South Africa produces an average of 154 million broilers (Gallus gallus domesticus) annually, arising to an estimated 886 million kg of broiler litter. The largest population of broilers are reared in the North West province. Various applications for this largely underexploited resource have been published, including forming part of ruminant diets and direct land application. This however has several disadvantages, as it could lead to eutrophication of fresh water sources and faecal contamination of produce. Windrow composting of broiler litter has previously been studied, and found to deliver a stabilized product free of pathogenic and phytotoxic effects, therefore making it an excellent soil conditioner. This study aimed to characterize the microbial community present during the windrow composting of broiler litter. Four different formulations of substrate were tested; these being broiler litter (Windrow 1), Windrow 1 with previously composted material (Windrow 2), Windrow 2 amended with woodchips (Windrow 3) and Windrow 3 with an additional 12.5% (w/w) zeolite (Windrow 4). Broiler litter used in this experiment had a C:N ration of 10.3:1, whilst the blue gum woodchips added as an amendment had a C:N ratio of 172:1. Windrow and environmental temperatures were monitored on a regular basis. Windrow 1 largely mimicked environmental temperature, and could not sustain a true thermophilic phase during the experimental period. Windrow 2 did achieve a short lived thermophilic phase during the first few days of the composting process, however could not sustain its temperature over the whole period. In contrast Windrows 3 and 4 sustained temperature above 40°C for the largest part of the experimental period, regardless of environmental temperature. No significant difference (p < 0.05) could be observed between average moisture levels in the 4 windrows. Internal moisture profiles were however found to differ significantly, especially on the surface of the windrows. Moisture was also lost faster in Windrows 1 and 2 compared to Windrows 3 and 4. Chemical analysis showed differences between the four windrows constructed. A higher amount of nitrogen was lost in Windrows 1 and 2, mostly due to a sub-optimal initial C:N ratio in these windrows. Windrow 2 contained the highest values for plant nutrients P, Mg, Ca, Mn and Cu. Microbial population dynamics were observed using PCR-DGGE of samples collected throughout the composting of various treatments. Various commercial DNA extraction kits where tested in a previous study for their ability to remove PCR inhibitory substances, such as humic acids. The Machery-Nagel Soil DNA isolation kit was used as it gave amplifiable DNA from all samples. Samples were amplified using a nested PCR approach primer sets 27f-1492r \ 341f(GC)-907r and EF3-EF4 \ EF4(GC)-fung5 (where “GC” indicates a GC-rich clamp) for prokaryotic and eukaryotic species respectively. The PCR products were analyzed by agarose gel electrophoresis, and equal amounts of product were subjected to denaturing gradient gel electrophoresis (DGGE). Bands obtained from these polyacrylamide gels where then re-amplified using the same secondary primer sets (without the GC-clamp), and sequenced. A total of 454 prokaryotic bands in 55 distinct rf-positions were observed. Seven distinct rf-positions were observed in eukaryotic DGGE profiles. Prokaryotic profiles were aligned and the microbial diversity was analyzed by means of Ward’s clustering algorithm and the dice coefficient of similarity, as well as Simpson’s reciprocal, Shannon-Weaver and Species richness indices. Canonical correspondence analysis (CCA) was also performed on both the banding patterns as well as the bands present, together with the physico-chemical results obtained. Several bands were successfully identified as being influenced by physico-chemical parameters. Temperature, C:N ratio, ash, and moisture showed a correlation on CCA bi-plots. Sixteen bands were sequence identified. These sequences were compared to two different databases. The 16S rRNA database for Bacteria and Archaea gave identities to genus level, however maximum identity scores were low. Of the 16 sequences, 12 sequences were identified as uncultured bacteria when compared to the nucleotide collection database. In comparing the sequences with sequences collected in the nucleotide collection database, 12 were either first described in composts and soils, or animal manures. Results indicated mostly members of the genus Bacillus and Paenibacillus. The addition of a carbon source greatly affected the microbial metabolism, resulting in a thermophilic phase being achieved in amended windrows. As no thermophilic phase was observed in windrows that were not amended with woodchips, it could be concluded that the use of a carbon source is irremissible when composting broiler litter. A zeolite amendment is also strongly advised, as this further increased temperatures within the windrow. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

South Africa produces an average of 154 million broilers (Gallus gallus domesticus) annually, arising to an estimated 886 million kg of broiler litter. The largest population of broilers are reared in the North West province. Various applications for this largely underexploited resource have been published, including forming part of ruminant diets and direct land application. This however has several disadvantages, as it could lead to eutrophication of fresh water sources and faecal contamination of produce. Windrow composting of broiler litter has previously been studied, and found to deliver a stabilized product free of pathogenic and phytotoxic effects, therefore making it an excellent soil conditioner. This study aimed to characterize the microbial community present during the windrow composting of broiler litter. Four different formulations of substrate were tested; these being broiler litter (Windrow 1), Windrow 1 with previously composted material (Windrow 2), Windrow 2 amended with woodchips (Windrow 3) and Windrow 3 with an additional 12.5% (w/w) zeolite (Windrow 4). Broiler litter used in this experiment had a C:N ration of 10.3:1, whilst the blue gum woodchips added as an amendment had a C:N ratio of 172:1. Windrow and environmental temperatures were monitored on a regular basis. Windrow 1 largely mimicked environmental temperature, and could not sustain a true thermophilic phase during the experimental period. Windrow 2 did achieve a short lived thermophilic