O objetivo deste estudo foi avaliar a influência da relação carbono/nitrogênio (C/N) e carbono/fósforo (C/P) na produção de hidrogênio (H2) em reator anaeróbio de leito fluidificado (RALF), contendo pneu triturado como material suporte para adesão microbiana. Foi utilizado substrato sintético contendo glicose (5.000 mg L-1) como fonte de carbono, com pH efluente de ±3,7. Foram operados quatro reatores com relações C/N=100 em R1, C/N=150 em R2, C/N=200 em R3 e C/N=250 em R4, sob condições mesofílicas. Os reatores foram operados por 18 meses e o experimento foi dividido em três fases: na 1º Fase a relação C/P foi mantida no valor de 300 e o tempo de detenção hidráulica (TDH) foi variado em 8, 6, 4, 2 e 1 h; na 2º Fase, o TDH foi mantido em 2 h e a relação C/P foi variada nos valores de 300, 500, 700, 900 e 1100; e na 3º Fase, o TDH foi mantido em 8 h e a relação C/P variada novamente nos valores de 300, 500, 700, 900 e 1100. Os maiores valores de produção volumétrica de hidrogênio (PVH) foram obtidos no R3 (C/N=200), TDH de 2 h e relação C/P=700 (0,70 L.h-1.L-1) e estiveram relacionados às rotas fermentativas de produção de etanol e ácido acético. Os maiores valores de rendimento de hidrogênio (YH) foram obtidos no TDH de 8 h, R3 (C/N=200) e relação C/P=300 (1,758 mol H2.mol glicose-1) e estiveram relacionados às rotas de produção de etanol, ácido butírico e ácido acético. O maior valor de rendimento de etanol (YEtOH) do estudo (1,8 mol EtOH. mol glicose-1) foi obtido na 2ª fase. A partir de análises de DGGE maior similaridade foi encontrada entre C/P=700 e C/P=900 para R1 (87%), R2 (97%) e R3 (92%) na 2ª fase. Por meio de afiliação filogenética do gene rRNA 16S de amostras das etapas de maior produção de H2 foram obtidos clones relacionados a espécie produtora de H2 e etanol Ethanoligenens sp. seguida da espécie produtora de ácido lático Lactobacillus sp. e da espécie produtora de ácido acético e butírico Clostridium sp.. / The aim of this study was to evaluate the influence of the carbon/nitrogen (C/N) and carbon/phosphorus (C/P) ratio in hydrogen (H2) production in anaerobic fluidized bed reactor (AFBR) containing grounded tire as support material for microbial adhesion. Synthetic substrate containing glucose (5,000 mg L-1) was used as the carbon source, and the effluent pH remained ±3.7. Four reactors were operated with C/N=100 (R1), C/N=150 (R2), C/N=200 (R3) and C/N=250 (R4), under mesophilic conditions. The reactors were operated for 18 months and the experiment was divided into three phases: in the 1st Phase, the C/P ratio was maintained at the value of 300 and hydraulic retention time (HRT) was varied in 8, 6, 4, 2 and 1 h; in the 2nd phase, the HRT was kept at 2 h and the C/P ratio was varied in 300, 500, 700, 900 and 1100; and in the 3rd Phase, the HRT was kept at 8 h and C/P ratio varied in the values of 300, 500, 700, 900 and 1100. The highest values of hydrogen production (HPR) were obtained in R3 (C/N=200), HRT of 2 h and C/P=700 (0.70 L.h-1.L-1) and they were related to fermentative pathways of ethanol and acetic acid production. The highest values of hydrogen yield (HY) were obtained in HRT of 8 h, R3 (C/N=200) and C/P=300 (1.758 mol H2.mol glucose-1) and they were related to fermentative pathways of ethanol, butyric and acetic acid production. The highest value of ethanol yield (EtOHY) (1.8 mol EtOH. mol glucose-1) was obtained in the 2nd Phase. By means of DGGE analyses was found highest similarity between C/P=700 and C/P=900 for R1 (87%), R2 (97%) and R3 (92%) in the 2nd Phase. Through phylogenetic affiliation of the 16S rRNA gene taken from samples of stages of highest H2 production it were obtained clones of hydrogen and ethanol producing specie related to Ethanoligenens sp., followed by the lactic acid bacteria Lactobacillus sp. and of the acetic and butyric acid producing Clostridium sp..

