia coli (ATCC 15224), via plate mating with Ralstonia eutropha JMP134 to create such a donor (E. coli D11). Transfer of Plasmid pJP4 to diverse indigenous populations was detected in soils, and under conditions, where it had not been observed previously. Plexiglass columns were used in the second study to evaluate dissemination of plasmid pJP4 under unsaturated or saturated flow conditions in a 2,4-D contaminated soil. In unsaturated soil, pJP4 was detected in both culturable donor and transconjugant cells extending to 10.5 cm from the inoculated layer. In soil subjected to saturated flow conditions, no transconjugants were detected; however, donors were found throughout the entire length of the column (30.5 cm). Thus, donor transport in conjunction with plasmid transfer to indigenous recipients allowed for significant dissemination of introduced genes through contaminated soil. The last study was conducted using soil contaminated with 2,4-D alone or co-contaminated with 2,4-D and cadmium (Cd). This study assessed the impact of introduction of the pJP4 genes via cell bioaugmentation (R. eutropha JMP134 donor), or via gene augmentation (E. coli D11 donor). Both introduced donors remained culturable and transferred plasmid pJP4 to diverse indigenous recipients. Cell bioaugmentation resulted in the most rapid 2,4-D degradation; however, upon a second exposure to 2,4-D, gene augmentation of indigenous populations was more successful. The presence of Cd (100 μg g dry soil⁻¹) had a minimal impact on 2,4-D degradation and transconjugant formation. The establishment of an array of stable indigenous plasmid hosts may be particularly useful in sites with potential for re-exposure or extensive, and thus, long term contamination.

Soil microorganisms play important roles in maintaining soil quality and ecosystem health. Development of effective methods for studying the composition, diversity, and behavior of microorganisms in soil habitats is essential for a broader understanding of soil quality. Forest management strategies and practices are of vital significance for sustainable forest production. How the different forest management measures will influence soil microbial communities is a widespread concern of forest industry and scientific communities. Only a small proportion (~0.1%) of the bacteria from natural habitats can be cultured on laboratory growth media. Direct extraction of whole-community DNA from soil, followed by polymerase chain reaction (PCR) and other analysis circumvents the problems of the culture-dependent methods and may shed light on a broader range of microbial communities in the soil. DNA-based molecular methods rely on high quality soil microbial DNA as template, and thus extraction of good quality DNA from soil samples has been a challenge because of the complex and heterogeneous nature of the soil matrix. The objectives of this research were to establish a set of DNA-based molecular methods and to apply them to investigate forest soil microbial composition and diversity. Soil samples were collected from different forest ecosystems, i.e., the natural forest (YNF) and the first rotation (~ 50 years) (Y1R) and the second rotation (~ 1 year) (Y2R) of hoop pine plantations at Yarraman, and from different forest residue management practices (the experiments had established 6.4 years before the samples were collected) at Gympie, two long-term experimental sites of the Queensland Department of Primary Industry-Forestry in subtropical Queensland, Australia. Some DNA-based molecular techniques, including DNA extraction and purification, PCR amplification, DNA screening, cloning, sequencing and phylogenetic analyses, were explored using Yarraman soil samples, which were high in organic matter, clay and iron oxide contents. A set of methods was assembled based on the recommendations of the method development experiments and applied to the investigations of the microbial composition and diversity of the Yarraman and Gympie soil samples. Four soil DNA extraction methods, including the Zhou method (Zhou et al., 1996), the Holben method (Holben, 1994), the UltraClean (Mo Bio) and FastDNA (Bio 101) soil DNA extraction kits, were explored. It was necessary to modify these methods for Yarraman soil. I designed and introduced a pre-lysis buffer washing step, to partially remove soil humic substances and promote soil dispersion. This modification greatly improved the quality of the extracted DNA, decreasing co-extracted humic substances