A Mata Atlântica é reconhecida como área de prioridade de conservação na América do Sul, devido ao grande número de espécies endêmicas e constantes ameças à sua biodiversidade em decorrência da substituição da vegetação natural. Embora várias informações sobre a diversidade vegetal e animal estejam disponíveis, pouco se sabe sobre a diversidade de microorganismos existentes no solo desse bioma. A diversidade de fungos do solo foi avaliada em três unidades de conservação da Mata Atlântica do estado de São Paulo: Parque Estadual de Carlos Botelho (PECB), Estação Ecológica de Assis (EEA) e Parque Estadual da Ilha do Cardoso (PEIC). Ao todo, foram analisadas 90 amostras de solo, coletadas em épocas de alta e baixa pluviosidade, e sob a copa de três espécies arbóreas: Cabralea canjerana, Guapira opposita e Maytenus robusta. Foram utilizados métodos independentes (PCR-DGGE e pirosequenciamento) e um método dependente do cultivo (lavagem de solo e filtração de partículas) para a análise da diversidade e estrutura das comunidades de fungos do solo. Os resultados obtidos foram analisados conjuntamente com os dados de atributos químicos do solo e frações da matéria orgânica do solo a fim de verificar suas possíveis relações com as comunidades de fungos. Os resultados obtidos sugerem uma grande diversidade de fungos no solo da Mata Atlântica. Através do método de cultivo, um total de 142 espécies de fungos foi identificado nas três áreas, sendo que a estrutura das comunidades de fungos não foi influenciada pelas espécies arbóreas, mas sim pelas áreas e épocas de amostragem. As comunidades de fungos cultiváveis do PECB e do PEIC foram mais similares entre si do que em relação às comunidades de fungos do solo da EEA, assim como os valores dos atributos químicos do solo dessas áreas foram mais semelhantes entre si. Pelo método de PCR-DGGE, as estruturas das comunidades de fungos das três áreas sofreram influência das espécies arbóreas sob a copas das quais as amostras foram coletadas. Somente a estrutura das comunidades de fungos no solo do PEIC não sofreu influência da época de amostragem. Usando pirosequenciamento, foram obtidas 39.152 sequências da região ITS de fungos, as quais foram agrupadas em 1.800 Unidades Taxonômicas Operacionais (UTOs). A diversidade de fungos na EEA e no PEIC variou em função das espécies arbóreas e épocas de coleta. A análise de NMS de cada área amostrada indicou que as comunidades de fungos do solo são muito homogêneas. O filo Ascomycota foi mais frequentemente detectado, tanto usando metodologia dependente quanto independente de cultivo. De maneira geral, as comunidades de fungos cultiváveis apresentaram maior relação com atributos químicos do solo, áreas e épocas de coleta, e as comunidades de fungos acessadas por PCRDGGE mostraram maior relação com as épocas de coleta e espécies arbóreas sob as quais as amostras de solo foram coletadas. A análise dos metadados usando rede neural revelou uma dependência da diversidade de fungos em relação às concentrações de ácidos húmicos, ácidos fúlvicos e humina no solo, além do pH e concentração de matéria orgânica total. / Brazilian Atlantic Forest is recognized as a top priority for conservation in South America because of its large number of innate species and the threats for the biodiversity due to vegetation changes. Although a plethora of data on plant and animal diversity in the Atlantic Forest is available, the diversity of soil microorganisms in this biome is still mostly unknown. Therefore, soil fungi diversity was evaluated in three Atlantic Forest conservation areas of São Paulo state: Estação Ecológica de Assis (EEA), Parque Estadual de Carlos Botelho (PECB) and Parque Estadual da Ilha do Cardoso (PEIC). A total of 90 soil samples were analyzed, collected in two different seasons (high and low precipitation seasons), and under the canopy projection of three tree species: Cabralea canjerana, Guapira opposita and Maytenus robusta. Two independent cultivation methods (PCR-DGGE and pyrosequencing) and one dependent method (soil washing and particles filtration method) were used for the analysis of soil fungi diversity and community structure. The results of these methods were analyzed together with the data on soil chemical attributes and organic matter fractions in the same samples, in order to verify the possible relations with soil fungi communities. The results suggest a great diversity of soil fungi in the Atlantic Forest. Through the cultivation method, a total of 142 fungi species were identified for the three areas. The structure of fungi communities was not affected by the tree species, but were affected by the sampling areas and seasons. Cultivable fungi community in PECB and PEIC were more similar to each other than in EEAs soil fungi community. In addition, the values of the chemical properties of these areas were more similar to each other. The structure of soil fungi community accessed by PCR-DGGE showed that the three areas were influenced by the tree species under the canopy where the soil samples were collected. Only the structure of PEICs fungi communities was not influenced by the season. By means of pyrosequencing, 39,152 sequences were retained from the ITS rDNA region, which were clustered in 1,800 Operational Taxonomic Unities (OTUs). Fungal diversity in EEA and PECB areas was influenced by the tree species and seasons. NMS analysis of each sampled area showed that soil fungi communities are very homogenous. Ascomycota was the most frequent phylum detected by both dependent and independent cultivation methods. Overall, the communities of cultivable fungi were more related to soil chemical attributes, sampling areas and seasons. Fungal communities accessed by PCR-DGGE showed greater relation with the seasons and tree species where the soil samples were collected. Analysis of metadata using neural network revealed fungal diversity dependence of humic acids, fulvic acids and humin concentrations in soil, as well as pH and organic matter concentrations.

