The soil is a complex system composed by live organisms, organic matter, gases, water and minerals parts that interact. In this sense, the soil organisms, besides inhabitants, are part of its composition. In the soil, the agricultural activity normally begin in a natural area, with several species of plants and animals living in equilibrium, may pass to a reduction of biodiversity, in function of agricultural practices. The present study was carried out in phases, aiming to evaluate and characterize the faunal composition, the population of nitrifying bacteria, and to develop a protocol to extract earthworms DNA. The faunal composition and nitrifying bacteria, population was done in an experiment with five years of no-till with the following treatments: a) witness (without lime); b) amount indicated by SMP method to pH 6.0 (100%), incorporated to soil in 20 cm depth; c) amount indicated by SMP method to pH 6.0 (100%), distributed in soil surface; d) half of the amount indicated by SMP method to pH 6.0 (50%), distributed in soil surface; and e) one quart of the amount indicated by SMP method to pH 6.0 (25%), distributed in soil surface. To evaluate the population of soil meso and macrofauna were collected samples in July (winter) and December (summer) of 2001. For epiedafic meso and macro organisms were installed traps for capture, and for euedafic macro organisms, were collected soil monoliths. The samples collected individually were placed in plastic bags, packed in thermo boxes and taken to Soil Biology Laboratory of UFSM Soils Department. Later, were performed separation, counting and classification of taxonomic groups in level of classes and order of the organisms? Were utilized the Shanon Diversity Index (H) to evaluate the population among the treatments and theTukey test (P=0.05) to compare the means of population in the different doses and mode of lime application. The doses and mode of lime application in no-till after 5 years were not a significant factor to affect the abundance and richness of soil fauna. The most representative population of organisms was Collembola. The quantitative determination of bacteria nitrifying population was performed with seeding of dispersed soil over a layer of silica gel and later addition of a mix composed of lime, alkaline silicates and an acid mixture, that became neutral forming a second layer of silica gel that contains the nutrients specific to the growth of the bacteria group studied. The experiment was performed in triplicates with 25 mg of soil samples per plaque. After 10 days of incubation in 28-30oC were done observations and counting of the colonies developed in each plaque, presuming that each bacteria formed one colony. The counting was done every 5 days for a period of 60 days. The application of 100% lime, incorporated or on soil surface, and 50% of lime on soil surface did not were significantly different in the number of colonies of nitrifying bacteria. The pH under 4.3 in the treatments witness and 25% of lime on soil surface affected negatively the bacteria population in depths 0-5 and 5-10 cm. To develop a protocol to extract DNA of earthworms, earthworms were collected in the experiment of doses and mode of lime application in the no-till system, in the experimental area of UFSM Soils Department. After visual characterization of identical groups, 10 individuals of each specie of Eisenia foetida and Pheretima spp were placed in alcohol 70% for later characterization after morphological patterns of each specie. Other 10 individuals were open and taken all materials from its interior. Before DNA extraction, the material stayed in sterilized water for 24 hours in refrigerator to eliminate any substances that may be stayed glued to the material. Later, the material was macerated and the DNA extracted. The DNA extracted was visualized in agarose gel 1.2%. The protocol developed was efficient to extract the DNA of Eisenia foetida and Pheretima spp. / O solo é um sistema complexo composto de seres vivos, ar, água, matéria orgânica e minerais que interagem. Neste sentido os organismos do solo, além de habitantes, são parte integrante de sua composição. No solo, a atividade agrícola que geralmente inicia como área natural, com muitas espécies de plantas e animais convivendo em equilíbrio, pode passar a uma redução da biodiversidade, decorrentes das práticas culturais. O presente estudo foi realizado em três etapas visando avaliar e caracterizar a composição faunística, a população de bactérias nitrificadoras e desenvolver um protocolo de extração de DNA para oligoquetas. A avaliação faunística e da população de bactérias nitrificadoras foi realizada em um experimento com cinco anos de plantio direto com os seguintes tratamentos: A) testemunha (sem calcário); B) quantidade recomendada pelo método SMP para pH 6,0 (100%), incorporada ao solo na profundidade de 20 cm; C) quantidade recomendada pelo método SMP para pH 6,0 (100%), distribuída na superfície do solo; D) metade da quantidade recomendada pelo método SMP para pH 6,0 (50%), distribuída na superfície do solo; E) um quarto da quantidade recomendada pelo método SMP para pH 6,0 (25%), distribuída na superfície do solo. Para avaliação da população da meso e macrofauna, foram coletadas amostras em julho (inverno) e