Hidrólise enzimática de biomassa lignocelulósica para a produção de etanol de segunda geração

Second generation ethanol production has great potential to be a sustainable reality, especially in Brazil due to the large amount of available sugarcane bagasse. Pretreatment methods and biomass hydrolysis continue to be the bottlenecks of the overall process, mainly this second step since the enzymes present high costs. Therefore, efforts have been taken to make the process more cost-effective with regards to the discovery of more effective enzymes. New sources of enzymes are continuously encountered and several strategies of enzyme prospection and production have been studied. One strategy used in the search for new and/or more efficient enzymes is comparative genomic analysis of different microorganisms which allows for the screening of several candidates of interest in a short period of time. Moreover, plant-degrading enzymes can be produced by fungi grown on abundantly available low-cost plant biomass. This work was divided in five chapters being the first chapter a current review about second generation ethanol production focused mainly on the saccharification step. Several strategies of enzyme prospection and production were discussed and detailed. In the second chapter, saccharification of acid- and alkali-pretreated sugarcane bagasse was compared using the enzymatic extract from the pathogen fungus Chrysoporthe cubensis and three commercial enzymatic mixtures. For the sugarcane bagasse studied in this work, the alkaline pretreatment promoted the best saccharification yields, where the C. cubensis extract was responsible for the higher release of glucose and xylose when compared to the commercial enzymatic mixtures Furthermore, the C. cubensis extract was able to produce high specific enzyme activities when compared to the commercial cocktails. In the third chapter, the genomic potential of the candidate fungi was evaluated and the most interesting enzymes for sugarcane bagasse hydrolysis were expressed in Aspergillus vadensis. Nine enzymes from three different fungi, Aspergillus terreus, Nectria haematoccoca and Phaeosphaeria nodorum, were successfully cloned and expressed by heterologous system and these enzymes represent a possibility for a better degradation of sugarcane bagasse. -xylosidases were biochemicallycharacterized and showed maxima activity in the pH range 4.5-5.0 and at temperatures of 55-60°C. In the fourth chapter, two xylanases from Aspergillus nidulans previously cloned in Pichia pastoris, here nominated as Xyn1818 and Xyn3613, were expressed, purified and characterized. The optima pH and temperature for Xyn1818 were 7.5 and 60°C while Xyn3613 achieved maximal activity at pH 6.0 and 50°C. Xyn1818 showed to be very thermostable, maintaining 50% of its original activity after 49 hours when incubated at 50°C. Xyn1818 presented higher activity against wheat arabinoxylan while Xyn3613 had the best activity against xylan from beechwood. Saccharification results showed that the commercial enzymatic cocktails were able to release more sugars (glucose and xylose) after supplementation with the xylanases Xyn1818 and Xyn3613 from A. nidulans. Finally, in the fifth chapter, Aspergillus niger and Trichoderma reesei were substrates: wheat straw and sugarcane bagasse. The fungi produced different sets of (hemi-)cellulolytic enzymes which was reflected in an overall strong synergistic effect in releasing sugars during saccharification using the enzyme blends from both fungi. It was observed that removing monosaccharides from the enzyme production media is very important when T. reesei and A. niger enzyme blends are combined to improve plant biomass saccharification.A produção de etanol de segunda geração apresenta grande potencial para ser uma realidade sustentável, especialmente no Brasil que prossui grandes quantidades de bagaço de cana-de-açúcar. Os maiores obstáculos deste processo são os pré- tratamentos e a hidrólise da biomassa, principalmente esta última etapa visto que as enzimas ainda apresentam custos muito elevados. Assim, esforços têm se concentrado em tornar o processo mais econômico com a descoberta de enzimas mais efetivas. Novas fontes de enzimas são continuamente encontradas e várias estratégias de prospecção e produção enzimática têm sido estudadas. Uma estratégia bastante utilizada na busca por novas enzimas e/ou enzimas mais eficientes é a análise de genômica comparativa de diferentes micro-organismos que permite a seleção de vários candidatos de interesse num curto período de tempo. Além disso, as enzimas podem ser produzidas por fungos quando estes são crescidos em biomassas que apresentam baixo custo e alta disponibilidade. Este trabalho foi dividido em cinco capítulos sendo que o primeiro consiste de uma revisão atual sobre a produção de etanol de segunda geração focada na etapa de sacarificação enzimática. Várias estratégias de prospecção e produção enzimáticas foram discutidas e detalhadas. No segundo capítulo, a sacarificação de bagaço de cana-de-açúcar após pré-tratamentos ácido e alcalino foi comparada usando o extrato enzimático do fungo fitopatógeno Chrysoporthe cubensis e três coquetéis comerciais. Para o bagaço de cana utilizado neste estudo, o pré-tratamento alcalino promoveu os melhores rendimentos de sacarificação sendo o extrato do fungo C. Cubensis o responsável pela maior liberação de glicose e xilose quando comparado às misturas enzimáticas comerciais. Além disso, o extrato de C. cubensis produziu maiores valores de atividade específica comparados aos dos coquetéis comerciais. No terceiro capítulo, o potencial genômico de fungos candidatos foi avaliado e as enzimas mais interessantes para a hidrólise de bagaço de cana-de-açúcar foram expressas em Aspergillus vadensis. Nove enzimas de três fungos diferentes, Aspergillus terreus, Nectria haematoccoca e Phaeosphaeria nodorum, foram viiclonadas e expressas por sistema heterólogo e representam uma nova possiblidade para a melhor degradação do bagaço de cana. Dentre estas enzimas, quatro - xilosidases foram bioquimicamente caracterizadas e apresentaram atividade máxima em valores de pH 4,5-5,0 e em temperaturas 55-60°C. No quarto capítulo, duas xilanases de Aspergillus nidulans previamente clonadas em Pichia pastoris, aqui denominadas Xyn1818 e Xyn3613, foram expressas, purificadas e caracterizadas. Xyn1818 apresentou ótima atividade em pH 7.5 e à 60°C enquanto Xyn3613 alcançou máxima atividade em pH 6.0 e à 50°C. Xyn1818 apresentou-se bastante termoestável à 50°C mantendo 50% de sua atividade original após 49 horas de incubação nesta temperatura. Xyn1818 apresentou maior atividade contra arabinoxilana de trigo enquanto o melhor substrato para Xyn3613 foi xilana beechwood. Testes de sacarificação mostraram que os coquetéis comerciais liberaram mais açúcares (glicose e xilose) quando suplementados com as xilanases Xyn1818 e Xyn3613 de A. nidulans. Finalmente, no quinto capítulo, os fungos Aspergillus niger e Trichoderma reesei foram avaliados quanto à produção de enzimas após crescimento em do e bagaço de cana-de-açúcar. Os fungos produziram diferentes tipos de enzimas (hemi)celulolíticas, o que foi refletido pelo forte efeito sinergístico na liberação de açúcares durante a sacarificação dos substratos utilizando o conjunto de enzimas dos dois microorganismos. Foi constatado que a remoção de monossacarídeos do meio de produção de enzimas é muito importante quando combinações de enzimas de T. reesei and A. niger são utilizadas para aprimorar a hidrólise de biomassas.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Synergistic effect of Aspergillus niger and Trichoderma reesei enzyme sets on the saccharification of wheat straw and sugarcane bagasse

