Orientador: Adriana Zerlotti Mercadante / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-16T15:01:11Z (GMT). No. of bitstreams: 1 Mandelli_Fernanda_M.pdf: 677502 bytes, checksum: 40f71e5be548712e1d9c40d24476ba1b (MD5) Previous issue date: 2010 / Resumo: Os carotenoides pertencem a um grupo de pigmentos naturais amplamente distribuídos na natureza, com grande diversidade de estruturas e funções. O interesse pelos carotenoides tem aumentado consideravelmente nos últimos anos pela evidência dos seus já conhecidos benefícios à saúde humana, devido às suas propriedades antioxidantes, anticarcinogênicas e imunomodulatórias. As principais fontes industriais de carotenoides são a síntese química e a extração a partir de plantas. Porém, são poucos os carotenoides que possuem métodos economicamente eficientes de produção. Uma alternativa para a obtenção natural destes pigmentos é a sua produção por micro-organismos, sendo uma das fontes as bactérias termófilas que, em sua maioria, possuem carotenoides com estrutura específica para fortalecimento da membrana celular. Assim sendo, o presente estudo tem como objetivos: identificar os carotenoides da linhagem Thermus filiformis ATCC 43280, avaliar o crescimento de massa celular, produção de carotenóides e porcentagem de proteção contra o oxigênio singlete através de diferentes condições de cultivo e verificar a atividade antioxidante do extrato rico em carotenoides na desativação do radical ABTS¿+ (2,2¿- azinobis-(3-etilbenzotiazolina-6-ácido sulfônico)) e do peróxido de hidrogênio. Para atingir os objetivos, os carotenoides foram caracterizados por cromatografia líquida de alta eficiência conectada aos detectores de arranjo de diodos e espectrômetro de massas (HPLCDAD-MS/MS), e as condições de cultivo e a interação entre as variáveis foram estudadas através de planejamento estatístico fatorial. Foram identificados nove carotenoides sintetizados pela bactéria Thermus filiformis, todos apresentando o mesmo comprimento de onda máximo a 449 ± 1 nm, que é um indicativo de um cromóforo de 11 ligações duplas conjugadas, sendo duas em anel ?. O carotenoide majoritário identificado em T. filiformis foi termozeaxantina-15 que consiste do carotenoide zeaxantina ligada a uma glicose acilada com ácido graxo ramificado de 15 carbonos ((3R,3'R)-3'-[(6-O-13-metiltetradecanoil-ß-Dglucopiranosil) oxi]-ß,ß-caroten-3-ol) o que representa 35,3 % do conteúdo total de carotenóides. Foram também identificados os carotenoides: zeaxantina (14,4 %), zeaxantina monoglicosilada (4,7 %) e termozeaxantinas com ácidos graxos ramificados, com cadeia de cadeia de 11 (2,3 %), 12 (5,2 %), 13 (25,4 %) e 14 (9,0 %) carbonos, além de derivados dos carotenoides termozeaxantina-13 (0,8 %) e termozeaxantina-15 (2,8 %). Dentre as variáveis estudadas no planejamento experimental (temperatura, pH, quantidade de triptona, extrato de levedura e elementos traços de Nitsch¿s), a temperatura foi a que mais influenciou a quantidade de biomassa, carotenoides totais e proteção proporcionada pelos carotenoides contra oxigênio singlete. Com a utilização desta ferramenta foi possível atingir valores de 658 mg/L de biomassa seca, 1517 µg/g de carotenoides totais e porcentagem de proteção conferida ao DMA (dimetilantraceno) frente a oxidação por oxigênio singlete na ordem de 85 %. O extrato rico em carotenoides de T. filfiormis apresentou capacidade de desativar o radical ABTS¿+ com valor de capacidade antioxidante equivalente a Trolox (TEAC) de 2,87, valor este superior aos relatados para outros carotenoides como ß-caroteno (1,9) e zeaxantina (1,4). / Abstract: Carotenoids are a group of natural pigments widely distributed in the nature with great diversity of structures and functions. The interest on carotenoids has considerably increased in recent years by the evidence of its known benefits to human health, due to its antioxidant, anticarcinogenic and immunomodulatory properties. The main industrial sources of carotenoids are chemical synthesis and extraction from plants. However, there are few carotenoids which production is economically feasible. An alternative to obtain these natural pigments is their production by microorganisms, and one of the sources is the thermophilic