Made available in DSpace on 2015-04-20T12:31:50Z (GMT). No. of bitstreams: 1 Eliandra de Freitas Sia.pdf: 1557550 bytes, checksum: e4d21c384795b53b83656a535e1bf31f (MD5) Previous issue date: 2012-09-04 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The filamentous fungi are an important group of microorganisms, being not only one of the most numerous among the microorganisms due to their potential in biotechnological processes such as the production of antibiotics, enzymes, vitamins and others. The fungi are also plant, animals and human pathogens, acting sometimes as toxins producers. The fungal growth is a routine practice at microbiological laboratories and the Potato Dextrose Agar (PDA) medium is the most used medium due to be a rich source of starch. However, in some regions of Brazil, the potato production has a quite high cost. For this reason the aiming of the present work was the search for new alternative sources of starch. Thus, initially several tubercles: cassava (Manihot esculenta), sweet potato (Ipomoea batatas), carrot (Daucus carota), ginger (Zingiber officinale) and yam (Dioscorea alata) were tested as a starch source to fungal growth. The cassava (Manihot esculenta) showed the best results to fungal growth. This species is a tropical plant with a great commercial importance to Brazil, Africa and other parts of the world. In brazilian north and northeast regions, the cassava is hugely cultivated by a low cost. After the first screening, it was optimized the utilization of Cassava Dextrose Agar (CDA) media. Many fungal species of biotecnological importance (Beauveria bassiana, Aspergillus niger, Penicillium sp., Fusarium sp., Phanerochaete chrysosporium) were evaluated to growth, development sporulation and hyphal formation. The BDA media was used as a control. The present results demonstrated a high efficiency of MDA to fungal growth. However, in MDA was observed a reduction of fungal sporulation comparing with BDA. Finally, both media BDA and MDA, were used to the endophytic fungal isolation from: guarana (Paullinia cupana) and olives (Olea europea). The fungal identification demonstrated that isolates varied according to host plant. The fungal diversity was higher in guaraná being higher using the MDA in comparison with the BDA media. To both media, it was obtained a low number of isolated fungi from O. europea. The present results clearly demonstrated that the cassava is an feasible important starch source, being a potential alternative media, mainly in tropical countries / Os fungos filamentosos são um importante grupo de micro-organismos, não apenas por serem os mais numerosos dentre todos os micro-organismos, mas devido ao seu potencial em processos biotecnológicos como a produção de antibióticos e enzimas, fontes de vitaminas, alimentação e outros. Os fungos também são conhecidos como patógenos de plantas, animais e humanos, e produtores de toxinas. Cultivar fungos é uma rotina em laboratórios de microbiologia e um dos meios muito utilizado é o Batata Dextrose Ágar (BDA), que utiliza a batata como uma fonte rica em amido. Em algumas regiões do país, como a Região Norte, a batata tem um elevado custo e por este motivo a busca por novas fontes de amido é uma alternativa. Assim, outros vegetais devem ser pesquisados visando a substituição da batata. No presente trabalho foram inicialmente utilizados mandioca (Manihot esculenta), batata-doce (Ipomoea batatas), cenoura (Daucus carota), gengibre (Zingiber officinale) e cará (Dioscorea alata), sendo demonstrado que a mandioca (Manihot esculenta) é uma alternativa relevante para o cultivo de fungos. Esta espécie vegetal é uma planta tropical de grande importância comercial no Brasil, na África e em outras regiões do mundo, sendo que para a região Norte a mandioca é muito cultivada e também de baixo custo. Assim, após a triagem inicial com outros tubérculos, foi realizada a avaliação e otimização do meio de cultura de Mandioca Dextrose Ágar (MDA). Para o ensaio do meio com mandioca foram utilizadas diversas espécies de fungos com importância biotecnológica (Beauveria bassiana, Aspergillus niger, Penicillium sp., Fusarium sp., Phanerochaete chrysosporium). Foi avaliada a taxa de crescimento, a esporulação e a formação de hifas no meio de cultura BDA (utilizado como controle) e MDA. Os resultados comprovaram a eficiência do MDA para o crescimento dos fungos analisados, destacando-se a taxa de crescimento radial em meio sólido e peso seco do micélio em meio líquido. Entretanto, em MDA há uma menor esporulação