A Mata Atlântica é reconhecida como área de prioridade de conservação na América do Sul, devido ao grande número de espécies endêmicas e constantes ameças à sua biodiversidade em decorrência da substituição da vegetação natural. Embora várias informações sobre a diversidade vegetal e animal estejam disponíveis, pouco se sabe sobre a diversidade de microorganismos existentes no solo desse bioma. A diversidade de fungos do solo foi avaliada em três unidades de conservação da Mata Atlântica do estado de São Paulo: Parque Estadual de Carlos Botelho (PECB), Estação Ecológica de Assis (EEA) e Parque Estadual da Ilha do Cardoso (PEIC). Ao todo, foram analisadas 90 amostras de solo, coletadas em épocas de alta e baixa pluviosidade, e sob a copa de três espécies arbóreas: Cabralea canjerana, Guapira opposita e Maytenus robusta. Foram utilizados métodos independentes (PCR-DGGE e pirosequenciamento) e um método dependente do cultivo (lavagem de solo e filtração de partículas) para a análise da diversidade e estrutura das comunidades de fungos do solo. Os resultados obtidos foram analisados conjuntamente com os dados de atributos químicos do solo e frações da matéria orgânica do solo a fim de verificar suas possíveis relações com as comunidades de fungos. Os resultados obtidos sugerem uma grande diversidade de fungos no solo da Mata Atlântica. Através do método de cultivo, um total de 142 espécies de fungos foi identificado nas três áreas, sendo que a estrutura das comunidades de fungos não foi influenciada pelas espécies arbóreas, mas sim pelas áreas e épocas de amostragem. As comunidades de fungos cultiváveis do PECB e do PEIC foram mais similares entre si do que em relação às comunidades de fungos do solo da EEA, assim como os valores dos atributos químicos do solo dessas áreas foram mais semelhantes entre si. Pelo método de PCR-DGGE, as estruturas das comunidades de fungos das três áreas sofreram influência das espécies arbóreas sob a copas das quais as amostras foram coletadas. Somente a estrutura das comunidades de fungos no solo do PEIC não sofreu influência da época de amostragem. Usando pirosequenciamento, foram obtidas 39.152 sequências da região ITS de fungos, as quais foram agrupadas em 1.800 Unidades Taxonômicas Operacionais (UTOs). A diversidade de fungos na EEA e no PEIC variou em função das espécies arbóreas e épocas de coleta. A análise de NMS de cada área amostrada indicou que as comunidades de fungos do solo são muito homogêneas. O filo Ascomycota foi mais frequentemente detectado, tanto usando metodologia dependente quanto independente de cultivo. De maneira geral, as comunidades de fungos cultiváveis apresentaram maior relação com atributos químicos do solo, áreas e épocas de coleta, e as comunidades de fungos acessadas por PCRDGGE mostraram maior relação com as épocas de coleta e espécies arbóreas sob as quais as amostras de solo foram coletadas. A análise dos metadados usando rede neural revelou uma dependência da diversidade de fungos em relação às concentrações de ácidos húmicos, ácidos fúlvicos e humina no solo, além do pH e concentração de matéria orgânica total. / Brazilian Atlantic Forest is recognized as a top priority for conservation in South America because of its large number of innate species and the threats for the biodiversity due to vegetation changes. Although a plethora of data on plant and animal diversity in the Atlantic Forest is available, the diversity of soil microorganisms in this biome is still mostly unknown. Therefore, soil fungi diversity was evaluated in three Atlantic Forest conservation areas of São Paulo state: Estação Ecológica de Assis (EEA), Parque Estadual de Carlos Botelho (PECB) and Parque Estadual da Ilha do Cardoso (PEIC). A total of 90 soil samples were analyzed, collected in two different seasons (high and low precipitation seasons), and under the canopy projection of three tree species: Cabralea canjerana, Guapira opposita and Maytenus robusta. Two independent cultivation methods (PCR-DGGE and pyrosequencing) and one dependent method (soil washing and particles filtration method) were used for the analysis of soil fungi diversity and community structure. The results of these methods were analyzed together with the data on soil chemical attributes and organic matter fractions in the same samples, in order to verify the possible relations with soil fungi communities. The results suggest a great diversity of soil fungi in the Atlantic Forest. Through the cultivation method, a total of 142 fungi species were identified for the three areas. The structure of fungi communities was not affected by the tree species, but were affected by the sampling areas and seasons. Cultivable fungi community in PECB and PEIC were more similar to each other than in EEAs soil fungi community. In addition, the values of the chemical properties of these areas were more similar to each other. The structure of soil fungi community accessed by PCR-DGGE showed that the three areas were influenced by the tree species under the canopy where the soil samples were collected. Only the structure of PEICs fungi communities was not influenced by the season. By means of pyrosequencing, 39,152 sequences were retained from the ITS rDNA region, which were clustered in 1,800 Operational Taxonomic Unities (OTUs). Fungal diversity in EEA and PECB areas was influenced by the tree species and seasons. NMS analysis of each sampled area showed that soil fungi communities are very homogenous. Ascomycota was the most frequent phylum detected by both dependent and independent cultivation methods. Overall, the communities of cultivable fungi were more related to soil chemical attributes, sampling areas and seasons. Fungal communities accessed by PCR-DGGE showed greater relation with the seasons and tree species where the soil samples were collected. Analysis of metadata using neural network revealed fungal diversity dependence of humic acids, fulvic acids and humin concentrations in soil, as well as pH and organic matter concentrations.