bacterium that produces carotenoids with specific structure for the strength of cell membrane. Therefore the aims of this work were to evaluate the biomass, carotenoid production and quenching of singlet oxygen by the strain Thermus filiformis ATCC 43280 through different cultivation conditions and to verify the antioxidant activity by scavenging of the ABTS¿+ (2,2'-azino-bis(3-ethylbenzthiazoline-6-Sulfonic Acid)) radical and hydroxy peroxide deactivation. In order to achieve these aims, the carotenoids were characterized by high-performance liquid chromatography connected to a photodiode array and mass spectrometry detectors (HPLC-DAD-MS/MS), and the cultivation conditions and the interactions between the variables were studied using a fractionated factorial design. Nine carotenoids synthesized by Thermus filiformis, were identified, all showing the same maximum absorbance wavelength, indicating the presence of a chromophore of 11 conjugated double bounds, being two in the ?-ring. The major carotenoid identified in T. filiformis was thermozeaxanthin-15 which is the carotenoid zeaxanthin linked to a glucose, acylated with a branched fatty acid of 15 carbons ((3R, 3'R) -3'- [(6-O-13- methyltetradecanoyl-ß-D-gluycopiranosyl) oxy]-ß,ß-carotene-3-ol) what represents 35.3 % of the total content of carotenoides. Zeaxanthin (14.4 %), zeaxanthin monoglucoside (4.7 %) and thermozeaxanthins with branched fatty acids chain with 11 (2.3 %), 12 (5.2 %), 13 (25.4 %) and 14 (90 %) carbons, besides thermozeaxanthin-13 (0,8 %) and thermozeaxanthina-15 (2,8 %) derivatives were also identified. Among the studied variables in the experimental design (temperature, pH, tryptone, yeast extract and Nitsch¿s trace elements) the temperature was the one that most influenced the biomass amount, total carotenoids and percentage of protection against singlet oxygen. Using this tool, it was possible to achieve values of 658 mg/L of dry biomass, 1517 µg/g of total carotenoids and percentage of protection of DMA (dimethylanthracene) against the oxidation by singlet oxygen about 85 %. The extract rich in carotenoids from T. filiformis showed capacity of scavenging ABTS¿+ radical with Trolox equivalent antioxidant capacity (TEAC) of 2.87, being this value higher than those reported in the literature for ß-carotene (1.9) and zeaxanthin (1.4) / Mestrado / Mestre em Ciência de Alimentos

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-01-13T10:47:42Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Janaina Costa Feistel - 2013.pdf: 840570 bytes, checksum: 815615a485820497e44d61ee8a9f1f17 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-01-13T10:47:59Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Janaina Costa Feistel - 2013.pdf: 840570 bytes, checksum: 815615a485820497e44d61ee8a9f1f17 (MD5) / Made available in DSpace on 2015-01-13T10:47:59Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Janaina Costa Feistel - 2013.pdf: 840570 bytes, checksum: 815615a485820497e44d61ee8a9f1f17 (MD5) Previous issue date: 2013-03-19 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Poultry products are recognized as sources of contamination by several pathogens. In the last decades the genus Campylobacter, particularly thermophilic species, stands out in developed countries, where it is considered the primary agent responsible for Foodborne Illnesses (DVA's). Due to the importance of this pathogen in public health and economics, researches are conducted in order to identify and characterize the isolated, as well as develop and improve techniques for the identification of the genus Campylobacter spp. with attention focused mainly to important thermophilic species related to food, allowing the determination of the prevalence of this pathogen in main placement sources, such as chicken. The objective of this study was to verify the occurrence of Campylobacter spp. and identify isolated thermophilic species from chicken carcasses in slaughterhouses in the state of Goiás submitted to the Federal Inspection Service which had proper authorization to internal and external trade. A total of 200 samples have been evaluated using the immunoenzymatic screening assay, with subsequent bacterial samples