by 31% and increasing DNA yield by 24%. The improved Holben method was recommended for fungal community studies, and the improved Zhou method for bacterial community studies. The extracted DNA was good in quality, with a consistent size of ~20 kb and a yield of 48-87 g g-1 soil, and could be successfully used for 16S (Zhou method) and 18S (Holben method) rDNA amplifications. For less difficult environmental samples, UltraClean kits could be a good option, because they are simple and fast and the extracted DNA are also of good quality. Screening of the DNA PCR products using TGGE, Heteroduplex-TGGE and SSCP was also explored. These methods were not so effective for the screening of the soil DNA PCR products, owing to the difficulty in interpretation of the results. Cloning was a necessary step to obtain a single sequence at species level in soil microbial community studies. The screening of the clone library by TGGE, Heteroduplex-TGGE and SSCP could only separate the clones into several major bands, although SSCP gave better separation. Sequencing of selected clones directly from the clone library obtained ultimate results of microbial taxonomic composition and diversity through well-established sequence analysis software packages and the databases. It was recommended that, in this project with the target of microbial community composition and diversity, soil DNA PCR products were directly cloned to construct clone libraries and a sample of clones were sequenced to achieve an estimate of the taxonomic composition of the soil. Fungal communities of the Yarraman soil samples under the natural forest (YNF) and the hoop pine plantations (YHP) were investigated using 18S rDNA based cloning and sequencing approaches. Twenty-eight clone sequences were obtained and analysed. Three fungal orders, i.e., Zygomycota, Ascomycota and Basidiomycota were detected from the YNF and YHP samples. By contrast, culture-based analyses of fungi in the literature were mostly Ascomycetes. YNF appeared to have more Ascomycota but less Zygomycota than YHP, and within the Zygomycota order, YHP had more unidentified species than YNF. Bacterial communities of Yarraman soil samples of YNF, Y1R and Y2R were investigated using 16S rDNA-based cloning and sequencing approaches. 305 16S rDNA clone sequences were analysed and showed an overall bacterial community composition of Unclassified bacteria (34.4%), Proteobacteria (22.0%), Verrucomicrobia (15.7%), Acidobacteria (10.2%), Chloroflexi (6.9%), Gemmatimonadetes (5.6%), and Actinobacteria (5.2%). There was a significant difference among YNF, Y1R and Y2R in the taxonomic group composition. YNF had a greater proportion of Acidobacteria (18.0%), Verrucomicrobia (23.0%) and Chloroflexi (9.0%) than Y1R and Y2R (corresponding to 6.3%, 12.1% and 5.9%, respectively), while Y1R and Y2R had a higher percentage of the Unclassified group (38.5% for Y1R and 46.5% for Y2R) than YNF (18.0%). For the Proteobacteria group, YNF had more Alpha-subdivision but Y1R and Y2R had more Delta-subdivision. From YNF to Y1R to Y2R, the clone sequence variable site ratios, 5% and 10% OTU numbers and Shannon's diversity index H' values tended to decrease, indicating the soil bacterial diversity decreased from the natural forest to the first and the second rotation hoop pine plantations. The large amount of unclassified clone sequences could imply a novel group of bacteria in the soil, particularly in the hoop pine soil samples. Alternatively they may result from artefacts during the PCR process. Bacterial communities of the Gympie soil under different residue management practices, i.e., residue (litter plus logging residue) removed (G0R), residue retained (G1R), and residue doubled (G2R), were also investigated using the 16S rDNA-based cloning and sequencing approaches. Acidobacteria (37.6%) and Proteobacteria (35.6%, including Alpha-subdivision of 29.9% and Gamma-subdivision of 5.7%) were dominant components of the communities, followed by Actinobacteria (14.7%), Verrucomicrobia (7.3%) and Unclassified bacteria. There was no significant difference among G0R, G1R and G2R in the bacterial community compositions and diversity. These findings provided an in-depth vision of the soil microbial communities under different forest management practices. Their combination with other soil analysis results, such as physical and chemical properties, and forest production data, could provide an improved understanding of sustainable forest management strategies.

