Liquid Perfluorodecalin Application for In Situ Extraction and Enhanced Naphthoquinones Production in Arnebia euchroma Cell Suspension Cultures

Abstract

Suspension cultures of Arnebia euchroma supported with liquid perfluorodecalin (PFD) degassed, aerated, or ethylene-saturated were investigated as a novel in situ extraction system for enhanced alkannin/shikonin production. Simultaneously, the effect of PFD applied as the liquid gas carrier on the growth of A. euchroma biomass was studied. The similar dry (4-fold) and fresh (7-fold) biomass increase was observed in the control (without PFD addition) and supplemented with PFD-degassed or PFD-aerated cultures while PFD-ethylene application impeded cell growth. The highest total of alkannin/shikonin production (23.23 mg flask−1) was observed when PFD-aerated has been used and it resulted in about 50 % higher yield of alkannin/shikonin compared with the control culture. Chiral HPLC analysis revealed that in cultures supported with PFD, both alkannin and shikonin were produced. Their mutual ratio varied depending on culture conditions, and the accumulation of alkannin prevailed under almost all culture conditions. PFD has proved to be exceptionally efficient and cell-safe solvent for the in situ extraction of naphthoquinone red pigments without exerting any detrimental effects on cell growth. Extracellularly secreted red naphthoquinones were easily dissolved and extracted from the PFD phase, which can be regenerated and reused (e.g., in continuous culture system).

Lignan accumulation in two-phase cultures of Taxus x media hairy roots

“…The results obtained in previous research performed by our group indicated that direct application of PFD to the culture system constitutes simple and efficient conditions for the enhancement of secondary metabolites production (Sykłowska-Baranek et al 2014, 2015a. In cultures of KT root line supplemented with PFD-aerated or degassed but without elicitors no lignan content was detected (Table 4).…”

“…PFD-aerated applied to the cell suspension cultures of Arnebia euchroma allowed to enhance naphthoquinone yield about 50% in comparison to control cultures (Sykłowska-Baranek et al 2014). The influence of PFD application at two time points: the day of inoculation (day 0) and the 14th day of culture together with elicitation (100 µM PHEN and MeJa) in ATMA transformed root cultures was studied (Sykłowska-Baranek et al 2015a).…”

Liquid Perfluorochemicals as Flexible and Efficient Gas Carriers Applied in Bioprocess Engineering: An Updated Overview and Future Prospects

“…by Percheron et al (1995) in the case of bacteria culture and by Sykłowska-Baranek et al (2014) in the case of plant cells) than the problem of oxygen supply. Nevertheless, accumulation of CO 2 which is gas continuously generated by cells and major waste product in culture media may cause detrimental effects in the cells, causing intracellular enzyme inhibition leading to death and further lysis of the cells.…”

“…Ethene has very poor solubility in water and liquid PFC-mediated supply of this compound has great potential of applicability in plant cell cultures. Up to date the effects of ethene-saturated PFD application have been studied only in the case of submerged cultures of Arneubia euchroma cells (Sykłowska-Baranek et al, 2014) in which 26 x higher value of the ratio between alkanin/shikonin, i.e. extracellularly secreted naphtoqinone-based plant secondary metabolites, has been achieved in comparison to cultures without PFD.…”

“…microorganisms, e.g. bacteria (Pilarek et al, 2011;Pilarek et al, 2013a), yeast (Pilarek and Szewczyk, 2008;Pilarek et al, 2006), fungi (Elibol, 2001) or microalgae (Hillig et al, 2013;Hillig et al, 2014), or plant cells (Davey et al 2005;Pilarek and Szewczyk 2008;Sykłowska-Baranek et al 2014) as wells as animal (Douglas et al 2014;Pilarek et al, 2013b;Pilarek et al, 2014;Rappaport, 2003;Shiba et al, 1998) to successfully obtain higher cell densities or to identify positive influence on metabolite excretion due to enhanced the mass transfer effects in culture systems, mainly.…”