Aliberti, Alberto (2015) New multifunctional degrading microorganisms and optimization of renewable source conversion in biofuels of second and third generation. [Tesi di dottorato]

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In recent years, climate change and energy issues have become prominent in public life. Governments have initiated extensive research into the large-scale production of alternative liquid transportation fuels from renewable resources to reduce the reliance on fossil fuels. Generally, biofuels used in the transportation sector are currently represented mainly by bioethanol and biodiesel. Second and third generation fuels are produce by non-edible crops, as Arundo donax, Populus nigra and Eucalyptus camaldulensis, and residues from industry, as sewage sludge. Biological catalysts (bacteria and yeasts) play significant roles in the conversion lignocellulosic biomass in bioethanol: degrade the cellulose, hemicellulose, lignin and pectin and ferment the monosaccharides. The pretreated biomass can be processed using a variety of process configurations: Prehydrolysis and simultaneous saccharification and fermentation (PSSF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SSCF). Advantages from bioethanol production: produced from a variety of raw materials; it is non-toxic; easily introduced into the existing infrastructure. Two principal techniques used for biodiesel production are acid esterification followed by basic transesterification and enzymatic esterification followed by basic transesterification. Moreover, the extraction and transformation of the lipids from organic wastewater sludge represent a cheap and readily available feedstock for biodiesel production. This research project was focused on the selection of new microorganisms able to hydrolyse and ferment renewable resource as lignocellulose biomass and on the evaluation and optimization of parameters in different configuration process for bioethanol and biodiesel production. The microbial isolation was performed from chipped vegetable biomass piles of Arundo donax, Eucalyptus camaldulensis and Populus nigra processed to degradation under natural conditions. Cellulolytic, pectinolytic, hemicellulolytic, ligninolytic and yeasts microorganisms were isolated by differential selective solid substrates. Qualitative and semi-quantitative assessments were performed to determinate endo- and exo- cellulase, ß-glucosidase, xylanase, pectinase, ligninase, peroxidase and laccase enzymatic activities. Moreover, xylose and glucose fermentation tests were performed. Furthermore, PSSF, SSF and SSCF experiments were carried out to evaluate and selected the optimal enzyme concentration, solid amount and process configuration for bioethanol production. In addition, different acid and enzymatic esterification, and basic transesterification were performed to biodiesel production using sewage sludge. 540 endo-cellulolytic, 678 exo-cellulolytic, 709 hemicellulolytic, 385 pectinolytic and 248 ligninolytic microorganisms were isolated (total 1291 isolates) of which 753 showed multi-enzymatic activities. Some isolates were able to convert all the principal components of vegetable biomasses such as cellulose, hemicelluloses, lignin and pectin. Moreover, Saccharomyces cerevisiae NA227, Pichia caribbica NS117 and Cyberlindnera maclurae E41L were selected to ability glucose and xylose ferment, respectively. The complex experiments of saccharification and fermentation carried out, allowed to define the best conditions for the second generation bioethanol production: 15% of pretreated A. donax biomass, 69.63 FPU g-1 of cellulose, temperature of 37°C, inocula separated of S. cerevisiae NA227 and P. caribbica NS117 (about 108 CFU mL-1 for each strain) and simultaneous saccharification and co-fermentation process (SSCF) as process configuration. The best performances, in biodiesel production from sewage sludge, were obtained using sulfuric acid and sodium hydroxide as catalysts. In conclusion: many procariotyc and eucariotic strains were characterised, identified and selected for their multi-enzymatic activities. They represent a precious biological and genetic source to upgrade the feasibility of lignocellulose conversion for the 'greener' technology of second-generation biofuel. The optimization of the bioethanol production required to find the best performances that were enhanced by modifying the biotechnological parameters of the fermentation process such as the temperature, the enzymes concentration, the amount of pretreated vegetable biomass, the configuration process as well as the selection of Saccharomyces and non-Saccharomyces yeast strains. Moreover, preliminary results in biodiesel production could give an important indication about the performances obtained using different catalysts.

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