The use of reduced oxygen-packaging techniques has resulted in anaerobic bacteria emerging as a problem for the brewing industry over the last twenty-five years. The genus Pectinatus, consisting of the species<i>P. cerevisiiphilus</i> and<i> P. frisingensis</i>, is a concern for producers of unpasteurized beer. As a result, there is an ongoing need to both understand this genus and develop rapid detection methodologies to combat its presence in the brewery. The objectives of this study were to sequence and characterize the flagellin genes from both Pectinatus species and evaluate the genes and proteins from a taxonomic and detection-suitability standpoint. <p>A combination of micro-protein sequencing, polymerase chain reaction (PCR) and Bubble-PCR was used to completely sequence one flagellin gene from each Pectinatus species. This knowledge was then utilized to sequence the flagellin gene from four additional Pectinatus isolates, two from each species. To confirm the identity of the flagellin genes, one flagellin gene from each species was cloned, expressed and detected with Pectinatus-specific antibodies. A discrepancy between of the predicted protein size and the actual protein size led to tests for glycosylation, a post-translational modification. Taxonomic analyses, based on the flagellin genes, were conducted at both the superkingdom and genus levels. Finally, genus- and species-specific PCR primer sets were designed and tested for the specific detection of Pectinatus in the brewery. <p>Cloning and expression data confirmed the identity of the sequenced genes as Pectinatus flagellin genes. Glycosylation was positively confirmed to be a post-translational modification for five of the six strains tested. Phylogenetic analysis revealed that both of the Pectinatus species grouped with the phylum Firmicutes (low G+C, Gram-positive bacteria) and that there was more diversity at the species level within the <i>P. frisingensis</i> flagellin gene than the <i>P. cerevisiiphilus</i> flagellin gene. As a final point, the detection of most Pectinatus isolates was achieved with the preliminary PCR primer sets designed, however, some non-Pectinatus beer spoilage organisms, primarily wort spoilage organisms, were also detected. Both the basic science and the applied results generated from this study will aid the brewing industry in its ongoing battle to control Pectinatus contamination.

The use of reduced oxygen-packaging techniques has resulted in anaerobic bacteria emerging as a problem for the brewing industry over the last twenty-five years. The genus Pectinatus, consisting of the species<i>P. cerevisiiphilus</i> and<i> P. frisingensis</i>, is a concern for producers of unpasteurized beer. As a result, there is an ongoing need to both understand this genus and develop rapid detection methodologies to combat its presence in the brewery. The objectives of this study were to sequence and characterize the flagellin genes from both Pectinatus species and evaluate the genes and proteins from a taxonomic and detection-suitability standpoint. <p>A combination of micro-protein sequencing, polymerase chain reaction (PCR) and Bubble-PCR was used to completely sequence one flagellin gene from each Pectinatus species. This knowledge was then utilized to sequence the flagellin gene from four additional Pectinatus isolates, two from each species. To confirm the identity of the flagellin genes, one flagellin gene from each species was cloned, expressed and detected with Pectinatus-specific antibodies. A discrepancy between of the predicted protein size and the actual protein size led to tests for glycosylation, a post-translational modification. Taxonomic analyses, based on the flagellin genes, were conducted at both the superkingdom and genus levels. Finally, genus- and species-specific PCR primer sets were designed and tested for the specific detection of Pectinatus in the brewery. <p>Cloning and expression data confirmed the identity of the sequenced genes as Pectinatus flagellin genes. Glycosylation was positively confirmed to be a post-translational modification for five of the six strains tested. Phylogenetic analysis revealed that both of the Pectinatus species grouped with the phylum Firmicutes (low G+C, Gram-positive bacteria) and that there was more diversity at the species level within the <i>P. frisingensis</i> flagellin gene than the <i>P. cerevisiiphilus</i> flagellin gene. As a final point, the detection of most Pectinatus isolates was achieved with the preliminary PCR primer sets designed, however, some non-Pectinatus beer spoilage organisms, primarily wort spoilage organisms, were also detected. Both the basic science and the applied results generated from this study will aid the brewing industry in its ongoing battle to control Pectinatus contamination.