isolation and identification through biotyping. In this study it was observed that the employed methods allowed the isolation of Campylobacter spp. in 24% (48/200) of the assessed samples. Among these samples, 21 (10.5%) were demonstrably positive for genre, having as basis the set of analytical screening method, sequential bacterial isolation for positive samples and evidence of enzymatic and biochemical assimilation and antimicrobial profile. C.coli was the most frequently isolated species (58.6%), followed by C.jejuni ssp. jejuni (17.2%), C.lari (13.8%) and Campylobacter jejuni ssp. doylei (3.4%). By using a screening method, conventional isolation system and numerical biotyping it was immediately revealed the characterization of the isolated Campylobacter. It was observed that some suggestive isolated, due to its positivity in the screening test, growth on selective sources, incubation temperature, motility and morphology, characteristics of the genus assessed, have not been identified by numerical system and thus were considered negative. From the results, it can be concluded that the association of screening method to conventional isolation and biotyping numerical profile allowed a greater understanding of the isolated. It can also be concluded that Campylobacter spp. is present in chicken meat for human consumption and three thermophilic species and two subspecies have been identified. The pathogen presence attests public health risk. / Os produtos de origem avícola podem veicular diversos microrganismos patogênicos. Nas últimas décadas, o gênero Campylobacter, em especial as espécies termofílicas, destaca-se como um dos principais agentes responsáveis por Doenças Veiculadas por Alimentos (DVA’s). Em decorrência da importância deste patógeno na saúde pública e na economia, pesquisas são realizadas com o objetivo de identificar e caracterizar os isolados, bem como desenvolver e aprimorar as técnicas para a identificação do gênero, permitindo a determinação da prevalência deste patógeno nas principais fontes de veiculação, como a carne de frango. O presente estudo teve como objetivo verificar a ocorrência de Campylobacter spp. e identificar as espécies termofílicas isoladas de carcaças de frango de abatedouros do estado de Goiás submetidos ao Serviço de Inspeção Federal e que possuíam autorização para o comércio interno e externo. Um total de 200 amostras foi avaliado utilizando o ensaio imunoenzimático como triagem, com posterior isolamento bacteriano e identificação por meio da biotipificação. Observou-se que os métodos empregados permitiram a identificação de Campylobacter spp. em 24% (48/200) das amostras avaliadas. Destas, 21 (10,5%) foram comprovadamente positivas para o gênero, tendo por fundamentação o isolamento bacteriano de amostras positivas à triagem e testes de assimilação enzimática, bioquímicas e de perfil antimicrobiano. Campylobacter coli foi a espécie mais isolada (58,6%), seguida por Campylobacter jejuni ssp. jejuni (17,2%), Campylobacter lari (13,8%) e Campylobacter jejuni ssp. doylei (3,4%). Observou-se que alguns isolados sugestivos, por apresentar positividade no teste de triagem, crescimento em meios seletivos, temperatura de incubação, morfologia e motilidade características do gênero em questão, não foram identificados pelo sistema numérico empregado neste estudo e, portanto, foram considerados negativos. Pelos resultados, pode-se concluir que a associação do método de triagem ao isolamento convencional e perfil numérico para biotipificação permitiram melhor identificação dos isolados. Assim, é possível afirmar que Campylobacter spp. foi identificada em carne de frango destinada ao consumo, havendo a descrição de três espécies termofílicas e duas subespécies. A presença deste patógeno confere risco à saúde pública por ingestão de alimentos contaminados e decorrência de doenças gastroentéricas e extraintestinais e em casos mais graves, porém não raros, a síndromes reumatológicas, oftalmológicas e neurológicas. Especificamente as espécies Campylobacter coli e Campylobacter jejuni descritas como de maior interesse em alimentos foram as de maior frequência e relacionam-se à maioria dos casos de campilobacteriose de origem alimentos.

