Differentiating Microbial Contributions to Soil Protease Activity.

Soil protein degradation is accomplished through the activity of protease enzymes that are produced by microorganisms in response to their need for nitrogen and/or carbon and excreted into the environment. Soil was subjected to five treatments: (1) non-amended positive control, (2) cycloheximide (2 mg/g) to inhibit fungal protein synthesis, (3) streptomycin (4 mg/g) to inhibit bacterial protein synthesis, (4) both cycloheximide and streptomycin, and (5) a chloroform-fumigated negative control. Soils were placed in canning jars with tops equipped with septa for gas sampling and incubated at 25^oC in laboratory. There were three replicates of each treatment. Samples for analysis were taken at 0, 1, 2, 4, 8, 16, and 32 days for measurement of soil respiration and protease activity. Protease activity was measured using two assays: casein degradation (combined exo- and endo-peptidase activity) and leucine aminopeptidase (LAP). Respired CO₂ was used as an index of microbial activity. Positive control respiration rates declined from 34.6 to 13.9 ng CO₂-C g^-1 soil d^-1 during the 32 days of incubation as soil carbon availability decreased. Chloroform depressed respiration of microbial community by 62 to 77% compared to positive control, indicating that it effectively inactivated respiration of bacterial and fungal populations. The bacterial inhibitor had little effect on respiration, possibly because this is a fungal-dominated soil. Surprisingly, the fungal inhibitor increased respiration, perhaps because of overflow respiration from fungi that were blocked in protein biosynthesis, or because bacteria used carbon from fungi that were killed. Soil protease activity was also suppressed by chloroform, depressing LAP activity from 74.4 to 24.4 nmol MC g^-1 soil h^-1and casein degradation activity from 106.7 to 71.9 nmol tyrosine g^-1 soil h^-1. There was little effect of the antibiotics on protease activity; however, this may be because soil conditions were not conducive to protease production. It would be useful to confirm these results with other bacterial and fungal inhibitors and under conditions known to stimulate protease production.