The influence of time, storage temperature, and substrate age on potential soil enzyme activity in acidic forest soils using MUB-linked substrates and l-DOPA

Abstract

The purpose of this experiment was to evaluate whether soil storage and processing methods significantly influence measurements of potential in situ enzyme activity in acidic forest soils. More specifically, the objectives were to determine if: (1) duration and temperature of soil storage; (2) duration of soil slurry in buffer; and (3) age of model substrates significantly influence the activity of six commonly measured soil extracellular enzymes using methylumbelliferone (MUB)-linked substrates and l-dihydroxyphenylalanine ( l-DOPA). Soil collected and analyzed for enzyme activity within 2 h was considered the best measure of potential in situ enzyme activity and the benchmark for all statistical comparisons. Sub-samples of the same soil were stored at either 4 °C or −20 °C. In addition to the temperature manipulation, soils experienced two more experimental treatments. First, enzyme activity was analyzed 2, 7, 14, and 21 days after collection. Second, MUB-linked substrate was added immediately (i.e. <20 min) or 2 h after mixing soil with buffer. Enzyme activity of soil stored at 4 °C was not significantly different from soil stored at −20 °C. The duration of soil storage was minimal for β-glucosidase, β-xylosidase, and peroxidase activity. N-acetyl-glucosaminidase (NAGase), phosphatase, and phenol oxidase activity appeared to change the most when compared to fresh soils, but the direction of change varied. Likewise, the activities of these enzymes were most sensitive to extended time in buffer. Fluorometric MUB and MUB-linked substrates generally had a 3-day shelf life before they start to significantly suppress reported activities when kept at 4 °C. These findings suggest that the manner in which acidic forest soils are stored and processed are site and enzyme specific and should not initially be trivialized when conducting enzyme assays focusing on NAGase, phosphatase, and phenol oxidase. The activities of β-glucosidase, β-xylosidase, and peroxidase are insensitive to storage and processing methods.