dos fungos quando comparado ao BDA. Finalmente, foi realizado, utilizando meio BDA e MDA, o isolamento de fungos endofíticos de duas plantas: guaraná (Paullinia cupana) e oliveira (Olea europea), nos quais o meio de cultura MDA também se mostrou eficiente. A identificação de fungos endofíticos revelou que eles variaram conforme a planta hospedeira. A diversidade de fungos endofíticos foi maior em guaraná no meio MDA que em BDA, e o número de isolados foi menor em oliveira nos dois meios utilizados. Assim, do ponto de vista econômico e social, o substrato de mandioca pode ser considerado uma alternativa de alta potencialidade na obtenção de meios de cultura para laboratórios de micologia, especialmente em países tropicais

Foram estudadas trinta e uma cepas fúngicas não identificadas, as quais foram denominadasX1 a X31. O potencial fotoprotetor foi avaliado pela medida espectrofotométrica da absorçãodos extratos na região do UV (280-400 nm). Os extratos com os melhores perfis de absorção em cultura estacionária foram X1, X2, X6, X12, X13, X18, X19, X22, X24 e X31 e, em cultura agitada X4 e X17. A reprodutibilidade do processo foi avaliada e as cepas fúngicas que apresentaram coeficiente de variação menor que 15% foram selecionadas para o estudo de fotoestabilidade. A fotoestabilidade dos extratos foi avaliada pela medida da viabilidade celular de fibroblastos L929 tratados com extratos previamente irradiados sob radiação UVA (11,2 J/cm2) e UVB (3,43 J/cm2) e extratos não irradiados, bem como, pela comparação das áreas sob as curvas de absorção na região do UV dos extratos irradiados e não irradiados. Os extratos selecionados para o estudo de fotoestabilidade foram X4, X12, X19, X22, X24 e X31. Os extratos não irradiados apresentaram os seguintes valores deIC50 para viabilidade celular (citotoxidade): X4-130µg/ml, X19-20µg/ml, X22-10 µg/ml e X24-60µg/ml. Após a radiação UVA e UVB, os extratos apresentaram redução significativa da viabilidade celular em relação ao IC50 dos extratos não irradiados. Sob luz UVB, os extratos X12 (IC50 35µg/ml) e X31 (IC50 70µg/ml) mantiveram a mesma porcentagem de redução da viabilidade celular quando comparado ao IC50 dos extratos não irradiados. No entanto após exposição à luz UVA, o extrato X12 aumentou a viabilidade celular de 50% (quando não irradiado) para 75% (irradiado). Enquanto que o extrato X31, mesmo após a radiação UVA, manteve a mesma redução de 50% da viabilidade celular. Nessa etapa os extratos selecionados foram os X12 e X31. O espectro de absorção na região do UV obtido para o extrato X12 mostrou uma redução da absorbância de 28,3% sob radiação UVB e de 60% sob radiação UVA em relação ao extrato não irradiado. O extrato X31 apresentou uma redução da absorbância de 17,6% e30% sob radiação UVB e UVA respectivamente, em relação ao extrato não irradiado. Os fungos selecionados foram identificados por PCR, sugerindo que o fungo X12 seja o Aspergillus terreus e o X31 seja o Talaromyces pinophilus. Por fim, foi feita a identificação da substância ativa do extrato X12 empregando a técnica de desreplicação, a qual fez o uso da instrumentação analítica acoplada UHPLC-DAD-(ESI)-HRMS associada ao banco de dados Chapman& Hall\'s Dictionary of Natural Products (DNP). No extrato X12 o composto majoritário foi identificado como sendo a citreoviridina. Assim, os resultados do presente trabalho permitiu estabelecer um procedimento para a seleção de fungos produtores de compostos absorvedores de radiação UV, que poderia ser aplicado na obtenção de novos filtros orgânicos naturais para protetores solares. / It were studied thirty-one fungal strains not identified, which were named X1 to X31. The photoprotective potential was assessed spectrophotometrically by measuring absorption of the extract in the UV region (280-400 nm). The extracts that presented the best absorption profiles in stationary culture were X1, X2, X6, X12, X13, X18, X19, X22, X24 and X31, and X4 and X17 in stirred culture. The reproducibility of the process was evaluated and fungal strains that showed a coefficient of variation lower than 15% were selected for the study of photostability. The photostability of the extracts was assessed by measuring cell viability of L929 fibroblasts treated with extracts previously irradiated under UVA light (11,2 J/cm2) and UVB (3,43 J/cm2) and not irradiated extracts, as well as by comparison of the areas under the curves of absorption in the UV region of the irradiated and non-irradiated extracts. The extracts selected for the study of