A cultura da cana-de-açúcar é a segunda maior cultura em valor de produção agrícola do país. Nos últimos anos ocorreu uma alteração no cenário canavieiro passando de colheita manual de cana queimada para mecanizada de cana crua. A colheita mecanizada da cana-de-açúcar acarreta muitos benefícios ambientais, porém o tráfego intenso de maquinários resulta na compactação do solo. Desse modo, buscamos avaliar como o efeito das alteração física do solo causado pelo tráfego de máquinas durante a colheita da cana interfere na composição das comunidades microbianas, especialmente as que participam das transformações do nitrogênio. As parcelas experimentais foram divididas em colheita manual (PDTR) e colheita mecanizada (PD). Todos os demais manejos e tratos culturais foram iguais, isolando a influência do impacto do maquinário durante a colheita. Para a avaliação da microbiota realizamos análise da estrutura da comunidade (DGGE ou T-RFLP) e análise de abundância (qPCR) de bactérias, fungos, arquéias, e do ciclo do nitrogênio envolvendo os genes marcadores para a nitrificação (amoA - AOA e AOB), fixação de nitrogênio (nifH) e desnitrificação (nirK, nirS, nosZ clado I e II). Observamos apenas a diferença dos parâmetros físicos na camada superficial por meio da resistência a penetração. A alteração no perfil da comunidade de bactérias e arquéias mostra que ambas são responsivas ao tratamento com tráfego do maquinário. Em relação aos microrganismos envolvidos nas transformações de nitrogênio, AOA foi altamente responsiva ao impacto do tráfego agrícola em ambos os tipos de textura, mas teve diferença na abundância apenas no solo arenoso. O gene nifH apresentou diferença na diversidade em solo argiloso e diferença de abundância em solo arenoso. E por fim, sugerimos que o gene nosZ em subsuperfície pode indicar uma possível campactação antes dos parâmetros físicos do solo. / In Brazil, sugarcane is the second most value of agricultural production. Lately, the harvest management in sugarcane fields has changed from manual harvesting methods with pre-harvest burns to mechanical harvests with green cane (unburnt harvest). The mechanized harvest brings many ambiental benefits. However, the intense traffic due to agricultural machines over the years is resulting in soil compaction. The purpose of this work was to evaluate how the physical effect of mechanized harvesting can interfere with the composition of soil microbial community, specially the microbial involved in the nitrogen cycle. Experimental plots were divided into manual harvest (PDTR) and mechanized harvest (PD). All aspects of crop management are the same, so we were able to study the impact of mechanical harvester traffic during the harvest in isolation. To evaluation of the microbiome we analyzed community structure (DGGE ou T-RFLP) along with their abundance (qPCR) for soil bacterial, fungal and archaeal, and microorganisms involved in the transformation of nitrogen that we used gene markers for nitrification (amoA - AOA and AOB), nitrogen fixation (nifH) and denitrification (nirK, nirS, nosZ clade I and II). We observed the difference between physical parameters only in topsoil by soil penetration resistance. Our results indicated structured community changes of bacterial and archaeal showed us that both are responsive to treatment of machinery traffic. Microorganisms involved in the nitrogen cycle, our results presented that AOA is highly responsive to the impact of agricultural traffic on both soil texture, but we observed the difference in abundance only in sandy soil. The nifH gene was different on community structure in clay soil and on abundance in sandy soil. Moreover, we suggest that nosZ gene could indicate a possible soil compaction before the physical parameters in a layer between 20 and 40 cm.

