There are more than three thousand landfills in the United States, in which approximately 55% (1998, U. S. EPA 1999) of the MSW generated in the US is buried. The majority of the landfills are conventional, but in the last two decades new types of landfills, called leachate recycle and bioreactor landfills, have been designed and tested as an enhanced environment for biochemical degradation of municipal solid waste. All the landfills are regulated under Subtitle D of the Resource Conservation and Recovery Act (RCRA). The shortage of time and money has limited the amount of research done on waste stability analysis. The purpose of this study was to evaluate the importance of lignocelluloses in biodegradation and the secondary settlement based on dry density and typical landfill evaluating parameters. Both parts of the study samples were collected and analyzed from eleven landfills. In the first part of the study, bioreactor landfills were found more effective, faster in the degradation of VS and cellulose as compared to conventional landfills. The time required for stabilization is reduced to about 1/3 that of conventional landfills. The lignocelluloses degradation that occurs in these landfills is happening in two phases. In the initial, rapid degradation phase, the primary degradation substrate is cellulose. In the second phase, after cellulose degraded to 15-20% of the waste, degradation of the remaining cellulose along with lignin and the hemicelluloses takes place. The start of lignin and hemicellulose degradation results in an increase in the biochemical methane potential (BMP). In the second part of the study, the addition of moisture to the landfills presented a contentious issue. Moisture is encouraged for MSW refuse degradation, but for settlement it reduces compressibility. In leachate recycle landfills, the dry density is higher than in conventional landfills; therefore there is more available room for further MSW load. The increase can reach up to 40 percent in total volume. / Master of Science

Zygomycetes is a class of fungi living worldwide as saprobes, as part of mycorrhizae, and as parasites. Humans have used some zygomycetes for centuries in the production of traditional foods, e.g. Indonesian tempe. In the present thesis, the experimental focus was on two zygomycetes strains, Mucor indicus CCUG 22424 and Rhizopus sp. IT. One of the distinguishing features of M. indicus is its dimorphism. The different cell forms were influenced by the culturing conditions. After inoculation, when the initial spore concentration was high (6-8×106 spores/ml), yeast-like growth dominated under anaerobic conditions. With a smaller inoculum, yielding 1-2×105 spores/ml, and access to oxygen, filamentous forms dominated. Only negligible differences in ethanol yield (390-420 mg/g hexoses), productivity (3-5 g/l/h), and inhibitor tolerance were observed. Differential expressions of probably four genes were observed between the yeast-like and filamentous growth forms. Lignocelluloses are a suitable substrate for cultivating zygomycetes, as they occur in abundance, particularly since zygomycetes, unlike Saccharomyces cerevisiae, can utilise pentoses. Lignocelluloses require pretreatment to achieve efficient hydrolysis of the cellulose. N-methylmorpholine-N-oxide (NMMO) was tested for pretreatment of spruce and birch. Reducing wood chip size and/or prolonged pretreatment, promoted hydrolysis yield. Best yields were achieved from &lt;2 mm chips and 5 h pretreatment. The hydrolysate was used for fermentation with M. indicus, resulting in 195 and 175 mg ethanol/g wood, and 103 and 86 mg fungal biomass/g wood, from spruce and birch respectively. Orange peel is another potential substrate. However, the hydrolysate contained 0.6 % (v/v) D-limonene, ten times higher than the concentration inhibiting S. cerevisiae. M. indicus was more