Submitted by dayse paz (daysepaz@hotmail.com) on 2016-04-06T11:50:24Z No. of bitstreams: 1 Disserta_o_Cristiane_Carvalho.pdf: 1091025 bytes, checksum: 71418d6d8927740542513191376e2254 (MD5) / Approved for entry into archive by cleuza maria medina dos santos (cleuzamai@yahoo.com.br) on 2016-05-09T14:26:19Z (GMT) No. of bitstreams: 1 Disserta_o_Cristiane_Carvalho.pdf: 1091025 bytes, checksum: 71418d6d8927740542513191376e2254 (MD5) / Made available in DSpace on 2016-05-09T14:26:19Z (GMT). No. of bitstreams: 1 Disserta_o_Cristiane_Carvalho.pdf: 1091025 bytes, checksum: 71418d6d8927740542513191376e2254 (MD5) Previous issue date: 2013 / Em lagos as macrófitas aquáticas são importantes fontes de matéria orgânica detrital, aumentam a complexidade do hábitat proporcionando vários nichos ecológicos a uma diversidade de espécies animais. Durante do processo de decomposição, a colonização dos detritos passa por uma sucessão ecológica entre fungos, bactérias e invertebrados, sendo seu papel fundamental para promover a circulação dos nutrientes. Assim, essa dissertação objetivou: (1) analisar os coeficientes de decomposição e a composição química dos detritos de duas macrófitas, a fim de verificar os efeitos dessas características sobre a biomassa fúngica e invertebrados; (2) analisar se o local de incubação das bolsas dos detritos influencia o recurso de hábitat e sobreposição espacial, interferindo na estrutura funcional da comunidade colonizadora. Para tanto foram realizados dois experimentos. Para o experimento 1 (setembro à dezembro de 2011), incubamos 24 bolsas de Potamogeton pectinatus e 24 de Chara zeylanica e retiradas em 1, 7, 20, 40, 60 e 80 dias. O material foi lavado, seco e triturado para análises químicas e determinação dos coeficientes de decomposição. A biomassa fúngica foi avaliada através do conteúdo de ergosterol. Para o experimento 2 (janeiro de 2012), foram incubadas 60 bolsas aleatoriamente divididas em 4 tratamentos, onde 15 bolsas contendo detritos de P. pectinatus foram incubadas em meio ao próprio estande (PP); 15 bolsas no estande de C. zeylanica (PC); 15 bolsas com detritos de C. zeylanica (CC) incubadas em meio ao seu estande e 15 no estande de P. pectinatus (CP), sendo retiradas em 5, 10 e 20 dias. Em ambos os estudos, os invertebrados foram classificados em grupos tróficos funcionais e determinadas a riqueza e abundância. As características químicas explicaram 59% da variação na abundância de invertebrados. A biomassa fúngica nos detritos não diferiu. A maior abundância e riqueza de invertebrados ocorreram nos detritos de P. pectinatus para o experimento 1 e nos detritos incubados em meio ao seu próprio estande (experimento 2). Houve diferença na perda de massa entre os tratamentos e entre detritos. A maior abundância na composição funcional foi de coletores - catadores e predadores, em ambos os estudos. Houve menor sobreposição espacial entre os grupos tróficos nos tratamentos em relação a PP. Nosso estudo demonstrou que os as características químicas e os coeficientes de decomposição interferem na colonização de invertebrados, relacionado ao tempo de incubação dos detritos. Além disso, o local de incubação afetou os coeficientes de decomposição e a estruturação da comunidade de invertebrados, relacionado à disponibilidade de hábitat oferecido pelos tratamentos. / In lakes the macrophytes are important sources of detrital organic matter, increase the complexity of providing various habitat niches to a variety of animal species. During the colonization process of decomposition of the waste undergoes an ecological succession among fungi, bacteria and invertebrates, and its key role in promoting the circulation of nutrients. Thus, this thesis aimed to: (1) analyze the decomposition rates and chemical composition of the detritus of two macrophytes in order to verify the effects of these characteristics on the fungal biomass and invertebrates, (2) examine if the site of the bags of incubation detritus influences the use and habitat overlap of space, affecting the functional structure of the settler community. Therefore, we performed two experiments. For experiment 1 (September to