Neste trabalho, investigou-se o crescimento e as interações de microorganismos, anaeróbios, em batelada com células planctônicas e em reatores diferenciais com biofilme, sob condições termofílicas (55ºC). Foram utilizadas técnicas clássicas para caracterizar os microorganismos anaeróbios estritos e biologia molecular (FISH), com sondas fluorescentes específicas, para caraterizar e quantificar os microrganismos pertencentes ao domínios Archea e Bacteria. Foram realizadas experimentos com meios de cultivo contendo acetato de sódio e propionato de sódio, na presença de sulfato de sódio. Os resultados do FISH mostraram, para os ensaios em batelada com acetato e sulfato, o predomínio de organismos pertencentes ao Domínio Archea (ARC915+MB1174) representados pelo gênero Methanosarcina sp. e bacilos fluorescentes. No biofilme, para a mesma condição, houve predomínio de células do Domínio Bacteria (EUB338) e relacionadas com a oxidação do acetato. Para a condição de cultivo com lactato e sulfato, as células bacterianas semelhantes a Desulfotomaculum sp. predominaram nos reatores em batelada e diferenciais, justificando a elevada porcentagem de células do Domínio Bacteria. Os resultados do NMP mostraram uma maior concentração de anaeróbios totais na condição lactato e sulfato (\'10 POT.11\' - \'10 POT.12\'). Para as arqueas metanogênicas essa concentração foi eveidenciada para a condição de cultivo com acetato e sulfato (\'10 POT.4\'). Os resultados desse trabalho sugeriram que em reatores em batelada, na condição de cultivo com acetato e sulfato, ocorreu equilíbrio entre metanogênese, oxidação de acetato e sulfatogênese, enquanto no biofilme, foram favorecidos os dois primeiros tipos de metabolismo. A condição lactato mais sulfato favoreceu a sulfetogênese em ambos os reatores. No reator diferencial, alimentado com proprionato e sulfato ocorreu fermentação com crescimento sintrófico entre oxidadoras de proprionato, metanogênicas e redutoras de sulfato hidrogenotróficas. / In this work, were investigated the growth and interactions of anaerobic microorganisms, in batch and differential reactors with planktonic cells and biofilm, respectively, in thermophilic conditions (55°C). Classic and molecular biology methods (FISH) were utilized to characterize strict anaerobes, with specific fluorescence probes, to characterize the Archaea and Bacteria Domain cells. Experiments with sodium acetate, s01ium lactate and sodium propionate plus sodium sulfate were realized. To the acetate and sulfate batch assays the results of FISH showed the predominance of Archaea Domain cells (ARC915+MB1174), represented by genera Methanosarcina sp. and fluorescents rods. Under the same conditions the biofilm results showed predominance of Bacteria Domain cells (EUB338) and those related to the acetate oxidation. In lactate plus sulfate condition the bacteria cells, as Desulfotomaculum sp., predominated in batch and differential reactors, justifying the high percentage of Bacteria Domain cells. The MPN results showed a high concentration of total anaerobes under lactate plus sulfate condition (\'10 POT.11\' - \'10 POT.12\'). For methanogenic arquea this concentration was obtained in the acetate plus sulfate condition (\'10 POT.4\'). These results suggest a process equilibrium may have occurred among methanogenesis, acetate oxidation and sulfidogenesis in batch reactors under acetate plus sulfate conditions. However, methanogenesis and acetate oxidation metabolisms predominated in the biofilm. Sulfidogenesis was easier in both reactors, under lactate plus sulfate conditions. In the differential reactors with propionate plus sulfate, the fermentation was in syntrophic co-culture among propionate oxidizing bacteria, methanogenic and hydrogenotrophic sulfate reducing bacteria.

