This dissertation demonstrates the development and application of a series of innovative phenotyping techniques and approaches to investigate polyphosphate-accumulating organisms (PAOs) and their presence and functions in natural and engineered systems. As the key bio-agents in enhanced biological phosphorus removal (EBPR) which is regarded among the most economic- and environment- friendly approaches, PAOs have this unique ability to efficiently remove large quantities of phosphorus (P) from wastes and store as the intracellular polymer polyphosphate (PolyP) which has been hypothesized with great potential in sustaining the worldwide increasingly agricultural P demand. However, such practices altering P cycling for P reclamation to alleviate P shortage have been challenged with many current knowledge gaps in a more comprehensive understanding of the biochemistry, microbiology, and ecology of PAOs. To fill in some of these knowledge gaps, this work is designed in three studies to explore the different perspectives of PAOs related in the P recycle practice through the development and advancement of a series of innovative phenotyping techniques. First, a thorough assessment of the bioavailability of PolyP – the key P pool possessed by PAOs – to plants has been performed. Enabled through the examination of plant growth conditions and responses to PolyP, this study provides direct and fundamental evidence to support the PAOs-centered P recovery strategy. Second, the study leverages the high-throughput phenotyping technique fluorescence-activated cell sorting (FACS) and 16s rRNA sequencing to develop a novel holistic approach in deciphering the diversified phenotypic characteristics of PAOs, which can be directly linked to their relevance and contribution in P cycling. Third, the study explores the effectiveness of PAOs bio-amendments to plants, and in addition to revealing their impacts onto plants and rhizomicrobiome, through the newly-developed single-cell Raman spectroscopy (SCRS)-based phenotyping techniques, it is also made possible to probe the structure and functions of PAOs in rhizosphere. Collectively, this work offers a series of cutting-edge microbial phenotyping techniques, which enables the higher-resolution, more in-depth characterization of PAOs and other applicable microbes, paving the path for more comprehensively understanding their biochemistry, microbiology, and ecology, but also provides initial but critical evidence in supporting the practical utilization of PAOs towards sustainable agriculture to mitigate the global P crisis.