Os microrganismos representam a forma de vida mais abundante e diversificada do planeta. A atividade agrícola leva a uma redução da biodiversidade do solo e a menor diversidade microbiana pode resultar na diminuição da ciclagem de nutrientes e no crescimento das plantas. Como forma de avaliar alterações na atividade microbiana e na estrutura das comunidades de bactérias do solo, decorrentes do cultivo da cana-de-açúcar, foram conduzidos dois experimentos. O primeiro, no município de Novo Horizonte (SP), com o objetivo de determinar ação da queima da cana-de-açúcar sobre a comunidade de bactérias do solo e o segundo experimento, nos municípios de Pirassununga (SP) e Jaboticabal (SP), com o objetivo de verificar o efeito da adubação nitrogenada sobre a comunidade bacteriana do solo. Amostras de terra foram coletadas nas profundidades 0-10 e 10-20 cm, na linha e entrelinha de plantio. No primeiro experimento foram utilizadas três cultivares de cana-de-açúcar (SP81-3250, SP80-1842 e RB72-454) sob os sistemas de manejo de colheita sem queima (mecanizada) e com queima (manual) prévia a colheita. Nesse experimento foram avaliadas a diversidade metabólica (Biolog) e a estrutura das comunidades bacterianas por meio da PCR-DGGE do gene rRNA 16S. A mudança no manejo de colheita da cana-de-açúcar provocou modificações no metabolismo heterotrófico do solo, alterando a diversidade metabólica. No entanto, não houve mudanças na estrutura das comunidades bacterianas do solo com e sem queima sob a variedade SP801842. Dessa forma, a primeira queima da cana-de-açúcar previamente à colheita alterou a capacidade e a diversidade metabólica microbiana, mas não mudou a estrutura das comunidades de bactérias em relação à área sem queima. No segundo experimento foram avaliadas amostras de terra, de duas áreas experimentais, sob cultivo de cana-de-açúcar (SP813250), com diferentes doses de N (0, 40, 80 e 120 kg de N ha-1) na forma de uréia, aplicadas no sulco de plantio. Para verificar possíveis alterações na comunidade bacteriana desses solos, foram avaliadas a estrutura das comunidades bacterianas por PCR-DGGE e a diversidade de bactérias oxidadoras de amônio (AOB), pelo seqüenciamento de bibliotecas do gene rRNA 16S, utilizando oligonucleotídeos iniciadores específicos. As doses de N alteraram a estrutura das comunidades bacterianas do solo nas duas áreas experimentais, determinadas por PCR-DGGE, entretanto, a adubação nitrogenada não alterou a diversidade de AOB no solo, das duas áreas. A estrutura da comunidade de AOB no solo da USA, sem adubação nitrogenada e com 80 kg de N ha-1 diferiu estatisticamente. Nas duas áreas, as unidades taxonômicas operacionais mais abundantes se relacionam filogeneticamente a Nitrosospira multiformes. / The microorganisms are the most abundant and diverse living creatures on earth. The agricultural practices reduce the soil biodiversity and a lower microbial diversity can result in a nutrient cycling and plant growth reduction. Two sugarcane crops experiments were investigated to evaluate modifications in the microbial activity and soil bacterial communities structure. The first of them was done at municipality of Novo Horizonte, Sao Paulo State (SP), and it has the aim to determine the sugarcane burn effects on soil bacterial community. The second experiment was introduced at municipalities of Pirassununga (SP) and Jaboticabal (SP) with the objective to verify the nitrogen fertilizing effect on soil bacterial community. The soil samples were taken in 0-10 cm and 10-20 cm depth, between and in the planting furrows. We used three sugarcane varieties (SP81-3250, SP80-1842 e RB72-454) in the first experiment under unburned and burned sugarcane pre-harvest. We evaluated metabolic diversity (Biolog) and the bacterial community structure performing PCR-DGGE of 16S rRNA gene in the first experiment. The sugarcane harvest management had modified the soil heterotrophic metabolism by altering its diversity. In spite of that, there is no difference between the burned and unburned soil bacterial communities under the variety SP80-1842. For that reason, the first year pre harvest burn altered the microbial metabolic diversity and capacity but did not change the bacterial community structure when related with unburned area. In the second experiment, soil samples under the SP81-3250 variety were analyzed from two sites. Each site received different levels of urea as nitrogen fertilization (0, 40, 80 e 120 kg de N ha-1) applied at planting furrows. The PCR-DGGE was applied to verify changes in the bacterial communities structure in these soils. The ammonia-oxidizing bacteria (AOB) diversity was evaluated by sequencing of 16S rRNA gene libraries after the amplification with specific primers. The levels of nitrogen fertilization altered the soil bacterial communities structure in both study sites, by PCRDGGE evaluation. However, the nitrogen application did not alter the soil AOB diversity in those two sites. The soil AOB community structure under no nitrogen and 80 kg N ha-1 application was different from the community structure under the other levels of fertilization in one of the two sites. The operational taxonomic unit are phylogenetically related to Nitrosospira multiformes in the two sites.

