The goal of this study was to undertake a microbial investigation of the Historic Huts areas on Ross Island, to gain knowledge of the fungal biodiversity and biochemical framework, focusing on the wood degrading potential of these fungi at both psychrophilic (cold) and mesophilic (moderate) temperatures. Eight hundred and forty nine samples were collected from three Heroic Era Historic Huts of Antarctica, from a variety of substrates but predominantly structural wood. The huts, Discovery Hut at Hut Point, Terra Nova Hut at Cape Evans and Nimrod Hut at Cape Royds, are located on Ross Island and were all assembled in the early 20th century by the Heroic Era explorers to house the expeditions, stores and animals. These wooden huts were abandoned when the expeditions left. The introduction of wood and other organic material to a pristine environment along with the creation of a microclimate within the harsh Antarctica environment created interesting sites for studying fungal diversity, wood decay and fungal cellulase enzymes in an extreme environment. Each hut can be classified as offering different conditions and circumstances for fungal propagules. Of the three huts, Terra Nova Hut is the only hut where there are visible fungal blooms within the hut and it, with Discovery Hut, had the greatest number of samples that contained fungi compared to Nimrod Hut which had the least. Discovery Hut, at less than 500 metres from the United States McMurdo Station, is the most visited by scientist and base staff and has been the most demonstrably affected by human impact of the three huts due to its closeness to the research stations on Ross Island To ensure a full understanding of the fungal diversity of the Historic Hut sites, a variety of sampling techniques were used along with a variety of culture media. Two thousand and seventy six isolates consisting of 1177 filamentous fungi and 899 single celled microorganisms (yeast and bacteria) were isolated; all these cultures were frozen and now form the University of Waikato Antarctic Culture Collection. Five genera dominated the fungal isolates that were identified and these were Cladosporium, Geomyces, Cadophora, Penicillium and Thelebolus. The fungal diversity of these Historic Huts' communities is low but the members present are metabolically active, consistent with other microbial communities in the Antarctic. The Historic Huts and surroundings contain a diverse array of provision in the way of wood and supplies, which provide nutrient sources for fungal growth. Endemic organisms present in the soil could have been enriched by using the introduced nutrient sources as primary and/or second metabolic substrates. In addition, fungi could have been introduced with the wooden huts and supplies when they were brought to Antarctica by the Heroic Era explorers, or introduced in the subsequent years with visitors and conservation work conducted at the sites. These introduced organisms, though, would have had to adapt to the change in climate and conditions posed by the Antarctic in order to survive and be subsequently isolated in this study. A screen for carboxymethylcellulase (CMCase) activity was done on a selection of the fungal isolates as the first step to understand the cellulytic potential of the Antarctica fungal community inhabiting the huts. One hundred and six fungal isolates from a total of 404, that were screened were deemed to be CMCase positive, 27 fungal isolates were chosen for further study including quantifying the activity of extracellular endo-1,4-β-glucanase at psychrophilic and mesophilic incubation temperatures. All but one isolate could produce endo-1,4-β-glucanase activity at 4 C and many produced more endo-1,4-β-glucanase activity at 4 C than at 15 C. Cadophora malorum 182, Cadophora malorum 242, Penicillium roquefortii 405, Penicillium roquefortii 408, Geomyces sp. 711, Geomyces sp. 824 and Cladosporium oxysporium 805 were selected for in-depth study of growth characteristics including growth temperature preferences, growth on a variety of cellulose substrates, water activity, and carbon sources, the latter done by using a commercially available microtitre plate containing 95 carbon sources. All seven of the fungal isolates were classified as psychrotolerant and produced, when cultured at either 4 C or at 15 C, cellulase, protease, amylase, xylanase, and pectinase and mannanase enzyme activities. The range of water activity that the Antarctic Penicillium roquefortii isolates could grow at was distinctive when compared with food Penicillium roquefortii isolates. The utilisation of different carbon sources showed that like many studies of Antarctica organism they have a diverse range of enzymatic activity, but interestingly the activity does not differ greatly with incubation temperature with most carbon sources being used or not used at both incubation temperatures tested. Although it took longer for the fungi to grow at the psychrophilic temperatures, the range of carbon sources they utilised was not reduced. The protein composition of the extracellular supernatants was visualised using various electrophoretic and staining techniques. The cellulase activity of the protein bands was visualised by cellulose-containing zymograms, which illustrated that the cellulase complex in all fungi tested was multi-enzyme and differed between species, isolates