phase during the first few days of the composting process, however could not sustain its temperature over the whole period. In contrast Windrows 3 and 4 sustained temperature above 40°C for the largest part of the experimental period, regardless of environmental temperature. No significant difference (p < 0.05) could be observed between average moisture levels in the 4 windrows. Internal moisture profiles were however found to differ significantly, especially on the surface of the windrows. Moisture was also lost faster in Windrows 1 and 2 compared to Windrows 3 and 4. Chemical analysis showed differences between the four windrows constructed. A higher amount of nitrogen was lost in Windrows 1 and 2, mostly due to a sub-optimal initial C:N ratio in these windrows. Windrow 2 contained the highest values for plant nutrients P, Mg, Ca, Mn and Cu. Microbial population dynamics were observed using PCR-DGGE of samples collected throughout the composting of various treatments. Various commercial DNA extraction kits where tested in a previous study for their ability to remove PCR inhibitory substances, such as humic acids. The Machery-Nagel Soil DNA isolation kit was used as it gave amplifiable DNA from all samples. Samples were amplified using a nested PCR approach primer sets 27f-1492r \ 341f(GC)-907r and EF3-EF4 \ EF4(GC)-fung5 (where “GC” indicates a GC-rich clamp) for prokaryotic and eukaryotic species respectively. The PCR products were analyzed by agarose gel electrophoresis, and equal amounts of product were subjected to denaturing gradient gel electrophoresis (DGGE). Bands obtained from these polyacrylamide gels where then re-amplified using the same secondary primer sets (without the GC-clamp), and sequenced. A total of 454 prokaryotic bands in 55 distinct rf-positions were observed. Seven distinct rf-positions were observed in eukaryotic DGGE profiles. Prokaryotic profiles were aligned and the microbial diversity was analyzed by means of Ward’s clustering algorithm and the dice coefficient of similarity, as well as Simpson’s reciprocal, Shannon-Weaver and Species richness indices. Canonical correspondence analysis (CCA) was also performed on both the banding patterns as well as the bands present, together with the physico-chemical results obtained. Several bands were successfully identified as being influenced by physico-chemical parameters. Temperature, C:N ratio, ash, and moisture showed a correlation on CCA bi-plots. Sixteen bands were sequence identified. These sequences were compared to two different databases. The 16S rRNA database for Bacteria and Archaea gave identities to genus level, however maximum identity scores were low. Of the 16 sequences, 12 sequences were identified as uncultured bacteria when compared to the nucleotide collection database. In comparing the sequences with sequences collected in the nucleotide collection database, 12 were either first described in composts and soils, or animal manures. Results indicated mostly members of the genus Bacillus and Paenibacillus. The addition of a carbon source greatly affected the microbial metabolism, resulting in a thermophilic phase being achieved in amended windrows. As no thermophilic phase was observed in windrows that were not amended with woodchips, it could be concluded that the use of a carbon source is irremissible when composting broiler litter. A zeolite amendment is also strongly advised, as this further increased temperatures within the windrow. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

The aim of this study was to determine the effects of the heavy metal pollution on microbial diversity along the gradient from a platinum mine tailings dam using culture-dependent (plating methods) and molecular methods. Tailings and soil samples were collected from seven sites (6 samples per site) at increasing distances from the tailings dam. Samples were collected over a two year period and included two rainy and two dry periods. Concentrations of various heavy metals were determined using an inductively coupled plasma mass spectrometer (ICP-MS). The results demonstrated that seasonal variations in metal concentrations occurred and also that concentrations were significantly different'(P < 0.05) between the experimental sites for each metal. The relative relationship between metals was in the following order: Al > Ni > Cu > Cr. Since soil metal concentration benchmarks for South Africa are lacking, the concentrations were compared to the Canadian microbial benchmarks (MB) and Netherlands maximum permissible concentrations (MPC). Concentrations of most of the heavy metals exceeded the MB and MPC. Levels and diversity of culturable fungi and bacteria at each site were determined using plate count methods. Results indicated that levels of bacteria and fungi were not suppressed by high concentrations of heavy metals. Significantly higher levels (P < 0.05) of fungi were found at the sites on the tailings dam (higher concentrations of heavy metals), compared to sites more than 300 m away. A commonly used soil health index (Shannon-Weaver diversity index) was used to compare microbial community diversity at each site and to evaluate whether or not the heavy metal contamination impacted negatively on these soil bacterial and fungal communities. Shannon-Weaver diversity indices were higher at sites on and close to the tailings dam than sites more than 300 m away. However, ratio of fungal to bacterial levels as determined by plate counts was inconsistent. Representatives of bacterial species that were grouped using colony morphology and whole cell protein profiles were identified by 16S rDNA sequences as Bacillus barbaricus (B. barbaricus) and -Paenibacillus lautus {P. Lautus). Restriction enzyme digest, SDS-PAGE and random amplified polymorphic DNA (RAPD) analyses provided supporting evidence that representatives were correctly grouped. Cluster analysis results demonstrated that the RAPD profiles of the metal tolerant P. lautus representatives were sufficiently dissimilar to discriminate between individuals from the spatially separated sites. The spatially separated sites also represented sites with high and low heavy metal concentrations. Observed genetic variability was thus also associated with varying levels of heavy metals. In conclusion, this study demonstrated the potential of using RAPD analysis as biomarkers for genotoxic effects of heavy metals on bacterial genomes. / Masters / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2009.