<p>Heterotrophic microorganisms are important for the flow of carbon and nutrients in the sea. Bacteria, nanoflagellates and ciliates are relevant components of the pelagic food web. In order to be able to predict the outcome of e.g. eutrophication or climate change we need to know how the different components of the pelagic food web are regulated. With the focus on the northern Baltic Sea food web, this thesis deals with limitation and control of heterotrophic protists, the effect of resource heterogeneity on food web efficiency and diversity of nanoflagellates.</p><p>In-situ microcosm experiments showed that the net growth of heterotrophic flagellates were resource limited throughout the year. Field data confirmed that the abundance of flagellates was bottom-up controlled. Furthermore, field data also showed that the annual average biomass of protists, flagellates and ciliates increased with primary productivity. On a smaller seasonal scale temperature and bacterial biomass were able to explain most of the variation in flagellate biovolume. The temporal variation in ciliate biovolume could not be explained by any bottom-up factors like bacterial biomass, flagellate biomass or chlorophyll a. This and an in-situ microcosm experiment implied that the seasonal dynamics of ciliates were more regulated by predators like mesozooplankton.</p><p>The food web efficiency i.e. how much of production at the resource level is converted to production at the top trophic level, may be affected by specific size or type of resource. Indoor mesocosms revealed that the food web efficiency was 11 times lower when heterotrophic bacteria dominated basal production instead of nano- and micro-sized phytoplankton. This was due to a lengthening of the food web when pico-sized bacteria constituted the main resource.</p><p>The PCR-DGGE molecular biological method was used to study the diversity of heterotrophic or mixotrophic chrysomonads. The focus was set on chrysomonads due to their relatively large contribution to the nanoflagellate community. Group-specific PCR primers were optimized for the target group. A field survey in the northern Baltic Sea showed that a handful of chrysomonad sequences were present throughout the year. Significantly more chrysomonads were recorded in the basin with higher primary productive and salinity. In total 15-16 different chrysomonad sequences were recorded. Most of them matched uncultured chrysomonad clones.</p>

Heterotrophic microorganisms are important for the flow of carbon and nutrients in the sea. Bacteria, nanoflagellates and ciliates are relevant components of the pelagic food web. In order to be able to predict the outcome of e.g. eutrophication or climate change we need to know how the different components of the pelagic food web are regulated. With the focus on the northern Baltic Sea food web, this thesis deals with limitation and control of heterotrophic protists, the effect of resource heterogeneity on food web efficiency and diversity of nanoflagellates. In-situ microcosm experiments showed that the net growth of heterotrophic flagellates were resource limited throughout the year. Field data confirmed that the abundance of flagellates was bottom-up controlled. Furthermore, field data also showed that the annual average biomass of protists, flagellates and ciliates increased with primary productivity. On a smaller seasonal scale temperature and bacterial biomass were able to explain most of the variation in flagellate biovolume. The temporal variation in ciliate biovolume could not be explained by any bottom-up factors like bacterial biomass, flagellate biomass or chlorophyll a. This and an in-situ microcosm experiment implied that the seasonal dynamics of ciliates were more regulated by predators like mesozooplankton. The food web efficiency i.e. how much of production at the resource level is converted to production at the top trophic level, may be affected by specific size or type of resource. Indoor mesocosms revealed that the food web efficiency was 11 times lower when heterotrophic bacteria dominated basal production instead of nano- and micro-sized phytoplankton. This was due to a lengthening of the food web when pico-sized bacteria constituted the main resource. The PCR-DGGE molecular biological method was used to study the diversity of heterotrophic or mixotrophic chrysomonads. The focus was set on chrysomonads due to their relatively large contribution to the nanoflagellate community. Group-specific PCR primers were optimized for the target group. A field survey in the northern Baltic Sea showed that a handful of chrysomonad sequences were present throughout the year. Significantly more chrysomonads were recorded in the basin with higher primary productive and salinity. In total 15-16 different chrysomonad sequences were recorded. Most of them matched uncultured chrysomonad clones.

No description available.

Esta tese foi dedicada à duas alternativas para tratamento dos lixiviados de aterros sanitários. A primeira delas, o tratamento consorciado do lixiviado com esgoto sanitário em sistemas aeróbios, teve como objetivo principal verificar se a remoção da matéria orgânica do lixiviado seria devida à biodegradação ou à simples diluição no esgoto sanitário. Para responder esse questionamento, propôs-se o método tentativo \"equivalente em ácido húmico\" (Eq.AH). Em ensaios com reatores de lodos ativados (10 L) operados em batelada, avaliaram-se as remoções de matéria orgânica e nitrogênio para diferentes misturas de lixiviado e esgoto sanitários brutos e pré-tratados, com proporções volumétricas de lixiviado de 0, 0,2, 2 e 5%. O tratamento foi mais eficiente para a mistura esgoto bruto e lixiviado pré-tratado por air stripping (para remoção da amônia) na proporção volumétrica de 2%. Essa condição foi avaliada em reatores piloto de lodos ativados (95 L) e biofiltro aerado submerso (178 L), alimentados continuamente com tempo de detenção hidráulica (TDH) de 24 h. O reator piloto de lodos ativados removeu 59% do COD e a eficiência de nitrificação foi de 60%; enquanto que o biofiltro removeu 83% do COD e eficiência de nitrificação foi de 77%. Com o método tentativo Eq.AH, verificou-se que a remoção da matéria orgânica do lixiviado pré-tratado se deu por biodegradação parcial com o esgoto sanitário, resultado confirmado por espectroscopia de infravermelho por transformada de Fourier. As remoções do COD Eq.AH nos reatores piloto de lodos ativados e biofiltro aerado submerso foram de, respectivamente, 47 e 71%. Adicionalmente, o monitoramento da remoção da matéria orgânica pôde ser realizado com eficácia pelo teste de coloração com nanquim, utilizando o método de classificação de tonalidades proposto nesta Tese. A segunda alternativa proposta nesta Tese consistiu no tratamento dos lixiviados bruto e pré-tratado com cal em reatores de lodos ativados. Os resultados, todavia, foram insatisfatórios: indicaram que essa alternativa se mostrou inviável: após 20 dias de aeração nos reatores de bancada alimentados em batelada, a remoção da matéria orgânica foi inferior a 40% e a remoção do nitrogênio foi devida à volatilização da amônia. / This thesis was dedicated to two alternatives for landfill leachate treatment. The first one, the aerobic co-treatment of leachate and domestic wastewater in aerobic systems, aimed to verify if the organic matter of leachate was removed by biodegradation or dilution with domestic wastewater. To answer this questioning, it was proposed the tentative method \"equivalent in humic acid\" (Eq.HA). In the experiments with bench-scale activated sludge reactors (10 L) operated in batch mode, it was evaluated the organic matter and nitrogen removal from mixtures containing raw and pre-treated leachate and domestic wastewater, at the volumetric ratios of 0, 0.2, 2 and 5%. The treatment was mostly efficient when it was used the mixture domestic wastewater and leachate pre-treated by air stripping (for ammonia removal), at a volumetric ratio of 2%. This condition was also evaluated in a pilot-scale activated sludge reactor (95 L) and a submerged aerobic biofilter (178 L), operated under continuous-flow regime with a 24-h hydraulic retention time. The pilot-scale activated sludge reactor removed 59% of DOC and nitrification efficiency was 60%; whereas the submerged aerobic biofilter removed 83% of DOC and nitrification efficiency was 77%. Using the Eq.HA approach, it was determined that pre-treated leachate organic matter removal was due to partial biodegradation with domestic wastewater, which was confirmed by Fourier transform infrared spectroscopy. The removals of COD Eq.HA were 47% for the pilot-scale activated sludge reactor and 71% for the submerged aerobic biofilter. Aditionally, organic matter could be assessed with the India ink reverse stain test, using the proposed method for tonalities classification. The second alternative proposed by this Thesis consisted in the treatment of raw and pre-treated leachate by lime addition, using activated sludge reactors. The results did not indicate the feasibility of this alternative: after 20 days of aeration, the organic matter removal was lesser than 40%, and nitrogen was mostly removed by ammonia volatilization.

