Periodic Reporting for period 1 - ProEMiBiL (Ethanol production from microalgae and lignocellulosic biomass.)
Période du rapport: 2019-06-01 au 2021-05-31
The European Union (EU) set-out an ambitious but achievable plan that by 2030 up to one-quarter of the total transport fuel demand should be met by clean and CO2-efficient biofuels to curb greenhouse gas emissions (GHG) from fossil fuels and its impact on global climate change. The EU 2006 Biofuel policy has clearly stated that the search for alternative pathways for renewable energy sources will result in considerable growth in biofuel technologies and industry sectors in the coming years. It is anticipated that biofuel production will be a major contributor to the European economy with the twin benefits of a large potential for job creation in both rural areas and industrial organizations and also a reduction in CO2 emissions. Therefore, sustainable, energy-efficient and innovative technologies are needed to produce biofuels from a wide range of raw materials feedstocks while adhering to the societal, economic and environmental norms of the EU. As an alternative to this conflict, the exploitation of new materials, such as residual biomass of lignocellulosic nature and aquatic (microalgae), can be an important strategy for the reconciliation of economic growth and environmental sustainability in the long term.
From the above, the overall research objective is the production of bioethanol from a biomass mixture of the microalgae Chlorella zofingiensis and residues from the production of sugarcane, focusing on the development of a fermentation technology to convert both the pentoses and hexoses present in the biomass.
1. To develop cultivation conditions to potentiate the production of Chlorella zofingiensis, for the greater recovery of carotenoids and lipids;
2. Pretreatment and enzymatic hydrolysis of carbohydrates from microalgal and lignocellulosic biomass;
3. Screening of yeast strains for fermentation of pentoses and hexoses;
4. Experimental planning to optimize bioethanol production by co-fermentation of pentoses and hexoses.
From the above, the overall research objective is the production of bioethanol from a biomass mixture of the microalgae Chlorella zofingiensis and residues from the production of sugarcane, focusing on the development of a fermentation technology to convert both the pentoses and hexoses present in the biomass.
1. To develop cultivation conditions to potentiate the production of Chlorella zofingiensis, for the greater recovery of carotenoids and lipids;
2. Pretreatment and enzymatic hydrolysis of carbohydrates from microalgal and lignocellulosic biomass;
3. Screening of yeast strains for fermentation of pentoses and hexoses;
4. Experimental planning to optimize bioethanol production by co-fermentation of pentoses and hexoses.
In general, the activities proposed in the workplan were fulfilled. The Chlorella zofingensis microalgae culture medium was optimized in order to maximize the production of carotenoids and lipids that were recovered for application in other segments. The residual biomass of the microalgae was saccharified resulting in a hydrolyzate rich in fermentable sugars. Sugarcane biomass treatment was also optimized using acid pretreatment and enzymatic hydrolysis. Enzymatic hydrolysis was performed using an endocellulase (E-CELBA) and a beta glycosidase (B-GOSPC), both commercial. Immobilization and stabilization studies of enzymes were carried out on different supports, resulting in stable and active derivatives. E-CELBA was immobilized on magnetic nanoparticles coated with dextran and activated with aldehyde groups, while B-GOSPC was immobilized on glyoxyl agarose. The saccharification of sugarcane biomass was optimized resulting in a lignocellulosic hydrolyzate rich in glucose.
The activities carried out so far have resulted in 5 publications in indexed scientific journals (OpenAccess), 3 other articles that are being finalized for submission, also in indexed and open-access scientific journals, and 2 book chapters to be published later this year.
The activities carried out so far have resulted in 5 publications in indexed scientific journals (OpenAccess), 3 other articles that are being finalized for submission, also in indexed and open-access scientific journals, and 2 book chapters to be published later this year.
A microalgae growing station was built with 12 photobioreactors, where several microalgae species can be grown. At the end of the project, further research involving the application of microalgae can be developed. This factor implies the emergence of new opportunities and development possibilities for future researchers and students of the Higher Institute of Engineering of Porto (ISEP).
A comprehensive review of the cultivation of the microalgae Chlorella zofingiensis was carried out to adopt the most appropriate technology to increase the production of carotenoids and lipids. The highest concentration of carotenoids and lipids occurred under stress conditions (less nitrogen source and high light intensity).
While Chlorella zofingiensis was cultivated under stress conditions to produce the desired compounds in this project (carotenoids and lipids), the concentration, harvest and extraction of these components were studied to be recovered and the residual biomass was saccharified to obtain a hydrolyzate rich in fermentable sugars.
In order to request the supply of sugarcane bagasse to be used as a feedstock, the project was presented to agroenergy sector of the Brazilian Agricultural Research Corporation (Embrapa). This contact was successfully made and will be donated by Embrapasugarcane bagasse.
From the acid hydrolysates analysis resulting from the sugarcane biomass pre-treatments, it was observed that the acid hydrolyzate obtained with H3PO4 presented a high glucose concentration and a low xylose and arabinose concentrations, this indicates that such treatment it is effective in breaking down the sugarcane biomass cellulose without need for a future enzymatic hydrolysis step to obtain fermentable sugars.
For HNO3, HCl and H2SO4, Central Composite Designs were performed and, based on the results, the conditions that maximize the achievement of total monosaccharides in the acid pre-treatment was chosen, in addition to the best acid.
