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Trento, Alberto (2013) Selection and genetic improvement of yeasts for the conversion of lignocellulose into second generation bioethanol. [Ph.D. thesis]

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Abstract (english)

Bioethanol produced from lignocellulosic biomass represents a promising alternative among biofuels. To date a cost-effective method for the industrial production of bioethanol from vegetal biomass has not been developed. One of the most attractive strategies is the construction of a CBP (Consolidated BioProcessing) microbe able both to hydrolyze the complex polymers of lignocellulosic biomass and to convert these into ethanol.
In this context, the present study focused on the development of an industrial CBP microbe for the conversion of cellobiose into ethanol. To this purpose, it was necessary to define a new screening method for the selection of a yeast strain, suitable for the industrial bioethanol production having high fermentative abilities and considerable tolerance to inhibitors commonly present in lignocellulosic hydrolysates. The selection started from a collection of oenological yeasts. These strains, although showing interesting fermentative abilities, did not exhibit a good tolerance to inhibitors such as furfural, acetic acid, formic acid and lactic acid.
Therefore, a new isolation programme was necessarily conducted in order to select efficient fermenting yeast strains able to tolerate high concentrations of inhibitory compounds. The isolation procedure, conducted in the presence of an inhibitors cocktail, allowed to obtain a wide collection of yeasts with interesting features for their future applications in the field of second generation bioethanol. Among them, few S. cerevisiae yeasts exhibited remarkable fermenting vigour at high temperature and promising inhibitors tolerance. In particular, S. cerevisiae T2 was selected as host for the development of a recombinant strain able to produce the BglI β-glucosidase of Saccharomycopsis fibuligera, one of the most efficient cellobiose hydrolyzing yeast species. For the first time, in this study, an industrial yeast strain secreting β-glucosidase BglI was described. However, the hydrolytic activity of the recombinant strain must be necessarily increased in order to produce an efficient cellulolytic CBP microbe.
On the basis of the preliminary results obtained, this multi-disciplinary work represents a first step towards the development of microbes for the single-step conversion of lignocellulosic biomass to ethanol.

Abstract (italian)

Il bioetanolo di origine lignocellulosica rappresenta una delle alternative più promettenti tra i biocarburanti. Dal punto di vista industriale, la produzione di bioetanolo da biomassa vegetale non è ancora sostenibile. Una delle strategie più interessanti proposte è la costruzione di un microganismo CBP (Consolidated BioProcessing) capace di idrolizzare i polimeri complessi della biomassa cellulosica e di convertirli efficacemente in etanolo.
In questa prospettiva, questo lavoro di tesi si è focalizzato sullo sviluppo di un microbo CBP di tipo industriale per la conversione di cellobiosio in alcol etilico. A tal scopo, è stato necessario mettere a punto un nuovo metodo per la selezione di un ceppo di lievito idoneo alla produzione di bioetanolo su scala industriale caratterizzato da elevate performance fermentative e da una notevole capacità di tollerare gli inibitori normalmente presenti negli idrolizzati lignocellulosici. La selezione di tale microrganismo è partita da una collezione di ceppi di lievito di origine enologica. I ceppi enologici saggiati, pur dimostrando elevate capacità fermentative, non si sono purtroppo rivelati tolleranti nei confronti di inibitori quali furfurale, acido acetico, acido formico ed acido lattico.
È stato quindi necessario eseguire un programma di isolamento mirato ad ottenere ceppi di lievito altamente fermentanti e capaci di tollerare elevate concentrazioni di inibitori. L’isolamento, eseguito in condizioni selettive per la presenza di un cocktail di inibitori, ha consentito di ottenere una ampia ceppoteca di lieviti con caratteristiche promettenti per la loro futura applicazione nel campo del bioetanolo di seconda generazione. Tra di essi, alcuni lieviti S. cerevisiae si sono distinti per vigore fermentativo ad elevata temperatura e per una consistente tolleranza agli inibitori.
In particolare, il ceppo S. cerevisiae T2 è stato selezionato come host strain per lo sviluppo di un ceppo ricombinante capace di secernere la betaglucosidasi BglI di Saccharomycopsis fibuligera, specie di lievito tra le più efficienti per l’idrolisi del cellobiosio. Per la prima volta in questo lavoro di tesi è stato descritto un ceppo di lievito industriale betaglucosidasico. In ogni caso, l’attività idrolitica del ceppo ricombinante dovrà essere necessariamente incrementata al fine di ottenere un efficiente microrganismo CBP cellulosolitico.
In base ai risultati ottenuti, questo studio rappresenta un primo passo verso lo sviluppo di microrganismi idonei alla conversione one-step di biomassa lignocellulosica in etanolo.

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EPrint type:Ph.D. thesis
Tutor:Casella, Sergio and Basaglia, Marina
Supervisor:Favaro, Lorenzo
Ph.D. course:Ciclo 25 > Scuole 25 > SCIENZE DELLE PRODUZIONI VEGETALI > AGROBIOTECNOLOGIE
Data di deposito della tesi:24 January 2013
Anno di Pubblicazione:24 January 2013
Key Words:Bioethanol/Bioetanolo, Yeast/Lievito, Cellobiose/Cellobiosio, Lignocellulose/Lignocellulosa
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > AGR/16 Microbiologia agraria
Struttura di riferimento:Dipartimenti > Dipartimento di Agronomia Animali Alimenti Risorse Naturali e Ambiente
Codice ID:5480
Depositato il:14 Oct 2013 11:47
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