photostability were X4, X12, X19, X22, X24 and X31. The non-irradiated extracts showed the following IC50 values for cell viability (cytotoxicity): X4- 130?g/ml X19-20?g/ml, X22-10/ml and X24-60?g/ml. After UVA and UVB radiation, the extracts showed significant reduction in cell viability compared to the IC50 of the unirradiated extracts. Under UVB light, the X12 extracts (IC50 35?g/ml) and X31 (IC50 70mg/ml) maintained the same percentage of cell viability reduction when compared to the IC50 of the unirradiated extracts. However after exposure to UVA light, X12 extract increased the cell viability from 50% (when not irradiated) to 75% (irradiated). While X31 extract even after the UVA irradiation, remained the same 50% of reduction in cell viability. At this stage the selected extracts were X12 and X31. The absorption spectrum in the UV region obtained for X12 extract showed a decrease in absorbance of 28.3% under UVB and 60% under UVA radiation relative to non-irradiated extract. The X31extract showed a reduction in absorbance of 17.6% and 30% in UVA and UVB radiation, respectively, compared to non-irradiated extract. The selected fungi were identified by PCR, suggesting that X12 fungus is Aspergillus terreus and X31 is the Talaromyces pinophilus. Finally it was identified the active substance of X12 extract employing dereplication technique which makes use of coupled analytical instrumentation UHPLC-DAD- (ESI) HRMS associated to the Chapman and Hall\'s Dictionary of Natural Products (DNP) database. The majority compound of X12 extract was identified as the citreoviridin. Thus, the results of this study allowed us to establish a procedure for the selection of producers of UV absorbing compounds from fungi, which could be applied in obtaining new natural organic filters for sunscreens.

Philosophiae Doctor - PhD / Biofuel production from microalgae is currently not economically competitive with fossil fuels due to high operational costs. A sustainable system needs to be developed which considers cultivation, harvesting and conversion to fuels as a single loop. The harvesting step has been identified as a major bottleneck within the biofuel production process, contributing to a significant proportion of the operational cost (20-30%). Chemical flocculation is a more affordable alternative to centrifugation and filtration. Chemical flocculants however negatively impact the quality of biomass and conversion efficiency to biofuel by increasing biomass ash content. Bioflocculation with biopolymers or microbes have a minimal impact on the quality of biomass. In this study, the interaction between the filamentous fungus Isaria fumosorosea and the microalgae C. sorokiniana is investigated. Under strict autotrophic conditions at pH 7-8, co-culture of microalgae (2-20 μm) with fungal blastospores resulted in theidevelopment of large pellets (1-2 mm) which may be easily harvested by sedimentation or filtration at 95% harvesting efficiency. Fungal assisted bioflocculation was compared to other harvesting methods with respect to cost and impact on the hydrothermal conversion process. Low cost carbon sources, including waste hydrothermal nutrients, minimal sugar concentrations and algal exudate may reduce fungal cultivation costs. Waste products, such as organic carbon, N, P, CO₂ and trace metals can be recycled and used for algae and fungal cultivation, closing the loop to make the system sustainable. / National Research Foundation; Swiss Government

Ochratoxin A is the most potent chlorinated derivative of the ochratoxin group, consisting of a 5'-chlorinated dihydroisocoumarin moiety linked by an amide bond to l-phenylalanine. Produced as a secondary fungal metabolite by several species of Aspergillus and Penicillium, ochratoxin A has been shown to readily contaminate a large variety of commodities including cereals, groundnuts, dried fruit, spices and coffee. This has led to widespread contamination of ochratoxin in wine, beer, milk and meat products. As ochratoxin A is a potent nephrotoxin exhibiting teratogenic and carcinogenic properties, the development of a rapid screening platform for the cost effective control of ochratoxin A content in foodstuffs is therefore required. The evaluation of metallophthalocyanine and carbon nanotube electrode modification toward the development of a nanostructured biosensor capable of enhancing the electrochemical detection of ochratoxin A in complex media is presented. Cyclic voltammetry at a glassy carbon electrode allowed for the optimization of