and temperatures of culturing. The cellulase activity of Cadophora malorum 182 was enriched by purification techniques including ion exchange chromatography and native preparative electrophoresis. The protein complex was not purified to homogeneity, but enriched for a mixture of proteins and the mixture was described as having the following properties; a temperature range of β-1,4-glucan cellobiohydrolase activity from 20 C to 80 C with the optimum activity seen at 60 C, β-1,4-glucan cellobiohydrolase activity that is stable at 4, 25 and 40 C for at least 24 hrs, lost at 50 C and 80 C within 24 hrs and 2 minutes respectively. Along with β-1,4-glucan cellobiohydrolase activity, the protein mixture contained Avicelase, CMCase, xylanase and mannanase activity. The thesis research showed that there was limited fungal diversity in the Historic Huts and artefacts (a total of five dominant genera were identified) but the fungi are actively growing and producing viable spores in the cold of Antarctica and producing the necessary enzymes for degradation of wood. Although the metabolism and growth rate is slower at psychrophilic temperatures, the fungal isolates studied as part of this thesis research could still function enzymatically at cold temperatures and this includes the degradation of wood as evidenced by in vitro wood decay studies examined by scanning electron microscopy where two isolates of one species demonstrated the ability to degrade wood. The cellulase complex of the investigated fungal isolate was multi-enzymed and although the components were not purified to homogeneity, an enriched mixture of proteins had enzyme activity and stability in a broad temperature range, and activity to a variety of cellulosic substrates. This thesis research adds to the knowledge of the fungal biodiversity in the Antarctic and increases the understanding of the biochemical framework, participating in relation to wood decay potential of these Antarctic fungal isolates.

Cellulosic biomass is the major organic matter produced in the biosphere. The biodegradation of this cellulosic material is achieved by enzymatic activities of the cellulose degrading microorganisms. These organisms usually express a complex extracellular or a membrane bound cellulolytic system comprising combination of several cellulase enzymes. Cellulases are the group of hydrolytic enzymes capable of hydrolysing insoluble cellulose to glucose. Phlebia gigantea is an aggressive white rot basidiomycete with ability to tolerate resinous extracts on freshly cut wood and higher growth rate. This helps the fungus to colonise the sapwood preventing other fungi from becoming established. Early research on the cellulase system of this organism reported the presence of a cellulase system composed of P-glucosidase, endoglucanase and a cellobiohydrolase. Based on these unpublished studies, our aim was to obtain a complete sequence of putative cellobiohydrolase I (CbhI) from this organism. Attempts to identify and isolate the cellulase gene resulted in an incomplete cDNA sequence of I 154 bp. To understand the cellulase system, expression and regulation of the cellulase enzymatic activity was examined for incubation of P. gigantea on substrates glucose, xylose, Avicel, carboxymethyl cellulose and cellobiose. The pH, total protein and biomass production results indicated that the capacity of P. gigantea to degrade cellulose is dependent upon the nature of the carbon source and the regulation of the cellulase synthesis is repressed in the presence of simple sugars like glucose and xylose. The study employed the highly effective method of purification by affinity adsorption and purified cellulase complex in large quantity. Characterisation of the kinetic properties of this cellulase complex revealed that the rate of cellulase catalysis were optimum at pH 5.0 and temperature 50GC. The purified complex was comprised of multiple proteins and demonstrated significant CMCase and CBHase activity on zymogram analysis. The purified cellulase complex was characterised by 2D gel electrophoresis and by peptide mass finger printing using MALDI-TOF massspectrometry analysis. The 2D gel analysis of the purified cellulase complex showed 15 spots within the range of pI 3.5 to pI 7 and the molecular weight between 20KDa to 100KDa. Three protein spots were selected based on the IEF and SDS zymogram and identified using MALDI-TOF MS analysis. These proteins were identified based on the peptide mass data belonging to the 6-phospho-a-glucosidase, p-glucosidase and glycosyl hydrolase family 13 a-amylase or pullulanases, suggesting the divergent evolution of specific cellulase proteins. This study showed P. gigantea as a potential cellulase source and the cellulase complex secreted by the induction of substrate, comprises a variety of enzymes related to hydrolysis of cellulose biomass. It is evident from this and previous studies that P. gigantea cellulase complex comprises of a specific set of enzymes that possess the ability to degrade crystalline cellulose and is one of the first organisms to colonise freshly cut wood. Further studies on the cellulase system of this primary colonist may open up the prospects to utilise this organism as the potential onsite bioreactor agent, pre-treating the biomass and increasing the economic feasibility of the industrial bioenergy processes.