resistant and successfully fermented the hydrolysate, producing 400 mg ethanol/g hexoses and 75 mg fungal biomass/g sugars. Both M. indicus and Rhizopus sp. grew in 1.0 % and 2.0 % D-limonene, although the latter was unable to grow in the hydrolysate. A third substrate was also used, spent sulphite liquor (SSL), which is a by-product from sulphite paper pulp mills. The SSL was diluted to 50 % and used for airlift cultivations of Rhizopus sp. In 1.0 vvm aeration, up to 340 mg biomass/g sugars was produced. Prolonged cultivations generally decreased the protein (from 500 to 300 mg/g) and lipid (from 70 to 20 mg/g) contents. In contrast, the cell wall fraction, measured as alkali-insoluble material (AIM), increased (160-280 mg/g), as did the glucosamine (GlcN) content (220-320 mg GlcN/g AIM). The produced fungal biomass could serve as animal feed, e.g. for fish. / <p>Akademisk avhandling som för avläggande av teknologie doktorsexamen vid Chalmers tekniska högskola försvaras vid offentlig disputation den 9 februari 2012, klockan 10.00 i KS101, Kemigården 4, Göteborg.</p>

The application of anaerobic digestion technology is growing worldwide, mainly because of its environmental benefits. Nevertheless, anaerobic degradation is a rather slow and sensitive process. One of the reasons is the recalcitrance nature of certain fractions of the substrate (e.g., lignocelluloses) used for microbial degradation; thus, the hydrolysis becomes the rate-limiting step. The other reason is that the degradation of organic matter is based on a highly dynamic, multi-step process of physicochemical and biochemical reactions. The reactions take place in a sequential and parallel way under symbiotic interrelation of a variety of anaerobic microorganisms, which all together make the process sensitive. The first stage of the decomposition of the organic matter is performed by fast growing (hydrolytic and acid forming) microorganisms, while in the second stage the organic acids produced are metabolized by the slow growing methanogens, which are more sensitive than the acidogens; thus, methanogenesis becomes the rate-limiting step. The first part of this work evaluates the effects of a pretreatment using an organic solvent, N-methylmorpholine-N-oxide (NMMO), on cellulose-based materials in order to overcome the challenge of biomass recalcitrance and to increase the rate of the hydrolysis. NMMO-pretreatment of straw separated from the cattle and horse manure resulted in increased methane yields, by 53% and 51%, respectively, in batch digestion tests. The same kind of pretreatment of the forest residues led to an increase by 141% in the methane production during the following batch digestion assays. The second part of this work evaluates the efficacy of a two-stage process to overcome the second challenge with methanogenesis as the rate-limiting step, by using CSTR (continuous stirred tank reactors) and UASB (up flow anaerobic sludge blanket) on a wide variety of different waste fractions in order to decrease the time needed for the digestion process. In the two-stage semi-continuous process, the NMMO-pretreatment of jeans increased the biogas yield due to a more efficient hydrolysis compared to that of the untreated jeans. The results indicated that a higher organic loading rate (OLR) and a lower retention time could be achieved if the material was easily degradable. Comparing the two-stage and the single-stage process, treating the municipal solid waste (MSW) and waste from several food processing industries (FPW), showed that the OLR could be increased from 2 gVS/l/d to 10 gVS/l /d, and at the same time the HRT could be decreased from 10 to 3 days, which is a significant improvement that could be beneficial from an industrial point of view. The conventional single stage, on the other hand, could only handle an OLR of 3 gVS/l/d and HRT of 7 days.

