A rapid assay for soil urease in the absence of bacteriostatic agents has been developed. The method comprises incubation of soil with an aqueous or buffered urea solution, extraction of ammonium with 1 N KCl and 0.01 NHCl and colorimetric NH4
+ determination by a modified indophenol reaction. The method is characterized by high sensitivity and stability of the coloured complex formed. Measurements obtained by this method showed that no change in urease activity occurred when field-moist samples of soils were stored at −20°C for as long as 5 months. Air-drying of field-moist soil samples may lead to an increase in urease activity.

Short-term assay of soil urease activity using colorimetric determination of ammonium

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.

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

Soil phosphatases, particularly acid and alkaline phosphomonoesterases, have been extensively studied [see reviews by Ramirez-Martinez (Folia Microbiol 13:161–174, 1968); Speir and Ross (Soil phosphatase and sulphatase. Soil enzymes. Academic, London, pp 197–250, 1978); Malcom (Soil Biol Biochem 15:403–408, 1983); and Tabatabai (Soil enzymes. Methods of soil analysis. Part 2. Microbiological and biochemical properties. Soil Science Society of America, Madison, pp 775–833, 1994)] because they mineralise organic phosphorus (P) to inorganic P. The effects of agricultural and forest managements, pollutants and any environmental factor on phosphatase activities of soil cannot be adequately interpreted because the currently available enzyme assays do not discriminate between the contribution of phosphatases associated with active microbial cells and that of extracellular phosphatases stabilised by soil colloids. Despite multiple evidence indicating that phosphatases can be adsorbed by surface-reactive particles such as clay minerals or entrapped by humic materials, the visualisation of the extracellular phosphatases in the soil matrix has never been achieved because ultracytochemical tests combined with electron microscopy cannot locate enzymes in electron-dense minerals such as clays, or in soil components such as humic materials, that react with counterstainers such as OsO4.

Role of Phosphatase Enzymes in Soil

Microorganisms are able to utilize nitrogen (N) from a wide range of organic and mineral compounds. In this paper, we review the current knowledge about the regulation of the enzyme systems involved in the acquisition of N and propose a conceptual model on the factors affecting the relative importance of organic and mineral N uptake. Most of the N input into soil is in the form of polymers, which first have to be broken down into smaller units by extracellular enzymes. The small organic molecules released by the enzymes can then be taken up directly or degraded further and the N taken up as ammonium (NH4+). When NH4+ is available at high concentrations, the utilization of alternative N sources, such as nitrate (NO3−) and organic molecules, is generally repressed. In contrast, when the NH4+ availability is low, enzyme systems for the acquisition of alternative N sources are de-repressed and the presence of a substrate can induce their synthesis. These mechanisms are known as N regulation. It is often assumed that most organic N is mineralized to NH4+ before uptake in soil. This pathway is generally known as the mineralization-immobilization-turnover (MIT) route. An advantage of the MIT route is that only one transporter system for N uptake is required. However, organic N uptake has the advantage that, in addition to N, it supplies energy and carbon (C) to sustain growth. Recent studies have shown that the direct uptake of organic molecules can significantly contribute to the N nutrition of soil microorganisms. We hypothesize that the relative importance of the direct and MIT route during the decomposition of residues is determined by three factors, namely the form of N available, the source of C, and the availability of N relative to C. The regulation system of soil microorganisms controls key steps in the soil N cycle and is central to determining the outcome of the competition for N between soil microorganisms and plants. More research is needed to determine the relative importance of the direct and MIT route in soil as well as the factors affecting the enzyme systems required for these two pathways.

Pathways of nitrogen utilization by soil microorganisms - a review.

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.

Soil enzymology: classical and molecular approaches

Urease activity in soil is persistent for long periods under low water, low temperature, and sterile regimes, and it has been suggested that some form of enzyme-protective mechanism exists in soil.Dublin soil was sonicated in water and extracted by adding a mixture of salts. Urease activity is associated with the organomineral complex thus obtained and is resistant to the activities of proteolytic enzymes. Clay-free soil organic matter prepared subsequently by filtration also exhibits urease activity which is resistant to proteolysis. Models consisting of enzymes with bentonite and lignin were found to mimic this resistance to proteolysis. Models consisting of enzymes with bentonite and lignin were found to mimic this resistance to proteolysis.A model system is presented which suggests both the origin and location of soil ureases and a reason for their persistence in nature.

A. H. Pukite

Papers

Concerning the Location and Persistence of Soil Urease