Consolidated Bioprocessing of rice starchy by-products into bioethanol: proof of concept and scale-up studies

Global awareness regarding greenhouse gas emissions is increasing as the earth’s surface temperatures and atmospheric pollution are rising. Countries are progressively adopting renewable energy sources, including biofuels. However, the biomass feedstock necessary to produce alternative fuels are mainly food and feed materials. Therefore, more sustainable raw starting materials are being sought, and new production processes are in development. Among emerging biofuels, bioethanol offers several advantages, including the possibility of blending with gasoline or being implemented at 100% in flexible fuel vehicles. In this regard, this study aims to optimize the fermentation parameters and scale up of bioethanol production using unripe rice, a starch-rich waste product from rice industry. Specifically, the recombinant Saccharomyces cerevisiae strain ER T12.7, previously engineered to express an α-amylase and a glucoamylase, was employed in a supplemented Consolidated BioProcessing (CBP) strategy. Key fermentation parameters were optimized to maximize ethanol production over set duration of 72 hours in a 1L scale reactor. Optimum combinations were tested at a 50L scale, achieving 100% of theoretical yields of ethanol after further optimization. Moreover, the impact of the nitrogen sources on the yeast growth and small-scale fermentation performance was assed. The results show that the latter are not critical for process yield and therefore allow for greater confidence in the scale-up process without nitrogen source supplements. Finally, regarding the yeast cultivation and amylases production, an alternative growth medium based on molasses, a waste product from sugar beet processing, was tested and optimized to reduce production costs increasing industrial feasibility. The second part of the thesis aimed to test the feasibility of recovering ER T12.7 enzymes from the liquid part of the spent slurry after fermentation and vacuum distillation of ethanol at 50 °C. This study, for the first time, reports the enzymes recovery from thin stillage and demonstrates that they are still active on a complex substrate as unripe rice. Furthermore, results show promising insights into the stability of enzymes over the period of one month both when conserved at 4 °C and room temperatures.