Mining robust in situ phosphorus-accumulating organisms via single-cell RACS-Culture for rational ecosystem engineering

Rational engineering of ecosystems is often hindered by the inability to rapidly identify, profile, culture and apply the microbes that underlie target metabolic activity in situ. Here, we developed an In-situ Metabolism driven Sorting, Culture and Augmentation (IMSCA) strategy via Raman-activated Cell Sorting coupled to single-cell culture (scRACS-Culture), and demonstrated it through the mining of in situ polyphosphate-accumulating organisms (PAOs) for wastewater treatment. Single-cell polyphosphate-accumulating activities in situ were quantitatively assessed directly from environmental samples via the polyphosphate band in Raman spectrum, revealing their remarkable distinction from those from pure cultures. Among cells with the highest in situ activities and then sorted for one-cell-one-well cultivation are Micrococcus luteum MCI5-8, which however shows very low activity as pure culture. This organism represents a new type of PAO due to its lack of anaerobic phosphate release, reliance on glycogen instead of polyhydroxyalkanoate as energy storage form, and incapability of denitrification. Process redesign based on these novel physiological traits showed that time- and location-specific introduction of MCI5-8 into actual wastewater elevated phosphorus removal efficiency from 45% to 89% in an anaerobic-anoxic-aerobic (AAO) reactor. Therefore, by label-freely profiling, sorting and cultivating individual cells based on in situ metabolism in a “screen-first culture-second” manner, IMSCA is a powerful and broadly applicable strategy for efficient bioresource mining and rational ecosystem engineering.