December, 2011), incubated 24 bags of Potamogeton pectinatus and 24 Chara zeylanica and withdrawn at 1, 7, 20, 40, 60 and 80 days. The material was washed, dried and ground for chemical analyzes and determination of the coefficients of decomposition. The fungal biomass was evaluated through the content of ergosterol. For experiment 2 (January, 2012), 60 bags were incubated randomly assigned to 4 treatments, where 15 bags of debris containing P. pectinatus were incubated in medium to own booth (PP); 15 scholarships in booth C. zeylanica (PC), 15 bags of detritus C. zeylanica (CC) incubated in the midst of its stand and 15 at the P. pectinatus (CP) and taken in 5, 10 and 20 days. In both studies, the invertebrates were classified into functional trophic groups and certain richness and abundance. The chemical characteristics explained 59% of variation in abundance of invertebrates. The fungal biomass in waste did not differ. The greatest abundance and diversity of invertebrates occurred in the debris of P. pectinatus for experiment 1 and incubated debris amidst its own booth (experiment 2). Was no difference in weight loss between treatments and among detritus. The highest abundance was in the functional composition of collectors - scavengers and predators, in both studies. There was a lower spatial overlap between trophic groups in treatments against PP. Our study demonstrated that the chemical characteristics and the coefficients of decomposition interfere with colonization of invertebrates, related to incubation time of detritus. Furthermore, the location of incubation affected the decomposition rates and invertebrate community structure, related to the availability of habitat offered by the treatments.

M.Sc. / Please refer to full text to view abstract

Investigations into the biology and control of the annual grassy weed Bromus pectinatus Thunb. were conducted at the National Plant Breeding Station, Njoro, Kenya, from 1982 to 1984. Pot growth of B. pectinatus was influenced by soil type and microclimate, but not by seed origin. B. pectinatus was germinated and grown in amended and untreated soils ranging in pH from 3.05 to 8.13. Soils with a pH near 3 could not support growth or germination of B. pectinatus. B. pectinatus grew best on a soil of pH 6.55 and when soil pH influenced germination the optimum soil pH was 6.0. Exposure to light inhibited the germination of B. pectinatus seeds. Germination of B. pectinatus seed was most rapid at a 17 C temperature. Germination of dormant B. pectinatus seeds was enhanced by seed hull removal or pricking the lemma or removing the rachilla segment. Germination of B. pectinatus seed in the soil was unaffected by depth of burial, whereas, emergence was reduced to 35, 19, 11, 4 and 0% from depths of 0, 1, 2, 4 and 8 cm, respectively. There was a relationship between field emergence of B. pectinatus and the precipitation pattern. After-harvest germination of B. pectinatus seed indicated that there was an innate dormancy in hulled seed which persisted for 8 months. Field measurements were used to develop an equation which related yield loss in wheat with B, pectinatus infestation. Delayed sowing of wheat and barley into a B. pectinatus infested site resulted in yield reductions that were correlated with length of delay. Replacement series studies were conducted using varying proportions of wheat : B. pectinatus and rapeseed : B. pectinatus. Rapeseed / canola was unaffected by B. pectinatus interference. A spatial interference study determined that B. pectinatus interfers with wheat mainly above ground. The herbicides isoproturon, pendimethalin and oxadiazon were found to be ineffective against B. pectinatus, The herbicides triallate, chlorsulfuron, metribuzin, trifluralin and EPTC achieved limited control of B, pectinatus. Superior control of B. pectinatus was achieved using fluazifop-butyl at 0.25 kg/ha and fenthiaprop-ethyl at 0.12 kg/ha, in rapeseed / canola. / May 1986

