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Rodriguez Gamero, Jesus Enrique (2018) Polyhydroxyalkanoates (PHAs) production from lipids containing agri-food wastes. [Ph.D. thesis]

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Abstract (italian or english)

In the last decades, economic and environmental concerns about oil shortage and fossil-based economy stimulated the need to shift from conventional plastics to bio-based options like polyhydroxyalkanoates (PHAs). PHAs are stored in many bacteria as intracellular carbon and energy source under limiting environmental conditions. PHAs have a great promise because of their material properties comparable to petrol-based plastomers. Although PHAs could have many applications, their replacement over the oil-based plastics is limited by their expensive production. Therefore, the selection of suitable resources as carbon feedstock for PHAs synthesis and extraction/recovery methods of PHAs are the main factors in the entire PHAs production chain, contributing up to 80% of the operating cost.
Cheap and abundant biomass waste streams have been considered as renewable substrates for the production of polymers, fuels, enzymes and bulk chemicals. The use of industrial or agricultural by-products can be a strategy to decrease also PHAs price. It has been calculated that in the European Union (EU), the slaughterhouses produce around 500,000 tons/year of fatty discards, which could be used efficiently for the production of PHAs, unfortunately, no bacteria with high lipolytic capacity and at the same time ability to accumulate high amounts of PHAs have been found.
Another bottleneck in the PHAs purification steps, is determined by the release of large amounts of chromosomal DNA that causes a dramatic viscosity increase and hampers the following filtration, centrifugation and PHAs recovery steps. High pressure homogenization (HPH) is one of the most widely known methods for large scale cell disruption. HPH is considered environmentally friendly since it does not need solvents to mediate an efficient microbial cells disintegration. After the HPH application, decrease of viscosity is generally achieved by the supplementation of hypochlorite, commercially available nucleases, or heat treatment. Although these methods may be applicable in small-scale fermentation systems, they are not environmentally and economically suitable for industrial PHAs manufacturing.
Looking for a cost-effective solution to the lipolytic activity issue, lipolytic genes (lipH-lipC) from Pseudomonas stutzeri BT3 have been integrated into Cupriavidus necator DSM 545, a well-known PHAs producer. The lipolytic enzymes have been proficiently expressed in the recombinant strain, greatly increasing the PHAs production from the slaughterhouses fatty wastes, indicating that the engineered strain can contribute to increase the economic efficiency of future PHAs upstream processing.
On the others hands, looking for a cost-effective solution to the viscosity issue, the staphylococcal nuclease gene nuc from Staphylococcus aureus has been integrated into the chromosomes of two efficient PHAs-producing bacteria, namely C. necator DSM 545 and Delftia acidovorans DSM 39. The viscosity of the lysates of C. necator recombinant cells was greatly reduced without affecting PHAs production, indicating that the engineered strain is expected to increase the economic efficiency of future PHAs downstream processing.

Abstract (a different language)

