Quantification of Marine Picocyanobacteria on Water Column Particles and in Sediments Using Real-Time PCR Reveals Their Role in Carbon Export

ABSTRACT Picocyanobacteria are the most abundant primary producers in the ocean and play a fundamental role in marine carbon cycling. Quantification of picocyanobacteria on sinking particles and in sediments is essential to understanding their contribution to the biological carbon pump. We designed a primer set targeting the 16S-23S rRNA internal transcribed spacer (ITS) sequence of cyanobacteria and established a quantitative PCR (qPCR) method for quantifying the ITS sequence abundance. High-throughput sequencing confirmed that this primer set can cover broad diversities of marine picocyanobacteria and avoid amplification of other marine cyanobacteria such as Trichodesmium and Crocosphaera. Amplification efficiencies were slightly different when seven marine Synechococcus and Prochlorococcus strains were assayed. The qPCR results were comparable with flow cytometry for water samples. Using this method, we found that, in the dark ocean, picocyanobacterial ITS sequence abundances were 10 to 100 copies/mL in the size fraction of 0.2 to 3 μm, which were 1 to 3 orders of magnitude more abundant than on the >3-μm particles. We also found that picocyanobacterial ITS abundance in sediment ranged from 105 to 107 copies/g along two nearshore-to-offshore transects in the northern South China Sea. These results further explain the important role of picocyanobacteria in carbon export. Collectively, we provide a qPCR method quantifying the total abundance of marine picocyanobacteria on water column particles and in sediments. Moreover, this newly designed primer set can be also applied to investigate the community of picocyanobacteria via high-throughput sequencing. IMPORTANCE Picocyanobacteria are the most abundant primary producers in the ocean. However, quantification of picocyanobacteria on the sinking particles and in sediments remains challenging using flow cytometry or epifluorescence microscopy. Here, we developed a real-time PCR method to quantify picocyanobacteria using a newly designed primer set specifically targeting the 16S-23S rRNA ITS sequence of cyanobacteria. We showed that in the dark ocean, picocyanobacteria are 1 to 3 orders of magnitude more abundant in small particles (0.2 to 3 μm) than in larger particles (>3 μm). This result supports the important role of direct sinking free-living picocyanobacteria cells in the carbon export to deep ocean. We also found that the picocyanobacterial ITS sequence abundance were 105 to 107 copies per gram in sediments, suggesting significant accumulation of sinking picocyanobacteria in the benthic ecosystem. This qPCR method can be used to quantify the contribution of picocyanobacteria to the biological carbon pump.