This master’s thesis discusses the useof enzymatic hydrolysis process of waste cardboard using simultaneous saccharification and fermentation (SSF) as a source of raw materials for production of liquid biofuels. This thesis is based on theses written by Ing. Brummer and Ing.Lepař.Thus, results gained in these works have been used and also further developed. The theoretical part summarizes the reasons for further development of SSF method and discusses, as well, the achievements reached in the processing of lignocellulosic waste materials by the SSF method so far.This section also discusses the general characteristics of lignocellulosic materials and also of the cellulolytic enzymes. It focusses also on individual pretreatment methods of lignocellulosic material and options of increasing the yield of the whole process. The experimental part verifies the particular results reached in previous theses and at the same time a further optimization of the method has been carried out because of the transfer of the whole process into a fermenter. Cardboard was set as the substrate for the experiments as it was evaluated by Ing. Brummer as the best one for enzymatic hydrolysis which was carried out by enzymes from Novozymes®. Parameters such as temperature, pH and kind of used buffer, the loading concentration of substrate and enzymes, were set according to the thesis of Ing. Lepař, which was aimed to their optimization. The SSF process done in fermenter of 2.0 l volume confirmed the previous results and furthermore it has been more effective through optimization of the added inoculum volume. It has been confirmed that the best substrate is cardboard finely grinded by vibrating mill. Also experiments with added nutrients had been done as an effort to increase the ethanol concentration, but these haven’t resulted insatisfying results. The maximal concentration of ethanol was 23,49 g/l, which was achieved after further optimization of various conditions. This result equals to experimental yield of 84,79 %.

Thesis (Ph. D. (Microbiology)) -- University of Limpopo, 2019 / In an effort to align the current research with the country’s biofuel strategy, the aim of the study was to utilize banana pseudostem in the production of fungal cellulolytic enzymes and bioethanol through fermentation of the banana pseudostem hydrolysate. The selection of microorganisms was based on the ability of the fungi to grow on agar containing Avicel (microcrystalline cellulose) followed by assaying for cellulases in the form of endoglucanase and total cellulase activity. Ten fungal isolates obtained from screening process showed positive endoglucanase activity on carboxymethyl cellulose – Congo Red agar plate. The six fungal isolates selected based on high cellulase activity belonged to Trichoderma and Aspergillus genera. In submerged fermentation (SmF), the maximum cellulase and endoglucanase production under optimal conditions by all fungal isolates was achieved in media with an initial of pH 6.5 at 30 °C. Under these conditions, the total cellulase activity was 9.79 filter paper units (FPU)/mL and endoglucanase activity 45.2 U/mL for Trichoderma longibrachiatum LMLUL 14-1 and total cellulase activity of 7.7 FPU/mL and endoglucanase activity of 32.7 U/mL for Trichoderma harzianum LMLUL 13-5. These cellulase activities were higher than in the crude enzymes system for all Aspergillus fumigatus. The production conditions for maximum β-glucosidase varied amongst the Aspergillus spp. For example, Aspergillus fumigatus LMLUL 13-4 had produced higher β-glucosidase activity in a medium with an initial pH of 6.5 and at an incubation temperature of 30 °C whereas A. fumigatus LMLUL 13-1 had produced higher β-glucosidase activity at an initial pH of 7.0 and at 35 °C. Solid state fermentation (SSF) to produce cellulase enzymes system was influenced by temperature, nature of the substrate (i.e. moisture, modification) and culturing technique/strategy (i.e. monoculture versus co-culture). Higher cellulase enzymes system was produced under the conditions of 30 °C, 75% moisture content of untreated (native) BPS and pH 6.5. All the fungi investigated, produced thermotolerant and acidophilic cellulase and endoglucanase, whilst β-glucosidase is both acidophilic and alkaliphilic. The cellulase enzymes complex of T. harzianum LMLBP07 13-5 is most stable, followed by A. fumigatus LMLPS 13-4 and the least stable cellulase enzymes complex was for T. longibrachiatum LMLULSA 14-1. For the pretreatment of BPS, the material was first subjected to three different pretreatment conditions; namely alkaline (3% NaOH), acid (5% H2SO4) and hot water (autoclave method) pretreatment to remove lignin and loosen the cellulose structure. After the pretreatments, alkaline method exposed more cellulose than other pretreatments methods. The alkaline pretreated BPS