Negli ultimi decenni, le preoccupazioni economiche e ambientali in merito alla carenza di petrolio e all'economia basata sui fossili, hanno stimolato la necessità di passare dalla plastica convenzionale a opzioni convenzionali fondate su biomassa come i poliidrossialcanoati (PHAs). I PHAs sono sintetizzati ed immagazzinati intracellularmente per alcuni batteri come fonti di carbonio ed energia in condizioni ambientali limitanti. I PHAs, per le loro proprietà materiali paragonabili ai plastomeri a base di petrolio, sono promettenti per sostituire le plastiche sintetiche. Sebbene i PHAs possano avere molte applicazioni, la loro sostituzione sulla plastica a base di petrolio è limitata dalla loro costosa produzione. Pertanto, la selezione di risorse idonee come materia prima di carbonio per la sintesi e il metodi di estrazione/recupero dei PHAs, sono i principali fattori dell'intera catena di produzione che contribuiscono con l'80% ai costi operativi.
Flussi di rifiuti di biomassa sono stati considerati come substrati rinnovabili per la produzione di polimeri, combustibili, enzimi e prodotti chimici. L'uso di sottoprodotti industriali o agricoli può essere una strategia per ridurre anche il prezzo di PHAs. È stato calcolato che nell'Unione europea (UE), i macelli producono circa 500.000 tonnellate/anno di scarti di materia grassa, che potrebbero essere utilizzati in modo efficiente per la produzione di PHAs, sfortunatamente, nessun batterio con elevate capacità lipolitiche e allo stesso tempo capacità per accumulare quantità elevate di PHAs è stato trovato.
Un altro ostacolo nelle fasi di estrazione del PHAs è determinato dal rilascio di grandi quantità di DNA cromosomico che provoca un notevole aumento della viscosità e difficolta le seguenti fasi di recupero di filtrazione e centrifugazione. L'omogeneizzazione ad alta pressione (HPH) è uno dei metodi più noti per l'interruzione cellulare su larga scala. HPH è considerato rispettoso dell'ambiente in quanto non ha bisogno di solventi per mediare un'efficiente disintegrazione delle cellule microbiche. Dopo l'applicazione di HPH, la diminuzione della viscosità viene generalmente ottenuta mediante l'integrazione di ipoclorito, nucleasi commerciale o trattamento termico. Sebbene questi metodi possano essere applicabili nei sistemi di fermentazione su piccola scala, non sono idonei dal punto di vista ambientale ed economico per la produzione industriale di PHAs.
Alla ricerca di una soluzione economicamente vantaggiosa al problema dell'attività lipolitica, i geni lipolitici (lipH-lipC) di Pseudomonas stutzeri BT3 sono stati integrati in Cupriavidus necator DSM 545, un noto produttore di PHAs. Gli enzimi lipolitici sono stati notevolmente espressi nel ceppo ricombinante, aumentando la produzione di PHAs dagli scarti grassi dei macelli, indicando che il ceppo ingegnerizzato può contribuire ad aumentare l'efficienza economica della futura elaborazione dei PHAs.
Ad ogni modo, alla ricerca di una soluzione economica al problema della viscosità, il gene della nucleasi (nuc) dello Staphylococcus aureus è stato integrato nei cromosomi di due batteri efficaci produttori di PHAs (C. necator DSM 545 e Delftia acidovorans DSM 39). La viscosità dei lisati delle cellule ricombinanti di C. necator DSM 545 è stata notevolmente ridotta senza influenzare la produzione di PHAs, indicando che il ceppo ingegnerizzato potrebbe aumentare l'efficienza economica dei futuri processi di estrazione di PHAs.

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EPrint type:Ph.D. thesis
Tutor:Casella, Sergio and Basaglia, Marina
Supervisor:Favaro, Lorenzo
Ph.D. course:Ciclo 31 > Corsi 31 > SCIENZE DELLE PRODUZIONI VEGETALI
Data di deposito della tesi:29 November 2018
Anno di Pubblicazione:28 November 2018
Key Words:Polyhydroxyalkanoates, Downstream processing, Viscosity, Nuclease, Cupriavidus necator, PHAs, Delftia acidovorans, Fatty waste, Lipase
Settori scientifico-disciplinari MIUR:Area 09 - Ingegneria industriale e dell'informazione > ING-IND/11 Fisica tecnica ambientale
Area 09 - Ingegneria industriale e dell'informazione > ING-IND/34 Bioingegneria industriale
Area 07 - Scienze agrarie e veterinarie > AGR/07 Genetica agraria
Area 09 - Ingegneria industriale e dell'informazione > ING-IND/22 Scienza e tecnologia dei materiali
Area 07 - Scienze agrarie e veterinarie > AGR/16 Microbiologia agraria
Area 05 - Scienze biologiche > BIO/19 Microbiologia generale
Struttura di riferimento:Dipartimenti > Dipartimento di Agronomia Animali Alimenti Risorse Naturali e Ambiente
Codice ID:11478
Depositato il:14 Nov 2019 13:43
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