Nitrification is the biological process whereby ammonia is converted first to nitrite by ammonia-oxidizing bacteria, and then the nitrite is subsequently converted to nitrate by nitrite-oxidizing bacteria. Ammonia and nitrite levels are closely monitored during treatment of wastewater due to their toxicity to other biological processes. Sybron Chemicals, Inc., is a company that manufactures a nitrifying bioaugmentation culture (1010N) that is used to enhance the naturally occurring levels of biological nitrification. The microbial population of the 1010N product has been examined using a combination of conventional bacteriological methods and modern molecular techniques, with the goal of developing nucleic acid probes that can be used to detect the product in an environmental sample. Small regions of the 16S rRNA genes of the bacteria in 1010N (and two new nitrifying enrichment cultures) were amplified via the polymerase chain reaction (PCR) and analyzed via temperature gradient gel electrophoresis (TGGE). TGGE is a procedure that allows for separation and visualization of individual PCR products that are the same size, based on differences in their sequence. Two of the predominant PCR products in 1010N were purified from the TGGE gel matrix, reamplified via PCR, and sequenced to allow for phylogenetic analysis and nucleic acid probe design. Coincidentally, two strains (NS500-9 and MPN2) that had been isolated from the 1010N mixed consortium and grown in pure culture were found, via TGGE, to have identical 16S rRNA sequences to the PCR products under investigation. Nearly the full-length 16S rRNA genes from these two organisms were PCR amplified, cloned, and sequenced in order to provide a basis for more accurate phylogenetic analysis. The two dominant organisms in the 1010N product, NS500-9 and MPN2, were thereby found to be most closely related to Nitrosomonas and Nitrobacter, respectively, in the existing database. Using the nucleic acid sequences of the cloned DNA, organism-specific DNA probes were designed for both NS500-9 and MPN2. Unfortunately, difficulties were encountered in using the probes to monitor 1010N activity levels via quantitative dot blot hybridizations (rRNA-DNA). Therefore, efforts were redirected to using the TGGE semi-quantitatively with an internal PCR standard (Brüggeman, et al., 2000) to estimate original cell numbers of 1010N within a mixed consortium. This method was not applicable to our system due to substantial preferential binding of the primers to template other than the standard. Samples from a laboratory-scale bioreactor, bioaugmented with 1010N, were used in an attempt to correlate an increase in activity with a detectable shift in population via TGGE. No detectable shift in population was detected in these samples even though the system exhibited increased levels of nitrification. Therefore, the sensitivity of the TGGE system was also examined by determining the limits of detection when 1010N was present in activated sludge. In both whole cell spiking experiments and genomic DNA spiking experiments, it was found that 1010N must be present at a level of at least 5% of the total population in order to be detected. While this provides some information about microbial populations, in order to evaluate the biological activity of a system, nucleic acid probes should be used in a rRNA based study. / Master of Science

BTX biodegradation rates, biochemical expression patterns and microbial ecology were studied under anoxic (denitrifying), anoxic/microaerobic/aerobic, and anoxic/microaerobic conditions in activated sludge sequencing batch reactors. The studies showed that toluene and m-xylene were denitrified via benzoyl-CoA reductase. Although benzene, o-, and p-xylene were recalcitrant under denitrifying conditions, they were biodegraded under microaerobic (< 0.2 mg/L dissolved oxygen) and nitrate or nitrite (NOx)-supplemented microaerobic conditions. The patterns of the specific enzymes associated with BTX biodegradation under microaerobic conditions indicated that the three compounds were metabolized by oxygen-dependent pathways. The expression levels of catechol 1, 2-dioxygenase and catechol 2, 3-dioxygenase under microaerobic conditions were induced to levels as high as under aerobic conditions (> 4 mg/L dissolved oxygen). Benzene, o-, and p-xylene biodegradation rates were twice as fast under NO_x-supplemented compared to NO_x-free microaerobic conditions, and the specific biodegradation rates under aerobic and NO_x-supplemented microaerobic conditions were comparable. 16S rRNA probes targeting representative toluene-degraders were used to investigate the microbial communities in the three sequencing batch reactors by using a dot blot hybridization technique. The hybridization results suggest that multiple redox environments fostered a more diverse microbial community and the activities of the target organisms in the reactors with multiple redox environments were higher than in the single redox reactor. Additionally, facultative toluene-degraders appeared to play a less significant role than the strict anoxic and aerobic toluene-degraders in all three SBRs. / Ph. D.