Soil microorganisms play important roles in maintaining soil quality and ecosystem health. Development of effective methods for studying the composition, diversity, and behavior of microorganisms in soil habitats is essential for a broader understanding of soil quality. Forest management strategies and practices are of vital significance for sustainable forest production. How the different forest management measures will influence soil microbial communities is a widespread concern of forest industry and scientific communities. Only a small proportion (~0.1%) of the bacteria from natural habitats can be cultured on laboratory growth media. Direct extraction of whole-community DNA from soil, followed by polymerase chain reaction (PCR) and other analysis circumvents the problems of the culture-dependent methods and may shed light on a broader range of microbial communities in the soil. DNA-based molecular methods rely on high quality soil microbial DNA as template, and thus extraction of good quality DNA from soil samples has been a challenge because of the complex and heterogeneous nature of the soil matrix. The objectives of this research were to establish a set of DNA-based molecular methods and to apply them to investigate forest soil microbial composition and diversity. Soil samples were collected from different forest ecosystems, i.e., the natural forest (YNF) and the first rotation (~ 50 years) (Y1R) and the second rotation (~ 1 year) (Y2R) of hoop pine plantations at Yarraman, and from different forest residue management practices (the experiments had established 6.4 years before the samples were collected) at Gympie, two long-term experimental sites of the Queensland Department of Primary Industry-Forestry in subtropical Queensland, Australia. Some DNA-based molecular techniques, including DNA extraction and purification, PCR amplification, DNA screening, cloning, sequencing and phylogenetic analyses, were explored using Yarraman soil samples, which were high in organic matter, clay and iron oxide contents. A set of methods was assembled based on the recommendations of the method development experiments and applied to the investigations of the microbial composition and diversity of the Yarraman and Gympie soil samples. Four soil DNA extraction methods, including the Zhou method (Zhou et al., 1996), the Holben method (Holben, 1994), the UltraClean (Mo Bio) and FastDNA (Bio 101) soil DNA extraction kits, were explored. It was necessary to modify these methods for Yarraman soil. I designed and introduced a pre-lysis buffer washing step, to partially remove soil humic substances and promote soil dispersion. This modification greatly improved the quality of the extracted DNA, decreasing co-extracted humic substances by 31% and increasing DNA yield by 24%. The improved Holben method was recommended for fungal community studies, and the improved Zhou method for bacterial community studies. The extracted DNA was good in quality, with a consistent size of ~20 kb and a yield of 48-87 g g-1 soil, and could be successfully used for 16S (Zhou method) and 18S (Holben method) rDNA amplifications. For less difficult environmental samples, UltraClean kits could be a good option, because they are simple and fast and the extracted DNA are also of good quality. Screening of the DNA PCR products using TGGE, Heteroduplex-TGGE and SSCP was also explored. These methods were not so effective for the screening of the soil DNA PCR products, owing to the difficulty in interpretation of the results. Cloning was a necessary step to obtain a single sequence at species level in soil microbial community studies. The screening of the clone library by TGGE, Heteroduplex-TGGE and SSCP could only separate the clones into several major bands, although SSCP gave better separation. Sequencing of selected clones directly from the clone library obtained ultimate results of microbial taxonomic composition and diversity through well-established sequence analysis software packages and the databases. It was recommended that, in this project with the target of microbial community composition and diversity, soil DNA PCR products were directly cloned to construct clone libraries and a sample of clones were sequenced to achieve an estimate of the taxonomic composition of