Dentre os contaminantes de água subterrânea de maior importância está o tetracloroeteno (PCE), o qual é altamente tóxico e potencialmente carcinógeno. As comunidades bacterianas de águas subterrâneas contaminadas com PCE e a diversidade de bactérias capazes de degradar esses organoclorados são pouco conhecidas. O objetivo deste trabalho é comparar a estrutura das comunidades de bactérias de amostras de água subterrânea em uma área contaminada com PCE e selecionar um consórcio microbiano capaz de degradar eficientemente o PCE em reator horizontal de leito fixo (RHLF). Amostras de água subterrânea de oito poços de monitoramento, instalados em uma área contaminada com PCE foram coletadas e analisadas para determinação de oxigênio dissolvido, potencial redox, condutividade elétrica, pH e concentração de tetracloroeteno, tricloroeteno, cis-dicloroeteno e cloreto de vinila (COVs). As amostras foram analisadas também para a determinação da estrutura das comunidades de bactéria por PCRDGGE e seqüenciamento de clones do gene rRNA 16S. Os parâmetros físico-químicos oscilaram consideravelmente ao longo do tempo em todos os poços de monitoramento (PM). Tetracloroeteno e tricloroeteno foram detectados apenas no PM6. As estruturas das comunidades bacterianas dos PMs analisados mostraram tanto variação temporal quanto espacial. As análises das comunidades bacterianas nos PM6 e PM8, contaminado e não-contaminado com PCE, revelaram resultados semelhantes aos obtidos por DGGE. Uma maior riqueza estimada de espécies bacterianas foi observada nas amostras do PM8, pelo menos em duas épocas de amostragem, sugerindo que a contaminação com PCE está associada com a redução da diversidade bacteriana em água subterrânea. Cultivos de enriquecimento e ensaios de degradação do PCE foram realizados utilizando-se um RHLF, o qual foi preenchido com sedimento do PM6 imobilizado em espuma de poliuretano e enriquecido com meio mineral básico suplementado com PCE. A análise das alterações nas comunidades de bactérias nos reatores foi feita por PCRDGGE e seqüenciamento parcial do gene rRNA 16S. No ensaio de degradação do PCE no RHLF foi utilizado meio com PCE suplementado ou não com lactato e acetato. Tanto pelo DGGE quanto pelo seqüenciamento, foi observada a seleção de bactérias específicas no reator. A partir das análises de seqüenciamento, essas bactérias foram identificadas como Alphaproteobacteria e Sphingobacteria. No ensaio de degradação do PCE, os parâmetros físico-químicos do meio não mostraram variações ao longo do comprimento dos reatores. As análises de COVs mostraram uma grande eficiência na degradação do PCE (98%), com um tempo de retenção de 12 horas, não havendo diferença significativa na percentagem de degradação em meio com lactato ou acetato, com relação ao controle sem fonte de carbono. No processo de degradação nenhum dos produtos da via de degradação do PCE foi detectado, o que sugere uma via alternativa de degradação do PCE, a qual ocorre em aerobiose. / Tetrachloroethene (PCE) is one of the most important contaminants of groundwater, since it is highly toxic and potentially carcinogenic. The bacterial communities of PCE contaminated groundwater and the diversity of bacteria capable of degrading this contaminant are barely known. The objective of this work is to compare the structure of bacterial communities from groundwater samples from a PCE contaminated site and select a microbial consortium capable to degrading efficiently PCE in a horizontal fixed bed reactor (HFBR). Groundwater samples from eight monitoring wells, installed in a PCE contaminated site were collected and analyzed for determination of dissolved oxygen, redox potential, electrical conductivity, pH, and concentrations of tetrachloroethene, trichloroethene (TCE), cis- and trans-dichloroethene, vinyl chloride (VOCs). The structure of the bacterial communities was determined by PCR-DGGE and 16S rRNA gene clone sequencing. The physical-chemical parameters oscillated considerately throughout time in all the monitoring wells (MW). PCE and TCE were detected only in MW6. The bacterial community structures in the groundwater from the MWs analyzed showed temporal and spatial variation. The analysis of the bacterial communities in MW6 and MW8, contaminated and non-contaminated with PCE, respectively, based on sequencing of 16S rRNA gene clones revealed results to the ones observed by DGGE. Estimated richness of bacterial species was higher in samples from MW8, at least in two sampling times, suggesting that the contamination with PCE is associated with reduction of bacterial diversity in groundwater. Enrichment cultures and PCE biodegradation assays were performed in a HFBR, which