Marginale Parodontitis, die multikausale Erkrankung des Parodonts ist eine Infektionskrankheit, modifiziert durch Wirtsfaktoren und äußere Einflüße. Die als pathogene Mischflora bezeichnete Kombination kommensaler Mikroorganismen spielt die primäre Rolle in der Ätiopathogenese der Parodontitis. In der Aufstellung des Studienziels wurden einzelne Bakterienarten (T. forsythensis, P. gingivalis, A. actinomycetemcomitans, C. rectus, F. nucleatum, Fusobacterium spp., P. intermedia, E. corrodens, V. parvula und C. ochracea) ausgewählt, die eventuell als "Markerkeime" in der aggressiven Form der Parodontitis betrachtet werden können. Dazu wurde eine Kontrollgruppe untersucht, die eine gesunde parodontale Flora besitzt. Die angewandte Nachweismethode basiert auf der PCR-Amplifikation von 16S rDNA und darauffolgender dot-blot Hybridisierung mit Oligonukleotidsonden. Die entsprechenden Sonden wurden hergestellt, optimiert und evaluiert. Für die epidemiologische Untersuchung wurde subgingivale Plaque von vier Parodontaltaschen und einer Kontrollstelle von 45 Patienten mit aggressiver Parodontitis, sowie an fünf Stellen von 21 Senioren entnommen. Die Prävalenz der einzelnen Bakterienarten wurde mit Hilfe des Chi-Quadrat Test verglichen. Obgleich eine hohe interindividuelle Variabilität der Kolonisationsmuster zu beobachten war, konnten T. forsythensis, P. gingivalis, C. rectus und F. nucleatum signifikant häufiger in den Parodontaltaschen als an den gesunden Stellen nachgewiesen werden und können deswegen als "Leitkeime" der aggressiven Parodontitis angesehen werden. A. actinomycetemcomitans konnte nur bei einzelnen Patienten mit aggressiver Parodontitis festgestellt werden. Die Ergebnisse für P. intermedia und E. corrodens ließen keine eindeutige Assoziation sowohl mit der aggressiven Parodontitis als auch mit dem gesunden Parodontalzustand zu. Bei Senioren wurde C. ochracea besonders häufig nachgewiesen. Die Ergebnisse dieser Studie bewiesen die erfolgreiche Einsetzbarkeit der hergestellten Oligonukleotidsonden. / A multifactorial risk pattern of periodontitis has been recognized, where in addition to host and environmental factors, a pathogenic microbiota plays a primary role. The purpose of the current research was to analyze the prevalence of periodontitis-associated microorganisms in patients with aggressive periodontitis and periodontally healthy elders by using molecular-biologic detection methods like eubacterial PCR-amplification of 16S rDNA in combination with dot-blot hybridization. The oligonucleotide probes for the detection of T. forsythensis, P. gingivalis, A. actinomycetemcomitans, C. rectus, F. nucleatum, Fusobacterium spp., P. intermedia, E. corrodens, V. parvula and C. ochracea were designed and evaluated. The PCR products of 42 cultivated target and closely related bacteria were used for the optimization of hybridization conditions. For the epidemiological study, subgingival plaque was sampled from four pockets and one healthy site of 45 aggressive periodontitis patients as well as from five sites of 21 elderly. The differences in the prevalence of bacterial species were analyzed by the chi-square test. The data revealed frequent colonization by T. forsythensis, P. gingivalis, F. nucleatum and C. rectus in patients with aggressive periodontitis, however individual variations were obvious. These species could be predominantly identified in periodontal pockets, but were significantly less common in the healthy sites of the periodontitis patients and in the elderly. These putative pathogens can be conclusively determined as the key-bacteria in patients with aggressive periodontitis. No direct association for P. intermedia and E. corrodens with aggressive periodontitis or periodontal health could be seen. A. actinomycetemcomitans could be detected in only a few patients, reducing its suspected importance in the etiology of aggressive periodontitis. C. ochracea was highly prevalent in the well-maintained elderly, suggesting its association with healthy flora. The results of the study confirmed the reliability of the oligonucleotide probes in a specific and sensitive detection of the respective oral species.