Chlorella vulgaris cold preservation (4∘C) as a means to stabilize biomass for bioreactor inoculation: A six-month study

Abstract

Chlorella vulgaris cells were maintained over six months (or tentatively) using three protocols: two-week subculturing (positive control), storage at 4^∘C, and simple abandonment (negative control). Cultures were monitored by their optical and cell densities over the trial period. Cells were characterized by their size, pigment profile, photosystem II status (OJIP test), electron transport rate assay (light curve), and lag phase duration when regrown. The abandoned cultures quickly showed cells deviating from their nominal state (increased size, a loss of their pigments, a negative alteration of their photosynthetic capacity, and an extended lag phase when inoculated into fresh medium). Frequent subculturing yielded reasonably stable performances. Yet, our experience showed that uncontrollable factors (human errors, lack of communication between teams) could expose the cultures to unfortunate incidents. 4^∘C preservation allowed the cells to have a constant size and a slightly increased, yet stable, pigment profile associated to a dark acclimation (+12 % total chlorophyll). Finally, regrowth tests demonstrated that 4^∘C preservation induces slightly improved performance (lag phase duration reduced by 9.5 %) than frequent subculturing. Those findings advocate for the use of 4^∘C preservation to reduce cell maintenance work and conserve a pool of cells in a similar state to be used as repeatable inoculum for larger-scale experiments while nullifying otherwise batch-to-batch variation effects. Subculturing work can be reduced from once every two weeks to once every six months at least.