G Peter van Walsum

Perkinsus marinus (Perkinsea) is an osmotrophic facultative intracellular marine protozoan responsible for “Dermo” disease in the eastern oyster, Crassostrea virginica. In 1993 in vitro culture of P. marinus was developed in the absence of host cells. Compared to most intracellular protozoan parasites, the availability of P. marinus to grow in the absence of host cells has provided the basis to explore its use as a heterologous expression system. As the genetic toolbox is becoming available, there is also the need for larger-scale cultivation and lower-cost media formulations. Here, we took an industrial approach to scaled-up growth from a small culture flask to bioreactors, which required developing new cultivation parameters, including aeration, mixing, pH, temperature control, and media formulation. Our approach also enabled more real-time data collection on growth. The bioreactor cultivation method showed similar or accelerated growth rates of P. marinus compared to culture in T-flasks. Redox measurements indicated sufficient oxygen availability throughout the cultivation. Replacing fetal bovine serum with chicken serum showed no differences in the growth rate and a 60% reduction in the medium cost. This study opens the door to furthering P. marinus as a valid heterologous expression system by showing the ability to grow in bioreactors. One-Sentence Summary Perkinsus marinus, a microbial parasite of oysters that could be useful for developing vaccines for humans, has been shown to grow well in laboratory equipment that can be expanded to commercial scale using a less expensive growth formula than usual laboratory practice.