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Os begomovÃrus, vÃrus da famÃlia Geminiviridae transmitidos por mosca branca, tÃm emergido como sÃrios patÃgenos de culturas agronÃmicas e hortÃcolas em regiÃes tropicais e subtropicais de muitos paÃses em todo o mundo. A partir da dÃcada de 80, tÃm aumentado os relatos da disseminaÃÃo da mosca branca, Bemisia argentifolii, e de begomovÃrus provocando impacto devastador nas regiÃes em que ocorrem. No Brasil, estes patÃgenos tÃm sido limitantes para a produÃÃo de tomate (Lycopersicon esculentum) em vÃrias Ãreas de cultivo com incidÃncia crescente desde 1994. No presente trabalho, plantas de tomate exibindo sintomas de infecÃÃo provocada por vÃrus como mosaico, clorose internerval, enrolamento do limbo foliar e reduÃÃo do crescimento, foram coletadas em lavouras de tomate indÃstria em AnÃpolis-GO. O vÃrus foi identificado como pertencente ao gÃnero Begomovirus mediante tÃcnica de PCR usando oligonucleotÃdeos especÃficos que amplificaram fragmentos dos componentes A e B do genoma viral. No capÃtulo I sÃo apresentados os resultados da caracterizaÃÃo molecular e no capÃtulo II, os dados da determinaÃÃo do cÃrculo de hospedeiros e da investigaÃÃo da relaÃÃo do begomovÃrus com o vetor Bemisia argentifolii. O isolado denominado GOANPL, foi clonado e parcialmente seqÃenciado tendo sido obtidas as seqÃÃncias nucleotÃdicas dos genes da capa proteica, Rep e de toda a regiÃo intergÃnica, em um total de 2.130 nucleotÃdeos. A anÃlise comparativa das seqÃÃncias revelou que, em geral, o GOANPL possui relacionamento genÃtico distante com begomovÃrus da Ãsia, Europa e Ãfrica sendo mais prÃximo das espÃcies do Brasil, particularmente, com os begomovÃrus identificados em Minas Gerais (TRMV) e no Distrito Federal (DF-BR2). Com este Ãltimo, apresentou alta homologia em todo o genoma podendo vir a constituir, com o mesmo, uma nova espÃcie. A determinaÃÃo do cÃrculo de hospedeiros do GO-ANPL foi realizada inoculando-se 46 espÃcies vegetais pertencentes a nove famÃlias botÃnicas, sob duas modalidades de inoculaÃÃo: mecÃnica e com a mosca branca. Constatou-se que o GO-ANPL infecta, preferencialmente, plantas da famÃlia Solanaceae como Nicotiana benthamiana, Datura stramonium e Nicandra physalodes. O nÃmero de espÃcies infectadas com o inseto vetor foi superior ao obtido pela inoculaÃÃo mecÃnica e diferiu dos resultados obtidos para outros isolados de begomovÃrus de tomate no Brasil. Os testes foram todos confirmados com hibridizaÃÃo com sondas moleculares, em \"dot blot\"No estudo da relaÃÃo vÃrus-vetor, foram investigados o perÃodo de acesso de aquisiÃÃo do vÃrus (PAA), o perÃodo de acesso de inoculaÃÃo do vÃrus (PAI), e o perÃodo de latÃncia do vÃrus na fase adulta do vetor, empregando-se cinco moscas/planta de tomate \'Santa Clara\' em todos os tratamentos. Para a definiÃÃo do PAA e do PAI, foram testados nove diferentes perÃodos de tempo: 0,25, 0,5, 1, 2, 4, 8, 16, 20 e 24 horas. Nos testes para determinaÃÃo do PAA, apÃs cada um desses perÃodos seguiu-se uma inoculaÃÃo de 48 horas e para definiÃÃo do PAI, antes de cada perÃodo antecedeu-se um perÃodo de acesso de aquisiÃÃo fixo de 72 horas. Constatou-se que o PAA mÃnimo da mosca branca foi de apenas 0,25 hora, com o qual foram obtidas 6% de plantas infectadas. O percentual de plantas aumentou de 6 para 65% com a extensÃo do PAA de 0,25 para 24 horas. Com relaÃÃo ao perÃodo de acesso de inoculaÃÃo do vÃrus, foram registrados 18% de plantas infectadas com o PAI de 0,5 hora. O percentual elevou-se para 67% quando 24 horas de PAI foram concedidos. Valores isolados de 90 e 100% na transmissÃo viral, tambÃm foram observados. O tÃrmino do perÃodo latente do vÃrus no vetor ocorreu 16h apÃs a aquisiÃÃo do mesmo em planta infectada, considerando os 3% de infecÃÃo observados nas plantas inoculadas. Os dados obtidos indicam que a interaÃÃo vÃrus-vetor à estabelecida desde a fase inicial de desenvolvimento do inseto. Como parte do estudo dessa interaÃÃo, avaliou-se a presenÃa do begomovÃrus GO-ANPL em todas as fases de desenvolvimento do inseto vetor (ovo, 1 ao 4 Ãnstar e adulto) na planta infectada, em adultos com diferentes PAA, na progÃnie de fÃmeas virulÃferas e em adultos cujos estÃgios ninfais desenvolveram-se em tomateiro infectado. A tÃcnica PCR foi empregada para a detecÃÃo do GO-ANPL em mais de 2.500 espÃcimens testados. O vÃrus foi detectado em ninfas do 1 ao 4 Ãnstar que se alimentaram em plantas de tomate infectada, contudo, em ovos provenientes de avirulÃferas, os quais foram ovipositados em planta infectada e coletados apÃs sete dias, o vÃrus nÃo foi detectado. A transmissÃo à progÃnie foi constatada pela detecÃÃo do vÃrus em ovos, ninfas e adultos que se desenvolveram em planta nÃo hospedeira do vÃrus. A transmissÃo transestadial ocorreu com Ãndice elevado e, ao lado da transmissÃo à progÃnie, indica que a retenÃÃo do vÃrus à uma etapa importante da interaÃÃo vÃrusâvetor. A transmissÃo do vÃrus para mudas de tomate, a partir de adultos da progÃnie de fÃmeas