Application of para-nitrophenol (pNP) enzyme assays in degraded tropical soils

Enzyme activities have been used as indicators of soil quality and changes in biogeochemical function due to management or perturbations. The objective of this study was to answer a number of methodological questions regarding sampling schemes, sample handling recommendations, and assay procedures to facilitate the use of enzyme assays in the tropical highlands of East Africa. We used para-nitrophenol (pNP) based substrates for five enzymes: β-glucosidase, cellobiohydrolase, chitinase, acid phosphatase, and alkaline phosphatase. In the first experiment, we examined sampling procedures and compared the results of determining enzyme activities on a plot using composite or discrete samples. Composite samples usually had higher activities than the means of individual cores (P<0.05), but relative ranking of sites was the same if analyses were based on composite or discrete samples. In the second experiment, we examined the effects of storage time and conditions on enzyme activity. Enzyme activity degraded rapidly in frozen samples, but was better maintained in samples stored at 4 °C. Phosphatase and cellobiohydrolase activity declined after 14 days of storage, while the activity of the other enzymes remained close to the values of fresh samples for 28 or more days. In the third experiment, we examined the effect of the addition of an antiseptic, toluene, to prevent bacterial growth during the assay. We found no consistent toluene effect (P>0.4), probably because the assays were of short duration and microbial growth was minimized. Finally, we looked at the incubation time necessary to produce reliable results. Phosphatases, with relatively high activities could reliably be determined in 2 h incubations, but the other enzymes had much lower activities and required longer incubation times for reliable determination. For the enzymes we looked at, 4 h was a good standard time for determining the activity of even the lowest activity enzymes. The results of this study provide practical guidelines for applying these enzyme assays in the degraded tropical soils.