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2004. / Thesis documentation sheet inserted. Errata sheet inserted. Appendix reprints four papers and manuscripts co-authored with others. Includes bibliographical references. Also issued electronically via World Wide Web in PDF format; online version lacks appendix.

Thèse de doctorat : Procédés biotechnologiques et alimentaires : INPL : 2006. / Titre provenant de l'écran-titre. Bibliogr.

Reproduction de : Thèse de doctorat : Biochimie : Lyon 1 : 2005. / Titre provenant de l'écran titre. 205 réf. bibliogr.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1962. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 57-64).

Thesis (MScAgric) -- Stellenbosch University, 2003. / ENGLISH ABSTRACT: Cellulose is by far the most abundant carbohydrate available from plant biomass. These biopolymers are therefore an important renewable source of food, fuels and chemicals. Cellulose is embedded in a matrix of hemicellulose, lignin and pectin and is composed of repeating glucose units linked by p-1,4-glycosidic bonds. The individual molecules are held together by hydrogen bonds, forming largely crystalline fibres. The hemicellulose, which is a low molecular weight heteropolysaccharide, coats and binds the cellulose microfibrils, preventing the cellulose from becoming too crystalline. Three predominant types of hemicelluloses are recognised, namely 1,3- and 1,4-p-D-galactans, 1,4-p-D-mannans and 1,4-p-D-xylans, which are named according to the sugar type that forms the polymer backbone. Pectic substances contain rhamnogalacturonan backbones in which 1,4-linked a-D-galacturonan chains are interrupted at intervals with a-L-rhamnopyranosyl residues carrying neutral side chains. Two groups of enzymes, cellulases and pectinases, are required for the microbial utilisation of crystalline cellulose and pectin. Cellulases are multicomponent complexes that are often composed of endoglucanases, exoglucanases and cellobiases. Cellobiose is the major end product of concerted endoglucanase and exoglucanase activity. Cellobiose is then hydrolysed to glucose by p-glucosidases. The enzymatic breakdown of pectic polymers occurs by the deesterifying action of the saponifying enzymes, pectinesterase, releasing the methyl groups of the pectin molecule, and by hydrolase or lyase action of the depolymerases (pectin lyase, pectate lyase and polygalacturonase), splitting the a- 1.4-glycosidic linkages in the polygalacturonate chain. The yeast Saccharomyces cerevisiae has been used extensively in the alcoholic beverage industry for fermentations of wine, beer and other alcoholic beverages for many years. However, it is unable to produce extracellular depolymerising enzymes that can efficiently degrade polysaccharides, which are the main cause of clarification and filtration problems. Enzyme preparations have been used in the alcoholic beverage industries to degrade haze-forming polysaccharides, thereby improving the filterability and quality of products such as beer and wine. An alternative would be to develop S. cerevisiae strains that produce extracellular polysaccharidases, enabling the yeast to degrade polysaccharides without the addition of commercial enzyme preparations. These strains can also be very useful in improving the quality of wine, as well as cutting the costs of the winemaking process. The objective of this study was to investigate the effects of two transformed S. cerevisiae strains on different wine