was filled with sediment from MW6 immobilized onto polyurethane foam and enriched with basic mineral medium supplemented with PCE. Shifts in bacterial community structure were analyzed using PCRDGGE and partial sequencing of 16S rRNA gene clones. In the PCE biodegradation assays in the HFBR, were performed in medium containing lactate or acetate. DGGE and 16S rRNA gene clone sequencing data suggest selection of specific bacteria in the reactor. Sequencing data showed that these bacteria belong to Alphaproteobacteria and Sphingobacteria. In the PCE biodegradation assays, media physical-chemical parameters did not show variation along the reactor length. VOC analyses showed a great efficiency in the degradation of PCE (98%) with a residence time of 12 hours in the reactor, and no significant differences were observed in the presence of lactate or acetate, as compared to the medium without a carbon source. During the biodegradation process, none of the products from the anaerobic pathway of PCE reductive dechlorination was detected, suggesting that an alternative PCE biodegradation pathway is occurring in aerobiosis.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-05-18T17:44:09Z No. of bitstreams: 1 2014 - Dayana da Silva Correia.pdf: 2038600 bytes, checksum: 031920d278558b902310d93f752920d7 (MD5) / Made available in DSpace on 2018-05-18T17:44:09Z (GMT). No. of bitstreams: 1 2014 - Dayana da Silva Correia.pdf: 2038600 bytes, checksum: 031920d278558b902310d93f752920d7 (MD5) Previous issue date: 2014-03-26 / CAPES / CNPq / The symbiosis between soil invertebrates and micro"organisms is a major ally in promoting the decomposition of plant residues in the soil. The micro"organisms in turn, have an immense genetic diversity and play crucial roles in maintaining ecosystems. One of these functions is the production of extracellular enzymes that assist the mineralization of organic matter. The possibility of developing new biotechnological processes based on the exploration of microbial diversity is immense, due to the great variability that exists between biological systems and it can be optimized to improve the agricultural production systems in a sustainable manner. The objective of this work was to study the profile of the bacterial community and cellulolytic potential of bacteria isolated from three different species of invertebrates saprophages. The experiments were performed at the Experimental Station of Embrapa Agrobiology, Serop?dica, Rio de Janeiro State. Millipede, of the Trigoniulus corallinus species, were collected in piles of plant compounds from local experimental field sites, which were subsequently incubated for 60 days under six different diets. Bacterial diversity in the intestinal tract of invertebrates was analyzed by PCR"DGGE of 16S rDNA gene amplification by PCR electrophoresis in denaturing gradient gel (DGGE); two domains were used, Bacteria and Actinobacteria. Some bands of the DGGE gel were extracted and sequenced. To assess the potential for production of cellulases in response to the presence of carboxy methyl cellulose (CMC) of isolates, the technique Congo red stain was used and the values were expressed as means (Ie) enzymatic index. From the highest values of (Ie) twenty" three bacteria were selected for the analysis of 16S rDNA. After the phylogenetic identification, the cellulolytic potential was rated, through cellulolytic activity of endoglucanase (CMCase), and endo" and exoglucanases (FPase) tests. To determine the molecular weight and activity of the enzymes polyacrylamide gels (SDS"PAGE) and zymography were performed. The results obtained in the technique of DGGE, the profiles of DGGE bands, showed that the intestinal microbiota of the invertebrates has distinct bacterial groups. It is possible to infer that, despite the communities having similar abundance, such as in the Trigoniulus corallinus and Cubaris murine species, the groups that make up this abundance were different among the invertebrate?s species. From the clones of the incised bands, three phyla members, Proteobacteria, Firmicutes and Bacteroidetes, were sequenced. Through phylogenetic analysis, it was possible to identify the 23 species of bacteria. Presenting two distinct Actinomycetes and Firmicutes phylum, the largest genus identified was Streptomyces, followed by an isolated Bacillus, Paenibacillus, and Staphylococcus. The intestinal tract of the three species of saprophages invertebrates showed to be an adequate environment for prospection of bacteria with cellulolytic efficiency, with high potential for future biotechnological studies. / A simbiose entre invertebrados do solo e microrganismos ? um grande aliado no auxilio