COMPARISON OF 2 METHODS FOR EXTRACTION OF ATP FROM SOIL

The main aim of this work was to investigate previously published repo rts that an acidic mixture of 0.67 M phosphoric acid, 20% dimethyl sul phoxide, 2 M urea, 20 mM EDTA, 0.75 mM adenosine and 32.5 mM Zwitterge nt (termed the PA reagent) extracted up to 7 times more adenosine 5'-t riphosphate (ATP) from soil than a reagent consisting of 0.5 M trichlo roacetic acid (TCA), 0.25 M phosphate and 0.10 M paraquat (termed the TCA reagent). Several extraction tests were performed using both fresh and air-dried soils at different soil-to-extractant ratios. The ATP c oncentrations in the soil extracts were determined by the fire-fly luc iferin-luciferase system. In 5 fresh U.K. arable soils the amounts of ATP extracted, corrected for incomplete extraction by measurement of t he recovery of an internal standard of added ATP (the spike), ranged f rom 1.29 to 7.80 and from 1.01 to 5.24 nmol ATP g(-1) soil, for the TC A and PA reagent, respectively. In air-dried soils the range was from 0.35 to 1.11 and from 0.35 to 1.24 nmol ATP g(-1) soil, respectively, for the TCA and PA reagent. Except for an acid soil, the amount of nat ive ATP extracted from the soils by the two reagents and uncorrected f or incomplete extraction was not significantly different within soils when a 1:5 soil-to-extractant ratio was used. However, the percentage recovery of added ATP as a spike was larger at higher soil-to-solution ratios with the TCA reagent but not with the PA reagent. The recoveri es of the spike with the TCA reagent were always lower than with PA re agent. However, soil ATP, corrected for incomplete extraction, was alw ays greater with the TCA reagent than with PA. The use of [C-14]ATP as a spike showed that no appreciable dephosphorylation of ATP added as the spike occurred with either reagents. The main difference between P A and TCA reagents was that with PA a constant and high recovery of sp ike ATP (ca. 90%) was obtained at all soil-to-solution ratios tested, while TCA gave higher recoveries of spike ATP at higher soil-to-soluti on ratios. Paradoxically, the amounts of native ATP extracted at each soil-to-solution ratio were generally similar between PA and TCA, alth ough increasing with increasing soil-to-solution ratio. Correction for incomplete extraction of the spike with the TCA reagent gave very sim ilar soil ATP concentrations while this was not the case with the PA r eagent. We suggest that this is the main difference between the two re agents. We can find no evidence to support other work which suggested that the PA reagent extracts more ATP from soil than the TCA reagent.