contained 52.3% cellulose, 10.8% hemicellulose and 8.7% lignin, which is 2.3-fold more cellulose and 0.48-fold less hemicellulose as well as 0.6-fold less lignin to the native BPS. The enzymatic saccharification of the alkaline pretreated BPS at different substrate loadings at 50 °C for 76 hours by an individual crude cellulase enzymes system from T. longibrachiatum LMLSAUL 14-1 and T. harzianum LMLBP07 13-5 cultures were used at a final concentration of 10 FPU/g. Saccharification released maximum glucose of 43.5 g/L and 20.1 g/L form alkaline pretreated BPS by crude cellulase enzymes from T. longibrachiatum LMLSAUL 14-1 and T. harzianum LMLBP07 13-5 measured at the highest solid loading. The production of bioethanol was carried out in separate hydrolysis and fermentation (SHF). Fermentation of nutrient supplemented BPS hydrolysate with an initial pH of 5.0 by S. cerevisiae UL01 occurred at 30 °C for 48 hours. The maximum ethanol concentration obtained after fermentation was 17.6 g/L corresponding to ethanol yield of 60% of the maximum theoretical yield. In conclusion, banana pseudostem is a suitable alternative substrate for the production of second-generation bioethanol. / National Research Foundation (NRF) and Vlaamse Interuniversitaire Raad (VLIR- UOS)

Both in-situ and ex-situ giant pandas (Ailuropoda melanoleuca), display shifts in bamboo species and part preference throughout the year. The effects of this shifting preference on gastrointestinal (GIT) microbiota were observed using traditional culturing methods to characterize normal GIT microflora from fecal samples and behavioral feeding data of adult male and female pandas over a fourteen-month period. Linear and quadratic fits were used to determine any significant relationships between the time of year and part preference on the GIT microflora (P<0.05). Significant values for time of year were observed with the linear fit in total aerobes (P-value=0.0368), streptococci (P-value=0.0120), and lactobacilli (P-value=0.0166) and quadratic fits in streptococci (P-value=0.0382) and Bacteroides spp. (P-value=0.0134) at á=0.05. Significant linear relationships were observed with part preference and lactobacilli and Bacteroides spp., P-values of 0.0028 and 0.0030, respectively, indicating that part preference and time of year may affect the flux of panda GIT microflora.

O presente trabalho teve por objetivo avaliar a atividade celulolítica de fungos isolados de bagaço de cana-de-açúcar e serapilheira, atuantes sobre bagaço de cana-de-açúcar e farelo de arroz como substratos. Dez isolados fúngicos de amostras de bagaço de cana-de-açúcar e material vegetal em decomposição foram avaliados quanto à atividade celulolítica, tendo como referências os fungos Trichoderma reesei QM9414 e T. reesei RUTC30. A atividade celulolítica foi estimada pela capacidade hidrolítica do extrato enzimático dos fungos crescidos em bagaço de cana-deaçúcar, com e sem pré-tratamento térmico e com diferentes proporções de farelo de arroz. A atividade celulolítica foi estimada tanto em papel de filtro, para celulase total, como a carboximetilcelulose sódica, para endoglucanase. Os isolados identificados por análise molecular através da região 26S rDNA foram: Aspergillus giganteus (S1), Aspergillus fumigatus (S2 e F4), Trichoderma viride/Trichoderma hamatum (S3 e S6), Trichoderma koningiopsis (TPB), Paecilomyces variotti (F1), Moniliophthora perniciosa (F2), Acremonium cellulyticus/Penicillium verruculosum (G3) e Trichoderma sp RA305 (MAD). O Trichoderma reesei QM9414 apresentou a maior atividade tanto para celulase total como para endoglucanase. O isolado F1 não diferiu estatisticamente para atividade de endoglucanase em relação à cepa QM9414 em substrato contento somente bagaço. O isolado MAD não diferiu estatisticamente para atividade de celulase total e endoglucanse em relação à cepa QM9414 em substrato contendo 10% de farelo de arroz. Os isolados TPB e S2 apresentaram as maiores atividades para endoglucanase em bagaço de cana-de-açúcar tratado. Tais resultados mostram que a biodiversidade em nichos, como o bagaço de cana-de-açúcar e materiais vegetais em decomposição, podem fornecer linhagens de fungos celulolíticos com grande potencial biotecnológico. / The objective of this work was to evaluate the cellulolytic activity of fungi isolated from sugarcane bagasse and decaying plant material, growing on sugarcane bagasse and rice bran as substrate. Ten isolates from sugarcane bagasse and decayed plant material were evaluated for their cellulolytic activity as compared to the fungi Trichoderma reesei QM9414 and T. reesei RUTC30. The cellulolytic activity was estimated by the hydrolytic capacity of the enzymatic extract of fungi grown on sugarcane bagasse, with and without pre-heat treatment and with different proportions of rice bran. It was used as substrates both