One-pot chemo-enzymatic synthesis and one-step recovery of length-variable long-chain polyphosphates from microalgal biomass

Abstract

Phosphate, an essential ingredient in fertilizers and detergents used daily worldwide, is a finite resource that may be exhausted within 70 years, while improper phosphate waste disposal into aquatic environments will result in eutrophication. Despite some chemical-based methods, biological phosphorus removal using polyphosphate-accumulating organisms, such as microalgae, is a sustainable alternative to reclaim phosphate from wastewater before wastewater enters aquatic environments, preventing ecosystem damage while recovering the phosphate for industrial use. Moreover, polyphosphates have profound biological functions and biomedical applications, serving as an energy stock, drug delivery vesicles, coagulation factors, and antiviral agents depending on the length of the polyphosphate chain, showing inherent value in polyphosphate recovery. However, before this study, there were no sustainable and efficient approaches to synthesizing purified polyphosphates enriched with different lengths, which limited industrial and biomedical applications. Here, by leveraging the power of thermodynamic coupling and phase transitions, we established a one-pot, two-step multi-enzyme cascade (comprising creatine kinase and two polyphosphate kinases) to transform heterogeneous polyphosphate in microalgae biomass to insoluble long-chain polyphosphate 1300-mers, allowing for further purification in a single step. In the cascade reactions, introducing creatine as the high-energy P-shuttle enables controlled manipulation of the creatine kinase reaction direction via pH modulation, effectively circumventing the competition between the two polyphosphate kinase-mediated reactions. Finally, we optimized a thermo-digestion approach to transform polyphosphate 1300-mers into shorter polyphosphates enriched with a specific narrow length range. Therefore, the processes established here create a sustainable P bioeconomy platform to refine microalgal biomass for biotechnological use.