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Gluconic Acid, Vitamin C and Ketones

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Gluconic Acid, Vitamin C and Ketones UNIVERSITY OF MODENA AND REGGIO EMILIA Ph.D school in: Food and agricultural science, technology and biotechnology ____________________________________________________________ Dean of Ph.D school: Prof. Alessandro Ulrici XXXIII Cycle Phenotypic and genomic analysis of Komagataeibacter strains to elucidate the cellulose biosynthesis mechanism Candidate: Salvatore La China Tutor: Dr. Maria Gullo Table of contents Abstract 3 Riassunto 4 Thesis outline 6 Chapter 1 9 Oxidative fermentations and exopolysaccharides production by acetic acid bacteria: a mini review Chapter 2 22 Biotechnological production of cellulose by acetic acid bacteria: current state and perspectives Chapter 3 39 Kombucha tea as a reservoir of cellulose producing bacteria: assessing diversity among Komagataeibacter isolates Chapter 4 51 Exploring K2G30 genome: A high bacterial cellulose producing strain in glucose and mannitol based media Chapter 5 65 Genome sequencing and phylogenetic analysis of K1G4: a new Komagataeibacter strain producing bacterial cellulose from different carbon sources Chapter 6 77 Characterization of bio-nanocomposite films based on gelatin/polyvinyl alcohol blend reinforced with bacterial cellulose nanowhiskers for food packaging applications Chapter 7 93 Mechanical and structural properties of environmental green composites based on functionalized bacterial cellulose 1 Chapter 8 106 General discussion and future perspectives Supplementary materials 111 References 124 Organizations 155 Acknowledgments 156 Curriculum vitae and list of publications 158 2 Abstract Abstract Acetic acid bacteria are versatile organisms converting a number of carbon sources into biomolecules of industrial interest. Such properties, together with the need to limit chemical syntheses in favor of more sustainable biological processes, make acetic acid bacteria suitable organisms for food, chemical, medical, pharmaceutical and engineering applications. At current, well-established bioprocesses by acetic acid bacteria are those derived from the oxidative pathways that lead to organic acids synthesis, such as acetic acid and gluconic acid, vitamin C and ketones. Others applications of acetic acid bacteria derive from their ability to produce exopolysaccharides. Among exopolysaccharides, bacterial cellulose is one of the most important biopolymer that actually is receiving more attention for sustainable industrial applications. The reasons why bacterial cellulose is an attractive biopolymer lies in its physical-chemical properties, and mechanical properties, as well as in the biodegradability and not toxic features. Among its physical-chemical properties stand out purity, considering that it consists of glucose monomers, high water-holding capacity, high crystallinity index and biocompatibility. All of these features make bacterial cellulose suitable for the use as native polymer or in composite materials. Within the Acetobacteriaceae family, the genus Komagataeibacter includes species of interest for the production of bacterial cellulose, especially the species Komagataeibacter xylinus, which is considered the model organism for the study of the biological e biochemical mechanisms of bacterial cellulose synthesis. Kombucha tea, a beverage originated from Asia continent, produced from black and green tea supplemented with sucrose, is a selective food matrix for recovering cellulose producing acetic acid bacteria. The occurrence of at least two microbial groups, yeasts and acetic acid bacteria, which establish a symbiotic relationship, allows the production of a sparkling, low alcoholic, acidic beverage, containing a layer of bacterial cellulose on the surface. The aim of this PhD thesis was to study the biological mechanisms of cellulose production in acetic acid bacteria. Bacterial strains from UMCC (Unimore Microbial Culture Collection) culture collection, isolated from Kombucha tea, were typed and selected for their ability to produce bacterial cellulose. A polyphasic study was performed on two strains, including the whole genome sequencing focusing the analysis of genes involved in cellulose synthesis, and a phenotypic characterization mainly aimed at the detection of the best carbon for cellulose production. This approach allowed also the taxonomic identification of the two strains, highlighting issues of taxonomic allocation within the Komagataeibacter genus. The bacterial cellulose obtained by the conditions set up in this study, was used to develop two bio-based materials. The first material is a combination of bacterial cellulose with gelatin and polyvinyl-alcohol that is a promising innovative biomaterial for food packaging uses; the second one is a bio-based composite material