filter paper, for total cellulolytic activity, and sodium carboxymethyl cellulose, for endoglucanase activity. The isolates identified by molecular analysis using 26S rDNA region were: Aspergillus giganteus (S1), Aspergillus fumigatus (S2 and F4), Trichoderma viride/Trichoderma hamatum (S3 and S6), Trichoderma koningiopsis (TPB), Paecilomyces variotti (F1), Moniliophthora perniciosa (F2), Acremonium cellulyticus/Penicillium verruculosum (G3) and Trichoderma sp RA305 (MAD). Trichoderma reesei QM9414 showed the highest total cellulolytic activity and endoglucanase activity among all isolates. Isolate F1 was not statistically different for endoglucanase activity in relation to strain QM9414 using bagasse as substrate. Isolate MAD was not statistically different for total cellulose activity and endoglucanase activity in relation to strain QM9414 using substrate containing 10% of rice bran. Isolates TPB and S2 had the highest endoglucanase activities using sugarcane bagasse treated as substrate. These results show that biodiversity in niches, such as bagasse and decayed plant material, can provide strains of cellulolytic fungi with great biotechnological potential.

O presente trabalho teve por objetivo avaliar a atividade celulolítica de fungos isolados de bagaço de cana-de-açúcar e serapilheira, atuantes sobre bagaço de cana-de-açúcar e farelo de arroz como substratos. Dez isolados fúngicos de amostras de bagaço de cana-de-açúcar e material vegetal em decomposição foram avaliados quanto à atividade celulolítica, tendo como referências os fungos Trichoderma reesei QM9414 e T. reesei RUTC30. A atividade celulolítica foi estimada pela capacidade hidrolítica do extrato enzimático dos fungos crescidos em bagaço de cana-deaçúcar, com e sem pré-tratamento térmico e com diferentes proporções de farelo de arroz. A atividade celulolítica foi estimada tanto em papel de filtro, para celulase total, como a carboximetilcelulose sódica, para endoglucanase. Os isolados identificados por análise molecular através da região 26S rDNA foram: Aspergillus giganteus (S1), Aspergillus fumigatus (S2 e F4), Trichoderma viride/Trichoderma hamatum (S3 e S6), Trichoderma koningiopsis (TPB), Paecilomyces variotti (F1), Moniliophthora perniciosa (F2), Acremonium cellulyticus/Penicillium verruculosum (G3) e Trichoderma sp RA305 (MAD). O Trichoderma reesei QM9414 apresentou a maior atividade tanto para celulase total como para endoglucanase. O isolado F1 não diferiu estatisticamente para atividade de endoglucanase em relação à cepa QM9414 em substrato contento somente bagaço. O isolado MAD não diferiu estatisticamente para atividade de celulase total e endoglucanse em relação à cepa QM9414 em substrato contendo 10% de farelo de arroz. Os isolados TPB e S2 apresentaram as maiores atividades para endoglucanase em bagaço de cana-de-açúcar tratado. Tais resultados mostram que a biodiversidade em nichos, como o bagaço de cana-de-açúcar e materiais vegetais em decomposição, podem fornecer linhagens de fungos celulolíticos com grande potencial biotecnológico. / The objective of this work was to evaluate the cellulolytic activity of fungi isolated from sugarcane bagasse and decaying plant material, growing on sugarcane bagasse and rice bran as substrate. Ten isolates from sugarcane bagasse and decayed plant material were evaluated for their cellulolytic activity as compared to the fungi Trichoderma reesei QM9414 and T. reesei RUTC30. The cellulolytic activity was estimated by the hydrolytic capacity of the enzymatic extract of fungi grown on sugarcane bagasse, with and without pre-heat treatment and with different proportions of rice bran. It was used as substrates both filter paper, for total cellulolytic activity, and sodium carboxymethyl cellulose, for endoglucanase activity. The isolates identified by molecular analysis using 26S rDNA region were: Aspergillus giganteus (S1), Aspergillus fumigatus (S2 and F4), Trichoderma viride/Trichoderma hamatum (S3 and S6), Trichoderma koningiopsis (TPB), Paecilomyces variotti (F1), Moniliophthora perniciosa (F2), Acremonium cellulyticus/Penicillium verruculosum (G3) and Trichoderma sp RA305 (MAD). Trichoderma reesei QM9414 showed the highest total cellulolytic activity and endoglucanase activity among all isolates. Isolate F1 was not statistically different for endoglucanase activity in relation to strain QM9414 using bagasse as substrate. Isolate MAD was not statistically different for total cellulose activity and endoglucanase activity in relation to strain QM9414 using substrate containing 10% of rice bran. Isolates TPB and S2 had the highest endoglucanase activities using sugarcane bagasse treated as substrate. These results show that biodiversity in niches, such as bagasse and decayed plant material, can provide strains of cellulolytic fungi with great biotechnological potential.

