Acetic acid bacteria are strictly aerobic, gram-negative bacteria belonging to Acetobacteraceae family. So far, 19 genera with 110 species of acetic acid bacteria have been reported, which are isolated from variety of sources such as flowers, fruits, and fermented beverages. Acetic acid bacteria are well known for their oxidative metabolism, producing a wide range of organic acids, thanks to membrane-bound dehydrogenases. Besides the oxidative metabolism, acetic acid bacteria are receiving great attention for their ability to produce biopolymers, such as cellulose and fructans, including levan. Biopolymer synthesis has received great attention in the recent years by the scientific community and industries, paving the way towards environmentally friendly green composites. Bacterial cellulose and levan, have widely been used for food, medical and industrial applications due to their mechanical and physical properties, bio-compatibility, and non-toxicity. The biosynthesis of polymers in acetic acid bacteria occurs through enzymatic complexes, able to polymerize sugars and sugar alcohols into well-structured polysaccharide chains. The biochemical pathways involved in the production of bacterial cellulose and levan have been extensively studied, with the main aim to improve the yield and to design scalable- processes. The regulation of the biochemical pathways and the molecular mechanisms that tune the entire metabolism to the environmental availability of carbon source are not completely known. The reprogramming of the metabolism is a strategy that was already applied to bacteria with the aim of optimize the production of the target compounds. Based on these considerations, the main aim of this PhD thesis was to study these aspects for both levan and bacterial cellulose production. Regarding levan, they are mainly used as a prebiotic and are highly exploited in food industries. The industrial application of acetic acid bacteria for levan production is constrained by a lack of understanding their metabolism. To address this gap, in this study, strains of Gluconobacter, Acetobacter, Komagataeibacter, Neoasaia and Kozakia genera were used to characterize the levan synthesis. Phenotypic behavior of the individual strains in terms of inter- and intra-specific variability was analyzed by the production kinetics of levan, related to biochemical changes. This study allowed to identify the best levan producers in the tested conditions and impacting factors that are limiting the production of levan. Bacterial cellulose was deeply studied in the last decades, with a particular emphasis on the carbon source utilization, cellulose modification, and genetic engineering, obtaining interesting results. Alternative methods, such as, the study of regulating mechanisms of the carbohydrate metabolism and the adaptation to the availability of a certain carbon source, were not elucidated, constraining the applicability of bacterial cellulose. With this aim, adaptation strategies to a new carbon source were studied in this thesis by applying the adaptive laboratory evolution approach and new sequencing techniques. The strain K. xylinus K2G30 from UMCC was continuously cultivated in mannitol as a sole-carbon source for different cycles (350 days). From phenotypic point of view, the bacterial cellulose yield was increased by two/fold by using mannitol. The transcriptome analysis was applied to understand the expression of key genes that regulates the metabolism in a different condition and the strategies adopted by these bacteria. Data obtained can be exploited to set new strategies for scale up, increasing the applicability at industrial level and testing different bio-wastes that are rich in mannitol for bacterial cellulose production.

I batteri acetici sono batteri strettamente aerobi, gram-negativi che fanno parte della famiglia delle Acetobacteraceae. Attualmente, il raggruppamento dei batteri acetici include 19 generi e 110 specie, le fonti di isolamento maggiormente descritte sono fiori, frutti e bevande fermentate. I batteri acetici sono conosciuti per il loro metabolismo ossidativo e produzione di acidi organici grazie alla presenza di deidrogenasi di membrana. Oltre al metabolismo ossidativo, i batteri acetici stanno ricevendo una grande attenzione da parte della comunità scientifica e industriale, grazie alla loro capacità di produrre biopolimeri come cellulosa e levani. I biopolimeri sono ampiamente studiati per la formulazione di composti a basso impatto ambientale. La cellulosa batterica e i levani sono tra i biopolimeri più studiati grazie alle loro proprietà meccaniche e fisiche, oltre alle caratteristiche di biocompatibilità e non tossicità. La biosintesi di polimeri da parte dei batteri acetici avviene ad opera di complessi enzimatici in grado di polimerizzare monomeri di zucchero in catene polisaccaridiche ben strutturate. La regolazione delle vie biosintetiche e dei meccanismi molecolari non sono tuttavia ben conosciuti. La riprogrammazione del metabolismo in base a fattori ambientali, tra cui la disponibilità di fonti di carbonio è una strategia che è stata applicata su altri generi batterici con lo scopo di aumentare la resa di produzione di composti target. In base a queste considerazioni, lo scopo principale di questa tesi di dottorato è stato quello di studiare i meccanismi molecolari che stanno alla base della sintesi di levani e cellulosa batterica. Per quanto riguarda i levani, questi polimeri sono utilizzati ampiamente in ambito alimentare come prebiotici. L’applicazione industriale dei batteri acetici per la sintesi di levani è limitata da una carenza di conoscenze nel loro metabolismo. Per colmare questa carenza, ceppi appartenenti ai generi Gluconobacter, Acetobacter, Komagataeibacter, Neoasaia and Kozakia sono stati utilizzati per caratterizzare la sintesi di levani. La cinetica di produzione di questi composti è stata studiata in questi ceppi con lo scopo di analizzare la variabilità fenotipica a livello inter- e intra-specie. Questo studio ha permesso di identificare i migliori produttori di levani e di stabilire le condizioni migliori, identificando i fattori che ne limitano la produzione. La cellulosa batterica è stata studiata approfonditamente negli ultimi decenni, ponendo particolare attenzione all’utilizzo delle fonti di carbonio da parte dei batteri, definendo strategie di modifica del polimero e applicando l’ingegneria genetica con lo scopo di migliorare la resa o disegnare nuovi polimeri. Metodi naturali alternativi come lo sfruttamento delle abilità dei batteri ad adattarsi alle condizioni ambientali, come ad esempio la disponibilità di una fonte di carbonio diversa, risultano poco studiati. Per colmare queste lacune, in questo lavoro di tesi è stato applicato un approccio di evoluzione in laboratorio che consente di studiare l’adattamento a una nuova fonte di carbonio. Il ceppo K. xylinus K2G30 è stato ripetutamente coltivato utilizzando mannitolo come fonte di carbonio per un totale di 350 giorni. Dal punto di vista fenotipico, è stato osservato un incremento della resa di cellulosa del 100%. L’analisi del trascrittoma è stata applicata per monitorare l’espressione di geni che regolano il metabolismo in condizioni diverse e consentono di capire le strategie adottate da ceppi in studio. I risultati ottenuti possono essere sfruttati per incrementare l’applicabilità di questi batteri in ambito industriale, disegnando nuovi processi, e valorizzando scarti agroalimentari contenenti fonti di carbonio idonee.

