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Evaluation of ethanol production from renewable cellulosic resources using process simulation tools
New Biotechnology · Volume 29S · September 2012
Poster 1.1.07 Bioprospecting lignocellulolytic enzymes from endophytic actinomycetes aiming at second generation ethanol production Carla Montanari Mergel 1,∗ , Diogo Robl 1,2 , Zita Maria de Oliveira Gregório 1 , Juan Diego Rojas 1 , Sindélia Freitas Azzoni 2 , Gabriel Padilla Maldonado 1 1
São Paulo University (USP), São Paulo, Brazil Brazilian Bioethanol Science and Technology Laboratory (CTBE), Campinas, Brazil 2
The use of lignocellulosic material which represents 50% of terrestrial biomass is an attractive alternative for the production of ethanol. This product called second generation ethanol using agroindustrial residues that after the enzymatic treatment generates fermentable sugars. Endophytic microorganisms are those able to colonize the interior tissues of plants and actinomycetes has received special attention, The endophytic actinomycetes are the subject of several studies involving the search for new bioactive compounds of great industrial interest. The aim of this study is to identify genes that encode lignocellulolytic enzymes for future application in second generation ethanol by enzymatic route. A collection of 50 strains of endophytic actinomycetes has been screened for lignocellulolytic activities, mainly for hemicellulases and acessory enzymes. At first approach, it was performed a screening by plate culture with specific substrates (Xylan, CMC and Lícor) and detection halos of hydrolysis by congo red. The second approach was performed by PCR screening using specific and degenerate primers for beta-glucosidase, arabinofuranosidase, endoglucanase, acetyl xylan esterase, pectinase and xylanase). Screening rounds by PCR for endoglucanase, arabinofuranosidase and beta-glucosidase have been performed. The positive genes isolated will be cloned into expression vectors and these enzymes will be produced using heterologous systems. Purified enzymes will be used for biochemical characterization and synergistic effect by formulating enzymatic mixtures and substrates hydrolysis. Preliminary results by the screening experiments showed that the majority of the strains present genes that codify for endoglucanase, beta-glucosidade and arabinofuranosidase enzymes, even that do not showed any substrate degradation by congo red method. As a conclusion, endophytic actinomycetes can be a important source of hydrolases. We expect identify and express these enzymes and be able to assembly recombinant enzymatic cocktail and contribute to second generation ethanol technology. http://dx.doi.org/10.1016/j.nbt.2012.08.106 Poster 1.1.08 Evaluation of ethanol production from renewable cellulosic resources using process simulation tools Demetri Petrides Intelligen, Inc., 2326 Morse Avenue, Scotch Plains, NJ 07076, USA
activity is driven primarily by the quest for fuel self-reliance and carbon oxides emission reductions. The main effort has been concentrated on bio-ethanol and bio-diesel which have been shown to give motor engine performance similar to that of conventional petroleum based fuels. In addition to product characteristics, however, process economics play an equally important role in any successful product commercialization. In this work, realistic process simulation models have been developed in order to analyze the economics of corn-stover to ethanol conversion. This presentation will illustrate how such models can guide R&D work and facilitate process optimization. http://dx.doi.org/10.1016/j.nbt.2012.08.107 Poster 1.1.09 Screening hemicellulolytic enzymes and accessory proteins from endophytic fungi for biomass degradation Diogo Robl 1,2,∗ , Carla Montanari Mergel 2 , Priscila da Silva Delabona 1 , José Geraldo da Cruz Pradella 1 , Gabriel Padilla Maldonado 2 1
Brazilian Bioethanol Science and Technology Laboratory (CTBE), Campinas, Brazil 2 São Paulo University (USP), São Paulo, Brazil In nature the lignocellulotics materials are degrading by many enzymes that are produced by consortium of microorganisms. Therefore, the diverse glycohydrolases could enable biomass deconstruction, reducing the costs related on enzymes production. This work aims to select potential hemicellulases and accessory proteins to form an efficient enzymatic cocktail for biomass degradation. A total of 114 endophytic fungi strains and 16 strains isolated from soil and spoiled books were tested on their ability to produce hemicellulases and accessory proteins. Two screenings were performed, one using the esculin gel diffusion assay (EGDA) with a 5 old days CMC culture broth supernatant. The other approach was the culture on agar plate media using beechwood xylan and liquor from sugar cane bagasse hydrotermic pretreatment. Then 50 promising isolates were grown in steam exploded/deslignificated sugar cane bagasse plus soyabean bran (3:1) in shake flasks at 29◦ C at 200 rpm. Activities of xylanase, pectinase, -glucosidase, endoglucanase and FPase were measured and the best ten isolates were selected and assayed for the capacity to degrade several substrates. High xylanase, -glucosidase and -glucanase were visualized in five strains extracts and the production of those activities seems to be associated. However, only few strains were able to produce arabinofuronosidase, xylosidase and mannosidase. Furthermore, none strain produced expressive activity against sugar beet arabinan, wheat arabinoxylan, CMC and filter paper under these conditions. In conclusion, the use of EGDA and agar plate media combined with shake flasks allowed selecting strains with a different hemicellulolytic and accessories enzymes profiles. Financial support: FAPESP. http://dx.doi.org/10.1016/j.nbt.2012.08.108
Over the past three decades there has been intense investigation on the development of fuel producing processes that are based on the use of renewable agricultural materials as feedstock. This www.elsevier.com/locate/nbt S39
