Ocean alkalinity enhancement (OAE) is proposed as a potential tool to remove atmospheric CO2 and mitigate climate change. However, the effects of OAE on marine protozoa remain poorly understood. In this study, we conducted acute and acclimated experiments on two heterotrophic nanoflagellates, i.e., Cafeteria burkhardae and Paraphysomonas longispina, to investigate their responses to two substances (NaHCO3 and NaOH) at low (set ~2,600 µmol L-1) and high (set ~4,000 µmol L-1) levels, respectively. Our results showed that the two species had similar negative reactions under acute exposure. However, the two species showed different tolerances after acclimation. The growth rates of C. burkhardae decreased under all OAE treatments, while its reactive oxygen species accumulated only at the high OAE level. The low-level OAE treatments had no significant effects, but high-level OAE was more adverse for P. longispina, which grew more slowly and grazed more, indicating lower growth efficiency. Underlying transcriptomic mechanisms were only analyzed for low-level OAE, which were consistent with the physiological responses. Replication and repair and metabolism-related pathways were significantly inhibited, with translation-related pathways stimulated in C. burkhardae. For P. longispina, in addition to translation-related pathways, replication and repair, and metabolism pathways were significantly upregulated. Overall, our findings suggest the potential negative effects of OAE on marine protozoa, and the effects can vary depending on the species, level, and substances. Given the critical role of protists in marine ecosystems, these adverse effects raise important concerns about the broader implications of OAE for marine biodiversity and ecosystem stability.IMPORTANCEOcean alkalinity enhancement (OAE) represents a novel approach to mitigate climate change by increasing the ocean's CO2 sequestration capacity. However, the potential ecological and environmental impacts of OAE on marine microorganisms, particularly protozoa, remain poorly understood. This study investigates the responses of two heterotrophic nanoflagellates, Cafeteria burkhardae and Paraphysomonas longispina, to varying levels of OAE treatments using NaHCO3 and NaOH. Our findings reveal significant species-specific differences in tolerance and physiological responses, with implications for microbial community dynamics in marine ecosystems. By employing transcriptomic analysis, we uncover the underlying molecular mechanisms. Ultimately, our study informs the development of sustainable ocean-based climate solutions, emphasizing the importance of considering microbial ecology in environmental management and policy.
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