composed of bacterial cellulose and inorganic compounds (titanium dioxide and clay), which properties outline the application as high performance membrane for biomedical use. 3 Riassunto Riassunto I batteri acetici sono microrganismi versatili in grado di convertire fonti di carbonio in biomolecole di interesse industriale. Nell’era in cui c’è una forte necessità di ridurre la sintesi chimica di materiali, a favore di produzioni mediate da processi biologici sostenibili, i batteri acetici rivestono un ruolo chiave per diverse applicazioni in campo chimico, biomedico, farmaceutico, alimentare e ingegneristico. Attualmente, i bioprocessi consolidati che vedono coinvolti i batteri acetici sono principalmente legati allo sfruttamento del loro potenziale ossidativo con produzione di acidi organici, come l’acido acetico e l’acido gluconico, la vitamina C e i chetoni. Altre applicazioni dei batteri acetici riguardano le loro abilità nella produzione di esopolisaccaridi. In particolar modo, la cellulosa batterica è, senza dubbio, uno dei biopolimeri che attualmente sta ricevendo molta attenzione per applicazioni industriali sostenibili. Quello che rende la cellulosa di origine microbica un polimero così attraente risiede sicuramente nelle proprietà chimico-fisiche e meccaniche, così come nella non tossicità e biodegradabilità. Tra le caratteristiche chimico-fisiche peculiari della cellulosa batterica spiccano la purezza, considerando che è composta esclusivamente da monomeri di glucosio, alto grado di cristallinità, elevata capacità di ritenzione idrica e biocompatibilità. Queste proprietà consentono l’utilizzo della cellulosa batterica sia nella forma nativa che in combinazione con altri materiali. All’interno della famiglia Acetobacteriaceae, il genere Komagataeibacter, include specie di grande interesse per la produzione di cellulosa, in modo particolare la specie Komagataeibacter xylinus, considerata il modello per lo studio dei meccanismi biologici e biochimici che regolano la produzione di cellulosa. Una delle matrici di origine alimentare da cui è possibile isolare ceppi del genere Komagataeibacter è una bevanda fermentata chiamata Kombucha tea, originaria del continente asiatico, e tradizionalmente prodotta fermentando te nero o te verde addizionato di saccarosio. La coesistenza di almeno due gruppi microbici, quali lieviti e batteri acetici in associazione simbiotica, è responsabile della trasformazione in una bevanda frizzante a basso contenuto alcolico, acidula e caratterizzata da una presenza di cellulosa sulla superficie. Lo scopo del presente lavoro di tesi è stato quello di approfondire i meccanismi biologici di produzione di cellulosa batterica. Ceppi batterici isolati da Kombucha tea e posseduti dalla collezione microbica UMCC (Unimore Microbial Culture Collection) sono stati genotipizzati e selezionati sulla base della capacità di produzione di cellulosa batterica. Due ceppi sono stati oggetto di uno studio polifasico che ha previsto sia il sequenziamento dell’intero genoma, finalizzato alla conoscenza dei geni coinvolti nel meccanismo di regolazione e produzione di cellulosa, che uno studio fenotipico di caratterizzazione delle migliori fonti di carbonio per la produzione di cellulosa batterica. Tale approccio ha permesso anche l’identificazione dei due ceppi, evidenziando alcune problematiche di collocazione tassonomica all’interno del genere Komagataeibacter. La cellulosa ottenuta da uno dei due ceppi, in condizioni ottimizzate nel presente studio, è stata utilizzata per lo sviluppo di due materiali, di cui uno ha dato risultati promettenti per applicazioni nel campo del confezionamento alimentare, in combinazione con gelatina-polivinil alcool; l’altra ha riguardato lo sviluppo di un materiale composito con composti 4 Riassunto inorganici (biossido di titanio e argilla) le cui caratteristiche ne delineano l’impiego come membrane ad alta prestazione in applicazioni biomedicali. 5 Thesis outline Thesis outline This PhD thesis was carried out at the Department of Life Sciences of the University of Modena and Reggio Emilia, between years 2017 and 2020. The manuscript deals with the research project performed during the three years focused on the development of new strategies for the production of bacterial cellulose. The main goal of the research project was to integrate different experimental approaches in order to understand the biological meaning and the molecular machinery that lead cellulose production in prokaryotes. This research project is an example of a multidisciplinary approach which considered different aspects related to environmental microbiology, food microbiology and material sciences, used to develop new materials with high impact in a wide range of applications, especially in biomedical field and food packaging. Furthermore, the use of bacterial
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