dezembro (verão) de 2001. Para meso e macro organismos epiedáficos foram instaladas armadilhas de captura, e para macrorganismos euedáficos foram coletados monólitos de solos. As amostras coletadas foram, acondicionadas em caixas de isopor e levadas ao Laboratório de Biologia do Solo do Departamento de Solos da UFSM. Posteriormente, foi realizada a separação, contagem e classificação de grupos taxonômicos em nível de classe e ordem. Utilizou-se o índice de diversidade de Shannon (H) para avaliação das populações e para comparação, as médias nos diferentes tratamentos entre as doses e modos e aplicação de calcário através do teste Tukey a 5%. As doses e modo de aplicação de calcário em sistema de plantio direto após 5 anos foram fatores menos determinantes do que as condições climáticas (inverno-verão) na abundância e riqueza da fauna edáfica. A população de organismo mais representativa foi de colembola. A determinação quantitativa de população de bactérias nitrificadoras foi através da semeadura do solo dispersado sobre uma camada de sílica gel e posterior adição de uma mistura composta por calcário, silicatos alcalinos e uma mistura ácida, que contem os nutrientes específicos para o desenvolvimento do grupo bacteriano em estudo. O experimento foi realizado em plaqueamento triplicado com 25 mg de amostra de solo por placa. Após 10 dias de incubação a 28-30o C foram realizadas observações e contagens das colônias desenvolvidas nas placas, realizadas a cada 5 dias por um período de 60 dias. As aplicações de calcário 100% na superfície, 100% incorporada e 50% na superfície não diferiram significativamente no número de colônias de bactérias nitrificadoras. O pH inferior a 4,3 nos tratamentos 0% e 25% de calcário na superfície diminuiu a população de bactérias nitrificadoras. As oligoquetas, para o trabalho de desenvolvimento de um protocolo de extração de DNA, foram coletadas no experimento citado acima. Após caracterização visual de grupos idênticos, 10 indivíduos de cada espécie de Eisenia foetida e Pheretima sp. foram caracterizadas segundo padrões morfológicos. Outros 10 indivíduos foram abertos e retirado todo o material existente no seu interior. Posteriormente o material foi submetido à maceração e extração do DNA. O DNA extraído foi visualizado em gel de agarose 1,2%. O protocolo desenvolvido foi eficiente para extração de DNA em Eisenia foetida e Pheretima sp.

Sorghum plants excrete phenolic acids which reduce subsequent crop yields. These acids accumulate in field soil by combining with soil and clay particles to form stable complexes which remain until degraded by bacterial metabolism. The amount of phenolic acids in soil samples were obtained by gas chromatography measurements, while Azotobacter populations were obtained by plate counts in 40 sorghum field samples from Denton County, Texas. One can conclude that increasing the Azotobacter population in the soil increased the degradation rate of phenolic acids proportionally. It is proposed that seed inoculation will introduce selected strains of Azotobacter into the soil. The presence of Azotobacter should increase crop size in subsequent plantings.

Thesis: Ph. D., Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution), 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references. / Nitrogen (N) species in hydrothermal vent fluids serve as both a nutrient and energy source for the chemosynthetic ecosystems surrounding deep-sea vents. While numerous pathways have been identified in which N-species can be produced and consumed in the context of submarine hydrothermal vent systems, their exact nature has been largely limited to interpretation of variations in concentrations. This thesis applies stable isotope approaches to further constrain the sources and fate of N-species in deep-sea vents across a variety of geological settings. First, I discuss isotope fractionation and reaction kinetics during abiotic reduction of nitrate (NO₃⁻) to ammonium ([sigma]NH₄⁺ = NH₃+NH₄⁺) under hydrothermal conditions. Results of lab experiments conducted at high temperatures and pressures revealed a wide degree of N isotope fractionation as affected by temperature, fluid/rock ratio, and pH-all which exert control over reaction rates. / Moreover, a clear pattern in terms of reaction products can be discerned with the reaction producing [sigma]NH₄⁺ only at high pH, but both [sigma]NH₄⁺ and N₂ at low pH. This challenges previous assumptions that NO₃⁻ is always quantitatively converted to NH₄⁺ during submarine hydrothermal circulation. Next, I report measurements of [sigma]NH₄⁺ concentrations and N isotopic composition ([delta]¹⁵N[subscript NH4]) from vent fluid samples, together with the largest compilation to date of these measurements made from other studies of deep-sea vent systems for comparison. The importance of different processes at sediment-influenced and unsedimented systems are discussed with a focus on how they ultimately yield observed vent [sigma]NH₄⁺ values. / Notable findings include the role that phase separation might play under some conditions and a description of how an unsedimented site from Mid-Cayman Rise with unexpectedly high NH4+ may be uniquely influenced by N₂ reduction to [sigma]NH₄⁺. Lastly, I explore [sigma]NH₄⁺ dynamics in the context of low-temperature vent sites at 9°50'N East Pacific Rise to investigate dynamics of microbially-mediated N transformations. Through both measurements of natural samples, as well as isotopic characterization of N species from incubation experiments and model simulations thereof, an exceptionally high variability observed in [delta]¹⁵N[subscript NH4] values emphasizes the complexity of these microbe-rich systems. / In sum, this thesis highlights the role of microbial processes in low temperature systems, demonstrates a more mechanistic understanding of lesser-understood abiotic N reactions and improves the coverage of available data on deep-sea vent [sigma]NH₄⁺ measurements. / by Chawalit "Net" Charoenpong. / Ph. D. / Ph.D. Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution)

