Kontinuirana proizvodnja bakteriofagov v sistemu dveh zaporednih bioreaktorjev

Nabergoj, Dominik (author), Podgornik, Aleš (mentor)

Bakteriofagi so bakterijski virusi, ki okužijo bakterijo, se v njej pomnožijo, ter se preko lize sprostijo v okolje. Proizvodnja bakteriofagov je v večini primerov tradicionalna, saj še vedno poteka v erlenmajericah na stresalnikih ali na industrijski ravni kot šaržni proces v bioreaktorjih. Kontinuirana proizvodnja bakteriofagov bi lahko predstavljala dragoceno alternativo. Osredotočili smo se na kontinuirano proizvodnjo bakteriofagov v sistemu dveh zaporednih bioreaktorjev, pri čemer smo preizkusili dve različici. Prva različica je bila sestavljena iz dveh zaporedno povezanih mešalnih bioreaktorjev ter je predstavljala t. i. »cellstat«, pri drugi različici pa je prvi predstavljal mešalni bioreaktor, drugi pa cevni bioreaktor. V obeh različicah se je v prvem bioreaktorju gojila bakterija brez prisotnosti bakteriofaga, v drugem bioreaktorju pa je potekala infekcija bakterije z bakteriofagom in pomnoževanje bakteriofaga. Za sistem bakterija-bakteriofag smo uporabili izključno litičen T4 bakteriofag ter fakultativno anaerobno bakterijo Escherichia coli K-12. Hitrost redčenja v mešalnem bioreaktorju ima pomemben vpliv na bakteriofagne rastne parametre bakteriofaga T4: konstanto adsorpcije, latentno periodo ter brstno število, obenem pa tudi pomembno vpliva na populacijsko hitrost rasti bakteriofagov. Pri študijah produktivnosti kontinuirane proizvodnje bakteriofagov (prva različica) se je izkazalo, da obstaja hitrost redčenja v drugem mešalnem bioreaktorju pri kateri je produktivnost maksimalna (velja za variabilno in konstantno fiziološko stanje bakterij), medtem ko sta se trenda koncentracije bakteriofagov razlikovala. Izvedli smo tudi teoretično analizo vpliva spremenljivk pri kateri se je izkazalo, da na produktivnost vplivajo procesni parametri (hitrost redčenja in koncentracija bakterij) ter tudi bakteriofagni rastni parametri (adsorpcija, latentna perioda in brstno število). Na optimalno hitrost redčenja, pri kateri se je dosegla maksimalna produktivnost, je imela največji vpliv latentna perioda. Na maksimalno produktivnost pa sta imela največji vpliv latentna perioda in brstno število. Z eksperimenti druge različice kontinuirane proizvodnje bakteriofagov smo pokazali, da je mogoče vzpostaviti robustno kontinuirano proizvodnjo bakteriofagov v sistemu dveh zaporednih bioreaktorjih, pri katerem je prvi predstavljal mešalni bioreaktor, drugi pa cevni bioreaktor.

• Language: Slovenian
• Keywords: bakteriofag T4, Escherichia coli, kontinuirana proizvodnja, produktivnost, bakteriofagni rastni parametri, rastna hitrost bakterije, cellstat, kemostat, cevni reaktor
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Publisher: [D. Nabergoj]
• Year: 2018
• PID: 20.500.12556/RUL-105728
• UDC: 602.3:578.347:602.1(043.3)
• COBISS.SI-ID: 9120633
• Publication date in RUL: 08.12.2018

Secondary language

• Language: English
• Title: Continuous production of bacteriohages in a system of two consecutive bioreactors

Abstract

Bacteriophages are bacterial viruses, which infect bacteria, amplify inside the host, and finally after lysis new bacteriophages are released. Phage production is in most of the cases still traditional, taking place in shaking flasks or on industrial level, in bioreactors as a batch process. Continuous production of phages could represent a valuable alternative. We focused on continuous bacteriophage production in system of two bioreactors connected in series and we tested two versions. The first version consisted of two connected stirred bioreactors in series, representing cellstat. Second version consisted of first bioreactor being stirred bioreactor and the other being a tubular bioreactor. In both versions, the bacteria was cultivated in the first bioreactor without presence of bacteriophages, in second bioreactor infection of bacteria with bacteriophage occured and bacteriophage multiplication was intiated. For bacteria-bacteriophage system we choosed obligately lytic bacteriophage T4 and facultative anaerobic Escherichia coli K-12 as host. Dilution rate in stirred bioreactor have a significant effect on bacteriophage growth parameters: adsorption constant, latent period and burst size, similarly has an important effect also on bacteriophage population growth rate. In studies to determine productivity of continuous bacteriophage production (first version), it was found that there exist dilution rate in the second stirred bioreactor at which the productivity is maximal (for variable and constant physiological state of bacteria), while the trends of concentration of bacteriophages differed significantly. Theoretical analysis was performed to determine influence of variables, the results showed that process parameters (dilution rate and concentration of bacteria) and bacteriophage growth parameters (adsorption, latent period and burst size), have an influence on productivity. Latent period have the most important effect on optimal dilution rate (dilution rate at which maximal productivity is achieved), while latent period and burst size have the most profound effect on maximal productivity. Experiments of second version showed that robust continuous bacteriophage production in a system of two consecutive bioreactors, the first being stirred bioreactor and the other being a tubular bioreactor, can be achieved.

• Keywords: bacteriophage T4, Escherichia coli, continuous production, productivity, bacteriophage growth parameters, bacterial growth rate, cellstat, chemostat, plug-flow reactor