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..

The microbial populations in the gastrointestinal tracts of poultry play an important role in normal digestive processes and in maintaining animal health. The purpose of this study was to evaluate the effects of probiotics, prebiotics and synbiotics on the growth parameters, gut ecosystem, histology and immune function. In this study, four experiments one in vitro and three in vivo were conducted using specific pathogen free (SPF) and Hubbard broiler chickens. The first experiment was designed to determine the influence of inulin as an effective prebiotic on lactic acid bacteria (LAB) strains, and to screen LAB for selection as a source of chicken probiotic. Eight strains of LAB were isolated from chicken caeca and three strains from the Plymouth University culture collection were screened for potential probiotic properties for growth in inulin from Jerusalem artichoke (Helianthus tuberosus) and commercial inulin (Frutafit® HD, Netherlands). Lactobacillus animalis JCM 8692 strain isolated from chicken caeca showed the highest auto-aggregation and co-aggregation ability, resistance to acidity and bile salts, strong suppression of pathogens and ability to adhere to epithelial cells compared with other isolated strains. The second experiment was conducted to investigate the influence of commercial inulin and Jerusalem artichoke tubers as prebiotic supplementation on the diversity of the caecal microflora, jejunum histology and immune organ of SPF chickens. This investigation has found that inulin which was extracted from JA had a similar result when compared with commercial inulin and could be a suitable candidate for an inulin source in broiler diets. The third experiment was conducted to investigate the influence of Bactocell® (PRO1) and Lb. animalis (PRO2) as probiotic supplements on broiler chickens. EPEF was significantly increased in probiotic1 and probiotic2 compared with control (311.03, 309.87 and 260.06) respectively. Both types of probiotics supported the growth of chicks healthy and could be a suitable candidate as a source of probiotic in broiler diet. The fourth experiment was conducted to investigate the influence of dietary supplementation of a probiotic (Lb. animalis), a prebiotic JA tuber and a combination of both (Synbiotic) in broiler chickens. Growth performance was improved in all additive supplementation compared with the control group. EPEF was increased in probiotic, prebiotic and synbiotic compared with control (290.8±11.8, 300.9±3.86, 322.1±7.09 and 262.3±5.94) respectively. Beneficial bacteria in the guts of chicks fed probiotic, prebiotic and synbiotic was increased compared with chicks fed control diet. The diversity of microbial population in the gastrointestinal tract of chickens improved due to additives. The intestinal villus lengths and microvilli density was improved in all additives supplementation in comparison with control. Overall, it was concluded that probiotic, prebiotic and synbiotics can positively affect production performance and can improve the gut health.

Magister Scientiae - MSc / 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 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. / South Africa

Visible lesions on coral colonies are potential indicators that environmental stressors are influencing a reef. To test this hypothesis, pairs of near-shore reefs on Taiwan were surveyed along an anthropogenically influenced gradient that included locations near the cities of Taipei and Taitung, and more remote reefs off Green Island. Two fringing reefs at Sanya, Hainan Island, a popular Chinese resort area, were also assessed. Field surveys were undertaken to detect, quantify and visually describe the occurrence of lesions at each site. Coral mucus samples were collected from both normal-appearing polyps and lesion-afflicted areas of colonies to assess carbon requirements of associated microbes. Tissue samples were also collected to identify bacterial communities inhabiting healthy tissue for comparison with those associated with lesions; denaturing gradient gel electrophoresis and 16S rRNA sequencing for bacterial identification were utilized in these analyses. In addition, tissue samples were collected in the vicinity of lesions and prepared for histological examination. At sites in Taiwan, lesions were encountered twice as often at the sites near Taipei and Taitung than at Green Island. The fewest (15/72 sightings) lesions were encountered at the reefs near Sanya, primarily because there has been nearly an 80% loss of coral cover at Sanya in recent decades. Overall, tissue loss was the most common lesion recorded (52%), followed by pink discoloration (27%) and color loss (i.e., bleaching, 15%). Porites was the taxon most commonly observed with one or more lesions (45% of sightings). Microbes within mucus from lesioned areas utilized similar carbon sources as microbes from mucus from healthy polyps, but utilized those sources more than twice as often. Examples of carbon sources utilized by microbes in >50% of the lesion samples were D-cellobiose, D-mannitol, N-acetyl-D-glucosamine, alpha-cyclodextrin, and glycogen. Bacterial assemblages on corals were significantly different between Taiwan and China, among sites, and between water samples and coral samples, but not between healthy samples and lesions. Bacterial sequences identified in tissue samples from lesions revealed the presence of well-known disease-related genera, such as Clostridium and Vibrio. Microbes specifically indicating anthropogenic sources, included Bacillus sp. (sewage sludge) and Geobacillus thermolevorans (irritable bowel syndrome). Histological examination of tissue samples, particularly those from lesions characterized as tissue loss, revealed fragmentation and detachment from the mesoglea of gastrodermis and epidermis, as well as brown granular material, and the presence of ciliates and small crustaceans. Corals are susceptible to a variety of diseases. For reefs in the western Atlantic and Caribbean, occurrences of lesions and characterization of coral diseases have been relatively well documented. In contrast, many areas in the vast Indo-Pacific, including the reefs of Taiwan and China, have received much less attention. This study of lesions and associated microbiomes on nearshore reefs of Taiwan and Hainan Island supports previous research that has revealed higher incidences of coral lesions and disease in reefs near extensive human populations. The results also support the hypothesis that many of the microbes associated with coral lesions are part of the natural coral microbiome and that some microbes can become opportunistic when the host corals are stressed.