the soil. Fungal communities of the Yarraman soil samples under the natural forest (YNF) and the hoop pine plantations (YHP) were investigated using 18S rDNA based cloning and sequencing approaches. Twenty-eight clone sequences were obtained and analysed. Three fungal orders, i.e., Zygomycota, Ascomycota and Basidiomycota were detected from the YNF and YHP samples. By contrast, culture-based analyses of fungi in the literature were mostly Ascomycetes. YNF appeared to have more Ascomycota but less Zygomycota than YHP, and within the Zygomycota order, YHP had more unidentified species than YNF. Bacterial communities of Yarraman soil samples of YNF, Y1R and Y2R were investigated using 16S rDNA-based cloning and sequencing approaches. 305 16S rDNA clone sequences were analysed and showed an overall bacterial community composition of Unclassified bacteria (34.4%), Proteobacteria (22.0%), Verrucomicrobia (15.7%), Acidobacteria (10.2%), Chloroflexi (6.9%), Gemmatimonadetes (5.6%), and Actinobacteria (5.2%). There was a significant difference among YNF, Y1R and Y2R in the taxonomic group composition. YNF had a greater proportion of Acidobacteria (18.0%), Verrucomicrobia (23.0%) and Chloroflexi (9.0%) than Y1R and Y2R (corresponding to 6.3%, 12.1% and 5.9%, respectively), while Y1R and Y2R had a higher percentage of the Unclassified group (38.5% for Y1R and 46.5% for Y2R) than YNF (18.0%). For the Proteobacteria group, YNF had more Alpha-subdivision but Y1R and Y2R had more Delta-subdivision. From YNF to Y1R to Y2R, the clone sequence variable site ratios, 5% and 10% OTU numbers and Shannon's diversity index H' values tended to decrease, indicating the soil bacterial diversity decreased from the natural forest to the first and the second rotation hoop pine plantations. The large amount of unclassified clone sequences could imply a novel group of bacteria in the soil, particularly in the hoop pine soil samples. Alternatively they may result from artefacts during the PCR process. Bacterial communities of the Gympie soil under different residue management practices, i.e., residue (litter plus logging residue) removed (G0R), residue retained (G1R), and residue doubled (G2R), were also investigated using the 16S rDNA-based cloning and sequencing approaches. Acidobacteria (37.6%) and Proteobacteria (35.6%, including Alpha-subdivision of 29.9% and Gamma-subdivision of 5.7%) were dominant components of the communities, followed by Actinobacteria (14.7%), Verrucomicrobia (7.3%) and Unclassified bacteria. There was no significant difference among G0R, G1R and G2R in the bacterial community compositions and diversity. These findings provided an in-depth vision of the soil microbial communities under different forest management practices. Their combination with other soil analysis results, such as physical and chemical properties, and forest production data, could provide an improved understanding of sustainable forest management strategies.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Australian School of Environmental Studies
Full Text

La décomposition des résidus de culture est un processus essentiel en sols de grandes cultures car elle représente la principale source carbone (C) exogène de ces systèmes. Bien que la dégradation des végétaux de diverses quantités et qualités soit bien connue, ses conséquences sur les communautés du sol et sur les fonctions qui en découlent sont moins maîtrisées. Pour mieux comprendre ces effets, nous avons suivi à des étapes clés de la décomposition l’évolution de composantes majeures de la diversité du sol (micro-organismes, nématodes, microarthropodes et macrofaune) en lien avec les principaux flux de C, d’azote (N) et les activités enzymatiques associées pour différentes quantités et qualités de litières. Les expériences ont été menées à différentes échelles spatiales et temporelles (champ, mésocosme et microcosme).La quantité et qualité de litières ont des effets distincts sur les communautés du sol. La quantité de litière impacte les détritivores, et la qualité de litière affecte plus généralement les principaux groupes fonctionnels. La litière labile affecte plus la voie bactérienne et les macro-détritivores en début de décomposition, et la litière récalcitrante la voie fongique et les méso-détritivores