Emerging tools for measuring and modeling the in situ activity of soil extracellular enzymes (original) (raw)

Soil enzymes in a changing environment: Current knowledge and future directions

Soil Biology and Biochemistry, 2013

This review focuses on some important and challenging aspects of soil extracellular enzyme research. We report on recent discoveries, identify key research needs and highlight the many opportunities offered by interactions with other microbial enzymologists. The biggest challenges are to understand how the chemical, physical and biological properties of soil affect enzyme production, diffusion, substrate turnover and the proportion of the product that is made available to the producer cells. Thus, the factors that regulate the synthesis and secretion of extracellular enzymes and their distribution after they are externalized are important topics, not only for soil enzymologists, but also in the broader context of microbial ecology. In addition, there are many uncertainties about the ways in which microbes and their extracellular enzymes overcome the generally destructive, inhibitory and competitive properties of the soil matrix, and the various strategies they adopt for effective substrate detection and utilization. The complexity of extracellular enzyme activities in depolymerising macromolecular organics is exemplified by lignocellulose degradation and how the many enzymes involved respond to structural diversity and changing nutrient availabilities. The impacts of climate change on microbes and their extracellular enzymes, although of profound importance, are not well understood but we suggest how they may be predicted, assessed and managed. We describe recent advances that allow for the manipulation of extracellular enzyme activities to facilitate bioremediation, carbon sequestration and plant growth promotion. We also contribute to the ongoing debate as to how to assay enzyme activities in soil and what the measurements tell us, in the context of both traditional methods and the newer techniques that are being developed and adopted. Finally, we offer our collective vision of the future of extracellular enzyme research: one that will depend on imaginative thinking as well as technological advances, and be built upon synergies between diverse disciplines.

Activation Energy of Extracellular Enzymes in Soils from Different Biomes

PLoS ONE, 2013

Enzyme dynamics are being incorporated into soil carbon cycling models and accurate representation of enzyme kinetics is an important step in predicting belowground nutrient dynamics. A scarce number of studies have measured activation energy (E a) in soils and fewer studies have measured E a in arctic and tropical soils, or in subsurface soils. We determined the E a for four typical lignocellulose degrading enzymes in the A and B horizons of seven soils covering six different soil orders. We also elucidated which soil properties predicted any measurable differences in E a. b-glucosidase, cellobiohydrolase, phenol oxidase and peroxidase activities were measured at five temperatures, 4, 21, 30, 40, and 60uC. E a was calculated using the Arrhenius equation. b-glucosidase and cellobiohydrolase E a values for both A and B horizons in this study were similar to previously reported values, however we could not make a direct comparison for B horizon soils because of the lack of data. There was no consistent relationship between hydrolase enzyme E a and the environmental variables we measured. Phenol oxidase was the only enzyme that had a consistent positive relationship between E a and pH in both horizons. The E a in the arctic and subarctic zones for peroxidase was lower than the hydrolases and phenol oxidase values, indicating peroxidase may be a rate limited enzyme in environments under warming conditions. By including these six soil types we have increased the number of soil oxidative enzyme E a values reported in the literature by 50%. This study is a step towards better quantifying enzyme kinetics in different climate zones.

Soil enzymology: classical and molecular approaches

Biology and Fertility of Soils, 2012

It is still problematic to use enzyme activities as indicators of soil functions because: (1) enzyme assays determine potential and not real enzyme activities; (2) the meaning of measured enzyme activities is not known; (3) the assumption that a single enzyme activity is an indicator of nutrient dynamics in soil neglects that the many enzyme activities are involved in such dynamic processes; (4) spatio-temporal variations in natural environments are not always considered when measuring enzyme activities; and (5) many direct and indirect effects make difficult the interpretation of the response of the enzyme activity to perturbations, changes in the soil management, changes in the plant cover of soil, etc. This is the first review discussing the links between enzyme-encoding genes and the relative enzyme activity of soil. By combining measurements of enzyme activity in soil with expression (transcriptomics and proteomics) of genes, encoding the relative enzymes may contribute to understanding the mode and timing of microbial communities' responses to substrate availability and persistence and stabilization of enzymes in the soil.