detection parameters including pH and type of supporting electrolyte. Britton-Robinson buffer was found to be the most suitable supporting electrolyte in terms of sensitivity and reproducibility obtaining a LOD of 0.28 μM as determined by differential pulse voltammetry. Subsequent analysis determined the dependence of OTA oxidation on pH in acidic media which proceeds with the transfer of two electrons to form a quinone/hydroquinone couple shown to adsorb to the electrode surface. Passivation of the electrode through adsorption of oxidation products was shown to severely limit the detection of OTA upon successive detection cycles. Comparison of various metallophthalocyanine modifiers showed an increase in sensitivity toward the detection of OTA at phthalocyanine complexes with metal based redox processes. However with the exception of NiPc and CoTCPc complexes, phthalocyanine modification was limited by the increase in deviation of current response and extent of fouling. NiPc modification showed an increase in sensitivity by two fold with fouling characteristics comparable to an unmodified electrode while low improvements in fouling was observed at CoTCPc modified electrodes with sensitivity in detection comparable to an unmodified electrode.Modification of the electrode with multi- and single walled carbon nanotubes produced a significant increase in sensitivity toward the detection of ochratoxin A. The electrocatalytic activity of nanotube modifiers was attributed to the increase in surface area and to the addition of oxygenated functional groups upon acid treatment as confirmed by Raman spectroscopy. Acid functionalization of the carbon nanotubes for a period of two hours produced the greatest increase in sensitivity obtaining a respective LOD of 0.09 μM and 0.03 μM for analysis of ochratoxin A at multi- and single walled carbon nanotube modified electrodes. Centrifugal purification of carbon nanotubes was deemed necessary to improve the electrocatalytic activity of the nanotube modifiers through the removal of carbonaceous impurities as visualized by atomic force microscopy. Furthermore, a crude lipase preparation, lipase A, was investigated as a potential biological recognition element for selective detection of ochratoxin A in complex media. Lipase A enabled the hydrolysis of ochratoxin A to the electroactive species ochratoxin α as confirmed by thin layer chromatography and voltammetric analysis. Additional isolation of a pure hydrolase from the lipase A preparation is required prior to utilization within a nanostructured biosensor platform capable of detecting ochratoxin A in complex media.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21829号 / 農博第2342号 / 新制||農||1068(附属図書館) / 学位論文||H31||N5201(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 田中 千尋, 教授 本田 与一, 准教授 刑部 正博 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Filamentösa svampar används inom många biotekniska områden. Svamparnas biomassa kan användas till föda eller djurfoder, medan biprodukter som svampar producerar kan användas inom områden som industri samt medicin. Kultiveringen av Neurospora intermedia och Aspergillius oryzae genomförs på smör, raps och frityrolja i detta arbete för att få en större förståelse för hur svamparna kan växa på olika lipider samt skillnaden som kan uppstå mellan olika arter av filamentösa svampar. Under arbetes studeras biomassakoncentration, proteinhalt samt pH. Även problem som att svamparna kan lagra lipider i cellerna och att det kan påverka resultaten genom en ökning av biomassakoncentrationen diskuteras.  Kultiveringarna genomfördes i Erlenmeyerflaskor med 20 g/L lipid samt saltblandning för att bestå med nödvändiga näringsämnen. För A.oryzae genomförs även kultivering i en airliftbioreaktor för att förstå hur svampen beter sig i större skala. Högst koncentrationen torrbiomassa som erhålls ifrån Erlenmeyerflaskorna är 18,49±1,90 g/L för A.oryzae samt17,31±1,14 g/L för N.intermedia med en proteinhalt för torr biomassa upp till ca 14%.Utbytetmellan torr biomassa och lipid som tillsatts i kultiveringen hamnade på 94,00%±0,06%(rapsolja) för A.oryzae samt 81,69%±0,007% (frityrolja) för N.intermedia. De höga utbytena kan förklaras med att svamparna kan lagra lipider i cellerna vilket även ger de höga koncentrationen av torr biomassa. För att förstå morfologin, lipidhalt i biomassan samt pH beteende behövs mer arbete utföras med ändring av parametrar som pH justeringar, temperatur, luftflöde samt analys av biomassan för att utreda hur mycket lipider som lagras. / Filamentous