Results Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue13.Nov2000:

Assays for b-glucosidase, b-xylosidase, b-NAGase and phenol oxidase enzymes were conducted at 208C in pH 5.0 acetate buffer, 50 mM, under previously determined substrate saturating conditions. For alkaline phosphatase, assays were conducted in pH 8.5 tris buffer 50 mM under previously determined substrate saturating conditions. In all cases there were three analytical replicates and duplicate controls. A more detailed description of the procedures used can be found in Sinsabaugh et al. 1994a. Activities for all enzymes were analysed monthly, except for phenol oxidase, which was analysed every two months. 2.4. Data analysis Data from the litter-bag samples were analysed to develop enzyme models establishing relationships between cumulative mass loss and cumulative enzyme activities Jackson et al., 1995. The first step in the analysis is to integrate the enzyme activities over time by multiplying the mean activity over each interval by the length of the interval. The values obtained are expressed as activity- day, where activities for all enzymes are expressed in m mol h 21 g 21 OM. Cumulative activity was calculated as the activity-day summed over all previous sample intervals. Simple linear regressions are performed relating initial mass loss as a function of cumulative activity-day for each enzyme and each size class and pond. Organic matter losses between treatments were compared using t-test …P , 0 : 05† : Enzyme activities among treatments were compared using Kruskal–Wallis tests, and Mann– Whitney tests were applied for a posteriori pairwise compar- isons, if appropriate. Relationships between different enzyme activities were determined by calculating Spear- man’s correlation coefficients Zar, 1996.

3. Results

3.1. Breakdown rates The physico-chemical characteristics of the two ponds differed considerably Table 1. Toro had higher alkalinity, pH and nutrient concentrations than Oro throughout the studied period. The results in Fig. 2 show a similarity between the two ponds in the percentages of total coarse and fine particulate organic matter CPOM, FPOM over the period of study. The percentage of total OM is similar between ponds with average values of 26 in Toro and 20 in Oro. However, these similarities are in terms of S. Alvarez, M.C. Guerrero Soil Biology Biochemistry 32 2000 1941–1951 1944 Table 1 Physico-chemical characteristics of Toro and Oro ponds. Values are means of all sampling dates, with ranges given in parentheses. TN ˆ total nitrogen; TP ˆ total phosphorous; SRP ˆ soluble reactive phosphorous Characteristic Toro Oro pH 7.28 8.4–6.37 6.4 6.91–6.03 Alkalinity meq l 21 3.33 4.66–1.646 0.75 0.961–0.48 Conductivity ms cm 21 2713 6130–731 211 380–139 Temperature 19.2 31.5–10 18.1 29–10.5 TN mg l 21 6.02 18.05–1.62 2.85 4.20–1.28 Nitrate mg l 21 4 20–0 Ammonium mg l 21 1108 4250–0 96.5 149–52 TP mg l 21 0.71 2.99–0.06 0.0975 0.26–0.02 SRP mg l 21 9.72 15.22–1.2 1.405 5.62–0 Fig. 2. Organic matter OM fractions of the two ponds over the sampling period; Coarse particulate organic matter — CPOM .1 mm; fine particulate organic matter — FPOM 0.063–0.5 mm. quantity but not quality. The coarse material in Toro pond was a heterogeneous mixture of roots of submerged macro- phytes, portions of Juncus spp., and Scirpus spp., seeds, and pellets from different herbivores deer, cattle. In Oro pond the coarse material consisted mainly of a mixture of leaf portions, wood fragments, seeds, stems, and bark of Euca- lyptus spp. Arthropod exuviae were also present. In both ponds the FPOM size fraction comprised fine amorphous particles. Mass loss of organic matter was linear for the two size fractions in both ponds during the studied period Fig. 3. Zero-order rate constants were calculated from the linear regression of OM remaining vs. time Table 2. Regres- sions were statistically significant …P , 0 : 01 ; n ˆ 14† : The highest rate of mass loss, 0.164 day 21 was observed in the coarse fraction at Toro pond. The lowest rate 0.019 d 21 was for the FPOM fraction in Oro pond. There were no significant differences in OM mass loss rates for CPOM between the ponds but there were signifi- cant differences in OM loss rates for FPOM …P , 0 : 001† : In general, activity patterns of enzymes associated with confined POM differed from those associated with in situ samples Figs. 4 and 5. 