en fin de décomposition. Ces évolutions sont reflétées par les dynamiques enzymatiques. Plus spécifiquement, la régulation top-down de la faune (manipulations de L.terrestris et de nématodes) a montré de forts impacts sur les enzymes. De manière similaire, la composition initiale du micro-réseau trophique du sol a fortement conditionné les dynamiques enzymatiques. Au contraire, la minéralisation du C de la litière demeure principalement dictée par sa qualité initiale. Cette thèse démontre l'importance de la gestion des litières sur les fonctions biologiques des sols agricoles. Faire varier la qualité et quantité des litières apparaît comme un levier pour moduler la santé des sols et certains services écosystémiques à long terme
Crop residues decomposition is an essential process in cultivated lands since it constitutes the main source of organic matter in these systems. Although the underlying mechanisms of residues degradation of varying qualities and quantities are well-known, their consequences on soil biota and the related functions are less understood. To better understand this process, we chose to follow at key steps of decomposition the evolution of major components of soil diversity (microorganisms, nematodes, micro-arthropods and macrofauna), the evolution of litter quality, the main carbon (C) and nitrogen (N) fluxes and enzyme activities associated, for different quantities and qualities of litter added. The experiments were set up at different spatial and temporal scales (field, mesocosm and microcosm experiments) in function of our objectives.Litter quantity and quality have distinct effects on soil biota. Litter quantity influences the detritivores, and litter quality influences more generally the main functional groups. Labile litter impacts more the bacterial energy channel and the macrodetritivores at the first stages of decomposition, and recalcitrant litter impacts more the fungal energy channel and the mesodetritivores at the latter stage of decomposition. These evolutions are mirrored by the enzymatic dynamics. More specifically, fauna top-down regulations (L.terrestris and nematodes manipulations) exhibit high effects on soil enzymes. Similarly, soil micro-food web initial composition drives enzymatic dynamics. Contrastingly, litter C mineralization depends mainly on its initial quality. This PhD shows the importance of litter management on the biological functions of cultivated soils. Varying litter quality and quantity appear as a mean to influence soil health and some ecosystem services on the long term

The reduction of pathogenic microorganisms is essential to minimize human health risks associated with the reuse, reclamation, and recycling of wastewater and the land application of biosolids (sewage sludge). The most advanced treatment technologies, as well as, the most representative methods and indicator organisms are necessary to ensure public safety. The goal of this dissertation is to assess advanced Bardenpho wastewater treatment technologies in regards to virus removal, suggest the most appropriate viral indicators of human fecal contamination and/or treatment process controls, and develop an updated method for enumerating Ascaris ova viability in land applied biosolids. Appendix A evaluates the incidence of 11 different virus types in sewage throughout a 12-month time period, and their subsequent reduction via advanced Bardenpho treatment processes. This study showed that wastewater treatment facilities utilizing advanced Bardenpho for secondary treatment are more effective at reducing viruses in wastewater than facilities utilizing conventional aeration basin and trickling filter processes. Appendix B develops a new method for determining the viability of Ascaris ova in land applied biosolids. In this method, early development stages prior to larval-development, are included in the estimation of potential viability. Comparisons between viability enumerations suggests that the conventional microscopy method, in which only ova containing motile larva are considered viable, underestimates the number of eggs that may progress to an infectious stage. Whereas, the method based on early-to-late stage development, considers the potential viability of all eggs, providing a more conservative approach.