>Magister Scientiae - MSc / The extreme conditions of Antarctic desert soils render this environment selective towards a diverse range of psychrotrophic microbial communities. Cracks and fissures in translucent quartz rocks permit an adequate amount of penetrating light, sufficient water and nutrients to support cryptic microbial development. Hypolithons colonizing the ventral surface of these quartz rocks have been classified into three types: cyanobacterial dominated (Type I),moss dominated (Type II) and lichenized (Type III) communities. Eukaryotic microbial communities were reported to represent only a minor fraction of Antarctic communities. In this study, culture independent techniques (DGGE, T-RFLP and clone library construction) were employed to determine the profile of the dominant eukaryotes, fungi and microalgae present in the three different hypolithic communities. The 18S rRNA gene (Euk for eukaryotes), internal transcribed spacer (ITS for fungi) and microalgal specific regions of the 18S rRNA gene, were the phylogenetic markers targeted for PCR amplification from hypolith metagenomic DNA. Results suggest that the three hypolith types are characterized by different eukaryotic, fungal and microalgal communities, as implied by nMDS analysis of the DGGE and T-RFLP profiles. Sequence analysis indicates close affiliation to members of Amoebozoa, Alveolata, Rhizaria (general eukaryote), Ascomycota (fungal) and Streptophyta (microalgal). Many of these clones may represent novel species. This study demonstrates that Dry Valley hypolithons harbour higher eukaryote diversity than previously recognised.Each hypolithon is colonized by specialized microbial communities with possible keystone species. The ecological role of the detected microorganisms in the hypolith environment is also theorized, and a trophic hierarchy postulated.

Entre as principais oleaginosas eleitas para a produção de biodiesel, encontra-se a mamona (Ricinus communis L.), por possuir elevado teor de óleo, conhecido como óleo de rícino, extraído pela prensagem das sementes, contendo 90% de ácido graxo ricinoléico, o qual confere ao óleo suas características singulares, possibilitando ampla gama de utilização industrial. A produção brasileira de mamona concentra-se na caatinga baiana. A agricultura desta região, embora seja feita sem o uso de insumos agrícolas mantém uma produtividade regular. Com isso, tornou-se importante avaliar o efeito ambiental desta cultura sobre os estoques de C (carbono) e N (nitrogênio) no solo e os mecanismos de sua manutenção. Este trabalho foi realizado em solos de uma fazenda na região de Irecê-BA, tendo como objetivos calcular os estoques de C e N no solo; calcular o fluxo de gases do efeito estufa dos resíduos de mamona; calcular os valores C e N da biomassa microbiana no solo (BMS) e analisar o perfil da comunidade de bactérias fixadoras de N2 no solo por PCR-DGGE. Os resultados indicaram a ausência de variações nos estoques de C entre os tratamentos de mamona com 10, 20 e 50 anos de cultivo (48 Mg ha-1) sendo superados pelos valores encontrados na caatinga (90 Mg ha-1); mesma condição para o estoque de N (5,3 Mg ha-1 nos tratamentos de mamona e 8 Mg ha-1 na caatinga). Já os valores de C-microbiano foram superiores na caatinga, mas não apresentaram diferenças entre os cultivos de mamona; para o N-microbiano, os valores não sofreram diferenças entre as camadas, porém foram inferiores no tratamento de mamona com 10 e 50 anos de cultivo. O fluxo de gases de N-N2O e C-CO2 foram maiores para o tratamento com aplicação de resíduos orgânicos de mamona chegando a 160 mg m-2 h-1 de C-CO2 e 600 mg m-2 h-1 de N-N2O. O teste de redução de acetileno (ARA) e a análise de DGGE indicaram que o perfil da comunidade de bactérias diazotróficas do solo nos diferentes tratamentos sofreu alteração e permitiu a estocagem de N no solo durante os 50 anos de cultivo viabilizando, ambientalmente, a produção de mamona nesta região. / Among the main oils elected for biodiesel production is the castor bean (Ricinus communis L.) for its high oil content, known as castor oil extracted by pressing the seeds containing 90% ricinoleic acid, which gives the oil its unique features, allowing a wide range of industrial use. Brazilian production of castor oil is concentrated in the tropical dry forest of Bahia. Farming in this region, although it is made without the use of agricultural inputs remains a regular productivity. With this, it became important to assess the \"environmental effect\" of this culture on the stocks of C (carbon) and N (nitrogen) in soil and the mechanisms for its maintenance. This work was carried out in soils of a farm in Irecê-BA, aimed to calculate the stocks of C and N in soil, calculated the flows of greenhouse gases from oil waste; to figure out the values of carbon (C) and nitrogen (N) of microbial biomass in soil (BMS and to analyze the profile of the community of N2-fixing bacteria in soil through denaturing gradient gel electrophoresis (DGGE). Results indicated the absence of changes in C stocks between treatments of castor oil with 10, 20 and 50 years of cultivation (about 48 Mg ha-1) being outweighed by values found in the tropical dry forest (90 Mg ha-1); same condition verified for the stock of N (5.3 Mg ha-1 treatments of castor and 8 Mg ha-1 in tropical dry forest). As for values of microbial-C, they were higher in the tropical dry forest and did not present differences between the cultures of castor oil; regarding microbial-N values, there have not been differences between the layers, but they were lower for castor oil with 10 and 50 years of cultivation. The gases flow accumulated N-N2O was significantly higher than that of CCO2 and higher for the treatment of soil with organic fertilizer castor reaching 160 mg m- 2 h-1 of C-CO2 and 600 mg m-2 h-1 of N-N2O. ARA analysis and DGGE indicated that the profile of the diazotrophic community of soil in different treatments had change, allowing the storage of N in soil during the 50 years of cultivation enabling, in environmental terms, the production of castor oil in this region.