da decomposi??o de res?duos vegetais presentes no solo. Os microrganismos por sua vez, apresentam uma imensa diversidade gen?tica e desempenham fun??es cruciais na manuten??o dos ecossistemas, uma dessas fun??es ? a produ??o de enzimas extracelulares que auxiliam na mineraliza??o da mat?ria org?nica. A possibilidade de desenvolver novos processos biotecnol?gicos com base na prospec??o da diversidade microbiana ? imensa, em decorr?ncia da grande variabilidade que existe entre os sistemas biol?gicos e que podem ser aperfei?oados para melhorar os sistemas de produ??o agr?colas de forma sustent?vel. O objetivo deste trabalho foi estudar o perfil da comunidade bacteriana e potencial celulol?tico de bact?rias isoladas de tr?s diferentes esp?cies de invertebrados sapr?fagos. Os experimentos foram montados no Campo Experimental da Embrapa Agrobiologia, Serop?dica, Rio de Janeiro. Gong?los, da esp?cie Trigoniulus corallinus, foram coletados em pilhas de compostos vegetais presentes em torno do campo experiemental, que posteriormente foram incubados durante 60 dias, sob seis diferentes dietas. A diversidade bacteriana do trato intestinal dos invertebrados foi analisada atrav?s da t?cnica de PCR"DGGE por amplifica??o do gene 16S rDNA PCR por eletroforese em gel com gradiente desnaturante (DGGE); foram utilizados dois dom?nios Bacteria e Actinobact?ria. Algumas bandas do gel de DGGE foram extra?das e sequenciadas. Para avaliar o potencial quanto ? produ??o de celulases em resposta ? presen?a de carboxi"metil"celulose (CMC) das bact?rias isoladas, foi utilizada a t?cnica de colora??o vermelho Congo, e os valores foram expressos atrav?s de (Ie) ?ndice enzim?tico. A partir dos maiores valores de (Ie), foram selecionadas vinte e tr?s bact?rias para a an?lise de sequenciamento do gene 16S rDNA. Ap?s a identifica??o filogen?tica, foi avaliado o potencial celulol?tico, atrav?s de testes de atividade celulol?tica de endoglucanases (CMCase) e endo e exoglucanases (FPase). Para determinar a massa molecular e atividade das enzimas foram realizados g?is de poliacrilamida (SDS"PAGE) e zimograma. Os resultados obtidos na t?cnica de DGGE, os perfis de bandas de DGGE mostrou que a microbiota intestinal dos invertebrados, det?m grupos bacterianos distintos. Pode"se inferir neste ponto, que apesar das comunidades possu?rem abund?ncia similar, como as esp?cies de Trigoniulus corallinus e Cubaris murina, os grupos que comp?em esta abund?ncia foram diferentes entre as esp?cies de invertebrados. A partir dos clones de bandas incisadas, foram sequ?nciados membros de tr?s filos, Proteobacteria, Firmicutes e Bacteroidetes. Atrav?s da an?lise filogen?tica, foi poss?vel identificar as 23 esp?cies de bact?rias. Apresentando dois filos distintos Actinomicetos e Firmicutes, o maior g?nero identificado foi Streptomyces, seguido de um isolado para Bacillus, Paenibacillus e Staphylococcus. O trato intestinal das tr?s esp?cies de invertebrados sapr?fagos revelou ser um ambiente h?bil ? prospec??o de bact?rias com efici?ncia celulol?tica, com alto potencial para futuros estudos biotecnol?gico.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-05-18T18:09:32Z No. of bitstreams: 1 2015 - Bruno Oliveira de Carvalho.pdf: 1725115 bytes, checksum: 78e6b6f127705b066a8a0f75bbb08e88 (MD5) / Made available in DSpace on 2018-05-18T18:09:32Z (GMT). No. of bitstreams: 1 2015 - Bruno Oliveira de Carvalho.pdf: 1725115 bytes, checksum: 78e6b6f127705b066a8a0f75bbb08e88 (MD5) Previous issue date: 2015-03-17 / CAPES / The knowledge of bacterial genetic information has enable advances in many areas allowing detecting microorganisms and evaluating the production of virulence factors and resistance in certain populations. This knowledge allows the detection of microorganisms, as well as the evaluation of virulence and resistance factors production in certain populations. More recently, molecular diagnosis based on the detection of specific fragments from these agents has been implemented in no viable bacterial cultivation environments or hampered by biotic or abiotic conditions. Besides specific agent detection, bacterial diversity analysis might be an important criterion, such as assessing soil quality. Cultivation has been the most common way for evaluating bacterial diversity. However, most of soil bacteria are not cultivable; therefore, the employment of molecular tools has been increasingly used on this sort of analysis. This survey aimed standardizing the use of cultivation independent techniques for granting three agriculture sectors demands. The first chapter standardized the employment of DGGE technique for analyzing milk bacterial diversity from mastitis and healthy teats belonging