Storage stability of Bacillus licheniformis after Shijian-19 satellite flight

To evaluate the long-term stability of Bacillus licheniformis live bacteria capsules (BL) carried by the Shijian-19 satellite (SJ19) underground storage at -80 °C.

BL was randomly divided into three groups: a spaceflight group (FS) exposed to 13.5-day orbital flight aboard SJ19 in single-dose packaging, a matched-packaging ground control (GS1), and an original commercial ground control (GS2). Bacterial growth curves, logarithmic-phase colony counts (LCC), acid-base tolerance, biofilm formation, adhesion ability, and amylase activity were measured immediately after recovery and after storage at -80 °C for 6 and 12 months. Treatment group and storage time effects were analyzed by two-way ANOVA or the Scheirer-Ray-Hare test, as appropriate.

All groups exhibited similar overall growth trends during storage. FS showed. comparable maximum cell densities (MCD) to GS1 throughout the experiment. MCD decreased significantly in all groups at month 6 (p < 0.001) and recovered to near baseline levels by month 12. FS had a significantly longer lag phase than GS1 at 0 and 6 months (p < 0.0001), while all groups showed pronounced lag-phase shortening at 12 months (p < 0.0001), and the intergroup difference gradually disappeared. The maximum specific growth rate declined over time, with no significant difference between FS and GS1. LCC in FS remained consistently lower than in GS1, reaching significance at months 6 (p = 0.01) and 12 (p = 0.0003). LCC increased over storage in all groups. Regarding enzyme activities, FS showed consistently lower α-amylase (α-AL) activity than GS1 at all time points (p < 0.01), and α-AL activity was significantly reduced in all groups at 6 and 12 months. FS also exhibited lower β-AL activity than GS1 at 6 and 12 months (p < 0.0001). Spaceflight significantly and persistently enhanced biofilm formation and adhesion ability relative to baseline. FS showed significantly higher biofilm formation (p < 0.01) and adhesion ability (p < 0.0001) than GS1 at months 0 and 6, with no significant difference in biofilm formation at month 12. Biofilm formation increased at month 6 (p < 0.0001) and decreased at month 12 (p ≤ 0.0001) in both FS and GS1. Adhesion ability decreased at month 6 (p < 0.0001) and recovered at month 12 (p < 0.0001) in all groups.

Spaceflight is associated with remodeled growth metabolism and surface properties of BL, inducing growth retardation, decreased LCC, and inhibited α-AL activity, while enhancing biofilm formation and adhesion ability. During the 12-month storage, space-exposed strain exhibited time-dependent growth recovery, continuous α-AL reduction, and upregulation of biofilm/adhesion. This provides experimental evidence for evaluating space-stressed probiotics after orbital exposure and ground recovery.