grape varieties. The following genes have been cloned and characterised previously: the Aspergillus niger endo-p-xylanase gene (xynC), the Butyrivibrio fibrisolvens endo-|3- 1.4-glucanase gene (endl), the Erwinia chrysanthemi pectate lyase gene (pelE) and the Erwinia carotovora polygalacturonase gene (p e h l). The yeast alcohol dehydrogenase I gene promoter (ADH1p), the alcohol dehydrogenase II gene terminator (ADH2j), the tryptophan synthase gene terminator (TRP5r) and the yeast mating-type pheromone a-factor secretion signal sequence (MFcrfs) were used to compile the following gene constructs: ADH1 p-MFa1 s-end1-TRP5r (designated END1), A DH1 p-xyn C-A DH2T (designated XYN4), ADH1 p-MFa1 s-peh1 -TRP5t (designated PEH1) and ADH1 p-MFa1 s-pelE-TRP5r (designated PELE). Two yeast integrating plasmids were constructed, one containing the END1 and XYN4 gene cassettes and the other containing the PEH1-PELE cassette. These two plasmids were then integrated into the URA3 locus of two separate industrial wine yeast strains of S. cerevisiae. To facilitate selection of the industrial yeast transformants in the absence of auxotrophic markers, the integrating plasmid containing the END1 and XYN4 gene cassettes was issued with the dominant selectable Geneticin G418-resistance {G f) marker. The integrating plasmid harbouring the PEH1-PELE gene cassette was issued with the dominant selectable sulphumetronmethyl resistance (SMR1) marker. The introduction of these plasmids into commercial wine yeast strains directed the synthesis of END1, XYN4, PELE and PEFI1 transcripts and the production of extracellular biologically active endo-P-1,4- glucanase, endo-(3-xylanase, pectate lyase and polygalacturonase. These recombinant yeasts were capable of extracting more colour from grape skins of certain varieties, as well as leading to more freeflow wine as a result of the more effective degradation of glucans, xylans and pectins in the skins. They also led to decreased turbidity in the wine, making it more filterable. Future work will entail further investigation of the effects of these recombinant yeasts on different white and red wine grape varieties. Another objective of this study was to screen non-Saccharomyces wine yeasts for the production of extracellular hydrolytic enzymes. The reason for this part of the thesis was to determine the types of extracellular hydrolytic enzymes that are produced and to determine which genera produce which kinds of extracellular enzymes. A total of 237 yeast isolates, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and Kluyveromyces, were screened for the production of extracellular pectinases, proteases, (3-glucanases, lichenases, p-glucosidases, cellulases, xylanases, amylases and sulphite reductase activity. These yeasts were all isolated from grapes and clarified grape juice to ensure that they were yeasts found in must during the initial stages of fermentation. This information can be used to pave the way to pinpoint the specific effects in wine of these enzymes produced by the so-called wild yeasts associated with grape must. This information can also be used to transform Saccharomyces wine yeasts with some of the genes from these non-Saccharomyces yeasts for the production of extracellular hydrolytic enzymes. However, future research will have to be done to determine the extent of the activity of these enzymes in wine fermentations and to obtain better knowledge of the physiological and metabolical features of