to the same animal. Results pointed to differences on bacterial profile from both groups. The second chapter evaluated the impact of the employment of drill cuttings with castor beans and cramble pies association as agricultural fertilizers by DGGE analysis. The application of this technique was efficient for evaluating treatments, as well as, confirming the treatments contribution for increasing bacterial diversity in soil samples. The third chapter compared the cultivation technique sensitivity for American Foul Brood (AFB) diagnosis, a bee disease caused by Paenibacillus larvae, to cultivation independent technique by PCR. This survey demonstrated that sensitivity of the technique was greater than that one by P. larvae cultivation technique. Thus, the present survey concluded that cultivation independent techniques were useful for granting agriculture sectors demands. / O conhecimento das informa??es gen?ticas bacterianas tem permitido avan?os em diversas ?reas do conhecimento. Esse conhecimento permite detectar microrganismos, bem como avaliar a produ??o de fatores de virul?ncia e resist?ncia em determinadas popula??es. Mais recentemente, o diagn?stico molecular pela detec??o de fragmentos de DNA espec?ficos desses agentes vem sendo implementado em situa??es onde o cultivo bacteriano n?o ? vi?vel ou ? dificultado por condi??es bi?ticas ou abi?ticas. Al?m da detec??o de agentes espec?ficos, o conhecimento da diversidade bacteriana presente em alguns ambientes pode ser um importante crit?rio, como, por exemplo, para avaliar a qualidade do solo. A forma mais comum de avaliar a diversidade bacteriana se d? atrav?s do cultivo. Por?m, a maior parte das popula??es bacterianas presentes no solo n?o s?o cultiv?veis, com isso, a utiliza??o de ferramentas moleculares s?o cada vez mais utilizadas nesse tipo de an?lise. O presente estudo teve como objetivo padronizar a utiliza??o de t?cnicas independentes de cultivo para atender demandas de tr?s setores da agricultura. O primeiro cap?tulo padronizou a utiliza??o da t?cnica de DGGE (Eletroforese em Gel de Gradiente Desnaturante) para an?lise da diversidade bacteriana do leite de tetos mast?ticos e sadios de um mesmo animal. Os resultados apontaram para diferen?as no perfil bacteriano dos dois grupos analisados. O segundo cap?tulo avaliou o impacto sobre a microbiota bacteriana em solos tratados com associa??es de cascalho de perfura??o e tortas de mamona e crambe como adubos agr?colas atrav?s da an?lise de DGGE. A aplica??o da t?cnica de DGGE foi eficiente na avalia??o e confirmou que os tratamentos contribu?ram para o aumento da diversidade bacteriana nas amostras de solos estudadas. O terceiro experimento comparou a sensibilidade da t?cnica de cultivo, oficial para diagn?stico da Cria P?trida Americana (CPA), uma doen?a ap?cola causada por Paenibacillus larvae, com t?cnica de independente de cultivo por PCR do material extra?do direto do mel. Este estudo constatou que a sensibilidade da t?cnica proposta foi superior ? obtida pela t?cnica de cultivo de P. larvae. Com isso, conclui-se que as t?cnicas independentes de cultivo foram ?teis no atendimento das demandas das quest?es dos setores agr?colas contemplados no estudo.

A cultura da cana-de-açúcar é a segunda maior cultura em valor de produção agrícola do país. Nos últimos anos ocorreu uma alteração no cenário canavieiro passando de colheita manual de cana queimada para mecanizada de cana crua. A colheita mecanizada da cana-de-açúcar acarreta muitos benefícios ambientais, porém o tráfego intenso de maquinários resulta na compactação do solo. Desse modo, buscamos avaliar como o efeito das alteração física do solo causado pelo tráfego de máquinas durante a colheita da cana interfere na composição das comunidades microbianas, especialmente as que participam das transformações do nitrogênio. As parcelas experimentais foram divididas em colheita manual (PDTR) e colheita mecanizada (PD). Todos os demais manejos e tratos culturais foram iguais, isolando a influência do impacto do maquinário durante a colheita. Para a avaliação da microbiota realizamos análise da estrutura da comunidade (DGGE ou T-RFLP) e análise de abundância (qPCR) de bactérias, fungos, arquéias, e do ciclo do nitrogênio envolvendo os genes marcadores para a nitrificação (amoA - AOA e AOB), fixação de nitrogênio (nifH) e desnitrificação (nirK, nirS, nosZ clado I e II). Observamos apenas a diferença dos parâmetros físicos na camada superficial por meio da resistência a penetração. A alteração no perfil da comunidade de bactérias e arquéias mostra que ambas são responsivas ao tratamento com tráfego do maquinário. Em relação aos microrganismos envolvidos nas transformações