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Journal of Visualized Experiments, 2013

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, Nor or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, Nor or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample). Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil type and temperature can influence enzyme kinetics. Video Link The video component of this article can be found at http://www.jove.com/video/50961/ 1. By producing EEs, soil microbes decompose and transform polymeric organic matter into smaller soluble molecules, thereby liberating previously bound micro-and macronutrients, which allows plants and microbes to assimilate available nutrients from the soil. EEs have been studied for decades, primarily by measuring their activities in laboratory assays 2-4 , since it is very difficult to directly detect and quantify enzymes. Extracellular enzyme activity (EEA) is most strongly controlled by the concentration of enzymes and corresponding substrates. The abundance of different C-, N-, and P-degrading enzymes in soils is controlled by numerous factors including microbial biomass, community composition, substrate availability, microclimate, and stoichiometric demands 5,6. However, in situ EEAs within the soil environment are also affected by temperature 7,8 , the binding of enzymes to soil clays and humic properties 2 , and diffusion constraints 9 , which ultimately regulate the active enzyme pool, in terms of size, substrate availability, and turnover rates 10-12. Therefore, acknowledging in situ soil conditions is critical when using laboratory enzyme assays to interpret soil microbial function across different environmental sites. Many different classes of EEA can be quantified in laboratory assays using a variety of synthetic substrates (please refer to "List of Reagents Table" for more detail). Some protocols utilize substrates in assays that are coupled to a colorimetric reaction that can be detected with a spectrophotometer, while others, including the protocol we describe here, utilize substrates that are bound to a fluorescent moiety. Fluorescence EE assays are typically more sensitive (by an order of magnitude) than colorimetric assays (which use a chromogenic moiety linked with a synthetic substrate) 12-14. Sensitivity in EEA detection includes two aspects: one is related to the quantity of the compound of interest detected and the other related to the lowest detectable potential enzyme activity. Methods for colorimetric P-nitrophenol (pNP)-based assays can be

Persistent Activities of Extracellular Enzymes Adsorbed to Soil Minerals

Microorganisms, 2020

Adsorption of extracellular enzymes to soil minerals is assumed to protect them against degradation, while modifying their activities at the same time. However, the persistence of the activity of adsorbed enzymes remains poorly understood. Therefore, we studied the persistence of cellulase and α-amylase activities after adsorption to soil amended with various amounts (+1, +5, and +10 wt.%) of three typical soil minerals, montmorillonite, kaolinite, and goethite. Soil without mineral addition (pure soil), pure minerals, and pure dissolved enzymes were used as references. Soil mineral–enzyme complexes were prepared and then incubated for 100 days; temporal changes in enzyme activities were analyzed after 0, 0.1, 1, 10, and 100 days. The specific enzyme activities (activities normalized to protein content) and their persistence (activities relative to activities at day 0) were compared to enzyme activities in solution and after sorption to the control soil. Amylase adsorption to pure m...

Control of Soil Extracellular Enzyme Activities by Clay Minerals—Perspectives on Microbial Responses

Soil Systems

Knowledge of how interactions of clay minerals and extracellular enzymes (EEs) influence organic matter turnover in soils are still under discussion. We studied the effect of different montmorillonite contents on EE activities, using two experiments—(1) an adsorption experiment with a commercially available enzyme (α-glucosidase) and (2) an incubation experiment (10 days) where microorganisms were stimulated to produce enzymes through organic carbon (OC) addition (starch and cellulose). Soil mixtures with different montmorillonite contents were created in four levels to a sandy soil: +0% (control), +0.1%, +1%, and +10%. The potential enzyme activity (pEA) of four enzymes, α-glucosidase, β-glucosidase, cellobiohydrolase, and aminopeptidase, involved in the soil carbon and nitrogen cycle were analysed. The adsorption experiment revealed a reduction in the catalytic activity of α-glucosidase by up to 76% with increasing montmorillonite contents. However, the incubation experiment showe...