The aim of this study was to determine the effects of the heavy metal pollution on microbial diversity along the gradient from a platinum mine tailings dam using culture-dependent (plating methods) and molecular methods. Tailings and soil samples were collected from seven sites (6 samples per site) at increasing distances from the tailings dam. Samples were collected over a two year period and included two rainy and two dry periods. Concentrations of various heavy metals were determined using an inductively coupled plasma mass spectrometer (ICP-MS). The results demonstrated that seasonal variations in metal concentrations occurred and also that concentrations were significantly different'(P < 0.05) between the experimental sites for each metal. The relative relationship between metals was in the following order: Al > Ni > Cu > Cr. Since soil metal concentration benchmarks for South Africa are lacking, the concentrations were compared to the Canadian microbial benchmarks (MB) and Netherlands maximum permissible concentrations (MPC). Concentrations of most of the heavy metals exceeded the MB and MPC. Levels and diversity of culturable fungi and bacteria at each site were determined using plate count methods. Results indicated that levels of bacteria and fungi were not suppressed by high concentrations of heavy metals. Significantly higher levels (P < 0.05) of fungi were found at the sites on the tailings dam (higher concentrations of heavy metals), compared to sites more than 300 m away. A commonly used soil health index (Shannon-Weaver diversity index) was used to compare microbial community diversity at each site and to evaluate whether or not the heavy metal contamination impacted negatively on these soil bacterial and fungal communities. Shannon-Weaver diversity indices were higher at sites on and close to the tailings dam than sites more than 300 m away. However, ratio of fungal to bacterial levels as determined by plate counts was inconsistent. Representatives of bacterial species that were grouped using colony morphology and whole cell protein profiles were identified by 16S rDNA sequences as Bacillus barbaricus (B. barbaricus) and -Paenibacillus lautus {P. Lautus). Restriction enzyme digest, SDS-PAGE and random amplified polymorphic DNA (RAPD) analyses provided supporting evidence that representatives were correctly grouped. Cluster analysis results demonstrated that the RAPD profiles of the metal tolerant P. lautus representatives were sufficiently dissimilar to discriminate between individuals from the spatially separated sites. The spatially separated sites also represented sites with high and low heavy metal concentrations. Observed genetic variability was thus also associated with varying levels of heavy metals. In conclusion, this study demonstrated the potential of using RAPD analysis as biomarkers for genotoxic effects of heavy metals on bacterial genomes. / Masters / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2009.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-24Bitstream added on 2014-06-13T21:05:09Z : No. of bitstreams: 1 brito_gnb_dr_sjc.pdf: 2623888 bytes, checksum: 9b38ae1778541597981089a1c5d9b974 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os Transtornos alimentares (TA) como Anorexia Nervosa (AN) e Bulimia Nervosa (BN) são acompanhados de inúmeras alterações sistêmicas e bucais relacionadas ao comprometimento do estado nutricional e às práticas compensatórias inadequadas para o controle do peso. O objetivo deste estudo foi avaliar diversidade microbiológica existente na cavidade bucal de pacientes com estes transtornos, por meio de técnicas de cultivo e utilizando métodos moleculares independentes de cultivo. Foram incluídos no estudo 32 pacientes anoréxicos e 27 bulímicos, pareados com 59 indivíduos controle. Amostras de enxágüe bucal foram semeadas para a avaliação da prevalência de leveduras do gênero Candida, estafilococos, enterococos, estreptococcos do grupo mutans (EGM), lactobacilos, enterobactérias/pseudomonas. Espécies de Candida, estafilococos, enterococos, enterobactérias/pseudomonas foram identificadas pelo sistema API. Amostras de biofilme supragengival foram coletadas e utilizadas somente nos procedimentos moleculares. As contagens de microrganismos nos grupos foram comparadas por ANOVA/Mann-Whitney (5%). Houve diferença estatisticamente significantes (p<0,05) para as contagem de leveduras do gênero Candida, estafilococos, enterococos, EGM e lactobacilos entre o grupo TA e controle, mas não houve diferenças significativas para a prevalência de enterobactérias/pseudomonas (p=0,312). Pequena diferença entre os grupos foi observada na diversidade de espécies dos microrganismos estudados pelo método de cultivo. Avaliação molecular foi realizada pela ribotipagem por seqüenciamento do 16S rRNA bacteriano e regiões D1/D2 do 28S rRNA. Foram avaliados cerca de 3000 clones do grupo TA e 1500 clones do controle. Sessenta e duas espécies ou filotipos de bactérias foram detectados... / Eating disorders such as nervous Anorexia and Bulimia Nervosa have several clinical and oral alterations related to the nutritional state involvement and the inadequate compensatory practices for weight control. The aim of this study was to evaluate the microbial diversity in the oral cavity of patients with Anorexia Nervosa and Bulimia nervosa by cultivation techniques and cultivationindependent molecular methods. The study included 32 patients and 27 bulimic anorexics, matched with 59 control subjects. Oral rinse samples were cultured to assess the prevalence of Candida species, staphylococci, enterococci, streptococci mutans (EGM), lactobacilli, Enterobacteriaceae / Pseudomonas. Candida species, staphylococci, enterococci, Enterobacteriaceae / Pseudomonas were identified by API systems. Supragingival biofilm samples were collected and used only in molecular procedures. Counts of microorganisms in the groups were compared by ANOVA / Mann-Whitney (5%). There was a statistically significant (p <0.05) for the counting of yeasts, staphylococci, enterococci, and lactobacilli EGM between TA and control groups, but there were no significant differences in the prevalence of Enterobacteriaceae / Pseudomonas (p = 0.312). Few differences between the groups were observed in the species diversity of organisms studied by the method of cultivation. Molecular analysis was performed by ribotyping by sequencing the 16S rRNA bacterial and D1/D2 regions of 28S rRNA. About 3000 clones of the TA group and 1500 clones of control were evaluated. Sixty-two species or filotypes of bacteria were detected, with 22 identifications were found only in the study group, only 6 in the control group and 34 in both groups. Microorganisms related to caries and periodontal diseases... (Complete abstract click electronic access below)