Thesis (MSc (Microbiology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Bio-fuels have been considered an ideal substitute for fossil fuels due to their availability and renewable nature. Bio-ethanol is currently of great market interest as an alternative fuel with the potential of supplementing petroleum as transportation fuel. Lignocellulosic biomass, a renewable energy source, can be "readily" converted to bio-ethanol. The main impediment in the conversion process is the recalcitrance of the main lignocellulosic components (cellulose, hemicelluloses and lignin) to enzymatic hydrolysis as well as the lack of available low-cost technology. Consolidated Bioprocessing (CBP) is a single process step which offers a cost-effective and economically feasible strategy for bio-ethanol production. The process requires micro-organisms that produce ethanol at high rates and titres. Saccharomyces cerevisiae has potential as a CBP candidate due to its high ethanol yield, robustness in industrial processes, well-developed gene expression system and its safety status. Unfortunately S. cerevisiae does not degrade polysaccharides and therefore requires heterologous expression of cellulases. Genetic engineering of S. cerevisiae for cellulose hydrolysis serves as an important step in yeast strain development for CBP, and serves as a stepping stone for the commercialisation of lignocellulosic bio-ethanol. Although cellulose- utilising S. cerevisiae strains have been constructed, the cellobiose conversion is slow, hampering optimal ethanol production. β-glucosidases have been shown to be the major rate-limiting factors in cellulose saccharification as their activity determines the extent of cellulose hydrolysis, by removing excess cellobiose which causes feed-back inhibition on endoglucanase and cellobiohydrolase activities (Du Plessis et al. 2009;Lynd et al. 2002). Therefore, insufficient supply of β-glucosidase activity is detrimental to CBP and can be addressed by increasing the enzyme supply or using highly active β-glucosidases to enhance cellobiose hydrolysis. In this study, several cellobiose fermenting S. cerevisiae strains were constructed. Extracellular fungal β-glucosidase-encoding genes were successfully expressed in S. cerevisiae under the transcriptional control of the ENO1 (enolase) promoter and terminator sequences. The recombinant enzymes produced were characterised based on pH and temperature optima as well as kinetic parameters. Bio-fuels have been considered an ideal substitute for fossil fuels due to their availability and renewable nature. Bio-ethanol is currently of great market interest as an alternative fuel with the potential of supplementing petroleum as transportation fuel. Lignocellulosic biomass, a renewable energy source, can be „readily‟ converted to bio-ethanol. The main impediment in the conversion process is the recalcitrance of the main lignocellulosic components (cellulose, hemicelluloses and lignin) to enzymatic hydrolysis as well as the lack of available low-cost technology. Consolidated Bioprocessing (CBP) is a single process step which offers a cost-effective and economically feasible strategy for bio-ethanol production. The process requires micro-organisms that produce ethanol at high rates and titres. Saccharomyces cerevisiae has potential as a CBP candidate due to its high ethanol yield, robustness in industrial processes, well-developed gene expression system and its safety status. Unfortunately S. cerevisiae does not degrade polysaccharides and therefore requires heterologous expression of cellulases. Genetic engineering of S. cerevisiae for cellulose hydrolysis serves as an important step in yeast strain development for CBP, and serves as a stepping stone for the commercialisation of lignocellulosic bio-ethanol. Although cellulose- utilising S. cerevisiae strains have been constructed, the cellobiose conversion is slow, hampering optimal ethanol production. β-glucosidases have been shown to be the major rate-limiting factors in cellulose saccharification as their activity determines the extent of cellulose hydrolysis, by removing excess cellobiose which causes feed-back inhibition on endoglucanase and cellobiohydrolase activities (Du Plessis et al. 2009;Lynd et al. 2002). Therefore, insufficient supply of β-glucosidase activity is detrimental to CBP and can be addressed by increasing the enzyme supply or using highly active β-glucosidases to enhance cellobiose