Neste trabalho, avaliou-se o uso do tratamento anaeróbio termofílico (55 graus Celsius) e mesofílico (35 graus Celsius) de vinhaça de cana-de-açúcar em um reator contendo biomassa imobilizada operado em bateladas seqüenciais (ASBBR). O reator foi preenchido com matrizes de espuma de poliuretano inoculado com lodo granular de um reator UASB que era operado a 35 graus Celsius e tratava água residuária de um abatedouro de aves. A agitação era promovida por agitador mecânico a 300 rpm. O tempo de ciclo do reator termofílico foi alterado nas diferentes fases operacionais em função das diferentes concentrações de vinhaça aplicadas, enquanto que o tempo de ciclo do reator mesofílico foi mantido em 24 horas. O monitoramento do reator foi feito determinando-se os teores de demanda química de oxigênio (DQO), acidez volátil total, alcalinidade a bicarbonato e valor do pH de amostras do afluente e efluente, bem como ao longo do ciclo. Foram efetuados também exames microscópicos para avaliar o desenvolvimento e alterações na composição da biomassa. Na fase de adaptação do lodo à temperatura termofílica, o reator foi alimentado com vinhaça com 0,3 a 1,0 gDQO/L, sendo que a biomassa do reator termofílico foi considerada adaptada 50 dias após a inoculação. O enriquecimento do reator mesofílico com biomassa metanogênica ocorreu em 21 dias. Nesse período, o reator foi alimentado com substrato à base de etanol (2,5 gDQO/L) e sais minerais. As cargas orgânicas aplicadas no reator termofílico foram 0,85, 2,54, 3,64, 4,49, 4,0, 5,7 e 5,24 gDQO/L.d e as eficiências médias de remoção de DQO foram de 43 \'+ OU -\' 7,9 %, 73 \'+ OU -\' 7,0 %, 68 \'+ OU -\' 19,2 %, 78 \'+ OU -\' 9,0 %, 55 \'+ OU -\' 10,0 %, 46 \'+ OU -\' 19,0 % e 75 \'+ OU -\' 7,5 %, respectivamente. O reator mesofílico foi submetido a cargas orgânicas de 2,85, 6,4, 7,9, 10,5, 22,2 e 36,0 gDQO/L.d com eficiências médias de remoção de 75 \'+ OU -\' 8,7 %, 76 \'+ OU -\' 9,6 %, 78 \'+ OU -\' 4,1 %, 78 \'+ OU -\' 7,7 %, 85 \'+ OU -\' 4,1 % e 79 \'+ OU -\' 2,1 %, respectivamente. Portanto, considerando-se a eficiência de remoção de DQO, o desempenho do ASBBR no tratamento de vinhaça de cana-de-açúcar foi superior quando operado sob temperatura mesofílica. Os exames microbiológicos permitiram constatar o predomínio de arquéias metanogênicas do gênero Methanosarcina e bacilos fluorescentes quando o reator foi operado a temperatura termofílica, enquanto que células semelhantes à arquéias do gênero Methanosaeta predominaram durante a operação a temperatura mesofílica. / In this work, the viability of the thermophilic (55 Celsius degrees) and mesophilic (35 Celsius degrees) anaerobic treatment of sugar cane vinasse in an anaerobic sequencing batch reactor containing immobilized biomass (ASBBR) was evaluated. The reactor was filled with polyurethane foam matrices inoculated with a granular sludge taken from an UASB reactor treating poultry slaughterhouse wastewater operating at 35 Celsius degrees. The mixing was provided by means of a mechanical mixer operated at 300 rpm. During the operation at thermophilic temperatures, the cycle time of the ASBBR was changed in the different operating phases according to the influent concentration. At the mesophilic temperatures, the reactor operated at a fixed cycle time of 24 hours. Monitoring analyses included the determinations of chemical oxygen demand (COD), total volatile acids, bicarbonate alkalinity and pH of influent and effluent samples, and along the cycles. The microbial community development inside the reactor was monitored during all the experimental phases by means of microscopic observations. During the adaptation period, the biomass of the thermophilic reactor was fed with low concentration vinasse with 0.3 to 1.0 gCOD/L. The biomass was considered adapted to the thermophilic temperature after 50 days. The enrichment of the mesophilic reactor with methanogenic biomass lasted 21 days. In this period the reactor was fed with ethanol based substrate (2.5 gDQO/L) and minerals. The applied organic loads in the thermophilic reactor were 0.85, 2.16, 4.59, 3.95, 4.07, 6.0 and 5.24 gDQO/L.d reaching average removal efficiencies of 43 \'+ OU -\' 7.9%, 73 \'+ OU -\' 7.0%, 68 \'+ OU -\' 19.2%, 78 \'+ OU -\' 9.0%, 55 \'+ OU -\' 10.0%, 46 \'+ OU -\' 19.0% and 75 \'+ OU -\' 7.5%, respectively. The mesophilic reactor was able to accommodate organic loads of 2.85, 6.4, 7.9, 10.5, 22.2 and 36.0 gCOD/L.d with average removal efficiencies of 75 \'+ OU -\' 8.7%, 76 \'+ OU -\' 9.6%, 78 \'+ OU -\' 4.1%, 78 \'+ OU -\' 7.7%, 85 \'+ OU -\' 4.1% and 79 \'+ OU -\' 2.1%, respectively. Therefore, the treatment of sugar cane vinasse in the ASBBR under mesophlic temperatures performed better than at thermophilic conditions. The microbiological observations of biomass samples indicated that methanogenic Methanosarcina-like archaea and fluorescent rods predominated at thermophilic temperatures, whereas the Methanosaeta-like archaea predominated in the reactor under mesophilic temperatures.

