Impact of electron acceptors on growth and phenotypic fingerprint of Clostridium kluyveri.

Ulčar, Barbara (author), Vodovnik, Maša (mentor), Ganigue, Ramon (co-mentor)

Carboxylate platform enables production of medium-chain carboxylates from short-chain carboxylates through chain-elongation process. Clostridium kluyveri is strictly anaerobic bacterium that is able to perform chain-elongation through reverse beta oxidation pathway. In this pathway, ethanol is used as an electron donor and different carboxylates, such as acetate and propionate, can serve as electron acceptors. Diverse transport proteins might be needed for the acetate and propionate uptake, which could alter phenotype of the cells. Phenotype can be measured using flow cytometry and obtaining phenotypic fingerprint. Beta diversity assessment is performed to detect differences in phenotypic fingerprints. The aim of this work is to gain knowledge about how C. kluyveri uses different electron acceptors and to address the question whether C. kluyveri has a preference for a specific electron acceptor. Batch experiments were performed with different electron acceptors supplied to the culture. The impact of an electron acceptor supplied and the impact of an electron acceptor used for pre-growing the culture on the growth, consumption profile, production profile and phenotypic fingerprint of the culture was assessed. Growth of the cultures was not affected by the electron acceptor supplied neither by the electron acceptor used for pre-growing the culture. End product profiles of acetate and propionate cultures were different between each-other, but the end product profiles of cultures supplied with both electron acceptors were similar, regardless of the electron acceptor used for pre-growing the culture. Analysis of substrate consumption and product formation indicated, that C. kluyveri might have a preference for some electron acceptors. This preference might be associated with differential affinity of the metabolic enzymes and is not influenced by the substrate uptake. Beta diversity comparison of the phenotypic fingerprints originating from different experimental conditions revealed, that separation of different phenotypic fingerprints is possible, but separation could not be explained by the electron acceptors supplied.

• Language: English
• Keywords: Clostridium kluyveri, electron acceptor, flow cytometry
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [B. Ulčar]
• Year: 2019
• PID: 20.500.12556/RUL-106457
• UDC: 602.3:579.852.13:57.086.8(043.2)
• COBISS.SI-ID: 9176697
• Publication date in RUL: 25.02.2019

Secondary language

• Language: Slovenian
• Title: Vpliv elektronskih akceptorjev na rast in fenotipski odtis bakterije Clostridium kluyveri.

Abstract

Karboksilatna platforma omogoča produkcijo srednje-verižnih karboksilatov iz kratko-veržnih karboksilatov preko procesa podaljševanja verige. Clostridium kluyveri je anaerobna bakterija, ki vrši reakcijo podaljševanja verige preko poti reverzne beta oksidacije. V reakciji kot donor elektronov nastopa etanol, sprejemniki elektronov so kratko-verižni karboksilati, kot sta acetat in propionat. Mogoče je, da so za privzem različnih sprejemnikov elektronov v celico potrebni različni transportni proteini, kar vpliva fenotip celic. Fenotip celic je mogoče opazovati z metodo pretočne citometrije preko določanja fenotipskega odtisa. Za primerjavo fenotipskih odtisov se uporablja ocena beta-raznolikosti. Namen tega dela je ugotoviti, ali ima bakterija C. kluyveri preferenco pri uporabi sprejemnika elektronov. Opazovali smo vpliv dodanega sprejemnika elektronov in vpliv sprejemnika elektronov uporabljenega za predhodno gojenje kulture na parametre rasti, profil porabe substratov in nastanka produktov ter na fenotipski odtis. Sprejemnik elektronov dodan kulturi in sprejemnik elektronov uporabljen za predhodno gojenje kulture ni vplival na parametre rasti. Končni profil produktov se je med kulturama, ki sta vsebovali le acetat ali propionat, razlikoval. Pri kulturah z dodanima obema sprejemnikoma elektronov je bil primerljiv, prejemnik elektronov uporabljen za predhodno gojenje kulture ni imel vpliva na končni profil produktov. Analiza porabe substratov in nastanka produktov je nakazovala, da bi bakterija C. kluyveri lahko imela preferenco za porabo določenih sprejemnikov elektronov. Rezultati so privedli do predvidevanja, da opažena preferenca ni posledica različnega privzema sprejemnikov elektronov v celico, njen vzrok bi lahko bila različna afiniteta metabolnih encimov do določenih sprejemnikov elektronov. Ocena beta raznolikosti fenotipskih odtisov kultur z dodanimi različnimi sprejemniki elektronov je pokazala, da je fenotipske odtise mogoče ločiti. Z rezultati ni bilo mogoče potrditi, da je razlikovanje posledica različnih sprejemnikov elektronov.

• Keywords: Clostridium kluyveri, sprejemnik elektronov, pretočna citometrija