Este trabalho avalia a utilização de fibras e cinzas de bagaço de cana-de-açúcar em compósitos cimentícios extrudados. Visa o estudo da potencialidade do emprego da fibra e cinza do bagaço da cana-de-açúcar na construção civil, o aproveitamento deste produto em matrizes cimentícias representa uma alternativa para diminuição de gastos com locais para deposição do resíduo evitando a degradação do meio ambiente. A cinza de bagaço de cana-de-açúcar apresenta características pozolânicas, além de garantir uma redução do consumo de cimento. Fibras de bagaço de cana-de-açúcar (FBC) foram classificadas por peneiramento, lavadas em água fervente para reduzir o açúcar residual e foi tratado quimicamente com silicato de sódio e sulfato de alumino com finalidade de proteger as fibras contra a agressão do meio alcalino proporcionado pelo cimento, imobilizar a matéria orgânica e reduzir a absorção de água, diminuindo as variações dimensionais e proporcionar melhor qualidade no compósito, tais como durabilidade e evitar o ataque de microorganismo. As cinzas de bagaço de cana-de-açúcar (CBC) foram queimadas com temperatura e tempo controlado para ter um ótimo grau de amorficidade e conseqüentemente maior reatividade. A moagem também foi efetuada a fim de melhorar a reatividade da cinza. Foi realizada uma série de ensaios de caracterização da CBC. A atividade pozolânica foi averiguada por ensaio condutividade elétrica. O desempenho mecânico e físico e na microestrutura dos compósitos de fibrocimento foram avaliados. Os compósitos foram avaliados antes e após ciclos de envelhecimento acelerado. A extrusão foi utilizada na produção de compósitos cimentícios com geometrias diferenciadas e na produção em pequena escala. Os resultados apresentaram que a fervura da FBC reduziu o açúcar residual e o tratamento químico mineralizou as FBC. As CBC apresentaram valores de sílica elevada. Foram produzidos placas com níveis de reforço entre 0,5% e 5,0% de FBC. Compósitos extrudados com 5% de reforço de FBC com 28 dias de cura apresentaram melhores resultados em tenacidade (TE), devido à maior introdução de FBC no compósito, mas não apresentou diferença entre o tratamento químico. Compósitos extrudados com 5% de reforço de FBC tratada após 200 ciclos de envelhecimento apresentaram maior absorção de água (AA) e porosidade aparente (PA). Esse fato se deve a explicado com a maior hidratação do cimento que ocasionou mineralização drástica da FBC e descolagem da fibra na interface fibra e matriz. Compósito extrudado com 5% de FBC tratada com 28 dias e após 200 ciclos com substituição de cimento por 30% de CBC apresentaram diminuição de módulo de ruptura (MOR) e maior absorção de água (AA). A substituição da matriz (agregado) e a maior relação água/cimento da mistura influenciaram neste resultado. Os resultados indicaram que os compósitos extrudados com reforço de fibra de bagaço de cana e 30% de cinza de bagaço de cana, em substituição ao cimento Portland, podem ser utilizados para produzir elementos construtivos. / This study evaluates the use of fibre and ash from sugarcane bagasse in extruded cementitious composites. Aims at studying the potentiality of using fibre and ash from sugarcane bagasse in construction, the use of this product in cementitious matrices represents an alternative to reduce expenses with the waste disposal sites for avoiding environmental degradation. The bagasse ash cane sugar has pozzolanic characteristics and ensures a reduction in cement consumption. Fibres from sugarcane bagasse (FSB) were classified by sieving, washed in boiling water to reduce the residual sugar and has been chemically treated with sodium silicate and aluminum sulphate in order to protect the fibres against the aggression of alkaline provided by the cement, immobilize the organic material and reduce the water absorption, reducing size variations and provide better quality in the composite, such as durability and prevent the attack of microorganisms. The ashes of sugarcane bagasse (ASB) were burned with controlled temperature and time to have a great degree of amorphicity and consequently higher reactivity. The grinding was also performed to improve the reactivity of the ash. We performed a series of tests to characterize the ASB. The pozzolanic activity was determined by electrical conductivity test. The mechanical and physical performance and microstructure of cement composites were evaluated. The composites were evaluated before and after ageing accelerated cycles. Extrusion was used in the production of cementitious composites with different geometries and small scale production. The results showed that boiling FSB reduced the residual sugar and chemically treated to mineralized FSB. The ASB had high amounts of silica. Plates were produced with enhanced levels of between 0.5% and 5.0% of FSB. Composites extruded with 5% of FSB with 28 days of healing showed better results in toughness (TE) due to the increased introduction of FSB in the composite, but no difference between the chemical treatments. Composites extruded with 5% strengthening of FSB treated after 200 ageing accelerated cycles had higher water absorption (AA) and apparent porosity (AP). This fact should be explained with the higher hydration of cement which caused drastic mineralization FSB and off the fibre and fibre-matrix interface. Extruded composite with 5% FBC treated with 28 days and after 200 cycles with replacement of cement by 30% of ASB showed a decrease of modulus of rupture (MOR) and higher water absorption (AA). The substitution matrix (aggregate) and the highest water / cement ratio of the mixture affected this result. The results indicated that the extruded composites reinforced with fibre sugarcane bagasse and 30% ash sugarcane bagasse in Portland cement mortars can be used to produce building elements.