Orientador: Munir Salomão Skaf / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-23T08:18:43Z (GMT). No. of bitstreams: 1 Prates_EricaTeixeira_D.pdf: 8470737 bytes, checksum: 319e6432b49bf0c2fb876c71f9e7fd49 (MD5) Previous issue date: 2013 / Resumo: A biomassa lignocelulósica proveniente do bagaço de cana-de-açúcar e de outras matérias-primas é um material altamente promissor para a geração de biocombustíveis renováveis e ambientalmente positivos. A melhor opção para a conversão dessa biomassa em açúcares solúveis fermentáveis a etanol, em termos de rendimento e de vantagens ambientais, é a catálise enzimática. Mas esta é também a etapa mais cara do processo de obtenção de etanol de segunda geração devido à baixa eficiência e alto custo dos coquetéis enzimáticos atualmente disponíveis para este fim. Para tornar estes processos mais eficientes e economicamente viáveis, é preciso aprofundar nossa compreensão dos mecanismos de hidrólise celulolítica. Grande investimento em pesquisa tem sido empregado com esta finalidade e, como parte disto, este trabalho consiste em um conjunto de pesquisas desenvolvidas na área de simulação computacional via dinâmica molecular de três enzimas celulolíticas: 1) Laminarinase de Rodhothermus marinus; 2) Endoglucanase 3 de Trichoderma harzianum e 3) b- glicosidase de Aspergillus niger. De modo geral, estes estudos visaram investigar a relação entre o arranjo estrutural e propriedades mensuráveis em laboratório interessantes na avaliação da performance destes biocatalisadores, como afinidade pelo substrato e estabilidade térmica. Como parte do Projeto Temático BioEn, financiado pela FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), estes estudos computacionais foram realizados em estreita colaboração com grupos de biofísicos estruturais e biólogos moleculares, sendo a escolha dos três temas embasada em resultados experimentais / Abstract: The lignocellulosic biomass from sugar cane bagasse and from other raw materials is a highly promising material for the generation of renewable and environmentally positive fuels. In terms of performance and environmental advantages, the best option for converting this biomass into soluble sugars to produce ethanol is the enzymatic catalysis. However, this is also the most expensive step of the second-generation ethanol production due to the low efficiency and high cost of the currently available enzyme cocktails. In order to make the process more efficient and economically viable, it is necessary to deepen the understanding of the cellulolytic hydrolysis mechanisms. Great investment in research has been employed for this purpose, and as part of these efforts, this work consists on a set of molecular dynamics studies of three cellulolytic enzymes, namely: 1) laminarinase from Rodhothermus marinus; 2) Endoglucanase 3 from Trichoderma harzianum and 3) b-glucosidase from Aspergillus niger. In general, these studies aimed to investigate the relationship between the structural arrangement and experimental data that are interesting for the biocatalyst performance evaluation, such as affinity to the substrate and thermal stability. As part of the BioEn Thematic Project, funded by FAPESP (Research Foundation of the State of São Paulo), these computational studies were carried out in close collaboration with structural biophysicists and molecular biologists. The choice of the three proteins considered here was based on these experimental studies / Doutorado / Físico-Química / Doutora em Ciências

Thesis submitted in fulfilment of Master's Degree in Technology: Biotechnology, Durban Institute of Technology, 2004. / M

Can composting save the world from its human generated biological waste problem and give a diversifying organic agriculture a much needed boost at the same time? The age old natural process of composting has been well researched and documented in scientific and other literature. Thermophilic composting transforms biological/organic waste into a stable plant and soil friendly material (compost) that has been sanitised through high temperature and humified at the same time – a true miracle of nature. Mixtures for thermophilic composting can be formulated fairly accurately from their water, C and N content. The pH needs to be within an acceptable range (5-8) but can be adjusted with agricultural lime if needed. The EC and ash content of sludge, together with the C and N content, are fairly reliable indicators of available bio-chemical energy for composting. Monitoring the temperature and water content gives sufficient feedback for proper management of the process. Bio-chemical transformations in composting material tell the tale of the microbial processes at the heart of composting and humification. The quality of compost depends largely on the quality of the raw materials used, even if mixture formulation and process management is effective. The study firstly evaluated the compostability of various types of