hydrolysis. In this study, several cellobiose fermenting S. cerevisiae strains were constructed. Extracellular fungal β-glucosidase-encoding genes were successfully expressed in S. cerevisiae under the transcriptional control of the ENO1 (enolase) promoter and terminator sequences. The recombinant enzymes produced were characterised based on pH and temperature optima as well as kinetic parameters. / AFRIKAANSE OPSOMMING: Biobrandstof word beskou as die ideale plaasvervanger vir fossielbrandstof weens die beskikbaarheid en herwinbare aard daarvan. Bio-etanol wek tans groot mark-verwante belangstelling as alternatiewe brandstof weens die potensiaal om petroleum as vervoerbrandstof aan te vul. Lignosellulose biomassa, 'n hernubare energiebron, kan "maklik" tot bio-etanol omgeskakel word. Die groot struikelblok in die omskakelingsproses is die weerstandbiedendheid van die lignosellulose komponente (sellulose, hemisellulose en lignien) teen ensiematiese hidroliese asook die gebrek aan beskikbaarheid van lae koste tegnologie. Gekonsolideerde Bioprosessering (KBP) is 'n enkel stap proses wat 'n koste-effektiewe en ekonomiesvatbare strategie voorstel vir bio-etanolproduksie. Die proses benodig 'n mikroorganisme wat daartoe instaat is om etanol teen hoë vlakke en tempo te kan produseer. Saccharomyces cerevisiae het potensiaal as 'n KBP kandidaat weens sy hoë vlakke van etanolproduksie, gehardheid in industriële prosesse, goed-ontwikkelde geenuitdrukking sisteme en veiligheidstatus. Ongelukkig kan S. cerevisiae nie polisakkariede afbreek nie en benodig derhalwe heteroloë uitdrukking van sellulases. Die genetiese manipulering van S. cerevisiae vir sellulose hidroliese dien as 'n belangrike stap in gisrasontwikkeling vir KBP en dien as 'n “stepping stone” vir die kommersialisasie van lignosellulose bio-etanol. Alhoewel sellulose-benuttende S. cerevisiae rasse reeds gekonstrueer is, is sellulose omskakeling stadig en belemmer dit optimale etanolproduksie. 'n Hoogs aktiewe glukosidase word derhalwe benodig om die hidroliese van sellobiose te versnel. Die studie behels die konstruksie van verskeie sellobiose-fermenterende S. cerevisiae rasse. Ektrasellulêre, fungiese -glukosidase-koderende gene was suksesvol in S. cerevisiae uitgedruk onderhewig aan die transkripsionele beheer van die ENO1 (enulase) promoter en termineerder DNS-volgordes. Die geproduseerde, rekombinante ensieme is gekarakteriseer op grond van optimale pH en temperatuur, asook kinetiese eienskappe. Die intrasellulêre benutting van sellobiose is 'n ideale benadering tot sellobiose hidroliese siende dat dit die risiko van kontaminasie verminder wat veroorsaak word deur die glukose wat vrygestel word in die ekstrasellulêre omgewing. Tog beskik S. cerevisiae nie oor 'n vervoer meganisme om sellobiose in die sel in te bring nie. Derhalwe is die intrasellulêre Phanaerochaete chrysosporium -glukosidase-koderende geen suksesvol saam met die Kluyveromyces lactis laktose permease uitgedruk. Alle rekombinante rasse is vir groei op sellobiose geevalueer. Die mees belowendste esktrasellulêre -glukosidase-produserende S. cerevisiae Y294[Pccbgl1] ras toon 'n aktiwiteit van 3.85 nkat.g-1, 1.85 keer meer die aktiwiteit van die S. cerevisiae Y294[SFB] ras (2.07 nkat.g-1). S. cerevisiae Y294[Pccbgl1] het ook 'n maksimum groei tempo van 0.25 h-1 onder anearobiese kondisies in vergelyking met die 0.064 h-1 van S. cerevisiae Y294[iPcbglB+lac12] toon. Onder anaërobe kondisies het S. cerevisiae Y294[Pccbgl1] 7.95 g.l-1 sellobiose verbruik en 4.05 g. l-1 etanol geproduseer oor 'n tydperk van 116 uur, terwyl S. cerevisiae Y294[iPcbglB+lac12] 0.41 g.l-1 sellobiose verbruik het en 0.21 g.l-1 etanol oor dieselfde tydperk geproduseer het. Die rekombinante rasse wat in die studie gekonstrueer is, is 'n belangrike stap in die ontwikkeling van S. cerevisiae as KBP sellulolitiese gis. / The South African National Research Institute (SANERI) for financial support