de nitrogênio, AOA foi altamente responsiva ao impacto do tráfego agrícola em ambos os tipos de textura, mas teve diferença na abundância apenas no solo arenoso. O gene nifH apresentou diferença na diversidade em solo argiloso e diferença de abundância em solo arenoso. E por fim, sugerimos que o gene nosZ em subsuperfície pode indicar uma possível campactação antes dos parâmetros físicos do solo. / In Brazil, sugarcane is the second most value of agricultural production. Lately, the harvest management in sugarcane fields has changed from manual harvesting methods with pre-harvest burns to mechanical harvests with green cane (unburnt harvest). The mechanized harvest brings many ambiental benefits. However, the intense traffic due to agricultural machines over the years is resulting in soil compaction. The purpose of this work was to evaluate how the physical effect of mechanized harvesting can interfere with the composition of soil microbial community, specially the microbial involved in the nitrogen cycle. Experimental plots were divided into manual harvest (PDTR) and mechanized harvest (PD). All aspects of crop management are the same, so we were able to study the impact of mechanical harvester traffic during the harvest in isolation. To evaluation of the microbiome we analyzed community structure (DGGE ou T-RFLP) along with their abundance (qPCR) for soil bacterial, fungal and archaeal, and microorganisms involved in the transformation of nitrogen that we used gene markers for nitrification (amoA - AOA and AOB), nitrogen fixation (nifH) and denitrification (nirK, nirS, nosZ clade I and II). We observed the difference between physical parameters only in topsoil by soil penetration resistance. Our results indicated structured community changes of bacterial and archaeal showed us that both are responsive to treatment of machinery traffic. Microorganisms involved in the nitrogen cycle, our results presented that AOA is highly responsive to the impact of agricultural traffic on both soil texture, but we observed the difference in abundance only in sandy soil. The nifH gene was different on community structure in clay soil and on abundance in sandy soil. Moreover, we suggest that nosZ gene could indicate a possible soil compaction before the physical parameters in a layer between 20 and 40 cm.

In this thesis, the application of molecular microbiology methods to understand wastewater treatment bio-reactions is described. Two different wastewater treatment systems were chosen for the experimental work. Firstly; the activated sludge processes at two different facilities in Sweden (Västerås and Eskilstuna) were investigated and compared in a context where low temperatures can affect the efficiency of the nitrogen removal performance in terms of nitrification. Initially, fluorescence in situ hybridization (FISH) was utilised in order to quantify some of the species involved in ammonia and nitrite oxidation at Västerås, providing information on how the different communities react to decreasing temperatures. Then, the polymerase chain reaction (PCR), cloning-sequencing method was employed in order to study the composition of the ammonia oxidizing bacteria (AOB) community at the same two wastewater treatment plants (WWTPs). Secondly; the potential use of constructed wetlands for the treatment of winery wastewater was studied. High ethanol concentration artificial wastewater with and without inorganic nutrients (nitrates and phosphates) was fed in a set of pilot-scale constructed wetlands. Pollutant removal performance and enzyme activity tests were carried out. Additionally, the bacterial community structure was investigated by means of denaturing gradient gel electrophoresis (DGGE). In the first set of studies it was shown that the AOB population which plays a major role in nitrifying reactors presented a seasonal shift and a higher diversity at Västerås during winter time, while the nitrification performance maintained stable levels and the ammonia removal efficiency increased. Thus, the higher ammonia removal efficiency at Västerås could be related to the diversity of the AOB population composition. Lastly, when constructed wetlands were in focus, the differential effects of ethanol and nutrients over the chemical oxygen demand (COD) removal performance were proven. In fact, the addition of nutrients on one of the experimental wetlands increased the COD (ethanol) removal and supported the maintenance of a bacterial population similar to the control wetland (no ethanol added). In conclusion, both studies proved a strong relationship between process performance (pollution removal) and the dynamics of the bacterial communities involved.