non-Saccharomyces yeasts. / AFRIKAANSE OPSOMMING: Sellulose is verreweg die volopste koolhidraat in plantbiomassa. Hierdie biopolimere is dus ‘n baie belangrike hernubare bron van voedsel, brandstof en chemikaliee. Sellulose is in 'n matriks van hemisellulose, lignien en pektien gebed en is uit herhaalde glukose eenhede, wat deur middel van (3-1,4-glukosidiese bindings geheg is, saamgestel. Die individuele molekules word deur waterstofbindings aan mekaar geheg, wat aanleiding gee tot die vorming van kristallyne vesels. Die hemisellulose, wat 'n lae molekulere gewig heteropolisakkaried is, bedek en bind die sellulose vesels en verhoed daarmee die vorming van vesels wat te kristallyn is. Drie predominante tipes hemisellulose word herken en sluit 1,3- en 1,4-p-D-galaktane, 1,4-p-D-mannane en 1,4-p-D-xylane in, wat vernoem word volgens die suikereenhede wat die polimeerruggraat vorm. Pektiene bestaan uit 'n rhamnogalakturonaanruggraat waarin 1,4-gekoppelde a-D-galakturonaankettings periodiek met a-L-rhamnopiranosiel residue, bevattende neutrale sykettings, onderbreek word. Twee groepe ensieme, nl. pektinase en sellulase, word deur mikrobes vir die benutting van kristallyne pektinase en sellulase vereis. Sellulase is multikomponent komplekse wat dikwels uit endoglukanase, ekso-glukanase en sellobiase saamgestel is. Sellobiose is die hoof eindproduk van die saamgestelde aktiwiteit tussen endoglukanase en ekso-glukanase en word verder gehidroliseer tot glukose deur |3-glukosidases. Die ensimatiese afbraak van pektien polimere vind deur die de-esterifiserings aksie van die versepings ensiem, pektienesterase, plaas. Dit lei tot die vrystelling van die metielgroepe van die pektienmolekuul. Deur die hidrolase of liase aksie van die depolimerase (pektien liase, pektaatliase en poligalakturonase), split die a-1,4-glukosidiese verbindings in die poligalakturonaatketting. Die gis Saccharomyces cerevisiae word al vir jare ekstensief in die alkoholbedryf vir die fermentasie van verskeie produkte, veral druiwe, gebruik. S. cerevisiae besit egter nie die vermoe om ekstrasellulere depolimiserende ensieme wat vir die effektiewe degradasie van polisakkariede verantwoordelik is, te produseer nie, wat die hoof oorsaak van die verhelderings- en filtreringsprobleme in onder andere wyn en bier is. Dit veroorsaak ook dat S. cerevisiae nie oor die vermoe beskik om waasvormende polisakkariede in wyn te degradeer nie. Tans word ensiempreparate in die alkoholiese bedryf vir die degradasie van die probleem polisakkariede gebruik. Sodoende word die filtreerbaarheid en kwaliteit van wyn en bier verbeter. ‘n Goeie alternatief is die ontwikkeling van S. cerevisiae-rasse wat oor die vermoe beskik om ekstrasellulere polisakkarase te produseer en dus polisakkariede self sonder die byvoeging van eksterne kommersiele ensiempreparate te degradeer. Hierdie rasse sal baie voordelig wees vir die verbetering van wynkwaliteit, sowel as vir die vermindering van die kostes verbonde aan die wynmaakproses. Die objektief van hierdie studie is dus om die uitwerking van twee getransformeerde S. cerevisiae rasse, wat ekstrasellulere polisakkarases produseer, op verskillende wyndruifvarieteite na te vors. Die volgende gene is reeds voorheen gekloneer en gekarakteriseer: die endo-pxylanase- geen (xynC) van Aspergillus niger, die endo-p-1,4-glukanase-geen (endl) van Butyrivibrio fibrisolvens, die pektaatliase-geen (pe/E) van Erwinia chrysanthemi en die poligalakturonase-geen (p e h l) van Erwinia carotovora. Die