## 4 Discussion
At the other end of the mission, sample return timelines may also be critical for some studies. One can expect a multi-day travel period from when the samples leave ISS until they are available for in-lab processing. Beyond the launch and landing time constraints, there is a fair amount of flexibility for sample processing during the mission. Overall mission length of the SpX CRS missions has been fairly consistent and range from 21-36 days, with an average of 28 days. Most experiments are likely to be conducted in a fraction of that time but they all need to be well choreographed, in terms of crew time and equipment availability, with the other research and priority activities being performed on the ISS. The combination of complex timing issues and the use of novel hardware and procedures requires significant planning and modeling beyond that usually employed for standard experiments. Although each of our payloads was largely developed independently, we embraced the opportunity to compare basic cellular characteristics of _C. albicans_ cultivated in microgravity across multiple missions (see Table 3 for a summary). The characteristics chosen for comparison are primarily those associated with virulence, including growth, cell and colony morphology, and antifungal resistance. In each of the payloads, yeast grew to a higher density when cultivated in microgravity. Surprisingly, the calculated generation time increased as the inoculum level rose, likely due to samples reaching a maximal density sooner, only to reside in stationary phase through the remainder of the growth period. Only in the FEP bags was the planktonic, ground-based, and generally accepted doubling time of 90-120 minutes approximated [51]. These data are limited to end-point analyses since kinetic studies were not performed; therefore, the contributions of rate of growth versus length of time in stationary phase cannot be distinguished. A potential secondary effect of yeast growth is the impact that metabolic activity might have on the surrounding microenvironment, amplified in microgravity due to low fluid shear and lack of convection and bulk flow. Further studies are required to determine whether the differences in growth between flight and terrestrial controls may be explained by a cellular impact on the surrounding microenvironment, including changes in the concentration of nutrients and waste products [34; 52]. In addition, _C. albicans_ produces significant levels of metabolic gas, such that the pressure within bioreactor units is substantially increased. The impact of such increased pressure on cell behavior has yet to be analyzed. That said, an increased cell density of nearly 10-fold was observed in the FPA hardware of Micro-14/FPA when compared to the Micro-6 FPAs. Whether the gas bubble incorporated into the Micro-14 FPAs impacted access to oxygen or provided a headspace to buffer the buildup of pressure is unclear. It is also worth noting that the mid-mission storage times and temperatures varied among the payloads and may have contributed to viability and ultimately cell density. Toinform the impacts of these variations, pre-flight, ground-based viability studies had been previously performed with yeast stored in water. Cells remain sufficiently viable during long term (~30 days) storage at ambient temperature, although viability was highest when stored at 12 \({}^{\circ}\)C and next highest at 4 \({}^{\circ}\)C (unpublished studies). From these results, it seems unlikely that the modest changes in storage temperature between flight and terrestrial samples contributed to the observed difference in cell density. These results also led to flexibility for the pre-growth storage temperature to simplify flight operations and alleviate the need for on-orbit temperature maintenance equipment, which is often in high demand. Unlike many cells, _C. albicans_ is fairly recalcitrant to temperature variations. As such, _C. albicans_ also retains reasonable viability when frozen in the absence of a cryopreservation agent.