THERMOPHILIC MICROORGANISMS AS SOURCE OF VALUE-ADDED MOLECULES AND BIOCATALYSTS FOR LIGNOCELLULOSIC BIOMASS CONVERSION

Feedstocks of lignocellulosic biomass represent a widely available and cheap organic material for the production of value added-products. In this context, thermophilic microorganisms are promising candidates both for the production of thermostable variants of carbohydrate-degrading enzymes, as well as for their own ability to thrive under harsh bioprocessing conditions. The first part of this PhD-thesis was focused on the characterization of new hemicellulolytic enzymes (TtGalA and DturCelB) from thermophilic source. TtGalA, a thermoactive and thermostable α-galactosidase from Thermus thermophilus HB27 was expressed in the native host, displaying an optimal hydrolytic activity at 90° C and pH 6.0 and long-term retained activity (30 hours) at 70°C. DturCelB is a b-mannanase from Dictyoglomus turgidum characterized in our research group exhibiting optimal catalytic activity at 70°C and pH 5.4. The heterosynergystic functional association between TtGalA and DturCelB was assayed on galactomannan polysaccharides in simultaneous and sequential assays. Our data showed that the two recombinant enzymes were able to work in synergy on all substrates tested, with TtGalA acting as first to remove galactose residues and therefore, exposing cleavage sites to DturCelB hydrolysis, thus increasing the degradation efficiency of complex substrates. Enzymatic immobilization techniques are suitable for enzyme recovery and recirculation. Several such techniques and support materials have been previously proposed, nevertheless the employment of viral particles has not been widely exploited so far. Because of our longstanding experience with archaeal thermophilic viral particles from Fuselloviridae, we aim at using them as self-assembling support to immobilize thermophilic carbohydrate-active enzymes. For this purpose, an UV induction protocol was successfully set up for the overproduction of thermophilic SSV1 virus particles. The second section of this thesis was addressed to the discovery and characterization of a new thermophilic strain (MA-13) of Bacillus coagulans. This microorganism was isolated from canned beans manufacturing residues for its ability to grow at 55°C, using carboxymethyl-cellulose and filter paper, as carbon sources. Noteworthy, MA-13 was tested for its ability to secrete soluble endo-1,4-β- glucanase enzymes into the culture supernatant. Moreover, it turned to be an efficient lactic acid producer on hydrolysate derived from the pre-treatment of wheat straw by acid- catalyzed hydrolysis and steam explosion. MA-13 was also used to set up the production of lactic acid from renewable lignocellulosic biomasses in SSF configuration