Fluorometric Quantification of Polyphosphate in Environmental Plankton Samples: Extraction Protocols, Matrix Effects, and Nucleic Acid Interference

Abstract

Polyphosphate (polyP) is a ubiquitous biochemical with many cellular functions and comprises an important environmental phosphorus pool. However, methodological challenges have hampered routine quantification of polyP in environmental samples. We tested 15 protocols to extract inorganic polyphosphate from natural marine samples and cultured cyanobacteria for fluorometric quantification with 4=,6-diamidino-2-phenylindole (DAPI) without prior purification. A combination of brief boiling and digestion with proteinase K was superior to all other protocols, including other enzymatic digestions and neutral or alkaline leaches. However, three successive extractions were required to extract all polyP. Standard addition revealed matrix effects that differed between sample types, causing polyP to be over-or underestimated by up to 50% in the samples tested here. Although previous studies judged that the presence of DNA would not complicate fluorometric quantification of polyP with DAPI, we show that RNA can cause significant interference at the wavelengths used to measure polyP. Importantly, treating samples with DNase and RNase before proteinase K digestion reduced fluorescence by up to 57%. We measured particulate polyP along a North Pacific coastal-to-open ocean transect and show that particulate polyP concentrations increased toward the open ocean. While our final method is optimized for marine particulate matter, different environmental sample types may need to be assessed for matrix effects, extraction efficiency, and nucleic acid interference. Inorganic polyphosphate (polyP) is a linear polymer of three to several hundred phosphate residues that appears to be found in cells of all organisms (1). It is required for many cellular functions, including bacterial virulence (2-4), biofilm formation (4, 5), quorum sensing (4), competitive fitness in the environment (6), microbial stress responses (3,7,8), and survival during stationary phase (7, 9-11). PolyP can also act as a phosphorus (P) store in microbes. PolyP breakdown allows cells to survive during P limitation (12, 13), although some microbes transiently accumulate polyP upon nitrogen and P stress (8,14,15). Massive accumulation of polyP by certain bacteria sequesters dissolved P during biological wastewater treatment (16)(17)(18).While polyP is studied in a range of microbiological and environmental fields, the lack of easy quantitative methods has hampered progress. For example, polyP has very rarely been measured in the oceans despite increasing interest in the physiological responses of marine microbes to the ultralow P concentrations frequently found at sea and their biogeochemical implications (14,(19)(20)(21)(22)(23)(24)(25). PolyP has been found at nanomolar concentrations, comprising around 10% of total particulate P (26-28). PolyP also plays a role in redox cycling and geological sequestration of P (26, 27) and potentially in phytoplankton iron storage (29).Common analytical approaches have significant drawbacks. Phosphorus-31 nuclear magnetic resonance ...

Accumulation and enhanced cycling of polyphosphate by Sargasso Sea plankton in response to low phosphorus

“…We present the ratio of polyP to total particulate P (polyP:TPP) although it is not a quantitative measure of the absolute molar quantity of TPP that is composed of polyP because polyP:TPP > 1 in some instances (detailed discussion of the enzymatic polyP extraction method and comparison with the more-established NaOH-based extraction are discussed in ref. 23 and SI Materials and Methods). SRP, TPP, APase activity, and membrane lipids were measured to examine how patterns in polyP related to biogeochemical P pools and known metrics of P stress.…”

“…This view is supported by observed changes in SQDG:PG (see following paragraph) as marine heterotrophic bacteria are PG-rich and do not substitute lipids under P stress (1). PolyP was extracted using an enzyme-based method, and fluorometric measurements were calibrated against synthetic polyP (23), which were corrected for background sample fluorescence and matrix effects, giving a relative measure of polyP concentration that is expressed as nanoequivalents per liter (neq·L −1 ) of the standard. We present the ratio of polyP to total particulate P (polyP:TPP) although it is not a quantitative measure of the absolute molar quantity of TPP that is composed of polyP because polyP:TPP > 1 in some instances (detailed discussion of the enzymatic polyP extraction method and comparison with the more-established NaOH-based extraction are discussed in ref.…”

“…Particulate organic carbon was determined on a stable isotope ratio mass spectrometer after fuming the samples with concentrated hydrochloric acid. PolyP was determined fluorometrically after boiling and enzymatic digestion, accounting for background fluorescence and matrix effects (23).…”

Phosphorus sequestration in the form of polyphosphate by microbial symbionts in marine sponges

“…This effect inflated the signal by 5-12%. Fluorescence data were adjusted accordingly, and polyP concentrations were calculated based on the equation in the supplemental information of the study by Martin and Van Mooy (12). Total P was determined using standard chemical techniques (38) by the Nutrient Analytical Service laboratory (Horn Point Laboratory, University of Maryland Center for Environmental Science).…”

“…The amount of polyP in the tissues of three sponge species was determined by extraction and measurement of fluorescent emission with DAPI staining (12). To make direct comparisons, we calculated the proportion of P in the form of polyp to the total P (Table 1).…”