In recent years considerable interest has been shown in the diversity of ammonia-oxidizing bacteria in soil communities. The majority of the research has been carried out in Northern Europe where soils have received high atmospheric inputs of nitrogen over the past two centuries. In contrast, although much work has been conducted on nitrogen cycling processes in nitrogen limited forest ecosystems in western North America, no studies have examined the characteristics of ammonia-oxidizing communities in those environments. I was interested in measuring nitrification potential along a high-elevation temperate meadow-to-forest gradient, and characterizing the ammonia-oxidizing communities along that gradient using both molecular and culturing methods. Two experimental sites (Lookout and Carpenter) were chosen in the H.J. Andrews Experimental Forest, located in the western Cascade Range of Oregon, at elevations of approximately 1500 meters. Although nitrification potential rates (NPRs) between sites were not significantly different (P=0.544), variation was observed both within and between sites for specific vegetation types. NPRs were significantly lower in forest (F) soil samples than in meadow (M) soil samples, averaging 5 and 2% of meadow NPRs at Lookout and Carpenter, respectively. In meadow soil samples, most probable number (MPN) population densities of ammonia-oxidizers ranged from 0.6 to 2.6 x 10⁴ cells gram⁻¹ of oven dry soil and 0.9 x 10³ to 1.1 x 10⁵ cells g⁻¹ OD soil at Lookout and Carpenter, respectively. In forest soil samples, population densities ranged from undetectable to 1.1 x 10⁴ cells g⁻¹ OD soil, and 0.9 x 10² to 2.3 x 10³ cells g⁻¹ OD soil at Lookout and Carpenter, respectively. Microbial community DNA was amplified using primers to the ammonia monooxygenase subunit A. Terminal restriction fragments polymorphism analysis with three different restriction enzymes (CfoI, TaqI, and AluI) revealed community profiles dominated by Nitrosospira species. One fragment from CfoI (66 bp) and one fragment from AluI (392-bp) were prominent in 47 soil samples from both sites, and represented between 32 to 100% of the Genescan fragment analyses of PCR products. A full length fragment from AluI digests (491-bp), and three fragments from CfoI (68, 100, and 135- bp) were found sporadically in fewer soil sample T-RFLPs, and within those samples represented smaller percentages of total peak areas. The CfoI 135-bp fragment length was associated primarily with M and meadow/forest (M/F) soils where it was observed in approximately 58 and 100% of the respective transect locations. Eight isolates recovered from soil samples were analyzed using the same molecular methods as the field samples. The T-RFLP patterns of the isolates corresponded with many of those found in the community fingerprints. Four unique amoA sequences were identified among these isolates, including one that possessed the dominant T-RFLP amoA fingerprint in soil samples. This sequence shared 99.8% similarity with Nitrosospira sp. Ka4, a cluster 4 ammonia oxidizer isolated in Norway. Sequence analysis phylogenetically associated the other three isolates (with unique amoA sequences) near Nitrosospira sp. Nsp 1 and Nitrosospira briensis, both cluster 3 ammonia oxidizers. Cloning and sequencing of soil DNA confirmed that ammonia oxidizers with these amoA sequences were present in the soil samples. Two additional amoA sequences were identified in clones that were 95% similar and paraphylogenetically positioned between representatives of clusters 3 and 4. So far, these sequences have not been found in any of the isolates analyzed. / Graduation date: 2003

Submitted in complete fulfillment for the degree of Doctor of Philosophy (Biotechnology), Durban University of Technology, Durban, South Africa, 2016. / Seasonal nitrification breakdown is a major problem in wastewater treatment plants which makes it difficult for the plant operators to meet discharge limits. The present study focused on understanding the seasonal impact of environmental and operational parameters on nitrifiers and nitrification, in a biological nutrient removal wastewater treatment works situated in the midlands of KwaZulu Natal. Composite sludge samples (from the aeration tank), influent and effluent water samples were collected twice a month for 237 days. A combination of fluorescent in-situ hybridization, polymerase chain reaction (PCR)-clone library, quantitative polymerase chain reaction (qPCR) were employed for characterizing