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Optimizacija in aplikacija metode za mikroskopsko kvantifikacijo morfoloških značilnosti bakterij iz rodu Streptomyces.
Slakan, Ana (Author), Petković, Hrvoje (Mentor) More about this mentor... This link opens in a new window, Štern, Alja (Co-mentor)

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Abstract
Bakterije iz rodu Streptomyces odlikuje izreden biosintezni potencial. Njihova morfologija naj bi bila tesno povezana s proizvodnjo sekundarnih metabolitov. Vzpostavili in optimizirali smo metodo priprave mikroskopskih preparatov iz zelo kompleksnih brozg bioprocesov s postopkom barvanja s kristal vijoličnim. V naslednji fazi smo razvili postopek digitalne slikovne analize morfoloških parametrov ter ga aplicirali na spremljanje morfologije Streptomyces clavuligerus pri proizvodnji klavulanske kisline. Pri tem smo pokazali, da se med rastjo in razvojem bakterije Streptomyces clavuligerus v laboratorijskih gojitvenih steklenicah pri nekaterih pogojih lahko pojavlja peletasta rast in lahko prihaja do procesov diferenciacije micelija. Rezultati kažejo tudi, da se v tekočih gojiščih na pilotnem nivoju pri iztrošenih kulturah Streptomyces clavuligerus pojavljajo sporam podobne strukture. Oboje nakazuje na to, da bi lahko bil razvojni cikel streptomicet v tekočih gojiščih bolj podoben razvojnemu ciklu na trdnih gojiščih, kot se trenutno domneva. Analiza različnih morfoloških parametrov kot so dolžina, debelina hif, razvejanost ter viabilnost micelija je omogočila ovrednotenje razlik med bioprocesi na pilotnem in na industrijskem nivoju. V standardnem pilotnem bioreaktorju smo tako opazovali razvoj bistveno krajših hif v primerjavi s pilotnim bioreaktorjem, ki je po geometriji in konfiguraciji mešal podoben industrijskemu. V bioreaktorju na industrijskem nivoju so bile hife še krajše, manj razvejane, vendar bistveno debelejše in viabilnejše od obeh pilotnih bioreaktorjev. Rezultati sicer kažejo, da je morfologija do določene mere povezana s produkcijo klavulanske kisline, vendar bi za potrditev potrebovali bistveno večjo količino podatkov. Vzpostavljena metoda ima pri spremljanju morfologije streptomicet izreden pomen in je primerna za rutinsko uporabo v industriji.

Language:Slovenian
Keywords:morfologija, filamentozne bakterije, Streptomyces clavuligerus, vzpostavitev metode, mikroskopska kvantifikacija, slikovna analiza, klavulanska kislina
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2017
Publisher:[A. Slakan]
UDC:602.3:579.873.7:601.2:576.31.08(043.2)
COBISS.SI-ID:8718201 Link is opened in a new window
Views:660
Downloads:223
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Secondary language

Language:English
Title:Optimization and application of a method for the quantification of morphological parameters of bacteria from the genus Streptomyces.
Abstract:
Members of the genus Streptomyces are recognized by their remarkable biosynthetic potential. It has been reported that the culture morphology strongly correlates with secondary metabolite production. In this work, we have established a method for sample preparation for microscopy from very complex fermentation broth samples, using crystal violet staining. Further, we developed a process of digital image analysis and applied it for morphological analysis of the clavulanic acid - producing Streptomyces clavuligerus. Our results show, that pelleted growth and differentiation processes can be observed during growth and development of S. clavuligerus cultures at specific conditions in shake flask cultures. Interestingly, spore-like structures of the S. clavuligerus culture have been observed in liquid media towards the end of bioprocess at the pilot scale, thus suggesting that the development of Streptomyces culture in liquid media is more similar to the development cycle on solid media. Further on, we analysed different morphological parameters such as length, thickness, branching and mycelium viability and compared the morphology at the pilot and industrial scale. The S. clavuligerus culture in the standard pilot bioreactor formed significantly shorter hyphae in comparison to the pilot bioreactor, which is by its geometry and impeller configuration similar to the industrial vessels. The S. clavuligerus culture grown at the industrial scale formed even shorter hyphae, less branched, but significantly thicker, and more viable compared to the cultures grown in bioreactors at the pilot scale. Our results indicate, there is a correlation between morphology and clavulanic acid production. However, more data is needed to reliably confirm this correlation. We have demonstrated that the methodology developed in this work is very useful for Streptomyces morphology observation and evaluation and it is suitable for routine use in the industrial environment.

Keywords:morphology, filamentous bacteria, Streptomyces clavuligerus, method development, microscopic quantification, image analysis, clavulanic acid

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