Les microorganismes jouent un rôle prépondérant dans le fonctionnement des écosystèmes aquatiques, où ils sont à la base de la minéralisation et du recyclage de la matière organique. Les« vrais » champignons, ou Eumycètes, font partie de ces microorganismes hétérotrophes qui permettent le renouvellement de la matière organique dans les écosystèmes. Pourtant, la diversité et l’importance quantitative et fonctionnelle des champignons restent très largement méconnues dans les milieux pélagiques. Récemment, l’utilisation de méthodes moléculaires pour étudier la diversité des picoeucaryotes (de taille < 5 μm) lacustres a mis en évidence l’importance des champignons microscopiques avec, notamment, la présence de chytridiomycètes (chytrides). Cette découverte, en conjonction avec le rôle important connu des Eumycètes dans d’autres écosystèmes naturels, nous a amené à poser l’hypothèse d’une diversité et d’un rôle fonctionnel importants des champignons dans les écosystèmes pélagiques. Ce travail vise à préciser la diversité globale, la structure génétique et l’importance quantitative des différentes divisions du règne des Eumycota dans les écosystèmes pélagiques lacustres, et à proposer des outils méthodologiques pour l’étude écologique de ces peuplements. La diversité phylogénétique et l’importance des champignons de taille comprise entre 0,6 et 150 μm ont été analysées dans trois milieux pélagiques différents. Une étude de clonage-séquençage de l’ADNr 18S et de l’ITS a été réalisée au printemps 2007 dans les lacs Pavin (oligomésotrophe), Aydat (eutrophe) et Vassivière (mésotrophe, humique). L’affiliation phylogénétique des séquences a permis, non seulement de confirmer la présence d’une importante diversité de chytridiomycètes parasites du phytoplancton, mais aussi de mettre en évidence la présence non négligeable d’ascomycètes et de basidiomycètes, potentiellement saprophytes. L’étude de la dynamique saisonnière de la structure des peuplements (par TRFLP) et de l’importance quantitative de différentes divisions (par PCR quantitative) de la communauté fongique ont permis de déceler des différences en fonction des saisons et de l’écosystème. Ces différences ont été reliées à la dynamique des peuplements phytoplanctoniques, avec une influence des apports allochtones, principalement dans le lac eutrophe d’Aydat. De plus, les séquences moléculaires générées au cours de ces dernières années ont permis l’élaboration d’amorces ciblant des clades de champignons microscopiques d’intérêt, pour une étude écologique de la dynamique des peuplements, par des approches PCR à temps réel et FISH (fluorescent in situ hybridization). Enfin, nous considérons que l’acquisition de données complémentaires permettra d’intégrer les champignons saprophytes et parasites dans les flux de matière et d’énergie qui transitent par les écosystèmes pélagiques et les cycles biogéochimiques associés. / Microorganisms play major roles in aquatic ecosystems, primarily as the main actors for organic matter mineralization and recycling. “True” fungi (i.e. Eumycota) are among heterotrophic microorganisms that are highly efficient in recycling organic materials in natural ecosystems. However, the overall diversity of fungi and their quantitative and functional importance remain largely unknown in typical pelagic ecosystems. Environmental 18S rDNA surveys have recently highlighted the importance of microscopic fungi in the diversity of picoeukaryotes (size < 5 μm) in lake ecosystems, including particularly the members of chytridiomycetes (i.e. chytrids) as the dominant phyla. These studies and the known major roles of fungi in natural ecosystems such as soils have leaded us to venture the hypothesis that fungal diversity and functional roles are important structuring factors in pelagic ecosystems. The main aims of the thesis were to examine the overall diversity, genetic structure and quantitative importance of various phyla belonging to the Kingdom Fungi in freshwater pelagic ecosystems. Methodological tools were also developed for ecological investigations of fungal populations of interest. Phylogenetic diversity and quantitative importance of fungi (size classe: 0.6 and 150 μm) were analysed in three contrasting pelagic lakes. Environmental 18S and ITS rDNA surveys were performed during spring 2007 in the oligomesotrophic Lake Pavin, the eutrophic Lake Aydat, and the mesotrophic and humic Lake Vassivière, all located in the French Massif Central. Phylogenetic affiliation of sequences confirmed the presence and the substantial diversity of chytridiomycetes, known as parasites of primarily phytoplankton. We also have unveiled a sizeable number of sequences belonging to the fungal lineages of ascomycetes and basidiomycetes, mainly known as saprophytes. The seasonal dynamics of fungal community structure (essayed by TRFLP),and the quantitative importance of various taxonomic divisions (estimates by real time quantitative PCR or qPCR), revealed significant differences with seasons and with ecosystems. These differences were linked to phytoplankton composition and population successions, with at times the influence of allochthonous inputs, primarily for the eutrophic Lake Aydat. Finally, molecular sequences obtained during the few past years allowed the development of primers for targeting microscopic fungal lineages of interest, and the ecological study of their in situ dynamics using qPCR and FISH (fluorescent in situ hybridization) approaches. Overall, we consider that the acquisition of complementary data is necessary to allow the inclusion of fungi and their main functions (i.e. saprophytisms and parasitism) in the energy and matter fluxes in pelagics ecosystems, and the related biogeochemical cycling.