alkoholdehidrogenase-geenpromotor (ADH1P), die alkoholdehidrogenase IIgeentermineerder (ADH2T), die gistriptofaansintase geen se termineerder (TRP5t) en die sekresiesein van die gisferomoon a-faktor (MFa1s) is gebruik om die volgende geenkonstrukte saam te stel: ADH1 p-MFa1 s-end1 -TRP5t (toekend as END1), ADH1 p-xynC-ADH2T (bekend as XYN4), ADH1 p-MFa1 s-peh1-TRP5T fbekend as PEH1), and ADH1 p-MFa1 s-pelE-TRP5T (bekend as PELE). Twee gisintegrerings plasmiede is gekonstrueer, een wat die END1- en XYN4- geenkassette bevat en die ander wat die PEH1-PELE-kasset besit. Hierdie twee plasmiede is daarna in twee aparte industriele wyngisrasse van S. cerevisiae by die URA3 lokus geintegreer. Vir die seleksie van die industriele wyngistransformante in die afwesigheid van ouksotrofiese merkers, is die dominante selekteerbare Geneticin G418 weerstandbiedende (G f) merker in die END1- en XYA/4-geenkassetbevattende plasmied geintegreer. Die dominante selekteerbare sulfumetronmetielweerstandbiedende (SMR1) merker is in die integreringsplasmied, wat die PEH1- PELE-geenkasset bevat, geintegreer vir seleksie. Transformasie van hierdie plasmiede in kommersiele wyngisrasse het tot die direkte sintese van die END1-, XYN4-, PELE- en PEH1-transkripte aanleiding gegee, sowel as tot die produksie van die biologies aktiewe ekstrasellulere endo-P-1,4-glukanase, endo-P-xylanase, pektaatliase en poligalaturonase. Tydens die wynmaakproses het bogenoemde rekombinante giste aanleiding gegee tot verhoogde kleurekstraksie uit die druifdoppe van sekere varieteite, asook tot verhoogde vryvloei wyn. Dit is verkry deur die effektiewe degradasie van die glukane, xilane en pektiene in die doppe. Die rekombinante giste het ook verlaagde turbiditeit in die wyn tot gevolg gehad, wat die wyne makliker filtreerbaar maak. Hierdie werk was net die eerste stap. In die toekoms sal verdere navorsing gedoen moet word om die presiese effekte van hierdie rekombinante giste op verskillende rooi en wit druifvarieteite te bepaal. ‘n Ander fokus van hierdie tesis was om nie-Saccharomyces wyngiste vir die produksie van ekstrasellulere hidrolitiese ensieme te selekteer. Die rede hiervoor is om te bepaal watter tipes ekstrasellulere hidrolitiese ensieme geproduseer word, asook watter ensieme deur watter genera geproduseer word, ‘n Totaal van 237 gisisolate wat tot die generas Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora en Kluyveromyces behoort, is vir die produksie van ekstrasellulere pektinase, protease, p-glukanase, lichenase, p-glukosidase, sellulase, xilanase, amilase en sulfiet reduktase-aktiwiteit getoets. Hierdie giste is almal vanaf druiwe en druiwesap geVsoleer om te verseker dat dit wel giste is wat gedurende die beginfases van fermentasie in die mos teenwoordig is. Hierdie inligting kan nou verder gebruik word om die spesifieke effekte wat hierdie ensieme, wat deur die sogenaamde wilde giste geproduseer word, tydens die beginfases van fermentasies op die mos het, te bepaal. Hierdie inligting kan ook in die toekoms gebruik word om Saccharomyces-wyngiste met gene van die ri\e-Saccharomycesgiste te transformeer om ekstrasellulere hidrolitiese ensieme vir die degradasie van die problematiese polisakkariede in wyn te produseer. Daar sal egter in die toekoms baie navorsing gedoen moet word om die omvang van hierdie ensiemaktiwiteite in wynfermentasies te bepaal, asook om meer kennis te bekom oor die fisiologiese en metaboliese samestelling van nie-Saccfraromyces wyngiste.

