Background: Phosphonates are the main components in the global phosphorus redox cycle. Little is known about phosphonate metabolism in freshwater ecosystems, although rapid consumption of phosphonates has been observed frequently. Cyanobacteria are often the dominant primary producers in freshwaters; yet, only a few strains of cyanobacteria encode phosphonate-degrading (C-P lyase) gene clusters. The phycosphere is defined as the microenvironment in which extensive phytoplankton and heterotrophic bacteria interactions occur. It has been demonstrated that phytoplankton may recruit phycospheric bacteria based on their own needs. Therefore, the establishment of a phycospheric community rich in phosphonate-degrading-bacteria likely facilitates cyanobacterial proliferation, especially in waters with scarce phosphorus. We characterized the distribution of heterotrophic phosphonate-degrading bacteria in field Microcystis bloom samples and in laboratory cyanobacteria “phycospheres” by qPCR and metagenomic analyses. The role of phosphonate-degrading phycospheric bacteria in cyanobacterial proliferation was determined through coculturing of heterotrophic bacteria with an axenic Microcystis aeruginosa strain and by metatranscriptomic analysis using field Microcystis aggregate samples. Results: Abundant bacteria that carry C-P lyase clusters were identified in plankton samples from freshwater Lakes Dianchi and Taihu during Microcystis bloom periods. Metagenomic analysis of 162 non-axenic laboratory strains of cyanobacteria (consortia cultures containing heterotrophic bacteria) showed that 20% (128/647) of high-quality bins from eighty of these consortia encode intact C-P lyase clusters, with an abundance ranging up to nearly 13%. Phycospheric bacterial phosphonate catabolism genes were expressed continually across bloom seasons, as demonstrated through metatranscriptomic analysis using sixteen field Microcystis aggregate samples. Coculturing experiments revealed that although Microcystis cultures did not catabolize methylphosphonate when axenic, they demonstrated sustained growth when cocultured with phosphonate-utilizing phycospheric bacteria in medium containing methylphosphonate as the sole source of phosphorus. Conclusions: The recruitment of heterotrophic phosphonate-degrading phycospheric bacteria by cyanobacteria is a hedge against phosphorus scarcity by facilitating phosphonate availability. Cyanobacterial consortia are likely primary contributors to aquatic phosphonate mineralization, thereby facilitating sustained cyanobacterial growth, and even bloom maintenance, in phosphate-deficient waters.

Lake Taihu is the third-largest freshwater lake in China and has been suffering from cyanobacterial blooms for over two decades. The northern part of the lake, Meiliang Bay, is known to be at high risk of dense and sustained Microcystis blooms and toxins. This study aimed to investigate and record the annual and seasonal dynamics of toxic genotype, Microcystis morphospecies succession and microcystin variation. It also aimed to find out the underlying driving factors influencing the dynamic changes. Microcystin (MC) and the Microcystis genotype were quantified using HPLC and quantitative real-time PCR, respectively. Our study, over three consecutive years, showed that the pattern of morphospecies succession was seasonally distinct and annually consistent. During the same period in 2012, 2013 and 2014, the average MC were, on dry weight basis, 733 μg·g⁻¹, 844 μg·g⁻¹, 870 μg·g⁻¹, respectively. The proportion of toxic Microcystis accounted for 41%, 44% and 52%, respectively. Cell bound microcystin was found to correlate with the percentage of toxic Microcystis. Based on historical and current data, we conclude that annual bloom toxicity was relatively stable or possibly increased over the last decade.

Accurately quantifying associated microbes is essential to understand the interactions between microplankton and their associated microbes. Most DNA-based methods, such as high-throughput sequencing, primarily assess the ratio of target objects to references in microplankton samples. However, simple random sampling (SRS) of individuals may lead to deviations in quantifying these ratios at the population level if these characteristics are associated with the reference content of individuals. This study considered group analysis, which involves detecting k groups with n individuals in each group, as an alternative approach and used simulated data based on the detection of Microcystis populations to evaluate the accuracy of different sampling plans. Our results indicate that increasing the number of individuals in each group could reduce sampling bias and improve the accuracy of comparisons between populations. Group analysis could also minimize the impact of the detection limit. This study demonstrated that, when detection methods only provide the ratio of target objects to references, group analysis is more appropriate than SRS for characterizing microplankton populations. Group analysis can be used not only for detecting associated microbes but also for identifying ingested organisms or the biochemical composition of microplankton. Our results also demonstrate how in situ individual-level studies support ecological investigations.