Le priming effect (PE) est la sur-minéralisation de la matière organique du sol (MOS) après un apport de matière organique fraiche. Ce phénomène serait généré par deux mécanismes distincts, la décomposition stœchiométrique et le « nutrient mining », ayant leur propre dynamique, leurs propres acteurs, leurs propres déterminants et leur propre stock de MO ciblés. Le premier serait plutôt lié à la séquestration de MO dans les sols et l’autre à sa déstabilisation. Comprendre comment piloter l’équilibre entre ces processus à travers les pratiques agricoles, permettrait d’améliorer durablement la fertilité des sols cultivés en milieu tropical dans un contexte de changement climatique. Pour identifier les déterminants, les acteurs et les effets du climat et des pratiques agricoles sur les différents processus générateurs de PE, nous avons combiné la caractérisation physicochimique des sols, la caractérisation des communautés microbiennes et le suivi de la minéralisation des MO par les techniques isotopiques. Ainsi, nous avons pu identifier différentes populations bactériennes et fongiques, associées à chacun des processus, que nous avons classées dans des guildes fonctionnelles. La taille de ces guildes déterminait l’équilibre entre les processus, et était corrélée à la qualité de la MO présente dans le sol. Plus précisément, nous avons montré que le PE stœchiométrique était favorisé dans les sols enrichis en matière organique peu évoluée et en nutriments N et P, entretenant donc une forte communauté de décomposeurs. Ces décomposeurs devaient ainsi limiter l’accès des mineurs à la nouvelle matière organique fraichement apportée et limiter le PE par « nutrient mining ». Sur parcelles agricoles, nos résultats suggéraient que le non labour, l’association légumineuses-céréales, et l’apport de compost favorisaient ces décomposeurs responsables du priming effect stœchiométrique et donc potentiellement la stabilisation durable de la matière organique dans les sols. / The priming effect (PE) is the supplementary mineralization of soil organic matter (MOS) after the addition of fresh organic matter. This phenomenon would be generated by two distinct mechanisms, stoichiometric decomposition and "nutrient mining", having their own dynamics, their own actors, their own determinants and their own MO stock targeted. The first would be related to the sequestration of MO in the soil and the other to its destabilization. Understanding how to manage the balance between these processes through agricultural practices, would allow to improve the fertility of soil cultivated in a tropical environment in a context of climate change.To identify the determinants, actors and effects of climate and agricultural practices on the various processes generating PE, we have combined soil physicochemical characterization, characterization of microbial communities and monitoring of mineralization of MO by isotopic techniques. Thus, we were able to identify different bacterial and fungal populations, associated with processes, which we classified in the functional guilds. The size of these guilds determined the balance between the processes, and was correlated with the quality of the MO present in the soil. Specifically, we showed that stoichiometric PE was favored in soils enriched with high quality organic matter and nutrients, N and P, thus maintaining a strong community of decomposers. These decomposers also limit the access of miners to the provided new organic matter hence limiting PE by "nutrient extraction". On agricultural plots, our results suggest that non-tillage, legume-cereal rotations and compost amendments favor these decomposers responsible for the stoichiometric priming effect and thus potentially the long-term stabilization of organic matter in soils.

