Gioiello, Alessia (2017) Isolation of new thermophiles from compost and their selection for the conversion of lignocellulosic biomass. [Tesi di dottorato]

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Composting, often described as nature’s way of recycling, is a self-heating, aerobic, solid-phase process, during which organic waste materials are biologically degraded into an extremely useful humus-like substance by means of various microorganisms. The main protagonists of this process are thermophilic microorganisms and their enzymes. In this work the compost was used as a starting material for the isolation of thermophilic microorganisms to verify the compost biodiversity; subsequently, the identification of enzymatic activities able to convert lignocellulosic biomass wastes to obtain products with several potential biotechnological applications in different types of sectors, was performed. The samplings were carried out in a different times of thermophilic phase of composting process, at the “Experimental Center of Composting” (CESCO), Cilento National Park, Laurino, Salerno, in which the residue of oil mills were used, and at the “Experimentation Center of Castel Volturno” (DISSPA), in which the compost derived from cowpat and straw. Two different approaches were used: isolation after enrichment (Method A) or isolation on selective agar plates method with xylan and carboxymethyl cellulose (0.2%, w/v) as substrates (Method B). The purification of isolates was then performed using the repeated serial dilution technique followed by re-streaking on solid medium. Two new Aeribacillus species, Aeribacillus strains N.8 and N.6B and two different strains of a new Geobacillus species, Geobacillus strains N.3BX and N.3BC, were isolated from CESCO. They showed an optimal temperature growth between 50 and 70°C in the pH values of 8.0-9.0. They were able to grow on medium containing up to 9% (w/v) NaCl with an optimum at 6-7 % (w/v) NaCl. The enzymatic activities of these new strains were studied in all three cellular compartments (extracellular, cytosolic and cell-bound fractions); in particular, Aeribacillus strain N.6B produced inulinase, pectinase and gelzan-lyase activity and strain N.8 showed gelzan-lyase activity. While, Geobacillus strains N.3BX and N.3BC produced xylanase, β-xylosidase and arabinofuranosidase activity. From the “Experimental Center of Composting” of Cilento National Park, it was also isolated a thermophilic microorganism named strain N.3TH2; based on the 16S rRNA gene sequence it possessed 100% of homology with Bacillus licheniformis. Strain N.3TH2 produced cellulase activity in the extracellular and cytosolic fractions. In particular, extracellular cellulase, that showed an optimal temperature activity of 60°C at pH 5.6, was studied. Moreover, six different strains, that fall within the genus Bacillus, were isolated from DISSPA. Their optimal temperature ranged between 50 and 60°C in the pH values of 7.0 at 5% (w/v) NaCl. They produced several enzymatic activities, such as xylanase, cellulase, amylase, protease, lypase and collagenase.

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