STUDIO DEI MECCANISMI DI PRODUZIONE DI ESOPOLISACCARIDI NEI BATTERI ACETICI UTILIZZANDO COME MODELLO LA BIOSINTESI DI CELLULOSA BATTERICA E LEVANI / Kavitha Anguluri , 2023 Apr 21. 35. ciclo, Anno Accademico 2021/2022.

STUDIO DEI MECCANISMI DI PRODUZIONE DI ESOPOLISACCARIDI NEI BATTERI ACETICI UTILIZZANDO COME MODELLO LA BIOSINTESI DI CELLULOSA BATTERICA E LEVANI

ANGULURI, KAVITHA
2023

Abstract

Acetic acid bacteria are strictly aerobic, gram-negative bacteria belonging to Acetobacteraceae family. So far, 19 genera with 110 species of acetic acid bacteria have been reported, which are isolated from variety of sources such as flowers, fruits, and fermented beverages. Acetic acid bacteria are well known for their oxidative metabolism, producing a wide range of organic acids, thanks to membrane-bound dehydrogenases. Besides the oxidative metabolism, acetic acid bacteria are receiving great attention for their ability to produce biopolymers, such as cellulose and fructans, including levan. Biopolymer synthesis has received great attention in the recent years by the scientific community and industries, paving the way towards environmentally friendly green composites. Bacterial cellulose and levan, have widely been used for food, medical and industrial applications due to their mechanical and physical properties, bio-compatibility, and non-toxicity. The biosynthesis of polymers in acetic acid bacteria occurs through enzymatic complexes, able to polymerize sugars and sugar alcohols into well-structured polysaccharide chains. The biochemical pathways involved in the production of bacterial cellulose and levan have been extensively studied, with the main aim to improve the yield and to design scalable- processes. The regulation of the biochemical pathways and the molecular mechanisms that tune the entire metabolism to the environmental availability of carbon source are not completely known. The reprogramming of the metabolism is a strategy that was already applied to bacteria with the aim of optimize the production of the target compounds. Based on these considerations, the main aim of this PhD thesis was to study these aspects for both levan and bacterial cellulose production. Regarding levan, they are mainly used as a prebiotic and are highly exploited in food industries. The industrial application of acetic acid bacteria for levan production is constrained by a lack of understanding their metabolism. To address this gap, in this study, strains of Gluconobacter, Acetobacter, Komagataeibacter, Neoasaia and Kozakia genera were used to characterize the levan synthesis. Phenotypic behavior of the individual strains in terms of inter- and intra-specific variability was analyzed by the production kinetics of levan, related to biochemical changes. This study allowed to identify the best levan producers in the tested conditions and impacting factors that are limiting the production of levan. Bacterial cellulose was deeply studied in the last decades, with a particular emphasis on the carbon source utilization, cellulose modification, and genetic engineering, obtaining interesting results. Alternative methods, such as, the study of regulating mechanisms of the carbohydrate metabolism and the adaptation to the availability of a certain carbon source, were not elucidated, constraining the applicability of bacterial cellulose. With this aim, adaptation strategies to a new carbon source were studied in this thesis by applying the adaptive laboratory evolution approach and new sequencing techniques. The strain K. xylinus K2G30 from UMCC was continuously cultivated in mannitol as a sole-carbon source for different cycles (350 days). From phenotypic point of view, the bacterial cellulose yield was increased by two/fold by using mannitol. The transcriptome analysis was applied to understand the expression of key genes that regulates the metabolism in a different condition and the strategies adopted by these bacteria. Data obtained can be exploited to set new strategies for scale up, increasing the applicability at industrial level and testing different bio-wastes that are rich in mannitol for bacterial cellulose production.
THE EXOPOLYSACCHARIDES MACHINERY IN ACETIC ACID BACTERIA BY EXPLORING CELLULOSE AND LEVAN SYNTHESIS
21-apr-2023
GULLO, Maria
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