Este trabalho avalia a utilização de fibras e cinzas de bagaço de cana-de-açúcar em compósitos cimentícios extrudados. Visa o estudo da potencialidade do emprego da fibra e cinza do bagaço da cana-de-açúcar na construção civil, o aproveitamento deste produto em matrizes cimentícias representa uma alternativa para diminuição de gastos com locais para deposição do resíduo evitando a degradação do meio ambiente. A cinza de bagaço de cana-de-açúcar apresenta características pozolânicas, além de garantir uma redução do consumo de cimento. Fibras de bagaço de cana-de-açúcar (FBC) foram classificadas por peneiramento, lavadas em água fervente para reduzir o açúcar residual e foi tratado quimicamente com silicato de sódio e sulfato de alumino com finalidade de proteger as fibras contra a agressão do meio alcalino proporcionado pelo cimento, imobilizar a matéria orgânica e reduzir a absorção de água, diminuindo as variações dimensionais e proporcionar melhor qualidade no compósito, tais como durabilidade e evitar o ataque de microorganismo. As cinzas de bagaço de cana-de-açúcar (CBC) foram queimadas com temperatura e tempo controlado para ter um ótimo grau de amorficidade e conseqüentemente maior reatividade. A moagem também foi efetuada a fim de melhorar a reatividade da cinza. Foi realizada uma série de ensaios de caracterização da CBC. A atividade pozolânica foi averiguada por ensaio condutividade elétrica. O desempenho mecânico e físico e na microestrutura dos compósitos de fibrocimento foram avaliados. Os compósitos foram avaliados antes e após ciclos de envelhecimento acelerado. A extrusão foi utilizada na produção de compósitos cimentícios com geometrias diferenciadas e na produção em pequena escala. Os resultados apresentaram que a fervura da FBC reduziu o açúcar residual e o tratamento químico mineralizou as FBC. As CBC apresentaram valores de sílica elevada. Foram produzidos placas com níveis de reforço entre 0,5% e 5,0% de FBC. Compósitos extrudados com 5% de reforço de FBC com 28 dias de cura apresentaram melhores resultados em tenacidade (TE), devido à maior introdução de FBC no compósito, mas não apresentou diferença entre o tratamento químico. Compósitos extrudados com 5% de reforço de FBC tratada após 200 ciclos de envelhecimento apresentaram maior absorção de água (AA) e porosidade aparente (PA). Esse fato se deve a explicado com a maior hidratação do cimento que ocasionou mineralização drástica da FBC e descolagem da fibra na interface fibra e matriz. Compósito extrudado com 5% de FBC tratada com 28 dias e após 200 ciclos com substituição de cimento por 30% de CBC apresentaram diminuição de módulo de ruptura (MOR) e maior absorção de água (AA). A substituição da matriz (agregado) e a maior relação água/cimento da mistura influenciaram neste resultado. Os resultados indicaram que os compósitos extrudados com reforço de fibra de bagaço de cana e 30% de cinza de bagaço de cana, em substituição ao cimento Portland, podem ser utilizados para produzir elementos construtivos. / This study evaluates the use of fibre and ash from sugarcane bagasse in extruded cementitious composites. Aims at studying the potentiality of using fibre and ash from sugarcane bagasse in construction, the use of this product in cementitious matrices represents an alternative to reduce expenses with the waste disposal sites for avoiding environmental degradation. The bagasse ash cane sugar has pozzolanic characteristics and ensures a reduction in cement consumption. Fibres from sugarcane bagasse (FSB) were classified by sieving, washed in boiling water to reduce the residual sugar and has been chemically treated with sodium silicate and aluminum sulphate in order to protect the fibres against the aggression of alkaline provided by the cement, immobilize the organic material and reduce the water absorption, reducing size variations and provide better quality in the composite, such as durability and prevent the attack of microorganisms. The ashes of sugarcane bagasse (ASB) were burned with controlled temperature and time to have a great degree of amorphicity and consequently higher reactivity. The grinding was also performed to improve the reactivity of the ash. We performed a series of tests to characterize the ASB. The pozzolanic activity was determined by electrical conductivity test. The mechanical and physical