Esta tese foi dedicada à duas alternativas para tratamento dos lixiviados de aterros sanitários. A primeira delas, o tratamento consorciado do lixiviado com esgoto sanitário em sistemas aeróbios, teve como objetivo principal verificar se a remoção da matéria orgânica do lixiviado seria devida à biodegradação ou à simples diluição no esgoto sanitário. Para responder esse questionamento, propôs-se o método tentativo \"equivalente em ácido húmico\" (Eq.AH). Em ensaios com reatores de lodos ativados (10 L) operados em batelada, avaliaram-se as remoções de matéria orgânica e nitrogênio para diferentes misturas de lixiviado e esgoto sanitários brutos e pré-tratados, com proporções volumétricas de lixiviado de 0, 0,2, 2 e 5%. O tratamento foi mais eficiente para a mistura esgoto bruto e lixiviado pré-tratado por air stripping (para remoção da amônia) na proporção volumétrica de 2%. Essa condição foi avaliada em reatores piloto de lodos ativados (95 L) e biofiltro aerado submerso (178 L), alimentados continuamente com tempo de detenção hidráulica (TDH) de 24 h. O reator piloto de lodos ativados removeu 59% do COD e a eficiência de nitrificação foi de 60%; enquanto que o biofiltro removeu 83% do COD e eficiência de nitrificação foi de 77%. Com o método tentativo Eq.AH, verificou-se que a remoção da matéria orgânica do lixiviado pré-tratado se deu por biodegradação parcial com o esgoto sanitário, resultado confirmado por espectroscopia de infravermelho por transformada de Fourier. As remoções do COD Eq.AH nos reatores piloto de lodos ativados e biofiltro aerado submerso foram de, respectivamente, 47 e 71%. Adicionalmente, o monitoramento da remoção da matéria orgânica pôde ser realizado com eficácia pelo teste de coloração com nanquim, utilizando o método de classificação de tonalidades proposto nesta Tese. A segunda alternativa proposta nesta Tese consistiu no tratamento dos lixiviados bruto e pré-tratado com cal em reatores de lodos ativados. Os resultados, todavia, foram insatisfatórios: indicaram que essa alternativa se mostrou inviável: após 20 dias de aeração nos reatores de bancada alimentados em batelada, a remoção da matéria orgânica foi inferior a 40% e a remoção do nitrogênio foi devida à volatilização da amônia. / This thesis was dedicated to two alternatives for landfill leachate treatment. The first one, the aerobic co-treatment of leachate and domestic wastewater in aerobic systems, aimed to verify if the organic matter of leachate was removed by biodegradation or dilution with domestic wastewater. To answer this questioning, it was proposed the tentative method \"equivalent in humic acid\" (Eq.HA). In the experiments with bench-scale activated sludge reactors (10 L) operated in batch mode, it was evaluated the organic matter and nitrogen removal from mixtures containing raw and pre-treated leachate and domestic wastewater, at the volumetric ratios of 0, 0.2, 2 and 5%. The treatment was mostly efficient when it was used the mixture domestic wastewater and leachate pre-treated by air stripping (for ammonia removal), at a volumetric ratio of 2%. This condition was also evaluated in a pilot-scale activated sludge reactor (95 L) and a submerged aerobic biofilter (178 L), operated under continuous-flow regime with a 24-h hydraulic retention time. The pilot-scale activated sludge reactor removed 59% of DOC and nitrification efficiency was 60%; whereas the submerged aerobic biofilter removed 83% of DOC and nitrification efficiency was 77%. Using the Eq.HA approach, it was determined that pre-treated leachate organic matter removal was due to partial biodegradation with domestic wastewater, which was confirmed by Fourier transform infrared spectroscopy. The removals of COD Eq.HA were 47% for the pilot-scale activated sludge reactor and 71% for the submerged aerobic biofilter. Aditionally, organic matter could be assessed with the India ink reverse stain test, using the proposed method for tonalities classification. The second alternative proposed by this Thesis consisted in the treatment of raw and pre-treated leachate by lime addition, using activated sludge reactors. The results did not indicate the feasibility of this alternative: after 20 days of aeration, the organic matter removal was lesser than 40%, and nitrogen was mostly removed by ammonia volatilization.