In order to study the immobilization and stabilization of two commercial enzymes: Endocellulase (E-CELBA) in dextran coated iron oxide magnetic nanoparticles activated with aldehyde groups (DIOMNP) and β-glucosidase (B-GOSPC) in glyoxyl agarose (GLA) so that their immobilized derivatives could be applied in the saccharification of pretreated sugarcane bagasse. This was the first time that the pretreated sugarcane bagasse was saccharified by cascade reaction using a endocellulase immobilized on dextran coated Fe2O3 with aldehyde groups combined with a β-glucosidase immobilized on glyoxyl agarose.
During sugarcane bagasse enzymatic hydrolysis, the results obtained using soluble enzymes and immobilized derivatives were similar, approximately 0.27 g glucose/g bagasse. However, the immobilized derivatives could be used for several consecutive hydrolysis cycles, without significant loss of activity. Specifically, DIOMNP endocellulase derivative maintained more than 60% of its enzymatic activity in seven reuse cycles, and GLA b-glucosidase derivative maintained more than 58% of its enzymatic activity after 8 reuse cycles.
In order to obtain better productivity and efficiency in the production of bioethanol by submerged fermentation of the lignocellulosic hydrolyzed sugarcane biomass, obtained in the previous steps the yeast screening was performed by evaluated by their potential for fermentation of pentoses and hexoses, and how they adapt to the medium containing inhibitors formed during the pretreatment of the biomass. The strains that best consume the sugars and resist the conditions of the medium was selected for the experiment of evaluation of the ethanol production was Saccharomyces cerevisiae and Spathaspora passalidarum.
The optimization of the ethanol production process was affected due to restrictions caused by the COVID-19. With mandatory confinement by the emergency state decreed by the Portugal government, practical activities were suspended for at least 3 months. In addition, material and equipment deliveries were affected, as many of them were purchased from international companies/suppliers. Even with the end of the project, activities will be continued with a view to obtaining the results expected in the original proposal.
A comprehensive review of the cultivation of the microalgae Chlorella zofingiensis was carried out to adopt the most appropriate technology to increase the production of carotenoids and lipids. The highest concentration of carotenoids and lipids occurred under stress conditions (less nitrogen source and high light intensity).
While Chlorella zofingiensis was cultivated under stress conditions to produce the desired compounds in this project (carotenoids and lipids), the concentration, harvest and extraction of these components were studied to be recovered and the residual biomass was saccharified to obtain a hydrolyzate rich in fermentable sugars.
In order to request the supply of sugarcane bagasse to be used as a feedstock, the project was presented to agroenergy sector of the Brazilian Agricultural Research Corporation (Embrapa). This contact was successfully made and will be donated by Embrapasugarcane bagasse.
From the acid hydrolysates analysis resulting from the sugarcane biomass pre-treatments, it was observed that the acid hydrolyzate obtained with H3PO4 presented a high glucose concentration and a low xylose and arabinose concentrations, this indicates that such treatment it is effective in breaking down the sugarcane biomass cellulose without need for a future enzymatic hydrolysis step to obtain fermentable sugars.
For HNO3, HCl and H2SO4, Central Composite Designs were performed and, based on the results, the conditions that maximize the achievement of total monosaccharides in the acid pre-treatment was chosen, in addition to the best acid.
In order to study the immobilization and stabilization of two commercial enzymes: Endocellulase (E-CELBA) in dextran coated iron oxide magnetic nanoparticles activated with aldehyde groups (DIOMNP) and β-glucosidase (B-GOSPC) in glyoxyl agarose (GLA) so that their immobilized derivatives could be applied in the saccharification of pretreated sugarcane bagasse. This was the first time that the pretreated sugarcane bagasse was saccharified by cascade reaction using a endocellulase immobilized on dextran coated Fe2O3 with aldehyde groups combined with a β-glucosidase immobilized on glyoxyl agarose.
During sugarcane bagasse enzymatic hydrolysis, the results obtained using soluble enzymes and immobilized derivatives were similar, approximately 0.27 g glucose/g bagasse. However, the immobilized derivatives could be used for several consecutive hydrolysis cycles, without significant loss of activity. Specifically, DIOMNP endocellulase derivative maintained more than 60% of its enzymatic activity in seven reuse cycles, and GLA b-glucosidase derivative maintained more than 58% of its enzymatic activity after 8 reuse cycles.
In order to obtain better productivity and efficiency in the production of bioethanol by submerged fermentation of the lignocellulosic hydrolyzed sugarcane biomass, obtained in the previous steps the yeast screening was performed by evaluated by their potential for fermentation of pentoses and hexoses, and how they adapt to the medium containing inhibitors formed during the pretreatment of the biomass. The strains that best consume the sugars and resist the conditions of the medium was selected for the experiment of evaluation of the ethanol production was Saccharomyces cerevisiae and Spathaspora passalidarum.
The optimization of the ethanol production process was affected due to restrictions caused by the COVID-19. With mandatory confinement by the emergency state decreed by the Portugal government, practical activities were suspended for at least 3 months. In addition, material and equipment deliveries were affected, as many of them were purchased from international companies/suppliers. Even with the end of the project, activities will be continued with a view to obtaining the results expected in the original proposal.