3.2. Enzyme activities Enzyme activities on the confined material in CPOM for Toro pond decreased with time Fig. 4. Enzyme FPOM activity values in Toro pond were relatively constant but for Oro pond more erratic Fig. 4. Phenol oxidase values in Toro were 10-fold greater than in Oro, for both CPOM and FPOM fractions Fig. 4E. In situ enzyme activities for the CPOM fraction in Toro pond showed two main activity peaks Fig. 5, one at 133– 162 days July, August, and the higher one at 312 days January coinciding with a consecutive flooding and drying period Fig.1. FPOM patterns remained constant over the study period. For CPOM material in Oro the pattern is less clear, but a decrease between days 101 and 160, when the pond dried up, can be observed, and then a sharp peak on day 310 when the pond became flooded Fig. 1. In general, activities were higher in Toro than in Oro pond for CPOM and maximum values were reached on day 310 124, 70, 23 and 456 mmol h 21 g 21 OM for b-glucosidase, b-xylosidase, b -NAGase and phenol oxidase, respectively. Phenol oxidase activities were again 10-fold higher in Toro than in Oro pond for both CPOM and FPOM fractions Fig. 5E. Statistically there were no differences between Toro and Oro ponds for b-glucosidase, b-NAGase and alkaline phos- phatase activities for both CPOM, FPOM and confined and in situ samples Mann–Whitney U-test, P . 0 : 1 for all treatments. b-xylosidase, and, especially, phenol oxidase activities did show statistical differences between ponds … P , 0 : 01 and P , 0 : 001 ; respectively. Phenol oxidase activities were over 10 times higher in Toro than in Oro pond. Within each pond, in situ b-glucosidase and b-NAGase activities were significantly …P , 0 : 001† higher than confined samples. b-xylosidase and phenol oxidase did S. Alvarez, M.C. Guerrero Soil Biology Biochemistry 32 2000 1941–1951 1945 Fig. 3. Organic matter OM vs. time for POM size fractions. CPOM .1 mm; FPOM 0.063–0.5 mm. Toro CPOM V; Toro FPOM B; Oro CPOM W; Oro FPOM × ; Toro CPOM lineal fitting — — ; Toro FPOM lineal fitting · · ·; Oro CPOM lineal fitting —; Oro FPOM linear fitting · · ·. Table 2 Linear regression statistics for relative mass loss with time for POM. CPOM .1 mm, FPOM 0.063–0.5 mm. n ˆ 14 for each size fraction and site Site Size fraction Slope r 2 P Toro CPOM 20.164 0.92 , 0.001 FPOM 20.041 0.42 , 0.05 Oro CPOM 20.118 0.71 , 0.001 FPOM 20.019 0.86 , 0.001 S. Alvarez, M.C. Guerrero Soil Biology Biochemistry 32 2000 1941–1951 1946 Fig. 4. Microbial extracellular activities over time associated with confined CPOM X and FPOM B at each site. Activity ˆ mmol substrate h 21 g 21 OM. X axis origin day 35 ˆ 22 April 1998. S. Alvarez, M.C. Guerrero Soil Biology Biochemistry 32 2000 1941–1951 1947 Fig. 5. Microbial extracellular activities over time associated with in situ CPOM X and FPOM B at each site. Activity ˆ mmol substrate h 21 g 21 OM. X axis origin day 35 ˆ 22 April 1998. not show differences between treatments …P . 0 : 1 in both cases. Between coarse and fine fractions b-glucosidase, b- xylosidase and b-NAGase showed significant differences … P , 0 : 001 for all treatments being higher in coarse than in fine fractions. Phenol oxidase activity was not signifi- cantly different between fractions …P . 0 : 1† : In Toro pond, b-glucosidase activity was significantly correlated with b-xylosidase …r ˆ 0 : 72 ; P , 0 : 001† ; b - NAGase …r ˆ 0 : 84 ; P , 0 : 001† and alkaline phosphatase … r ˆ : 42 ; P , 0 : 05† in both CPOM and FPOM fractions, in situ and in confined material. Significant correlations were also found between b-NAGase and b-xylosidase activities …r ˆ 0 : 52 ; P , 0 : 001† and b-NAGase and alka- line phosphatase activities …r ˆ 0 : 54 ; P , 0 : 001† : Phenol oxidase activity did not show any significant correlation with any other enzyme, except b-xylosidase …r ˆ 0 : 56 ; P , : 01† : In Oro pond, b-glucosidase activity showed significant correlations in both CPOM and FPOM fractions and in situ and confined material with b-xylosidase …r ˆ 0 : 30 ; P , : 05† ; b -NAGase …r ˆ 0 : 89 ; P , 0 : 001† and alkaline phos- phatase activities …r ˆ 0 : 37 ; P , 0 : 01† : Significant correla- tions were also found between b-NAGase and b-xylosidase … r ˆ : 35 ; P , 0 : 05† and b -NAGase and alkaline S. Alvarez, M.C. Guerrero Soil Biology Biochemistry 32 2000 1941–1951 1948 Fig. 6. Instantaneous mass loss rates for in situ POM estimated from enzymatic decomposition models. A Toro. B Oro. CPOM X; FPOM B; water depth W; water polyphenol concentration ; sediment polyphenol concentration ; eucalyptus leaves input .Lateral litter inputs