performance and microstructure of cement composites were evaluated. The composites were evaluated before and after ageing accelerated cycles. Extrusion was used in the production of cementitious composites with different geometries and small scale production. The results showed that boiling FSB reduced the residual sugar and chemically treated to mineralized FSB. The ASB had high amounts of silica. Plates were produced with enhanced levels of between 0.5% and 5.0% of FSB. Composites extruded with 5% of FSB with 28 days of healing showed better results in toughness (TE) due to the increased introduction of FSB in the composite, but no difference between the chemical treatments. Composites extruded with 5% strengthening of FSB treated after 200 ageing accelerated cycles had higher water absorption (AA) and apparent porosity (AP). This fact should be explained with the higher hydration of cement which caused drastic mineralization FSB and off the fibre and fibre-matrix interface. Extruded composite with 5% FBC treated with 28 days and after 200 cycles with replacement of cement by 30% of ASB showed a decrease of modulus of rupture (MOR) and higher water absorption (AA). The substitution matrix (aggregate) and the highest water / cement ratio of the mixture affected this result. The results indicated that the extruded composites reinforced with fibre sugarcane bagasse and 30% ash sugarcane bagasse in Portland cement mortars can be used to produce building elements.

A parede celular das plantas inclui componentes polissacarídeos complexos, e a sacarificação destes polímeros necessita da ação de conjuntos de enzimas que atuem em sinergia. Enzimas podem formar complexos multi-enzimáticos que possuem mais de uma atividade catalítica derivada de domínios distintos de uma mesma cadeia polipeptídica. O objetivo deste trabalho foi construir uma enzima bifuncional com os domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) para desconstrução de material lignocelulósico de cana-de-açúcar. Para isso, dois diferentes domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) oriundos de Clostridium thermocellum foram fundidas para criar a quimera feruloil esterase/acetil-xilano esterase (FaeAxe). O desenho racional da quimera foi feito utilizando-se de métodos computacionais, que permitiram a criação de um modelo estrutural da enzima. A construção da quimera foi feita por overlap PCR, clonada em vetor pET-SUMO e expressa em Escherichia coli. As duas enzimas parentais (Fae e Axe) foram clonadas em vetor pET28 e expressas em E. coli. Durante a etapa de expressão, observou-se que todas as enzimas foram expressas na forma solúvel. As enzimas feruloil esterase e acetilxilano esterase têm como substrato o polímero arabinoxilano, cuja degradação é uma etapa chave na sacarificação de biomassa. Dessa forma, as atividades da quimera, bem como das enzimas parentais foram testadas contra polímeros arabinoxilano de trigo e arabinoxilano de cana-de-açúcar após a hidrólise pela endoxilanase GH11 de Bacillus Subtilis e analisadas por meio de espectrometria de massas. A atividade desacetilase da enzima parental acetil-xilano esterase e da quimera FaeAxe foram confirmadas, evidenciando que a quimera preservou essa atividade catalítica. A atividade da enzima feruloil esterase e da quimera FaeAxe na remoção de ácido ferúlico dos oligossacarídeos gerados pela endoxilanase GH11 não foi observada / The plant cell wall is comprised of a matrix of polysaccharides and saccharification of these polymers requires the joint action of diverse enzymes. Enzymes may form multi-enzymatic complexes that have more than one catalytic activity derived from different domains of a single polypeptide chain. The aim of this work was to construct a bifunctional enzyme with two catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) for degradation of sugar cane lignocellulosic material. The two different catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) from Clostridium thermocellum were fused to generate a bifunctional chimera feruloyl esterase/acetylxylan esterase (FaeAxe). A molecular model was created by rational design using a 3D-structure guided strategy. The fusion was created using overlap PCR, and the resulting product was cloned into the pETSUMO vector. The chimeric protein and the parental enzymes were expressed in Escherichia coli