WWS and eucalyptus sawdust bulking agent with the focus on the known requirements and conditions for thermophilic aerobic composting. This entailed a composting experiment during which the sludge and bulking agent were characterised, feedstock mixtures formulated and certain composting conditions (temperature and water content) and feedstock characteristics (pH, EC and ash content) monitored over time as piles were managed through turning, sampling and watering. Secondly, the study evaluated the quality of compost produced from various types of WWS and eucalyptus sawdust bulking agent as influenced by the raw material and initial feedstock characteristics, microbial dynamics and environmental conditions during composting and as indicated by certain chemical and biochemical transformations (including OM decomposition) during composting and end product (compost) characteristics. Tracking the chemical and biochemical evolution during composting in the four treatments entailed the laboratory analyses of a variety of signature characteristics for samples taken at the start of composting, at the end of the thermophilic phase when pile temperature decreased to below 40ºC and at the end of the curing phase of composting when the temperature of piles did not exceed ambient temperature. Finally, a seedling emergence bioassay was performed as a direct test of compost maturity and quality. The main findings of this study are that dewatered, activated sludge has a high level of thermophilic compostability with Eucalyptus sawdust as bulking agent. Dried, digested sludge has a low level of compostability with Eucalyptus sawdust as bulking agent mainly because of the anaerobic digestion that depleted the C and bio-chemical energy in the sludge. Little value is added to digested sludge through composting with eycalyptus sawdust except that it probably facilitated a further “curing” of the already stable material. Overall, dewatered ACT-MUN sludge and eucalyptus sawdust produced the compost of highest quality and stability/maturity in a volume ratio of 1:4 (sludge:sawdust). Dewatered ACT-IND sludge and eucalyptus sawdust produced an interesting compost of high stability/maturity with a high level of humification and medium fertility status. Increasing the sludge:sawdust ratio closer to 1:4 (volume) will enhance the chemical quality of this good compost. Dried DIG-MUN sludge and eucalyptus sawdust produced a stable compost of very low pH, relatively low fertility, poor physical character and high risk of phytotoxicity. Composting is a great option for WWS treatment that deserves a lot more attention in South Africa. / Dissertation (MSc Agric)--University of Pretoria, 2013. / gm2014 / Plant Production and Soil Science / unrestricted

Masters of Science / Thermophilic bacteriophages provide simple model systems for understanding biochemical and biological adaptation mechanisms at elevated temperatures. The essential objectives of this study were to characterise the physicochemical properties of select Geobacillus bacteriophages and to sequence their complete genomes. The later objective is believed to be an essential prerequisite to the engineering of a sitespecific integration vector for the stable cloning of exogenous genes into host bacteria. Bacteriophages were assayed at 55oC by the agar overlay technique using dry Karoo soils as source material. A pure strain of bacteriophage called GV1 (for Geobacillus stearothermophilus virus 1) was isolated with the strain Geobacillus stearothermophilus TAU3A1. Plaques were medium sized (2 to 4 mm diameters), with regular contour, clear, and without resistant cells. Host range specificity study showed that GV1 was lytic on thirteen thermophilic Bacillus-like strains tested, including strains of Geobacillus stearothermophilus, G. thermoglucosidasius, B. licheniformis, Anoxybacillus idirlerensis, and A. kuwalawohkensis. However, GV1 failed to infect a mesophilic strain of Bacillus megaterium. TEM analysis of semipurified particles revealed that the phage belongs to the family of Siphoviridae. Morphological characteristics included a long tail of approximately 100 nm and a hexagonal head of approximately 50 nm diameter. Viability and stability studies showed that the phage was best maintained at -80oC in PMN buffer supplemented with 20% glycerol. It was stable at a pH range of 5.5 to 7.5 and MgCl2 and CaCl2 concentration of 0.001 M. hermostability experiments, conducted over short periods of time, showed that GV1 was stable over the temperature range 50 to 75oC, with optimum at 55oC. The study of phage-host interactions showed that phage articles inhibited the initial growth of infected cultures in the first six hours post-infection, presumably while mature phages were released. This was followed by a steady recovery of the growth rate. Atempts to obtain pure particles and to extract and sequence phage DNA were unsuccessful due to the low titer nature of the phage.