As celulases e as xilanases são enzimas que hidrolisam a celulose e a xilana, respectivamente, contidas nos resíduos lignocelulósicos. A possibilidade de aplicar estas enzimas na produção de etanol vem sendo objeto de diversos estudos. No entanto, ainda não há uma tecnologia economicamente viável para a produção deste biocombustível a partir da biomassa lignocelulósica. Entre os microrganismos que apresentam altos títulos para estas enzimas, incluem-se linhagens de Penicillium echinulatum; porém, ainda faltam dados de sua fisiologia e estudos da produção de enzimas em biorreator. Neste trabalho, empregou-se a linhagem mutante celulolítica desreprimida S1M29 de P. echinulatum e o meio de cultivo foi composto por celulose, sacarose, solução de sais, Tween 80, farelo trigo e farelo de soja. Avaliou-se o efeito de diferentes temperaturas e pHs na produção das enzimas. O efeito da concentração da celulose sobre as atividades enzimáticas foi avaliada em regime descontínuo (RD) e regime descontínuo alimentado (RDA). Verificou-se que a temperatura mais apropriada para a produção de celulases e xilanases é de 28ºC e dentre os valores de pHs avaliados, o pH 6,0 apresentou a maior produção das enzimas. O aumento da concentração da celulose no RD proporcionou maiores atividades para endoglicanases, porém o mesmo não foi obtido para xilanases. Para FPA (Filter Paper Activity), aumentos proporcionais nas atividades foram obtidos somente com concentrações de até 3% de celulose em RD, condição que também proporcionou as maiores atividades de - glicosidases. O RDA incrementou as atividades de FPA, endoglicanases e xilanases, mas não de -glicosidases. Estes resultados contribuem para a otimização de processos e para a produção econômica de enzimas por P. echinulatum, visando o desenvolvimento de tecnologias economicamente viáveis para produção de etanol a partir de materiais lignocelulósicos. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-06-11T13:29:22Z No. of bitstreams: 1 Dissertacao Laisa dos Reis.pdf: 1870988 bytes, checksum: 956ace97d10d44f22cb5eba7cea275d1 (MD5) / Made available in DSpace on 2014-06-11T13:29:23Z (GMT). No. of bitstreams: 1 Dissertacao Laisa dos Reis.pdf: 1870988 bytes, checksum: 956ace97d10d44f22cb5eba7cea275d1 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cellulases and xylanases are enzymes that hydrolyze cellulose and xylan respectively, which are found in lignocellulosic residues. Although the applicability of these enzymes in the ethanol production has been the subject of several studies, an economically viable technology for the production of biofuel from lignocellulosic biomass is currently not available. Strains of Penicillium echinulatum are among the microorganisms that have high titers of these enzymes. However, data related to physiology and enzyme production in bioreactor for such strains are still missing. A cellulolytic mutant strain of P. echinulatum S1M29 and a culture medium composed of cellulose, sucrose, salt solution, Tween 80, wheat bran and soybean meal were used in this study. The effect of different temperatures and pHs during the enzymes production was evaluated. The effect of cellulose concentration in the enzymatic activity was evaluated in batch cultivation (BC) and fed-batch cultivation (FBC). It was found that the appropriate temperature for the production of cellulases and xylanases is 28°C, while the higher enzyme production occurred at pH 6.0. The high cellulose concentration in BC provided the greatest activities for endoglicanases, but the same result was not obtained for xylanases. For Filter Paper Activity (FPA), proportional increases in activity were obtained only with concentrations up to 3% of cellulose in BC, which is also linked to the highest activities for -glucosidases. FBC increased the activities of FPA, endoglucanases and xylanases, but it did not increase the -glucosidases activities. Such results contribute towards the optimization of enzyme production using P. echinulatum and the development of economically viable technologies for the production of ethanol from lignocellulosic materials.