Plant-degrading enzymes can be produced by fungi on abundantly available low-cost plant biomass. However, enzymes sets after growth on complex substrates need to be better understood, especially with emphasis on differences between fungal species and the influence of inhibitory compounds in plant substrates, such as monosaccharides. In this study, Aspergillus niger and Trichoderma reesei were evaluated for the production of enzyme sets after growth on two "second generation" substrates: wheat straw (WS) and sugarcane bagasse (SCB). A. niger and T. reesei produced different sets of (hemi-)cellulolytic enzymes after growth on WS and SCB. This was reflected in an overall strong synergistic effect in releasing sugars during saccharification using A. niger and T. reesei enzyme sets. T. reesei produced less hydrolytic enzymes after growth on non-washed SCB. The sensitivity to non-washed plant substrates was not reduced by using CreA/Cre1 mutants of T. reesei and A. niger with a defective carbon catabolite repression. The importance of removing monosaccharides for producing enzymes was further underlined by the decrease in hydrolytic activities with increased glucose concentrations in WS media. This study showed the importance of removing monosaccharides from the enzyme production media and combining T. reesei and A. niger enzyme sets to improve plant biomass saccharification

The influence of pretreatment methods on saccharification of sugarcane bagasse by an enzyme extract from Chrysoporthe cubensis and commercial cocktails: A comparative study

Biomass enzymatic hydrolysis depends on the pretreatment methods employed, the composition of initial feedstock and the enzyme cocktail used to release sugars for subsequent fermentation into ethanol. In this study, sugarcane bagasse was pretreated with 1% H2SO4 and 1% NaOH and the biomass saccharification was performed with 8% solids loading using 10 FPase units/g of bagasse of the enzymatic extract from Chrysoporthe cubensis and three commercial cocktails for a comparative study. Overall, the best glucose and xylose release was obtained from alkaline pretreated sugarcane bagasse. The C. cubensis extract promoted higher release of glucose (5.32g/L) and xylose (9.00g/L) than the commercial mixtures. Moreover, the C. cubensis extract presented high specific enzyme activities when compared to commercial cocktails mainly concerning to endoglucanase (331.84U/mg of protein), β-glucosidase (29.48U/mg of protein), β-xylosidase (2.95U/mg of protein), pectinase (127.46U/mg of protein) and laccase (2.49U/mg of protein)

The influence of pretreatment methods on saccharification of sugarcane bagasse by an enzyme extract from Chrysoporthe cubensis and commercial cocktails : A comparative study

Biomass enzymatic hydrolysis depends on the pretreatment methods employed, the composition of initial feedstock and the enzyme cocktail used to release sugars for subsequent fermentation into ethanol. In this study, sugarcane bagasse was pretreated with 1% H2SO4 and 1% NaOH and the biomass saccharification was performed with 8% solids loading using 10 FPase units/g of bagasse of the enzymatic extract from Chrysoporthe cubensis and three commercial cocktails for a comparative study. Overall, the best glucose and xylose release was obtained from alkaline pretreated sugarcane bagasse. The C. cubensis extract promoted higher release of glucose (5.32g/L) and xylose (9.00g/L) than the commercial mixtures. Moreover, the C. cubensis extract presented high specific enzyme activities when compared to commercial cocktails mainly concerning to endoglucanase (331.84U/mg of protein), β-glucosidase (29.48U/mg of protein), β-xylosidase (2.95U/mg of protein), pectinase (127.46U/mg of protein) and laccase (2.49U/mg of protein)