Cultivation of Marine Microorganisms in Single-Use Systems

Abstract

: Marine cultures are an important source of novel substances and enzymes. As efforts to isolate strains from (deep) sea environments increase, the demand for methodology platforms to cultivate these organisms is also rising. Due to the high salt concentration and the shear sensitivity exhibited by some heterotrophic microalgae, single-use systems originally designed for the cultivation of mammalian cell lines can be a valuable alternative. Using the cultivation of the heterotrophic marine microalgae Crypthecodinium cohnii as an example, this chapter makes suggestions for experimental design, for improving process development by integrating parallel experiments, and for scaling-up and scaling-down methodologies. It describes how to identify suitable single-use systems and how to integrate a two-layer system with perfluordecalin to improve the gas transfer in deep-well plates. The process is also scaled up in several single-use systems. We also describe challenges in the process development to achieve sufficient oxygen transfer, monitoring, and control, and we discuss limitations such as corrosion, long-term stability, and leachables in single-use systems. Finally, we demonstrate a method for cheap, fast, and consistent process development for marine microorganisms.

Antioxidant activity of alcohol aqueous extracts of Crypthecodinium cohnii and Schizochytrium sp.

“…C. cohnii, distributed world-wide, is a marine heterotrophic microalga (dinoflagellate) (Beam and Himes, 1982). Until recently, studies of C. cohnii have focused on cultivation (Pleissner and Eriksen, 2012;Hillig et al, 2013), DHA production (Gong et al, 2015), the biological activity of DHA (Fedorova-Dahms et al, 2014), and DHA applications in the breeding and aquatic industries (Schiavone et al, 2007;Ganuza et al, 2008). Schizochytrium sp.…”

Perfluorodecalin-supported system enhances taxane production in hairy root cultures of Taxus x media var. Hicksii carrying a taxadiene synthase transgene

“…From the bioprocess engineering point of view, PFCs are recognized as the most flexible liquid carriers/scavengers of gaseous compounds (O 2 and CO 2 , mainly) and were successfully applied in culture systems of various types of cells, i.e. microorganisms such as bacteria (Pilarek et al 2011;2013a), yeast (Pilarek et al 2006), fungi (Elibol and Ozer 2000) and microalgae (Hillig et al 2013(Hillig et al , 2014, animal (Shiba et al 1998;Rappaport 2003;Pilarek et al 2013bPilarek et al , 2014Douglas et al 2014) as well as plant cells (Wardrop et al 1996;Lowe et al 2003;Davey et al 2005;Pilarek and Szewczyk 2008;Sykłowska-Baranek et al 2014). …”

Modelling of a hybrid culture system with a stationary layer of liquid perfluorochemical applied as oxygen carrier

“…The second type of PFCsupported cell culture systems utilises a liquid perfluorinated oxygen carrier as a stationary layer in a hybrid culture system developed with two immiscible liquid phases: aqueous-based culture medium and hydrophobic PFC. Such culture systems are exceedingly suitable when a 3-D structure of fragile aggregates, spheroids or sheets of cells, as well as cell-consisting biomaterial-based implant constructs must be preserved to eliminate negative effects caused by hydrodynamic shear forces generated in typical intensively-stirred/agitated culture systems (Davey et al, 2005;Hillig et al, 2014;Pilarek et al, 2013;Pilarek et al, 2014). It should be emphasised that the data available in open literature for such a system are still rather limited, with most quantitative data just omitted.…”