The interplay between geology and biology has shaped the Earth during billions of years. Microbe-mineral interactions are prime examples of this interplay and underscore the importance of microorganisms in making Earth a suitable environment for all forms of life. The present thesis takes an interdisciplinary approach to obtain an integrated understanding of microbe-mineral interactions. More specifically it addresses how the composition and distribution of biogenic weathering agents (siderophores) differ with regard to soil horizon and mineral type in situ, what siderophore type soil microorganisms produces under laboratory conditions, what role microbial surface attachment plays in mineral weathering reactions and what central roles and applications siderophores have in the environment. Podzol, the third most abundant soil in Europe, and most abundant in Scandinavia, was chosen for a field experiment, where three minerals (apatite, biotite and oligoclase) were inserted in the organic, eluvial and upper illuvial soil horizons. The study started with an investigation of the siderophore composition in the bulk soil profile and on the mineral surfaces (paper I), which was followed by a study of the siderophore producing capabilities of microorganisms isolated from the soil profile under laboratory conditions (paper II). Subsequently, a study was done on the impact of microbial surface attachment on biotite dissolution (paper III). Finally, the roles of siderophores in nature and their potential applications were reviewed (paper IV). The major findings were that the concentration of hydroxamate siderophores in the soil attached to the mineral surfaces was greater than those in the surrounding bulk soil, indicating that the minerals stimulate the microbial communities attached to their surfaces to produce more siderophores than the microorganisms in the bulk soil. Each mineral had a unique assemblage of hydroxamate siderophores, that makes the mineral type one of the main factors affecting siderophore composition in the natural environment. Siderophore production varied between the microbial species originating from different soil horizons, suggesting that the metabolic properties of microbes in deep soil horizons function differently from those at upper soil horizons. Microbial surface attachment enhanced the biotite dissolution, showing that attached microbes has a greater influence on weathering reactions in soil than planktonic populations. In conclusion, our findings reflected that the complicated relationship between microorganisms and mineral surfaces reinforces the central theme of biogeochemistry that the mineral controls the biological activity in the natural environments. However, the importance of these relationships to the biogeochemical systems requires further investigation.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: In press.

 

The goal of my dissertation research was to investigate the structure and function of ammonia-oxidizing microbial communities in temperate forest soils. Accomplishing this goal required a hybrid approach: I used modern molecular biology techniques alongside soil biogeochemical measurements and framed my research using ecological theory largely developed in plant systems. All of my field work was done at Coweeta Hydrologic Laboratory, a Forest Service Station and Long Term Ecological Research Site near Otto, NC. Watershed-level land use manipulations have been performed at Coweeta since the 1930s, including clear-cutting, fertilizing, liming, burning, grazing by cattle, and replanting entire watersheds in white pine. While these treatments were originally imposed to assess the effects of land use on water yield, they have resulted in changes in soil characteristics as well. Working at Coweeta has therefore allowed me to sample ammonia-oxidizer communities across a gradient of soil variables, such as pH and nitrogen (N) availability, within the geographically-constrained area of the Coweeta Basin. First, I used amplicon-based pyrosequencing to independently assess the diversity of ammonia-oxidizing archaea (AOA) and bacteria (AOB) at several sites within Coweeta. I found that AOA and AOB diversity were a function of both resource availability (i.e. N availability) and environmental harshness (i.e. soil pH) in line with general ecological theory developed for plant systems by Tilman and Grime, respectively. Next, I tested whether AOA and AOB were substrate or nutrient limited in this system by adding either N or a nutrient solution containing both potassium and phosphorus to soil incubations and assessing the growth response of AOA and AOB using quantitative polymerase chain reaction (qPCR). I found strong evidence for substrate limitation by AOB and a marginally-significant positive effect of nutrient addition on growth of AOA. Another intriguing finding from this study was that both AOA and AOB grew during unamended soil incubations. Unamended (buried-bag) incubations have been used to estimate in situ rates of nitrification for over 50 years. By measuring the growth of AOA and AOB alongside nitrification during buried-bag incubations, I discovered that AOA are the dominant ammonia-oxidizers in temperate forest soils. However, I found that AOA are much less efficient at using the energy from ammonia oxidation to create biomass than AOB in the forest soils I sampled. Overall, I found that temperate forest soils contain low abundances of AOA and AOB, with relatively low diversity in both groups. This is especially true for the diversity of AOA, where a single taxon dominated the community at every site. Soil pH and N availability seem to be major selective forces for forest soil ammonia oxidizers, though other nutrients such as potassium and phosphorus may regulate the activity of AOA as well. AOA are most-likely the dominant ammonia oxidizers in temperate forest systems, though this may change with increased disturbance. In a broader sense, I found that ecological theory developed for plant communities was applicable to chemoautotrophic microbes despite the large differences in life history between these groups of organisms.
Ph. D.