fungi are used in many different biotechnological fields. The fungal biomass can be used for food or feed, while by-products that the fungi produce can be used in different industries or medical applications. The cultivations of Neurospora intermedia and Aspergillius oryzae implemented on butter, rapeseed and frying oil that is used in this work is for the larger understanding of how the fungi can grow on different lipids and the difference between species of fungi. Biomass concentration, protein content and pH are the main focus during this work, but problems like lipids storage in the biomass that can affect the results by increase of the total biomass concentration is discussed. The cultivations were implemented in Erlenmeyerflasks with 20 g/L lipid with added saltsolution with necessary nutrients. For A.oryzae cultivation in an airlift bioreactor was carried out for the understanding of larger scale cultivation. Highest concentration of dry biomass obtained from Erlenmeyerflasks are 18,49±1,90 g/L for A.oryzae and 17,31±1,14 g/L for N.intermedia with protein content for dry biomass up to 14%.The yield for dry biomass perlipid added during the cultivation ended at 94,00%±0,06% (rapeseed oil) for A.oryzae and 381,69%±0,007% (frying oil) for N.intermedia. During cultivation the cells can store lipids and that explains the high yields and biomass concentrations. For the understanding of morphology, the amount of lipids in the biomass and pH behaviour needs more work that includes changes of parameters like pH adjustment, temperature, airflow and analysis of the biomass for lipid content.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: It is known that many hyphomycetous fungi are dispersed by wind, water and insects. However, very little is known about how these fungi may differ from each other regarding their ability to be disseminated by different environmental vectors. Consequently, to obtain an indication of the primary means of spore dispersal employed by representatives of the genera Acremonium, Aspergillus and Penicillium, isolated from soil and indoor environments, we monitored spore liberation of cultures representing these genera in an airflow cell. The experimental data obtained, of plate counts conducted of the air at the outlet of the airflow cell, were subjected to an appropriate analysis of variance (ANOVA), using SAS statistical software. Intraspecific differences occurred regarding aerial spore release. Under humid conditions, however, Penicillium species were more successful in releasing their spores than Aspergillus and the Acremonium strain. Under desiccated conditions the Aspergillus took longer to release their spores than representatives of Acremonium and Penicillium. The taxa that were investigated did not differ from each other regarding the release of spores in physiological salt solution (PSS). Although not proven, indications are that water may act as an important dispersion agent for these fungi, because washing of cultures with PSS resulted in all cases in an immediate massive release of colony forming units. Subsequently, using standard plate count techniques, conidial adhesion of the fungi mentioned above to synthetic membranes, leaf cuttings and insect exoskeletons differing in hydrophobicity and electrostatic charge were investigated. We found that the different genera showed different adhesion profiles for the series of test surfaces, indicating differences in physico-chemical characteristics of the fungal spore surfaces. In general, the Penicillium strains showed a greater ability to adhere to the test surfaces, than the aspergilli, while the representative of Acremonium showed the least adherence. No significant difference in the percentage spore adhesion was found between hydrophobic and hydrophilic materials. Furthermore, evidence was uncovered supporting the contention that, under dry conditions, electrostatic surface charges play a role in the adherence of fungal spores to surfaces, because adherence was positively correlated (Correlation coefficient = 0.70898, p = 0.001) to positive electrostatic charges on the lamellar surfaces. In the next part of the study, standard plate count methods were used to determine the relative adhesion of the above mentioned hyphomycetous fungi, as well as a polyphyletic group of yeasts, to the test surfaces submerged in 10 mM sodium phosphate buffer (pH 7.0). As was found with the experiments with the dry surfaces, both intraspecific and intergenus differences were uncovered. Overall, the