Soil Enzymes as Bioindicators of Soil Ecosystem Status

Applied Ecology and Environmental Research, 2015

A variety of methods were developed to measure soil biological activity. All these methods are not suited to produce generally accepted results, but they give relative information about the ecological status of soil. Soil enzymatic activity assays is only one way to measure the ecosystem status of soils. The technique is quite simple and produces reproducible results, and is nowadays of practical importance because the influence of agro-chemicals, industrial waste, heavy metals, as well as soil fertility management can be measured. Especially the search for urease inhibitor is of particular interest in order to reduce ammonia losses from soils. Soil enzymes have been reported as useful soil quality indicators due to their relationship to soil biology, being operationally practical, sensitive, integrative, ease to measure and described as "biological fingerprints" of past soil management, and relate to soil tillage and structure. The focus of this article is to provide a review of soil enzyme activity as a biological, process-level indicator for impacts of natural and anthropogenic activities on soils. This knowledge of soil enzymology can be applicable as bioindicator to human endeavour of ecosystem perturbation, agricultural practices and xenobiotic pollution.

Stoichiometry of soil enzyme activity at global scale

Ecology Letters, 2008

Extracellular enzymes are the proximate agents of organic matter decomposition and measures of these activities can be used as indicators of microbial nutrient demand. We conducted a global‐scale meta‐analysis of the seven‐most widely measured soil enzyme activities, using data from 40 ecosystems. The activities of β‐1,4‐glucosidase, cellobiohydrolase, β‐1,4‐N‐acetylglucosaminidase and phosphatase g−1 soil increased with organic matter concentration; leucine aminopeptidase, phenol oxidase and peroxidase activities showed no relationship. All activities were significantly related to soil pH. Specific activities, i.e. activity g−1 soil organic matter, also varied in relation to soil pH for all enzymes. Relationships with mean annual temperature (MAT) and precipitation (MAP) were generally weak. For hydrolases, ratios of specific C, N and P acquisition activities converged on 1 : 1 : 1 but across ecosystems, the ratio of C : P acquisition was inversely related to MAP and MAT while the ...

Methodologies for Extracellular Enzyme Assays from Wetland Soils

Wetlands, 2014

25 Measurement of extracellular enzymic activity in wetland soils can give an 26 indication of the ecosystems biogeochemical processes, and rates of nutrient and 27 carbon cycling. Analysis of these have allowed researches to gain an understanding of 28 the ecosystems' microbial ecology and how it can be affected by environmental 29 factors. Here we give a detailed description of the assays necessary to determine the 30 activity of a suite of key hydrolase enzymes and phenol oxidases. These enzymes 31 control the rates of decomposition and consequently the production of biogenic 32 greenhouse gases. Knowing the processes responsible for the breakdown of organic 33 matter is therefore essential if it becomes necessary to curb these emissions. Our 34 protocols allow for cost effective analysis of a large number of samples and provide 35 sufficient accuracy to determine differences between soil types. When coupled with 36 contemporary microbial techniques these enzyme assays permit entire biochemical 37 pathways to be determined, giving unparalleled knowledge on the processes involved 38 in wetland ecosystems. 39 40 41 42 43 44 45 46 47 48

Relationships Between Enzyme Activities and Microbial Growth and Activity Indices in Soil1

Soil Science Society of America Journal, 1983

Soil enzyme activities are often used as indices of microbial growth and activity in soils. Quantitative information concerning which soil enzymes most accurately reflect microbial growth and activity is lacking. Relationships between the activities of 11 soil enzymes and microbial respiration, biomass, viable plate counts, and soil properties were determined in surface samples of 10 diverse soils. Correlation analyses showed that alkaline phosphatase, amidase, a-glucosidase, and dehydrogenase activities were significantly (P < 0.01) related to microbial respiration as measured by CO 2 evolution in soils which had received glucose amendments. Phosphodiesterase, arylsulfatase, invertase, a-galactosidase, and catalase activities were correlated at the 5% level while acid phosphatase and urease activities were not significantly correlated to microbial respiration. There was no significant correlation between the 11 soil enzymes assayed and CO 2 evolution in the 10 unamended soils. Only phosphodiesterase and a-galactosidase activities were significantly (P < 0.05) related to microbial numbers obtained on some selective culture media. Alkaline phosphatase, amidase, and catalase were highly correlated (P < 0.01) with microbial biomass as determined by CO 2