Characterization of extracellular enzyme activities involved in organic matter decomposition and nutrient cycling in forest soils

Introduction

Forests cover roughly 31% of Earth's land surface and store an estimated 45% of terrestrial carbon, yet the biological machinery cycling that carbon through the soil remains poorly mapped in many regions [1] . Compared with extensive cataloguing of above-ground plant diversity, enzymatic processes controlling below-ground decomposition have received less attention in South American forest ecosystems [2] . Extracellular enzymes produced by soil microorganisms and plant roots are the main agents of organic matter breakdown [3] . They catalyze hydrolysis of complex polymers cellulose, proteins, and organic phosphorus compounds into molecules available for uptake [4] . Among the most commonly measured are β-glucosidase (carbon cycle), acid phosphatase (phosphorus cycle), urease (nitrogen cycle), and dehydrogenase as a general microbial activity indicator [5] . Their combined profiles serve as sensitive indicators of soil health [6] . Land use change is a major driver of enzyme alteration. Conversion of native forests to monoculture plantations has been linked to 20-55% reductions in key activities [7] . Argentina's Chaco region experienced rapid deforestation over recent decades, with native dry forest replaced by eucalyptus plantations and cropland [8] . But few investigations have tracked enzymatic consequences in the Chaco-Pampa transition zone [9] . Seasonal moisture also regulates enzyme production, with wet-dry cycles shifting microbial communities and expression patterns [10] . This research characterized six extracellular enzyme activities across three forest soil types in central Argentina over a full annual cycle, aiming to determine how land use and seasonal moisture affect nutrient cycling capacity [11,12] .

Material and Methods

Material

This research was conducted at the Environmental Biochemistry Laboratory, Department of Biological Sciences, Córdoba Institute of Applied Sciences, Córdoba, Argentina, from June 2023 to May 2024. Soil samples were collected bimonthly from nine plots (three sites × three replicates). At each plot, five cores (0-20 cm depth) were taken along a diagonal transect using a 5 cm auger and composited. Samples were sieved through 2 mm mesh within 24 hours, stored at 4°C for enzyme assays (within one week), and at -20°C for chemical analyses. All reagents were analytical grade from Sigma-Aldrich Argentina [13] . Methods β-Glucosidase and acid phosphatase were measured by incubating 1 g soil with p-nitrophenol-linked substrates at 37°C for 1 hour, read at 410 nm [14] . Cellulase used carboxymethylcellulose with DNS reagent at 540 nm after 24-hour incubation at 50°C [15] . Urease was assessed by ammonium release from urea (2 h, 37°C) using indophenol blue at 630 nm. Dehydrogenase measured TTC reduction to TPF (24 h, 37°C) at 485 nm [16] . Protease used casein hydrolysis (2 h, 50°C) with Folin-Ciocalteu at 700 nm. Organic carbon was determined by Walkley-Black oxidation, total nitrogen by Kjeldahl, and available phosphorus by Bray-1. Microbial biomass carbon was estimated by chloroform fumigation-extraction [17] . All measurements were triplicated. Data were analyzed by twoway ANOVA (land use × season), Tukey's HSD, Pearson correlations, and PCA using R v4.3.1 [18] .

Seasonal sampling conditions

Sampling spanned six bimonthly campaigns covering dry season (June-September 2023) and wet season (October 2023-March 2024), with final collection in May 2024. Soil moisture ranged from 8.3% during peak dry (August) to 27.6% during peak wet (January). Soil temperature at 10 cm varied between 9.2°C (July) and 24.8°C (January), recorded using portable TDR probes and thermocouples. Figure 1 shows each enzyme's proportional contribution. β-Glucosidase was largest at 28.4%, followed by acid phosphatase at 22.1%, indicating carbon and phosphorus cycling dominated.

Result

Comprehensive Interpretation

PCA separated soil types along axes explaining 71.3% of variance. PC1 (46.8%) reflected carbon-cycling enzymes, PC2 (24.5%) phosphorus-cycling. Pearson correlations showed organic carbon-β-glucosidase r = 0.84 (p<0.001), nitrogen-urease r = 0.78 (p<0.01), and microbial biomassdehydrogenase r = 0.81 (p<0.001).

Discussion

Higher enzyme activities in native Chaco forest align with reports from subtropical dry forests showing undisturbed ecosystems maintain more active microbial communities [1,3] . The 31-39% reduction under eucalyptus likely reflects lower quality leaf litter containing elevated phenolic compounds that slow microbial colonization [7] . The strong organic carbon-β-glucosidase correlation (r = 0.84) reinforces established links between substrate availability and hydrolytic activity. β-Glucosidase catalyzes the final cellulose breakdown step and responds directly to carbon input quality [5] . Native forest's highest values for both organic carbon and enzyme activity support a positive feedback between litter inputs and decomposition capacity. Seasonal moisture markedly affected all enzymes, with wet season values 18-47% higher. This matches findings from semi-arid forests where soil rewetting triggers microbial growth pulses [10] . Protease showed the largest seasonal contrast (47%), possibly because protein mineralization depends on moisture-mediated substrate diffusion [16] . PCA separation of land use types along functional axes suggests enzyme profiling can serve as a diagnostic tool for assessing soil degradation after forest conversion [9] .

Conclusion

References

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