and purified and the enzymes were expressed in soluble form. Xylanases, feruloyl esterases and acetylxylan esterases degrade arabinoxylan polymers and their activity is a key step in the saccharification of biomass. The catalytic properties of the chimera and of the parental enzymes were tested against wheat and sugarcane arabinoxylan polymers after hydrolysis by GH11 endoxylanase from Bacillus subtilis and analyzed by mass spectroscopy. The deacetylase activity of acetyl-xylan esterase parental enzyme and FaeAxe chimera were confirmed, showing that the chimera kept the deacetylase activity. After hydrolysis by GH11 endoxylanase from Bacillus subtilis the feruloyl esterase and FaeAxe chimera activities on ferulic acid removal were not observed

Ethanol from lignocellulosic biomass is a promising alternative to rapidly depleting oil reserves. However, natural recalcitrance of lignocelluloses to biological and chemical treatments presents major engineering challenges in designing an ethanol conversion process. Current methods for pretreatment and hydrolysis of lignocelluloses generate a mixture of pentose (C5) and hexose (C6) sugars, and several microbial growth inhibitors such as acetic acid and phenolic compounds. Hence, an efficient ethanol production process requires a fermenting microorganism not only capable of converting mixed sugars to ethanol with high yield and productivity, but also having high tolerance to inhibitors. Although recombinant bacteria and yeast strains have been developed, ethanol yield and productivity from C5 sugars in the presence of inhibitors remain low and need to be further improved for a commercial ethanol production. The overarching objective of this work is to transform Zymomonas mobilis into an efficient whole-cell biocatalyst for ethanol production from lignocelluloses. Z. mobilis, a natural ethanologen, is ideal for this application but xylose (a C5 sugar) is not its 'natural' substrate. Back in 1995, researches at National Renewable Energy Laboratory (NREL) had managed to overcome this obstacle by metabolically engineering Z. mobilis to utilize xylose. However, even after more than a decade of research, xylose fermentation by Z. mobilis is still inefficient compared to that of glucose. For example, volumetric productivity of ethanol from xylose fermentation is 3- to 4- fold lower than that from glucose fermentation. Further reduction or complete inhibition of xylose fermentation occurs under adverse conditions. Also, high concentrations of xylose do not get metabolized completely. Thus, improvement in xylose fermentation by Z. mobilis is required. In this work, xylose fermentation in a metabolically engineered Z. mobilis was markedly improved by applying the technique of adaptive mutation. The adapted strain was able to grow on 10% (w/v) xylose and rapidly ferment xylose to ethanol within 2 days and retained high ethanol yield. Similarly, in mixed glucose-xylose fermentation, the strain produced a total of 9% (w/v) ethanol from two doses of 5% glucose and 5% xylose (or a total of 10% glucose and 10% xylose). Investigation was done to identify the molecular basis for efficient biocatalysis. An altered xylitol metabolism with reduced xylitol formation, increased xylitol tolerance and higher xylose isomerase activity were found to contribute towards improvement in xylose fermentation. Lower xylitol production in adapted strain was due to a single mutation in ZMO0976 gene, which drastically lowered the reductase activity of ZMO0976 protein. ZMO0976 was characterized as a novel aldo-keto reductase capable of reducing xylose, xylulose, benzaldehyde, furfural, 5-hydroxymethyl furfural, and acetaldehyde, but not glucose or fructose. It exhibited nearly 150-times higher affinity with benzaldehyde than xylose. Knockout of ZMO0976 was found to facilitate the establishment of xylose fermentation in Z. mobilis ZM4. Equipped with molecular level understanding of the biocatalytic process and insight into Z. mobilis central carbon metabolism, further genetic engineering of Z. mobilis was undertaken to improve the fermentation of sugars and lignocellulosic hydrolysates. These efforts culminated in construction of a strain capable of fermenting glucose-xylose mixture in presence of high concentration of acetic acid and another strain with a partially operational EMP pathway.