virulÃferas, nÃo foi constatada. Contudo, transmissÃo para tomateiro em um percentual de 33% foi verificado nos casos em que a inoculaÃÃo das plantas foi realizada pelos adultos que retiveram o vÃrus da sua fase imatura (transestadial) / The whitefly-transmitted viruses from the family Geminiviridae, genus Begomovirus, have been reported as an economically important pathogen group that affect important crops in tropical and subtropical countries. Since the beginning of the 1980 decade, the occurrence of the whitefly associated to Begomovirus infection has drastically increased worldwide. In Brazil, these pathogens have been responsable for severe economical losses in tomato (Lycopersicon esculentum) orchards and the production has hampered since 1994. In this work, infected tomato plants showing symptoms, such as mosaic, intervein clearing, leaf curling and growth reduction were collected in tomato orchards in AnÃpolis, State of GoiÃs. The virus was identified as a member of the genus Begomovirus by PCR reaction, using specific primers to amplify fragments of A and B components of the virus DNA genome. The Chapter I of this thesis presents the results of the molecular characterization of the virus and the Chapter II shows the determination of its host range and the relationship with its natural vector Bemisia argentifolii. The virus isolate denoted GO-ANPL was cloned and partially sequenced. Part of the sequenced genome (2.180 nucleotides long) corresponded to the coat protein and Rep genes and comprised the entire intergenic region. Sequence comparison revealed that the GO-ANPL isolate is distantly related to the begomoviruses found in Asia, Europe and Africa, and it is related to other begomoviruses reported in Brazil. The virus isolate showed to be more closely related to viruses found in the State of Minas Gerais (TRMV isolate) and in the Federal District (isolate DF-Br2). The highest homology was observed with the isolate DF-Br2 and it may represent a new specie of the genus Begomovirus. In order to determine the virus host range, 46 plant species from nine different botanical families were mechanically and using the virus vector inoculated. The GO-ANPL isolate preferentially infected plants of the family Solanaceae as Nicotiana benthamiana, Datura stramonium and Nicandra physalodes. The number of infected plants was higher when they were inoculated by the virus vector, and the results were distinct from those obtained for other begomoviruses reported in Brazil. Viruses infections were all confirmed by dot blot hybridization using specific molecular probes to the virus. 4 To study virus/vector interaction, the acquisition access period (AAP), inoculation access period (IAP), and the latent period were determined transfering five whiteflies per plant and using tomato cv. Santa Clara as the host. For the AAP and IAP, nine different time periods were tested: 0.25, 0.5, 1, 2, 4, 8, 16, 20 and 24 h. The minimal AAP determined was 0.25 h, after which, 6% of the tested plants became infected. The number of infected plants increased to 65% with an AAP of24h.Afteran IAP of 0.5 h, 18% of the plants were infected and their number increased to 67% after an IAP of 24 hours. The latent period was considered to be 16 h, after which, 3% of the inoculated plants became infected. The results of AAP, IAP and latent period seem to indicate an early interaction between virus and vector starting at early stages of vector development. The presence of the GOANPL was determined in all stages of the vector (eggs, 1st to 4th instar and adults) in infected plants, in adults under different AAPs, in the progenies of viruliferous females, and in adults originated from nymphs developed from infected plants. More than 2.500 insects were tested by PCR to detect the GO-ANPL isolate. The virus was detected in nymphs from the 1st to 4th instar that had fed in infected plants and no virus was found in eggs from aviruliferous female that had been laid in infected plants. Transmission to the progenies was observed, since the virus was detected in all stages of insect development from eggs to adults. High level transmission was also observed in newly emerged adults that had acess to virus-infected plants as immatures. This fact, in addition to the transmission to the progenies, suggests that virus retention is an important part of virus/vector interaction. No transmission was observed from adults originated from viruliferous females. However, 33% of virus transmission was obtained when adults that retained virus from their early larval stages were employed