MUF and AMC substrate enzymatic analysis was measured using a Cytofluor

600TM (PerSeptive Biosystems, Inc., Framingham, MA) automated spectrofluorimeter

with KineticalcTM software at 360 nm excitation and 460 nm emission at 200 C. Assays

were performed using Coming 48-well culture plates in which 400 [iL of sample, 360

[IL of 10 mM Tris-HCI pH 8.5, and 40 [iL of substrate was added. Stock substrate

concentrations were 2000 [iM for MUF-P-D glucoside, 1000 [iM for MUF-phosphate,

and 6000 [iM for L-Leucine aminomethylcoumarin resulting in well concentrations of

100 [iM, 50 [iM, and 300 [iM, respectively. Each sample run was performed in

quadruplicate. Initial and final fluorescence measurements as well as measurements

every five minutes were taken during the 1 hour incubation. Changes in fluorescence

were determined by subtracting the initial from the final fluorescence. Graphs produced

from the readings taken every five minutes were analyzed to ensure that linear kinetics

was being observed. Concentrations of MUF and AMC released were calculated by the

application of standard curves to the initial and final fluoresences. The absolute

difference in concentrations yielded the substrate released during the incubation period.

 The effects of quenching on MUF and AMC substrates were determined in order to

account for fluorescence blocking or absorption effects caused by coloring, particle

suspension, humic matter, self-quenching, or other inhibitions in the suspensions. MUF

and AMC standards were placed in each sample suspension to determine the quench

percentage of each matrix. The final and initial fluoresences were then converted using

the appropriate quench percentage.

 Phenol oxidase and peroxidase analysis was performed by adding 2.0 mL soil

suspension to 2.0 mL 10 mM L-dihydroxyphenylalanine (L-DOPA) dissolved in 10 mM