Investigations into the biology and control of the annual grassy weed Bromus pectinatus Thunb. were conducted at the National Plant Breeding Station, Njoro, Kenya, from 1982 to 1984. Pot growth of B. pectinatus was influenced by soil type and microclimate, but not by seed origin. B. pectinatus was germinated and grown in amended and untreated soils ranging in pH from 3.05 to 8.13. Soils with a pH near 3 could not support growth or germination of B. pectinatus. B. pectinatus grew best on a soil of pH 6.55 and when soil pH influenced germination the optimum soil pH was 6.0. Exposure to light inhibited the germination of B. pectinatus seeds. Germination of B. pectinatus seed was most rapid at a 17 C temperature. Germination of dormant B. pectinatus seeds was enhanced by seed hull removal or pricking the lemma or removing the rachilla segment. Germination of B. pectinatus seed in the soil was unaffected by depth of burial, whereas, emergence was reduced to 35, 19, 11, 4 and 0% from depths of 0, 1, 2, 4 and 8 cm, respectively. There was a relationship between field emergence of B. pectinatus and the precipitation pattern. After-harvest germination of B. pectinatus seed indicated that there was an innate dormancy in hulled seed which persisted for 8 months. Field measurements were used to develop an equation which related yield loss in wheat with B, pectinatus infestation. Delayed sowing of wheat and barley into a B. pectinatus infested site resulted in yield reductions that were correlated with length of delay. Replacement series studies were conducted using varying proportions of wheat : B. pectinatus and rapeseed : B. pectinatus. Rapeseed / canola was unaffected by B. pectinatus interference. A spatial interference study determined that B. pectinatus interfers with wheat mainly above ground. The herbicides isoproturon, pendimethalin and oxadiazon were found to be ineffective against B. pectinatus, The herbicides triallate, chlorsulfuron, metribuzin, trifluralin and EPTC achieved limited control of B, pectinatus. Superior control of B. pectinatus was achieved using fluazifop-butyl at 0.25 kg/ha and fenthiaprop-ethyl at 0.12 kg/ha, in rapeseed / canola.

Investigations into the biology and control of the annual grassy weed Bromus pectinatus Thunb. were conducted at the National Plant Breeding Station, Njoro, Kenya, from 1982 to 1984. Pot growth of B. pectinatus was influenced by soil type and microclimate, but not by seed origin. B. pectinatus was germinated and grown in amended and untreated soils ranging in pH from 3.05 to 8.13. Soils with a pH near 3 could not support growth or germination of B. pectinatus. B. pectinatus grew best on a soil of pH 6.55 and when soil pH influenced germination the optimum soil pH was 6.0. Exposure to light inhibited the germination of B. pectinatus seeds. Germination of B. pectinatus seed was most rapid at a 17 C temperature. Germination of dormant B. pectinatus seeds was enhanced by seed hull removal or pricking the lemma or removing the rachilla segment. Germination of B. pectinatus seed in the soil was unaffected by depth of burial, whereas, emergence was reduced to 35, 19, 11, 4 and 0% from depths of 0, 1, 2, 4 and 8 cm, respectively. There was a relationship between field emergence of B. pectinatus and the precipitation pattern. After-harvest germination of B. pectinatus seed indicated that there was an innate dormancy in hulled seed which persisted for 8 months. Field measurements were used to develop an equation which related yield loss in wheat with B, pectinatus infestation. Delayed sowing of wheat and barley into a B. pectinatus infested site resulted in yield reductions that were correlated with length of delay. Replacement series studies were conducted using varying proportions of wheat : B. pectinatus and rapeseed : B. pectinatus. Rapeseed / canola was unaffected by B. pectinatus interference. A spatial interference study determined that B. pectinatus interfers with wheat mainly above ground. The herbicides isoproturon, pendimethalin and oxadiazon were found to be ineffective against B. pectinatus, The herbicides triallate, chlorsulfuron, metribuzin, trifluralin and EPTC achieved limited control of B, pectinatus. Superior control of B. pectinatus was achieved using fluazifop-butyl at 0.25 kg/ha and fenthiaprop-ethyl at 0.12 kg/ha, in rapeseed / canola.