Consolidated bioprocessing (CBP) provides a cost effective cellulose processing strategy, in which enzyme production, substrate hydrolysis, and fermentation of sugars to ethanol are all carried out in a single step by microorganisms. For industrial-scale bioethanol production, CBP-enabling microbes must be able to both efficiently degrade lignocellulosic material to fermentable sugars and synthesize bioethanol with high yields. Microbes with these properties have so far not been identified. Developing naturally occurring cellulolytic isolates with CBP-relevant properties requires a comprehensive understanding of their lignocellulosic hydrolysis mechanism and metabolism. In my quest to find a suitable organism for potential use in CBP, I took to investigate the under-characterized anaerobic bacterium, Clostridium termitidis strain CT1112. C. termitidis produces fermentative hydrogen and ethanol from a variety of lignocellulose derived substrates. I sought to investigate the metabolism of C. termitidis on different substrates and the mechanisms of substrate hydrolysis using a combination of microscopy, comparative bioinformatics, and ‘Omic (transcriptomic and proteomic) analyses. Comparative bioinformatics analyses revealed higher numbers of genes encoding carbohydrate active enzymes (CAZymes) with the potential to hydrolyze a wide-range of carbohydrates, and ‘Omic analyses were used to quantify the levels of expression of CAZymes, including endoglucanases, exoglucanases, hemicellulases and cellulosomal components. While cellulases and cellulosome components were highly expressed on cellulose, xylanases and glucosidases were predominantly expressed on pentoses, and chitinases (as well as cellobiose phosphorylases) were significantly up-regulated on cellobiose. In addition to growth on xylan, the simultaneous consumption of two important lignocellulose constituents, cellobiose and xylose was also observed. The ability to metabolize both hexose and pentose sugars is a highly desirable feature of CBP-relevant organisms. Metabolic profiles in association with ‘Omics analyses showed that hexoses and pentoses are consumed via the Embden-Meyerhof-Parnas and Pentose-Phosphate pathways, respectively, and that the genome content and expression profiles dictate end-product synthesis patterns. Genes and gene-products of enzymes in central metabolism and end-product synthesis were detected in high abundance under all substrate conditions, regardless of the amounts of end-products synthesized. The capabilities described thus far, identifies C. termitidis as a strain of interest for CBP. Further studies are, however, required for its development in to an industry-ready strain for biofuel production. / February 2016

Os micro-organismos apresentam uma imensa diversidade genética e desempenham funções únicas e cruciais na manutenção de ecossistemas. Uma dessas funções é a produção de enzimas extracelulares, que ajudam na degradação da matéria orgânica e são cada vez mais procuradas e exploradas pela indústria. Essa propriedade aumenta a busca por enzimas que possam ser utilizadas nos diversos setores industriais com maior aproveitamento e baixo custo. A celulase pertence a essa classe de enzimas e é formada por um complexo multienzimático capaz de hidrolisar celulose por meio da quebra da ligação &beta;,1-4. A partir dessa característica da celulase, foi realizada uma expressão heteróloga relacionada com a hidrólise da celulose em biblioteca metagenômica de solo da Caatinga. Foram realizados testes de produção enzimática por meio dos quais selecionamos os clones 283/A8 e 307/E11 como melhores produtores de endoglicanases. Com o objetivo de analisar a cinética de produção de celulases pelos clones, estes foram inoculados em diferentes fontes de celulose, pH e temperatura. A faixa ideal de pH foi 5,0 e de temperatura, 50º C, verificada para as enzimas Celulase Total, Endoglicanase e &beta;-glicosidase. Quanto à termoestabilidade, as enzimas presentes mantiveram mais de 60% da atividade inicial após 2 horas de incubação a 50º C. O perfil de proteínas analisado por SDS-PAGE demonstrou que os clones secretam um conjunto de enzimas celulolíticas com 25 a 100 KDa, quando cultivado em farelo de trigo, e 30 a 60 KDa, quando cultivados em CMC. No ensaio de cromatografia para o clone 307/E11, foram selecionadas 5 frações que obtiveram melhores resultados na dosagem enzimática e testados frente ao pH e à temperatura. O resultado obtido foi que o complexo enzimático bruto, extraído do sobrenadante produzido pelo clone, possui melhor atividade frente ao pH e à temperatura do que as frações parcialmente purificadas. / Microorganisms are distinguished by a wide genetic diversity and they perform unique and crucial functions concerning the maintenance of ecosystems. One of those functions is the production of extracellular enzymes, which help in the degradation of organic matter and which are increasingly wanted and explored by industry. Such feature