Made available in DSpace on 2015-04-01T12:09:01Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 4073387 bytes, checksum: 8309cf98c379c11892e9d5cd2fae29dd (MD5) Previous issue date: 2011-12-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Soil samples of native pasture (site A) and of soil cultivated with grass Paspalum conjugatum, Bergius (site B) collected from Caatinga vegetation in the semi-arid region in Paraíba state (07°23‟27 S 36°31‟58 W) were utilized for constructing four metagenomic libraries, aiming the evaluation of microbial diversity through amplification of gene 16S rRNA of domains Bacteria and Archaea. The metagenomic DNAs were extracted by utilizing FastDNA® SPIN Kit for Soil (BIO 101), which were amplified by PCR, by using universal primers 27F / 1525R (Bacteria) and 20F / 958R (Archaea). The purified fragments were linked to vector pGEM Teasy and transformed by thermal shock in chemically competent Escherichia coli DH10B. Transformants were cultivated in LB/Ampicillin medium (100 μM/ml), IPTG (800 μg/mL) and XGal (80 μg/mL) at 37ºC/18-20 h. A selection of 250 clones of each library was performed, sequenced and after discarding the low quality sequences and chimerics, 64 and 68 sequences were obtained (Bacteria) and 89 and 141 sequences (Archaea) from soils of sites A and B, respectively, which were compared to public bank of data RDB and NCBI (similarity >95%). In site A the phylum Acidobacteria (48.4%) was the most abundant, followed by phyla Bacteroidetes (10.9%), Proteobacteria (10.9%), and Firmicutes (6.3%). In site B Proteobacteria (45.6%) was the most abundant, followed by Firmicutes (10.3%), Acidobacteria (8.8%), Bacterioidetes (7.3%); and also Cyanobacteria (1.5%) and Planctomycetes (1.5%) which were not found in site A. Among the sequences obtained, 23.4% (site A) and 25.0% (site B) were not classified (similarity <95%). In the domain Archaea the phyla found were Euryarchaeota (3.4 and 45.4%) and Crenarchaeota (2.2 and 3.5%), in sites A and B, respectively; it should be observed that 94.4% and 51.1% of the sequences were not classified (similarity <95%), between sites A and B, respectively. Larger diversity (Shannon‟s índex), richness (Chao 1), and distribution (equity index) of communities were observed at species level, in the phyla Bacteria and Archaea, in both sites. The metagenomic libraries 16S rRNA of Bacteria and Archaea, when compared by using the LIBSHUFF program, differed significantly (p<0.0001). The results of the present study showed the occurrence of a great diversity of bacteria and archaea in that semi-arid environment, with peculiar features of elevated temperature and hydric limitations, emphasizing the possibility of investigations on search of new genes and/or microbial isolates with biotechnological potential. / Amostras do solo da pastagem nativa (sítio A) e sob cultivo do capim marrequinho (Paspalum conjugatum, Bergius) (sítio B), coletadas na região semi-árida do bioma Caatinga, Paraíba, (07°23‟27 S 36°31‟58 O), foram utilizadas para construção de quatro bibliotecas de clones metagenômicos, para avaliação da diversidade microbiana pela amplificação do gene 16S rRNA dos domínios Bacteria e Archaea. Os DNA metagenômicos foram extraídos utilizando FastDNA® SPIN Kit for Soil (BIO 101), os quais foram amplificados por PCR utilizando primers universais, 27F / 1525R (Bacteria) e 20F / 958R (Archaea). Os fragmentos purificados foram ligados ao vetor pGEM Teasy e transformados por choque térmico em Escherichia coli DH10B quimicamente competente. Os transformantes foram cultivados em meio Agar LB/Ampicilina (100 μ/mL), IPTG (800 μg/μL) e XGal (80 μg/μL), a 37ºC/18-20 h. Foram selecionados 250 clones de cada biblioteca os quais foram sequenciados e após descarte das sequências de baixa qualidade e quiméricas, foram obtidas 64 e 68, 89 e 141 sequências para Bacteria e Archaea, nos solos dos sítios A e B, respectivamente, as quais foram comparadas em banco de dados públicos RDB e NCBI (≥95% de similaridade). No sítio A o filo Acidobacteria (48,4%) foi o mais abundante, seguido dos filos Bacteroidetes (10,9%), Proteobacteria (10,9%), e Firmicutes (6,3%). No sítio B Proteobacteria (45,6%) foi o de maior destaque, seguido de Firmicutes (10,3%), Acidobacteria (8,8%), Bacterioidetes (7,3%); e ainda Cyanobacteria (1,5%) e Planctomycetes (1,5%), que não foram encontrados no sítio A. Entre as sequências geradas, 23,4% (sítio A) e 25,0% (sítio B) não foram classificadas (similaridade <95%). No domínio Archaea foram encontrados os filos Euryarchaeota (3,4 e 45,4%) e Crenarchaeota (2,2 e 3,5%), nos sítios A e B, respectivamente; destacando-se que 94,4% e 51,1% das sequências não foram classificadas (similaridade <95%), entre os sítios A e B, respectivamente. Uma maior diversidade (índice de Shannon), riqueza (índice Chao 1) e distribuição (índice de equidade) das comunidades foram observadas no nível de espécies, tanto para Bacteria como para Archaea, nos dois sítios. As bibliotecas de clones metagenômicos 16S rRNA de Bacteria e Archaea, quando comparadas, utilizando-se o programa LIBSHUFF, diferiram significativamente (p<0,0001). Os resultados desse estudo mostraram a ocorrência de uma grande diversidade de bactérias e arqueas, nesse tipo de ambiente pouco estudado e com características peculiares de temperatura elevada e limitações hídricas, com possibilidade de busca de novos genes e/ou isolados microbianos, com potencial biotecnológico.

