High throughput method for extracting polyphosphates from diatoms

Abstract

Polyphosphates (polyP) are widely distributed in living organisms and have crucial cellular functions. Little is known about the physiological roles of polyP in algae, especially in diatoms. Therefore, we have developed a standardized method for polyP extraction and quantification from diatoms, using the freshwater model organism A. minutissimum and further marine diatoms to validate the method. Investigating the efficiency of polyP extraction methods, we found that a commercial DNA isolation kit yielded best results. The method is based on separation of polyP from lower molecular weight compounds using gel filtration spin columns. Moreover, we demonstrate that self-made gel filtration spin columns and extraction buffers can also extract polyP from A. minutissimum efficiently. Under defined conditions, samples spiked with polyP of chain-lengths between 45 to 700 Pi moieties consistently resulted in a recovery of more than 70% of the initially loaded polyP. PolyP was quantified with an ascorbate-antimony-molybdate assay that detects phosphates (Pi) by hydrolysis of a specific exopolyphosphatase (PPX). The method detects as little as 3 μM polyP in high throughput analyses using 96-well plates. This sensitive, rapid and straightforward method is ideal to characterize the physiological role of polyP in diatoms.