Growth inhibition of dinoflagellate algae in shake flasks: Not due to shear this time!

Abstract

Large scale algae cultures present interesting challenges in that they exhibit characteristics of typical bacterial and animal cell cultures. One current commercial food additive, docosahexaenoic acid (DHA), is produced using the dinoflagellate algae, Crypthecodinium cohnii. Like animal cell culture, the perceived sensitivity of algae culture to hydrodynamic forces has potentially limited the agitation and aeration applied to these systems. However, the high density cultivation of C. cohnii required for an economically feasible process inevitably results in high oxygen demand. In this study, we demonstrated what first appeared to be a problem with shear sensitivity in shake flasks is most probably a mass transfer limitation. We subsequently demonstrated the limit of chronic and rapid energy dissipation rate, EDR, that C. cohnii cells can experience. This limit was determined using a microfluidic device connected in a recirculation loop to a stirred tank bioreactor, which has been previously used to repeatedly expose animal cells to high levels of EDR. Inhibition of cell growth was observed when C. cohnii cells were subjected to an EDR of 5.9 x 10(6) W/m(3) with an average frequency of 0.2/min or more. This level of EDR is sufficiently high that C. cohnii can withstand typically encountered hydrodynamic forces in bioprocesses. This result suggests that at least one dinoflagellate algae, C. cohnii, is quite robust with respect to hydrodynamic forces and the scale-up of process using this type of algae should be more concerned with providing sufficient gas transfer given the relatively high oxygen demand.

Growth and docosahexaenoic acid production performance of the heterotrophic marine microalgae Crypthecodinium cohnii in the wave‐mixed single‐use reactor CELL‐tainer

“…These shear forces were predominantly induced on the edges of the stirrer and by bursting bubbles on the free surface of the STR . In contrast, Hu et al claimed that C. cohnii can withstand the shear forces, which usually appear in STR cultivations, and also de Swaaf et al applied STRs for their cultivations. However, the results obtained in this study clearly underline the negative impact of shear forces on the growth and production performance of C. cohnii , which should not be neglected during process development.…”

“…Hu et al reported a specific oxygen uptake rate of 10 −7 mg cells −1 h −1 , which corresponds to 75 pmol cells −1 day −1 and exceeds the oxygen demand of mammalian cells in perfusion cultures, which reaches values of 5.5 pmol cells −1 day −1 . Morphological changes of the cells were reported under oxygen limitation .…”

Cultivation of Marine Microorganisms in Single-Use Systems

“…They observed that this damage is reversible and that cells undergo a recovery process in which they first begin to spin and then move in a straight line once the shear forces have ceased [64]. It was demonstrated that the algae are not harmed by shear stress that occurs in shake flasks [60]. However, Yeung et al showed that cell proliferation is inhibited by the occurrence of high shear stress but that the culture is able to recover [66].…”

“…Hu et al [60] estimated that a volumetric oxygen transfer coefficient (k L a) of at least 33 h -1 is necessary for the growth of C. cohnii cells. The bioreactor experiments in stirred bioreactors described in the literature maintained k L a values of approximately 600-800 h -1 in order to ensure a sufficient oxygen supply at lab scale [61].…”

“…As C. cohnii exhibits a high demand for oxygen [60], Ultra Yield TM flasks (UYF, Thomson Instrument Company, Oceanside, CA, USA) were used because they are characterized by comparably high k L a values of up to 400 h -1 [40]. A microscopic examination of the culture revealed a high number of oil droplets, which we believe to be an indicator of cell lysis.…”