A parede celular das plantas inclui componentes polissacarídeos complexos, e a sacarificação destes polímeros necessita da ação de conjuntos de enzimas que atuem em sinergia. Enzimas podem formar complexos multi-enzimáticos que possuem mais de uma atividade catalítica derivada de domínios distintos de uma mesma cadeia polipeptídica. O objetivo deste trabalho foi construir uma enzima bifuncional com os domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) para desconstrução de material lignocelulósico de cana-de-açúcar. Para isso, dois diferentes domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) oriundos de Clostridium thermocellum foram fundidas para criar a quimera feruloil esterase/acetil-xilano esterase (FaeAxe). O desenho racional da quimera foi feito utilizando-se de métodos computacionais, que permitiram a criação de um modelo estrutural da enzima. A construção da quimera foi feita por overlap PCR, clonada em vetor pET-SUMO e expressa em Escherichia coli. As duas enzimas parentais (Fae e Axe) foram clonadas em vetor pET28 e expressas em E. coli. Durante a etapa de expressão, observou-se que todas as enzimas foram expressas na forma solúvel. As enzimas feruloil esterase e acetilxilano esterase têm como substrato o polímero arabinoxilano, cuja degradação é uma etapa chave na sacarificação de biomassa. Dessa forma, as atividades da quimera, bem como das enzimas parentais foram testadas contra polímeros arabinoxilano de trigo e arabinoxilano de cana-de-açúcar após a hidrólise pela endoxilanase GH11 de Bacillus Subtilis e analisadas por meio de espectrometria de massas. A atividade desacetilase da enzima parental acetil-xilano esterase e da quimera FaeAxe foram confirmadas, evidenciando que a quimera preservou essa atividade catalítica. A atividade da enzima feruloil esterase e da quimera FaeAxe na remoção de ácido ferúlico dos oligossacarídeos gerados pela endoxilanase GH11 não foi observada / The plant cell wall is comprised of a matrix of polysaccharides and saccharification of these polymers requires the joint action of diverse enzymes. Enzymes may form multi-enzymatic complexes that have more than one catalytic activity derived from different domains of a single polypeptide chain. The aim of this work was to construct a bifunctional enzyme with two catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) for degradation of sugar cane lignocellulosic material. The two different catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) from Clostridium thermocellum were fused to generate a bifunctional chimera feruloyl esterase/acetylxylan esterase (FaeAxe). A molecular model was created by rational design using a 3D-structure guided strategy. The fusion was created using overlap PCR, and the resulting product was cloned into the pETSUMO vector. The chimeric protein and the parental enzymes were expressed in Escherichia coli and purified and the enzymes were expressed in soluble form. Xylanases, feruloyl esterases and acetylxylan esterases degrade arabinoxylan polymers and their activity is a key step in the saccharification of biomass. The catalytic properties of the chimera and of the parental enzymes were tested against wheat and sugarcane arabinoxylan polymers after hydrolysis by GH11 endoxylanase from Bacillus subtilis and analyzed by mass spectroscopy. The deacetylase activity of acetyl-xylan esterase parental enzyme and FaeAxe chimera were confirmed, showing that the chimera kept the deacetylase activity. After hydrolysis by GH11 endoxylanase from Bacillus subtilis the feruloyl esterase and FaeAxe chimera activities on ferulic acid removal were not observed