and quantifying the dominant nitrifiers in the plant. In order to have more insight into the activated sludge community structure, pyrosequencing was used in profiling the amoA locus of ammonia oxidizing bacteria (AOB) community whilst Illumina sequencing was used in characterising the plant’s total bacterial community. The nonlinear effect of operating parameters and environmental conditions on nitrification was also investigated using an adaptive neuro-fuzzy inference system (ANFIS), Pearson’s correlation coefficient and quadratic models. The plant operated with higher MLSS of 6157±783 mg/L during the first phase (winter) whilst it was 4728±1282 mg/L in summer. The temperature recorded in the aeration tanks ranged from 14.2oC to 25.1oC during the period. The average ammonia removal during winter was 60.0±18% whereas it was 83±13% during summer and this was found to correlate with temperature (r = 0.7671; P = 0.0008). A significant correlation was also found between the AOB (amoA gene) copy numbers and temperature in the reactors (α= 0.05; P=0.05), with the lowest AOB abundance recorded during winter. Sanger sequencing analysis indicated that the dominant nitrifiers were Nitrosomonas spp. Nitrobacter spp. and Nitrospira spp. Pyrosequencing revealed significant differences in the AOB population which was 6 times higher during summer compared to winter. The AOB sequences related to uncultured bacterium and uncultured AOB also showed an increase of 133% and 360% respectively when the season changed from winter to summer. This study suggests that vast population of novel, ecologically significant AOB species, which remain unexploited, still inhabit the complex activated sludge communities. Based on ANFIS model, AOB increased during summer season, when temperature was 1.4-fold higher than winter (r 0.517, p 0.048), and HRT decreased by 31% as a result of rainfall (r - 0.741, p 0.002). Food: microorganism ratio (F/M) and HRT formed the optimal combination of two inputs affecting the plant’s specific nitrification (qN), and their quadratic equation showed r2-value of 0.50. This study has significantly contributed towards understanding the complex relationship between the microbial population dynamics, wastewater composition and nitrification performance in a full-scale treatment plant situated in the subtropical region. This is the first study applying ANFIS technique to describe the nitrification performance at a full-scale WWTP, subjected to dynamic operational parameters. The study also demonstrated the successful application of ANFIS for determining and ranking the impact of various operating parameters on plant’s nitrification performance, which could not be achieved by the conventional spearman correlation due to the non-linearity of the interactions during wastewater treatment. Moreover, this study also represents the first-time amoA gene targeted pyrosequencing of AOB in a full-scale activated sludge is being done. / D

Symbioses between microbes and multicellular eukaryotes are found in all biomes, and encompass a spectrum of symbiotic lifestyles that includes parasitism and disease, commensalism, and mutually beneficial interdependent host-microbe relationships. Regardless of outcome, these symbiotic lifestyles are governed by a complex molecular "courtship" between microbe and potential host. This courtship is the primary determinant of the host range of a given microsymbiont. Host immunity poses a formidable barrier to the establishment of host-microbe relationships, and the majority of microbial suitors will be thwarted by it. Only by successfully "wooing" the host cell's immune defenses with the appropriate molecular signals can a microsymbiont successfully colonize its host. A strategy common to microsymbionts across the spectrum of symbiotic lifestyles and host organisms is the delivery of microbial-encoded effector proteins into the cytoplasm of host cells to manipulate the host cell's molecular machinery for the purposes of subverting host immunity. Bacteria, in particular, have adapted a number of secretion systems for this purpose. The most well-characterized of these is the type III secretion system (T3SS), a molecular apparatus that specializes in injecting type III effector (T3Es) proteins directly into host cells. The work in this thesis focuses on T3Es of plant-associated bacteria, with particular emphasis on mutualistic bacteria. We present evidence that collections of T3Es from Sinorhizobium fredii and Bradyrhizobium japonicum are, in stark contrast to those of phytopathogenic bacteria, in a co-evolutionary equilibrium with their hosts. This equilibrium is characterized by highly conserved T3E collections consisting of many "core" T3Es with little variation in nucleotide sequence. The T3Es of Mesorhizobium loti MAFF303099 suggest a completely different picture of the evolution of T3Es. MAFF303099 recently acquired its T3SS locus, and the work in this thesis provides an evolutionary snapshot of a mutualist that is innovating a T3E collection primarily through horizontal gene transfer. Collectively, this work represents the first comprehensive catalog of T3Es of rhizobia and, in the case of Sinorhizobium and Bradyrhizobium, the first evidence of purifying selection for T3Es. / Graduation date: 2012