Montella, Salvatore (2016) Lignocellulosic biomasses as sources of fermentable sugars and biocatalysts for biorefinery. [Tesi di dottorato]

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The lignocellulosic biomasses constitute the Earth’s most abundant repository of carbon, thus the future development of processes that exploit this raw materials as sources of fermentable sugars represents a key challenge for a bio-based economy. Because of the complex macromolecular structure, the lignocellulose conversion requires a pretreatment step and an effective hydrolysis. The attention is mainly focusing on the enzymatic hydrolysis, carried out by a tailor-made enzyme cocktail, due to the increasing concerns regarding the environmental impact. A several number of works exploit the autochthonous microbial community involved in lignocellulose decomposition as reservoir of novel lignocellulose-degrading enzymes. The overall goal of this PhD project was the valorization of selected lignocellulosic biomasses as source of both fermentable sugars and novel biocatalysts for the production of biobased products via fermentation. Two different lignocellulosic biomasses (the perennial crop Arundo donax and the Newspaper Waste – NW – fraction of Municipal Solid Waste) were tested as source of monosaccharides by enzymatic hydrolysis after Ammonia Fiber Expansion (AFEX) or Extractive Ammonia (EA) pretreatment. The ability of the recombinant endocellulase rCelStrep, α-L-arabinofuranosidase rPoAbf and its evolved variant rPoAbf F435Y/Y446F to improve the saccharification yields was evaluated. For the AFEX pretreated A. donax, the substitution of rPoAbf F435Y/Y446F to the corresponding enzymatic activity in a mixture of purified enzymes led to obtain higher values (62, 63 and 80% for glucan, xylan and arabinan conversion respectively) than those achieved by using a commercial mixture. The addition of the same enzyme to the commercial mixture enhanced both xylan and arabinan conversions (up to 80%) after 6 days of saccharification. The maximum glucan conversion yield (45.61%) obtained for EA pretreated NW by adding rCelStrep to the commercial mix was higher than or comparable to those reported in the recent manuscripts adopting similar conditions to those used in this study. The microbial diversity present in three different ecosystems (chipped wood of Arundo donax, Eucalyptus camaldulensis and Populus nigra subjected to natural biodegradation in open field or underwood) was explored to identify novel lignocellulose-degrading enzymes. By using traditional microorganisms cultivation- based methods, one Pediococcus acidilactici strain and five Streptomyces strains were identified as producers of novel endo-xylanase(s) and endo-cellulases respectively. The cellulases were tested in the hydrolysis of pretreated A. donax. Interestingly, by using the cellulase(s) from the AE-T0-58P (10) strain in substitution of the corresponding activity in a commercial mix, the glucose and xylose yields obtained (4.47±0.5 g/L and 5.87±0.2 g/L respectively after 72 h) were 82% and 85% respectively of the values obtained by using the commercial mix. Moreover, the DNA from the microorganisms adherent to the abovementioned biomasses were subjected to metagenomic analysis. The functional clustering of the open reading frames (ORFs) predicted by the shotgun sequencing of the extracted DNAs showed a prevalence of poorly characterized genes, suggesting the three detected biomasses as potential sources of not yet known proteins. The Glycoside Hydrolases (GHs) abundance in the sample from P. nigra (1.85% on total ORFs) was higher than that detected in environments well-known as reservoires of GHs. The in-depth KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway mapping of the putative genes coding for enzymes involved in the polysaccharides hydrolysis confirmed that the three analyzed samples were a valuable source of a full set of (hemi)cellulases and accessory enzymes required for an effective lignocellulose hydrolysis.

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