A Floresta Ombrófila Mista também chamada de Floresta de Araucária representa um dos mais ricos remanescentes de florestas pluviais subtropicais brasileiras, tendo como principal representante a Araucaria angustifolia, espécie considerada ameaçada de extinção. A diversidade microbiana possui um importante papel no funcionamento e manutenção do equilíbrio dos ecossistemas florestais, mas é desconhecida em solos com araucária. O presente estudo teve como objetivo avaliar a diversidade, funcionalidade e estrutura das comunidades microbianas em florestas de Araucaria angustifolia natural, introduzida e impactada pela queima acidental. O estudo foi realizado no Parque Estadual de Campos de Jordão, SP, tendo como áreas de estudo: floresta nativa com predomínio de araucária (FN), reflorestamento de araucária (RF), e reflorestamento de araucária com queima acidental em julho de 2001 (RQ). Em cada área, foram escolhidas ao acaso dez árvores de araucária e ao redor de cada árvore coletou-se uma amostra composta de três subamostras. Foram avaliados atributos químicos, microbiológicos, estrutura das comunidades de Bacteria e Archaea por PCR-DGGE e seqüenciamento parcial do gene rRNA 16S de Bacteria, análise da capacidade de utilização de substratos de carbono (Biolog) e perfis de ácidos graxos ligados a ésteres de fosfolipídios (PLFAs). As áreas são caracterizadas por solos ácidos, com elevado conteúdo de matéria orgânica (MO) e baixa disponibilidade de cátions metálicos básicos. A área FN apresentou maiores teores de carbono da biomassa microbiana (CBM), atividade respiratória basal (C-CO2) e relação carbono da biomassa microbiana: carbono orgânico total (CBM:COT), comparada ao RF e RQ. Os maiores valores de quociente metabólico (qCO2) foram encontrados no RQ quando comparado a FN e RF. A análise canônica discriminante identificou o atributo microbiano qCO2 e químicos, Mg e pH, como sendo responsáveis pela discriminação das áreas, seguidos do teor de P. A análise de PCRDGGE revelou que as estruturas das comunidades bacterianas de FN e RQ foram mais similares entre si do que em RF. A análise de escala multidimensional (NMDS) com ANOSIM baseada nos perfis de amplicons de Bacteria mostrou que as três áreas são diferentes entre si, enquanto para Archaea não houve diferença entre as áreas estudadas. A afiliação taxonômica das seqüências de clones do gene rRNA 16S mostrou que o solo de FN apresenta uma maior diversidade de táxons. Os filos Proteobacteria e Actinobacteria foram os mais freqüentes nas três áreas. A maior diversidade estimada pelo índice de Shannon foi encontrada em RQ, em comparação a FN e RF. A análise por Biolog mostrou que área FN apresenta a maior taxa de utilização de substratos, em relação RF e RQ, as quais não diferiram entre si. Os perfis de PLFAs não apresentaram diferenças entre as áreas estudadas. Observou-se uma maior biomassa bacteriana, principalmente de Gram-positivas, quando comparada a biomassa fúngica de PLFAs das áreas estudadas. As análises multivariadas apresentaram-se como importantes ferramentas no estudo de diversidade microbiana, sendo os atributos químicos e microbiológicos do solo úteis para a interpretação dos resultados obtidos. / The Ombrophilic Mixed Forest, also called Araucaria Forest, represents one of the richest remainders of subtropical pluvial forests in Brazil. Its main representative species is the endangered Araucaria angustifolia. The microbial diversity plays an important role in functioning of forest ecosystems. However, the microbial diversity in soils with araucaria forests is mostly unknown. The aim of this work was to evaluate the diversity, structure of microbial communities in their possible functions in a natural preserved araucaria forest (FN), a planted araucaria forest (RF) and a planted araucaria forest impacted by accidental fire (RQ). The study was carried out at the State Park of Campos of Jordão (SP). For each area, ten araucaria trees were randomly selected and a sample composed of three sub-samples was collected at approximately one meter from the trunk of each tree. Chemical and microbiological attributes, as well as structures of bacterial and archaeal communities were evaluated using PCR-DGGE and the partial sequencing of the 16S rRNA gene from Bacteria, community level physiological profiles using Biolog, the phospholipid fatty acids profiles (PLFAs). The studied areas were characterized by acidic soils, with high content of organic matter (OM) and low availability of basic metallic cations. The area FN presented the highest contents of carbon in the microbial biomass (CBM), higher basal respiration activity (C-CO2) and higher microbial biomass carbon: total organic carbon ratio (CBM:TOC), compared to RF and RQ. The highest values of metabolic quotient (qCO2) were observed in RQ, when compared to FN and RF. Using canonical discriminant analysis (CDA), qCO2, Mg concentration and pH were identified as the main attributes responsible for the discrimination of the areas, followed by the P concentration. The PCR-DGGE analysis revealed that the bacterial community structures in FN and RQ share higher levels of similarity, as compared to RF. Non-metric multidimensional scale analysis (NMDS) and ANOSIM based on the profiles of bacterial 16S rRNA gene amplicons showed that the all three areas had different bacterial communities, whereas archaeal communities were similar, based on 16S rRNA genes amplicon profiles. The phylogenetic affiliation of 16S rRNA gene clone sequences showed that soil from FN presents higher taxa diversity, as compared to RF and RQ. The phyla Proteobacteria and Actinobacteria were the most frequent in the three areas studied. Higher Shannon index was observed in RQ soil than FN and RF soils. Biolog analysis showed that FN has the highest substrate utilization rates, when compared to RF and RQ, which did not show significant differences. In general, PLFAs profiles did not show differences for the areas studied. Estimated bacterial biomass was higher than fugal biomass, with predominance of Gram-positive bacteria. Integration of chemical and microbial attributes through multivariate analyses is essential for identifying the factors determining microbial community structure in forest soils.