in Toro are negligible. phosphatase activities …r ˆ 0 : 37 ; P , 0 : 001† : Phenol oxidase activity did not show any significant correlation with any other enzyme, except with b-xylosidase …r ˆ : 47 ; P , 0 : 05† : b -Glucosidase and b-NAGase activities were similar with higher values in CPOM fraction and in in situ samples, and with no significant differences between the ponds. b- xylosidase presented a slightly different pattern, with statis- tically higher values in Toro pond, and with no apparent differences between in situ and confined samples, although it showed more activity on the CPOM fraction. There were no differences between phenol oxidase activities in CPOM and FPOM fractions and in situ or confined samples, but there were significant differences between the ponds, with values much lower in Oro than in Toro pond. Alkaline phosphatase showed the least consistent pattern and did not appear to be related to particle size or site. 3.3. Enzyme decomposition models Linear regressions were calculated between cumulative enzyme activity-day and OM remaining for each combi- nation of enzyme size fraction and site in confined samples. All regressions were significant …P , 0 : 001† : The slope of the relative organic mass remaining vs. cumulative activity- day can be viewed as the apparent enzyme efficiency AEE of a particular enzyme in the degradation process. This slope is analogous to a zero-order rate constant with units of activity-day act-day 21 Sinsabaugh et al., 1994b. Following the procedures of Sinsabaugh and co-workers Sinsabaugh et al., 1994a; Jackson et al., 1995 a simple index model to incorporate all the enzyme data into one pool without the bias associated with the different ranges of activity was developed. The enzyme data were stan- darised to a 0–1 scale by dividing the activity recorded for a specific enzyme on a sampling date by the maximum value obtained for that enzyme during the study. Average enzyme activity for each sampling date was calculated by summing the relative activities of each enzyme and dividing the total by five the number of enzymes assayed. This mean activity was integrated over time to obtain estimates of cumulative activity-day. Linear regressions of organic mass remaining vs. average cumulative activity-day were performed. The slope obtained is the global AEE for each size fraction. For more details on the calculations see Jack- son et al. 1995. Apparent enzyme efficiencies for CPOM and FPOM were 21.87 and 20.085 act-day 21 for Toro pond and 20.805 and 20.0525 act-day 21 for Oro pond, respectively …r 2 ˆ : 99 ; P , 0 : 001 ; n ˆ 11 for all cases. The models generated for confined CPOM and FPOM from the litter-bag study were used to estimate mass loss rates for in situ POM. The enzyme activity data for each in situ sample cores were transformed to a relative scale as described for the confined samples, and the resulting values were averaged to calculate mean relative activity. Instanta- neous mass loss rates were calculated by multiplying this value by the apparent enzymatic efficiency for that size class: 21.87 and 20.085 act-day 21 for Toro pond and 20.805 and 20.0525 act-day 21 for Oro pond for CPOM and FPOM, respectively Fig. 6. By this calculation mass loss rates for in situ POM were higher for CPOM and FPOM in Toro pond in relation to Oro. Rates of FPOM decomposi- tion were much lower than CPOM loss rates. CPOM rates fluctuated throughout the sampling period. In Toro two clear peaks can be distinguished. The first coincided with the summer season and higher temperatures. The second peak came in January, when the pond became flooded again for a few days Fig. 6A. In Oro pond, a decrease in mass loss rates can be seen in July, a few weeks before the pond dried up, which also coincided with the maximum inputs of euca- lyptus leaves and fruits and minimum water depth. Polyphe- nol concentrations also peaked at this time Fig. 6B. Mass loss rates increased sharply in January, which coincided with a rainy period and, as in Toro, a brief period of flood- ing. Over the sampling period in situ organic mass loss rates averaged 20.667 and 20.036 day 21 in Toro and 20.261 and 20.023 day 21 in Oro for CPOM and FPOM, respec- tively. Based on these estimates the processing rates for in situ CPOM and FPOM, were 149 and 2778 days in Toro pond and 382 and 4347 days in Oro, respectively, compared with values of 578 and 2109 days in Toro and 778 and 5296 days in Oro pond determined by confined POM in bags.

4. Discussion