Thermophilic composting (TC) and vermicomposting (V) are the two most common methods used for biological stabilization of solid organic wastes. Both have their advantages and disadvantages but the proposed method of combining composting and vermicomposting (CV) borrows pertinent attributes from each of the two methods and combines them to enhance overall process and product qualities. Dairy manure and waste paper are two wastes produced in large quantities at the University of Fort Hare. The study was carried out to address the following specific objectives, to determine (i) the effectiveness of combined thermophilic composting and vermicomposting on the biodegradation and sanitization of mixtures of dairy manure and paper waste, (ii) an optimum precomposting period for dairy manure paper waste mixtures that results in vermicomposts of good nutritional quality and whose use will not jeopardize human health, (iii) the effectiveness of phosphate rock (PR) in increasing available P and degradation and nutrient content of dairy manure-paper vermicomposts, (iv) the physicochemical properties of vermicompost substituted pine bark compost and performance of resultant growing medium on plant growth and nutrient uptake. Results of this study revealed that wastes with a C: N ratio of 30 were more suitable for both V and CV as their composts were more stabilized and with higher nutrient contents than composts made from wastes with a C: N ratio of 45. Both V and CV were effective methods for the biodegradation of dairy manure and paper waste mixtures with C: N ratio of 30 but the latter was more effective in the biodegradation of waste mixtures with a C: N ratio of 45. The combinination of composting and vermicomposting eliminated the indicator pathogen E. coli 0157 from the final composts whereas V only managed to reduce the pathogen population. iv A follow up study was done to determine the effects of precomposting on pathogen numbers so as to come up with a suitable precomposting period to use when combine composting dairy manure-waste paper mixtures. Results of this study showed that over 95% of fecal coliforms, E. coli and of E. coli 0157 were eliminated from the wastes within one week of precomposting and total elimination of these and protozoan (oo)cysts achieved after 3 weeks of precomposting. The vermicomposts pathogen content was related to the waste’s precomposting period. Final vermicomposts pathogen content was reduced and varied according to precomposting period. Vermicomposts from wastes precomposted for over two weeks were less stabilized, less humified and had less nutrient contents compared to vermicomposts from wastes that were precomposted for one week or less. The findings suggest that a precomposting period of one week is ideal for the effective vermicomposting of dairy manure-waste paper mixtures. Results of the P enrichment study indicated an increase in the inorganic phosphate and a reduction in the organic phosphate fractions of dairy manure-waste paper vermicompost that were enriched with PR. This implied an increase in mineralization of organic matter and or solubilization of PR with vermicomposting time. Applying PR to dairy manure-waste paper mixtures also enhanced degradation and had increased N and P contents of dairy manure-waste paper vermicomposts. Earthworms accumulated heavy metals in their bodies and reduced heavy metal contents of vermicomposts. A study to determine the physicochemical properties of vermicompost substituted pine bark compost and performance of resultant growing medium on plant growth and nutrient uptake was done. Results obtained revealed that increasing proportions of dairy manure vermicomposts in pine bark compost improved tomato plant height, stem girth, shoot and root dry weights. v Tomatoes grew best in the 40 to 60% CV substituted pine bark and application of Horticote (7:2:1 (22)) fertilizer significantly increased plant growth in all media. Progressive substitution pine bark with dairy manure vermicomposts resulted in a decrease in the percentage total porosity, percentage air space whilst bulk density, water holding capacity, particle density, pH, electrical conductivity and N and P levels increased. Precomposting wastes not only reduced and or eliminated pathogens but also improved the stabilisation and nutrient content of dairy manure waste paper mixtures. The application of PR to dairy manure waste paper mixtures improved the chemical and physical properties of vermicomposts. Earthworms bio-accumulated the heavy metals Cd, Cr, Cu, Pb and Zn whilst the contents of these in the vermicomposts declined. It is, therefore, recommended that dairy manure waste paper mixtures be precomposted for one week for sanitization followed by PR application and vermicomposting for stabilization and improved nutrients contents of resultant vermicomposts. Substitution of pine bark compost with 40 to 60 % PR-enriched vermicompost produced a growing medium with superior physical and chemical properties which supported good seedling growth. However, for optimum seedling growth, supplementation with mineral fertilizer was found to be necessary.

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