Resumo: Os Transtornos alimentares (TA) como Anorexia Nervosa (AN) e Bulimia Nervosa (BN) são acompanhados de inúmeras alterações sistêmicas e bucais relacionadas ao comprometimento do estado nutricional e às práticas compensatórias inadequadas para o controle do peso. O objetivo deste estudo foi avaliar diversidade microbiológica existente na cavidade bucal de pacientes com estes transtornos, por meio de técnicas de cultivo e utilizando métodos moleculares independentes de cultivo. Foram incluídos no estudo 32 pacientes anoréxicos e 27 bulímicos, pareados com 59 indivíduos controle. Amostras de enxágüe bucal foram semeadas para a avaliação da prevalência de leveduras do gênero Candida, estafilococos, enterococos, estreptococcos do grupo mutans (EGM), lactobacilos, enterobactérias/pseudomonas. Espécies de Candida, estafilococos, enterococos, enterobactérias/pseudomonas foram identificadas pelo sistema API. Amostras de biofilme supragengival foram coletadas e utilizadas somente nos procedimentos moleculares. As contagens de microrganismos nos grupos foram comparadas por ANOVA/Mann-Whitney (5%). Houve diferença estatisticamente significantes (p<0,05) para as contagem de leveduras do gênero Candida, estafilococos, enterococos, EGM e lactobacilos entre o grupo TA e controle, mas não houve diferenças significativas para a prevalência de enterobactérias/pseudomonas (p=0,312). Pequena diferença entre os grupos foi observada na diversidade de espécies dos microrganismos estudados pelo método de cultivo. Avaliação molecular foi realizada pela ribotipagem por seqüenciamento do 16S rRNA bacteriano e regiões D1/D2 do 28S rRNA. Foram avaliados cerca de 3000 clones do grupo TA e 1500 clones do controle. Sessenta e duas espécies ou filotipos de bactérias foram detectados... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Eating disorders such as nervous Anorexia and Bulimia Nervosa have several clinical and oral alterations related to the nutritional state involvement and the inadequate compensatory practices for weight control. The aim of this study was to evaluate the microbial diversity in the oral cavity of patients with Anorexia Nervosa and Bulimia nervosa by cultivation techniques and cultivationindependent molecular methods. The study included 32 patients and 27 bulimic anorexics, matched with 59 control subjects. Oral rinse samples were cultured to assess the prevalence of Candida species, staphylococci, enterococci, streptococci mutans (EGM), lactobacilli, Enterobacteriaceae / Pseudomonas. Candida species, staphylococci, enterococci, Enterobacteriaceae / Pseudomonas were identified by API systems. Supragingival biofilm samples were collected and used only in molecular procedures. Counts of microorganisms in the groups were compared by ANOVA / Mann-Whitney (5%). There was a statistically significant (p <0.05) for the counting of yeasts, staphylococci, enterococci, and lactobacilli EGM between TA and control groups, but there were no significant differences in the prevalence of Enterobacteriaceae / Pseudomonas (p = 0.312). Few differences between the groups were observed in the species diversity of organisms studied by the method of cultivation. Molecular analysis was performed by ribotyping by sequencing the 16S rRNA bacterial and D1/D2 regions of 28S rRNA. About 3000 clones of the TA group and 1500 clones of control were evaluated. Sixty-two species or filotypes of bacteria were detected, with 22 identifications were found only in the study group, only 6 in the control group and 34 in both groups. Microorganisms related to caries and periodontal diseases... (Complete abstract click electronic access below) / Orientador: Cristiane Yumi Koga Ito / Coorientador: Francisco Gorgônio da Nóbrega / Banca: Antonio Olavo Cardoso Jorge / Banca: Janete Dias Almeida / Banca: Flaviana Bombarda de Andrade Ferreira / Banca: Mário Henrique de Barros / Doutor

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No. of bitstreams: 1 Tese - Solange Pires de Araújo.pdf: 3515735 bytes, checksum: b93126b2f4b53b8defbc853c668965cc (MD5) Previous issue date: 2014-10-20 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / The Amazon, which owns the largest fauna and flora in the world presents unparalleled wealth of biological diversity, however, keep intact this megadiversity requires scientific and technological knowledge. In this context, there is an important biotechnological tool that is the bioremediation of impacted environments with petroleum hydrocarbons and derivatives. In the present study samples of microbial communities of fungi and bacteria associated with aquatic macrophytes Eichhornia crassipes (Mart.) Solms, Ichnanthus calvescens Döll and Cyperus ligularis L., were researched. These host plants common at Rio Negro were collected in contaminated environments by oil and oil products at Waterway Station from Manaus. From the species selected, 155 bacterial strains were isolated, being 97 epiphytic and 58 endophytic, and 54 fungi strains, being 30 epiphytic and 24 endophytic. Selective media were used for isolation of microorganisms such as BH liquid medium (Bushhnell Haas) plus oil. The oil and diesel used are from the Base Oil Urucu, Amazonas. The biodegradability tests were performed on selective medium (BH), with the addition of oil as the carbon source known as Medium I This test was repeated with the addition to the medium I the redox indicator 2,6-dichlorophenol indophenol (DCPIP), called medium II. After the evaluationof microbial isolates were selected 6 bacteria and 7 fungi. Molecular identification of bacteria was performed by the 16S ribosomal DNA region and revealed the presence of Bacillus pumilus (endophytic / epiphytic), Lysinibacillus fusiform (epiphytic), Pseudomonas aeruginosa (epiphytic) and Acinetobacter junii (epiphytic/epiphytic). For molecular identification of fungi was performed by the ITS1 and ITS2 region and revealed the presence of Curvularia trifolii (epiphytic), Curvularia clavata (endophytic / epiphytic), Gibberella intermedia (epiphytic/endophytic), Phoma herbarum (epiphytic) and Dothideomycetes sp (epiphytic). These microorganisms were selected for composition of microbial consortium that were used for hydrocarbon biodegradation tests. The measurement of biodegradation of oil and diesel activities was estimated by chromatography and mass spectrometry. In tests we used water of Rio Negro with the aim of approaching research the environment that are being studied. Degradation of hydrocarbons by consortia of fungi and bacteria had significant average values (98.7 to 100%), but did not show any statistical difference between the degradation of the control containing water of the Rio Negro (97.3%). In the experiment with the mixed consortium (FB), there were significant differences, because although the control containing water of the Rio Negro has promoted degradation of diesel by wild microbiota (81.7%), this degradation was lower and statistically different from the mixed consortium (97,5%). Analysis were carried out for degradation of the compounds naphthalene and phenanthrene of diesel by consortia . It was observed that phenanthrene was the best that has been degraded by the mixed consortium (F / B), however the naphthalene was better degraded by the control containing only water from the Rio Negro, highlighting the potential of wild microorganisms that deserve attention in future research, the isolation of these ones in waters from Rio Negro. In the experimental design with the consortia, the results showed that mixed consortia (FB) have potential for use in future bioremediation. / A Amazônia, detentora da maior fauna e flora do mundo apresenta riqueza inigualável de diversidade biológica, entretanto, manter intacta essa megadiversidade requer conhecimentos científicos e tecnológicos. Nesse contexto, situa-se uma importante ferramenta biotecnológica que é a biorremediação de ambientes impactados por hidrocarbonetos de petróleo e derivados. No presente trabalho realizou-se estudo de amostras das comunidades microbianas de fungos e bactérias associadas às macrófitas aquáticas Eichornia crassipes (Mart.) Solms, Ichnanthus calvescens Döll e Cyperus ligularis L. Essas plantas hospedeiras comuns nas águas do rio Negro foram coletadas em ambientes contaminados por petróleo e derivados na Estação Hidroviária de Manaus. Das espécies vegetais selecionadas foram isoladas 155 linhagens de bactérias, sendo 97 epifíticas e 58 endofíticas e 54 cepas de fungos, sendo 30 epifíticos e 24 endofíticos. Foram empregados meio seletivo para isolamento dos microrganismos tal como meio liquido BH (Bushhnell Haas) acrescido de petróleo. O petróleo e o diesel utilizados foram provenientes da Base Petrolífera de Urucu, Amazonas. Os ensaios de biodegradabilidade foram realizados em meio seletivo (BH), com a adição de petróleo como fonte de carbono denominado Meio I. Este ensaio foi repetido com a adição ao meio I do indicador redox 2,6-diclorofenol indofenol (DCPIP), denominado meio II. Após a avaliação dos isolados microbianos foram selecionados 6 bactérias e 7 fungos. A identificação molecular das bactérias foi realizada por meio da região do DNA ribossomal 16S e revelou a presença de Bacillus pumilus (Endofítica/epifítica), Lysinibacillus fusiformes (Epifítica), Pseudomonas aeruginosa (Epifítica) e Acinetobacter junii (Epifítica/epifítica). A identificação molecular dos fungos foi realizada por meio da região TS1 e ITS2 e revelou a presença das seguintes espécies: Curvularia trifolii (epifítica), Curvularia clavata (endofítica/epifítica), Gibberella intermedia (epifítica/endofitica), Phoma herbarum (epifítica) e Dothideomycetes sp (epifítica). Estes microrganismos foram selecionados para composição do consórcio microbianos que foram utilizados em ensaios de biodegradação de hidrocarbonetos. A mensuração das atividades de biodegradação de petróleo e diesel foi estimada por cromatografia e espectrometria de massa. Nos ensaios utilizou-se água do rio Negro com o objetivo de aproximar a pesquisa ao ambiente de estudo. A degradação dos hidrocarbonetos pelos consócios de fungos e bactérias apresentaram médias significativas (98,7-100%), mas não apresentaram diferenças estatísticas entre a degradação do controle contendo água do rio Negro (97,3%). No experimento com o consórcio misto (F/B), houve diferenças significativas, pois embora o controle contendo água do rio Negro tenha promovido degradação do diesel pela microbiota selvagem (81,7%), esta degradação foi inferior e diferente estatisticamente do consórcio misto (97,5%). Foram realizadas análises de degradação dos compostos naftaleno e fenantreno do óleo diesel pelos consórcios Observou-se que o composto fenantreno foi o que melhor foi degradado pelo consórcio misto (F/B). Entretanto, o naftaleno foi melhor degradado pelo controle contendo somente água do rio Negro, destacando o potencial dos microrganismos selvagens que merecem atenção nas futuras pesquisas, no isolamento destes em águas do rio Negro. O índice de toxicidade dos extratos microbianos foram avaliados como toxicidade moderada para o consórcio misto (F/B), já para o consórcio de fungos e consórcios de bactérias não apresentou toxicidade. No planejamento experimental com os consórcios, os resultados obtidos demonstraram que consórcios mistos (F/B) apresentaramm potencial para uso em futuros processos de biorremediação.