A Deep-Sea Multi-Sequence Sampling System Integrating In Situ Microbial Filtration with Rapid RNA Stabilization

Rapid depressurization and warming during recovery can trigger stress in deep-sea microbes and accelerate RNA degradation. We developed a remotely operated vehicle (ROV)-oriented multi-sequence microbial sampler for 2000 m sampling (20 MPa, 2 °C) that integrates in situ filtration with immediate RNAlater injection (an RNA stabilization reagent), collecting up to 12 samples per dive. A Dirichlet sampling–B-spline–SVM framework was used to optimize the cam profile of the sequence trigger for robust actuation under geometric constraints and realistic tolerances in both manufacturing and assembly. Relative to the baseline 3-4-5 motion law, the optimized design reduces nominal peak driving torque by ~18–20% and lowers the maximum torque under tolerance perturbations; tests show a further ~10–25% reduction using a SiC ball–ZrO2 block pair versus a MoS2-lubricated titanium pushrod–ZrO2 block pair. A Darcy–Forchheimer porous-media computational fluid dynamics (CFD) model predicts earlier clogging on the lower membrane and a fast-to-slow RNAlater displacement process; greater membrane resistance mismatch delays 95% displacement and increases RNAlater loss. Simulations and Rhodamine B tests suggest an RNAlater consumption of 0.9 L per parallel filter (one membrane per side), and 20 MPa chamber tests confirm stable operation and membrane retrieval.