boosts the search of enzymes that can be exploited in several industrial sectors with improved full use and low cost. Cellulase belongs to such class of enzymes and it is composed of a multi-enzymatic complex which is able to hydrolyse cellulose through the breaking of the chemical bond &beta;,1-4. Considering such attribute of the cellulase, a heterologous expression, related to cellulose hydrolysis in a metagenomic inventory of the soil of Caatinga, was realized. Tests of enzymatic production were performed, and through them, we could elect the clones 283/A8 and 307/E11 as the best endoglicanase producers. Those clones were inoculated in different cellulose sources, pH and temperature, so that we could analyse the kinetic of cellulase production between them. The ideal pH range was 5.0 and the ideal temperature range was 50º C (122º F), verified for the enzymes Total Cellulase, Endoglicanase and &beta;-glucosidase. With regard to thermostability, the present enzymes kept more than 60% of the initial activity after 2 hours of incubation at 50º C (122º F). The proteins profile analysed with the help of SDS-PAGE proved that the clones secrete a group of cellulolytic enzymes with a weight average of 25 to 100 KDa, when cultivated in wheat bran, and of 30 to 60 KDa, when cultivated in CMC. Five fractions with the best results, regarding enzymatic dosage and tested before pH and temperature, were chosen by the chromatography research for the clone 307/E11. The achieved result proved that the raw enzymatic complex, extracted from the supernatant produced by the clone, develops a better activity before pH and temperature than the fractions partially purified.

O álcool, considerado um subproduto de grande importância da cultura de cana-deaçúcar, tem apresentado grande interesse nos últimos anos devido a questões econômicas e ambientais. A estimativa de produção para a safra 2007-2008, de acordo com a Companhia Nacional de Abastecimento (Conab), é de 251,59 milhões de toneladas de álcool. Por ser considerada uma fonte de energia alternativa (em substituição aos combustíveis fósseis) e renovável, muitos estudos estão sendo direcionados à cultura da cana-de-açúcar como, por exemplo, o aproveitamento do bagaço considerado um resíduo do setor sucroalcooleiro. O intuito deste trabalho foi o de realizar a hidrólise do bagaço de cana-de-açúcar por meio de enzimas celulolíticas comerciais visando identificar e quantificar açúcares fermentescíveis. Com essa finalidade, primeiramente, foram selecionadas as seguintes enzimas comerciais HPL1800, CL3708, P1250 e P4500; as quais apresentaram maior atividade celulolítica total em papel de filtro. Posteriormente foram testados dois pré-tratamentos do bagaço (ácido ou alcalino) e verificadas a atuação dessas enzimas em cada pré-tratamento do bagaço de cana-de-açúcar pelas metodologias do ácido dinitrosalicílico (DNS) e da cromatografia líquida (HPAEC - PAD). Os dados obtidos por ambas as metodologias foram analisados estatisticamente e concluiu-se que a ação conjunta do pré-tratamento ácido 0,5%, autoclave a 121ºC por 30 minutos e a enzima P4500 foram as melhores formas de tratamento para a obtenção de açúcares. / Ethanol is considered an important by-product from sugar cane culture. Nowadays it has been shown great importance in economics and environmental questions. The estimate ethanol production 2007-2008 is about 251.59 millions of tons, according to Companhia Nacional de Abastecimento (Conab). Alcohol is considered an alternative and renewable source of energy; this has lead several new studies on development of sugarcane culture and its derivatives, such as bagasse which is considered a residue from sugarcane industry. The aim of this research is the enzymatic hydrolysis of sugarcane bagasse for sugar production and its quantification and identification. The first step consisted at the selection of higher cellulolytic activity commercial enzymes in filter paper. The four enzymes selected were: HPL1800, CL3708, P1250 and P4500. After that, their performances were tested with two different pre-treated (acid and alkali) bagasse. The total sugars presents in the hydrolyzed were measured by dinitrosalicilic acid (DNS) and liquid chromatography (HPAEC - PAD) methodologies. The results were analyzed with statistics program. The datelines showed that joint action of 0.5% acid pre-treatment, 121ºC per 30 minutes and enzyme P4500 were the best treatment to sugars attainment.