Ecosystems currently face widespread biodiversity losses and other environmental disturbances, such as climate warming, related to increased anthropogenic activities. Within this context, scientists consider the effects of such changes on the biodiversity, and hence on the activity, of soil microorganisms. Indeed, soil microorganisms mediate a wide range of soil processes. Currently, knowledge on soil microbial diversity is still limited, partially due to technical limitations. The advent of molecular-based analyses now allows studying the soil microbial diversity. These advances in the study of soil microbial communities have lead to a growing evidence of the critical role played by the microbial community in ecosystem functioning. This relationship is supposed to be relevant for narrow processes, regulated by a restricted group of microorganisms, such as the nitrification process. This PhD thesis aimed at studying ammonia oxidizing bacteria (AOB) community structure and richness as an integrated part of soil functioning. This research aimed at investigating the effect of aboveground plant diversity on ammonia oxidizing bacteria diversity and function in forest and grassland soils with focus on the influence of (a) functional group identity of grassland plants (legumes, grasses, forbs), (b) grassland plant species richness and (c) tree species, on AOB diversity and function. Another objective of this research was to study the effect of a 3°C increase in air temperature on AOB diversity and function. The link between AOB diversity and function (potential nitrification) is also investigated. For grassland ecosystems, a microcosm experiment was realized. An experimental platform containing 288 assembled grassland communities was established in Wilrijk (Belgium). Grassland species were grown in 12 sunlit, climate controlled chambers. Each chamber contained 24 communities of variable species richness (S) (9 S=1, 9 S=3 and 6 S=9).The grassland species belonged to three functional groups: three species of each grasses (Dactylis glomerata L., Festuca arundinacea SCHREB., Lolium perenne L.), forbs (non-N-fixing dicots; Bellis perennis L., Rumex acetosa L., Plantagolanceolata L.), and legumes (N-fixing dicots; Trifolium repens L., Medicago sativa L., Lotus corniculatus L.). Half of these chambers were exposed to ambient temperature and the other half were exposed to (ambient +3°C) temperature. One ambient and one (ambient+3°C) chambers were destructively harvested 4, 16 and 28 months after the start of the experiment. The influence of plant functional group identity on the nitrification process and on AOB community structure and richness (AOB diversity) was assessed in soils collected from the first two destructive amplings (chapter 2). The effect of plant species richness on AOB diversity and function was considered for soils sampled after 16 and 28 months (chapter 3). AOB function was determined by potential nitrification. AOB community structure and richness were assessed by polymerase chain reaction followed by denaturing gradient gel electrophoresis (DGGE) and sequencing of excised DGGE bands. I found that functional group identity can affect AOB community structure. In particular, the presence of legumes, both in monoculture or in mixture with forbs and grasses, lead to AOB community composition changes towards AOB clusters tolerating higher ammonium concentrations. This change in AOB community structure was only linked to increased potential nitrification under monocultures of legumes, when ammonium was supposed to be not limiting. This study revealed that physiological attributes of AOB and resource availability may be important factors in controlling the nitrification process. This research showed that the impact of plant species richness on the nitrification process could be mediated by the interactions between plants and AOB, through competition for substrate. A 3°C increase in air temperature did not affect AOB community structure, richness or function. In forest ecosystems, we studied the effect of tree species in forest sites located in Belgian and in the Grand-Duchy of Luxembourg covered each by several deciduous or coniferous tree species (Fagus sylvatica L., Quercus petraea (Mattuschka) Lieblein, Picea abies (L.) Karst, Pseudotsuga menziesii (Mirbel) Franco). We investigated the influence of these tree species on microbial processes (chapter 5) related to C and N cycling, particularly with emphasize on the nitrification process and on the diversity of AOB (chapter 6). The results showed that the effect of tree species on net N mineralization was likely to be mediated through their effect on soil microbial biomass, reflecting their influence on organic matter content and carbon availability. Influence of tree species on nitrification (potential and relative) might be related to the presence of ground vegetation through its influence on soil ammonium and labile C availability. AOB community structure was more site-specific than tree specific. However, within sites, AOB community structure under broadleaved trees differed from the one under coniferous trees. The effect on tree species on AOB was likely to be driven by the influence of tree species on net N mineralization, which regulates the substrate availability for AOB. The results also demonstrated that the relationship between AOB diversity and function might be related both to AOB abundance and AOB community structure and richness. This thesis showed no clear relationship between AOB community structure or richness and AOB function. However, we revealed that aboveground grassland plant richness, grassland plant functional groups and tree species influence AOB community structure and richness. Actuellement, les écosystèmes sont soumis à dimportantes pressions anthropiques et environnementales, pouvant aboutir à des pertes massives de biodiversité. Les scientifiques sinterrogent sur limpact de ces perturbations sur la diversité et, par conséquent, sur lactivité des microorganismes du sol. En effet, ceux-ci régulent de nombreux processus du sol. Actuellement, de nombreuses lacunes subsistent dans la connaissance de la diversité microbienne du sol. Celles-ci peuvent être partiellement attribuées aux