fungi adhered better to hydrophilic surfaces than to hydrophobic surfaces. This indicated that the fungal surfaces were covered with relatively hydrophilic compounds such as carbohydrates. Subsequently, it was demonstrated that all the fungi adhered to plasma membrane glycoprotein coated polystyrene and the presence of fungal carbohydrates on the surfaces of the fungal propagules was confirmed using epi-fluorescence microscopy. Differences in the strategy of the fungal genera to release their airborne spores, as well as differences in their adhesion profiles for the series of test materials, may be indicative of a unique environmental niche for each genus. In future, this phenomenon should be investigated further. / AFRIKAANSE OPSOMMING: Hifomisete fungi is daarvoor bekend om te versprei deur middel van wind, water, en insek vektore. Maar nietemin, daar is bykans geen kennis m.b.t. hoe hierdie fungi van mekaar verskil t.o.v. hul vermoë om versprei te word deur omgewings vektore nie. Gevolglik was spoorvrystelling van kulture, verteenwoordigend van die genera Acremonium, Aspergillus en Penicillium gemoniteer om ‘n aanduiding te kry van primêre wyse van spoorverspreiding waardeur verteenwoordigers van die onderskeie genera ingespan word. Eksperimentele data ingewin, vanaf plaat tellings wat uitgevoer was op lug afkomstig vanuit die uitlaat-klep van die lugvloei kapsule, was onderwerp aan ‘n toepaslike analise van afwyking (ANOVA), deur gebruik te maak van ‘n SAS statistiese pakket. Intraspesie verskille is waargeneem t.o.v. lug spoorvrystelling. Desnieteenstaande was Penicillium meer suksesvol onder vogtige kondisies t.o.v. spoorvrystelling in vergelyking met Aspergillus en die Acremonium stam. Onder droë kondisies het verteenwoordigers van Aspergillus langer geneem om hul spore vry te stel as verteenwoordigers van onderskeidelik, Penicillium en Acremonium. Geen verskille was waargeneem m.b.t. spoorvrystelling in fisiologiese soutoplossing (FSO) tussen die verskillende filogenetiese stamme nie. Alhoewel dit nie bewys is nie, wil dit voorkom asof water as belangrike verspreidingsagent van die betrokke fungi dien, aangesien die spoel van kulture met FSO tot ‘n oombliklike enorme vrystelling van kolonie-vormende eenhede gelei het. Gevolglik, deur gebruik te maak van standaard plaattellings tegnieke, was spoor aanhegting van bogenoemde fungi aan sintetiese membrane, blaar snitte en insek eksoskelette wat verskil in terme van hidrofobisiteit en elektriese lading, ondersoek. Daar was gevind dat die aanhegtingsprofiele m.b.t. hierdie reeks toetsoppervlaktes van die verskillende genera verskil, wat op sigself ‘n aanduiding was van verskille in fisieschemiese eienskappe van die swamspoor oppervlaktes. Penicillium stamme het ‘n hoër aanhegtings vermoë aan die toetsoppervlaktes getoon as die aspergilli, terwyl die verteenwoordiger van Acremonium die laagste aanhegting getoon het. Geen betekenisvolle verskille i.t.v. persentasie spoor aanhegting was gevind tussen hidrofobiese en hidrofiliese oppervlakte nie. Daarbenewens was die argument dat spoorvrystelling onder droë kondisies beïnvloed word deur elektrostatiese oppervlak ladings, bevestig deur ons bevindinge, want aanhegting het positief gekoreleer (Korrelasie koëffisient = 0.70898, p = 0.001) met positiewe ladings op die oppervlaktes. ‘n Standaard plaattellingstegniek was aangewend in die volgende fasset van die studie om die relatiewe aanhegting van bogenoemde hifomisete fungi, sowel as ‘n polifilitiese groep giste aan die toetsoppervlaktes, gedompel in 10 mM natrium fosfaat buffer (pH 7.0) vas te stel. Intraspesie en intragenus verskille was weereens waargeneem, net soos in die geval van die eksperimente met die droë oppervlakte. In die algemeen het die swamme baie beter geheg aan hidrofiliese oppervlaktes in vergelyking met hidrofobiese oppervlakte. Dit was ‘n aanduiding dat die swamspoor oppervlaktes bedek was met relatiewe hidrofiliese verbindings bv. koolhidrate. Verder was daar bewys dat alle swamme ingesluit in hierdie studie die vermoë het om plasmamembraan glikoproteïn bedekte polistireen te bind, en gevolglik was die teenwoordigheid van van koolhidrate op die swamspore bevestig m.b.v epi-fluoresensie mikroskopie. Verskille in die strategie van swamme om spore in die lug vry te stel, sowel as verskille in die aanhegtingsprofiele vir ‘n reeks toetsmateriale, mag net ‘n aanduiding wees van ‘n unieke omgewings nis vir elke genus wat in hierdie studie ondersoek is. Hierdie verskynsel moet dus in die nabye toekoms nagevors word.

