<![CDATA[The development of microalgae-based biofuels through co-culture systems holds great potential; however, the scale-up process is often hindered by issues related to inoculum stability during storage. This study aimed to evaluate the effect of storage duration at -20 °C (for 2, 4, 6, 8, 10, and 12 weeks) on cell size and cell density of Chlorella vulgaris in a monoculture system compared with a co-culture system with an indigenous bacterial consortium (Bacillus paramycoides, Lysinibacillus fusiformis, and Delftia sp.). Optical density was measured using a UV-Vis spectrophotometer at 675 nm, while cell morphology was evaluated through microscopic observation in five fields of view. Statistical analysis using ANOVA revealed a significant decline in cell density over storage time (p < 0.05) throughout the 12 weeks in both systems. This structural preservation is presumed to be mediated by Extracellular Polymeric Substances (EPS) forming microalgae-bacteria flocs (MaB-Flocs) that serve as a protective physical buffer. However, viability assays conducted at week 4 showed no cell proliferation after thawing in either system, accompanied by distinct color degradation pathways. Therefore, storage at -20 °C without cryoprotectants is ineffective as a cryopreservation method and unsuitable for delaying cell density measurements, although co-cultivation effectively stabilizes cell morphology evaluations.]]>
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