A biomassa lignocelulósica é uma alternativa atrativa para o aumento na oferta de biocombustíveis, uma vez que é constituída de celulose e hemicelulose. Esses polímeros são constituídos principalmente de unidades menores de glicose e xilose, os quais por meio de bactérias anaeróbias termófilas, podem ser metabolizados em etanol. Portanto, estabeleceu-se o objetivo desse trabalho, em utilizar as principais fontes de carbono da biomassa lignocelulósica (celulose, glicose e xilose), e produzir etanol por meio da ação de consórcio microbiano selecionado a partir de inóculo termófilo e anaeróbio. O inóculo foi submetido a condição de crescimento com variação de pH (2,3,4,5,6,e 7) e variação de dois meios de cultivo em reatores em batelada, visando favorecer bactérias celulolíticas e fermentativas produtoras de etanol. Para a produção de etanol, o pH e meio de cultivo mais adequados foram 7,0 e Meio Thermoanaerobacter ethanolicus, respectivamente. A partir do inóculo enriquecido nas condições nutricionais de pH e meio de cultivo, prosseguiu-se a realização dos ensaios de produção de etanol a partir de celulose, glicose e xilose (1g/L de cada substrato), em pH 7 e meio T. ethanolicus. Os ensaios foram realizados em reator em batelada, em triplicata, a 55 &#186;C, ambos seguidos de um reator controle, sem adição desses substratos orgânicos. Os rendimentos de etanol foram de 1,73 mol etanol/mol glicose e 1,33 mol de etanol /mol de xilose. Para o substrato celulose obteve-se 1,88 mmol de etanol/g de celulose. Para os reatores controle de glicose, celulose e xilose, no qual o extrato de levedura foi a única fonte orgânica adicionada, a produção de etanol foi 1,27 mmol/L, 0,39 mmol/L e 1,65 mmol/L, respectivamente. Em todos os reatores foi detectado produção de ácido acético, ácido butírico e ácido propiônico. A produção de ácido acético foi de 5,73 mmol/L, 9,73 mmol/L e 14,45 mmol/L, para os reatores de glicose, celulose e xilose, respectivamente. No reator com glicose, observou-se baixo rendimento de hidrogênio (0,31 mol hidrogênio/mol glicose), e nos demais reatores não foi constatado produção desse gás. Em contrapartida, observou-se rendimentos de 6,6 mmol de metano/g de celulose e 0,68 mol de metano/mol de xilose para os respectivos reatores. Dessa forma, pode-se mencionar que em função das características do consórcio microbiano foi possível obter a degradação da celulose e metabolização da glicose e xilose em etanol. / Lignocellulosic biomass is an attractive alternative to increase biofuels proposal, as its composed of cellulose and hemicellulose. These polymers are consisted in individual molecules of glucose and xylose, through some thermophilic bacteria, can metabolize these carbohydrates in ethanol. Therefore, this study reports on using the principals carbon sources of lignocellulosic biomass (cellulose, glucose, and xylose), and producing ethanol through microbial consortium from anaerobic and thermophilic inoculum. The biomass was submitted to variation of pH (2,3,4,5,6, and 7) and two kinds of medium, due to ethanol production in batch reactors. For ethanol production, the optimized pH and medium were 7,0 and Thermoanaerobacter ethanolicus medium, respectively. The enriched culture was being cultivated in pH and medium experiments were used to ethanol production experiments that carried out in batch reactors, from cellulose, glucose and xylose were realized in triplicate and were maintained at 55 &#176;C, in both batches had a control reactor (without these organics substrates). Positive results in ethanol yields were 1,73 mol ethanol/ mol glucose and 1,33 mol ethanol/ mol xylose. In celluloses reactors the microbial consortium was efficient in substrate degradation, however, was obtained lower ethanol yields (1,88 mol ethanol/ g cellulose). In control reactors from glucose, cellulose and xylose, that yeast extract was the unique organic source, ethanol production was 1,27 mmol/L, 0,39 mmol/L e 1,65 mmol/L, respectively. In all reactors were detected acetic, butyric and propionic acids. The acetic acid production was 5,73 mmol/L, 9,73 mmol/L e 14,45 mmol/L in glucose, cellulose and xylose reactors, respectively. For glucoses reactors were observed lower hydrogen production (0,31 mol hydrogen/ mol glucose), in the other reactors did not observed gases production. Instead of the following yields were obtained: 6,6 mmol methane/ g cellulose and 0,68 mol methane/ mol xylose. Taking this into account, microbial consortium enriched had characteristics to degrade cellulose and metabolize glucose and xylose to ethanol.