This work was focused on DNA isolation from cheeses for the use in polymerase chain reaction. First, there was optimised the procedure of homogenisation of different types of cheeses from commercial sources, cell lysis and DNA isolation. DNA was isolated using magnetic microspheres and phenol extraction. It was shown that the DNA was amplified in PCR for domain Bacteria after dilution. Next, there was optimised the procedure of DNA isolation from fresh cheeses and from contaminated fresh cheeses and their pickles. DNA from all samples was amplified in PCR. The presence of DNA of domain Bacteria and yeast DNA was demonstrated. In the last part of the work, there were optimised the preparation of PCR mixtures and bacterial DNA amplification in PCR with primers with clamp (F357-GC and R518). Synthetized PCR products were analysed using DGGE. It was shown that amplicons of DNA isolated from cheeses and pickles differ in positions and numbers. Larger number of bands of different intensities was detected after amplification of DNA isolated from contaminated pickles.

This diploma thesis deals with the molecular characterization of carotenogenic yeasts. The techniques used for the analysis of the conserved regions of the D1/D2 rDNA region of the 26S ribosomal large subunit region and the ITS1 and 5,8-ITS2 regions were nested PCR and DGGE. The results of DGGE show that all analyzed yeast strains have very similar sequences of these regions The yeast Rhodotorula mucilaginosa with the collection number CCY 20-7-28 showed differences from the other carotenogenic yeast strains. As a part of melucular characterisation using ribosomal gene sequences, eight yeast strains were examinated for substrate utilisation tests using different substrates. Characterisation of growth and metabolite production was tested in each strain too. The next aim of this thesis was to prepare a carotenoid yeast strain characterized by overproduction of metabolites, in particular carotenoids and lipids,. Yeasts were subjected to a random mutation caused by UV irradiation and the influence of this mutantagen onthe production of metabolites was evaluated. As a candidate yeast strain C. capitatum CCY 10-1-2 was selected. This selection was based on previous studies due to its good production of lipids using waste glycerol as asubstrate. This strain was subsequently adapted to waste whey, glycerol, and a glucose as a basic carbon source.

Ziel der vorliegenden Arbeit war es, einen Beitrag zur Aufklärung der Ätiologie der Graskrankheit mit Hilfe der DGGE, besonders im Hinblick auf in vitro unkultivierbare Bakterien der Darmflora zu leisten. Es sollte ebenfalls geprüft werden, ob die DGGE die Diagnose der Graskrankheit erleichtern kann. Weiterhin sollte der Einfluß von C. botulinum auf die Erkrankung durch den Nachweis von Toxin, Bakterien und Antikörpern untersucht werden. Es standen zur Untersuchung Proben des Colons, Caecums und Kotes von erkrankten Pferden und Kontrolltieren, Kotproben von klinisch gesunden Pferden, die aus denselben Beständen wie die erkrankten Tiere stammen sowie Serum aller drei Gruppen zur Verfügung. Wegen der hohen individuellen Variabilität der Darmflora war kein eindeutiges Merkmal der Graskrankheit im Profil der mikrobiellen Gemeinschaft des Darmes oder Kotes nachweisbar. Allerdings ließ sich anhand der Clusteranalyse ein Abgrenzung der Flora des Caecums und besonders des Colons der erkrankten und gesunden Tiere erkennen. Für eine Diagnose der Graskrankheit am lebenden Tier anhand der Kotflora ist die DGGE jedoch wegen ihrer geringen Aussagekraft und methodischen Probleme nicht geeignet. Der Verdacht, dass C. botulinum an der Ätiologie der Graskrankheit beteiligt ist, konnte durch die Ergebnisse im Tierversuch und ELISA weiter untermauert werden.

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