Highly Effective Polyphosphate Synthesis, Phosphate Removal, and Concentration Using Engineered Environmental Bacteria Based on a Simple Solo Medium-Copy Plasmid Strategy

Microbial polyphosphate (polyP) production is vital to the removal of phosphate from wastewater. However, to date, engineered polyP synthesis using genetically accessible environmental bacteria remains a challenge. This study develops a simple solo medium-copy plasmid-based polyphosphate kinase (PPK1) overexpression strategy for achieving maximum intracellular polyphosphate accumulation by environmental bacteria. The polyP content of the subsequently engineered Citrobacter freundii (CPP) could reach as high as 12.7% of its dry weight. The biomass yield of CPP was also guaranteed because of negligible metabolic burden effects resulting from the medium plasmid copy number. Consequently, substantial removal of phosphate (P_i) from the ambient environment was achieved simultaneously. Because of the need for exogenous P_i for in vivo ATP regeneration, CPP could thoroughly remove P_i from synthetic municipal wastewater when it was applied for the "one-step" removal of P_i with a bench-scale sequence batch membrane reactor. Almost all the phosphorus except for that assimilated by CPP for cellular growth could be recovered in the form of more concentrated P_i. Overall, engineering environmental bacteria to overexpress PPK1 via a solo medium-copy plasmid strategy may represent a valuable general option for not only biotechnological research based on sufficient intracellular polyP production but also removal of P_i from wastewater and P_i enrichment.