difficultés méthodologiques associées à l'étude des micro-organismes du sol. Lavènement des techniques moléculaires nous permet de combler ces lacunes. Les avancées réalisées dans l'étude des communautés microbiennes du sol ont mis en évidence le rôle crucial joué par les communautés microbiennes dans le fonctionnement des écosystèmes. De plus, il semblerait que les processus régulés par un groupe restreint dorganismes, tel le processus de nitrification, soient plus sensibles à toute altération de la communauté. Lobjectif de cette thèse était détudier la structure de la communauté ainsi que la richesse (nombre de bandes DGGE) des bactéries oxydant lammoniac (AOB) comme une partie intégrante du fonctionnement des sols. Notre étude se focalisait sur linfluence de (a) différents groupes fonctionnels de plantes (graminées, légumineuses, dicotylédones), (b) communautés de plantes présentant une richesse spécifique croissante et (c) différentes essences forestières, sur la diversité (structure de la communauté et richesse des AOB) et la fonction des AOB. Cette recherche étudiait également limpact dune augmentation de température de 3°C sur ces paramètres. Létablissement dun lien éventuel entre la diversité et la fonction (nitrification potentielle) des AOB a aussi été envisagé. Concernant les écosystèmes prairiaux, nous avons réalisé une étude en microcosmes. Une plateforme expérimentale comprenant 288 communautés artificielles de plantes a été établie à Wilrijk (Belgique). Cette plateforme consistait en 12 chambres, dont une moitié était à température ambiante et la seconde était à température ambiante augmentée de 3°C. Chaque chambre contient 24 communautés de plantes de richesse spécifique variable (9 S=1, 9 S=3 et 6 S=9). Les communautés de plantes sont créées avec 9 espèces de plantes appartenant à trois groupes fonctionnels : 3 espèces de graminées (Dactylis glomerata L., Festuca arundinacea SCHREB., Lolium perenne L.), de légumineuses (dicotylédones fixatrices dazote ;Trifolim repens L., Medicago sativa L., Lotus corniculatus L.), et de dicotylédones non fixatrices dazote (Bellis perennis L., Rumex acetosa L., Plantago lanceolata L.). Les sols issus dune chambre à température ambiante et dune chambre à température ambiante augmentée de 3°C ont été échantillonnés, respectivement, 4, 16 et 28 mois après le début de lexpérimentation. Linfluence des groupes fonctionnels de plantes sur le processus de nitrification ainsi que sur la structure de la communauté et la richesse des AOB a été mesuré sur les sols issus des deux premiers échantillonnages (chapitre 2). Nous avons mesuré leffet de la richesse croissante en plantes sur la diversité et lactivité des AOB sur les sols échantillonnés après 16 et 28 mois dexpérimentation (chapitre 3). La structure de la communauté ainsi que la richesse des AOB ont été évaluées à laide dune amplification spécifique par réaction de polymérisation en chaîne (PCR) de lADN génomique extrait du sol suivie par une séparation par électrophorèse sur gel dacrylamide en présence dun gradient dénaturant (DGGE). Nous avons identifié les différentes AOB présentes par séquençage des bandes DGGE excisées. Nos résultats ont montré que les différents groupes fonctionnels peuvent affecter la structure de la communauté des AOB. En particulier, la présence de légumineuses, aussi bien en monoculture quen mélange avec des graminées ou des dicotylédones non fixatrices dazote, provoque des changements au sein de la structure de la communauté des AOB, favorisant la présence de clusters tolérants des concentrations en ammonium plus élevées. Ces changements de la structure de la communauté des AOB sont liés à des augmentations de la production potentielle de nitrates (nitrification potentielle) quand lammonium est supposé être non limitant. Cette étude révèle que la physiologie des AOB ainsi que la disponibilité en substrat peuvent être des facteurs majeurs intervenant dans le contrôle du processus de nitrification. Cette recherche montre que linfluence de la richesse spécifique des plantes sur la nitrification pourrait dépendre des interactions entre les plantes et les AOB via la compétition pour le substrat. Une augmentation de la température de lair de 3°C na pas influencé les richesse, structure de la communauté ou les fonctions des AOB. Pour les écosystèmes forestiers, nous aborderons leffet de différentes essences forestières (Picea abies (L.) KARST, Fagus sylvatica L., Quercus petraea LIEBLEIN ; Pseudotsuga menziezii (MIRB.) FRANCO) dans différents peuplements au Grand Duché du Luxembourg et en Belgique. Nous avons étudié l'influence de ces essences forestières sur les processus microbiens (chapitre 5) liés aux cycles du carbone et de lazote, en particulier leur effet sur le processus de nitrification et la diversité des AOB (chapitre 6). Notre étude révèle que linfluence des essences forestières sur la minéralisation nette de lazote pourrait être attribuable à leur effet sur la biomasse microbienne, reflétant ainsi leur effet sur la teneur en matière organique et la disponibilité en carbone. Limpact des essences forestières sur la nitrification (à la fois sur la nitrification relative et sur la nitrification potentielle) serait conditionné par la présence de végétation au sol, en raison de linfluence de celle-ci sur la disponibilité en ammonium et en carbone labile. Nous avons observé que la structure de la communauté des AOB était plus spécifique aux sites quaux essences forestières. Cependant, au sein dun site, elle différait sous feuillus et sous conifères. Les essences forestières influenceraient la structure de la communauté des AOB au travers de limpact quelles ont sur la minéralisation nette de lazote qui régule, quant à elle, la disponibilité en ammonium. Cette recherche démontre que le lien observé entre la diversité et la fonction dépendrait la fois de labondance, de la structure de la communauté et de la richesse des AOB. Cette thèse na révélé aucune relation claire entre la structure de la communauté ou la richesse des AOB et leur fonction. Par contre, nous avons observé que la richesse spécifique et les groupes fonctionnels de plantes prairiales et les essences forestières affectent la structure de la communauté et la richesse des AOB.