Poster 1.1.07 Bioprospecting lignocellulolytic enzymes from endophytic actinomycetes aiming at second generation ethanol production Carla Montanari Mergel 1,∗ , Diogo Robl 1,2 , Zita Maria de Oliveira Gregório 1 , Juan Diego Rojas 1 , Sindélia Freitas Azzoni 2 , Gabriel Padilla Maldonado 1 1
São Paulo University (USP), São Paulo, Brazil Brazilian Bioethanol Science and Technology Laboratory (CTBE), Campinas, Brazil 2
The use of lignocellulosic material which represents 50% of terrestrial biomass is an attractive alternative for the production of ethanol. This product called second generation ethanol using agroindustrial residues that after the enzymatic treatment generates fermentable sugars. Endophytic microorganisms are those able to colonize the interior tissues of plants and actinomycetes has received special attention, The endophytic actinomycetes are the subject of several studies involving the search for new bioactive compounds of great industrial interest. The aim of this study is to identify genes that encode lignocellulolytic enzymes for future application in second generation ethanol by enzymatic route. A collection of 50 strains of endophytic actinomycetes has been screened for lignocellulolytic activities, mainly for hemicellulases and acessory enzymes. At first approach, it was performed a screening by plate culture with specific substrates (Xylan, CMC and Lícor) and detection halos of hydrolysis by congo red. The second approach was performed by PCR screening using specific and degenerate primers for beta-glucosidase, arabinofuranosidase, endoglucanase, acetyl xylan esterase, pectinase and xylanase). Screening rounds by PCR for endoglucanase, arabinofuranosidase and beta-glucosidase have been performed. The positive genes isolated will be cloned into expression vectors and these enzymes will be produced using heterologous systems. Purified enzymes will be used for biochemical characterization and synergistic effect by formulating enzymatic mixtures and substrates hydrolysis. Preliminary results by the screening experiments showed that the majority of the strains present genes that codify for endoglucanase, beta-glucosidade and arabinofuranosidase enzymes, even that do not showed any substrate degradation by congo red method. As a conclusion, endophytic actinomycetes can be a important source of hydrolases. We expect identify and express these enzymes and be able to assembly recombinant enzymatic cocktail and contribute to second generation ethanol technology. http://dx.doi.org/10.1016/j.nbt.2012.08.106 Poster 1.1.08 Evaluation of ethanol production from renewable cellulosic resources using process simulation tools Demetri Petrides Intelligen, Inc., 2326 Morse Avenue, Scotch Plains, NJ 07076, USA
activity is driven primarily by the quest for fuel self-reliance and carbon oxides emission reductions. The main effort has been concentrated on bio-ethanol and bio-diesel which have been shown to give motor engine performance similar to that of conventional petroleum based fuels. In addition to product characteristics, however, process economics play an equally important role in any successful product commercialization. In this work, realistic process simulation models have been developed in order to analyze the economics of corn-stover to ethanol conversion. This presentation will illustrate how such models can guide R&D work and facilitate process optimization. http://dx.doi.org/10.1016/j.nbt.2012.08.107 Poster 1.1.09 Screening hemicellulolytic enzymes and accessory proteins from endophytic fungi for biomass degradation Diogo Robl 1,2,∗ , Carla Montanari Mergel 2 , Priscila da Silva Delabona 1 , José Geraldo da Cruz Pradella 1 , Gabriel Padilla Maldonado 2 1
Brazilian Bioethanol Science and Technology Laboratory (CTBE), Campinas, Brazil 2 São Paulo University (USP), São Paulo, Brazil In nature the lignocellulotics materials are degrading by many enzymes that are produced by consortium of microorganisms. Therefore, the diverse glycohydrolases could enable biomass deconstruction, reducing the costs related on enzymes production. This work aims to select potential hemicellulases and accessory proteins to form an efficient enzymatic cocktail for biomass degradation. A total of 114 endophytic fungi strains and 16 strains isolated from soil and spoiled books were tested on their ability to produce hemicellulases and accessory proteins. Two screenings were performed, one using the esculin gel diffusion assay (EGDA) with a 5 old days CMC culture broth supernatant. The other approach was the culture on agar plate media using beechwood xylan and liquor from sugar cane bagasse hydrotermic pretreatment. Then 50 promising isolates were grown in steam exploded/deslignificated sugar cane bagasse plus soyabean bran (3:1) in shake flasks at 29◦ C at 200 rpm. Activities of xylanase, pectinase, -glucosidase, endoglucanase and FPase were measured and the best ten isolates were selected and assayed for the capacity to degrade several substrates. High xylanase, -glucosidase and -glucanase were visualized in five strains extracts and the production of those activities seems to be associated. However, only few strains were able to produce arabinofuronosidase, xylosidase and mannosidase. Furthermore, none strain produced expressive activity against sugar beet arabinan, wheat arabinoxylan, CMC and filter paper under these conditions. In conclusion, the use of EGDA and agar plate media combined with shake flasks allowed selecting strains with a different hemicellulolytic and accessories enzymes profiles. Financial support: FAPESP. http://dx.doi.org/10.1016/j.nbt.2012.08.108
Over the past three decades there has been intense investigation on the development of fuel producing processes that are based on the use of renewable agricultural materials as feedstock. This www.elsevier.com/locate/nbt S39