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2006. / Filamentous fungi have been employed for production of heterologous proteins such as enzymes, antibiotics and vaccines due to their good secretion capacities and effective posttranslational modifications of these proteins. With an improvent in recombinant DNA technologies it has become possible to express many useful proteins in species such as the Aspergilli. However the submerged cultivation of filamentous fungi is complicated by the difficulties in mixing and oxygen and nutrient transfer in the highly viscous culture fluids that result. The purpose of the project was to investigate the potential of simultaneous control of morphology and production of enzymes in the dimorphic fungus, Mucor circinelloides, in order to overcome problems associated with the submerged cultivation of filamentous fungi. Dimorphic M. circinelloides, a zygomycete in the order Mucorales, occurs in a filamentous form or a yeast-like morphology in response to environmental conditions. Recently, advances were made in transformation of Mucor, and it has become possible to transform M. circinelloides to express heterologous proteins. The first example of a strong, regulated promoter from M. circinelloides being used for recombinant protein production was the expression of the glucose oxidase gene (from Aspergillus niger) under the control of the glyceradehyde-3-phosphate dehydrogenase (gpd1) promoter. Glucose oxidase (GOX) is an enzyme used to prevent oxidation of foods to extend shelf-life, to produce low-kilojoule beverages and to measure glucose levels in medical diagnostic applications. The scope of this project was to establish the conditions for yeast and filamentous growth of M. circinelloides in order to allow control of morphology, and to evaluate enzyme production under these conditions. Enzyme production of the GOX producing mutant strain, that was recently constructed, was compared to that of a wild type M.circinelloides strain. M. circinelloides was cultured in two-stage batch fermentations, firstly a yeast stage and then a filamentous stage. The yeast morphology was induced by anaerobic conditions while the filamentous morphology was achieved by exposure to air. The enzyme, biomass and metabolite production of the glucose-oxidase producing mutant strain and the wild type were monitored during the two-stage fermentations. GOX from the mutant and native amylase activity levels from the wild type were compared to each other and to other production systems for these enzymes. The morphology could be maintained in a yeast form under N2 with addition of ergosterol and Tween 80. The GOX activity levels in the culture fluid were comparable to some of the unoptimized GOX production systems in literature, but much lower than the optimized, recombinant GOX production systems that employ certain yeasts, or Aspergilli or Penicillium. The intracellular GOX levels were almost 6-fold higher than the extracellular levels which was unexpected as GOX is usually well-secreted. The morphological control improved the morphology for the initial yeast-stage of the fermentation but did not improve the morphology during the filamentous, enzyme-producing stage and it decreased the biomass yield and enzyme production by 50%. The constraint of Mucor to its yeast-like form did not improve the broth homogeneity or enzyme production and increased the time required for enzyme production. In this study M. circinelloides did not perform that well against other species already used to produce these enzymes. However, M. circinelloides could be used to produce enzymes from zygomycetes that systems such as A. niger do not produce well.

Les procédés de bioraffinerie, et notamment les agrocarburants, sont aujourd'hui reconnus comme essentiels pour sortir de l'économie actuelle basée sur le pétrole. Dans le cas du bioéthanol produit à partir de biomasse lignocellulosique, l'hydrolyse enzymatique par les enzymes de Trichoderma reesei est le principal point faible du procédé et doit être améliorée. Ces travaux de thèse s'intègrent dans le cadre du projet Futurol, et ont pour objectif d'identifier de nouvelles enzymes capables d'améliorer l'activité de T. reesei sur la lignocellulose. Une analyse post-génomique réalisée sur les secrétomes de vingt souches fongiques s'est révélée particulièrement prometteuse pour l'identification d'enzymes lignocellulolytiques d'intérêt. Une approche de génomique comparative a également abouti à la sélection de deux endo-mannanases de famille GH5 et GH26 chez le champignon Podospora anserina. Ces hémicellulases ont permis d'améliorer significativement la libération de glucose par T. reesei à partir d'épicéa. Une étude fondamentale approfondie a permis de résoudre les structures cristallographiques et de mettre en évidence les relations entre les spécificités enzymatiques de chaque enzyme et leurs caractéristiques structurales. La structure tridimensionnelle de la mannanase GH26 couplée à son CBM35 présente un linker court et rigide et une organisation du site actif atypique. Les deux mannanases ont également fait l'objet d'un travail d'ingénierie aléatoire qui a abouti à des variants des deux enzymes présentant une amélioration de l'efficacité catalytique et/ou une modification de spécificité. / Biorefineries such as biofuels are nowadays considered as essential to reduce our dependence on oil products. In the production process of bioethanol from lignocellulosic biomass, enzymatic hydrolysis performed by Trichoderma reesei enzymes is the main bottleneck of the process and requires improvements.The present work is part of the Futurol project, and aims at identifying new enzymes to improve the activity of T. reesei toward lignocellulose. Post-genomic analyses on twenty fungal strains have revealed the potential of this approach to identify lignocellulolytic enzymes of interest. Comparative genomics also led to the selection of two endo-mannanases from families GH5 and GH26 from the fungus Podospora anserina. These hemicellulases significantly improved glucose release upon T. reesei hydrolysis of spruce. An in-depth fondamental study allowed the solving of cristallographic structures and revealed the relationships between enzymatic specificities and structural characteristics. The structure of GH26 catalytic module appended to CBM35 highlighted a short and rigid linker and an atypical active site organization. The two mannanases were subjected to molecular engineering. Variants displaying improved catalytic efficiency and/or modified specificity were identified for both enzymes.

