Increase in (hemi)cellulolytic potential of Penicillium oxalicum applied to the deconstruction of biomass

Author(s)

Thaís Gabrielle Silva

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2020-08-07

Examining board members

Gonçalo Amarante Guimarães Pereira; César Rafael Fanchini Terrasan; Fernanda Mandelli

• Advisor: Gonçalo Amarante Guimarães Pereira

Abstract

Obtaining renewable fuels such as ethanol is highly desirable to reduce the emission of greenhouse gases into the atmosphere. Ethanol can be produced by the first (1G) and second generation (2G) technologies. Despite the advantages of 2G production, this is still considered costly, mainly due to the saccharification stage which requires a high dosage of enzymes for the conversion of biomass polymers into fermentable sugars. In addition to the costs inherent to the production of these enzymes, the logistics and stabilization also contribute to the high value dispensed at this stage. Therefore, the increase in enzymatic activities of cellulases, hemicellulases and accessory activities produced by microrganisms is highly desirable. By this way, the filamentous fungus Penicillium oxalicum, due to its wide spectrum of CAZymes (enzymes related to the breakdown of polysaccharides) secreted under certain growth conditions, emerges as a promising alternative for the production of these enzymes. According to that describe above, the aim of this work aim was to optimize the production of enzymes related to the deconstruction of biomass by a wild-type strain of P. oxalicum by adopting alternative sources of carbon and nitrogen and obtain lineages with improved enzyme-activity properties by applying random mutagenesis methodologies. The adoption of the design of experiments (DOE) allowed to observe the effects of some culture medium variables in the production of enzymes by P. oxalicum. Among them, the concentration of the nitrogen source (peptone) and agitation had a significant effect on filter paper activity (FPase, U/mL) and the maximum activity was reached in the following conditions: peptone, 0.72 g/L and agitation, 180 rpm. Subsequently, in order to reduce the costs inherent to nitrogen sources, peptone was totally or partially replaced by soybean meal or soybean hulls, which did not affect the FPAse titles, but impact the process costs. This is the first study to explore energy cane as a carbon source to promote the production of CAZymes by fungi strains, what contributes to the spectrum of biomass to be applied in the field of biomass deconstruction. The random mutagenesis strategies such as physical (UV light) and chemical (EMS) mutagenesis was also adopted in this study to obtain P. oxalicum lineages with improved CAZymes activities. The mutant EMS19, obtained after two rounds of mutagenesis, resulted in 100% more FPAse activity and secreted 50% more proteins when grown in avicel and xylan as carbon sources. The xylanases and ß-glucanases activities were also found improved in EMS 19 compared to parental strain (WT). The secretomes comparison of WT and EMS19 secretome allowed to understand the pivotal CAZymes for lignocellulosic degradation in EMS19, which affected the enzymatic activity displayed by this fungus (AU)

• FAPESP's process: 17/12546-9 - Construction of strain Penicillium oxalicum with potential increased biomass saccharification in order to produce the second generation ethanol
• Grantee: Thaís Gabrielle Silva
• Support Opportunities: Scholarships in Brazil - Master