<p>The metagenomic approach to gene discovery circumvents conventional gene and gene product acquisition by exploiting the uncultured majority of microorganisms in the environment. It was demonstrated in this study that metagenomic methods are suitable for gene mining in extreme environments that harbor very high levels of unculturable microorganisms. DNA was extracted from Antarctic mineral soil samples taken from the Miers Valley, Antarctica. The metagenomic DNA was also used to construct a fosmid library comprising over 7900 clones with an average insert size of 29 kb. PCR amplification using bacterial and archaeal 16S rRNA gene specific primers and subsequent denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rDNA amplicons showed that a small percentage of bacterial diversity (&gt / 1%) was captured in the metagenomic fosmid library. Activity-based screening for lipase and esterase genes using a tributyrin plate assay yielded twelve positive clones. LD1, a putative, novel cold-active GDSL lipase/esterase was identified and sequenced. The C-terminal domain of the ORF was found to be an autotransporter similar to those associated with type V secretion systems in Gram negative bacteria. Sub-cloning of the gene resulted in lipolytic activity in E. coli. Preliminary enzyme assays have determined that LD1 hydrolyses p-nitrophenyl esters with chain lengths shorter than C10, an indication that the enzyme is an esterase. Complete purification and characterisation of this enzyme is subject to further study.</p>

Bacteria play important roles in the transformation of nutrients in wetlands, but few studies have examined parameters affecting variation in bacterial community composition between wetlands. We compared the composition of eubacterial and denitrifying bacterial biofilm communities in 32 agricultural constructed wetlands in southern Sweden, and the extent to which wetland environmental parameters could explain the observed variation. Structure and richness of the eubacterial 16S rRNA gene and three denitrifying bacterial enzyme genes (nirK, nirS and nosZ), analysed by molecular fingerprinting methods, varied among the constructed wetlands, which could be partly explained by different environmental parameters. Results from the enzyme gene analyses were also compared to determine whether the practice of using a single denitrifying bacterial gene could characterize the overall community composition of denitrifying bacteria. We found that nirK was more diverse than both nirS and the nosZ, and the band structure and richness of the three genes were not related to the sam environmental parameters. This suggests that using a single enzyme gene may not suffice to characterize the community composition of denitrifying bacteria in constructed agricultural wetlands. / <p>Included in doctoral thesis: Milenkovski, Susann. Structure and Function of Microbial Communities in Constructed Wetlands - Influence of environmental parameters and pesticides on denitrifying bacteria. Lund University 2009.</p>

Geosmin is an odorous secondary metabolite produced by some cyanobacteria during growth and released from the cells. Little is known about the biosynthesis of geosmin and the gene(s) required for its production have not been characterized. During late August and early September geosmin episodes due to planktonic cyanobacteria frequently occur in the northwest basin of Lake Ontario waters resulting in taste and odour episodes in drinking water that serves more than 5 million people. At high concentrations geosmin evades traditional drinking water treatment and reaches the tap. These episodes often elicit consumer concern and are wrongly construed to reflect impaired drinking water safety. Water quality managers in the region have generally been unable to prevent or control taste and odour episodes via a proactive approach due to the lack of knowledge of cyanobacterial communities in offshore waters as well as the inability to predict when geosmin will reach intake pipes due to downwelling, the process by which the surface waters mix with the hypolimnion. This study evaluated denaturing gradient gel electrophoresis (DGGE) as a molecular tool for proactive monitoring of potential taste and odour-causing cyanobacteria in environmental samples. The 16S rRNA gene was assessed for its ability to distinguish among geosmin-producing and non-producing strains. This study also examined the evolutionary relationships among geosmin-producing cyanobacteria using the full-length 16S rRNA gene and compared phylogenies with current taxonomy. <br /><br /> A DGGE standard using the V3 hypervariable region of the 16S rRNA gene was developed using geosmin-producing and non-producing isolates of cyanobacteria. Included in the standard was the suspected primary contributor to Lake Ontario taste and odour, <em>Anabaena lemmermannii</em> Richter. This standard was then applied to various environmental collections from Lake Ontario (August 2005) to examine the cyanobacterial community composition. DGGE profiles were consistent with the presence of <em>An. lemmermannii</em> at locations with increased geosmin concentrations (determined using gas chromatography-mass spectrometry), supporting hypothesis that <em>An. lemmermannii</em> is the primary contributor to northwestern Lake Ontario taste and odour. In addition, the application of DGGE in the identification of potential geosmin-producing species of cyanobacteria was deemed to be a potentially useful approach to monitoring cyanobacterial communities in source waters. The 16S rRNA-V3 region alone did not distinguish among geosmin-producing and non-producing strains, however with additional data (actual geosmin concentration) it was showed relationships. <br /><br /> In the phylogenetic analyses, geosmin-producing cyanobacteria did not group monophyletically and it was not possible to state that a single evolutionary event has led to the acquisition of the geosmin-producing trait. Phylogenies also showed that the taxonomy of the Cyanobacteria is largely unresolved. All five Sections (bacteriological classification)/four orders (Komárek & Anagnostidis classification) were paraphyletic, however the heterocystous cyanobacteria (Sections IV and V/Nostocales and Stigonematales) grouped separately from the non-heterocystous cyanobacteria (Sections I, III/Chroococcales and Oscillatoriales). Although both systems of classification compared in this study were similar, nomenclature and groupings were occasionally different among the groups. This demonstrates the incongruity between bacteriologists and phycologists and emphasizes the need for a consensus system of classification for the Cyanobacteria.