O estudo de fungos celulolíticos tem-se mostrado relevante, tendo em vista o interesse econômico do complexo celulases, especialmente na indústria têxtil e, mais recentemente, para propósitos energéticos. No presente trabalho, a fusão de protoplastos foi utilizada para combinar genótipos de mutantes parcialmente desreprimidos para produção de celulases de Penicillium echinulatum (9A02S1B9) e richoderma harzianum (AS5CH3), utilizando a técnica do doador morto, buscando-se obter recombinantes com maior produção de celulases. Nesta estratégia, ambas as linhagens tiveram seu micélio tratado com Glucanex 0,01 g/mL, para quebra da parede celular. Os protoplastos resultantes da linhagem portadora de marca de resistência ao benomil (9A02S1B9) foram inativados por calor (técnica do doador morto) de 60oC antes da etapa de fusão, a qual após foi induzida por PEG4000 e Ca2+, com protoplastos da linhagem sensível ao benomil (AS5CH3). A partir de um produto de fusão, foram selecionados 24 sub-clones, após estratégias de estabilização e seleção para precocidade e eficiência na formação de halo de hidrólise de celulose em placas de Petri. Os produtos de fusão apresentaram morfologia e esporulação semelhantes a um dos parentais, sendo treze semelhantes à Penicillium, nove semelhantes à Trichoderma e dois mostrando formas alteradas. Os produtos de fusão que segregaram para morfologia de T. harzianum apresentaram a característica de resistência ao benomil, sendo capazes de crescer e esporular em meios contendo até 100 μg/mL deste inibidor. A morfologia, o perfil de bandas, obtidos por RAPD, e o padrão de secreção de celulases dos produtos de fusão foram sempre mais semelhantes a um dos parentais. Os clones apresentaram variação quanto ao halo de hidrólise de celulose em placas de Petri e na atividade sobre papel filtro FPAases, -glicosidase ou endoglicanase, quando crescidas em cultivo submerso ou em estado sólido. Desta variabilidade, verificaram-se aumentos significativos para algumas das linhagens em relação aos parentais. A aplicação da metodologia de fusão de protoplastos para obter recombinantes entre P. echinulatum e T. harzianum, empregando a técnica do doador morto, mostrou-se adequada na geração de variabilidade para produção de celulases. / Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2015-02-12T12:22:13Z No. of bitstreams: 1 Dissertacao Barbara Lizandra Perini de Souza.pdf: 2248330 bytes, checksum: 2a20339f50d031a74d2a889ddbe2435e (MD5) / Made available in DSpace on 2015-02-12T12:22:13Z (GMT). No. of bitstreams: 1 Dissertacao Barbara Lizandra Perini de Souza.pdf: 2248330 bytes, checksum: 2a20339f50d031a74d2a889ddbe2435e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The study of cellulolytic fungi has proved to be important, considering economic interest of the cellulase complex, especially in the textile industry and, more recently, for energy purposes. In this work, the protoplast fusion was used to combine genotypes of mutants partially non repressed for cellulases production of Penicillium echinulatum (9A02S1B9) and Trichoderma harzianum (AS5CH3) using the technique dead donor, intending to obtain recombinants with higher cellulases production. In this strategy, both strains had their mycelium treated with Glucanex  0,01 g/mL, to lyse the cell wall. The protoplast obtained from the benomyl-resistant (9A02S1B9) were heat-inactivated (technique of dead donor) at 60ºC, before the step of fusion, induced by PEG4000 and Ca2+, with protoplast of the sensitive-benomyl strain (AS5CH3). Twenty four sub-clones were selected from one fusion product, after stabilization and selection strategies for precocity and efficiency in the formation clearing zones of by cellulose hydrolysis in Petri plates. The fusion products showed similar morphology and sporulation to one of parents, thirteen similar to Penicillium, nine similar to Trichoderma and two showed altered forms. The fusion products which segregate to the morphology of T. harzianum resistance to benomyl, being able to grow and sporulate in media containing up to 100 μg/mL of this inhibitor. The morphology, the profile of bands, obtained by RAPD, and the pattern of cellulase secretion by fusion products were ever more similar to one of parents. The fusants presented variation in the halo of cellulose hydrolysis in Petri plates, and in the activity on filter paper (FPAases), - glicosidase or endoglicanase, when grown submerged cultivation or solid state. From this variability, significant improvement was verified for some of the parental strains. The application of the protoplast fusion methodology to obtain recombinant between P. echinulatum and T. harzianum, using the technique of dead donor, has proved to be adequate to generate variability in the production of cellulases.