Les contaminations par les moisissures représentent un problème majeur au sein de l'industrie agroalimentaire, pharmaceutique, cosmétique, et dans le secteur médical. Actuellement, l'identification des champignons filamenteux est basée sur l'analyse des caractéristiques phénotypiques, nécessitant une expertise et pouvant manquer de précision, ou sur les méthodes moléculaires, coûteuses et fastidieuses. Dans ce contexte, l'objectif de cette étude a consisté à développer un protocole simple et standardisé à l'aide de la spectroscopie infrarouge à transformée de Fourier (IRTF) combinée à une méthode d'analyse chimiométrique, proposant une méthode alternative pour l'identification rapide des moisissures. Au total, 498 souches de champignons filamenteux (45 genres et 140 espèces) ont été analysées à l'aide d'un spectromètre IRTF à haut débit. L'analyse discriminante des moindres carrés partiels (PLS -DA), méthode chimiométrique supervisée, a été appliquée à chaque spectre dans les gammes spectrales 3200-2800 et 1800-800 cm-1. Différents modèles de calibration ont été construits à partir de 288 souches, ceci en cascade de la sous-division jusqu'à l'espèce en se basant sur la taxonomie actuelle. La prédiction des spectres en aveugle, obtenus à partir de 105 souches, au niveau du genre et de l'espèce est respectivement de 99,17 % et 92,3 %. La mise en place d'un score de prédiction et d'un seuil a permis de valider 80,22 % des résultats. L'implémentation d'une fonction de standardisation (SF) a permis d'augmenter le pourcentage de spectres bien prédits, acquis sur un autre instrument, de 72,15 % (sans fonction) à 89,13 %, validant la transférabilité de la méthode. Puisqu'une biomasse mycélienne suffisante peut être obtenue après 48h de culture et que la préparation des échantillons implique l'utilisation d'un protocole simple, la spectroscopie IRTF combinée à la PLS-DA apparaît comme une méthode rapide et peu coûteuse, ce qui la rend particulièrement attractive pour l'identification des champignons filamenteux au niveau industriel. Les résultats obtenus placent la spectroscopie IRTF parmi les méthodes analytiques prometteuses et avant-gardistes, possédant un haut pouvoir discriminant et une forte capacité d'identification, en comparaison avec les techniques conventionnelles. / Mold contaminants represent a major problem in various areas such as food and agriculture, pharmaceutics, cosmetics and health. Currently, molds identification is based either on phenotypic characteristics, requiring an expertise and can lack accuracy, or on molecular methods, which are quite expensive and fastidious. In this context, the objective was to develop a simple and standardized protocol using Fourier transform infrared (FTIR) spectroscopy combined with a chemometric analysis, allowing to implement an alternative method for rapid identification of molds. In total, 498 fungal strains (45 genera and 140 species) were analyzed using a high-throughput FTIR spectrometer. Partial Least Squares Discriminant Analysis (PLS-DA), a supervised chemometrics method, was applied to each spectrum in the spectral ranges 3200-2800 and 1800-800 cm-1 for the identification process. Using 288 strains, different calibration models were constructed in cascade and following the current taxonomy, from the subphylum to the species level. Blind prediction of spectra from 105 strains at the genus and species levels was achieved at 99.17 % and 92.3% respectively. The establishment of a prediction score and a threshold permitted to validate 80.22% of the obtained results. The implementation of a standardization function (SF) permitted to increase the percentage of well predicted spectra from strains analyzed using another instrument from 72.15% (without SF) to 89.13% and permitted to verify the transferability of the method. Since sufficient mycelial biomass can be obtained at 48h culture and sample preparation involved a simple protocol, FTIR spectroscopy combined with PLS-DA is a very rapid and cost effective method, which could be particularly attractive for the identification of moulds at the industrial level. The results obtained places FTIR spectroscopy among the avant-garde promising analytical approaches, with high discriminant power and identification capacity, compared to conventional techniques.