Short communicationMeasuring the effect of freezing on hydrolytic and oxidative extracellular enzyme activities associated with plant litter decomposition

- Freezing is a viable solution to storing litter samples prior to most EEA assays.
- Most carbon cycling EEAs tested were not affected by freezing.
- All oxidative EEAs tested were not influenced by freezing.
- Only N-acetyl-β glucosaminidase (NAG) responded to freezing.
- NAG activity increased by 35% following freezing.

Extracellular enzymes are the proximate drivers of biogeochemical cycling in ecosystems. In terrestrial environments, studies of extracellular enzyme activities (EEAs) in soils far outpace studies in substrates such as leaf litter. Despite recent methodological advances that have made EEA assays feasible for researchers conducting large-scale studies, there are still a number of methodological uncertainties that exist, particularly when working with less studied substrates (i.e. grassland leaf litter). These uncertainties are of particular importance when considering the logistics of sample processing after collecting samples in large numbers and/or from distant field sites. Studies of soil handling suggest that while assaying biochemical and molecular markers in fresh samples is ideal, that freezing samples is often a viable solution if the former is not feasible. Due to the inherent differences of litter and soil materials and associated microenvironments, we can draw only limited conclusions from the results of studies on sample handling using soil. As a result, we investigated the effect of two typical freezing temperatures (−20° and −80 °C) on subsequent EEAs in decomposing leaf litter material. We incubated litter material from six grassland plant communities for 12 weeks at 25 °C, then measured the EEA of five hydrolytic and two oxidative enzymes in fresh litter, and in litter that was frozen for four weeks at either −20° or −80 °C. Results indicate that freezing only affected the activity of N-acetyl-β glucosaminidase (NAG), an enzyme that was affected by freezing in prior soil studies. We observed an approximate 35% increase in NAG activity with freezing. The remaining six EEAs measured were not observed to be affected by freezing, and the level of freezing (i.e. temperature) did not influence any of the seven EEAs measured, suggesting that freezing is a suitable alternative when laboratory analyses on fresh litter samples are not logistically possible.